Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

Summary

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

Abstract

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug delivery and immune response. The present study stemmed from the observation that the effects of charge, size, and shape of solid particles on cell affinity are usually considered, but we seldom realize the essential role of softness, dynamic restructuring phenomenon, and complex interface interaction in cellular affinity. Here, we developed poly-lactic-co-glycolic acid (PLGA) nanoparticle-stabilized Pickering emulsion (PNPE) that overcame the shortcomings of rigid forms and simulated the flexibility and fluidity of pathogens. A method was set up to test the affinity of PNPE to cell surfaces and elaborate on the subsequent internalization by immune cells. The affinity of PNPE to bio-mimetic extracellular vesicles (bEVs)-the replacement for bone marrow dendritic cells (BMDCs)-was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed real-time monitoring of cell-emulsion adhesion. Subsequently, the PNPE was used to deliver the antigen (ovalbumin, OVA) and the uptake of the antigens by BMDCs was observed using confocal laser scanning microscope (CLSM). Representative results showed that the PNPE immediately decreased frequency (ΔF) when it encountered the bEVs, indicating rapid adhesion and high affinity of the PNPE to the BMDCs. PNPE showed significantly stronger binding to the cell membrane than PLGA microparticles (PMPs) and AddaVax adjuvant (denoted as surfactant-stabilized nano-emulsion [SSE]). Furthermore, owing to the enhanced cellular affinity to the immunocytes through dynamic curvature changes and lateral diffusions, antigen uptake was subsequently boosted compared with PMPs and SSE. This protocol provides insights for designing novel formulations with high cell affinity and efficient antigen internalization, providing a platform for the development of efficient vaccines.

Introduction

To combat epidemic, chronic, and infectious diseases, it is imperative to develop effective adjuvants for prophylactic and therapeutic vaccinations^1,2. Ideally, the adjuvants should possess excellent safety and immune activation^3,4,5. Effective uptake and process of antigens by antigen-presenting cells (APCs) are thought to be an essential stage in the downstream signaling cascades and initiation of the immune response^6,7,8. ....

Protocol

All methods described in this protocol have been approved by the Institute of Process Engineering, Chinese Academy of Sciences. All animal experiments were performed in strict accordance with the Regulations for the Care and Use of Laboratory Animals and Guideline for Ethical Review of Animal (China, GB/T35892-2018).

1. Preparation and characterization of PLGA nanoparticles

1. Preparation of PLGA nanoparticles (PNPs)
   1. Add 0.5 g of polyvinyl alcohol (PVA) to 120 mL of deionized water at 90 °C and stir until completely dissolved to prepare the PVA solution. Store the solution in the refrigerator (4 °C) aft....

Results

A simple one-step sonification was used to obtain PNPE. First, we prepared uniform PNPs for use as the solid stabilizer (Figure 1A). The morphology of PNPs were observed though SEM, showing that they are mostly uniform and spherical (Figure 1B). The hydrodynamic size and zeta potential of the formulations were detected via DLS. The diameter of the PNPs was 187.7 ± 3.5 nm and the zeta potential was -16.4 ± 0.4 mV (Figure 1C .......

Discussion

We developed PLGA nanoparticle-stabilized oil/water emulsion as a delivery system for enhanced antigen internalization. The prepared PNPE possessed a densely packed surface to support the landing spot and unique softness and fluidity for potent cellular contact with the immune cell membrane. Furthermore, the oil/water interface offered high-content antigen loading, and amphiphilic PLGA conferred PNPE with high stability for the transportation of antigens to immune cells. The PNPE could rapidly adhere to the surface of th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AddVax	InvivoGen	Vac-adx-10
Cell Strainer	Biosharp	BS-70-CS	70 μm
Confocal Laser Scanning Microscope (CLSM)	Nikon		A1
Cy3 NHS Ester	YEASEN	40777ES03
DAPI Staining Solution	Beyotime	C1005
Fetal Bovine Serum (FBS)	Gibco	16000-044
FITC Phalloidin	Solarbio	CA1620
Mastersizer 2000 Particle Size Analyzer	Malvern
Micro BCA protein Assay Kit	Thermo Science	23235
Membrane emulsification equipment	Zhongke Senhui Microsphere Technology		FM0201/500M
Mini-Extruder	Avanti Polar Lipids, Inc
NANO ZS	Malvern		JSM-6700F
Polycarbonate membranes	Avanti Polar Lipids, Inc
Poly (lactic-co-glycolic acid) (PLGA)	Sigma-Aldrich	26780-50-7	Mw 7,000-17,000
Poly-L-lysine Solution	Solarbio	P2100
Poly (vinyl alcohol) (PVA)	Sigma-Aldrich	9002-89-5
QSense Silicon dioxide sensor	Biolin Scientific	QSX 303	Surface roughness < 1 nm RMS
Quartz Crystal Microbalance	Biosharp		Q-SENSE E4
RPMI Medium 1640 basic	Gibco	C22400500BT	L-Glutamine, 25 mM HEPES
Scanning Electron Microscopy (SEM)	JEOL		JSM-6700F
Squalene	Sigma-Aldrich	111-02-4