(W1230-01-01) Trafficking of Acetalated Dextran Microparticles after Subcutaneous Injection

Purpose: Acetalated Dextran (Ace-DEX) microparticles (MPs) have been utilized for the delivery of therapeutics for the antigen-specific prevention of infectious disease1 and treatment of autoimmune disorders^2,3. Advantages of the Ace-DEX platform include acid sensitivity, pH neutral degradation products, intracellular delivery of cargo to antigen presenting cells (APCs), and promotion of antigen cross presentation. To achieve effective delivery, it is imperative to characterize uptake by APCs and in vivo trafficking. In this work, MP trafficking is assessed following the subcutaneous delivery of Alexa Fluor 647 (AF647) surface conjugated Ace-DEX MPs. The goal of this work is 1) to assess systemic MP trafficking and 2) determine the cells responsible for the phagocytosis of MPs.

Methods: Ace-DEX-Biotin was made by the acetalation of 3% 70K Dextran-Biotin mixed with 70K Dextran according to the established protocol4. MPs were made by homogenization and incubated for 2 hrs. with streptavidin AF647 for surface conjugation. To evaluate MP uptake in vitro, green fluorescent protein (eGFP) expressing bone marrow derived dendritic cells (BMDCs) from C57BL/6 mice were plated in chamber slides at 200,000 cells/well and incubated at 37oC overnight. AF647 surface conjugated MPs were added at a dose of 12.5 µg per well. Cells were fixed at 3 h, 6 h, 24 h, and 48 h and visualized via confocal microscopy. To evaluate in vivo trafficking of AF647 labeled MPs, non-obese diabetic (NOD) mice were subcutaneously administered 2 mg of AF647 labeled MPs in the right flank. Signal at the injection depot was measured over the course of the experiment by in vivo imaging system (IVIS) and on day 7 organs were harvested (liver, heart, spleen, kidneys, lungs, draining LN, pancreatic LN, and distal LN) and imaged on IVIS.

Results: Increased association between cells and MPs and internalization of MPs were observed overtime. Fluorescence was maintained within cells after 48 h treatment (Figure 1). MP signal at the depot decreased overtime (Figure 2). Majority of the signal at 168 h is measured in the draining lymph node (LN) (Figure 3).

Conclusion: Frequency of MP internalization by BMDCs increased over time. MPs are leaving the subcutaneous depot and trafficking to the draining LN within a week. Future work is planned to assess cell recruitment to MP depot and identify cell populations taking up MPs.

References: [1] Johnson B, et al. The Journal of Immunology 2021. [2] Peine et al., Mol Pharmaceutics 2014. [3] Chen N, et al. Adv. Healthcare Mater 2018. [4] Kauffman KJ, et al. ACS Appl Mater Interfaces (2012), 4 (8), 4149–4155.

Acknowledgments: We would like to thank the Chapel Hill Analytical and Nanofabrication Laboratory (CHANL) for helping with molecular characterization, the Biomedical Research Imaging Center (BRIC) for in vivo imaging, Microscopy Services Laboratory (MSL) in the Department of Pathology and Laboratory Medicine for confocal microscopy, the National Institute of Health (NIH) for funding this project with the grant R01DK130225, and to the Eshelman School of Pharmacy at UNC-Chapel Hill for providing funding and the facilities for this project.

Figure 1: eGFP expressing BMDCs take up AF647 labeled Ace-DEX MPs at various timepoint



Figure 2: A) AF647 labeled Ace-DEX MP signal measured at the particle depot at various timepoints. B) Systemic trafficking measure of AF647 labeled Ace-DEX MPs.