Fluorinated redox-responsive poly(amidoamine) as a vaccine delivery

Subscriber access provided by University of Rhode Island | University Libraries

Controlled Release and Delivery Systems

Fluorinated redox-responsive poly(amidoamine) as a vaccine delivery system for anti-tumor immunotherapy Hongyuan Yuan, Zonghua Liu, Wei Xue, and Yong Yang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b00945 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Fluorinated redox-responsive poly(amidoamine) as a vaccine delivery system for anti-tumor immunotherapy Hongyuan Yuan, Yong Yang, Wei Xue, Zonghua Liu* Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical Engineering, Jinan University, West Huangpu Road 601, Guangzhou, 510632, China

* Corresponding author: Zonghua Liu ([email protected])

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 27

Abstract: As a potential method for tumor treatment, tumor vaccine immunization induces the tumor-specific cellular immune response by immunization with tumor antigens. The delivery of exogenous antigen proteins into the cytoplasm of antigen-presenting cells is well accepted to induce an intensive cellular immune response for tumor treatment. In this work, we fluorinated a redox-responsive hyperbranched poly(amidoamine) (HPAA) with heptafluorobutyric anhydride to prepare a fluorinated HPAA (HPAA-F7) for use as a vaccine delivery system for anti-tumor therapy. The immunization results show that HPAA-F7 as a vaccine carrier could effectively promote the intracellular uptake and cytoplasmatic delivery of antigen proteins and induce a potent anti-tumor cellular immunity. The novel vaccine carrier HPAA-F7 could be further developed for anti-tumor immunotherapy.

Keywords: poly(amidoamine), fluorination, cellular immune response, anti-tumor immunotherapy.


Page 3 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Tumor vaccines, which induce specific anti-tumor immunity by injecting specific tumor antigens into the patient, are widely applied in tumor immunotherapy.1-2 Numerous studies have demonstrated that inducing the specific killing of tumor cells and simultaneously avoiding damage to normal tissues is the key aim of tumor treatment. Because tumor vaccines can trigger a tumor-specific immune response to exclusively kill tumors without damaging normal tissues, they are considered an ideal method for tumor treatment.3 Nevertheless, when tumor antigens are used alone, they cannot induce strong anti-tumor immunity, and consequently, immune adjvants or vaccine delivery systems are used along with the antigens to elicit a more potent tumor-specific immune response.4 Tumor vaccine-induced cellular immunity (not humoral immunity) can achieve the desired anti-tumor therapeutic effect. One of the current challenges in developing tumor vaccines lies in finding effective immune adjuvants or vaccine delivery systems to assist tumor antigens to induce anti-tumor cellular immunity. When traditional immune adjuvants are taken up by the antigen-presenting cells (APCs) and then degraded and processed in the acidic environment of the endosomes/lysosomes, the cellular immune response is ineffective for tumor immunotherapy.5 In contrast, when exogenous protein antigens are delivered into the cytosol of APCs and then presented via the major histocompatibility complex (MHC) I pathway (“cross-presentation”), the introduction of the antigen-specific cellular immunity is expected.6-7 Recently, designing safe and effective vaccine carriers to elicit effective antigen cross-presentation and cellular immunity has gained attention for tumor vaccine-based immunotherapy.8 With this aim in mind, intelligent vaccine carriers have been designed to deliver tumor antigen proteins into the cytoplasm of APCs to trigger cellular immunity by the cross-presentation of the antigens.4 In recent years, cationic polymers have attracted widespread attention as vaccine carriers to promote antigen cross-presentation.9 Cationic polymers can cause the endosomal/lysosomal escape of antigens via a proton sponge effect and then deliver the antigens into the cytoplasm to elicit cross-presentation and cellular immunity. For example, the positively charged natural polymer chitosan can induce a highly specific cellular immune response.10 Ma et al. prepared a nanogel with a positively charged surface for vaccine delivery that had an effective immunotherapy effect.11 Currently, polycation gene carriers modified by fluorination have been found to greatly



Page 4 of 27

promote cellular uptake and the endosomal/lysosomal escape of their siRNA cargos12. Cheng et al. reported that fluorinated dendrimers could greatly improve the efficiency of gene transfection due to their high affinity to the cell surface and the subsequent then cellular internalization.13-14 Gu et al. reported that a biodegradable fluorinated cationic peptide dendrimer used as a gene carrier could successfully promote cellular internalization, endosomal escape, and the release of siRNA into the cytosol.15 Thus, fluorinated polycation carriers have been widely used in the delivery of siRNA to induce potent RNAi-based cancer therapeutic efficacy.16-17 From the above reports, we hypothesized that fluorinated cationic polymers could be used as tumor antigen carriers with enhanced intracellular uptake and cytoplasmic delivery of tumor antigens to induce cross-presentation and cellular immunity. Here, we report modifying a disulfide bond-containing hyperbranched poly(amidoamine) (HPAA) using heptafluorobutyric anhydride to prepare fluorinated HPAA (HPAA-F7) for use as vaccine delivery system for anti-tumor immunotherapy. The redox-responsive polycation HPAA has been widely used as a gene carrier. The cationic polymer HPAA with its proton sponge effect has been reported cause endosome rupture and promote the cytoplasmic delivery of gene cargos.18 HPAA as an siRNA vector with its proton sponge effect and redox responsiveness could achieve a better gene therapy efficacy.19 HPAA has also been used as a vaccine delivery system to control the release of antigen into the cytoplasm after APC uptake.4, 19 In this work, we fluorinated HPAA was further to combine the individual advantages of HPAA as a gene/vaccine carrier with fluorination for the gene/vaccine delivery. The prepared HPAA-F7 is expected to facilitate the cellular uptake and cytoplasmic delivery of exogenous antigens to induce cellular immune responses for anti-tumor immunotherapy.

Experimental Materials Acryloyl chloride, 1-(2-aminoethyl)-piperazine (AEPZ, 99%), cystamine dihydrochloride were furnished by Aladdin (Shanghai, China) for the preparation of HPAA. Heptafluorobutyric anhydride and triethylamine were purchased from Aladdin (Shanghai, China). Ovalbumin (OVA), freund's adjuvant (FA) and lysis buffer for red blood cell were supplied by Sigma-Aldrich (MO, USA). Roswell park memorial institute-1640 medium (RPMI-1640), fetal bovine serum (FBS) and


Page 5 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


penicillin-streptomycin were bought from Gibco (CA, USA) as the medium of cell culture formulating. Cy5.5-conjugated OVA was provided by Shanghai Universal Biotech Co, Ltd (Shanghai, China). The fluorochrome-conjugated anti-mouse antibodies for the conduct of flow cytometry experiments were offered by eBioscience (CA, USA). Ultrapure milli-Q water was used throughout the experiment. Animals Female Balb/c mice and female C57BL/6 mice aged 6-8 weeks old were obtained from HFK Biological Technology Co, Ltd (Beijing, China). All animal experiments conducted in line with the “ Guidelines for Care and Use of Laboratory Animals ”

and acquire consent from the

Experimental Animal Ethics Committee in Jinan University. Preparation and characterization of nanocomposites as delivery vehicles For all formulations, the intermediate product N,N’-cystamine bisacrylamide (CBA) was synthesized with acryloyl chloride and cystamine dihydrochloride. Briefly, the intermediate product CBA was synthesized with, 3.304 mL acryloyl chloride (0.04 mol) dissolved in 3.33 mL of dichloromethane (DCM) and a NaOH solution (0.4 g/mL, 0.04 mol) was added alternately dropwise to a cystamine dihydrochloride solution (0.225 g/mL, 0.016 mol) in an ice bath. The mixture was allowed to react at room temperature under stirring for 8 h. The product was then extracted with DCM, and purged using a sequence of saturated NaHCO3, NaCl, and ultrapure water. CBA was then collected as the DCM was eliminated with a low pressure rotary evaporator. Afterwards, the cationic compound HPAA was synthesized according to the following procedure.19 Briefly, the calcium chloride aqueous solution (0.04 g/mL, 13.2 mL) was mixed with CBA (2.57 g) that was dissolved in a calcium chloride-methanol solution (0.04 g/mL, 40 mL). AEPZ (0.64 mL, 4.9 mmol) was then added dropwise into the solution, with the solution mentioned previously being heated to 50ºC. After 48 h, AEPZ (1.4 mL, 10.7 mmol) was added and reacted for 8 h. The HPAA was obtained after dialysis and lyophilization. The fluorinated HPAA-F7 was then synthesized via the anhydride reaction. Briefly, 0.05 g of HPAA and 0.25 g of heptafluorobutyric anhydride (0.9 mmol) were dissolved in 10 mL of methanol, and then 0.147 mL of triethylamine (1.06 mmol) was immediately added to the methanol solution. The reaction mixture was stirred for 1 day and dialyzed for 2 days against ultrapure water. The product was lyophilized to generate fluorinated HPAA-F7.



Page 6 of 27

Then, the nanocomposites suspension, which was prepared by mixing the same volume of the polymer solution (150 μg/mL) with OVA solution (30 μg/mL), was incubated for 30 min at room temperature. The hydrodynamic sizes and zeta potentials of the obtained OVA nanocomposites were measured by using a nano laser particle size analyzer. OVA uptake by DC2.4 cells in vitro DC2.4 cells were seeded in 24-well plates for 1 day, and washed with the phosphate buffer solution (PBS). Then the free OVA-Cy5.5, HPAA/OVA-Cy5.5 and HPAA-F7/OVA-Cy5.5 were added slowly to the wells and incubated at 37ºC with 5% CO2 for 6 h. After washed with PBS three times, the fluorescence intensity of the collected cells was detected by flow cytometry (Beckman). Meanwhile, the confocal laser scanning microscopy (CLSM) was used to visualize the intracellular localization of OVA. As mentioned above, free OVA and polymer nanocomposites were cultivated with DC2.4 cells for 6 h in the cell culture incubator respectively. The acidic vesicles in DC2.4 cells were stained with Lyso-Tracker Green DND-26 (0.5 mL) for 2 h. Hereafter the plates were washed three times and 0.5 mL 4% paraformaldehyde was added to the plates for 10 min. Afterwards, the DAPI (4’,6-diamidino-2-phenylindole) was used to stain the nuclei of DC2.4 cells for 5 min. Subsequently, the cells were observed by CLSM. Immunohistochemistry assay The available antigen delivered into the spleen was researched by immunohistochemical assays. Female Balb/c mice were randomly divided into 4 groups (4 mice in each group). Different vaccine preparations of free OVA, FA/OVA, HPAA/OVA and HPAA-F7/OVA (30 μg OVA for each preparation, 100 μL per mice) were injected subcutaneously in the inguinal region. On the 2nd and 7th day after immunization, the mice were euthanized. The spleens were collected, fixed with 4% paraformaldehyde, and then subjected to immunohistochemical analysis. Immunization of Balb/c mice Balb/c mice aged 8-weeks were divided to four groups (n = 5) stochastically, which were subcutaneously injected with 100 μL (50 μL per inguinal region of the hind legs) of the various formulations (30 μg OVA for each formulation): OVA alone, FA/OVA, HPAA/OVA and HPAA-F7/OVA on day 0, 7 and 14. The specific formulations are presented in Table 1. Preparation of splenocyte culture The mice were sacrificed on day 21. The spleens were collected and mechanically digested


Page 7 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


into single-cell suspensions in the complete RPMI 1640 medium. The residual tissue fragments of the obtained cell suspensions were removed by filtered through a 200 μm cell strainer. Then the cells were lysed using red blood cell lysate for 10 min. Subsequently, the splenocytes cells were re-suspended and then centrifuged at 1000 rpm at 4ºC for 5 min. Finally, the precipitated cells were re-suspended in the complete RPMI 1640 culture medium. Antibody titer Blood samples were collected from the mice at the seventh day after the third injection. Then the supernatant sera were collected and stored at -80ºC for the analysis of IgG and IgG subtypes. The OVA-specific IgG, IgG1 and IgG2a antibody titer in the serum were quantified by the enzyme-linked immunosorbent assay (ELISA). Specific details have been described in previous studies of our group.20 Splenocyte proliferation assay The splenocytes (5×106 cells per mL) were seeded (100 μL per well) in a 96-well plate and restimulated with OVA (50 μg/mL) for 72 h. Meanwhile, the other batch of splenocytes were incubated in the same 96-well plate without OVA restimulated. Subsequently, 20 μL of cell counting kit-8 solution was added into the plates and incubated for 4 h at 37ºC. The absorbance was measured by a multiskan MK3 photometric microplate reader at 450 nm. Proliferation index (PI) was calculated according to the following formula: PI = OD (450 nm) for stimulated samples/OD (450 nm) for non-stimulated samples. Cytokine secretion The splenocytes (2.5×106 cells per mL) were cultured in 12-well plates and restimulated with OVA (20 μg/mL) for 60 h. Then, the cell suspensions were collected and centrifuged at 1000 rpm for 5 min at room temperature. Afterwards, the supernatants were collected and stored at -80ºC for the analysis of interferon gamma (IFN-γ), interleukin-6 (IL-6), interleukin-4 (IL-4) and interleukin-10 (IL-10) by using ELISA MAXTM Deluxe Sets (Biolegend, San Diego, CA, USA). In addition, the pellet cells were collected for analysis of memory T cell assays following the manufacturer’s instruction. Determination of OVA-specific memory T cells As mentioned in section 2.10, the centrifuged splenocytes were stained with 100 μL of different fluorochrome-conjugated anti-mouse antibodies dyes (FITC-anti-CD4, Cy5.5-anti-CD8a,



Page 8 of 27

PE-anti-CD44, and APC-anti-CD62L (1 : 200 v/v in PBS)) for 30 min at 4ºC. The cell suspensions were then centrifuged at 1000 rpm for 5 min and washed with PBS three times to remove the unbound fluorescent antibody dyes. Finally, the splenocytes were detected by the flow cytometry. In vivo tumor treatment The right backs of C57BL/6 mice were injected with E.G7-OVA cells (5×105 cells) under the anesthesia on day 1, which were subsequently immunized with PBS, free OVA, HPAA/OVA or HPAA-F7/OVA on the 8th,15th and 22nd day respectively. The size of the tumor and the time of survival of the mice were recorded to assess the anti-tumor ability of HPAA/OVA and HPAA-F7/OVA nanoparticles. The tumor size was detected with the vernier caliper and calculated by using the following formula: tumor volume (mm3) = length × (width2) × 0.5. Notably, the mice were sacrificed once the tumor volumes reached to 1500 mm3. Statistical analysis Graph Pad Prism software was used to deal with all the statistical analysis. And the data in this paper were shown as mean ± standard deviation (SD). The significance of the data was evaluated by using Student’s t-test: P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

Results and discussion Preparation and characterization of HPAA and HPAA-F7 In this work, redox-responsive cationic HPAA and fluorinated HPAA-F7 were prepared as carriers for the vaccine delivery system. The hyperbranched cationic polymer HPAA was synthesized by a Michael addition reaction with N,N’-cystaminebisacrylamide (CBA) and 1-(2-aminoethyl)-piperazine (AEPZ). HPAA-F7 was also produced by the reaction of the cationic polymer HPAA and heptafluorobutyric anhydride. The preparation routes are shown in Figure 1. The chemical structures of HPAA and HPAA-F7 were identified by 1H NMR and

19F

NMR,

respectively. As shown in Figure 2a, the peaks at chemical shifts of 3.024 ppm and 2.760 ppm were attributed to hydrogens on CBA. And the peaks at chemical shifts of 2.531 ppm and 2.624 ppm were attributed to AEPZ. These results verified the successful preparation of HPAA. In addition, 19F

NMR was used to further confirm the smooth introduction of fluorine in HPAA-F7. As shown

in Figure 2b, fluorine atoms in the HPAA-F7 were revealed at peaks (1-3), showing that fluorine was contained in the HPAA-F7 polymer. The fluorine content in HPAA-F7 was measured by


Page 9 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


inductively coupled plasma mass spectrometry (ICP-MS). It is found that 1 mg of HPAA-F7 contained 0.026 mg of fluorine. After calculation, there was 3.82 mol of fluorine atom on 1 mol of HPAA-F7. In addition, the redox-responsiveness of HPAA was characterized by measuring the different elution time of untreated and glutathione-treated HPAA by using gel permeation chromatography (GPC), as shown in Figure S1 of the supporting information. The molecular weight of HPAA was determined to be 26.891 kDa by GPC analysis. Furthermore, the result indicates that glutathione treatment caused the breakage of the disulfide bonds of HPAA and the degradation of HPAA. It indicates that the redox-responsive vaccine carrier HPAA could degrade in the presence of glutathione in the cytoplasm of dendritic cells and hence release vaccine cargo to the cytoplasm to promote antigen cross-presentation and anti-tumor cellular immune response. Finally, the zeta potentials and hydrodynamic sizes of HPAA, HPAA-F7, HPAA/OVA and HPAA-F7/OVA were measured in aqueous solutions by DLS. The results suggested that the zeta potentials of HPAA and HPAA-F7 were 35.75 ± 0.264 mV and 45.35 ± 2.475 mV, respectively. The sizes of HPAA and HPAA-F7 were 426.45 ± 79.974 nm and 329.8 ± 11.879 nm, respectively. OVA and HPAA or HPAA-F7 merged into nanocomposites via electrostatic adsorption and the zeta potentials of the HPAA/OVA and HPAA-F7/OVA nanocomposites dropped to 28.85 ± 0.778 mV and 25.15 ± 0.778 mV, respectively, while the sizes increased to 594.6 ± 75.236 nm and 571.85 ± 31.608 nm, respectively (Table 2). OVA uptake by DC2.4 cells in vitro The antigen intracellular release behavior of the nanocomplexes was evaluated in the endosomal/lysosomal and cytoplasmic environments of DCs. As shown in Figure 3, the internalization efficiency of the free OVA was about 3.62%, which was significantly lower than that of the HPAA/OVA and HPAA-F7/OVA that could deliver antigens to DCs and promote antigen uptake to some degree (Figure 3a). This could be attributed to the positively charged surface of HPAA/OVA and HPAA-F7/OVA that could promote their interaction with the negatively charged cell membranes. Flow cytometry analysis show that the enhancement of mean fluorescence intensity of DC2.4 cells incubated with fluorinated HPAA-F7/OVA was higher than HPAA/OVA through (Figure 3b). Thus, HPAA-F7/OVA promoted a higher cellular uptake of OVA than HPAA/OVA. In addition, the uptake process of OVA by DC2.4 cells was detected in vitro by CLSM. In the Figure 4, the red spots represent the antigen ingested by DC2.4 cells and the green



Page 10 of 27

spots represent the lysosomes. DC2.4 cells treated with HPAA-F7 had a large number of red spots, demonstrating that HPAA-F7 improved the uptake of antigen. In addition, compared to other groups, a larger portion of red spots in HPAA-F7/OVA were at a distance from the green spots, indicating that the antigenic proteins of HPAA-F7/OVA were delivered more efficiently into the cytoplasm. These results showed that the OVA-containing nanoparticles (HPAA-F7/OVA) in vitro led to a higher cellular uptake and cytoplasmic delivery, which is attributed to the high affinity of the fluorinated HPAA-F7 with the cell membrane and then cellular internalization. Immunohistochemistry assay After antigen is delivered to the spleen, it can be detected by immunohistochemistry (Figure 5). Examination of spleen tissue sections obtained two days after immunization, showed that the groups with HPAA-F7/OVA and FA/OVA had more antigen, in comparison to free OVA and HPAA/OVA. After seven days, only antigen in the HPAA-F7/OVA group was largely detected in spleens tissues, to a greater extant than HPAA/OVA; A minor amount of antigen, however, was detected in the free OVA group. All groups at two days post-immunization showed more protein antigen that at seven days. This findings illustrates that the HPAA-F7/OVA nanocomposite can prolong the presentation of antigens, resulting in a stronger immune response. Systemic antibody responses in vaccinated mice The titers of OVA-specific IgG and related subtypes, which provide an efficient activation of antibodies and immune protection, were detected in mouse serum by the enzyme-linked immunosorbent assay (ELISA). As shown in Figure 6, various groups immunized with OVA-formulated nanocarriers showed the production of OVA-specific IgG, IgG2a, and IgG1, especially the HPAA-F7/OVA group that induced a higher expression of IgG, IgG2a, and IgG1 than the free OVA and HPAA/OVA groups. In addition, the titer ratio of IgG2a and IgG1, which represent Th1 and Th2 immune responses, respectively, can be used to evaluate the type of immune response.21-23 Compared to the free OVA and HPAA/OVA groups, the titer ratio of IgG2a and IgG1 was higher level in the HPAA-F7/OVA group. These results suggest that HPAA-F7/OVA induced a higher level of cellular immune response. Splenocyte proliferation When facing the re-invasion of a pathogen, organisms can rapidly generate an immune memory effect in the immune response. As shown in Figure 7, the group vaccinated with


Page 11 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


HPAA-F7/OVA and HPAA/OVA had higher splenocyte proliferation indexes than free OVA group. Both groups can induce a significant and effective splenocyte proliferation. The splenocytes gathered from mice in the HPAA-F7/OVA group displayed a higher level of proliferation than those gathered from the HPAA/OVA group, indicating that the HPAA-F7/OVA induced a higher OVA-specific immune memory effect. Cytokine levels secreted by ex vivo restimulated splenocytes The detection of cytokine levels is an important aspect of basic and clinical immunity research. As shown in Figure 8, the fluorinated HPAA-F7/OVA group led to more secretion of proinflammatory cytokine IL-6, when compared to the free OVA and HPAA/OVA groups. In addition, Th-1 cytokine IFN-γ was produced at a higher level with fluorinated HPAA-F7/OVA and in the FA/OVA groups, illustrating the high levels of Th-1 polarized cellular immunity. Furthermore, HPAA-F7/OVA nanoparticles induced the highest levels of IL-10 and IL-4, compared to free OVA and HPAA/OVA vaccine formulations. In summary, HPAA-F7/OVA had stronger immune-stimulation effects than free OVA and HPAA/OVA, which is attributed to the higher cellular uptake and cytoplasmic delivery which led to the sustained release of OVA from HPAA-F7/OVA nanoparticles. Memory T cell responses The immunoprophylaxis of vaccines is aimed to induce an immunological memory function after vaccination. The memory T cells directly produce an antigen-specific immune response when the body encounters the same antigen again, leading to effective immunoprotection that is critical for disease prevention. The immune memory effect caused by the antigen delivery system needs to be evaluated. In this research, the percentage of memory T cells in CD4+T cells and CD8+T cells from mice spleen was determined after immunization. As shown in Figure 9, the specifically expressed proteins of the central memory T cells (CD44+CD62L+) and effector memory T cells (CD44+CD62L−) had higher levels of expression in the CD4+T cells and CD8+T cells from mice immunized with HPAA/OVA and HPAA-F7/OVA nanoparticles, in comparison to free OVA. Thus, HPAA/OVA and HPAA-F7/OVA nanoparticles had induced an effective memory T cell response. In particular, the percentage of CD44+CD62L+ and CD44+CD62L− in CD8+T cells from the HPAA-F7/OVA group displayed a significantly higher level than that of the free OVA and HPAA/OVA groups. These results indicate that HPAA-F7/OVA induced a stronger memory T cell



Page 12 of 27

response, which could be used effectively in vaccine formulations to enhance the immune memory function. In vivo tumor treatment To further evaluate the efficacy of anti-tumor immunity, we used a tumor challenge assay. E.G7-OVA tumor-bearing mice were closely monitored after being immunized with vaccine nanocomposites. As shown in Figure 10a, injection of HPAA/OVA and HPAA-F7/OVA significantly inhibited tumor growth, compared to injection with PBS alone or free OVA. Furthermore, HPAA-F7/OVA worked better than HPAA/OVA. The sustained inhibition was observed unchanged for 26 days. In addition, until day 40, 80% of the mice injected with HPAA-F7/OVA were alive, which was far higher than any of the other test groups (Figure 10b). Thus, the OVA-specific cellular immune responses triggered by HPAA-F7/OVA could clearly prolong the survival of mice and enhance the efficiency of cancer immunotherapy. Conclusion In contrast to other immunotherapeutic strategies, we synthesized fluorinated HPAA-F7, with the combined advantages of a fluorination strategy and the redox-response of HPAA. Our immunization study demonstrated that the cationic polymer HPAA-F7 had an improved immune effect after being fluorinated (including higher IgG antibody titers; spleen cell proliferation; higher secretion levels of cytokines IFN-γ, IL-6, IL-4, and IL-10; improved immune memory function; and a greater tumor suppressive effect). The improved effectiveness can be explained by the increased intracellular uptake of antigen and increased cytoplasmic delivery, leading to more effective humoral and cellular immune responses. Our results suggest that the fluorination strategy could be used as a general approach to improve tumor immunotherapy with hyper-branched polymer-based vaccine vectors.

Acknowledgements This work was financially supported by the Fundamental Research Funds for the Central Universities and Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering. Supporting information


Page 13 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure S1, gel permeation chromatography (GPC) analysis of the HPAA.

Conflict of interest There are no conflicts of interest to declare.



Page 14 of 27

References: 1.

Liu, Q.; Chen, X.; Jia, J.; Zhang, W.; Yang, T.; Wang, L.; Ma, G., pH-Responsive Poly(D,L ‑

lactic-co-glycolic acid) Nanoparticles with Rapid Antigen Release Behavior Promote Immune Response. Acs Nano 2015, 9 (5), 4925-4938. DOI: 10.1021/nn5066793. 2.

Goldberg, M. S., Immunoengineering: how nanotechnology can enhance cancer immunotherapy. Cell 2015,

161 (2), 201-4. DOI: 10.1016/j.cell.2015.03.037. 3.

Leleux, J.; Roy, K., Micro and nanoparticle-based delivery systems for vaccine immunotherapy: an

immunological

and

materials

perspective.

Advanced

healthcare

materials

2013,

2

(1),

72-94.

DOI:

10.1002/adhm.201200268. 4.

Li, D.; Sun, F.; Bourajjaj, M.; Chen, Y.; Pieters, E. H.; Chen, J.; van den Dikkenberg, J. B.; Lou, B.; Camps,

M. G.; Ossendorp, F.; Hennink, W. E.; Vermonden, T.; van Nostrum, C. F., Strong in vivo antitumor responses induced by an antigen immobilized in nanogels via reducible bonds. Nanoscale 2016, 8 (47), 19592-19604. DOI: 10.1039/c6nr05583d. 5.

Petrovsky, N.; Aguilar, J. C., Vaccine adjuvants: Current state and future trends. Immunology and Cell

Biology 2004, 82, 488-496. DOI: 10.1111/j.1440-1711.2004.01272.x. 6.

Rock, K. L.; Shen, L., Cross-presentation: underlying mechanisms and role in immune surveillance.

Immunological Reviews 2005, 207, 166-183. DOI: 10.1111/j.0105-2896.2005.00301.x. 7.

Molino, N. M.; Anderson, A. K. L.; Nelson, E. L.; Wang, S. W., Biomimetic Protein Nanoparticles Facilitate

Enhanced Dendritic Cell Activation and Cross Presentation. Acs Nano 2013, 7, 9743-9752. DOI: 10.1021/nn403085w. 8.

Xu, L.; Xiang, J.; Liu, Y.; Xu, J.; Luo, Y.; Feng, L.; Liu, Z.; Peng, R., Functionalized graphene oxide serves

as a novel vaccine nano-adjuvant for robust stimulation of cellular immunity. Nanoscale 2016, 8 (6), 3785-95. DOI: 10.1039/c5nr09208f. 9.

Storni, T.; Kundig, T. M.; Senti, G.; Johansen, P., Immunity in response to particulate antigen-delivery

systems. Adv Drug Deliv Rev 2005, 57 (3), 333-55. DOI: 10.1016/j.addr.2004.09.008. 10. Rauw, F.; Gardin, Y.; Palya, V.; Anbari, S.; Gonze, M.; Lemaire, S.; van den Berg, T.; Lambrecht, B., The positive adjuvant effect of chitosan on antigen-specific cell-mediated immunity after chickens vaccination with live Newcastle disease vaccine. Veterinary immunology and immunopathology 2010, 134 (3-4), 249-58. DOI: 10.1016/j.vetimm.2009.10.028. 11. Li, P.; Luo, Z.; Liu, P.; Gao, N.; Zhang, Y.; Pan, H.; Liu, L.; Wang, C.; Cai, L.; Ma, Y., Bioreducible alginate-poly(ethylenimine) nanogels as an antigen-delivery system robustly enhance vaccine-elicited humoral and cellular immune responses. Journal of controlled release : official journal of the Controlled Release Society 2013, 168 (3), 271-9. DOI: 10.1016/j.jconrel.2013.03.025. 12. Hai., W. L.; Cheng., W. D.; Xun., X. H.; Zi., Y. Y., High DNA-Binding Affinity and Gene-Transfection Efficacy of Bioreducible Cationic Nanomicelles with a Fluorinated Core Angewandte Chemie International Edition 2016, 55, 755-759. DOI: 10.1002/anie.201508695, 10.1002/ange.201508695. 13. Wang, M.; Liu, H.; Li, L.; Cheng, Y., A fluorinated dendrimer achieves excellent gene transfection efficacy at extremely low nitrogen to phosphorus ratios. Nature communications 2014, 5, 3053. DOI: 10.1038/ncomms4053. 14. Lv, J.; Chang, H.; Wang, Y.; Wang, M.; Xiao, J.; Zhang, Q.; Cheng, Y., Fluorination on polyethylenimine allows efficient 2D and 3D cell culture gene delivery. Journal of Materials Chemistry B 2015, 3 (4), 642-650. DOI: 10.1039/c4tb01447b. 15. Cai, X.; Zhu, H.; Zhang, Y.; Gu, Z., Highly Efficient and Safe Delivery of VEGF siRNA by Bioreducible Fluorinated Peptide Dendrimers for Cancer Therapy. ACS applied materials & interfaces 2017, 9 (11), 9402-9415. DOI: 10.1021/acsami.6b16689. 16. Chen, G.; Wang, K.; Hu, Q.; Ding, L.; Yu, F.; Zhou, Z.; Zhou, Y.; Li, J.; Sun, M.; Oupicky, D., Combining


Page 15 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fluorination and Bioreducibility for Improved siRNA Polyplex Delivery. ACS applied materials & interfaces 2017, 9 (5), 4457-4466. DOI: 10.1021/acsami.6b14184. 17. Chen, G.; Wang, K.; Wang, Y.; Wu, P.; Sun, M.; Oupicky, D., Fluorination Enhances Serum Stability of Bioreducible Poly(amido amine) Polyplexes and Enables Efficient Intravenous siRNA Delivery. Advanced healthcare materials 2018, 7 (5). DOI: 10.1002/adhm.201700978. 18. Lin, C.; Engbersen, J. F., Effect of chemical functionalities in poly(amido amine)s for non-viral gene transfection. Journal of controlled release : official journal of the Controlled Release Society 2008, 132 (3), 267-72. DOI: 10.1016/j.jconrel.2008.06.022. 19. Li, M.; Zhou, X.; Zeng, X.; Wang, C.; Xu, J.; Ma, D.; Xue, W., Folate-targeting redox hyperbranched poly(amido amine)s delivering MMP-9 siRNA for cancer therapy. Journal of Materials Chemistry B 2016, 4 (3), 547-556. DOI: 10.1039/c5tb01964h. 20. Lv, M.; Li, S.; Zhao, H.; Wang, K.; Chen, Q.; Guo, Z.; Liu, Z.; Xue, W., Redox-responsive hyperbranched poly(amido amine) and polymer dots as a vaccine delivery system for cancer immunotherapy. Journal of Materials Chemistry B 2017, 5 (48), 9532-9545. DOI: 10.1039/c7tb02334k. 21. Yuba, E.; Tajima, N.; Yoshizaki, Y.; Harada, A.; Hayashi, H.; Kono, K., Dextran derivative-based pH-sensitive

liposomes

for

cancer

immunotherapy.

Biomaterials

2014,

35

(9),

3091-101.

DOI:

10.1016/j.biomaterials.2013.12.024. 22. Xing, J.; Liu, Z.; Huang, Y.; Qin, T.; Bo, R.; Zheng, S.; Luo, L.; Huang, Y.; Niu, Y.; Wang, D., Lentinan-Modified Carbon Nanotubes as an Antigen Delivery System Modulate Immune Response in Vitro and in Vivo. ACS applied materials & interfaces 2016, 8 (30), 19276-83. DOI: 10.1021/acsami.6b04591. 23. Yoshizaki, Y.; Yuba, E.; Sakaguchi, N.; Koiwai, K.; Harada, A.; Kono, K., Potentiation of pH-sensitive polymer-modified liposomes with cationic lipid inclusion as antigen delivery carriers for cancer immunotherapy. Biomaterials 2014, 35 (28), 8186-96. DOI: 10.1016/j.biomaterials.2014.05.077.



Page 16 of 27

Table 1. Components of the different vaccine formulations Groups

Ovalbumin (μg)

OVA alone

PBS (μL)

30

100

150 μg/mL HPAA

30

100

150 μg/mL HPAA-F7

30

100

Freund’s adjuvanta (50 μL)

30

100

aWe

used completed Freund’s adjuvant for the first injection and used incompleted Freund’s

adjuvant for the second and third injections according to the protocol.

Table 2. Zeta potentials and sizes of HPAA, HPAA-F7, HPAA/OVA and HPAA-F7/OVA Groups

Size (nm)

PDI

Zeta (mV)

HPAA

426.45 ± 79.974

0.504 ± 0.035

35.75 ± 0.264

HPAA/OVA

594.6 ± 75.236

0.424 ± 0.042

28.85 ± 0.778

HPAA-F7

329.8 ± 11.879

0.508 ± 0.074

45.35 ± 2.475

HPAA-F7/OVA

571.85 ± 31.608

0.424 ± 0.018

25.15 ± 0.778


Page 17 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents Use Only

Fluorinated redox-responsive poly(amidoamine) as a vaccine delivery system for anti-tumor immunotherapy Hongyuan Yuan, Yong Yang, Wei Xue, Zonghua Liu*



Figure 1. Schematic illustration of synthesis routes to HPAA and HPAA-F7.

60x82mm (300 x 300 DPI)


Page 18 of 27

Page 19 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. 1H NMR spectra of the HPAA (a) and 19F NMR spectra of the HPAA-F7 (b). 177x230mm (96 x 96 DPI)



Figure 3. (a&b) Internalized OVA-Cy5.5 in DC2.4 cells after being incubated for 6 h with OVA-Cy5.5, or the HPAA/OVA-Cy5.5 and HPAA-F7/OVA-Cy5.5 nanocomposites. OVA-Cy5.5-positive cells were measured using flow cytometry.


Page 20 of 27

Page 21 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Intracellular localization of OVA-Cy5.5 in DC2.4 cells after being incubated for 6 h with OVA-Cy5.5 or the HPAA/OVA-Cy5.5 and HPAA-F7/OVA-Cy5.5 nanocomposites. The scale bar is 5 μm. 146x110mm (96 x 96 DPI)




Page 22 of 27

Page 23 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Antigen-specific (a) IgG, (b) IgG2a, and (c) IgG1 titers in sera from the immunized mice with different vaccine formulations, and (d) the ratio of IgG2a to IgG1. 414x378mm (300 x 300 DPI)



Figure 7. Proliferation index of the splenocytes from the immunized mice with different vaccine formulations. 273x229mm (96 x 96 DPI)


Page 24 of 27

Page 25 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. Cytokine secreting levels of the splenocytes from the immunized mice with different vaccine formulations: (a) IFN-γ, (b) IL-6, (c) IL-4, and (d) IL-10. 419x365mm (300 x 300 DPI)



Figure 9. Frequencies of central/effector memory CD4+ and CD8+ T cells in the splenocytes from the immunized mice with different vaccine formulations. The frequencies of CD44+CD62L+ central memory CD4+T cells (a), CD44+CD62L− effector memory CD4+T cells (b), CD44+CD62L+ central memory CD8+T cells (c), and CD44+CD62L− effector memory CD8+ T cells (d). FACS graphs of the representative percentage in each group (e&f). 204x322mm (300 x 300 DPI)


Page 26 of 27

Page 27 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 10. Anti-tumor effect induced by the subcutaneous administration of OVA, HPAA/OVA and HPAAF7/OVA. (a) Tumor volumes and (b) survival curve are shown. 337x128mm (96 x 96 DPI)


Fluorinated redox-responsive poly(amidoamine) as a vaccine delivery

Recommend Documents