Huayu Tian

Stimuli‐Sensitive Synthetic Polypeptide‐Based Materials for Drug and Gene Delivery

Stimuli-sensitive synthetic polypeptides are unique biodegradable and biocompatible synthetic polymers with structures mimicking natural proteins. These polymers exhibit reversible secondary conformation transitions and/or hydrophilic-hydrophobic transitions in response to changes in environmental conditions such as pH and temperature. The stimuli-triggered conformation and/or phase transitions lead to unique self-assembly behaviors, making these materials interesting for controlled drug and gene delivery applications. Therefore, stimuli-sensitive synthetic polypeptide-based materials have been extensively investigatid in recent years. Various polypeptide-based materials, including micelles, vesicles, nanogels, and hydrogels, have been developed and tested for drug- and gene-delivery applications. In addition, the presence of reactive side groups in some polypeptides facilitates the incorporation of various functional moieties to the polypeptides. This Review focuses on recent advances in stimuli-sensitive polypeptide-based materials that have been designed and evaluated for drug and gene delivery applications. In addition, recent developments in the preparation of stimuli-sensitive functionalized polypeptides are discussed.

RGD targeting hyaluronic acid coating system for PEI-PBLG polycation gene carriers

Hyaluronic acid (HA), a natural anionic mucopolysaccharide, was used to coat polyethylenimine-poly(γ-benzyl L-glutamate)/DNA (PEI-PBLG/DNA) complexes. HA was further modified by introducing RGD peptide with grafting density of one RGD in every 1.9 HA repeating units. HA can coat the cationic surface of PEI-PBLG/DNA complexes without destroying them even at high weight ratio of HA/PEI-PBLG/DNA=40/10/1. Coating the complexes by HA and HA-RGD caused lower surface charges and little bigger size than the naked PEI-PBLG/DNA. HA/PEI-PBLG/DNA has little lower transfection efficiency compared with naked PEI-PBLG/DNA, while the transfection efficiency of HA-RGD/PEI-PBLG/DNA is 9.7 times of HA/PEI-PBLG/DNA for the RGD target bonding affinity to the receptors on the cell surface. HA coating on PEI-PBLG/DNA reduced the electrostatic binding affinity to the cells while the RGD binding affinity for integrin on HeLa cells can not only compensate the reduced binding affinity but also enhance the affinity for HA-RGD/PEI-PBLG/DNA. RGD and RDG competition assay and lactate dehydrogenase (LDH) release studies further confirmed the specific target functions of RGD on HA. Cell viability measurements confirmed the high viability (above 70% viability) of the cells treated with HA-RGD and HA coated complex particles. These results would show that HA-RGD coated PEI-PBLG/DNA complexes have an attractive feature to a targeting in vivo non-viral gene delivery system.

Nanoparticles for Gene Delivery

Nanocarriers are a new type of nonviral gene carriers, many of which have demonstrated a broad range of pharmacological and biological properties, such as being biodegradable in the body, stimulus-responsive towards the surrounding environment, and an ability to specifically targeting certain disease sites. By summarizing some main types of nanocarriers, this Concept considers the current status and possible future directions of the potential clinical applications of multifunctional nanocarriers, with primary attention on the combination of such properties as biodegradability, targetability, transfection ability, and stimuli sensitivity.

Formation of Reversible Shell Cross-Linked Micelles from the Biodegradable Amphiphilic Diblock Copolymer Poly(l-cysteine)-block-Poly(l-lactide)

A novel biodegradable diblock copolymer, poly(L-cysteine)-b-poly(L-lactide) (PLC-b-PLLA), was synthesized by ring-opening polymerization (ROP) of N-carboxyanhydride of beta-benzyloxycarbonyl-L-cysteine (ZLC-NCA) with amino-terminated poly(L-lactide) (NH 2-PLLA) as a macroinitiator in a convenient way. The diblock copolymer and its precursor were characterized by (1)H NMR, Fourier transform infrared (FT-IR), gel permeation chromatography (GPC), and X-ray photoelectron spectroscopy (XPS) measurements. The length of each block polymer could be tailored by molecular design and the ratios of feeding monomers. The cell adhesion and cell spread on the PZLC-b-PLLA and PLC-b-PLLA films were enhanced compared to those on pure PLA film. PLC-b-PLLA can self-assemble to form micelles in aqueous media. A pyrene probe is used to demonstrate the micelle formation of PLC-b-PLLA in aqueous solution. Due to the ease of disulfide exchange with thiols, the obtained micelles are reversible shell cross-linked (SCL) micelles. The morphology and size of the micelles are studied by dynamic light scattering (DLS) and environmental scanning electron microscopy (ESEM).

Hydrophobic poly (amino acid) modified PEI mediated delivery of rev-casp-3 for cancer therapy

Recent studies in amphiphilic cationic polymers have demonstrated their potential as gene carriers with high transfection efficiency and low cytotoxicity in the in vitro settings to deliver drug, siRNA and plasmid DNA. Yet their safety and efficacy in vivo remain to be a challenge, and require further investigation. In our previous work, PP80 was synthesized as a novel amphiphilic cationic polymer by grafting hydrophobic polyphenylalanine segment on PEI, which displayed higher transfection efficiency than PEI in a number of cell lines in vitro. Here, we reported the favorable biocompatibility displayed by PP80/pDNA complex both in vitro and in vivo. Furthermore, when therapeutic gene rev-casp-3 was conjugated to PP80 and administered intratumorally to a HeLa xenograft model, significant tumor apoptosis was induced with concurrent tumor growth inhibition, indicating that PP80 mediated expression of rev-casp-3 gene in solid tumors with not detectable side effects on the tumor-bearing mice. These data demonstrated that PP80 warrants further investigation as a promising cancer gene delivery vehicle.

PLK1shRNA and doxorubicin co-loaded thermosensitive PLGA-PEG-PLGA hydrogels for osteosarcoma treatment.

Combination cancer therapy has emerged as crucial approach for achieving superior anti-cancer efficacy. In this study, we developed a strategy by localized co-delivery of PLK1shRNA/polylysine-modified polyethylenimine (PEI-Lys) complexes and doxorubicin (DOX) using biodegradable, thermosensitive PLGA-PEG-PLGA hydrogels for treatment of osteosarcoma. When incubated with osteosarcoma Saos-2 and MG-63 cells, the hydrogel containing PLK1shRNA/PEI-Lys and DOX displayed significant synergistic effects in promoting the apoptosis of osteosarcoma cells in vitro. After subcutaneous injection of the hydrogel containing PLK1shRNA/PEI-Lys and DOX beside the tumors of nude mice bearing osteosarcoma Saos-2 xenografts, the hydrogels exhibited superior antitumor efficacy in vivo compared to the hydrogels loaded with PLK1shRNA/PEI-Lys or DOX alone. It is noteworthy that the combination treatment in vivo led to almost complete suppression of tumor growth up to 16 days, significantly enhanced PLK1 silencing, higher apoptosis of tumor masses, as well as increased cell cycle regulation. Additionally, ex vivo histological analysis of major organs of the mice indicated that the localized treatments showed no obvious damage to the organs, suggesting lower systemic toxicity of the treatments. Therefore, the strategy of localized, sustained co-delivery of PLK1shRNA and DOX by using the biodegradable, injectable hydrogel may have potential for efficient clinical treatment of osteosarcoma.

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.

Synergistic co-delivery of doxorubicin and paclitaxel by porous PLGA microspheres for pulmonary inhalation treatment.

PLGA porous microspheres loaded with doxorubicin (DOX) and paclitaxel (PTX) were developed for in situ treatment of metastatic lung cancer. The synergistic effect of the combined drugs was investigated against B16F10 cells to obtain the optimal prescription for in vivo studies. The combination therapy showed great synergism when DOX was the majority in the combination therapy, while they showed moderate antagonism when PTX is in major. The combination of DOX and PTX at a molar ratio of 5/1 showed the best synergistic therapeutic effect in the free form. However, the drugs exhibited more synergism in the PLGA microspheres at a molar ratio of 2/1, due to the difference in drug release rate. The in vivo study verified the synergism of DOX and PTX at the optimal molar ratio. These results suggested that dual encapsulation of DOX and PTX in porous PLGA microspheres would be a promising technology for long effective lung cancer treatment.

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.

Biodegradable mPEG-b-P(MCC-g-OEI) copolymers for efficient gene delivery.

Cationic polymers play an important role in gene delivery. To search for potential non-viral gene carriers, a series of biodegradable cationic polymers, poly(ethylene glycol)-block-poly(carbonates-graft-oligoethylenimine) [mPEG-b-P(MCC-g-OEI), PPO] copolymers, were synthesized by grafting different kinds of oligoethylenimine (OEI) to the same biodegradable backbone, a derivative of poly(ethylene glycol)-block-polycarbonates. The as-synthesized PPO copolymers were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and gel permeation chromatography. Two of the as-synthesized PPO copolymers (PPO600 and PPO1800) could efficiently condense DNA into nanosized particles (100-140 nm) with positive surface charges when the PPO/DNA mass ratio is above 10:1. The cell toxicity and gene transfection evaluations show that PPO copolymers, especially PPO1800, which exhibits lower cytotoxicity and higher gene transfection efficiency than PEI25K in the absence and the presence of serum in the CHO and COS-7 cell lines, have great potential as non-viral gene carriers. The PPO1800 copolymer images obtained by confocal laser scanning microscopy prove that PPO copolymers could efficiently mediate the entry of plasmid DNA into cells. These results show that PPO copolymers may be potential non-viral gene carriers in future applications of gene therapy.