Enhanced Transfection Efficiency Research Articles

Deciphering the Role of Chondroitin Sulfate in Increasing the Transfection Efficiency of Amphipathic Peptide-Based Nanocomplexes.

Glycosaminoglycans, both cell-surface and exogenous, can interfere with DNA delivery efficiency of nonviral carrier systems. In this work, we report an extensive comparative study to explore the effect of exogenously added chondroitin sulfate on biophysical characteristics, cellular uptake, transfection efficiency, and intracellular trafficking of nanocomplexes formed using primary and secondary amphipathic peptides developed in our laboratory. Our results indicate that the presence of exogenous chondroitin sulfate exhibits differential enhancement in transfection efficiency of the amphipathic peptides depending upon their chemical nature. The enhancement was more pronounced in primary amphipathic peptide-based nanocomplexes as compared to the secondary counterpart. This difference can be attributed to possible alteration of the intracellular entry pathway in addition to increased extracellular stability, less cellular toxicity, and assistance in nuclear accumulation. These results imply potential use of glycosaminoglycans such as chondroitin sulfate to improve the transfection efficiency of primary amphipathic peptides for possible in vivo applications.

Inhibition of intrinsic coagulation improves immune checkpoint-targeted gene delivery of cationic solid lipid nanoparticles

Abstract Tumor-targeted delivery of CRISPR/Cas9 plasmid or siRNA to inhibit immune checkpoint protein expression in tumor cells has the potential to improve anticancer immune responses. Cationic solid lipid nanoparticles (cSLNs) are promising carrier for the delivery of negatively charged nucleic acids. cSLNs with high surface charge and low PEG density have been demonstrated to enhance transfection efficiency in vitro, but for unknown reasons fail to improve gene delivery in vivo. We prepared 9 kinds of cSLNs with varying surface charge and PEG density and detected the interactions of these cSLNs with blood cells. We found that increasing surface charge and/or reducing PEG density present a risk for systemic platelet aggregation and activation. Thrombosis was clearly detected in liver, lung and kidney from mice treated with cSLNs with high surface charge and low PEG density. Mechanistic studies revealed that the intrinsic coagulation pathway was responsible for cSLN-induced thrombus formation. Furthermore, pretreatment of recipient mice with heparin, a clinically-approved intrinsic coagulation inhibitor, was highly effective in preventing toxicity, prolonging the circulation time, improving tumor accumulation and enhancing the tumor cell uptake in tumor-bearing mice. These results indicate that the thrombotic toxicity of cSLNs is an important factor affecting their drug delivery efficiency in vivo, and that heparin treatment offers a useful strategy for improving cSLN-based cancer immunotherapies.

Tailoring the Supramolecular Structure of Guanidinylated Pullulan toward Enhanced Genetic Photodynamic Therapy.

In the progress of designing a gene carrier system, what is urgently needed is a balance of excellent safety and satisfactory efficiency. Herein, a straightforward and versatile synthesis of a cationic guanidine-decorated dendronized pullulan (OGG3P) for efficient genetic photodynamic therapy was proposed. OGG3P was able to block the mobility of DNA from a weight ratio of 2. However, G3P lacking guanidine residues could not block DNA migration until at a weight ratio of 15, revealing guanidination could facilitate DNA condensation via specific guanidinium-phosphate interactions. A zeta potential plateau (∼+23 mV) of OGG3P complexes indicated the nonionic hydrophilic hydroxyl groups in pullulan might neutralize the excessive detrimental cationic charges. There was no obvious cytotoxicity and hemolysis, but also enhancement of transfection efficiency with regard to OGG3P in comparison with that of native G3P in Hela and HEK293T cells. More importantly, we found that the uptake efficiency in Hela cells between OGG3P and G3P complexes was not markedly different. However, guanidination caused changes in uptake pathway and led to macropinocytosis pathway, which may be a crucial reason for improved transfection efficiency. After introducing a therapeutic pKillerRed-mem plasmid, OGG3P complexes achieved significantly enhanced KillerRed protein expression and ROS production under irradiation. ROS-induced cancer cells proliferation suppression was also confirmed. This study highlights the guanidine-decorated dendronized pullulan could emerge as a reliable nonviral gene carrier to specifically deliver therapeutic genes.

Co-transfection of star-shaped PDMAEMAs enhance transfection efficiency of protamine/pDNA complexes in the presence of serum

In previous studies, we have found that un-complexed free cationic polymers greatly promote gene transfection efficiency of pDNA complexes. Dividing the gene transfection system into two parts, bound chains and free chains, we exploited mixing different biomaterials as the second component of our designed system to improve gene transfection outcome. In this study, we investigated the effectiveness of star-shaped poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) with different arm numbers as free chains (i.e., co-transfection agent) while using natural salmon protamine as bound chains. We explored the gene transfer ability of the system in both serum free and complete cell culture medium. Our results reveal that the star-shaped PDMAEMAs enhance gene transfection efficiency of protamine/pDNA complexes, in the same range and even better than the gold standard polyethyleneimine (PEI, branched type with 25 kg/mol molar mass) in serum-free and serum containing medium conditions, respectively. From 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Lactate Dehydrogenase (LDH) release data, it was found that the PDMAEMA stars have tolerable cytotoxicity but with significant membrane disruption level, especially when increasing the arm number. Flow cytometry experiments showed that PDMAEMA stars clearly improve the uptake rate as well as the number of protamine/pDNA complexes taken up by the target cells. Using Taqman Real-time Polymerase Chain Reaction (PCR), further exploration of the effect of PDMAEMAs during different gene transfection stages reflects that these un-bound star-shaped polymers mainly act upon the cellular internalization step. Our results indicate that the star-shaped PDMAEMAs are promising vectors for gene delivery.

Comparative Study of Diethylaminoethyl-Chitosan and Methylglycol-Chitosan as Potential Non-Viral Vectors for Gene Therapy.

In this paper, we compared the transfection efficiency and cytotoxicity of methylglycol-chitosan (MG-CS) and diethylaminoethyl-chitosan (DEAE-CSI and DEAE-CSII with degrees of substitution of 1.2 and 0.57, respectively) to that of Lipofectamine (used as a reference transfection vector). MG-CS contains quaternary amines to improve DNA binding, whereas the DEAE-CS exhibits pH buffering capability that would ostensibly enhance transfection efficiency by promoting endosomal escape. Gel retardation assays showed that both DEAE-CS and MG-CS bound to DNA at a polysaccharide:DNA mass ratio of 2:1. In Calu-3 cells, the DNA transfection activity was significantly better with MG-CS than with DEAE-CS, and the efficiency improved with increasing polysaccharide:DNA ratios. By contrast, the efficiency of DEAE-CSI and DEAE-CSII was independent of the polysaccharide:DNA ratio. Conversely, in the transfection-recalcitrant JAWSII cells, both Lipofectamine and MG-CS showed significantly lower DNA transfection activity than in Calu-3 cells, whereas the efficiency of DEAE-CSI and DEAE-CSII was similar in both cell lines. The toxicity of DEAE-CS increased with increasing concentrations of the polymer and its degree of substitution, whereas MG-CS demonstrated negligible cytotoxicity, even at the highest concentration studied. Overall, MG-CS proved to be a more efficient and less toxic transfection agent when compared to DEAE-CS.

Development of Fully Degradable Phosphonium-Functionalized Amphiphilic Diblock Copolymers for Nucleic Acids Delivery.

To expand the range of functional polymer materials to include fully hydrolytically degradable systems that bear bioinspired phosphorus-containing linkages both along the backbone and as cationic side chain moieties for packaging and delivery of nucleic acids, phosphonium-functionalized polyphosphoester-block-poly(l-lactide) copolymers of various compositions were synthesized, fully characterized, and their self-assembly into nanoparticles were studied. First, an alkyne-functionalized polyphosphoester-block-poly(l-lactide) copolymer was synthesized via a one pot sequential ring opening polymerization of an alkyne-functionalized phospholane monomer, followed by the addition of l-lactide to grow the second block. Second, the alkynyl side groups of the polyphosphoester block were functionalized via photoinitiated thiol–yne radical addition of a phosphonium-functionalized free thiol. The polymers of varying phosphonium substitution degrees were self-assembled in aqueous buffers to afford formation of well-defined core–shell assemblies with an average size ranging between 30 and 50 nm, as determined by dynamic light scattering. Intracellular delivery of the nanoparticles and their effects on cell viability and capability at enhancing transfection efficiency of nucleic acids (e.g., siRNA) were investigated. Cell viability assays demonstrated limited toxicity of the assembly to RAW 264.7 mouse macrophages, except at high polymer concentrations, where the polymer of high degree of phosphonium functionalization induced relatively higher cytotoxicity. Transfection efficiency was strongly affected by the phosphonium-to-phosphate (P+/P–) ratios of the polymers and siRNA, respectively. The AllStars Hs Cell Death siRNA complexed to the various copolymers at a P+/P– ratio of 10:1 induced comparable cell death to Lipofectamine. These fully degradable nanoparticles might provide biocompatible nanocarriers for therapeutic nucleic acid delivery.

Polyplex Particles Based on Comb-Like Polyethylenimine/Poly(2-ethyl-2-oxazoline) Copolymers: Relating Biological Performance with Morphology and Structure.

The present contribution is focused on feasibility of using comb-like copolymers of polyethylenimine with poly(2-ethyl-2-oxazoline) (LPEI-comb-PEtOx) with varying grafting densities and degrees of polymerization of PEI and PEtOx to deliver DNA molecules into cells. The copolymers form small and well-defined particles at elevated temperatures, which are used as platforms for binding and condensing DNA. The electrostatic interactions between particles and DNA result in formation of sub-100 nm polyplex particles of narrow size distribution and different morphology and structure. The investigated gene delivery systems exhibit transfection efficiency dependent on the copolymer chain topology, shape of the polyplex particles, and internalization pathway. Flow cytometry shows enhanced transfection efficiency of the polyplexes with elongated and ellipsoidal morphology. The preliminary biocompatibility study on a panel of human cell lines shows that pure copolymers and polyplexes thereof are practically devoid of cytotoxicity.

Dual-stimuli-responsive albumin-polyplex nanoassembly for spatially controlled gene release in metastatic breast cancer

Stimuli-responsive polymeric nanoparticles are useful for overcoming challenges such as transfection efficiency and the specific and safe delivery of genes to cancer cells. Transfection outcomes can be improved through spatially and temporally controlled gene release. We formulated a nanoassembly comprising a disulfide-crosslinked polyethylenimine (ssPEI) conjugated with a tumor-specific cell-penetrating peptide (DS 4-3) (SPD) polyplex and bovine serum albumin (BSA)-loaded IR780 (BI) nanoparticle, thereby forming a dual-stimulus-triggered, tumor-penetrating and gene-carrying nanoassembly (BI-SPD) via electrostatic complexing. BI-SPD nanoassembly were composed of highly stable nanosized complexes with an average size of 457 ± 27.5 nm, exhibiting an up to two-fold enhanced transfection efficiency with no sign of potential cytotoxicity in breast cancer cells. Moreover, upon laser irradiation, a four-fold increase in transfection efficiency was achieved due to the rapid endosomal escape of polyplexes triggered by the local heat induced by the BI-SPD nanoassembly. Additionally, the high redox environment in tumor cells facilitated the disassembly of the SPD polyplex for efficient plasmid release in the cytosol. The BI-SPD nanoassembly also exhibited high penetration and enhanced photothermally triggered gene expression in the 4T1 spheroid model. This BI-SPD nanoassembly has the potential to enhance the expression of therapeutic genes in tumor models without causing significant toxicity to surrounding healthy tissues, since it has shown higher tumor targeting and accumulation in the 4T1 tumor in mice model.

Viral vector mimicking and nucleus targeted nanoparticles based on dexamethasone polyethylenimine nanoliposomes: Preparation and evaluation of transfection efficiency.

Non-viral vectors such as polymers and liposomes have been used as gene delivery systems to overcome intrinsic problems of viral vectors, but transfection efficiency of these vectors is lower than viral vectors. In the present study, we tried to design non-viral gene delivery vectors that mimic the viral vectors using the benefits of both cationic liposomes and cationic polymer vectors along with targeting glucocorticoid receptors to enhance cellular trafficking of vectors. Cationic liposomes containing DOTAP and cholesterol were prepared by thin-film hydration following extrusion method. Dexamethasone mesylate was synthesized and then conjugated to polyethylenimine through a one-step reaction. A novel gene delivery system, Lipopolyplex was developed by premixing liposome and different molecular weight of bPEI-Dexa as carriers followed by addition of plasmid at three different carrier/pDNA (C/P) weight ratios. The resulted complexes were characterized for their size, zeta potential and ability of DNA condensation. Transfection efficiency of vectors in neuro2A was determined by Luciferase reporter gene assay. Also, the toxicity of gene carriers was investigated in this cell line. Mean particle size of prepared complexes was less than 200 nm and there was no significant difference in their size by increasing the molecular weight of PEIs. All complexes had positive surface charge. Complete condensation of DNA was occurred at C/P ratio of one for all complexes. Lipopolyplexes were more efficient than polyplexes and lipoplexes alone and transfection efficiency was improved by adding dexamethasone. The complexes containing liposome, PEI 10 kDa and dexamethasone (PEI10:Lipo:Dexa(0.05)) had the highest transfection activity about 40-fold and 3.6-fold in comparison with PEI10 and PEI10:Lipo, respectively. Furthermore, the non-viral vectors described in this study showed low cytotoxicity. The results of this study confirmed that PEI in combination with liposome forms lipopolyplex with low toxicity and enhanced transfection efficiency. Moreover, using dexamethasone, in combination with lipopolyplex might be useful to increase the gene delivery potential of these lipopolyplexes.

PEGylated dendrimer-entrapped gold nanoparticles with low immunogenicity for targeted gene delivery.

The high efficiency and specificity of gene therapy are mainly ascribed to the excellent transfection ability of the gene carrier. Non-viral polymer vectors have attracted extensive attention because of their low cytotoxicity and outstanding genetic loading capacity compared with viral vectors. For safe and efficient transfection of nuclear acids, here we report a novel gene delivery system, dendrimer-entrapped gold nanoparticles modified with a folate-conjugated poly (ethylene glycol) (Au DENPs-PEG-FA), possessing superior gene transfection efficiency than that of partially hydrophilic methoxy poly(ethylene glycol) (mPEG)-modified dendrimer-entrapped gold nanoparticles (Au DENPs-mPEG). The prepared Au DENPs-PEG-FA were well characterized, and our data revealed that the vector showed good cytocompatibility. Additionally, the quantification of inflammatory cytokines detected by qRT-PCR showed that the vectors displayed low innate immune response. The efficiency of nucleic acid (encoding enhanced green fluorescent protein (EGFP) and luciferase (Luc) reporter) transfection evaluated via flow cytometry and confocal microscopic imaging suggested that the Au DENPs-PEG-FA were able to transfect nucleic acid into HeLa cells with enhanced transfection efficiency. Furthermore, the existence of FA rendered the Au DENPs with excellent targeting performance via FA receptor-ligand binding interaction. The designed Au DENPs-PEG-FA with low immunogenicity and enhanced gene transfection efficiency may hold a great promise to be a superior vector for gene therapy.

The stochastic effect of polydispersity on polymeric DNA delivery vectors

ABSTRACTThe molecular composition and architecture of polymeric DNA delivery reagents govern their transfection efficiency. One parameter that is understudied is the influence of polydispersity on transfection efficiency. Here we report the synthesis of two molecularly identical polymer series, but with different polydispersities (1.3 vs. 2.0) and quantify the effect polydispersity on transfection efficiency. The results show that the effect of polydispersity on transfection efficiency in cell culture is not predictable and is stochastic in nature. In general terms, polymers with high polydispersity mediated higher levels of transfection in vitro compared to low polydispersity polymers with the same side chain distribution and composition. However, at higher molecular weights, the effect of polydispersity was polymer specific, with enhanced transfection efficiency in some cases, but negligible in others. An interesting outcome is that the higher polydispersity polymers generally enhanced the stability of polymer:DNA complexes and this enhancement generally correlated with enhanced transfection efficiency. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 45965.

Exploring the role of peptides in polymer-based gene delivery.

Polymers are widely studied as non-viral gene vectors, but suffer from high cytotoxicity and low transfection efficiency. Incorporating short, bioactive peptides into polymer-based gene delivery systems can address this issue. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we highlight the peptides' roles in polymer-based gene delivery, and elaborate how to utilize various functional peptides to enhance the transfection efficiency of polymers. The optimized peptide-polymer vectors should be able to alter their structures and functions according to biological microenvironments and utilize inherent intracellular pathways of cells, and consequently overcome the barriers during gene delivery to enhance transfection efficiency.

Preparation, characterization, and transfection efficiency of low molecular weight polyethylenimine-based nanoparticles for delivery of the plasmid encoding CD200 gene.

Various strategies have been utilized to improve both gene transfer efficiency and cell-induced toxicity of polyethylenimine (PEI), the most extensively investigated cationic polymeric vector. In this study, we sought to enhance transfection efficiency of low molecular weight PEI (LMW PEI) while maintaining its low toxicity by cross-linking LMW PEI via succinic acid linker. These modifications were designed to improve the hydrophilic–hydrophobic balance of the polymer, by enhancing the buffering capacity and maintaining low cytotoxic effects of the final conjugate. Decreased expression of CD200 in the central nervous system has been considered as one of the proposed mechanisms associated with neuroinflammation in multiple sclerosis; therefore, we selected plasmid-encoding CD200 gene for transfection using the modified PEI derivatives. Dynamic light scattering experiments demonstrated that the modified PEIs were able to condense plasmid DNA and form polyplexes with a size of approximately 130 nm. The highest level of CD200 expression was achieved at a carrier to plasmid ratio of 8, where the expression level was increased by 1.5 fold in the SH-SY5Y cell line, an in vitro model of neurodegenerative disorders. Furthermore, the results of in vivo imaging of the LMW PEI-based nanoparticles in the mouse model of multiple sclerosis revealed that fluorescently labeled plasmid encoding CD200 was distributed from the injection site to various tissues and organs including lymph nodes, liver, brain, and finally, kidneys. The nanoparticles also showed the ability to cross the blood–brain barrier and enter the periventricular area.

Functional nanosome for enhanced mitochondria-targeted gene delivery and expression

Mitochondria dysfunction plays a role in many human diseases. Therapeutic techniques for these disorders require novel delivery systems that can specifically target and penetrate mitochondria. In this study, we report a novel nanosome composed of dequalinium-DOTAP-DOPE (1,2 dioleoyl-3-trimethylammonium-propane-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) (DQA80s) as a potential mitochondria-targeting delivery vector. The functional DQAsome, DQA80s, showed enhanced transfection efficiency compared to a vector DQAsomes in HeLa cells and dermal fibroblasts. In addition, DQA80s/pDNA complexes exhibited rapid escape from the endosome into the cytosol. We observed the delivery of pDNA to mitochondria in living cells using flow cytometry, confocal microscopy, and TME imaging. More specifically, we confirmed our results by co-localization of hmtZsGreen constructs to mitochondria when delivered via DQAsomes and DQA80s in living cells. The mitochondria-targeting DQAsomes and DQA80s induced mitochondrial dysfunction through depolarization of mitochondrial membrane potential. Our data demonstrate that DQA80s show promise for use as a mitochondria-targeted carrier system for treatment of mitochondria diseases in vivo.

Design of pH‐sensitive peptides from natural antimicrobial peptides for enhancing polyethylenimine‐mediated gene transfection

Poor endosomal release is a major barrier of polyplex-mediated gene transfection. Antimicrobial peptides (AMPs) are commonly used to improve polyethylenimine (PEI)-mediated gene transfection by increasing endosomal release. In the present study, we designed novel pH-sensitive peptides that highly enhance transfection efficiency compared to their parent peptides. Two analogues of melittin (Mel) and RV-23 (RV) were synthesized by replacing the positively-charged residues in their sequences with glutamic acid residues. The pH-sensitive lysis ability of the peptides, the effect of the peptides on physicochemical characteristics, the intracellular trafficking, the transfection efficiency, and the cytotoxicity of the polyplexes were determined. The acidic peptides showed pH-sensitive lytic activity. The hemolytic activity of acidic peptides at pH 5.0 was higher than that at pH 7.4. The incorporation of acidic peptides did not affect the DNA binding ability of PEI but affected the physicochemical characteristics of the PEI/DNA polyplexes, which may be beneficial for endosomal release and gene transfection. The incorporation of acidic peptides into PEI/DNA polyplexes enhanced the PEI-mediated transfection efficiency corresponding to up to 42-fold higher luciferase activity compared to that of PEI alone. The results of the present study indicate that replacement of positively-charged residues with glutamic acid residues in the AMP sequence yields pH-sensitive peptides, which enhance the transfection efficiency of PEI/DNA polyplexes in various cell lines.

Zeta-potential-changing nanoparticles conjugated with cell-penetrating peptides for enhanced transfection efficiency.

The aim of this study was to develop zeta-potential-changing nanoparticles (NPs) combining cell-penetrating peptides for gene delivery. NPs were formed using phosphorylated carboxymethyl cellulose-glucosamine 6-phosphate (CMC-G6P) and polyethylene imine-polyarginine conjugates. Phosphate release was evaluated using intestinal alkaline phosphatase and cell lines. Transfection studies with plasmid DNA were then performed. The zeta potential of CMC-G6P/branched PEI NPs was -3 mV and switched to +4 mV after intestinal alkaline phosphatase cleavage. The released phosphate in human colon adenocarcinoma cell line was more pronounced than human embryonic kidney cell line 293. Transfection studies demonstrated the greatest expression of plasmid DNA when being incorporated into CMC-G6P/polyethylene imine-polyarginine NPs. Novel zeta potential changing NPs combining cell-penetrating peptides are a promising tool to deliver DNA drugs to target cells.

Megalin-targeted enhanced transfection efficiency in cultured human HK-2 renal tubular proximal cells using aminoglycoside-carboxyalkyl- polyethylenimine -containing nanoplexes

Non-viral vectors are of interest as therapeutic gene delivery agents in gene therapy, because they are simple to prepare, easy to modify and have definable safety profiles compared to viral vectors. The potential of gene therapy in the treatment of renal diseases is limited by a lack of effective kidney-targeted gene delivery systems. Aminoglycoside antibiotics gentamicin and neomycin were connected by amide linkages to carboxyl groups on carboxyalkylated-PEI25 (25kDa PEI) or carboxyalkylated-PEI10 (10kDa PEI). Aminoglycoside-carboxyalkylated-PEI conjugates were characterized with respect to size, surface charge density, DNA condensation ability, and buffering capacity. Polyplexes prepared by electrostatic interaction between aminoglycoside-carboxyalkylated-PEIs and enhanced green fluorescent protein-expressing (EGFP) plasmid DNA had appropriate nano-scale size (143–173nm). Their targeting potential was investigated in cultured HK-2 immortalized human cortex/proximal tubule kidney epithelial cells, which expresses megalin, a scavenger receptor that mediates endocytosis of a diverse group of ligands, including aminoglycoside antibiotics. Aminoglycoside-carboxyalkylated-PEIs significantly increased EGFP gene transfection efficiency in HK-2 cells by ∼13-fold for aminoglycoside-carboxyalkylated-PEI25 and ∼7-fold increase for aminoglycoside-carboxyalkylated-PEI10 relative to the corresponding PEIs without aminoglycosides. The transfection efficiency of polyplexes was dependent on the weight ratio of aminoglycoside-containing ligand in the carrier. In the presence of a range of concentrations of human serum albumin, which competes for megalin binding, aminoglycoside-carboxyalkylated-PEI-mediated transfection was reduced to background levels. These results suggest that aminoglycoside-carboxyalkylated-PEI polyplexes can target megalin-expressing kidney-derived cells in vitro resulting in improved transfection efficiency with low cytotoxicity.

Supramolecular block copolymers for gene delivery: enhancement of transfection efficiency by charge regulation.

A class of cationic supramolecular block copolymers with readily controlled charges has been exploited. Upon post-synthetic structural optimization, this copolymer exhibits comparable biocompatibility, greatly improved pDNA condensation capability and biostability, and further enhanced transfection efficiency in vitro. This work provides valuable insight into the creation of advanced nonviral vectors for gene delivery.

Multi-functional nanotracers for image-guided stem cell gene therapy.

Stem cell therapy based on human mesenchymal stem cells (hMSCs) has shown great promise for various disease treatments. However, traditional stem cell-mediated therapy is limited due to their multipotent differentiation ability (uncontrolled spontaneous differentiation) and the difficulty in monitoring cells after implantation in vivo. Here, we report a new multi-functional stem cell nanotracer (M-NT) for directing controlled differentiation through gene delivery, as well as tracking stem cells with dual-modal imaging (optical and CT imaging). The M-NT was prepared through a facile surface modification process of ∼100 nm-sized gold nanoparticles with catechol-functionalized branched polyethylenimine (C-bPEI). The C-bPEI-functionalized M-NT exhibited greatly enhanced long-term colloidal stability in aqueous solution and a capability to complex with plasmid DNA (pDNA; i.e., pEGFP) through electrostatic interaction for gene delivery and transfection to control differentiation. M-NT/pEGFP complexes showed an enhanced transfection efficiency into hMSCs with low cytotoxicity compared with branched polyethylenimine/pDNA complexes. Accordingly, successful in vitro chondrogenic differentiation was achieved in hMSCs treated with M-NT/pSOX9 complexes. Finally, hMSCs transfected with M-NT/pEGFP complexes were transplanted into Balb/c nude mice and successfully visualized through dual-modal optical fluorescence and computed tomography (CT) imaging. We believe that this approach could represent a promising platform for genetic material-mediated direction of differentiation and cell tracking in stem cell therapy.

양이온 펩타이드가 컨쥬게이트된 수용성 키토산의 유전자 전달체로서의 특성

Recently gene delivery has been designed newly using bioactive biomaterial and applied in the various field by many researchers. In this study, we proposed a new gene delivery system which has the capability of targeting effect in the specific tissue and remarkably enhanced transfection efficiency. We investigated <TEX>$^1H-NMR$</TEX> spectroscopy, particle size analyzer and gel retardation to confirm the correct preparation of gene delivery. Also, we identified the hemo-compatibility of gene delivery by hemolysis assay, non-cytotoxicity by MTT test and transfection efficiency. The uptake mechanism of the gene carrier was confirmed using inhibitor agent such as sodium azide, indomethacin, quercetin, colchicine, and chloropromazine. As a results, it was identified that gene carrier prepared by in this study entered in the cell by the microtubule-dependent, energy-dependent and clathrin-mediated endocytosis pathway.