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.

Covalently functionalized poly(etheretherketone) implants with osteogenic growth peptide (OGP) to improve osteogenesis activity.

Polyetheretherketone (PEEK), as the most promising implant material for orthopedics and dental applications, has bone-like stiffness, excellent fatigue resistance, X-ray transparency, and near absence of immune toxicity. However, due to biological inertness, its bone conduction and bone ingrowth performance is limited. The surface modification of PEEK is an option to overcome these shortcomings and retain most of its favorable properties, especially when excellent osseointegration is desired. In this study, a simple reaction procedure was employed to bind the osteogenic growth peptide (OGP) on the surface of PEEK materials by covalent chemical grafting to construct a bioactive interface. The PEEK surface was activated by N,N'-disuccinimidyl carbonate (DSC) after hydroxylation, and then OGP was covalently grafted with amino groups. The functionalized surface of PEEK samples were characterized by X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FT-IR), water contact angle measurement and biological evaluation in vitro. OGP-functionalized PEEK surface significantly promoted the attachment, proliferation, alkaline phosphatase (ALP) activity and mineralization of pre-osteoblast cells (MC3T3-E1). The in vivo rat tibia implantation model is adopted and micro-CT analyses demonstrated that the OGP coating significantly promoted new bone formation around the samples. The in vitro and in vivo results reveal that the modification by covalent chemical functionalization with OGP on PEEK surface can augment new bone formation surrounding implants compared to bare PEEK and PEEK implant modified by covalently attached OGP is promising in orthopedic and dental applications.

Micro-porous polyetheretherketone implants decorated with BMP-2 via phosphorylated gelatin coating for enhancing cell adhesion and osteogenic differentiation.

Polyetheretherketone (PEEK) is a high-performance semicrystalline thermoplastic polymer that is widely used in the orthopedics treatment. However, due to its biological inertness, the surface modification of PEEK using different methods to improve the biocompatibility remains a significant challenge. Herein, we attempted to use the covalently coating of phosphorylated gelatin loaded with bone morphogenetic protein 2 (BMP-2) on hydroxylated micro-porous PEEK films for enhancing the biological activity. Environmental scanning electron microscope (ESEM), fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and water contact angle measurements were applied to characterize the surface of modified or untreated PEEK films. The influence on cell adhesion, proliferation and differentiation was evaluated by culturing of mouse pre-osteoblasts (MC3T3-E1) on different modified PEEK substrates in vitro. Surface characterization showed that the modification was successfully performed on PEEK films. The biological results indicated that surface modification of micro-porous PEEK using phosphorylated gelatin significantly promoted cell adhesion and proliferation. And the osteogenic differentiation was effectively improved while loading with different amounts of BMP-2. Findings from this study indicated that this novel biological modification on PEEK films might be helpful for altering its biological inertness and further expand its medical applications as a kind of orthopedic implants.

Codelivery of antitumor drug and gene by a pH-sensitive charge-conversion system.

In the present study, a gene and drug codelivery system was developed by electrostatic binding of polyethylenimine-poly(l-lysine)-poly(l-glutamic acid) (PELG), polyethylenimine (PEI), cis-aconityl-doxorubicin (CAD), and DNA. Zeta potential and drug release analysis confirmed the pH-responsive charge conversion and acid-sensitive drug release functional properties of the PELG/PEI/(DNA+CAD) system. Gel retardation assay and transfection experiment showed the codelivery system had effective DNA binding ability and good transfection efficiency on HepG2 cells. The therapeutic gene p53 was further employed to study its combinational effects with CAD. Cytotoxicity assay showed the half inhibitory concentration (IC50) of the PELG/PEI/(p53+CAD) codelivery system was lower than that of the gene or the drug delivery system. Confocal laser scanning microscopy (CLSM) showed that the drug and gene could be delivered into the cells simultaneously. A significant increase of p53 gene expression was achieved after HepG2 cells treated by PELG/PEI/(p53+CAD) codelivery system. The apoptosis experiment indicated clearly that the codelivery system could lead an effective apoptosis on tumor cells, which was beneficial for the treatment of cancer. The biodistribution and tumor accumulation of the codelivery system was explored via in vivo imaging in subcutaneous xenograft and in situ tumor models. The tumor and some major organs were excised and imaged, and the results showed that the codelivery system can accumulate efficiently in tumor for both tumor models. It can be suggested from the above results that the PELG/PEI/(DNA+CAD) codelivery system will have great potential applications in cancer therapy.

pH-responsive zwitterionic copolypeptides as charge conversional shielding system for gene carriers.

A novel rapid pH-responssive polymer polyethylenimine-poly(l-lysine)-poly(l-glutamic acid) (PELG) was designed as the shielding system. The zwitterionic copolypeptide PELG with negatively charged at physical pH can act as the shielding system to shield positively charged polyplexes. PELG was used to shield PEI25k/DNA to form ternary polyplex, the polyplex surface zeta potential can change from a negative to positive nearly pH value of 6.9. Because the pH value of tumor extracellular environment is about 6.5, the positive charges on the polyplexes could be restored in tumors, which is beneficial to the electrostatic interactions between positive polyplexes and negative tumor cells, leading to high cell uptake efficiency and high transfection efficiency.

A pH-sensitive charge-conversion system for doxorubicin delivery.

A novel pH-sensitive charge-conversion shielding system was designed by the electrostatic binding of polyethylenimine (PEI)-poly(l-lysine)-poly(l-glutamic acid) (PELG), PEI, and cis-aconityl-doxorubicin (CAD). Doxorubicin (DOX) was modified by cis-aconityl linkage to form acid-sensitive CAD, which was then adsorbed by the positively charged PEI. The PEI/CAD complexes were subsequently shielded with the pH-responsive charge-conversion PELG. In normal tissues, the PELG/PEI/CAD complexes were negatively charged; in acidic tumor tissues, the shielding PELG was positively charged and detached from the PELG/PEI/CAD complexes. The resulting positively charged PEI/CAD complexes thus became exposed and were endocytosed. CAD was then cleaved in the acidic intracellular environment of endosomes and lysosomes, and converted back into DOX. The charge reversal of the PELG/PEI/CAD complexes was verified by zeta potential analysis at different pH values. Moreover, DOX release increased with decreasing pH. Cell uptake and confocal laser scanning microscopy analyses showed that, at pH 6.8, PELG/PEI/CAD had the highest endocytosis rate and more DOX entered cell nuclei. More importantly, the system showed remarkable cytotoxicity against cancer cells. These results revealed that the combination of pH-sensitive charge-conversion shielding with pH-sensitive drug release is a potential drug delivery system for tumor treatment.

Effective tumor treatment by VEGF siRNA complexed with hydrophobic poly(amino acid)-modified polyethylenimine.

Two copolymers are designed and synthesised for siRNA delivery based on polyethylenimine by grafting hydrophilic acrylamide segments and hydrophobic poly(γ-benzyl L-glutamate). The amphiphilic PEI-PBLG/siRNA complex demonstrates high gene silencing efficiency in the absence or presence of 10 vol% and 50 vol% sera in vitro. The anti-tumor effects in vivo are evaluated in luciferase-bearing mice expressing CT26 tumors. PEI-PBLG/siVEGF complex provides a higher and more sustained suppressive effect by reducing VEGF mRNA expression in the tumors, leading to higher tumor growth inhibition efficacy. Further studies on the potential use of the PEI-PBLG carrier system in mediating the silencing of genes other than VEGF or in other tumor models are recommended.

Polylysine-modified polyethylenimine inducing tumor apoptosis as an efficient gene carrier.

Polyethylenimine (PEI) is receiving increasing attention as a gene carrier with high transfection efficiency. However, its high charge density and cytotoxic effects limit its application. Polylysine (PLL) is another polymeric gene carrier with good biodegradability and biocompatibility, although its lack of endosomal escape ability strongly impairs its transfection efficiency. In this study, PLL was introduced to PEI by ring-opening polymerization of ε-benzyloxycarbonyl-l-lysine N-carboxyanhydride, followed by deprotection of carbobenzyloxy groups. As-prepared PEI-PLL multiarm hyperbranched copolymers were characterized as gene carriers in vitro by measuring their particle size, zeta potential, cytotoxicity, transfection efficiency, and cell internalization. The optimum transfected efficiency of PEI-PLL was nearly seven times higher than that of PEI with a molecular weight of 25kDa. Furthermore, pKH3-rev-casp-3 plasmid DNA was used as a gene for anti-tumor treatment in a xenograft model using nude mice. Compared with 25kDa PEI, PEI-PLL exhibited better tumor inhibition effects in 23days. In addition, terminal deoxynucleotidyl transferase dUTP nick end labeling, immunohistochemistry, and western blot analysis were used to determine the anti-tumor mechanism of PEI-PLL. The results showed that tumor cell apoptosis led to tumor inhibition, which could be attributed to pKH3-rev-casp-3 inducing poly(ADP-ribose) polymerase-1 cleavage. PEI-PLL is a promising gene carrier candidate for further application in vivo.