A general strategy towards personalized nanovaccines based on fluoropolymers for post-surgical cancer immunotherapy

Xu, Jun; Lv, Jia; Zhuang, Qi; Yang, Zongjin; Cao, Zhiqin; Xu, Ligeng; Pei, Pei; Wang, Chenya; Wu, Hanfei; Dong, Ziliang; Chao, Yu; Wang, Chao; Yang, Kai; Peng, Rui; Cheng, Yiyun; Liu, Zhuang

doi:10.1038/s41565-020-00781-4

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Published: 02 November 2020

A general strategy towards personalized nanovaccines based on fluoropolymers for post-surgical cancer immunotherapy

Jun Xu¹,
Jia Lv²,
Qi Zhuang¹,
Zongjin Yang¹,
Zhiqin Cao¹,
Ligeng Xu¹,
Pei Pei³,
Chenya Wang¹,
Hanfei Wu¹,
Ziliang Dong¹,
Yu Chao¹,
Chao Wang ORCID: orcid.org/0000-0002-8054-3472¹,
Kai Yang³,
Rui Peng ORCID: orcid.org/0000-0001-8001-520X¹,
Yiyun Cheng ORCID: orcid.org/0000-0001-6019-5645^4,2 &
…
Zhuang Liu ORCID: orcid.org/0000-0002-1629-1039¹

Nature Nanotechnology volume 15, pages 1043–1052 (2020)Cite this article

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Abstract

Cancer metastases and recurrence after surgical resection remain an important cause of treatment failure. Here we demonstrate a general strategy to fabricate personalized nanovaccines based on a cationic fluoropolymer for post-surgical cancer immunotherapy. Nanoparticles formed by mixing the fluoropolymer with a model antigen ovalbumin, induce dendritic cell maturation via the Toll-like receptor 4 (TLR4)-mediated signalling pathway, and promote antigen transportation into the cytosol of dendritic cells, which leads to an effective antigen cross-presentation. Such a nanovaccine inhibits established ovalbumin-expressing B16-OVA melanoma. More importantly, a mix of the fluoropolymer with cell membranes from resected autologous primary tumours synergizes with checkpoint blockade therapy to inhibit post-surgical tumour recurrence and metastases in two subcutaneous tumour models and an orthotopic breast cancer tumour. Furthermore, in the orthotopic tumour model, we observed a strong immune memory against tumour rechallenge. Our work offers a simple and general strategy for the preparation of personalized cancer vaccines to prevent post-operative cancer recurrence and metastasis.

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Main

Cancer immunotherapy has brought great hope to the fight against cancer in recent years^1,2. Different types of cancer immunotherapy strategies, have been extensively studied or tested in the clinic³, alone or in combination with other conventional therapeutic strategies, such as chemotherapy^4,5 or radiotherapy^6,7. Moreover, it has been reported that post-surgery immunotherapy, such as immune checkpoint blockade (ICB) therapy, may be able to reduce the risk of cancer recurrence and metastasis^8,9. However, its therapeutic benefits remain to be improved.

Among various types of cancer immunotherapy, cancer vaccines can induce tumour-specific immune responses^10,11,12. To activate and expand tumour-specific T cells, recent advances in nanoparticle (NP)-based vaccines (nanovaccines) have focused on the co-delivery of tumour-specific antigens and adjuvants to antigen-presenting cells (APCs), such as dendritic cells (DCs)^13,14,15. In this process, cytosolic delivery and cross-presentation of antigens by professional DCs via the major histocompatibility complex class I (MHC-I) antigen-presenting pathway are essential to trigger robust antitumour responses^16,17. Therefore, to fabricate efficient cancer nanovaccines, it is critical to develop antigen carriers that act as immune adjuvants, and are capable of shuttling antigens directly into the cytosol of APCs.

Recently, fluoropolymers, such as fluorinated dendrimers and polyethyleneimines (PEIs), were reported to be promising gene and protein delivery carriers^18,19,20. Fluorocarbon chains grafted on these cationic polymers could enhance their abilities to mediate the transmembrane delivery of cargoes into the cell cytosol, owing to the low surface energy and lipophobicity of fluoroalkyl chains^21,22. Here we hypothesized that such fluoropolymers might also be employed as antigen carriers to fabricate tumour nanovaccines with an efficient antigen cross-presentation. We found that two types of fluoroalkane-grafted polyethyleneimines (F-PEIs), F₇-PEI and F₁₃-PEI, both showed reduced cytotoxicity compared with that of bare PEI, and, when mixed with a model protein antigen ovalbumin (OVA), would self-assemble to form F-PEI/OVA NPs. Such F-PEI/OVA NPs, especially the formulation with higher fluorine content, could promote high levels of antigen cross-presentation due to the efficient cytosolic delivery of OVA. In vivo, such a formulation elicits high levels of antitumour T-cell responses, which leads to an effective inhibition of established B16-OVA tumours. We further demonstrate that the fluoropolymer could be employed to fabricate personalized cancer vaccines by mixing it with tumour cell membranes collected from the surgically resected autologous tumours. Such fluoropolymer-based personalized nanovaccines, when combined with ICB, are able to prevent post-operative tumour recurrence and tumour metastases, as shown in two subcutaneous tumour models and an orthotopic breast cancer tumour. Our work presents a simple and reliable strategy to fabricate cancer nanovaccines based on fluoropolymers, promising for an effective patient-specific post-surgery immunotherapy.

Fluoropolymer for antigen delivery

F-PEIs were synthesized via an amine–epoxide reaction (Fig. 1a)²⁰ to obtain F₇-PEI and F₁₃-PEI. The average number of 3-(perfluoro-n-hexyl)-1,2-propenoxide ligands conjugated per PEI was determined to be ~30 in F₁₃-PEI (fluorine content = 20.49%), whereas the average number of 3-(perfluoro-n-propyl)-1,2-propenoxide ligands conjugated per PEI was ~27 in F₇-PEI (fluorine content = 12.02%) (Supplementary Fig. 1). The obtained F₇-PEI and F₁₃-PEI were mixed with OVA at different weight ratios (w/w, pH 7.0) (Fig. 1b). The formed NPs showed hydrodynamic sizes in the range of 157–250 nm (Fig. 1b,c) and similar zeta potentials at around +50 mV (Fig. 1d). As the control sample, intact branched PEI (25 kDa) was also able to prepare NPs on mixing with OVA, and the obtained PEI/OVA NPs showed sizes and surface charges comparable to those of F-PEI/OVA NPs (Supplementary Fig. 2a,b).

**Fig. 1: Preparation, characterization and DC activation of nanovaccines.**

We then studied the interactions of our F-PEI/OVA NPs with mouse bone marrow-derived dendritic cells (BMDCs). First, our tested samples had rather low levels of endotoxin (Supplementary Table 1) and F-PEI/OVA NPs showed no striking cytotoxicity to BMDCs in our tested concentration range, in contrast to PEI/OVA NPs prepared with non-modified PEI, which appeared to be highly toxic (Fig. 1e and Supplementary Fig. 3). Such a high cytotoxicity of unmodified PEI excluded the possibility of using PEI/OVA for further DC activation experiments.

The maturation of DCs is critical for antigen presentation and the initiation of subsequent immune responses^23,24,25,26. Interestingly, BMDCs pulsed with either F₁₃-PEI/OVA or F₇-PEI/OVA at the F-PEI:OVA ratio of 1:1 showed the highest upregulation in co-stimulatory molecules (CD80, CD86, CD40 and MHC-II) compared with those treated with F-PEI/OVA at other ratios (Fig. 1f–h and Supplementary Fig. 4a). Consistent with DC maturation data, F₁₃-PEI/OVA and F₇-PEI/OVA (F-PEI:OVA = 1:1 w/w) induced the highest production of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-12p70 (Supplementary Fig. 4b–d) by treated BMDCs.

We then examined the cross-presentation efficiency for DCs treated with F-PEI/OVA NPs. Treating BMDCs by F₁₃-PEI/OVA NPs (F₁₃-PEI:OVA = 1:1 w/w) resulted in the highest level of the SIINFEKL peptide presented on the BMDCs’ surface (Fig. 1i and Supplementary Fig. 5a,b). We hypothesized that at the higher F-PEI:OVA ratios, such as 2:1 and above, the fluoropolymer would have a stronger binding with the antigen, and thus hinder its release for further antigen cross-presentation. Together, our data suggest that both F₁₃-PEI/OVA and F₇-PEI/OVA at the optimal F-PEI:OVA ratio of 1:1 could trigger an efficient DC maturation, but the former, F₁₃-PEI/OVA, appeared to be more effective in inducing antigen cross-presentation.

Using an in vitro OT-I CD8⁺ T-cell priming assay, BMDCs pre-treated with F₁₃-PEI/OVA NPs could induce a significant proliferation of OT-I CD8⁺ T cells to a level much higher than those induced by BMDCs pre-treated with OVA or F₇-PEI/OVA (Fig. 1j,k). Meanwhile, greatly increased secretions of TNF-α and interferon-γ (IFN-γ) (Supplementary Fig. 6) were also observed for those CD8⁺ T cells activated by F₁₃-PEI/OVA-pre-treated BMDCs. These data suggest that F₁₃-PEI/OVA could effectively activate BMDCs and trigger subsequent OVA-specific T-cell responses.

The underlying mechanisms of such fluoropolymer-based NPs to promote cross-presentation and DC maturation were subsequently studied. The model antigen OVA was labelled with fluorescein isothiocyanate (FITC) to facilitate intracellular tracking. Both F₁₃-PEI/OVA-FITC NPs and F₇-PEI/OVA-FITC NPs showed a greatly enhanced uptake by BMDCs compared with that of OVA-FITC alone (Fig. 2a).

**Fig. 2: In vitro DC activation by nanovaccines.**

A recent study demonstrated that PEI could re-polarize myeloid-derived suppressor cells (MDSCs) from M2- to M1-type through the Toll-like receptor 4 (TLR4)-mediated signalling pathway²⁷. Interestingly, the cellular uptakes of F₇-PEI/OVA-FITC and F₁₃-PEI/OVA-FITC NPs by BMDCs from Tlr4 gene knockout mice (Tlr4^–/–) were found to be significantly lower than that by BMDCs from wild-type (wt) mice (C57BL/6), which suggests the possible involvement of TLR4-dependent endocytosis in the internalization of F-PEI-based NPs (Fig. 2b). In addition, Tlr4^–/– BMDCs appeared to be much more inert to treatments with F₇-PEI, F₁₃-PEI, F₇-PEI/OVA, F₁₃-PEI/OVA or a classical TLR4 agonist lipopolysaccharide (LPS), which indicates that F-PEI and F-PEI/OVA may possibly activate DCs via the TLR4 signalling pathway (Fig. 2c). Using murine TLR4 (NF-κB-SEAP/KI-[IL-8]Lucia) dual-reporter HEK293 cells with a stable transfection of mouse TLR4 (mTLR4) MD-2/CD14 genes, we further confirmed that our fluoropolymers and fluoropolymer-based NPs could, indeed, trigger TLR4 activation in a dose-dependent manner, especially when carrying OVA (Fig. 2d and Supplementary Fig. 7). Additional experiments further indicated that, beyond activating TLR4, the DC maturation triggered by F-PEI/OVA NPs may also be related to other pathways, such as the inflammasome signalling pathway (Supplementary Fig. 8a)²⁸.

Besides the TLR4-dependent endocytosis, we also investigated other endocytosis pathways for these fluoropolymer-based NPs by using dynasore, a dynamin inhibitor of clathrin-mediated endocytosis, or genistein, an inhibitor of caveolae-dependent endocytosis, and uncovered that both pathways should be involved in the internalization of these F-PEI-based NPs (Supplementary Fig. 9). Meanwhile, most of the OVA-FITCs delivered by F-PEI were not co-localized with lysosomes and instead evenly distributed within cells after 4–6 h, which indicates an efficient endosomal escape of the protein antigen delivered by our fluoropolymers (Fig. 2e, Supplementary Fig. 10). In contrast, the intracellular protein delivery by bare PEI appeared to be much less efficient. Furthermore, the F-PEI/OVA NPs after cell entry would dissociate to release the protein antigen, a behaviour favourable for antigen presentation by DCs (Supplementary Fig. 11).

The intracellular delivery of OVA-FITC by fluoropolymers F₇-PEI and F₁₃-PEI in the presence of bafilomycin A1, which prevents the acidification of endosomes²⁹, was also studied. We observed trapped F-PEI/OVA NPs in endosomes and/or lysosomes, which suggests that the pH buffering mechanism would, indeed, occur during the endosomal escape of F-PEI-based NPs (Supplementary Fig. 12). Notably, besides the pH buffering effect attributed to the PEI backbone, the fluoroalkyl chains may also play a unique role here in enhancing the cellular uptake and endosomal escape of F-PEI/OVA NPs. With hydrophobic and lipophobic characteristics, such fluoroalkyl chains would be easily fused with membrane lipids via hydrophobic interactions, and their lipophobic characteristic could ensure a rapid diffusion of the fluoroalkyl chains across the lipid membranes^20,30.

Fluoropolymer-based nanovaccines exhibit robust immune responses in vivo

In vivo experiments were next carried out to evaluate the immunization efficacy of F-PEI/OVA nanovaccines (Fig. 3a). The fluoropolymer-based NPs were similarly able to enhance the antigen uptake by DCs in the immunized mice (Supplementary Fig. 13). Consistent with in vitro results, vaccination with F₁₃-PEI/OVA triggered the highest level of CD86⁺CD80⁺ BMDCs in the inguinal lymph nodes (LNs) of treated mice compared with that induced by OVA alone or F₇-PEI/OVA treatment on day 3 after immunization (Fig. 3b–d and Supplementary Fig. 14). Notably, F₁₃-PEI/OVA also resulted in the highest level of CD11c⁺SIINFEKL-H-2Kb⁺ antigen cross-presented DCs in draining LNs five-day post-immunization (Fig. 3e). Furthermore, F₇-PEI and F₁₃-PEI could elicit Th1-polarized immune responses, as shown by an OVA-specific immunoglobulin (IgG)_2a/IgG₁ ratio of >1 (Fig. 3f), and the F₁₃-PEI/OVA nanovaccine could induce an effective production of IFN-γ (Fig. 3g).

**Fig. 3: In vivo immune stimulation by F-PEI/OVA nanovaccines.**

F-PEI/OVA-induced OVA-specific T-cell immune responses were then investigated on day 7 (Fig. 3b). Notably, the frequency of SIINFEKL-MHC-I tetramer⁺CD8⁺ T cells presented a sixfold increase in the F₁₃-PEI/OVA NP group compared with that in the OVA group, and a twofold increase compared with that in the F₇-PEI/OVA group (Fig. 3h,i and Supplementary Fig. 15). Typically, stronger antigen-specific T-cell mediated immune responses occur on the second exposure to the same antigen³¹. During this process, T cells rapidly eliminate any cells that express this surface antigen by secreting IFN-γ. On the second exposure to the same antigen, OVA peptide (SIINFEKL), IFN-γ ELISPOT (enzyme-linked immune absorbent spot) responses (Fig. 3j,k) and the relative ratios of IFN-γ⁺CD8⁺ T cells (Fig. 3l and Supplementary Fig. 16) in the splenocytes from the mice immunized with F₁₃-PEI/OVA were both found to be significantly increased, which demonstrates the robust in vivo antigen-specific T-cell immune responses triggered by the F₁₃-PEI/OVA nanovaccine. Notably, the adaptive T-cell responses triggered by F₁₃-PEI/OVA appeared to be much lower in Tlr4^–/– mice (Supplementary Fig. 17), which further demonstrates the TLR4-mediated immune activation pathway for our F-PEI-based nanovaccine.

To further assess the immune protection effect induced by the F-PEI/OVA nanovaccines, immunized mice were challenged with OVA-expressing B16-OVA melanoma tumour cells seven days post the final immunization (Fig. 4a). For mice injected with F₁₃-PEI/OVA NPs, their average tumour volume was considerably smaller than that in the other groups at all time points (Fig. 4b), and their survivals were significantly prolonged (Fig. 4c). Consistent with our previous observation (Fig. 3j,k), ELISPOT responses and the relative ratios of IFN-γ⁺CD8⁺ T cells (Supplementary Fig. 18) in the splenocytes of F₁₃-PEI/OVA-immunized mice collected on day 21 were found to be the highest among all the tested groups.

**Fig. 4: F₁₃-PEI/OVA nanovaccine inhibits tumour growth and prolongs survival in tumour-bearing mice.**

The therapeutic effect of the F-PEI-based nanovaccines was also evaluated using the B16-OVA melanoma tumour model. CpG oligonucleotide, a TLR-9 agonist, and aluminium (alum) were both chosen as the control adjuvant. In the PBS and OVA control groups, all the animals died within 25 days. The OVA + CpG, OVA + alum and F₇-PEI/OVA groups conferred a minor degree of immune therapeutic effect. In contrast, F₁₃-PEI/OVA-immunized mice maintained an evidently high survival rate, with 37.5% up to 60 days (Fig. 4d–g), which demonstrates the potential of F₁₃-PEI/OVA as a therapeutic cancer vaccine.

To facilitate in vivo tracking, F-PEI/OVA NPs were radiolabelled with ¹²⁵I (labelling on OVA) and injected at the tail base of mice for biodistribution studies at different time points. Significant retention of the nanovaccines was observed in the draining LN (Supplementary Fig. 19). Major organs, which included the liver, spleen, kidney, heart and lung, were collected and sliced for haematoxylin and eosin staining. No obvious sign of organ damage or inflammatory lesion was observed for mice seven days after the intradermal injection with F₁₃-PEI/OVA three times (Supplementary Fig. 20), which suggests that F₁₃-PEI/OVA NPs may be used as a safe and effective nanovaccine.

Fluoropolymer-based personalized nanovaccine for treating post-surgical distant tumours

Residual microtumours or circulating tumour cells may cause lethal tumour recurrence and metastasis months or even years post-operation. Cancer vaccines developed from lysates of surgically collected tumour cells represent a promising avenue for cancer immunotherapy^32,33,34. We carried out a more clinically relevant study by using the cancer cell membrane collected from resected tumours as the source of tumour antigens and F₁₃-PEI as the antigen carrier (F₁₃-PEI/Mem) to fabricate the F₁₃-PEI-based nanovaccine, which was then used together with ICB therapies to treat leftover tumours post-surgery (Fig. 5a).

**Fig. 5: F₁₃-PEI/Mem nanovaccine synergy with ICB therapies to treat distant tumours post-surgery.**

By simply mixing collected Mem with F₁₃-PEI, positively charged F₁₃-PEI/Mem NPs with an average size of ~50 nm could be easily obtained (Supplementary Fig. 21). Here, in our experiments, each C57BL/6 mouse was subcutaneously inoculated with two B16F10 melanoma tumours on both right and left flanks. Next, each mouse after surgical resection of its tumour on one side was intradermally injected at the site of the tail base with F-PEI-based vaccines that contained tumour cell membranes collected from the same mouse (Fig. 5a,b). The tumour growth on the opposite side of each mouse was then closely monitored (Fig. 5c and Supplementary Fig. 22). Notably, the tumour growth on the mice immunized with F₁₃-PEI/Mem was significantly delayed, compared that on unimmunized mice and mice immunized with Mem alone. Anti-PD1 (programmed cell death protein 1) therapy could further promote the antitumour efficacy of F₁₃-PEI/Mem. The therapeutic response of F₁₃-PEI/Mem + anti-PD1 appeared to be much better than that achieved by anti-PD1 alone or by Mem + anti-PD1. In fact, after the treatment with F₁₃-PEI/Mem + anti-PD1, 4 of the 6 mice became tumour free and survived for over 60 days (Fig. 5d), which demonstrates the therapeutic benefit of such combination therapy.

In addition to the B16F10 melanoma tumour, we then tested our strategy to treat CT26 murine colon tumours, which display a partial response to anti-CTLA4 blockade in preclinical experiments³⁵. Similar to the results in treating B16F10 tumours, F₁₃-PEI/Mem NPs + anti-CTLA4 treatment resulted in the most significant tumour growth suppression and 50% of those mice survived up to 60 days, much better than the therapeutic responses achieved in other control groups (Fig. 5e–g). The body weights of mice were not impacted by the treatment (Supplementary Fig. 23).

Next, we sought to verify that the observed antitumour immune responses triggered by such membrane-based nanovaccines were, indeed, related to the tumour-specific neoantigens expressed on the tumour cell membrane. The murine CT26 colon carcinoma model was recently reported to harbour a single-point mutation within the Slc20a1 protein (DKPLRRNNSYTSY[T/I]MAICGMPLDSFRA), and the neoepitope presented on MHC-I molecules^36,37. The IFN-γ⁺ ELISPOT responses (Supplementary Fig. 24) in the splenocytes from mice treated with F₁₃-PEI/Mem (or together with anti-CTLA4) were significantly increased after this neoantigen resimulation, which demonstrates a successful establishment of the neoantigen-specific immune responses after vaccination with our F-PEI-based nanovaccines combined with tumour cell membranes.

Furthermore, the number of tumour-infiltrating CD8⁺ T cells, which play a critical role in the antitumour immunity, was dramatically increased as the CD4⁺Foxp3⁺ T regulatory cells decreased in tumours on mice after the combined F₁₃-PEI/Mem + anti-CTLA4 treatment (Supplementary Fig. 25a–c). Meanwhile, more effector memory T cells (T_EM) were detected in the peripheral blood mononuclear cells of immunized mice (Supplementary Fig. 25d). To confirm the significance of T cells in the abscopal therapeutic response, T-cell blocking experiments were conducted using anti-CD8 antibody. After treatment with the CD8a monoclonal antibody (anti-CD8a), all the CD8⁺ T cells in the blood were blockaded by anti-CD8a, whereas the CD8⁺ T cells remained unaffected in mice treated with mouse monoclonal IgG (Fig. 5h). As expected, the blocking of CD8⁺ T cells dramatically impaired the therapeutic efficacy of F₁₃-PEI/Mem vaccination + anti-CTLA4 treatment in inhibiting opposite tumours, which indicates that the adoptive immune responses through tumour-infiltrating CD8⁺ T cells are important to the abscopal effect in our treatment strategy (Fig. 5i).

Fluoropolymer-based personalized nanovaccine for treating post-surgical orthotopic breast tumour metastases

Here, we propose a personalized cancer vaccine combined with ICB therapy for post-surgical immunotherapy of the recurrent tumours. Therefore, an orthotopic breast tumour model with spontaneous metastases was created by inoculating 4T1 tumour cells that expressed firefly luciferase (4T1-Luc) into the breast pad of each BALB/c mouse (Fig. 6a). To track the metastases of 4T1-Luc tumour cells, in vivo bioluminescence imaging was carried out every 5 days after day 10 (Fig. 6b). Compared with mice vaccinated with Mem only, vaccination with F₁₃-PEI/Mem could partly delay the tumour metastases. Of the 8 mice, 2 became tumour free and survived up to 120 days (Fig. 6c). Moreover, although anti-CTLA4 treatment alone appeared to be not so effective, F₁₃-PEI/Mem vaccination combined with anti-CTLA4 showed impressive therapeutic effects in inhibiting tumour metastases. The survival rate in this group also showed a dramatic increase to 62.5% (5 of the 8 mice) within 120 days (Fig. 6c), which demonstrates the great promise of combining fluoropolymer-based personalized cancer vaccines with ICB therapy to treat post-surgical tumour metastases, a major challenge in clinical cancer treatment.

**Fig. 6: F₁₃-PEI/Mem nanovaccine synergy with ICB therapy and the long-term memory immune response to treat orthotopic 4T1 tumours post-surgery.**

To assess the immune memory effects, mice that survived on day 120 after the first 4T1-Luc tumour-cell challenge in the F₁₃-PEI/Mem + anti-CTLA4 group were rechallenged with 4T1-Luc cells. Compared with the untreated mice, the tumour growth on the mice that survived after combination therapy (F₁₃-PEI/Mem + anti-CTLA4) appeared to be much slower (Fig. 6d). In fact, 2 of the 5 mice in this group showed no reoccurrence of the secondary tumour and all the mice exhibited extended survival compared to that of the age-matched control mice (Fig. 6e).

To further verify the immunological memory responses induced by our vaccination-based combination therapy, on day 120, peripheral blood mononuclear cells were collected to assess the memory CD8⁺ T cells. Intriguingly, for the mice that survived to 120 days after the first 4T1-Luc tumour cell challenge (F₁₃-PEI/Mem + anti-CTLA4), the percentage of T_EM and central memory T cells (T_CM) in the CD3⁺CD8⁺ T cells showed a significant increase compared with that in the control mice (Fig. 6f–h and Supplementary Fig. 26). Furthermore, one week after the mice were rechallenged with 4T1-Luc tumour cells, the serum levels of both TNF-α and IFN-γ (Fig. 6i,j) were significantly increased in this group (F₁₃-PEI/Mem + anti-CTLA4).

Conclusions

In this work, we report a new strategy to fabricate tumour nanovaccines based on fluoroalkane-grafted cationic polymers, F-PEI. Such F-PEIs self-assemble with protein antigens or tumour cell membrane antigens on simple mixing to form NPs with uniform sizes. Importantly, such fluoropolymer-based nanovaccines showed superior efficiencies in triggering antitumour immune responses compared those of vaccines using conventional immune adjuvants, such as CpG and alum, probably owing to their cytosolic antigen delivery ability to facilitate antigen cross-presentation, in addition to their ability to trigger DC activation, such as by stimulating the TLR4 signalling pathway. Therefore, F-PEI, by utilizing the unique hydrophobic and lipophobic chemical properties of fluoroalkane chains, is a unique type of antigen carrier and immune adjuvant to construct nanovaccines.

We found in our study that the tumour-specific immune responses triggered by fluoropolymer-based nanovaccines could be further amplified by ICB therapy. In particular, by utilizing the ability of fluoropolymer to package tumour cell membranes, personalized antitumour vaccines could be prepared by mixing F₁₃-PEI with cancer cell membranes separated from the resected tumour. As demonstrated in two subcutaneous tumour models and an orthotopic tumour model with spontaneous metastasis, such F₁₃-PEI/Mem personalized nanovaccines in combination with ICB therapy were able to treat metastatic tumours that cannot be completely removed by surgery. Furthermore, we observed a strong immune-memory effect to effectively protect the cured mice from tumour rechallenge. Such an enhanced personalized anticancer vaccine synergy with ICB therapy may have great value in personalized post-surgical cancer immunotherapy for clinical translation.

In addition to the fabrication of personalized cancer vaccines using surgically resected tumours, the local administration of F₁₃-PEI into tumours post-tumour ablation therapies, such as high-intensity focused ultrasound (HIFU) therapy (Supplementary Fig. 27), may enable in situ generation of cancer vaccines from tumour debris to achieve a strong abscopal effect to inhibit the growth of distant tumours. Beyond protein-based and cell-membrane-based vaccines demonstrated in this work, these fluoropolymers may be used in the development of nucleic acid-based vaccines (for example, mRNA vaccines), for which cross-membrane delivery, DC activation and antigen presentation are essential processes. Moreover, beyond vaccines against cancer, such fluoropolymer may be further extended to the fabrication of other types of important vaccines, for example, vaccines against viral infections such as COVID-19.

Methods

Materials

Branched PEI with a molecular weight of 25 kDa was obtained from Sigma-Aldrich. 3-(perfluoropropyl)-1,2-propenoxide and 3-(perfluorohex-1-yl)-1,2-propenoxide were obtained from J&K Scientific. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was purchased from Sangon Biotech. OVA, OVA_257–263 (SIIEFNKL) peptide and CpG oligodeoxynucleotides were obtained from InvivoGen. Injective alum and bicinchonic acid kits were obtained from Thermo Scientific. LPS was purchased from Sigma-Aldrich. The inhibitors, which included dynasore and genistein, were purchased from MedChemExpress. Phycoerythrin (PE) -labelled SIINFEKL-MHC I tetramer was purchased from MBL. Antibodies used in this study are listed in Supplementary Table 2. The Slc20a1 peptide (DKPLRRNNSYTSYIMAICGMPLDSFRA) was synthesized by GenScript.

Synthesis of F₇-PEI and F₁₃-PEI

Functionalized PEIs were synthesized by the dropwise addition of epoxides that bore fluoroalkanes into PEI in methanol at a feeding ratio of 36: 1. The solutions were stirred at room temperature for 48 h. The products were purified by intensive dialysis against methanol and double-distilled water (molecular weight cutoff 3,500 Da), and then lyophilized for characterization.

Preparation and characterization of F₇-PEI/OVA and F₁₃-PEI/OVA NPs

The synthesized F₇PEI and F₁₃-PEI amphiphiles were mixed with OVA in deionized water at different weight ratios for 30 min. The sizes and zeta potentials of the formed F₇-PEI/OVA and F₁₃-PEI/OVA NPs were measured with a Zetasizer Nano ZS (Malvern Instruments). The morphology of the NPs was observed with an FEI Tecnai F20 transmission electron microscope.

Animals and cells

Female C57BL/6 mice (6–8 weeks) and female BALB/c mice (6–8 weeks) were purchased from Nanjing Pengsheng Biological Technology Co., Ltd. Female OT-I transgenic mice (6–8 weeks) were a gift from X. Wang, Soochow University. Female gene knockout Tlr4^–/– mice were purchased from GemPharmatech Co., Ltd. Mice were housed in groups of 5 mice per individually ventilated cage in a 12 h light–dark cycle (8:00–20:00 light; 20:00–8:00 dark), with constant room temperature (21 ± 1 °C) and relative humidity (40–70%). All the mice had access to food and water ad libitum. The animal procedures were performed with ethical compliance and approval by the Institutional Animal Care and Use Committee at Soochow University. DC2.4 cells (a gift from C. Wang, Soochow University), B16-OVA cells (a gift from H. Liu, Soochow University) and B16F10 cells (Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured in DMEM (HyClone) with high glucose that contained 10% fetal bovine serum (FBS; Gibco) and 1% penicillin sulfate and streptomycin (PS, HyClone) at 37 °C in 5% CO₂. CT26 cells (Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) and 4T1-Luc cells (a gift from PerkinElmer Co.) were cultured in Roswell Park Memorial Institute (RPMI) 1640 media (HyClone) that contained 10% FBS (Gibco) and 1% PS at 37 °C in 5% CO₂. HEK-Dual Null (NF/IL8) cells (InvivoGen) were cultured with DMEM that contained 4.5 g l^–1 glucose, 2 mM l-glutamin, 10% FBS and 1% PS supplemented with 100 μg ml^–1 Normocin (Invitrogen) and 50 mg ml^–1 Zeocin (Invitrogen) at 37 °C in 5% CO₂. HEK-Dual mTLR4 (NF/IL8) cells (InvivoGen) were cultured with DMEM that contained 4.5 g l^–1 glucose, 2 mM l-glutamin, 10% FBS and 1% PS supplemented with 100 μg ml^–1 Hygromycin B Gold (InvivoGen), 100 μg ml^–1 Normocin and 50 μg ml^–1 Zeocin at 37 °C in 5% CO_2. The identity of the cell lines was frequently checked by their morphological features. All the cell lines were tested to be mycoplasma-free by their suppliers. No mycoplasma contamination was found before use.

In vitro cytotoxicity assessment, cross-presentation and DC maturation assay

BMDCs were differentiated from bone marrow cells isolated from the femurs and tibias of 6-week-old C57BL/6 mice according to an established method³⁸. Non-adherent and loosely adherent cells with a maturation level of about 20% (percentage of CD86⁺CD80⁺ cells in CD11c⁺ cells) on day 7 were collected and used for the studies. The cytotoxicities of PEI/OVA, F₇-PEI/OVA and F₁₃-PEI/OVA NPs were first measured by a well-established MTT assay. Generally, BMDCs incubated with F₇-PEI/OVA and F₁₃-PEI/OVA NPs at various concentrations were seeded in a 96-well plate at a density of around 5 × 10⁴ cells for 24 h. Only the OVA, F₇-PEI/OVA and F₁₃-PEI/OVA NPs at various concentration formulations without BMDCs are shown as the controls. Then, a standard MTT assay was used to determine the relative cell viability.

To assess DC cross-presentation, 10⁶ ml^–1 BMDCs were pulsed with OVA (10 μg ml^–1), F₇-PEI/OVA NPs ([OVA] = 10 μg ml^–1) or F₁₃-PEI/OVA NPs ([OVA] = 10 μg ml^–1) with different F-PEI:OVA ratios for 12 h. After various treatments, BMDCs were harvested, resuspended in flow cytometry (fluorescence-activated cell sorting (FACS)) buffer (PBS that contained 1% FBS), incubated with anti-CD16/32 at room temperature and stained with anti-SIINFEKL-H-2Kb-APC and anti-CD11c-FITC before analysis by flow cytometry (BD Accurit TM C6 Plus).

For in vitro DC maturation experiments, 10⁶ ml^–1 immature DCs were plated into 24-well non-treated dishes (Nunc) and simulated with OVA (10 μg ml^–1), F₇-PEI/OVA NPs ([OVA] = 10 μg ml^–1) or F₁₃-PEI/OVA NPs ([OVA] = 10 μg ml^–1) with different F-PEI:OVA ratios for 12 h. After various treatments, BMDCs were harvested, resuspended in flow cytometry (FACS) buffer (PBS that contained 1% FBS), incubated with anti-CD16/32 at room temperature and then stained with fluorophore-labelled antibodies against CD11c, CD86, CD80, CD40 and MHC-II for 30 min before analysis by flow cytometry (BD Accurit C6 Plus) and by FlowJo software. The supernatant was collected to measure cytokine TNF-α, IL-6 and IL-12p70 levels an using enzyme-linked immunosorbent assay (ELISA) assay according to the manufacturer’s protocols (Invitrogen).

In vitro CD8⁺ T-cell priming assay

Briefly, BMDCs were incubated with OVA ([OVA] = 10 μg ml^–1), F₇-PEI/OVA ([F₇-PEI] = 10 μg ml^–1, [OVA] = 10 μg ml^–1) or F₁₃-PEI/OVA ([F₇-PEI] = 10 μg ml^–1, [OVA] = 10 μg ml^–1) NPs at 37 °C for 12 h. The treated BMDCs were then washed by PBS that contained 0.1% BSA. CD8⁺ T lymphocytes from OT-I mice negatively selected by magnetic separation (MACS system, Miltenyi Biotec), according to the manufacturer’s instructions, were stained with the CellTrace CFSE Cell Proliferation Kit (Invitrogen) according to the vendors’ protocol. Treated DCs (10⁵ ml^–1) were then mixed with CFSE-stained splenic CD8⁺ T cells at a DC:T-cell ratio of 1:10 and incubated in round-bottom 96-well plates (Beyotime) for 72 h. Cells were washed by PBS that contained 1% FBS, incubated with anti-CD16/32 at room temperature and stained with anti-CD3-PE and anti-CD8a-APC, and finally analysed by FACS. The supernatant was collected and analysed for cytokine TNF-α and IFN-γ by ELISA according to the manufacturer’s protocols (Invitrogen).

In vitro cellular uptake and intracellular localization of antigens

To assess the cellular uptake of OVA, F₇-PEI/OVA and F₁₃-PEI/OVA by BMDCs, 10⁶ ml^–1 immature wt BMDCs or Tlr4^–/– BMDCs were plated in 24-well dishes and stimulated with FITC-labelled OVA (10 μg ml^–1), F₇-PEI/OVA-FITC NPs ([F₇-PEI] = 10 μg ml^–1, [OVA-FITC] = 10 μg ml^–1) or F₁₃-PEI/OVA-FITC NPs ([F₁₃-PEI] = 10 μg ml^–1, [OVA-FITC] = 10 μg ml^–1) for 12 h. BMDCs were harvested and trypan blue was added to quench the extracellular fluorescence. After washing three times, the treated BMDCs were incubated with anti-CD16/32 for 30 min at 4 °C before being stained with anti-CD11c-FITC-APC, to analyse the cellular uptake using a flow cytometer (BD Accurit C6 Plus). To elucidate the endocytosis pathways, wild BMDCs were incubated with endocytosis inhibitors dynasore (80 μM) or genistein (700 μM) for 30 min before the addition of F₇-PEI/OVA-FITC or F₁₃-PEI/OVA-FITCNPs. The untreated cells were tested as the control.

For the endosomal escape test, DC2.4 cells were treated with the F₇-PEI/OVA-FITC or F₁₃-PEI/OVA-FITC complexes for 2, 4 or 6 h, and then washed with PBS three times. The lysosome and nuclei were stained with Lyso-tracker red (200 nM) and Hoechst 33342, respectively. After being washed with PBS, the cells were observed with a laser scanning confocal microscope (LSM 880, Carl Zeiss). The weight ratio of F₇-PEI or F₁₃-PEI to OVA-FITC was 1:1.

The fluorinated polymer (F₇-PEI or F₁₃-PEI) was mixed with rhodamine B isothiocyanate (RB) at a molar ratio of 1:3 for fluorescent labelling. DC2.4 cells were incubated with F₇-PEI-RB /OVA-FITC or F₁₃-PEI-RB/OVA-FITC complexes for 30 min and the excess F-PEI-RB/OVA-FITC complexes were washed away with PBS. After that, the treated cells were incubated at 37 °C and observed with a laser scanning confocal microscope (LSM 880, Carl Zeiss) at indicated time intervals. The weight ratio of F₇-PEI-RB or F₁₃-PEI-RB to OVA-FITC was 1:1. Bafilomycin A1 (50 nM) (Yuanye Bio-Technology) was used to treat the DC2.4 cells for 1 h before adding the F-PEI/OVA-FITC complex to further investigate the endosomal escape behaviours of the vectors.

Endotoxin-level measurement

Endotoxin levels of various F-PEI-based formulations without dilution were detected according to the manufacturer’s protocols using the chromogenic LAL endotoxin assay kit (GenScript). According to US Food and Drug Administration guidelines, sterile water for injection and irrigation has an allowable endotoxin limit of 0.25 EU ml^–1.

LN analysis

The inguinal LNs were dissected from the vaccinated mice, and then processed through mechanical disruption before digestion for 30 min at 37 °C in RPMI-1640 (10% heat-inactivated FBS and 1% PS) that contained 1.5 mg ml^–1 collagenase IV(Sigma), 1.5 mg ml^–1 collagenase I (Sigma), 1.5 mg ml^–1 hyaluronidase (Sigma) and 0.2 mg ml^–1 DNase I (Sigma). The samples were then passed through 200-mesh nylon mesh filters to obtain single-cell suspensions, which were incubated with anti-CD16/32 for 30 min at 4 °C before being stained with anti-CD11c-APC, anti-CD86-PE and anti-CD80-PE/Cy7 to analyse DC maturation. For the analysis of DC cross-presentation, the obtained single-cell suspensions were incubated with anti-CD16/32 at room temperature before staining with anti-CD11c-FITC and anti-SIINFEKL/H-2Kb-APC. Flow data were acquired on a flow cytometer (BD Accurit C6 Plus) and analysed by FlowJo software.

ELISA assay

IL-1β from the supernatant of BMDCs after treatment with F-PEI/OVA and IFN-γ from serum of the vaccinated mice were determined by ELISA kits (Invitrogen) following standard protocols. The levels of OVA-specific IgG₁ and IgG_2a in the serum were also measured by ELISA following a literature protocol³⁸.

Tetramer analysis and peptide re-stimulation of splenocytes

The splenocytes were isolated by mincing the spleen from the immunized mice through 200-mesh cell strainers and the red blood cells (RBCs) were then lysed using RBC lysis buffer. Splenocytes stained with phycoerythrin-labelled SIINFEKL-MHC I tetramer, anti-CD8-APC and anti-CD3-FITC were analysed for the percentages of OVA-specific CD8⁺ T cells using the tetramer staining assay following the standard protocol.

For ELISPOT evaluations (BD Biosciences), 5 × 10⁵ splenocytes were seeded into each well in duplicate and incubated with antigens (10 µg ml^–1 OVA_257–264 peptides) or Slc20a1 peptide (DKPLRRNNSYTSYIMAICGMPLDSFRA). The plates were kept at 37 °C in 5% CO₂ for 24 h. Following the vendor’s protocol, an automated ELISPOT Plate Reader (AID iSpot) was used to determine the amount of IFN-γ SFCs and the data presented as SFC per one million cells or SFC per 0.5 million cells.

To demonstrate the cellular responses, 10⁶ splenocytes collected from immunized mice were incubated with OVA_257–264 peptide (10 μg ml^–1) in the presence of brefeldin A and monensin for 6 h. Then, the cells were stained with anti-CD3-FITC, anti-IFN-γ-APC and anti-CD8-PE according to the intracellular staining protocol before the flow cytometry measurement (BD Accurit C6 Plus).

Prophylactic and therapeutic studies with OVA-based nanovaccines

For the prophylactic study, different vaccines were intradermally injected at the tail base into the female C57BL/6 mice of each group at one week intervals three times. Seven days after the final vaccination, the vaccinated mice were subcutaneously challenged with 3 × 10⁵ B16-OVA cells. The tumour volume was measured with a caliper every other day and calculated according to following the formula: V = length × width × width/2 (mm³). Mice were euthanized when the tumour volume reached 1,500 mm³.

For the therapeutic study, female C57BL/6 mice (6 weeks old) were subcutaneously injected with 3 × 10⁵ B16-OVA cells. Female C57BL/6 mice were intradermally vaccinated with different kinds of vaccines, as described in the main text, on days 4, 11 and 18. Mice were euthanized when the tumour volumes reached 1,500 mm³.

Biodistribution of F₁₃-PEI/OVA NPs

To investigate their biodistribution, F₁₃-PEI/OVA NPs were radiolabelled with ¹²⁵I (labelling on OVA) following the standard protocol and injected at the tail base of the mice. At different time points, major organs, such as the heart, liver, spleen, lung, kidney, inguinal LN and the injection site, were collected and weighed, and the radioactivity was measured with a gamma counter (LB211, Berthold Technologies GmbH & Co KG).

The in situ B16F10, CT26 and 4T1-Luc cell membrane derivation

Tumours with a weight of about 600 mg were resected from B16F10, CT26 and 4T1-Luc tumour-bearing mice. These tumour tissues were then processed through mechanical disruption and digested by 1.5 mg ml^–1 collagenase IV (Sigma), 1.5 mg ml^–1 collagenase I (Sigma), 1.5 mg ml^–1 hyaluronidase (Sigma) and 0.2 mg ml^–1 DNase I (Sigma) for 45 min at 37 °C. The cells were filtered through nylon 200-mesh filters and washed with FACS buffer. RBCs were lysed using RBC lysis buffer. The CD45⁺ leukocyte cells were then removed from the obtained single-cell suspension using CD45⁺ magnetic beads (Miltenyi Biotec)³⁹ and about 6 × 10⁶ tumour cells could be collected. The obtained cell membranes were prepared by mechanical disruption in the presence of phosphatase inhibitor and protease inhibitor cocktails, as previously reported³³. Then, the membranes were obtained by centrifugation at 14,800 r.p.m. for 25 min to remove the cell nuclei and cytoplasm. The resulting packed membrane was washed in 0.2 mM ethylenediaminetetraacetic acid in DNase free/RNase free water (Invitrogen). The total membrane protein content was quantified with a bicinchonic acid protein assay kit (Thermo Scientific).

Preparation and characterization of F₁₃-PEI/Mem NPs

The synthesized F₁₃-PEI amphiphiles were mixed with the obtained cancer cell membrane in deionized water at a weight ratio of 1:1 for 30 min. The sizes and zeta potentials of the formed F₁₃PEI-MemNPs were measured with a Zetasizer Nano ZS (Malvern Instruments). The morphology of the NPs was observed with a FEI Tecnai G20 TWIN cytotransmission electron microscope.

Membrane-based nanovaccine synergy with ICB therapeutic study post-surgery

To study the therapeutic effects of the F₁₃-PEI/Mem nanovaccine after surgery, B16F10 cells were transplanted into the left flanks of C57BL/6 mice. After 9 days, a second tumour as the distant tumour (5 × 10⁵ B16F10) was inoculated into the right flank of each mouse. At day 10, mice were randomly divided into six groups for different treatments: blank (1), anti-PD1 (2), membrane (3), membrane + anti-PD1 (4), F₁₃-PEI/Mem (5) and F₁₃-PEI/Mem + anti-PD1 (6). Tumours in the left flanks of all the mice were resected by surgery to prepare the tumour cell membranes. On 4 (day 14) and 11 (day 21) days after surgery, the mice in groups 3 and 4 were intradermally vaccinated with membranes using a one-to-one correspondence. By simply mixing with the F₁₃-PEI, the obtained F₁₃-PEI/Mem formulations were intradermally injected into the surgical resection mice in groups 5 and 6 using one-to-one correspondence 4 (day 14) and 11 (day 21) days post-surgery. For groups 2, 4 and 6, the mice were intravenously administrated with anti-PD1 (20 µg per mouse for each injection) 5 (day 15), 8 (day 18), 12 (day 22) and 15 (day 25) days post-surgery. The sizes of the left tumour were measured every other day. Mice were euthanized when the tumour volumes reached 1,500 mm³. Similar experiments were conducted for the CT26 tumour model, except that anti-CTLA4 (20 µg per mouse for each injection) was used to replace anti-PD1 for the treatment study. The sizes of the left tumour were measured every other day. Mice were euthanized when the tumour volumes reached 1,000 mm³.

Tumour-infiltrating lymphocytes analysis

For analyses of the immune cells in distant tumours after the above immune combination therapy, tumours were resected from the CT26 tumour-bearing mice on day 21 (for the distant tumour, 10⁶ CT26 tumour cells were injected on day 9). These tumour tissues were then processed through mechanical disruption, digested and filtered to obtain single-cell suspensions, which were then stained with antibodies according to our previous report for flow cytometry analysis⁴⁰. For instance, a single-cell suspension was incubated with anti-CD16/32 and then stained with anti-CD45-Alexa Fluor 488, anti-CD3e-APC, anti-CD8a-Percp/Cy5.5 and anti-CD4-APC/Cy7 for 30 min at 4 °C before being fixed and permeabilized by eBioscience Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Cells were then stained with anti-Foxp3-PE for 30 min at 4 °C and analysed using flow cytometry (BD FACS Verse).

T-cell blocking experiments

BALB/c mice that bore CT26 tumours were treated with F₁₃-PEI/Mem + anti-CTLA4 after surgery, as described before. The treated mice were intravenously injected with anti-mouse-CD8 (for CD8⁺ T-cell depletion, 2.43, BioXcell) or mouse IgG (as Control, Southern Biotech) (20 μg per mouse), which were given three times, on days 0 (day 10), 5 (day 15) and 10 (day 20) after surgical removal of their first tumours. On day 3 (day 13) post-surgery, the blood T cells were collected to confirm the blockade of the CD8 T cells. The sizes of the tumours on the opposite side without surgery were measured every other day.

Orthotopic tumour model for F₁₃-PEI/Mem-triggered immunotherapy

To build the spontaneous metastatic 4T1 orthotopic murine breast cancer model, 2 × 10⁶ 4T1-Luc cells dispersed in PBS were injected into the breast pad of each BALB/c mouse. After ten days, we randomly divided the mice into six groups (n = 8): (1) surgery, (2) surgery + anti-CTLA4, (3) membrane, (4) membrane + anti-CTLA4, (5) F₁₃-PEI/Mem and (6) F₁₃-PEI/Mem + anti-CTLA4. Tumours with the weight of 600 mg in the orthotopic breast pad of mice were resected. Membranes could be collected by hypotonic lysing, mechanical membrane disruption and differential centrifugation. On days 14 and 21, mice were vaccinated with membranes or F₁₃-PEI/Mem by intradermal injection. Mice in groups 2, 4 and 6 were intravenously administrated with anti-CTLA4 (10 µg per mouse for each injection) on days 15, 18, 22 and 25. Spontaneous metastases of tumour cells were monitored by an IVIS spectrum imaging system analysed by Living Image software (Perkin Elmer). On day 120, the mice that survived were subcutaneously injected 5 × 10⁵ 4T1-Luc cells to evaluate the immunological memory effect.

Immunological memory responses triggered by our combination therapy

For the analysis of memory T cells, circulating peripheral blood mononuclear cells from the peripheral blood were harvested from the survived mice and stained with anti-CD3-FITC, anti-CD8-PerCP-Cy5.5, anti-CD62L-APC and anti-CD44-PE. Data analysis was carried out using FlowJo software. One week after the 4T1-Luc cells inoculation (day 127), the cytokines, which included TNF-α (Invitrogen) and IFN-γ (Invitrogen) from sera, were determined by ELISA kits following the standard protocols.

Statistical analysis

All the values in the present study are presented as the mean ± s.d. unless otherwise indicated in the figure captions. One-way analysis of variance was used for multiple comparisons when more than two groups were compared, and Student’s t-test was used for two-group comparisons. All the statistical analyses were carried out with GraphPad Prism software package (PRISM 8.0; GraphPad Prism Software), Excel 2016 or Excel 2019. Survival curves were obtained using the Kaplan–Meier method and compared by the log-rank test. The threshold for statistical significance was P < 0.05.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The main data supporting the results in this study are available within the paper and Supplementary Information. The raw and analysed datasets generated during the study are too large to be publicly shared, yet they are available for research purposes from the corresponding authors upon reasonable request.

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Acknowledgements

This work was partially supported by the National Key Research and Development (R&D) Program of China (2016YFA0201200, 2017YFE0131700 and 2019YFA0904500), the National Natural Science Foundation of China (51525203, 21725402 and 51761145041), the 111 Project, the Jiangsu Social Development Project (BE2019658), the Natural Science Foundation of Jiangsu Higher Education Institutions of China (19KJA310008) and the Collaborative Innovation Center of Suzhou Nano Science and Technology (Nano-CIC). The authors thank H. Liu for B16-OVA tumour cells and X. Wang for the OT-I mice. Our gratitude also goes to Y. Huang for valuable comments and suggestions.

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Authors and Affiliations

Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, China
Jun Xu, Qi Zhuang, Zongjin Yang, Zhiqin Cao, Ligeng Xu, Chenya Wang, Hanfei Wu, Ziliang Dong, Yu Chao, Chao Wang, Rui Peng & Zhuang Liu
South China Advanced Institute for Soft Matter Science and Technology, South China University of Technology, Guangzhou, China
Jia Lv & Yiyun Cheng
State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection and School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, China
Pei Pei & Kai Yang
Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, China
Yiyun Cheng

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Zhuang Liu
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Contributions

Z.L., Y.C., R.P. and J.X. conceived the project and designed the experiments. Z.L. and R.P. supervised the project. Z.L., R.P. and J.X. wrote the manuscript. J.X., J.L., Q.Z., Z.Y., Z.C., P.P., C.W., H.W., Z.D. and Y.C. carried out the experiments. J.L. assisted in taking and analysing the microscope images. C.W. and K.Y. supported the operation and analysis of the flow cytometer. All the authors discussed the results and commented on the manuscript.

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Correspondence to Rui Peng, Yiyun Cheng or Zhuang Liu.

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The authors declare no competing interests.

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Peer review information Nature Nanotechnology thanks Jeffrey Hubbell, Helder A. Santos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Figs. 1–27 and Tables 1 and 2.

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Xu, J., Lv, J., Zhuang, Q. et al. A general strategy towards personalized nanovaccines based on fluoropolymers for post-surgical cancer immunotherapy. Nat. Nanotechnol. 15, 1043–1052 (2020). https://doi.org/10.1038/s41565-020-00781-4

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Received: 27 July 2019
Accepted: 16 September 2020
Published: 02 November 2020
Issue Date: December 2020
DOI: https://doi.org/10.1038/s41565-020-00781-4

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