Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation

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Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation Fiona Fernandes1,#, Pooja Kotharkar1,#, Adrija Chakravorty1,#, Meenal Kowshik1 and Indrani Talukdar1,* 1

Dept. of Biological Sciences, BITS Pilani, K. K. Birla Goa campus, Zuarinagar, Goa-403726

ARTICLE HISTORY Received: August 06, 2019 Revised: September 16, 2019 Accepted: November 12, 2019 DOI: 10.2174/1574888X14666191202095041

Abstract: Stem cell-based regenerative medicine holds exceptional therapeutic potential and hence the development of efficient techniques to enhance control over the rate of differentiation has been the focus of active research. One of the strategies to achieve this involves delivering siRNA into stem cells and exploiting the RNA interference (RNAi) mechanism. Transport of siRNA across the cell membrane is a challenge due to its anionic property, especially in primary human cells and stem cells. Moreover, naked siRNA incites immune responses, may cause off-target effects, exhibits low stability and is easily degraded by endonucleases in the bloodstream. Although siRNA delivery using viral vectors and electroporation has been used in stem cells, these methods demonstrate low transfection efficiency, cytotoxicity, immunogenicity, events of integration and may involve laborious customization. With the advent of nanotechnology, nanocarriers which act as novel gene delivery vehicles designed to overcome the problems associated with safety and practicality are being developed. The various nanomaterials that are currently being explored and discussed in this review include liposomes, carbon nanotubes, quantum dots, protein and peptide nanocarriers, magnetic nanoparticles, polymeric nanoparticles, etc. These nanodelivery agents exhibit advantages such as low immunogenic response, biocompatibility, design flexibility allowing for surface modification and functionalization, and control over the surface topography for achieving the desired rate of siRNA delivery and improved gene knockdown efficiency. This review also includes discussion on siRNA co-delivery with imaging agents, plasmid DNA, drugs etc. to achieve combined diagnostic and enhanced therapeutic functionality, both for in vitro and in vivo applications.

Keywords: Stem cell, differentiation, nanoparticles, nanocarriers, transfection, siRNA, regenerative medicine. 1. INTRODUCTION Stem cells (SCs) are unspecialised cells that are capable of long-term self-renewal in their undifferentiated state and possess cell potency to differentiate into various specialised cell types. These unique properties of SCs can be explored in the field of regenerative medicine and as a cell source for tissue engineering and stem cell based therapy [1-3]. Thus, it is necessary to develop effective methods of controlling the differentiation of SCs for exploiting their therapeutic potential and broad clinical applications. Change in gene expression profiles directs SC differentiation [4, 5]. Overexpression of lineage-specific genes inserted by plasmid DNA, knockdown or modulation of pluripotency or multi-potency markers by noncoding RNAs (especially by small interfering or micro RNAs) or by *Address correspondence to this author at the Department of Biological Sciece, BITS Pilani, K.K. Birla Goa Camus, P.O. Box: 403726, Goa, India; Tel: +9108322580359; E-mail: [email protected] #These authors contributed equally to this manuscript. 1574-888X/20 $65.00+.00

small molecules would trigger differentiation of stem cells into specific lineages. In this review, the nanoparticle-mediated delivery of double-stranded RNA (dsRNA) to SCs will be discussed which can be used to control their differentiation by gene silencing, using the highly conserved natural mechanism of RNAi [6]. Long double-stranded or short hairpin-loop RNA (shRNA) which are synthesised by endogenous transcription inside the nucleus, are eventually processed by an RNase type-III endonuclease, dicer, in the cytoplasm into 21-23 nucleotide (nt) long siRNA (small interference RNA). The siRNA, composed of a sense or passenger strand and an antisense or guide strand, is then recruited into the RNAInduced Silencing Complex (RISC), which is mainly composed of Argonaut (Ago) proteins [7, 8]. Among other Ago proteins, Ago2 contains an enzymatically competent RNase H-like domain, which cleaves and degrades the sense strand of the siRNA sharing the identical sequence of the target mRNA. On the other hand, the antisense or the guide © 2020 Bentham Science Publishers

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strand with the complementary sequence to the target mRNA remains in the RISC and directs the recognition and cleavage of the target mRNA by Ago2 [9, 10]. Taking advantage of this in vivo phenomenon of regulating target mRNA transcripts by a non-coding RNA, various methods of in vitro synthesis and delivery of siRNA into different cell lines have been developed. Introduction of a 2123 nt long siRNA designed to target a specific mRNA, would bypass the dicer⁠⁠-mediated processing and is directly be loaded and processed by RISC. Although long dsRNA has been used to silence target genes in many organisms like worms and plants, when introduced in mammalian cells, long dsRNAs greater than 30nts, have shown to activate the anti-viral immune response, leading to non-specific RNA degradation [11]. ⁠Synthetic siRNA, on the other hand, can not only bypass the antiviral mechanisms but can also be easily customised for any target gene, and therefore has attracted a lot of attention for gene manipulation studies [12, 13]. ⁠ The discovery of siRNA revolutionized the field of cell and molecular biology. However, the delivery of naked siRNA suffers several challenges. Naked siRNA is easily degraded by endonucleases, exhibits low serum stability, may cause off-target effects and may invoke an immune response in cells. Furthermore, siRNA is anionic, and therefore it is harder to transport across the cell membrane. Compared to plasmid DNA, siRNA has a stiff structure and relatively low spatial charge density. Thus, it is difficult to formulate a compact complex of siRNA, which is imperative to ensure its stability in the bloodstream [14, 15].⁠

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Developing a safe and effective delivery system for successful siRNA therapy is, therefore, crucial. Although viral vectors have been successfully used for siRNA delivery into various cells, including SCs [16-18], their clinical application is limited by the risk of causing mutations in cells and inciting immune responses [19-22]⁠. Electroporation has also been a successful siRNA delivery method, but it may cause cell damage and ion imbalances [23-27]. Voltage pulse mediated delivery of siRNA is associated with high cell mortality rate making this option limited for less abundant cells (adult stem cells, for example). Prolonged steps of optimization of various parameters in different cell types and the high cost of the equipment are some of the added disadvantages of this technique. To address such challenges, a lot of research has been focussed on the development of various non-viral methods [28, 29], such as nanocarriers and commercial transfection agents (for example lipofectamine, RNAi-Max, DharmaFECT, etc.). Most if not all commercial transfection agents show cytotoxicity and thus, are a poor choice, especially when a long term effect of siRNA is desired by repeated attempts of transfection. On the other hand, nanocarriers are relatively safer, less toxic, less immunogenic [30, 31]. Moreover, the typical nano-size enables their movements through micro-capillaries [32]⁠. Nanocarriers could be classified as organic or inorganic, based on their physical and chemical properties. Organic siRNA nanocarriers (Fig. 1) are composed of self-aggregating lipid-based or liposome inspired nanoparticles, protein or

Fig. (1). Organic Nanoparticles. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

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Fig. (2). Inorganic Nanoparticles. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

peptide-based nanoparticles or polymeric nanoparticles. On the other hand, inorganic nanocarriers (Fig. 2) are insoluble and non-biodegradable and may require additional modification with polymers to improve their solubility. Such nanocarriers are a hybrid of both organic and inorganic materials, which may comprise of gold nanoparticles, magnetic nanoparticles, quantum dots, calcium phosphate nanoparticles or carbon-based nanostructures [31]. A system to track the biological distribution, migration and proliferation after transplantation of SCs, would be an added advantage. Nanocarriers that perform both diagnostic and therapeutic functions, termed as theranostic agents are designed to achieve imaging as well as siRNA delivery [33]. During endocytosis of siRNA, multiple mechanisms (Fig. 3) may be used for the internalization of the nanodelivery vehicle [34]. Knowledge of the internalization mechanism is essential for designing highly efficient therapeutic nanocarriers necessitating the understanding of various possible endocytic pathways and their role in transfection. In general, endocytosis may be receptor-mediated (clathrincoated), receptor-independent, or it may involve macropinocytosis. Receptor independent endocytosis includes caveolin-mediated endocytosis, clathrinindependent endocytosis and clathrin- and caveolinindependent endocytosis. Once the siRNA nanocarrier is internalized, it is entrapped in an endosome. The contents of the endosome would typically be degraded by lysosomal enzymes and therefore another aspect to keep in mind while designing siRNA nanocarriers is to create an endosomal escape strategy to ensure cytosolic delivery and to maximise the gene knockdown efficiency [35, 36].

Compared to other cell lines, SCs are a lot harder to transfect due to limited cellular uptake [37] making it difficult to deliver siRNA using the conventional non-viral vectors [38, 39] . In the cell membrane of primary cells, especially SCs, the structural variations in lipid molecules that mediate transfection may be the cause of low transfection efficiencies [40]. Therefore, the development of efficient siRNA delivery methods for transfection in SCs with the aim of gene manipulation for potential therapeutic applications is warranted. In this review article, various organic, inorganic, hybrid and theranostic nano-carriers and nano-topography based transfection strategies that have been developed to increase the transfection efficiency of siRNA with the aim of lineagespecific differentiation of stem cells have been discussed. 2.1. Organic Nanocarriers In this section, various nanoparticles used to design organic nanocarriers to deliver siRNAs in stem cells for their differentiation into specific lineages have been discussed. These nano carriers are broadly classified as lipid-based and liposome-inspired nanoparticles, protein and peptide nanocarriers and polymeric nanoparticles. A schematic diagram depicting various organic nanocarriers is shown in Fig. (1). 2.1.1. Lipid-Based and Liposome-Inspired Nanoparticles Liposomes are used as vesicle based siRNA carriers. They are spherical vesicles composed primarily of a lipid bilayer with cholesterol [41]. The siRNA delivery system based on a cationic liposome DOTAP (N-(1-(2, 3-

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Fig. (3). Endocytosis Mechanism. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

dioleoyloxy))-N, N, N-trimethylammonium propane methylsulfate) targeting cathepsin S gene was used to transfect CD34+ cells. CD34+ is a well-known marker for blood and bone marrow-derived progenitor cells, especially for hematopoietic and endothelial stem cells. Knockdown of cathepsin S leads to in vitro differentiation of the CD34+ cells into dendritic cells. The DOTAP-mediated transfection of siRNA showed more than 60% knockdown efficiency, which is comparable to that of lentiviral vectors, with minimal cytotoxicity. Further, knockdown was performed on the cells throughout the differentiation process i.e. for 14 days and repeated transfection had no adverse effect on cell development [42]. Sterosomes are non-phospholipid liposomes and are formulated with single-chain amphiphiles and high content of sterols. These particles have an additional positive charge and therefore increased stability. SA/Chol (stearylamine (SA) and cholesterol (Chol)) sterosome and siRNA complexes were shown to have increased cellular uptake and gene knockdown efficiency in mesenchymal stem cells as compared to commercial transfection agents. This complex was further used for knocking down the expression of noggin, which is a specific antagonist of bone morphogenetic proteins preventing its interaction with the receptor. siRNA transfection using SA/Chol resulted in more than 45% knockdown compared to Lipofectamine 2000 which showed 25% knockdown of the gene expression [43]. Further studies with cationic sterosome formulated with stearylamine and cholesterol also proved that in addition to forming complexes with siRNA, it also solubilizes small molecules like phenamil in a single-vehicle. Synergistic effects were observed in terms of knockdown of noggin expression by siRNAs as well as stimulation of BMP signaling by Phenamil.

The SA/Cholsterosomes loaded with phenamil and noggin siRNA proved efficient in inducing osteogenic differentiation of Mesenchymal stem cells (MSCs) in both 2D monolayer culture and 3D hydrogel environment in in vitro as well as in vivo model [44]. Novel lipid-like nanoparticles called lipidoids are shown to have high efficiency in terms of siRNA delivery. Lipidoids are small lipid-like molecules which possess cationic behavior by virtue of having an amine backbone. Natural lipids have only 2 tails, but lipidoids can have one to seven tails depending on the amine used to synthesize them. These lipidoids possess high transfection efficiency and have reduced cytotoxicity in human embryonic stem cell-derived cells (hESCds). In a study carried out by F. Yang et.al, co-delivery of osteoinductive DNA and siRNA directing the osteogenic differentiation of human adipose-derived stem cells (hADSCs) was carried out using a combination of poly (β-amino esters) and lipidoids. The poly (β-amino esters) served as a vehicle for delivering osteoinductive DNA (BMP2) and lipidoid as a delivery vehicle for siRNAs (siGNAS and siNoggin). A significant increase in the expression of BMP2 gene along with the downregulation of GNAS and Noggin in hADSCs was observed. Co-delivery of the siRNAs along with the plasmid DNA, resulted in a significant increase in the expression of osteogenic markers and accelerated differentiation of hADSCs [45]. Another class of lipid-based nanoparticles is Niosomes, which are non-ionic surfactant based vehicles, structurally similar to liposomes with the incorporation of lipids. Niosomes, which exhibit higher stability and loading capacity are cost-effective and easier to synthesize as compared to

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traditional liposomes and thus are more suitable for drug and gene delivery. Yang C. et al developed a novel niosome system which acted as a theranostic platform comprising of a cationic lipid DOTAP, a PEGylated lipid TPGS (d-αTocopheryl polyethylene glycol 1000 succinate), a nonionic surfactant sorbitanmono-oleate (Span 80) and encapsulating indocyaninegreen (ICG). This noisome exhibited dual functions of efficient siRNA/miRNA delivery as well as the labeling of stem cells in the near infra-red range. Efficient delivery of siRNA, as well as miRNA in stem cells, was demonstrated by inhibition of miR-138, a negative regulator of osteoblast differentiation by iSPN (ICG containing noisome system)/anti-miR-138, leading to the promotion of osteogenesis. Silencing efficiency of ~88% was achieved which was much higher compared to Lipofectamine. The internalization of iSPN/siRNA involved macropinocytosis and caveolae-mediated pathways, and conferred fluorescence to the cells [46].

dependent of the endosomal pathway. While entering the cell membranes, these peptides adopt a helical conformation that exposes the positively charged residues that mask the negative charge of siRNA on one side and the tryptophan groups on the other to favor cellular uptake [49].

2.1.2. Protein and Peptide Nanocarriers

Synthetic and natural polymers have drawn active interest as nanocarriers for siRNA delivery onto stem cells because they display a wide diversity in their structure and physico-chemical properties. There are various examples of polymeric carriers developed for siRNA delivery such as polyethyleneimine (PEI), poly-dimethylaminoethyl methacrylate, poly-amidoamine, chitosan, poly-lysine and poly -β-amino esters [51].

Proteins and peptides are being explored as ideal delivery agents for various biological molecules into hard to transfect cells such as stem cells [47]. The various peptide-based strategies for siRNA delivery include cell-penetrating peptide (CPP), CADY, Peptide Transduction Domain-dsRNA Binding Domain (PTD-DRBD) etc. which will be discussed in the following section: Enhancement in the cellular delivery of siRNA can be done with the help of CPP. It consists of five or more molecules of peptide linked to each other, forming a chain-like structure which aids in cellular delivery. The CPPs explored so far including Tat, Oligo-Arg, Transportan and Pentratin, bind to the biological moieties via covalent bonds. These molecules improve the cellular internalization of siRNA and other biological moieties by an endocytic pathway (micropinocytosis). For in vitro delivery of siRNA to cultured cells, covalent attachment of the siRNA to the cargo is preferred, whereas for in vivo delivery, the siRNA is usually bound non-covalently to short amphipathic CPPs like MPG peptide (an amphipathic peptide, consisting of a hydrophobic N terminal and a hydrophilic C-terminal domain). To prove the role of Oct-3/4 as a key player in the process of mesodermal and cardiac commitments of the embryonic epiblast and embryonic stem cells (ESCs), a siRNA targeting this gene was delivered via MPG. The siRNA mediated inhibition of Oct-3/4 in ES cells prevents mesodermal specification and their differentiation into cardiomyocytes. Likewise, Oct-3/4 siRNA injected in the inner cell mass of blastocysts affects early embryonic cardiogenesis [48].⁠ For challenging and hard-to-transfect cell lines, a secondary amphipathic peptide CADY of 20 residues comprised of aromatic tryptophan and cationic arginine residues which formed stable complexes with siRNAs was used. The balance between the stability of the complex, concentration of the CPP at the cell membrane and the interaction of the CPP with the lipid phase was critical in order to achieve efficient gene delivery. Sub-nanomolar concentrations of siRNA delivered via CADY exhibited a significant knockdown of the target gene at mRNA and protein levels. The entry of CADY/siRNA complexes inside the cells was rapid and in-

In another successful attempt to transfect the hard-to transfect cell lines that include T cells, human umbilical vein endothelial cells and hESCs, a Peptide Transduction Domain-dsRNA Binding Domain (PTD-DRBD) fusion protein was used as a siRNA delivering vehicle. These particles bind siRNA with high affinity and mask the overall negative charge of the nucleic acid. While PTD facilitates cellular uptake of siRNA, DRBD encompasses the siRNA and prevents the aggregate formation and, thereby cytotoxicity. Thus, this approach could be used for the efficient transfection of RNAi in difficult to transfect cell lines [50]. 2.1.3. Polymeric Nanoparticles

PEI has exhibited high transfection efficiencies because it causes endosomal osmotic swelling and rupture, which prevents lysosomal degradation of siRNA. However, it is unsuitable for in vivo drug delivery because it can have toxic effects as it causes membrane damage, activates the complement system and interacts with blood cells. Even in easyto-transfect cell lines, high dose of siRNA is required for transfection. Modification with poly-ethylene glycol (PEG) has been attempted as a strategy to overcome this drawback [52]. Poly-β-amino esters (PBAEs) are easy to synthesize, chemically modify and are hydrolytically degradable. To optimize them for siRNA delivery into hMSCs, Tzeng et al. evaluated an array of modified PBAE nanocarriers synthesized by insertion of carbons either between acrylate groups/ amine group and the alkyl groups in the side chain of the base polymers. Cystamine-terminated polymers achieved up to 91% of knockdown after 20 days of transfection. The low weight ratio of polymer to siRNA facilitated its tight binding with the polymer, which leads to an environmentally triggered release. For example, release of the siRNA in the cytoplasm is triggered by its reducing property. These particles were further used to deliver siRNA against ‘B-cell lymphoma (Bcl)-like protein’ (BCL2L2), an osteogenesis inhibitor, thereby triggering osteogenesis in hMSCs [53]. A further study by Núnez et al. was performed where osteogenic differentiation of pluripotent-like stem cells from the dental pulp (DPPSC) was carried out by oligopeptide modified PBAEs; which was used for simultaneous delivery of anti-Oct-3/4 siRNA, anti-NANOG siRNA and Runx2 plasmid. The results of this study indicated that inhibition of Oct 3/4 and the increased expression of the Runx2 enhanced the expression of an osteogenic marker and increased matrix mineralization. These effects were attributed to the double

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Fig. (4). Dual delivery of siRNA and pDNA by polymeric nanoparticle. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

transfection strategy of co-delivering siRNA and plasmid simultaneously (Fig. 4) which did not induce any chromosomal instability or alteration of the cell viability [54]⁠. Diblock copolymers are comprehensively evaluated in the study carried out by Benoit et al. Diblock copolymers are composed of cationic blocks for complexation of siRNA and pH-responsive blocks for endosomal escape. These copolymers confer advantages of protecting siRNA from nucleases, increase uptake efficiency and facilitate escape from endosomal trafficking. The siRNA delivery efficiency of diblock copolymer using fluorescently-labeled siRNA (FAMsiRNA) was 96% as opposed to 64% by commercially available DharmaFECT in MSCs with minimal cytotoxicity. Protein knockdown was sustained for a period of 6 days [55]. pH-responsive diblock copolymers have been reported to provide tailorable nanoparticle architecture and chemistry critical for siRNA delivery. Malcolm et.al. investigated how varying the hydrophobicity, molecular weights and ratio of the corona (first block) and core (second block) affects the delivery of siRNA in murine and human MSCs. The corona was composed of poly-dimethylaminoethyl methacrylate (pDMAEMA) which allowed complexation with siRNA by electrostatic interaction. The core was composed of DMAEMA, 2-propylacrylic acid, and butyl methacrylate (BMA). It facilitates endosomal escape and mediates selfassembly at neutral pH. Uptake of siRNA in hMSCs positively correlated with the molecular weight of the first block; whereas gene silencing and cytocompatibility correlated with percent BMA in all cell types [56].⁠ pH-dependent tripartite polyionic complex (PIC) micelles were also explored as a nonviral method for delivering siRNA in MSCs. The mechanism of micelle formation was based on the spontaneous association of a double-hydrophilic block copolymer with a cationic homopolymer for the complexation with siRNA. Studies on internalization of the mi-

celles with tagged siRNA in primary bone marrow derived stem cells showed caveolae/lipid-raft dependent mechanism and disassembly at acidic pH to escape lysosomal degradation. However, the gene knockdown efficiency assessed by downregulation of Runx2 gene in mMSC was less efficient (40%) as compared to Lipofectamine 2000 (60%) [57]. A family of synthetic polymers called cationic dendrimers represents a highly promising class of siRNA nanocarriers. They are multivalent, have a very precisely constructed structure and a high cargo payload. Dendrimers can be classified by their generation- the number of repeated branching cycles performed during dendrimer synthesis. More exposed functional groups are found on higher generation dendrimers which makes them desirable for siRNA delivery [58]. A study was conducted to compare the transfection efficiency of siRNA against Oct-4 in mouse ESCs using a high generation dendrimer, poly-amidoamine, (PAMAM G5), DOTAP and a commercially available transfection reagent Arrest-In. It was found that the uptake efficiency of siRNA in conjugation with DOTAP was highest (60%)for N/P ratio of 10 compared to that of naked siRNA (1.9%), PAMAM G5 (26% for N/P ratio of 10 and 35% for N/P ratio of 20) and Arrest-In/siRNA complex (39%). However, when the functional effects were analyzed, PAMAM G5 was more than 100-fold better than DOTAP as it could not escape the endosomal degradation pathway [59]. These results highlighted the importance of the endosomal processing of the siRNA upon uptake. Efficient transfection of highly refractory human hematopoietic CD34+ stem cells was achieved using an amphiphilic arginine decorated dendrimer made up of a hydrophilic PAMAM Dendron modified by a hydrophobic alkyl chain and functionalized by polyarginine. The poly arginine moiety is a widely used cell-penetrating peptide, as it possesses positively charged functional groups at physiological

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pH. The modified dendrimers exhibit enhanced siRNA transfection and gene silencing efficiency in human prostate cancer and hematopoietic SCs, due to an increase in association with the cell membrane facilitated by H bonding. Knockdown of ~60% of CD4 (primary receptor for HIV 1) mRNA was observed when anti-CD4 siRNA was delivered by the modified dendrimer as opposed to dendrimer alone and RNAi Max, both of which showed no effect. It was also found that this nanoparticle formed stable complexes with siRNA and showed no evident cytotoxicity [60]⁠.

Another class of adaptive amphiphilic dendrimer nano vehicle that self-assembles in water into vesicle-like dendrimer some nanostructures of ~200nm was designed for siRNA delivery using click chemistry. This dendrimer was able to form a monolayer film at the air/water interface which could swell up to make giant vesicles indicating a lipid-like behavior. Additionally, when these dendrimer’s were complexed with siRNA, they formed many highly ordered small spherical micelles in the range of 6-8 nm with positively charged surface enabling stronger electrostatic interaction with the siRNA. The self-assembly (Fig. 5) of the dendrimer into adaptive supramolecular structures along with a combination of lipid and vector features implies a virus-like delivery property of these dendrimers. The gene delivery capability of dendrimer conjugated with antitat/rev-dsiRNA was assessed in human peripheral blood mononuclear cells (PBMC-CD34+), hematopoietic stem cells (HS-CD34+) and glioblastoma stem cells (GSCs). On transfection, more than 50% reduction in the viral infection was observed whereas, Lipofectamine-RNAiMAX was not able to deliver the siRNA in human primary and stem cells and Trans IT-TKO showed cytotoxicity. The amphiphilic dendrimer also exhibited the potential for in vivo delivery of siRNA. Macropinocytosis was found to be the dominant uptake pathway. Using this delivery system which outperformed commercially available reagents, effective gene knockdown was observed in hematopoietic stem cells (HSCCD34+) and glioblastoma stem cells (GSCs) [60].

Structure-activity relationships (SAR) of ligand-modified dendrimers were screened for their gene delivery capacity in hard to transfect cell lines using generation 5 (G5) polyamidoamine (PAMAM) dendrimers with 128 surface primary amine groups conjugated with 129 different ligands. The conjugated ligands included several groups like alkyl, fluoroalkyl chains, amino acids, benzene derivatives, heterocyclic chains, etc. An efficient triphenylphosphonium-modified G5 dendrimer carrier of siRNA which was found by screening a second-generation library of dendrimers showed efficient knockdown of the Smurf1 gene (SMAD Specific E3 Ubiquitin Protein Ligase 1; involved Hedgehog signaling pathways) and downregulation of the Smurf1 protein in primary mouse mesenchymal stem cells. Thus this study enables one to design a future siRNA delivery vehicle depending upon the ligand group [61]. A series of combinatorial and computational approaches were used for designing amphiphilic dendrons capable of self-assembly (Fig. 5) into supramolecular dendrimer micelles. Dendrons with shorter aliphatic/hydrophilic chain formed large aggregates when complexed with siRNA, whereas dendrons with alkyl chains (C16-C22) formed nanometer range complexes. SiRNA delivery was most efficient for C18 dendron; which was also used to carry siRNA targeting Hsp27 in a prostate cancer xenografted nude mice model and in human CD34+ stem cells. The lower efficiency of the other dendrons was attributed either to the intrinsic incapability of self-assembly or the inability to form a stable micelle. These approaches showed that the right balance between the hydrophobic chain length and the hydrophilic dendritic portion of the amphiphilic dendrimers plays a crucial role in their self-assembly and siRNA delivery activity [62]⁠.

Cyclodextrins are cyclic natural-polymer based nanomaterials which are made up of α-1-4-D-glucose or amylose, are derived from the enzymatic conversion of starch. Cyclodextrin being a natural polymer exhibits low immunogenicity and when used as a siRNA delivery agent, shows extended gene silencing in various cell lines including stem cells [7]. A cationic star polymer composed of a β-cyclodextrin core, poly-(2-dimethyl aminoethyl methacrylate, DMAEMA) as the cationic component and poly-(2-hydroxyethylmethacrylate, HEMA) as the non-toxic stealth component was studied for siRNA delivery. The formulation of polymerH3 (with DMAEMA/P (HEMA) weight content of 50: 50) showed maximum transfection efficiency in mouse ESCs in comparison to Lipofectamine [63]⁠.

Fig. (5). Self Assembly of Amphiphilic Dendrons to form Supramolecular Dendrimer Micelles. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

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DexAM, a cyclodextrin modified dendritic polyamine construct complex was used in dual delivery of siRNAs and solubilizing hydrophobic small molecules (various drugs for example). Such a delivery vehicle would help in eliciting high cellular viability over a long period of time and avoid the use of corrosive solvents such as DMSO. The βcyclodextrin in DexAM acts as a solubilizer for the hydrophobic drugs and the dendritic polyamine backbone provides high-density positive charge for complementing with nucleic acids to form cationic complexes. When genetically modified Neural Stem Cells (NSCs), stably expressing green fluorescent protein (GFP), were treated with DexAM complexed with GFP siRNA, about 80% decrease in intensity of GFP, after 96 hours of siRNA transfection was observed as opposed to 33% of knockdown with commercial transfection agents (Lipofectamine, Liofecatmine 2000 and FuGENE). The high efficiency was attributed to the highly dense surface primary amines and the three-dimensional spherical structure. DexAM carrying the siRNA targeting the SOX9 (sex-determining region Y-box 9) gene, and retinoic acid, led to ~71% differentiation of the NSCs into neurons [64]. 2.1.4. Other Nanoparticles Delivery from a three-dimensional scaffold using a substrate is another strategy of gene delivery technique using nanostructures. These nanostructures provide advantages over conventional nanoparticles in terms of increased surface area, minimal escape of cargo molecules in the medium and higher internalization efficiency. Among these nanostructures, vertically aligned nanowires are being considered as a novel biomaterial for gene delivery. In one such study carried out by Yoshihiro et al. vertically aligned high-density silicon nanowire substrate was used as a three-dimensional topological feature for cellular modification. Here a polydopamine (PD) coated silicon nanowire (PDSiNWs) was used for siRNA delivery in A549luc cells. The gene knockdown assay was carried out by measuring the production of luciferase. It was observed that PDSiNWs exhibited a higher efficiency of siRNA delivery leading to better knockdown efficiency of the target gene. The interaction of PDSiNWs with the cellular membrane perturbed the lateral diffusion of lipids promoting the cellular internalization of siRNA. The coating of various substrates on to the nanostructures can improve the biomolecule carrying capacity which may find potential applications in the field of tissue engineering [65]. 2.2. Inorganic Nanocarriers This section discusses different inorganic nanoparticles which are functionalized to make siRNA delivery vehicles into stem cells. These nanocarriers involve gold nanoparticles, magnetic nanoparticles, quantum dots, calciumphosphate based nanoparticles and carbon-based nanocarriers. Fig. (2) summarizes the broad categories as well as subcategories of the inorganic nanocarriers discussed in this article. 2.2.1. Gold Nanoparticles Gold nanoparticles (AuNPs) have unique optical [66, 67] and catalytic properties [68, 69], they are biocompatible and

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nontoxic [70, 71], and have a range of applications like diagnostics, therapeutics and photonics. The surface of AuNPs can be easily functionalized to suit the application purpose. AuNPs have been extensively used for transfection studies in cancer cells [72, 73]; however, naked AuNPs lack the ability to successfully transfect primary cells and SCs. The transfection efficiency can be enhanced by conjugation of AuNPs with molecules which target and enhance the interaction with cell membrane as well as the genetic material [74]. Gold nanorods (AuNR) coated with PSS (poly-sodium 4styrenesulfonate) for negative charge and PAH (polyallylamine hydrochloride) for a positive charge, interacted electrostatically and exhibited positive charge on the surface allowing efficient binding of siRNA. The AuNR-PSS-PAH nanocarrier for FAM labelled Lysine-specific demethylase 1 (LSD1) siRNA showed efficient cellular uptake (70 %) compared to treatment with AuNR alone (1.1%). LSD1 expression is needed to maintain pluripotency in embryonic and cancer stem cells. There was more than 50% downregulation of the LSD1 at the mRNA level in the hMSCs transfected with siLSD1 compared to the control cells. The mRNA levels of stem cell marker genes SOX2 &Oct-4 were downregulated while the mRNA level for differentiation gene FOXA2 and BMP2 showed upregulation. Thus, the siRNA-nanocarrier complex was successful in the knocking down of LSD1, regulation of the pluripotency genes and promoting differentiation. The transfected cells grown in hepatocyte growth factor showed upregulation of the liver marker genes hepatic nuclear factor 1 (HNF1-α) and CK-8 within 3 days and maintained it up to 7 days [75]. The efficient usage of gold nanocarriers for siRNA delivery has been shown in various systems other than stem cells. Spherical nucleic acid nanoparticle conjugates, SNA-NCs, designed by densely coating inorganic gold nanoparticles with highly oriented oligonucleotides, exhibit the potential for topical delivery of siRNA. Primary normal human keratinocytes, hKCs, when transfected with these gold nanoparticles conjugated with siRNAs, showed an efficient uptake, successful (about 90%) and prolonged (up to 96 hrs) gene silencing effect, low cytotoxicity and low off-target effect compared to a cationic lipid-based transfection agent DharmaFECT1®. This method also showed a remarkably low concentration of the siRNA requirement for a similar amount of gene knockdown efficiency by DharmaFECT. The SNANC nanocarriers conjugated with EGFR siRNA when mixed with a commonly used petrolatum-based moisturizer and administered on different strains of mice, showed penetration into the epidermis within 3 hours. The EGFR mRNA expression was reduced by 65% and protein expression was completely suppressed without any sign of inflammation or toxicity when 50 nM SNA-NCs was applied for 3 weeks. Human skin equivalents (3D organotypic raft cultures) as human skin models were also tested for penetration and knockdown efficiency for SNA-NCs-EGFR siRNA conjugate. The uptake was observed in as less as 2 hours with 25nM of SNA-NCs concentration. Although the uptake was only 4.7% of the applied SNA-NCs, the EGFR mRNA and protein showed 52 and 75% knockdown, respectively [76]. This highly successful gold nano-carrier as an efficient delivery agent of siRNA could be explored in stem cells.

Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation

2.2.2. Magnetic Nanoparticle Magnetic nanoparticles (MNPs) have a unique ability to provide Magnetic Resonance Imaging (MRI) [77-79] as well as to carry a therapeutic payload. Magnetofection is a new method of transfection in which superparamagnetic iron peroxide conjugated with the genetic payload is delivered to the cells by application of the magnetic field [80]. MNPs or their different functional derivatives have been used to deliver siRNAs into stem cells. For example, ZnFe2O4 MNP core with an outer Au shell stabilized by 11mercaptoundecanoic acid (MUA) were proficient in delivering siGFP to NSCs genetically manipulated for stable expression of GFP (NSC-GFP). The MNP showed better transfection efficiency as well as cell viability compared to the commercially available transfection agent X-tremeGENE. The incubation time needed for MNP transfection under the magnetic field was much less than that required for XtremeGENE. The process of multifection (multiple transfections) helped to achieve much higher transfection efficiency without affecting the cell viability. siRNAs targeting the genes CAVEOLIN-1 and SOX9 showed a substantial increase in the percentage of differentiated oligodendrocytes and neurons, respectively [81]. A successful study on the delivery of siRNA into bone marrow-derived hMSCs was achieved using spherical PEIcoated Fe3O4 nanoparticles with an average diameter of 12nm and charge +44.0 mV. When 4ng/ml of these nanoparticles was used with siRNA at a weight ratio of 4, high transfection efficiency was achieved with low cytotoxicity and a high silencing efficiency (>60%). Endocytosis was found to be the mechanism of internalization in these cells. This delivery system could be a good fit for stem cell therapy applications since these nanoparticles possess imaging properties [82]. Apart from the above studies, the MNPs were also explored for the delivery of micro RNA and plasmid DNAs in stem cells. MNPs conjugated with PEI were used for the delivery of the micro RNA, miR-335 into hMSCs. Encoded in the second intron of the mesoderm-specific transcript (MEST), miR-335 regulates the genes responsible for proliferation, differentiation and migration in hMSCs. The cells transfected with miR/PEI and miR/PEI/MNP showed 1000 fold enhancement of miR expression compared to the control and by 72 hrs the miR level of cells transfected with miR/PEI/MNP was 4 fold higher than miR/PEI. Also, the miR/PEI/MNP resulted in sustained expression of miR and the complex remained in the cytoplasm (as opposed to the miR/PEI complex, which was found in the nucleus) enhancing the efficiency of the miR-mediated down-regulation of the target gene expression [83]. ⁠ A magnetofection agent PolyMAG was developed to deliver plasmid DNA in to stem cells. The transfection efficiency of this magnetofection for eGFP was found to be higher in mESCs (45%) compared to FuGENE6 (15%). The PolyMAG mediated transfection of eGFP in mESC was stable and exhibited high levels of GFP expression without interfering with the expression pattern in the stem cell markers and retained the property of differentiation into the specific germ layers under appropriate differentiation condition while continuing the expression of GFP [80].

Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 9

Magnetofection-mediated transfection of plasmid DNA into hair follicle mesenchymal stem cells (hHF-MSCs) was optimized by using low MNP: DNA ratio and multifection method. An increase in incubation time and use of OptiMEM instead of serum-free DMEM also helped to achieve better transfection efficiency and low cytotoxicity as compared to lipofectamine 2000. Nanog gene pDNA transfected into hHF-MSCs by this optimized procedure expressed about 6 fold higher Nanog mRNA level compared to the control. Nanog in adult MSCs enhanced the rate of proliferation of these cells and completely restored the diminished myogenic differentiation potential [84]. Transfection efficiency of MNP by the magnetofection method was found to be higher for oscillating magnetic field compared to the static magnetic field in neurospheres. The most significant increase was seen in the oscillating magnetic field of 4Hz. The transfected NSCs differentiated mainly to astrocytes but not to neuron or oligodendrocytes when different magnetic fields were applied [85].⁠When magneto-multifection was tried in NSCs with an optimal concentration of MNP, transfection efficiency was found to be slightly better than lipofectamine, with no cytotoxic effect and similar differentiation potential. The MNP could also codeliver different plasmids with better transfection efficiency (>80%) compared to sequential delivery (40%) [86]. Taken together these reports highlight the importance of MNPs in delivering siRNA, micro-RNA and plasmid DNAs into stem cells with high efficiency and the potential to differentiate them into specific lineages. 2.2.3. Quantum Dots Fluorescent molecular probes play an important role in biomolecular imagining both in vivo and in vitro. Conventional fluorescent probes like fluorescent proteins and other fluorescent organic molecules exhibit challenges due to scattering, absorbance and inhibition caused by auto fluorescence in the body. Quantum dots (QD) exhibit broad excitation spectra and a narrow, sharply defined emission peak with large Stokes shift are stable and show reduced photobleaching. These physiochemical properties are advantageous over conventional probes like organic dyes and fluorescent proteins. QDs can also serve as theranostic nanocarriers which can achieve high sensitivity and resolution for long duration at low costs [87, 88]. However, in spite of these excellent and advantageous properties, the usages of QDs in cell-based applications are limited due to their cytotoxicity. Various methods overcoming this difficulty have been adopted. Some examples are discussed below: The sonochemical synthetic method was used to prepare a library of biocompatible QD with the empirical formula ZnxS-AgyIn1-yS2 (ZAIS). These QDs prepared by varying the concentration of the organometallic precursor element, retain the strong fluorescence primarily in the cytoplasm and exhibit low cytotoxicity and phototoxicity as compared to conventional CdSe/ZnS QDs when transfected in hMSCs and human brain tumor cells (U87 glioblastoma cell line). 80% siRNA mediated knockdown of EGFP mRNA was observed in U87 cells genetically modified for GFP expression. Similar results could also be expected from stem cells, and these

10 Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00

QDs could be explored as a viable option for simultaneous delivery and imaging probe in stem cells [89]. Another method to reduce the toxicity of CdSe QD is by incorporation of ZnS shell and PEG coating. Heterobifunctional cross-linker, sulfosuccinimidyl-4-(N-maleimidomethyl) and/or cyclohexane-1-carboxylate (sulfo-SMCC) can be used for bioconjugation on selective sites of the protein through stable thioether bond with a sulfhydryl-exposed antibody. Within the concentration range of 20-120pmol/L, the cytotoxic effect of QD-SMCC was lower than PEI. When exposed to the MSCs isolated from the femoral marrow cavity of the rat models, the cells produced a large number of filopodia which engulfed the QD-SMCC within 3 hrs of transfection. QD-SMCC-were released into the cytoplasm after rupturing the endosomes, 12hrs post-transfection. Both transfection and knockdown efficiency were better when SOX9 siRNA was conjugated as QD-SMCC-siSOX9 rather than PEI-siSOX9 for delivery into these cells. The downregulation of SOX resulted in the down-regulation of type II collagen and aggrecan. SOX9 acts as a transcriptional activator of both these genes causing accelerated differentiation of adult MSCs toward chondrocytes [90]. QD conjugated with β cyclodextrin and positively charged peptides like RGD have also shown better biocompatibility and negligible cytotoxicity as compared to QD alone. RGD serves the dual purpose of binding with the siRNA and facilitating better uptake by binding with integrin receptors on the cell surface. The RGD-β-CD-QD nanocarrier served as a drug delivery vehicle and also showed significant uptake in hMSCs. The hMSCs developed filopodia around the complex, and uptake was mediated by micropinocytosis (Fig. 3). This nanocarrier was also used to deliver a small molecule kartogenin (KGN), capable of inducing chondrogenesis in hMSCs. It was found that the upregulation of chondrogenic markers was higher when the small molecule was delivered via the nanocarrier as compared to the administration of KGN by directly supplementing it in the media. A similar result was obtained for a 3D mass culture. This method to induce chondrogenesis was compared with the siRNA mediated knockdown of the Runx2 gene, delivered by the QD nanoparticles complexed with KGN and siRunx2. Runx2 suppresses chondrocyte hypertrophy and the associated pathogenic calcification in hyaline cartilage. The expression level of aggrecan, type II collagen, and Sox 9 indicative of chondrocyte differentiation were found to be similar to that of lipofectamine-mediated delivery of the siRNA and the nanocarrier mediated KGN delivery. The expression of Runx2 and type X Collagen was found to be lower than the KGN nanocarrier group but equivalent to the lipofectamine-siRNA group. Thus, the nanoparticle-siRNA did not inhibit chondrocyte differentiation but suppressed the hypertrophy of chondrogenically induced hMSCs [91]. In a follow-up study by the same group, it has been confirmed that the QD- nanocarrier is capable of co-delivering small molecules as well as siRNAs into stem cells in contrast to Lipo2000, which is limited to the delivery of oligonucleotide-based cargoes. The QD was shown to successfully codeliver a hydrophobic and osteogenic small molecule; dexamethasone (Dex) and a siRNA against peroxisome proliferator-activated receptor gamma (PPAR ϒ) in hMSCs.

Fernandes et al.

PPAR ϒ is a regulator for adipogenesis and its downregulation leads to osteogenesis. The co-delivery resulted in higher expression of Runx2 compared to the delivery of the individual molecule by the QD conjugated siPPARϒ or Dex separately (more than 90% expression compared to approximately 32% and 57%, respectively). The QD could be fluorescently tracked for up to 14 days [92]. 2.2.4. Calcium Phosphate Nanoparticle Calcium phosphate (Ca:P) nanoparticles mimic the natural bone material. They are biocompatible, non-toxic, inexpensive and easy to prepare [93]. The release of the encapsulated nucleic acids into the cellular cytoplasm is facilitated by the pH-dependent solubility of Ca:P and endosomal acidification. Despite these advantages, a major drawback in these nanoparticles is their tendency to form aggregates due to their uncontrollable size. This causes inconsistent transfection efficiencies in various cell lines. Aggregation can be prevented by using a stabilizing agent which further helps in improving the transfection efficiency [94, 95]. For example, Glutamine (Gln)-conjugated oligochitosan (OChi) was used to stabilize these nanoparticles. The modified Ca:P nanoparticles in the presence of OChi or Gln-OChi were spherical in shape and did not form large agglomerates. The modification improved the cellular uptake of the nanoparticles with minimal cytotoxicity. The Gln-OChiCa:P exhibited higher transfection efficiency (more than 90%) compared to Ca:P nanoparticle (about 65%) alone which was comparable with that of lipofectamine2000 mediated transfection in HeLa cell lines. The knockdown of the GFP gene expressed in Hela cells was yet again similar to that of the lipofectamine-mediated effect. When compared to the chitosan-based nanoparticles, Ca:P nanoparticles stabilized by chitosan showed similar transfection efficiency and higher gene knockdown efficiency. In adipose-derived MSCs (ADSCs), the transfection efficiency of the Ca:P nanoparticle was much lower as compared to that in HeLa cells. However, when modified by chitosan, the transfection efficiency became comparable. The bioactivity of the Ca:P nanoparticles in a photocross linkable chitosan 3D hydrogel were evaluated by transfection with fluorescently labelled siRNA against Noggin. The transfected ADSCs showed a decrease in NOG mRNA level to 49%, similar to the lipofectamine, and the knockdown led to bone formation in vivo and osteogenesis in vitro [96]. 2.2.5. Potential Carbon based Nanocarriers Carbon Nanotubes (CNT) contain covalently bonded sp2-hybridized carbon atoms which form six-membered rings. They are elongated, tube-like nanostructures which have high conductivity, surface area, tensile strength, and high aspect ratio [97]. They can be used as scaffold materials to promote proliferation and differentiation of stem cells [98]. The CNTs could be single-walled (SWCNT) or multiwalled (MWCNT). While the SWCNTs have a smaller diameter, exhibit more flexibility and offer photoluminescence which could be exploited for imaging, the MWCNTs have a wider surface area for attachment of the cargo and functionalization supporting more efficient internal encapsulation.

Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation

Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 11

Pristine CNTs lack solubility, and form thick and inhomogeneous bundles, exhibit short circulation half-lives, immunogenicity and low biocompatibility. Surface modification of the CNT, for example by functionalization with PEG, enhances the bio-distribution and eventual elimination of CNT after uptake into the cells. SWCNTs functionalized with PEG, have been used to deliver siRNA in tumor cell lines. It had been observed that the complexes of siRNA with SWCNT alone (siRNA / SWCNT), or with SWCNTfunctionalized with PEG (siRNA / linear-PEG-5000 / SWCNT or siRNA / branched-PEG-SWCNT) did not displace/release the siRNA from the complex, for approximately 4 hours in the presence of 1% BSA. This result showed that in vivo delivery of siRNA by these complexes would not release the payload immediately in the blood, indicating their potential as in vivo transfection agents with high knockdown efficiency due to better stability [99].

tein expression with 200 nM siRNA. Survivin. It is a novel apoptosis inhibitor encoded by BIRC5 (baculoviral inhibitor of apoptosis repeat-containing 5) gene [106].

SWCNT can be used as scaffold materials to promote proliferation and differentiation of human, rat, canine MSCs, and hESCs. Scaffolds of CNT functionalized with polyacrylic acid (PAA), a weak acid with an adverse effect on neuronal differentiation and neuronal cell attachment, showed improved cell adhesion and viability as compared to poly-L-ornithine (PLO), conventionally used for neuron growth. The scaffold showed two-fold higher neuron differentiation from hESCs as compared to PLO [100].

The cellular environment can be modulated to achieve greater transfection efficiencies for SCs. Nanotopography of a surface can have great impact on cell morphology and adherence. For SCs, it has been proven that it is, in fact, nanotopography of a surface and not its microtopography that acts as the primary influencing factor [108]. Physical interaction with some specific nanotopographical cues can be used to regulate the functions of SCs, particularly MSCs. The nanotopography can be manipulated as per requirement by changing various nano-scale surface features to induce more efficient transfection and differentiation in SCs.

Apart from the above carbon-based nanocarriers, few other examples are there which could potentially be useful for siRNA delivery in stem cells as well as in stem cell based therapy. Nanodiamonds with facile surface functionalization and low cytotoxicity are an attractive option for the delivery of drugs or siRNA to different cell types. They can also be used as a scaffold for the differentiation of stem cells [101].Fluorescent nanodiamonds reported by several groups have been used for the tracking and labelling of stem cells and can be proposed as a theranostic nanocarrier of siRNA for stem cell based therapy [102, 103]. 2.2.6. 2D Nanomaterials 2D nanomaterials are ultrathin layers of a nanomaterial with a minimum thickness of one atomic layer. 2D nanomaterial exhibits high surface-to-volume ratio and thus shows high loading and transfer efficiency. Graphene Oxide (GO) is a 2D carbon-based nanomaterial with promising gene delivery capability. GO particles loaded with siRNA targeting Casein kinase-2 interacting protein-1 (Ckip-1) and dual functionalized with PEI and PEG were used to improve osteogenic differentiation in pre-osteoblast MC3T3-E1 cells [104]. GO functionalized with folic acid (FA), NH2-mPEG-NH2 (5k) and Poly-allylamine hydrochloride (PAH) was capable of co-delivering HDAC1 and K-Ras siRNA to MIA PaCa-2 cells thereby inhibiting tumor growth and proliferation [105]. Black phosphorus (BP) nanosheets are novel 2D nanomaterial with promising biomedical applications. BP functionalized with PEI successfully delivered survivin siRNA in MCF-7 cells. The complex showed endosomal escape, cytoplasmic retention and 80% suppression of the survivin pro-

MoS2 (molybdenum disulfide) is another 2D nanomaterial capable of gene delivery. Positively charged MoS2-PEGPE successfully delivered PLK1 siRNA in HepG2 cells. FACS analysis showed higher apoptotic cells with increase in N/P ratio. Decrease in PLK1 protein level in cells treated with MoS2-PEG-PEI/siPLK1 was comparable to that of lipofectamine/siPLK1treated cells [107]. 2D nanomaterials have shown promising results in siRNA delivery in cancer cell lines and can be further tested for transfection of stem cells. 3. NANOTOPOGRAPHY AND NANOSTRUCTURED SCAFFOLDS

A novel nanotopography-mediated reverse uptake (NanoRU) system was developed wherein siRNA delivery in NSCs was facilitated by the nanotopographical features of the extracellular microenvironment, without the requirement of exogenous delivery vehicles. NanoRU was formed by self-assembling, positively-charged (amine-terminated) silicon oxide nanoparticles (SiNPs) of different sizes (100-700 nm) on a glass substrate. A monolayer of siNPs was then coated with a solution of laminin, an extracellular matrix protein that binds the integrin receptors on NSCs influencing their differentiation, and siRNA to be transfected. 100 nm SiNPs in NanoRUs showed the most efficient knockdown in NSCs compared to larger sizes when used against GFP in genetically modified GFP-labelled NSCs. The same system was used to knockdown neural switch gene (SOX9) expression of NSCs, and resulted in a significant enhancement in neuronal differentiation. The fluorescently labelled siNPs showed that the NaoRUs only deliver the siRNA into the cell and not the silica nanoparticle. Caveolae mediated endocytosis rather than the less efficient clathrin-mediated endocytosis was found to be the pathway followed for the uptake mechanism. Thus, by using this efficient delivery system, the use of cytotoxic cationic delivery agents for siRNA can be avoided. This study shows that nanotopography has a great influence on efficient siRNA uptake by SCs [109]. A 3D siRNA environment modified with lipid-like materials (lipidoids) was developed which showed up to 90% transfection efficiency, a significant increase as compared to similar 2D growth environments. Lipidoids (in particular, 98N12(5)) can self-assemble into nanoparticles and form complexes with siRNA based on electrostatic interaction and provides an efficient siRNA delivery system both in vitro

12 Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00

and in vivo. These nano-complexes are internalized by endocytosis and released from the endosome to the cytoplasm leading to intracellular gene delivery. A porous 3D cell support or scaffold made of biodegradable polymers (poly-Llactide and poly(lactic-co-glycolic acid)) was seeded with a mixture of matrigel, siRNA-lipidoid nanoparticles and hESCs. A siRNA targeting KDR gene (a type III tyrosine kinase receptor for vascular endothelial growth factor, VEGF), was used to direct the differentiation pattern in hESCs towards a specific lineage. The delivery system was successful in knocking down 72% of the KDR gene expression which resulted in significant upregulation in mesodermal and ectodermal markers, and down-regulation in the endodermal differentiation markers. This result was in contrast to the control cells, which spontaneously differentiated into all the three germ layers when cultured on a scaffold without lipidoid-siRNA complexes. Thus, this 3D system was shown to be effective in not only specific gene silencing, but also in triggering differentiation into a specific lineage [110]. A novel method for spatially controlled development of multiple cell types was engineered by adhering nanoparticles containing different siRNAs into the nanostructured scaffolds. Due to the difference in spatial localization of distinct nanoparticle-containing siRNAs, different tissue types within a single implant was aimed to be developed (Fig. 6). The delivery system was engineered by siRNA-enhanced scaffolds (siRESs; composed of biodegradable nanostructured poly-ε-caprolactone scaffolds) functionalized with a lyophilized polymer/lipid-based nanoparticulate siRNA delivery system. The scaffold with various sized pores and enhanced surface area was filled with siRNA nanoparticles which showed a high amount of transfection efficiency when seeded with hMSCs. Knockdown efficiency was tested against various genes, which showed 40-60% efficiency between 24-72 h post-transfection. SiRNAs targeting tribbles homolog 2 (TRIB2) and BCL2 like 2 (BCL2L2) genes, in-

Fernandes et al.

duced adipogenic and osteogenic differentiation, respectively. To construct a tissue comprising of two cell types, the scaffold cylinders were cut in half, and each part was coated with siRNA, either TRIB2 or BCL2L2. HMSCs were seeded after joining the two sides together. This system was tested both in vitro and in vivo for tissue-specific differentiation. In the in vitro culture, an adipogenic (AP2) and an osteogenic (ALP) differentiation marker were upregulated in the TRIB2 siRNA-coated and BCL2L2 siRNA-coated part, respectively. On the other hand, when the combined scaffold was implanted subcutaneously for 2 weeks in mice, the cells on the BCL2L2 siRNA-coated side showed morphology distinct from the TRIB2 siRNA-coated side. Staining with Sirius red confirmed the extensive deposition of organized birefringent collagen in the BCL2L2 siRNA-coated but not in the TRIB2 siRNA-coated side. This approach demonstrated that customized cell specialization can be achieved in distinct locations within a composite scaffold by precise deposition of specific siRNAs [111]. A safe method for sustained and long term delivery of siRNA using mesoporous silica nanoparticle (MSN) complexes was explored to avoid delivery by viral vectors and/or delivery by multiple doses which often cause toxicity. SiRNA incorporation in MSN complexed with PEI was evaluated by two methods: 1) surface absorption method by Poly (e-caprolactone) (PCL) fibre using mussel-inspired bioadhesive and 2) direct encapsulation method by poly (caprolactone-co-ethyl ethylene phosphate) (PCLEEP) nanofibers. The surface absorption method with siRNA/MSN-PEI showed a burst release of siRNA within the first one hour and almost 100% release in 30days. The encapsulated scaffolds of siRNA/MSN-PEI, on the other hand, showed a sustained release of siRNA over 5 months. In vitro gene silencing study targeting collagen type I expression in human dermal fibroblast (HDF) showed sus-

Fig. (6). Spatial control of siRNA-NP leading to enhanced differentiation. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation

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tained availability of siRNA for 30 days using surface adsorbed siRNA/MSN-PEI. Subcutaneous implantation of encapsulated siRNA/MSN-PEI scaffolds revealed that siRNA remained localized within the implants and caused fibrous capsule reduction after 4 weeks. Thus, such nanofiber scaffold-mediated non-viral long term siRNA delivery method could have potentially useful applications of stem cell-based therapy and regenerative medicine [112].

extensively discussed. The roles of dual delivery systems which can deliver both drugs/imaging agents and siRNA to SCs are also included. These advances are expected to pave the way for the future state of the art cargo delivery technologies, essential for, but not limited to applications in regenerative medicine.

4. CLINICAL APPLICATION Worldwide about 500 MSC based clinical trials were carried out for cardiovascular, neurological, orthopedic, autoimmune disease and organ transplantation as per the National Institutes of Health, USA [113]. However, trials involving stem cells transfected using siRNA-nanoparticle complex are restricted due to challenges associated with the toxicity of the nanoparticles, although such systems have been demonstrated to be highly effective gene delivery agents with the capability of being modified to achieve control of the rate of release of the genetic material. Some of the currently approved nanoparticle-mediated siRNA delivery strategies for clinical trials include Liposomes, biodegradable polymers, gold nanoparticles etc. for treatment of diseases such leukemia, Pachyonychia congenita, Glioblastoma etc. [114]. Hence, there is a pressing need for development of various biodegradable siRNA nanocarriers as discussed in this article, showing the property of less cell toxicity, demonstrating enhanced survival rate of the transfected stem cells, enabling the multipotent and pluripotent stem cells to differentiate into specific lineages in both 2D and 3D cultures and exhibiting the property of coupled delivery with drug molecules. Such nanocarriers would be the candidates of choice for clinical applications and their potential could be explored in growing patient-specific organoids in 3D culture, in screening for drug molecules for combinatorial treatment for lineage specific differentiation, as well as to treat various diseases by regenerative medicines [115] CONCLUSION Regenerative medicine is an emerging area amalgamating cutting edge technology in healthcare and personalized treatment for application of stem cell-based therapy and thus research in this field is highly warranted. Some of the areas in which extensive studies are being carried out include cardiac tissue regeneration, spinal cord injury, muscular degeneration etc. Manipulating gene expression to drive differentiation of pluripotent or multipotent stem cells into lineagespecific manner is the key to this technology. RNAi is a powerful tool which has been used to cater to this need. However, the safe and efficient delivery of siRNA into SCs has been a major challenge. This review describes the recent advances in the area of nanotechnology-based approaches that have made it possible to deliver siRNA into hard-totransfect stem cells, achieve improved transfection efficiencies and increased gene knockdown, compelling lineagespecific differentiation. Nanocarriers that are currently being explored and are covered in this review include organic, inorganic and hybrid nanoparticles, both in 2D and 3D environments. Nanotopography of these delivery agents, which governs factors such as duration of release for long term effects both for in vitro as well as in vivo applications, are also

LIST OF ABBREVIATION siRNA

=

Small Interfering RNA

SC

=

Stem Cell

DNA

=

Deoxy Ribonucleic Acid

RNA

=

Ribonucleic Acid

dsRNA

=

Double Stranded RNA

RNAi

=

RNA Interference

shRNA

=

Short Hairpin-Loop RNA

Nt

=

Nucleotide

RISC

=

RNA-Induced Silencing Complex

Ago

=

Argomaut Proteins

mRNA

=

Messenger RNA

DOTAP

=

(N-(1-(2, 3-dioleoyloxy))-N, N, NTrimethylammonium Propane MethylSulfate)

CD34+

=

Cluster of Differentiation 34

SA

=

Stearylamine

Chol

=

Cholestrol

MSC

=

Mesenchymal Stem Cell

hESCds

=

Human Embryonic Stem Cell Derived Cells

hADSCs

=

Human Adipose Derived Stem Cells

BMP2

=

Bone Morphogenetic Protein2

GNAS

=

Guanine Nucleotide Binding Protein, Alpha Stimulating

PEG

=

Polyethylene Glycol

TPGS

=

(d-α-Tocopheryl Polyethylene Glycol 1000 Succinate)

Span 80

=

Sorbitan Mono-Oleate

ICG

=

Indocyanine Green

iSPN

=

ICG Containing Noisome

Anti-miR-138 miRNA =

microRNA

CPP

=

Cell Penetrating Peptide

PTD-DRBD

=

Peptide Transduction Domain-dsRNA

TAT

=

Transactivator of Transcription

Oct-3/4

=

Octamer-Binding Transcription Factor

ESCs

=

Embryonic Stem Cells

HUVECs

=

Human Umbilical Vein Endothelial Cells

14 Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00

Fernandes et al.

PEI

=

Polyethyleneimine

MUA

=

11-Mercaptoundecanoic Acid

PBAEs

=

Poly-β-amino esters

SOX9

=

BCL2L2

=

B-Cell Lymphoma (Bcl)-Like Protein

SRY[Sex Determining Region Y]-Box 9

DPPSC

=

Dental Pulp Derived Pluripotent-Like Stem Cells

MEST

=

Mesoderm-Specific Transcript

hHF-MSCs

=

Human Hair Follicle Mesenchymal Stem Cells

QD

=

Quantum Dots

NANOG

=

Homeobox Transcription Factor

Runx2

=

Runt-Related Transcription factor 2

Sulfo-SMCC =

Sulfosuccinimidyl-4-(Nmaleimidomethyl)-cyclohexane-1carboxylate

Butyl Methacrylate

RGD

=

Arginylglycylaspartic Acid

=

Polyionic Complex

KGN

=

Kartogenin

PAMAM G5

=

Fifth Generation Poly-Amidoamine Dendrimer

Dex

=

Dexamethasone

N/P ratio

=

Nanoparticle to Plasmid Ratio

PPAR ϒ

=

Peroxisome Proliferator-Activated Receptor Gamma

SAR

=

Structure-Activity Relationships

Ca:P

=

Calcium Phosphate

SMAD

=

Specific E3 Ubiquitin Protein Ligase 1; Involved Hedgehog Signaling Pathways

Glu

=

Glutamine

Hsp27

=

Heat Shock Protein 27

Ochi

=

Oligochitosan

Gln-Ochi

=

Glutamine-Conjugates Oligochitosan

CNT

=

Carbon Nanotubes

SWCNT

=

Single-Walled Carbon Nanotubes

MWCNT

=

MULTI-Walled Carbon Nanotubes

BSA

=

Bovine Serum Albumin

PAA

=

POLY ACRYLIC ACID

PLO

=

POLY-L-ORNITHINE

GO

=

Graphene Oxide

MC3T3-E1

=

Mouse OSTEOBLASTIC CELLS

Ckip-1

=

Casein KINASE-2 INTERACTING PROTEIN-1

FA

=

Folic ACID

HDAC1

=

Histone DEACETYLASE1

K-Ras

=

Kirsten RAT SARCOMA 2 VIRAL ONCOGENE HOMOLOG

MIA PaCa-2

=

HUMAN PANCREATIC CARCINOMA CELL LINE

BP

=

Black PHOSPHORUS

MCF-7

=

Michigan Cancer Foundation-7(Breast CANCER CELL LINE)

BIRC5

=

BACULOVIRAL INHIBITOR OF APOPTOSIS REPEAT-CONTAINING 5

FAM-siRNA =

Fluorescein Amidites Labeled siRNA

pDMAEMA

=

poly-Dimethylaminoethyl Methacrylate

BMA

=

PIC

PBMC-CD34+ =

Human Peripheral Blood Mononuclear Cells

HS-CD34+

=

Hematopoietic Stem Cells

GSCs

=

Glioblastoma Stem Cells

HEMA

=

poly-(2-Hydroxyethylmethacrylate)

DMSO

=

Dimethyl Sulfoxide

NSCs

=

Neural Stem Cells

GFP

=

green Fluorescent Protein

PD

=

Polydopamine

PDSiNWs

=

Polydopamine (PD) Coated Silicon Nanowire

AuNPs

=

Gold Nanoparticles

AuNR

=

Gold Nanorods

PSS

=

poly-Sodium 4-Styrenesulfonate

PAH

=

poly-Allylamine Hydrochloride

LSD1

=

Lysine-Specific Demethylase 1

SOX2

=

SRY[Sex Determining Region Y]-Box 2

FOXA2

=

Forkhead Box A2

HNF1-α

=

Hepatic Nuclear Factor 1

CK-8

=

Cytokeratin-8

SNA-NCs

=

Spherical Nucleic Acid Nanoparticle Conjugates

MoS2

=

molybdenum disulfide

hKCs

=

Primary Normal Human Keratinocytes

HepG2

=

EGFR

=

Epidermal Growth Factor Receptor

HEPATOMA G2(HUMAN LIVER CANCER CELL LINE)

MNPs

=

Magnetic Nanoparticles

Plk1

=

Polo-LIKE KINASE 1

MRI

=

Magnetic Resonance Imaging

NanoRU

=

NANOTOPOGRAPHY-MEDIATED REVERSE UPTAKE

Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation

SiNPs

=

SILICON OXIDE NANOPARTICLES

KDR

=

Kinase Insert Domain Receptor

VEGF

=

Vascular Endothelial Growth Factor

Sires

=

siRNA-Enhanced Scaffolds

TRIB2

=

Tribbles Homolog 2

BCL2L2

=

Bcl-2[B-Cell Leukemia/Lymphoma 2]like Protein 2

AP2

=

Adipocyte Protein 2

ALP

=

Alkaline Phosphatase

MSN

=

Mesoporous Silica Nanoparticles

PCL

=

Poly (e-caprolactone)

PCLEEP

=

Poly (Caprolactone-co-ethyl Ethylene Phosphate)

HDF

=

Human Dermal Fibroblast

Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 15 [8]

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CONSENT FOR PUBLICATION Informed consent was obtained from all individual participants included in the study.

[15]

FUNDING

[16]

None. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise.

[17]

[18]

ACKNOWLEDGEMENTS Declared none. [19]

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