Shengrong Guo

Recent progress of cell-penetrating peptides as new carriers for intracellular cargo delivery.

The plasma membrane as a selectively permeable barrier of living cells is essential to cell survival and function. In many cases, however, the efficient passage of exogenous bioactive molecules through the plasma membrane remains a major hurdle for intracellular delivery of cargoes. During the last two decades, the potential of peptides for drug delivery into cells has been highlighted by the discovery of numerous cell-penetrating peptides (CPPs). CPPs serving as carriers can successfully intracellular transport cargoes such as siRNA, nucleic acids, proteins, small molecule therapeutic agents, quantum dots and MRI contrast agents. This review mainly introduces recent advances of CPPs as new carriers for the development of cellular imaging, nuclear localization, pH-sensitive and thermally targeted delivery systems. In particular, we highlight the exploiting of the synergistic effects of targeting ligands and CPPs. Whats more, the classification and cellular uptake mechanisms of CPPs are briefly discussed as well.

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.

Quaternized Chitosan Inhibits icaA Transcription and Biofilm Formation by Staphylococcus on a Titanium Surface

ABSTRACT Our previous study (Z. X. Peng et al., Carbohydr. Polym. 81:275-283, 2010) demonstrated that water-soluble quaternary ammonium salts, which are produced by the reaction of chitosan with glycidyl trimethylammonium chloride, provide chitosan derivatives with enhanced antibacterial ability. Because biofilm formation is believed to comprise the key step in the development of orthopedic implant-related infections, we further evaluated the efficacy of hydroxypropyltrimethyl ammonium chloride chitosan (HACC) with different degrees of substitution (DS; referred to as HACC 6%, 18%, and 44%) in preventing biofilm formation on a titanium surface. We used a tissue culture plate method to quantify the biomass of Staphylococcus epidermidis and Staphylococcus aureus biofilms and found that HACC, especially HACC 18% and 44%, significantly inhibited biofilm formation compared to the untreated control, even at concentrations far below their MICs (P < 0.05). Scanning electron microscopy showed that inhibition of biofilm formation on titanium increased dramatically with increased DS and HACC concentrations. Confocal laser scanning microscopy indicated that growth of a preexisting biofilm on titanium was inhibited by concentrations of HACC 18% and 44% below their minimum biofilm eradication concentrations. We also demonstrated that HACC inhibited the expression of icaA, which mediates the production of extracellular polysaccharides, both in new biofilms and in preexisting biofilms on titanium. Our results indicate that HACC may serve as a new antibacterial agent to inhibit biofilm formation and prevent orthopedic implant-related infections.

Characterization and in vitro release of praziquantel from poly(ɛ-caprolactone) implants

Poly(epsilon-caprolactone) (PCL) implants containing praziquantel (PZQ), a broad-spectrum antiparasite drug, are fabricated by injection molding and characterized in terms of content uniformity, morphology, drug physical state and stability. In vitro drug release from the implants is also studied. It is found that drug is dispersed uniformly in all implants and keeps stable over 365 days at 4 degrees C/60% RH. X-ray diffraction analysis reveals that PZQ exists primarily in its crystalline state in implants with high drug contents (50% and 25%). All implants exhibit similar release behaviors and about 70% of the drug is released after 365 days. The cross-sections of all implants present two distinct zones (i.e. peripheral white zone and inner pink zone) and the boundary between the two zones changes as time progresses. Drug content in the white zone is very low (less than 1%), but drug content in the pink zone is almost the same as the predefined value. Porous structures in the white zone but dense structures in the pink zone are observed by SEM. Obvious PCL degradation occurs till up to 365 days. These results show that the release process of PZQ is a gradual diffusion from the exterior to the interior of the implants.

In vitro and in vivo evaluation of praziquantel loaded implants based on PEG/PCL blends.

In the present study, a series of praziquantel (PZQ) loaded implants based on PEG/PCL blends are fabricated by a combination of twin-screw mixing and hot-melt extrusion. In vitro drug release from these implants and the performance of the implants after implantation in rats are evaluated. XRD and DSC analysis results exhibit each component in the implants is mainly in its crystalline state. Dissolution test shows that the higher PEG content there is in the implants, the faster the drug will be released. Interestingly, PEG release from all implants is far faster than PZQ release, and complete PEG release occurs in 72 h. SEM result displays that after the in vitro drug release test, the cross-sections of implants with low PEG contents (0-5%) primarily consist of discrete pores; while those of implants with high PEG contents (10-30%) consist of interconnected pores or channels. The fitting results of drug release data with kinetic models reveal that PZQ release is governed by diffusion. After implantation, drug release becomes more moderate compared with in vitro drug release, and it tends to follow zero-order in the later stage. These results suggest that changing the composition of the PEG/PCL blends is an effective tool to adjust in vitro/in vivo drug release from the implants.

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.

5-Fluorouracil-loaded multilayered films for drug controlled releasing stent application: Drug release, microstructure, and ex vivo permeation behaviors

A series of poly(epsilon-caprolactone) (PCL)-based multilayered films containing antitumor 5-fluorouracil as stent struts were produced. A backing layer and a surface drug layer were applied to the main drug layer to realize unidirectional, controlled drug release and enhance the mechanical properties. The intricate multilayered structure endowed the films with flexibility to regulate drug release. The in vitro release results showed that drug release was dependent on the drug loading and environmental pH, and could also be effectively regulated by addition of hydrophilic PEG as pore forming agent. Drug release from the multilayered film was found to be dominated by diffusion mechanism. Rapid PEG release led to the formation of pores and the resulting fast drug release. SEM images revealed that the 5-FU crystalline particles were homogeneously dispersed in the film, and there emerged many pores on the surface and in the bulk of film after PEG and drug release, indicating that the porous structure of film was formed and gradually evolved with drug and PEG release. Ex vivo permeation behaviors of drug from the film through porcine esophageal mucosa were in high correlation with the in vitro release behaviors, and the permeation rate was determined by the release of drug from film. With the virtue of releasing drug in a unidirectional and controlled manner, the multilayered films provide an attractive mode to produce polymeric drug delivery stents for localized treatment of stenosis or occlusion.

Smart gelation of chitosan solution in the presence of NaHCO3 for injectable drug delivery system

In situ gelling systems are attractive as injectable vehicles for drug delivery. The present work described a novel gelation process of acidic chitosan solution in the presence of sodium bicarbonate (NaHCO(3)). The NaHCO(3) concentration played an important role in this gelling system. When it came within the appropriate range, the chitosan/NaHCO(3) system would stay at sol state in certain condition and showed sol-gel transition from the top to the bottom after heating. The rheological properties of the gelling system, as well as the morphology and erosion behavior of the formed chitosan hydrogels were evaluated as a function of the NaHCO(3) concentration in sols. The hydrogels showed porous morphologies with some diversification depending on the NaHCO(3) concentration, which also affected their erosion behaviors and drug release rates. Moreover, the gelation mechanism of such chitosan/NaHCO(3) system was studied and proposed as the formation of three-dimensional chitosan network with physical junctions thanks to the deprotonation of -NH(3)(+) in chitosan accompanying with the gradual neutralization between HCO(3)(-) and acid. In vivo gelation test was also performed by the dorsal subcutaneous injection of chitosan/NaHCO(3) solution in rat. The formation of in situ gels suggested such system promising applications in injectable drug delivery system.

PH and near-infrared light dual-stimuli responsive drug delivery using DNA-conjugated gold nanorods for effective treatment of multidrug resistant cancer cells

A thiolated pH-responsive DNA conjugated gold nanorod (GNR) was developed as a multifunctional nanocarrier for targeted, pH-and near infrared (NIR) radiation dual-stimuli triggered drug delivery. It was further passivated by a thiolated poly(ethylene glycol)-biotin to improve its cancer targeting ability by specific binding to cancer cell over-expressed biotin receptors. Doxorubicin (DOX), a widely used clinical anticancer drug, was conveniently loaded into nanocarrier by intercalating inside the double-stranded pH-responsive DNAs on the GNR surface to complete the construction of the multifunctional nanomedicine. The nanomedicine can rapidly and effectively release its DOX payload triggered by an acidic pH environment (pH~5) and/or applying an 808nm NIR laser radiation. Compared to free DOX, the biotin-modified nanomedicine displayed greatly increased cell uptake and significantly reduced drug efflux by model multidrug resistant (MDR) breast cancer cell lines (MCF-7/ADR). The application of NIR radiation further increased the DOX release and facilitated its nuclear accumulation. As a result, this new DNA-GNR based multifunctional nanomedicine exerted greatly increased potency (~67 fold) against the MDR cancer cells over free DOX.

Surface characterization of blood compatible amphiphilic graft copolymers having uniform poly(ethylene oxide) side chains

Abstract The surface characterization of poly(methyl methacrylate)-graft-poly(ethylene oxide) (PMMA-g-PEO) was investigated by XPS and contact-angle measurement, and its in vitro blood compatibility was assessed by plasma recalcification time measurement. The surface and bulk composition of different PMMA-g-PEO graft copolymer showed that PMMA segments were always enriched at the copolymer–air interface, but surface enrichment of PEO segments could occur in films of copolymers with longer PEO side chain (Mn of PEO, 3200) and a higher bulk PEO content. Contact-angle studies indicated that the surface hydrophilicity increased as the surface PEO content increased. The contact angles of water on the copolymer decreased linearly with contact time until they reached a balance value θe or 0, and the relationship between θ (from the static contact angle, θs to θe) and contact time(t) can be expressed by: θ=−kt. The proportionality constant k and Δθ (θs−θe) depended on the bulk structural parameters (the bulk composition and PEO side chain length). The microphase-separated structure of copolymers was also observed by transmission electron microscopy (TEM). The relationship between the surface properties of PMMA-g-PEO graft copolymer and its blood compatibility was addressed.