Poster J10 - Functionalized poly-lactic acid nanoparticles for brain drug delivery

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

DA

Daniel Alday

Undergraduate Research Assistant
Louisiana State University
Baton Rouge, Louisiana, United States

Primary Investigator(s)

QC

Qi Cai

Assistant Professor
Louisiana State University, United States

Co-Author(s)

CA

Carlos Astete

Assistant Professor
Louisiana State University, United States
CS

Cristina Sabliov

Professor
Louisiana State University, United States

Introduction: Formed by the tight-junction (TJ) complex at the interfacial leaflets of brain endothelial cells (ECs) and the low level of transcytosis through the endothelium, the blood-brain barrier (BBB) maintains brain homeostasis and represents a formidable challenge for brain drug delivery. Many therapeutic agents, such as hydrophilic and macromolecular drugs, cannot cross the BBB. One promising solution is the employment of polymeric nanoparticles (NPs) as a drug carrier. In this study, we developed brain microvascular-targeting poly-lactic acid (PLA) nanoparticles for drug delivery into the brain. Specifically, we engineered PLA NPs functionalized with Lycopersicon Esculentum (Tomato) Lectin (LEL) and Albumin (Alb). PLA is a biodegradable, biocompatible polymer approved by the FDA for use in immunocompromised people to correct nasolabial fold deficiencies and other facial wrinkles. Lectin binds to the endothelial glycocalyx, which is a brushlike structure comprising glycoproteins on the inner lining of the microvasculature. Tomato lectin is a common plant lectin that is non-toxic and non-immunogenic and has been safely used in oral drug delivery systems. We demonstrated that PLA-LEL-Alb nanoparticles exhibit a high targeting efficiency towards human brain microvascular endothelial cells (D3 cells). Moreover, incubating functionalized PLA NPs with D3 cells does not influence cell viability and BBB integrity. Therefore, these NPs have excellent translational potential for developing effective drug-delivery strategies for brain diseases.

Materials and

Methods: We first synthesized PLA-LEL, PLA-Alb, PLA-TRITC, and PLA-PEG conjugates and then prepared the functionalized PLA NPs. PLA was dissolved in Dichloromethane and mixed with Oxalyl Chloride and Dimethylformamide. Dry ALB, LEL, TRITC, and PEG were dissolved in Dimethyl Sulfoxide and added to each PLA solution respectively. In the synthesis of PLA-LEL-Alb-TRITC, PLA-ALB: PLA-TRITC: PLL-LEL (70:7:20 by mass) was combined through microfluidization. Using the same method, PLA-PEG-TRITC NPs were synthesized as a control. The particle size range was determined using transmission electron microscopy (TEM), and the zeta potential was measured using electrophoretic motion tracking (EMT). To prepare the in vitro BBB model, 9*10^4 human brain microvascular endothelial cells (D3 cells) were cultured on transwell inserts (0.45 μm pore size) in a 12-well plate for 7 days. The transepithelial electrical resistance (TEER) and dye permeability (FITC-Dextran 3 kDa) were monitored to confirm the formation of an intact BBB. To visualize the NP’s binding efficiency, 0.1-10 mg/mL of the NPs were incubated with the D3 cells for 1 hour, followed by rinsing with PBS and fixed with 4% PFA. The cell nuclei were stained with Hoechst. To determine the cytotoxicity of the NPs, 1-10 mg/mL of the NPs were incubated with the D3 cells for 1 hr, and the TEER changes were monitored. Moreover, 3*10^4 of the D3 cells were seeded in a 96-well plate. 1-10 mg/mL of the PLA-LEL-Alb NPs were incubated with the D3 cells for 1 day, and the WST-1 assay was used to determine the cell viability.

Results, Conclusions, and Discussions: The current techniques used to functionalize polymeric NPs for targeting purposes are based on protein adsorption/reaction over the surface of preformed NPs or conjugation of peptides, proteins, and aptamers to the polymer followed by nanoparticle formation. However, surface functionalization of preformed NPs is challenging as the presence of surfactants on the surface of the NPs can mask the carboxyl groups that are available to react. Hence, we focused on polymer conjugation before NP synthesis. TEM images with uranyl acetate negative staining revealed the PLA-LEL-ALB-TRITC particles are spherical and mostly ranged from a size of 150±45 nm, as shown in Figure 1A. EMT analysis showed the particles had a slightly negative potential of -2.64 FWHM 5.12 mV . In this study, we exploited a simple and cost-effective transwell monolayer model, which allows us to evaluate the toxicity of nanoparticles on brain endothelial cells and investigate nanoparticle-cell interactions. The in vitro BBB model has a TEER value of 14.4±0.5 Ω∙cm2 measured by a cell culture cup chamber and a permeability of (2.33±0.7)*10^-6 cm/s measured with FITC Dextran 3kDa, indicating the formation of the intact BBB. Incubating the D3 cells with 1 mg/mL of the PLA-LEL-Alb-TRITC nanoparticles for 1 hr shows intracellular uptake of the NPs (Figure 1B). In contrast, incubating the D3 cells with PLA-PEG-TRITC does not show cellular uptake. No significant decrease in the BBB integrity was observed after incubating the nanoparticles with the in vitro BBB model (Figure 1C). Moreover, WST-1 assay results demonstrated that incubating 1 mg/mL of the PLA-LEL-Alb nanoparticles does not influence the viability of the D3 cells (Figure 1D). These results suggest that PLA-LEL-Alb NPs have a high affinity to brain endothelial cells with low cytotoxicity. We previously demonstrated that LEL increases nanoparticle targeting to the brain microvasculature in mice. Moreover, the expression of albumin receptor GP60 on vascular endothelial cells enables albumin-functionalized NP to cross the BBB. Taken together, our nanoparticle delivery platform has the promise of being used in future brain disease therapy.

Acknowledgements (Optional):