Improved Gene Transfection Efficiency Research Articles

Magnetic Polymer Microcarriers for Cell Engineering Applications with Enzyme‐Free Cell Harvesting and Rapid Recovery in Large‐Scale Cell Culture

AbstractStimulus‐responsive large‐scale cell culture microcarriers are promising biological materials for large‐scale cell production. Herein, multifunctional thermal and magnetic microcarriers (TMMs) made of poly(N‐isopropylacrylamide) (PNIPAM), polylysines (PLLs), gelatin methacryloyl (GelMA), poly(ethylene glycol) diacrylate (PEGDA), N,N‐methylenebis(acrylamide) (MBA), Fe3O4 and SiO2 are prepared to achieve large‐scale enzyme‐free cell harvesting and also can be used as cells stereogrowth scaffold to mimic cells suspension state to develop novel application, such as recombinant proteins produced. In TMMs, NIPAM and other materials are mixed and then underwent in situ radical polymerization by UV irradition with 2‐hydroxy‐2‐methylpropiophenone as initiator, in which GelMA provides cell adhesion sites and PLLs enable a positive microenvironment with SiO2 as filler. As enzyme‐free cell microcarriers, cell harvesting from TMMs by PNIPAM are achieved at 25 °C through vortex oscillation within 15 min and TMMs can be recycled at least for four times by magnetic recovery. Moreover, TMMs can be used as stereoculture scaffold to let cells in suspension as adherent state to improve gene transfection efficiency. Combined with magnetothermal effect, TMMs can further improve gene transfer to obtain industrialization of proteins expression. Therefore, TMMs are potential for the development of cell manufacturing and the expansion of cell stereogrowth application for recombinant proteins production.

Chondroitin sulfate modified calcium phosphate nanoparticles for efficient transfection via caveolin-mediated endocytosis

Efficient transfection remains a challenge for gene delivery in both cell biological scientific research and gene therapeutic fields. Existing transfection strategies rarely pay attention to altering the endocytosis pathway of nanocarriers for transfection efficiency improvement. In this work, we innovatively postulated that calcium phosphate nanoparticles coated with glycosaminoglycan could be internalized by cells mainly through caveolin-mediated endocytosis pathway allowing genes to bypass lysosome route, and hence enhance the transfection efficiency. To achieve this, we developed calcium phosphate nanoparticles (CP-ALN-CS) coated with chondroitin sulfate (CS) and alendronate (ALN) in a modular manner. The CP-ALN-CS had a hydrodynamic size of 131.0 ± 8.7 nm and exhibited favorable dispersity, stability, and resistance to nuclease degradation. Unlike conventional calcium phosphate and PEI-based transfection, CP-ALN-CS exhibited efficient cellular uptake with co-localization in Golgi apparatus and endoplasmic reticulum. Through bypassing the lysosome involved cellular uptake route, CP-ALN-CS can effectively protect genes from degradation and relieve cytotoxicity. After loading plasmid DNA, CP-ALN-CS showed extraordinary transfection efficiency in HEK 293T cells, outperforming the PEI which is considered as the gold standard. The current work provides a novel and facile approach to improve gene transfection efficiency and is valuable for the design of next-generation in vitro transfection reagents.

Selectively Fluorinated PAMAM-Arginine Conjugates as Gene Delivery Vectors.

Polyamidoamine (PAMAM) dendrimers are among the most studied cationic polymers as non-viral gene delivery vectors. However, an "ideal" PAMAM-based gene delivery vector is still missing due to the high manufacturing costs and non-negligible cytotoxicity associated with the use of high-generation dendrimers, whereas low-generation dendrimers are far from displaying efficient gene transfection. In order to cover this gap in the literature, in this study, we propose the functionalization of the outer primary amines of PAMAM G2 and PAMAM G4 with building blocks bearing fluorinated moieties along with a guanidino functional group. We have designed and synthetized two fluorinated arginine (Arg)-based Michael acceptors which were straightforwardly "clicked" to PAMAM dendrimers without the need for coupling reagents and/or catalysts. The obtained conjugates, in particular, derivative 1 formed starting from the low-cost PAMAM G2 and a building block bearing two trifluoromethyl groups, were able to efficiently complex plasmid DNA, had negligible cytotoxicity, and showed improved gene transfection efficiency as compared to undecorated PAMAM dendrimers and a corresponding unfluorinated PAMAM-Arg derivative, with derivative 1 being two orders of magnitude more efficient than the gold standard branched polyethylenimine, bPEI, 25 kDa. These results highlight the importance of the presence of trifluoromethyl moieties for both gene transfection and a possible future application in 19F magnetic resonance imaging.

Manganese-doped biostimulatory nanoneedle for MRI-visual bispecific antibody gene delivery and immunosuppression reversal as a cancer immunotherapy strategy

Bispecific antibody (BsAb) is a new strategy being widely investigated in hematological cancer; however, its immunotherapy effect still exhibits poor prognosis in patients within solid tumors due to the rapid metabolism and short circulation in vivo. In this study, manganese oxide doped silicate nanosystem (Mn-MSN) was prepared for BsAb encoded minicircle DNA delivery. By transforming the Mn-MSN system’s morphology from spherical to needle-like, it markedly improved gene transfection efficiency through the small “wounds” bio-stimulation, which was readily monitored by the magnetic resonance imaging and achieved high rate of antibody secretion for more than 5 days. This strategy also triggered the activation of cGAS-STING signaling pathway and redirected host lymphoid cells towards cancers. Therefore, a broad-spectrum T-cell responses was significantly enhanced in two kinds of immunologically cold solid tumors, resulted in potent anti-tumor immunotherapy. Our immunomodulation nanoplatform presented a powerful gene delivery vehicle that could directly activate native T cells, and gave a new insight into the development of genetic immunotherapy.

Emerging 2D pnictogens: a novel multifunctional photonic nanoplatform for cutting-edge precision treatment.

The elements of the pnictogen group, known as the 15th (VA) family in the periodic table, including phosphorus (P), arsenic (As), antimony (Sb) and bismuth (Bi), have been widely used by alchemists to treat various diseases since ancient times and hold a pivotal position in the history of medicine, owing to their diverse pharmacological activities. Recently, with the development of modern nanotechnology, pnictogen group elements appear in a more innovative form, namely two-dimensional (2D) pnictogens (i.e. phosphorene, arsenene, and bismuthene) with a unique layered crystal structure and extraordinary optoelectronic characteristics, which endow them with significant superiority as a novel multifunctional photonic nanoplatform for cutting-edge precision treatment of various diseases. The puckered layer structure with ultralarge surface area make them ideal drug and gene delivery vectors that can avoid degradation and reduce target effects. The anisotropic morphology allows their easier internalization by cells and may improve gene transfection efficiency. Tunable optoelectronic characteristics endow them with excellent phototherapy performance as well as the ability to act as an optical switch to initiate subsequent therapeutic events. This review provides a brief overview of the properties, preparation and surface modifications of 2D pnictogens, and then focuses on its applications in cutting-edge precision treatment as a novel multifunctional photonic nanoplatform, such as phototherapy, photonic medicine, photo-adjuvant immunotherapy and photo-assisted gene therapy. Finally, the challenges and future development trends for 2D pnictogens are provided. With a focus on 2D pnictogen-based multifunctional photonic nanoplatforms, this review may also provide profound insights for the next generation innovative precision therapy.

Endosomal Escapable and Nuclear Localizing Cationic Polyaspartate-Based CRISPR Activation System for Preventing Respiratory Virus Infection by Specifically Inducing Interferon-λ.

Global pandemics caused by viruses cause widespread panic and economic losses. The lack of specific antivirals and vaccines increases the spreading of viral diseases worldwide. Thus, alternative strategies are required to manage viral outbreaks. Here, we develop a CRISPR activation (CRISPRa) system based on polymeric carriers to prevent respiratory virus infection in a mouse model. A polyaspartate grafted with 2-(diisopropylamino) ethylamine (DIP) and nuclear localization signal peptides (NLS-MTAS fusion peptide) was complexed with plasmid DNA (pDNA) encoding dCas9-VPR and sgRNA targeting IFN-λ. The pH-sensitive DIP and NLS-MTAS groups were favor of endo-lysosomal escape and nuclear localization of pDNA, respectively. They synergistically improved gene transfection efficiency, resulting in significant reporter gene expression and IFN-λ upregulation in lung tissue. In vitro and in vivo prophylactic experiments showed that the non-viral CRISPRa system could prevent infection caused by H1N1 viruses with minimal inflammatory responses, presenting a promising prophylactic approach against respiratory virus infections.

Development of a non-viral gene vector for enhancing gene transfection efficiency

Construction of a safe and high transfection efficiency non-viral gene delivery vector to improve the effectiveness of gene delivery is vital for cancer treatment. Herein, we used 1, 2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol to prepare cationic liposomes in combination with poly(aspartate-graft-PEI423) (PAE) and plasmid DNA to prepare Liposomes-poly(aspartate-graft-PEI423)/DNA (LP-PAE/DNA) to improve gene transfection efficiency. LP-PAE/DNA particle size was about 220 nm, the zeta potential was about 20 mV and the morphology was spherical. The LP-PAE/DNA lipopolyplexes showed higher transfection efficiency at a weight ratio of 10:1:1 in HeLa, A549 and SPC-A1 cell line. LP-PAE/DNA lipopolyplexes toxicity in different cell lines and the main endocytic pathways through which LP and LP-PAE mediate DNA into cells was examined. In addition, the results of hemolysis test, red blood cell morphology observation and blood routine experiments showed that LP-PAE/DNA would not cause systemic hematological toxicity. The histological analysis including hematoxylin-eosin (HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay confirmed lipopolyplexes/liposomes mediate the entry of therapeutic genes into the body not causing damage to normal organs and enhancing tumor tissue apoptosis relative to more traditional approaches. Therefore, it could be hoped the feasibility of LP-PAE as an efficient and promising platform for gene delivery.

Preparation and characterization of polyamidoamine dendrimers conjugated with cholesteryl-dipeptide as gene carriers in HeLa cells

Improving the transfection efficiency of non-viral carriers by using cationic polymers is a useful approach to addressing several challenges in gene therapy, such as cellular uptake, endosomal escape, and toxicity. Among the various cationic polymers, polyamidoamine (PAMAM) dendrimers have been widely utilized because of the abundance of terminal functional groups, thereby enabling further functionalization and enhancing DNA condensation and internalization into cells. The combination of various functional groups is required for these PAMAM dendrimer derivatives to function appropriately for gene delivery. Herein, we synthesized PAMAM G2-HRChol by conjugating dipeptide (histidine-arginine) and cholesterol at different ratios (6% or 23%) on the surface of PAMAM dendrimer generation 2 (PAMAM G2). Both PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% have buffering capacity, leading to improved endosomal escape after entering the cells. PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% dendrimers were condensed with pDNA to form nano-polyplexes at a weight ratio of 4 (polymer/pDNA). Polyplexes are positively charged, which facilitates cellular uptake. The transfection efficiency of PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% dendrimers was similar to that of PEI 25 kDa under optimum conditions, and the cytotoxicity was much lower than that of PEI 25 kDa in HeLa cells. In addition, after apoptin gene transfection was performed, cell death ratios of 34.47% and 22.47% were observed for PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23%, respectively. The results show that a suitable amount of cholesterol can improve gene transfection efficiency, and the PAMAM G2-HRChol 6% dendrimer could be a potential gene carrier in HeLa cells.

Type I collagen scaffold with WNT5A plasmid for in situ cartilage tissue engineering.

Cartilage tissue lacks the ability to heal. Cartilage tissue engineering using cell-free scaffolds has been increasingly used in recent years. This study describes the use of a type I collagen scaffold combined with WNT5A plasmid to promote chondrocyte proliferation and differentiation in a rabbit osteochondral defect model. Type I collagen was extracted and fabricated into a collagen scaffold. To improve gene transfection efficiency, a cationic chitosan derivative N,N,N-trimethyl chitosan chloride (TMC) vector was used. A solution of TMC/WNT5A complexes was adsorbed to the collagen scaffold to prepare a WNT5A scaffold. Osteochondral defects were created in the femoral condyles of rabbits. The rabbits were divided into defect, scaffold, and scaffold with WNT5A groups. At 6 and 12 weeks after creation of the osteochondral defects, samples were collected from all groups for macroscopic observation and gene expression analysis. Samples from the defect group exhibited incomplete cartilage repair, while those from the scaffold and scaffold with WNT5A groups exhibited "preliminary cartilage" covering the defect. Cartilage regeneration was superior in the scaffold with WNT5A group compared to the scaffold group. Safranin O staining revealed more proteoglycans in the scaffold and scaffold with WNT5A groups compared to the defect group. The expression levels of aggrecan, collagen type II, and SOX9 genes were significantly higher in the scaffold with WNT5A group compared to the other two groups. Type I collagen scaffold showed effective adsorption and guided the three-dimensional arrangement of stem cells. WNT5A plasmid promoted cartilage repair by stimulating the expression of aggrecan, type II collagen, and SOX9 genes and proteins, as well as inhibiting cartilage hypertrophy.

Ultrasound molecular imaging-guided tumor gene therapy through dual-targeted cationic microbubbles.

The success of gene therapy depends largely on the development of gene vectors and effective gene delivery systems. It has been demonstrated that cationic microbubbles can be loaded with negatively charged plasmid DNA and thus improve gene transfection efficiency. In this study, we developed dual-targeting cationic microbubbles conjugated with iRGD peptides(Cyclo(Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys)) and CCR2 (chemokine (C-C motif) receptor 2) antibodies (MBiRGD/CCR2) for ultrasound molecular imaging and targeted tumor gene therapy. The ultrasound molecular imaging experiments showed that there were significantly enhanced ultrasound molecular imaging signals in the tumor that received MBiRGD/CCR2, compared with those that received MBiRGD, MBCCR2, or MBcontrol. As a therapy plasmid, pGPU6/GFP/Neo-shAKT2, carrying an expression cassette for the human AKT2 RNA interference sequence, was used. Our results demonstrated that MBiRGD/CCR2 had a significantly higher gene transfection efficiency than MBiRGD, MBCCR2, or MBcontrol under ultrasound irradiation, resulting in much lower AKT2 protein expression and stronger tumor growth inhibition effects in vivo and in vitro. In conclusion, our study demonstrated a novel gene delivery system via MBiRGD/CCR2 for ultrasound molecular-imaging-guided gene therapy of breast cancer.

Development of A2G80 peptide-gene complex for targeted delivery to muscle cells

Therapeutic strategies based on antisense oligonucleotides and therapeutic genes are being extensively investigated for the treatment of hereditary muscle diseases and hold great promise. However, the cellular uptake of these polyanions to the muscle cells is inefficient. Therefore, it is necessary to develop more effective methods of gene delivery into the muscle tissue. The A2G80 peptide (VQLRNGFPYFSY) from the laminin α2 chain has high affinity for α-dystroglycan (α-DG) which is expressed on the membrane of muscle cells. In this study, we designed a peptide-modified A2G80 with oligoarginine and oligohistidine (A2G80-R9-H8), and prepared peptide/plasmid DNA (pDNA) complex, to develop an efficient gene delivery system for the muscle tissue. The peptide/pDNA complex showed α-DG-dependent cellular uptake of the A2G80 sequence and significantly improved gene transfection efficiency mediated by the oligohistidine sequence in C2C12 myoblast cells. Further, the peptide/pDNA complex promoted efficient and sustained gene expression in the Duchenne muscular dystrophy mouse models. The A2G80-R9-H8 peptide has the potential for use as a specific carrier for targeting muscle in gene therapy in muscular dystrophy.

Dual-Location Dual-Acid/Glutathione-Degradable Cationic Micelleplexes through Hydrophobic Modification for Enhanced Gene Silencing.

Gene therapy holds great promise for the treatment of acquired genetic disorders such as cancer with reduced side effects compared to chemotherapy. For gene therapy to be successful, it is crucial to develop efficient and nontoxic gene carriers to overcome the poor in vivo stability and low cellular uptake of nucleic acid-based therapeutic agents. Here, we report a new and versatile approach exploring a combination of hydrophobic modifications and dual-stimuli-responsive degradation (SRD) for controlled gene delivery with amphiphilic block copolymer-based nanocarriers. The block copolymer, synthesized by atom transfer radical polymerization, is designed with an acid-labile acetal linkage at the block junction and a pendant disulfide group in the hydrophobic block. The incorporation of labile linkages enables both disulfide-core-cross-linking and dual-location dual-acid/reduction-responsive degradation (DL-DSRD). Furthermore, the disulfide linkages integrated as hydrophobic moieties facilitate the nucleic acids to condense into nanometer-sized micelleplexes through electrostatic interactions of pendant dimethylamino groups with the anionic phosphate groups of the nucleic acids. Our preliminary results demonstrate that the DL-DSRD approach through hydrophobic modification is a robust platform in the development of gene delivery systems with enhanced colloidal stability, reduced cytotoxicity, and improved gene transfection efficiency.

Fabrication of aminated poly(glycidyl methacrylate)-based polymers for co-delivery of anticancer drugs and the p53 gene.

Aminated poly(glycidyl methacrylate)s-based polymers for gene delivery not only can reduce toxicity and improve solubility, but can improve gene transfection efficiency and reduce protein aggregation. In this study, we first prepared poly(glycidyl methacrylate) (PGMA) via reversible addition-fragmentation chain transfer (RAFT) polymerization, and then the obtained PGMA homopolymer was post-modified with ethanol amine (EA), 1-amino-2-propanol (AP), 3-(dibutylamino)propylamine (DA) and N-(2-hydroxyethyl)ethylenediamine (HA), respectively, to yield four kinds of PGMA-based gene vectors containing hydroxyl groups (abbreviated as PGEA, PGAP, PGDA and PGHA). The effects of the different side chains and hydroxyl groups on the biological properties of these four cationic polymers were investigated. We found that the transfection efficiency of the PGHA/p53 complex was higher than those of the other three polymer/gene complexes through MTT assay and laser scanning confocal microscopy. Hence, we chose HA for further post-modification to fabricate a cationic copolymer, PCL-ss-P(PEGMA-co-GHA) (abbreviated as PGHAP), via a combination of ring opening polymerization (ROP) and RAFT copolymerization. The PCL-ss-P(PEGMA-co-GHA) amphiphilic copolymer could self-assemble into nanoparticles, which could be used to encapsulate anticancer drug doxorubicin (DOX) and compress the p53 gene to form the DOX-loaded PCL-ss-P(PEGMA-co-GHA)/p53 complex (abbreviated as DPGHAP/p53). The gel retardation assay showed that p53 gene could be well immobilized and remained stable under the electronegative conditions. MTT assay showed that the DPGHAP/p53 complex had a significant antitumor effect on A549 cells and H1299 cells compared with free DOX or/and p53 gene therapy alone. Furthermore, the test results from live cell imaging systems revealed that the DPGHAP/p53 complexes could effectively deliver DOX and the p53 gene into A549 cells. Therefore, the constructed cationic polymer PCL-ss-P(PEGMA-co-GHA) has potential application prospects as a co-vector of anticancer drugs and genes.

Biosynthetic nanobubbles for targeted gene delivery by focused ultrasound.

Ultrasound-targeted microbubble destruction (UTMD) has recently drawn considerable attention in biomedicine applications due to its great potential to locally enhance gene delivery. However, conventional microbubbles have a microscale particle size and polydisperse particle size distribution, which makes it difficult for them to directly come into contact with tumor cells and to efficiently deliver therapeutic genes via ultrasound cavitation effects. In the current study, we developed a kind of novel cationic biosynthetic nanobubble (CBNB) as an ultrasonic gene delivery carrier through coating PEI on the surface of these biosynthetic nanobubbles (BNBs). The BNBs, produced from an extremely halophilic archaeon (Halobacterium NRC-1), possess a nanoscale size and can produce stable contrast signals both in vitro and in vivo. Surface modification with PEI polymer greatly increased the DNA loading capability of BNBs, leading to significantly improved gene transfection efficiency when combining with ultrasound. To our knowledge, this is the first report to apply biosynthetic bubbles as non-viral gene carriers which can effectively deliver genes into tumor cells with the aid of ultrasound cavitation. Our study provides a powerful tool for image-guided and efficient gene delivery using biosynthetic nanoscale contrast agents.

Preparation of gene drug delivery systems of cationic peptide lipid with 0G-PAMAM as hydrophilic end and its biological properties evaluation.

As an efficient gene delivery, non-viral vectors should have high transfection efficiency, excellent endosomal escape, low cytotoxicity, and the ability to rapidly release the gene into the cytoplasm.Cationic liposome have been widely used as efficient gene carriers, but the cytotoxicity, rapid degradation and low cellular uptake are major drawback impeding its further appolication. Herein, with double lauric acid as hydrophobic chains, tartaric acid as skeleton, 0 generation PAMAM modified with lysine as hydrophilic head, a new type cationic peptide lipid was synthetised. The alkyl chain promote lipid across cell membranes and with membrane fusion, 0 generation PAMAM modified with lysine hydrophilic end amino can contain a large number of protons which can change into ammonium and combine with the DNA negatively charge phosphate groups. It is expected that this carrier has low toxicity, high transfection efficiency and targeting property. By adjusting the cationic liposome/gene weight ratio, the transfection system was optimized to improved gene transfection efficiency, reduce cytotoxicity, and increase property and stability, etc.

Asymmetric polyplex-nanocapsules loaded with photosentisizer for light-assisted gene transfer

Inefficient intracellular gene delivery is still a limitation for the clinical translation of gene therapy. Recently, photochemical internalization (PCI) has emerged with the opportunity to overcome endo-lysosomal sequestration in gene delivery, which utilizes photosensitizer (PS) plus light generating reactive oxygen species (ROS) at sub-lethal level to facilitate intracellularly targeted drug delivery. In this work, asymmetric polyplex-nanocapsules were prepared based on the triblock copolymers of PEG-PCL-PEI by using the simple solvent-injection method. Subsequently, the hydrophobic PS was encapsulated in the hydrophobic layer of polyplex-nanocapsules through hydrophobic interaction. The results from agarose gel electrophoresis and fluorescence scanning spectroscopy show that DNA could be condensed effectively and the PS was encapsulated, resulting in the stable polyplex-nanocapsules. The obtained polyplex-nanocapsules were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements with the average size ranging from 200 to 280nm and the negatively charged surface. Importantly, these polyplex-nanocapsules can be uptaken by Hela cells, resulting in improved gene transfection efficiency in comparison with the case without laser treatment due to the assistance of PCI effect. This work demonstrates a promising strategy to build the light-assisted gene delivery system containing PS and transporting genes simultaneously in one platform.

Synthesis of a Cationic Supramolecular Block Copolymer with Covalent and Noncovalent Polymer Blocks for Gene Delivery.

The design and fabrication of safe and highly efficient nonviral vectors is the key scientific issue for the achievement of clinical gene therapy. Supramolecular cationic polymers have unique structures and specific functions compared to covalent cationic polymers, such as low cytotoxicity, excellent biodegradability, and smart environmental responsiveness, thereby showing great application prospect for gene therapy. However, supramolecular gene vectors are facile to be degraded under physiological conditions, leading to a significant reduction of gene transfection efficiency. In order to achieve highly efficient gene expression, it is necessary for supramolecular gene vectors being provided with appropriate biostability to overcome various cell obstacles. To this end, a novel cationic supramolecular block copolymer composed of a conventional polymer and a noncovalent polymer was constructed through robust β-cyclodextrin/ferrocene host-guest recognition. The resultant supramolecular block copolymer perfectly combines the advantages of both conventional polymers and supramolecular polymers ranging from structures to functions. This supramolecular copolymer not only has the ability to effectively condense pDNA for enhanced cell uptake, but also releases pDNA inside cancer cells triggered by H2O2, which can be utilized as a prospective nonviral delivery vehicle for gene delivery. The block polymer exhibited low cytotoxicity, good biostability, excellent biodegradability, and intelligent responsiveness, ascribing to the dynamic/reversible nature of noncovalent linkages. In vitro studies further illustrated that the supramolecular block polymer exhibited greatly improved gene transfection efficiency in cancer cells. This work offers an alternative platform for the exploitation of smart nonviral vehicles for specific cancer gene therapy in the future.

SRL-Coated PAMAM Dendrimer Nano-Carrier for Targeted Gene Delivery to the Glioma Cells and Competitive Inhibition by Lactoferrin.

Glioma, as a primary tumor of central nervous system, is the main cause of death in patients with brain cancer. Therefore, development of an efficient strategy for treatment of glioma is worthy. The aim of the current study was to develop a SRL peptide-coated dendrimer as a novel dual gene delivery system for targeting the LRP receptor, an up-regulated gene in both BBB and glioma cells. To perform this investigation, our newly developed nanocarrier (PAMAM-PEG-SRL) was used for gene delivery to C6 glioma cell lines. DNA (GFP) was loaded in these functionalized nanoparticles and their cellular uptake/distribution and gene transfection efficacy was evaluated by fluorescence and confocal microscopy. In vitro studies showed that SRL-modified nanoparticles have good transfection efficacy. Results revealed improved gene transfection efficiency of newly-synthesized delivery system. We also found that lactoferrin, as a LRP ligand, reduced the gene transfection efficacy of the delivery system due to its higher affinity compared to SRL peptides (Competitive inhibition). The present results suggest that the synthesized delivery system has the potential to be used as an alternative targeted drug delivery system for brain tumors.

Efficient delivery of C/EBP beta gene into human mesenchymal stem cells via polyethylenimine-coated gold nanoparticles enhances adipogenic differentiation.

The controlled differentiation of stem cells via the delivery of specific genes encoding appropriate differentiation factors may provide useful models for regenerative medicine and aid in developing therapies for human patients. However, the majority of non-viral vectors are not efficient enough to manipulate difficult-to-transfect adult human stem cells in vitro. Herein, we report the first use of 25 kDa branched polyethylenimine-entrapped gold nanoparticles (AuPEINPs) and covalently bound polyethylenimine-gold nanoparticles (AuMUAPEINPs) as carriers for efficient gene delivery into human mesenchymal stem cells (hMSCs). We determined a functional application of these nanoparticles by transfecting hMSCs with the C/EBP beta gene, fused to EGFP, to induce adipogenic differentiation. Transfection efficacy with AuPEINPs and AuMUAPEINPs was 52.3% and 40.7%, respectively, which was 2.48 and 1.93 times higher than that by using Lipofectamine 2000. Luciferase assay results also demonstrated improved gene transfection efficiency of AuPEINPs/AuMUAPEINPs over Lipofectamine 2000 and polyethylenimine. Overexpression of exogenous C/EBP beta significantly enhanced adipogenesis in hMSCs as indicated by both of Oil Red O staining and mRNA expression analyses. Nanoparticle/DNA complexes exhibited favorable cytocompatibility in hMSCs. Taken together, AuPEINPs and AuMUAPEINPs potentially represent safe and highly efficient vehicles for gene delivery to control hMSC differentiation and for therapeutic gene delivery applications.

Achieving high gene delivery performance with caveolae-mediated endocytosis pathway by (l)-arginine/(l)-histidine co-modified cationic gene carriers

Developing new amphiphilic polymers with natural product moieties has been regarded as a promising way to achieve biocompatibility and certain biological functions. In prior work, we developed some natural (l)-arginine modified cationic polymers (PAHMAA-Rs) as cationic gene carriers. For the sake of continuing optimize the gene delivery performance, herein, a new series of (l)-arginine and (l)-histidine co-modified cationic poly (ω-aminohexyl methacrylamide)s (PAHMAA-R-H) were synthesized and characterized with 1H NMR, GPC-SLS and FT-IR. Their proton buffering capacities were studied by acid-base titration assay. pDNA binding affinity and self-assembly properties of the polyplexes were analyzed by agarose gel retardation assay, DLS and AFM, respectively. In vitro cytotoxicity of the PAHMAA-R-H was determined by MTT and LDH assays in H1299 cells, the gene transfection efficacy and intracellular uptake capability were evaluated by luciferase assay and FACS, respectively. Moreover, the endocytosis pathways and intracellular distribution of the polyplexes were investigated by using specific endocytic inhibitors and fluorescent co-localization techniques. The results demonstrated that co-modification of (l)-arginine and (l)-histidine onto the PAHMAA polymer could enhance proton buffering capacity, shield surface charge, decrease cytotoxicity, and improve gene transfection efficiency and serum-compatibility. Moreover, the gene transfection and intracellular uptake behaviors were disclosed strongly rely on the (l)-arginine/(l)-histidine modification ratios. The polyplexes tend to be internalized through caveolae-mediated endocytosis gateway and localized with endosomes/lysosomes in H1299 cells. Notably, among the polymers, the PAHMAA-R18-H6 exhibited remarkable gene delivery efficiency and serum compatibility, which made it promising gene transfection agent for practical application.