Adjuvanticity Regulation by Biodegradable Polymeric Nano

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The Adjuvanticity Regulation by Biodegradable Polymeric Nano-/microparticles Size Jilei Jia, Weifeng Zhang, Qi Liu, Tingyuan Yang, Lianyan Wang, and Guanghui Ma Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00434 • Publication Date (Web): 17 Nov 2016 Downloaded from http://pubs.acs.org on November 22, 2016

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Molecular Pharmaceutics

The

Adjuvanticity

Regulation

by

Biodegradable

Polymeric

Nano-/microparticles Size †‡‖

Jilei Jia, , , †

†‡‖

Weifeng Zhang, , ,

†‡

†

†

†§

Qi Liu, , Tingyuan Yang, Lianyan Wang,*, Guanghui Ma*, ,

State Key Laboratory of Biochemical Engineering, PLA Key Laboratory of Biopharmaceutical

Production & Formulation Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China. ‡

§

University of Chinese Academy of Sciences, Beijing, 100049, PR China Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing,

210023, PR China ‖

*

These authors contributed equally. Corresponding authors.

Tel/fax: 8610-82544931, E-mail addresses: wanglianyan@ ipe.ac.cn (L.Y. Wang) Tel/fax: 8610-82627072, E-mail addresses: ghma@ ipe.ac.cn (G.H. Ma)

1

ACS Paragon Plus Environment


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Abstract Polymeric nano-/microparticles as vaccine adjuvants have been researched in experimental and clinical studies. A more profound understanding of how the physicochemical properties regulate specific immune responses has become a vital requirement. Here we prepared poly (D,L-lactic-co-glycolic acid) (PLGA) nano-/microparticles with uniform sizes (500 nm, 900 nm, 2.1 µm, and 4.9 µm) and the size effects on particle uptake, activation of macrophages, and antigen internalization were evaluated. Particle uptake kinetic studies demonstrated that 900 nm particles were the easiest to accumulate in cells. Moreover, they could induce macrophages to secrete NO and IL-1β ， and facilitate antigen internalization. Furthermore, 900 nm particles, mixed with antigen, could exhibit superior adjuvanticity in both humoral and cellular immune responses in vivo, including offering the highest antibody protection, promoting the maximum secretion levels of IFN-γ and IL-4 than particles with other sizes. Overall, 900 nm might be the optimum choice for PLGA particle-based vaccine adjuvants especially for recombinant antigens. And understanding the effect of particle size on the adjuvanticity based immune responses might have important enlightenments for rational vaccine design and applications. Key words: PLGA nano-/microparticles; particle size; adjuvanticity; vaccine delivery

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1. Introduction Polymeric nano-/microparticles (NP/MPs) have been extensively investigated, especially biodegradable materials, such as chitosan, poly (lactic acid) (PLA) and poly (D,L-lactic-co-glycolic) acid (PLGA). These particles are used in the cosmetics industry, diagnostic imaging, tissue engineering, separations and purification of biological product, drug delivery and as vaccine adjuvants.1-5 Previous studies by others have shown that biodegradable particles have a high biosafety and biocompatibility and are able to control the release rate and duration of entrapped cargoes.6-9 They can also function as potent vaccine adjuvants, owe to their abilities to improve antigen uptake by antigen-presenting cells (APCs) and further promote the expression of co-stimulatory molecules and major histocompatibility complex (MHC) molecules.10, 11 These excellent properties would be extremely beneficial for various new vaccines, such as protein subunits, recombinant proteins, and synthetic oligopeptides, which have been brought about with the development of biotechnology and genetic engineering.12 While the antigens we use today have great safety, the weak immunogenicity makes them require adjuvant to elicit powerful humoral and cellular immune response for preventing infectious diseases.13

The adjuvanticity of biodegradable polymeric nano-/microparticles has been significantly influenced by various physicochemical characteristics (including morphology, particle size, hydrophobicity and surface charge), vaccine formulation and administration route among others. Previous work has shown that the kinetics and magnitude of the IgG2a response could be strongly affected by the administration 3



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route. For example, intralymphatic administration induced a robust IgG2a response, yet the intradermal and intramuscular routes enhanced a Th1-type response.14 Moreover, the combined vaccine formulation would elicit stronger antigen-specific immune responses than antigen-encapsulated or antigen-mixed with nanoparticles formulation.15 Additionally, high hydrophobicity of polymer-based particles surface could promote antigen internalization and elevate cytokine secretion levels.16-18 Regard to surface charge, Foged et al. reported that the MPs with positive surface charge could greatly enhance dendritic cells (DCs) uptake19, and Chen et al. similarly demonstrated it20. Chitosan-based microparticles with abundant amino groups were favorable for enhancement of antibody titers and activation of complement system.21 However, there has been some controversy about the effect of particle size. Wang et al. reported the 300 nm ursolic acid NPs possessed higher delivery efficiency than the 100 nm particles.22 Meanwhile, Yue et al. suggested that 430 nm chitosan particles could elevate the cell response in macrophages when compared with microsized particles.23 Nixon et al. showed that peptide-entrapped particles of mean size smaller than 500 nm could induce stronger cytotoxic T lymphocyte (CTL) response than larger MPs.24 Nevertheless, several studies suggested that 1-2 µm polystyrene (PS) and phenylated polyacrolein (PPA) MPs were more readily phagocytosed.18 Others reported that ~5 µm PLGA microspheres elicited an enhanced immune response after pulmonary immunization.25 Therefore, particle size might be one of the important factors to determine the entire adjuvanticity including humoral and cellular immune responses.26 After investigating these experiment results, we found that the optimized 4


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size dimension for polymeric MPs-based vaccine adjuvants was unclear, which might be partly due to the difference of the polymer materials to display diverse surface charge and hydrophobicity20, 27. However, the main reason would be attributed to the uncontrolled particle size with a broad distribution. These polymeric particles with varying

sizes,

prepared

by

ultrasonication29, spray drying30,

mechanical 31

stirring22,

, ionic gelation14,

32

28

,

homogenization10,

and emulsification/solvent

evaporation25, 33, resulted in inaccurate and confused situations. To fully clarify the size-dependent adjuvanticity of polymeric particles, PLGA, a FDA-approved biodegradable polymer, was chosen to prepare uniform spherical nano-/microparticles with the diameters of (500 nm, 900 nm, 2.1 µm, and 4.9 µm) using premix membrane emulsification technique. The particles with similar surface characteristics and extremely narrow size distribution were obtained, which could also exclude the impact of shape, surface charge and hydrophobicity. We systematically evaluated internalization of particles with different sizes by macrophages and the ability to activate macrophages in vitro. Next, the adjuvanticity of particles was measured in vivo including the production of antigen-specific antibody and secretion of cytokines. These results would be expected, in principle, to found the basis of future design for polymeric particle-based vaccine and their biomedical applications.

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2. Experimental 2.1 Materials and mice PLGA (molar ratio for lactide/glycolide=75:25, Mw≈13 000 Da) was obtained from Lakeshore Biomaterials (Birmingham, AL, USA). Poly (vinyl alcohol) (PVA-217) was purchased from Kuraray (Tokyo, Japan)，in which the degree of polymerization was 1700 and degree of hydrolysis was 88.5%. Premix membrane emulsification equipment was kindly provided by the National Engineering Research Center for Biotechnology (Beijing, China). DMEM, RPMI 1640 medium and fetal bovine serum (FBS) were ordered from Gibco (Carlsbad, CA, USA). A Cell Counting Kit-8 (CCK-8) was supplied by the Dojindo Laboratories (Kyoto, Japan). Ovalbumin (OVA) as model antigen was purchased from Sigma-Aldrich, and imiquimod (IMQ) used

as

immunostimulator

was

obtained

from

Invivogen

(USA).

All

fluorochrome-conjugated anti-mouse antibodies and mouse cytokine ELISA kits were purchased from eBioscience (San Diego, CA, USA). Specific pathogen-free female BALB/c mice (4-6 weeks of age) were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), kept in a pathogen-free facility. All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals, and approved by Experimental Animal Ethics Committee. 2.2 Preparation and characterization of different PLGA NP/MPs Uniform varisized PLGA particles were prepared by combining premix membrane emulsification technique and the emulsion-solvent evaporation method 6


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(Fig. 1).15 Briefly, 650 mg PLGA was dissolved in 13 mL dichloromethane (O), then was poured into 50 mL 1.9 wt% PVA solution (W) with magnetic stirring (600 rpm, 90 s) to obtain coarse O/W emulsion. Subsequently, the emulsion was transferred into the premix reservoir and extruded through the Shirasu porous glass membrane (SPG Technology Co. Ltd., Japan) with different pore sizes of 1.4 µm, 2.8 µm, 7.2 µm and 16.8 µm under high pressure for four times. The resultant uniform-sized emulsion droplets were solidified into particles by solvent evaporation. PLGA particles with different sizes were centrifuged at 5,000×g for 5 min, and further washed three times with ultrapure (UP) water. Finally, PLGA NP/MPs were lyophilized for storage at 4°C. Similarly, fluorescent dye (Nile Red) was dissolved in dimethylsulfoxide (DMSO), 0.5 mL DMSO solution and PLGA were poured into dichloromethane (O), and Nile Red-labeled NP/MPs were obtained as the steps above for subsequent experiments. The fluorescence intensity of Nile Red-labeled NP/MPs with different sizes was measured and showed in Fig. S1. The morphology of the PLGA particles with different sizes was evaluated using JSM-6700F scanning electron microscopy. They were sprinkled on the sample holder and subsequently coated with platinum at room temperature using an ion vacuum sputter (JFC-1600, Japan) prior to observation. The size distribution and zeta potential of PLGA NA/MPs were also analyzed with the Zetasizer Nano ZS (Malven Instruments Ltd., UK). The formulations were prepared by simply mixing soluble antigen with various particles under gentle shaking for 2 h. The antigen adsorption efficiency was 7



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determined using Micro-BCA protein assay kit by indirect detection of protein concentration in the supernatant.

Fig. 1 Schematic illustration of PLGA nano-/microparticles preparation

2.3 Endotoxin levels The endotoxin levels of different formulations were all analyzed by the Tachypleus Amebocyte Lysate (TAL) assay kit (Pyrosate 0.25 EU/mL) from Zhanjiang A&C Biological Ltd. (Guangdong, China). All materials used were sterile and the endotoxin levels of all formulations were assessed according to the manufacturer’s instructions, and the values were less than 0.05 EU/mg NP/MPs, which excluded the effect of endotoxin contamination. 2.4 Macrophage culture The RAW 264.7 mouse macrophages was obtained from American Type Culture Collection and cultured in DMEM supplemented with inactivated FBS (10% v/v), penicillin (100 U/mL) and streptomycin (100 U/mL), which were incubated in a humidified 5% CO2 incubator at 37°C.34, 35 Mouse peritoneal macrophages were collected from BALB/c mice (female, 6-8 weeks of age) by lavage of their abdominal cavity with 5 ml PBS. After centrifugation 8


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(500×g, 5 min) and resuspension in complete medium, the isolated cells (5 × 105 cells/mL) were then added into 24-well plates and cultured for 6 h. The culture plates were washed with fresh medium to remove non-adherent cells, and the vigorous macrophages were applied for subsequent studies in vitro.20 2.5 Cytotoxicity analysis of different PLGA particles To analyze the cytotoxicity and bio-safety of PLGA nano-/microparticles with different sizes, macrophage viability was tested using CCK-8. The RAW 264.7 macrophages (2.0 × 105 cells/well) were seeded into 96-well plates in 100 µL growth medium. After 24 h incubation with serial dilutions of PLGA particles, the plates were added with CCK-8 reagent (10 µL/well) and further incubated for another 4 h. Absorbance was measured at 450 nm （with 620 nm as reference） and the results were calculated as the ratio of the OD450 values of wells containing particles-stimulated cells with those containing only cells with medium, which were performed in triplicate. 2.6 PLGA particles uptake by macrophages in vitro The RAW 264.7 macrophages and mouse peritoneal macrophages were cultured with Nile Red-labeled NP/MPs in 24-well plates. Cells were collected at various time points as 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h, washed twice with sterile PBS and then stained with the fluorochrome-labeled antibody against CD11b for 30 min to specifically identify macrophages. The percentage of NP/MPs-positive cells in CD11b+ cells and the mean fluorescence intensity (MFI) were acquired on a CyAnTM ADP flow cytometer. 9



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2.7 Activation of macrophages by PLGA particles in vitro Mouse peritoneal macrophages (1.0 × 106 cells/mL) were seeded into 24-well plates in 1 mL growth medium per well and cultured overnight in a humidified CO2 incubator. Afterwards, various particles (125 µg/mL) co-administrated with/without IMQ R837 (5 µg/mL) were added in plates to activate cells for 24 h. Macrophages were centrifuged (500×g, 5 min) and concentrations of NO and IL-1β in supernatants were measured with Platinum ELISA Kits. 2.8 Antigen uptake by macrophages Antigen uptake behaviors by macrophages were investigated through FITC-labeled OVA in vitro using flow cytometry. Cells were incubated with different formulations (NP/MPs, 125 µg/mL; Alexa Fluor 488-labelled OVA, 6.25 µg/mL) in 24-well plates and were collected at the indicated time points (2 h, 4 h, 12 h). Subsequently, they were stained with anti-mouse CD11b antibody and detected with flow cytometer to figure out the amount of OVA-positive macrophages. 2.9 Immunization studies Four BALB/c mice groups (n=6) were intramuscularly immunized twice at 2-week intervals with 100 µL suspensions (Fig. S2). Different vaccine formulations contained 25 µg of OVA or 3 µg of influenza split vaccine antigen (H5N1) and mixed with 500 µg of particles with different sizes. 2.10 Determination of serum IgG and IgG subclasses by ELISA According to a protocol described previously36, we detected quantitatively the antigen-specific IgG and IgG subclasses in sera. Briefly, ELISA plates were coated 10


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with 20 µg OVA (or 2 µg H5N1 antigen) per well in carbonate buffer (0.05 M, pH 9.6) for 12 h at 4°C. Plates were washed four times with 0.01 M PBS containing 0.05% (m/v) Tween 20 (PBST) and then blocked with 2% (m/v) BSA in PBST for 1 h at 37°C. After washing another four times, appropriate sera dilutions (100 µL/well) were added with a two-fold dilution to incubate for 40 min. Thereafter, plates were then washed with PBST and stained with 100 µL HRP-conjugated anti-mouse antibodies (diluted 1:10000) for 30 min. The ELISA plates were washed again and 200 µL of TMB substrate was pipetted to all wells for 20-mimute incubation at room temperature. After stopping the reaction by adding 50 µL of 2 M H2SO4, OD450 was measured and antibody titers were described as the reciprocal sample dilution corresponding to twice higher OD value than that of the negative sera. 2.11 Determination of cytokine production For mice immunized with ovalbumin, splenocytes (5×106 cells/mL) were harvested on day 28 and re-stimulated with 50 µg/mL OVA for 65 h. Similarly, for mice immunized with H5N1, splenocytes were cultured with hemagglutinin (HA, 2.5 µg/mL) in a humid incubator (37°C, 5% CO2). The supernatant containing cytokines was collected by centrifugation (500×g, 5 min) and concentrations of IFN-γ and IL-4 were measured by Ready-to-use Sandwich ELISA kits.37 2.12 Statistical analysis Statistical analysis was performed using GraphPad Prism 5.0 software and all data were expressed as the means ± SEM. Differences among groups were determined by one-way ANOVA and Tukey’s multiple comparison. 11



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3. Results and discussion 3.1 Preparation and characterization of different PLGA NP/MPs In this study, uniform-sized PLGA nano-/microparticles with four kinds of diameters

were

successfully

fabricated

by

combining

premix

membrane

emulsification technique and the emulsion-solvent evaporation method. As shown in Table 1, their respective diameters were ~538.5 nm, 972.5 nm, 2126 nm and 4934 nm, measured by dynamic light scattering size measuring systems, and these particles were named MPs500 nm, MPs900 nm, MPs2.1 µm and MPs4.9 µm, respectively. The negative surface charge of PLGA particles was similar (approximately -20 mV). SEM graphs (Fig. 2A) also revealed that all NP/MPs were smooth and spherical with narrow size distributions. Meanwhile, the residual content of PVA and DCM were measured (Supporting Information, S1), which excluded the unfavorable impact of other impurities to further investigate the size effect of PLGA particles. Table 1 The physicochemical characterization of different PLGA NP/MPs Particles

Diameter (nm)

Zeta potential (mV)

MPs500 nm

538.5±16.5

-19.6±0.95

MPs900 nm

972.5±15.4

-22.3±2.21

MPs2.1 µm

2126±10

-16.8±0.96

MPs4.9 µm

4934±93

-17.4±2.43

To determine the cytotoxicity of PLGA particles with different sizes, we next assessed the viability of the RAW 264.7 cells after co-cultured with particles for 24 h

in vitro. The cell viability of all groups retained more than 80% (Fig. 2B), which referred to their negligible cytotoxicity regardless of the increase of the particles 12


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amount (up to 1250 µg/mL). Hence, the PLGA particles were safe as vaccine delivery and adjuvant system.

Fig. 2 Characteristics of various PLGA particles. (A) Scanning electron micrographs (SEM) of four kinds of nano-/microparticles; (B) Viability of RAW 264.7 macrophages after incubation with different concentrations of nano-/microparticles.

3.2 Evaluation of PLGA particles uptake by macrophages in vitro Antigen-presenting cells (APCs) play an essential role in capturing and processing antigen, activating T/B lymphocytes and inducting the immune response. We assessed the interaction between PLGA particles and macrophages (the typical kind of professional APCs), which would determine the adjuvanticity of various NP/MPs. First, the RAW 264.7 macrophages were utilized to investigate the internalization of NP/MPs with several concentrations (Fig. 3A), which indicated that almost all of the RAW 264.7 cells (>90%) took in various particles with a high concentration of 250 µg/mL. When moderate numbers of particles (50 and 125 µg/mL) were added in the medium, there was an obvious distinction among different treatment groups: almost all the macrophages easily captured the MPs900 nm, nearly 80% cells internalized the MPs500 nm, but only about 40% particle-positive cells existed in the MPs2.1

µm

and MPs4.9

µm

groups. This might be due to the distinction of 13



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internalization pathways19, 23, which revealed that 900 nm particles were probably taken up by macropinocytosis and phagocytosis, however, the smaller spheres mostly by receptor-mediated endocytosis and microsized particles mainly by phagocytosis. Those would result in MPs900 nm uptake by macrophages more easily. We further investigated the uptake kinetic results of different PLGA particles in the concentration of 125 µg/mL (Fig. 3B). MPs900 nm and MPs500

nm

were rapidly

internalized up to 80% within 0.5 h, and the amount of particle-positive macrophages reached peaks and kept saturated state at 4 h. Then the ratios of MPs-positive cells had a slight decline, which could be caused by exocytosis38. MPs in cytoplasm could move to the outside of cells with the integration of cell membrane and endosome/lysosome.39, 40 It would be a dynamic balance owing to endocytosis and exocytosis in the later time, and the levels of Nile Red-positive macrophages were as follows: MPs900

nm

> MPs500

nm

> MPs2.1

µm

> MPs4.9

µm.

The same results were

obtained in mouse primary peritoneal macrophages (Fig. S4), which also indicated that compared to MPs with other sizes (500 nm, 2.1 µm, and 4.9 µm), MPs900 nm could be more easily and rapidly internalized by APCs.

14


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Fig. 3 PLGA particles uptake by Raw 264.7 macrophages. (A) Cellular uptake profiles of different nano-/microparticles in various concentrations; (B) Kinetics of nano-/microparticles uptake by macrophages.

3.3 Activation of macrophages by PLGA particles in vitro Activation of APCs is essential for antigen presentation and initiation of adaptive immune response. Secreting nitric oxide (NO) is a major marker of activated macrophages, so that NO production indicates the activation degree of macrophages31, 33

. As shown in Fig. 4A, NO concentration in supernatants of MPs900

nm

group

co-cultured with macrophages was much higher than other groups (both p < 0.05, for MPs2.1 µm and MPs4.9 µm), which revealed that 900 nm particles could significantly activate APCs. To further explore the activation ability of various particles, we next detected the secretion of IL-1β in supernatants of macrophages and MPs cultured with/without Imiquimod (IMQ). As one of the significant pro-inflammatory cytokines, IL-1β played a major role in infection process to mediate APCs activation. The process of IL-1β secretion were divided into two steps: first, pathogen associated molecular 15



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patterns (PAMPs) induced APCs to generate the inactive pro-IL-1β trapped inside cells; then, under the action of inflammatory protease caspase-1, pro-IL-1β is cut into active IL-1β to release into the extracellular environment.41-43 The IL-1β concentration of all treatment groups became a little higher than the blank control group, but there was no obvious difference of each group (Fig. 4B, left). The introduction of IMQ (Toll-like receptor 7 ligands, as PAMPs) induced much more pro-IL-1β and the IL-1β concentrations were also greatly increased (Fig. 4B, right). Particulate adjuvants co-cultured with IMQ resulted in more IL-1β secretion, up to approximate 250% - 360% of blank group. Sharp, F. A. et al. reported that PLGA particles could activate caspase-1 and promote its secretion.44 With the effect of the two aspects, particles synergized with IMQ to produce more IL-1β to release out of cells. This also might be owing to the augment of internalized IMQ amount when PLGA particles were phagocytosed, which should be further confirmed. Hereinto, MPs900 nm could induce the highest secretion level of IL-1β, which possessed the strongest ability to enhance the activation of APCs. These results were consistent with the MPs uptake levels by macrophages which would be conducive to generating a potent immune response.

16


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Fig. 4 Activation effects of different PLGA nano-/microparticles on peritoneal macrophages. (A)

nitric oxide (NO) production by macrophages exposed to different particles; (B) Secretion of IL-1β by macrophages (+ means added, - means not added).

3.4 Evaluation of antigen uptake in vitro The induction of immune responses would inevitably require the antigen internalization into APCs. Macrophages were a kind of primary APCs to sense pathogens and antigens in the body circulation45. Therefore, we next assessed antigen uptake of FITC-OVA-MPs by mouse peritoneal macrophages in vitro. First, the antigen adsorption efficiency was measured by less than 5% which testified that NP/MPs surface adsorbed little antigen, and also, the surface zeta potential of particles after antigen adsorption had little change. Compared to macrophages co-cultured with FITC-OVA alone, antigen uptake was significantly augmented in macrophages with all FITC-OVA-MPs groups (Fig. 5). Macrophages treated with FITC-OVA-MPs900 nm exhibited the highest percentage of OVA+ cells than MPs with other sizes. These data suggested that improvement of antigen internalization by MPs900 nm might be contributed by the enhancement of particle uptake (Fig. 3) and 17



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their strongest induction of macrophage activation (Fig. 4).

Fig. 5 Effects of different microparticles on antigen (Ag) uptake by peritoneal macrophages

3.5 The adjuvanticity of different NP/MPs for OVA model vaccine in mice After confirming antigen internalization, we further determined the efficacy of NP/MPs with different sizes including humoral and cellular immune responses. We measured antigen-specific IgG and IgG subclass profiles (IgG1, IgG2a, and IgG2b) in sera collected from immunized mice (Fig. 6). On day 14, MPs-based adjuvants improved IgG production, and MPs900 nm produced the highest IgG titers significantly higher than then soluble antigen (p

Adjuvanticity Regulation by Biodegradable Polymeric Nano

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