Category: Formulation and Quality
Purpose: Spinal Cord Injury (SCI) is considered as one of the most challenging problems clinically. The pathophysiology of SCI includes a primary phase, which is immediately followed by the secondary phase. Both these phases have been shown to cause an oxidative stress, leading to the formation of reactive oxygen species (ROS). Oxidative stress plays a significant role in deteriorating the injury state. The current treatment options include the use of antioxidants and anti-inflammatory agents to the site of the SCI injury. Delivery of these therapies to the site of the injury is limited and requires administration of extremely high doses to achieve therapeutic activity. The goal of the current study focuses on the preparation, characterizations and in vitro evaluation of polymeric PLGA nanoparticles containing a naturally occurring antioxidant (α- tocopherol). We hypothesize that the delivery of the antioxidant to the injury site is higher when administered as nanoparticles as compared to in solution.
Methods: PLGA nanoparticles were prepared by emulsion and solvent evaporation technique. The nanoparticles were optimized for their size using various polymer and stabilizer concentrations. The prepared freeze-dried nanoparticle characterization includes particle size, zeta potential, powder x-ray diffraction (XRD) and Fourier – Transform InfraRed (FTIR) Spectroscopy. Nanoparticle morphology was evaluated using Scanning Electron Microscopy (SEM). An UPLC method using a fluorescence detection was developed and validated for the quantification of α- tocopherol. This method was used to determine the drug load, entrapment efficiency as well as the in vitro drug release from these nanoparticles. Cytotoxicity studies for blank and drug loaded nanoparticles were carried out in In vitro cell culture model using HA-sp cell lines. Oxidative stress condition was induced using hydrogen peroxide (H2O2). Cell cytotoxicity was evaluated using MTT assay. The cells were pre-treated with 0.01, 0.1, 0.25, 1, 10 and 100 μM concentrations of α-tocopherol solution and PLGA nanoparticles containing the drug for 30 minutes, followed by induction of oxidative stress for 24 and 48 hours. The comparative study between the drug in solution and PLGA nanoparticles was conducted to assess the degree of protection offered by both the systems tested. All studies were conducted in triplicate.
Results: PLGA nanoparticles sizes were 152 ± 10.6 nm with zeta potential of -10.68 ± 0.43. The drug loading in the nanoparticles was 7.47 ± 0.57% with an entrapment efficiency of 76.6 ± 11.5%. The FTIR data confirmed that there was no interaction between the drug and the polymer. The XRD data revealed that the drug is present in the dissolved state in the formulation. The UPLC detection method used an emission wavelength of 292 nm and excitation wavelength at 324 nm. The α- tocopherol retention was at 3.8 minutes. The method validation included specificity, linearity with a concentration range of 15 ng/mL – 1 μg/mL (R2 = 0.999), accuracy ( > 95%) and precision (% RSD < 10 %). With an oxidative stress condition on Human Astrocyte-spinal cord (HA-sp) cells with 400 μM of H2O2 resulted in 40% cell cytotoxicity. Upon treatment for 30 minutes with the drug solution the cells showed a protection from up to 5% and 15% protection at 24 and 48 hours, respectively. The PLGA loaded nanoparticles showed nearly 35% and 38% protection at 24 and 48 hours, respectively. All experiments were analyzed for statistical significance using ANOVA and defined as P< 0.05.
Conclusion: PLGA nanoparticles containing α- tocopherol was prepared and characterized. The UPLC method developed and validated allowed the quantification of α- tocopherol in these nanoparticles. In vitro studies indicated that both, the pure drug solution as well as the PLGA nanoparticles, showed protection from the oxidative stress induced by H2O2. However, the PLGA nanoparticles, showed an enhanced protection as compared to drug in solution. This delivery approach may overcome the limitation of current mode of drug administration in SCI treatment.