Solid core-shell drug nanoparticles for therapeutic delivery

Solid core-shell drug nanoparticles are poised to offer a solution to the challenges faced with the conventional administration of therapeutic agents. Engineering drug delivery carriers that can selectively target the diseased tissue, reduce systemic toxicity, increase drug absorption, and locally control the release of the drug are current unmet medical needs. One area that has generated much interest in the past 25 years is the delivery of anti-cancer agents. Polymeric drug delivery systems, such as nanoparticles (10 to 1000 nm) have many potential advantages for the delivery of anti-cancer agents including improving bioavailability, reducing adverse side effects, enhancing permeability across physiological barriers, and controlling drug release. Although encapsulating anti-cancer agents within polymeric nanoparticles have shown efficacy in vitro, in vivo therapeutic efficacy has proven more difficult. It has been hypothesized that engineering a biocompatible and targeted nanoparticle surface can help overcome the difficulties experienced with current polymeric nanoparticles.;New approaches in the delivery of anti-cancer therapeutics are continuously being developed with the intent to meet the medical requirements in increasing efficacy and reducing adverse side effects. The goal for any drug delivery carrier that treats cancer or other diseases is to increase the efficacy per dose by selectively delivering the therapeutic agent to the active site and by designing a carrier that can overcome biological barriers that may prevent it from reaching the targeted tissue. Core-shell drug nanoparticles can provide a new method in encapsulating anti-cancer agents within a polymeric nanometer scale shell while retaining the therapeutic agents' biological activity. In this dissertation, the fabrication, encapsulation, characterization, and in vitro delivery of core-shell drug nanoparticles is discussed.;The focus of this dissertation was twofold: to fabricate solid core drug nanoparticles from water insoluble therapeutic agents and to encapsulate the nanoparticles within a polymeric nanometer scale shell, the nanoshell. The polymeric nanoshell composed of multilayer polyelectrolytes serves two purposes; first the surface of nanoshell can be chemically modified with targeting ligands to assist the carrier in overcoming biological barriers, and secondly, the nanoshell can control the release of the drug at the active site. Nanoparticles of dexamethasone and paclitaxel, the chosen therapeutic agents, were prepared by a modified emulsification-solvent evaporation technique. Size analysis of the solid core nanoparticles was determined by laser light scattering, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). These there methods confirmed that the size of the core was within a range of 100-200 nm and with approximately an average size of 150 nm. Optimization studies illustrated that a monodisperse suspension of dexamethasone nanoparticles within a desired size range can be fabricated by adjusting the surfactant concentration in the aqueous phase and homogenization speed. This method in preparing solid core nanoparticles can be extended to additional therapeutic agents with limited water solubility for the treatment of other aliments.;Electrostatic Layer-by-Layer self-assembly is a versatile technique used for coating planar substrates, colloidal particles, and other surfaces with various geometries. This technique was used as a method for encapsulating solid core drug nanoparticles within nanoshells composed of multilayer polyelectrolytes. Polyelectrolyte pairs including poly (allylamine hydrochloride)/poly (styrene-4-sulfonate), and poly (L-lysine)/heparin sulfate were implemented to establish a proof-of-concept in using this self-assembly technique to create nanoshells of desired thickness and composition with nano-scale precision. To confirm the LbL self-assembly of the nanoshell the &zgr;-potential was measured at each adsorption step. A charge reversal upon subsequent polyelectrolyte deposition confirmed the successful encapsulation of the solid core drug nanoparticles. The surface and bulk morphologies of the encapsulated drug nanoparticles were determined by TEM and SEM, respectively. TEM images indicated that a nanoshell composed of two polyelectrolyte layers of poly (allylamine hydrochloride) and poly (styrene sulfonate) was approximately 10 nm thick. The nanoscale precision achieved with LbL assembly provides control of surface properties of the core-shell carrier, which is important in the design of an efficacious delivery system.;The exterior surface of the nanoshell was functionalized with biocompatible polymer poly (ethylene glycol) (PEG) by the covalent attachment to the amine terminated polyelectrolyte carrier. Surface chemical analysis was performed with x-ray photoelectron spectroscopy (XPS) to confirm the successful medication of the nanoshell. Covalent attachment of PEG to the nanoshell resulted in an increase in atomic percent of oxygen and an increase in the peak area percent of (C-C-O)n, which represents the repeat unit in the backbone of a PEG molecule. Also, a neutral &zgr;-potential re-confirmed the presence of non-ionic PEG grafted at the surface of the nanoshell. In vitro studies were performed to study the phagocytic uptake of core-shell fluorescent nanoparticles (200 nm) using a flow cytometric assay. Results showed that a neutral and hydrophilic nanoshell can reduce the uptake of core-shell nanoparticles after 24 hours of incubation with suspension macrophages. This key result was demonstrated with core-shell nanoparticles chemically modified with PEG 2, 5, and 20 kDa. The results to date hold promise in using the LbL technique to control the surface chemistry when fabricating a nanoshell for drug delivery applications.;The in vitro therapeutic efficacy of paclitaxel core-shell nanoparticles was retained upon fabrication and encapsulation in nanoshells composed of PAH/PSS/PAH and PLL/HS/PLL/PEG 20 kDa. Breast carcinoma cells, MCF-7 were treated with 22 μg/mL of paclitaxel core and core-shell nanoparticles for 24, 48, and 72 hours. At the end of each incubation period, MCF-7 cells were arrested in the G2/M phase of the cell cycle and displayed abnormal microtubule morphologies. Prolonged treatment with paclitaxel led to cell death by apoptosis or necrosis as evidenced by images collected by confocal microscopy. The results suggested that the anti-tumor mechanism of paclitaxel was maintained within this new drug delivery system.;A targeted drug delivery system of core-shell nanoparticles was achieved by the chemical modification of a lipophilic nanoshell with the epidermal growth factor (EGF) protein. This protein was chosen because its receptor has been found to be overexpressed in many disease states including high grade brain tumors. The nanoshell was composed of biocompatible polyelectrolytes, PLL/HS and an electrostatically adsorbed phospholipid bilayer containing PEG and an amine functionality. Endocytosis of the targeted delivery system into the cellular cytoplasm of gliomas expressing the epidermal growth factor receptor was demonstrated by fluorescent confocal microscopy and transmission electron microscopy.;Finally, a multi-functional colloidal carrier that can provide targeted delivery and imaging to diseased tissue was designed as a proof-of-concept. By utilizing the particle lithography technique and LbL assembly a multi-functional carrier with unique surface chemical regions was designed. Site-specific targeting of an imaging particle lithographed with an arginine-glycine-aspartic acid (RGD) motif to integrin cell surface receptors on 3T3 fibroblast was demonstrated after a 24 and 48 hour study. Confocal images suggested that covalent attachment of RGD to the lithographed imaging particle remains active and promotes 3T3 cellular adhesion and spreading opposed to multifunctional carriers modified with inactive RAD motif.;In summary, this dissertation describes the fabrication, encapsulation, characterization, and implementation of core-shell drug nanoparticles for the delivery of therapeutic agents selective to diseased tissue, such as tumors. The work presented in this thesis advances the field of nanotechnology by providing new strategies towards engineering surfaces on colloidal carriers for the selective and stealth delivery of water-insoluble therapeutic agents to diseased tissue.