L. Gildea, A.C.N. Sipowe, V. Tadjuidje, O.A. Forte, V.A. Dennis
Alabama State University,
United States
Keywords: MOMP (major outer membrane protein), PLGA, microfluidics, dendritic cells, innate immune responses
Summary:
Biodegradable polymeric nanoparticles (BPNs) that encapsulate antigens are being used as a strategy for therapeutic delivery. However, the effects of nanoparticle size and homogeneity on innate immune responses have only been briefly studied. Recent advancements in nanotechnology, particularly microfluidic techniques for producing size-tunable nanoparticles, are now being applied to create effective therapeutic delivery targets. We hypothesized that homogeneous-sized nanoparticles would lead to greater activation of antigen-presenting cells (APCs) compared to heterogeneous-sized conventional BPNs. In this study, we aimed to fabricate size-tunable BPNs using PLGA(poly D, L-lactide-co-glycolide) to encapsulate a modeled chlamydial recombinant major outer membrane protein (rMOMP), referred to as PLGA-rMOMP. To prepare the homogeneous-sized BPNs, we optimized fabrication parameters, including total flow rate (TFR), flow rate ratio (FRR), and concentrations of both the organic phase (PLGA) and the aqueous phase (either rMOMP or PBS). These components were mixed within the staggered herringbone micromixer (SHM) channels of a microfluidics system. We successfully fabricated both the encapsulated (PLGA-rMOMP) and control (PLGA-PBS) BPNs in three size ranges: sub-100 nm (small), 150-170 nm (medium), and 220-270 nm (large). The SHM-BPNs were physiochemically characterized for various parameters, including zeta-size, zeta-potential, UV-visible spectroscopy, encapsulation-efficiency (EE%), Fourier transform-infrared spectroscopy (FT-IR), and morphology via scanning electron microscopy (SEM). The characterization data indicated that the SHM-BPNs were within the desired size ranges, had stable surface charges of -1 mV (small), -2 mV (medium), and -4 mV (large), and exhibited homogeneity as evidenced by a low polydispersity index (PDI) of less than 0.1. In contrast, the conventional BPNs had an average size of 180 nm, a surface charge of -12 mV, and a PDI greater than 0.1. Electron micrographs displayed the smooth surface and size homogeneity of SHM-BPNs compared to conventional BPNs. UV-visible analysis revealed minimal absorption of protein on the surface of the BPNs, confirming encapsulation through FT-IR scans that indicated functional group peaks at 1654 cm⁻¹ and 1563 cm⁻¹. The encapsulation-efficiency of rMOMP was approximately 65% for SHM-BPNs and 90% for conventional BPNs. In vitro characterization studies involved stimulating bone marrow-derived dendritic cells (BMDCs) with varying concentrations (0.1, 1, and 5 µg/ml) of encapsulated-rMOMP within either SHM or conventional BPNs. After 24 hours, cell-free supernatants were collected for cytokine-specific ELISAs, and the cells were processed for gene-expression analysis using TaqMan real-time PCR or were stained with fluorochrome-conjugated antibodies specific for co-stimulatory and antigen-presentation molecules for flow-cytometry assessment. Our results indicated that SHM-BPNs were endocytosed via caveolin pathway in a dose-dependent manner. The data also showed that higher concentrations (1 and 5 µg/ml) led to enhanced activation of pathogen pattern recognition receptors (TLR-2), co-stimulatory molecules (CD40, CD80, CD86), antigen-presentation molecules (MHC-II), and significantly increased production of cytokines (IL-6, IL-12p40), particularly TNF-α, compared to the heterogeneous conventional BPNs. The control BPNs (both conventional and SHM) exhibited lower gene-expression and activation and did not induce any cytokine production. In summary, microfluidics-based BPNs demonstrated that delivering encapsulated-rMOMP within homogeneous-sized BPNs is more effective at activating APCs for antigen-presentation than heterogeneous conventional BPNs, likely due to the uniform activation of endocytosis pathways.