The formulation of gelatin nanoparticles and their effect on melanoma growth ‘in vivo’

Abstract

Malignant melanoma is one of the leading causes of cancer in the United States. The outcome of the disease is closely related to the degree of progression. Research efforts have been directed at identifying an effective treatment that would improve survival rates in patients diagnosed with early stage melanoma. Bacillus Calmette-Guerin (BCG) vaccine is a potent immunostimulant that is currently used in the clinical treatment of human bladder carcinoma and has been evaluated for use as adjuvant therapy in melanoma with varying success. The aim of this project was to relate the antimelanoma activity of BCG to its fibronectin-binding properties and to formulate a gelatin nanoparticle preparation capable of mimicking this activity of BCG. An attempt was made to repeat and scale-up a literature method for the production of gelatin nanoparticles using desolvation techniques. However, the methodology produced dispersions of aggregates, the size of which varied with pH and amount of desolvating agent added. The effects of time, temperature, pH and desolvation on the molecular weight profile of gelatin obtained by size exclusion chromatography were evaluated. The molecular weight profile was arbitrarily sub-divided into fractions based on the available literature and on the above chromatograms. These represented consecutive states of polymerization of the gelatin sub-units. The relative abundance of the fractions was successfully validated by a light scattering technique. Dilute aqueous gelatin solutions were denatured when the incubation temperature was increased above 37°C, while the rate of renaturation was optimized over a temperature range of 7°C to 20°C at a pH of 5.0, equivalent to the approximate isoelectric point (IEP). The molecular weight profiles remained unchanged at around 37°C in the pH range of 5 to 7. When the gelatin solutions were mixed with ethanol, higher molecular weight tractions (microgel, delta and zeta tract10ns, >700kDa) were observed to precipitate at lower percentage ethanol concentrations than the lower molecular weight material (<700 kDa), with maximum precipitation occurring close to the isoelectric point of pH ).0. Thus, the molecular weight profile of gelatin is critically affected in a time-dependent manner by both the pH and temperature of the solution. This is due to the effect of these two factors on changes m the noncovalent interactions responsible for the molecular structure of gelatin. The molecular weight profiles, in tum, affect the phase behavior of gelatin in hydroalcoholic solutions. A robust method for the production of colloidal dispersions of non-aggregated gelatin nanoparticles was developed utilizing this information. The nanoparticles had mean diameters of 220 to 250 nm, as measured by photon correlation spectroscopy and validated by scanning electron microscopy. The particles exhibited mild aggregation when stored over a period of two weeks in water or in phosphate-buffered saline, but were stable following lyophilization without any cryoprotectant when stored at room temperature for two weeks. The fibronectin-binding properties of aqueous gelatin solutions, gelatin nanoparticles, Tice® BCG vaccine, and PSI (an antineoplastic glycan extracted from the Tice® BCG vaccine), were compared by an enzyme-linked immunosorbent assay. PSI did not exhibit fibronectin-binding properties. However, aqueous gelatin solution, gelatin nanoparticles and BCG vaccine all bound to fibronectin but to different extents. The ability of BCG to bind to fibronectin is probably related to the presence of cell surface fibronectin-binding proteins, namely the Antigen 85 complex and other proteins such as the fibronectin attachment protein (FAP). Fibroneotin-integrin interactions in vivo provide growth and motility signals for melanoma tumor cells. Consequently, the ability of the formulations tested above to interfere with the incidence of a murine D16~l'O melanoma model in vivo was investigated. DCG vaccine exhibited dose-dependent antimelanoma activity. PS 1 exhibited considerable antincoplastic activity against a murine S 180 sarcoma model and other tumors ‘in vivo’ but was ineffective against melanoma. This indicates that the ability of the BCG vaccine to inhibit melanoma growth is not related to the presence of immunostimulatory cell surface glycans. Furthermore, certain cytokines involved in macrophage stimulation, an observed effect of PS1, are known to cause upregulation of fibronectin-binding integrins on melanoma cells. Gelatin nanoparticles exhibited a marked ability to interfere with the development of primary B16-FO melanoma tumors, indicating that the ability of BCG to prevent melanoma formation is, at least in part, related to its ability to bind to fibronectin. Dilute aqueous B225 gelatin solutions, however, exhibited no such activity. Since the gelatin binding sites and cell-binding sites of fibronectin are located in different domains, these results suggest a relationship between the spatial dimensions of the fibronectin-binding particles/molecules and their ability to effectively interfere with binding of melanoma cells to the fibronectin component of the extracellular matrix. The ability of unloaded gelatin nanoparticles to physically inhibit fibronectin-integrin binding deprives the melanoma cells of the growth and cytoskeletal signals necessary for the development of primary melanoma tumors, and represents a potentially effective dosage form for prevention of the dissemination of melanoma tumors following surgical primary tumor resection.