Cancer vaccines designed to stimulate tumor-specific immunity are expected to be used in tumor treatment. Suitable cytoplasmic delivery of tumor antigens, stimulation of the innate immune system, and cross-presentation of tumor antigens by antigen-presenting cells are essential for inducing strong tumor-specific immunity. The nanocarrier system is a promising non-viral agent that can facilitate the cytoplasmic delivery of many different cargoes. At present, strategies including the use of "proton sponge" polymers, cell penetrating peptides, charge reversible molecules and pore-forming molecules have been developed to promote the cytoplasmic delivery of vaccines. The co-delivery of tumor antigens and adjuvants in a single nanoparticle has also been achieved to enhance the poor immunogenicity of tumor antigens.

In addition to this, endosomal capture and low immunogenicity of tumor antigens usually limit the efficiency of vaccination strategies. Therefore, the development of highly effective anti-tumor vaccines, especially personalized vaccines that can effectively induce human T cells to start, is still a difficult challenge to overcome.

Brief introduction:
In view of this, Liang Xingjie of the National Nanoscience Center and Li Jinghong of Tsinghua University and others have reported a proton-driven nano-deformation-based vaccine (NTV) for cancer immunotherapy. The results were published on Nature Nanotechnology with the title "Proton-driven transformable nanovaccine for cancer immunotherapy".

The overall process (focusing)
As shown in the figure below, the vaccine includes a nano deformer based on a polymer-peptide conjugate and a loaded antigen peptide (AP). In an acidic endosomal environment, vaccines based on nanomorphs have undergone drastic morphological changes from nanospheres (about 100 nm in diameter) to nanosheets (a few microns in length or width), thereby mechanically destroying the endosomal membrane and causing antigenicity. The peptide is directly delivered to the cytoplasm, and activates the immune system through the NLRP3 inflammasome pathway, thereby inducing strong and safe anti-tumor immunity.

Figure | Schematic diagram of proton-driven NTV for cancer immunotherapy

Specific design of nano vaccine
First, the polymer p(DMAEMA22-OGEMA4)-b-p(MAVE)30 was synthesized using reversible addition-fragmentation chain transfer polymerization (RAFT). Then two D peptides are selected: naphthalene-binding D peptide (NDP), pyrene-binding D peptide (PDP), which is hydroxylated and then combined with the hydroxyl group on the polymer through an acetal bond. The two self-assembled into spherical nanostructures at pH 7.4, but in an acidic environment (pH 5.6), the acetal bond is rapidly cleaved and peptides are released. The released NDP is reassembled into nanofibers (NT1), while PDP peptides are assembled into nanosheets (NT2). This may be because PDP has a larger aromatic structure than NDP, which may result in a stronger π- between molecules. π stacking interaction.

Then use chicken ovalbumin-derived peptide (OVA241-270) as the model antigen peptide, and use the double emulsion method for drug loading, and more than 80% of the peptides are released within 24 hours.

Figure | Design and Characterization of NTV

Can promote DC maturation without adjuvant
In cell experiments, both NTV1 and NTV2 can induce significant enhancement of cytoplasmic peptide fluorescence signal. For the surface presentation experiment of SIINFEKL epitope peptides, it was found that the level of SIINFEKL-H-2Kb complex in NTV2-treated cells at 24 h was much higher than that of cells in other groups, and this difference was still high even at 48 h .

The researchers also found that NTV2 effectively stimulates the maturation of BMDC by enhancing the expression levels of cytokines (IL-1β, TNF-α, IL-10 and IL-12p70) and DC maturation markers (such as CD40, CD80 and CD86). These results indicate that NTV2 effectively delivers AP to the cytoplasm and promotes the proliferation of DCs, thereby promoting long-term surface presentation of peptides in vitro and cross-presentation to CD8+ T cells.

It is worth noting that even without an adjuvant, NTV2 will promote DC maturation. NTV2-induced BMDC maturation may occur by activating the NLRP3 inflammasome pathway, and NTV2 enhances the antigen processing in DC.

Figure | NTV2 induces strong and continuous cross-presentation to CD8+ T cells

Obvious anti-tumor effect in a variety of tumor models
In vivo experiments in mice, the results showed that NTV2 promotes AP delivery to draining lymph nodes, which may be the result of the effective NLRP3-inflammasome pathway activation, and the delivered AP is mainly located in antigen presenting cells such as DC and macrophages. In addition, the NTV2 nano-vaccine showed an antigen-specific killing effect of about 87.3% in the body, which proved that the NTV2 nano-vaccine caused a strong tumor-specific immune response in the body.

In the evaluation of anti-tumor efficiency in the in vivo tumor B16F10-OVA model, NTV2 led to an improvement in total T cell infiltration, an increase in central memory and effect memory T cell populations, and a reduction in Treg population and PD-1 expression, thereby providing effective in vivo The tumor growth inhibitory effect and long-term anti-tumor immunity also indicate that the mechanism of DCs activation in vivo is the activation of the NLRP3-inflammasome pathway.

The other two tumor models (human papillomavirus (HPV)-E6/E7 tumor model and B16F10 neoantigen model) also have good curative effects, and in combination with anti-PD-L1 therapy, mice can survive for more than 83 days These results all indicate that the NT-based platform is a potential tool for personalized cancer immunotherapy in the future.

Figure | NTV2 inhibits tumor growth and prolongs survival in tumor-bearing mice

More than tumors!
In summary, the developed proton-driven deformable nano-vaccine not only promotes the cytoplasmic delivery and cross-presentation of AP, but also activates the NLRP3 inflammasome pathway, thereby enhancing anti-tumor immunity. Similarly, NT-based carriers can be easily adapted to encapsulate tumor neoantigens or be administered in combination with other checkpoint blockers or therapeutic agents. Researchers predict that when loaded with pathogenic antigens, NTV2 will help it be used to prevent and treat many infectious diseases.

Extended learning:
In recent years, many studies have continued to optimize the application of nano-medicine in the body through nano-deformation. Wonder Theory has previously reported many papers on nano-deformation. For details, please click on the following headings:

1. Top issue trend丨72 changes in nanomedicine
2. Imitate nature, better than nature | Wang Lei and others use nano deformation to trap bacteria! The effect exceeds the gold standard treatment
3. Nature Nanotech.: Nanoparticles deformed, staying in the tumor for a longer time
4. AM: Using shape-deforming hydrogels to guide cell network assembly
5. Chinese medicine Dayao Jing et al. Nano Lett.: Instant deformation! Nano assembly improves tumor drug delivery

references:
Gong, N., et al. Proton-driven transformable nanovaccine for cancer immunotherapy. Nat. Nanotechnol. (2020).
https://doi.org/10.1038/s41565-020-00782-3

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