In Vivo Imaging Tracking and Immune Responses to Nanovaccines

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Biological and Medical Applications of Materials and Interfaces

In vivo imaging tracking and immune responses to nanovaccines involving combined antigen nanoparticles with a programmed delivery Xia Dong, Jie Liang, Afeng Yang, Chun Wang, Deling Kong, and Feng Lv ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04867 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 14, 2018

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ACS Applied Materials & Interfaces

In Vivo Imaging Tracking and Immune Responses to Nanovaccines Involving Combined Antigen Nanoparticles with a Programmed Delivery

Xia Dong1#, Jie Liang1#, Afeng Yang1, Chun Wang1,2, Deling Kong1, Feng Lv1*

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 Department of Biomedical Engineering, University of Minnesota, Minnesota 55455, United States

# 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)

Keywords:

Nanovaccines; antigen nanoparticles; fluorescence imaging; vaccine delivery;

combined vaccine; ovalbumin

1

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Abstract Combined nanovaccine can generate robust and persistent antigen-specific immune responses. A combined

nanovaccine

was

developed

genipin-crosslinked-polyethyleneimine-antigen

based

nanoparticles

on and

in

antigen vivo

loaded

multispectral

fluorescence imaging tracked the antigen delivery of combined nanovaccine. The inner layer antigen nanoparticles carried abundant antigens by self-crosslinking for persistent immune response, while the outer antigen on the surface of antigen nanoparticles provided initial antigen exposure. The delivery of combined nanovaccine was tracked dynamically and objectively by the separation of inner genipin crosslinked antigen nanoparticle and the outer fluorescent antigen. The immune responses of the combined nanovaccine were evaluated including antigen-specific CD4+ and CD8+ T-cell responses, IgG antibody level, immunological memory and CD8+ cytotoxic T lymphocyte (CTL) responses. The results indicated that the inner and outer antigen of combined vaccine can be tracked in real time with a programmed delivery by the dual fluorescence imaging. The programmed delivery of inner and outer antigen induced strong immune responses with a combination of a quick delivery and persistent delivery. With the adequate antigen exposure, dendritic cells were effective activated and matured, and following T cells were further activated for immune response. Compared with a single nanoparticle formulation, the combined nanovaccine exactly elicited stronger antigen specific immune response.

Keywords:

Nanovaccines; antigen nanoparticles; fluorescence imaging; vaccine delivery;

combined vaccine; ovalbumin

2


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1. Introduction Vaccines have tremendous potential to fight with infectious disease for immunotherapy, because they can elicit antigen-specific immune responses with less side effects. However, limited immunotherapy efficacy of vaccine is in part attributed to the low vaccine delivery efficiency. With the development of nanotechnology, the combination of immunotherapy and nanotechnology has been paid much attention

1-3

. Owing to the unique characteristics, nanovaccines exhibit

remarkable vaccine efficiency on stimulating or modulating the immune response in vivo 4. Moreover, nanovaccines can adjust their shape, scale, surface potential, hydrophilcity and antigen loading using different materials and manufacturing conditions

5-7

. They can induce long-term

immune response by conjugation, encapsulation, adhesion or mixing of the antigen with nanoparticles for vaccine delivery

8-10

. Notably, protein-based nanoparticles guide a novel design

strategy and show great potential because of the huge loading capacity

11-14

. The protein-based

nanoparticles exhibit fascinating features owing to dual function of bioactive proteins. The proteins can not only stimulate the immune response as the antigens, but also serve as a carrier for adjuvant agents using antigens themselves. Previous studied have proved that protein particles could strengthen immune response in vivo compared to the same soluble protein in the lack of immunologic adjuvant

15,16

. Our previous report demonstrated that genipin crosslinked functional

ovalbumin nanoparticles significantly triggered antigen-specific immune responses14. To improve the immunotherapy efficacy of nanovaccine, the design and development of an efficient combined nanovaccine is essential and important to the immune therapy. Combined nanovaccine can induce more intense and durable antigen-specific immune responses. They can provide a robust original immunity by the abundant initial antigen exposure and a long-term immunity through the sustained release of antigen. Two kinds of activate mode both can efficiently induce the activation of dendritic cells (DCs) and T cell differentiation in lymph nodes 17,18

. Ma et al reported a combined nanovaccine with antigen loaded nanoparticles mixed with

soluble antigen for improving immune responses

19

. They found the combined nanovaccine

induced stronger antigen-specific immune responses compared to single-component nanoparticles. Ma et al also investigated the kinetics of different antigen nanoparticles formulations and the effect on stimulating the immune system by comparison of combined nanovaccine and single 3



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nanovaccine using a cationic lipid-PLGA hybrid nanoparticle as the delivery carrier

20

. The

combined nanovaccine elicited remarkable higher antigen-specific immune response compared with antigen-adsorbed nanoparticles or free antigen

21

. All these results demonstrated that a

combination of a quick delivery and persistent delivery for antigen ensured initial antigen exposure and antigen duration, and nanoparticle adjuvant also played a significant role for induce strong immune responses. Thus, a more effective nanovaccine is our goal based on novel nanoparticles and methods. Along with the development of the novel combined nanovaccine, understanding the effect of each antigen component of combined nanovaccine is crucial for rational vaccine design. Imaging tracking visualization in vivo can provide an accurate vaccine delivery with a noninvasive and real time method22,23. Compared to other medical imaging technology, fluorescence imaging generates an advanced and unique ability to track the vaccine delivery because of its advantageous including non-invasion, low radiation as well as high sensitivity

24-26

. In some vaccine tracking reports,

fluorescence imaging either tracked the DCs migration, or monitored the antigen vaccine delivery. 18,27

. Notably, multispectral fluorescence imaging can monitor each component of the combined

delivery system simultaneously by a different labeling on the basis of the separation of the difference fluorescence signals

28

. Our previous studies have reported the successful tracking of

several drug delivery systems by multispectral fluorescence imaging

29-31

. In order to reveal the

programmed delivery of the combined nanovaccines, the multispectral fluorescence imaging tracking the vaccine delivery will be the first try with an innovative strategy. Herein, a novel combined nanovaccine was developed based on ovalbumin antigen loaded genipin-crosslinked-polyethyleneimine-antigen

nanoparticle

and

in

vivo

multispectral

fluorescence imaging tracked each antigen delivery of combined nanovaccine with a programmed delivery. The combined nanovaccine was constructed by the self-crosslinked antigens nanoparticles coated with antigens. The inner layer of combined nanovaccine was consisted of genipin-crosslinked- polyethyleneimine -antigen nanoparticle, carrying abundant antigens by self-crosslinking for persistent immune response. Polyethyleneimine (PEI) can enhance the immune response as an adjuvant of nanovaccine, but also provide a platform for absorb the outer antigen with electrostatic forces. The outer antigen can exert the fast delivery to induce initial antigen exposure. The programmed delivery of inner and outer antigen will induce strong immune 4


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responses with a combination of a quick delivery and persistent delivery (Figure 1). Apart from that, the inner and outer antigen can be tracked simultaneously by the dual fluorescence imaging. Genipin afforded the accurate imaging tracking of inner antigen with self-fluorescence. Better than the external tags, the intrinsic fluorescent labels would afford the information of carriers or antigens more stably and accurately in vitro or in vivo 32. The delivery of combined nanovaccine can be tracked dynamically and objectively by the separation of inner genipin crosslinked antigen nanoparticle and the outer fluorescent antigen. Multispectral fluorescence imaging technology can reveal the effect of each antigen component of the combined nanovaccine. Moreover, the immune responses of the combined nanovaccine were evaluated including antigen-specific CD4+ and CD8+ T-cell responses, IgG antibody level, immunological memory and CD8+ cytotoxic T lymphocyte (CTL) responses. Therefore, our objectives were to design an efficient combined vaccine and investigate how the combined nanovaccine elicited robust and persistent antigen-specific immune responses with a programmed delivery.

Figure 1

5



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2. Materials and methods 2.1 Materials, Cell culture, animals, and preparation and characterization of OVA NPs and combined OVA NP They were described in detail in the supporting information. 2.2. Cell viability assay of OVA NPs and combined OVA NPs

BMDCs viability was measured by WST-1 assay by various concentrations of free OVA, OVA NPs and combined OVA NPs. After culture in 96-well plates (1 × 105 cells/well) for 2h, DCs were incubated with different concentrations of free OVA or nanoparticles for 24 h to test the cell viability. After that, cells were added 10 µL of WST-1 solution and incubated for 4 h at 37℃, OD450 was measured by a Multifunction Microplate Reader (Varioskan™ Flash; Thermo Fisher Scientific, USA). 2.3. Cellular uptake and intracellular degradation of OVA NPs and combined OVA NPs

To investigate the location of OVA nanoparticles in DCs, on day 6 of the culture, cells were cultured (1 × 105 cells per well) in confocal image dishes and incubated for 2 h. Free OVA-RB, OVA NPs and combined OVA NPs were added to incubate for different times. After washing with PBS for 3 times, 200ul of LysoTracker Red DND working solution was added to the cells and incubated for 2 h. Then the cells were fixed for 20 minutes, followed by labeling of DAPI (Beyotime Institute of Biotechnology, Shanghai, China). The Confocal luminescence images of DCs were taken on the CLSM (CLSM 410; Zeiss, Jena, Germany) and free OVA-RB and OVA NPs excited at 514 nm and 633 nm, respectively. All the artificial color processed from various dyes was observed by using Fluoview FV500 imaging software. Besides, in order to analyse the cellular uptake quantitatively, DC cells were seeded (1 × 105 cells per well) in six-well plates for 2 h. Then, the cells were harvested after incubation with free OVA-RB, OVA NPs and combined OVA NPs respectively and washed for further FACS analysis using a Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). To observe the cellular uptake of OVA NPs and combined OVA NPs, DCs were cultured (1 × 105 cells per well) in six-well plates for 2 h. Then, the cells were harvested after incubation with free OVA-RB, OVA NPs and combined OVA NPs for 4 h, 12 h and 24 h. FACS analysis was execute by a Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). 6


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2.4. Maturation of BMDCs in vitro and vivo To measure the maturity of DCs in vitro, DCs were incubated with free OVA, OVA NPs and combined OVA NPs (each containing 40 µg OVA) for 24h and harvested and stained with antibodies against MHC I, CD11c, CD86 and CD80 for 30 min at 4℃. After washing with PBS, the expression of MHC I, CD86 and CD80 on DCs was measured by a flow cytometry (BD Biosciences, San Jose, CA). All samples were done in triplicate. For in vivo study, mice (3 mice/group) were intramuscularly injected with 50 µL suspension of free OVA, OVA NPs and combined OVA NPs in physiological saline (each containing 50 µg OVA) to measure MHC I, CD86 and CD80 expression on CD11c+DCs in the lymph nodes. After 24 h post-immunization, inguinal lymph nodes of treated mice were grinded to single cell suspension and stained with anti-CD11c, anti-MHC I, anti-CD86 and anti-CD80 for 30 min at 4℃. MHC I, CD86 and CD80 expression on CD11c+ DCs was determined by a BD Biosciences flow cytometry. 2.5. Determination of T-cell responses by flow cytometry Splenocytes (2 × 106 cells) were harvested from each group on aseptic procedures after three immunizations, and retreated with OVA (50 µg/mL) for 72 h at 37 °C. Then, cells were collected and stained with anti-CD4, anti-CD8a, anti-CD44, and anti-CD62L and anti-IFN-γ for 30 min at 4 °C. T-cell proliferation in spleen was evaluated by the proportion of CD4+ and CD8+ cells. The percentages of effector memory T cells (CD44Hi CD62LLo), central memory T cells (CD44Hi CD62LHi) and CD4+ IFN-γ+ and CD8+ IFN-γ+ were detected by flow cytometer (BD Biosciences, San Jose, CA). 2.6. ELISA analysis of cytokine and levels of OVA-specific IgG and isotypes ELISA analysis of cytokine and levels of OVA-specific IgG and isotypes was performed according to the methods of our previous publication14. The detailed description was provided in the supporting information. 2.7. CTL assay The mice were immunized three times weekly with 50 µL suspension of free OVA, OVA NPs and combined OVA NPs. At 7 d after last immunization, the splenocytes from different groups were collected and cultured in medium containing OVA (50 µg/mL) and 10 ng/ml recombinant mouse IL-2 for 72 h at 37 °C. CTL activity was detected by LDH Assay against 1 × 104 7



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E.G7-OVA cells. After washing three times, effector and target cells were added in round bottom 96-well culture plates (Costar, Corning, New York, USA) to incubate for 4 h at the proper E:T ratio. Then, 100 µL of supernatant was taken out to detect the LDH leakage level. Specific lysis was calculated by the equation33 [ (experimental LDH release - effector cells spontaneous LDH release - target cells spontaneous LDH release)/(target cells maximum LDH release - target cells spontaneous LDH release) ] × 100% (n = 4). 2.8. Dual fluorescence tracking for the draining lymph nodes and degradation of free OVA-RB, OVA NPs and combined OVA NPs. In order to ensure the distinguish between OVA-RB and OVA NPs by the multispectral fluorescence imaging in vivo, the fluorescence imaging in vitro was produced by imaging system (Maestro 2，CRI，USA) firstly. Multispectral fluorescence imaging can extract the respective signals of the OVA NPs and OVA-RB to track the draining lymph nodes and degradation of free OVA-RB, OVA NPs and combined OVA NPs in vivo. Parallel experiment was designed by 3 groups of C57BL/6 mice, which were removed back hair and labeled separately. After intraperitoneal injection with chloral hydrat (4%), free OVA-RB, OVA NPs and combined OVA NPs (50 µg of OVA) was injected subcutaneously to the base of tail of mice, respectively. The signal of OVA NPs was extract at an excitation wavelength of 595 nm , while that of OVA-RB was 523 nm. Meanwhile, fluorescence tracking for the draining lymph nodes was recorded after administration from 0 h to 24 h and tracking for the degradation of the different groups for 20 days. The fluorescence signals were also calculated quantitatively over time by the Maestro software. Besides, the degradation fluorescent signal at different time is presented as the percentage of the initial recorded fluorescent intensity value. 2.9. Statistical Analysis. All the values in the study were presented as the mean ± standard error (SD). Statistical difference was performed using one-way ANOVA followed by Tukey’s multiple comparison. Significance was set at α=0.05. All statistical analysis was carried out in GraphPad Prism 5.0 software (San Diego, CA, USA).

3. Results and discussion 3.1. The characterization of nanoparticles 8


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Figure 2 9



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The OVA nanoparticles (OVA NPs) were successfully prepared by cross-linked reaction with genipin between the amino groups of OVA and PEI. The reaction mechanism of genipin is that the amine moiety attached genipin to form the crosslinking between primary amine groups

32

.The

crosslinking degree of OVA NPs was measured by free amino content analysis. The free amino group content of OVA/PEI mixed solution is 4.84 times to the OVA NPs, suggesting a crosslinking degree of approximate 80%. The secondary structure of OVA NPs was reflected by CD spectra (Figure 2A). The covalent crosslinking would not change the structure and function of OVA antigen as free OVA and OVA NPs did not significantly exhibit the spectral difference. The combined OVA nanoparticles were prepared by the electrostatic interaction with OVA antigen on the surface of the OVA nanoparticles. The SDS-PAGE bands indicated the difference of free OVA, the OVA NPs and the combined the OVA NPs (Figure 2B). OVA NPs have a higher molecular weight than free ova molecules due to the cross-linking. It is difficult for OVA NPs to move in the SDS-PAGE. However, the combined OVA NPs gave the band of OVA because the external OVA antigen can be broken away from the combined OVA NPs under the electric field in SDS-PAGE. The surface morphology of OVA nanoparticles and combined OVA nanoparticles were studied using AFM (Figure 2C). No obvious difference was detected in the morphology of OVA-NPs and combined OVA NPs. The suitable size of nanoparticles is propitious to vaccine delivery systems and easier to be taken up by immunological cell to promote antigen specific immunity. The mean size of OVA-NPs was 216.4 nm (PdI = 0.371) and the zeta potential was 23.9 mv, while the combined OVA NPs had an increased size of 302.2 nm (PdI = 0.454) and the zeta potential dramatically decreased to 16.2 mv after the electrostatic adsorption of free OVA (Figure 2D). Although OVA is negatively charged at neutral pH, the OVA NPs still show the positive zeta potential due to the crosslinking with the high positive PEI. Based on the opposite charge of OVA NPs (positive charge) and free OVA (negative charge), the combined OVA NPs was further prepared by electrostatic interaction with OVA antigen on the surface of the OVA NPs. These data showed that free OVA on the surface of OVA NPs prospectively increased the size and decreased the zeta potential of the nanoparticles. Genipin is a colorless crosslinking agent without fluorescence itself. Only when it crosslinked with the amino compound did it generated the obvious fluorescence. The OVA NPs and combined OVA NPs emitted self-fluorescence because of the crosslinking of genipin. Optical intensity of 10


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OVA NPs and combined OVA NPs are very important for fluorescence tracking in vivo. The UV-Vis and fluorescent properties of OVA NPs and combined OVA NPs were evaluated. OVA NPs and combined OVA NPs all had a characteristic absorption peak at about 600 nm in the UV-Vis spectrum (Figure 2E), which indicated that the combination of free OVA and OVA NPs did not result in the peak shift. Fluorescence spectrum of OVA NPs and combined OVA NPs (Figure 2F) indicated that the maximum emission at 610 nm was excited at the wavelength of 585nm. In addition, the fluorescence stability of organic dyes is the basis for the long-time monitoring. In order to investigate the self fluorescence decay from the quenching or bleaching effect of organic dyes, the fluorescence stability of Rb-OVA and genipin crosslinked OVA NPs was measured for 7 days by CRI imaging system (Figure S1). The results approved their beneficial fluorescence stability without obvious self fluorescence decay (Figure S2). The suitable fluorescence emission and stability met the demand for in vitro or in vivo fluorescence tracking. 3.2. BMDCs viability assay

Figure 3 To investigate the effect of the nanoparticles on BMDCs viability, the cytotoxicity was measured by WST-1 assay after incubating BMDCs with a series of concentrations of free OVA, OVA NPs or combined OVA NPs (Figure 3). No cytotoxicity was observed for free OVA treated BMDCs with the concentration range from 1 µg/mL to 50 µg/mL, while the nanoparticles did not exhibit significant influence on BMDCs growth at a lower concentration range (1~20 µg/mL), with 70~80% of viability remained for OVA NPs or combined OVA NPs treated cells. But an 11



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obvious cytotoxicity appeared when the concentration of OVA was raised to 50 µg/mL in the OVA NPs group. In the meanwhile, a significant toxic difference could be found between the cell viability of OVA NPs (10%) and combined OVA NPs (70%) treated BMDCs at this concentration because the high positive charge of PEI exerted a toxic effect 34 and inhibit the growth of BMDCs to some extent. Despite the impact of the PEI, the combined nanovaccine formulation significantly decreased the cytotoxicity because of the package of outer OVA. Compared with OVA NPs, the higher biocompatibility of combined OVA NPs make it exhibit great application potential as an attractive antigen delivery vehicle. In consideration of cytotoxicity of high concentration nanoparticles and the requirement of sufficient antigen for stimulating immune responses, the concentration of 20 µg/mL was used for the subsequent cell experiments. 3.3. Cellular uptake and intracellular trafficking of OVA-nanoparticle in BMDCs

12


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Figure 4 13



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Effective uptake of antigen by DCs is essential for DC activation, antigen processing and presentation35. The application of the nanoparticles could significantly enhance intracellular antigen uptake36. Generally, antigen or nanoparticles would be labeled fluorescence molecules for tracking in order to quantify the cellular uptake. In this work, the influence signal of genipin from genipin-PEI-crosslinked OVA nanoparticles was used to track the inner OVA antigen, and outer OVA absorbed on the surface of genipin-PEI-crosslinked OVA nanoparticles was labeled with RB. The two types of fluorescence signals were monitored for the location of each component of the combined nanoparticles in BMDCs by confocal fluorescent microscope. As shown in Figure 4A, free OVA could be observed by the fluorescence of RB (magenta), OVA NPs could also be detected based on the intrinsic fluorescence signal of genipin(red). What’s more, combined OVA NPs could be simultaneously monitored through the inner red fluorescence and outer magenta fluorescence. The intensity of each type of fluorescence signal gradually increased with extended incubation time. In comparison, the mean fluorescent intensity of free OVA RB treated cells was the weakest under the same condition, magenta fluorescence signal from outer OVA of combined nanoparticles remarkable enhanced owing to the application of nanoparticles. The enhanced positively-charged of OVA NPs also play an important role with regard to the increased cellular uptake due to the addition of PEI, which make the OVA NPs or combined OVA NPs more stable and more apt to enter the negative charged cell membrane37. As shown in Figure 4, the cellular uptake efficiency was significantly enhanced after incubation with OVA NPs for 24 hours compared with free OVA treated group, while the red fluorescence intensity of combined OVA NPs group was approximately equivalent to that of OVA NPs group. Moreover, it has been observed that nanoparticles were mainly concentrated in lysosomes (green). The cellular uptake of combined OVA NPs, single OVA NPs and free OVA RB was also quantitatively determined by flow cytometry (Figure 4B). The fluorescence signals of single OVA NPs and combined OVA NPs strengthened gradually after incubating with the cells for 4 h, 12 h or 24 h. But the fluorescence intensity of free OVA RB has no significant change with time owing to the low uptake of soluble antigen. These data suggested that nanoparticles induced more effective antigen uptake and could successfully carry soluble antigen adsorbed on the surface of nanoparticles into the antigen presenting cell. The genipin-PEI-crosslinked OVA nanoparticles themselves are not only effective antigen carriers, meanwhile, meanwhile the optical property 14


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make cell intrinsic imaging more convenient for visible tracking.

Figure 5 To further observe the intracellular behavior of genipin-PEI-crosslinked OVA within the cytosol of DCs, the high magnification confocal images were obtained after incubation for 12 h (Figure 5). The overlay yellow fluorescence indicated that OVA was co-localized with endo/lysosomes. But a small portion of red fluorescence was still visible within green vesicles in combined OVA NPs and single OVA NPs-treated DCs, while free OVA totally located in endolysosomes. The result indicated the possible escape of OVA antigen in nanoparticles from the endo/lysosomes. Futhermore, it was found that combined OVA NPs escaped more efficiently compared to single OVA NPs. Though the mechanism is still unknown, antigen-specific CD8+T cell immune response could be enhanced by lysosome escape, thereby, it is supposed that genipin-PEI-crosslinked OVA nanoparticles can induce cell immune more efficiently. Based on the findings that combined OVA NPs and single OVA NPs have stronger cellular uptake compared with free OVA RB, we are highly interested whether or not the degradation of OVA nanoparticles inside the cells is closely related with the improved DCs function. To observe the morphological changes of the internalized OVA particles, BMDCs were imaged after incubation with combined OVA NPs and single OVA NPs for 2 h, 6 h, 12 h and 34 h, respectively (Figure 6). No significant changes were observed on the degradation of the two kinds of nanoparticles in the first 12 hours. But smaller volumes fragments began to appear at 34 h incubation, it indicated that genipin-PEI-crosslinking OVA nanoparticles gradually degraded over time. The process of intracellular degradation of nanoparticles was related to the efficient antigen epitope presentation and had a direct impact on the following immune responses. Herein, the 15



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degradation of the covalent cross-linking OVA nanoparticles suggested the chemical processing would not influence the effect of OVA antigen, which was a very important precondition for antigen presentation of DCs. Taken together, all the data showed that OVA nanoparticles could exert a stronger immune effect in cells due to its antigen uptake, escape performance and favorable stability.

Figure 6 3.4. Multispectral fluorescence resolution in vitro and vivo Although fluorescence imaging skills have been widely applied for cellular imaging including flow cytometry and confocal laser scanning imaging 38-40, it is still a tremendous challenge beyond of the cellular imaging because of the interference of the biotissues and the resolution of imaging instrument. High sensitive fluorescence imaging can reveal the microscope structure and function in cellular levels, while a large-scale imaging needs to be acquired for in vivo imaging. In addition, the cells samples can be optimized to reduce the interference of other fluorescent signals, which increase the imaging quality with high sensitivity and resolution. Whereas, the biotissues can generate some fluorescent signals including hemoglobin and other component. Accordingly, these factors lead to the increased difficulty of multispectral fluorescence imaging in vivo. 16


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Figure 7 The in vivo separation of several fluorescent signals is a vital factor for the imaging tracking. In the combined OVA NPs system, a dual fluorescent labeling was perform with RB and genipin as fluorescent tags. For tracking inner OVA NPs and outer free OVA RB components of combined OVA NPs, multispectral fluorescence labeling signals in vitro and vivo were distinguished according to the fluorescence spectrum of two constituent of combined OVA NPs. Based on different emission of OVA NPs and OVA RB，a multicolor image distinguished different labels from combined OVA NPs. As shown in Figure 7, the signals of OVA NPs were labeled with red, while that of OVA RB were green. The combined OVA NPs gave an obvious coincidence signal of OVA NPs and OVA RB in yellow label for in vitro or in vivo fluorescence imaging. The separated fluorescent signals of OVA NPs and OVA RB in vitro can be well recognized by multispectral fluorescence imaging, which afforded the reference for the dual fluorescence imaging of combined OVA NPs in vivo. In Figure 7D, the composite signal of combined OVA NPs can locate in the mouse back with yellow, suggesting the overlay signals of OVA NPs and OVA RB. The OVA NPs (Figure 7E) and OVA RB (Figure 7F) signals can be extracted with multi spectral resolution method by CRI imaging system, respectively. Multispectral fluorescence resolution is necessary for tracking of multi-component of vaccine. They can distinguish difference labels and confirm the combination of multiple components. In vivo multispectral fluorescence imaging increases the sensitivity and usefulness for vaccine 17



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development, including the degradation and delivery in vivo defense system. OVA NPs can be combined with OVA RB for real time monitoring, so that the nanovaccine can be delivered accurately with visualization method by multispectral fluorescence imaging. 3.5. Fluorescence imaging tracking of the draining lymph nodes and degradation of OVA nanoparticles To track antigen presentation or vaccine delivery with visual and noninvasive method, fluorescence imaging is considerable important to monitor. OVA nanoparticles could be observed in the injection site or lymph node by imaging system owing to the fluorescent signals of Genipin or RB from the nanoparticles system in vivo. As shown in Figure 8A and Figure S3, the inner OVA NPs and outer OVA of combined OVA NPs group were presented separately with clear fluorescent signal in tail-base injection site for 20 days real-time tracking. The degradation of free OVA RB was significantly faster than single OVA NPs and combined OVA NPs, which suggested that OVA nanoparticles formation could prevent the quick antigen-release. Furtherly, the quantitative fluorescence intensity of different groups was analyzed by the ratio of initial fluorescence. As shown in Figure 8B, fluorescence intensity of free OVA RB group descended rapidly, only about 10% remained after 3 days, but the outer OVA RB in the combined OVA NPs group decayed to 40%. The nanoparticles carrier extended the release of the OVA antigen in the combined OVA NPs. On the other hand, single OVA NPs and combined OVA NPs group did not give obvious difference by monitoring the fluorescent signals of genipin. They had a longer retention time up to 20 days, in which around 10% of fluorescence intensity was still reserved after 20 days. All the results indicated that single OVA NPs and combined OVA NPs group would have a longer retention time for stimulating a continuous immune response.

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Figure 8 Lymph node plays an important role in specific immune response including humoral immunity and cellular immunity. When maturated DCs migrated to the lymph node, the lymphatic drainage in vivo quickly received stimulation of antigen, then specific immune response was triggered. Figure 8C recorded the fluorescent signals in the lymph nodes of combined OVA NPs, single OVA NPs and free OVA RB treated mice within 12h. The fluorescent signals in draining lymph nodes were compared by Maestro software at different time points (Figure S4). As expected, no obvious 19



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fluorescent signal was observed in lymph nodes of free OVA treated mice because of the rapid dissipation of soluble antigen from the injection site. In contrast, single OVA NPs or combined OVA NPs gradually accumulated in the draining lymph nodes, and a peak appeared at 9 hours for single OVA NPs and 3 hours for combined OVA NPs. In addition, a clear fluorescent signal from outer OVA RB of combined OVA NPs could be observed in the lymph nodes, which indicated that the outer soluble antigen in the combined vaccine could effectively migrate to immune organ and afford sufficient initial antigen to motivate the immune system. In other words, we described the dynamic process of antigen exposure to the immune system through imaging tracking pattern in the present study. 3.6. Activation of BMDCs in vitro and vivo To initiate T cell immune responses, the transform of DCs from immaturity to maturity is necessary. The level of DC maturation could be indicated by the upregulation of costimulatory molecules and histocompatibility complex (MHC) complex on the membrane18. Thus, the expression of costimulatory molecules (CD80, CD86) and MHC class I molecules (MHC-1) on DCs were analyzed by flow cytometry after incubating DCs with free OVA and OVA nanoparticles in vitro. LPS significantly stimulate DCs to mature and all expression of costimulatory molecules (CD80, CD86) and MHC class I molecules (MHC-1) on DCs surface were remarkably elevated than other three groups. Flow cytometry data demonstrated that combined OVA NPs and single OVA NPs induced higher level of CD80, CD86 and MHC-I expression than free OVA group (Figure 9A). In vivo, after capturing the antigen, DCs will process the antigen into peptides during the migration to lymph nodes and present peptide to T cells for initiating immune response. So the expression of CD80, CD86 and MHC-I on DCs in draining lymph nodes will more directly reflect the DCs activation in vivo. As shown in Figure 9B, the data in vivo were consistent with that in vitro, illustrating that combined OVA NPs induced stronger DCs maturation. Moreover, the expression of CD80 and CD86 significantly increased in combined OVA NPs group compared with that in single OVA NPs group, which illustrated that combined OVA NPs might lead a stronger immune response in vivo than in vitro. It was possible that the outer antigen of combined nanoparticles absorbed by electrostatic effect provided an adequate initial antigen exposure and inner OVA nanoparticles supported a long-term antigen sustain. 20


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Figure 9 21



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3.7. Antigen-specific immune response The efficacy of nanovaccines is closely related with various characteristics including nanoparticle size41, surface charge42, administration route43 or antigen release rate44. The antigen release kinetics is a key factor by controlling antigen exposure time. Kanchan et al. confirmed that antigen loaded polylactide (PLA) polymer particles can induce lasting primary antibody response 45

. Our previous study also illustrated that functional OVA-NPs could strengthen the

antigen-specific immune responses compared to soluble OVA including CD4+ and CD8+ T-cell activation, higher antibody level and enhanced immunological memory function14. In the present study, the kinetics of antigen exposure of combined OVA-NPs to the immune system has been investigated in detail by imaging tracking. Next, whether the combined nanoparticles could induce an enhanced antigen-specific immune response in vivo would be verified. Cellular immunity is the primary way of preventing endogenous pathogen infectious disease and treating tumor. Generally, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) are important index of cellular immunity. In this study, splenocytes were restimulated with OVA after harvest from different vaccinated mice, intracellular cytokine staining was executed to measure the cellular secretion of IFN-γ. As shown in Figure 9C, the percentage of IFN-γ+ CD4+ and IFN-γ+CD8+ T cells in combined OVA NPs treated group significantly increased compared to the other groups. To further detect cytokine secretion of different antigen nanoparticles immunized mice in vivo, serum was collected from immunized mice to measure cytokines release of IFN-γ and TNF-α. The secretion of cytokines was determined by ELISA assay. The result showed that the secretion of IFN-γ (Figure 9D) and TNF-α (Figure 9E) significantly increased in the combination of OVA NPs and free OVA (combined OVA NPs) treated mice compared to that in single OVA NPs or free OVA immunized mice, which would further cause a robust T cell response. 3.8. Cytotoxic T lymphocyte responses Cytotoxic T lymphocytes (CTL) mainly derived from CD8+ T cells, they can kill cancer cells or infected cells via releasing cytotoxins such as perforin and granzymes46. In this study, it has been demonstrated OVA nanoparticles could effectively migrate to the draining lymph node, so the OVA-specific CTL response in vivo was further confirmed by evaluating the cytotoxicity of CTL to tumor cells. Herein, splenocytes from different treated mice was re-stimulate with OVA and 22


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incubated with E.G7-OVA cells after immunization for 7 days, then the cytotoxicity was performed. It was obvious that an enhanced cytotoxicity response was induced by CTL in OVA nanoparticles treated T cells compared to free OVA treated ones at a 50:1 ratio (Figure 9F). Moreover, combined OVA NPs showed greater cytotoxicity against EG7-OVA than single OVA NPs. All these results indicated that robust antigen-specific immune response could be induced to kill specific tumor cells with the combined OVA NPs, suggesting the important potential in the treatment of cancer immunotherapy. 3.9. Systemic antibody responses in vaccinated mice The level of IgG, IgG1 and IgG2a in the serum after immunization was used to evaluate the effect of combined OVA NPs on antibody response. As shown in Figure 9G, significantly higher antigen-specific serum IgG titer was elicited by nanoparticles antigen formulation compared to soluble antigen. Notable, combined OVA NPs induced the highest level of IgG response. The result indicated that the nanovaccine could induce a higher level of antibody production than the soluble antigen. In comparison with free OVA, the PEI in OVA NPs should also exert adjuvant activity in the antibody-producing process besides the difference of nanostructure between them47,48. For combined OVA NPs, the outer layer OVA and inner OVA nanoparticles generated different dynamic behavior for antigen exposure to the immune system, which might be the most important factor to induce the most powerful antibody response for combined nanoparticles19. Furthermore, the level of IgG2a and IgG1 were determined to reveal whether it was a Th1-polarized immune response. Figure 9H demonstrated that combined OVA NPs and single OVA NPs induced higher value of IgG2a/IgG1 compared with free OVA, suggesting that OVA self-crosslinked nanoparticles favored a Th1 response. Taken together, mice immunized with combined OVA NPs not only have a higher level of antigen-specific IgG antibody production, but also might induce an enhanced cellular immune response in vivo, which exhibited great possibility for the application of anti-tumor immunotherapy. 3.10. OVA nanoparticles induced memory T-cell responses in vivo

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Figure 10 Immunological memory is an important mark of the adaptive immune and memory T cells are the key components. When antigen invades the body again, memory T cells will more rapidly respond and provide better clearance of pathogens. So a long-time effective immunological memory is necessary for nanovaccine to offer persistent protection of reinfection. As well known, CD44highCD62Llow and CD44highCD62Lhigh were markers of effector-memory and central-memory T cells. Herein, the percentages of memory T-cell in antigen stimulated splenocytes was evaluated by flow cytometry. Figure 10 provided the proportion of effector-memory and central-memory CD4+ T and CD8+ T cells. It was obvious that the frequency of CD44highCD62Lhigh central-memory and CD44highCD62Llow effector-memory CD4+ T and CD8+ T cells increased 24


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significantly from the mice immunized with combined OVA NPs and single OVA NPs than that of free OVA. A significant improvement for memory CD4+ T cells was detected in combined OVA NPs group compared to single OVA NPs group. Over all, combined OVA NPs generated the strongest immunological memory responses, providing better protection from pathogens.

4. Conclusions In summary, a combined nanovaccine was successfully designed and prepared based on OVA NPs combined with OVA. Multispectral fluorescence imaging tracked the vaccine delivery accurately both in vitro and in vivo. The combination pattern of OVA nanoparticles and soluble OVA exhibited higher antigen load efficiency and better biocompatibility. With the adequate antigen exposure, DCs were effective activated and matured, following T cells were further activated for immune response, including cytokine secretion, antibody production, CTL and memory immune response. Although the exact mechanism is unknown, the combined nanovaccine exactly elicited stronger antigen-specific immune responses than single nanoparticles. Therefore, combined OVA NPs provided a new strategy for building visible antigen delivery system.

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81601595), the Natural Science Foundation of Tianjin, China (Nos.16JCYBJC27800), the Science and Technology Support Program of Tianjin (14RCGFSY00146) and the CAMS Innovation Fund for Medical Sciences(CIFMS 2017-I2M-3-020).

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Figure legends Figure 1. Schematic illustration of the preparation of combined nanovaccine and immune responses to nanovaccines involving combined antigen nanoparticles with a programmed delivery Figure 2. Characterization of different OVA formulation. (A) Far-UV CD spectra, (B) Polyacrylamide gel electrophoresis, (C) Atomic force microscopy images, (D) Size and zeta potential distribution, (E) UV-vis spectra, (F) Fluorescence emission spectra. Figure 3. Relative viabilities of BMDCs after 24h of incubation with free OVA, OVA-NPs and combined OVA-NPs. (n=6). Figure 4. Cellular uptake and intracellular location after incubation with BMDCs. (A) BMDCs images after 4 hour, 12 hour and 24 hour treatment with different OVA NPs formulation by CLSM. Red: OVA-NPs, magenta: OVA RB, green: lysotracker, blue:DAPI, (B) flow cytometry analysis for the cellular uptake with different OVA NPs formulation. Figure 5. CLSM images of DC cells for the lysosomal escape of OVA nanoparticles after incubated with different OVA NPs formulation at 12h. Red: OVA-NPs, magenta: OVA RB, green: lysotracker, blue:DAPI. Figure 6. Intracellular degradation of OVA-NPs and combined OVA-NPs visualized by confocal microscopy after incubation for 2 h, 6h, 12h and 34h.Red: OVA-NPs, magenta: OVA RB. Figure 7. Multicolor imaging of merged fluorescence in the combined OVA-NPs of red OVA-NPs and green OVA RB. (A) Merged imaging of OVA-NPs and free OVA RB in vitro, (B) Separated imaging of OVA-NPs with red in vitro, (C) Separated imaging of free OVA RB with green in vitro, (D) Merged imaging of OVA-NPs and free OVA RB in vivo, (E) Separated imaging of OVA-NPs with red in vivo, (F) Separated fluorescence of free OVA RB with green in vivo. Figure 8. Imaging visualization in vivo of the different OVA formulation in the injection area and lymph nodes on the basis of the fluorescence of rhodamine and genipin. (A) Imaging monitoring of free OVA RB, OVA-NPs or combined OVA-NPs with rainbow color as a representative of 30


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three for different groups, (B) Quantitative fluorescent analysis of OVA RB and OVA-NPs was calculated by the Maestro software. The change on fluorescent signal was presented as a ratio of initial intensity as the mean ± SD (n=3), (C) Fluorescence imaging of draining lymph nodes with antigen migration from 0h to 12 h with rainbow color. Figure 9. (A) Quantification of MHC I+, CD86+ and CD80+ expression in DCs after 24h of treatment with the different OVA formulation in vitro, (B) Quantification of MHC I+, CD86+ and CD80+ expression in DCs (CD11c+) in vivo, (C) The percentage of IFN-γ+ CD4+ T and IFN-γ+ CD8+ cells with intracellular staining by flow cytometry, (D) TNF-α cytokines secretion by ELISA, (E) IFN-γ cytokines secretion by ELISA, (F) In vitro cytotoxic T lymphocyte responses. The killing activity was measured by LDH assay after incubating restimulated splencytes with EG7.OVA tumor cells. E:T means refers to the effector:target ratio, (G) Antigen-specific total IgG levels in sera from immunized mice, (H) IgG1/ IgG2a value in sera from immunized mice with the different OVA formulation. Results are presented as the mean ± SD (n = 6) (*p< 0.05). Figure 10. (A) The representative flow cytometry images for memory T cells analysis. The percentage of CD44highCD62Lhigh central memory (B) and CD44highCD62Llow effector memory (C) of CD4+ and CD8+ T by flow cytometry after restimulating with OVA (50 µg/mL) for 7 days post immunization. Results are presented as the mean ± SD (n = 6) (*p< 0.05).

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In Vivo Imaging Tracking and Immune Responses to Nanovaccines

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