A Visible Codelivery Nanovaccine of Antigen and Adjuvant with Self

Jan 10, 2019 - Codelivery nanovaccines of antigens and adjuvants have achieved positive therapy for cancer immunotherapy. The insufficient immunogenic...
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Biological and Medical Applications of Materials and Interfaces

A Visible Co-delivery Nanovaccine of Antigen and Adjuvant with Self-carrier for Cancer Immunotherapy Xia Dong, Jie Liang, Afeng Yang, Zhiyong Qian, Deling Kong, and Feng Lv ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20364 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 11, 2019

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A Visible Co-delivery Nanovaccine of Antigen and Adjuvant with Self-carrier for Cancer Immunotherapy

Xia Dong1#, Jie Liang1#, Afeng Yang1, Zhiyong Qian2, Deling Kong1, Feng Lv*

1 Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300192, PR China 2 State Key Laboratory of Biotherapy, West China Hospital, and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu 610041, Sichuan, P R China.

# These authors contributed equally to this work and should be considered co-first authors. *Corresponding author: Feng Lv Institute of Biomedical Engineering Chinese Academy of Medical Sciences & Peking Union Medical College Tianjin 300192, PR China Tel/Fax: 86-22-87893236, E-mail: [email protected] (Lv F)

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Abstract Co-delivery nanovaccine of antigen and adjuvant has achieved positive therapy for cancer immunotherapy. The insufficient immunogenicity of these vaccines leads to the difficulty of eliciting robust immune effect for immune clearance due to the inadequate loading efficiency, complex preparation process, low safety concerns and weak immune response. Herein, a visible co-delivery nanovaccine of antigen and adjuvant based on self-crosslinked antigen nanoparticles (ovalbumin nanoparticles) combined with adjuvant (CpG) for cancer immunotherapy was prepared using antigens themselves as carriers. Ovalbumin nanoparticles (ONPs) not only provide sufficient antigen for continuous simulation of immune response with high antigen loading efficiency, but also serve as a natural carrier of CpG. In vitro and in vivo experiments proved that ONPs-CpG can elicit robust immune response including DC maturity, T cell activation and IFN-γ production. ONPs-CpG induced strong tumor-specific immunity and exhibit remarkable antitumor immunotherapy effect in vivo using mouse models of lymphoma. Furthermore, to perform the precise vaccine delivery, the dual fluorescent co-delivery nanovaccine was real-time monitored in vivo by visible imaging method. With regard to migration tracking, fluorescence imaging allowed for both high resolution and sensitivity of visible detection based on the fluorescence of ONPs and CpG. The multi-functional nanovaccine could function as a robust platform for cancer immunotherapy and a visible system for antigen-adjuvant tracking.

Keywords:

nanovaccine; co-delivery; cancer immunotherapy; fluorescence imaging;

self-carrier

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1. Introduction Cancer immunotherapy, as a promising treatment strategy relative to traditional therapy, has attracted dominant attention in recent years1-5. It can activate the immune system of patients themselves to resist or eliminate cancer cells. As a representative immunotherapy method, cancer vaccines show tremendous potential in cancer immunotherapy owing to the antigen-specific immune response and the long-term immunological memory induced by them6. Among them, protein or peptide based vaccines show more advantages in terms of safety, stability, specificity or reproducibility7,8. Nevertheless, the insufficient immunogenicity of these vaccines leads to the difficulty of eliciting robust immune effect for immune clearance. With the development of adjuvant, co-delivery of antigen and adjuvant can significantly promote the effectiveness of cancer vaccines and even overcome the body's immune tolerance9-11. Some reports have indicated that sustained release of antigen and adjuvant from delivery system can generate “antigen reservoirs” effect that further enhanced tumor-specific cytotoxic T lymphocytes response12. Co-delivery of antigen and adjuvant has achieved positive role for cancer immunotherapy9,10 . The application of carrier system for co-delivery of antigen and adjuvant would provide a suitable strategy for further enhancing tumor therapy effect. Vaccine delivery system is widely accepted to improve the vaccine efficiency for cancer immunotherapy. Nanoparticles as carriers for cancer vaccine delivery provide a biomedical platform for enhancing adaptive T cell responses in cancer immunotherapy10,13-15. While they still have some limitations including the inadequate antigen loading efficiency, complex preparation process, low safety concerns and weak immune response. It is an urgent need to construct high-efficient nano-carriers. At present, antigen-based nanovaccines triggered an innovative strategy for immunotherapy through the self-assembly or self-crosslinking of antigens6,16,17. They ensured ultrahigh antigen loading efficiency without extra carriers by simple antigen-directed strategy. It can enhance the antigen delivery efficiency and induce long-term antitumor immunity using antigens themselves as carriers. Our research group has previously reported an antigen-based

nanovaccine

consisting

of

functional

ovalbumin

(OVA)

nanoparticles,

demonstrating the advantages as a novel nanovaccine to induce efficient immune response17,18. Therefore, we deduce that a co-delivery system of antigen and adjuvant based on the ovalbumin 3

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nanoparticles would generate a more powerful antigen-specific T-cell response to cancer immunotherapy.

Among

immunoadjuvants,

oligodeoxynucleotides

(ODNs)

containing

cytosine-phosphate-guanine (CpG) is one of effective modulators for cancer immunotherapy, which can evoke an immunostimulatory cascade and potentiate antigen-specific immune response through TLR 9 binding and activation19-22. A co-delivery system including ovalbumin and CpG could introduce antigen and adjuvant to the same antigen present cells and execute a dual-function of CpG adjuvant and antigen simultaneously23,24. Thereby, loading CpG on the OVA nanoparticles would achieve as a novel co-delivery system by the merging of antigens and immunoadjuvants. Imaging-guided vaccine delivery provides a novel strategy for precise immunotherapy. A visible vaccine delivery system is crucial for real-time tracking of the in vivo process of antigens or immunoadjuvants25,26. By monitoring the vaccine delivery process, the vaccine could be constantly optimized from various aspects including cellular uptake, targeted migration or promoting the maturation. Fluorescence imaging tracking of vaccine delivery has attracted fargoing research interests owing to its unique optical properties and superior stability of noninvasive tracking27-30. It is noteworthy that multispectral fluorescence imaging can make a distinction between different fluorescent signals by a polychromatic composite images26,31-34, which can simultaneously track the various components of the dual florescence vaccine system by different markers. It is an effective tool for the accurate monitoring of the vaccine delivery, revealing the location and degradation of each component in the visible vaccine17. For precise co-delivery of antigen and adjuvant, multispectral fluorescent tracking of antigen and adjuvant disclosed the accurate delivery of both components. Fluorescence imaging monitoring represents a potential non-interfering approach to separate composite fluorescence signals of the antigens or adjuvants

29,35.

The dual fluorescent adjuvants/antigens-based visible vaccine delivery system not

only induces a potent antigen-specific immune response, but also has an advantage of real-time tracking the distribution and degradation of vaccine for precise delivery. Herein, a visible co-delivery nanovaccine of antigen and adjuvant based on ovalbumin nanoparticles combined with CpG for cancer immunotherapy was prepared. The CpG loaded OVA nanoparticles consisted of CpG and self-crosslinking of OVA antigens by electrostatic forces. OVA antigen nanoparticles were used as self-carriers for the nanovaccine instead of other external synthetic nanoparticles. OVA nanoparticles (ONPs) were used not only as antigens for 4

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inducing innate and adaptive immunity, but also as delivery carriers for CpG to improve the cellular uptake. The activity of CpG could be further enhanced when OVA nanoparticles deliver CpG to immune cells via the endocytic pathway due to the biological characteristics of TLR 9 signaling. All of these effects will induce high level of DCs maturity for antigen presentation and the following antigen-specific CD8+ T cells response. With the simplest OVA nanoparticles-based strategy for building nanovaccines, the co-delivery system of antigen and adjuvant will remarkably improve immunogenicity and exert antitumor therapeutic effects. Meanwhile, based on our previous study with visible nanovaccines17, it is supposed that the visible co-delivery nanovaccine of ovalbumin nanoparticles and CpG would achieve imaging-guided dual fluorescence tracking in vivo. For precise co-delivery of antigen and adjuvant, multispectral fluorescent tracking of antigen and adjuvant would disclose the accurate delivery of both components. This multi-functional nanovaccine could function as a robust platform for tumor immunotherapy and a visible system for antigen-adjuvants tracking.

2. Materials and methods OVA nanoparticles combined with CpG (ONPs-CpG) were prepared with free CpG on the surface of OVA nanoparticles by electrostatic interaction. The structure and properties were measured by agarose gel electrophoresis, atomic force microscope, particle size analyzer, Circular Dichroism Chiroptical Spectrometer, UV−vis and fluorescence spectrum photometer. For the cell viability assay, a Cell Counting Kit 8 was used to evaluate the cells proliferation. Cellular imaging of ONPs and ONPs-CpG was observed by confocal microscope. For further evaluation quantitatively, DC cells treated with TAMRA-labeled CpG, ONPs or ONPs-CpG were analyzed by flow cytometer. Maturation of BMDCs in vitro was analyzed by co-staining with antibodies labeled with fluorescence using flow cytometry analysis. Fluorescence imaging tracking of DCs migration and nanovaccine degradation were investigated based on the fluorescence of TAMRA-labeled CpG, ONPs and ONPs-CpG. The fluorescence signals were monitored and analyzed by the Maestro software during the whole imaging process. T cell proliferation assay was performed by flow cytometer with CFSE staining. Cytokine detection was measured by ELISA. After the tumor model was established, the animals were grouped and administered. Body 5

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weight and tumor size of each mouse were monitored to assess the anti-tumor activity in vivo at a regular interval of every 2 days for 12 days. Histochemical staining of tumor tissue sections was performed to evaluate the histological change of different treatments. Statistical analysis was expressed by mean ± standard error (SD) and Student's t test. The detailed experimental materials and methods were provided in the Supporting Information.

3. Results and discussion 3.1. Preparation and characterization of nanovaccines

Figure 1 6

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ONPs-CpG was fabricated with CpG and self-crosslinked OVA antigens nanoparticles by electrostatic forces. First, self-crosslinked ONPs was formed with the cross-linking of genipin, and then CpG was absorbed on the outer layer of ONPs based on the opposite charge (Figure 1A ). With different amount of CpG during the encapsulation process, the adsorptivity of CpG with ONPs was performed at various ratios using agarose gel electrophoresis (Figure 1B). Compared to free CpG, the migration bands of CpG became gradually weak with CpG/NPs ratios at 3:1 to 1:3, indicating the loading efficiency of CpG increased accordingly. The suitable CpG/NPs ratio at 1:3 was used for the following experiments due to the high loading efficiency. The internal morphology of ONPs and ONPs-CpG were analyzed by AFM, revealing no obvious structural difference between ONPs and ONPs-CpG (Figure 1C). The mean diameter of ONPs and ONPs-CpG was 145.6 nm (PdI = 0.371) and 158.8 nm (PdI = 0.256), while that of ONPs and ONPs-CpG was 142.1 nm (PDI = 0.333) and 157.9 nm (PDI = 0.192) after 24h later, respectively (Figure 1D). Besides, the zeta potential of ONPs and ONPs-CpG were 41.0 mv and 38.4mv, respectively. The absorption of CpG on the surface of ONPs led to the decrease of the potential and the increase of size. Furthermore, ONPs-CpG can retain the uniform and dispersed status in PBS and serum without aggregation after 24h (Figure 1E). To ensure the optical properties of ONPs and ONPs-CpG, UV-Vis and fluorescence spectrum were performed to realize the feasibility of in vivo visualized fluorescence tracking. The ONPs and ONPs-CpG gave a similar absorption appearance at ~592 nm due to the crosslinking of genipin (Figure 1F). The maximum emission peak at 630 nm of ONPs and ONPs-CpG affirmed the ability for in vivo tracking with an excitation wavelength of 580nm (Figure 1G). 3.2. In vitro cell viability evaluation The impact of free OVA, ONPs and ONPs-CpG on the growth of DCs was investigated (Figure 1H). No cytotoxicity was observed for any examined concentration of OVA for BMDCs, while a continuous increase in the cytotoxicity was evoked when incubation BMDCs with ONPs and ONPs-CpG of the increasing concentrations. It suggested an inhibitory action to the growth of BMDCs on a higher concentration range of nanoparticles because PEI with higher molecular weight gave certain cytotoxicity. In our previous study, genipin crosslinked OVA-PEI nanoparticles had been applied as a nanovaccine17. When CpG was loaded on ONPs, the cytotoxicity can be reduced to some extent. In vitro cytotoxicitiy assay showed that ~70% to ~100% 7

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viability of BMDCs maintained a normal profile at ONPs-CpG concentrations below 20 μg/mL. Therefore, the subsequent cell studies were performed to incubate with free OVA, ONPs and ONPs-CpG at the moderate concentration of 20 μg/mL. 3.3. Antigen uptake and distribution

Figure 2 To explore the intracellular behavior of nanoparticles, ONPs-CpG, ONPs or TAMRA-labeled CpG were added to DCs and incubation for analysis by CLSM and flow cytometry. Figure 2A illustrated the intracellular distribution of different formulations. First of all, only a small proportion of free CpG oligodeoxynucleotide could enter into the DCs. When combined with ONPs, CpG was more effectively delivered into cells with significantly increased fluorescence intensity (magenta). The quantitative data from flow cytometry (Figure 2B ) further verified that 8

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the mean fluorescence intensities of CpG in ONPs-CpG group improved approximate 4-fold compared to that in free CpG treated group after 4h of incubation. The result indicated that ONPs could be applied as an effective carrier for CpG. Besides, it was noted that there was no significant difference in red fluorescence from genipin between ONPs and ONPs-CpG groups, indicating that their cellular uptake efficiency was consistent. Moreover, both LSCM images and FCM analysis data illustrated that the intracellular red fluorescence intensity of self-crosslinking OVA nanoparticles (ONPs and ONPs-CpG) significantly enhanced after treatment for 12 h compared with that treated for 4 h, suggesting a gradually increased antigen uptake with the incubation time. All of the above results illustrated that the visible ONPs were not only applied as a specific antigen with ultrahigh loading capacity, but also as a natural carrier for the immunostimulator CpG. Herein, co-delivery of protein antigen and adjuvant was achieved by the simplest pattern, which provided a new strategy for the design of multifunctional nanovaccines using antigen itself as a carrier. 3.4. Maturation of BMDCs Dendritic cells (DCs) as antigen-capturing cells are responsible for initiating native T cells activation. Upon capture of the antigen by immature DCs, the mature DCs mainly present antigen into nearby draining lymph nodes to activate the immune responses. The level of DC maturation is a quite important perquisite for assessing the immune response, as indicated by up-regulation of some typical markers like costimulatory molecules CD40, CD80, CD86 and CCR7 on DCs membrane. In vitro experiments (Figure 3A) indicated that the percentage of mature DCs was significantly more in ONPs-CpG treated group than that in ONPs treated ones. Especially, stimulation with ONPs-CpG remarkably improved the CCR7 expression, with an average percentage of CCR7+DCs of 76.34% in the ONPs-CpG treated group whereas 57.58% in ONPs treated ones (Figure 3B). Besides, CD 40+DCs and CD80/CD86 DCs were also significantly improved after incubation with ONPs-CpG than that of other treated groups. The difference on inducing DCs maturation ability of ONPs and ONPs-CpG indicated the loading of CpG on ONPs-CpG played a key role in the process, further demonstrating that ONPs could enhance the delivery of CpG to stimulate DCs maturation for activating the following immune responses. Furthermore, DCs maturation in ONPs or ONPs-CpG treated mice was markedly improved 9

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compared to that in free OVA treated ones. This phenomenon was consistent with our previous reports17, illustrating that both the structured change from soluble protein to nanovaccine and the introduction of PEI had important impact on promoting DCs to mature. PEI would play the underlying adjuvant role for cancer immunetheropy 36.

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Figure 3 11

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Figure 4 12

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Meanwhile, the in vivo expression levels of CD40, CD80, CD86 and CCR7 of DCs from lymph nodes were measured after treatment by PBS, free OVA, ONPs or ONPs-CpG for 3d (Figure 4). Consistent with in vitro results, ONPs-CpG induced a much higher level of DCs maturation compared to ONPs and free OVA. Taken together, compared to previous study about co-delivery of antigen and adjuvant37,38, the ONPs-CpG nanovaccine successfully merged the function of antigens and vehicles, and effectively exerted the functions of OVA and immunologic adjuvant CpG on promoting DCs maturation. This provided a crucial prerequisite for inducing an enhanced immunological effect in vivo. 3.5 In vivo fluorescence imaging Efficient delivery of antigens to immune cells could exert a strong immunotherapeutic effect on tumor treatment. Fluorescence imaging in vivo was used to track antigen presentation by an optical noninvasive avenue as a visible tool for accurate monitoring. For fluorescence imaging of the ONPs-CpG system, a dual labeling with fluorescence was conducted with TAMRA-labeled

CpG and genipin crosslinked ONPs. To observe the ONPs-CpG in vivo, multispectral fluorescence imaging exerted the signal tracking of TAMRA-labeled CpG and ONPs. In vitro and in vivo multicolor composite images were acquired to distinguish between two constituent of ONPs and CpG with non-overlapping emission (Figure 5A). ONPs-CpG presented a yellow-labeled fluorescent signal with an obvious coincidence of ONPs by red and TAMRA-labeled CpG by green. The multispectral fluorescence imaging could discriminate ONPs and TAMRA-labeled CpG with non-interfering signals successfully, suggesting the feasibility of simultaneous tracking individual component in vivo. To further assess the antigen and adjuvant delivery after injection in vivo, fluorescence images of free CpG, ONPs and ONPs-CpG groups with tail-base injections were observed for 20 days in Figure 5B. The degradation of ONPs and ONPs-CpG groups was far more durable. ONPs enhanced the persistence of OVA antigen and CpG as carriers. Consistent with the images, the quantitative fluorescence intensity with parallel experiments was also employed to confirm the sustained release of the OVA nanoparticles (Figure 5C). The fluorescence intensity of CpG reduced very fast in free CpG treated mice after 1 day, while it could remain to 20 days with approximate 5% of relative fluorescence intensity in ONPs-CpG group. By analyzing the fluorescence decay of genipin from antigen, they gave the same trend of sustained degradation for 13

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both ONPs and ONPs-CpG group. Fluorescence imaging tracking demonstrated that ONPs-CpG could achieve long-term antigen and adjuvant delivery for improving the tumor immunotherapy effect.

Figure 5 14

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Efficient migration of nanoparticles to lymph nodes is very important for activating local naïve T cells. Herein, whether ONPs and ONPs-CpG could facilitate migration to popliteal lymph nodes was observed by imaging tracking (Figure 5D). In the ONPs and ONPs-CpG groups, the fluorescent signals were observed in the popliteal lymph nodes at 3h post-injection. However, no migration was presented for the free CpG group during antigen delivery. The combination of CpG and ONPs resulted in more efficient migration due to the delivery of the nanoparticles carriers. The nanoparticles offered great potential for migration to the draining lymph nodes, which improved the ability of delivering antigen molecules to antigen present cells. With regard to migration tracking, fluorescence imaging allowed for both high resolution and sensitivity of visible detection by the fluorescence of ONPs and CpG. Therefore, these features rendered our nanoparticles system a tremendous prospect for cancer immunotherapy because of their efficient antigen and adjuvant delivery. 3.6. In vivo anti-tumor therapeutic effects of ONPs-CpG

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In order to explore the anti-tumor efficacy of ONPs-CpG, E.G7-OVA tumor-bearing mice were subcutaneously administrated with OVA, ONPs and ONPs-CpG nanovaccine or PBS individually. The tumor growth and body weight were monitored. As shown in Figure 6A, ONPs and ONPs-CpG showed significant effect in inhibiting tumor growth compared to free OVA or PBS. Unlike OVA nanoparticles with sustained antigen release, soluble antigen rapidly diffused and metabolized in vivo, so free OVA only caused a weak suppression of tumor growth than PBS. For OVA nanoparticles treated groups, the tumor treated with ONPs-CpG showed the most powerful tumor growth suppression owing to the addition of CpG compared with the ones treated ONPs, with mean tumor volume of 100.64 mm3 and 200.64 mm3 on day 12, respectively. In other word, the result indicated that ONPs-CpG elicited strong tumor-specific immunity and exhibited remarkable antitumor ability in vivo. Moreover, no obvious body weight change was found in ONPs-CpG treated groups, indicating good biocompatibility in vivo (Figure 6B). Histological examination was also employed with HE stained-tumor tissue (Figure 6C). For the PBS control group, the tumor tissues presented infiltrating growth tendency apparently with tight cell arrangement. Compared with the PBS and free OVA group, the ONPs-CpG administrated group reduced the percentage of proliferating tumor cells and appeared a significant difference in morphological characteristics, inducing cell death in tumor cells. Moreover, ONPs also inhibited the proliferation in tumor cells that became infrequently or dissolving partially. All of these results implied the potential of ONPs-CpG for immunoprophylaxis and immunotherapeutic during cancer treatment. Many reports have proved that despite the inhibition of the immune cells activity in the tumor microenvironment, intratumoral injection of nanovaccine was still an efficient approach of inducing antitumor immunity20. Based on these findings, more and more research focused on the co-delivery of antigens and immunologic adjuvants to further induce stronger immune response. Han et al. describe that a phospholipidbased phase separation gel loaded with OVA and CpG induced potent immune memory response with no systemic toxicity24. Chintan et al. prepared a nanovaccine for controlled release of antigen peptide and adjuvant, leading to stronger effect on BMDCs maturation and 2 times higher antitumor efficacy23. Yang et.al reported a cancer cell membranes-based nanovaccine encapsulated with immune-adjuvant nanoparticles, triggering efficient antitumor immune responses39. Nevertheless, designing ideal nanovaccines with a simple 16

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producing process, higher antigen loading efficiency, favorable biocompatibility and a powerful immune response remains a huge challenge. Compared to other co-delivery nanovaccines, our co-delivery system of antigen and adjuvant was designed with the simple antigens nanoparticles-based strategy to improve immunogenicity and exert antitumor therapeutic effects with self-carriers. The advantages of self-crosslinking ONPs have been fully discussed in our previous report40,41. In the present study, ONPs not only provided sufficient antigen to stimulate immune response, but also served as a natural carrier for sustained release of CpG. Meanwhile, the antigen could be tracked through signals of genipin in real-time without any additional fluorescence labeling. Thus, the co-delivery of antigen and adjuvant was successfully achieved by the application of visible ONPs for cancer immunotherapy, which provided a novel therapeutic strategy for constructing imaging-guided multifunctional nanovaccines using antigen itself as a carrier. 3.7 Immunotherapy mechanistic study of ONPs-CpG

Figure 7 T-cell activity is important parameters for evaluating the vaccine-induced anti-tumor immune responses

42,43.

In the process of cellular immune response, active effector CD8+ T cells will

infiltrate the tumor tissue with followed release of granzymes or perforin to eliminate tumor cells. Accordingly, the final immunotherapeutic effect directly depends on the activity of 17

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tumor-infiltrating lymphocytes. In order to explore whether ONPs-CpG induced a rapid T cell mediated immune responses in vivo, CD8+ T cells in lymphocytes, splenocytes and tumor tissues was measured respectively in tumor-bearing mice treated with ONPs-CpG, ONPs or free OVA, with PBS injection as control. ONPs-CpG induced the largest percentage of CD8+ T cells in total CD3+ T cells compared to other groups both in draining lymph nodes and in spleen. More importantly, a robust production of effector CD8+ T cells in tumor was developed in ONPs-CpG immuned mice (~7.12%), which was over 2-fold higher than that of ONPs treated mice (3.25%) (Figure7). It was well known that different types of immune cells in the tumor play different roles during the occurrence and development of tumor, as well as in the prognosis stage after surgery. Cytotoxic lymphocytes and CD4+ T cells with a Th1 phenotype can usually control spontaneous or chemically induced tumors growth, while regulatory T-cells (Treg) and several myeloid populations such as neutrophils or macrophages commonly contribute to tumors outgrowth44. Especially, clinical data reveals that the amount and distribution of CD8+ T cells in tumor microenvironment is a major determinant of immunotherapeutic efficiency45,46. In our study, a significant improvement of CD8+ T cells was a result from the delivery of CpG, further leading to the antigen-specific cytotoxic T lymphocyte immunity for better tumor suppression.

Figure 8 Effective T-cell proliferation is another important aspect that reflects cell activation

47.

To

evaluate the proliferation capability, CD8+ T cells from spleen of different immunized mice were stained with carboxyfluoresceinsuccinimidyl ester (CFSE) and re-stimulated with antigen. Based on flow cytometry data, CD8+ T cells from ONPs-CpG immunized mice showed the highest proliferation capability in all groups (Figure 8). Meanwhile, moderate CD8+ T cell proliferation was detected in the ONPs-treated mice compared to free OVA-treated ones. Besides, the interferon gamma (IFN-γ) level in serum analyzed by ELISA assay indicated that ONPs-CpG 18

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treated mice could induce much more IFN-γ secretion than ONPs or free OVA treated ones. Overall, the co-delivery system ONPs-CpG successfully inhibited the tumor growth by enhancing CD8+ T cell activation. The immunotherapy process of the co-delivery nanovaccine was revealed by the above measurement. A dual fluorescent co-delivery nanovaccine exerted the delivery and imaging of antigen and adjuvant based on ovalbumin nanoparticles combined with CpG for cancer immunotherapy (Figure 9). The co-delivery of antigen and adjuvant remarkably promoted DCs activation and maturation due to the high antigen loading efficiency and depot effect. Then, mature DCs effectively migrated and presented MHC I-antigen peptide compound to native T cells. Activated T cells continuously proliferated and secreted cytokine IFN-r. Meanwhile, the increase of the CD8+ T cells percentage in the tumor suggested that more cytotoxic T lymphocytes infiltrated tumor tissue, which further led to an enhanced antitumor effect.

Figure 9

4. Conclusion In summary, a visible co-delivery nanovaccine of antigen and adjuvant (ONPs-CpG) with high 19

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antigen loading efficiency and real-time tracking feature was established for cancer immunotherapy. Meanwhile, real-time fluorescence imaging can track the co-delivery process. In vitro and in vivo experiments proved that ONPs-CpG can elicit robust immune response including DC maturity, T cell activation and IFN-γ production. All of these effects enabled ONPs-CpG exhibit powerful cancer immunotherapy effects. However, it was just a preliminary study and still kept a long distance from clinical applications. Further research is needed from the aspects of material optimization, efficacy improvement, and biosafety evaluation. It is believe that this visible antigen-based co-delivery strategy will elicit a novel approach to the design of multifunctional nanovaccines for cancer immunotherapy.

Supporting Information. Detailed experimental materials and methods.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 81601595, 31870951),

the

Natural

Science

Foundation

of

Tianjin,

China

(Nos.16JCYBJC27800,

18JCYBJC17400) and the CAMS Innovation Fund for Medical Sciences (CIFMS 2017-I2M-3-020).

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Figure legends Figure 1. Preparation and characterization of co-delivery nanovaccine. (A) Synthesis scheme of ONPs-CpG; (B) Agarose gel electrophoresis analysis of ONPs-CpG at different CpG/ONPs ratios; (C) Atomic force microscope images; (D) Size distribution; (E) the stability of ONPs-CpG over time in PBS and serum; (F) UV-vis spectra; (G) Fluorescence emission spectra of ONPs and ONPs-CpG with an excitation wavelength at 580 nm; (H) Cellular viabilities after incubation with OVA, ONPs and ONPs-CpG (n=6). Figure 2. Intracellular distribution of co-delivery nanovaccine. (A) LSCM images of BMDCs cells treated with free CpG, ONPs and ONPs-CpG. Red: ONPs, Magenta: TAMRA-labeled CpG, Green: lysosome, Blue: cell nucleus; (B) FCM analysis for the cellular uptake with free CpG, ONPs and ONPs-CpG. Figure 3. Maturation of BMDCs (CD11c+) in vitro induced by co-delivery nanovaccine. (A) Flow cytometric analysis of CD40+, CD80+, CD86+ and CCR7+ expression in DCs after treatment with free OVA, ONPs and ONPs-CpG for 24h (n=3); (B) Quantification of activated dendritic cells with different OVA formulation. (n = 3) (*p < 0.05). Figure 4. Maturation of DCs (CD11c+) in vivo induced by co-delivery nanovaccine. (A) Flow cytometric analysis of CD40+, CD80+, CD86+ and CCR7+ expression in draining lymph nodes after treatment by PBS, free OVA, ONPs and ONPs-CpG for 3d (n=3). (B) Quantification of activated dendritic cells with different OVA formulation. (n = 3) (*p < 0.05). Figure 5. Imaging visualization of co-delivery nanovaccine based on the fluorescence signals of TAMRA-labeled CpG and ONPs. (A) Multispectral fluorescence imaging of ONPs-CpG. Dual fluorescence of ONPs and TAMRA-labeled CpG in vitro (a) and in vivo (d), Separated fluorescence of ONPs in vitro (b) and in vivo (e) with red, Separated fluorescence of TAMRA-labeled CpG in vitro (c) and in vivo (f) with green; (B) fluorescence imaging in vivo for TAMRA-labeled CpG, ONPs and ONPs-CpG with rainbow color (n = 3); (C) Quantitative fluorescent analysis of TAMRA-labeled CpG, ONPs and ONPs-CpG by the Maestro software. Fluorescent signal at the injection site was presented as a percentage of initial signal (n = 3). (D) 24

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Nanovaccine migrated into draining lymph nodes from the injection sites from 0h to 12 h with rainbow color. Figure 6. In vivo immunotherapeutic effects of co-delivery nanovaccine in tumor-bearing mice. (A) Tumor growth in E.G7-OVA tumor-bearing mice treated with co-delivery nanovaccine. (n = 6); (B) Body weight change in E.G7-OVA tumor-bearing mice. (n = 6); (C) HE staining of tumor tissue. Figure 7. Percentage analysis of CD3+CD8+ T cells of lymphocyte, splenocyte and tumor tissue. (n = 3) (*p < 0.05). Figure 8. Effective T-cell proliferation and IFN-γ secretion. (A) Flow analysis of CFSE labeled CD8+ T cell proliferation in spleen; (B) Quantitative analysis of CFSE labeled CD8+ T cell proliferation. (n = 3) (*p < 0.05); (C) IFN-γ cytokines production detection by ELISA. (n = 6) (*p < 0.05). Figure 9. Scheme of cancer immunotherapy on the basis of the dual fluorescent co-delivery nanovaccine of antigen and adjuvant

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