FGF receptor-mediated gene delivery using ligands coupled to polyethylenimine

To obtain new nonviral vectors with high gene delivery efficiency and special cell targeting ability, an attractive strategy is to link ligands to polyethylenimine (PEI). Fibroblast growth factor receptors (FGFRs) are highly expressed on a variety of human cancer cells and are potential targets for cancer gene therapy. In this study, the peptides NH2-Met-Gln-Leu-Pro-Leu-Ala-ThrGly-Gly-Gly-Cys-COOH (MC11) which have been proved to combine specially with the FGFR on cell membrane are coupled to PEI using N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP) as a linker with different molar ratios (1 : 0.3, 1 : 0.75, 1 : 1.5, and 1 : 3.0) and the new polymer PEI-MC11 is verified by a series of physicochemical methods including 1H-NMR and FTIR. The agarose gel electrophoresis assay, particle size test, zeta potential test, and electron microscope observation show that PEI-MC11 can efficiently condense plasmid DNA into nanoparticles with about 200 nm in diameter and with positive surface charge at the suitable N/P ratio. The MTT assay suggests the decreased toxicity of the polymers. The results of the gene delivery efficiency in vitro show that PEI-MC11/pDNA polyplexes have significantly greater transgene activity than PEI/pDNA in COS-7 and HepG2 cells which express FGFR positively, while no such effect is observed in PC3 cells which have negative FGFR. The enhanced gene delivery efficiency of PEI-MC11 can be blocked by the co-culture of free peptides MC11 before the gene delivery procedure. The synthesized nonviral vector based on PEI with the targeting peptides MC11 for binding FGFR has improved efficiency of gene delivery and targeting specificity in FGFR positive cells. It may have potential application in cancer gene therapy.     

Thyroid hormone (T3)-modification of polyethyleneglycol (PEG)-polyethyleneimine (PEI) graft copolymers for improved gene delivery to hepatocytes

Targeting of gene vectors to liver hepatocytes could offer the opportunity to cure various acquired and inherited diseases. Efficient gene delivery to the liver parenchyma has been obscured from efficient targeting of hepatocytes. Here we show that the thyroid hormone, triiodothyronine (T3), can be used to improve the gene transfer efficiency of nonviral gene vectors to hepatocytes in vitro and to the liver of mice in vivo. T3 conjugated to the distal ends of fluorescent labeled PEG-g-dextran resulted in T3-specific cellular endosomal uptake into the hepatocellular cell line HepG2. PEG-g-PEI graft copolymers with increasing molar PEG-ratios were synthesized, complexed with plasmid DNA, and transfected into HepG2 or HeLa cells. Gene transfer efficiency decreased as the number of PEG blocks increased. T3 conjugation to PEI and the distal ends of PEG blocks resulted in T3 specific gene transfer in HepG2 cells as evidenced by reduction of gene transfer efficiency after pre-incubation of cells with excess of T3. In vivo application of T3-PEG-g-PEI based gene vectors in mice after tail vein injection resulted in a significantly 7-fold increase of gene expression in the liver compared with PEG-g-PEI based gene vectors.     

Characterization of a multifunctional PEG-based gene delivery system containing nuclear localization signals and endosomal escape peptides

Endosomal escape and nuclear localization are two barriers to gene delivery that need to be addressed in the design of new nonviral gene delivery vehicles. We have previously synthesized low-toxicity polyethylene glycol (PEG)-based vehicles with endosomal escape functionalities, but it was determined that the transfection efficiency of PEG-based vehicles that escaped the endosome was still limited by poor nuclear localization. Two different nuclear localization signal (NLS) peptides, SV40 and TAT, were coupled to PEG-based vehicles with DNA-binding peptides (DBPs) to determine the effect of NLS peptides on the transfection efficiency of PEG-based gene delivery vehicles. Coupling one SV40 peptide, a classical NLS, or two TAT peptides, a nonclassical NLS, to PEG-DBP vehicles increased the transfection efficiency of PEG-DBP/DNA particles 15-fold and resulted in similar efficiency to that of a common cationic polymer vehicle, polyethylenimine (PEI). The transfection efficiency of both types of PEG-DBP-NLS particles was further increased 7-fold in the presence of chloroquine, suggesting that the transfection efficiency of PEG-DBP-NLS particles is limited by their ability to escape the endosome. To develop particles that could escape the endosome and target the nucleus, a mixture of PEG-DBP-NLS vehicles and PEG-based vehicles with DBPs and endosomal escape peptides were complexed with plasmid DNA to form multifunctional particles that had a transfection efficiency 2-3 times higher than that of PEI. Additionally, the PEG-based vehicles were less toxic and more resistant to nonspecific protein adsorption than PEI, making them an attractive alternative for nonviral gene delivery.     

A novel transfecting peptide comprising a tetrameric nuclear localization sequence

The transport of exogenous DNA into the nucleus of eukaryotic cells is a prerequisite for successful gene delivery. To favor nuclear transport we synthesized a tetramer of the nuclear localization signal (NLS) of the SV40 large T-antigen as a novel nonviral gene delivery vector. This 4.4-kDa lysine-rich peptide (NLSV404) binds and compacts DNA by electrostatic interaction and forms stable polyplexes. Apart from its sequence-specific potency to mediate nuclear accumulation of conjugated albumin, NLSV404 also displays properties of nuclear transport for plasmid DNA as confirmed by fluorescence in situ hybridization. Further, NLSV404 polyplexes are shown to efficiently transfect various cell lines such as 16HBE14o–, HeLa S6, and Cos7 cells. NLSV404 polyplexes displayed at least 20-fold higher transfection rates than analogous polyplexes formed by the nuclear transport-deficient mutant sequence cNLS. Using growth-arrested cells, NLSV404 complexes were at least 100-fold more efficient than cNLS complexes. Combination of NLSV404 peptide but not of cNLS peptide with preformed polyethylenimine and dendrimer DNA complexes resulted in a strong increase in transfection efficiency. Incubation of cells prior to transfection with NLSV404 polyplexes with excess free peptide NLSV404 but not with cNLS resulted in a dose-dependent dramatic decrease in the transfection rate, suggesting a sequence-specific competitive inhibition. These results indicate that NLSV404 mediates nuclear accumulation of transfected plasmid DNA and that it can be a highly useful component of nonviral gene vectors.Abbreviations BSA-BODIPY Fluorescence-labeled bovine serum albumin - EGFP Enhanced green fluorescent protein - FISH Fluorescent in situ hybridization - NLS Nuclear localization signal - PEI Polyethylenimine - SV40 Simian virus 40     

Tumor-Targeted Gene Transfer with DNA Polyplexes

Systemic gene delivery systems are needed for therapeutic applications; in some situations, target cells might be spread throughout the organism, as in the case of cancer metastases, which can be reached only via the systemic route. Within the class of nonviral vectors, polymer-based transfection particles named DNA polyplexes and lipid-based systems named DNA lipoplexes are being developed for this purpose. For systemic circulation, masking the surface charge of DNA complexes has to be accomplished to avoid interactions with plasma components, erythrocytes, and the reticuloendothelial system. Among other vector formulations, polyplexes based on polyethylenimine (PEI), shielded with polyethylene glycol (PEG), and linked to the receptor binding ligands transferrin (Tf) or epidermal growth factor (EGF) have been developed. Complexes were found to mediate efficient gene transfer into tumor cell lines in a receptor-dependent and cell-cycle-dependent manner. Systemic administration of surface-shielded Tf-PEI polyplexes into the tail vein of mice resulted in preferential gene delivery into distantly growing subcutaneous tumors. In contrast, application of positively charged PEI polyplexes directed gene transfer primarily to the lung.     

Gene delivery to differentiated neurotypic cells with RGD and HIV Tat peptide functionalized polymeric nanoparticles

A number of neurodegenerative disorders may potentially be treated by the delivery of therapeutic genes to neurons. Nonviral gene delivery systems, however, typically provide low transfection efficiency in post-mitotic differentiated neurons. To uncover mechanistic reasons for this observation, we compared gene transfer to undifferentiated and differentiated SH-SY5Y cells using polyethylenimine (PEI)/DNA nanocomplexes. Differentiated cells exhibited substantially lower uptake of gene vectors. To overcome this bottleneck, RGD or HIV-1 Tat peptides were attached to PEI/DNA nanocomplexes via poly(ethylene glycol) (PEG) spacer molecules. Both RGD and Tat improved the cellular uptake of gene vectors and enhanced gene transfection efficiency of primary neurons up to 14-fold. RGD functionalization resulted in a statistically significant increase in vector escape from endosomes, suggesting it may improve gene delivery by more than one mechanism.     

Cationic star polymers consisting of alpha-cyclodextrin core and oligoethylenimine arms as nonviral gene delivery vectors

A series of novel cationic star polymers were synthesized by conjugating multiple oligoethylenimine (OEI) arms onto an alpha-cyclodextrin (alpha-CD) core as nonviral gene delivery vectors. The molecular structures of the alpha-CD-OEI star polymers, which contained linear or branched OEI arms with different chain lengths ranging from 1 to 14 ethylenimine units, were characterized by using size exclusion chromatography, 13C and 1H NMR, and elemental analysis. The alpha-CD-OEI star polymers were studied in terms of their DNA binding capability, formation of nanoparticles with plasmid DNA (pDNA), cytotoxicity, and gene transfection in cultured cells. All the alpha-CD-OEI star polymers could inhibit the migration of pDNA on agarose gel through formation of complexes with pDNA, and the complexes formed nanoparticles with sizes ranging from 100 to 200 nm at N/P ratios of 8 or higher. The star polymers displayed much lower in vitro cytotoxicity than that of branched polyethylenimine (PEI) of molecular weight 25K. The alpha-CD-OEI star polymers showed excellent gene transfection efficiency in HEK293 and Cos7 cells. Generally, the transfection efficiency increased with an increase in the OEI arm length. The star polymers with longer and branched OEI arms showed higher transfection efficiency. The best one of the star polymers for gene delivery showed excellent in vitro transfection efficiency that was comparable to or even higher than that of branched PEI (25K). The novel alpha-CD-OEI star polymers with OEI arms of different chain lengths and chain architectures can be promising new nonviral gene delivery vectors with low cytotoxicity and high gene transfection efficiency for future gene therapy applications.     

Efficient transfection method using deacylated polyethylenimine-coated magnetic nanoparticles

Low efficiencies of nonviral gene vectors, such as transfection reagent, limit their utility in gene therapy. To overcome this disadvantage, we report on the preparation and properties of magnetic nanoparticles [diameter (d) = 121.32 ± 27.36 nm] positively charged by cationic polymer deacylated polyethylenimine (PEI max), which boosts gene delivery efficiency compare with polyethylenimine (PEI), and their use for the forced expression of plasmid delivery by application of a magnetic field. Magnetic nanoparticles were coated with PEI max, which enabled their electrostatic interaction with negatively charged molecules such as plasmid. We successfully transfected 81.1 ± 4.0% of the cells using PEI max-coated magnetic nanoparticles (PEI max-nanoparticles). Along with their superior properties as a DNA delivery vehicle, PEI max-nanoparticles offer to deliver various DNA formulations in addition to traditional methods. Furthermore, efficiency of the gene transfer was not inhibited in the presence of serum in the cells. PEI max-nanoparticles may be a promising gene carrier that has high transfection efficiency as well as low cytotoxicity.     

Arginine-rich polyplexes for gene delivery to neuronal cells

Neuronal gene therapy potentially offers an effective therapeutic intervention to cure or slow the progression of neurological diseases. However, neuronal cells are difficult to transfect with nonviral vectors, and in vivo their transport across the blood–brain barrier (BBB) is inefficient. We synthesized a series of arginine-rich oligopeptides, grafted with polyethyleneimine (PEI) and modified with a short-chain polyethylene glycol (PEG). We hypothesized that the arginine would enhance cellular uptake and transport of these polyplexes across the BBB, with PEG imparting biocompatibility and “stealth” properties and PEI facilitating DNA condensation and gene transfection. The optimized composition of the polyplexes demonstrated hemocompatibility with red blood cells, causing no lysis or aggregation, and showed significantly better cytocompatibility than PEI in vitro. Polyplexes formulated with luciferase-expressing plasmid DNA could transfect rat primary astrocytes and neurons in vitro. Confocal imaging data showed efficient cellular uptake of DNA and its sustained intracellular retention and nuclear localization with polyplexes. Intravenous administration of the optimized polyplexes in mice led to gene expression in the brain, which upon further immunohistochemical analysis demonstrated gene expression in neurons. In conclusion, we have successfully designed a nonviral vector for in vitro and in vivo neuronal gene delivery.     

Dual-targeting non-viral vector based on polyethylenimine improves gene transfer efficiency

Polyethylenimine (PEI) is the polymer most commonly used for transferring plasmids into eukaryotes, but its gene-transfer efficiency is lower compared to viral vectors. Receptors targeting PEI combined with ligands can enhance efficiency of gene transfer into the corresponding receptor-positive cells. Using the double-receptor-mediated pathway of viral infection, in this study we synthesized a novel non-viral vector based on PEI combined with two peptides recognizing FGF receptors (peptide YC25) and integrins (peptide CP9) on the cell surface. The dual targeting vector showed a physicochemical character similar to that of PEI, such as pDNA formation, particle size, zeta potential and lower toxicity. In vitro gene transfer showed that the dual-receptor targeted vector (YC25-PEI-CP9) exhibited a markedly higher transgene efficiency in cell lines with positive expression of FGF receptors and integrins, compared with single-peptide-modified PEI or unmodified PEI. In the cells with only integrin-positive expression, YC25-PEI-CP9 mediated a higher transgene expression than PEI but lower than CP9-PEI. The corresponding free peptides could inhibit the transgene efficiency of the peptide-coupled PEI. In vivo gene transfer in tumor-bearing nude mice also demonstrated that the dual-targeting vectors showed a significantly enhanced transfection efficiency in tumors with positive expression of FGF receptors and integrins. The synthesized polymer YC25-PEI-CP9 has the prospect to act as a novel kind of non-viral vector in gene therapy.     

Gene delivery of PEI incorporating with functional block copolymer via non-covalent assembly strategy

A novel functional diblock polymer P(PEGMA-b-MAH) is prepared and incorporated to improve the gene delivery efficiency of poly(ethyleneimine) PEI via non-covalent assembly strategy. First, P(PEGMA-b-MAH) is prepared from l-methacrylamidohistidine methyl ester (MAH) by reversible addition fragmentation chain transfer polymerization, with poly[poly(ethylene glycol) methyl ether methacrylate] (P(PEGMA)) as the macroinitiator. Then P(PEGMA-b-MAH) is assembled with plasmid DNA (pDNA) and PEI (M_w = 10 kDa) to form PEI/P(PEGMA-b-MAH)/pDNA ternary complexes. The agarose gel retardation assay shows that the presence of P(PEGMA-b-MAH) does not interfere with DNA condensation by the PEI. Dynamic light scattering tests show that PEI/P(PEGMA-b-MAH)/pDNA ternary complexes have excellent serum stability. In vitro transfection indicates that, compared to the P(PEGMA-b-MAH) free PEI-25k/pDNA binary complexes, PEI-10k/P(PEGMA-b-MAH)/pDNA ternary complexes have lower cytotoxicity and higher gene transfection efficiency, especially under serum conditions. The ternary complexes proposed here can inspire a new strategy for the development of gene and drug delivery vectors.     

Dual-targeting non-viral vector based on polyethylenimine improves gene transfer efficiency

Polyethylenimine (PEI) is the polymer most commonly used for transferring plasmids into eukaryotes, but its gene-transfer efficiency is lower compared to viral vectors. Receptors targeting PEI combined with ligands can enhance efficiency of gene transfer into the corresponding receptor-positive cells. Using the double-receptor-mediated pathway of viral infection, in this study we synthesized a novel non-viral vector based on PEI combined with two peptides recognizing FGF receptors (peptide YC25) and integrins (peptide CP9) on the cell surface. The dual targeting vector showed a physicochemical character similar to that of PEI, such as pDNA formation, particle size, zeta potential and lower toxicity. In vitro gene transfer showed that the dual-receptor targeted vector (YC25-PEI-CP9) exhibited a markedly higher transgene efficiency in cell lines with positive expression of FGF receptors and integrins, compared with single-peptide-modified PEI or unmodified PEI. In the cells with only integrin-positive expression, YC25-PEI-CP9 mediated a higher transgene expression than PEI but lower than CP9-PEI. The corresponding free peptides could inhibit the transgene efficiency of the peptide-coupled PEI. In vivo gene transfer in tumor-bearing nude mice also demonstrated that the dual-targeting vectors showed a significantly enhanced transfection efficiency in tumors with positive expression of FGF receptors and integrins. The synthesized polymer YC25-PEI-CP9 has the prospect to act as a novel kind of non-viral vector in gene therapy.     

Long-term efficient gene delivery using polyethylenimine with modified Tat peptide

Polyethylenimine (PEI), a cationic polymer, has been widely studied and shown great promise as an efficient gene delivery vehicle. Likewise, the HIV-1 Tat peptide, a cell-permeable peptide, has been successfully used for intracellular gene delivery. To improve the favorable properties of these two vectors, we combine PEI with the modified Tat peptide sequence bearing histidine and cysteine residues (mTat). In vitro mTat/PEI-mediated transfection was evaluated by luciferase expression plasmid in two cell types. mTat/PEI produced significant improvement (≈5-fold) in transfection efficiency of both cell lines with little cytotoxicity when compared to mTat alone, PEI alone, or four commercial reagents. The particle size of mTat/PEI/DNA complex was significantly smaller than mTat or PEI alone, and it was correlated with higher transfection efficiency. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI transfection. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not. This suggested caveolae-mediated endocytosis as the transfection mechanism. Furthermore, the results of in vivo studies showed that animals administered mTat/PEI/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, PEI/DNA, or DNA alone, without any associated toxicity. The combination of mTat with PEI could significantly improve transfection efficiency, expanding the potential use as a non-viral gene vector both in vitro and in vivo.     

Cellular uptake pathways of lipid-modified cationic polymers in gene delivery to primary cells

Hydrophobic modifications have emerged as a promising approach to improve the efficiency of non-viral gene delivery vectors (GDV). Functional GDVs from non-toxic polymers have been created with this approach but the mechanism(s) behind lipid-mediated enhancement in transfection remains to be clarified. Using a linoleic acid-substituted 2?kDa polyethylenimine (PEI2LA), we aimed to define the cellular uptake pathways and intracellular trafficking of plasmid DNA in normal human foreskin fibroblast cells. Several pharmacological compounds were applied to selectively inhibit uptake by clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis (CvME) and macropinocytosis. We found that PEI2LA complexes were taken up predominantly through CME, and to a lesser extent by CvME. In contrast, its precursor molecule, PEI2 complexes was internalized primarily by CvME and macropinocytosis. The commonly used 25?kDa PEI 25 complexes utilized all endocytic pathways, suggesting its efficiency is derived from a different set of transfection pathways than PEI2LA. We further applied several endosome disruptive agents and found that hypertonic media enhanced the transfection of PEI2LA by 6.5-fold. We infer that lipid substitution changes the normal uptake pathways significantly and transfection with hydrophobically modified GDVs may be further enhanced by incorporating endosome disruptive elements into vector design.     

Characterization of lactoferrin as a targeting ligand for nonviral gene delivery to airway epithelial cells

In this study lactoferrin (Lf) was investigated as a targeting ligand for receptor-mediated gene delivery to human bronchial epithelial cells. A high number of lactoferrin receptors (LfRs) were detected on bronchial epithelial (BEAS-2B), but not on alveolar epithelial (A549) cells by fluorescence microscopy and FACS measurements, suggesting potential targeting selectivity for bronchial epithelial cells. Molecular conjugates with ratios of Lf to branched polyethylenimine 25 kDa (PEI) ranging from 4:1 to 1:40 (mol/mol) were synthesized and analyzed for complexation of plasmid DNA (pDNA), transfection efficiency, and cytotoxicity. Whereas particle size increased with the degree of Lf coupling from 45 to 225 nm, surface charge was not significantly influenced. Transfection studies on BEAS-2B cells revealed that Lf-PEI 1:20 exhibited the highest luciferase gene expression which was 5-fold higher at an N/P ratio (molar ratio of PEI nitrogen to pDNA phosphate) of 4 than PEI and could be inhibited by an excess of free Lf. With A549 cells, no significant enhancement in transfection efficiency between Lf-PEI/pDNA and PEI/pDNA complexes could be observed. Increasing the degree of Lf coupling to PEI resulted in reduced transfection efficiency in both alveolar and bronchial epithelial cells. Cell viability assays resulted in significantly lower cellular toxicity of Lf-PEI/pDNA compared with PEI/pDNA complexes. We suggest that Lf represents a potent targeting ligand for receptor-mediated gene delivery to bronchial epithelial cells and might be a promising candidate for lung gene transfer in vivo.     

Arginine functionalized peptide dendrimers as potential gene delivery vehicles

The quest for highly efficient and safe gene delivery systems has become the key factor for successful application of gene therapy. Peptide dendrimers are currently investigated as excellent candidates for non-viral gene delivery vectors. In this study, we report the synthesis and characterization of arginine functionalized peptide dendrimer-based vectors ranging from 5th generation (G5A) to 6th generation (G6A) via click chemistry, and their use for gene transfection in vitro and in vivo. The dendrimers can condense plasmid DNA (pDNA) and protect pDNAs from nuclease digestion. Both atomic force microscopy (AFM) and dynamic light scattering (DLS) revealed that the sizes of dendrimer/DNA particles were within 180-250 nm range. In vitro studies showed that the functionalized peptide dendrimers provided serum independent and high transfection efficiency on all studied cells, as over 2 fold higher than that of branched polyetherimide (PEI) in the presence of serum. Dendrimer G5A with molecular weight of 17 kDa demonstrated 6-fold transfection activity than PEI in breast tumor models, as well as good biosafety proved by in vitro and in vivo toxicity evaluation. However, G6A with molecular weight of 46 kDa showed much higher cytotoxicity. The functionalized dendrimer G5A with optimal generation may be therefore a potential candidate for gene delivery vehicle.     

Maximizing gene delivery efficiencies of cationic helical polypeptides via balanced membrane penetration and cellular targeting

The application of non-viral gene delivery vectors is often accompanied with the poor correlation between transfection efficiency and the safety profiles of vectors. Vectors with high transfection efficiencies often suffer from high toxicities, making it unlikely to improve their efficiencies by increasing the DNA dosage. In the current study, we developed a ternary complex system which consisted of a highly membrane-active cationic helical polypeptide (PVBLG-8), a low-toxic, membrane-inactive cationic helical polypeptide (PVBLG-7) capable of mediating mannose receptor targeting, and DNA. The PVBLG-7 moiety notably enhanced the cellular uptake and transfection efficiency of PVBLG-8 in a variety of mannose receptor-expressing cell types (HeLa, COS-7, and Raw 264.7), while it did not compromise the membrane permeability of PVBLG-8 or bring additional cytotoxicities. Because of the simplicity and adjustability of the self-assembly approach, optimal formulations of the ternary complexes with a proper balance between membrane activity and targeting capability were easily identified in each specific cell type. The optimal ternary complexes displayed desired cell tolerability and markedly outperformed the PVBLG-8/DNA binary complexes as well as commercial reagent Lipofectamine™ 2000 in terms of transfection efficiency. This study therefore provides an effective and facile strategy to overcome the efficiency-toxicity poor correlation of non-viral vectors, which contributes insights into the design strategy of effective and safe non-viral gene delivery vectors.     

Enhanced gene delivery to PC12 cells by a cationic polypeptide

Targeted gene delivery to diseased subtypes of neurons will be beneficial to the success of gene therapy of neurological disorders. We designed a recombinant cationic polypeptide to facilitate gene delivery to neuronal-like PC12 cells that express the nerve growth factor (NGF) receptors. The recombinant polypeptide was composed of a targeting moiety derived from loop 4-containing hairpin motif of NGF and a DNA-binding moiety of 10-lysine sequence and expressed in Escherichia coli. It activated NGF receptor, TrkA and its downstream signaling pathways in PC12 and promoted the survival of neuronally differentiated PC12 cells deprived of serum. The polypeptide could also bind plasmid DNA and enhance polycation-mediated gene delivery in NGF receptor-expressing PC12 cells, but not in COS7 cells lacking NGF receptors. The enhancement of gene transfer in PC12 was inhibited by pretreatment of free, unbound polypeptides, suggesting a NGF-receptor-specific effect of the polypeptide. These observations demonstrated the concept of using receptor-mediated mechanism for targeted gene delivery to neurons.