Visualizable Delivery of Nanodisc Antigen-Conjugated Adjuvant for Cancer Immunotherapy

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Apr 2022Visualizable Delivery of Nanodisc Antigen-Conjugated Adjuvant for Cancer Immunotherapy Yangyun Wang†, Subin Lin†, Hanqiu Jiang, Yuan Gu, Yanxian Wu, Jie Ma, Yubin Ke, Leshuai W. Zhang, Yong Wang and Mingyuan Gao Yangyun Wang† State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123 , Subin Lin† Department of Orthopedic, The Second Affiliated Hospital of Soochow University, Suzhou 215004 , Hanqiu Jiang China Spallation Neutron Source, Dongguan Branch, Institute of High Energy Physics, Chinese Academy of Sciences, Dongguan 523803 , Yuan Gu State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123 , Yanxian Wu State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123 , Jie Ma State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123 , Yubin Ke China Spallation Neutron Source, Dongguan Branch, Institute of High Energy Physics, Chinese Academy of Sciences, Dongguan 523803 , Leshuai W. Zhang State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123 , Yong Wang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123 and Mingyuan Gao *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123 Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 https://doi.org/10.31635/ccschem.021.202000670 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Engineering synthetic vaccines is promising for improving the efficacy of cancer immunotherapy. One of the major challenges in the development of vaccines is achieving controllable codelivery of antigen and adjuvant to lymph nodes for maximizing the antitumor immune responses. To address this issue, we herein developed an innovative visualizable nanodisc vaccine based on ovalbumin (OVA) and short-stranded oligodeoxynucleotides containing unmethylated cytosine-phosphate-guanine (CpG) motifs. The nanovaccine was fabricated by covalently attaching CpG onto the surface of a nanodisc antigen formed upon self-assembly of amphiphilic molecular conjugates of OVA and cypate (Cy), a near-infrared (NIR) fluorescent dye, for noninvasively visualizing the delivery of the resulting nanovaccines. Systematic in vitro experiments demonstrated that the engineered nanovaccines can specifically locate to dendritic cells (DCs) via toll-like receptor 9 and membrane thiols, and then efficiently activate DCs. The animal experiments combining NIR fluorescence imaging with the lymphatic T-cell phenotype and key cytokine secretion analyses revealed that targeted lymphatic homing of the mature DCs and consequent priming of CD8+ T cells were enabled to initiate strong tumor-specific T-cell responses with robust immune memory effects. Thus, this study offers a visualizable platform for optimizing the efficacy of nanovaccines toward cancer immunotherapies. Download figure Download PowerPoint Introduction Vaccines hold enormous potential to stimulate systemic immune responses in cancer immunotherapy, but the response rate of clinical vaccines is unfortunately <30%.1 The challenge of vaccine development is not only achieving controllable codelivery of antigen and adjuvant to lymph nodes (LNs),2 but also maximizing the functions of the two major components of a given vaccine by spatiotemporally manipulating their immunoregulatory pathways.3,4 Because of the attenuated immune effects of traditional adjuvants such as aluminum salts and water–oil emulsions, new adjuvant formulations are being developed for engineering synthetic vaccines.5,6 As a potent molecular adjuvant, short-stranded oligodeoxynucleotides containing unmethylated cytosine-phosphate-guanine (CpG) motifs can considerably enhance antigen-specific immune responses by binding to toll-like receptor 9 (TLR9) in antigen-presenting cells (APCs).7,8 Nonetheless, small-molecule CpG-based vaccines have not fully realized clinical applications.9 Nanomaterials, including polymers,10–12 lipid,13,14 silica,15 and gold nanomaterials,16 can provide an alternative carrier for the fabrication of nanovaccines and codelivering the antigen and CpG to draining LNs. These current nanovaccines attempt to activate APCs in the draining LNs,17 but the nonconjugated components in these nanovaccines also drain into the systemic circulation and access APCs in distal tissues, which might cause serious systemic toxicity.18 Hence, well-defined molecular structure and payload will be attractive for improving the LNs-targeting delivery and biosafety of nanovaccines.19 Moreover, the in vivo fate and function of vaccines after their delivery can also determine whether they can finally effectively prime cancer-specific cytotoxic CD8+ T cells.20 Current predictors of vaccine efficacy, which are invasive and take months to develop, usually depend on the host response.21,22 The gap between administration and evaluation may lead to potential pathogen infection before verifying vaccine prevention.23 Advanced bioimaging technology opportunely provides powerful tools to noninvasively visualize the in vivo spatiotemporal fate of administrated vaccines.24 It is believed that visualizable delivery of vaccines is desirable to evaluate the efficacy of candidate formulations and improve their rationality of design for both preclinical and translational studies.25 Therefore, in combination with the design of nanovaccines with precise targeting and optimal responses, there is an increasing need to integrate imaging functions in nanovaccines to understand how they will activate immune response and prevent tumors in vivo.26,27 Herein, we report a novel ovalbumin (OVA)/CpG-based nanovaccine for cancer immunotherapy. The nanovaccine is comprised of a nanodisc antigen core formed upon self-assembly of amphiphilic conjugates of OVA and near-infrared (NIR) fluorescent dye cypate (Cy), and a shell of CpG molecules covalently attached on the surface of the core. The resulting nanovaccine, denoted as [email protected], is characterized by high antigen-loading efficiency, small size (approximately 30 nm), adjustable ratio between antigen and adjuvant, and visualizability in vivo. The engineering nanovaccines can specifically deliver to dendritic cells (DCs) via the TLR9 and membrane thiols, and then efficiently activate DCs. Owing to the inherent NIR fluorescence, the spatiotemporal trafficking, particularly the accumulation of the nanovaccines in sentinel LNs, was noninvasively monitored in vivo after delivery. Systematic animal experiments were then carried out to show the robust tumor-specific T-cell responses induced by the nanovaccines and the corresponding mechanisms. Experimental Section General procedure for nanoantigen and nanovaccines synthesis The nanoantigen was synthesized according to the dye-induced assembly strategy of antigen molecules.28 A 50 mL OVA solution (10 mg mL−1, pH = 10) was added into 5 mL of 20 mM cypate sulfo-NHS (NHS = N-hydroxysulfosuccinimide). Sulfhydrylation stirring (500 rpm) for 24 h, the solution was dialyzed (8000–14,000 MW) against ultrapure water for 48 h. To synthesize the nanovaccines based on the nanoantigen (Cy-OVA), nanoantigen (0.4 μmol) was initially conjugated with N-succinimidyl 3-maleimidopropionate (0.8 μmol) to produce maleimide-grafted nanoantigen (Cy-OVA-M), and then the 3′-end sulfhydrylation CpG (5-TCCATGACGTTCCTGACGTT-3) was reduced with tri(2-carboxyethyl) phosphine hydrochloride; the molar ratio was 1:10. Reduced CpG (12 nmol) was finally reacted with the Cy-OVA-M for the preparation of the nanovaccines. The quantitation of conjugated CpG was monitored with the Cy5.5-labeled CpG. The concentration of the obtained nanoantigen and nanovaccines was calculated with the absorbance performance of the Cy molecule. The morphology, structure, and hydrodynamic diameter were characterized by transmission electron microscopy (TEM), small-angle neutron scattering (SANS), and dynamic light scattering (DLS), respectively. More details are available in the Supporting Information. Animals and cells All animal experiments were approved by the Animal Ethics Committee of Soochow University (Suzhou, China). This study was performed in strict accordance with the national guidelines for the care and use of laboratory animals (Certificate no. 20020008, Grade II) in the Soochow University Laboratory Animal Center (Suzhou, China). C57/BL6 and BALB/c mice were obtained from Suzhou JOINN Clinical Co., Ltd. (Suzhou, China). The orthotopic osteosarcoma model was established using 4-week-old female BALB/c mice based on the literature. All animal experiments were performed under anesthesia with 2.5% isoflurane. B16F10-OVA+ cells were kindly provided by Professor Xueguang Zhang at Soochow University. K7M2 cells were obtained from National Infrastructure of Cell Line Resource of China (Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China). Cells were cultured in minimum essential medium [MEM, 10% fetal bovine serum, 100 U/mL penicillin G sodium and 100 μg/mL streptomycin, MEM sodium pyruvate (1 mM), NaHCO3, MEM vitamins, MEM nonessential amino acids, and 20 μM β-mercaptoethanol]. More details are available in the Supporting Information. Results and Discussion Preparation and characterization of nanovaccines The conjugate of Cy and OVA was initially prepared by covalently linking carboxylated Cy to OVA molecule via amidation reaction mediated by EDC and sulfo-NHS (EDC = N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide). Owing to the hydrophobicity of the Cy dye, the resulting conjugates are amphiphilic and tend to self-assemble in aqueous systems into nanodisc with OVA-rich surface structures. Therefore, the resulting nanoparticles are also called nanoantigens and are subsequently denoted as Cy-OVA. Then, the thiolated CpG molecules were covalently attached via a sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) linker to the surface of the nanoantigens,29 as shown in Figure 1a, to form a quasi-core–shell-structured nanovaccine with both antigen and adjuvant simultaneously accessible, while the NIR dye molecules are embedded deep inside. According to the TEM results (Figures 1b and 1c), the average size of the resulting [email protected] nanovaccines is 18.9 ± 5.3 nm. The hydrodynamic size of the nanovaccines is approximately 27 nm as determined by DLS (Figure 1d), and the zeta potential is approximately −26.7 mV. SANS studies suggest that [email protected] particles possess a core–shell structure with an overall disc-like shape (Figure 1e), similar to the high-density lipoprotein-based nanovaccines previously reported.13 According to the SANS fitting results, the mean diameter is 28.2 nm, and the shell thickness is around 1.2 nm. To further characterize the resulting nanovaccines, Cy5.5-labeled CpG was prepared and used to construct a control vaccine for quantitatively estimating the coupling efficiency of CpG with UV–vis absorbance spectroscopy and fluorescence spectroscopy. As Figure 1f shows, the absorbance of [email protected] ranging from 550 to 850 nm is dramatically increased, showing the major characteristic of the absorption of Cy5.5, in comparison with Cy-OVA. The fluorescence spectroscopy results (Figure 1g) also support an effective coupling of CpG molecules onto the surface of the nanoantigen core, with a coupling efficiency up to 78% ( Supporting Information Figure S1) and antigen to CpG ratio up to 15:1. Given that any possible cytotoxicity of the nanovaccines may impair the immunomodulatory effects,30 they were evaluated in vitro for effects on cell viability. The cell survival rate results ( Supporting Information Figure S2) suggest that the current nanovaccines hardly affect the viability of mouse embryonic fibroblasts (3T3 cells) and DCs. Figure 1 | Preparation and characterization of nanovaccines. (a) The fabrication strategy of nanovaccines. (b) The TEM image of [email protected] (c) Size distribution of nanovaccines from TEM image. (d) The hydrodynamic size of [email protected] (e) SANS and fitting model of Cy-OVA. (f) The absorbance spectra of [email protected] and Cy-OVA. (g) The fluorescence spectra of [email protected], Cy-OVA, and CpG-Cy5.5. Download figure Download PowerPoint Target and activation of DCs in vitro In the current design, the [email protected] nanovaccines are expected to specifically target the DCs via TLR9 and cell surface thiol moieties.12,31 To show such ability, DCs incubated with the nanovaccines were subjected to analyses with flow cytometry and confocal microscopy. According to the flow cytometry results in Figure 2a, the DCs’ uptake of the Cy-OVA nanoantigens is 13.7%. However, it is as high as 70.0% for CpG labeled with Cy5.5, and further increases to 80.4% for the [email protected] nanovaccines. The remarkably high uptake of the nanovaccines by DCs can reasonably be attributed mainly to TLR9-mediated endocytosis and partially to the generally enhanced cellular uptake for nano-objects. Confocal microscopy results in Figure 2b also confirm that the DCs’ uptake of [email protected] is much higher than that for Cy-OVA. In addition, the nanovaccines present relatively uniform signals across the cytoplasm of the DCs. Figure 2 | Uptake pathway of DCs. (a) Flow cytometry for TLR9-directed targeted delivery. (b) Confocal microscopy for TLR9-directed targeted delivery. (c) Confocal microscopy for thiols on the DCs surface and TLR9-directed targeted delivery. Download figure Download PowerPoint According to the current nanovaccine design, the surface maleimide groups of the nanoantigen particles have two functions. Some of them are used to covalently attach CpG molecules to form the nanovaccines and the remaining maleimide groups are expected to enhance the binding affinity of the nanovaccines to DCs through the cell surface thiol residues.32,33 As Figure 2c demonstrates, the DCs incubated with the Cy-OVA nanoantigens bearing surface maleimide groups (denoted as Cy-OVA-M) exhibit comparable fluorescence with those incubated with [email protected] nanovaccines, and they both show stronger fluorescence than the cells incubated with the nanoantigen particles (i.e., Cy-OVA), suggesting that CpG and the remaining maleimide residues on the surface of [email protected] nanovaccines synergistically enhance the specific codelivery of the antigen and adjuvant into the DCs. In addition to effective DC targeting, the following activation of DCs is another key issue for priming immune response. To show the DC activation effect of the nanovaccines, flow cytometry and the Meso Scale Discovery (MSD) multiplex assay were carried out to analyze the change of DC phenotype and the release of different types of cytokines. Three phenotypic markers, including CD40, CD80, and CD86, were selected for showing DCs’ maturation upon coincubation with [email protected] Although both Cy-OVA nanoantigens and CpG adjuvant can stimulate DC maturation( Supporting Information Figures S3a–S3f), the DCs incubated with [email protected] present the highest activation level. After incubation with the nanovaccines for 24 h, the expressions of all three phenotypic markers in DCs increased by factors of 2–3 in comparison with those of the nontreated controls. Four proinflammatory cytokines secreted by mature DCs, including IL-1β, IL-6, TNF-α, and IL-12p70, were also quantitatively determined. According to the secretion levels of these cytokines in Supporting Information Figure S3g, the CpG adjuvant group shows higher activation ability than the Cy-OVA nanoantigens. This may be due to the excellent phagocytosis and immune activation properties of CpG. Nonetheless, the DCs incubated with [email protected] generate much higher levels of cytokines than those treated with CpG, showing enhancement factors of 1.53, 1.28, and 1.49, for IL-12p70, IL-6, and TNF-α, respectively. With respect to IL-1β, the enhancement factor reaches as high as 3.6. All above results suggest that the [email protected] nanovaccines can specifically target, induce maturation, and enhance proinflammatory cytokines secretion of DCs. Antigen presentation and hypermobility of DCs To investigate the antigen presentation ability of DCs upon stimulation with [email protected] nanovaccines in vitro, the expression of epitope-determining peptide (SIINFEKL) was assayed by flow cytometry. The SIINFEKL level is the highest for [email protected] stimulated DCs ( Supporting Information Figure S4a). Although Cy-OVA also exhibits a remarkable effect, the adjuvant and antigen apparently synergistically enhance the antigen presentation. The migration ability of mature DCs is very important for DCs homing to LNs to initiate adaptive immune responses.23 Once DCs become mature upon stimulation with antigen and adjuvant, they will upregulate the expression of biomarkers associated with the migration ability, such as CC-chemokine receptor 7 (CCR7). CCR7 enables DCs to migrate to and retain in regional LNs to expand the adaptive immune response.34,35 Therefore, CCR7 is considered an important biomarker for evaluating the migration ability of mature DCs. As shown in Supporting Information Figure S4b, the percentage of DCs expressing CCR7 is 22.8% upon incubation with [email protected], while it is only 5.7% and 18.6% for DCs incubated with free CpG and Cy-OVA, respectively. Apart from CCR7, the dynamics of the cytoskeleton are another indispensable indicator for DC motility.36 As the two major components of cytoskeleton, microfilaments and microtubules were analyzed via immunofluorescence assays. The results given in Supporting Information Figure S4c reveal that the DCs incubated with [email protected] nanovaccines exhibit a typical motility phenotype showing strong signals from filamentous actin and extended veils, in contrast to those in the control groups that show weak filament-based phenotype. Regarding β-tubulin, no significant difference is shown among different groups of DCs incubated with CpG, Cy-OVA, and [email protected], respectively. These results indicate that [email protected] nanovaccines can significantly enhance the motility of DCs, which is very conducive to the homing of mature DCs to the secondary lymphatic system. In vivo lymphatic homing and immune responses Timely and efficient delivery of vaccines to the secondary lymphatic system, for example, spleen and LNs, is essential for inducing strong and continuous immune responses against tumors.2 Therefore, the NIR Cy dye was embedded in the nanovaccines to allow for real-time tracking of the migration and estimating of the LNs’ retention of the current nanovaccines in vivo.25 NIR fluorescence imaging studies were carried out after subcutaneously injecting [email protected] into the foot paws of mice, with the Cy-OVA nanoantigens serving as control. Figure 3a reveals that the lymphatic homing of both nanovaccine and its control can be visualized well. But the axillary and inguinal nodes of mouse receiving [email protected] nanovaccines present much stronger NIR fluorescence signals than those of the control. Moreover, these signals last for more than 6 days, suggesting that the [email protected] nanovaccines have potential for providing long humoral and cellular immune responses. The spatiotemporal dynamics of mature DCs stimulated by vaccines, particularly in terms of DCs’ movement from peripheral tissues to the draining LNs, will be very important for regulating the DC-driven immune responses and vaccination.21 For DC-based vaccines, their continuous retention in the secondary lymphatic system can be attributed to the uptake and transport of the vaccines by DCs. Therefore, to demonstrate that DCs become mature and gain homing ability upon stimulation by the current nanovaccines in vivo, DCs were incubated with [email protected] nanovaccine prior to the subcutaneous foot paw injection, as shown in Figure 3b. The results in Figure 3c show that the DCs treated with [email protected] not only migrate to the inguinal LNs, but also accumulate in the axillary LNs and spleen of mice 24 h postinjection of the DCs loaded with the nanovaccines. Moreover, the signals of inguinal LNs and axillary LNs are much stronger than those of the control mice receiving DCs loaded with Cy-OVA. Apart from that, the signal of the axillary LNs remains strong even 3 days postinjection. All these results indicate that the [email protected] nanovaccine is more conducive than the corresponding nanoantigen in promoting the DCs’ migration to the secondary lymphatic system, quite probably in consequence of a series of positively regulated events, including chemotaxis, cytoskeletal rearrangement, and hypermobility. Figure 3 | (a) In vivo real-time NIR fluorescence tracking of Cy-OVA and [email protected] in the LNs for 6 days. The fluorescence imaging of necropsy was performed after 6 days. (b) Schematic diagram of DCs-incubated with Cy-OVA and [email protected] prior to the subcutaneous foot paw injection. (c) The dynamic migration over 3 days of DCs-loaded with Cy-OVA and [email protected] using in vivo NIR fluorescence imaging. Download figure Download PowerPoint After showing the positive effects of the nanovaccines, a series of immune responses caused by [email protected] homing to the secondary lymphatic system was then investigated by injection of the nanovaccines into the tail root (Figure 4a). Both [email protected] and its control, Cy-OVA, were found to in the major including and LNs of mice at 24 h postinjection (Figure Although the signals of [email protected] and Cy-OVA are comparable from both axillary and inguinal nodes (Figure the signal of the from spleen is as high as that of the To further the uptake of the nanovaccines by cells of specific types in the and secondary tissues, LNs were and into a cell for flow cytometry Figure reveals that accumulation of the nanovaccines is associated with DCs and T cells with from Figure | In vivo lymphatic homing and immune responses of nanovaccines. (a) Schematic diagram of lymphatic (b) After 24 h, vivo NIR fluorescence imaging of and LNs of the mice subcutaneously with nanovaccines. (c) The distribution of nanovaccines in the secondary tissues, including and inguinal (d) The distribution of nanovaccines in was analyzed by flow cytometry. (e) The distribution of nanovaccines and T cells in LNs was analyzed by (f) The expression level of specific on the surface of CD8+ T Download figure Download PowerPoint The following immunofluorescence of draining LNs that the [email protected] nanovaccine and its control, Cy-OVA are mainly in the T cells are as shown in Figure The results in Figure indicate that the nanovaccines can further activate T cells to into CD8+ T cells that specifically on the cell approximately of T cells after the mice injection of [email protected], while it is for the Cy-OVA nanoantigen and they both are much higher than that for the CpG that of draining LNs is an indicator for [email protected] nanovaccines reduced systemic in comparison with free CpG, as by the size of from different groups ( Supporting Information Figure All these results support that the targeting of the [email protected] nanovaccines can effectively induce immune responses while simultaneously showing systemic effects. memory by the nanovaccines Owing to the immune memory effect of the by the memory T cells can be upon by the efficient lymphatic homing and effective activation of CD8+ T we further evaluated the ability of [email protected] in cytotoxic T response with the given in Figure The spleen of mouse was to Then, of the was with free OVA for 2 h by weak with mM while the of the was not but subjected to with 5 mM Then, the resulting cells were and into mice 7 days After h, the was and subjected to flow cytometry to determine the of and cells for further estimating the The results shown in Figures and reveal that [email protected] nanovaccines to the highest of approximately three stronger than that with Cy-OVA suggesting that nanovaccines can remarkably enhance the response. Figure 5 | The immune memory effect of the stimulated with nanovaccines. (a) Schematic diagram of immune memory effect (b) Flow cytometry for spleen (c) The in (d) Schematic diagram of immune memory effect (e) Flow cytometry with for the of CD8+ T cells in the T (f) Flow cytometry of of CD8+ T (g) assay of in memory CD8+ T of in memory CD8+ T Download figure Download PowerPoint The CD8+ T cell response is generally associated with the major antigen presentation pathway in the immune memory and the molecules on the of CD8+ T cells T cell To the of cellular and show the of the immune memory effect, the were on the from

Topics & Concepts