Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation PDF
Preview Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation
Send Orders for Reprints to [email protected] Current Stem Cell Research & Therapy, 2020, 15, 000-000 1 REVIEW ARTICLE Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation Fiona Fernandes1,#, Pooja Kotharkar1,#, Adrija Chakravorty1,#, Meenal Kowshik1 and Indrani Talukdar1,* 1Dept. of Biological Sciences, BITS Pilani, K. K. Birla Goa campus, Zuarinagar, Goa-403726 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 mem- brane 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 vec- tors and electroporation has been used in stem cells, these methods demonstrate low transfection effi- ciency, cytotoxicity, immunogenicity, events of integration and may involve laborious customization. A R T I C L E H I S T O R Y 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 nano- Received: August 06, 2019 materials that are currently being explored and discussed in this review include liposomes, carbon Revised: September 16, 2019 Accepted: November 12, 2019 nanotubes, quantum dots, protein and peptide nanocarriers, magnetic nanoparticles, polymeric nanopar- ticles, etc. These nanodelivery agents exhibit advantages such as low immunogenic response, biocom- DOI: 10.2174/1574888X14666191202095041 patibility, 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 knock- down 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 small molecules would trigger differentiation of stem cells into specific lineages. Stem cells (SCs) are unspecialised cells that are capable of long-term self-renewal in their undifferentiated state and In this review, the nanoparticle-mediated delivery of possess cell potency to differentiate into various specialised double-stranded RNA (dsRNA) to SCs will be discussed cell types. These unique properties of SCs can be explored in which can be used to control their differentiation by gene the field of regenerative medicine and as a cell source for silencing, using the highly conserved natural mechanism of tissue engineering and stem cell based therapy [1-3]. Thus, it RNAi [6]. is necessary to develop effective methods of controlling the Long double-stranded or short hairpin-loop RNA differentiation of SCs for exploiting their therapeutic (shRNA) which are synthesised by endogenous transcription potential and broad clinical applications. inside the nucleus, are eventually processed by an RNase Change in gene expression profiles directs SC type-III endonuclease, dicer, in the cytoplasm into 21-23 differentiation [4, 5]. Overexpression of lineage-specific nucleotide (nt) long siRNA (small interference RNA). The genes inserted by plasmid DNA, knockdown or modulation siRNA, composed of a sense or passenger strand and an of pluripotency or multi-potency markers by noncoding antisense or guide strand, is then recruited into the RNA- RNAs (especially by small interfering or micro RNAs) or by Induced Silencing Complex (RISC), which is mainly composed of Argonaut (Ago) proteins [7, 8]. Among other Ago proteins, Ago2 contains an enzymatically competent *Address correspondence to this author at the Department of Biological RNase H-like domain, which cleaves and degrades the sense Sciece, BITS Pilani, K.K. Birla Goa Camus, P.O. Box: 403726, Goa, India; strand of the siRNA sharing the identical sequence of the Tel: +9108322580359; E-mail: [email protected] #These authors contributed equally to this manuscript. target mRNA. On the other hand, the antisense or the guide 1574-888X/20 $65.00+.00 © 2020 Bentham Science Publishers 2 Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 Fernandes et al. strand with the complementary sequence to the target mRNA Developing a safe and effective delivery system for remains in the RISC and directs the recognition and cleavage successful siRNA therapy is, therefore, crucial. Although of the target mRNA by Ago2 [9, 10]. viral vectors have been successfully used for siRNA delivery into various cells, including SCs [16-18], their clinical Taking advantage of this in vivo phenomenon of regulating application is limited by the risk of causing mutations in target mRNA transcripts by a non-coding RNA, various cells and inciting immune responses [19-22]. Electroporation methods of in vitro synthesis and delivery of siRNA into has also been a successful siRNA delivery method, but it different cell lines have been developed. Introduction of a 21- may cause cell damage and ion imbalances [23-27]. Voltage 23 nt long siRNA designed to target a specific mRNA, would pulse mediated delivery of siRNA is associated with high bypass the dicer-mediated processing and is directly be loaded cell mortality rate making this option limited for less and processed by RISC. Although long dsRNA has been used abundant cells (adult stem cells, for example). Prolonged to silence target genes in many organisms like worms and steps of optimization of various parameters in different cell plants, when introduced in mammalian cells, long dsRNAs types and the high cost of the equipment are some of the greater than 30nts, have shown to activate the anti-viral added disadvantages of this technique. immune response, leading to non-specific RNA degradation [11]. Synthetic siRNA, on the other hand, can not only bypass To address such challenges, a lot of research has been the antiviral mechanisms but can also be easily customised for focussed on the development of various non-viral methods any target gene, and therefore has attracted a lot of attention [28, 29], such as nanocarriers and commercial transfection for gene manipulation studies [12, 13]. agents (for example lipofectamine, RNAi-Max, DharmaFECT, etc.). Most if not all commercial transfection The discovery of siRNA revolutionized the field of cell agents show cytotoxicity and thus, are a poor choice, and molecular biology. However, the delivery of naked especially when a long term effect of siRNA is desired by siRNA suffers several challenges. Naked siRNA is easily repeated attempts of transfection. On the other hand, degraded by endonucleases, exhibits low serum stability, nanocarriers are relatively safer, less toxic, less immunogenic may cause off-target effects and may invoke an immune [30, 31]. Moreover, the typical nano-size enables their response in cells. Furthermore, siRNA is anionic, and movements through micro-capillaries [32]. therefore it is harder to transport across the cell membrane. Compared to plasmid DNA, siRNA has a stiff structure and Nanocarriers could be classified as organic or inorganic, relatively low spatial charge density. Thus, it is difficult to based on their physical and chemical properties. Organic formulate a compact complex of siRNA, which is imperative siRNA nanocarriers (Fig. 1) are composed of self-aggregating to ensure its stability in the bloodstream [14, 15]. 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). Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 3 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 Compared to other cell lines, SCs are a lot harder to the other hand, inorganic nanocarriers (Fig. 2) are insoluble transfect due to limited cellular uptake [37] making it and non-biodegradable and may require additional difficult to deliver siRNA using the conventional non-viral modification with polymers to improve their solubility. Such vectors [38, 39] . In the cell membrane of primary cells, nanocarriers are a hybrid of both organic and inorganic especially SCs, the structural variations in lipid molecules materials, which may comprise of gold nanoparticles, that mediate transfection may be the cause of low magnetic nanoparticles, quantum dots, calcium phosphate transfection efficiencies [40]. Therefore, the development of nanoparticles or carbon-based nanostructures [31]. A system efficient siRNA delivery methods for transfection in SCs to track the biological distribution, migration and proliferation with the aim of gene manipulation for potential therapeutic after transplantation of SCs, would be an added advantage. applications is warranted. Nanocarriers that perform both diagnostic and therapeutic In this review article, various organic, inorganic, hybrid functions, termed as theranostic agents are designed to achieve and theranostic nano-carriers and nano-topography based imaging as well as siRNA delivery [33]. transfection strategies that have been developed to increase During endocytosis of siRNA, multiple mechanisms (Fig. the transfection efficiency of siRNA with the aim of lineage- 3) may be used for the internalization of the nanodelivery specific differentiation of stem cells have been discussed. vehicle [34]. Knowledge of the internalization mechanism is essential for designing highly efficient therapeutic 2.1. Organic Nanocarriers nanocarriers necessitating the understanding of various In this section, various nanoparticles used to design or- possible endocytic pathways and their role in transfection. In ganic nanocarriers to deliver siRNAs in stem cells for their general, endocytosis may be receptor-mediated (clathrin- differentiation into specific lineages have been discussed. coated), receptor-independent, or it may involve These nano carriers are broadly classified as lipid-based and macropinocytosis. Receptor independent endocytosis liposome-inspired nanoparticles, protein and peptide nano- includes caveolin-mediated endocytosis, clathrin- carriers and polymeric nanoparticles. A schematic diagram independent endocytosis and clathrin- and caveolin- depicting various organic nanocarriers is shown in Fig. (1). independent endocytosis. Once the siRNA nanocarrier is internalized, it is entrapped in an endosome. The contents of 2.1.1. Lipid-Based and Liposome-Inspired Nanoparticles the endosome would typically be degraded by lysosomal enzymes and therefore another aspect to keep in mind while Liposomes are used as vesicle based siRNA carriers. designing siRNA nanocarriers is to create an endosomal They are spherical vesicles composed primarily of a lipid escape strategy to ensure cytosolic delivery and to maximise bilayer with cholesterol [41]. The siRNA delivery system the gene knockdown efficiency [35, 36]. based on a cationic liposome DOTAP (N-(1-(2, 3- 4 Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 Fernandes et al. 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 methyl- The SA/Cholsterosomes loaded with phenamil and noggin sulfate) targeting cathepsin S gene was used to transfect siRNA proved efficient in inducing osteogenic differentia- CD34+ cells. CD34+ is a well-known marker for blood and tion of Mesenchymal stem cells (MSCs) in both 2D mono- bone marrow-derived progenitor cells, especially for hema- layer culture and 3D hydrogel environment in in vitro as well topoietic and endothelial stem cells. Knockdown of cathep- as in vivo model [44]. sin S leads to in vitro differentiation of the CD34+ cells into Novel lipid-like nanoparticles called lipidoids are shown dendritic cells. The DOTAP-mediated transfection of siRNA to have high efficiency in terms of siRNA delivery. Lipi- showed more than 60% knockdown efficiency, which is doids are small lipid-like molecules which possess cationic comparable to that of lentiviral vectors, with minimal cyto- behavior by virtue of having an amine backbone. Natural toxicity. Further, knockdown was performed on the cells lipids have only 2 tails, but lipidoids can have one to seven throughout the differentiation process i.e. for 14 days and tails depending on the amine used to synthesize them. These repeated transfection had no adverse effect on cell develop- lipidoids possess high transfection efficiency and have re- ment [42]. duced cytotoxicity in human embryonic stem cell-derived Sterosomes are non-phospholipid liposomes and are for- cells (hESCds). mulated with single-chain amphiphiles and high content of In a study carried out by F. Yang et.al, co-delivery of os- sterols. These particles have an additional positive charge teoinductive DNA and siRNA directing the osteogenic dif- and therefore increased stability. SA/Chol (stearylamine ferentiation of human adipose-derived stem cells (hADSCs) (SA) and cholesterol (Chol)) sterosome and siRNA com- was carried out using a combination of poly (β-amino esters) plexes were shown to have increased cellular uptake and and lipidoids. The poly (β-amino esters) served as a vehicle gene knockdown efficiency in mesenchymal stem cells as for delivering osteoinductive DNA (BMP2) and lipidoid as a compared to commercial transfection agents. This complex delivery vehicle for siRNAs (siGNAS and siNoggin). A sig- was further used for knocking down the expression of nog- nificant increase in the expression of BMP2 gene along with gin, which is a specific antagonist of bone morphogenetic the downregulation of GNAS and Noggin in hADSCs was proteins preventing its interaction with the receptor. siRNA observed. Co-delivery of the siRNAs along with the plasmid transfection using SA/Chol resulted in more than 45% DNA, resulted in a significant increase in the expression of knockdown compared to Lipofectamine 2000 which showed osteogenic markers and accelerated differentiation of 25% knockdown of the gene expression [43]. Further studies hADSCs [45]. with cationic sterosome formulated with stearylamine and cholesterol also proved that in addition to forming com- Another class of lipid-based nanoparticles is Niosomes, plexes with siRNA, it also solubilizes small molecules like which are non-ionic surfactant based vehicles, structurally phenamil in a single-vehicle. Synergistic effects were ob- similar to liposomes with the incorporation of lipids. served in terms of knockdown of noggin expression by siR- Niosomes, which exhibit higher stability and loading capac- NAs as well as stimulation of BMP signaling by Phenamil. ity are cost-effective and easier to synthesize as compared to Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 5 traditional liposomes and thus are more suitable for drug and dependent of the endosomal pathway. While entering the cell gene delivery. Yang C. et al developed a novel niosome sys- membranes, these peptides adopt a helical conformation that tem which acted as a theranostic platform comprising of a exposes the positively charged residues that mask the nega- cationic lipid DOTAP, a PEGylated lipid TPGS (d-α- tive charge of siRNA on one side and the tryptophan groups Tocopheryl polyethylene glycol 1000 succinate), a nonionic on the other to favor cellular uptake [49]. surfactant sorbitanmono-oleate (Span 80) and encapsulating In another successful attempt to transfect the hard-to - indocyaninegreen (ICG). This noisome exhibited dual func- transfect cell lines that include T cells, human umbilical vein tions of efficient siRNA/miRNA delivery as well as the la- endothelial cells and hESCs, a Peptide Transduction Do- beling of stem cells in the near infra-red range. Efficient de- main-dsRNA Binding Domain (PTD-DRBD) fusion protein livery of siRNA, as well as miRNA in stem cells, was dem- was used as a siRNA delivering vehicle. These particles bind onstrated by inhibition of miR-138, a negative regulator of siRNA with high affinity and mask the overall negative osteoblast differentiation by iSPN (ICG containing noi- charge of the nucleic acid. While PTD facilitates cellular some system)/anti-miR-138, leading to the promotion of uptake of siRNA, DRBD encompasses the siRNA and pre- osteogenesis. Silencing efficiency of ~88% was achieved vents the aggregate formation and, thereby cytotoxicity. which was much higher compared to Lipofectamine. The Thus, this approach could be used for the efficient transfec- internalization of iSPN/siRNA involved macropinocytosis tion of RNAi in difficult to transfect cell lines [50]. and caveolae-mediated pathways, and conferred fluorescence to the cells [46]. 2.1.3. Polymeric Nanoparticles 2.1.2. Protein and Peptide Nanocarriers Synthetic and natural polymers have drawn active inter- est as nanocarriers for siRNA delivery onto stem cells be- Proteins and peptides are being explored as ideal delivery cause they display a wide diversity in their structure and agents for various biological molecules into hard to transfect physico-chemical properties. There are various examples of cells such as stem cells [47]. The various peptide-based polymeric carriers developed for siRNA delivery such as strategies for siRNA delivery include cell-penetrating pep- polyethyleneimine (PEI), poly-dimethylaminoethyl tide (CPP), CADY, Peptide Transduction Domain-dsRNA methacrylate, poly-amidoamine, chitosan, poly-lysine and Binding Domain (PTD-DRBD) etc. which will be discussed poly -β-amino esters [51]. in the following section: PEI has exhibited high transfection efficiencies because it Enhancement in the cellular delivery of siRNA can be causes endosomal osmotic swelling and rupture, which pre- done with the help of CPP. It consists of five or more mole- vents lysosomal degradation of siRNA. However, it is un- cules of peptide linked to each other, forming a chain-like suitable for in vivo drug delivery because it can have toxic structure which aids in cellular delivery. The CPPs explored effects as it causes membrane damage, activates the com- so far including Tat, Oligo-Arg, Transportan and Pentratin, plement system and interacts with blood cells. Even in easy- bind to the biological moieties via covalent bonds. These to-transfect cell lines, high dose of siRNA is required for molecules improve the cellular internalization of siRNA and transfection. Modification with poly-ethylene glycol (PEG) other biological moieties by an endocytic pathway (micropi- has been attempted as a strategy to overcome this drawback nocytosis). For in vitro delivery of siRNA to cultured cells, [52]. covalent attachment of the siRNA to the cargo is preferred, whereas for in vivo delivery, the siRNA is usually bound Poly-β-amino esters (PBAEs) are easy to synthesize, non-covalently to short amphipathic CPPs like MPG peptide chemically modify and are hydrolytically degradable. To (an amphipathic peptide, consisting of a hydrophobic N ter- optimize them for siRNA delivery into hMSCs, Tzeng et al. minal and a hydrophilic C-terminal domain). evaluated an array of modified PBAE nanocarriers synthe- sized by insertion of carbons either between acrylate groups/ To prove the role of Oct-3/4 as a key player in the proc- amine group and the alkyl groups in the side chain of the ess of mesodermal and cardiac commitments of the embry- base polymers. Cystamine-terminated polymers achieved up onic epiblast and embryonic stem cells (ESCs), a siRNA to 91% of knockdown after 20 days of transfection. The low targeting this gene was delivered via MPG. The siRNA me- weight ratio of polymer to siRNA facilitated its tight binding diated inhibition of Oct-3/4 in ES cells prevents mesodermal with the polymer, which leads to an environmentally trig- specification and their differentiation into cardiomyocytes. gered release. For example, release of the siRNA in the cy- Likewise, Oct-3/4 siRNA injected in the inner cell mass of toplasm is triggered by its reducing property. These particles blastocysts affects early embryonic cardiogenesis [48]. were further used to deliver siRNA against ‘B-cell lym- For challenging and hard-to-transfect cell lines, a secon- phoma (Bcl)-like protein’ (BCL2L2), an osteogenesis inhibi- dary amphipathic peptide CADY of 20 residues comprised tor, thereby triggering osteogenesis in hMSCs [53]. of aromatic tryptophan and cationic arginine residues which A further study by Núnez et al. was performed where os- formed stable complexes with siRNAs was used. The bal- teogenic differentiation of pluripotent-like stem cells from ance between the stability of the complex, concentration of the dental pulp (DPPSC) was carried out by oligopeptide the CPP at the cell membrane and the interaction of the CPP modified PBAEs; which was used for simultaneous delivery with the lipid phase was critical in order to achieve efficient of anti-Oct-3/4 siRNA, anti-NANOG siRNA and Runx2 gene delivery. Sub-nanomolar concentrations of siRNA de- plasmid. The results of this study indicated that inhibition of livered via CADY exhibited a significant knockdown of the Oct 3/4 and the increased expression of the Runx2 enhanced target gene at mRNA and protein levels. The entry of the expression of an osteogenic marker and increased matrix CADY/siRNA complexes inside the cells was rapid and in- mineralization. These effects were attributed to the double 6 Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 Fernandes et al. 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 celles with tagged siRNA in primary bone marrow derived simultaneously (Fig. 4) which did not induce any chromo- stem cells showed caveolae/lipid-raft dependent mechanism somal instability or alteration of the cell viability [54]. and disassembly at acidic pH to escape lysosomal degrada- tion. However, the gene knockdown efficiency assessed by Diblock copolymers are comprehensively evaluated in downregulation of Runx2 gene in mMSC was less efficient the study carried out by Benoit et al. Diblock copolymers are (40%) as compared to Lipofectamine 2000 (60%) [57]. composed of cationic blocks for complexation of siRNA and pH-responsive blocks for endosomal escape. These copoly- A family of synthetic polymers called cationic dendrim- mers confer advantages of protecting siRNA from nucleases, ers represents a highly promising class of siRNA nanocarri- increase uptake efficiency and facilitate escape from en- ers. They are multivalent, have a very precisely constructed dosomal trafficking. The siRNA delivery efficiency of di- structure and a high cargo payload. Dendrimers can be clas- block copolymer using fluorescently-labeled siRNA (FAM- sified by their generation- the number of repeated branching siRNA) was 96% as opposed to 64% by commercially avail- cycles performed during dendrimer synthesis. More exposed able DharmaFECT in MSCs with minimal cytotoxicity. Pro- functional groups are found on higher generation dendrimers tein knockdown was sustained for a period of 6 days [55]. which makes them desirable for siRNA delivery [58]. pH-responsive diblock copolymers have been reported to A study was conducted to compare the transfection effi- provide tailorable nanoparticle architecture and chemistry ciency of siRNA against Oct-4 in mouse ESCs using a high critical for siRNA delivery. Malcolm et.al. investigated how generation dendrimer, poly-amidoamine, (PAMAM G5), varying the hydrophobicity, molecular weights and ratio of DOTAP and a commercially available transfection reagent the corona (first block) and core (second block) affects the Arrest-In. It was found that the uptake efficiency of siRNA delivery of siRNA in murine and human MSCs. The corona in conjugation with DOTAP was highest (60%)for N/P ratio was composed of poly-dimethylaminoethyl methacrylate of 10 compared to that of naked siRNA (1.9%), PAMAM G5 (pDMAEMA) which allowed complexation with siRNA by (26% for N/P ratio of 10 and 35% for N/P ratio of 20) and electrostatic interaction. The core was composed of Arrest-In/siRNA complex (39%). However, when the func- DMAEMA, 2-propylacrylic acid, and butyl methacrylate tional effects were analyzed, PAMAM G5 was more than (BMA). It facilitates endosomal escape and mediates self- 100-fold better than DOTAP as it could not escape the en- assembly at neutral pH. Uptake of siRNA in hMSCs posi- dosomal degradation pathway [59]. These results highlighted tively correlated with the molecular weight of the first block; the importance of the endosomal processing of the siRNA whereas gene silencing and cytocompatibility correlated with upon uptake. percent BMA in all cell types [56]. Efficient transfection of highly refractory human hema- pH-dependent tripartite polyionic complex (PIC) micelles topoietic CD34+ stem cells was achieved using an am- were also explored as a nonviral method for delivering phiphilic arginine decorated dendrimer made up of a hydro- siRNA in MSCs. The mechanism of micelle formation was philic PAMAM Dendron modified by a hydrophobic alkyl based on the spontaneous association of a double-hydrophilic chain and functionalized by polyarginine. The poly arginine block copolymer with a cationic homopolymer for the com- moiety is a widely used cell-penetrating peptide, as it pos- plexation with siRNA. Studies on internalization of the mi- sesses positively charged functional groups at physiological Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 7 pH. The modified dendrimers exhibit enhanced siRNA trans- Another class of adaptive amphiphilic dendrimer nano fection and gene silencing efficiency in human prostate can- vehicle that self-assembles in water into vesicle-like den- cer and hematopoietic SCs, due to an increase in association drimer some nanostructures of ~200nm was designed for with the cell membrane facilitated by H bonding. Knock- siRNA delivery using click chemistry. This dendrimer was down of ~60% of CD4 (primary receptor for HIV 1) mRNA able to form a monolayer film at the air/water interface was observed when anti-CD4 siRNA was delivered by the which could swell up to make giant vesicles indicating a modified dendrimer as opposed to dendrimer alone and lipid-like behavior. Additionally, when these dendrimer’s RNAi Max, both of which showed no effect. It was also were complexed with siRNA, they formed many highly or- found that this nanoparticle formed stable complexes with dered small spherical micelles in the range of 6-8 nm with siRNA and showed no evident cytotoxicity [60]. positively charged surface enabling stronger electrostatic Structure-activity relationships (SAR) of ligand-modified interaction with the siRNA. The self-assembly (Fig. 5) of the dendrimers were screened for their gene delivery capacity in dendrimer into adaptive supramolecular structures along hard to transfect cell lines using generation 5 (G5) polyami- with a combination of lipid and vector features implies a doamine (PAMAM) dendrimers with 128 surface primary virus-like delivery property of these dendrimers. The gene amine groups conjugated with 129 different ligands. The delivery capability of dendrimer conjugated with anti- conjugated ligands included several groups like alkyl, fluoro- tat/rev-dsiRNA was assessed in human peripheral blood alkyl chains, amino acids, benzene derivatives, heterocyclic mononuclear cells (PBMC-CD34+), hematopoietic stem chains, etc. An efficient triphenylphosphonium-modified G5 cells (HS-CD34+) and glioblastoma stem cells (GSCs). On dendrimer carrier of siRNA which was found by screening a transfection, more than 50% reduction in the viral infection second-generation library of dendrimers showed efficient was observed whereas, Lipofectamine-RNAiMAX was not knockdown of the Smurf1 gene (SMAD Specific E3 Ubiq- able to deliver the siRNA in human primary and stem cells uitin Protein Ligase 1; involved Hedgehog signaling path- and Trans IT-TKO showed cytotoxicity. The amphiphilic ways) and downregulation of the Smurf1 protein in primary dendrimer also exhibited the potential for in vivo delivery of mouse mesenchymal stem cells. Thus this study enables one siRNA. Macropinocytosis was found to be the dominant to design a future siRNA delivery vehicle depending upon uptake pathway. Using this delivery system which outper- the ligand group [61]. formed commercially available reagents, effective gene A series of combinatorial and computational approaches knockdown was observed in hematopoietic stem cells (HSC- were used for designing amphiphilic dendrons capable of CD34+) and glioblastoma stem cells (GSCs) [60]. self-assembly (Fig. 5) into supramolecular dendrimer mi- Cyclodextrins are cyclic natural-polymer based nanoma- celles. Dendrons with shorter aliphatic/hydrophilic chain terials which are made up of α-1-4-D-glucose or amylose, formed large aggregates when complexed with siRNA, are derived from the enzymatic conversion of starch. Cyclo- whereas dendrons with alkyl chains (C16-C22) formed dextrin being a natural polymer exhibits low immunogenicity nanometer range complexes. SiRNA delivery was most effi- and when used as a siRNA delivery agent, shows extended cient for C18 dendron; which was also used to carry siRNA gene silencing in various cell lines including stem cells [7]. targeting Hsp27 in a prostate cancer xenografted nude mice A cationic star polymer composed of a β-cyclodextrin core, model and in human CD34+ stem cells. The lower efficiency poly-(2-dimethyl aminoethyl methacrylate, DMAEMA) as of the other dendrons was attributed either to the intrinsic the cationic component and poly-(2-hydroxyethylme- incapability of self-assembly or the inability to form a stable thacrylate, HEMA) as the non-toxic stealth component was micelle. These approaches showed that the right balance studied for siRNA delivery. The formulation of polymerH3 between the hydrophobic chain length and the hydrophilic (with DMAEMA/P (HEMA) weight content of 50: 50) dendritic portion of the amphiphilic dendrimers plays a cru- showed maximum transfection efficiency in mouse ESCs in cial role in their self-assembly and siRNA delivery activity comparison to Lipofectamine [63]. [62]. 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). 8 Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 Fernandes et al. DexAM, a cyclodextrin modified dendritic polyamine nontoxic [70, 71], and have a range of applications like diag- construct complex was used in dual delivery of siRNAs and nostics, therapeutics and photonics. The surface of AuNPs solubilizing hydrophobic small molecules (various drugs for can be easily functionalized to suit the application purpose. example). Such a delivery vehicle would help in eliciting AuNPs have been extensively used for transfection studies in high cellular viability over a long period of time and avoid cancer cells [72, 73]; however, naked AuNPs lack the ability the use of corrosive solvents such as DMSO. The β- to successfully transfect primary cells and SCs. The transfec- cyclodextrin in DexAM acts as a solubilizer for the hydro- tion efficiency can be enhanced by conjugation of AuNPs phobic drugs and the dendritic polyamine backbone provides with molecules which target and enhance the interaction with high-density positive charge for complementing with nucleic cell membrane as well as the genetic material [74]. acids to form cationic complexes. When genetically modi- Gold nanorods (AuNR) coated with PSS (poly-sodium 4- fied Neural Stem Cells (NSCs), stably expressing green fluo- styrenesulfonate) for negative charge and PAH (poly- rescent protein (GFP), were treated with DexAM complexed allylamine hydrochloride) for a positive charge, interacted with GFP siRNA, about 80% decrease in intensity of GFP, electrostatically and exhibited positive charge on the surface after 96 hours of siRNA transfection was observed as op- allowing efficient binding of siRNA. The AuNR-PSS-PAH posed to 33% of knockdown with commercial transfection nanocarrier for FAM labelled Lysine-specific demethylase 1 agents (Lipofectamine, Liofecatmine 2000 and FuGENE). (LSD1) siRNA showed efficient cellular uptake (70 %) The high efficiency was attributed to the highly dense sur- compared to treatment with AuNR alone (1.1%). LSD1 ex- face primary amines and the three-dimensional spherical pression is needed to maintain pluripotency in embryonic structure. DexAM carrying the siRNA targeting the SOX9 and cancer stem cells. There was more than 50% downregu- (sex-determining region Y-box 9) gene, and retinoic acid, led lation of the LSD1 at the mRNA level in the hMSCs trans- to ~71% differentiation of the NSCs into neurons [64]. fected with siLSD1 compared to the control cells. The mRNA levels of stem cell marker genes SOX2 &Oct-4 were 2.1.4. Other Nanoparticles downregulated while the mRNA level for differentiation Delivery from a three-dimensional scaffold using a sub- gene FOXA2 and BMP2 showed upregulation. Thus, the strate is another strategy of gene delivery technique using siRNA-nanocarrier complex was successful in the knocking nanostructures. These nanostructures provide advantages down of LSD1, regulation of the pluripotency genes and over conventional nanoparticles in terms of increased surface promoting differentiation. The transfected cells grown in area, minimal escape of cargo molecules in the medium and hepatocyte growth factor showed upregulation of the liver higher internalization efficiency. Among these nanostruc- marker genes hepatic nuclear factor 1 (HNF1-α) and CK-8 tures, vertically aligned nanowires are being considered as a within 3 days and maintained it up to 7 days [75]. novel biomaterial for gene delivery. The efficient usage of gold nanocarriers for siRNA deliv- In one such study carried out by Yoshihiro et al. verti- ery has been shown in various systems other than stem cells. cally aligned high-density silicon nanowire substrate was Spherical nucleic acid nanoparticle conjugates, SNA-NCs, used as a three-dimensional topological feature for cellular designed by densely coating inorganic gold nanoparticles modification. Here a polydopamine (PD) coated silicon with highly oriented oligonucleotides, exhibit the potential nanowire (PDSiNWs) was used for siRNA delivery in A549- for topical delivery of siRNA. Primary normal human kerati- luc cells. The gene knockdown assay was carried out by nocytes, hKCs, when transfected with these gold nanoparti- measuring the production of luciferase. It was observed that cles conjugated with siRNAs, showed an efficient uptake, PDSiNWs exhibited a higher efficiency of siRNA delivery successful (about 90%) and prolonged (up to 96 hrs) gene leading to better knockdown efficiency of the target gene. silencing effect, low cytotoxicity and low off-target effect The interaction of PDSiNWs with the cellular membrane compared to a cationic lipid-based transfection agent Dhar- perturbed the lateral diffusion of lipids promoting the cellu- maFECT1®. This method also showed a remarkably low lar internalization of siRNA. The coating of various sub- concentration of the siRNA requirement for a similar amount strates on to the nanostructures can improve the biomolecule of gene knockdown efficiency by DharmaFECT. The SNA- carrying capacity which may find potential applications in NC nanocarriers conjugated with EGFR siRNA when mixed the field of tissue engineering [65]. with a commonly used petrolatum-based moisturizer and administered on different strains of mice, showed penetration 2.2. Inorganic Nanocarriers into the epidermis within 3 hours. The EGFR mRNA expres- sion was reduced by 65% and protein expression was com- This section discusses different inorganic nanoparticles pletely suppressed without any sign of inflammation or tox- which are functionalized to make siRNA delivery vehicles icity when 50 nM SNA-NCs was applied for 3 weeks. Hu- into stem cells. These nanocarriers involve gold nanoparti- man skin equivalents (3D organotypic raft cultures) as hu- cles, magnetic nanoparticles, quantum dots, calcium- man skin models were also tested for penetration and knock- phosphate based nanoparticles and carbon-based nanocarri- down efficiency for SNA-NCs-EGFR siRNA conjugate. The ers. Fig. (2) summarizes the broad categories as well as sub- uptake was observed in as less as 2 hours with 25nM of categories of the inorganic nanocarriers discussed in this SNA-NCs concentration. Although the uptake was only article. 4.7% of the applied SNA-NCs, the EGFR mRNA and pro- tein showed 52 and 75% knockdown, respectively [76]. This 2.2.1. Gold Nanoparticles highly successful gold nano-carrier as an efficient delivery Gold nanoparticles (AuNPs) have unique optical [66, 67] agent of siRNA could be explored in stem cells. and catalytic properties [68, 69], they are biocompatible and Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 9 2.2.2. Magnetic Nanoparticle Magnetofection-mediated transfection of plasmid DNA into hair follicle mesenchymal stem cells (hHF-MSCs) was Magnetic nanoparticles (MNPs) have a unique ability to optimized by using low MNP: DNA ratio and multifection provide Magnetic Resonance Imaging (MRI) [77-79] as well method. An increase in incubation time and use of Opti- as to carry a therapeutic payload. Magnetofection is a new MEM instead of serum-free DMEM also helped to achieve method of transfection in which superparamagnetic iron per- better transfection efficiency and low cytotoxicity as com- oxide conjugated with the genetic payload is delivered to the pared to lipofectamine 2000. Nanog gene pDNA transfected cells by application of the magnetic field [80]. into hHF-MSCs by this optimized procedure expressed about MNPs or their different functional derivatives have been 6 fold higher Nanog mRNA level compared to the control. used to deliver siRNAs into stem cells. For example, Nanog in adult MSCs enhanced the rate of proliferation of ZnFe O MNP core with an outer Au shell stabilized by 11- these cells and completely restored the diminished myogenic 2 4 mercaptoundecanoic acid (MUA) were proficient in deliver- differentiation potential [84]. ing siGFP to NSCs genetically manipulated for stable ex- Transfection efficiency of MNP by the magnetofection pression of GFP (NSC-GFP). The MNP showed better trans- method was found to be higher for oscillating magnetic field fection efficiency as well as cell viability compared to the compared to the static magnetic field in neurospheres. The commercially available transfection agent X-tremeGENE. most significant increase was seen in the oscillating mag- The incubation time needed for MNP transfection under the netic field of 4Hz. The transfected NSCs differentiated magnetic field was much less than that required for X- mainly to astrocytes but not to neuron or oligodendrocytes tremeGENE. The process of multifection (multiple transfec- when different magnetic fields were applied [85].When tions) helped to achieve much higher transfection efficiency magneto-multifection was tried in NSCs with an optimal without affecting the cell viability. siRNAs targeting the concentration of MNP, transfection efficiency was found to genes CAVEOLIN-1 and SOX9 showed a substantial in- be slightly better than lipofectamine, with no cytotoxic effect crease in the percentage of differentiated oligodendrocytes and similar differentiation potential. The MNP could also co- and neurons, respectively [81]. deliver different plasmids with better transfection efficiency A successful study on the delivery of siRNA into bone (>80%) compared to sequential delivery (40%) [86]. marrow-derived hMSCs was achieved using spherical PEI- Taken together these reports highlight the importance of coated Fe O nanoparticles with an average diameter of 12nm 3 4 MNPs in delivering siRNA, micro-RNA and plasmid DNAs and charge +44.0 mV. When 4ng/ml of these nanoparticles into stem cells with high efficiency and the potential to dif- was used with siRNA at a weight ratio of 4, high transfection ferentiate them into specific lineages. efficiency was achieved with low cytotoxicity and a high si- lencing efficiency (>60%). Endocytosis was found to be the 2.2.3. Quantum Dots mechanism of internalization in these cells. This delivery sys- tem could be a good fit for stem cell therapy applications since Fluorescent molecular probes play an important role in these nanoparticles possess imaging properties [82]. biomolecular imagining both in vivo and in vitro. Conven- tional fluorescent probes like fluorescent proteins and other Apart from the above studies, the MNPs were also ex- fluorescent organic molecules exhibit challenges due to scat- plored for the delivery of micro RNA and plasmid DNAs in tering, absorbance and inhibition caused by auto fluores- stem cells. MNPs conjugated with PEI were used for the cence in the body. Quantum dots (QD) exhibit broad excita- delivery of the micro RNA, miR-335 into hMSCs. Encoded tion spectra and a narrow, sharply defined emission peak in the second intron of the mesoderm-specific transcript with large Stokes shift are stable and show reduced pho- (MEST), miR-335 regulates the genes responsible for prolif- tobleaching. These physiochemical properties are advanta- eration, differentiation and migration in hMSCs. The cells geous over conventional probes like organic dyes and fluo- transfected with miR/PEI and miR/PEI/MNP showed 1000 rescent proteins. QDs can also serve as theranostic nanocar- fold enhancement of miR expression compared to the control riers which can achieve high sensitivity and resolution for and by 72 hrs the miR level of cells transfected with long duration at low costs [87, 88]. However, in spite of miR/PEI/MNP was 4 fold higher than miR/PEI. Also, the these excellent and advantageous properties, the usages of miR/PEI/MNP resulted in sustained expression of miR and QDs in cell-based applications are limited due to their cyto- the complex remained in the cytoplasm (as opposed to the toxicity. Various methods overcoming this difficulty have miR/PEI complex, which was found in the nucleus) enhanc- been adopted. Some examples are discussed below: ing the efficiency of the miR-mediated down-regulation of the target gene expression [83]. The sonochemical synthetic method was used to prepare a library of biocompatible QD with the empirical formula A magnetofection agent PolyMAG was developed to de- Zn S-Ag In S (ZAIS). These QDs prepared by varying the liver plasmid DNA in to stem cells. The transfection effi- x y 1-y 2 concentration of the organometallic precursor element, retain ciency of this magnetofection for eGFP was found to be the strong fluorescence primarily in the cytoplasm and ex- higher in mESCs (45%) compared to FuGENE6 (15%). The hibit low cytotoxicity and phototoxicity as compared to con- PolyMAG mediated transfection of eGFP in mESC was sta- ventional CdSe/ZnS QDs when transfected in hMSCs and ble and exhibited high levels of GFP expression without in- human brain tumor cells (U87 glioblastoma cell line). 80% terfering with the expression pattern in the stem cell markers siRNA mediated knockdown of EGFP mRNA was observed and retained the property of differentiation into the specific in U87 cells genetically modified for GFP expression. Simi- germ layers under appropriate differentiation condition while lar results could also be expected from stem cells, and these continuing the expression of GFP [80]. 10 Current Stem Cell Research & Therapy, 2020, Vol. 15, No. 00 Fernandes et al. QDs could be explored as a viable option for simultaneous PPAR ϒ is a regulator for adipogenesis and its downregula- delivery and imaging probe in stem cells [89]. tion leads to osteogenesis. The co-delivery resulted in higher expression of Runx2 compared to the delivery of the indi- Another method to reduce the toxicity of CdSe QD is by vidual molecule by the QD conjugated siPPARϒ or Dex incorporation of ZnS shell and PEG coating. Heterobifunc- separately (more than 90% expression compared to approxi- tional cross-linker, sulfosuccinimidyl-4-(N-maleimidomethyl) mately 32% and 57%, respectively). The QD could be fluo- and/or cyclohexane-1-carboxylate (sulfo-SMCC) can be used rescently tracked for up to 14 days [92]. for bioconjugation on selective sites of the protein through stable thioether bond with a sulfhydryl-exposed antibody. 2.2.4. Calcium Phosphate Nanoparticle Within the concentration range of 20-120pmol/L, the cy- Calcium phosphate (Ca:P) nanoparticles mimic the natu- totoxic effect of QD-SMCC was lower than PEI. When ex- ral bone material. They are biocompatible, non-toxic, inex- posed to the MSCs isolated from the femoral marrow cavity pensive and easy to prepare [93]. The release of the encapsu- of the rat models, the cells produced a large number of filo- lated nucleic acids into the cellular cytoplasm is facilitated podia which engulfed the QD-SMCC within 3 hrs of trans- by the pH-dependent solubility of Ca:P and endosomal acidi- fection. QD-SMCC-were released into the cytoplasm after fication. Despite these advantages, a major drawback in rupturing the endosomes, 12hrs post-transfection. Both trans- these nanoparticles is their tendency to form aggregates due fection and knockdown efficiency were better when SOX9 to their uncontrollable size. This causes inconsistent trans- siRNA was conjugated as QD-SMCC-siSOX9 rather than fection efficiencies in various cell lines. Aggregation can be PEI-siSOX9 for delivery into these cells. The downregula- prevented by using a stabilizing agent which further helps in tion of SOX resulted in the down-regulation of type II colla- improving the transfection efficiency [94, 95]. gen and aggrecan. SOX9 acts as a transcriptional activator of both these genes causing accelerated differentiation of adult For example, Glutamine (Gln)-conjugated oligochitosan MSCs toward chondrocytes [90]. (OChi) was used to stabilize these nanoparticles. The modi- fied Ca:P nanoparticles in the presence of OChi or Gln-OChi QD conjugated with β cyclodextrin and positively were spherical in shape and did not form large agglomerates. charged peptides like RGD have also shown better biocom- The modification improved the cellular uptake of the patibility and negligible cytotoxicity as compared to QD nanoparticles with minimal cytotoxicity. The Gln-OChiCa:P alone. RGD serves the dual purpose of binding with the exhibited higher transfection efficiency (more than 90%) siRNA and facilitating better uptake by binding with integrin compared to Ca:P nanoparticle (about 65%) alone which was receptors on the cell surface. The RGD-β-CD-QD nanocar- comparable with that of lipofectamine2000 mediated trans- rier served as a drug delivery vehicle and also showed sig- fection in HeLa cell lines. The knockdown of the GFP gene nificant uptake in hMSCs. The hMSCs developed filopodia expressed in Hela cells was yet again similar to that of the around the complex, and uptake was mediated by micropino- lipofectamine-mediated effect. When compared to the chito- cytosis (Fig. 3). This nanocarrier was also used to deliver a san-based nanoparticles, Ca:P nanoparticles stabilized by small molecule kartogenin (KGN), capable of inducing chitosan showed similar transfection efficiency and higher chondrogenesis in hMSCs. It was found that the upregulation gene knockdown efficiency. In adipose-derived MSCs of chondrogenic markers was higher when the small mole- (ADSCs), the transfection efficiency of the Ca:P nanoparti- cule was delivered via the nanocarrier as compared to the cle was much lower as compared to that in HeLa cells. How- administration of KGN by directly supplementing it in the ever, when modified by chitosan, the transfection efficiency media. A similar result was obtained for a 3D mass culture. became comparable. The bioactivity of the Ca:P nanoparti- This method to induce chondrogenesis was compared with cles in a photocross linkable chitosan 3D hydrogel were the siRNA mediated knockdown of the Runx2 gene, deliv- evaluated by transfection with fluorescently labelled siRNA ered by the QD nanoparticles complexed with KGN and si- against Noggin. The transfected ADSCs showed a decrease Runx2. Runx2 suppresses chondrocyte hypertrophy and the in NOG mRNA level to 49%, similar to the lipofectamine, associated pathogenic calcification in hyaline cartilage. The and the knockdown led to bone formation in vivo and os- expression level of aggrecan, type II collagen, and Sox 9 teogenesis in vitro [96]. indicative of chondrocyte differentiation were found to be similar to that of lipofectamine-mediated delivery of the 2.2.5. Potential Carbon based Nanocarriers siRNA and the nanocarrier mediated KGN delivery. The expression of Runx2 and type X Collagen was found to be Carbon Nanotubes (CNT) contain covalently bonded lower than the KGN nanocarrier group but equivalent to the sp2-hybridized carbon atoms which form six-membered lipofectamine-siRNA group. Thus, the nanoparticle-siRNA rings. They are elongated, tube-like nanostructures which did not inhibit chondrocyte differentiation but suppressed the have high conductivity, surface area, tensile strength, and hypertrophy of chondrogenically induced hMSCs [91]. high aspect ratio [97]. They can be used as scaffold materials to promote proliferation and differentiation of stem cells In a follow-up study by the same group, it has been con- [98]. firmed that the QD- nanocarrier is capable of co-delivering small molecules as well as siRNAs into stem cells in contrast The CNTs could be single-walled (SWCNT) or multi- to Lipo2000, which is limited to the delivery of oligonucleo- walled (MWCNT). While the SWCNTs have a smaller di- tide-based cargoes. The QD was shown to successfully co- ameter, exhibit more flexibility and offer photoluminescence deliver a hydrophobic and osteogenic small molecule; dex- which could be exploited for imaging, the MWCNTs have a amethasone (Dex) and a siRNA against peroxisome prolif- wider surface area for attachment of the cargo and function- erator-activated receptor gamma (PPAR ϒ) in hMSCs. alization supporting more efficient internal encapsulation.
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