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.
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.
To combat epidemic, chronic, and infectious diseases, it is imperative to develop effective adjuvants for prophylactic and therapeutic vaccinations1,2. Ideally, the adjuvants should possess excellent safety and immune activation3,4,5. 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 response6,7,8. Hence, gaining a clear understanding of the mechanism of interaction of immune cells with antigens and designing adjuvants to enhance internalization are efficient strategies to enhance the efficiency of vaccines.
Micro-/nanoparticles with unique properties have been previously investigated as antigen delivery systems to mediate the cellular uptake of antigens and the cellular interaction with pathogen-associated molecular patterns9,10. Upon contacting with cells, delivery systems begin to interact with the extracellular matrix and cell membrane, which led to internalization and subsequent cellular responses11,12. Previous studies have brought to light that the internalization of particles takes place through cell membrane-particle adhesion13, followed by flexible deformation of the cell membrane and diffusion of the receptor to the surface membrane14,15. Under these circumstances, the properties of the delivery system depend on the affinity to APCs, which subsequently affect the uptake quantity16,17.
To gain insights into the design of the delivery system for improved immune response, extensive efforts have been focused on the investigation of the relationship between the properties of particles and cellular uptake. The present study stemmed from the observation that solid micro-/nanoparticles with various charges, sizes, and shapes are often studied in this light, while the role of fluidity in antigen internalization is seldom investigated18,19. In fact, during adhesion, the soft particles demonstrated dynamic curvature changes and lateral diffusions to increase the contact area for multivalent interactions, which can hardly be replicated by the solid particles20,21. In addition, cell membranes are phospholipid bilayers (sphingolipids or cholesterol) at the site of uptake, and hydrophobic substances can alter the conformational entropy of lipids, reducing the amount of energy required for cellular uptake22,23. Thus, amplifying mobility and promoting hydrophobicity of the delivery system may be an effective strategy for strengthening antigen internalization to enhance immune response.
Pickering emulsion, stabilized by solid particles assembled at the interface between two immiscible liquids, have been widely used in the biological field24,25. In fact, the aggregating particles on the oil/water interface determine the formulation of multi-level structures, which promote multi-level delivery system-cellular interactions, and further induce multi-functional physiochemical properties in drug delivery. Because of their deformability and lateral mobility, Pickering emulsions were expected to enter in multi-valent cellular interaction with the immunocytes and be recognized by the membrane proteins26. In addition, as oily micelle cores in Pickering emulsions are not completely covered with solid particles, Pickering emulsions possess gaps of different sizes between particles on the oil/water interface, which cause higher hydrophobicity. Thus, it is crucial to explore the affinity of Pickering emulsions to APCs and elaborate on the subsequent internalization to develop efficient adjuvants.
Based on these considerations, we engineered a PLGA nanoparticle-stabilized Pickering emulsion (PNPE) as a fluidity vaccine delivery system that also helped to gain valuable insights in the affinity of the PNPE to BMDCs and cellular internalization. The real-time adhesion of bio- mimetic extracellular vesicles (bEVs; a replacement of BMDCs) to PNPE was monitored via a label-free method using a quartz crystal microbalance with dissipation monitoring (QCM-D). Following characterization of the affinity of PNPE to BMDCs, confocal laser scanning microscopy (CLSM) was used to determine the antigen uptake. The result indicated the higher affinity of PNPE to BMDCs, and the efficient internalization of the antigen. We anticipated that the PNPE would exhibit higher affinity to APCs, which may better stimulate the internalization of antigens to enhance immune responses.
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
2. Preparation and characterization of PNPE
3. Isolate and culture BMDCs 28
NOTE: Make sure that all reagents and samples are placed on ice, as this has a positive effect on cell activity. To maintain sterility, perform all steps on an ultra-clean bench using sterile utensils.
4. Preparation of bio-mimetic extracellular vesicles (bEVs)
5. bEVs adhere to the PNPE
NOTE: The SiO2 sensor was modified via spin-coating method.
6. CLSM analysis of antigen uptake
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 and Supplementary Table 1). To prepare PNPE, the mixture of PNP solution (0.4%, w/v) and squalene was simply sonicated at 100 w for 5 min (Figure 1D). After optimization of the continuous phase, the obtained PNPE was internally composed of squalene and externally adsorbed with PNPs (Supplementary Figure 1). An optical microscope was employed to observe the emulsion droplets, and the Mastersizer particle size analyzer was used to determine the size distribution. PNPE exhibited no less particles to prevent the coalescence of the emulsions and no more particles to excess in the continuous phase that cause the larger aggregates (Figure 1E). As shown in Figure 1F and Supplementary Table 1, the emulsion size was 2100 ± 300 nm and the zeta potential was -27.1 ± 0.5 mV, indicating that the particles aggregated on the oil/water interface. To observe the cellular uptake of antigen, the Cy5-labled OVA was adsorbed on the PNPE. To start with, 500 µL of PNPE was mixed with the 500 µL Cy5-labeled OVA at room temperature for 1 h. The absorption of antigens on the formulations was tested using BCA assay. Owing to its high specific surface area and hydrophobicity, more than 70.6% of the fluidic antigens were adsorbed on the PNPE within 1 h, indicating substantial potential for loading large quantities of antigens in a short time. As the control group, PMPs and SSE were also characterized. The PMPs were comparable in size (1987 ± 310 nm) to the PNPE. The SSE was negatively charged emulsion (-15.9 ± 0.8 mV) with a diameter size of 147.2 ± 0.5 nm. Additionally, the loading efficiency of PMPs and SSE was only 25.6% ± 0.6% and 23.4% ± 0.2%, respectively due to the limited adsorption with antigens (Supplementary Table 1). Additionally, to evaluate the stability of PNPE, the prepared PNPE were stored at 4 °C and 25 °C, respectively. The size and zeta potential of the stock solution were determined on days 0-6 (1:50 dilution). As shown in Supplementary Figure 2, the droplets remained similar in size and zeta potential during storage at 4 °C and 25 °C suggested higher stability of the PNPE for the delivery of antigens. Then the QCM-D was used to measure the flexibility of PNPE. As shown in Figure 1G, as a result of its rigid structure, the PMPs exhibited lower dissipation (ΔD). At the same time, PNPE had a significantly higher ΔD, demonstrating its excellent viscoelasticity and flexibility, which may be one of the reasons for improved cell attachment and uptake.
To validate the affinity of PNPE to the BMDCs membrane, the bEVs were used to replace the intact cells because of their fitting size for the accurate QCM-D detection. In this process, the mature BMDCs were harvested and serially extruded through polycarbonate membrane filters that contained 10, 5, and 1 µm pore sizes to prepared nano-sized vesicles. The obtained bEVs were collected and purified via ultracentrifugation and re-suspended in HEPES buffered saline (Figure 2A). The morphology of the negatively stained bEVs was then explored using transmission electron microscopy (TEM). A nanoparticle tracking analysis (NTA) was used to test the size of bEVs. The results revealed that bEVs were closed lipid-bilayered vesicular forms and the distribution was homogeneous (Figure 2B). NTA showed that the size distribution of the bEVs with peak diameter of the purified bEVs was 131 nm (Figure 2C). We thus concluded that the bEVs were a typical membrane structure with uniform distribution, which allowed for more accuracy in the QCM-D assay as a substitute for BMDCs.
Next, the adhesion of bEVs to PNPE was tracked using QCM-D. First, the QCM-D SiO2 sensor was modified with PLL to confer a positive charge to subsequently fix PNPE on the surface and simulate the bio-interphase process. It was immediately followed by adding the bEV solution into the chamber to elucidate the interaction between PNPE and bEVs (Figure 3A). As shown in Figure 3B, the immediate decrease in frequency (ΔF) indicated a rapid adhesion of bEVs to PNPE after encounter. Moreover, ΔF decreased with increasing bEV concentration, reflecting a concentration-dependent effect. PNPE has a densely packed surface to support the landing spot; moreover, it exhibited dynamic curvature changes and lateral diffusion for potent cellular contact, resulting in high affinity to BMDCs. In contrast, PMPs and SSE were weakly bound to bEVs, even at high concentration (80 µg/mL), which probably resulted from a lack of contact sites with the immune cells (Figure 3C).
After confirming the process of adhesion to BMDCs, PNPE was used to deliver antigen to the BMDCs. To directly visualize the antigen internalization profiles in vitro, Cy5-labeled OVA was mixed with PMPs, SSE, and PNPE to treat with BMDCs, respectively. After 6 h post-treatment, CLSM was performed to analyze the cellular uptake of Cy5-labeled OVA in BMDCs. As shown in Figure 4A, the Cy5-OVA fluorescence signal indicated that the total amount of antigen internalized into cells is significantly higher in the PNPE-treated group compared to that in the PMPs and SSE-treated group. The quantitative analysis of cellular uptake was further performed, showing a significantly higher relative intensity of fluorescence in BMDCs treated with PNPE than in those treated with PMPs and SSE (p < 0.001), which corroborated the observation above (Figure 4B). The obtained results demonstrated that the PNPE promoted the antigen internalization and effectively delivered the antigen intracellularly. Consequently, the uptake efficiency of antigens was positively associated with the delivery system affinity to targeted cells, and PNPE effectively increased the antigen cellular uptake via multi-level contact with the cell membrane.
Figure 1: Characterization of PLGA nanoparticle-stabilized Pickering emulsion. (A) Schematic of the experimental procedure used for the preparation of PLGA nanoparticles (PNPs). (B) Scanning electron microscopy (SEM) images of PNPs. Scale bar = 200 nm. (C) Size distribution of PNPs. (D) Schematic of the experimental procedure used for the preparation of nanoparticle-stabilized Pickering emulsion (PNPE). (E) Optical micrographs of PNPE. Scale bar = 20 µm. (F) Size distribution of PNPE. (G) Quartz crystal microbalance with dissipation monitoring (QCM-D) analysis on the softness of PNPE, PLGA microparticles (PMPs), and surfactant-stabilized emulsion (SSE) through detection of dissipation (ΔD). Please click here to view a larger version of this figure.
Figure 2: Characterization of bio-mimetic extracellular vesicles. (A) Schematic of the experimental procedure used for the preparation of bio-mimetic extracellular vesicles (bEVs). (B) Transmission electron microscopy (TEM) images of bEVs. Scale bar = 200 nm. (C) Size distribution of bEVs measured using nanoparticle tracking analysis (NTA) (n = 3). Please click here to view a larger version of this figure.
Figure 3: Affinity of PNPE to bEVs analyzed using QCM-D. (A) Schematic of the modification of the PLL surface through PNPE coating. (B) Changes in frequency (ΔF) of PNPE after encountering with different concentrations of bEVs. (C) Comparison of changes in ΔF in PLGA microparticles (PMPs), surfactant-stabilized emulsion (SSE), and PNPE. Please click here to view a larger version of this figure.
Figure 4: Uptake of antigens. (A) Representative diagram of the cellular uptake of antigens. Scale bar = 20 µm. (B) Relative intensity fluorescence of cellular uptake of antigens. The graph displays mean ± SEM from three independent experiments. One-way ANOVA was used to analyze the significance of the observed differences. *** p < 0.001. Please click here to view a larger version of this figure.
Supplementary Figure 1: The optimization of continuous phase. (A) Optical micrographs of PNPE constructed by different continuous phases. Scale bar = 20 µm. (B) The sizes of the emulsions were detected after the preparation. Results were expressed as mean ± SEM (n = 3). Please click here to download this File.
Supplementary Figure 2: The stability of PNPE. Size (A) and zeta potential (B) of PNPE after 0, 3, and 6 days of storage at the indicated temperatures. Data were demonstrated as mean ± SEM (n = 3). Please click here to download this File.
Supplementary Table 1: Characterizations of the formulations. PNPs, PNPE, and SSE were prepared according to the indicated protocol. The size distributions and zeta potential were determined by dynamic light scattering analyzer (DLS) or Mastersizer particle size analyzer. The loading efficiency was evaluated using the BCA assay. Data were demonstrated as mean ± SEM (n = 3). Please click here to download this File.
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 the cells, indicating that the PLGA nanoparticle-stabilized emulsion has a strong affinity to the cell membrane for cellular uptake. Furthermore, PNPE had a high safety profile because both squalene and PLGA are Food and Drug Administration (FDA) approved ingredients30, which is expected to leverage safe transfer into the clinic.
The molecular weight of the PLGA and the type of continuous and dispersed phases affected the PNPE properties, including stability and hydrophobicity. In this research, 17 kDa molecular weight PLGA nanoparticles were selected as the stabilizer and squalene as the disperse phase, resulting in increased stability. Additionally, deionized water as a continuous phase simplified the composition of the formulation. Under this condition, PNPE allowed efficient assembly of antigens and promoted the delivery of antigens to immune cells. Furthermore, the method of one-step sonication was used to prepare PNPE, which eliminated the tedious process and avoided the possibility of contamination. It is essential to note that the delivery system composed of PNPs and squalene needs to be a uniform force, otherwise no emulsion is formed or the prepared PNPE is not uniform in size.
While studying the affinity of the delivery system to cells was an extraordinary challenge performed in vivo, in vitro studies could help to elucidate the process involved in cellular adhesion and internalization. Recently, this problem has gotten much attention in the biomedical field. Substantial efforts have been made to shed light on particles’ affinity to cells using flow cytometry and CLSM analysis31,32. While these methods provide an average readout at the cellular level, the specific dynamic binding processes between cells and delivery systems are not easily monitored. In contrast, QCM primarily depends on a sensitive piezoelectric crystal that the ΔF changes with mass. The ability to detect minute mass changes allows QCM-D to monitor the targeted molecular interactions. The technique can be used to monitor real-time events, allowing the study of affinity of PNPE with BMDCs under different conditions33.
The adhesion to PNPE was investigated using bEVs instead of intact cells. As the well-controlled simple APCs, bEVs are believed to inherit most of the characteristics of the parent cells. In general, extracellular vesicles with small and homogeneous particle sizes perform better in adhesion assays. Thus, the bEVs were prepared via extrusion through polycarbonate filters with 10, 5, and 1 µm pore sizes34. The diameter and yield of bEVs could be controlled by regulating the number of extrusions and the pore size of polycarbonate membranes. It is recommended to make 30 passes through the polycarbonate membranes. However, this method also had one limitation in that part of membrane may be inversed, causing the proteins on the DC surface to be encapsulated in the bEVs, which slightly decreased the affinity to emulsions.
It was shown that the modification of the SiO2 sensor surfaces using positively charged proteins was suitable for the subsequent PNPE coating. The described implementation of PLL (Polycation polypeptide) as the intermediate layer between SiO2 sensor surfaces and PNPE proved to be an improvement for the fast-coating method. Changes in mass caused by any event, for instance, nonspecific adsorptions of any component from the flow of solution on the SiO2 sensor, might affect the accuracy of experimental results35. Therefore, it was necessary to ensure that the PLL retained a consistent quality on the chip after spin coating, and their surface would be completely covered by PNPE to avoid the measurement error caused by nonspecific adsorption. At the same time, when ΔF no longer changed during the contact with the PNPE-containing mobile phase, the chip surface was considered completely covered. This ensured that the detected signals were generated by the adhesion of bEVs to the PNPE. We demonstrated that QCM-D was a good method for showing the adhesion of bEVs to PNPE in real-time, reflecting the high affinity of PNPE to APCs. Unfortunately, this method could not reflect the interaction between individual cells and the delivery system. Therefore, a more accurate determination will require further design of the protocol and optimization of the measure procedure.
After assaying the high affinity of PNPE to bEVs, enhanced internalization was further verified. We demonstrated that the potent cellular uptake of antigens correlated with the high affinity of PNPE. PNPE possessed the multi-level structure for effective loading of antigens and flexibility for multi-level contact with the cell membrane to enhance delivery. Therefore, the rationally designed Pickering emulsion stimulated cellular internalization and intracellular pathways through the robust interaction with the cell membrane. Having demonstrated these advantages, PNPE may shed light on the development of novel, safe, and efficient antigen delivery systems for enhancing vaccines.
This protocol successfully demonstrated the high affinity of PNPE to the phospholipid bilayer of immune cells and subsequent intracellular delivery of the antigen to immune cells in vitro. Therefore, various types of antigens from different pathogens can be delivered and characterized using the proposed protocol to confer protection against infectious diseases.
The authors have nothing to disclose.
This work was supported by Project supported by the National Key Research and Development Program of China (2021YFE020527, 2021YFC2302605, 2021YFC2300142), From 0 to 1 Original Innovation Project of Basic Frontier Scientific Research Program of Chinese Academy of Sciences (ZDBS-LY-SLH040), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant No. 21821005).
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 |