Xiaofen Xu

The use of quaternised chitosan-loaded PMMA to inhibit biofilm formation and downregulate the virulence-associated gene expression of antibiotic-resistant staphylococcus.

Biomaterial-associated infections remain a serious complication in orthopaedic surgery. Treatments, including the local use of antibiotic-loaded polymethylmethacrylate (PMMA) bone cement, are not always successful because of multiantibiotic-resistant organisms. In this study, we synthesised a new quaternised chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) that contains a series of substitutions of quaternary ammonium and demonstrated that HACC with a 26% degree of substitution (DS; referred to as 26%HACC) had a strong antibacterial activity and simultaneously good biocompatibility with osteogenic cells. We loaded 26%HACC at 20% by weight into PMMA bone cement to investigate whether HACC in PMMA prevents bacterial biofilm formation on the surface of bone cements. Chitosan-loaded PMMA (at the same weight ratio), gentamicin-loaded PMMA and PMMA with no antibiotic were also investigated and compared. Two clinical isolates, Staphylococcus epidermidis 389 and methicillin-resistant S. epidermidis (MRSE287), and two standard strains, S. epidermidis (ATCC35984) and methicillin-resistant Staphylococcus aureus (ATCC43300), were selected to evaluate the bacterial biofilm formation at 6, 12 and 24 h using the spread plate method, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results showed that 26%HACC-loaded PMMA inhibited biofilm formation on its surface, while the PMMA control and chitosan-loaded PMMA were unable to inhibit biofilm formation. The gentamicin-loaded PMMA decreased the number of viable methicillin-resistant Staphylococcus strains, but its ability to inhibit biofilm formation was lower than 26%HACC-loaded PMMA. Real-time PCR demonstrated that 26%HACC-loaded PMMA markedly downregulated the expression of icaAD, which encodes essential enzymes for polysaccharide intercellular adhesion (PIA) biosynthesis, upregulated the expression level of icaR, which negatively mediates icaAD expression, and also downregulated the expression of MecA, which encodes membrane-bound enzymes known to be penicillin-binding proteins. Our study indicates that 26%HACC-loaded PMMA prevents biofilm formation of Staphylococcus, including antibiotic-resistant strains, on the surface of bone cement, and downregulates the virulence-associated gene expression of antibiotic-resistant staphylococcus, thus providing a promising new strategy for combating implant infections and osteomyelitis.

Physical characterization and osteogenic activity of the quaternized chitosan-loaded PMMA bone cement.

Gentamicin-loaded polymethylmethacrylate (PMMA), widely used for primary cemented arthroplasty and revision surgery for preventing or treating infections, may lead to the evolution of antibiotic-resistant bacteria and dysfunction of osteogenic cells, which further influence the osteointegration of bone cement. In a previous study, we reported that a new quaternized chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) that was loaded into PMMA significantly inhibited the formation of biofilms caused by methicillin-resistant Staphylococcus strains. In the present study, we further investigated the surface morphology, hydrophilicity, apatite formation ability and osteogenic activity of HACC-loaded PMMA. Chitosan-loaded PMMA, gentamicin-loaded PMMA and PMMA without antibiotic were also investigated and compared. The results showed that, compared to other PMMA-based cements, HACC-loaded PMMA had improved properties such as a lower polymerization temperature, prolonged setting time, porous structures after immersion in phosphate-buffered saline, higher hydrophilicity, more apatite formation on the surface after immersion in simulated body fluid, and better attachment and spreading of the human-marrow-derived mesenchymal stem cells. We also found better stem cell proliferation, osteogenic differentiation, and osteogenesis-associated genes expression on the surface of the HACC-loaded PMMA compared to the gentamicin-loaded PMMA. Therefore, this new anti-infective bone cement had improved physical properties and osteogenic activity, which may lead to better osteointegration of the bone cement in cemented arthroplasty.

Evaluation of antibacterial activity of N-phosphonium chitosan as a novel polymeric antibacterial agent

N-phosphonium chitosans (NPCSs) with different degrees of substitution (3%, 13% and 21%) were synthesized and evaluated as novel polymeric antibacterial agents. Their antibacterial activities compared with hydroxypropyltrimethyl ammonium chloride chitosan (HACC), parent chitosan and (5-carboxypentyl) triphenylphosphonium bromide (CTPB) were tested against Escherichia coli and two strains of drug-resistance Staphylococcus aureus by minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and biofilm prevention assays. The results show that the NPCS with 3% or 13% substitution has lower MIC and MBC values and stronger ability to inhibit biofilm formation of all the three bacteria than HACC, chitosan and CTPB. In addition, the antibacterial activity of NPCSs increases with their substitution decreasing from 21% to 3%. Overall, the antibacterial activity of NPCS with 3% or 13% substitution is better than that of NPCS with 21% substitution, HACC with 22% substitution, chitosan and CTPB. It can be considered that NPCS with appropriate degree of substitution has favorable antibacterial activity and is a potential polymeric antibacterial agent.

Novel water soluble phosphonium chitosan derivatives: synthesis, characterization and cytotoxicity studies.

Novel water soluble phosphonium chitosan derivatives (WSPCSs) with two different degrees of substitution (3.6% and 4.2%) of quaternary phosphonium were synthesized in a homogeneous system at 25°C. The chemical structures of the WSPCS were characterized by (31)P NMR (nuclear magnetic resonance), (1)H NMR, FT-IR (Fourier-transformed infrared) spectroscopy and WAXD (wide-angle X-ray diffraction). Their solubility in water and several organic solvents and cytotoxicity to L929 cells were also evaluated. The degree of substitution (DS) of WSPCS was calculated by (1)H NMR. WAXD analysis showed that the derivatives had low crystallinity. The derivatives could be easily dissolved in water. MTT (methyl thiazolyl tetrazolium) assay indicated that they had low cytotoxicity to L929 cells.

Selective tissue distribution and long circulation endowed by paclitaxel loaded PEGylated poly(ɛ-caprolactone-co-l-lactide) micelles leading to improved anti-tumor effects and low systematic toxicity

High tumor targeting and sustained drug concentration are key points for successful anti-tumor therapy, however, it is a challenging task. In this work, a novel micelle formulation of paclitaxel (PTX) has been prepared for the purpose of prolonging the blood circulation time as well as improving the accumulation of the drug within the tumor tissue. PEGylated P(CL-co-LLA) (poly(ε-caprolactone-co-L-lactide)) micelles containing PTX were prepared by solid dispersion-sonication method with a higher drug-loading efficiency and encapsulation ratio (28.4% and 94.7%, respectively). Pharmacokinetic study revealed that the drug-loading micelles exhibited a higher AUC values and a prolonged residence time of drug in the blood circulation than those of PTX injection. As demonstrated by tissue distribution and anti-tumor study in S180 tumor-bearing mice, the PEG-P(CL-co-LLA)/PTX micelles displayed modified tissue distribution of PTX and increased accumulation of PTX in tumor, therefore, resulted in anti-tumor effects enhancement and drug concentration in the normal tissues reduction. Furthermore, the preliminary safety tests were performed by measuring the body weight, histopathology, blood cell counts and clinical chemistry parameters, and the results showed no subacute toxicity to hematological system, major organs or tissues in mice. Taken together, our valuation shows that PEG-P(CL-co-LLA) micelles is a potential drug delivery system of PTX for the effective treatment of the tumor and systematic toxicity reduction, thus, the micellar formulation can provide a useful alternative dosage form for i.v. administration of PTX.

Synthesis and preliminary cellular evaluation of phosphonium chitosan derivatives as novel non-viral vector.

In this study, N-phosphonium chitosans (NPCSs) with two degrees of substitution were synthesized in a homogeneous system as nonviral gene vectors. Grafted polymer/DNA complexes at various charge ratios were formulated and characterized. Particle sizes of NPCS/DNA complexes were between 110 and 160 nm as determined by dynamic light scattering. Accordingly, scanning electron microscopy photo of NPCS/DNA complexes exhibited a compact morphology. Zeta potentials of these complexes changed as the charge ratio and pH varied. The cytotoxicity assay showed that NPCS polymers were less toxic than branched PEI-25K. Furthermore, gene transfection efficiencies of NPCS/DNA complexes showed that the gene transfection ability of the grafted polymer was much better than chitosan and NPCS with the degree of substitution of 21.5% had comparative gene transfection efficiency to branched PEI-25K. Together, these results suggest that the low toxic NPCS grafted polymers could be used as effective gene delivery vectors.

Synthesis, in vitro and in vivo evaluation of new norcantharidin-conjugated hydroxypropyltrimethyl ammonium chloride chitosan derivatives as polymer therapeutics.

New norcantharidin-conjugated hydroxypropyltrimethyl ammonium chloride chitosan derivatives (NCTD-HACCs) were synthesized and characterized by (1)H NMR, Fourier-transform infrared spectroscopy (FT-IR), and wide-angle X-ray diffraction (WAXD). Two NCTD-HACCs with different degrees of substitution (DS) (12.2% and 24.8%) were obtained, which had good water solubility. NCTD was released from the NCTD-HACCs via hydrolysis, faster in pH 5.0 than pH 7.4 and presenting one biphasic drug release pattern with rapid release at the initial stage and slow release later. Fluorescence microscope and flow cytometry analysis demonstrated that the NCTD-HACC was endocytosized into MGC80-3 cells and the uptaken amount increased as incubation time. Compared with free NCTD, the NCTD-HACCs showed lower in vitro anti-tumor activity against human gastric cancer MGC80-3 cells, but higher in vivo tumor growth inhibition in S180 tumor-bearing mice. The in vivo near-infrared (NIR) fluorescence real-time imaging result showed the fluorescence intensity in tumor was much higher than that in heart, liver, spleen and lung (except kidney) after i.v. injection of the FITC-labeled NCTD-HACC2, indicating specific accumulation of the NCTD-HACC in tumor.

Novel 4-Arm Poly(Ethylene Glycol)-Block-Poly(Anhydride-Esters) Amphiphilic Copolymer Micelles Loading Curcumin: Preparation, Characterization, and In Vitro Evaluation

A novel 4-arm poly(ethylene glycol)-block-poly(anhydride-esters) amphiphilic copolymer (4-arm PEG-b-PAE) was synthesized by esterization of 4-arm poly(ethylene glycol) and poly(anhydride-esters) which was obtained by melt polycondensation of α-, ω-acetic anhydride terminated poly(L-lactic acid). The obtained 4-arm PEG-b-PAE was characterized by 1H-NMR and gel permeation chromatography. The critical micelle concentration of 4-arm PEG-b-PAE was 2.38 μg/mL. The curcumin-loaded 4-arm PEG-b-PAE micelles were prepared by a solid dispersion method and the drug loading content and encapsulation efficiency of the micelles were 7.0% and 85.2%, respectively. The curcumin-loaded micelles were spherical with a hydrodynamic diameter of 151.9 nm. Curcumin was encapsulated within 4-arm PEG-b-PAE micelles amorphously and released from the micelles, faster in pH 5.0 than pH 7.4, presenting one biphasic drug release pattern with rapid release at the initial stage and slow release later. The hemolysis rate of the curcumin-loaded 4-arm PEG-b-PAE micelles was 3.18%, which was below 5%. The IC50 value of the curcumin-loaded micelles against Hela cells was 10.21 μg/mL, lower than the one of free curcumin (25.90 μg/mL). The cellular uptake of the curcumin-loaded micelles in Hela cell increased in a time-dependent manner. The curcumin-loaded micelles could induce G2/M phase cell cycle arrest and apoptosis of Hela cells.

Primary in vitro and in vivo evaluation of norcantharidin-chitosan/poly (vinyl alcohol) for cancer treatment

Abstract Two novel polymer–drug conjugates norcantharidin-poly(vinyl alcohol) and norcantharidin-chitosan (NCTD-PVA and NCTD-CS) were synthesized via alcoholysis reaction and characterized by 1H-NMR and FTIR. NCTD was released from the conjugates via hydrolysis, faster in PBS (pH 5.0) than that in PBS (pH 7.4). NCTD-PVA and NCTD-CS inhibited human esophageal carcinoma ECA-109 cell and murine breast cancer EMT6 cell growth in a dose-dependent manner. The IC50 values of NCTD, NCTD-PVA and NCTD-CS on ECA-109 cell at 48 h were 9.4 ± 0.9, 55.3 ± 3.0 and 168.8 ± 8.9 μg/ml, respectively, and the IC50 values of the three compounds on EMT6 cell were 3.1 ± 0.3, 30.5 ± 5.4 and 90.7 ± 8.1 μg/ml, respectively. The two conjugates both induced esophageal carcinoma ECA-109 cell apoptosis and arrested cell cycle at the S phase. Caspase-8 and caspase-3 were activated in the ECA-109 cell after incubating with NCTD-PVA or NCTD-CS. The primary in vivo antitumor activity was assessed in the EMT6 tumor-bearing mouse model. NCTD-PVA and NCTD-CS displayed higher tumor inhibition rates than that of free NCTD.

Investigation on formulation and preparation of adenovirus encoding human endostatin lyophilized powders

A recombinant adenovirus encoding human endostatin gene, E10A, has finished phase II trials for head and neck cancer. However, the rigid storage temperature (-80°C) and the toxicity of glycerol in the E10A liquid preparation limited its clinical application. In this study, lyophilization was applied to develop a stable E10A lyophilized powder without glycerol that is able to maintain biological activity at 4°C and suitable for intravenous administration. The E10A lyophilized formulations composed of nontoxic and already clinically used excipients were characterized in terms of the pH change during freezing, the eutectic melting temperature (T(eu)) and the collapse temperature (T(c)). Freeze thawing tests were carried out to examine the protective effect of various excipients during freezing. Mannitol and its combinations with sucrose or inulin showed effective protection of E10A. The E10A lyophilized powders were analyzed by particle size measurement, residual humidity quantification, infectivity assay and gene expression level. An optimized formulation (formulation I1) yielded a good recovery of 76% of the starting infectivity after lyophilization and 89% of the original infectivity after storage at 4°C for 180 days. Also the gene expression capability of E10A in formulation I1 was maintained after lyophilization. In addition, it was found that the matrix of amorphous excipients, mannitol combinations with sucrose or inulin, was indispensible in protecting E10A against the stress of freezing and dehydration. Hereby, the E10A lyophilized powder with eliminated glycerol toxicity and improved stability could enhance the applicability of E10A for cancer gene therapy through intravenous administration.