Subcutaneous vaccination using injectable biodegradable hydrogels for long-term immune response

Nanomedicine: Nanotechnology, Biology, and Medicine 21 (2019) 102056

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Subcutaneous vaccination using injectable biodegradable hydrogels for long-term immune response Ashlynn L.Z. Lee a,⁎, Chuan Yang a , Shujun Gao a, 1 , James L. Hedrick b,⁎, Yi Yan Yang a,⁎ a

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore b IBM Almaden Research Center, 650 Harry Road, San Jose, CA 95120, USA Revised 27 June 2019

Abstract Prolonged vaccine release enables gradual immunostimulation, providing long-term immunity. Herein, Vitamin E-PEG-Vitamin E triblock ‘ABA’ hydrogel, which is formed through physical cross-linking of flower-shaped micelles and can reside in vivo for N17 weeks, was employed for delivery of cancer preventive vaccines to provide sustained anticancer immunity. Mice vaccinated with hydrogel formulations produced a significantly higher quantity of antibodies compared to solution formulations. OVA was used to study EG.7-OVA tumor rejection in vaccinated mice. Among all formulations, OVA-loaded hydrogel containing aluminum-based adjuvant had the best therapeutic outcome, and only 2/10 mice developed solid tumors with significantly smaller tumor size. Moreover, no adverse effect on liver and kidney was detected with the hydrogel formulation. In a lymphoma metastasis mouse model, vaccination with the OVA-loaded hydrogel and adjuvant resulted in increased survival (66.7%) compared to other formulations (12.5-50%) over 100 days. This hydrogel is a promising formulation for sustained delivery of vaccines. © 2019 Elsevier Inc. All rights reserved. Key words: Injectable hydrogel; Immunization; Vaccine; Ovalbumin; Hepatitis B

Conventional vaccination strategies that are intended to provide antibody-mediated prophylaxis typically require repeated dosing administered over several months to evoke adequate immune response against the targeted disease. One major pitfall of such regimen of multiple vaccine doses is non-compliance to the vaccination schedule due to poor patients’ access to healthcare services and the cost involved in such vaccination program. A well-studied example is the human papillomavirus (HPV) vaccine that has been reported to give effective prevention against HPVinduced cervical intraepithelial neoplasia. However, complete vaccination would require 3 doses, spanning across 6 months. Acknowledgements: This work is funded by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore), and IBM Almaden Research Center. The authors have no competing conflicts of interest related to this work. ⁎Corresponding authors. E-mail addresses: [email protected], (A.L.Z. Lee), [email protected], (J.L. Hedrick), [email protected]. (Y.Y. Yang). 1 Current address: NanoBio Lab, 31 Biopolis Way, #09-01 The Nanos, Singapore 138669. https://doi.org/10.1016/j.nano.2019.102056 1549-9634/© 2019 Elsevier Inc. All rights reserved.

To reduce the need to administer multiple vaccine doses for improved patient compliance and to boost vaccine completion rates, research efforts have been devoted to developing sustained vaccine delivery systems. Prolonged vaccine release allows for gradual immunostimulation, which can reduce the requirement of multiple vaccine shots. Such formulations can also provide longterm immunity with buffered impact on the immune system of the vaccinated individuals. Materials such as particulate delivery systems including liposomes 1, polymeric nanoparticles 2 and microspheres 3 as well as hydrogels 4 have been evaluated for long term vaccine delivery. Taking into account that vaccines have highly intricate structures that are delicate and essential for their activity, particulate formulations are typically less favorable since the antigens are exposed to stress conditions during entrapment processes, including increased temperatures, forceful perturbation and contact with organic solvents or extreme pH 5. Pertaining to hydrogel delivery systems, chitosan has been frequently used 6,7,8, conceivably due to its abundance in the natural environment and inherent immune stimulating activity 9. However, there are major hurdles to cross in attaining clinical approval of chitosan-based hydrogel as a drug delivery matrix due to its potential to cause allergic reactions and hypersensitivity 10.

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Figure 1. Schematic showing the formation of vaccine loaded-hydrogels.

Studies on peptide hydrogels formed either via self-assembly 11 or enzyme-catalysis 12 have been reported in spite of drawbacks such as high material costs and undefined immunogenicity of synthetic peptides 13. On the contrary, polymeric hydrogels are attractive because of the versatility in synthetic strategies to create a broad range of biocompatible polymers. Thermosensitive hydrogels based on poly(lactide-co-glycolide), polylactide or polycaprolactone 14 have been studied but the main drawbacks of these systems include acidic degradation products 15 and inflammatory complications 16. Furthermore, high concentration (17% w/v) and hydrophobic content of the polymers were required to form gels. Hydrogels prepared using acrylic acid polymers have also been used 17, but are deemed unsuitable to be applied as an in vivo drug delivery matrix as it is non-biodegradable for polymers with molecular weights N700 Da 18. Recently, a vaccine delivery matrix developed from a thermogelling pentablock copolymer comprising of Pluronics, poly(diethylaminoethylmethacrylate (PDEAEM) blocks flanked by tertiary amine groups has been reported 19. The gel was almost completely degraded in vivo by 47 days (b7 weeks) post-injection and although a higher amount of antibody was produced with the use of this delivery system at 6 weeks postimmunization, immune response at longer time points was not investigated. Previously, we reported a novel approach in delivering protein therapeutics using ‘ABA’ triblock copolymers of vitamin E and vitamin D-functionalized polycarbonate and poly(ethylene glycol) (PEG) to form hydrogels via physically cross-linkage, which enabled sustained delivery of the proteins and delayed cancer progression in animal models 20,21. Very recently, with the aim to produce hydrogels with greater stability and slower rate of biodegradation, we introduced a carbamate bond to the block junction of ‘ABA’ copolymers through a facile synthesis method, which formed gels with a greater in vivo stability, offering more sustained release of proteins 22. In this study, this hydrogel was used to deliver ovalbumin (OVA) as a model antigen Hepatitis B vaccine to provide sustained antigen release and superior immune response. Effective delivery of cancer preventive vaccine will allow protective immunity against cancer development. This ‘ABA’ triblock copolymer arranges itself to form flower-like micelles with PEG block being exposed to the aqueous environment. At higher polymer concentrations, bridging units are formed between the micelles through the insertion into the hydrophobic cores of neighboring micelles,

which in turn results in the formation of hydrogel network. OVA was entrapped into the hydrogel network during the gel formation process (Figure 1). Viscoelastic properties of the hydrogel and OVA release profiles were characterized. In vitro biocompatibility was investigated using NIH3T3 murine fibroblast cell line, and the in vivo biocompatibility and biodegradability of the hydrogel was studied via histological examination. The efficiency of antibody production and EG.7-OVA tumor rejection in mice that were vaccinated with different OVA formulations was evaluated.

Methods Polymer synthesis Vitamin E-functionalized carbamate-based polymer VEPEG-VE was synthesized using PEG of 20 kDa according to the method described previously 22. Methods on rheological and scanning electron microscopic (SEM) characterization, in vitro and in vivo release of vaccineloaded hydrogels, cytotoxicity, in vivo biocompatibility and gel degradation, detection of mouse anti-OVA IgG1 and anti-HBsAg IgG in blood plasma via ELISA, cytotoxic T lymphocyte induction and assay following vaccination with OVA-loaded hydrogel and T cell proliferation assay following vaccination with HBsAg-loaded hydrogel are described in Supporting Information. Biodistribution of fluorescently-labeled OVA delivered using different formulations Female C57/BL6 mice (6-8 weeks old, weighing between 18 and 20 g) were used for this experiment. The mice were randomly assigned into 4 groups and injected subcutaneously (s. c.) with 200 μg of Alexa Fluor750-labeled OVA in different formulations: (1) hydrogel, (2) hydrogel with the adjuvant Alum, (3) solution and (4) solution with Alum. All animal experiments were performed in accordance with the approved protocol from the Institutional Animal Care and Use Committee (IACUC) at the Biological Resource Centre of Singapore. Anesthetized animals were placed on an animal plate maintained at 37 oC. The near-infrared fluorescence was imaged using the ICG filter pairs (exposure time: 1 s). Scans were performed at 0, 1, 4, 7, 10, 16

A.L.Z. Lee et al / Nanomedicine: Nanotechnology, Biology, and Medicine 21 (2019) 102056

and 21 days post administration using IVIS (Caliper Life Science, U.S.A.). At 7 and 21 days, organs including lymph nodes, heart, spleen, lungs, kidneys, liver and skin were harvested for imaging under the same conditions. In vivo tumor challenge Subcutaneous tumor model Female C57BL/6 mice weighing between 20 and 25 g were injected s.c. with 150 μL of hydrogel or solution containing 200 μg of OVA. In some formulations, Alum was mixed with OVA in the volume ratio of 1:3. On Day 14 or 28 post-immunization, murine lymphoma E.G7-OVA and EL-4 were propagated in the mice by subcutaneous inoculation of 5 × 10 6 in vitro-cultured cells in 200 μL of cell suspension at equal volume with Geltrex (Thermofisher, USA)]. Subsequently, the tumors were measured at 14 days from the day when the tumor cells were injected into the mice by calipers in two orthogonal diameters. The tumor volumes were calculated as 0.5×L×W 2, where L and W are the major and minor diameters, respectively. Data reported are average ± standard error of the mean values. Fisher’s Exact test was used to compare tumor incidence. Student's t test was used to compare the tumor volume or weight between experimental groups. P ≤ 0.05 was reflected as statistically significant. Metastasis model Female C57BL/6 mice weighing between 20 to25 g were injected s.c. with 150 μL of hydrogel or solution containing 200 μg of OVA. In some formulations, Alum was mixed with OVA in the volume ratio of 1:3. On Day 28 (Day 0), 57 (Day 29) and 91 (Day 63) post-immunization, murine lymphoma E.G7-OVA were administered into the mice by intravenous inoculation of 2 × 10 6, 1 × 10 7 and 1 × 10 7 in vitro-cultured cells in 200 μL of cell suspension on respective days. Subsequently, the survival of the mice was recorded. The data obtained was analyzed via Student’s t test and P ≤ .05 was reflected as statistically significant. Results

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Table 1 Mechanical properties of VE-PEG-VE hydrogels with different OVA concentrations with and without Alum at 25°C. Sample

Ovalbumin (g/L)

Alum

G′ (Pa)

G″ (Pa)

1 2 3 4 5 6

0 0.33 1.33 0 0.33 1.33

0 0 0 + + +

902 ± 47 633 ± 31 688 ± 20 2932 ± 30 1111 ± 84 1267 ± 59

395 366 201 117 580 479

± 48 ± 70 ± 19 ± 15 ± 14 ± 40

increased mechanical strength. For example, G′ was ~2000 Pa (at 6-10 rad/s), while in the presence of Alum, the mechanical strength was increased to ~3500 Pa (Figure S1, A). Both OVA- and HBsAg-loaded hydrogels exhibit shearthinning properties regardless of the presence of Alum (Figure S1, B, Figure S2, A). These findings show that Alum resulted in the increase of both the mechanical strength and viscosity of the hydrogels. To serve as an injectable drug delivery carrier, it is also necessary for the disrupted hydrogel network to recover its mechanical strength after being exposed to shear stress during/after the injection process. A dynamic step strain amplitude test was performed to study this property. The test shows that at a low strain, the G′ of the OVA-loaded hydrogel without Alum was ~650 Pa (γ = 2%) (Figure S2, B). At G′ N G″, the gel mainly displayed a solid-like behavior. At a high strain (γ = 100%), the G′ value was immediately reduced by more than 30 times to ~20 Pa and the G′ became lower than G″, showing that the gel became more liquid-like. After 200 s of exposure to high strain, the strain was decreased to a low value (γ = 2%) and the G′ values gradually increased to be higher than G″, showing the recovery to a more solid-like behavior 23. A similar trend was also observed for OVAloaded hydrogel containing Alum (Figure S2, C) and HBsAgloaded hydrogels (Figure S1, C). This reversibility of mechanical strength of the hydrogel is advantageous for use as an injectable matrix for the sustained release of vaccine.

Physical properties of hydrogels

SEM imaging of antigen-loaded hydrogels

Hydrogels were formed via the formation of physical crosslinkage of VE-PEG-VE triblock copolymers at concentration of 4 wt.% (Figure 1). Cancer preventive vaccines were incorporated into the hydrogel matrix during the gelation process. Viscoelastic properties of the hydrogels containing different concentrations of antigens with and without the presence of Alum was studied. While the blank hydrogel has storage modulus G′ value of 902 Pa, the incorporation of OVA in the absence of Alum results in significantly lower storage moduli values (Table 1). This suggests that the presence of OVA within the polymer network might have disrupted the intermolecular interactions between the polymer chains, thereby resulting in lower gel stiffness. Interestingly, the incorporation of Alum had the opposite effect, where the storage moduli values were significantly increased. Hydrogel containing the adjuvant without OVA had high G′ of 2932 Pa while the addition of OVA reduced the gel stiffness to slightly above 1000 Pa. Similarly, the presence of Alum in HBsAg-load hydrogel

To study the cross-section morphology of the gels, samples were lyophilized and examined through SEM imaging. From Figure 2 and Figure S3, it was observed that the hydrogels exhibited porous structure and the porosity was decreased by addition of antigens and Alum. In particular, the inclusion of Alum resulted in more densely packed hydrogels. This finding is in agreement with the mechanical strength data (Table 1, Figure S1, A). No aggregation or clustering was seen for all samples, showing homogenous mixing of the antigens, Alum and the polymer. In vitro biocompatibility of hydrogels To study the in vitro biocompatibility of hydrogel (4 wt.%), NIH3T3 was used as a model cell line, and incubated with various hydrogel formulations (Figure S4). Incubating the cells with the blank hydrogel and those containing OVA with and without Alum for 24 h showed greater than 90% cell viability, suggesting excellent cytocompatibility.

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Figure 2. SEM images of cryo-fixed hydrogel containing (A) 0.33 g/L OVA, -Alum, (B) 1.33 g/L OVA, -Alum, (C) 0.33 g/L OVA, +Alum and (D) 1.33 g/L OVA, +Alum.

In vivo degradation and biocompatibility of hydrogels In vivo degradation of the hydrogel was evaluated by administering the OVA-loaded hydrogels (4 wt.%) with and without Alum into mice via subcutaneous injection. At various time points post injection, the hydrogel and its surrounding tissue was excised and examination of the histological specimens was performed. Figure S5 shows that during the first week post injection, the hydrogel (bracketed regions) was considerably intact. By 17 weeks, the hydrogel still remain visible. The longevity of the hydrogel can allow for prolonged in vivo release and protection of the antigens against proteolytic factors. To investigate the effect of HBsAg-loaded hydrogel on kidney functions, the amount of creatinine was measured at 1 and 3 months post-vaccination. The levels of creatinine in blood plasma are an important indication of nephrotoxicity 24. From Figure S6, A, the level of creatinine was similar cross all treatment groups, thereby showing that there was no impedance to kidney functions with the polymeric hydrogels used. Similarly, the effect of the various vaccine formulations on liver function was determined by measuring the plasma levels of alanine aminotransferase (ALT). This ubiquitous enzyme is most commonly associated with the liver and the measurement of the enzyme level has been conventionally used as a clinical parameter for the diagnosis of liver function. At 1 and 3 months post-vaccination, no change to the ALT level was observed (Figure S6, B), indicating no adverse effects on the liver 25. In vivo biodistribution of vaccine delivered by using different formulations The biodistribution of Alexa Fluor 750 labeled-OVA was examined in C57/BL6 mice by non-invasive fluorescent imaging

after the injection of hydrogel and solution formulations (Figure 3). OVA remained longer within the injection site to a greater extent when it was given in hydrogel formulations as compared to the solution formulations. The presence of Alum adjuvant prolonged the residency of OVA, possibly due to the adsorption and aggregation of OVA on the surface and/or inside Alum particles 26. No OVA in lymph nodes was seen, which suggests that immunostimulation occurred at non-lymphoid tissue 27,28. Immunization and antibody production IgG1 antibodies can activate complement to mediate complement-dependent cytotoxicity (CDC) and recruit effector cells for antibody-dependent cellular cytotoxicity (ADCC) against cancer cells 29. Immunization of C57BL/6 mice triggers the production of anti-OVA antibody and studies have shown that OVA can induce vigorous IgG1 antibody responses in mice 30,31. In Figure 4, A, mice that were vaccinated with OVA-loaded hydrogel produced a higher plasma level of anti-OVA IgG1 compared to those injected with the solution formulations (P b 0.05) by Day 28 post-immunization. Antibody response was dosedependent, and increased as a function time. Additionally, OVA delivered together with Alum induced significantly more antiOVA IgG1 antibodies than those without Alum for both hydrogel and solution formulations (Figure 4, A). The immunostimulatory effects of aluminum-based adjuvants have been widely studied and the mode of action includes the repository effect, pro-phagocytic effect, and activation of the pro-inflammatory NLRP3 pathway. Throughout the study period of 28 days, the mice showed good acceptance to all treatment formulations as they did not show any symptoms caused by toxicity such as lethargy, muscle loss,

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Figure 3. Biodistribution of OVA using non-invasive bioimaging. (A) Mice injected with Alexa Fluor 750-labeled OVA delivered using different formulations and (B) organs extracted on Day 7 and 21 post-injection.

dehydration or anorexia 32. Moving forward, the OVA-loaded hydrogel was tested for its ability to provide long-term immune response against OVA with one-time administration. At 6 months post injection, the mice had a higher concentration of anti-OVA IgG1 antibodies in blood plasma than at Day 28 (Figure 4, B). In the case of HBsAg-loaded hydrogel, the plasma level of HBsAG–specific IgG antibodies was analyzed at 1 and 3 months post-immunization. The amount of antigen given to the mice and the presence of Alum can influence the production of anti-HBsAg IgG. With 40 ng HBsAg (0.266 mg/L) given per mouse, those that were vaccinated with hydrogel formulation produced higher quantity of anti-HBsAg IgG compared to those injected with the solution formulations (P b 0.05) at 3 months post-immunization (Figure 4, C). This was independent of the incorporation of Alum. When a higher amount of HBsAg (400 ng) was given in the absence of Alum, the plasma level of anti-HBsAg IgG produced

was higher for those vaccinated using HBsAg-loaded hydrogel as compared to those with solution at 1 month post-immunization (Figure 4, D). At 1 month post-immunization, the amount of antiHBsAg IgG produced was also significantly higher when a larger amount of vaccine was used (400 ng vs. 40 ng of HBsAg) for all formulations (Figure 4, C and D). At a higher dose of 400 ng, the presence of Alum did not affect the plasma level of anti-HBsAg IgG in both hydrogel and solution formulations. At 3 months postimmunization, the amount of anti-HBsAg IgG produced in mice that were given hydrogel formulation containing a lower amount of vaccine (40 ng HBsAg) with Alum was similar to those vaccinated with solution formulation containing 400 ng HBsAg. The same was not observed for the solution formulations. We postulate that this is as a result of the slow degradation of the hydrogel, whereby HBsAg present within the matrix network was released gradually. This delayed the consumption of HBsAg and prolonged stimulation of the immune system.

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Figure 4. Production of anti-OVA IgG1 and anti-HBsAg IgG in mouse plasma as a function of time after vaccination with different formulations. Quantification of anti-Ova IgG1 at (A) 14-28 days and (B) 6 months after vaccination. Quantification of anti-HBsAg in mouse plasma with vaccination using hydrogel or solution formulations containing (C) 40 ng and (D) 400 ng of HBsAg at 1 and 3 months post-immunization. (n = 3-4) 3-4 mice were tested per condition per time point. Each error bar represents the standard deviation of each tested group. The data obtained was analyzed via Student’s T-Test and P≤0.05 was reflected as statistically significant.

Cytotoxic T lymphocyte induction following OVA immunization Immunization was carried out using hydrogel and solution formulations containing OVA in the presence or absence of Alum. Comparing against solution formulations, the immunization with OVA-loaded hydrogel induced significantly higher cytotoxic T lymphocyte (CTL) response against E. G7-OVA cells (a derivative of murine lymphoma EL-4 cells transfected with OVA cDNA) but not against EL4 (OVAepitope non-expressing) cells (Figure 5). This trend was independent of Alum. CTLs are cells that are responsible for killing the cancer cells and eradicating tumors 33 . These results indicate that the immunization using OVA-loaded hydrogel was much more effective in inducing CTL response as compared to the solution formulations. In both formulations, the incorporation of Alum boosted CTL responses against EG.7-OVA cells.

Effect of OVA immunization on tumor regression To investigate the therapeutic efficacy and specificity of OVA immunization on tumor regression, C57/BL6 mice that were vaccinated with OVA in various formulations were challenged with OVA epitope-expressing EG.7-OVA and OVA-epitope non-expressing parental EL4 tumor cells. When tumor challenge was performed at 14 days post-vaccination, there was no significant difference in tumor incidence found between the various treatment groups or between the treatment groups and the control. The tumor volume of mice vaccinated with the OVA-loaded hydrogels was significantly smaller than control but similar to those treated with solution formulations. In contrast, when vaccination was performed at 28 days prior to the tumor cell inoculation, significant subcutaneous tumor regression was observed in the EG.7-OVA model (Figure 7). In particular, the mice that were injected with OVA-loaded

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Figure 5. Cytotoxic T lymphocyte (CTL) response against E.G7-OVA and EL-4 cells of mice immunized with OVA-loaded hydrogel and solution formulations with and without Alum. (n = 4) 4 mice were tested per condition. Each error bar represents the standard deviation of each tested group. The data obtained was analyzed via Student’s T-Test and P≤0.05 was reflected as statistically significant.

hydrogel containing Alum had the best therapeutic outcome where only 2 out of 10 mice developed solid tumors (tumor incidence: 20%) and this was significantly lower compared to tumor incidence in mice vaccinated with OVA-loaded hydrogel without Alum (p = 0.007) and other treatment groups (p = 0.001). For example, 9 out of 10 mice that were immunized with OVA-loaded hydrogel without Alum developed EG.7-OVA tumors (tumor incidence: 90%) and the tumors were significantly smaller than the control group but larger compared to those treated with gels containing Alum. The mice that were treated with solution formulation (n = 10/10) all developed EL4 tumors regardless of the presence of Alum (tumor incidence: 100%) (Figure 7, B, D and F). These results demonstrate the effectiveness of 28-day OVA immunization via OVA-loaded hydrogel against OVA-expressing tumor. Effects of OVA immunization on mice survival after i.v. inoculation of cancer cells A mouse model with lymphoma metastasis was developed by administrating EG.7-OVA cells into the tail vein of C57BL/6 mice. Therapeutic vaccination with the OVA-loaded hydrogel and Alum resulted in a final increased survival (66.7%) compared to other formulations (ranging between 12.5-50%) over 100 days (Figure 8). Survival of mice following vaccination with the OVA-loaded hydrogels in the absence of adjuvant (50%) was also higher compared to solution formulations with Alum (44%) and without Alum (12.5%). In formulations without Alum, the survival of mice that were vaccinated with the hydrogel was significantly higher compared to those vaccinated with the solution (P = 0.03582). Discussion A biodegradable hydrogel system for subcutaneous vaccination was prepared using an ‘ABA’-type triblock copolymer of the center PEG flanked by a terminal Vitamin E moiety on each end

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Figure 6. Relative tumor volume of mice immunized with OVA-loaded hydrogel and solution formulations and subsequently challenged with EG.7OVA tumor cells at 14 days post-vaccination. Tumor size was measured at 14 days from the day the tumor cells were injected into the mice. Percentage values refer to the population of mice with tumors within the treatment group. (n = 8 or 9) 8 or 9 mice were tested per condition. Each error bar represents the standard deviation of each tested group. Fisher’s Exact test was used to compare tumor incidence. Student's t test was used to compare the tumor volume between experimental groups. P≤0.05 was reflected as statistically significant.

via carbamate bond, i.e. VE-PEG-VE. A low polymer concentration of 4 wt% was sufficient to create a hydrogel that was injectable and could release cancer preventive vaccines in a sustained manner. From Figure S7, A, sustained in vitro release of HBsAg was observed for up to 2 weeks with 80% of the antigen being released within 10 days. As for OVA-loaded hydrogel, in vitro release of OVA was sustained for close to 2 weeks with ~ 65% of the OVA being released (Figure S7, B). As with our earlier study, we anticipate that the diffusion barrier encompasses primarily of the physical interactions between the polymer and OVA molecules such as hydrogen bonding and van der Waals forces 21. When the OVA solution was administrated into mice, a much higher amount of OVA was released into the blood at early time points within the first 60 min of administration (Figure S8, A). The hydrogel provided more sustained antigen release with a higher amount of OVA (0.12 μg/mL) being present in the blood as compared to the solution formulation (0.01 μg/mL) at 21 days post-injection (Figure S8, B). This was likely attributed to the gel matrix that could act as a diffusion barrier against the release of OVA, while at the same time, protecting OVA from proteolytic damage. In vivo degradation of the hydrogel was slow and remained in mice for N 17 weeks. The stability of the hydrogel provides an added advantage for the hydrogel to modulate the release speed of OVA. Subsequently, the depletion rate of OVA is lessened and the duration of which the immune system is stimulated increases. In turn, this translates to stronger immunity against OVA as the mice that were vaccinated with OVA-loaded hydrogel resulted in significantly larger quantity of anti-OVA IgG1 than those injected with the solution formulations both at Day 28 and 6 months postimmunization (Figure 4, A and B). Similarly, for those vaccinated using HbsAg-loaded hydrogel, a higher level of antibodies was

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Figure 7. Relative volume, weight and images of tumors resected from mice immunized with OVA-loaded hydrogel and solution formulations and subsequently challenged with EG.7-OVA (A, C and E) and EL4 (B, D and F) tumor cells at 28 days post-vaccination. Percentage values refer to the population of mice with tumor within the treatment group. (n = 10) 10 mice were tested per condition. Fisher’s Exact test was used to compare tumor incidence. Student's t test was used to compare the tumor volume and weight between experimental groups. P≤0.05 was reflected as statistically significant. Tumor weight (C and D) and images of tumors (E and F) were taken at Day 19 from the day the tumor cells were injected into the mice. Blue scale bars in tumor micrographs represent 1 cm.

produced as compared to those vaccinated using the solution formulations (Figure 4, C). This was independent of the incorporation of Alum but was dependent more on the amount of antigen used and time post-immunization (Figure 4, C and D). Furthermore, we also demonstrated that it is possible to use much lower amount of vaccine to achieve similar antigen specific IgG production with the hydrogel formulation.

The hydrogel was effectively used as a matrix for sustained delivery of OVA to elicit immune protection against the lymphoma EG.7-OVA cells. In the subcutaneous tumorbearing mice, a one-time s.c. injection of OVA-loaded hydrogel yielded significantly lower tumor incidence and tumor volume against EG.7-OVA cells as compared to OVA solution formulations. Similarly, in the intravenously disseminated

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Figure 8. Survival record of mice after vaccination with various OVA formulations. Vaccination with OVA-loaded hydrogels provides protection against metastatic EG.7-OVA cells in a murine model of lymphoma. (n = 8-9) 8-9 mice were tested per condition. The data obtained was analyzed via Student’s t test and P ≤ 0.05 was reflected as statistically significant.

EG7.OVA model, one-time s.c. injection of OVA-loaded hydrogel drastically increased mouse survival when compared to OVA solution formulations. Vaccination can induce cytotoxic T cells against antigenspecific cells. Cytotoxic T cells kill their targets by inducing them to go through apoptosis. When brought into contact with target cells, they can program antigen-specific target cells to die within several minutes, although death may take hours to become fully evident 34. Significantly higher cytotoxic T lymphocyte (CTL) response against OVA epitope expressing (EG.7-OVA) cells was demonstrated when mice were vaccinated using OVA-loaded hydrogel as compared to solution formulations (Figure 6). As for the HBsAg-loaded formulations, the hydrogel formulations gave rise to significanltly larger population of CD4+ and CD8+ T cells (Fig. S9A and B). Importantly, the OVA-loaded hydrogels were significantly more effective in preventing tumor regression as they resulted in the best therapeutic outcome where only 2 out of 10 mice injected with OVA-loaded hydrogel containing Alum developed EG.7-OVA solid tumors and the tumors were also significantly smaller than the other treatment groups. The high immunization efficacy of the hydrogel formulation was probably a result of the sustained release of OVA from the hydrogel matrix that enabled a continuous supply of the antigen. Specificity of the treatment was also demonstrated as all mice developed EL4 tumors, which do not express OVA epitopes. In a separate study, EG.7-OVA cells were injected intravenously into mice to mimic cancer metastasis. Final survival of the mice at the end of the 100-day study period was drastically increased by therapeutic vaccination with the OVA-loaded hydrogel as compared to solution formulations. The gel presented in this study is superior compared to other hydrogels reported for vaccine delivery. Firstly, when compared to a study conducted by Mallapragada et al. 19, the in vitro

release of OVA from our gel is significantly longer (close to 2 weeks) compared to the gel based on copolymers of Pluronics and PDEAEM where the in vitro release of OVA lasted for about 5 days. With regards to in vivo persistence of the hydrogels, our hydrogel also persisted much longer (N17 weeks) compared to the hydrogel reported by Mallapragada et al. (~7 weeks). Secondly, in another study 12 where peptide hydrogels were used for the delivery of OVA, the preparation/formation of the hydrogels was more intricate and involves the use of alkaline phosphatase, which adds to both the cost and complexity of the formulation. The persistence of the peptide hydrogels is not reported in this study, but is widely known that such materials degrade relatively quickly in vivo, ranging between 4 and 8 weeks 35,36,37. Our findings establishes the prospective that the carbamatebased hydrogel can serve as a delivery matrix for prophylaxis of cancer with sustained release of cancer vaccines. Furthermore, the need for only one injection to provide protection against cancer renders this immunization regimen undoubtedly appealing. With this hydrogel, we will be able to extend its application in delivering vaccines against human infectious diseases such as hepatitis and human papillomavirus.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.nano.2019.102056.

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