(T1030-11-76) The Physicochemical Properties and Morphological Changes of PLGA-PEG Nanoparticles During Long-Term In Vitro Release

Purpose: Nanoparticles have numerous applications in localized drug delivery due to their unique physical and chemical properties. Localized drug delivery can be especially advantageous in situations where systemic drug delivery may not be effective or may lead to unwanted side effects. Localized nanoparticle drug delivery can also be used in infections, inflammation, cardiovascular diseases, regenerative medicine to promote tissue repair and regeneration. [1] However, prolonged retention of nanoparticles in a localized area can have potential side effects such as toxicity, inflammation, and immune responses, which may arise from both the drug and the polymer used to load the drug.[2] To study the changes that occur during the long-term local release of nanoparticles, a batch of nanoparticles was prepared for this experiment and the in vivo release environment was simulated using PBS and culture medium at 37℃. Over the course of a month, the physicochemical properties and morphological changes of the nanoparticles were monitored and recorded, while drug release was measured.

Methods: To facilitate observation of drug properties within nanoparticles using electron microscopy, a soluble salt crystal was utilized as a payload, and the oil-in-water (O/W) emulsion method was used to coat the salt crystals with PLGA-PEG. The resulting nanoparticles were dispersed in 1 mL of PBS or culture medium (DMEM with 10% FBS， ascorbic acid, sodium β-glycerophosphate, and antibiotics) in 1.5 mL centrifuge tubes, and the tubes were shaken at 110 rpm and 37 °C. At predetermined intervals, 1 mL of the suspension was withdrawn and replaced with fresh medium, and the supernatant and pellet were collected after centrifugation for measurement. The size distribution and morphology of the nanoparticles were studied using scanning electron microscopy (SEM), while dynamic light scattering (DLS) was used to analyze the size distribution and zeta potential at different time points. Inductively coupled plasma mass spectrometry (ICP-MS) was employed to measure drug release, and the cumulative release rate was calculated.

Results: During the initial release stage, dynamic light scattering (DLS) data showed a sharp increase in nanoparticle size from 500nm (size at T0) to 1000nm (Figure 1B). However, scanning electron microscope (SEM) images (Figure 2,3) revealed that the actual particle size was smaller than measured in the medium. This clustering phenomenon can be explained by the zeta potential data, which indicated a value close to 0 at the start of the release, leading to direct particle aggregation. As drug release occurred, the zeta potential decreased, possibly due to the charge of the drug released with drug molecules. While the particle size of PBS nanoparticles decreased continuously, that of the culture medium group increased and then decreased, reaching a minimum on day 8 (175nm and 332nm, respectively), corresponding to complete drug release. Notably, SEM images did not show drug crystals from the 8th day onwards (Figure 2). On day 29, the particle size of the PBS group increased to 690nm and that of the culture medium group to 3000nm. The SEM images suggested self-assembly of the polymer in the culture medium group. Different from the nanoparticles reached around 4,000 nm on the first day of release, which looked more like formed protein corona. Since the complex composition of the culture medium, especially the high protein concentration (10%, v/v), further investigation is required to understand the mechanism causing the above phenomenon.

Conclusion: In conclusion, this experiment showed that nanoparticles underwent changes in size and morphology over 29 days, including aggregation, protein corona formation, drug release, and polymer reassembly. The exact mechanism behind these changes remains unclear. As PLGA-PEG degrades in vivo over several weeks to months, careful consideration of the fate of the polymer over time is crucial when designing nanoparticles.

References: 1. Patra, J.K., et al., Nano based drug delivery systems: recent developments and future prospects. Journal of Nanobiotechnology, 2018. 16(1): p. 71.
2. Sharma, S., R. Parveen, and B.P. Chatterji, Toxicology of Nanoparticles in Drug Delivery. Curr Pathobiol Rep, 2021. 9(4): p. 133-144.

Acknowledgements: NoStopharm Inc. and Maryland Industrial Partnerships (MIPS) for technical assistance and financial support.

Figure. 1. Characterization of nanoparticles during the 29 days release period. (A) Drug accumulate release curve; (B) Particle size distribution curve; (C) Zeta potential curve.

Figure. 2. SEM images of nanoparticles during the 29 days release period in PBS medium

Figure. 3. SEM images of nanoparticles during the 29 days release period in culture medium