Xiangrong Song

Dual agents loaded PLGA nanoparticles: systematic study of particle size and drug entrapment efficiency.

PLGA nanoparticles simultaneously loaded with vincristine sulfate (VCR) and quercetin (QC) were prepared via O/W emulsion solvent evaporation. Six independent processing parameters and PLGA characteristics were assessed systematically to enhance the incorporation of the dual agents with different properties (VCR and QC, hydrophilic and hydrophobic molecule, respectively) into PLGA nanoparticles and control particle size. Approaches investigated for the enhancement of drug entrapment efficiencies and the controlling of particle size included the influence of the molecular weight (MW) of PLGA and the lactide-to-glycolide (L:G) ratio of PLGA, PLGA concentration, PVA concentration, initial QC content, acetone-to-dichloromethane (A/D) volume ratio, aqueous phase pH and aqueous to organic phase (W/O) volume ratio. The nanoparticles produced by optimal formulation were submicron size (139.5+/-4.3 nm, n=3) with low polydispersity index (0.095+/-0.031, n=3). Nanoparticles observed by transmission electron microscopy (TEM) showed extremely spherical shape. The entrapment efficiencies determined by high performance liquid chromatography (HPLC) by ultracentrifuge method were 92.84+/-3.37% for VCR and 32.66+/-2.92% for QC (n=3). The drug loadings were 0.0037+/-0.0001% for VCR and 1.36+/-0.12% for QC (n=3).

Receptor mediated gene delivery by folate conjugated N-trimethyl chitosan in vitro

Folate conjugated N-trimethyl chitosan (folate-TMC) that was used for intracellular delivery of protein before was studied as a gene delivery carrier in this study using N-trimethyl chitosan (TMC) as a reference. MTT assay indicated that the two polymers were much less toxic than PEI. Agarose gel electrophoresis indicated that the two polymers effectively condensed pDNA. TMC/pDNA complex and folate-TMC/pDNA complex were nano-scale spherical particles confirmed by atomic force microscopy. Cellular uptake of the folate-TMC/pDNA complex containing YOYO-1 labeled pDNA in KB cells was enhanced compared with that of the TMC/pDNA complex and was inhibited by free folate (1 mM) in the medium. Transfection efficiency of the folate-TMC/pDNA complex in KB cells and SKOV3 cells (folate receptor over-expressing cell lines) increased with increasing N/P ratio and were enhanced up to 1.6-fold and 1.4-fold compared with those of the TMC/pDNA complexes, however, there was no significant difference between transfection efficiencies of the two complexes in A549 cells and NIH/3T3 cells (folate receptor deficient cell lines). It was concluded that the increase in transfection efficiencies of the folate-TMC/pDNA complexes were attributed to folate receptor mediated endocytosis. Subcellular distributions of both of the complexes at different time points in the process of cellular uptake were examined by confocal laser scanning microscope, which suggested that different intracellular trafficking pathways were employed by the two complexes.

Novel thienopyridine derivatives as specific anti-hepatocellular carcinoma (HCC) agents: synthesis, preliminary structure-activity relationships, and in vitro biological evaluation.

Novel thienopyridine derivatives 1b-1r were synthesized, based on a hit compound 1a that was found in a previous cell-based screening of anticancer drugs. Compounds 1a-1r have the following features: (1) their anticancer activity in vitro was first reported by our group. (2) The most potent analog 1g possesses hepatocellular carcinoma (HCC)-specific anticancer activity. It can specifically inhibit the proliferation of the human hepatoma HepG2 cells with an IC(50) value of 0.016μM (compared with doxorubicin as a positive control, whose IC(50) was 0.37μM). It is inactive toward a panel of five different types of human cancer cell lines. (3) Compound 1g remarkably induces G(0)/G(1) arrest and apoptosis in HepG2 cells in vitro at low micromolar concentrations. These results, especially the HCC-specific anticancer activity of 1g, suggest their potential in targeted chemotherapy for HCC.

Preparation and characterization of folate conjugated N-trimethyl chitosan nanoparticles as protein carrier targeting folate receptor: in vitro studies.

Folate conjugated N-trimethyl chitosan (folate-TMC) was synthesized and characterized using Fourier transform infrared (FTIR) and 1H spectroscopy. The fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) loaded TMC-nanoparticle (FB-TMC-NP) and FITC-BSA loaded folate-TMC-nanoparticle (FB-f-TMC-NP) were prepared by ionic cross-linking of TMC or folate-TMC with sodium alginate. Single factor analysis method was used to optimize the formulation of nanoparticles. The encapsulating efficiencies of FB-TMC-NP and FB-f-TMC-NP produced by optimal formulation were 98.3 ± 1.9% and 98.7 ± 2.7% (n=3), respectively. In addition, the mean diameters of FB-TMC-NP and FB-f-TMC-NP were 184.3 ± 8.3 nm and 176.1 ± 5.0 nm (n = 3), respectively. Transmission electron microscope (TEM) showed that the nanoparticles were of spherical shapes. The intracellular uptake of FB-f-TMC-NP by SKOV3 cells (folate receptor overexpressing cells) was 3.7-fold more than that of FB-TMC-NP and could be inhibited by the presence of 1 mM folate in the culture medium, although there was no significant difference between the intracellular uptake of FB-f-TMC-NP in A549 cells (folate receptor–deficient cells) and that of FB-TMC-NP in the same cells. In conclusion, the enhancement of cellular uptake of FB-f-TMC-NP by SKOV3 cells in a specific way was attributed to the folate receptor–mediated endocytosis. FB-TMC-NP was a promising carrier for protein.

Calcium phosphate embedded PLGA nanoparticles: a promising gene delivery vector with high gene loading and transfection efficiency.

In the purpose of increasing incorporation efficiency and improving the release kinetics of plasmid DNA (pDNA) from poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles, a facile method for the fabrication of calcium phosphate (CaPi) embedded PLGA nanoparticles (CaPi-pDNA-PLGA-NPs) was developed. The effect of several preparation factors on the particle size, incorporation efficiency, pDNA release and transfection efficiency in vitro was studied by Single Factor Screening Method. These preparation factors included the molecular weight (MW), hydrolysis degree (HD) of polyvinyl alcohol (PVA), sonication power and time, composition of organic phase, initial concentration of calcium phosphate and calcium (Ca) to phosphate ion (P) ratio (Ca/P ratio), etc. The CaPi-pDNA-PLGA-NPs made according to the optimal formulation were spherical in shape observed by transmission electron microscopy (TEM) with a mean particle size of 207±5 nm and an entrapment efficiency of 95.7±0.8%. Differential scanning calorimetry (DSC) suggested that there existed interaction between the DNA-calcium-phosphate (CaPi-pDNA) complexes and the polymeric matrices of PLGA. X-ray diffractometry (XRD) further proved the conclusion and indicated that the CaPi-pDNA was in weak crystallization form inside the nanoparticles. The Brunauer-Emmett-Teller (BET) surface area measurement demonstrated that the CaPi-pDNA-PLGA-NPs are mesoporous with specific surface area of 57.5m(2)/g and an average pore size of 96.5 Å. The transfection efficiency of the CaPi-pDNA-PLGA-NPs on human embryonic kidney 293 (HEK 293) cells in vitro was 22.4±1.2%, which was much higher than those of both the pDNA loaded PLGA nanoparticles (pDNA-PLGA-NPs) and the CaPi-pDNA embedded PLGA microparticles (CaPi-pDNA-PLGA-MPs). The CaPi-pDNA-PLGA-NPs are promising vectors for gene delivery.

Development of Lipid-Shell and Polymer Core Nanoparticles with Water-Soluble Salidroside for Anti-Cancer Therapy

Salidroside (Sal) is a potent antitumor drug with high water-solubility. The clinic application of Sal in cancer therapy has been significantly restricted by poor oral absorption and low tumor cell uptake. To solve this problem, lipid-shell and polymer-core nanoparticles (Sal-LPNPs) loaded with Sal were developed by a double emulsification method. The processing parameters including the polymer types, organic phase, PVA types and amount were systemically investigated. The obtained optimal Sal-LPNPs, composed of PLGA-PEG-PLGA triblock copolymers and lipids, had high entrapment efficiency (65%), submicron size (150 nm) and negatively charged surface (−23 mV). DSC analysis demonstrated the successful encapsulation of Sal into LPNPs. The core-shell structure of Sal-LPNPs was verified by TEM. Sal released slowly from the LPNPs without apparent burst release. MTT assay revealed that 4T1 and PANC-1 cancer cell lines were sensitive to Sal treatment. Sal-LPNPs had significantly higher antitumor activities than free Sal in 4T1 and PANC-1 cells. The data indicate that LPNPs are a promising Sal vehicle for anti-cancer therapy and worthy of further investigation.

Efficient inhibition of ovarian cancer by short hairpin RNA targeting claudin-3.

Ovarian cancer is one of the most lethal gynecologic neoplasms. Even though various new chemotherapeutics have been developed for the treatment of ovarian cancer, drug resistance and undesired serious side effects remain unavoidable obstacles for chemotherapeutic approaches. New strategies to overcome the therapeutic dilemma are needed. Claudin-3 (CLDN3) is a recently discovered gene generally overexpressed in human ovarian cancers but not in normal ovarian tissue. Its high expression has been identified to associate with the invasion, proliferation and survival of cancer cells, making it a promising target for gene therapy of ovarian cancer. However, in gene therapy, traditional gene carriers such as virus or cationic liposomes suffer from distressing shortcomings of potential carcinogenicity, obvious cytotoxicity and immunogenicity. Nanoparticles (NPs) based on PLGA are a novel gene delivery system with good biodegradability, excellent biocompatibility and low toxcity for in vivo gene delivery compared with traditional gene carriers. We constructed a plasmid expressing shRNA targeted CLDN3 (pshCLDN3) encapsulated with PLGA-NPs, and administered it by i.p. injection to nude mice bearing intraperitoneal SKOV3 ovarian cancer, to investigate the antitumor potential of knocking down CLDN3. After 12 times of administration, the tumors of each group were compared. The underlying antitumor mechanisms were revealed by immunostaining of CD31, Ki-67 and TUNEL assay, to exhibit possible alterations in microvessel density, cell proliferation and cell apoptosis. Our study demonstrated that i.p. administration of pshCLDN3 effectively suppressed the expression of CLDN3 and, thus, inhibited the growth of ovarian tumors, significantly reducing tumor weight by 67.4% compared with blank controls (p<0.05). Immunostaining of CD31, Ki-67 and TUNEL assay demonstrated decreased angiogenesis (p<0.05), reduced proliferation (p<0.05) and increased apoptosis (p<0.05) in the pshCLDN3 treated group compared with controls. No obvious toxicity of PLGA-NPs was observed either in vitro or in vivo. Our results indicated that knockdown of CLDN3 by pshCLDN3 encapsulated in PLGA NPs may provide a promising approach for the treatment of ovarian cancer.

Preparation and characterization of folate-poly(ethylene glycol)-grafted-trimethylchitosan for intracellular transport of protein through folate receptor-mediated endocytosis.

To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins to specific tumor cells, folate-poly(ethylene glycol)-grafted-trimethylchitosan (folate-PEG-g-TMC) was synthesized. Nano-scaled spherical polyelectrolyte complexes between the folate-PEG-g-TMC and fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) were prepared under suitable weight ratio of copolymer to FITC-BSA by ionic interaction between the positively charged copolymers and the negatively charged FITC-BSA. Intracellular uptake of FITC-BSA was specifically enhanced in SKOV3 cells (folate receptor over-expressing cell line) through folate receptor-mediated endocytosis compared with A549 cells (folate receptor deficient cell line). Folate-PEG-g-TMC shows promise for intracellular transport of negatively charged therapeutic proteins into folate receptor over-expressing tumor cells.

Self-Assembled Methoxy Poly(Ethylene Glycol)-Cholesterol Micelles for Hydrophobic Drug Delivery

To promote the application of methoxy poly(ethylene glycol)-cholesterol (mPEG-Chol), mPEG-Chol was used to prepare core-shell micelles encapsulating poorly water-soluble docetaxel (DTX-PM) by modified cosolvent evaporation method. Approaches to enhance DTX entrapment efficiency (EE) and minimize particle size were investigated in detail, including organic and aqueous phase composition, organic/aqueous phase ratio, and polymer concentration. In optimal formulation, micelles had higher EE (97.6%) and drug loading (4.76%) with the diameter of 13.76 ± 0.68 nm and polydispersity index of 0.213 ± 0.006. Transmission electron microscopy (TEM) showed that the micelles were spherical, and differential scanning calorimetry (DSC) analysis proved that DTX was successfully entrapped into mPEG-Chol micelles. The in vitro cytotoxicity experiments displayed that blank micelles had no effect on the growth of SKOV-3, BXPC-3, A549, and HepG-2 cells, demonstrating that mPEG-Chol was one of the biocompatible biomaterials. The half inhibition concentration of DTX-PM on SKOV-3, BXPC-3, A549, and HepG-2 cells were 10.08, 7.6, 28.37, and 125.75 ng/mL, respectively. DTX-PM had the similar antitumor activity to free DTX, indicating that mPEG-Chol was a promising micellar vector for hydrophobic drug delivery. In addition, this work provided a new and facile approach to prepare drug-loaded micelles with controllable performances.

Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts☆

Ovarian cancers highly overexpress folate receptor α (FRα) and claudin3 (CLDN3), both of which are associated with tumor progression and poor prognosis of patients. Downregulation of FRα and CLDN3 in ovarian cancer may suppress tumor growth and promote benign differentiation of tumor. In this study, F-P-LP/CLDN3, a FRα targeted liposome loading with short hairpin RNA (shRNA) targeting CLDN3 was prepared and the pharmaceutical properties were characterized. Then, the antitumor effect of F-P-LP/CLDN3 was studied in an in vivo model of advanced ovarian cancer. Compared with Control, F-P-LP/CLDN3 promoted benign differentiation of tumor and achieved about 90% tumor growth inhibition. In the meantime, malignant ascites production was completely inhibited, and tumor nodule number and tumor weight were significantly reduced (p<0.001). FRα and CLDN3 were downregulated together in tumor tissues treated by F-P-LP/CLDN3. The antitumor mechanisms were achieved by promoting tumor cell apoptosis, inhibiting tumor cell proliferation and reducing microvessel density. Finally, safety evaluation indicated that F-P-LP/CLDN3 was a safe formulation in intraperitoneally administered cancer therapy. We come to a conclusion that F-P-LP/CLDN3 is a potential targeting formulation for ovarian cancer gene therapy.