ABSTRACT
Fluorescent coupling of bovine serum albumin (BSA) and extracellular antigen of melanoma (ECA) using modified carbodiimide method was evaluated enabling BSA to serve as model protein to evaluate the release profiles of different microsphere formulations and uptake in B16 melanoma cells. Properties of the fluorescent labeled BSA (FBSA) including stability, transition temperature and fluorescent intensity were evaluated. It was found that FBSA produced using this method showed comparable transition temperature and fluorescent intensity compared to the commercially available FITC-labeled BSA and demonstrated stability of the fluorescent-protein linkage after overnight treatment with both trypsin and human plasma using fluorescent microscope. This study showed that the modified carbodiimide labeling method can serve as an alternative method for fluorescent labeling of target protein at reasonable cost particularly when sufficient amount of target protein is required for microsphere formulation screening.
Subject(s)
Cross-Linking Reagents/chemistry , Ethyldimethylaminopropyl Carbodiimide/chemistry , Fluorescent Dyes/metabolism , Melanoma, Experimental/metabolism , Melanoma-Specific Antigens/metabolism , Serum Albumin, Bovine/metabolism , Animals , Calorimetry, Differential Scanning , Cell Line, Tumor , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/chemistry , Humans , Melanoma, Experimental/immunology , Melanoma-Specific Antigens/chemistry , Melanoma-Specific Antigens/immunology , Mice , Microscopy, Fluorescence , Microspheres , Phagocytosis , Protein Stability , Serum Albumin, Bovine/chemistry , Time Factors , Trypsin/metabolismABSTRACT
For many years, various cancer vaccines have been widely evaluated, however clinical responses remain rare. In this review, we attempt to address the question of which delivery strategies and platforms are feasible to produce clinical response and define the characteristics of the strategy that will induce long-lasting antitumor response. We limit our analysis and discussion to microparticles/nanoparticles, liposomes, heat-shock proteins, viral vectors and different types of adjuvants. This review aims to provide an overview of the specific characteristics, strengths and limitations of these delivery systems, focusing on their impacts on the development of melanoma vaccine. To date, only adoptive T-cell transfer has shown promising clinical outcomes compared to other treatments.
Subject(s)
Cancer Vaccines/administration & dosage , Melanoma/immunology , Adenoviridae/genetics , Adjuvants, Immunologic/administration & dosage , Animals , Cancer Vaccines/immunology , Genetic Vectors , Heat-Shock Proteins/administration & dosage , Humans , Liposomes , Nanoparticles , Plasmids , Toll-Like Receptors/administration & dosage , Vaccines, DNA/administration & dosageABSTRACT
The purpose of this study was to construct a human M cell model to evaluate the transport of polystyrene and albumin microparticles via this model. The integrity of the M cell model was evaluated by determining the transepithelial electrical resistance (TEER), and the alkaline phosphate activity was determined as an indicator of the presence of M-cell-like cells in the coculture. Both Fluoresbrite microparticles and albumin microparticles were transported by both Caco-2 monoculture and M cell models. The albumin microparticles showed a significantly greater transport via the M-cell-like cells compared to Caco-2 cells monoculture (p < 0.05). The coculture showed comparable TEER and reduction of 15-36% of alkaline phosphatase (AP) activity compared to the Caco-2 cell monoculture. This human M cell model was successfully constructed using 3 and 8 microm transwell inserts and it will serve as a useful tool in formulations screening and optimization of microparticle for oral vaccine delivery.
Subject(s)
Biological Transport, Active/physiology , Nanoparticles , Peyer's Patches/metabolism , Albumins/metabolism , Alkaline Phosphatase/metabolism , Caco-2 Cells , Coculture Techniques , Electric Impedance , Fluorescent Dyes , Humans , Models, Biological , Peyer's Patches/cytology , Polystyrenes , Vaccines/administration & dosageABSTRACT
The purpose of this study was to formulate and evaluate the physicochemical properties and efficacy of an oral melanoma vaccine. Blood, feces and vaginal wash were collected weekly and analysed by ELISA. The mortality and diameter of the tumors were determined using a vernier caliper. The oral melanoma vaccine microparticles demonstrated desirable particle size, product yield, and zeta potentials. In addition, FT-IR and DSC studies revealed that there was no significant degradation in microencapsulated extra-cellular antigen (ECA). The oral vaccine group showed 25% greater survival rate compared to the control in the efficacy and challenge studies.
