Dual-Ligand Modified Polymer-Lipid Hybrid Nanoparticles for Docetaxel Targeting Delivery to Her2/neu Overexpressed Human Breast Cancer Cells.

In this study, a dual-ligand polymer-lipid hybrid nanoparticle drug delivery vehicle comprised of an anti-HER2/neu peptide (AHNP) mimic with a modified HIV-1 Tat (mTAT) was established for the targeted treatment of Her2/neu-overexpressing cells. The resultant dual-ligand hybrid nanoparticles (NPs) consisted of a poly(lactide-co-glycolide) core, a near 90% surface coverage of the lipid monolayer, and a 5.7 nm hydrated polyethylene glycol shell. Ligand density optimization study revealed that cellular uptake efficiency of the hybrid NPs could be manipulated by controlling the surface-ligand densities. Furthermore, the cell uptake kinetics and mechanism studies showed that the dual-ligand modifications of hybrid NPs altered the cellular uptake pathway from caveolae-mediated endocytosis (CvME) to the multiple endocytic pathways, which would significantly enhance the NP internalization. Upon the systemic investigation of the cellular uptake behavior of dual-ligand hybrid NPs, docetaxel (DTX), a hydrophobic anticancer drug, was successfully encapsulated into dual-ligand hybrid NPs with high drug loading for Her2/neu-overexpressing SK-BR-3 breast cancer cell treatment. The DTX-loaded dual-ligand hybrid NPs showed a decreased burst release and a more gradual sustained drug release property. Because of the synergistic effect of dual-ligand modification, DTX-loaded dual-ligand hybrid NPs exerted substantially better therapeutic potency against SK-BR-3 cancer cells than other NP formulations and free DTX drugs. These results demonstrate that the dual-ligand hybrid NPs could be a promising vehicle for targeted drug delivery to treat breast cancer.

Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency.

We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.

Micelles of enzymatically synthesized PEG-poly(amine-co-ester) block copolymers as pH-responsive nanocarriers for docetaxel delivery.

A series of PEGylated poly(amine-co-ester) terpolymers were successfully synthesized in one step via lipase-catalyzed copolymerization of ω-pentadecalactone (PDL), diethyl sebacate (DES), and N-methyldiethanolamine (MDEA) comonomers in the presence of poly(ethylene glycol) methyl ether as a chain-terminating agent. The resultant amphiphilic poly(ethylene glycol)-poly(PDL-co-MDEA-co-sebacate) (PEG-PPMS) block copolymers consisted of hydrophilic PEG chain segments and hydrophobic random PPMS chain segments, which self-assembled in aqueous medium to form stable, nanosized micelles at physiological pH of 7.4. Upon decreasing the medium pH from 7.4 to 5.0, the copolymer micelles swell significantly due to protonation of the amino groups in the micelle PPMS cores. Correspondingly, docetaxel (DTX)-encapsulated PEG2K-PPMS copolymer micelles showed gradual sustained drug release at pH of 7.4, but remarkably accelerated DTX release at acidic pH of 5.0. The drug-loaded micelle particles were readily internalized by SK-BR-3 cancer cells and, compared to free DTX drug, DTX-loaded micelles of the copolymers with optimal compositions exhibited enhanced potency against the cells. Biodegradable PEG-PPMS copolymer micelles represent a new type of promising, pH-responsive nanocarriers for anticancer drug delivery, and the drug release rate from the micelles can be systematically controlled by both pH and the copolymer composition.

PEGylated poly(amine-co-ester) micelles as biodegradable non-viral gene vectors with enhanced stability, reduced toxicity and higher in vivo transfection efficacy.

Nanosized micelles based on cationic, amphiphilic poly(ethylene glycol)-poly(ω-pentadecalactone-co-N-methyldiethyleneamine-co-sebacate) (PEG-PPMS) block copolymers have been successfully developed to serve as a new type of biodegradable non-viral vectors for DNA delivery. PEG-PPMS copolymers with various compositions were synthesized in one step via lipase-catalyzed copolymerization of ω-pentadecalactone (PDL), N-methyldiethanolamine (MDEA) and diethyl sebacate (DES) with poly(ethylene glycol) methyl ether (MeO-PEG-OH). The effects of PEG molecular weight, PEG and PDL contents on the biological properties (including the gene transfection efficiency) of the copolymer micelles were investigated. The LucDNA-loaded micelles formed from the copolymers with 30-50 wt% PEG showed high stability in serum-containing aqueous medium, which is in sharp contrast to rapid aggregation of LucDNA/PPMS polyplex particles. The conjugation of PEG to PPMS chains significantly reduces the cytotoxicity and hemolysis activity of the PEG-PPMS micelles. Compared to PEG-free PPMS, the micelles of PEG-PPMS copolymers with optimal compositions (e.g., 42%PEG5K-PPMS-10%PDL and 42%PEG5K-PPMS-20%PDL) exhibited enhanced capability to condense and protect DNA. Although the optimized micelles showed comparable or slightly lower gene transfection efficacy than the reference PPMS in vitro, the efficiency of LucDNA/42%PEG5K-PPMS-20%PDL micelles in transfecting tumor cells in mice was twice as high as that of LucDNA/PPMS polyplex particles due to their strong DNA condensation ability and excellent stability under physiological conditions. The PEG-PPMS micelle system with improved properties is a family of potentially promising non-viral vectors for in vivo delivery of therapeutic genes to treat tumors.

Targeted delivery of 10-hydroxycamptothecin to human breast cancers by cyclic RGD-modified lipid-polymer hybrid nanoparticles.

Lipid-polymer hybrid nanoparticles (NPs) combining the positive attributes of both liposomes and polymeric NPs are increasingly being considered as promising candidates to carry therapeutic agents safely and efficiently into targeted sites. Herein, a modified emulsification technique was developed and optimized for the targeting lipid-polymer hybrid NPs fabrication; the surface properties and stability of the hybrid NPs were systematically investigated, which confirmed that the hybrid NPs consisted of a poly (lactide-co-glycolide) core with ∼90% surface coverage of the lipid monolayer and a ∼4.4 nm hydrated polyethylene glycol (PEG) shell. Optimization results showed that the lipid:polymer mass ratio and the lipid-PEG:lipid molar ratio could affect the size, lipid association efficiency and stability of hybrid NPs. Furthermore, a model chemotherapy drug, 10-hydroxycamptothecin, was encapsulated into hybrid NPs with a higher drug loading compared to PLGA NPs. Surface modification of the lipid layer and the PEG conjugated targeting ligand did not affect their drug release kinetics. Finally, the cytotoxicity and cellular uptake studies indicated that the lipid coverage and the c(RGDyk) conjugation of the hybrid NPs gained a significantly enhanced ability of cell killing and endocytosis. Our results suggested that lipid-polymer hybrid NPs prepared by the modified emulsion technique have great potential to be utilized as an engineered drug delivery system with precise control ability of surface targeting modification.

[Purification of chymopapain and its effect on nucleus pulposus in vitro].

OBJECTIVE: To establish a simple and effective procedure for purification of chymopapain and study about its effect on nucleus pulposus in vitro. METHODS: Chymopapain was purified by ion exchange chromatograph and tested its effect on nucleus pulposus in vitro. RESULTS: A simple and effective procedure for purification of chymopapain was established and the chymopapain did degrade nucleus pulposus. CONCLUSION: The ion exchange chromatograph was a simple and effective procedure for purification of chymopapain. It is necessary to study its effect on nucleus pulposus in vivo.