Design of a thrombin resistant human acidic fibroblast growth factor (hFGF1) variant that exhibits enhanced cell proliferation activity.

Acidic fibroblast growth factors (FGF1s) are heparin binding proteins that regulate a wide array of key cellular processes and are also candidates for promising biomedical applications. FGF1-based therapeutic applications are currently limited due to their inherent thermal instability and susceptibility to proteases. Using a wide range of biophysical and biochemical techniques, we demonstrate that reversal of charge on a well-conserved positively charged amino acid, R136, in the heparin binding pocket drastically increases the resistance to proteases, thermal stability, and cell proliferation activity of the human acidic fibroblast growth factor (hFGF1). Two-dimensional NMR data suggest that the single point mutations at position-136 (R136G, R136L, R136Q, R136K, and R136E) did not perturb the backbone folding of hFGF1. Results of the differential scanning calorimetry experiments show that of all the designed R136 mutations only the charge reversal mutation, R136E, significantly increases (ΔTm = 7 °C) the thermal stability of the protein. Limited trypsin and thrombin digestion results reveal that the R136E mutation drastically increases the resistance of hFGF1 to the action of the serine proteases. Isothermal titration calorimetry data show that the R136E mutation markedly decreases the heparin binding affinity of hFGF1. Interestingly, despite lower heparin binding affinity, the cell proliferation activity of the R136E variant is more than double of that exhibited by either the wild type or the other R136 variants. The R136E variant due to its increased thermal stability, resistance to proteases, and enhanced cell proliferation activity are expected to provide valuable clues for the development of hFGF1- based therapeutics for the management of chronic diabetic wounds.

The effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells.

Particle-based vaccine delivery systems are under exploration to enhance antigen-specific immunity against safe but poorly immunogenic polypeptide antigens. Chitosan is a promising biomaterial for antigen encapsulation and delivery due to its ability to form nano- and microparticles in mild aqueous conditions thus preserving the antigenicity of loaded polypeptides. In this study, the influence of chitosan encapsulation on antigen uptake, activation and presentation by antigen presenting cells (APCs) is explored. Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) and ovalbumin (OVA) were used as model protein antigens and encapsulated in chitosan particles via precipitation-coacervation at loading efficiencies >89%. Formulation conditions were manipulated to create antigen-encapsulated chitosan particles (AgCPs) with discrete nominal sizes (300 nm, 1 µm, and 3 µm). Uptake of AgCPs by dendritic cells and macrophages was found to be dependent on particle size, antigen concentration and exposure time. Flow cytometry analysis revealed that uptake of AgCPs enhanced upregulation of surface activation markers on APCs and increased the release of pro-inflammatory cytokines. Lastly, antigen-specific T cells exhibited higher proliferative responses when stimulated with APCs activated with AgCPs versus soluble antigen. These data suggest that encapsulation of antigens in chitosan particles enhances uptake, activation and presentation by APCs.

Temperature-sensitive polymer-coated magnetic nanoparticles as a potential drug delivery system for targeted therapy of thyroid cancer.

The objective of this work was to develop and investigate temperature-sensitive poly(N-isopropylacrylamide-acrylamide-allylamine)-coated iron oxide magnetic nanoparticles (TPMNPs) as possible targeted drug carriers for treatments of advanced thyroid cancer (ATC). These nanoparticles were prepared by free radical polymerization of monomers on the surface of silane-coupled iron oxide nanoparticles. In vitro studies demonstrated that TPMNPs were cytocompatible and effectively taken up by cancer cells in a dose-dependent manner. An external magnetic field significantly increased nanoparticle uptake, especially when cells were exposed to physiological flow conditions. Drug loading and release studies using doxorubicin confirmed the temperature-responsive release of drugs from nanoparticles. In addition, doxorubicin-loaded nanoparticles significantly killed ATC cells when compared to free doxorubicin. The in vitro results indicate that TPMNPs have potential as targeted and controlled drug carriers for thyroid cancer treatment.

Multifunctional particles for melanoma-targeted drug delivery.

New magnetic-based core-shell particles (MBCSPs) were developed to target skin cancer cells while delivering chemotherapeutic drugs in a controlled fashion. MBCSPs consist of a thermo-responsive shell of poly(N-isopropylacrylamide-acrylamide-allylamine) and a core of poly(lactic-co-glycolic acid) (PLGA) embedded with magnetite nanoparticles. To target melanoma cancer cells, MBCSPs were conjugated with Gly-Arg-Gly-Asp-Ser (GRGDS) peptides that specifically bind to the α(5)ß(3) receptors of melanoma cells. MBCSPs consist of unique multifunctional and controlled drug delivery characteristics. Specially, they can provide dual drug release mechanisms (a sustained release of drugs through degradation of PLGA core and a controlled release in response to changes in temperature via thermo-responsive polymer shell), and dual targeting mechanisms (magnetic localization and receptor-mediated targeting). Results from in vitro studies indicate that GRGDS-conjugated MBCSPs have an average diameter of 296 nm and exhibit no cytotoxicity towards human dermal fibroblasts up to 500 µg ml(-1). Further, a sustained release of curcumin from the core and a temperature-dependent release of doxorubicin from the shell of MBCSPs were observed. The particles also produced a dark contrast signal in magnetic resonance imaging. Finally, the particles were accumulated at the tumor site in a B16F10 melanoma orthotopic mouse model, especially in the presence of a magnet. Results indicate great potential of MBCSPs as a platform technology to target, treat and monitor melanoma for targeted drug delivery to reduce side effects of chemotherapeutic reagents.

Development of multiple-layer polymeric particles for targeted and controlled drug delivery.

The purpose of this work was to develop multilayered particles consisting of a magnetic core and two encompassing shells made up of poly(N-isopropylacrylamide) (PNIPAAm) and poly(D,L-lactide-co-glycolide) (PLGA) for targeted and controlled drug delivery. Transmission electron microscopy confirmed that multilayered particles were obtained with PNIPAAm magnetic nanoparticles embedded within the PLGA shell. Factorial analysis studies also showed that the particle size was inversely proportional to the surfactant concentration and sonication power and directly proportional to the PLGA concentration. Drug-release results demonstrated that these multilayer particles produced an initial burst release and a subsequent sustained release of both bovine serum albumin (BSA) and curcumin loaded into the core and shell of the particle, respectively. BSA release was also affected by changes in temperature. In conclusion, our results indicate that the multilayered magnetic particles could be synthesized and used for targeted and controlled delivery of multiple drugs with different release mechanisms. FROM THE CLINICAL EDITOR: Authors demonstrate the synthesis of multilayered particles consisting of a magnetic core and two encompassing shells made up of poly (N-isopropylacrylamide) (PNIPAAm) and poly(D, L-lactide-co-glycolide) (PLGA) for targeted and controlled drug delivery. The presented results indicate successful synthesis and application for targeted and controlled delivery of multiple drugs with different release mechanisms.