Polydopamine as the Antigen Delivery Nanocarrier for Enhanced

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Polydopamine as the antigen delivery nanocarriers for enhanced immune response in tumor immunotherapy wang ning, Ying Yang, Xiaoli Wang, Xinxin Tian, Wenjuan Qing, Xiaoxiao Wang, Jiayi Liang, Hailing Zhang, and Xigang Leng ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00359 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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ACS Biomaterials Science & Engineering

Polydopamine as the antigen delivery nanocarriers for enhanced immune response in tumor immunotherapy Ning Wang, Ying Yang, Xiaoli Wang, Xinxin Tian, Wenjuan Qin, Xiaoxiao Wang, Jiayi Liang, Hailing Zhang*, Xigang Leng* Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College , Tianjin Key Laboratory of Biomaterials, Tianjin 300192, China * Corresponding author. E-mail address: [email protected] (H.L. Zhang), [email protected] (X.G. Leng). ABSTRACT:

This study was aimed to investigate the efficacy of polydopamine

nanoparticles (Pdop-NPs) as a subcutaneous antigen delivery vehicle in anti-tumor therapy. The nanoparticles were prepared by self-polymerization of dopamine in an aerobic and weak alkaline solution, and the tumor model antigen-ovalbumin (OVA) was grafted onto the nanoparticles to form OVA@Pdop nanoparticles (OVA@Pdop-NPs). The particle size of OVA@Pdop-NPs was 232.8 nm with a zeta potential of -23.4 mV, and the loading capacity of OVA protein was 754 µg mg-1. OVA@Pdop-NPs were essentially non-cytotoxic, and even demonstrated a slightly viability effect on bone marrow-derived dendritic cells (BMDCs). As compared to free OVA, OVA@Pdop-NPs exhibited higher cellular uptake and were easier to migrate to lymph nodes in vivo. Both in vitro and in vivo experiments showed that OVA@Pdop-NPs promoted the maturation

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of DCs with up-regulated expression of major histocompatibility complex (MHC), costimulatory molecules and cytokines. When used to treat the mice bearing OVA-MC38 colon tumor, OVA@Pdop-NPs could effectively activate OVA-specific cytotoxic CD8+ T cells, induce the production of memory CD4+ and CD8+ T cells, and thus led to significantly suppressed tumor growth. All those preliminary data demonstrated the application potential of OVA@Pdop-NPs as a vaccine vector in cancer immunotherapy. Keywords cancer immunotherapy, polydopamine, nanoparticles, colon cancer INTRODUCTION In the context of cancer vaccines based on new antigens, mRNA, DNA or synthetic long peptides (SLPs) are commonly used. Antigenic peptides are widely utilized for developing cancer vaccines due to their advantageous properties, which include direct functional T cell epitopes, low toxicity, low cost, and ease of synthesis.1 Unformulated peptide vaccine administered subcutaneously can rapidly move into peripheral blood vessels, and thus lead to systemic spread. As such, antigen presenting cells (APCs) captures only a small fraction of the administered SLPs that can reach the cytoplasm for subsequent antigen presentation, the ultimate therapeutic effects of these peptide vaccines usually do not meet expectations, 2 and thus limit their application in cancer therapy. Effective antigen presentation is a fatal prerequisite for antitumor immunity, which requires the activation of APCs. Most antigens are usually unable to escape from


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lysosomes, and finally presented by the MHC II molecule that only elicits humoral immunity.

3-4

In order to induce specific and highly active cytotoxic T lymphocytes

(CTLs) which is vital for anti-tumor immunity, the antigen must be presented together with MHC I molecule along with the simultaneous expression of the corresponding costimulatory molecules by dendritic cells (DCs). Therefore, it is necessary to design a tumor

vaccine

that

can

retain

tumor

antigens

in

the

cytoplasm,

promote

cross-presentation within APCs to activate the MHC I mediated presentation, and finally induce cellular immunity. Nanoparticulated vaccine formulations have been receiving increasing attention, which are easier to be absorbed by DCs for more efficient antigen presentation,5 providing the possibility of making up for vaccine restrictions.6-7 The efficacy of nanoparticulated peptide vaccine to induce immune response depends on the choice of vector, antigen and adjuvant. At present, most of the nanoparticulated vaccine formulation are designed following two strategies: the antigens are either encapsulated in nanoparticles (NPs) or displayed on the surface of NPs. It was reported that the antigen encapsulated in NPs by self-assembly process induced a stronger antigen specific immune response than the soluble antigen.8-9 More recently, the applications of antigen-on-nanoparticle surface formulations, such as virus-like particles10 or protein particles,11 has become increasingly attractive, with numerous studies demonstrating that APC engagement with nanoparticles themselves triggers inflammatory responses.12-14



Although a variety of nanomaterials have been used for antigen delivery, surface modification of those nanomaterials remained to be challenging. Because of the inherent chemical inertness of poly(lactic-co-glycolic acid) (PLGA) or poly(lactic acid) (PLA) NPs, some coupling agents or activated connectors are necessarily required for antigen modification. In addition, the modification process was generally a multi-step reaction, and the subsequent purification was usually inefficient and troublesome.15 The biosafety issues are also a major concern in the development and medical application of nanomaterials. Cationic polymers, such as chitosan (CS) and polyethyleneimine (PEI), were effective in delivering antigens and eliciting immune response,16 but they may be cytotoxic due to damage to the cell membrane.

17

As such, development of non-toxic

biomaterials as a biomimetic antigen delivery system remains a bottleneck. Polydopamine nanoparticles (Pdop-NPs) has been rapidly applied to many biological areas such as bioimaging, photothermotherapy and drug delivery systems due to their excellent biocompatibility.18-19 Dopamine is not only one of the key neurotransmitters, but also an important connector between the nervous and immune systems. As an extracellular messenger, dopamine regulates the immune system by interacting with dopamine receptors on the immune cells.20-21 More and more investigations have shown that dopamine plays a pivotal role in regulating the immune system. Dopamine can activate resting effector T cells and suppress regulatory T cells, thus touch off multiple key T cell functions. Moreover, dopamine receptors are widely distributed in the brain and peripheral tissues, such as the blood vessel, various immune


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cells and tumors.22 Previous studies discovered that dopamine had antiangiogenic and anti-cancer activity via activation of dopamine receptor on endothelial and tumor cells.23 Therefore, dopamine could be an excellent material as antigen delivery carrier for tumor immunotherapy. As a self-polymer of dopamine, Pdop-NPs have abundant active catechol group, making it easily to further modify biomacromolecule on its surface by chemical conjugation.24 Through their surface rich reaction sites, Pdop-NPs could easily bind to the free amino and thiol groups of protein, and likely to serve as efficient antigen delivery vectors. In this study, in vitro and in vivo experiments were conducted to evaluate the effects of Pdop-NPs loading model antigen OVA as a vaccine in anti-tumor therapy. A simple synthetic route was designed to load OVA protein on Pdop-NPs under a mild condition that avoids utilization of any organic solvent. The resultant nanovaccine abbreviated as OVA@Pdop-NPs, with a uniform size of about 232.8 nm, was assessed for its cytotoxicity and cellular uptake by bone marrow-derived dendritic cells ( BMDC ) in vitro. The effects of OVA@Pdop-NPs on BMDC maturation, antigen presentation, and in vivo translocation were also investigated. In addition, their impacts on tumor growth, CD8+ T immune cell activation and memory T cell responses as well as the intratumoral immune cell subpopulation were explored with the tumor-bearing mice.

EXPERIMENTAL SECTION Materials and animals. Dopamine hydrochloride (Dop, MW=189.64) was purchased



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from Yuancheng Technology Development Co. Ltd. (Wuhan, China). Trypsin and phosphate buffer saline (PBS) were obtained from Solarbio Science &Technology Co. Ltd (Beijing, China). OVA was a product of Sigma-Aldrich (St Louis, MO, USA ). The Enhanced BCA Protein Assay Kit, MTT Cell Proliferation and Cytotoxicity Assay Kit, penicillin, streptomycin and Lyso-Tracker Red DND-99 were purchased from Beyotime (Tianjin, China). Near-Infrared Cy7 NHS ester (Excitation: 730 nm, Emission: 790 nm) was purchased from ApexBio Technology (Houston, TX, USA). RPMI-1640 medium and heat inactivated fetal bovine serum (FBS) were purchased from Biological Industries (Beit HaEmek, Israel). Ploy (I:C) was purchased from InvivoGen (San Diego, CA, USA). Recombinant mouse GM-CSF and IL-4 were purchased from PeproTech (Rocky Hill, NJ, USA). IFN-γ, TNF-α and IL-6 ELISA kits, and fluorochrome-labelled monoclonal antibodies against mouse CD11c-PerCP-Cyanine5.5, MHCI-APC, MHCII-FITC, CD80-APC, CD86-FITC, CD40-PE, CD3e-PerCP-Cyanine5.5, CD8a-APC, CD4-FITC, CD69-PerCP-Cyanine5.5, CD62L-PerCP-Cyanine5.5, CD44-PE, Gr1-FITC, F4/80-FITC, CCR7-PE,

CD11b-APC,

CD45-PerCP-Cyanine5.5,

CD206-APC,

Foxp3-PerCP-

Cyanine5.5, CD25-PE were purchased from eBioscience (San Diego, CA, USA). Ovalbumin-expressing MC38 (OVA-MC38), a murine colon cancer cell line of C57BL/6 origin, was purchased from BioVector NTCC Inc (Beijing, China). C57BL/6 mice ( 6-8 weeks) were obtained from SPF Biotechnology Co. Ltd. (Beijing, China). All animal procedures were reviewed and ethically approved by the Center of Tianjin Animal Experiment Ethics Committee and Authority for Animal Protection .


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Synthesis of OVA@Pdop-NPs. In brief, 20 mg of dopamine hydrochloride was dissolved in 10 mL deionized (DI) water, and 84 μL of 1 mol L-1 sodium hydroxide was added with subsequent stirring at 50℃ for 3 hours. The resultant Pdop-NPs were collected by centrifugation at 8500×g for 10 min and washed seven times with DI water, and resuspended in 2 mL DI water. Subsequently, 5 mg of OVA was dissolved in 5 mL DI water, followed by addition of 0.5mL Pdop solution with stirring at room temperature. Five hours after that, the resulted OVA@Pdop-NPs were collected by centrifugation at 19000×g for 10 min and washed three times with DI water. Then OVA@Pdop-NPs were resuspended with water and stored at 4 °C. Characterization of OVA@Pdop-NPs. The average particle size, size distribution, and zeta potential were measured using the Particle Size and Zeta Potential Analyzer (Nano-ZS 90, Malvern Instrument, UK). Morphology of OVA@Pdop-NPs was observed with a scanning electron microscope (SEM). The amount of OVA grafted on the OVA@Pdop-NPs was determined by detecting the sulfur content using the Inductively Coupled Plasma Emission Spectrometer (ICP-OES, SPECTROBLUE, Kleve, Germany). Cytotoxicity assay. The cytotoxicity of OVA@Pdop-NPs was assessed using bone marrow derived dendritic cells (BMDCs). Briefly, BMDCs were isolated from hind limb bone of C57BL/6 mouse, and then cultured in RPMI 1640 medium containing 10% FBS, 1% penicillin and streptomycin, GM-CSF (20 ng mL-1) and IL-4 (10 ng mL-1) at 37 °C for 6 days to acquire immature DCs. At day 2 and day 4, two-thirds of the medium was



removed and replaced with fresh media containing GM-CSF (20 ng mL-1) and IL-4 (10 ng mL-1). After culture for 6 days, the cells were seeded in the 96-well plate at a density of 1×104 cells per well and incubated overnight at 37 °C, and then co-incubated with OVA, Pdop or OVA@Pdop-NPs at different concentrations (0.5 - 100 μg mL-1 based on the content of OVA) for 48 hours at 37 °C. Thereafter, MTT assay was conducted to assess the cell viability. In brief, 10 μL of 5 mg mL-1 MTT was added to each well and allowed for 4 hours' incubation at 37 °C. After that, 100 μL of solvent was added to solubilize the formazan crystals. The optical absorbance (OD) at 570 nm was measured with a microplate reader. The cell viability was calculated as follows: cell viability(%) = (OD of sample – OD of the blank) / ( OD of the control - OD of the blank) ×100%. Cellular uptake of OVA@Pdop-NPs. Both flow cytometry and confocal laser scanning microscopy were employed to investigate the cellular uptake of the nanoparticles using FITC-labeled OVA (FOVA) fabricated OVA@Pdop-NPs. BMDCs obtained as aforementioned were seeded on the coverslips inside the Petri dishes at a density of 1×106 cells per dish and subsequently incubated for 12 hours with free FOVA and FOVA@Pdop-NPs (with the content of OVA equivalent to 50 μg mL-1), respectively. After that, the cells were collected for flow cytometry analysis (BD FACSCalibur, BD Biosciences, San Jose, CA). To conduct morphological observation, the culture medium was replaced with the red lysosomal fluorescent probe-containing medium after co-incubation with the nanoparticles and allowed for additional 2 hours' incubation at


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37°C, followed by washing with PBS for three times. Finally, the nucleus was stained with DAPI for 20 minutes, followed by washing three times with PBS. The cell morphology was observed with a confocal laser scanning microscope (CLSM410, Zeiss, Germany). Maturation of BMDCs sensitized with OVA@Pdop-NPs in vitro. BMDCs were obtained as mentioned above. On the 6th day after the primary culture, the immature DCs were stimulated with OVA + ploy I:C, Pdop and OVA@Pdop-NPs (with the content of OVA equivalent to 20μg mL-1), respectively. After 12 h, the DCs were harvested and incubated with appropriately diluted anti-CD11c-PerCP-Cyanine5.5, anti-CD40-PE, anti-MHCI-APC, anti-MHCII-FITC, anti-CCR7-PE, and anti-CD86-FITC monoclonal antibodies on 4°C for 30 min. The expression of MHC I, MHC II , CD40, CCR7 and CD86 on BMDCs was measured by flow cytometry. The concentration of cytokines released by BMDCs into the culture medium was measured by enzyme linked immunosorbant assay (ELISA). In vivo trafficking of OVA@Pdop-NPs. C57BL/6 mice were subcutaneously vaccinated with Cyanine 7 ( Cy7) labeled OVA (Cy7-OVA) or Cy7-OVA@Pdop-NPs, dispersed in 100 μL PBS with the content of OVA being 100 μg per mice. The fluorescence intensity at the injection site was quantified using the Maestro EX In Vivo Imaging System (Cambridge Research & Instrumentation, Boston, MA, USA) at predetermined time intervals to evaluate the in the vivo distribution and ability to migrate



to the draining lymph nodes. The obtained images were analyzed with CRI Maestro analysis software. Animal vaccination. Six-to-eight-week-old female C57BL/6 mice were injected subcutaneously with 1 × 106 OVA-MC38 cells in 0.1 mL PBS at the right flank and were divided into 4 groups (six mice in each group), which respectively received injection of PBS, OVA + ploy I:C, Pdop and OVA@Pdop -NPs, with the content of OVA being 100 μg per mice. The mice were vaccinated three times on day 4, 11 and 18. The tumor volume was calculated as 1/2× tumor length (mm)×[ tumor width (mm)]2. T cell subpopulation analysis in tumor-bearing mice. The vaccinated mice were sacrificed 3 days after the last immunization, the spleen, lymph nodes and tumor tissues were then harvested and developed into single-cell suspension. The cells were then stained with anti-CD3e- PE-Cyanine5.5, anti-CD8a-APC and anti-CD4-FITC antibodies according to the manufacturer’s instructions. The subpopulation of T cells in the tumor-bearing mice was analyzed by flow cytometry. The proliferation of CD8+ T cells in vitro. Splenocytes were isolated from the C57BL/6 mice of all the groups and labeled with 5, 6-carboxyfluorescein acetate N-succinimidyl ester (CFSE) according to the manufacturer’s instructions. The CFSE-labeled splenocytes cells (4×106 cells mL-1) were re-stimulated with 20 μg mL-1 soluble OVA for 5 days in a 24-well plate. Subsequently, the cells were collected and stained with anti-CD8-APC and


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anti-CD3e- PE-Cyanine5.5 antibodies according to the manufacturer’s instructions. The proliferation of CD8+ T cells was assessed by flow cytometry.25-26 T cell response in vivo. Six-to-eight-week-old female C57BL/6 mice were immunized with PBS, Pdop, OVA + poly I:C or OVA@Pdop-NPs ( with 100 μg OVA per mice) on day 0, 7 and 14. At the 3th day after the last immunization, the splenocytes were harvested from the mice and 4×106 cells were re-stimulated with 20 μg mL-1 OVA in a 24-well plate for 72 h at 37℃. The splenocytes were then stained with anti-CD8-APC, anti-CD4-FITC, anti-CD62L- PerCP-Cyanine5.5 and anti-CD44-PE antibodies according to the manufacturer’s instructions. The number of central memory T cell (CD44HiCD62LHi) was measured by flow cytometry. The concentration of cytokine IFN-γ released by T cells into the culture medium was determined by ELISA. Moreover, the splenocytes were stained with anti-CD8-APC, anti-CD4-FITC and anti-CD69-PerCPCyanine5.5, and the number of CD4+ or CD8+ T cells within activated T cells (CD69+) were analyzed by flow cytometry. Effect of OVA@Pdop-NPs on the intratumoral immune cells. Six-to-eight- week-old female C57BL/6 mice were injected subcutaneously with 1×106 OVA-MC38 cells dispersed in 100 μL PBS at the right flank on day 0. The mice were immunized with PBS, Pdop, OVA + poly I:C, OVA@Pdop-NPs ( 100 μg OVA per mice) three times on day 4, 11, and 18. At the 3th day after the last immunization, the tumor tissues were harvested from the mice and prepared into single cell suspensions, which were subsequently stained



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with anti-F4/80-FITC, anti-CD206-PE, anti-Gr1-FITC, anti-CD11b-PerCP-Cyanine 5.5, anti-CD45-APC,

anti-CCR7-PE,

anti-Foxp3-APC,

anti-CD4-FITC

and

anti-CD25-PerCP-Cyanine5.5 antibodies according to the manufacturer’s instructions. The number of MDSCs (CD45+CD11b+Gr1+), Tregs (Foxp3+CD4+CD25+) and TAM (M1-type: F4/80+CCR7+; M2-type: F4/80+CD206+) was measured by flow cytometry. Statistical Analysis. The quantitative data collected were expressed as means ±S.D. Statistical significance was analyzed using one-way ANOVA followed by Tukey’s post-test. Statistical significance is denoted by *p < 0.05, **p < 0.01 and ***p < 0.001.

RESULTS AND DISCUSSION Polymer Synthesis and Structural Characterization. First, Pdop-NPs with a uniform size of 216.3 nm were produced by polymerization of dopamine monomer in an aerobic alkaline solution. As a catecholamine polymer, Pdop-NPs contain catechol and amine groups on their surface, and could be easily covalently bound to the tumor model antigenOVA or any other proteins, which contain primary amine and thiol groups as the nucleophilic ligands (Fig. 1A). The process of OVA@Pdop-NPs synthesis was rapid under mild condition, which only took several hours, while avoiding any organic solvent and complex purification. Table 1 summarized the size and surface charge of Pdop-NPs and OVA@Pdop-NPs, and the antigen loading capacity ( LC ). Under this simple and mild reaction condition, Pdop-NPs demonstrated a strong antigen loading capacity, with the LC yielding as high as 754±70.1 μg mg-1, which was significantly higher than that


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reported using Pdop coated NPs or other materials as the carriers.24,

27-28

The antigen

grafted on the surface of Pdop-NPs simply mimicked the structure of pathogens, together with their size of about 200 nm, facilitated DC cell uptake and presentation.29 The size of OVA@Pdop-NPs was 232.8 nm, which was slightly larger than that of Pdop-NPs (216.3 nm) due to the loading of OVA (Fig. 1B), while their surface charge slightly increased to -25.17 mV. In a word, the data showed that OVA@Pdop-NPs and Pdop-NPs had the similar dimension and surface charge. In addition, the particle size and zeta potential of OVA@Pdop-NPs maintained stable during 30 days of observation (Fig. 1C, D), suggesting their significant storage stability.



Figure 1. Characterization of OVA@Pdop-NPs. Schematic diagram of Pdop synthesis and OVA loading. The size distribution (A) and SEM image of OVA@Pdop-NPs (B). The particle size (C) and zeta potential (D) of OVA@Pdop-NPs during 30 days of observation. The data is expressed as mean ± SD (n=6).


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Table 1. Characterization of Pdop and OVA@Pdop-NPs. NPs

Size (nm)

PDI

Zeta potential (mV)

LC ( μg mg -1)

Pdop

216.3±1.63

0.107±0.010

-27.88±0.24

—

OVA@Pdop-NPs

232.8±1.59

0.173±0.040

-25.17±0.41

754±70.1

PDI, polydispersity. LC, loading capacity ( μg mg -1) = bound OVA protein / Pdop-NPs. Cytotoxicity

of

OVA@Pdop

-NPs

against

BMDCs.

The

cytotoxicity

of

OVA@Pdop-NPs was assessed with primary bone marrow dendritic cells (BMDCs) derived

from

C57BL/6

mouse

femur

using

the

standard

methylthiazolyl-

diphenyl-tetrazolium bromide (MTT) assay. As a surface modification material, Pdop exhibited excellent biocompatibility and low toxicity 30. Our experiments further confirm that co-incubation of BMDCs with OVA@Pdop-NPs for 48 hours at the concentrations ranging from 0.5 to 100 μg mL-1 were essentially non-toxic to BMDCs, and even demonstrated some extent of cell viability stimulating effect (Fig. 2).



Figure 2. The viability of BMDCs after cocultured for 48 hours with various concentrations of free OVA, Pdop and OVA@Pdop-NPs, respectively. The data is presented as mean ± SD (n=3). OVA@Pdop-NPs enhanced antigen uptake by DC in vitro. DCs are the most powerful APCs that are composed of a variety of cells including macrophages, DCs and non-professional APCs such as endothelial cells, fibroblasts, and epithelial cells. Uptake of antigen by DCs is the very beginning of vaccine-induced adaptive immunity and a key step in initiating tumor immunity.31 It has been reported that the particle size might affect the DCs uptake15, and nanosized formulations were easier to be endocytosed by DCs,25, 32-33

In our study, confocal laser scanning microscopy (CLSM) and flow cytometry were

employed to assess the cellular uptake of FITC-labeled OVA (FOVA) by primary BMDCs. The rate of FOVA@ Pdop-NPs by BMDCs (98.2% ± 0.4%) was significantly higher than that of free FOVA (76.2% ± 2.9%). As shown in Fig. 3A and B, the average


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fluorescence intensity of OVA in the OVA@Pdop-NPs group was 2.7-fold higher than that in the free OVA group. These results suggested that OVA@ Pdop-NPs dramatically enhanced the uptake of OVA by BMDCs. The location of the antigen taken up in the DC cytoplasm has a crucial influence on the antigen processing and presentation pathway. As shown in Figure 3C, almost all of the FOVA were colocalized with the lysosomes in the free OVA group (yellow color), whereas some FOVA@Pdop-NPs were found in the cytoplasm (green color). This lysosome escape phenomenon was also reported wherein Pdop-coated gold nanoparticles were employed.34 This experimental results hints that OVA@Pdop -NPs might promote the cross-presentation ,14, 35 and thus simultaneously activate CD4+ and CD8+ T cells.



Figure 3. Antigen uptake by BMDC. (A) Single-parameter histogram plot for uptake of OVA@Pdop-NPs by BMDCs as measured by flow cytometry after 12 hours. CON = untreated cells, OVA = soluble free OVA, OVA@Pdop = OVA@Pdop NPs. (B) The histogram of mean fluorescence intensity (MFI) of BMDCs determined by flow cytomery. (C) Confocal photo- micrographs of BMDC after 12 hours of co-incubation with FOVA


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or FOVA@ Pdop-NPs, wherein OVA was labeled with FITC (green), lysosomes were labeled with Lyso-Tracker (red) and nucleus was stained with DAPI (blue). The OVA particles, which did not colocalize with lysosomes, were shown by white arrow. The data are presented as mean ± SD (n=3). The differences between the groups were determined using one-way ANOVA followed by Tukey’s post-test. *P

Polydopamine as the Antigen Delivery Nanocarrier for Enhanced

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