(102f) Poly(Lactide-Co-Glycolide) Nanoparticles for Targeted and Controlled Delivery of Doxorubicin for the Treatment of Cancer

Current cancer treatment methods have a number of limitations that lead to poor therapeutic results.  Chemotherapeutic treatments lack specificity and lead to the damage of healthy tissue, especially of the normally dividing cells of the bone marrow, skin, and gastro-intestinal mucosa, among other tissues.  Some chemotherapeutic agents, including the one here utilized, can extravasate into the perivascular spaces and cause severe localized tissue damage or necrosis because of their vesicant properties.  In addition, neoplastic cells readily mutate and many cancers have developed resistance to chemotherapeutic agents.  The need for improved therapies for the treatment of cancer is great, and one of the ways to overcome the deficiencies of current treatment methods is to develop delivery systems that significantly improve the pharmacological characteristics of the drug in vivo.

The novel drug delivery system here utilized consists of biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles.  These nanoparticles are used as vehicles for the targeted and controlled delivery of doxorubicin, a commonly used and potent chemotherapeutic agent.  PLGA nanoparticles provide protection to the therapeutic agent from degradation by physiological conditions, and release the drug in a controlled manner so that its concentration is maintained within therapeutic levels for longer periods of time.  In addition, these nanoparticles are small enough to circulate through capillaries, cross the highly-permeable vasculature supplying blood to the tumor, and enter tumor cells through endocytosis or receptor-mediated transport.  PLGA nanoparticles can be potentially targeted to specific tissues by including targeting moieties in the formulation, and modified to include poly(ethylene glycol) pendant chains for increased circulation time in the vasculature.  These favorable pharmacological characteristics result in improved therapeutic efficacy, better use of the pharmaceutical agent, and increased patient compliance and quality of life.

Nanoparticles were prepared through a modified oil-in-water emulsion?solvent evaporation method.  Approximately 6mg of doxorubicin were dissolved in methanol, and mixed with a solution of 100mg of poly(lactide-co-glycolide) (PLGA) in acetone to form the organic phase.  This phase was emulsified in an aqueous solution containing bovine serum albumin as a stabilizer.  The emulsion was sonicated, and stirred under vacuum for 45 minutes to remove the solvent.  Nanoparticles were recovered through centrifugation, washed to remove unencapsulated doxorubicin, and lyophilized.  Particle morphology was studied with scanning electron microscopy.  Particle size was determined with a Coulter Nanosizer, and confirmed with transmission electron microscopy.  Release studies were performed in vitro in buffered and non-buffered saline at 37^oC to determine the release kinetics of these particles.  Absorbance at 480nm was utilized to measure doxorubicin concentrations based on standard curves of known concentration.  MDA-MB-231 breast cancer cells were used to determine cellular uptake and intracellular distribution of the nanoparticles through confocal microscopy, as well as cytotoxicity induced by these particles in comparison to free drug.

Nanoparticles were observed to be spherical, and have an average diameter of approximately 220nm.  The maximum drug loading obtained was of 4mg of doxorubicin per 100mg of nanoparticles, and the average encapsulation efficiency was approximately 70%.  Significant differences were observed between the release profiles of doxorubicin nanoparticles in buffered and non-buffered saline.  The release of doxorubicin from the nanoparticles in non-buffered saline occurred at a rate of approximately 27% per day.  In buffered saline the release rate was almost constant during the first week at 9.8% of the drug per day and no burst release was observed (Figure 1).  Particles were observed to enter breast cancer cells as early as 2 hours after exposure (Figure 2).   

PLGA nanoparticles were successfully formulated, characterized and evaluated in vitro.  These nanoparticles promise to be an effective system for targeted and controlled release of doxorubicin or other chemotherapeutic agents with reduced systemic toxicity, increased therapeutic efficiency, and increased patient compliance. 

Figure 1.  In vitro release of doxorubicin from PLGA nanoparticles in buffered and non-buffered saline.  Values represent release data from 5 nanoparticle batches, each in triplicate.  Error bars represent the standard deviation of all data. 

Figure 2.  Brightfield and fluorescence microscopy images of corresponding MDA-MB-231 cells after 48 hours (top two images) and 2 hours (bottom two images) of exposure to 62.5 μg/ml of doxorubicin-loaded nanoparticles. 

This work was supported by an NSF Integrative Graduate Education and Research Traineeship (IGERT).