(M1430-08-49) Preparation, Characterization, and Evaluation of Cellular Uptake of Spray-Dried Albumin Nanoparticles

Purpose: Spray drying is a one-step process of preparing solid nanoparticles from a solution suspension or emulsion. Recent advancements in spray drying technology have led to increased attempts in formulating solid particles within the nanometer range. These particles can be further functionalized and targeted to different parts in the body. This study demonstrates the development of solid polymeric nanoparticles in a single step, a process previously achieved in only a few studies. For the formulation of the nanoparticles, a method was developed to prepare a polymeric matrix by chemically crosslinking bovine serum albumin (BSA). The suitability of spraying the crosslinked matrix in the spray dryer was studied by determining the effect of viscosity. These particles were physically characterized based on their size, charge, and swelling behavior, and were further evaluated in-vitro for their cellular uptake.

Methods: BSA solutions of different concentrations between 0.50 % w/v to 5.00% w/v were crosslinked using a chemical crosslinking agent, glutaraldehyde. An HPLC method was developed to estimate the time of crosslinking. This included the derivatization of glutaraldehyde with 2,4-dinitrophenylhydrazine (2,4-DNPH) and quantifying the derived product. The viscosity of the cross-linked BSA solutions was determined, providing insights into the pumpability and suitability for spray drying. The solutions with appropriate viscosities were spray dried using a Büchi B-90 HP spray dryer. Two sizes of nebulizers were used to carry out the spray drying process: medium and small.  Process parameters in a spray dryer influence the formation of particles and are important because the quality of the final product is dependent upon these. After various optimizations, a spray drying method was developed using a combination of parameters that yielded particles in the nano range. The particles were collected manually using a silicon spatula and stored in an air-tight glass vial. To determine the size of the particles, a DLS particle size analyzer was used. The particles were dispersed in a 0.01% Tween 80 solution prepared using deionized water for the analysis. Using the same method of particle size determination, the swelling of particles was studied. The particles were dispersed in a 0.01% Tween 80 solution, and the size was determined at fixed time points over the period of 6 hours. The zeta potential was measured using the electrophoretic light scattering (ELS) method. To evaluate the cellular uptake of the nanoparticles, Human umbilical vein endothelial cells (HUVECs) labeled with red fluorescent protein (RFP) were used. The cells were plated in a monolayer in a 6 well plate, and after the cells reached 90% confluency, they were treated with 0.125 mg of blank nanoparticles (dispersed in 1 mL endothelial cell growth medium) in each well. The uptake was observed using a fluorescent microscope at different time points over a period of 10 hours.

Results: HPLC analysis of the cross-linked BSA solutions indicated a minimum of 12 hours for complete cross-linking of BSA with glutaraldehyde. The viscosity studies suggested that crosslinked BSA solutions of up to 5.00 % w/v concentration were within the desired viscosity range of < 10 cps; however, spray-drying studies indicated that BSA solutions greater than 1.50% w/v in concentration clogged the nebulizer in the spray head. Particle size of the prepared nanoparticles was found to be dependent on the concentration of the BSA solution. Lower concentrations produced smaller particles between 500-700 nm of range. Higher concentrations produced particles that were in the micrometer range. It was also observed that the addition of surfactants helped in obtaining particles of smaller size. Furthermore, swelling studies indicated no significant increase in particle size of the nanoparticles up to 6 hours. The blank nanoparticles exhibited the zeta potential of -23.920 ± 0.601 mV, indicating their colloidal stability. The uptake of nanoparticles by HUVECS was confirmed by the appearance of the yellow fluorescence in the merged images, indicating the intracellular localization of the green fluorescence from the BSA nanoparticles with the red fluorescence of the cells. Additionally, cells retained their normal morphology and exhibited no signs of abnormal behavior, suggesting that the nanoparticles did not induce cellular stress.

Conclusion: Solid nanoparticles were successfully prepared using a one-step spray drying technique. The particles were characterized based on their size, charge, and zeta potential. The co-localization of nanoparticles confirmed their cellular uptake. Future studies will focus on loading these nanoparticles with antisense oligonucleotides targeting VEGF and evaluating their potential to inhibit angiogenesis. In the future, these nanoparticles can serve as a platform technology to deliver various therapeutic agents. Facile surface modifications can be employed to enhance the binding affinity of the drug-loaded polymeric albumin nanoparticles to their target.

Fluorescence Microscopy Images Showing Nanoparticle Internalization in HUVECs. (Left to right): HUVECs labeled with RFP appear red. Green fluorescence indicates the presence of nanoparticles. Merged image showing yellow regions where red-labeled HUVECs and green nanoparticles overlap, confirming successful internalization of nanoparticles by the cells.