Gene Delivery Carriers Research Articles

Enzyme-Responsive Charge-Reversal Polymer-Mediated Effective Gene Therapy for Intraperitoneal Tumors

Gene therapy has demonstrated effectiveness in many genetic diseases, as evidenced by recent clinical applications. Viral vectors have been extensively tested in clinical gene-therapy trials, but nonviral vectors such as cationic polymers or lipids are much less used due to their lower gene-transfection efficiencies. However, the advantages of nonviral vectors, such as easily tailored structures, nonimmunogenetics, and relatively low cost, still drive great efforts to improve their transfection efficiencies. A reverse question asks if nonviral vectors with current gene transfection efficiency can find application niches. Herein, we synthesized a cationic polymer, poly{ N-[2-(acryloyloxy)ethyl]- N-[ p-acetyloxyphenyl]- N, N-diethylammonium chloride} (PQDEA), as a gene-delivery carrier and compared it side by side with chemotherapy drugs for cancer treatment. PQDEA is rapidly hydrolyzed by intracellular esterases into anionic poly(acrylic acid) to give low cytotoxicity and fast release of DNA for expression. PQDEA formed stable complexes with DNA (PQDEA/DNA polyplexes), which were further coated with a lipid layer to make serum-stable lipidic polyplexes, LPQDEA/DNAs, for in vivo use. In an intraperitoneal tumor xenograft model mimicking late-stage metastatic cervical cancer, the LPQDEA/DNA vector with TRAIL suicide gene exerted strong tumor inhibition as effective as paclitaxel, the first-line anticancer drug, but gave much less tumor relapse and much longer survival than the clinical chemotherapy drugs, irinotecan and paclitaxel. Equally important, the gene therapy showed much fewer adverse effects than the chemotherapy drugs. This work shows that nonviral vectors with current transfection efficiencies may produce therapeutic advantages and may be safe and worthy of clinical translation in, for example, intraperitoneal cancer therapy.

Membrane structural change of dimyristoylphosphatidylcholine liposome on the interaction with polyethyleneimine.

Polyethyleneimine (PEI) has long been considered as "golden standard" for polymeric gene delivery carriers. To get a better understanding on the molecular basis of PEI cytotoxicity, dynamic light scattering, zeta-potential measurement, fluorescence emission, Fröster resonance energy transfer and anisotropy measurement were conducted to reveal the interaction between PEI and dimyristoylphosphatidylcholine (DMPC) liposome and the influence on the structural properties of the membrane. PEI was found to bind onto the surface of the liposome, inducing an aggregation of the vesicle and an increase in surface potential at low PEI concentration up to 0.05 mg mL-1. A further increase in PEI concentration made little change on the surface potential, however reduced the aggregation of the vesicle due to the repulsion between the adsorbed PEI chains. PEI binding slightly increased the fluidity of lipid in interface region and decreased its packing density, and thus resulted in an enhanced leakage of calcein through the membrane. The polymer size played an important role in PEI-DMPC liposome interaction. PEI of higher molecular weight was more favorable to interact with DMPC and more efficient to perturb the structural properties of the membrane.

Design of PEI-conjugated bio-reducible polymer for efficient gene delivery

The poly(cystaminebis(acrylamide)-diaminohexane) (poly(CBA-DAH)) was designed previously as a bio-reducible efficient gene delivery carrier. However, the high weight ratio required to form the polyplexes between poly(CBA-DAH) with pDNA is still a problem that needs to be addressed. To solve this problem and increase the transfection efficiency, poly(ethylenimine) (PEI, 1.8 kDa) was conjugated to poly(CBA-DAH) via disulfide bond. The PEI conjugated poly(CBA-DAH) (PCDP) can bind with pDNA at a very low weight ratio of 0.5 and above, like PEI 25 kDa, and form the polyplexes with nano-size (102–128 nm) and positive surface charge (27–34 mV). PCDP and PCDP polyplexes had negligible cytotoxicity and indicated similar or better cellular uptake than the comparison groups such as PEI 25 kDa and Lipofectamine® polyplexes. To confirm the transfection efficiency, the plasmid DNA (pDNA) encoded with the luciferase reporter gene (gWiz-Luc) and green fluorescent protein reporter gene (GFP) were used and treated with PCDP into the A549, Huh-7, and Mia PaCa-2 cells. PCDP/pDNA polyplexes showed highest transfection efficiency in all tested cell lines. In the luciferase assay, PCDP polyplexes showed 10.2 times higher gene transfection efficiency than Lipofectamine® polyplexes in mimic in vivo conditions (30% FBS, A549 cells). The VEGF siRNA expressing plasmid (pshVEGF), which is constructed as a therapeutic gene by our previous work, was delivered by PCDP into the cancer cells. The VEGF gene expression of PCDP/pshVEGF polyplexes was dramatically lower than control and the VEGF gene silencing efficiencies of PCDP/pshVEGF (w/w; 10/1) polyplexes were 54% (A549 cells), 77% (Huh-7 cells), and 66% (Mia PaCa-2 cells). In addition, PCDP/pshVEGF had reduced cell viability rates of about 31% (A549 cells), 39% (Huh-7 cells), and 42% (Mia PaCa-2 cells) and showed better results than all comparison groups. In the transfection efficiency and VEGF silencing assay, PCDP polyplexes showed better results than poly(CBA-DAH) at 4-fold lower weight ratio. The data of all experiments demonstrate that the synthesized PCDP could be used for efficient gene delivery and could be widely applied.

Intracellular distribution and internalization pathways of guanidinylated bioresponsive poly(amido amine)s in gene delivery

Guanidinylated bioresponsive poly(amido amine)s polymers, CAR-CBA and CHL-CBA, were synthesized by Michael-type addition reaction between guanidine hydrochloride (CAR) or chlorhexidine (CHL) and N,N′-cystaminebisacrylamide (CBA). Previous studies have shown that both polymers had high transfection efficiencies as gene delivery carriers. In this study, we investigated the nucleolus localization abilities and cellular internalization pathways of these two polymers in gene delivery. Each polymer condensed plasmid DNA (pDNA) and formed nanoparticle complexes, and then their transfection studies were performed in MCF-7 cells. Both complexes were found enriched in nucleolus after cellular transfection, and their transfection efficiencies were significantly improved when transfection was performed on MCF-7 cells arrested at M phase. The transfection efficiency of CAR-CBA-pDNA was inhibited by chlorpromazine, and cell endosomes were disrupted after being exposed to CAR-CBA-pDNA. In regards to CHL-CBA-pDNA, its transfection efficiency was not affected by three types of endocytosis inhibitors used in the study, and CHL-CBA-pDNA showed no effect on endosomes. Cellular lactate dehydrogenase release and membrane morphology were changed after cells were transfected by the two complexes. The results indicated that both CAR-CBA and CHL-CBA polymers demonstrated good nucleolus localization abilities. It was beneficial for transfection when cells were arrested at M phase. CAR-CBA-pDNA cellular internalization was involved with clathrin-mediated endocytosis pathway, and escaping from endosomal entrapment, while the cellular uptake of CHL-CBA-pDNA occurs via clathrin- and caveolae-independent mechanism.

Polyethylenimine-Mediated CCR5 Gene Knockout Using Transcription Activator-Like Effector Nucleases.

CCR5 acts as one of the key coreceptors for human immunodeficiency virus infection, which leads to acquired immune deficiency syndrome. CCR5 gene knockout comes up as an alternative method for treatment of the disease. Transcription activator-like effector nuclease is a powerful gene editing tool characterized by the ease of design, high rates of cleavage activity, and the accessibility of all ranges of transcription activator-like effector nucleases. In this study, transcription activator-like effector nuclease plasmids specifically targeting to knock out CCR5 gene were designed, constructed and then transfected to Hela and HEK293T cell lines with the assistance of the well-established gene delivery carrier polyethylenimine. The transfection efficiency of polyethylenimine reached 50-60% at the optimized N/P ratio of 6/1. The genomic CCR5 sequencing results suggested an estimated knockout efficiency of around 50% under the optimized condition. Thus, CCR5 gene knockout was achieved in cell lines by the polyethylenimine-mediated transfection of transcription activator-like effector nuclease plasmids of interest. More efforts such as the improvement of transfection efficiency of gene delivery carrier could be made to achieve higher gene knockout efficiency of transcription activator-like effector nucleases.

Inhibition of cell proliferation and migration through nucleobase-modified polyamidoamine-mediated p53 delivery.

IntroductionThe nucleobase 2-amino-6-chloropurine-modified polyamidoamine (AP-PAMAM) was used as a carrier for p53 gene delivery to achieve the antitumor effects.Methods and materialsThe condensation of p53 plasmid was studied through gel retardation assay, and the transfection efficiency was evaluated through the transfection assay of pEGFP-N3 and pGL-3 plasmids. Using human cervical carcinoma cell line HeLa as a model, the inhibition of cell proliferation and migration was studied through flow cytometry, wound healing and Transwell migration assays, respectively. The p53 expression level was detected through quantitative polymerase chain reaction and Western blotting analyses.ResultsThe carrier could condense p53 plasmid into stable nanoparticles at N/P ratios of 2.0, and higher transfection efficiency than polyamidoamine (PAMAM) could be obtained at all the N/P ratios studied. AP-PAMAM-mediated p53 delivery could achieve stronger antiproliferative effect than PAMAM/p53. The antiproliferative effect was identified to be triggered by the induction of cell apoptosis (apoptotic ratio of 26.17%) and cell cycle arrest at S phase. Additionally, AP-PAMAM/p53 transfection has been found to suppress the cell migration and invasion of cancer cells. Finally, the enhanced p53 expression level could be detected after p53 transfection at mRNA and protein levels.ConclusionThe PAMAM derivative-mediated p53 delivery could be a promising strategy for achieving tumor gene therapy.

2D Nanostructured Metal Hydroxides with Gene Delivery and Theranostic Functions; A Comprehensive Review.

Layered double hydroxides (LDHs) with two dimensional structure have been attracted considerable interest in exploring new intercalative nanohybrids, such as inorganic-LDHs, organic-LDHs and bio-LDHs ones, which often exhibit extraordinarily synergetic effects and complementary performances. More recently, bio-related nanotechnology becomes one of the most essential research field in the viewpoint of the health and safety of human being. In this regard, LDHs have been focused as an important inorganic material for gene and drug delivery carriers with imaging and targeting functions. In the present review, an attempt has been made to describe gene delivery systems based on LDH nanoparticles in terms of synthetic routes of gene-LDH nanohybrids, their physico-chemical properties, intercellular uptake mechanisms, intracellular trafficking pathways and drug resistance, and passive and active targeting functions in in-vitro and in-vivo, and finally imaging functions. And recent studies of gene-therapies with LDHs are also discussed from the viewpoint of state-of-the-art nanohybrids technology.

Precisely Defined Polymers for Efficient Gene Delivery.

Gene therapy requires successful delivery of therapeutic nucleic acids into target cells; thus, efficient and safe gene delivery carriers are crucial to its success. Although many polymeric materials have shown their potential as effective nucleic acid carriers, the inherent heterogeneity and polydispersity of these polymers hinder their application in clinical studies because of difficulties in their further precise modification, structure-activity relationship study, as well as consistent manufacturing. Therefore, precisely defined polymers, with potential for site-specific optimization according to structure-activity relationship information and highly controllable production, have been extensively investigated. In this review, we focus on the design and development of precisely defined polymers for efficient gene delivery, illustrated with examples including dendrimers, peptide-based polymers, and sequence-defined oligoaminoamide oligomers.

Chemoenzymatic synthesis of a cholesterol-g-poly(amine-co-ester) carrier for p53 gene delivery to inhibit the proliferation and migration of tumor cells

An amphiphilic cholesterol-g-poly(amine-co-ester) synthesizedviaa chemoenzymatic route has been successfully applied as a carrier in p53 gene delivery.

2-Amino-6-chloropurine-modified polyamidoamine-mediated p53 gene transfection to achieve anti-tumor efficacy

The modification of 2-amino-6-chloropurine on polyamidoamine was performed to synthesize a derivative, AP-PAMAM, which was then employed as a carrier for p53 gene delivery to achieve anti-tumor efficacy.

Transferrin-functionalized chitosan-graft-poly(l-lysine) dendrons as a high-efficiency gene delivery carrier for nasopharyngeal carcinoma therapy

Transferrin-functionalized chitosan-graft-poly(l-lysine) dendrons (CS-PLLD-Tf) were synthesized in this work and then used to deliver the MMP-9 shRNA plasmid to HNE-1 cells for gene therapy. The obtained CS-PLLD-Tf displayed excellent gene transfection in vitro, verifying the strategy of constructing a high-efficiency gene carrier by conjugating the multiple low generation PLLDs to one molecule of CS to form a copolymer. Particularly, CS-PLLD-Tf showed much higher gene transfection efficiency than PEI-25k and CS-PLLD in the presence of serum, and more than 40% HNE-1 cells were transfected. This result led to an obvious decrease in MMP-9 protein expression and the apoptosis of HNE-1 cells as well as inhibition of the growth and invasion of HNE-1 tumors. The in vivo anti-tumor assay showed that CS-PLLD-Tf delivered the MMP-9 plasmid effectively and the tumor volume decreased more than 77% compared with the PBS control group. Moreover, CS-PLLD-Tf displayed higher bioavailability and good biocompatibility such as lower cytotoxicity, excellent blood compatibility and non-toxicity to organs, suggesting a potential application in gene therapy of tumors.

Polyplex Micelles with Phenylboronate/Gluconamide Cross-Linking in the Core Exerting Promoted Gene Transfection through Spatiotemporal Responsivity to Intracellular pH and ATP Concentration.

Polyplexes as gene delivery carriers require integrated functionalities to modulate intracellular trafficking for efficient gene transfection. Herein, we developed plasmid DNA (pDNA)-loaded polyplex micelles (PMs) from poly(ethylene glycol)-based block catiomers derivatized with 4-carboxy-3-fluorophenylboronic acid (FPBA) and d-gluconamide to form pH- and ATP-responsive cross-linking in the core. These PMs exhibited robustness in the extracellular milieu and smooth endosomal escape after cellular uptake, and they facilitated pDNA decondensation triggered by increased ATP concentration inside of the cell. Laser confocal microscopic observation revealed that FPBA installation enhanced the endosomal escapability of the PMs; presumably, this effect resulted from the facilitated endo-/lysosomal membrane disruption triggered by the released block catiomers with hydrophobic FPBA moieties in the side chain from the PM at lower pH condition of endo-/lysosomes. Furthermore, the profile of intracellular pDNA decondensation from the PMs was monitored using Förster resonance energy transfer measurement by flow cytometry; these observations confirmed that PMs optimized for ATP-responsivity exerted effective intracellular decondensation of loaded pDNA to attain promoted gene transfection.

Receptor-mediated targeted delivery of DNA using Lactoferrin nanoparticles

Efficient gene delivery facilitated by non-viral vectors, is comparatively safer alternative than viral carriers. Current approaches to gene delivery largely depends on methods that overcome cellular and tissue barriers impeding efficient DNA delivery. While the conventional delivery systems have the drawback of low cellular uptake and off target effects, the receptor-mediated gene delivery system has shown remarkable breakthrough. In that context exploiting the specific receptor targeting properties of lactoferrin protein, we have developed pDNA loaded lactoferrin protein nanoparticles (pDNA-LfNPs). pDNA-LfNPs were spherical in shape within the size range of 140±20nm. They were found to be resistant against nuclease digestion and stable under long storage. Additionally, LfNPs were biocompatible and have targeting ability to facilitate gene uptake by receptor mediated internalization in lactoferrin receptor expressing cell lines. It is also evident from our studies that Lf nanoparticles did not exhibit any cytotoxicity even at the highest concentration. Here we first time report the use of lactoferrin nanoparticles as a gene delivery carrier with targeting abilities.

Revealing cooperative binding of polycationic cyclodextrins with DNA oligomers by capillary electrophoresis coupled to mass spectrometry

Gene delivery is critical for the development of nucleic acid-based therapies against a range of severe diseases. The conception of non-viral (semi)synthetic vectors with low cytotoxicity and virus-like efficiency is gathering a lot of efforts, but it represents a fantastic challenge still far from accomplishment. Carbohydrate-based scaffolds offer interesting features towards this end, such as easy availability, relatively cheap cost, tuning properties and a good biocompatibility. The lack of analytical methods providing quantitative and qualitative data on their binding properties with oligonucleotides (DNA/RNA), with a minimal time and sample consumption, represents a limitation for these channels. Here, we attempted to fill the gap by hyphenation of capillary electrophoresis with mass spectrometry (CE-MS). This coupling strategy allows discriminating free and complexed DNA oligomers with cationic cyclodextrins (CDs), determining the stoichiometry where the highest observed is always DNAn: n/3(CD), and unambiguously assigning the partners through m/z detection. Very reliable data were obtained with migration time within 5.5 (standard deviation < 0.5%) and 25 min (standard deviation < 1.1%) for UV and MS detection, respectively. Furthermore, varying the nitrogen/phosphorus ratio (N/P), key parameters relating to the thermodynamics e.g. the micro and macroscopic dissociation constants Kd and KD, respectively (both in low μM range) and the Gibbs free energy ΔG (−16.3 to −26.9 kJ mol−1), and also the cooperativity as Hill number (nH between 0.98 and 15.75) of the supramolecular process can be delineated, providing a unique tool for the high throughput screening and selection of efficient gene delivery carriers.

Induction of a robust immune response against avian influenza virus following transdermal inoculation with H5-DNA vaccine formulated in modified dendrimer-based delivery system in mouse model.

This study was aimed to evaluate the immunogenicity of recombinant plasmid deoxyribonucleic acid (DNA), pBud-H5-green fluorescent protein (GFP)-interferon-regulatory factor (IRF)3 following delivery using polyamidoamine (PAMAM) dendrimer and transactivator of transcription (TAT)-conjugated PAMAM dendrimer as well as the effect of IRF3 as the genetic adjuvant. BALB/c mice were vaccinated transdermally with pBud-H5-GFP, PAMAM/pBud-H5-GFP, TAT-PAMAM/pBud-H5-GFP, and TAT-PAMAM/pBud-H5-GFP-IRF3. The expression analysis of H5 gene from the blood by using quantitative real-time reverse transcriptase polymerase chain reaction confirmed the ability of PAMAM dendrimer as a carrier for gene delivery, as well as the ability of TAT peptide to enhance the delivery efficiency of PAMAM dendrimer. Mice immunized with modified PAMAM by TAT peptide showed higher hemagglutination inhibition titer, and larger CD3+/CD4+ T cells and CD3+/CD8+ T cells population, as well as the production of cytokines, namely, interferon (IFN)-γ, interleukin (IL)-2, IL-15, IL-12, IL-6, and tumor necrosis factor-α compared with those immunized with native PAMAM. These results suggest that the function of TAT peptide as a cell-penetrating peptide is able to enhance the gene delivery, which results in rapid distribution of H5 in the tissues of the immunized mice. Furthermore, pBud-H5-GFP co-expressing IRF3 as a genetic adjuvant demonstrated the highest hemagglutination inhibition titer besides larger CD3+/CD4+ and CD3+/CD8+ T cells population, and strong Th1-like cytokine responses among all the systems tested. In conclusion, TAT-PAMAM dendrimer-based delivery system with IRF3 as a genetic adjuvant is an attractive transdermal DNA vaccine delivery system utilized to evaluate the efficacy of the developed DNA vaccine in inducing protection during challenge with virulent H5N1 virus.

Cationic starch/pDNA nanocomplexes assembly and their nanostructure changes on gene transfection efficiency

This study aims at developing biocompatible starch based gene carriers with good gene delivery and transfection efficacy. By controlling the molecular weight and aggregation behavior of spermine modified cationic starch (CS) molecules, nanocomplexes spontaneously formed through electrostatic interaction using CS and plasmid pAcGFP1-C1 (pDNA) displaying different structural changes (particle size, zeta potential, shape, compactness) response to the simulated intracellular pH variation. Results indicated that CS2 with weight average molecular weight (Mw) of 6.337 × 104 g/mol displayed relatively higher transfection efficacy (~30%) in HepG2 cells than others and revealed significantly low cytotoxicity. By simulating the intracellular pH variation, Dynamic Light Scattering (DLS) and Small Angle X-ray Scattering (SAXS) results demonstrated that CS2 could bind to pDNA tightly and form nanocomplexes with smaller and compact internal aggregate structure at acidic conditions, which facilitated the effective pDNA protection under endosome pH change, while larger and loose internal aggregate structure at physiological pH which promoted the disintegration of CS2/pDNA nanocomplexes. Therefore, CS with suitable Mw of around 6.0 × 104 g/mol represents a potential gene carrier for gene delivery. This study also demonstrated that controlling the internal nanostructure change of polymer/gene nanocomplexes could provide guidance in designing effective starch based gene carriers.

Molecular genetics and emerging therapies for retinitis pigmentosa: Basic research and clinical perspectives

Retinitis Pigmentosa (RP) is a hereditary retinopathy that affects about 2.5 million people worldwide. It is characterized with progressive loss of rods and cones and causes severe visual dysfunction and eventual blindness in bilateral eyes. In addition to more than 3000 genetic mutations from about 70 genes, a wide genetic overlap with other types of retinal dystrophies has been reported with RP. This diversity of genetic pathophysiology makes treatment extremely challenging. Although therapeutic attempts have been made using various pharmacologic agents (neurotrophic factors, antioxidants, and anti-apoptotic agents), most are not targeted to the fundamental cause of RP, and their clinical efficacy has not been clearly proven. Current therapies for RP in ongoing or completed clinical trials include gene therapy, cell therapy, and retinal prostheses. Gene therapy, a strategy to correct the genetic defects using viral or non-viral vectors, has the potential to achieve definitive treatment by replacing or silencing a causative gene. Among many clinical trials of gene therapy for hereditary retinal diseases, a phase 3 clinical trial of voretigene neparvovec (AAV2-hRPE65v2, Luxturna) recently showed significant efficacy for RPE65-mediated inherited retinal dystrophy including Leber congenital amaurosis and RP. It is about to be approved as the first ocular gene therapy biologic product. Despite current limitations such as limited target genes and indicated patients, modest efficacy, and the invasive administration method, development in gene editing technology and novel gene delivery carriers make gene therapy a promising therapeutic modality for RP and other hereditary retinal dystrophies in the future.

Mussel-inspired polydopamine-polyethylenimine conjugated nanoparticles as efficient gene delivery vectors for mammalian cells

Efficient delivery of DNA to cells is the primary concern to address the objective of gene therapy. Many attempts have been made to develop polymeric carriers for gene delivery. To have an efficient carrier, it is vital to understand the properties of the vector for better stability, transfection efficiency and minimal toxicity. Branched polyethylenimine (bPEI) has been considered as the ‘gold standard’ for gene delivery but suffers a major drawback of exhibiting high cytotoxicity. Here, we have attempted to develop a mussel-derived polymer, polydopamine (PDA), conjugated polyethylenimine nanoparticles in such a way that the toxic nature of bPEI is suppressed by the conversion of free primary amine groups to secondary and tertiary amines. Keeping the amount of PDA fixed, varying amounts of bPEIs of different molecular weights (25, 10 and 1.8kDa) were conjugated via Michael addition and/or Schiff base. A trend in hydrodynamic size of the conjugated nanoparticles was observed in the range from 160 to 300nm and zeta potential from +12–30mV in the projected three series, viz., (i) PDA1-25bPEI0.5, PDA1-25bPEI1, PDA1-25bPEI2; (ii) PDA1-10bPEI0.5, PDA1-10bPEI1, PDA1-10bPEI2; and (iii) PDA1-1.8bPEI0.5, PDA1-1.8bPEI1, PDA1-1.8bPEI2. A visible trend in the DNA condensation ability and buffering capacity was also noticed. Further, cell cytotoxicity assays revealed that pDNA complexes of PDA-bPEI nanoparticles were non-toxic to mammalian cells and these complexes exhibited several folds higher transfection efficiency than the complexes of native bPEIs as demonstrated by fluorescence measurements and flow cytometry. Altogether, the results advocate the promising potential of these conjugated nanoparticles for future in vivo applications.

Folic Acid Decorated Chitosan Nanoparticles and its Derivatives for the Delivery of Drugs and Genes to Cancer Cells

Nanotechnology offers a number of nanoscale implements for medicine. Among these, nanoparticles are revolutionizing the field of drug and gene delivery. Chitosan is a natural polymer which provides a profitable tool to an innovative delivery system due to its inherent physicochemical and biological characteristics. Chitosan nanoparticles are promising drug and gene delivery carriers because of small size, better stability, low toxicity, inexpensiveness, simplicity, easy fabrication and versatile means of administration. Chitosan can also be easily modified chemically due to the presence of reactive functional hydroxide and amine groups. Folic acid is commonly engaged as a ligand, for targeting cancer cells, as its receptor, that transports folic acid into the cells through endocytosis and is over-expressed on the surface of several human epithelial cancer cells. Integrating folic acid into chitosan-based drug delivery inventions directs the systems with a well-organized targeting ability. The present review outlines several illustrations of this versatile system based on folate decorated chitosan, which have shown potential as auspicious delivery systems published over the past few years. In addition, it is probable to formulate chitosan nanocarriers that exhibit manifold usage beyond targeted delivery, such as nanotheranostics and cancer stem cell therapy.

Synthesis and Characterization of Fatty Acid Grafted Chitosan Polymer and Their Nanomicelles for Nonviral Gene Delivery Applications.

The aim of this study was to synthesize and characterize fatty acid-grafted-chitosan (fatty acid-g-CS) polymer and their nanomicelles for use as carriers for gene delivery. CS was hydrophobically modified using saturated fatty acids of increasing fatty acyl chain length. Carbodiimide along with N-hydroxysuccinimide was used for coupling carboxyl group of fatty acids with amine groups of CS. Proton nuclear magnetic resonance and Fourier transform infrared spectroscopy were used to quantify fatty acyl substitution onto CS backbone. The molecular weight distribution of the synthesized polymers was determined using size exclusion high performance liquid chromatography and was found to be in range of the parent CS polymer (∼50 kDa). The critical micelle concentration (cmc) of the polymers was determined using pyrene as a fluorescent probe. The cmc was found to decrease with an increase in fatty acyl chain length. The amphiphilic fatty acid-g-CS polymers self-assembled in an aqueous environment to form nanomicelles of ∼200 nm particle size and slightly positive net charge due to the cationic nature of free primary amino groups on CS molecule. These polymeric nanomicelles exhibited excellent hemo- and cytocompatibility, as evaluated by in vitro hemolysis and MTT cell viability assay, respectively, and showed superior transfection efficiency compared to unmodified chitosan and naked DNA. The surface of these nanomicelles can be further modified with ligands allowing for selective targeting, enhanced cell binding, and internalization. These nanomicelles can thus be exploited as potential nonviral gene delivery vectors for safe and efficient gene therapy.