Targeted Co-Delivery of Antigen and Dual-Agonists by Hybrid

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Targeted Co-Delivery of Antigen and Dual-Agonists by Hybrid Nanoparticles for Enhanced Cancer Immunotherapy Linhua Zhang, Shengjie Wu, Yu Qin, Fan Fan, Zhiming Zhang, Chenlu Huang, Weihang Ji, Lu Lu, Chun Wang, Hongfan Sun, Xigang Leng, Deling Kong, and Dunwan Zhu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b00030 • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019

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Targeted Co-Delivery of Antigen and Dual-Agonists by Hybrid Nanoparticles for Enhanced Cancer Immunotherapy

Linhua Zhang1, Shengjie Wu1, Yu Qin1, Fan Fan1, Zhiming Zhang1, Chenlu Huang1, Weihang Ji2, Lu Lu3, Chun Wang2, Hongfan Sun1, Xigang Leng1, Deling Kong4,5, Dunwan Zhu1,* 1Tianjin

Key Laboratory of Biomedical Materials, Institute of Biomedical

Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, China 2Department

of Biomedical Engineering, University of Minnesota, 7-116 Hasselmo

Hall, 312 Church Street S.E, Minneapolis, MN 55455, USA 3Institute

of Radiation Medicine, Chinese Academy of Medical Sciences and Peking

Union Medical College, Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Tianjin 300192, China 4State

Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China

5Jiangsu

Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer

Institute, Xuzhou Medical University, Xuzhou 221004, Jiangsu, China *Corresponding

author at:

Dunwan Zhu, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, China. E-mail address: [email protected]. Tel: 0086-022-87891191 1


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ABSTRACT Among approaches of current cancer immunotherapy, dendritic cells (DCs)targeted vaccine based on nanotechnology could be a promising way to efficiently induce potent immune responses. To enhance DCs targeting and vaccine efficiency, we included Imiquimod (IMQ), a toll-like receptor 7/8 (TLR 7/8) agonist and Monophosphoryl Lipid A (MPLA), a TLR4 agonist to synthesize lipid-polymer hybrid nanoparticles using PCL-PEG-PCL and DOTAP (IMNPs) as well as DSPE-PEGmannose (MAN-IMNPS). The spatio-temporal delivery of MPLA (within outer lipid layer) to extracellular TLR4 and IMQ (in the hydrophobic core of NPs) to intracellular TLR7/8 can activate DCs synergistically to improve vaccine efficacy. Ovalbumin (OVA) as a model antigen was readily absorbed by positively charged DOTAP and showed a quick release in vitro. Our results demonstrated that this novel nanovaccine enhanced cellular uptake, cytokines production and maturation of DCs. Compared with quick metabolism of free OVA-agonists, depot effect of OVA-IMNPs was observed whereas MAN-OVA-IMNPs promoted trafficking to secondary lymphoid organs. After immunization with subcutaneous injection, nanovaccine, especially MAN-OVAIMNPs induced more antigen-specific CD8+ T cells, greater lymphocyte activation, stronger cross-presentation, and more generation of memory T cells, antibody, IFN-γ, and Granzyme B. Prophylactic vaccination of MAN-OVA-IMNPs significantly delayed tumor development and prolonged survival in mice. Therapeutic tumor challenge indicated that MAN-OVA-IMNPs prohibited tumor progression more efficiently than other formulations and the combination with immune checkpoint 2


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blockade further enhanced antitumor effects. Hence, DCs-targeted vaccine co-delivery with IMQ and MPLA adjuvants by hybrid cationic nanoparticles in a spatio-temporal manner is a promising multifunctional antigen delivery system in cancer immunotherapy. KEYWORDS: hybrid nanoparticles; antigen; TLR agonists; vaccine delivery; cancer immunotherapy

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Immunotherapy functioned by vaccination is one of the most useful and costeffective methods to prevent human infectious disease or fight against cancer. Since Dr. William presented the initial idea of combating cancer using immune system in the early 1800s, cancer immunotherapy has evolved drastically and brought about a lot of crucial breakthroughs.1,

2

Compared with traditional inactivated or live-attenuated

vaccines, protein/peptide subunit vaccines has attracted higher attention because of the improved safety, ease of manufacturing, as well as enhanced quality control.3 Nevertheless, protein or peptide antigens in their soluble form are often rapidly cleared and poorly immunogenic, thus stimulating weak and short-lived immune responses.3 To overcome these problems, one promising strategy is to formulate nanoparticlesbased vaccine, which can protect antigen from degradation, facilitate antigen internalized by dendritic cells (DCs), and allow co-delivery of antigen and immunostimulatory adjuvant to insure activation of immune system.4-6 Among various nanoparticles, lipid-polymer hybrid nanoparticles (LPNPs), consisting of a polymeric core coated with a single layer or multiple layer lipid shell, have emerged as effective vesicles to combine the advantages of liposomes and polymeric NPs while mitigate their intrinsic limitations.7, 8 The LPNPs exhibit (1) high stability, improved encapsulation of hydrophobic drugs, and controlled release owned to the polymer core, and (2) high biocompatibility and reduced leakage of the encapsulated hydrophobic drugs attributed to the lipid layer.9 With these combined advantages, LPNPs have been rapidly developed as an effective drug or subunit vaccine delivery platform.10-12 In addition, LPNPs offer potential as novel vaccine adjuvants 4


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since their structure is similar to the viral architecture. Particularly, cationic lipid layer composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) can complex anionic protein/peptide for intracellular antigen delivery and processing to elevate cellular and humoral immune responses.13 The ‘sponge effect’ of DOTAP has the potential to promote cross-presentation, allowing exogenous antigen escape from the endosomes and enter into the cytosol for inducing both CD4+ T cell responses via major histocompatibility complex class Ⅱ (MHC Ⅱ) pathway and CD8+ T cell responses via MHC Ⅰ pathway.14-16 Under appropriate stimulation, CD8+ T cells proliferate and differentiate into specific cytotoxic T lymphocytes (CTLs), which is critical for effective vaccination. DCs are the most crucial and potent antigen presenting cells (APCs) to take up and process antigens, initiate immune responses, and regulate both adaptive humoral and cellular immunity.17-19 Upon vaccination, nanoparticles-based vaccine can form ‘depot effect’ at the injection site, thus enable prolonged and sustained antigen exposure to DCs and result in enhanced antigen uptake and processing. Meanwhile, promoting vaccine delivery to the draining lymph nodes (LNs), where T- and B- cell activation is initiated, is another important process to generate effective and long-term antitumor immunity.20, 21 The lymphatic targeting can be achieved by decorating the nanoparticles with antibodies or ligands specific to DCs.22 Mannose receptor (MR) which is highly expressed on APCs allows nanoparticle design to endow lymphatic targeting.23, 24 Wang and co-workers showed that the incorporation of mannosylated DSPE-PEG (DSPEPEG-Man) into DOTAP promoted vaccine-elicited long-lasting immunological 5


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response.21 After immunization, APCs, especially DCs, capture antigens and migrate into draining LNs, to present antigens through MHC I and MHC II pathway to activate T- and B-lymphocyte as well as memory cell differentiation.25-27 However, evidence indicated that antigen delivery without proper maturation stimuli to DCs would lead to immune tolerance.28 Thus, MR-targeted vaccines might be most effective in vivo via co-delivery of immunostimulants with supplementary activating signals to induce complete mature of DCs. Emerging evidence of study on some successful vaccines, such as yellow fever vaccine (YF-17D), indicates that their efficacy may result from robust activation of DCs via multiple toll-like receptors (TLRs) and subsequently increased secretion of proinflammatory cytokines.29, 30 TLRs are the main sensors of innate immunity and can activate a variety of immune cells. Using TLRs agonists as adjuvants via delivery system has been shown to significantly enhance immune response to administered antigens and markedly improved antigen cross-presentation.31-35 Recently, further improvements on the vaccine delivery have indicated the importance of delivering multiple TLRs and antigen to the same DCs to activate synergistic immune responses via triggering different signaling pathways.36-39 As is known, TLRs are sub-divided into two types based on their localization: cell membrane-associated receptors (TLRs 1, 2, 4, 5, and 6) which recognize bacterial cell wall components and endosome-associated receptors (TLRs 3, 4, 7, 8, and 9) which specially sense bacterial or viral nuclei acids.40 Stimulating DCs by TLR3 or TLR4 combined with TLR7/8 or TLR9 have synergistic effect, leading to upregulated secretion of several inflammatory cytokines, amplified 6


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innate immune response, and enhanced adaptive Th1-cell immunity.41, 42 In addition, the localization of TLR receptors can influence the ligand accessibility, thus affecting the downstream signaling pathways. Siefert et al. demonstrated that spatial orientation of multiple TLR agonist on or in nanoparticles influenced the direction and magnitude of immune responses.38 Therefore, to achieve effective cancer immunotherapy, nanoparticles-based vaccines should be rationally designed to co-deliver multiple TLRs and antigen in a programmed manner and can trigger the immune responses in a spatiotemporal and synergistic manner. Herein, we constructed a novel versatile and mannose-targeting LPNPs vaccine system from biodegradable polymer PCL-PEG-PCL and cationic lipid DOTAP for programmed co-delivery of antigen and dual TLR agonists. Our nanoparticles-based vaccine (MAN-OVA-IMNPs) consists of four distinct components: (1) a hydrophobic inner core self-assembled by PCL-PEG-PCL for the encapsulation of water-insoluble TLR7/8 agonist imiquimod (IMQ), (2) a lipid layer for the incorporation of TLR4 agonist MPLA which is a clinically approved Th-1-skewing adjuvant, (3) a cationic DOTAP lipid for the electrostatic adsorption of anionic OVA antigen, and (4) mannosetargeting moiety decorated on the outer lipid layer via functionalized PEGylated lipid. After vaccination, the MAN-OVA-IMNPs can be efficiently internalized by immature DCs via mannose-targeting to MR and MPLA-ligating to TLR4 expressed on the DCs surface.

Then,

the

‘sponge

effect’

of

DOTAP

can

facilitate

endosomal escape of exogenous antigens for cross-presentation by DCs, leading to antigen peptide presented by both MHC-Ⅰ and MHC-Ⅱ pathway. Meanwhile, MAN7


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OVA-IMNPs spatio-temporally deliver MPLA to extracellular TLR4 and IMQ to intracellular TLR7/8, activating DCs synergistically through both myeloid differentiation primary-response gene 88 (MyD88) and TIR-domain-containing adaptor protein inducing INF-β (TRIF) pathways to improve vaccine efficacy. Furthermore, mannose-decoration can enhance LNs targeting and promote induction of antigen-specific CD4+ and CD8+ T cells for both humoral and cellular immune response. The targeted co-delivery of antigen and dual agonists by cationic lipid hybrid PCLPEG-PCL nanoparticles for cancer immunotherapy was shown as Scheme 1. We conducted a series of experiments to characterize the particles including size and zeta potential, morphology, in vitro OVA and IMQ release. Mannose receptor mediated DCs uptake was evaluated by confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorter (FACS). Additionally, DCs maturations were both evaluated in vitro and in vivo using FACS. By labeling OVA with Rhodamine, we visualized the delivery behavior in real-time, showing that the mannose-targeting NPs facilitate lymphatic trafficking. After subcutaneous injection with different formulations, MAN-OVA-IMNPs rather than OVA-IMNPs promoted lymphocyte activation, cross-presentation, cytokines production, CD4+ and CD8+ T cell response, and long-lasting immunological memory, thus significantly delayed tumor development and prolonged survival on preventive E.G7-OVA tumor model. Therapeutic vaccination by MAN-OVA-IMNPs prohibited tumor progression and the combination with anti-PD-1 further enhanced antitumor effects. First, using thin-film hydration as well as ultrasonic dispersion method, we 8


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successfully fabricated OVA-IMNPs and MAN-OVA-IMNPs by reaction of DSPEPEG-NH2 and α-d-mannopyranosylphenyl isothiocyanate, which exhibited uniform size distribution. Figure 1A shows schematic illustration of the MAN-OVA-IMNPs structure. PCL-PEG-PCL tri-block polymers formed spherical nanoparticles with hydrophobic core and hydrophilic corona, and coated with lipid layer on the surface of the NPs. Two TLR ligands, IMQ and MPLA, were in the hydrophobic core or on the surface, respectively. Meanwhile, model antigen ovalbumin (OVA) was absorbed by positively charged DOTAP on the surface companied with surface modification by mannose to target DCs. To directly observe the structure of lipid-polymer hybrid NPs, fluorescent rhodamine-PE was added and then large particles were formulated by sonicating for only 1 min instead of 10 min. CLSM image clearly showed red fluorescence of rhodamine-PE lipid shell, demonstrating that polymer particles with lipid coating were successfully fabricated (Figure 1B). Then, to observe hydrophobic drug loading and OVA adsorption, rhodamine-PE labelled lipid-polymer hybrid particles delivering hydrophobic coumarin-6 and FITC-labeled OVA were formulated. CLSM image showed red lipid shell with green fluorescence of FITC-OVA and coumarin-6, confirming that lipid-polymer hybrid particles can encapsulate hydrophobic molecule in the polymer core and adsorb OVA antigen on the lipid surface (Figure 1C). TEM image of IMNPs further demonstrated that the NPs were spherical ‘core-shell’ morphology with an obvious lipid layer coating on the surface of the NPs (Figure 1D). After incubating with OVA, the TEM and AFM image of OVA-IMNPs clearly showed that OVA was adsorbed on the surface of the NPs (Figure 1E and 1F). 9


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Both OVA-IMNPs and MAN-OVA-IMNPs were of similar size and zeta potential with narrow size distribution (Table 1 and Figure 1G). Antigen release behavior of MANOVA-IMNPs was found to be up to 92 % in total within 24 h (Figure 1H). IMQ was released in a slow and continuous manner, probably due to the entrapment in the inner core of the NPs (Figure 1I). Faster IMQ release was observed at pH 5.5, which is beneficial for the binding of IMQ with TLR7/8 within acidic endosomal compartment. The accelerated IMQ release in response to reduced pH value might result from the protonation of aromatic amines in IMQ under acidic condition.43 We then measured whether the constructed LPNPs vaccine can facilitate antigen uptake by DCs. First, the purity of cultured DCs was measured by FACS, showing that DCs population is up to 79.3 % of the total population (Figure 2A). After that, the biocompatibility of the LPNPs vaccine was examined by incubating DCs with OVAIMNPs or MAN-OVA-IMNPs at various concentrations for 48 h and then the viability of DCs was determined by MTS Kits. As shown in Figure 2B, the DCs viability was about 90 % and unaffected by concentration, indicating excellent biocompatibility of the formulated vaccine. Next, the antigen uptake and intracellular distribution were observed by CLSM. Confocal images clearly showed that DCs took up more OVAFITC-IMNPs or MAN-OVA-FITC-IMNPs than OVA-FITC-agonists (Figure 3). What’s more, antigen OVA from OVA-IMNPs and MAN-OVA-IMNPs was observed both in the lysosome and cytoplasm, indicating lysosome escape, whereas free OVAagonists (free OVA plus IMQ and MPLA solution) taken up by DCs were trapped in lysosomes. The co-localization of OVA and lysosomes within a larger number of cells 10


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was then analyzed using Zeiss LSM software. As shown in Figure S1, the colocalization of free OVA-agonists, OVA-IMNPs, and MAN-OVA-IMNPs was 73.6, 44.4, and 28.8 %, respectively, demonstrating that the formulated vaccines facilitated antigen escape from lysosomes. MAN-OVA-IMNPs further accelerated the escape, probably due to that the MR constitutive recycling mechanisms enable quick and successive accumulation of antigens for subsequent MHC presentation.44, 45 Only after endosomal escape can exogenous antigen be presented to MHC I molecules and activate CD8+ T cells to elicit robust CTL response.46 Then, the ability of LPNPs vaccine in promoting antigen uptake by DCs was quantitatively evaluated by flow cytometry. As shown in Figure 2C, OVA-FITC-IMNPs significantly enhanced the DCs uptake of OVA antigen, with an increase of 4-5 folds compared with OVA-FITCagonists. In addition, MAN-OVA-FITC-IMNPs further increased the uptake of OVA about 2-3 folds, indicating that mannose receptor mediated cellular uptake greatly elevated internalization of MAN-OVA-FITC-IMNPs. Once met with DCs, mannose and MPLA on the surface of NPs immediately engaged mannose receptor and TLR4 on the cell membrane which increased cellular uptake directly. Meanwhile, TLR4 triggered by MPLA can initiate both My88 and TRIF pathways leading to activation of DCs. In addition, encapsulation of IMQ within NPs may confer TLR 7/8 ligand protection during endocytosis by APCs and then IMQ released in the endosome can ligate intracellular TLR 7/8 to activate DCs through MyD88 pathways. We further analyzed the effect of OVA-IMNPs or MAN-OVA-IMNPs on DCs maturation. The activation of DCs involves several cell surface markers, such as 11


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adhesion molecules CD40, co-stimulatory molecules CD86/80, which offered obligatory signals to prime T cells.47 DCs were incubated with free OVA-agonists and OVA encapsulated nanoparticles for 24 h at 37 oC and examined by FACS. To ensure DCs maturation, we incorporated MPLA, a TLR4 agonist, and IMQ, a TLR 7/8 agonist when fabricating nanoparticles. Representative flow cytometry histogram plots of CD86, CD80 and CD40 were shown in Figure S2 as the supporting information. As expected, all NPs significantly up-regulated the expression of CD86, CD80, and CD40 compared with free OVA-agonists and PBS group. Due to in vivo quick degradation, free OVA-agonists marginally elevated the expression of co-stimulatory molecules. On the contrary, MAN-OVA-IMNPs displayed the greatest capacity to stimulate DCs maturation and up-regulated the expression of CD86, CD80, and CD40 on DCs (Figure 2D-F). Overall, our in vitro results presented in Figure 2 and Figure 3 indicated that MAN-OVA-IMNPs had significant superiority in DCs uptake and maturation to OVAIMNPs and free OVA-agonists. Lymph node is the site where immune cells settled and initiated responses. Thus, it is necessary to measure the ability of NPs migrating to draining lymph nodes and inducing immune response. To distinguish the signal of injection site from lymph nodes clearly, we chose tail-base, which specifically drains to the inguinal lymph nodes, to subcutaneously (SC) inject the labeled NPs. MAN-OVA-Rhodamine-IMNPs were the fastest NPs to drain to the inguinal lymph nodes within 6 h after injection, while OVARhodamine-IMNPs within 9 h and free OVA-Rhodamine-agonists within 24 h. Figure 4A showed the image of MAN-OVA-Rhodamine-IMNPs in draining inguinal lymph 12


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nodes 24 h after SC injection. 9 days after injection, when hardly to monitor the fluorescent signal on the inguinal lymph nodes in vivo, the draining lymph nodes were isolated and imaged, and the ex vivo results were consistent with the results in vivo. As shown in Figure 4B, no detectable fluorescence was observed in mice treated with free OVA-Rhodamine-agonists group, whereas strong fluorescence signal was observed for NPs treated mice. Notably, the fluorescent intensity of MAN-OVA-Rhodamine-IMNPs group on the inguinal lymph nodes was greatly higher than that of free OVARhodamine-agonists group. The above results indicated that OVA adsorbed on nanoparticles had stronger ability to migrate from peripheral tissue to inguinal lymph nodes where immune cells initiate responses than free OVA. And MAN-OVA-IMNPs were the best from this perspective. Figure 4C-D showed the presence of the OVAencapsulated NPs at the injection site at different time points, compared with free OVAagonists. As expected, the fluorescence of free OVA-agonists weakened rapidly, showing no signal at 48 h post-injection. In contrast, both OVA-IMNPs and MANOVA-IMNPs were gradually cleared with detectable fluorescence at 336 h and 144 h post-injection, respectively, demonstrating antigen protection and depot was achieved for the formulated vaccine. It should be noted that OVA-IMNPs were cleared more gradually than MAN-OVA-IMNPs, which drained fast to inguinal lymph nodes due to the mannose modified on the surface. Generally speaking, immature DCs capture exogenous antigen, become mature via a complex process and highly express adhesion (CD40), co-stimulatory (CD86), MHC class I and MHC class II molecules. Then, mature DCs migrate from peripheral tissues 13


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to lymph nodes where rich T-lymphocytes reside to trigger adaptive immune responses.48 We already assessed different OVA formulations on DCs maturation in vitro. Whereas, DCs generated in vitro differed from in vivo situation where numerous types of cells exit. It would be interesting to see whether the different OVA formulations could influence DCs in vivo the same way as in vitro. We treated C57BL/6 mice with PBS, free OVA-agonists, OVA-IMNPs or MAN-OVA-IMNPs and compared the expression of CD40, CD86, MHC class I and II molecules on DCs from inguinal lymph node. Figure 4E-H showed that surface markers expressed on DCs by OVA-IMNPs and MAN-OVA-IMNPs were 2-3 folders higher than those by free OVAagonists excepted MHC class I molecules. Among all groups, MAN-OVA-IMNPs showed the highest activity to stimulate DCs maturation. Notably, MAN-OVA-IMNPs produced 2.01-times higher expression of MHC class I molecules than OVA-IMNPs did. It has been reported that mannose receptor (MR) can target antigen into a distinct endosomal compartment, where the antigen is protected from degradation by lysosomal proteases and processed to be loaded on MHC class I molecules for eliciting antigenspecific CTLs.49 Before running the in vivo anti-tumor effect experiment, we would like to know first the potential of our MAN-OVA-IMNPs to enhance cellular immune responses. CD3 molecule is the co-receptor of the TCR on CD8+ T cells (CD3+CD8+), called cytotoxic T lymphocytes (CTLs) which directly kill cancer cells, or on CD4+ T cells (CD3+CD4+) which are helper T (Th) cells with a major roles in helping B cells to regulate adaptive immunity.50 Both CTLs and Th cells are pivotal to induce remarkable 14


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immune response and achieve immune prevention and protection. Therefore, we examine the immune responses to vaccines, including OVA-IMNPs, MAN-OVAIMNPs, free OVA-agonists, and PBS in vivo. C57BL/6 mice were immunized twice with one-week interval. Seven days after the boosting immunization, splenocytes were isolated, harvested and stained with CD8, CD4, CD3 antibodies. FACS data showed that OVA-IMNPs and MAN-OVA-IMNPs led to effective proliferation both in Th cells and in CTLs. Taking MAN-OVA-IMNPs for example, the percentage of Th cells (CD3+CD4+) climbed up to 35.5 % compared with OVA-IMNPs (31.2 %), free OVAagonists (28.0 %) and PBS (20.5 %) (Figure 5A). Meanwhile, MAN-OVA-IMNPs elevated the CTLs to 24.0 % which was 1.22-, 1.28-, and 1.73-fold higher than OVAIMNPs, free OVA-agonists, and PBS did, respectively (Figure 5B). Statistical analysis indicated that MAN-OVA-IMNPs induced significantly higher Th cells and CTLs than OVA-IMNPs (CD3+CD4+, p

Targeted Co-Delivery of Antigen and Dual-Agonists by Hybrid

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