Injectable Hydrogels Coencapsulating Granulocyte-Macrophage

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Biological and Medical Applications of Materials and Interfaces

Injectable hydrogels coencapsulating granulocytemacrophage colony-stimulating factor and ovalbumin nanoparticles to enhance antigen uptake efficiency Zhiting Sun, Jie Liang, Xia Dong, Chun Wang, Deling Kong, and Feng Lv ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04312 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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ACS Applied Materials & Interfaces

Injectable

hydrogels

coencapsulating

granulocyte-macrophage

colony-stimulating factor and ovalbumin nanoparticles to enhance antigen uptake efficiency 1

1

1

1,2

1

1

Zhiting Sun , Jie Liang , Xia Dong *, Chun Wang , Deling Kong , 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 Department of Biomedical Engineering, University of Minnesota, Minnesota 55455, United States

*Corresponding author: Xia Dong 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] (Dong X)

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: antigen delivery; hydrogel; GM-CSF; nanoparticle; in vivo fluorescence tracking

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Abstract The combination of an antigen and adjuvant has synergistic effects on an immune response. Coadministration of an antigen and a granulocyte-macrophage colony-stimulating factor (GM-CSF) hydrogel delivery system will afford a novel strategy for enhancement of an immune response because of the dual role of the hydrogel as a vaccine carrier with a sustained release and a platform for recruiting dendritic cells (DCs). Herein, an injectable poly(caprolactone)-poly(ethylene glycol)poly(caprolactone) [PCL-PEG-PCL] thermosensitive hydrogel coencapsulating GM-CSF and ovalbumin nanoparticles (OVA-NPs) was developed to enhance antigen uptake efficiency. The GM-CSF released from the hydrogel ensured accumulation of DCs; this effect improved the antigen uptake efficiency with the targeted delivery to antigen-presenting cells. Furthermore, the dual delivery system induced a stronger immune effect including higher CD8+ T proportion, interferon γ secretion, and a greater cytotoxic-T-lymphocyte response, which may benefit from the recruitment of DCs, increasing antigen residence time, and the controllable antigen release owing to the combined effect of the hydrogel and nanoparticles. Meanwhile, the real-time antigen delivery process in vivo was revealed by a noninvasive fluorescence imaging method. All the results indicated that the visible dual-delivery system may have a greater potential for the efficient and trackable vaccine delivery.

Keywords: antigen delivery; hydrogel; GM-CSF; nanoparticle; in vivo fluorescence tracking

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1.

Introduction

As one of the most effective therapies for combating infectious diseases, vaccination has by far saved a large number of lives all over the world

1,2

. Nonetheless, limited immunotherapeutic efficacy of

vaccines is in part attributed to the low efficiency of vaccine delivery. Administering antigens with adjuvants can elicit antigen-specific immune responses with less adverse effects. Some important strategies can be further developed to improve immunotherapy including sustained antigen provision, recruitment of antigen-presenting cells (APCs), enhanced migration of APCs to lymph node, and inducing high level of immunomodulatory cytokines and chemokines secretion3,4. Notably, codelivery of an antigen and adjuvant can dramatically increase immune responses for the development of vaccine delivery systems5,6. They can generate stronger and more prolonged adjuvant-induced, antigen-specific immune responses. Thus, novel vaccine delivery systems need to be developed to enhance immunotherapy via the combination of an antigen and adjuvant.

The weak delivery efficiency of soluble protein antigens leads to a significant reduction of immunization efficacy. The use of a vaccine delivery system is important for antigen delivery, which is related to immune responses7,8. The delivery systems include microparticles or nanoparticles that target immune cells as well as depotlike delivery systems that support a sustained release9,10. Among these vaccine delivery systems, nanoparticles and a thermosensitive hydrogel attract our attention. Nanoparticles can effectively deliver an antigen to a targeted site and improve the therapeutic immune response via beneficial cellular uptake and bioavailability and milder adverse effects11-13. Nanoparticle-based vaccine delivery systems have shown substantial advances in the field of immunotherapy including immunotherapy of tumors and infectious diseases14-16. Hydrogel-based vaccine delivery systems can yield a high local concentration of an antigen and reduce the adverse effects by means of excellent hydrophilicity and biocompatibility17,18. In particular, a thermosensitive hydrogel can be injected into a targeted site by noninvasive administration. These factors can induce a lasting immune response via the sustained release of the antigen. Because of the advantages of nanoparticles and the hydrogel, the combined vaccine delivery system can prevent the rapid phagocytosis of a nanodelivery system and extend the antigen release process via two-stage delivery from the hydrogel and nanoparticles. The hydrogel–nanoparticle system can achieve desired spatiotemporal delivery for on-demand vaccine delivery, which has a tremendous potential for 3



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immunotherapy.

Vaccine

adjuvants

play

an

important

complementary

role

in

immune

responses.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) can activate APCs like dendritic cells (DCs) or macrophages19,20. It can significantly improve the antigen delivery efficiency by recruiting DCs in the site of antigen injection; this approach strengthens and prolongs adjuvant-induced, antigen-specific immune responses21. A promising strategy involves the use of a GM-CSF–loaded biomaterial scaffold that enriches and programs immune cells in a localized manner22,23. Hydrogels have excellent permeability and structural similarity to extracellular matrices due to their three-dimensional semisolid polymer networks with abundant water. As a carrier of GM-CSF, a hydrogel can afford a platform for the cell growth with minimal invasion to surrounding tissues24. GM-CSF–loaded hydrogels can regulate accumulation of abundant immature DCs to improve an immune response22. The vaccine consisted of GM-CSF loaded hydrogel also guided a simple and effective strategy for the development of novel hepatitis B virus vaccines

24

. Via the combination of

antigen delivery and the GM-CSF adjuvant, an efficient cancer vaccine has been developed based on a two-step hybrid strategy consisted of a prior recruiting of DCs by GM-CSF loaded mPEG−PLGA hydrogel and a latter injection of vaccine vectors carrying antigens

25

. In order to enhance the

synergistic effect on the immune response, the coadministration of an antigen and of the GM-CSF hydrogel delivery system represents a novel strategy for eliciting an immune response via the dual role of the hydrogel as a vaccine carrier with a sustained release and a platform for recruiting DCs.

Herein, an injectable hydrogel coencapsulating GM-CSF and ovalbumin nanoparticles (OVA-NPs) was developed to enhance antigen uptake efficiency. The poly(caprolactone)-poly(ethylene glycol)poly(caprolactone) [PCL-PEG-PCL] thermosensitive hydrogel has been applied as a drug carrier with beneficial injectability26,27. It can be studied in terms of vaccine delivery as a carrier coencapsulating the antigen and adjuvant. A genipin–cross-linked OVA-NP–loaded hydrogel can exert a lasting immune response with the sustained delivery of the antigen via a dual effect of the nanoparticles and hydrogel. Antigen-based protein nanoparticles can enhance an immune response better than other nanovaccines can because such nanoparticles can promote the efficiency of antigen-loading without extra carriers because of their self-assembly or cross-linking. Nonetheless, the combined system consisting of the nanoparticles and hydrogel will further extend biological half-life of the antigen. Moreover, the 4


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sustained release of GM-CSF from the hydrogel can recruit abundant DCs, and these resident DCs may provide a great opportunity for antigen intake and presentation to T cells in lymph nodes. These enhanced phenomena of DC recruitment and of the sustained antigen release should further enhance the immune response (Figure 1). Thus, the injectable hydrogel coencapsulating GM-CSF and OVA-NPs has a significant potential for immunotherapeutic clinical application.

Figure 1 2. Materials and methods 2.1. Materials Ovalbumin (OVA) was purchased from Sigma-Aldrich, Inc. (USA). The fetal bovine serum (FBS) was supplied by Grand Island Gibco, Inc. (USA). Grand Island Life Technologies (USA) provided LysoTracker probes. IL-4 and Recombinant mouse GM-CSF were all purchased from Rocky Hill Peprotech (USA). Mouse cytokine ELISA kits and fluorescence-labeled antibodies against CD3e, CD40, CD80, CD86, CCR7, and CD11c were supplied by San Diego eBioscience (USA). Genipin (with a purity of 98%) and chitosan (with a Mw from 5,000 Da to 8,000 Da and a degree of deacetylation of 90%) was acquired from Xi’an Plant Bio-engineering Co., Ltd. (China) and Zhejiang Golden-Shell Biochemical Co., Ltd. China), respectively. All other chemical reagents were provided with a highest quality of commercial availability. 2.2. Preparation and characterization of the OVA-NP hydrogel system 5



Chitosan-modified genipin–cross-linked OVA-NPs were prepared via a cross-linking reaction and surface modification according to previous report28. The PCL-PEG-PCL copolymer (Mn=3011) was synthesized according to our previous report. 26. Then, the OVA-NP hydrogel system (GEL/OVA-NPs) was simply prepared by the mixing of OVA-NPs and the PCL-PEG-PCL copolymer solution Morphology of OVA-NPs was examined by atomic force microscopy (AFM; Multi Mode8, Veeco Instruments, USA). The fluorescent spectrum of OVA-NPs was scanned by a spectrophotometer (Varioskan TM Flash, Thermo Fisher Scientific, USA). Rheological properties of the OVA-NP hydrogel system were tested using a rheometer (MCR 302, Anton Paar Instrument, Austria). The temperature response of the hydrogel loaded with OVA-NPs was recorded over the range of 10–60 °C at a frequency of 1 Hz and 1% strain. Fluorescence imaging of the OVA-NP hydrogel system in vitro was performed by an imaging system (Maestro, CRI, USA) 28. 2.3. Cells and mice C57BL/6 mice (Male, 6 weeks old) were chosen to perform the in vivo experiments under proper conditions at Peking Union Medical College (PUMC). All animal procedures were approved by the PUMC IACUC. DCs were generated from C57BL/6 mice according to a previously described method29. Activation and maturation of BMDCs was evaluated with free OVA, OVA-NPs, or GEL/OVA-NPs (OVA: 20 µg/mL). The DCs were harvested after incubation for 24 h and stained with a different antibody for 30 min at 4 °C shielded from light. Then, the DCs were washed twice, and the amounts of these surface markers on CD11c+ DCs were monitored by flow cytometry (BD Biosciences, San Jose, CA). Each sample was analyzed in triplicate. 2.4. In vivo tracking of labeled nanoparticles C57BL/6 mice were subcutaneously injected with free OVA, OVA-NPs, or GEL/OVA-NPs (50 mg of OVA) into the back with hair removed (3 mice/group). The fluorescent signals of genipin could be used to track OVA-NPs and GEL/OVA-NPs, whereas free OVA was monitored via additional labeling (with Rhodamine B). The in vivo fate of GEL/OVA-NPs and OVA-NPs was studied at different time points on an imaging system (Maestro 2, CRI, USA) (Ex:595nm, exposure time: 200 ms). Migration of maturated DCs to a lymph node was examined simultaneously by the in vivo imaging. 2.5. Expression of costimulatory molecules and CCR7 on DCs in draining lymph nodes 6


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Animals were subcutaneously injected with free OVA, OVA-NPs, or GEL/OVA-NPs (50 mg of OVA) into the back with hair removed (6 mice/group). The draining lymph nodes were harvested at 3 days after immunization. A single-cell suspension was incubated with an anti-mouse CD16/CD32 Fc blocking antibody. The expression on CD11c+ cells was determined by flow cytometry after stained with an antibody against a mouse protein. 2.6. Release characteristics of GM-CSF from the hydrogel in vivo by fluorescence imaging To investigate the release characteristics in vivo of GM-CSF from the hydrogel, CRI fluorescence imaging system (Maestro 2，CRI, USA) was applied to monitor Rhodamine B labeled GM-CSF when loaded in gels until day 14 after injection. GEL+GM-CSF was subcutaneously injected into the back of mice (n=3) and the fluorescence signal was obtained with the excitation wavelength of 523nm by the Maestro software. Meanwhile, the fluorescence signal of GM-CSF at different time with degradation was calculated as the percentage of the original fluorescent intensity on day 0. 2.7. In vivo DC recruitment by the GM-CSF–loaded hydrogel A GM-CSF–loaded hydrogel (GM-CSF: 5 µg ) was subcutaneously injected using an empty hydrogel as a control (6 mice/group). Mice immunized with PBS served as controls. At 3, 7, and 14 days after injection, single-cell suspensions from a hydrogel or the surrounding tissues were incubated with an anti-mouse CD16/CD32 Fc blocking antibody for blockage of FcR in DCs, then stained with anti-CD11c and anti-CCR7 antibodies. CD11c+ cells were counted to quantify recruited DCs by flow cytometry. 2.8. The immune response induced by the OVA-NPs/GM hydrogel system Mice were immunized 3 times with PBS (control), OVA-NPs, OVA-NPs/GM, GEL/OVA-NPs, or GEL/OVA-NPs/GM (each containing 50 µg OVA) by subcutaneous injection at 1-week intervals (6 mice/group). The draining lymph nodes were collected to analyze the proportion of CD8+ T cells 3 days after the first injection. Splenocytes (2 × 106 cells) were harvested 14 days after the last immunization. Then they were restimulated with 50 µg/mL OVA for 72 h to detect interferon (IFN)-γ secretion by an ELISA (eBioscience). For a cytotoxic T lymphocyte assay, E.G7-OVA target cells (5 × 103 cells/well) treated with mitomycin were added to splenocytes from each experimental group and cultured at a proper effector:target (E:T) ratio. After incubation for 4 h, the LDH leakage level in the suspensions 7



was detected to record the percentage of specific lysis of target cells by effector cells 28 . 2.9. Statistical analysis The mean ± standard deviation (SD) was applied to indicate the results. Significance of differences was evaluated by ANOVA or Student’s t test and statistically significant were considered when P value was less than 0.05. 3. Results and discussion 3.1. The design and characterization of the OVA-NP hydrogel system Prolonging the residence time of vaccines and achieving a controllable antigen release via application of carriers will significantly improve vaccination efficacy because vaccine carriers can not only protect an antigen from enzymatic degradation but also prevent the initial burst of the antigen release during the process of stimulating the immune system. The functional OVA self–cross-linked nanoparticles may promote DC activation and maturation in vitro and next trigger an antigen-specific immune response in vivo. Herein, it was expected that the PCL-PEG-PCL hydrogel would further increase the immune response through the extension of antigen exposure because the antigen would undergo a two-stage delivery from the hydrogel and nanomaterial. The OVA-NPs combined with the hydrogel may have the advantage of dual carriers as compared to a single carrier.

Figure 2 8


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The surface morphology of OVA-NPs was studied by AFM, which indicated that the diameter of OVA-NPs was approximately 150 ± 4.75 nm (Figure 2A). Analysis of fluorescent spectra of OVA-NPs confirmed the maximum emission at 615 nm at an excitation wavelength of 596 nm (Figure 2B). To quantify the fluorescence of GEL/OVA-NPs, an acceptable fluorescence signal was acquired at the excitation wavelength of 595 nm (Figure.2C). Next, the thermosensitive behavior of the OVA-NP hydrogel system was investigated. The OVA-NP hydrogel system was a flowable sol at 25 °C, then formed a stable hydrogel as the temperature increased to 37 °C (Figure 2D). After that, the rheological analysis evaluated the sol–gel–sol transition of GEL/OVA-NPs during a temperature increase from 10 to 60 °C (Figure 2E). The loss modulus (G'') is higher than storage modulus (G') at room temperature, meaning that the system is a sol before the phase transition. Later, G' rapidly increased and exceeded storage modulus; this finding indicated that the nanoparticle–hydrogel system was stabilized in a gel state at phisical temperature. The rheological assay of GEL/OVA-NPs was consistent with the sol–gel transition during examination in a tube. This result proved the injectability of the OVA-NP hydrogel system. 3.2. In vivo tracking of labeled NPs To reveal the effects of the dual controlled-release system, the OVA-NP–loaded PCL-PEG-PCL hydrogel, OVA-NPs, or free OVA were injected subcutaneously. The fluorescence of genipin was used to track the nanoparticles at the injection site or in a lymph node instead of any additional fluorescent labeling. Free OVA was labeled with rhodamine B owing to the lack of cross-linking with genipin. The changes in fluorescence intensity reflected the process of the controlled release of OVA (Figure 3A). The fluorescence intensity rapidly decayed in the free-OVA group, until finally no fluorescent signal could be detected on day 8. In contrast, the fluorescent signal of OVA-NPs was still present even at 13 days after injection in the groups treated with OVA-NPs or GEL/OVA-NPs. Furthermore, the fluorescent signals of GEL/OVA-NPs decreased continuously at a slower rate than those of OVA-NPs. By quantitative analysis in different groups, the release process of OVA was revealed more clearly (Figure 3B). Approximately 84% or 63% of the fluorescence stayed in groups GEL/OVA-NPs and OVA-NPs at 24 h respectively, whereas only 28% remained in the free-OVA group. When the fluorescent signal was not visible in the free OVA group on day 8, 41% and 25% of that remained in groups GEL/OVA-NPs and OVA-NPs, respectively. What is more, on day 13, the fluorescent signal in 9



group OVA-NPs became weak (11% of the original fluorescence), but a relatively strong fluorescent signal could still be detected (30%) at the injection site in GEL/OVA-NP–treated mice. These data indicated that self–cross-linked OVA-NPs protected OVA from a quick release because of the extended process of the sustained release, and introduction of the hydrogel further prolonged the process.

Figure 3 The activated DCs migration into lymph is crucial for the subsequent T-cell responses. Fluorescence of draining lymph nodes revealed the antigen depot effects (Figure 3C). Significantly strong fluorescence could be detected at 24 h in groups GEL/OVA-NPs and OVA-NPs. By comparison, no fluorescent signal was observed in a draining lymph node in the free-OVA group. They suggested that the dual function of the nanoparticles and hydrogel ensured the antigen persistence at the injection site, and the dual delivery system of OVA-NPs combined with the hydrogel may hold greater promise for the vaccine delivery. 3.3. Activation of DCs

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DCs are some of the most important classes of APCs, and their main function is the recognition and capture of antigens, which are key steps in innate and adaptive immunity30,31. Immature DCs take up antigens, process them into peptides, and then transform into mature DCs. The latter with a high T-cell–stimulatory activity express upregulated levels of CD40, CD80, CD86, and chemokine receptor 7 (CCR7)32. Therefore, to investigate the immunological influence of GEL/OVA-NPs, upregulation of these costimulatory molecules as markers of DCs was measured by flow cytometry. A significant high expression of costimulatory molecules was observed in DCs stimulated with GEL/OVA-NPs after 24 h of culturing (Figure 4A). It was found that the difference in the percentage of CD80+ DCs was remarkable, the average percentage of CD80+ DCs in GEL/OVA-NPs stimulated group was 76.77%, significantly higher than OVA-NPs(67.12%) or free OVA(57.63%) treated ones. Nevertheless, CD40 expression did not generate marked difference between groups GEL/OVA-NPs and OVA-NPs. It is worth noting that significant upregulation of CCR7 was detected in groups GEL/OVA-NPs(50.26%) or OVA-NPs(47.79%) compared with groups free OVA(19.94%) and PBS groups(19.12%). CCR7 can mediate migration of DCs to regional lymph nodes and perform an important function in the activation of B and T lymphocytes33. The promotion of CCR7 expression in DCs indicated that antigen nanoparticles were more available for stimulating the chemotaxis characteristics of DCs as compared with the soluble antigen.

Figure 4 Fluorescence imaging in vivo revealed that GEL/OVA-NPs prolonged the antigen retention and effectively transported the antigen to the draining lymph nodes. In our previous study, it has also been demonstrated that OVA-NPs induce a stronger immune response than soluble OVA28. Herein, whether the dual delivery system based on a hydrogel with nanoparticles can further strengthen an immune 11



response in vivo was the key issue to clarify. First, the maturation of DCs in lymph nodes must be confirmed. For this purpose, the mice were immunized with different OVA groups, while the PBS group served as a control. Next, the expression of the costimulatory molecules was detected in the DCs of the draining lymph nodes. As depicted in Figure 4B, a similar result was observed relative to the detection of in vitro DC maturation, and the highest level of maturation of DCs was induced in GEL/OVA-NPs. All the results indicated that the combined delivery system (the nanoparticles combined with the hydrogel) had stronger in vitro and in vivo immunostimulatory effects than antigen nanoparticles. 3.4. In vivo recruitment of DCs In contrast to traditional biomaterials, degradable thermosensitive hydrogels are usually used as drug delivery systems owing to their minimal invasiveness and good injectability34,35. In recent years, thermosensitive polymeric materials were used as intranasal vaccine carriers to improve vaccination efficacy by prolonging the sustained release of a vaccine and by enhancing uptake efficiency or controlling the release of vaccines36,37. On the other hand, the major function of thermosensitive hydrogels is to protect antigens from the initial burst release at the injection site. Besides retention, many other factors should also be considered for triggering a more effective immune response like increasing the number of DCs at the injection site. DCs are closely related to the regulation of T- and B-cell immunity owing to their remarkable function to process antigens38. Although the application of vaccine carriers has significantly improved the effect on the immune response, the DCs deficiency at the injection site is still another limitation for priming the immune response.

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Figure 5 GM-CSF plays an important role in inducing DC differentiation, proliferation, migration and cytokines secreation in vitro39. Especially, many studies have shown that DCs treated with GM-CSF may improve the activation of T cells both in vitro and in vivo 40,41. In our study, to enhance antigen uptake by DCs and to maximally enrich the area around the injection site with host DCs, biocompatible injectable thermosensitive hydrogels were introduced. A hydrogel served as a physical platform, which not only provided a microenvironment to recruit DCs at injection site but also facilitated the controlled release of GM-CSF. It was hypothesized that the sustained release of GM-CSF would recruit a significant number of DCs for the following antigen intake and presentation process. The fluorescence imaging of GEL+GM-CSF reflected the in-situ release character of GM-CSF (Figure 5). The visible GM-CSF release was tracked and monitored by fluorescence imaging for 14 days and the release of GM-CSF was accelerated gradually (Figure 5A).. Also, the fluorescent signal of GM-CSF was analyzed quantitatively by measuring relative fluorescent intensity (Figure 5B). The fluorescent signal of GM-CSF had a decay of ~13% within 24h and the signal retained at ~ 45% at day 13



5. With continued tracking over 11 days, the signal of GM-CSF decreased to less than 5 % compared to the original fluorescence. The remnant GM-CSF in the hydrogel decreased gradually with a sustained release process. The continued release of GM-CSF can ensure the effective DCs recruitment.

Figure 6 As seen in Figure 6A and C, CD11c+ DCs increased with a significant rate in the implanted hydrogels or surrounding tissue of GM-CSF hydrogel–injected mice compared to the empty hydrogel group. Remarkably, it was observed that GM-CSF recruited the highest number of DCs on day 7 after injection. The percentage of DCs in implanted hydrogels in the GEL+GM-CSF group (17%) significantly exceeded that in the empty-GEL group (6%). Then, the number of DCs modestly decreased at 14 days. The same phenomenon was also observed on day 7 in the tissue surrounding the injection site. Besides, it was found that the number of DCs was higher in the tissue peripheral toward the injected hydrogel (~7% at 7 days and ~6% at 14 days) than inside the hydrogel (~2% at 7 days and ~1% at 14 days) in the empty-GEL–injected group. By contrast, in the GEL+GM-CSF–injected group, the result was opposite: the number of recruited cells inside the implanted hydrogel was higher. This finding indicated that GM-CSF made it easier for DCs to infiltrate the interior of the implanted 14


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hydrogels. In addition, the GM-CSF hydrogel not only recruited DCs but also significantly stimulated DCs to mature. The percentage of CCR7+ CD11c+ DCs increased remarkably either inside the hydrogel or in the surrounding tissue (Figure 6B and 5D). Unlike DC recruitment, DC maturation was the highest on the 14th day both inside the hydrogel (~47%) and in the surrounding tissues (~35%). All these data suggested that the hydrogel containing GM-CSF may lead to recruitment and maturation of DCs at the hydrogel injection site via a sustained release of GM-CSF.

Figure 7 Owing to the increased DCs amount after recruitment, more DCs will migrate to lymph nodes and present antigen to B- and T-cell cells in the lymph nodes for the subsequent immune response. As presented in Figure 7A, the number of DCs significantly increased in the lymph nodes after injection at 3 and 7 days in the GEL+GM-CSF–injected group; the highest count of DCs in the lymph nodes was observed on day 3. Aside from the DC count in lymph nodes, the GM-CSF hydrogel induced the highest DC maturation on days 3 and 7 in vivo (Figure 7B). Moreover, it was found that the recruitment by the GM-CSF hydrogel peaked after 7 days in comparison with the number of DCs in the implanted hydrogel and the tissue surrounding the hydrogel. This result is consistent with the data from another study25, where a two-step hybrid strategy was devised. The first step was the recruitment of DCs, then viral or nonviral vectors carrying the antigen were introduced. Herein, a dual-release hydrogel system was prepared to simultaneously carry antigen nanoparticles and stimulating factor GM-CSF. The enhanced migratory behavior of recruited DCs to lymph nodes has been proved. All these data revealed the remarkable potential of the hydrogel 15



containing GM-CSF to recruit DCs and promote their maturation. Next, whether GEL+ GM-CSF can enhance an antigen-specific immune response will be discussed. 3.5. The GM-CSF hydrogel combined with chitosan-modified OVA-NPs enhanced an antigen-specific immune response

Figure 8 As known that CD8+ T cells are crucial for cancer immunotherapy. Mature DCs will migrate to draining lymph nodes after acquiring and processing pathogen antigens, where they present antigen epitope peptide to CD8+ T cells with specific receptors. Subsequently, T-cells are activated and rapidly proliferated and differentiated, the resultant effector CD8+ T cells will clear pathogens by killing infected cells or releasing cytotoxins such as IFN-γ or granzymes42,43. Our previous study suggests that OVA-NPs can trigger an antigen-specific immune response as compared to soluble OVA28. Herein, it is reasonable to suppose that PCL-PEG-PCL hydrogel as a dual carrier may further improve vaccination efficacy by controlling the sustained release of the vaccine or by enhancing uptake efficiency. Meanwhile, the recruitment of DCs by GM-CSF loaded onto the hydrogel helped a larger number of DCs to take up and present the antigen in vivo. Consequently, a stronger immune response could be 16


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induced.

Figure 9 The activity of CD8+ T cells can be reflected by the cell percentage and the specific cytokines expression such as IFN-γ. The PCL-PEG-PCL hydrogel further strengthened the T-cell activation in vivo (Figure 8) on the basis of our another study where the positive effect of OVA-NPs on CD8+ T cells had been verified

28

. The proportion of CD8+ T cells from GEL/OVA-NP–treated mice (23.5%)

increased significantly compared with that from OVA-NP–treated mice (19.2%). No significant difference was observed between groups GEL/OVA-NPs and OVA-NPs/GM. Furthermore, the highest proportion of CD8+ T cells in GEL/OVA-NPs/GM group implied that the GM-CSF from the hydrogel played a key role for recruiting DCs. Similarly, the highest expression of IFN-γ was induced in group GEL/OVA-NPs/GM (Figure 9). IFN-γ is closely related with tumor control and can directly improve the immunogenicity of tumor cells44. It can also enhance the sensitivity of CTLs to pathogens, which improves the ability of CTLs to eliminate pathogens45. Therefore, the improved expression of IFN-γ induced by GEL/OVA-NPs/GM will be useful for tumor immunotherapy.

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Figure 10 Primary CTL responses participate in the inhibition of tumor growth. CTLs mainly derive from CD8+ T cells and kill cancer cells via release of cytotoxins such as perforin and granzymes. To confirm whether GEL/OVA-NPs/GM can induce OVA-specific CTLs in vivo, splenocytes from different treated mice were stimulated with OVA and then incubated with E.G7-OVA cells. After that, the CTL-mediated cytotoxicity was measured. As depicted in Figure 10, the splenocytes from the GEL/OVA-NPs/GM group manifested stronger CTL responses to E.G7-OVA cells at an E/T ratio of 20:1 compared to that from GEL/OVA-NPs and OVA-NPs/GM-CSF treared groups. Our results suggested that GEL/OVA-NPs/GM induced the strongest immune response according to such parameters as the CD8+ T-cell proportion, IFN-γ secretion, and the CTL response. Nevertheless, only modestly improved immunity was observed in the OVA-NPs/GM-CSF group compared to group GEL/OVA-NPs. This finding was probably due to the lack of a sustained GM-CSF release from the hydrogel. Because a stable and enduring microenvironment for DC recruitment was lacking, it is difficult to collect a large number of cells because of the quick dissipation of GM-CSF from the injection site; this effect directly influenced the subsequent process of immunity development. Overall, the dual delivery system of the GM-CSF hydrogel combined with OVA-NPs induced a stronger immune response including DC maturation and a CD8+ T response, which may benefit from the recruitment of DCs, from an increase in antigen residence time, and from the controllable antigen release owing to the combined effect of the hydrogel and nanoparticles. In comparison with the two-step hybrid strategy, the combined hydrogel system consisting of nanoparticles and an adjuvant can enhance the specific immune response through the recruitment of DCs and the sustained antigen release without the second injection.

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4. Conclusions In summary, an injectable hydrogel coencapsulating GM-CSF and OVA-NPs was developed here to enhance antigen uptake efficiency. The combined system consisting of nanoparticles and the hydrogel can generate consecutive recruitment of DCs and promote DC maturation through the sustain release of GM-CSF. By enriching the hydrogel and tissues with DCs, we further improved antigen uptake efficiency, and then the CD8+ T-cell response was strengthened. The combined system consisting of the nanoparticles and hydrogel may represent a new alternative method for the combined application of a vaccine and adjuvant for induction of antitumor immunity.

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

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Figure legends

Figure 1. Schematic illustration of the in vivo processing of the injectable hydrogels coencapsulating GM-CSF and OVA-NPs. (The sustainable release of GM-CSF from the injectable hydrogel recruits host dendritic cells [DCs] to the injection site and surrounding tissues. The large number and percentage of DCs enhances antigen uptake efficiency for the immune response in vivo.) Figure 2. Characterization of the OVA-NP hydrogel system. (A) The surface morphology of OVA-NPs according to AFM; (B) the fluorescence spectrum of OVA-NPs; (C) in vitro fluorescence imaging of the OVA-NP hydrogel system; (D) thermosensitive behavior of the OVA-NP hydrogel system according to the tube-inversion method; (E) rheological behavior of the OVA-NP hydrogel system. Figure 3. In vivo visualization of free OVA, OVA-NPs and GEL/OVA-NPs in the injection site and draining lymph nodes. (A) Fluorescence imaging for in vivo tracking of OVA antigen on the basis of the fluorescent signals of genipin or Rhodamine B, and (B) quantitative analysis of fluorescence intensity. The fluorescent signal at the injection site is expressed as a percentage of the initial signal. Statistical analysis was performed; n = 3 mice per group. (C) Antigen transport into draining lymph nodes. Figure 4. DC maturation induced by different nanoparticles in vitro (A) and in vivo (B). Statistical analysis of the results on CD40, CD80, CD86, and CCR7 that were obtained by flow cytometry. Data are expressed as the mean ± SD (n = 6; *p < 0.05) Figure 5. Release characteristics of GM-CSF from the hydrogel in vivo by fluorescence imaging. (A) Fluorescence imaging for in vivo tracking of release. (B) quantitative analysis of fluorescence intensity. The fluorescent signal at the injection site is expressed as a percentage of the initial signal. Statistical analysis was performed; n = 3 mice per group. Figure 6. In vivo recruitment of DCs in response to the hydrogels loaded with GM-CSF. Flow cytometric analysis of the number of DCs (CD11c+) recruited by a GM-CSF–loaded hydrogel and DC maturation (CCR7+CD11c+) in the implanted hydrogels (A,B) or surrounding tissue (C,D) after injection of an empty hydrogel or GM-hydrogel. The statistical significance was analyzed by Student’s t test; *p < 0.05. Figure 7. The DC number (A) and maturation (B) in the draining lymph nodes after injection of the 23



empty hydrogel or GM-hydrogel on days 3, 7, and 14 after injection. The statistical significance was analyzed by Student’s t test; *p < 0.05. Figure 8. Typical flow cytometry profiles (phenotype analysis) of CD3+CD8+ T cells in the draining lymph nodes. Data are expressed as the mean ± SD (n = 6; *p < 0.05). Figure 9. IFN-γ expression was measured by an ELISA; after that, splenocytes were restimulated ex vivo with OVA (50 µg/mL). Data are representative of three independent experiments, *p < 0.05. Figure 10. The CTL response was measured by incubating restimulated splenocytes with E.G7-OVA cells. The activity is represented by the mean percentage of specifically lysed tumor cells. E:T means effector:target. Data are expressed as the mean ± SD (n = 6; *p < 0.05).

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A graphic for the Table of Contents

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Injectable Hydrogels Coencapsulating Granulocyte-Macrophage

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