A Targeted Nano Drug Delivery System of AS1411 Functionalized Graphene Oxide Based Composites.

A novel method for the preparation of antitumor drug vehicles has been optimized. Biological materials of chitosan oligosaccharide (CO) and γ-polyglutamic acid (γ-PGA) have previously been employed as modifiers to covalently modify graphene oxide (GO), which in turn loaded doxorubicin (DOX) to obtain a nano drug delivery systems of graphene oxide based composites (GO-CO-γ-PGA-DOX). The system was not equipped with the ability of initiative targeting, thus resulting into toxicity and side effects on normal tissues or organs. In order to further improve the targeting property of the system, the nucleic acid aptamer NH2 -AS1411 (APT) of targeted nucleolin (C23) was used to conjugate on GO-CO-γ-PGA to yield the targeted nano drug delivery system APT-GO-CO-γ-PGA. The structure, composition, dispersion, particle size and morphology properties of the synthesized complex have been studied using multiple characterization methods. Drug loading and release profile data showed that APT-GO-CO-γ-PGA is provided with high drug loading capacity and is capable of controlled and sustained release of DOX. Cell experimental results indicated that since C23 was overexpressed on the surface of Hela cells but not on the surface of Beas-2B cells, APT-GO-CO-γ-PGA-DOX can target Hela cells and make increase toxicity to Hela cells than Beas-2B cells, and the IC50 value of APT-GO-CO-γ-PGA-DOX was 3.23±0.04 µg/mL. All results proved that APT-GO-CO-γ-PGA can deliver antitumor drugs in a targeted manner, and achieve the effect of reducing poison, which indicated that the targeted carrier exhibits a broad application prospect in the field of biomedicine.

Poly(l-glutamic acid)-Based Zwitterionic Polymer in a Charge Conversional Shielding System for Gene Therapy of Malignant Tumors.

Recently, pH-sensitive polymers have received extensive attention in tumor therapy. However, the rapid response to pH changes is the key to achieving efficient treatment. Here, a novel shielding system with a rapidly pH-responsive polymer (PAMT) is synthesized by click reaction between poly(γ-allyl-l-glutamate) and thioglycolic acid or 2-(Boc-amino)ethanethiol. The zwitterionic biodegradable polymer PAMT, which is negatively charged at physiological pH, can be used to shield positively charged nanoparticles. PAMT is electrostatically attached to the surface of the positively charged PEI/pDNA complex to form a ternary complex. The zwitterionic PAMT-shielded complex exhibits rapid charge conversion when the pH decreases from 7.4 to 6.8. For the in vivo tumor inhibition experiment, PAMT/PEI/shVEGF injected intravenously shows a more significant inhibitory effect on tumor growth. The excellent results are mainly attributed to introduction of the zwitterionic copolymer PAMT, which can shield the positively charged PEI/shVEGF complex in physiological conditions, while the surface potential of the shielded complexes changes to a positive charge in the acidic tumor environment.

In situ synthesis of poly (γ- glutamic acid)/alginate/AgNP composite microspheres with antibacterial and hemostatic properties.

In the present work, a poly(γ-glutamic acid)/alginate/silver nanoparticle (PGA/Alg/AgNP) composite microsphere with excellent antibacterial and hemostatic properties was prepared by the in situ UV reduction and emulsion internal gelation method, and its potential application for antibacterial hemostatic dressing was explored. Well dispersed AgNPs were in situ synthesized by a UV reduction method with alginate as stabilizer and reductant. The AgNPs showed excellent antibacterial activities against both gram-negative and gram-positive bacteria. Additionally, the AgNPs prepared by the in-situ UV reduction exhibited better biocompatibility and antibacterial effects than those prepared by the conventional chemical reduction method. PGA/Alg/AgNP composite microspheres were then prepared with the AgNPs by an emulsion internal gelation method. Such microspheres were found to be a porous and hollow network with pH-sensitive swelling properties and excellent hemostatic performance, indicating its application potentials as an advanced antibacterial hemostatic material.

Development of Zwitterionic Polypeptide Nanoformulation with High Doxorubicin Loading Content for Targeted Drug Delivery.

Much attention has been drawn to targeted nanodrug delivery systems due to their high therapeutic efficacy in cancer treatment. In this work, doxorubicin (DOX) was incorporated into a zwitterionic arginyl-glycyl-aspartic acid (RGD)-conjugated polypeptide by an emulsion solvent evaporation technique with high drug loading content (45%) and high drug loading efficiency (95%). This zwitterionic nanoformulation showed excellent colloidal stability at high dilution and in serum. The pH-induced disintegration and enzyme-induced degradation of the nanoformulation were confirmed by dynamic light scattering and gel permeation chromatography. Efficient internalization of DOX in the cells and high antitumor activity in vitro was observed. Compared with the free drug, this nanoformulation showed higher accumulation in tumor and lower systemic toxicity in vivo. The DOX-loaded zwitterionic RGD-conjugated polypeptide vesicles show potential application for targeted drug delivery in the clinic.

Poly(γ-Glutamic Acid) as an Exogenous Promoter of Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells.

Cartilage damage and/or aging effects can cause constant pain, which limits the patient's quality of life. Although different strategies have been proposed to enhance the limited regenerative capacity of cartilage tissue, the full production of native and functional cartilaginous extracellular matrix (ECM) has not yet been achieved. Poly(γ-glutamic acid) (γ-PGA), a naturally occurring polyamino acid, biodegradable into glutamate residues, has been explored for tissue regeneration. In this work, γ-PGA's ability to support the production of cartilaginous ECM by human bone marrow mesenchymal stem/stromal cells (MSCs) and nasal chondrocytes (NCs) was investigated. MSC and NC pellets were cultured in basal medium (BM), chondrogenic medium (CM), and CM-γ-PGA-supplemented medium (CM+γ-PGA) over a period of 21 days. Pellet size/shape was monitored with time. At 14 and 21 days of culture, the presence of sulfated glycosaminoglycans (sGAGs), type II collagen (Col II), Sox-9, aggrecan, type XI collagen (Col XI), type X collagen (Col X), calcium deposits, and type I collagen (Col I) was analyzed. After excluding γ-PGA's cytotoxicity, earlier cell condensation, higher sGAG content, Col II, Sox-9 (day 14), aggrecan, and Col X (day 14) production was observed in γ-PGA-supplemented MSC cultures, with no signs of mineralization or Col I. These effects were not evident with NCs. However, Sox-9 (at day 14) and Col X (at days 14 and 21) were increased, decreased, or absent, respectively. Overall, γ-PGA improved chondrogenic differentiation of MSCs, increasing ECM production earlier in culture. It is proposed that γ-PGA incorporation in novel biomaterials has a beneficial impact on future approaches for cartilage regeneration.

Quaternary complexes modified from pDNA and poly-l-lysine complexes to enhance pH-buffering effect and suppress cytotoxicity.

We developed a modified complex of pDNA and poly-l-lysine (PLL) by the addition of poly-l-histidine (PLH) and γ-polyglutamic acid (γ-PGA) to enhance its pH-buffering effect and suppress cytotoxicity. The binary and ternary complexes of pDNA with PLL or/and PLH showed particle sizes of approximately 52-76 nm with cationic surface charge. The ternary complexes showed much higher gene expression than the binary complexes with PLL. The mixed solution of PLL and PLH showed higher buffering capacity than PLL solution. The high gene expression of ternary complexes was reduced by bafilomycin A1 . These results indicated the addition of PLH to PLL complexes promoted endosomal escape by enhancing the pH-buffering effect. The binary and ternary complexes showed cytotoxicity and blood agglutination because of their cationic surface charge. We therefore developed quaternary complexes by the addition of anionic γ-PGA, which was reported to decrease the toxicity of cationic complexes. In fact, quaternary complexes showed no cytotoxicity and blood agglutination. Also, quaternary complexes showed higher gene expression than ternary complexes regardless of their anionic surface charge. Quaternary complexes showed selectively high gene expression in the spleen after their intravenous administration. Thus, we successfully developed the quaternary complexes with high gene expression and no toxicity.

Redox-responsive, reversibly-crosslinked thiolated cationic helical polypeptides for efficient siRNA encapsulation and delivery.

Cationic helical polypeptides, although highly efficient for inducing membrane penetration, cannot stably condense siRNA molecules via electrostatic interactions, which greatly limit the gene knockdown efficiency. By developing and crosslinking the thiolated polypeptide via formation of disulfide bonds post formation of the polypeptide/siRNA complexes, we were able to obtain stable complexes without compromising the helical secondary structure as well as the membrane activity of the polypeptide. As such, the stable polypeptide/siRNA complex was able to notably protect the siRNA cargo from nuclease digestion in the extracellular environment, while the functions of the polypeptide/siRNA complex for effective cellular internalization and endosomal escape are still largely preserved. Because the disulfide is susceptible to cleavage in response to intracellular redox triggers, siRNA release from the complex is expected upon redox triggering by glutathione (GSH) intracellularly and was actually observed upon redox triggers mediated by glutathione (GSH). With the collective contribution of the potent membrane activity and redox-responsive cargo release profiles, the crosslinked complexes enable efficient gene silencing without appreciable cytotoxicity, thus providing a potential strategy for polypeptide-based intracellular siRNA delivery.

Infection-mimicking poly(γ-glutamic acid) as adjuvant material for effective anti-tumor immune response.

Bio-derived low molecular weight poly(γ-glutamic acid) (γ-PGA) was suggested as a novel adjuvant material for use in cancer vaccines. When the infection-mimicking γ-PGA was immunized with ovalbumin (OVA) as a model antigen, increase in the dendritic cell (DC)-mediated functions such as activation, maturation, antigen uptake, migration to lymph nodes, and priming of lymphocytes, which included cross-presentation, was observed. These DC-mediated functions were found to be facilitated by γ-PGA in a dose-dependent manner, with stimulation of toll-like receptor 4 (TLR4) being one of the underlying mechanisms. The in vivo efficacy of γ-PGA was tested in a mouse tumor model where both arms of adaptive immunity (humoral and cell-mediated) were found to be significantly enhanced in the presence of γ-PGA, indicating efficient priming of B and T cells. Moreover, immunization of mice with γ-PGA followed by EG7-OVA tumor challenge led to dramatic inhibition of tumor growth. After 71 days, the cured mice were rechallenged with tumor cells at a distant site in order to test the memory effect. No tumor growth was observed, which indicates the presence of a systemic, long-lasting immune response. Based on these results, low molecular weight γ-PGA is expected to have tremendous potential for applications in cancer immunotherapy.

γ-PGA-coated mesoporous silica nanoparticles with covalently attached prodrugs for enhanced cellular uptake and intracellular GSH-responsive release.

Poor cellular uptake of drug delivery carriers and uncontrolled drug release remain to be the major obstacles in cancer therapy due to their low delivery efficiency. In this study, a multifunctional intracellular GSH (glutathione)-responsive silica-based drug delivery system with enhanced cellular uptake capability is developed. Uniform 50 nm colloidal mesoporous silica nanoparticles (MSNs) with mercaptopropyl-functionalized core and silanol-contained silica surface (MSNs-SHin ) are designed and fabricated as a platform for drug covalent attachment and particle surface modification. Doxorubicin (DOX) with primary amine group as an anticancer model drug is covalently conjugated to the mesopores of MSNs-SHin via disulfide bonds in the presence of a heterobifunctional linker (N-Succinimidyl 3-(2-pyridyldithio) propionate). Poly(γ-glutamic acid) (γ-PGA) can be coated onto the particle surface by sequential electrostatic adsorption of polyethyleneimine (PEI) and γ-PGA. The constructed delivery system exhibits enhanced cellular uptake via a speculated γ-glutamyl transpeptidase (GGT)-mediated endocytosis pathway and controlled drug release capacity via intracellular GSH-responsive disulfide-bond cleavage, and thus significantly inhibits the growth of cancer cells. The multifunctional delivery system paves a new way for developing high-efficient particle-based nanotherapeutic approach for cancer treatment.

[Preparation, characterization and Calu-3 cellular uptake of three kinds of poly(b-benzyl-L-amino)block-poly(ethylene glycol) nanoparticles].

The aim of this paper is to compare the cytotoxicity and cellular uptake efficiency of three kinds of poly(b-benzyl-L-amino) block-poly(ethylene glycol) nanoparticles (PXA-PEG-NPs) using Calu-3 cells, and select one as a nasal drug delivery vector for curcumin (Cur). Poly(gamma-benzyl-L-glutamate) block-poly(ethylene glycol) nanoparticles (PBLG-PEG-NPs), poly(gamma-benzyl-L-lysine) block-poly(ethyleneglycol) nanoparticles (PZLL-PEG-NPs) and poly(gamma-benzyl-L-aspartate) block-poly(ethylene glycol) nanoparticles (PBLA-PEG-NPs) were prepared by emulsion-solvent evaporation method. MTT assays were used to evaluate the cytotoxicity of PXA-PEG-NPs against Calu-3 cells. The cellular uptake of nanoparticles was visualized by an inverted fluorescence microscope and quantified by a flow cytometer. The results indicated that even at high concentration of 2 mg x mL(-1) the three nanoparticles had no cytotoxicity on Calu-3 cells. Compared to the curcumin solution, the three curcumin-loaded PXA-PEG-NPs showed significantly higher cellular uptake efficiency on Calu-3 cells (at equal concentration of curcumin with 5 microg x mL(-1) Cur solution), PBLG-PEG-NPs group was the highest. The cellular uptake increased with incubation time, and has positive correlation with nanoparticle concentration. In brief, PXA-PEG-NPs are conducive to delivery Cur into cells, and PBLG-PEG-NPs might be provided as a good nasal drug delivery carrier.

A new potential nano-oncological therapy based on polyamino acid nanocapsules.

A critical objective in cancer therapy is to reduce the systemic toxicity through the modification of the biodistribution of anticancer drugs. Herein, we disclose a new biodegradable nanocarrier, polyglutamic acid (PGA) nanocapsules, and present the in vivo pharmacokinetics/toxicity proof-of-concept for the anticancer drug plitidepsin. These novel nanocapsules were prepared using a modified solvent displacement technique where the polyamino acid was electrostatically deposited onto the lipid core. The nanocapsules exhibited an average size of 200 nm, a negative zeta potential and a great capacity for the encapsulation of plitidepsin (encapsulation efficiency above 90%). In addition, the nanocapsules could be freeze-dried and showed an adequate stability profile upon storage. Finally, the in vivo proof-of-concept studies performed in mice indicated that the encapsulation provided the drug with a prolonged blood circulation and a significantly reduced toxicity. In fact, the maximum tolerated dose of the nanoencapsulated drug was more than 3 times that of the reference formulation (Cremophor® EL plitidepsin solution). Overall, beyond the value of this specific formulation, the work reported here represents the evidence of the potential of polyamino acid nanocapsules in nano-oncological therapy.

Reduced in vitro and in vivo toxicity of siRNA-lipoplexes with addition of polyglutamate.

We previously designed a new siRNA vector that efficiently silences genes in vitro and in vivo. The vector originality is based on the fact that, in addition to the siRNA molecule, it contains two components: 1) a cationic liposome that auto-associates with the siRNA to form particles called "lipoplexes" and, 2) an anionic polymer which enhances the lipoplex's efficiency. This anionic polymer can be a nucleic acid, a polypeptide or a polysaccharide. We show here how the nature of the added anionic polymer into our siRNA delivery system impacts the toxic effects induced by siRNA lipoplexes. We first observed that: (i) siRNA lipoplexes-induced toxicity was cell line dependent, tumoral cell lines being the more sensitive; and (ii) plasmid DNA-containing siRNA lipoplexes were more toxic than polyglutamate-containing ones or cationic liposomes. We next determined that the toxicity induced by plasmid-containing lipoplexes is a long-lasting effect that decreased cell survival capacity for several generations. We also found that treated cells underwent death following apoptosis pathway. Systemic injection to mice of siRNA lipoplexes, rather than of cationic liposome, triggered a production of several cytokines in mice and replacement of plasmid by polyglutamate reduced the elevation of all assayed cytokines. In order to enhance siRNA lipoplexes efficiency, the addition of polyglutamate as anionic polymer should be preferred to plasmid DNA as far as in vitro as well as in vivo toxicity is concerned.

Decisive role of hydrophobic side groups of polypeptides in thermosensitive gelation.

Thermosensitive hydrogels based on PEG and poly(l-glutamate)s bearing different hydrophobic side groups were separately synthesized by the ring-opening polymerization (ROP) of l-glutamate N-carboxyanhydrides containing different alkyl protected groups, that is, methyl, ethyl, n-propyl, and n-butyl, using mPEG(45)-NH(2) as macroinitiator. The resulting copolymers underwent sol-gel transitions in response to temperature change. Interestingly, the polypeptides containing methyl and ethyl showed significantly lower critical gelation temperatures (CGTs) than those bearing n-propyl and butyl side groups. Based on the analysis of (13)C NMR spectra, DLS, circular dichroism spectra, and ATR-FTIR spectra, the sol-gel transition mechanism was attributed to the dehydration of poly(ethylene glycol) and the increase of ß-sheet conformation content in the polypeptides. The in vivo gelation test indicated that the copolymer solution (6.0 wt %) immediately changed to a gel after subcutaneous injection into rats. The mass loss of the hydrogel in vitro was accelerated in the presence of proteinase K, and the MTT assay revealed that the block copolymers exhibited no detectable cytotoxicity. The present work revealed that subtle variation in the length of a hydrophobic side group displayed the decisive effect on the gelation behavior of the polypeptides. In addition, the thermosensitive hydrogels could be promising materials for biomedical applications due to their good biocompatibility, biodegradability, and the fast in situ gelation behavior.

PEG/RGD-modified magnetic polymeric liposomes for controlled drug release and tumor cell targeting.

Polymeric liposomes (PEG/RGD-MPLs), composed of amphiphilic polymer octadecyl-quaternized modified poly (γ-glutamic acid) (OQPGA), PEGylated OQPGA, RGD peptide grafted OQPGA and magnetic nanoparticles, was prepared successfully. These PEG/RGD-MPLs could be used as a multifunctional platform for targeted drug delivery. The results showed that PEG/RGD-MPLs were multilamellar spheres with nano-size (50-70 nm) and positive surface charge (28-42 mV). Compared with magnetic conventional liposomes (MCLs), PEG/RGD-MPLs exhibited sufficient size and zeta potential stability, low initial burst release and less magnetic nanoparticles leakage. The cell uptake results suggested that the PEG/RGD-MPLs (with RGD and magnetic particles) exhibited more drug cellular uptake than non RGD and non magnetism carriers in MCF-7 cells. MTT assay revealed that PEG/RGD-MPLs showed lower in vitro cytotoxicity to GES-1cells at ≤ 100 µg/mL. These data indicated that the multifunctional PEG/RGD-MPLs may be an alternative formulation for drug delivery system.

Micellization of cisplatin (NC-6004) reduces its ototoxicity in guinea pigs.

Nanocarriers potentially reduce or prevent chemotherapy-induced side effects, facilitating the translation of nanocarrier formulation into the clinic. To date, organ-specific toxicity by nanocarriers remains to be clarified. Here, we studied the potential of polymeric micelle nanocarriers to prevent the ototoxicity, which is a common side effect of high-dose cisplatin (CDDP) therapy. In this study, we evaluated the ototoxicity of CDDP-incorporating polymeric micelles (NC-6004) in guinea pigs in comparison with that of cisplatin. Their auditory brainstem responses (ABRs) to 2, 6, 12, 20, and 30kHz sound stimulation were measured before and 5 days after the drug administration. Groups treated with NC-6004 showed no apparent ABR threshold shifts, whereas groups treated with CDDP showed dose-dependent threshold shifts particularly at the higher frequencies. Consistent with the ABR results, groups treated with NC-6004 showed excellent hair-cell preservation, whereas groups treated with CDDP exhibited significant hair-cell loss (P<0.05). Synchrotron radiation-induced X-ray fluorescence spectrometry imaging demonstrated that the platinum distribution and concentration in the organ of Corti were significantly reduced (P<0.01) in guinea pigs treated with NC-6004 compared with guinea pigs treated with CDDP. These findings indicate that micellization of CDDP reduces its ototoxicity by circumventing the vulnerable cells in the inner ear.

Polyglutamic acid-based nanocomposites as efficient non-viral gene carriers in vitro and in vivo.

A series of polyethylenimine (PEI) and γ-polyglutamic acid (PGA) nanocomposites (PPGA) was prepared and evaluated in terms of their cell viability and transfection efficiency in vitro and in vivo. On complexion with pDNA, the positively charged PPGA/DNA nanocomposites resulted in a higher level of in vitro reporter gene transfection (2.7-7.9-fold) as compared to native PEI, and selected commercial reagents and >95% cell viability in HEK293, HeLa and HepG2 cell lines. Further, PPGA-5 nanocomposite (the best working system in terms of transfection efficiency among the series) was found to efficiently transfect primary mouse keratinocytes up to 22% above the control level. PPGA-5, when tested for in vivo cytotoxicity in Drosophila, did not induce any stress in the exposed larvae in comparison with control. In vivo gene expression using PPGA-5 showed the highest transfection efficiency in spleen of mouse closely followed by heart tissues after intravenous injection through tail vein. Besides, these nanocomposites also delivered siRNA efficiently into mammalian cells, resulting in ∼ 80% suppression of EGFP expression. These results together demonstrated the potential of the projected nanocomposites for in vivo gene delivery.

The poly-γ-D-glutamic acid capsule of Bacillus anthracis enhances lethal toxin activity.

The poly-γ-D-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguises B. anthracis from immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

A novel GAP460 biopolymer for use as a carrier in drug-delivery applications.

We synthesized a new non-toxic biopolymer (GAP460) containing γ,L-glutamic acid and aspartate (Asp). Conjugates of GAP460 and cisplatin exhibited a drug-carrying capacity of nearly 40%, 3-times higher than γ-PGA and dramatically decreasing the amount of biopolymer required for high-dose delivery. Treatment with GAP460-cisplatin conjugate (PACC) not only effectively inhibited tumor growth in nude mice, but also resulted in extended survival and lower nephrotoxicity, suggesting that GAP460 could be used as an effective carrier for drug delivery and that PACC may have potential therapeutic applications in the clinical treatment of cancer.

Synthesis, characterization, and biological evaluation of poly(L-γ-glutamyl-glutamine)- paclitaxel nanoconjugate.

The purpose of this study was to develop a novel, highly water-soluble poly(L-γ-glutamyl-glutamine)-paclitaxel nanoconjugate (PGG-PTX) that would improve the therapeutic index of paclitaxel (PTX). PGG-PTX is a modification of poly(L-glutamic acid)- paclitaxel conjugate (PGA-PTX) in which an additional glutamic acid has been added to each glutamic side chain in the polymer. PGG-PTX has higher water-solubility and faster dissolution than PGA-PTX. Unlike PGA-PTX, PGG-PTX self-assembles into nanoparticles, whose size remains in the range of 12-15 nm over the concentration range from 25 to 2,000 µg/mL in saline. Its critical micellar concentration in saline was found to be ~25 µg/mL. The potency of PGG-PTX when tested in vitro against the human lung cancer H460 cell line was comparable to other known polymer-PTX conjugates. However, PGG-PTX possesses lower toxicity compared with PGA-PTX in mice. The maximum tolerated dose of PGG-PTX was found to be 350 mg PTX/kg, which is 2.2-fold higher than the maximum tolerated dose of 160 mg PTX/kg reported for the PGA-PTX. This result indicates that PGG-PTX was substantially less toxic in vivo than PGA-PTX.

Gamma-polyglutamic acid-coated vectors for effective and safe gene therapy.

In the present study, we developed some novel gene delivery vectors, coated cationic complexes with gamma-polyglutamic acid (gamma-PGA) for effective and safe gene therapy. Cationic complexes were constructed with pDNA and cationic vectors, such as poly-L-arginine hydrochloride (PLA), poly-L-lysine hydrobromide (PLL), N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA)-cholesterol (Chol) liposomes, and DOTMA-dioleylphosphatidylethanolamine (DOPE) liposomes. The cationic complexes showed high gene expression with strong cytotoxicity in melanoma B16-F10 cells. The cationic complexes were also strongly toxic to erythrocytes. On the other hand, the gamma-PGA was able to coat all cationic complexes and form stable nano-sized particles with negative charges. These gamma-PGA-coated complexes had high gene expression without cytotoxicity and toxicities to the erythrocytes. In in vivo transfection experiments, polyplexes showed high transfection efficiency over 10(5) RLU/g in the lung tissue after intravenous injection, although gamma-PGA-coated polyplexes showed a high value in the spleen. High transfection efficiency in lipoplexes and gamma-PGA-coated lipoplexes was observed in the spleen and lung. Thus, gamma-PGA-coated vectors are useful for clinical gene therapy.