Polyphosphazenes for Medical Applications [2nd, completely revised and updated edition] 9783110654189, 9783110652536

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Polyphosphazenes for Medical Applications [2nd, completely revised and updated edition]
 9783110654189, 9783110652536

Table of contents :
Preface
Contents
Chapter 1. Synthetic procedures
Chapter 2. Degradable poly(organo)phosphazenes
Chapter 3. Nanomedicine
Chapter 4. Tissue engineering
Chapter 5. Opportunities and challenges
Abbreviations
Index

Citation preview

Ian Teasdale, Oliver Brüggemann, Helena Henke Polyphosphazenes for Medical Applications

Also of Interest Organophosphorus Chemistry. Novel Developments Keglevich (Ed.),  ISBN ----, e-ISBN ----

Organocatalysis. Stereoselective Reactions and Applications in Organic Synthesis Benaglia (Ed.),  ISBN ----, e-ISBN ----

Organoselenium Chemistry Ranu, Banerjee (Eds.),  ISBN ----, e-ISBN ----

Phosphorus Chemistry. The Role of Phosphorus in Prebiotic Chemistry Zhao, Liu, Gao, Xu,  ISBN ----, e-ISBN ---- Solubility in Pharmaceutical Chemistry Saal, Nair,  ISBN ----, e-ISBN ----

Ian Teasdale, Oliver Brüggemann, Helena Henke

Polyphosphazenes for Medical Applications 2 ED

Authors Assoc. Prof. Dr. Ian Teasdale Institute of Polymer Chemistry Johannes Kepler University Linz Altenbergerstrasse 69 4040 Linz Austria [email protected] Prof. Dr. Oliver Brüggemann Institute of Polymer Chemistry Johannes Kepler University Linz Altenbergerstrasse 69 4040 Linz Austria [email protected] Dr. Helena Henke Institute of Polymer Chemistry Johannes Kepler University Linz Altenbergerstrasse 69 4040 Linz Austria [email protected]

ISBN 978-3-11-065253-6 e-ISBN (PDF) 978-3-11-065418-9 e-ISBN (EPUB) 978-3-11-065291-8 Library of Congress Control Number: 2020935451 Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.dnb.de. © 2020 Walter de Gruyter GmbH, Berlin/Boston Cover image: everythingpossible / iStock / Getty Images Plus Typesetting: Integra Software Services Pvt. Ltd. Printing and binding: CPI books GmbH, Leck www.degruyter.com

Preface Polyphosphazenes are a family of polymers based on the repeat unit structure of phosphorus and nitrogen, connected by alternating double and single bonds. The phosphorus atoms are thus pentavalent and the remaining two substituents (usually organic) can be chosen from a vast selection to give poly(organo)phosphazenes with a broad spectrum of properties and hence a wide range of applications. Indeed, polyphosphazenes with a vast array of properties and suggested applications have been described in the scientific literature, predominantly pioneered by the group of H.R. Allcock since the 1960s. Of the many poly(organo)phosphazenes reported, those relevant for medical applications are particularly promising due to the unique and tunable properties for highly demanding and evermore complex applications. Synthetic polymer materials are now commonplace in medicine and can fulfill a host of functions, from fixation devices to wound healing and from pharmaceutical formulations to polymer therapeutics. The objective of this volume is to bring the reader up to date on the state of the art for poly(organo)phosphazenes designed specifically for use in medical applications. In doing so, we review the progress made in polyphosphazene preparation methods and review our present understanding of their essential properties. A further objective is in reviewing the latest developments in this ever-expanding field to highlight the main areas of strength and weakness and thus decipher the most hopeful future prospects for polyphosphazenes as biomedical materials. The book is divided into four main chapters, each chapter containing a short introduction to the topic, followed by the latest research highlights in that particular area. The aim of Chapter 1 is to bring the reader up to date with the current synthetic procedures available for poly(organo)phosphazenes. The reader is informed how structure–property relationships can be controlled to design novel poly(organo)phosphazenes with specific properties for the desired application, and how polymers with the required molecular weights and architectures can be prepared. Chapter 2 looks into the degradability and bioerodability of poly(organo)phosphazenes. The rate of hydrolytic degradation of poly(organo)phosphazenes can be tuned by the choice of organic substituent, such that the entire spectrum from longterm biostable to rapidly eroding polymers can be prepared. Special attention is given here to the degradation properties and how they can be controlled, since bioerosion and degradability are essential properties for many medical applications, especially those discussed in Chapters 3 and 4, namely nanomedicine and tissue engineering. In these two chapters, the structural characteristics, preparation techniques and application studies in medical fields are detailed, with a particular focus on the most recent and most promising applications. This includes their use as immunoadjuvants and for the stabilization and transport of proteins and DNA, as polymer therapeutics for targeted drug delivery, and their development as injectable hydrogels for controlled drug-release devices. Furthermore, a chapter is devoted to the use of poly(organo) https://doi.org/10.1515/9783110654189-202

VI

Preface

phosphazenes as degradable scaffolds for tissue regeneration. In Chapter 5, the most important features are summarized and a critical assessment is given into the current state of play and future prospects for polyphosphazenes in medicine. This volume should not only provide a useful and critical summary for researchers already working in the field or looking to enter the field, but it is also hoped that the content is of interest to those working in the biomedical fields in which these polymers can be applied, to give a basic understanding of the materials available and highlight the recent developments, possibilities and unanswered questions with regard to their use in medical applications.

Contents Preface

V

Chapter 1 Synthetic procedures 1 1.1 Poly(dichloro)phosphazene 1 1.2 Macromolecular substitution 3 1.3 Ring-opening polymerization 4 1.4 Chain growth polycondensation 6 1.5 Macromolecular architecture 10 1.5.1 P=N backbone branching 10 1.5.2 Grafting 13 1.5.3 Block copolymers 16 1.5.4 Self-assembly 18 1.6 Conclusion 21 References 22 Chapter 2 Degradable poly(organo)phosphazenes 27 2.1 Bioerodible polymers for biomedicine 27 2.1.1 Bioerodible solid biomaterials and polymer matrices 2.1.2 Water-soluble, degradable polymers 30 2.2 Poly(organo)phosphazene degradation 31 2.2.1 Side-group influence on degradation kinetics 32 2.2.2 Amino acid ester-derived polyphosphazenes 35 2.2.3 The effect of pH 38 2.2.4 Stimuli-responsive degradation 39 2.3 Degradable molecular-level hybrids 41 2.4 Blends of poly(organo)phosphazenes 43 2.5 Bulk versus surface erosion 45 2.6 Degradation product cytotoxicity 46 2.7 Conclusion 46 References 47

28

Chapter 3 Nanomedicine 53 3.1 Polyphosphazenes in immunology 54 3.1.1 Vaccine adjuvants and delivery systems 54 3.1.2 Polyphosphazene electrolytes as immunological adjuvants 3.1.3 Structure–activity relationships 57 3.1.4 Safety considerations 60

56

VIII

3.1.5 3.1.6 3.1.7 3.2 3.3 3.3.1 3.3.2 3.3.3 3.4 3.4.1 3.5 3.5.1 3.5.2 3.5.3 3.5.4 3.6 3.7 3.7.1 3.7.2 3.7.3 3.8 3.9

Contents

Immunological activity 61 Polyelectrolyte microsphere formulations 63 Alternative delivery routes 64 Protein delivery 66 Cationic polyphosphazenes and their polyplexes 67 Gene delivery 67 Gene silencing 70 Charged polyphosphazenes for enteral drug delivery 71 Controlled release from polyphosphazene matrices 72 Polyphosphazene-based drug depot devices 72 Polymer therapeutics 74 Macromolecular drug carriers 74 Polyphosphazene drug conjugates 75 Polyphosphazene carriers for photodynamic therapy 81 Colon-specific azo-based drug conjugates 82 Self-assembled micelles and polymersomes 84 Thermosensitive poly(organo)phosphazenes 87 Thermosensitive polymers 87 Thermosensitive polyphosphazene drug carriers 88 Injectable hydrogels 89 Theranostics 93 Conclusion 95 References 96

Chapter 4 Tissue engineering 107 4.1 Introduction to tissue engineering 107 4.2 Architecture of polyphosphazene scaffolds for tissue engineering 107 4.2.1 Formats 109 4.2.1.1 Linear polyphosphazenes 109 4.2.1.2 Cross-linked polyphosphazenes 112 4.2.2 Properties 116 4.3 Applications of polyphosphazene scaffolds in tissue engineering 120 4.3.1 Bone tissue engineering 121 4.3.2 Endothelial tissue engineering 126 4.3.3 Neural tissue engineering 128 4.4 Degradation of polyphosphazenes developed for tissue engineering 129 4.5 Conclusion 135 References 135

Contents

Chapter 5 Opportunities and challenges 139 5.1 From laboratory to clinic 5.2 Future prospects 140 References 141 Abbreviations Index

147

143

139

IX

Chapter 1 Synthetic procedures The polyphosphazene structure is based on an inorganic backbone of alternating phosphorus and nitrogen atoms (Figure 1.1). The inorganic backbone is responsible for the many unique features of polyphosphazenes, including its high flexibility, high thermal stability as well as hydrolytic degradability. The remaining substituents of the pentavalent phosphorus atoms are most commonly of an organic nature, leading to poly(organo)phosphazenes. These side groups are decisive in determining the resulting properties of the polymer. The properties can thus be systematically varied through a combination of substituents, resulting in a large number of different polyphosphazenes with a wide range of properties and hence applications [1].

R P R'

N n

Inorganic–organic hybrid polymers Figure 1.1: The inorganic polyphosphazene backbone can be substituted with a variety of organic substituents to give polymers with a broad spectrum of chemical and physical properties and access to advanced materials for a host of applications.

1.1 Poly(dichloro)phosphazene The most commonly applied route to prepare poly(organo)phosphazenes remains via the inorganic macromolecular precursor poly(dichloro)phosphazene, [NPCl2]n. Poly(dichloro)phosphazene comprises two highly labile chlorine atoms per repeat unit, which induces a synthetic flexibility by allowing the facile substitution of a whole host of organic side groups and thus the preparation of a wide array of physical and chemical properties. The [NPCl2]n backbone can be substituted by essentially any given nucleophile (Figure 1.2), including H2O, which produces hydroxyphosphazenes (with P–OH moieties), which cannot only lead to cross-linking via intermolecular condensation but is also a degradation intermediate (see Chapter 2). Furthermore, the by-product of this hydrolysis, HCl, would be expected to further accelerate backbone degradation. Hydrolysis is exceptionally rapid, with extensive crosslinking reported within days for solutions of [NPCl2]n stored in even extremely dry

https://doi.org/10.1515/9783110654189-001

2

Chapter 1 Synthetic procedures

Cl P N n Cl

H2O –HCl

OH P N n Cl

Cl P N m Cl –HCl

Cl P N m O P N n Cl

Crosslinking

Degradation

Figure 1.2: Hydrolysis pathway of poly(dichloro)phosphazene leading to degradation and crosslinking.

solvents [2]. Hence, the hydrolytic sensitivity of the precursor becomes a primary concern and indeed this conundrum has accompanied polyphosphazene synthesis since its very beginnings [1]. Thus, the reproducible synthesis of this hydrolytically sensitive precursor, as well as its characterization, stabilization and storage are major stepping stones in the development of commercially viable materials. Synthetic reproducibility for any application, not least for medical applications, where regulatory approval must be met, is of critical importance. Furthermore, direct chromatographic analysis of this hydrolytically unstable precursor is extremely difficult, with degradation and crosslinking occurring in the column, meaning usually only indirect analysis of substituted derivatives can be applied. To this end, Andrianov and coworkers have developed a stabilized route using diglyme (Figure 1.3). The precise nature of the stabilization effect is unclear, although it is feasible that either diglyme coordination of water and/or the stabilization of cationic degradation intermediates is responsible [2]. Using this method it is possible to store [NPCl2]n for several years, without any detrimental effects of hydrolysis and/or cross-linking being observed. Just as importantly, this stabilization procedure has allowed the direct analysis of [NPCl2]n via size exclusion chromatography [2], which may be important for procedure standardization. Indeed, good manufacturing practices have been developed for this precursor [3] and the method has been used for the preparation of polymers used in clinical trials [4]. Furthermore, the storage in diglyme enables direct macromolecular substitution without prior removal of the solvent.

1.2 Macromolecular substitution

3

Diglyme : THF, Vol/Vol 100 : 0

No Cross-Linking (4 years)

75 : 25

No Cross-Linking (4 years)

50 : 50

Cross-Linked

25 : 75

Cross-Linked

10 : 90

Cross-Linked

0 : 100 Cross-Linked 0

100

200

300

400

500

Cross-linking, hours Figure 1.3: Stabilization of poly(dichloro)phosphazene in diglyme. THF, tetrahydrofuran. Reproduced with permission from A.K. Andrianov, J. Chen and M.P. LeGolvan, Macromolecules, 2004, 37, 2, 414. ©2004, American Chemical Society [2].

1.2 Macromolecular substitution Once prepared, the [NPCl2]n precursor is then substituted to give (more) hydrolytically stable polymers (Figure 1.4). This macromolecular substitution is a relatively unique procedure and has a decisive influence on the properties of the polymers. As previously mentioned, in this sense the (problematic) hydrolytic instability of [NPCl2]n can be regarded as a double-edged sword, as it simultaneously facilitates the macromolecular substitution of the polymer backbone and hence the variety of

Cl

RNH2

P N Cl

n

Cl

–HCl

RONa

P N

n

NaCl

n

NHR

OR P

P N Cl

NHR

OR

N n

Figure 1.4: Most common routes for the macromolecular substitution of poly(dichloro)phosphazene.

4

Chapter 1 Synthetic procedures

poly(organo)phosphazenes that can be produced. The highly labile chlorine atoms can be readily replaced by a host of nucleophiles, in particular, amines and alkoxides. In this manner, a wide variety of organic side groups can be coupled onto the polyphosphazene backbone, and hundreds of poly(organo)phosphazenes have been reported with wide-ranging properties (see [1] for a summary of many of those reported up to 2003). This simple macrosubstitution would also lend itself in theory to high-throughput synthesis, with the possibility of preparing a library of polymers from a single [NPCl2]n chain [3]. Despite the high reactivity of the [NPCl2]n backbone, care must be taken to ensure complete substitution, which consists of multiple parallel substitution reactions on a single macromolecule. Incomplete chlorine replacement and thus residual P–Cl bonds would not only lead to structural irregularities and inconsistent polymer functionality but impact the degradation rates of the polymers and/or lead to cross-linking. The presence of residual chlorine atoms has been shown to considerably accelerate backbone degradation rates [8]. As this process is inherently irregular, the composition and stability of the resulting poly(organo)phosphazene is unpredictable and thus must be avoided. The use of “forcing conditions,” that is, an excess of the nucleophile, long reaction times and appropriate reaction conditions ensure the complete removal of chlorine atoms and thus reproducible polymers. Such reproducibility considerations apply in particular when producing polymers for medical applications, as the effect of this unpredictability could be highly detrimental.

1.3 Ring-opening polymerization The traditional and most widely used route to prepare high-molecular-weight (Mw) poly(dichloro)phosphazene is the thermally induced ring-opening polymerization (ROP) of hexachlorophosphazene [5]. This is most commonly carried out in the molten state under vacuum, in a sealed tube at 250 °C (Figure 1.5). In this regard, a host of variations have been attempted, with varying degrees of improvement over the basic technique (for a comprehensive review of these see [1]). Importantly, the basic procedure can also be scaled up to pilot plant or manufacturing level [1]. The preparation of [NPCl2]n in solution, with the added convenience that solutionstate synthesis offers, has also been achieved, for example, in 1,2,4-trichlorobenzene (TCB) at 214 °C [6]. Furthermore, [NPCl2]n can also be synthesized via a convenient direct solution state preparation from phosphorus pentachloride (PCl5) and ammonium chloride (NH4Cl) in the presence of sulfamic acid and calcium sulfate dihydrate [7] in refluxing TCB. Both the molten and solution-state synthetic routes provide high Mw [NPCl2]n and for both routes catalysts such as OP(OPh)3/BCl3 or BCl3 [8] can be added to achieve some Mw control. A convenient and widely applied route to control the Mw of [NPCl2]n is the use of anhydrous aluminum chloride (2–10%) [9].

5

1.3 Ring-opening polymerization

Cl

Cl P

n

Cl

Δ

N N Cl P P Cl N Cl Cl

P N n Cl

ROP

Δ

Cl

Cl P

N Cl P N Cl

N Cl P+

Cl



Cl

P

P

N N P Cl Cl P N Cl Cl

Cl

Cl

Cl Cl Cl Cl Cl Cl P N N P P + N N P Cl P N N Cl – Cl Cl

Cl

Figure 1.5: Commonly accepted mechanism for the ring-opening polymerization of hexachlorophosphazene.

Generally, the ring-opening procedure requires a high purity of the hexachlorophosphazene [NPCl2]3 monomer for reproducible results. High temperatures are also required [10] although an ambient temperature approach in 1,2-dichlorobenzene with a weakly coordinating anionic trialkylsilylium carborane [10] has also been carried out. A further inherent drawback of ROP is the tendency to produce branching and subsequently crosslinked substances at higher conversions. This could be caused by traces of moisture and thus the formation of hydroxyphosphazenes, but there is growing evidence that this is a polymerization-based phenomenon (Figure 1.6), that is, cannot be attributed purely to hydrolysis with the consequence that no amount of drying or purification will prevent this [2].

CI



+

N P N N

CI CI

CI P CI P CI CI

N P Δ

CI P N CI P CI CI

CI CI

N CI P N CI N P P N CI CI CI CI

CI P+ N P CI CI

CI CI P

N CI P CI

N CI

N

P N

CI



CI P CI

N

Figure 1.6: Proposed inherent cause of branching and (at higher conversions) cross-linking during the synthesis of [NPCl2]n via ROP [2].

6

Chapter 1 Synthetic procedures

Furthermore, despite still being the route able to prepare the highest Mw, ROP inherently produces polymers with broad dispersities (Mu/Mn) >2 due to its initiation mechanism, in which the formation of new chains can occur throughout. Although high Ð values are perfectly tolerable for many medical applications, for example, as inert biomaterials, the method is less suitable for some biomedical applications in which precise molecular size is often an essential property. Furthermore, advanced polymer architectures and macromolecular constructs cannot be readily attained via this method, due to the absence of end-group control, and hence the development of poly(dichloro)phosphazene with controlled Mw has been important in order to broaden the spectrum of available applications.

1.4 Chain growth polycondensation The development of a living polymerization route to poly(dichloro)phosphazenes is of importance for many future medical applications of polyphosphazenes as it facilitates the synthesis of [NPCl2]n with controlled Mw and opens the door to the preparation of advanced macromolecular constructs. Thus the conception and development of living cationic polymerization of phosphoranimines by Allcock and Manners [11, 12] can be regarded as a major advancement in polyphosphazene science. As shown in Figure 1.7, trichlorophosphoranimine (Cl3PNSi(CH3)3) is initiated with PCl5 and the addition of further monomer molecules proceeds via a living, chain growth polycondensation, which itself is a relatively rarely observed polymerization mechanism [13], with the elimination of trimethylchlorosilane ((CH3)3SiCl) until monomer conversion is complete. The reaction can be carried out in solution (usually CH2Cl2) at ambient temperatures, and the Mw can be controlled by the ratio of PCl5 to Cl3PNSi(CH3)3. Analysis of macrosubstituted derivatives shows a linear increase in Mw with respect to conversion, a clear indicator of a living polymerization [11] (Figure 1.8), and the Ð is generally low (Mw/Mn = 1.01–1.4). Furthermore, in contrast to the ring-opening route, branching of the [NPCl2]n is not generally observed, indeed the absence of reactions between the phosphazene cations with internal P–Cl bonds has been ruled out in small-molecule model studies [15]. Poly(dichloro)phosphazene obtained via a cationic polymerization method is a living polymer with cationic chain ends that can be used, for example, for controlled termination or reactions with another phosphoranimine and because the chain ends remain active, block copolymers can be prepared via

2 eq. PCl5 Cl3P N SiMe3

–Me3SiCl

Cl + – Cl P N PCl3 PCl6 Cl

n Cl3P N Si(CH3)3 –n Me3SiCl

Figure 1.7: Living cationic polymerization of Cl3PNSi(CH3)3 initiated by PCl5.

Cl + – Cl3P N P N PCl3 PCl6 n Cl

1.4 Chain growth polycondensation

(a)

7

(b) Interior [Cl2P=N]n

Monomer consumption

1.0

44

Time after initi ati

37

on (m

51

in)

57

Remaining monomer

Adjacent PCl2 groups to PCl3+

0.8 0.6 0.4 0.2 0.0 0 10 20 30 40 50 60 Time after monomer addition (min)

24 17 9 Initiation with PCl5

3 Monomer

0 –10 –20 –30 –40 –50 –60 –70 δ (ppm)

(c)

1

In (Mt/M0)

30

–1

0

–2 –3 –4 0

10 20 30 40 50 Time after monomer addition (min)

Figure 1.8: PCl5 initiated polymerization of Cl3PNSi(CH3)3 in CH2Cl2 monitored by 31P{1H} nuclear magnetic resonance spectroscopy shows consumption of the monomer accompanied by linear chain growth (a). The amount of remaining monomer can be plotted (b), showing its consumption and a plot of ln(Mt/M0) over time (c) showing a linear relationship indicating the living nature of the polymerization. Mt, monomer concentration at time t; M0, initial monomer concentration. Reproduced with permission from Sandra Wilfert in “Novel and Functional Polyphosphazenes for Biomedical Applications,” Johannes Kepler University, Linz, Austria, 2014 [PhD Thesis]. ©2014, Johannes Kepler University [14].

sequential addition [16]. The living cationic polymerization pathway also allows access to a variety of polymer architectures (see Section 2.5) The precise mechanism is still a matter of investigation, although it would appear from both experimental observations of the monomer to initiator (M:I) ratios to Mw attained [11], as well as from model studies [15], that two PCl5 molecules are needed to form the initiating species with a PCl3+ cationic end group and PCl6− as the counterion [17]. Other initiators and solvents have also been reported [11], but PCl5 in CH2Cl2 appears to offer the best combination in terms of reaction kinetics and initiator solubility. The PCl6− counterion, however, has also been reported to initiate chain growth of Cl3PNSi(CH3)3 and thus could potentially cause competing chain growth [15]. Furthermore, bidirectional chain growth has also been observed due to delocalization of the charge on the propagating [Cl3PN–PCl3]+ species. As the two chain ends may react at different rates, this could lead to broad dispersity [15] and, furthermore, the precise control of molecular architecture is hindered. Trialkoxyphosphoranimines can be used to ensure monodirectional growth, although the effectiveness appears heavily dependent on the nature of the R groups [18] and more recently similar monoend-capped initiators of the type [R3P = N = PCl3][X] (X = Cl, PCl6) have been shown to

8

Chapter 1 Synthetic procedures

ensure monodirectional chain growth [19]. Similarly, the use of chlorinated phosphine groups, R3PCl2, known to exist in their ionic form [R3PCl][Cl] in CH2Cl2 [20] can be used to initiate the polymerization [21–23] (Figure 1.9) ensuring monodirectional growth and thus can be used for the control of molecular architecture (see Section 1.5). One limitation to this chain growth polycondensation method is the loss of control at higher M:I ratios (higher n). The origin of this could be competing initiating species at lower concentrations of PCl5 and an upper limit has been experimentally observed at approximately n = 100, above which it becomes difficult to precisely control Mn and Ð [11, 24]. To achieve polymers with a much higher Mw, a solvent-free bulk polymerization of Cl3PNSi(CH3)3 with either PCl5 [11, 12] or R3PCl2 [25] can yield polymers with n = 400. Another disadvantage is that the living cationic routes discussed are reliant on the prior synthesis of the trichlorophosphoranimine monomer Cl3PNSi(CH3)3, and thus the reliable preparation of this air and moisture-sensitive monomer is crucial to the polymerization route. A relatively high-yielding monomer preparation can be achieved (up to 80%) with good purity [26]. The complex preparation of this monomer remains, however, the major bottleneck in terms of up-scaling of the preparation of polyphosphazenes via living cationic polymerization. The subsequent polymerization is a facile ambient temperature procedure, but the repeated vacuum distillations required to prepare Cl3PNSi(CH3)3 in sufficient purity are not conducive to industrial manufacture. In order to circumvent the tiresome preparation of Cl3PNSi(CH3)3, a one-pot in situ synthesis of poly(dichloro)phosphazene directly from PCl3 has also been developed [27]. Although some loss in control of Ð is unavoidable, it is still superior to that of a ring-opening procedure and may offer significant advantages in terms of future scale-up of the preparation of [NPCl2]n via chain growth polycondensation. An alternative route to [NPCl2]n via the thermal condensation polymerization of Cl3P=(O)Cl2 is also possible, although this requires high temperatures and produces [NPCl2]n with relatively broad Ð [28]. Poly(organo)phosphazenes can also be achieved directly, without the need for the [NPCl2]n precursor, via an anionic polymerization of N-silylphosphoranimines with fluoride ion initiators at 180 °C [29, 30], although without quite achieving the control of the cationic route. Alternatively, it has been shown that it is possible to prepare poly(alkyl/aryl) phosphazenes, with a P–C bond connecting the organic component, directly from N-silylphosphoranimines using (usually thermal) condensation methods [31]. Such poly(alkyl/aryl)phosphazenes differ significantly in that they contain direct carbon linkages on the phosphorus atom, not the oxygen or nitrogen atom most commonly attained from the macrosubstitution of [NPCl2]n, and as such can be considered isoelectronic analogues of silicones [32]. Although the R groups chosen here are somewhat limited compared with the macrosubstitution route, a large selection of poly (alkyl/aryl)phosphazenes have been reported [32–34]. Furthermore, it has been shown

1.4 Chain growth polycondensation

9

ROP Cl Cl P N N P P Cl Cl Cl N Cl

250°C ROP

Cl P N n Cl

Mw/Mn = 2–7, n = 10,000

Thermal condensation R

n RO P N SiMe3

180°C

R P N n R

Mw/Mn = 1.5–2.5, n = 200

250°C

Cl P N n Cl

Mw/Mn = 1.5–2.5, n = 600

125°C TBAF

R P N n R

R

Cl n Cl P N POCl2 Cl Anionic condenzation R

n RO P N SiMe3 R

Mw/Mn = 1.5–2.5, n = 200

Cationic polymerization

RT

n Cl3P N SiMe3

PCl5

RT

n Cl3P N SiMe3

[R3PCl][Cl]

RT

n Cl3P N SiMe3

[R3P=N=PCl3][PCl6]

R n Br P N SiMe3 R

RT (MeO)3P

Cl P N n Cl

Mw/Mn = 1.01–1.4, n = 100

Cl P N n Cl

Mw/Mn = 1.01–1.4, n = 100

Cl P N n Cl

Mw/Mn = 1.01–1.4, n = 100

R P N n R

Mw/Mn = 1.2–1.8, n = 100

Figure 1.9: Summary of the major synthetic routes to polyphosphazenes, with approximate values for the maximum chain lengths (n) normally prepared by this route and reported values for Ð (M /M ). w n RT, room temperature; TBAF, tetrabutylammonium fluoride.

10

Chapter 1 Synthetic procedures

that simple poly(alkyl/aryl)phosphazenes can undergo further macromolecular functionalization, for example, electrophilic substitution of aromatic substituents [35] or acidic methyl groups attached to the phosphorus can undergo deprotonation with nBuLi to give essentially macromolecular organolithium reagents which can undergo organometallic additions to give a variety of functional groups [36]. Phosphine azides have also been used but the explosive nature of some intermediates renders this a less attractive route [37]. More recently, the ambient temperature polymerization of P-bromo(alkyl/aryl)phosphoranimines, initiated by organic phosphites, has been shown to be an effective route to poly(alkyl/aryl)phosphazenes, ensuring monodirectional chain growth and relatively narrow Ð [38, 39].

1.5 Macromolecular architecture 1.5.1 P=N backbone branching Organic-based dendrimers using hexachlorophosphazene as a multiplying linker are relatively well known [40], exploiting the six functional groups of the phosphazene core to multiply functionality and reduce the steps required for high generation dendrimers. However, tailoring the polyphosphazene (P=N) backbone is an altogether more difficult task and to the best of the author’s knowledge, no known controlled branching of the P=N backbone has been achieved. A phosphazene “dendrimer” has been prepared via living cationic polymerization [41], although elegant, this chemistry is essentially reliant on the polyamidoamine (PAMAM) core to provide the structural dendritic basis (Figure 1.10) and is effectively a PAMAM-grafted-polyphosphazene. To achieve this, trialkoxyphosphoranimines are reacted with one equivalent of PCl5 to create the initiating species, which upon the addition of Cl3PNSi(CH3)3 forms a polymer with a living chain end. This chain end can be terminated with a previously prepared phosphoranimine-capped polymer; in this case, a PAMAM dendrimer. This chemistry is also the basis for a number of organic–inorganic block copolymers (see Section 1.5.3). A triarmed star polymer has also been prepared from a core functionalized with three phosphoranimines [42], although this divergent approach appears to be less widely applicable [18] than the quenching procedure performed for the synthesis of the dendrimers. The development of the phosphine-mediated polymerization has opened the door to star-shaped polymers, where polyphosphazene arms are grown from a central core (Figure 1.11). To achieve three-arm stars, 1,1,1-tris(diphenylphosphino)methane is used (Figure 1.11 (a)) [43], while the phosphazene trimer substituted with 3-(diphenylphosphino)-1-propanamine yields a hexadiphenylphosphine subsequentially functioning as the core of a six-arm star [44]. These star-shaped polyphosphazenes substituted with propargylamine are reacted with 1-thioglycerol in a thiol-yne photoreaction to further increase the number of functional groups per repeat unit to 8 [44].

Figure 1.10: The synthesis of dendrimeric structures with poly(organo)phosphazene side-arms. DAB, diaminobutane; PN, polyphosphazene. Reproduced with permission from S.Y. Cho and H.R. Allcock, Macromolecules, 2007, 40, 9, 3115. ©2007, American Chemical Society [41].

1.5 Macromolecular architecture

11

Figure 1.11: Synthesis of three-arm stars (a) and star-branched molecular brushes (b). Schematic representation of the intricate structure of these polymers (c). Reproduced from H. Henke, S. Posch, O. Brüggemann, I. Teasdale, Macromolecular Rapid Communications, 2016, 37, 769 © 2016 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim [43].

(c)

(b)

(a)

12 Chapter 1 Synthetic procedures

1.5 Macromolecular architecture

13

Poly(dichloro)phosphazene can also be substituted with this diphenylphosphine amine, resulting in a macroinitiator, where polyphosphazene arms can be grown from a polyphosphazene backbone, yielding intricate structures of star dendritic molecular brushes, and up to 30,000 end groups on one macromolecule (Figure 1.11(b) and (c)) [43], which might be increased even higher with the above-mentioned thiolyne chemistry. Very recently, a method has been developed to functionalize these three- and six-arm stars at the ω-end of each polymer chain via end-capping of the living chain ends with vinyl group bearing N-organophosphoranimines achieved by azidation of 4-(diphenylphosphino)styrene (Figure 1.12) [45].

1.5.2 Grafting The simplest manner to alter the macromolecular architectures of poly(organo)phosphazenes is through macromolecular addition, that is, grafting of structured polymers onto the P=N backbone. Examples here include decorating of [NPCl2]n with multiple mono-end-functionalized oligomers of an organic polymer (Figure 1.13) [24]. Since the backbone of [NPCl2]n already possesses two substitutable groups, the density of branching is inherently high, further increasing this can effectively give highly branched polymers with a large number of end groups [24], analogous to dendronized structures, recently described as barbwire bottlebrushes [46]. If both the [NPCl2]n and side groups are prepared via living polymerization, then controlled architectures with defined dimensions can be attained. With the rapid expansion in controlled radical polymerization chemistry, for example, atom-transfer radical polymerization (ATRP) [47], it is clear that combinations with the inorganic polyphosphazene backbone, and its many unique properties, can add extra dimensions and multiply the opportunity for new hybrid materials. As the inorganic component in such polymers is low, often below 5%, the resultant polymers often possess the solution, chemical and biological properties of the attached organic component and can, in effect, be viewed as highly branched versions on an inorganic (potentially degradable) backbone. A grafting-to approach, whereby end-functionalized polymers are grafted onto a polyphosphazene backbone, could pose steric difficulties for longer side chains, particularly as it is paramount that the [NPCl2]n is completely substituted. A grafting-from approach, however, allows the grafting of longer side chains. A recent example is the functionalization of [NPCl2]n with tertiary bromide moieties and subsequent ATRP, with the dual functionality of the repeat units leading to extremely dense molecular brushes of various organic polymers, including polystyrene (PS), poly(tert-butyl acrylate) and poly(N-isopropylacrylamide) (Figure 1.14) [48]. A variety of similar molecular brush-type polymers, for example, poly(methylphenylphosphazene)-graft-polymethylmethacrylate [49] and poly(methylphenylphosphazene)-

PPh2

N CI P CI N n PCI4

Ph2P

NHNH P N N N P P NH H N HN HN PPh2 N CI P CI N n PCI4

CI P N P N nPCI4 Ph2 CI

RHN

RHN

N P n N P NHR Ph N P RHN NHR Ph

Ph SiMe 3 P N Ph

PPh2

N RHN P NHR N n RHN P NHR RHN N Ph P Ph

Ph2P

NHR Ph NHR NHR P N P N nP N P Ph2 NHR NHR Ph

Ph

P

Ph

PPh2 N RHN P NHR N n RHN P NHR RHN N

NHNH P N N N P NH P H N HN HN

P Ph N RHN P NHR RHN N NHR P RHN N n PPh 2

Ph P Ph RHN N RHN P NHR N RHN P NHR N n PPh2

Figure 1.12: Synthetic approach to ω-chain end six-arm stars via end-capping with N-organophosphoranimines [45].

N P n N CI4P CI

CI

N

CI P n CI N=PPh 2

CI4P

PCI4 N CI P CI N n PPh2

Ph

14 Chapter 1 Synthetic procedures

15

1.5 Macromolecular architecture

O

CI P N n CI

(i)

P N

HO

n

O 9

1.0

NHAc S

AcHN

O x

y

NH2

P N

(iii) 9

O

O

x

y

O

O (ii)

O

H N

WF / dLog M

O

S

S

10 11

4.0

4.5 Log MW

0.8

HN AcHN

O

9

y

O

x

O

0.6 0.4 0.2

n

O

O S

S

N H NHAc

O y

O

AcHN HN

O x

0.0 3.5

5.0

10 (M-1000) 11 (M-2070)

O y

O x

Figure 1.13: Multiarm, water-soluble polyphosphazenes with controlled dimensions. Reagents and conditions: (i) NaH, THF, 16 h; (ii) DMAP, CHCl3, 16 h and (iii). DMPA, CHCl3, hν, 1 h. DMAP, 4-(dimethylamino)pyridine; DMPA, 2,2-dimethoxy-2-phenylacetophenone; THF, tetrahydrofuran. Reproduced with permission from H. Henke, S. Wilfert, A. Iturmendi, O. Brüggemann and I. Teasdale, Journal of Polymer Science, Part A: Polymer Chemistry Edition, 2013, 51, 20, 4467. ©2013, Wiley Periodicals, Inc. [24].

O O N

O

Br

O

Star ATRP

P 3 or n

O

O

O

Br O

Comb

Cyclic trimer or linear polymer Figure 1.14: An example of a molecular brush hybrid poly(organo)phosphazene. Reproduced with permission from X. Liu, Z. Tian, C. Chen and H.R. Allcock, Macromolecules, 2012, 45, 3, 1417. ©2012, American Chemical Society [48].

16

Chapter 1 Synthetic procedures

graft-poly(dimethylsiloxane) [50] have also been prepared using ATRP initiation starting from poly(alkyl)phosphazenes. Furthermore, brush polymers have also been reported in which, rather than the backbone, polyphosphazenes form the side chains [51]. These were prepared via termination of the living cationic [NPCl2]n with norbornenyl phosphoranimines, forming brush-type polymers upon subsequent ring-opening metathesis of the norbornenyl groups.

1.5.3 Block copolymers Grafting to the end groups of polyphosphazenes prepared via living polymerization techniques allows access to block copolymers, which, with their carefully controlled segment lengths and chemistry, are invaluable tools in controlling morphology, as well as the formation of complex or hierarchical assemblies and nanostructures [52]. Because the chain ends remain active during the polymerization of phosphoranimines, polyphosphazene blocks can be prepared with other inorganic or organic polymers, either by (i) terminating with phosphoranimine end-functionalized polymers, (ii) using the polyphosphazene end groups to initiate the polymerization of a second polymer block or (iii) to incorporate end-functionalized groups capable of initiating polyphosphazene polymerization (Figure 1.15). Route (i) has been most

OR O P=N–SiMe3 OR

(i) OR RO P=N OR

(ii)

Cl P=N Cl

+



PCl3 PCl6 n

–Me3Sicl

OR O P=N SiMe3 OR

(iii) P

Cl Cl

n Cl3P=N–SiMe3

PCl5 –Me3Sicl

n Cl3P=N–SiMe3 –Me3SiCl

OR RO P=N OR

Cl P=N Cl

Cl P nCl

OR N=P O OR

OR O P=N OR

Cl P=N Cl

P=N

+ – PCl3 PCl6 n

Cl P=N PCl4 n Cl

Figure 1.15: Examples of the three most common routes to hetero poly(dichloro)phosphazene block copolymers with organic or inorganic polymers.

1.5 Macromolecular architecture

17

widely used by the Allcock group to create an array of block copolymers, including polyester [53], polycarbonate [53] and PS [54]. Of particular interest for drugdelivery applications could be triblock polyethylene glycol [55] and polypropylene glycol (PPG) [56] derivatives, some of which were shown to self-assemble into micelles [57] (see Section 1.5.4). Also of considerable interest for biomedical applications are fully hydrolysable polylactide-polyphosphazene block copolymers [58]. Route (ii) employing a polyphosphazene phosphoranimine-capped macroinitiator is also reported [55], although this has been less widely used, possibly due to reports that such phosphoranimines function much more efficiently as chain terminators than initiators [18], thus lending a preference to route (i). The third major route involves the use of alternative macroinitiators. As an example of this, it has recently been shown that dichlorophosphoranes can also initiate the polymerization of Cl3P=N−Si(CH3)3 [22, 23]. The same concept has also been exploited to prepare organometallic-inorganic block copolymers [21, 59]. Diblock copolymers in which both segments are based on polyphosphazenes are also of interest here as both blocks can be varied to give, in theory, a wide range of properties. For the living cationic polymerization method [NPCl2]n, this can be achieved purely by a second addition of Cl3PNSi(CH3)3. However, clearly a second different phosphoranimine monomer must be prepared, as the ensuing macrosubstitution would render both blocks identical. This has, for example, been achieved with the successive addition of ClR2PNSi(CH3)3 [16] or RCl2PNSi(CH3)3 [60] type phosphoranimines. Polyphosphazene-block-polyphosphazene diblock copolymers in which the second block is based on [NPCl2]n have, to the best of the author’s knowledge, not yet been prepared, as the macromolecular substitution renders the living end groups unreactive. In contrast, polyalkylphosphazene diblock copolymers can and have been prepared by the subsequent addition of an alkylphosphoranimine. Matyjaszewski and coworkers prepared AB block polyphosphazene copolymers with alkyl phosphoranimine monomers [30] by the addition of a second monomer after consumption of the first, using the anionically initiated polymerization of phosphoranimines. As mentioned earlier (see Section 1.4), the living polymerization of Cl3PNSi(CH3)3 catalyzed by PCl5 has some bidirectional growth character, which may have consequences for preparing well-defined diblock polyphosphazenes from [NPCl2]n. To counter this, Suárez-Suárez and coworkers showed that polyphosphazeneblock-polyphosphazenes may be obtained from [Ph3P = N = PCl3][Cl], hence avoiding bidirectional growth and thus enabling the synthesis of well-defined blocks (Figure 1.16) [19].

18

Chapter 1 Synthetic procedures

Monodirection chain growth polycondensation by sequential monomer addition Ph

Ph

+ P=N=PCl3

Ph

+ PCl3

P Ph

Ph

Ph P Ph

Ph

+ PMePhCl

Ph

Macromolecular substitution Ph

Ph P

Ph

+ PMePhCl

Nucleophile (O)

Ph

= Cl3P=N-SiMe3 = ClPhMeP=N-SiMe3

P Ph Ph

= [N=PCl2]n

= [N=PMePh]n

= [N=P(Nu)2]n

= Nucleophile Figure 1.16: Synthesis of polyphosphazene-block-polyphosphazene copolymers. Reproduced with permission from S. Suárez-Suárez, G.A. Carriedo, M.P. Tarazona and A. Presa Soto, Chemistry – A European Journal, 2013, 19, 18, 5644. ©2013, Wiley-VCH [61].

1.5.4 Self-assembly Macromolecular engineering is a key and rapidly advancing field with the ability to build self-assembled nanostructures having wide-reaching consequences in many fields, not least biomedical applications and nanomedicine [62]. An example is the plethora of micelle- and polymersome-type structures designed for use in drug encapsulation and transport. In this context, it has been reported that polydisperse, randomly substituted poly(organo)phosphazenes from ROP can self-assemble to form nanosized aggregates such as micelles and polymersomes [63] (Figure 1.17). This phenomenon is presumably due to the high flexibility of the polyphosphazene backbone, allowing the macromolecule to twist into conformations allowing aggregation of the hydrophobic moieties. A number of these and similar systems have been investigated for their drug-delivery capabilities and will be discussed later (see Section 3.4). Furthermore, such amphiphilic, randomly substituted poly (organo)phosphazenes have been reported to undergo thermoresponsive selfassembly (see also Section 3.6).

1.5 Macromolecular architecture

19

N =P ˗N =P ˗N =P ˗N =P ˗N =P ˗

Figure 1.17: Polymersome forming, amphiphilic randomly substituted poly(organo)phosphazenes. Reproduced with permission from C. Zheng, L.Y. Qiu and K.J. Zhu, Polymer, 2009, 50, 5, 1173. ©2009, Elsevier [63].

More precisely defined self-assembly systems than those described in the previous section can be prepared from block copolymers. For example, an amphiphilic polyphosphazene diblock based on roughly equal proportions of [N=P(OCH2CH2OCH2CH2OCH3)2]n, as a hydrophilic block, and [N=PPh(OCH2CH2OCH2CH2OCH3)]m showed a critical micelle concentration (CMC) of 80 mg/L in an aqueous solution [60], whilst the lower CMC values of 12.4 and 5.2 mg/L are reported for polyethylene oxide-block-poly [bis(trifluoroethoxy)phosphazene]s depending on the block length. Amphiphilic triblock polymers with PPG of the same polyphosphazene showed similar selfassembly behavior with CMC values in a comparable range [56]. Cyclodextrin host guest inclusion complexes provide a clear example of how the synthetic flexibility of poly(organo)phosphazenes can be utilized to form supramolecular constructs (Figure 1.18). Polyphosphazenes decorated with adamantyl

Hydrophobic Hydrophilic Hydrophobic polyphosphazene polyphosphazene polystyrene block block block Cyclodextrin inclusion

Adamantyl side group β-Cyclodextrin

Hydrophobic polystyrene block

Micelle Formation

Figure 1.18: Polyphosphazenes decorated with adamantyl groups to form inclusion complexes with βCD and show self-assembly behavior in aqueous media. Reproduced with permission from S.Y. Cho and H.R. Allcock, Macromolecules, 2009, 42, 13, 4484. ©2009, American Chemical Society [64].

20

Chapter 1 Synthetic procedures

groups have been shown to form inclusion complexes with β-cyclodextrin (β-CD) [64]. Hydrophobic poly(organo)phosphazenes can thus be rendered hydrophilic through noncovalent binding of the β-CD. Using this method, block copolymers with PS could be switched from hydrophobic to amphiphilic upon β-CD complexation. Furthermore, the amphiphilic β-CD-bound polyphosphazene-co-PS constructs self-assemble to form micelles with low CMC values of 0.925 mg/L that can be simply manipulated by changes to the β-CD concentration in an aqueous medium. More advanced structures have also been prepared through attachment of adamantyl groups to polyphosphazene chain termini, as well as supramolecular gels, through the combination with polymers bearing multiple β-CD units [65]. Well-defined polyphosphazene block copolymers with poly(ferrocenylsilane) (PFS) have also been reported, where the combination of crystallinity of the PFS block and versatility of the polyphosphazene block crystallization-directed living supramolecular polymerizations lead to spatially defined and controllable nanostructures [59]. Although not designed specifically for medical applications, they show a prime example of how the tunability of polyphosphazenes can be exploited for advanced macromolecular engineering. Certain bidendate substituents, in particular 2,2ʹ-dioxybiphenyl-based groups, have been shown to react with [NPCl2]n in a geminal manner, that is, both alcohol groups substitute the same phosphorus atom forming phosphorus heterocycles [66], thus not leading to cross-linking, as would be expected with the majority of bifunctional nucleophiles. This produces a unique type of polyphosphazene spirophosphazenes [67] which may have wide-ranging applications, including catalysis [68], and can now be directly functionalized [69]. Furthermore, if enantiomeric binaphthyl groups are attached, helicity is observed [70]. Helical polymers are important molecules in nature and such synthetic, chiral and/or optically active polymers could have wide-reaching applications. For example, it was shown that polyphosphazenes with a chiral block have an intrinsic preferred helical sense (Figure 1.19) [61, 72]. Furthermore, this helicity can be transferred to nonchiral blocks and to the bulk morphology of prepared films [61] influencing their selfassembly behavior and forming chiral macroporous films which could be decorated with gold nanoparticles [73] A way to stabilize gold nanoparticles was found by the same group that made use of the pyridinyl functionalities of poly(bistrifluoroethoxy phosphazene)-b-poly(2-vinylpyridine) [74]. For a more in-depth discussion of polyphosphazene block copolymers for self-assembly the reader is referred to the work of the Soto group, especially their recent review on that topic [71].

1.6 Conclusion

FG

OO 1-x

FG

O

Ph

P N

P=N

N

21

P N x

FG

Me

n N

m

N S

O

Figure 1.19: Helical polyphosphazene block copolymers. Reproduced with permission from Carriedo, G. A. de la Campa, R. Soto, A. P., Polyphosphazenes – Synthetically Versatile Block Copolymers (“Multi-Tool”) for Self-Assembly. Eur. J. Inorg. Chem. 2018, 2018 (22), 2484–2499. © 2018 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim [71].

1.6 Conclusion Recent advances in the controlled synthesis of the polyphosphazene backbone, added to the long-known synthetic flexibility through side-group manipulation, represent significant progress for polyphosphazene chemistry. The ability to carefully control the main chain length, dispersity and architecture additionally leads the way to polymer self-assembly and defined nanostructures, which could provide the future of advanced materials for use in medicine. Polyphosphazene synthesis poses many more challenges than other standard polymers, for example, for controlled radical polymerizations. However, the synthetic flexibility is enormous and thus polyphosphazenes offer the potential to prepare precise structures with unique properties tailored to the desired application. Although this alone may provide many advanced materials, with properties unachievable with standard carbon-based chemistry, the combination of the tunable biodegradability of polyphosphazenes (see Chapter 2) could be significant for medical applications, as biodegradable polymers with the same controlled and tunable synthetic properties are few and far between.

22

Chapter 1 Synthetic procedures

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[11]

[12] [13]

[14] [15]

[16]

[17]

[18]

[19]

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[39] Huynh K, Lough AJ, Manners I. Reactions of P-donor ligands with N-silyl(halogeno) organophosphoranimines: formation of cations with P−P coordination bonds and poly(alkyl/ aryl)phosphazenes at ambient temperature. J Am Chem Soc 2006;128:14002–14003. [40] Maraval V, Caminade A-M, Majoral J-P, Blais J-C. Dendrimer design: How to Circumvent the dilemma of a reduction of steps or an increase of function multiplicity? Angew Chem Int Ed 2003;42:1822–1826. [41] Cho SY, Allcock HR. Dendrimers derived from polyphosphazene-poly(propyleneimine) systems: Encapsulation and triggered release of hydrophobic guest molecules. Macromolecules 2007;40:3115–3121. [42] Nelson JM, Allcock HR. Synthesis of triarmed-star polyphosphazenes via the “living” cationic polymerization of phosphoranimines at ambient temperatures. Macromolecules 1997;30: 1854–1856. [43] Henke H, Posch S, Brueggemann O, Teasdale I. Polyphosphazene based star-branched and dendritic molecular brushes. Macromol Rapid Commun 2016;37:769–774. [44] Linhardt A, König M, Iturmendi A, Henke H, Brüggemann O, Teasdale I. Degradable, dendritic polyols on a branched polyphosphazene backbone. Ind Eng Chem Res 2018;57:3602–3609. [45] Henke H, Plavcan O, Teasdale I. Telechelic α,ω-chain end-functionalized polyphosphazenes as building blocks for degradable soft materials. unpublished results2019. [46] Zhulina EB, Sheiko SS, Borisov OV. Solution and melts of barbwire bottlebrushes: Hierarchical structure and scale-dependent elasticity. Macromolecules 2019;52:1671–1684. [47] Siegwart DJ, Oh JK, Matyjaszewski K. ATRP in the design of functional materials for biomedical applications. Prog Polym Sci 2012;37:18–37. [48] Liu X, Tian Z, Chen C, Allcock HR. Synthesis and characterization of brush-shaped hybrid inorganic/organic polymers based on polyphosphazenes. Macromolecules 2012;45: 1417–1426. [49] Cambre J, Wisian-Neilson P. Poly(methylphenylphosphazene)–Graft–Poly(methyl Methacrylate) copolymers via atom transfer radical polymerization. J Inorg Organomet Polym Mater 2006;16:311–318. [50] Wisian-Neilson P, Islam MS. Poly(methylphenylphosphazene)-graft-poly(dimethylsiloxane). Macromolecules 1989;22:2026–2028. [51] Allcock HR, de Denus CR, Prange R, Laredo WR. Synthesis of norbornenyl telechelic polyphosphazenes and ring-opening metathesis polymerization reactions. Macromolecules 2001;34:2757–2765. [52] Mai Y, Eisenberg A. Self-assembly of block copolymers. Chem Soc Rev 2012;41:5969–5985. [53] Krogman NR, Steely L, Hindenlang MD, Nair LS, Laurencin CT, Allcock HR. Synthesis and characterization of polyphosphazene-block-polyester and polyphosphazene-blockpolycarbonate macromolecules. Macromolecules 2008;41:1126–1130. [54] Allcock HR, Powell ES, Chang YY, Kim C. Synthesis and micellar behavior of amphiphilic polystyrene-poly[bis(methoxyethoxyethoxy)phosphazene] block copolymers. Macromolecules 2004;37:7163–7167. [55] Nelson JM, Primrose AP, Hartle TJ, Allcock HR, Manners I. Synthesis of the first organic polymer polyphosphazene block copolymers: Ambient temperature synthesis of triblock poly (phosphazene ethylene oxide) copolymers. Macromolecules 1998;31:947–949. [56] Allcock HR, Cho SY, Steely LB. New amphiphilic poly[bis(2,2,2-trifluoroethoxy)phosphazene]/ poly(propylene glycol) triblock copolymers: Synthesis and micellar characteristics. Macromolecules 2006;39:8334–8338. [57] Chang Y, Prange R, Allcock HR, Lee SC, Kim C. Amphiphilic Poly[bis(trifluoroethoxy) phosphazene]−Poly(ethylene oxide) block copolymers: Synthesis and micellar characteristics. Macromolecules 2002;35:8556–8559.

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[58] Weikel AL, Cho SY, Morozowich NL, Nair LS, Laurencin CT, Allcock HR. Hydrolysable polylactide-polyphosphazene block copolymers for biomedical applications: synthesis, characterization, and composites with poly(lactic-co-glycolic acid). Polym Chem 2010;1: 1459–1466. [59] Soto AP, Gilroy JB, Winnik MA, Manners I. Pointed-oval-shaped micelles from crystalline-coil block copolymers by crystallization-driven living self-assembly. Angew Chem Int Ed 2010;49: 8220–8223. [60] Chang Y, Lee SC, Kim KT, Kim C, Reeves SD, Allcock HR. Synthesis and micellar characterization of an amphiphilic diblock copolyphosphazene. Macromolecules 2000;34: 269–274. [61] Suárez-Suárez S, Carriedo GA, Tarazona MP, Presa Soto A. Twisted morphologies and novel chiral macroporous films from the self-assembly of optically active helical polyphosphazene block copolymers. Chem–Eur J 2013;19:5644–5653. [62] Matyjaszewski K. Macromolecular engineering: From rational design through precise macromolecular synthesis and processing to targeted macroscopic material properties. Prog Polym Sci 2005;30:858–875. [63] Zheng C, Qiu LY, Zhu KJ. Novel polymersomes based on amphiphilic graft polyphosphazenes and their encapsulation of water-soluble anti-cancer drug. Polymer 2009;50:1173–1177. [64] Cho SY, Allcock HR. Synthesis of adamantyl polyphosphazene−polystyrene block copolymers, and β-cyclodextrin-adamantyl side group complexation. Macromolecules 2009;42:4484–4490. [65] Tian Z, Chen C, Allcock HR. Synthesis and assembly of novel Poly(organophosphazene) structures based on noncovalent “Host–Guest” inclusion complexation. Macromolecules 2014;47:1065–1072. [66] Carriedo GA, Fernández-Catuxo L, García Alonso FJ, Gómez-Elipe P, González PA. Preparation of a new type of phosphazene high polymers containing 2,2‘-dioxybiphenyl groups. Macromolecules 1996;29:5320–5325. [67] Carriedo GA. Polyphosphazenes with p(dioxy-biaryl) cycles in the repeating units: a review. J Chil Chem Soc 2007;52:1190–1195. [68] Carriedo GA, Crochet P, Alonso FJG, Gimeno J, Presa-Soto A. Synthesis and catalytic activity of (eta(6)-p-cymene)(phosphane)ruthenium(II) complexes supported on poly (biphenoxyphosphazene) or chiral poly(binaphthoxyphosphazene) copolymers. Eur J Inorg Chem 2004:3668–3674. [69] de la Campa R, García D, Rodríguez S, Carriedo GA, Presa Soto A. Direct functionalization of poly(spirophosphazene)s via the regioselective lithiation of the aromatic rings using a cooperative superbase. Macromol Rapid Commun 2017;38:1700039. [70] Carriedo GA, Alonso FJG, Presa-Soto A. High molecular weight phosphazene copolymers having chemical functions inside chiral pockets formed by (R)-(1,1 ‘-binaphthyl-2,2 ‘-dioxy) phosphazene units. Eur J Inorg Chem 2003:4341–4346. [71] Carriedo GA, de la Campa R, Soto AP. Polyphosphazenes – synthetically versatile block copolymers (“Multi-Tool”) for self-assembly. Eur J Inorg Chem 2018;2018:2484–2499. [72] Suarez-Suarez S, Carriedo GA, Presa Soto A. Tuning the chirality of block copolymers: From twisted morphologies to nanospheres by self-assembly. Chem Eur J 2015;21:14129–14139. [73] Suárez-Suárez S, Carriedo GA, Presa Soto A. Gold-decorated chiral macroporous films by the self-assembly of functionalized block copolymers. Chem Eur J 2013;19:15933–15940. [74] Cortes MdlA, de la Campa R, Valenzuela ML, Díaz C, Carriedo GA, Presa Soto A. Cylindrical micelles by the self-assembly of crystalline-b-coil polyphosphazene-b-P2VP block copolymers. stabilization of gold nanoparticles. Molecules 2019;24:1772.

Chapter 2 Degradable poly(organo)phosphazenes 2.1 Bioerodible polymers for biomedicine As many clinical applications, both current and proposed, require temporary, rather than permanent materials, synthetic bioerodible polymers have an important position among biomaterials and can fulfill many functions. Bioerosion can be defined as the transformation of a solid material into a water-soluble material [1], which can be due to simple solubilization over time, but also frequently by actual polymer degradation, that is, breaking of chemical bonds to form small molecules. In this regard, all polymers are degradable to some degree and thus the definition is somewhat discretionary, but in this book we refer to the definition of “degradable” as polymers which degrade within their expected lifetime, or shortly thereafter, under the conditions applied [2]. In fact, relatively few polymers are genuinely degradable in a suitable time frame for medical applications and furthermore, in a controlled manner to nontoxic products, and despite much research, as of 2006, only seven distinct synthetic polymer classes in a small range of applications have gained the US Food and Drugs Administration approval [1]. For a detailed review of the degradable polymers currently in clinical use and development, the reader is referred to [3]. It is important to note at this point that the terms biodegradation/degradation/bioerosion/bioresorption are used interchangeably in the scientific literature. Herein, we refer to the following definitions as suggested by Treiser and coworkers [1], that is, degradation being a chemical process by which covalent bonds are cleaved, biodegradation being degradation as a consequence of a biological agent, bioerosion is used to describe the conversion of a solid material into a water-soluble one under physiological conditions, regardless of the mechanism of conversion, and bioresorption is referred to as the removal of the polymer by cellular activity. Due to the inherent complexity and extremely wide range of biomedical applications requiring bioerodible polymers, each with its individual requirements, a one-polymer-fits-all strategy is impossible. Thus, an extensive range of materials with wide-ranging properties are essential and, where possible, materials with adaptable properties. The currently available, degradable synthetic polymers do not cover the range of material properties required and thus considerable development is required in this area. Additionally, the demands of degradable polymers, in terms of biocompatibility, are much higher than nondegradable materials, since not only the polymers and their components (residual monomer, stabilizers and so on) must be biocompatible, but any degradation products, intermediates and metabolites thereof must also be biologically benign.

https://doi.org/10.1515/9783110654189-002

28

Chapter 2 Degradable poly(organo)phosphazenes

2.1.1 Bioerodible solid biomaterials and polymer matrices Of many biomedical applications, surgical sutures (stitches) represent the oldest and possibly best known use of degradable polymers. For sutures and other fixation devices, suitable mechanical strength and a controllable rate of degradation are clearly of critical importance to allow gradual strength transfer as the natural tissue heals. Poly(lactic-co-glycolic acids) (PLGA) (Figure 2.1a) in its various homo- and copolymer combinations are commonly used for such applications, as are polydioxanone (Figure 2.1c) and polycaprolactone (Figure 2.1b) among others [4]. Fixation devices, for example, panels and screws, prepared from bioerodible polymers can also be used in place of nondegradable metals and thus eliminate the need for secondary interventions. An example here is Lactosorb® (82% poly(lactic acid) PLA and 18% poly(glycolic acid) PGA) used clinically for low load-bearing fractures such as facial injuries [5].

O

O

n O

m

n

O

N C n

(E)

RO (F)

O

O O R n O

n (C)

(B)

R

O

O

(A)

O O

O

O

O

O

n

R P N n R (G)

(D)

O ¨ P O

O n

O R (H)

Figure 2.1: Basic structures for some groups of synthetic polymers commonly employed as degradable materials in medical applications: (a) polylactide-co-polyglycolides (also commonly used as their homopolymers (i.e., n/m = 0)), (b) polycaprolactones, (c) polydioxanones, (d) polyorthoesters, (e) polyanhydrides, (f) polyalkylcyanoacrylates, (g) poly(organo)phosphazenes and (h) polyphosphoesters.

Various derivatives of polyalkylcyanoacrylates, well known as household adhesives, are also used successfully as surgical glues for wound repair [6]. Bioerosion occurs mainly via hydrolysis of the ester side groups to produce the water-soluble poly (acrylic acid). The toxicity of the short alkyl chain derivatives can be overcome to some extent by longer alkyl chains, leading to biocompatible polymers with hydrolysis rates dependent on the hydrophobic fraction. Furthermore, the inherent instability of the C–C backbone, particularly in the presence of bases [7], may lead to a fairly unique backbone depolymerization and thus degradation of the actual polymer

2.1 Bioerodible polymers for biomedicine

29

main chains via unzipping upon deprotonation of the C–H chain terminus which may broaden their potential usage [8]. Bioerodible, implantable/injectable drug-delivery devices that can transport and release drugs in a controlled manner have also been extensively investigated. One relative success story here is the use of degradable polyanhydride-based wafers, GliadelTM, which is approved in many countries for use in local chemotherapy and has been shown to be well tolerated, offering survival benefits to patients with newly diagnosed malignant glioma [9]. These poly[carboxyphenoxy-propane/ sebacic acid]anhydride wafers are implanted after brain tumor surgery and slowly release the chemotherapeutic drug carmustine over a 2- to 3-week period. Of the many degradable polymer systems investigated for drug-release depots, degradable polyphosphazenes have also been investigated and will be discussed in more detail in Section 3.2. A further major field of investigation for bioerodible polymers is their use as scaffolds for tissue engineering. Tissue engineering is the use of a bioerodible polymer as an artificial extracellular matrix, supporting cell growth and organization and will be discussed in Chapter 4, as it represents a major focus of polyphosphazene research. Most of the degradable polymers degrade via either enzymolysis (common for biopolymers) or hydrolysis. As enzymolysis often depends on enzyme concentration, and availability and activity varies between tissue types and individuals, hydrolytically unstable polymers can potentially offer more control in terms of the predictability of their degradation profile and thus in vivo behavior. Rates and type of erosion of hydrolytically unstable polymers are governed predominantly by the polymer chemistry, its hydrophobicity and hydrolytic stability; however, device formulation and morphology are also important factors. Solid bioerodible polymers are frequently described in terms of surface and/or bulk erosion profiles [1]. During bulk erosion, the rate of water penetration into the device exceeds the rate of transformation into soluble materials. This inherently causes defects throughout the system and hence rapid mechanical failure which may be a disadvantage for some applications, for example, if controlled drug release is required from the polymer implant which would thus have a nonlinear release profile. On the other hand, surface erosion, as the name suggests, occurs only at the exterior of the device, due to the slower rate of water penetration than erosion, and hence a steady rate of transformation is observed and structural integrity at the interior of the device is maintained. Surface erosion tends to be observed for hydrophobic materials with very hydrolytically labile linkages, the best known examples being polyanhydrides (Figure 2.1e) and polyorthoesters (Figure 2.1f). Enzymatic erosion, whereby enzymes are sterically hindered from entering the device, may also lead to surface erosion [1].

30

Chapter 2 Degradable poly(organo)phosphazenes

2.1.2 Water-soluble, degradable polymers Another ever-expanding field for the application of macromolecules is polymer therapeutics and so-called nanomedicines, that is, enhancement of the bioavailability and biodistribution of drugs via macromolecular carriers [10, 11]. However, there are a number of significant safety concerns over the long-term clinical use of nondegradable polymers, for example, polyethylene glycol (PEG) or poly(N-(2-hydroxypropyl) methacrylamide) that have been used up to now for such intravenous applications [12]. Although when used enterally, nondegradable polymers can be readily consumed and excreted without concern, parenterally administered high molecular weight (Mw) polymers cannot be eliminated by glomerular filtration in the kidneys [13, 14]. Even when biocompatibility is often well proven, the effect of long-term administration of high Mw materials is thus problematic [15]. Even the ubiquitous PEG has been shown to have potential long-term consequences [16, 17]. The long-term accumulation of biopersistent polymers can lead to iatrogenic illnesses, for example, symptoms similar to liposomal storage disease [18]. Indeed this has been known for many years, since the use of polyvinylpyrrolidone (PVP) as a plasma expander which was administered to many soldiers during the Second World War [19]. Although effective as a plasma expander, it was noticed that only a certain percentage of the polydisperse PVP was excreted from the body. Low Mw fractions (120 kDa) were found to be retained by the reticuloendothelial system with PVP observed in the spleen, bone marrow and lymph nodes. Although no deleterious effects of this long-term retention were observed at the time, concerns were voiced and it was proposed to limit PVP Mw to 25–40 KDa. Indeed PVP with Mw above approximately 10,000 g/mol are currently not recommended for repeated use in procedures for which excessive storage may be an issue, for example subcutaneous or intravenous administration [20]. The fate of nondegradable polymers that enter the bloodstream must therefore be carefully considered [13]. This would include, for example, bioerodible polymer implants, which erode into nondegradable polymers, as well as intravenous drugdelivery applications. Nevertheless, 60 years since this information came to light, many nanomedicines based on nondegradable polymers are still being proposed. This is of particular concern for modern nanomedicines where high dosages and/or repeated parenteral administration are required, as is the case, for instance, in chemotherapy, a commonly investigated therapy in nanomedicine. As nanomedicines in future years become more widespread, the biopersistence of the polymeric building blocks is expected to become an ever-increasing problem. Since many of the desired properties, indeed their raison d’être, stem from the high Mw of macromolecular

2.2 Poly(organo)phosphazene degradation

31

carriers, it is apparent that the development of high Mw synthetic polymers that degrade to benign small molecules is essential. Two types of water-soluble, degradable polymers are well known in the polymer literature: semidegradable or completely degradable. Semidegradable polymers degrade to smaller polymers, which must be under the renal clearance limit [21, 22] (including supramolecular structures, for example, nanogels [23] and micelles [24]). However, it should be noted here that lysosomal accumulation is also a risk for polymers with a Mw below the renal clearance limit and hence where possible, polymers used should degrade to small molecules [15]. Many natural polymers, for example, collagen and hyaluronic acid are known to completely degrade to benign small molecules which are subsequently absorbed in the biochemical pathways of the body. However, the synthetic flexibility and property manipulation of natural polymers is often limited. Some synthetic polymers, such as poly(L-glutamic acid) and poly(aspartic acid), are also known to degrade to monomeric amino acids and would seem to currently represent the most promising options for future materials in nanomedicine [12, 13]. Degradable poly(organo)phosphazenes designed for such applications will be discussed in Chapter 3.

2.2 Poly(organo)phosphazene degradation The inherent degradability of the inorganic backbone is a major feature of many poly(organo)phosphazenes, in particular, with a view to their application in biomedicine. Polyphosphazenes are known to degrade to a predictable and nontoxic biologically buffered mixture of the organic side groups, as well as phosphates and ammonia, resulting from the backbone [25–27]. The near-neutral pH of the degradation products is in contrast to the plethora of polyesters currently used or investigated for biomedical applications, which tend to produce acidic degradation products upon hydrolytic degradation. Since phosphates can be metabolized and ammonia can be excreted by the organism, assuming the organic side group is carefully chosen, biocompatible degradation products of the corresponding poly(organo)phosphazene are thus easily achieved. The most plausible degradation pathway of poly(organo)phosphazenes is shown below (Figure 2.2). In the presence of water, organic side groups are first exchanged on the phosphorus atom in the polymer backbone leading to the formation of hydroxyphosphazenes and, after proton transfer, phosphazenes [26, 28]. These intermediates are extremely hydrolytically sensitive and undergo further chain cleavage, eventually leading to low Mw fragments. This has also been substantiated using mass spectrometry of the degradation intermediates [29], which confirmed the progressive shortening of the backbone until 7-membered units were reached, which also hydrolyzed, but at a slower rate. Consequently, the complete hydrolytic breakdown of poly(organo)phosphazenes yields the corresponding organic side groups, together with

32

Chapter 2 Degradable poly(organo)phosphazenes

R

NH P N n HN R

+H2O –RNH2

OH P N n HN R Hydroxyphosphazene

O H P N n HN R

+H2O –RNH2

O H P N n OH

Phosphazane +H2O

O P OH NHR

+

H2N

Phosphates and ammonia

Figure 2.2: The proposed mechanism for the hydrolytic degradation of poly(organo)phosphazenes.

phosphates and ammonia as the final degradation products of the polymer main chain (Figure 2.3). The stability and rate of hydrolysis is highly dependent on the nature of the organic side-group substituent and hence can be adjusted by careful choice of suitable side groups coupled to the polyphosphazene backbone.

2.2.1 Side-group influence on degradation kinetics The nature of the organic side groups has a decisive influence on the degradation rate of the resulting poly(organo)phosphazene, and indeed a full range of hydrolysis can be achieved, starting from the highly hydrophobic CF3CH2O-, which is completely resistant for many years to all but the strongest bases, and ending with poly(organo) phosphazenes, which are so unstable that they cannot be reliably prepared. The most hydrolysis-susceptible side-substituent is chlorine and hence if preparing polymers from a poly(dichloro)phosphazene precursor, knowledge of the residual chlorines is required [27, 30]. Not only is the P–Cl moiety itself extremely labile, its hydrolysis leads directly to the degradation intermediate hydroxyphosphazene [31] and also produces HCl as a by-product, which is known to further catalyze hydrolysis (see Section 1.1). Although through the partial substitution of poly(dichloro) phosphazene it is thus possible to prepare degradable materials, in practice, however, full substitution is usually strived for, not least for the sake of reproducibility. As hydrolysis occurs via hydrolytic attack of the phosphazene backbone, hydrophobicity and steric bulk represent the simplest factors when designing poly(organo) phosphazenes with tailored degradation rates; the larger, and/or more hydrophobic groups essentially protecting the phosphorus center from nucleophilic attack by water. A common and simple route to tailor the degradation rate is thus to combine hydrophilic and hydrophobic moieties onto a single chain. An early example of this

33

2.2 Poly(organo)phosphazene degradation

pH 2 Before degradation

Detector response

Partial hydrolysis of side groups

1 day 7 days Degradation products with -P-OH and -P=O moieties

4 days 1 day Before degradation 3

4

5

4 days 6 7 8 9 10 Retention volume (mL)

11

12

Phosphate

7days 50

0 δ (ppm)

–50

Figure 2.3: Degradation of a water-soluble poly(organo)phosphazene under accelerated conditions (pH 2): (a) size exclusion chromatography of a water-soluble degradable poly(organo) phosphazene; it can be seen that main chain degradation is accompanied by an initial increase in 31 the free side groups (retention volume 11−12 mL) and (b) P-nuclear magnetic resonance spectroscopy of the same polymers shows evidence of the production of phosphates as the major degradation products. Reproduced from S. Wilfert, A. Iturmendi, W. Schoefberger, K. Kryeziu, P. Heffeter, W. Berger, O. Brüggemann and I. Teasdale, Journal of Polymer Science, Part A: Polymer Chemistry Edition, 2014, 52, 2, 287. ©2014, Wiley [25].

is the combination of imidazole and 4-methylphenol [32], in which the hydrophilic imidazole group was shown to sensitize the polymer toward hydrolysis, while the hydrophobic, bulky phenol hindered hydrophilic attack. However, although general tendencies can be observed, it would appear that hydrophilicity alone cannot explain hydrolytic sensitization for all polyphosphazenes. For example, oligoethyleneoxy side chains, including poly[bis(2-(2-methoxyethoxy) ethoxy)phosphazene] and its many structural variations [33], used as solid polymer electrolytes, are reported to be biostable, whereas structurally similar polymers with P-NH-R attachments, instead of the P-O-R are known to undergo hydrolytic degradation [34]. Also, ethyl esters of serine and threonine substituted onto polyphosphazenes via the N-terminus are reported to degrade faster than their more hydrophilic counterparts, connected via the O-terminus [35]. Thus, the type of linkage to the

34

Chapter 2 Degradable poly(organo)phosphazenes

phosphorus is critical, indeed many of the groups reported to sensitize polyphosphazenes toward hydrolysis are based on nitrogen linkages, suggesting a higher hydrolytic lability of this bond. It is also feasible that proton migration from the NHR group to the backbone aids degradation, in particular, when one considers the structural similarity to proton-scavenging phosphazene bases [36]. The precise detail of all possible mechanisms requires more investigation, as not all contributions to the degradation pathway are known. This is a difficult task using macromolecules, made harder by the extremely wide variety of substituents used and their possible contributions. Small molecule studies are helpful, and have been reported [28], although directly assessing their bearing on macromolecules is difficult due to the superior stability of the cyclic ring system. Furthermore, many sidegroup functionalities may be involved in the degradation pathway, and side-group participation may explain the hydrolytic sensitivity of some polyphosphazenes in which the side groups are connected by oxygen atoms. For example, Luten and coworkers showed a hitherto unexpected rapid degradation of dimethylaminoethanolsubstituted polyphosphazenes [37], and attributed this to a possible intramolecular reaction of the amine to directly produce phosphazene. However, they were not able to rule out incomplete chlorine substitution, which would lead to unstable polymers, as discussed earlier. Many hydrolysis sensitizing side groups which are attached to the polyphosphazene via oxygen atoms also contain functional groups with extractable protons, for example, free alcohol or acid groups, for which proton migration may explain the enhanced degradation rates. The addition of sugars, for example, glucose, produces water-soluble polymers with reported hydrolytic degradation (albeit slow and over many years) [38], whereas water-soluble glycerol substituents cause a rapid degradation to glycerol, phosphates and ammonia [39] and thus are interesting as degradable, water-soluble polymers, or indeed in their crosslinked form, hydrogels for biomedical applications. Relatively few investigations have been carried out on these materials since the original work in the 1980s, possibly due to the protection group strategies required for their preparation, but in recent years, hyperbranched polyglycerols have been proven to be excellent materials for polymer drug delivery [40], and hence a reinvestigation of hydrolytically degradable poly(diglyceryl)phosphazenes for such purposes may be warranted. Aromatic polyacids, for example, poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) (Figure 2.4c) and its ethyl derivative poly[di(sodium carboxylatoethylphenoxy)phosphazene] are probably the most well studied, degradable watersoluble polyphosphazenes and have been investigated for their use in vaccine delivery and immunology (see Section 3.1) [31]. These polyacids are reported to degrade in pH neutral, aqueous conditions [31], making them suitable for parenteral administration, despite the presence of the hydrophobic phenol side groups, which would normally impart hydrolytic stability. This is presumably due to the acid functionalities which can transfer protons to the backbone and cause hydrolytic instability. The rate

35

m

2.2 Poly(organo)phosphazene degradation

O

O–Na+

O

O OH N O P N n O

N O P N n O

O P N n O

HN

NH P N n NH

P N n HN m

O

O

HO

N HO

OH O–Na+

O

(A)

(B)

O

N

(C)

O (D)

(E)

Figure 2.4: Some water-soluble, degradable poly(organo)phosphazenes.

of degradation can be further enhanced by cosubstitution with more hydrophilic groups (Figure 2.5) [41]. PYRP were also observed to undergo hydrolytic cleavage [41]. This is a hydrophilic, water-soluble polymer, with a P–O–R backbone linkage and with no obvious proton donors to assist chain cleavage. Despite detailed investigations, no ring opening was observed and hence intramolecular catalysis was ruled out as only direct cleavage of the side groups was observed. Whether the lactam moiety contributes in some way to degradation was not investigated at the time, but since tertiary amines, [37] esters (see Section 2.2.2) and amides [25] in close proximity to the phosphazene backbone all lead to accelerated degradation, this should possibly be investigated in the future.

2.2.2 Amino acid ester-derived polyphosphazenes Amino acid esters as side groups for polyphosphazenes represent the most extensively researched degradable poly(organo)phosphazenes, leading to hydrophobic, biocompatible polymers with variable hydrolytic stabilities and thus degradation rates [26, 28, 42]. The various structures available are summarized in Figure 2.6. Since the various “R” groups can be diverse, it is clear to see that even within the “amino acid ethyl ester” class of poly(organo)phosphazenes, a large array of constellations are possible, giving rise to a plethora of available polymers with varied rates of degradation and other properties [26]. This, in addition to the high reactivity of the amino groups toward polydichlorophosphazene, makes them ideal substituents for the preparation of libraries of degradable polyphosphazenes. Furthermore, the structural

36

Chapter 2 Degradable poly(organo)phosphazenes

Residual MW, % 100

80

4

60 2

3

40

20 1 0 0

5

10

15

20 Time, days

Figure 2.5: Degradation profiles for the water-soluble PYRP (polymer 1) and PCPP (polymer 4). PYRP: Poly[bis(2-(2-oxo-1-pyrrolidinyl)ethoxy phosphazene]. Combinations of the two side groups produce polymers 2 and 3 with intermediate degradation rates. Conditions: pH 7.4 at 55 °C. Reproduced with permission from A.K. Andrianov, A. Marin and P. Peterson, Macromolecules, 2005. 38, 19, 7972. ©2005, American Chemical Society [41].

O R2

O R2

O

O

O

O

O

NH

O

O

HN

R1

HN

R1

HN

R1

P N n R3 (A)

P N n R3 (B)

P N n R3 (C)

Figure 2.6: Generic structures for amino-acid-ester-based poly(organo)phosphazenes: (a) amine acid ester, (b) dipeptide and (c) depsipeptide. R1 and R2 = amino acid alkyl moiety, for example, H, CH3, CH(CH3)2 and so on; and R3 = either the same amino acid, another amino acid ester or indeed any other organic substituent.

similarity, varying mostly only at the α-carbon and ester moiety, make them ideal for studies into the properties of degradable polyphosphazenes. In addition, amino acid esters have known biocompatibility, a wide variation of structures and are readily

2.2 Poly(organo)phosphazene degradation

37

available. Ethyl esters are generally favored rather than methyl or other esters, as they should hydrolyze to benign degradation products, an important consideration when designing bioerodible polymers. Amino acid esters, when substituted onto polyphosphazenes possess both P-NH-R linkages, as well as functional groups (esters), both of which may participate in degradation and indeed most poly(organo)phosphazenes with amino acid ester substituents are hydrolytically unstable, to varying degrees. Hydrolysis of the (ethyl) ester, although expected to be slow in pH neutral conditions, is clearly a possible degradation route. The resulting acid would be expected to accelerate degradation by proton transfer to the nitrogen on the polyphosphazene backbone. Since similar structures containing the less hydrolysis-sensitive amide units instead of esters in this position also enhance hydrolysis rates [25], it is not clear how large an influence ester hydrolysis has on the degradation. This would point the way to either a simple leaving group effect, or perhaps an intramolecular attack of the backbone from the ester/ amide, as discussed in Section 2.2.1, although more detailed studies are required to confirm this. Several other mechanisms have also been proposed [26, 43], although mechanistic investigations do not appear to have been carried out for all proposed routes. Strong evidence has been earlier reported that glycine ethyl esters are the main degradation product of the respectively substituted polymers, again suggesting that cleavage of the side group from the backbone, rather than ester cleavage, is the major degradation route [44]. Degradation studies on tyrosine-substituted polyphosphazenes also suggested cleavage as the major degradation pathway [45], although again ester hydrolysis cannot be ruled out. It should be noted here that ester cleavage is also likely to be dependent upon degradation conditions, above all the pH, further complicating matters. It has been clearly shown in many detailed studies that the substitution of adjacent bulky side groups, for example, at the α-C position of amino acid esters, shields the polyphosphazene backbone from the attack of water molecules resulting in more hydrolytically stable polymers (Figure 2.7). This provides a very simple

H2O RO

O

HN P

RO

O

HN N

P

N

n NHR’

n NHR’

Figure 2.7: Hydrolysis inhibition by α-substituted amino acid esters. The substituents at the α-carbon have a decisive effect on the hydrolytic stability of poly(organo)phosphazenes, shown here for valine.

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Chapter 2 Degradable poly(organo)phosphazenes

method to tailor the rate of degradation of amino-acid-ester-substituted polyphosphazenes. For example, the glycine ethyl ester derivative is reported to degrade in around 3 months (half-life), the alanine derivative in 6 months, while the valine ethyl ester requires approximately 1 year to reach this level of degradation [26]. Cosubstitution of small amounts of depsipeptides such as ethyl 2-(o-glycyl)lactate (GlyLacOEt) or ethyl p-(o-alanyl)lactate (AlaLacOEt) lead to drastic increases in backbone degradation rates in comparison to the simple amino acid ester derivatives [34, 46, 47]. Dipeptides offer a similar structure and can also be used to rapidly enhance degradation rates and such polymers have been developed as degradable scaffolds for tissue engineering [48], in particular, due to their blend forming properties (see Section 2.4). Glycolic and lactic acid derivatives are also important, especially since currently, PLGA and their various combinations are debatably the most important degradable polymers for biomedical applications. In this regard, a series of polyphosphazenes were prepared with esters of lactic and glycolic acid [49], with the methyl group in the α-position of the lactate, lowering hydrolysis rates as a result of hindering water access to the phosphorus. Comparisons to PLA and PGA showed faster degradation rates but, as may be expected, the polymers lack the crystalline properties of PLA, imparting inferior mechanical properties on the hybrid polymers. Clearly, the ability to cosubstitute the polyphosphazene backbone with substituents, rendering different hydrolytic stabilities, gives a wide scope of variability in degradation rates [26, 50]. Using such methods, rates of degradation can be easily tuned, although one must keep in mind that even small compositional changes will not only affect the hydrolytic stability, but also all other polymer properties, such as the mechanical properties. Often, the same structural properties which increase mechanical stability also increase hydrolytic stability, and vice versa, the key is, therefore, finding a compromise to tune the properties for the desired application.

2.2.3 The effect of pH Mildly basic conditions (pH 7–10) generally tend to slow the degradation rate of poly(organo)phosphazene in comparison to neutral pH, an observation consistent for a range of different organic substituents [41, 51, 52]. This is somewhat surprising for a hydrolysis reaction, but is reported for a number of different poly(organo) phosphazenes. Contradictory reports showed the more rapid hydrolysis of glycineester-substituted polyphosphazene at higher pH values, although these were tested at the extreme value of pH 12 [53]. At lower pH values ( 1

Unimer

Polymersome

Spherical micelle

85

PEG Unimer Oligopeptide

Hydrophobicity

Morphology

Figure 3.23: Hexachlorophosphazene can be substituted with amphiphilic substituents with the unhindered rotation around the substituted phosphorus allowing the hydrophobic substituents to agglomerate in aqueous media. Reproduced with permission from Y.J. Jun, M.K. Park, V.B. Jadhav, J.H. Song, S.W. Chae, H.J. Lee, K.S. Park, B. Jeong, J.H. Choy and Y.S. Sohn, Journal of Controlled Release, 2010, 142, 1, 132. ©2010, Elsevier [137].

(Taxotere®) and although the plasma half-life times could not be extended, it was observed that the micelle stability was superior and that the inherent photoinstability of Taxotere® could be overcome. Furthermore, a comparative acute toxicity study showed a more than twofold higher median lethal dose value for the phosphazene micelle compared with the docetaxel in Taxotere®, presumably due to the improved encapsulation. This docetaxel-loaded phosphazene-based micelle is reported to have been taken forward for more detailed preclinical studies [139]. The same tactic of cosubstitution with a short-chain PEG and a smaller hydrophobic unit can also be achieved with the significantly larger and hydrolytically degradable linear polyphosphazenes [140]. The backbone flexibility allows these to fold and thus self-assemble, for example, in aqueous solutions the hydrophobic segments of the amphiphilic copolymers can agglomerate to give micelle-type structures. Micelles through random cosubstitution with PEG and hydrophobic ethyl tryptophan units have been loaded with the anticancer drug DOX and investigated as nanomedicines [141]. Through tuning of the structural properties, polymersomes can also be prepared with ethyl-p-benzoate as the hydrophobic component [142]. The polymersomes are capable of encapsulating the chemotherapeutic DOX·HCl

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and are shown to improve its therapeutic effect against tumor cells (IC50 values were 17-fold lower than the free drug) due to an increased cellular uptake of the nano-sized carrier. These polymersomes have since undergone in vivo investigations for use as carriers for the anticancer drug DOX and are proposed for the treatment of breast cancer (Figure 3.24) [143]. The authors noted a high payload and capacity encapsulation efficiency of the polymersomes. DOX is transported in the lipophilic bilayer while it is proposed that the more hydrophilic DOX·HCl salt is transported in the polymersome interior cavity. The DOX·HCl salt performed better during in vivo studies in mice, perhaps not unexpectedly due to its better postrelease aqueous solubility. This formulation could be a clinically useful approach for the delivery of DOX, demonstrating similar efficacy but less toxicity than comparative administration of the free drug (Figure 3.24).

PEP-5-D 800

Saline DOX·HCI (2 mg/kg) PEP-5-DH (2 mg/kg) PEP-5-D (2 mg/kg)

Tumor volume (mm3)

700 600

D

Hydr ialysis opho b DOX ic (D) c PEP-5-DH hili rop ) Hyd CI (DH ·H DOX Dialysis

500 400 300 200

PEP graft copolymer

100 0

10

20

30

Time (day)

Figure 3.24: Schematic depiction of the preparation of graft polyphosphazene-based polymersomes. The systems can be loaded with hydrophobic DOX, as well as its more hydrophilic HCl salt. The DOX·HCl-loaded polymersome (PEP-5-DH) was able to suppress MCF-7 xenograft tumors in nude mice with a comparable efficacy to that of the free drug. Reproduced with permission from J. Xu, Q. Zhao, Y. Jin and L. Qiu, Nanomedicine: Nanotechnology, Biology and Medicine, 2014, 10, 2, 349. ©2014, Elsevier [143].

Similar polymersomes have also been prepared with a pH-triggered release profile designed to selectively release its payload after endocytosis. These were prepared through incorporation of N,N-diisopropylethylenediamine groups, which become protonated at lower pH values causing disruption to the vesicle and thus payload release [144]. Polyphosphazene-based polymersomes have also been successfully applied to microRNA therapy (see also Section 3.3.2) as a potential new therapeutic option for chemotherapeutic-resistant human lung cancer [145] Cancer immunotherapy has been one of the biggest breakthroughs in cancer research in the last decade [146], but for safe, systemic delivery of immune response

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87

modifiers (IRM), nanocarriers are of importance. In this context, self-assembled polyphosphazenes have also been shown to be outstanding carriers for this application. For example, Qiu et al. constructed polymersomes for the delivery of IL-2 plasmid cytokines [147]. In vivo experiments showed significant suppression of tumor growth in BALB/c mice bearing CT-26 colon carcinoma. Endosomal targeted release of macromolecular prodrugs based on amphiphilic polyphosphazenes have also been shown to undergo self-assembly and formation of nanoaggregates (Figure 3.25) [148]. The polymers were able to selectively release IRM molecules in the endosomes of cancer cells. The result was a strong activation of T-cells in murine splenocytes as shown by increased proliferation and expression of the IL‐2 receptor on CD8+ T-cells accompanied by strong IFN-γ release. These macromolecular prodrugs could thus form the basis of novel cancer vaccines [148].

H2O

Polymer

Intramolecular self-assembly

Intermolecular self-assembly

Figure 3.25: Drug-loaded amphiphilic polyphosphazenes undergo self-assembly and formation of nanoaggregates. Reproduced with permission from Aichhorn, S. et al., Chemistry – A Eur. J. 2017, 23 (70), 17,721 [148].

3.7 Thermosensitive poly(organo)phosphazenes 3.7.1 Thermosensitive polymers Thermosensitive polymers, that is, materials that can undergo conformational changes during a temperature-driven response are of interest in a number of modern-day applications. When the change is from a more soluble to a less soluble state, the polymer is said to have a lower critical solution temperature (LCST), whereby an entropically driven expulsion of solvent above the LCST causes collapse of the polymer chains leading to precipitation and/or gelation (Figure 3.26). Of particular interest for biomedical applications are polymers that undergo an LCST transition in aqueous environments at near or below body temperature, allowing them to be useful as triggered materials for controlled drug-release devices [149], as well as matrices for tissue engineering [150] and biomimetic materials [151]. An essential property for materials that show thermosensitive behavior in aqueous environments is the amphiphilicity of the polymers

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Hydrophilic

Hydrophobic

H O H

O

H

O H

H

H

H

O

O

H

H

H H O H H HH O O O

H O H H H H O O HH H H H H H HO H H O O H H H O H H O H O O H H H H H O O H O O H H O H H H H H O H O H H O H H H O O H H H H O H O H H O H H O H H H H O H

O

Loss of bound water

Change in temperature or pH

H

H O H O H

Hydrated polymer chains

H O

H

H

O

O H

H

H

O H

H

H

H

O H

O H

O H H

H O H H

O H

Collapse of polymer

Figure 3.26: Schematic representation of a thermosensitive polymer in water. Upon increasing the temperature above the LCST, entropic loss of water causes collapse and agglomeration of the polymer chains. Reproduced with permission from S. Pennadam, K. Firman, C. Alexander and D. Gorecki, Journal of Nanobiotechnology, 2004. 2, 1, 8. ©2004, Springer [155].

used, namely the ratio of hydrophobic to hydrophilic proportions, also known as the hydrophilic–lipophilic balance (HLB). If this can be precisely controlled, polymers can be designed to undergo sharp transitions at the required temperature. One of the oldest and well-known thermosensitive polymers is poly(N-isopropyl acrylamide) (PNIPAm), consisting of hydrophilic (amide) and hydrophobic (isopropyl) groups on an aliphatic carbon backbone. PNIPAm has well-investigated LCST properties [152] and has been proposed for a host of applications, including its use in biomedicine [153]. Another commonly used group include pluronics [154], also known under the nonproprietary name “poloxamers,” which commonly consist of triblock copolymers of PEO and PPO, the ratio of which determines the HLB.

3.7.2 Thermosensitive polyphosphazene drug carriers Cosubstitution of the poly(organo)phosphazene backbone with the required hydrophobic and hydrophilic substituents is a relatively simple task rendering this group of polymers ideal for the preparation of amphiphilic thermosensitive materials. Indeed, simple grafting of PNIPAm [156] or PEO–PPO copolymers [157] onto a polyphosphazene backbone has been shown to provide hybrid, degradable, thermosensitive poly(organo)phosphazenes. A wide variety of amphiphilic poly(organo)phosphazenes with thermosensitive properties have in fact been reported for many biomedical applications, the most important being detailed in the following section.

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89

Some of the micellar and polymersome structures based on poly(organo)phosphazenes discussed in Section 3.6, which by their nature are amphiphilic, have also been reported to show thermosensitive behavior, that is, to aggregate into nanoscale structures upon surpassing their LCST in water. For example, PNIPAm-grafted poly(organo)phosphazenes have been shown to have a temperature-triggered self-aggregation to micellar structures with an LCST around 30 °C [158]. Furthermore, it has, for example, been shown that at lower temperatures the hydrophobic drugs can be loaded and have a prolonged plasma lifetime and a slow, diffusion-controlled release.

3.7.3 Injectable hydrogels A significant amount of work has also been carried out in the development of poly(organo)phosphazene-based injectable hydrogels, that is, polymers that form highly viscous materials or gels due to noncovalent interactions above the LCST, with particular interest in their application as injectable drug depot devices. One way this can be achieved is through polyphosphazene cosubstitution with hydrophilic oligomeric PEG units and hydrophobic amino acid esters [159–161] (Figure 3.27). The proportion of incorporated amino acid ester side groups can be used to fine-tune the properties of the resulting polymers, for example, determining the amphiphilicity

O O a

NH [N

N

P ( NHR )

O

HN P ]

CH3 c

n

NH

b

O O

AMPEG 550 : m=11 AMPEG 750 : m=16

a

a+b+c=2 CH3

NHR= NH

m

CH2 COOCHCOOCH2CH3 GlyLacOEt

O

O

O

NH CH2 CONHCH2COH3 NH CH2 COH GlyGlyOH

GlyOH

Figure 3.27: Hydrophilic oligomeric PEG units in combination with hydrophobic amino acid esters lead to grafted poly(organo)phosphazenes with interesting LCST properties. AMPEG, α-amino-ωmethoxy-polyethylene glycol. Reproduced with permission from M-R. Park, C-S. Cho and S-C. Song, Polymer Degradation and Stability, 2010, 95, 6, 935. ©2010, Elsevier [159].

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and thus LCST of the polymers. Furthermore, gelation of the polymers above the LCST would appear to be imparted by hydrophobic interactions between the sidechain fragments of amino acid esters in aqueous solutions; hence, they also have a significant impact on the viscosity and modulus of the resulting gel. As may be expected, hydrolysis rates and thus degradability of the thermosensitive poly(organo)phosphazenes can also be tailored by the type and amount of amino acid ester side groups. The hydrolysis rate of these polymers can also be enhanced by cosubstitution with depsipeptide esters [161], a commonly applied tactic for other applications discussed in detail in Chapter 4. The bioerosion profiles have been investigated in detail both in vitro and in vivo, and while the more hydrophilic polymers underwent bioerosion by dissolution, not degradation, the more hydrophobic gels underwent hydrolytic degradation with the side group depsipeptide accelerating hydrolysis rates compared with the nonsubstituted polymer under physiological conditions [162]. As would be expected, even faster hydrolysis rates were observed for polymers with free carboxylic acid groups (see Chapter 2 for a detailed discussion on the factors affecting degradation rates). Some of the early poly(organo)phosphazenes designed as injectable hydrogels suffered from a weak mechanical gel strength. If used for drug depot devices, for example, this has the disadvantage of faster dissolution after gelation. Several attempts have been made to overcome this via the combination of thermosensitive gelation with a postgelation chemical cross-linking. This has been achieved, for example, for thiolated thermosensitive poly(organo)phosphazenes, which can undergo a reductive chemical cross-linking after thermogelling [163]. Alternatively, the cosubstitution of poly(organo)phosphazenes with methacrylate groups [164] or acrylate functional groups [165] gives rise to photo cross linkable polymers. After thermogelling, irradiation of the gel produces a chemically cross-linked system with improved mechanical properties compared with gelation alone. Thermosensitive poly(organo)phosphazenes can also be prepared using a cyclic trimer as a basis. The region and structural control that is achievable results in different properties to that of the linear variants, for example, a more rapid and responsive phase transition is reported to be possible [166]. Indeed, the cosubstitution of cyclotriphosphazene with PEG oligomers and hydrophobic Pt prodrugs has been used to prepare thermosensitive conjugates for local intratumoral delivery of anticancer agents. The dissolved polymer–drug conjugates are shown to solidify after injection due to their LCST being below body temperature and thus act as a depot for the sustained release of the Pt conjugate. Similarly, thermosensitive cyclotriphosphazenePt-1,2-diaminocyclohexane (DACH) conjugates have also been successfully prepared (Figure 3.28). These conjugates not only showed promising cytotoxicity against a number of cell lines but were also reported to be hydrolytically degradable [167]. These platinum drug carriers are examples of the many, similar, injectable and biodegradable poly(organo)phosphazene hydrogels that have been investigated in recent years as delivery systems for chemotherapeutics. The anticancer drug DOX is

3.7 Thermosensitive poly(organo)phosphazenes

91

NH2

H2N

Pt O

O

O

O O O

O

O O

O H2N

Pt

NH O O

O

P

N H

NH2

N O

NH

HN N P

P

O

O

O

N NH O O

HN O

H2 N

O Pt

O

O

N H2

O

Figure 3.28: Example of a Pt-DACH-thermosensitive cyclotriphosphazene-injectable drug depot.

another drug that has been well investigated for its intratumoral delivery [168] (Figure 3.29). DOX can be noncovalently loaded into the hydrogel and was shown to provide sustained and localized drug distribution in tumor tissue for up to 49 days, with a twofold reduction in systemic drug exposure [169], minimizing toxicity but still inhibiting tumor growth compared with administration of the free drug. Alternatively, DOX can be covalently linked via an amide bridge [170]. The covalent linkage has the advantage of a slower and more sustained release. The same tactic of covalently binding the payload via an amide bridge to the hydrolytically degradable thermosensitive poly(organo)phosphazene has also been successfully applied to the anticancer drug PTX [170]. The covalent conjugate showed excellent antitumor activity in vivo and was observed to be as effective as the free drug for the suppression of tumor growth after local injection. Achieving sufficient dosage was proved to be difficult with the covalently bound drugs; hence, the authors suggested that a combination of simple physical mixing of free PTX with the polymer–PTX conjugate may be required to obtain an optimally effective therapeutic dose. Indeed, subsequent work by the same authors showed that a purely noncovalently bound mixture can accommodate a large dose of the drug, but still reduce systemic exposure by limiting the biodistribution to mainly inside the tumor tissue.

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1,800 1,600

(37 °C, 1,600)

Viscosity (Pa.s)

1,400 1,200 1,000 800 (37 °C, 430)

600 400 200 0

(37 °C, 175)

10

20

(a)

(b)

30

40

50

60

Temperature (°C)

10 °C

37 °C

Figure 3.29: Viscosity profile showing the reversible gelation behavior of a DOX-loaded thermosensitive poly(organo)phosphazene at various concentrations. Reproduced with permission from G.D. Kang, S.H. Cheon and S.C. Song, International Journal of Pharmaceutics, 2006, 319, 1–2, 29. ©2006, Elsevier [168].

Similar thermosensitive poly(organo)phosphazenes have also been assessed in combination with camptothecin [171], 5-fluorouracil [172] and silibinin [173], as well as the breast cancer drug 2-methoxyestradiol [174], thus proving to be a versatile, effective and safe delivery system. Furthermore, as well as the delivery of standard, lipophilic chemotherapeutic agents, the approach has been extended to other, noncancer therapies, for example, injectable polyplex hydrogels for the localized and long-term delivery of siRNA (discussed in Section 3.3.2) and growth hormone delivery. Deprotection of some of the ethyl ester groups in these polymers leads to free carboxylic acid groups and thus anionic polymers that can be used to form polyplexes with cationic species. Alternatively, the substitution of some carboxylic acid moieties with cationic groups gives rise to cationic thermosensitive polymers that are suitable for the loading of negatively charged proteins. This is reported, for example, with the human growth hormone (hGH), which is anionic under physiological conditions [175]. A balance of interactions provides the optimum sustained-release profiles, which can be readily achieved using this flexible polyphosphazene-based system [176]. A careful balance between binding ability and

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subsequent hormone release is essential, with strong binding leading to an incomplete payload release. For this reason, dual ionic systems have also been developed, with anionic poly(organo)polyphosphazenes binding to a previously prepared, slightly positive, complex of the hormone and protamine sulfate [177]. This way it was possible to achieve both a controlled and sustained release as well as a satisfactory bioavailability of hGH. As well as drug-delivery and theranostic applications (see below), bioerodible, injectable hydrogels also hold potential as scaffolds for tissue engineering. Indeed, thermosensitive poly(organo)phosphazenes have also been investigated for their ability to support cell differentiation [178]. Tissue engineering using degradable poly(organo)phosphazenes is a topic dealt with in more detail in Chapter 4.

3.8 Theranostics Polymer theranostics aim to combine therapeutics and imaging into single polymer-based systems [179]. The ability to load multiple active agents opens the door for the injectable hydrogels described in Section 3.7.3 to be used in theranostics. An example of this is the loading of cobalt ferrite (CoFe2O4) nanoparticles into thermosensitive polyphosphazene-based hydrogels [180]. This was developed to prepare a contrast platform, utilizing the slow release of the magnetic nanoparticles from the injected hydrogel to be detected by magnetic resonance imaging (MRI). Furthermore, the combination of this diagnostic tool with therapeutic drugs gives rise to theranostics, for example, in combination with the anticancer agent PTX [181]. The polymer–nanoparticle combination has also been proposed and investigated for its use as a therapy for malignant brain tumors, by physically loading the brain cancer drug SN-3 into the injectable hydrogel (Figure 3.30). Such a tactic is shown to be highly promising, potentially eliminating the requirement for invasive surgery. Indeed the multifunctional nature of polyphosphazenes lends itself to the cofunctionalization with labels, as has been reported by Sohn and coworkers who conjugated docetaxel via pH-cleavable linkers only pegylated polyphosphazenes [182]. The resultant polyphosphazene–DTX conjugate named “Polytaxel” by the authors could easily be labeled with fluorescent labels to give a theranostic polymer which, as well as showing long blood circulation times, was demonstrated to accumulate preferentially in tumor tissue while being cleared from all major organs after approximately 6 weeks postinjection. This highly promising theranostic polymer by the authors has now entered preclinical studies. While gold AuNPs are excellent contrast agents for CT imaging, among other biomedical applications, there exists a dilemma in that larger sizes (~50–100 nm) are required for extended blood circulation times, but such particles cannot be used safely as they cannot be excreted by the kidneys and hence may accumulate in the

Gel

Sol

Body temp.

Reversible Sol-to-gel phase transition

Room Temp.

Physical mixing

7-Ethyl-10-hydroxycamptothecin

Pre-scan 1 day after injection

8 days after injection

15 days after injection

Long-team biodegradation and sustained drug release

MRI-monitored long-term therapy(MLT)

Glycyl lactate ethyl ester AMPEG750

Phosphazene back bone

Anticancer drug

Figure 3.30: MRI-monitored long-term therapeutic hydrogel (MLTH) system for brain tumors without surgical resection. Reproduced with permission from J. Il Kim, B. Kim, C. Chun, S.H. Lee and S-C. Song, Biomaterials, 2012, 33, 19, 4836. ©2012, Elsevier [180].

Stereotactic injection

Thermosensitive/magnetic poly(organophosphazene) hydrogel system

MRI-monitored long-term therapeutic hydrogel (MLTH) system

94 Chapter 3 Nanomedicine

3.9 Conclusion

95

body. Biodegradable polyphosphazenes have been demonstrated to offer a solution to this dilemma, whereby small, excretable AuNPs are encapsulated with PCPP to give nanoagglomerates (~100 nm). These larger nanoparticles are excellent contrast agents, but can subsequently disassemble into for their smaller components for renal excretion (Figure 3.31) [183].

Sub-5 nm AuNP

Biodegradable polyphosphazene

AuNP encapsulated in polymer nanoparticle

Polymer degradation in vivo leads to AuNP release for excretion

Figure 3.31: Sub-5 nm god nanoparticles are bound with PCPP to form larger nanoparticles in the region of 100 nm suitable for CT imaging, which can also degrade allowing for renal excretion of the gold nanoparticles. Reproduced with permission from Cheheltani, R.; Ezzibdeh, R. M.; Chhour, P.; Pulaparthi, K.; Kim, J.; Jurcova, M.; Hsu, J. C.; Blundell, C.; Litt, H. I.; Ferrari, V. A.; Allcock, H. R.; Sehgal, C. M.; Cormode, D. P., Tunable, biodegradable gold nanoparticles as contrast agents for computed tomography and photoacoustic imaging. Biomaterials 2016, 102, 87–97 ©2016, American Chemical Society [183].

The same tactic using the polyphosphazenes with oxidation-stimulated degradation (described in Section 2.2.4) have recently been used to provide signaling for ROS, a hallmark of inflammation and cancer [184]. The AuNPs displayed strong photoacoustic signals, thanks to interparticle plasmon coupling when loaded in the polyphosphazene spheres. However, the PA signal decreased significantly, as the nanoagglomerates disassembled upon exposure to ROS. This effect can be used for biomolecule imaging as the concentration of ROS correlated with the decrease in PA signal. Since the CT signal is independent on the local environment of the gold, this was used in combination with PAI to permit the imaging of endogenous ROS [184].

3.9 Conclusion The many examples for the use of polyphosphazenes in nanomedicine presented in this chapter give a clear indication to the wide-ranging capability of this group of polymers and their potential in this field. These mostly look to utilize the inherent unique combination of properties offered by the inorganic polyphosphazene backbone, including the flexible backbone for solubility and complexation with

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biomolecules, the lability and high density of exchangeable functional groups for drug loading and property optimization, as well as the hydrolytic degradability of the backbone for tailored release and/or safe parenteral administration. Furthermore, due to the wide range of organic side groups that can be attached, a broad spectrum of chemical, physical and biological properties are achievable, ranging from water soluble to amphiphilic to hydrophobic, and a full range of surface charges from cationic, neutral or anionic, depending on the desired application. Furthermore, once the desired characteristics have been determined, fine-tuning is also possible to achieve, for example, the preferred LCST temperature of the thermosensitive polymers, charge of polyplexes or the required degradation rate of the nanomedicine. This structural variability can also be disadvantageous in some aspects, meaning broad structure/property relationships cannot be applied to poly(organo)phosphazenes due to the stark differences in polymer properties, depending on the attached side groups. For example, biocompatibility has been proven for many, but small differences can have a large impact meaning every new polymer must be taken on merit. The degradation products of the polyphosphazene backbone have been proven many times over to be essentially nontoxic, although this is still clearly dependent on the organic components of any poly(organo)phosphazene and thus are predictable only if known and studied organic components are applied. The success of PCPP in clinical trials bodes well for other polyphosphazenebased nanomedicines, as does the multitude of data from in vitro and animal studies presented. These promising results, coupled with recent advances in synthetic approaches (see Chapter 1), should mean it is possible to provide wellcharacterized reproducible materials required for a successful transition to the clinic.

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[145] Peng Y, Zhu X, Qiu L. Electroneutral composite polymersomes self-assembled by amphiphilic polyphosphazenes for effective miR-200c in vivo delivery to inhibit drug resistant lung cancer. Biomaterials 2016;106:1–12. [146] Couzin-Frankel J. Cancer Immunotherapy. Science 2013;342:1432–1433. [147] Gao M, Zhu X, Wu L, Qiu L. Cationic polyphosphazene vesicles for cancer immunotherapy by Efficient in Vivo Cytokine IL-12 plasmid delivery. Biomacromolecules 2016;17:2199–2209. [148] Aichhorn S, Linhardt A, Halfmann A, et al. A pH-sensitive macromolecular prodrug as TLR7/8 targeting immune response modifier. Chem Eur J 2017;23:17721–17726. [149] Schmaljohann D. Thermo- and pH-responsive polymers in drug delivery. Adv Drug Delivery Rev 2006;58:1655–1670. [150] Chan G, Mooney DJ. New materials for tissue engineering: towards greater control over the biological response. Trends Biotechnol 2008;26:382–392. [151] Kouwer PHJ, Koepf M, Le Sage VAA, et al. Responsive biomimetic networks from polyisocyanopeptide hydrogels. Nature 2013;493:651–655. [152] Schild HG. Poly(N-isopropylacrylamide): experiment, theory and application. Prog Polym Sci 1992;17:163–249. [153] Abulateefeh SR, Spain SG, Aylott JW, Chan WC, Garnett MC, Alexander C. Thermoresponsive polymer colloids for drug delivery and cancer therapy. Macromol Biosci 2011;11:1722–1734. [154] Batrakova EV, Kabanov AV. Pluronic block copolymers: Evolution of drug delivery concept from inert nanocarriers to biological response modifiers. J Controlled Release 2008;130: 98–106. [155] Pennadam S, Firman K, Alexander C, Gorecki D. Protein-polymer nano-machines. Towards synthetic control of biological processes. J Nanobiotechnology 2004;2:8. [156] Zhang JX, Qiu LY, Wu XL, Jin Y, Zu KJ. Temperature-triggered nanosphere formation through self-assembly of amphiphilic polyphosphazene. Macromol Chem Phys 2006;207:1289–1296. [157] Wilfert S, Iturmendi A, Henke H, Brüggemann O, Teasdale I. Thermoresponsive polyphosphazene-based molecular brushes by living cationic polymerization. Macromol Symp 2014;337:116–123. [158] Zhang JX, Qiu LY, Zhu KJ, Jin Y. Thermosensitive micelles self-assembled by novel N-isopropylacrylamide oligomer grafted polyphosphazene. Macromol Rapid Commun 2004;25: 1563–1567. [159] Park M-R, Cho C-S, Song S-C. In vitro and in vivo degradation behaviors of thermosensitive poly(organophosphazene) hydrogels. Polym Degrad Stabil 2010;95:935–944. [160] Lee BH, Lee YM, Sohn YS, Song SC. A thermosensitive poly(organophosphazene) gel. Macromolecules 2002;35:3876–3879. [161] Lee BH, Lee YM, Sohn YS, Song S-C. Thermosensitive and hydrolysis-sensitive poly (organophosphazenes). Polym Int 2002;51:658–660. [162] Lee BH, Song SC. Synthesis and characterization of biodegradable thermosensitive poly (organophosphazene) gels. Macromolecules 2004;37:4533–4537. [163] Potta T, Chun C, Song SC. Chemically crosslinkable thermosensitive polyphosphazene gels as injectable materials for biomedical applications. Biomaterials 2009;30:6178–6192. [164] Potta T, Chun C, Song S-C. Dual cross-linking systems of functionally photo-cross-linkable and thermoresponsive polyphosphazene hydrogels for biomedical applications. Biomacromolecules 2010;11:1741–1753. [165] Potta T, Chun C, Song S-C. Rapid photocrosslinkable thermoresponsive injectable polyphosphazene hydrogels. Macromol Rapid Commun 2010;31:2133–2139. [166] Lee SB, Song SC, Jin JI, Sohn YS. Thermosensitive cyclotriphosphazenes. J Am Chem Soc 2000;122:8315–8316.

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[167] Cho YW, Choi M, Lee K, Song S-C. Cyclotriphosphazene-Pt-DACH conjugates with dipeptide spacers for drug delivery systems. J Bioact Compat Polym 2010;25:274–291. [168] Kang GD, Cheon SH, Song SC. Controlled release of doxorubicin from thermosensitive poly (organophosphazene) hydrogels. Int J Pharm 2006;319:29–36. [169] Al-Abd AM, Hong K-Y, Song S-C, Kuh H-J. Pharmacokinetics of doxorubicin after intratumoral injection using a thermosensitive hydrogel in tumor-bearing mice. J Controlled Release 2010;142:101–107. [170] Chun C, Lee SM, Kim CW, et al. Doxorubicin-polyphosphazene conjugate hydrogels for locally controlled delivery of cancer therapeutics. Biomaterials 2009;30:4752–4762. [171] Cho J-K, Chun C, Kuh H-J, Song S-C. Injectable poly(organophosphazene)-camptothecin conjugate hydrogels: Synthesis, characterization, and antitumor activities. Eur J Pharm Biopharm 2012;81:582–590. [172] Lee SM, Chun CJ, Heo JY, Song SC. Injectable and thermosensitive poly(organophosphazene) hydrogels for a 5-Fluorouracil delivery. J Appl Polym Sci 2009;113:3831–3839. [173] Cho JK, Park JW, Song SC. Injectable and biodegradable poly(organophosphazene) gel containing silibinin: Its physicochemical properties and anticancer activity. J Pharm Sci 2012;101:2382–2391. [174] Cho J-K, Hong K-Y, Park JW, Yang H-K, Song S-C. Injectable delivery system of 2methoxyestradiol for breast cancer therapy using biodegradable thermosensitive poly(organophosphazene) hydrogel. J Drug Targeting 2011;19:270–280. [175] Park M-R, Chun C, Ahn S-W, Ki M-H, Cho C-S, Song S-C. Sustained delivery of human growth hormone using a polyelectrolyte complex-loaded thermosensitive polyphosphazene hydrogel. J Controlled Release 2010;147:359–367. [176] Seo B-B, Park M-R, Chun C, Lee J-Y, Song S-C. The biological efficiency and bioavailability of human growth hormone delivered using injectable, ionic, thermosensitive poly(organophosphazene)-polyethylenimine conjugate hydrogels. Biomaterials 2011;32: 8271–8280. [177] Park M-R, Seo B-B, Song S-C. Dual ionic interaction system based on polyelectrolyte complex and ionic, injectable, and thermosensitive hydrogel for sustained release of human growth hormone. Biomaterials 2013;34:1327–1336. [178] Yoon J-Y, Park K-H, Song S-C. A thermosensitive poly(organophosphazene) hydrogel for injectable tissue-engineering applications. J Biomater Sci Polym Ed 2007;18:1181–1193. [179] Krasia-Christoforou T, Georgiou TK. Polymeric theranostics: using polymer-based systems for simultaneous imaging and therapy. J Mater Chem B 2013;1:3002–3025. [180] Il Kim J, Kim B, Chun C, Lee SH, Song S-C. MRI-monitored long-term therapeutic hydrogel system for brain tumors without surgical resection. Biomaterials 2012;33:4836–4842. [181] Kim JI, Lee BS, Chun C, Cho J-K, Kim S-Y, Song S-C. Long-term theranostic hydrogel system for solid tumors. Biomaterials 2012;33:2251–2259. [182] Jun YJ, Park JH, Avaji PG, et al. Design of theranostic nanomedicine (II): synthesis and physicochemical properties of a biocompatible polyphosphazene-docetaxel conjugate. Int J Nanomedicine 2017;12:5373–5386. [183] Cheheltani R, Ezzibdeh RM, Chhour P, et al. Tunable, biodegradable gold nanoparticles as contrast agents for computed tomography and photoacoustic imaging. Biomaterials 2016;102:87–97. [184] Bouché M, Pühringer M, Iturmendi A, et al. Activatable hybrid polyphosphazene-AuNP nanoprobe for ROS Detection by bimodal PA/CT imaging. ACS Appl Mater Interfaces 2019;11: 28648–28656.

Chapter 4 Tissue engineering 4.1 Introduction to tissue engineering For the replacement of tissue, such as skin or bones, or even organs, cells have to be induced to generate complex structures by using prefabricated scaffolds. These scaffolds are applied typically as three-dimensional (3D) structures for the directed cultivation of tissue-forming cells. The use of polymers as scaffold materials in tissue engineering (TE) is becoming more and more of interest since polymers allow the preparation of a variety of structures with pore sizes fitting perfectly to the selected cell types [1–4]. Table 4.1 gives an overview of the different possibilities of attaining polymeric scaffolds for the later application in TE [5]. From several processes, one can choose the appropriate method of fabricating the polymer matrices, depending on the specific requirements of the products, and the demand of controllability of the method. Typical processes may lead to fibers, films, foams or membranes using, for instance, phase separation, solvent casting, lamination, templating and melt-molding procedures; in some cases, composite materials are used. Problems with the scaffolds are often based on potentially harmful solvent residues, lack of mechanical strength and difficulties in controlling the desired morphologies of the 3D structures. It is a prerequisite that after implantation of the newly established tissue into an organism, the scaffold, as a foreign material, should show clear bioerosion and bioresorption after a short period of time. A few polymers exhibit this behavior, such as polyesters like poly(lactic acid) (PLA), poly(glycolic acid) or their copolymers poly (lactic-co-glycolic acid) (PLGA), however, leading to acidic pH during decomposition. Polyphosphazenes, however, are known to be converted into harmless phosphates and ammonia salts resulting in near-neutral pH conditions and, together with residues of organic side arms, should be excreted easily from the body. Furthermore, the tailorable properties of polyphosphazenes lead to defined bioresorption kinetics, pore sizes and additional chemical functionalities. Thus, polyphosphazenes can be considered as extraordinary materials for the synthesis of scaffolds to be applied in TE [3, 4, 6–10].

4.2 Architecture of polyphosphazene scaffolds for tissue engineering TE scaffolds made from polymers are available in different formats as already mentioned in Section 4.1. Polymeric scaffolds can be based on fibers, films, foams, membranes and 3D bulk materials, using simple linear polymers with or without

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Table 4.1: Processing of polymer scaffolds for applications in tissue engineering. Adapted from [1]. Processing

Advantages

Disadvantages

Phase separation





Nondecreased activity of the molecule

– Solvent casting and particulate leaching

– – –

Controllable porosity, up to 93% Independent controllability of porosity and pore size Crystallinity can be tailored



Difficult to control precisely scaffold morphology Potential harmful solvent residues



Limit to membranes up to thickness of 3 mm Lack of required mechanical strength for the load-bearing tissues Potential harmful solvent residues



Fiber felts

– –

Easy process High porosity of scaffold



Lack of structural stability

Fiber bonding



High porosity of scaffold

– – –

Limit application to other polymers Lack of mechanical strength Potential harmful solvent residues

Membrane lamination



3D matrix

– –

Lack of mechanical strength Potential harmful solvent residues

Melt molding



Independent controllability of porosity and pore size Macroscopic shape control



High temperature required for nonamorphous polymer

– Polymeric/ ceramic fiber composite foam

– –

Superior compressive strength Independent controllability of porosity and pore size



Potential harmful solvent residues

High-pressure processing



No organic solvents required

– –

Mostly nonporous surfaces Closed-pore structure inside the polymer matrix

Hydrocarbon templating

– –

No limit for thickness Enhanced controllability of pore structure, porosity, etc.



Potential harmful solvent residues

cross-linking, leading to elastomeric or even thermoplastic structures. In general, the usage of polyphosphazenes should also allow the realization of similar concepts. In the past few years, more and more examples are given in the literature for the fabrication of polyphosphazene-based scaffolds for TE. It is even expected that such scaffolds will be obtainable via 3D-bioprinting with polyphosphazene containing bioinks [8].

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4.2.1 Formats 4.2.1.1 Linear polyphosphazenes One way to introduce linear polyphosphazenes into the world of TE is the combination with other materials, in other words, to form blends [11]. For bone TE, the combination of polyphosphazenes with hydroxyapatite nanoparticles (nHAp) is an example, leading to, for example, cylindrical- or disk-shaped forms after sintering the composite microspheres (Figures 4.1a and b). Nukavarapu and coworkers demonstrated that osteoblast cells adhere and grow on such composite scaffolds based on poly[bis(ethyl phenylalaninato)phosphazene] (PNEPhA) (Figure 4.1c) [12].

1 cm

500 μm

250 μm

Figure 4.1: Macro, micro and nanostructure of PNEPhA 20 nHAp composite microsphere scaffolds. (a) Optical image showing cylindrical (10 mm length and 4.5 mm diameter) and disk-shaped scaffolds (2 mm thick and 8 mm diameter) fabricated using the dynamic solvent sintering method. Cylindrical scaffolds were used for mechanical testing and disk-shaped scaffolds were used for porosity and in vitro cell studies. (b) Scanning electron microscopy (SEM) showing the microstructure of the scaffolds, where the adjacent microspheres are fused via the dynamic solvent sintering method. (c) Cytoskeletal actin distribution of primary rat osteoblast cells grown on a composite microsphere matrix for 12 days. Reproduced with permission from S.P. Nukavarapu, S.G. Kumbar, J.L. Brown, N.R. Krogman, A.L. Weikel, M.D. Hindenlang, L.S. Nair, H.R. Allcock and C.T. Laurencin, Biomacromolecules, 2008, 9, 7, 1818. ©2008, American Chemical Society [12].

Alternatively, electrospinning of the polymer solution as well as their blends can be used for the formation of nonwoven nanofiber (NF) mats. Carampin et al. formed flat and tubular biocompatible poly[(ethyl phenylalanato)1.4 (ethyl glycinato)0.6 phosphazene-based scaffolds by electrospinning [13]. Greish et al. prepared biodegradable alanine-substituted polyphosphazene nanofibrous scaffolds by electrospinning [14]. Deng and coworkers have combined PLGA with poly[(glycine ethyl glycinato)1 (phenylphenoxy)1 phosphazene] and, after electrospinning the cosolution, rolled up 3D fiber-layered concentric structures of the fiber-based mats [15]. Whereas Figure 4.2 explains the general concept of this approach, Figure 4.3 shows as an example

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Central cavity Fiber layer Ln+1 Ln

C

Central cavity Fiber Gap between layer fiber layers L C Ln+1n

Intermolecular hydrogen bonding

PLAGA Electrospinning

Controlled rolling

Ln+1 Ln C

Structure shrinking

Gap between fiber layers

Ln+1 Ln C

PPHOS Miscible blend

3D fiber-layered concentric structure 2D Blend nanofiber sheets (~250 μm thick)

3D biomimetic scaffold

C:central cavity Ln:fiber layer Ln+1:fiber layer adjacent to layer Ln

Figure 4.2: Schematic of 3D biomimetic scaffold design and fabrication. Intermolecular hydrogen bonding interactions between polyphosphazene and PLGA result in a miscible blend system. Electrospinning of the polymer blend solution creates a nonwoven NF mat. Rectangular polymer sheets are then cut from the NF mat (∼250 μm thick) and rolled up into a 3D fiber-layered concentric structure in a controlled fashion. Finally, incubation in the cell media drives away the air within the structure and leads to structure shrinkage resulting in the formation of a 3D intact nanostructured scaffold. During shrinkage, the scaffold structure including the gap space between the fiber layers (Ln and Ln + 1) is significantly reduced. However, the dimensional stability of the open central cavity (C) is maintained to encourage nutrient transport. Reproduced with permission from M. Deng, S.G. Kumbar, L.S. Nair, A.L. Weikel, H.R. Allcock and C.T. Laurencin, Advanced Functional Materials, 2011, 21, 2641. ©2011, Wiley-VCH [15].

results of the 3D-bone-mimicking silk-like scaffold structures. For this purpose, the authors used a Teflon rod as a core to roll it around the polyphosphazene–PLGA composite mats. Nair and coworkers employed electrospinning technology to form polyphosphazene fibers for TE purposes [16]. They found that the nature of the solvent, the diameter of the electrospinning needle, the solution concentration and the applied voltage all had significant effects on the resulting polyphosphazene fibers. For instance, a change from tetrahydrofuran (THF) to chloroform as the solvent led from highly nonuniform to uniform fibers (Figure 4.4). A specific group of functionalized polyphosphazenes was presented by Krogman and coworkers describing the synthesis of polylactide side chains on polyphosphazene backbones using the N-linked amino acids serine or threonine ethyl esters as anchors for the grafted side groups (Figure 4.5) [17]. It was found that the graft density of PLA could be governed by the concentration of the sodium/naphthalene complex which was used for the initiation of the polymerization [18].

4.2 Architecture of polyphosphazene scaffolds for tissue engineering

(a)

(b)

111

(c)

(d) 3D biomimetic scaffold (f)

350 μm

(e)

500 μm

Figure 4.3: Fabrication of polyphosphazene-PLGA 3D biomimetic scaffolds. Optical microscopy images: (a) 250 μm thick electrospun BLEND NF matrices exhibiting bead-free silk-like morphology (indicated by the arrows) with an average fiber diameter of 50–500 nm were cut into (b) rectangular strips (40 mm × 10 mm, L × W) and rolled around a 1 mm thick Teflon rod in a controlled manner to produce (c) bone-mimicking concentric structures. These concentric structures were immersed in cell culture media for 10 min to produce (d) the compact scaffold with an open central cavity. (e) and (f) SEM images at two different locations of the scaffold illustrating the morphologies of the open central cavity and fiber lamella structures of 3D biomimetic scaffolds. Reproduced with permission from M. Deng, S.G. Kumbar, L.S. Nair, A.L. Weikel, H.R. Allcock and C.T. Laurencin, Advanced Functional Materials, 2011, 21, 2641. ©2011, Wiley-VCH [15].

Honeycomb patterned films (Figure 4.6) based on linear poly(glycine ethyl ester-co-alanine ethyl ester)phosphazene were developed by Duan et al. by casting the polyphosphazene solutions on polytetrafluoroethylene sheets. Although such systems showed osteoblast cell adhesion and proliferation initially inferior to control PLGA scaffolds, these specially structured polyphosphazene materials were found to be bone-binding bioactive polymers allowing an enhancement of protein adsorption, apatite deposition as well as osteogenic differentiation [19]. Huang et al. generated degradable polyphosphazene films with photoluminescent moieties by solution-casting allowing not only the regeneration of tissue but also a long-term in vivo tracking and observing the degradation. It was shown that the degradation rate and fluorescent intensity could be tailored by changing the ratio of hydrolyzable amino acid esters to the photoluminescent side group substituents [20]. This concept was transferred to nanoparticle formats in combination with the delivery of simvastatin, a drug for improving the osteogenic differentiation of bone marrow mesenchymal stromal cells [21].

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(a)

1,000 20KV 20mmSpot: 12 Title: Comment:

10u SS: P3 Date: 08–11–2003 Time: 21:52 Filename: SAMPLE5SAMPLE5S

(b)

X1,000 20KV 26mm Spot: 9 Title: Comment:

10u SS: P3 Date: 07–15–2003 Time: 11:15 Filename: PNMP3A6PNMP3A6I

Figure 4.4: (a) SEM of electrospun poly[bis(p-methylphenoxy)phosphazene] (PNmPh) fibers from THF at a polymer concentration of 8% (w/v) at 33 kV using an 18-gauge needle, showing the formation of highly nonuniform distorted fibers. (b) SEM of electrospun PNmPh fibers from chloroform at a polymer concentration of 8% (w/v) at 33 kV using an 18-gauge needle, showing the formation of distinct uniform fibers. Reproduced with permission from L.S. Nair, S. Bhattacharyya, J.D. Bender, Y.E. Greish, P.W. Brown, H.R. Allcock and C.T. Laurencin, Biomacromolecules, 2004, 5, 6, 2212. ©2004, American Chemical Society [16].

4.2.1.2 Cross-linked polyphosphazenes Cross-linking of linear polyphosphazenes was expected to lead to a drastic change of the material’s properties, at least from a mechanical point of view, since this modification results in elastic or even nonflexible and nonsoluble scaffolds. Krogman and coworkers have further developed their concept of linking amino acids to polyphosphazene backbones to an alternative derivatization, where the serine and threonine were both protected at their N- and C-termini and linked via their free hydroxyl functions to the polyphosphazene main chain. After deprotection, the free carboxy functionalities of the amino acid side groups were able to interact with calcium ions.

4.2 Architecture of polyphosphazene scaffolds for tissue engineering

O O R

OH

NHCHC(O)OC2H5 N P n NHCHC(O)OC2H5 R

OH

CH3

O

+

O

H3C O

Na/Naphthalene THF, RT

H 10 CH3

O

R

O

113

NHCHC(O)OC2H5 N P n NHCHC(O)OC2H5 O

R O

CH3 O H 10

Polymer 4a, 4b, 4c: R = H Polymer 5a, 5b, 5c: R = CH3 Figure 4.5: Synthesis of polymers 4a−c and 5a−c that contain poly(L-lactide) grafts grown from the alcohol function of serine or threonine ethyl ester on a polyphosphazene backbone. RT, room temperature. Reproduced with permission from N.R. Krogman, A.L. Weikel, N.Q. Nguyen, L.S. Nair, C.T. Laurencin and H.R. Allcock, Macromolecules, 2008, 41, 7824. ©2008, American Chemical Society [17].

Calcium could simultaneously bind to two polymer chains resulting in a noncovalent and reversible cross-linking effect. With sodium chloride, the calcium ions could be displaced, disrupting the crosslinks and allowing the polymeric hydrogel to dissolve (Figure 4.7) [17, 18]. A completely different type of cross-linking was realized by the use of polyphosphazenes equipped with ferulic acid (hydroxycinnamic acid) as side groups. When exposed to ultraviolet light, the aliphatic double bonds were photo-crosslinked via a [2 + 2]-cycloaddition (Figure 4.8) [22]. By this means, degrees of crosslinking up to 62% were achieved after only 60 s. Investigation of the uncross-linked polymers, in terms of their susceptibility to hydrolytic degradation, led to the conclusion that with less ferulic acid and with smaller amino acid residues, a higher accessibility of the polyphosphazene backbones to hydrolytic attack was obtained, leading to faster degradation rates (5b > 6b > 7b > 8b) (Figure 4.9) [22]. In total, the degree of hydrolysis reached up to 25% after 8 weeks. However, after cross-linking, only one polymer (5b) showed a measurable mass loss with 5% after 8 weeks, whereas the other investigated polymers (6b, 7b and 8b) remained intact in this period of time. A complex system of a degradable polyphosphazene in combination with αcyclodextrin was designed to be applied as injectable photo-cross-linkable hydrogel. The cured hydrogel’s surface could be tailored from cell-philic to cell-phobic by choosing either glycine ethyl ester or monoacrylic-terminated polyethylene glycol (PEG) groups, respectively, as substituents of the polyphosphazene backbone. It was demonstrated that exemplary HeLa cells adhered better to the cell-philic

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Chapter 4 Tissue engineering

a1

a2

10μm

100μm SE

16–sep–10

10μm

WD16.2mm 5.0kV x500 100um

b1

SE

09–sep–10

WD17.9mm 5.0kV x5.0k 10um

b2

10μm

100μm SE

16–sep–10

WD15.9mm 5.0kV x500 100um

C

10μm SE

09–sep–10

d

100μm SE

WD16.8mm 5.0kV x5.0k 10um

16–sep–10

WD16.3mm 5.0kV x500 100um

100μm SE

16–sep–10

WD16.3mm 5.0kV x500 100um

Figure 4.6: SEM images of various poly(glycine ethyl ester-co-alanine ethyl ester)phosphazenes (a1), (b1), (c) and PLGA (d) films are shown. SEM images (a2) and (b2) show the cross-section morphologies of (a1) and (b1). Reproduced with permission from S. Duan, X. Yang, J. Mao, B. Qi, Q. Cai, H. Shen, F. Yang, X. Deng and S. Wang, Journal of Biomedical Materials Research, 2013, 101A, 2, 307. ©2012, Wiley Periodicals, Inc. [19].

hydrogel [23]. The same research group further developed this approach by linking citronellol to the polyphosphazene backbone, and with that, forming a hybrid hydrogel in combination with a photo-cross-linkable methacrylate gelatin including incorporated calcium phosphate. These hydrogels were found to be injectable, deformable

4.2 Architecture of polyphosphazene scaffolds for tissue engineering

115

Ca2+ R

NH2

R

– OCHCHC(O)O N

OCHCHC(O)ONa

P

N

n – OCHCHC(O)O R

NH2

P

n OCHCHC(O)ONa

NH2

R

NH2

R

NH2

NaCl Ca2+ R

NH2

– OCHCHC(O)O N

OCHCHC(O)ONa

P

N

n – OCHCHC(O)O R

P n OCHCHC(O)ONa R

NH2

NH2

Ca2+

Figure 4.7: Calcium cross-linked serine- and threonine-containing polyphosphazenes. Displacement of the cross-linking calcium ions with sodium ions resulting in disruption of the crosslinks. Reproduced with permission from N.R. Krogman, A.L. Weikel, N.Q. Nguyen, L.S. Nair, C.T. Laurencin and H.R. Allcock, Macromolecules, 2008, 41, 7824. ©2008, American Chemical Society [17].

O O

O

Polymer O

Polymer O

OCH3

OCH3

HO

Polymer

OH

UV Exposure 0–60s

O

O

OH

HO

H3CO OCH3 Polymer

O

Figure 4.8: Photo-cross-linking of ferulic-acid-containing polyphosphazenes by a [2 + 2]cycloaddition. Reproduced with permission from N.L. Morozowich, J.L. Nichol, R.J. Mondschein and H.R. Allcock, Polymer Chemistry, 2012, 3, 778. ©2012, The Royal Society of Chemistry [22].

and fatigue resistant, when tested with bone mesenchymal stromal cells, allowing cell migration and growth [24].

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O

(5b) × =1.5, y = 0.5, R =H (6b) × =1.5, y = 0.5, R =CH3 (7b) × =1.6, y = 0.4, R =CH(CH3)2 (8b) × =1.6, y = 0.4, R =CH2C6H5

OH O NP HN

OCH3 nO O y

R

100 95 90 %Mass loss

85 80

5b

75

6b

70

7b

65

8b

60 55 50 0

2

4 Weeks

6

8

Figure 4.9: (a − b) Antioxidant-containing polyphosphazenes with differing types and ratios of side groups. (c) Percent weight loss of uncross-linked polymers 5b–8b. Reproduced with permission from N.L. Morozowich, J.L. Nichol, R.J. Mondschein and H.R. Allcock, Polymer Chemistry, 2012, 3, 778. ©2012, the Royal Society of Chemistry [22].

Rothemund et al. created cross-linked polyphosphazene based scaffolds by applying thiol-ene click chemistry (Figure 4.10). For this means, the polyphosphazene was substituted with allyl moieties for further functionalization, foremost for clickreactions with the cross-linker, a trithiol. A divinylester was used as an additional component for cross-linking, offering an easy way of altering the polymer properties, especially the degradation rate, which were shown to be dependent on the ratio of the glycine-substituted polyphosphazene to the divinylester [25, 26].

4.2.2 Properties For the application of polyphosphazenes in the area of TE, the mechanical stability of such polymers is a major demand. For example, during bone formation a lasting stability is required to allow the cells to populate the scaffold homogeneously and

4.2 Architecture of polyphosphazene scaffolds for tissue engineering

(a)

(b)

117

(c)

(d) 200μm

200μm

Figure 4.10: SEM images of polymer 2 (polyphosphazene to trithiol, 51:49 wt%) (a) and polymer 5 (polyphosphazene to divinylester to trithiol, 11:34:55 wt%) (b), respectively, as well as X-ray CT images of polymer 2 (c) 3D imaging and (d) in cross section. The highly porous structure with interconnected pores and pore sizes in the range of 100–200 mm (average pore sizes 144 and 150 mm for polymer 2 and polymer 5, respectively, according to X-ray CT analysis). Reproduced with permission from S. Rothemund, T.B. Aigner, A. Iturmendi, M. Rigau, B. Husár, F. Hildner, E. Oberbauer, M. Prambauer, G. Olawale, R. Forstner, R. Liska, K.R. Schröder, O. Brüggemann and I. Teasdale, Macromolecular Bioscience, 2015, 15, 3, 351. ©2014 Wiley-VCH [25].

to grow in the desired structures. Deng and coworkers have shown that in the presence of cells the scaffolds maintain their mechanical stabilities longer compared with cell-free scaffolds in the same medium, since the growing cells compensate the loss of stability caused by erosion of the polymers [15]. The mass loss of the polyphosphazene-based scaffolds is a general phenomenon and, of course, explainable by the desired final degradability or erosion of the polymers in the absence or presence, respectively, of cells. However, during cultivation of the tissues, it is important to understand how, and how fast, the degradation or erosion takes place. For this purpose, Nykänen and coworkers investigated the change of the fiber dimensions of an electrospun polyphosphazene coupled with proline groups poly[bis(L-proline methyl ester)phosphazene] (PProP) in the absence of cells in a simulated body fluid (SBF), which biomimics the composition of human blood plasma. They clearly demonstrated the reduction of the fibers’ diameters after incubation with the SBF. After only 2 days, a significant shift toward smaller diameters was determined (Figure 4.11b) [27]. Morozowich and coworkers also investigated the effects of polyphosphazenes exposed to SBF. After linking ferulic acid as antioxidant and nucleation sites for calcium, as well as ethyl esters of amino acids as sides group to the polyphosphazene backbone, followed by solution casting, the polymers in the form of films were treated dynamically with an SBF solution for 4 weeks, that is, the solution was changed every 24 h. Polymer 2, containing ethyl glycinato side groups and a slightly lower content of ferulic acid, was found to show the fastest mineralization of monocalcium phosphate

0

(a)

200

600

800

1000 1200 0

Fiber diameter (nm)

400

number of fibers 151 x–= 794 ± 11 nm x͂ = 800 nm s = 133 nm

Before mineralization (b)

200

600

800

1000 1200 0

Fiber diameter (nm)

400

number of fibers 160 – x = 445 ± 17 nm x͂ = 435 nm s = 215 nm

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Figure 4.11: Statistical study of the diameter of PProP fibers produced via the electrospinning method with average (x-bar), median (x-tilde) and standard deviation s calculated. (a) Fibers before incubation into 1× SBF, (b) after 2 days of incubation and (c) after 2 weeks of incubation. Reproduced with permission from V.P.S. Nykänen, M.A. Puska, A. Nykänen and J. Ruokolainen, Journal of Polymer Science Part B: Polymer Physics Edition, 2013, 51, 1318. ©2013, Wiley Periodicals, Inc. [27].

Normalized appearance frequency

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monohydrate (MCPM) with the highest weight gain of 27% in 4 weeks, compared with 19% with P3, 17% with P4 and 14% with P5 (Figure 4.12) [28]. 30 P2 P3 P4

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Another important factor in TE is the potential of the scaffold to allow for a strong cell adhesion on its surface, since this is a requirement to form higher order cell structures, that is, tissues. Barrett and coworkers have screened different polyphosphazenes in terms of their cell adhesion behavior. For that purpose, they synthesized poly[bis (trifluoroethoxy)phosphazene] (TFE), poly[bis(2-(2methoxyethoxyethoxy)-phosphazene] (MEEP), poly-[(methoxyethoxyethoxy)1.0-(carboxylatophenoxy)1.0phosphazene] (PMCPP), poly[(methoxyethoxyethoxy)1.0-(cinnamyloxy)1.0phosphazene] (PMCP) and poly[(methoxyethoxyethoxy)1.0(p-methylphenoxy)1.0phosphazene] (PMPP) and patterned films of these polymers onto glass substrates. They found that the use of micropatterned films of PMCPP, PMCP and PMPP, with their positive cellular adhesive properties (+CAP), led to selective neuroblastoma cell adhesion, contrary to –CAP films of TFE and MEEP. Furthermore, the authors compared the polyphosphazene-coated slides with glass slides either uncoated or coated with PEG, indium tin oxide, polystyrene, poly(d-lysine) or collagen (biocoat) (Figure 4.13). They stated that TFE and MEEP were not significantly different from PEG, at least after 24 h. After 48 h, it was clearly

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Figure 4.13: Confluency after 24 and 48 h in culture, expressed on a percent basis for the tested materials. The whole cell culture substrate was coated with the specific cell culture material, and the confluency measurements were performed under a microscope with a 10× objective. The SK-NBE(2c) human neuroblastoma cell line was used. N = 15 for each material tested. Reproduced with permission from E.W. Barrett, M.V.B. Phelps, R.J. Silva, R.P. Gaumond and H.R. Allcock, Biomacromolecules, 2005, 6, 1689. ©2005, American Chemical Society [29].

observed that the polyphosphazenes PMCPP, PMCP and PMPP were more adherent than all the other coatings, except for the collagen-based film which was the second best performing material, PMPP [29].

4.3 Applications of polyphosphazene scaffolds in tissue engineering This section highlights the predominant applications of polyphosphazenes in TE for the cultivation of osteoblasts for bone regeneration [4, 15, 16, 30–36], as well as some examples of endothelial [16, 37] and neural TE [38]. Furthermore, citronellolcontaining cross-linked polyphosphazenes with an elastomeric character were proposed as putative scaffolds for ligament and tendon TE [39, 40].

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4.3.1 Bone tissue engineering Laurencin and coworkers generated, besides a two-dimensional (2D) format, 3D scaffolds using poly[(ethyl glycinato)(methylphenoxy)phosphazene] (PNEGmPh) in the presence of salt crystals. After solvent removal, the salt crystals were removed from the polymer matrices by simple leaching. The different scaffolds were incubated with MC3T3 osteoblast cells. It was observed that the 3D scaffold allowed a continuous growth of the cells throughout the matrix, whereas the 2D format led to a decrease of cell adhesion after 1 week (Figure 4.14) [30, 31].

No. of osteoblasts adhered per Cm2 x 10e4

200 TCPS 2D-P(PHOS 3D-P(PHOS

180 160 140 120 100 80 60 40 20 0 0

5

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15

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Time (days) Figure 4.14: Osteoblast proliferation on 2D and 3D polyphosphazene (PNEGmPh) matrices. Cells were found to proliferate on 3D matrices over the 21-day culture time whereas a decline in cell number was found for 2D films after 7 days. TCPS: tissue-culture polystyrene. Reproduced with permission from C.T. Laurencin, S.F. El-Amin, S.E. Ibim, D.A. Willoughby, M. Attawia, H.R. Allcock and A.A. Ambrosio, Journal of Biomedical Materials Research, 1996, 30, 133. ©1996, John Wiley & Sons, Inc. [31].

The osteocompatibility of 2D NF sheets made out of electrospun polyphosphazenes and PLGA as blends was investigated by Deng and coworkers. Figures 4.15a, c and d demonstrate an almost complete coverage of the polymeric sheets with osteoblasts, and thus, indicate the desired compatibility. Furthermore, a higher phenotypic expression of alkaline phosphatase (ALP) was clearly determined with the blend materials compared with pure PLGA, at least after 3 days (Figure 4.15f), indicating the polyphosphazene-based early mature osteoblast phenotype expression. With these 2D sheets, 3D bone hierarchy mimicking concentric structures were developed (see

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Figure 4.15: Osteocompatibility of 2D BLEND NF matrices. (a)–(d) SEM images presenting the osteoblast morphologies on NF matrices at various time points during culture. (a)–(b) Surface morphologies of PLGA and BLEND NF 24 h post cell seeding, (c) osteoblasts almost covered the entire surface with a well-spread morphology by day 3 on BLEND NF surfaces. Inset: representative live/dead assay fluorescent image showing spindle-like osteoblast morphology on BLEND NF at day 3, scale bar 20 μm and (d) osteoblasts formed cell multilayers on BLEND NF by day 7. (e) Comparable cell proliferation ([3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2 H-tetrazolium] (MTS) assay) was observed on both PLGA and BLEND NF surfaces indicating good osteocompatibility. (f) Significantly higher amount of ALP expression on BLEND NF at day 3 indicating early mature osteoblast phenotype expression due to the polyphosphazene component. (∗) denotes significant difference (p < 0.05). Reproduced with permission from M. Deng, S.G. Kumbar, L.S. Nair, A.L. Weikel, H.R. Allcock and C.T. Laurencin, Advanced Functional Materials, 2011, 21, 2641. ©2011, Wiley-VCH [15].

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Figure 4.16: In vitro MC3T3-E1 cell growth on ethyl glycinato-substituted polyphosphazenes (polymers 5−8). The results are from one of three similar experiments and each line represents the mean from triplicate samples. The SD of the mean at each point is less than 4%. SD, standard deviation. Reproduced with permission from C.T. Laurencin, M.E. Norman, H.M. Elgendy, S.F. ElAmin, H.R. Allcock, S.R. Pucher and A.A. Ambrosio, Journal of Biomedical Materials Research, 1993, 27, 963. ©1993, John Wiley & Sons, Inc. [32].

Section 4.2.1.1, as well as Figures 4.2 and 4.3). Such 3D forms of polyphosphazene fibers showed a mechanical behavior similar to that of native bone [15]. In an early work, Laurencin and coworkers also cultivated an osteogenic cell line, that is, MC3T3-E1 on PNEGmPh in the form of disks fabricated via solution casting [34]. They observed an increase of cell growth with a higher content of the ethyl glycinato side group (only above 10% ethyl glycinato), with 75%, demonstrating the highest cell growth (Figure 4.16) [32]. Interestingly, when using polyphosphazenes with imidazolyl side groups, the cells did not adhere to the scaffolds (Figure 4.17a and b) as well as with the ethyl glycinato-substituted (imidazolyl-free) polyphosphazenes (Figure 4.17c–f) [32].

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(a)

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Figure 4.17: MC3T3-El cells on polyphosphazene surfaces after 2 days in culture. (a) Poly[(20% imidazolyl) 80% methylphenoxy) phosphazene], (b) poly[(54% imidazolyl) (46% methylphenoxy) phosphazene], (c) poly[(10% ethyl glycinato) (90% methylphenoxy) phosphazene], (d) poly[(25% ethyl glycinato) (75% methylphenoxy) phosphazene], (e) poly[(50% ethyl glycinato) (50% methylphenoxy) phosphazene] and (f) poly[(75% ethyl glycinato) (25% methylphenoxy) phosphazene]. Magnification = 40×. Reproduced with permission from C.T. Laurencin, M.E. Norman, H.M. Elgendy, S.F. El-Amin, H.R. Allcock, S.R. Pucher and A.A. Ambrosio, Journal of Biomedical Materials Research, 1993, 27, 963. ©1993, John Wiley & Sons, Inc. [32, 34].

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Nair and coworkers also investigated the osteocompatibility of, this time, alanine-containing polyphosphazenes. They synthesized poly[(ethyl alanato)1.0 (ethyl oxybenzoate)1.0phosphazene] (PN-EA/EOB) and poly[(ethyl alanato)1.0 (propyl oxybenzoate)1.0phosphazene] (PN-EA/POB) and compared films of these two polymers made via solution casting with TCPS as a control. In the initial phase of the experiments, the cells did not adhere to PN-EA/POB to the same extent as to PN-EA/EOB (Figure 4.18), since PN-EA/POB showed higher swelling and a more irregular surface than PN-EA/EOB hindering the adherence of the cells substantially. However, this difference changes after a few days leading to comparable numbers of cells adhering to PN-EA/POB and PN-EA/EOB. It was also determined that the values did not change for both polyphosphazene supports after 14 days [33]. 1200

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Figure 4.18: Proliferation kinetics of primary rat osteoblast cells cultured on PN-EA/EOB, PN-EA /POB and TCPS. Initial seeding density was 50,000 cells/well. Reproduced with permission from L.S. Nair, D.A. Lee, J.D. Bender, E.W. Barrett, Y.E. Greish, P.W. Brown, H.R. Allcock and C.T. Laurencin, Journal of Biomedical Materials Research Part A, 2006, 76, 1, 206. ©2006, Wiley Periodicals, Inc. [33].

PNmPh-based fiber mats were also used by Nair and coworkers and applied for the cultivation of osteoblasts MC3T3-E1 cells. It was shown that the cells’ adhesion and proliferation were promoted by the polyphosphazene scaffolds. After 4 days, the cells had migrated through the matrix and adhered to the fibers (Figure 4.19a). After 7 days the polymer matrix was completely covered with cells (Figure 4.19b) [16]. A poly(dopamine)-coated electrospun polyphosphazene fibrous matrix was presented by Li et al., showing superior adhesion and proliferation of osteoblasts relative to noncoated polyphosphazene fibers [34]. For enhancing the osteogenic differentiation of mesenchymal stem cells, Chun et al. developed an injectable, degradable and

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Figure 4.19: (a) SEM showing MC3T3-E1 cells on electrospun PNmPh fiber matrices after 4 days in culture and (b) matrices after 7 days in culture. Reproduced with permission from L.S. Nair, S. Bhattacharyya, J.D. Bender, Y.E. Greish, P.W. Brown, H.R. Allcock and C.T. Laurencin, Biomacromolecules, 2004, 5, 6, 2212. ©2004, American Chemical Society [16].

thermosensitive cross-linked polyphosphazene matrix with covalently linked RGD moieties. These peptide units acted as activating cell adhesion anchors, enhancing biofunctionality, and by this means, accelerating the regeneration of bone defects [35]. Huang et al. showed that amino acid ester substituted polyphosphazenes induced osteogenic differentiation of bone mesenchymal stromal cells, originating from the phosphorus containing surfaces of the polyphosphazene film-like scaffolds allowing to strengthen the cell–scaffold interaction [36]. An injectable nanoparticle hydrogel based on a polyphosphazene loaded with bone morphogenetic protein-2 (BMP-2) as a growth factor was developed by Seo et al. for bone regeneration. Due to the sustained release of this protein, a higher osteocalcin secretion in C2C12 mouse myoblast cells was observed [41]. In mice tests using a similar polyphosphazene/BMP-2 system, it was found that there is crucial period for bone regeneration in which a sufficient BMP-2 release is required that could be tuned by changing the physical properties of the hydrogels [42]. A successful bone regeneration with BMP-2 containing polyphosphazene hydrogels was also observed in canine models [43]. In an even more complex approach, a polyphosphazene/calcium phosphate scaffold containing chitosan microspheres also loaded with BMP-2 was able to induce an osteoblast proliferation (Figure 4.20) [44].

4.3.2 Endothelial tissue engineering The applicability of polyphosphazenes in TE has been tested several times with osteoblasts (see Section 4.3.1); such evaluations have also been performed with endothelial cells (EC). Nair and coworkers chose bovine coronary artery endothelial cells (BCAEC) and investigated the usability of nonwoven NF meshes based on electrospun

4.3 Applications of polyphosphazene scaffolds in tissue engineering

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Figure 4.20: Mesenchymal stem cell adhesion and spreading on scaffold based on calcium phosphate/polyphosphazene/chitosan microspheres loaded with BMP-2 after (a) 3 days, (b) 7 days, and (c) 14 days of cell culture observed by SEM. Reproduced with permission from A. Sobhani, M. Rafienia, M. Ahmadian and M.-R. Naimi-Jamal, Tissue Engineering and Regenerative Medicine, 2017, 14, 5, 525. ©2017, Springer [44].

PNmPh as scaffolds (see Section 4.2.1.1 and Figure 4.4). Figure 4.21 demonstrates clearly that the NF mats support the adhesion of the BCAEC [16]. Nonwoven fiber-based matrices of poly[(ethyl phenylalanate)0.8(ethyl alanate)(ethyl glycinate)0.4 phosphazene] (PPAGP) and poly[bis(ethyl alanate) phospha0.8 zene] (PAlaP) were evaluated together with poly(D,L-lactic acid) (PDLLA) in terms of their applicability for TE with rat neuromicrovascular EC. It was found that all three polymers allowed the EC to adhere to the scaffolds (Figure 4.22a), however, only the polyphosphazene containing materials exhibited improved cell growth, showing the highest increase with the PPAGP support (Figure 4.22b) [37]. Furthermore, the EC did not only adhere in a flattened way to PPAGP (Figure 4.23a) but formed bridges between the PPAGP fibers (Figure 4.23b) and, astonishingly, even

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Figure 4.21: SEM showing the adhesion of BCAEC on electrospun PNmPh fiber matrices after 24 h in culture. Reproduced with permission from L.S. Nair, S. Bhattacharyya, J.D. Bender, Y.E. Greish, P.W. Brown, H.R. Allcock and C.T. Laurencin, Biomacromolecules, 2004, 5, 6, 2212. ©2004, American Chemical Society [16].

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Figure 4.22: Rat neuromicrovascular EC adhesion (a) and growth (b) on different substrates, PDLLA, PAlaP and PPAGP. Reproduced with permission from M.T. Conconi, S. Lora, S. Baiguera, E. Boscolo, M. Folin, R. Scienza, P. Rebuffat, P.P. Parnigotto and G.G. Nussdorfer, Journal of Biomedical Materials Research Part A, 2004, 71, 669. ©2004, Wiley Periodicals, Inc. [37].

formed tubular or capillary-like structures (Figure 4.23c). This led to the conclusion that such a system may be used for engineering blood vessels [37].

4.3.3 Neural tissue engineering Thin films of electrically conductive poly[(glycine ethyl ester)0.65(aniline pentamer)0.35 phosphazene] (PGAP) were fabricated by Zhang and coworkers and evaluated as scaffolds for tissue formation based on RSC96 Schwann cells, which are the main supportive cell type of the peripheral nervous system. The vision was to develop polymeric supports to be applied in the regeneration of peripheral nerves, since it is

4.4 Degradation of polyphosphazenes developed for tissue engineering

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Figure 4.23: Environmental SEM of EC seeded on PPAGP fibers after 24 h of culture. Magnification: (a) 435×, (b) 570× and (c) 285×. Reproduced with permission from M.T. Conconi, S. Lora, S. Baiguera, E. Boscolo, M. Folin, R. Scienza, P. Rebuffat, P.P. Parnigotto and G.G. Nussdorfer, Journal of Biomedical Materials Research Part A, 2004, 71, 669. ©2004, Wiley Periodicals, Inc. [37].

known that electroactive materials stimulate the attachment and proliferation especially of nerve cells and, thus, enhance nerve regeneration [45]. A comparison of PGAP and PDLLA demonstrated that the RSC96 cells adhered to both supports after 3 days of incubation. However, cells on the PGAP scaffold were elongated and spread with a higher total cell density (Figure 4.24a) compared with PDLLA where the cells developed only single filopodia (Figure 4.24b) [38].

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Figure 4.24: SEM images of RSC96 cells seeded on the PGAP film (a) and PDLLA film (b) after incubation for 3 days (original magnification 250×). Reproduced with permission from Q. Zhang, Y. Yan, S. Li and T. Feng, Materials Science and Engineering C, 2010, 30, 160. ©2010, Elsevier [38].

4.4 Degradation of polyphosphazenes developed for tissue engineering One of the major advantages of polyphosphazenes is their ability to degrade at a range of different rates, depending on the requirement. The general concept of using polyphosphazenes for the fabrication of scaffolds in TE is the possibility of

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removing the polymer supports in the later stages of tissue formation. Kumbar and coworkers have formulated, as an example, the general degradation pathway of poly[(amino acid ester)phosphazene] which is often used specifically for tissue regeneration. It is shown that the degradation products consist of amino acids stemming from the side groups and simple phosphates and ammonia salts derived from the polyphosphazene backbone (Figure 4.25; see chapter 2 for more details) [46]. COOEt HC

COOEt HC

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Figure 4.25: General degradation pathway for poly[(amino acid ester)phosphazene].

Singh and coworkers systematically investigated the role of amino acids as side groups in polyphosphazenes with respect to their degradation behavior. For this purpose, they synthesized four different polyphosphazenes: (1) poly[bis(ethyl alanato) phosphazene] (PNEA), (2) poly[(ethyl alanato)1 (ethyl glycinato)1 phosphazene] (PNEAEG), (3) poly[(ethyl alanato)1 (p-methylphenoxy)1 phosphazene] (PNEAMPH) and (4) poly[(ethyl alanato)1 (p-phenylphenoxy)1 phosphazene] (PNEAPHPH) (Figure 4.26) [47]. For all four polymers, a strong decline of the molecular weight (Mw) was determined in the hydrolysis experiments in phosphate buffered saline (PBS) at 37 °C, with the strongest effect shown for polymer 2, followed by 1, then 3 and 4, which showed similar behavior (2 > 1 > 3, 4). (Figure 4.27a) [47]. This trend was explained by the bulkiness and hydrophobicity of the side groups; with a more hydrophobic, here an aromatic, side group, the hydrolytic degradation was slowed down. When recording the mass loss, a substantial change in weight was only observed for polymer 2. In this case, the mass loss was 90% in 7 weeks. For the other polyphosphazenes, 1, 3 and 4, less than 5% mass loss was determined (Figure 4.27b), explained by, again, their higher hydrophobicities and Mw [47].

4.4 Degradation of polyphosphazenes developed for tissue engineering

NHCH(CH3)COOC2H5 N

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Figure 4.26: Polymer structures of L-alanine-cosubstituted polyphosphazenes: (1) PNEA, (2) PNEAEG, (3) PNEAMPH and (4) PNEAPHPH. Reproduced with permission from A. Singh, N.R. Krogman, S. Sethuraman, L.S. Nair, J.L. Sturgeon, P.W. Brown, C.T. Laurencin and H.R. Allcock, Biomacromolecules, 2006, 7, 3, 914. ©2006, American Chemical Society [47].

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Figure 4.27: (a) Mw decline for polymers PNEA (1), PNEAEG (2), PNEAMPH (3) and PNEAPHPH (4) in PBS solution at 37 °C. Mw for polymer 2 could not be recorded beyond 3 weeks due to rapid hydrolysis, (b) mass loss recorded for polymers 1−4 in PBS solution at 37 °C. Reproduced with permission from A. Singh, N.R. Krogman, S. Sethuraman, L.S. Nair, J.L. Sturgeon, P.W. Brown, C.T. Laurencin and H.R. Allcock, Biomacromolecules, 2006, 7, 3, 914. ©2006, American Chemical Society [47].

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Some amino acid side groups seem clearly to aid the degradation rates of the polyphosphazenes. This was found as well for PNEGmPh, which was used as a scaffold material for the cultivation of MC3T3-El osteoblasts (see Section 4.3.1). It was shown that when increasing the content of amino acid side groups in the polyphosphazene, for example, from 10% to 75%, the rate of polyphosphazene degradation in a phosphate buffer at pH = 7.4 and 37 °C was drastically enhanced (Figure 4.28) [30, 32].

Percent mass loss (%)

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Figure 4.28: Percentage degradation versus time (h) of PNEGmPh with a different content of amino acid side groups (10%, 25%, 50% and 75%) in 0.1 M sodium phosphate buffer at 37 °C and pH = 7.4. PPHOS-EG10: poly[(10% ethyl glycinato)(90% p-methylphenoxy) phosphazene]; PPHOS-EG25: poly[(25% ethyl glycinato)(75% p-methylphenoxy) phosphazene]; PPHOS-EG50: poly[(50% ethyl glycinato)(50% p-methylphenoxy) phosphazene] and PPHOS-EG75: poly[(75% ethyl glycinato)(25% p-methylphenoxy) phosphazene]. Reproduced with permission from M. Deng, S.G. Kumbar, Y. Wan, U.S. Toti, H.R. Allcock and C.T. Laurencin, Soft Matter, 2010, 6, 3119. ©2010, The Royal Society of Chemistry [30].

However, it is relevant whether the amino acids substituents are N-linked or Olinked to the polyphosphazene backbone. Morozowich et al. found that polymers with amino ester side groups bound to the polymer via their N-terminus showed degradation effects after weeks following a bulk erosion process, whereas polyphosphazenes with O-linked amino acid substituents were water soluble and hydrolyzed more rapidly. Nevertheless, both variants of polymers were not completely decomposed after 6 weeks [48]. When investigating the degradability of PN-EA/EOB and PN-EA/POB which were used for osteoblast cultivation (see Section 4.3.1), higher degradation rates were determined in PBS at 37 °C for PN-EA/POB leading to a weight loss of 25% in 12 weeks, compared with less than 15% in the case of PN-EA/EOB (Figure 4.29) [33].

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Time of degradation (weeks) Figure 4.29: Percentage weight loss of PN-EA/EOB and PN-EA/POB in PBS at 37 °C over a 12-week study. Reproduced with permission from L.S. Nair, D.A. Lee, J.D. Bender, E.W. Barrett, Y.E. Greish, P.W. Brown, H.R. Allcock and C.T. Laurencin, Journal of Biomedical Materials Research Part A, 2006, 76, 1, 206. ©2006, Wiley Periodicals, Inc. [33].

Zhang and coworkers have developed a novel electrically conductive PGAP for neural TE (see Section 4.3.3). This polymer, in the form of membranes, was tested in terms of degradability in PBS at pH = 7.4 and 37 °C. The authors measured a weight loss of 50% for PGAP after 70 days, in comparison to 70% determined for poly(glycine ethyl ester) phosphazene (PGEE) over the same period (Figure 4.30) [38]. The extent of degradation of PGAP within 70 days became quite obvious when monitoring the progress of the degradation with SEM. Whereas after 20 days only minor changes are observable (Figure 4.31b), the scaffold showed a relatively high degree of disintegration after 50 days (Figure 4.31c) and even more after 70 days with an increase in pore size (Figure 4.31d) [38]. Ogueri et al. found that poly[(glycineethylglycinato)x (phenylphenoxy)y phosphazene] as well as poly[(ethylphenylalanato)x (glycineethylglycinato)y phosphazene] were successfully tested for preosteoblast MC3T3-E1 cell growth, hydrolyzed to near neutral pH media, whereas the control matrix PLGA decomposed comparably faster leading to a putatively tissue-irritating pH of 2.2, a clear advantage of the polyphosphazenes [49]. Interestingly, blending of polyphosphazenes with PLGA allows to reduce this low pH effect of the degradation products of PLGA due to their simple neutralization by the polyphosphazene fragments [50].

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Figure 4.30: Weight loss of PGEE and PGAP polymer membranes in 0.1 mol/L PBS at 37 °C and pH = 7.4. Results represent means ± SD (n = 3). Reproduced with permission from Q. Zhang, Y. Yan, S. Li and T. Feng, Materials Science and Engineering C, 2010, 30, 160. ©2010, Elsevier [38].

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Figure 4.31: SEM images of the PGAP after degradation in PBS at 37 °C for various times. (a) nonincubated, (b) 20 days, (c) 50 days and (d) 70 days. Reproduced with permission from Q. Zhang, Y. Yan, S. Li and T. Feng, Materials Science and Engineering C, 2010, 30, 160. ©2010, Elsevier [38].

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4.5 Conclusion In several publications, a number of variations of polyphosphazenes in terms of different side groups have been demonstrated to be applicable for TE. The desired scaffolds can be fabricated in different appropriate formats such as 2D objects, for example, films and membranes or in the form of 3D matrices. For that purpose, even 3D printing of polyphosphazene-based inks is in grasp. A few examples of cell types are described in the literature, which have been used for TE on polyphosphazene scaffolds like osteoblasts and endothelial or Schwann cells. Most of the polyphosphazenes tested appear to be quite compatible with the cells they are intended to support in terms of adhesion and proliferation. They are can be degradable and with careful design the degradation products tend to be toxicologically harmless. It can be expected that some future implanted bones, blood vessels or even nerves will have been made based on tailored, highly advanced polyphosphazenes.

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Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chem Rev 2001;101:1869–1880. Tang X, Thankappan SK, Lee P, et al. Chapter 21 - Polymeric Biomaterials in Tissue Engineering and Regenerative Medicine. In: Kumbar SG, Laurencin CT, Deng M, eds. Natural and Synthetic Biomedical Polymers. Oxford: Elsevier; 2014:351–371. [3] Guo B, Ma PX. Synthetic biodegradable functional polymers for tissue engineering: a brief review. Sci China Chem 2014;57:490–500. [4] Ogueri KS, Jafari T, Escobar Ivirico JL, Laurencin CT. Polymeric biomaterials for scaffold-based bone regenerative engineering. Regener Eng Transl Med 2019;5:128–154. [5] Yang S, Leong K-F, Du Z, Chua C-K. The design of scaffolds for use in tissue engineering. part I. Tissue Eng 2001;7:679–689. [6] James R, Deng M, Kumbar SG, Laurencin CT. Chapter 11 - Polyphosphazenes. In: Kumbar SG, Laurencin CT, Deng M, eds. Natural and Synthetic Biomedical Polymers. Oxford: Elsevier; 2014:193–206. [7] Baillargeon AL, Mequanint K. Biodegradable polyphosphazene biomaterials for tissue engineering and delivery of therapeutics. Biomed Res Int 2014:2014. [8] Donderwinkel I, van Hest JCM, Cameron NR. Bio-inks for 3D bioprinting: recent advances and future prospects. Polym Chem 2017;8:4451–4471. [9] Ogueri KS, Ivirico JLE, Nair LS, Allcock HR, Laurencin CT. Biodegradable polyphosphazenebased blends for regenerative engineering. Regener Eng Transl Med 2017;3:15–31. [10] Ogueri KS, Laurencin CT. Polyphosphazene-Based Biomaterials for Regenerative Engineering. In: Polyphosphazenes in Biomedicine, Engineering, and Pioneering Synthesis. American Chemical Society; 2018:53–75. [11] Weikel AL, Cho SY, Morozowich NL, Nair LS, Laurencin CT, Allcock HR. Hydrolysable polylactide-polyphosphazene block copolymers for biomedical applications: synthesis, characterization, and composites with poly(lactic-co-glycolic acid). Polym Chem 2010;1: 1459–1466.

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[12] Nukavarapu SP, Kumbar SG, Brown JL, et al. Polyphosphazene/nano-hydroxyapatite composite microsphere scaffolds for bone tissue engineering. Biomacromolecules 2008;9: 1818–1825. [13] Carampin P, Conconi MT, Lora S, et al. Electrospun polyphosphazene nanofibers for in vitro rat endothelial cells proliferation. J Biomed Mater Res Part A 2007;80A:661–668. [14] Greish YE, Bender JD, Singh A, et al. Hydrolysis of Ca-deficient hydroxyapatite precursors in the presence of alanine-functionalized polyphosphazene nanofibers. Ceram Int 2013;39: 519–528. [15] Deng M, Kumbar SG, Nair LS, Weikel AL, Allcock HR, Laurencin CT. Biomimetic structures: Biological implications of dipeptide-substituted polyphosphazene–polyester blend nanofiber matrices for load-bearing bone regeneration. Adv Funct Mater 2011;21:2641–2651. [16] Nair LS, Bhattacharyya S, Bender JD, et al. Fabrication and optimization of methylphenoxy substituted polyphosphazene nanofibers for biomedical applications. Biomacromolecules 2004;5:2212–2220. [17] Krogman NR, Weikel AL, Nguyen NQ, Nair LS, Laurencin CT, Allcock HR. Synthesis and characterization of new biomedical polymers: Serine- and threonine-containing polyphosphazenes and poly(l-lactic acid) grafted copolymers. Macromolecules 2008;41: 7824–7828. [18] Krogman NR. Polyphosphazenes for Advanced Biomedical Applications [PhD Thesis]. Pennsylvania, USA: The Pennsylvania State University; 2008. [19] Duan S, Yang X, Mao J, et al. Osteocompatibility evaluation of poly(glycine ethyl ester-coalanine ethyl ester)phosphazene with honeycomb-patterned surface topography. J Biomed Mater Res Part A 2013;101A:307–317. [20] Huang Z, Yang L, Zhang X, et al. Synthesis and fluorescent property of biodegradable polyphosphazene targeting long-term in vivo tracking. Macromolecules 2016;49:8508–8519. [21] Huang Z-H, Wei P-F, Jin L, Hu X-Q, Cai Q, Yang X-P. Photoluminescent polyphosphazene nanoparticles for in situ simvastatin delivery for improving the osteocompatibility of BMSCs. J Mater Chem B 2017;5:9300–9311. [22] Morozowich NL, Nichol JL, Mondschein RJ, Allcock HR. Design and examination of an antioxidant-containing polyphosphazene scaffold for tissue engineering. Polym Chem 2012;3:778–786. [23] Huang Z, Liu X, Chen S, Lu Q, Sun G. Injectable and cross-linkable polyphosphazene hydrogels for space-filling scaffolds. Polym Chem 2015;6:143–149. [24] Huang Z, Gao C, Huang Y, et al. Injectable polyphosphazene/gelatin hybrid hydrogel for biomedical applications. Mater Des 2018;160:1137–1147. [25] Rothemund S, Aigner TB, Iturmendi A, et al. Degradable glycine-based photo-polymerizable polyphosphazenes for use as scaffolds for tissue regeneration. Macromol Biosci 2015;15: 351–363. [26] Teasdale I, Rothemund S, Aigner T, et al., inventors. Polymer for tissue engineering patent US 2017/0183453 A1. 2017. [27] Silva Nykänen VP, Puska MA, Nykänen A, Ruokolainen J. Synthesis and biomimetic mineralization of l-proline substituted polyphosphazenes as bulk and nanofiber. J Polym Sci, Part B: Polym Phys 2013;51:1318–1327. [28] Morozowich NL, Nichol JL, Allcock HR. Investigation of apatite mineralization on antioxidant polyphosphazenes for bone tissue engineering. Chem Mater 2012;24:3500–3509. [29] Barrett EW, Phelps MVB, Silva RJ, Gaumond RP, Allcock HR Patterning poly (organophosphazenes) for selective cell adhesion applications. Biomacromolecules 2005;6: 1689–1697.

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[30] Deng M, Kumbar SG, Wan Y, Toti US, Allcock HR, Laurencin CT Polyphosphazene polymers for tissue engineering: an analysis of material synthesis, characterization and applications. Soft Matter 2010;6:3119–3132. [31] Laurencin CT, El-Amin SF, Ibim SE, et al. A highly porous 3-dimensional polyphosphazene polymer matrix for skeletal tissue regeneration. J Biomed Mater Res 1996;30:133–138. [32] Laurencin CT, Norman ME, Elgendy HM, et al. Use of polyphosphazenes for skeletal tissue regeneration. J Biomed Mater Res 1993;27:963–973. [33] Nair LS, Lee DA, Bender JD, et al. Synthesis, characterization, and osteocompatibility evaluation of novel alanine-based polyphosphazenes. J Biomed Mater Res Part A 2006;76A: 206–213. [34] Li Y, Shi Y, Duan S, et al. Electrospun biodegradable polyorganophosphazene fibrous matrix with poly(dopamine) coating for bone regeneration. J Biomed Mater Res Part A 2014;102: 3894–3902. [35] Chun C, Lim HJ, Hong K-Y, Park K-H, Song S-C. The use of injectable, thermosensitive poly (organophosphazene)–RGD conjugates for the enhancement of mesenchymal stem cell osteogenic differentiation. Biomaterials 2009;30:6295–6308. [36] Huang Z, Yang L, Hu X, et al. Molecular mechanism study on effect of biodegradable amino acid ester–substituted polyphosphazenes in stimulating osteogenic differentiation. Macromol Biosci 2019;19:1800464. [37] Conconi MT, Lora S, Baiguera S, et al. In vitro culture of rat neuromicrovascular endothelial cells on polymeric scaffolds. J Biomed Mater Res Part A 2004;71A:669–674. [38] Zhang Q, Yan Y, Li S, Feng T. The synthesis and characterization of a novel biodegradable and electroactive polyphosphazene for nerve regeneration. Mater Sci Eng C 2010;30:160–166. [39] Nichol JL, Morozowich NL, Decker TE, Allcock HR. Crosslinkable citronellol containing polyphosphazenes and their biomedical potential. J Polym Sci Part A: Polym Chem 2014;52: 2258–2265. [40] Nichol JL, Allcock HR. Polyphosphazenes with amino acid citronellol ester side groups for biomedical applications. Eur Polym J 2015;62:214–221. [41] Seo B-B, Choi H, Koh J-T, Song S-C. Sustained BMP-2 delivery and injectable bone regeneration using thermosensitive polymeric nanoparticle hydrogel bearing dual interactions with BMP-2. J Controlled Release 2015;209:67–76. [42] Seo B-B, Koh J-T, Song S-C. Tuning physical properties and BMP-2 release rates of injectable hydrogel systems for an optimal bone regeneration effect. Biomaterials 2017;122:91–104. [43] Seo B-B, Chang H-I, Choi H, et al. New approach for vertical bone regeneration using in situ gelling and sustained BMP-2 releasing poly(phosphazene) hydrogel system on peri-implant site with critical defect in a canine model. J Biomed Mater Res Part B: Appl Biomater 2018;106:751–759. [44] Sobhani A, Rafienia M, Ahmadian M, Naimi-Jamal M-R. Fabrication and characterization of polyphosphazene/calcium phosphate scaffolds containing chitosan microspheres for sustained release of bone morphogenetic protein 2 in bone tissue engineering. J Tissue Eng Regener Med 2017;14:525–538. [45] Kotwal A, Schmidt CE. Electrical stimulation alters protein adsorption and nerve cell interactions with electrically conducting biomaterials. Biomaterials 2001;22:1055–1064. [46] Kumbar SG, Bhattacharyya S, Nukavarapu SP, Khan YM, Nair LS, Laurencin CT. In vitro and in vivo characterization of biodegradable poly(organophosphazenes) for biomedical applications. J Inorg Organomet Polym Mater 2006;16:365–385. [47] Singh A, Krogman NR, Sethuraman S, et al. Effect of side group chemistry on the properties of biodegradable L-alanine cosubstituted polyphosphazenes. Biomacromolecules 2006;7: 914–918.

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[48] Morozowich NL, Mondschein RJ, Allcock HR. Comparison of the synthesis and bioerodible properties of N-linked versus O-linked amino acid substituted polyphosphazenes. J Inorg Organomet Polym Mater 2014;24:164–172. [49] Ogueri KS, Escobar Ivirico JL, Li Z, Blumenfield RH, Allcock HR, Laurencin CT. Synthesis, physicochemical analysis, and side group optimization of degradable dipeptide-based polyphosphazenes as potential regenerative biomaterials. ACS Appl Polym Mater 2019;1: 1568–1578. [50] Ogueri KS, Ogueri KS, Allcock HR, Laurencin CT. A regenerative polymer blend composed of glycylglycine ethyl ester-substituted polyphosphazene and poly(lactic-co-glycolic acid). ACS Appl Polym Mater 2020.

Chapter 5 Opportunities and challenges The numerous examples presented in this book for the medical use of polyphosphazenes, developed and ongoing, are testament to the useful properties of these unique polymers. Striking is the breadth in the spectrum of applications that have been investigated within this field, with poly(organo)phosphazenes proving to be an extremely versatile synthetic platform. Generally, tunable properties are critical for medical applications in complex and diverse biological environments and few polymers can offer the range of synthetic versatility, while at the same time proving over and over again the ability to prepare biocompatible materials which, when required, degrade in physiological conditions to benign degradation products. These applications utilize the unique combination of properties offered by poly(organo)phosphazenes, which can be fine-tuned to give the precisely desired characteristics required by a specific application, be it, for example, an overall charge, required critical solution temperature (LCST), degradation rate or degree of lipophilicity. Indeed, due to the most commonly applied postpolymerization substitution method, libraries of materials are readily available with a broad spectrum of properties.

5.1 From laboratory to clinic Despite the obvious potential shown, due to many years of innovative new developments [1], transfer to the clinic has been somewhat sluggish. There may be a number of reasons for this, not always of a scientific nature, but the synthetic versatility, portrayed as one of the main virtues of polyphosphazenes, could conversely also be a hindrance in their further development. This versatility leads to an inherent complexity and confusion due to the multifaceted nature of the materials produced. A new researcher to the field, perhaps looking to use polyphosphazenes for their own biomedical application, will find little directly comparable data, not to mention any database detailing their structure–property relationships. The data that is available is often inconsistently characterized, due in part to the nature of the polymers in hand, for which small structural changes result in drastic changes in property, but also the broadness of applications, for which the necessary properties (and thus reported properties) differ widely. Particularly unhelpful in this regard are the generalizations often encountered in the scientific literature such as “polyphosphazene, a biocompatible, biodegradable polymer,” which is not only misleading but misconceived. The multifaceted nature of polyphosphazenes means that such comments cannot be applied the same way one could make this comment about, for example, a poly(lactic acid) (PLA) homopolymer.

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Some polyphosphazenes can be made to be degradable [2], but majority of them are not degradable in a suitable time frame for most medical applications, and many are indeed biocompatible, but this is highly dependent on the organic substituents. More careful wording would be helpful to those less familiar with the properties of these polymers, for example, medical researchers or industrial scientists searching for a suitable polymer as a tool for their latest development. In this sense, polyphosphazenes should only be referred to as “a broad group/family of polymers,” such as polyesters or polyamides, not a single polymer in the same way a PLA homopolymer can be viewed. Furthermore, although the polyphosphazene backbone degradation products are essentially nontoxic, this depends on the organic components, which although allowing certain predictability means every new polymer must be taken on merit. This extra complication could also be conceived as a disadvantage in reaching the clinic for more complexly substituted polymers, since minor structural changes lead to a novel polymer that must be reassessed. A further limiting factor is the challenges of producing this synthetic class of polymer and the requirement for robust, reproducible procedures. Recent efforts in this regard have resulted in significant advances, including the good manufacturing practices method developed for ring-opening polymerization [3] (Chapter 1). Progress has also been made in the living polymerization approaches [4–6], allowing preparation of the well-defined materials required for a successful transition to the clinic. The tricky monomer synthesis required for the controlled polymerization routes remains, however, despite some improvements [7, 8], a bottleneck in terms of their robust, reproducible synthesis.

5.2 Future prospects The relative high cost and synthetically challenging procedures mean that polyphosphazenes are always going to find it difficult to compete as (inert) engineering materials for medical applications against polymers with more robust synthetic pathways and cheaper sources. However, for advanced applications requiring unique specialty polymers, with precisely tailored properties, polyphosphazenes can be extremely valuable. Indeed, it is in certain unique niches where polyphosphazenes have come to the fore by exhibiting superior properties. An example of this is the commercially available fluorinated polyphosphazene-based denture liners [9], where the patient comfort is reported to be superior to most other materials. A further, highly encouraging example is this of trifluoroethoxypolyphosphazene (marketed as Cobra Pz-F or Polyzene-F) [10]. Two products coated with this polymer have received FDA approval in recent years, and clinical trials in Europe have been highly promising due to their unique properties above and beyond its competitors. In particular, the ability of Pz-F to bind albumin would appear to be superior to noncoated stents as well as other polymer-coated stents. This provides a protective coating, which produces an antiinflammatory effect and unique thromboresistant properties [11–13].

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In terms of future applications, a niche field that appears highly promising is its use as immunoadjuvants [14] (Section 3.1), whereby they have consistently been shown to perform better than most of the other tested materials for this application, with its unique dual-functionality integrating delivery and immunostimulating modalities in one water-soluble macromolecule. Similarly, when used as tissue engineering scaffolds, the precise tuning of degradation rates, facile cosubstitution and, importantly, the nonacidic degradation products represent significant advantages over, for example, commonly used degradable polyesters [15]. The ability to fine-tune not only the LCST but also degradability makes them ideal for injectable hydrogels [16, 17] (Section 3.5.2). For polymer therapeutics (Section 3.4) controlled molecular weights are essential, but although this is readily achievable for many polymers (via atom-transfer radical polymerization and so on), the carbon backbone generated by these procedures is inherently nondegradable. The unique combination of controlled polymerization and degradability, in combination with the high functional group density for drug loading [18, 19], also makes polyphosphazenes highly attractive candidates for parenteral drug-delivery applications. The synthetic flexibility of poly(organo)phosphazenes combined, when required, with a tunable degradability can be used to prepare specialty materials with precisely designed functions. With intelligent design and structural modifications, it is envisaged that many advanced biomedical materials of the future could be derived from polyphosphazenes. As many reports summarized in this book confirm, progress in this direction is indeed already well underway!

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[4]

[5]

[6]

[7]

Allcock HR. Chemistry and Applications of Polyphosphazenes. Hoboken, USA: Wiley; 2003. Allcock HR, Morozowich NL. Bioerodible polyphosphazenes and their medical potential. Polym Chem 2012;3:578–590. Andrianov AK, Chen J, LeGolvan MP. Poly(dichlorophosphazene) as a precursor for biologically active polyphosphazenes: Synthesis, characterization, and stabilization. Macromolecules 2004;37:414–420. Suárez Suárez S, Presa Soto D, Carriedo GA, Presa Soto A, Staubitz A. Experimental and theoretical study of the living polymerization of N-silylphosphoranimines. Synthesis of new block copolyphosphazenes. Organometallics 2012;31:2571–2581. Blackstone V, Lough AJ, Murray M, Manners I. Probing the mechanism of the PCl5-initiated living cationic polymerization of the phosphoranimine Cl3P = NSiMe3 using model compound chemistry. J Am Chem Soc 2009;131:3658–3667. Wilfert S, Henke H, Schoefberger W, Brüggemann O, Teasdale I. Chain-end-functionalized polyphosphazenes via a one-pot phosphine-mediated living polymerization. Macromol Rapid Commun 2014;35:1135–1141. Wang B. Development of a one-pot in situ synthesis of poly(dichlorophosphazene) from PCl3. Macromolecules 2005;38:643–645.

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Wang B, Rivard E, Manners I. A new high-yield synthesis of Cl3P=NSiMe3, a monomeric precursor for the controlled preparation of high molecular weight polyphosphazenes. Inorg Chem 2002;41:1690–1691. Razavi R, Khan Z, Haeberle CB, Beam D. Clinical applications of a polyphosphazene-based resilient denture liner. J Prosthodont 1993;2:224–227. Bates MC, Yousaf A, Sun L, Barakat M, Kueller A. Translational research and early favorable clinical results of a novel polyphosphazene (Polyzene-F) nanocoating. Regener Eng Transl Med 2019;5:341–353. Hiroyuki J, Hiroyoshi M, Qi C, et al. Thromboresistance and functional healing in the COBRA PzF stent versus competitor DES: implications for dual antiplatelet therapy. EuroIntervention 2019;15:e342–e53. Sakakura K, Cheng Q, Otsuka F, et al. TCT-806 thrombogenicity of novel polyphosphazene surface-modified coronary stent compared to standard bare metal stent In Swine Shunt Model. J Am Coll Cardiol 2013;62:B244–B5. Henn C, Satzl S, Christoph P, et al. Efficacy of a polyphosphazene nanocoat in reducing thrombogenicity, In-stent stenosis, and inflammatory response in porcine renal and iliac artery stents. J Vasc Interventional Radiol 2008;19:427–437. Andrianov AK. Polyphosphazenes as Vaccine Adjuvants. Vaccine Adjuvants and Delivery Systems. John Wiley & Sons, Inc.; 2006:355–378. Deng M, Kumbar SG, Wan Y, Toti US, Allcock HR, Laurencin CT. Polyphosphazene polymers for tissue engineering: an analysis of material synthesis, characterization and applications. Soft Matter 2010;6:3119–3132. Kim JI, Lee BS, Chun C, Cho J-K, Kim S-Y, Song S-C. Long-term theranostic hydrogel system for solid tumors. Biomaterials 2012;33:2251–2259. Lee BH, Lee YM, Sohn YS, Song SC. A thermosensitive poly(organophosphazene) gel. Macromolecules 2002;35:3876–3879. Teasdale I, Wilfert S, Nischang I, Brüggemann O. Multifunctional and biodegradable polyphosphazenes for use as macromolecular anti-cancer drug carriers. Polym Chem 2011;2: 828–834. Jun YJ, Kim JI, Jun MJ, Sohn YS. Selective tumor targeting by enhanced permeability and retention effect. Synthesis and antitumor activity of polyphosphazene-platinum (II) conjugates. J Inorg Biochem 2005;99:1593–1601.

Abbreviations β-CD 2D 3D ALP AMPEG APC ATRP BCAEC BSA CAP CMC DAB DACH DIEA DMAEA DMAP DMPA DNA DOX EC EPR HAI HBSAg Hgh HIV HLB ICP-MS IDR IgG ITO LCST LOD M:I M0 MCPM MEEP MLT MLTH Mn M0 MRI Mt Mw NF nHAp O/N OVA

β-Cyclodextrin Two-dimensional Three-dimensional Alkaline phosphatase α-Amino-ω-methoxy-polyethylene glycol Antigen-presenting cells Atom-transfer radical polymerization Bovine coronary artery endothelial cells Bovine serum albumin Cellular adhesive properties Critical micelle concentration Diaminobutane 1,2-Diaminocyclohexane N,N-Diisopropylethylamine 2-Dimethylaminoethylamine 4-(Dimethylamino)pyridine 2,2-Dimethoxy-2-phenylacetophenone Deoxyribonucleic acid Doxorubicin Endothelial cells Enhanced permeation and retention Hemagglutination inhibition Hepatitis B surface antigen Human growth hormone Human immunodeficiency virus Hydrophilic–lipophilic balance Inductively coupled plasma mass spectrometry Innate defense regulator Immunoglobulin G Indium tin oxide Lower critical solution temperature Limit of detection Monomer to initiator ratio Initial monomer concentration Monocalcium phosphate monohydrate Poly[bis(2-(2-methoxyethoxy)ethoxy)phosphazene] Monitored long-term therapeutic Monitored long-term therapeutic hydrogel Number average molecular weight Initial monomer concentration Magnetic resonance imaging Monomer concentration at time t Weight average molecular weight Nanofiber(s) Hydroxyapatite nanoparticles Overnight Ovalbumin

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144

PAEP PAlaP PAMAM PBS PCEP PCPP PDL PDLLA PDT PEG PEI PEO PFS PGA PGAP PGEE PLA PLGA PMAP PMCP PMCPP PMPP

Abbreviations

Poly[bis(2-(2-aminoethoxy)ethoxy)phosphazene] Poly[bis(ethyl alanate) phosphazene] Polyamidoamine Phosphate-buffered saline Poly[di(sodium carboxylatoethylphenoxy) phosphazene] Poly[di(sodium carboxylatophenoxy)phosphazene] Poly(D-lysine) Poly(D,L-lactic acid) Photodynamic therapy Polyethylene glycol Polyethylenimine Polyethylene oxide Poly(ferrocenylsilane) Poly(glycolic acid) Poly[(glycine ethyl ester)0.65(aniline pentamer)0.35 phosphazene] Poly(glycine ethyl ester) phosphazene Poly(lactic acid) Poly(lactic-co-glycolic acid) Poly(methylaminophosphazene) Poly[(methoxyethoxyethoxy)1.0-(cinnamyloxy)1.0 phosphazene] Poly[(methoxyethoxyethoxy)1.0 (carboxylatophenoxy)1.0phosphazene] Poly[(methoxyethoxyethoxy)1.0

(p-methylphenoxy)1.0 phosphazene] PN PNEA PN-EA/EOB PN-EA/POB PNEAEG PNEAMPH PNEAPHPH PNEGmPh PNEPhA PNIPAm PNmPh PPAGP PPG PPHOS PPHOS-EG10 PPHOS-EG25 PPHOS-EG50 PPHOS-EG75 PPO PProP PS PTX PVP PYRP RNA ROP

Polyphosphazene Poly[bis(ethyl alanato)phosphazene] Poly[(ethyl alanato)1.0(ethyl oxybenzoate)1.0 phosphazene] Poly[(ethyl alanato)1.0(propyl oxybenzoate)1.0 phosphazene] Poly[(ethyl alanato)1 (ethyl glycinato)1 phosphazene] Poly[(ethyl alanato)1 (p-methylphenoxy)1 phosphazene] Poly[(ethyl alanato)1 (p-phenylphenoxy)1 phosphazene] Poly[(ethyl glycinato)(methylphenoxy) phosphazene] Poly[bis(ethyl phenylalaninato)phosphazene] Poly(N-isopropyl acrylamide) Poly[bis(p-methylphenoxy)phosphazene] Poly[(ethyl phenylalanate)0.8(ethyl alanate)0.8(ethyl glycinate)0.4 phosphazene] Polypropylene glycol Poly[(glycine ethyl glycinato)1 (phenylphenoxy)1 phosphazene] Poly[(10% ethyl glycinato)(90% p-methylphenoxy) phosphazene] Poly[(25% ethyl glycinato)(75% p-methylphenoxy) phosphazene] Poly[(50% ethyl glycinato)(50% p-methylphenoxy) phosphazene] Poly[(75% ethyl glycinato)(25% p-methylphenoxy) phosphazene] Polypropylene oxide Poly[bis(L-proline methyl ester)phosphazene] Polystyrene Paclitaxel Polyvinylpyrrolidone Poly[bis(2-(2-oxo-1-pyrrolidinyl)ethoxy) phosphazene] Ribonucleic acid Ring-opening polymerization

Abbreviations

RT SBF SD SEM siRNA TBAF TCB TCPS TE TFE THF UV

Room temperature Simulated body fluid Standard deviation Scanning electron microscopy Short interfering ribonucleic acid Tetrabutylammonium fluoride 1,2,4-Trichlorobenzene Tissue-culture polystyrene Tissue engineering Poly[bis(trifluoroethoxy)phosphazene] Tetrahydrofuran Ultraviolet

145

Index Absorption 82 Acid(s) 4, 10, 28, 29, 31, 34–38, 41, 44–46, 55–58, 60, 63, 66, 67, 69, 70, 73, 75, 89, 90, 92, 107, 110–113, 114, 115, 117, 126, 127, 130, 132, 139 – Acidic 10, 31, 39, 43–46, 82, 107, 141 – Acidity 43 Acrylic acid 28, 56 Adhesion 111, 119, 121, 125–128, 135 Adhesive 28, 119 Administration 27, 30, 34, 60, 61, 64–66, 68, 74, 81, 86, 91, 96 Agglomerate 78, 85, 95 – Agglomeration 41, 68, 88 Aggregation 18, 89 Albumin 56, 64, 140 Aluminium chloride 4 α-Amino-ω-methoxy-polyethylene glycol 89 Amphiphilic 18–20, 41, 84, 85, 87–89, 96 Anionic 5, 9, 56, 62, 66, 69, 93, 96 – polymerisation 8, 17, 67, 71, 92 Antibacterial 61 Anticancer 53, 73, 75, 76, 80, 84–86, 90, 91, 93, 94 Antigen 54–58, 60–65 – presenting cell(s) 55 Antimicrobial 58 Antioxidant 116, 117 Antitumor 84 – Activity 75, 77, 91 – agent 73 Architecture 6–8, 10–21, 54, 74, 107–119 Aromatic 10, 34, 58, 130 Arthritis 73 Atom-transfer radical-polymerisation 13, 141 Binding 20, 56–59, 62, 68, 69, 71, 74, 91–93, 111 Bioactive 77, 111 Biocompatible 27, 28, 31, 35, 109, 139, 140 Biodegradable 21, 68, 90, 95, 109, 139 Bioerodible 27–31, 37, 46, 93 Biomaterial 6, 27–31, 43, 47 Biomimetic 84, 87, 110, 111 Blend 38, 43–45, 109–111, 121, 122 – Blending 43, 44, 46, 47, 133 Block copolymer 6, 10, 16–21, 44, 54, 84, 88 Bonding 44, 57, 82, 108, 110 https://doi.org/10.1515/9783110654189-007

Bone 30, 109–111, 115, 116, 120, 121, 123, 126 – tissue engineering 29, 38, 87, 93, 109, 121–126 Bottlebrush polymers 13 Bovine serum albumin 56 Branched 12, 13, 34, 39, 41, 70 – Branching 5, 6, 10–13, 41 Brush-type polymers 13, 16 Buffer 31, 43, 44, 46, 69, 130, 132 Bulk 8, 20, 29, 32, 33, 37, 43, 45, 46, 107, 130, 132 Burst release 80 By-product 1, 32, 43 Calcium phosphate 55, 114, 117, 126, 127 Calcium sulfate 4 Camptothecin 92, 94 Capacity 66, 69, 86 Carrier 30, 31, 53–55, 67, 68, 74–75, 77, 78, 80, 81, 86–88, 90 Catalysis 20, 35 Cationic polymer 67 – polymerisation 6–10, 17, 78 Cavity 86, 110, 111 Cell 29, 40, 55, 56, 61, 63, 64, 67–69, 75, 77, 81–83, 86, 87, 90, 93, 107, 109–111, 113, 115–117, 119–129, 133, 135 – growth 29, 115, 121, 123, 127, 133 – proliferation 87, 111, 122, 125, 135 – viability 83 Cellular 27, 62, 64, 69, 77, 84, 86 – adhesive properties 119 Chain 4, 13, 14, 16, 17, 20, 21, 28, 29, 31–33, 35, 39, 41, 42, 68, 78, 81, 84, 85, 87, 88, 90, 110, 112, 113, 114 – end 6, 7, 10, 13, 16, 68 – growth polycondensation 6–10, 18 – length 9, 21 Chemical 1, 13, 27, 40, 41, 53–56, 58, 62, 74, 77, 79, 90, 96, 107 – composition 54 – properties 1, 13, 41, 96 – structure 55, 56, 58, 62, 77, 79 Chemotherapy 29, 30 Chiral 20 Chitosan 84, 126, 127 Chromatography 2, 33

148

Index

Cleavage 31, 35, 37, 40, 42 Coated 65, 66, 119, 120, 125, 140 – Coating 63, 65, 120, 140 Collagen 31, 119, 120 Colon 82–84, 87 Colorectal cancer 82 Complexation 20, 66, 95 Compliance 71 Component 8, 13, 27, 41, 44, 46, 47, 53, 54, 58, 74, 85, 95, 96, 116, 122, 140 Composite 107–110 Composition 4, 38, 54, 107, 117 Compound 40, 56, 77, 84 Concentration 7, 8, 19, 20, 29, 41, 46, 69, 78, 92, 95, 110 – polymerisation 112 Condensation 1, 8 Conformational 57, 87 Conjugation 55, 59, 75, 77 Controlled 6, 10, 13, 15, 16, 21, 27, 29, 53, 55, 59, 63, 70, 72–74, 78, 80, 84, 87–89, 93, 110, 111, 140, 141 – drug release 29, 53, 72, 74, 87 – polymerisation 13, 21, 140, 141 – release 29, 53, 55, 72–74, 78, 84, 87, 89 Conversion 5, 6, 27 Copolymerization 47 Covalent 27, 55, 62, 66, 74, 81, 82, 91 – Covalently 53, 74, 91, 126 Critical micelle concentration 19 Critical solution temperature 87, 139 Crosslinked 5, 34, 113 – Crosslinking 1, 2, 113 Crystalline 38 – Crystallinity 20, 108 – Crystallisation 20 Cyclic 15, 34, 78, 90 Cyclodextrin 19 – β-Cyclodextrin 19, 20 Cylindrical 109 Cytotoxicity 46, 77, 81, 90 Degradability 1, 31, 43, 54, 72, 80, 83, 90, 96, 117, 132, 133, 141 – Degradable 13, 27–47, 55, 60, 67, 69, 72–74, 81–83, 85, 88, 90, 91, 93, 111, 113, 125, 135, 140, 141

– Degradation 1, 2, 4, 27–29, 31–47, 56, 60, 63, 68–74, 78, 80, 81, 89, 90, 95, 96, 111, 113, 116, 117, 129–135, 139–141 – Degrade 27, 29, 31, 33, 34, 38, 39, 69, 72, 75, 80, 95, 129, 139 Delivery System 54–55, 90, 92 Dendrimer 10, 11, 84 Density 13, 56, 57, 65, 83, 96, 110, 125, 129, 141 Deoxyribonucleic acid 67 Depolymerisation 28 Deposition 65, 111 Derivatisation 112 – Derivative 2, 6, 17, 28, 34, 38, 46, 53, 77 Diaminobutane 11 Diffusion 72, 73, 89 Dimension 13, 15, 44, 53, 68, 73, 80, 110, 117 – Dimensional stability 110 2-Dimethylaminoethylamine 68 4-(Dimethylamino)pyridine 15 Disease 30, 41, 53, 67, 70, 73, 81 Displacement 115 Dissolution 65, 70, 71, 90 Distribution 63, 74, 91, 109 Dosage 30, 61, 91 – Dose 61, 62, 65, 85, 91 Doxil 53 Doxorubicin 85, 86, 90–92 Drug 18, 27, 29, 30, 53, 54, 70–78, 80–82, 84–93, 111 – conjugate 41, 53, 75, 82, 83, 90 – delivery 17, 18, 29, 34, 53, 71–75, 80, 84, 93, 141 – depot 72, 89, 90 – loading 96, 141 – release 29, 53, 72, 73, 74, 79, 80, 87 – resistance 77 – targeting 74 Elastic 112 Electroactive 129 Electrophilic 10 Electrostatic 58, 67, 69 Enantiomeric 20 Encapsulation 18, 63, 64, 73, 85, 86 Endothelial cells 126 Endothelial tissue engineering 126–128 Enhanced permeation and retention 74

Index

Enteral 71, 82 Environment 40, 71, 72, 82, 87, 95, 139 – Environmental 129 Enzyme 29, 41, 45, 66, 78 Erosion 29, 44, 45, 73, 117, 132 Extracellular matrix 29 Fabrication 108, 110, 111, 129 Failure 29, 62 Fibre 107–112, 117, 118, 121, 123, 125–129 Film(s) 20, 39, 64, 65, 73, 107, 111, 114, 117, 119, 120, 121, 125, 126, 128, 129, 135 Flexibility 1, 18, 19, 21, 31, 57, 58, 67, 74, 85, 141 – Flexible 55, 57, 67, 92, 95 Fluorescent 64, 93, 111, 122 Fluorouracil 92 Foam 107, 108 Formulation 29, 53, 55, 59, 60, 61, 63–65, 73, 81, 84, 86 Functional group 10, 34, 37, 58, 67, 69, 74, 90, 96, 141 Functionalisation 10, 13, 55, 93, 116 Functionality 4, 10, 13, 54, 65, 66, 74, 80, 126, 141 Galactose 69 Gastric 71 Gastrointestinal tract 71, 82 Gel(s) 20, 55, 67, 70, 71, 89, 90, 94 Gelation 70, 71, 87, 90, 92 Gene 54, 67–71 – delivery 54, 67–70 – silencing 70–71 – therapy 67 Glucose 34, 74 Gold 20, 93, 95 Graft 84, 86, 110, 113 – Grafted 13, 41, 84, 89, 110 – Grafting 13–16, 88 Hemagglutination inhibition 59 Hepatitis B surface antigen 62, 65 High density 56, 57, 65, 96 High-molecular weight 4, 30, 39 High-throughput 4, 58 Human growth hormone 92 Human immunodeficiency virus 55 Hybrid 1, 13, 15, 38, 41, 43, 88, 114

149

Hydrogel 34, 63, 70, 71, 89–94, 113, 114, 126, 141 Hydrogen bonding 44, 82, 110 Hydrolysable 17 Hydrolysis 1, 2, 5, 28, 29, 32–34, 37–39, 43, 57, 72, 73, 90, 113, 130, 131 Hydrolytic stability 29, 34, 35, 37, 38, 41, 47, 58, 68, 72 Hydrophilic 19, 20, 32, 33, 35, 73, 84, 86, 88, 89, 90 Hydrophobic 18–20, 28, 29, 32–35, 53, 84–86, 88–90, 96, 130 – Hydrophobicity 29, 32, 41, 45, 55, 58, 83, 84, 130 Hydroxyapatite 109 Imaging 64, 93, 95, 117 Immunoglobulin G 61 Immunological activity 61–62 Immunology 34, 54–66 Implantable 29, 73 In situ 8, 69 In vitro 56, 68–70, 75, 77, 80–84, 90, 96, 109, 123 In vivo 29, 44, 56, 58, 60, 61, 64, 68, 69, 71, 73, 75, 77, 78, 80, 84, 86, 87, 90, 91, 95, 111 Indium tin oxide 119 Inflammation 41, 73, 95 – Inflammatory 53, 74 Inhibition 37, 59, 77 Initiation 6, 7, 16, 110 – Initiator 7, 8, 17 Injectable 29, 67, 70, 71, 73, 89–93, 113, 125, 126, 141 – hydrogel 71, 89–93, 113, 114, 126, 141 Injection 60–62, 65, 70, 71, 73, 90, 91, 93, 94 Insulin 71–74 Intramuscular 61, 65, 66 Ionic 8, 69, 71, 93 Irradiation 40, 81, 83, 90 Leaching 71, 108, 121 Leaving group 37 Light 30, 40, 58, 62, 81, 113 Lipid 53 Lipophilic 86, 88, 92, 139

150

Index

Living polymerization 6, 13, 16, 17, 59, 80, 140 Lower critical solution temperature 87 Macroinitiator 13, 17 Macromolecular 1–3, 6, 30, 56, 63, 73–75, 77–80, 82 – architecture 10–21 – engineering 18, 20 – prodrugs 79, 82, 87 – substitution 2–4, 17, 41, 57 Macroporous 20 Macroscopic 108 Magnetic 7, 33, 93, 94 – resonance imaging 93 Manufacturing 2, 4, 60, 140 Matrices 28, 44, 55, 72, 73, 87, 107, 111, 121, 122, 125–128, 135 Matrix 29, 45, 72, 73, 108, 109, 121, 125, 126, 133 Mechanical properties 38, 41, 43, 90 Mechanical strength 28, 107, 108 Mechanism 5–7, 27, 32, 34, 37, 39, 41, 45, 55, 62, 74, 79, 81 Metallodrug 78 – Platinum 75, 77, 90 – Ruthenium 77, 79 Micellar 84, 89 Micelle 17–20, 31, 53–55, 84–87 Microsphere 53, 63, 64, 72–74, 109, 126, 127 Microstructure 109 Mitomycin C 73 – Modulus 90 Molecular weight 4, 30, 39, 130, 141 Monitored long-term therapeutic 94 Monocalcium phosphate monohydrate 117, 119 Monomer 5–8, 17, 18, 27, 31, 140 Morphology 16, 20, 29, 85, 108, 111, 122 Molding 107, 108 Multifunctional 54, 55, 74, 75, 80, 93 N,N-diisopropylethylamine 82 Nanofiber(s) 109, 110 Nanomedicine 18, 30, 31, 53–96 Nanoparticle 20, 53, 55, 59, 73, 77, 78, 93, 95, 109, 111, 126 Nanostructure 16, 18, 20, 21, 84, 109, 110 Nanotechnology 86 Neural tissue engineering 128–129 N-isopropyl acrylamide 88, 89

Nuclear magnetic resonance spectroscopy 7, 33 Nucleation 117 Nucleophilic 32, 38 – substitution 1, 20 Oligomeric 41, 79, 89 Oral 64, 71, 72 Ovalbumin 64 Overnight 82 Oxygen 8, 34, 41, 81 Paclitaxel 78, 91, 93 Particle 55, 93 – Particulate 55, 59, 64, 108 PEGylation 68 Penetration 29, 40, 44 pH 31, 33, 34, 36–39, 44–46, 66, 69, 72, 74, 77–81, 86, 88, 93, 107, 132–134 Pharmacokinetic 54, 75 Pharmacology 73 Phase 56, 60, 61, 90, 125 – separation 107, 108 – transition 90, 94 Phosphate 31–34, 39, 43, 46, 55, 107, 114, 117, 126, 127, 130 – buffer 130, 132 Phosphine 8, 10 Photodynamic therapy 81–82 Plasma 30, 75, 85, 89, 117 Platinum 75–77, 84, 90 Poly(D,L-lactic acid) 127–129 Poly(dichloro)phosphazene 1–4, 6, 8, 13, 16, 32, 82 Poly(D-lysine) 119 Poly(ferrocenylsilane) 20 Poly(glycine ethyl ester) phosphazene 133, 134 Poly(glycine ethyl ester)0.65(aniline pentamer)0.35phosphazene] 128, 129, 133, 134 Poly(glycolic acid) 28, 38, 107 Poly(lactic acid) 28, 38, 107, 110, 139, 140 Poly(lactic-co-glycolic acid) 28, 38, 43–45, 55, 74, 107, 109–111, 114, 121, 122, 133 Poly(methylaminophosphazene) 69, 71, 72 Poly(N-isopropyl acrylamide) 88, 89 Poly(organo)phosphazene 1, 4, 8, 11, 13, 15, 18–20, 27–47, 54–56, 62, 67–76, 80–84, 87–93, 96, 139, 141

Index

Poly[(10% ethy glycinato)(90% p-methylphenoxy)phosphazene) 124, 132 Poly[(25% ethy glycinato)(75% p-methylphenoxy)phosphazene) 124, 132 Poly[(50% ethy glycinato)(50% p-methylphenoxy)phosphazene) 42, 124, 132 Poly[(75% ethy glycinato)(25% p-methylphenoxy)phosphazene) 124, 132 Poly[(ethyl alanato)1.0(ethyl glycinato)1.0phosphazene] 130, 131 Poly[(ethyl alanato)1.0(ethyl oxybenzoate)1.0phosphazene] 121, 125, 132, 133 Poly[(ethyl alanato)1.0(p-methylphenoxy) 1.0phosphazene] 130, 131 Poly[(ethyl alanato)1.0(p-phenylphenoxy) 1.0phosphazene] 130, 131 Poly[(ethyl alanato)1.0(propyl oxybenzoate)1.0phosphazene] 125, 132, 133 Poly[(ethyl glycinato)(methylphenoxy) phosphazene 121, 123, 132 Poly[(ethyl phenylalanate)0.8(ethyl alanate)0.8 (ethyl glycinate)0.4phosphazene] 127–129 Poly[(glycine ethy glycinato)1.0 (phenylphenoxy)1.0phosphazene) 44, 110 Poly[(methoxyethoxyethoxy)1.0(carboxylatophenoxy)1.0phosphazene] 119, 120 Poly[(methoxyethoxyethoxy)1.0(cinnamyloxy)1.0phosphazene] 119, 120 Poly[(methoxyethoxyethoxy)1.0-(p-methylphenoxy) 1.0phosphazene] 119, 120 Poly[bis(2-(2-aminoethoxy)ethoxy) phosphazene] 68–70 Poly[bis(2-(2-methoxyethoxy)ethoxy) phosphazene] 33, 69, 119, 120 Poly[bis(2-(2-oxo-1-pyrrolidinyl)ethyoxy phosphazene] 35, 36, 82 Poly[bis(ethyl alanate)phosphazene] 127, 128 Poly[bis(ethyl alanato)phosphazene] 130, 131 Poly[bis(ethyl phenylalaninato) phosphazene] 109 Poly[bis(L-proline methyl ester) phosphazene 117, 118 Poly[bis(p-methylphenoxy)phosphazene] 112, 125–128 Poly[bis(trifluoroethoxy)phosphazene] 119, 120

151

Poly[di(sodium carboxylatophenoxy) phosphazene] 34, 36, 56–66, 72–74, 95, 96 Polyamidoamine 10, 67 Polycaprolactone 28 Polycarbonate 17 Polycondensation 6–10, 18 Polydioxanone 28 Polydispersity index 41 Polyester 17, 31, 43, 44, 46, 74, 107, 140, 141 Polyethylene 17, 19, 30, 67, 68, 79, 89, 115 – glycol 17, 30, 41, 68, 73, 76, 78, 84, 85, 89, 90, 113, 115, 119, 120 – oxide 19, 79 Polyethyleneimine 67–71 Polylactide 110 Polymersome 18, 19, 83–89 Polymerization 4–10, 13, 16, 17, 20, 21, 28, 47, 59, 78, 80, 110, 139, 140, 141 – mechanism 6 Polymethylmethacrylate 13 Polyplexes 53, 54, 67–74, 92, 96 Polypropylene 17, 19, 41, 79, 88 – glycol 17, 19, 41 – oxide 79, 88 Polystyrene 13, 17, 19, 20, 119, 121 Polyvinylpyrrolidone 30, 41, 81 Pore 107, 108, 117, 133 Porosity 108, 109 Precursor 1–3, 8, 32, 57, 74 Proliferation 87, 111, 121, 122, 125, 126, 129, 135 Protein 53–58, 60, 62–67, 92, 111, 126 Protonated 39, 86 Protonation 10, 29, 38, 66, 81 Purification 5 – Purity 5, 8 Quenching 10 Radical 13, 21, 141 Reactive oxygen species 41, 81, 95 Reactivity 4, 35, 74 Regeneration 44, 111, 120, 126, 128–130 Release system(s) 63, 73 Reproducibility 1, 4, 32, 55 Resonance 7, 33, 93 Retention 30, 33, 42 Ribonucleic acid 70, 71

152

Index

Ring opening polymerization 4–6, 59, 140 Ruthenium 77, 79

Synthesis 1, 4–6, 8, 10–12, 17, 18, 21, 58, 60, 66, 75, 77, 107, 110, 113, 140

Safety 30, 60 Sample 39, 59, 61, 80, 112, 123 Scaffold 28, 38, 93, 107–129, 132, 133, 135, 141 Scale 4, 8, 57, 89, 122 Scanning electron microscopy 65, 109, 111, 112, 114, 117, 122, 126, 127–129, 133, 134 Self-assembly 17–21, 77, 83–87 Sequential addition 7 Serum 56, 66 Short interfering ribonucleic acid 70, 71, 92 Side-group 21, 32–35 Simulated body fluid 117–119 Small intestine 82 Solubilisation 27, 76 – Solubility 7, 41, 58, 75, 78, 80–82, 86, 95 – Soluble 15, 27–36, 43, 46, 56, 60, 63–65, 67, 69, 72, 73, 77, 81, 82, 87, 96, 132, 141 – Solution 4, 6, 13, 19, 41, 46, 71, 87, 95, 109–111, 117, 123, 125, 131, 139 Stabilisation 2, 61, 67 – Stability 1, 3, 4, 28, 29, 32, 34, 37, 38, 41, 43, 44, 46, 47, 58, 66, 68, 70, 72, 85, 89, 108, 110, 116, 117 Star 10, 12–16 – Star poylmers 10, 77, 84 Stomach 76, 77, 83 Structure 1, 11, 12, 13, 16, 18, 20, 21, 28, 31, 35–38, 42, 44, 47, 55–60, 62, 72, 76–79, 81, 83–85, 89, 96, 107–111, 117, 119, 121, 128, 131, 139 Surface 29, 55, 62, 68, 73, 74, 96, 108, 113, 119, 122, 124–126 – erosion 29, 45 Swelling 125

Target 53, 67, 70, 71, 81 – Targeted 53, 55, 58, 77, 80, 81, 87 Termination 6, 16 Tetrabutylammonium fluoride 9 Tetrahydrofuran 3, 13, 15, 82, 110, 112, 113 Therapeutic 40, 41, 53, 66, 67, 70, 71, 74, 80, 82, 86, 91, 93, 94 Thermal stability 1 Thermoplastic 107 Thermoresponsive 18, 70, 84 Thermosensitive 41, 70, 84, 87–94, 96, 126 Thiol 10 – Thiol-ene 116 Tissue engineering 29, 38, 87, 93, 107–135, 141 1,2,4-Trichlorobenzene 4 Trimer 10, 15, 78, 84, 90 Ultraviole 40, 115 Unstable 1, 29, 32, 34, 37, 41 Urocanic acid 69 Vaccine 34, 59, 61, 62, 64, 66, 72, 87 – adjuvant 54–56, 61 Viscosity 90, 92 Water 2, 29, 31, 37–39, 44, 45, 88, 89 – soluble 15, 27–31, 33–36, 43, 46, 56, 60, 63–65, 67, 69, 72, 73, 77, 81, 82, 87, 96, 132, 141 – soluble polymer 34, 35, 46, 60, 63, 81 Wavelength 40, 82