Unimolecular chiral poly(amino acid)s as adjuvants of nanovaccines for augmented cancer immunotherapy
Qi Wei , Hua Xin , Xiaolong Wang , Changjuan Qin , Yuanzhen Su , Di Li , Jianxun Ding
Chirality, an intrinsic geometric property characterized by mirror-image configurations, is a universal feature of matter in nature [1]. Chiral molecules exist as two enantiomers with distinct spatial arrangements. Biomacromolecules essential to life, such as amino acids and nucleic acids, inherently possess chirality [2,3]. As a result, chirality plays a crucial role in regulating biological and physiological processes. Notably, enantiomers often exhibit different pharmacological, toxicological, and other biological effects [4,5], which has motivated extensive research into their biomedical applications [6,7].
Chiral biomaterials have emerged as a key research area, particularly in drug delivery and inflammation modulation [8–10]. However, most current studies focus on exogenous strategies, such as chiral surface ligand engineering [11,12], leaving the intrinsic chiral effects of biomaterials underexplored. Amino acids, representative of natural chiral molecules, spontaneously form polymer systems with inherent chirality through stereoselective polymerization processes [13,14]. Our previous studies have reported that hydrogels based on D-type methoxy poly(ethylene glycol)-polyalanine hydrogel induce a stronger inflammatory response in subcutaneous tissue [15]. Additionally, a nanovaccine composed of poly(L-phenylalanine)-block-poly(D-lysine) complexed with ovalbumin (OVA) outperformed its L-lysine counterpart in activating dendritic cells (DCs) and enhancing anti-cancer efficacy [13]. These findings suggest that the immunomodulatory effects of poly(amino acid)s may be closely related to their chirality. Nevertheless, the mechanisms by which amino acid chirality modulates immune cell behavior remain poorly understood.
In this study, we synthesized dendritic polylysine with different chirality and loaded them with OVA to form unimolecular chiral nanovaccines. Prior to this, dendritic D-polylysine with varying branching density and degrees of polymerization were screened to assess their intrinsic immunostimulatory effects and physicochemical characteristics, including particle size distribution, zeta potential, and antigen loading efficiency. This optimization identified G0-PD-Lys50 as the most effective nanovaccine adjuvant. G0-PD-Lys50-OVA and its L-type counterpart, G0-PL-Lys50-OVA, were then compared focusing on their ability to activate DCs in lymph nodes (LNs) and stimulate cytotoxic T lymphocyte (CTL) responses. G0-PD-Lys50-OVA exhibited superior immunostimulatory effects. Additionally, in the LLC-OVA tumor model, the nanovaccine significantly enhanced tumor suppression and promoted stronger anti-cancer immune responses (Scheme 1). These results highlight chiral poly(amino acid)s as promising vaccine adjuvants capable of improving cancer immunotherapy by modulating immune responses stereoselectively.
To synthesize dendritic poly(D-lysine), polyamidoamine (PAMAM) macromolecules of different generations (G0, G1, and G2) were used as initiators to trigger the ring-opening polymerization (ROP) of N(ε)-benzyloxycarbonyl-L-lysine N-carboxyanhydride (Cbz-D-Lys NCA), yielding nine unimolecular dendritic poly(D-lysine) with varying branching density and polymerization degrees (Fig. 1A and Fig. S1 in Supporting information) [16]. The successful synthesis of these polymers was confirmed by proton nuclear magnetic resonance (1H NMR) and Fourier transform-infrared (FT-IR) spectra (Figs. 1B and C, and Figs. S2–S5 in Supporting information). As shown in Fig. 1B, the 1H NMR spectrum before deprotection (down) displayed characteristic resonances at 7.31 ppm (g), corresponding to the aromatic protons of the benzene ring (−C6H5). The resonances at 5.19 ppm (f) were attributed to the protons of the CH2 group in the benzyloxylcarbonyl moiety. The resonance at 4.60 ppm (a) was attributed to the −NH−CH−CO− bonds in the polylysine, while the resonances at 1.50–1.84 ppm (b–d) were attributed to the methylene groups (−CH2−CH2−CH2−). As illustrated in Fig. 1C and Fig. S4, the FT-IR displayed absorption peaks at 3315 cm−1 (νN−H), 3033 cm−1 (νC−H), 1658 cm−1 (νC=O), and 1248 cm−1 (νC−O), further confirmed the successful polymer formation (down). Following deprotection, the disappearance of the 7.31 ppm signal (g) in the 1H NMR spectrum and the 3033 cm−1 (νC−H) peak in the FT-IR spectrum (up) confirmed the effective removal of the benzyloxycarbonyl group (Figs. 1B and C, and Figs. S3 and S5). Notably, the molecular weights of the dendritic poly(D-lysine) increased with polymerization generation, as indicated by the progressive shift to earlier elution peaks in gel permeation chromatography (GPC). As demonstrated in Fig. 1D and Table S2 (Supporting information), nine structurally defined dendritic poly(D-lysine) with precisely controlled branching densities were successfully synthesized through optimized reaction conditions.
Subsequently, systematic in vitro screening of dendritic poly(D-lysine) was conducted to evaluate how branching density and polymerization degree affect immunostimulatory activity. Initial validation confirmed that dendritic PAMAM poly(D-lysine) induced potent immunostimulatory responses. Specifically, G0-PD-Lys25, G0-PD-Lys50, and G2-PD-Lys10 significantly enhanced interferon-gamma (IFN-γ) secretion in murine DC2.4 cells, exhibiting 26.9-, 28.6-, and 27.3-fold increases, respectively, compared to the control (Fig. 1E). Beyond DC activation, efficient antigen loading and delivery are essential for vaccine efficacy. The physicochemical characterization of dendritic PAMAM poly(D-lysine)-OVA nanocomposites revealed key structure–activity relationships. Antigen loading assays identified G0-PD-Lys50 and G1-PD-Lys50 as top performers, with loading efficiencies exceeding 80% (Fig. 1F), suggesting strong OVA-binding capacity. Furthermore, the changes in particle size and zeta potential of unimolecular polylysine micelles before and after complexation with OVA were investigated. The results showed that before complexation, the size distribution of polylysine nanoparticles ranged from 5.1 ± 0.9 nm to 21.6 ± 2.3 nm, with a zeta potential between 16.3 ± 0.5 mV and 21.3 ± 0.5 mV. After complexation with OVA, the size distribution of the polylysine nanovaccine ranged from 7.1 ± 0.3 nm to 22.8 ± 3.5 nm, and the zeta potential decreased to a range of 6.5 ± 0.4 mV to 19.1 ± 0.6 mV (Fig. S6 and Table S3 in Supporting information). The increase in particle size and the decrease in the zeta potential of the polylysine nanovaccine after OVA loading indirectly demonstrate the successful incorporation of OVA. The effectiveness of these nanovaccines also depends on lymphatic drainage. Recent studies have established that hydrodynamic diameter, particularly in the range of 10–200 nm, is a key determinant of interstitial transport and LN migration [17,18]. Nanovaccines smaller than this threshold are rapidly cleared, reducing LN accumulation. Accordingly, G0-PD-Lys10-OVA, G0-PD-Lys25-OVA, and G1-PD-Lys10-OVA were excluded from further consideration due to suboptimal diameters of 7.1 ± 0.3, 9.6 ± 0.1, and 9.2 ± 0.2 nm, respectively (Fig. 1G and Table S3). By integrating criteria of DC activation potency (> 25-fold IFN-γ induction), antigen loading efficiency (> 80%), and optimal nanoparticle size range (10–200 nm), G0-PD-Lys50 emerged as the lead candidate for further investigation into chirality-mediated immune modulation.
The chiral poly(L-lysine) (G0-PL-Lys50) was synthesized via PAMAM-G0-initiated the ROP of Cbz-L-Lys-NCA. GPC showed nearly identical elution profiles for G0-P(Cbz-L-Lys)50 and G0-P(Cbz-D-Lys)50, confirming similar molecular weight distributions (Fig. S7 in Supporting information). Circular dichroism (CD) spectra further confirmed the enantiomeric characteristics of G0-PL-Lys50, which exhibited a negative Cotton effect at 190 nm and a positive peak at 220 nm, while G0-PD-Lys50 displayed precisely inverted optical rotation patterns (Fig. S8 in Supporting information).
To investigate chirality-dependent immunomodulation, stable nanovaccines (G0-PL-Lys50-OVA and G0-PD-Lys50-OVA) were formulated through electrostatic complexation of OVA with respective chiral polylysine carriers (Figs. 2A–D). Dynamic light scattering (DLS) analysis showed hydrodynamic diameters of 11.7 ± 0.1 nm for G0-PL-Lys50-OVA and 12.2 ± 0.6 nm for G0-PD-Lys50-OVA in phosphate-buffered saline (PBS), respectively (Fig. S9A in Supporting information), with corresponding zeta potentials of 20.0 ± 0.8 and 20.4 ± 0.1 mV (Fig. S9B in Supporting information). The minimal differences in size, charge, and morphology ruled out physicochemical confounders, thereby isolating chirality as the critical factor in evaluating immune modulation.
Cell uptake, activation, and antigen processing by DCs constitute pivotal steps in bridging innate and adaptive immunity [19]. The uptake of Cy5.5-labeled nanovaccines was examined in DC2.4 cells. At 4 h post-incubation, both G0-PL-Lys50-OVA-Cy5.5 and G0-PD-Lys50-OVA-Cy5.5 exhibited similar endocytic efficiencies, achieving 2.3- and 1.9-fold higher uptake, respectively, compared to free OVA-Cy5.5. Notably, at 24 h, G0-PD-Lys50-OVA-Cy5.5 showed a 1.6-fold increase in intracellular accumulation relative to G0-PL-Lys50-OVA-Cy5.5 and a 6.8-fold increase over free OVA-Cy5.5 (Fig. S10 in Supporting information). This time-dependent chiral selectivity was further supported by fluorescence imaging, which revealed the preferential perinuclear localization of D-form nanovaccines (Fig. S11 in Supporting information). These findings underscore two key insights: (1) Chiral nanocarriers significantly enhance antigen delivery to DCs, and (2) D-form topology amplifies uptake in a time-resolved manner [20,21].
Free antigens internalized by antigen-presenting cells (APCs) are typically routed to lysosomes for enzymatic degradation, severely limiting their immunogenicity. Our study demonstrated that cationic nanovaccines overcome this limitation through pH-responsive endosomal escape [22]. As shown in Fig. 2E and Fig. S12 in Supporting information, the co-localization analysis showed enhanced lysosomal trafficking of both G0-PL-Lys50-OVA-Cy5.5 and G0-PD-Lys50-OVA-Cy5.5 in DC2.4 cells compared to free OVA-Cy5.5, with Pearson's correlation coefficients of 0.58 at 4 h and ≥0.7 at 8 h. However, from 8 h to 12 h, lysosomal signals diminished, indicating partial lysosomal escape and cytoplasmic antigen release (Fig. 2F). To investigate DC activation, immature bone marrow-derived dendritic cells (BMDCs) were treated with different formulations for 24 h, and the surface expression of co-stimulatory markers was analyzed. As illustrated in Figs. 2G–I, G0-PD-Lys50-OVA significantly upregulated the expression levels of CD80, CD40, and major histocompatibility complex class Ⅱ (MHC Ⅱ) compared to other groups. Specifically, relative to G0-PL-Lys50-OVA, G0-PD-Lys50-OVA increased CD80 by 2.0-fold, CD40 by 1.2-fold, and MHC II by 2.2-fold. These results demonstrate that G0-PD-Lys50-OVA more effectively promotes DC uptake, antigen processing, and activation in vitro, underscoring its superior immunostimulatory potential.
LNs serve as key sites for adaptive immune activation, particularly in the paracortical zone, which is enriched with DCs and T cells [23]. LN-migrating efficiency was evaluated using a footpad injection model. All experimental procedures involving mice were conducted following guidelines approved by the Ethics Committee of the Changchun Institute of Applied Chemistry (IACUC Issue No. CIAC 2022[0094]). In vivo fluorescence imaging revealed comparable accumulation of Cy5.5-labeled G0-PL-Lys50-OVA-Cy5.5 and G0-PD-Lys50-OVA-Cy5.5 in popliteal LNs at 12 and 24 h post-injection (Fig. 3A). Spatial profiling of LN sections demonstrated that both nanovaccines penetrated deeper into the paracortex region than free OVA-Cy5.5 at 12 h (Fig. 3B), localizing antigens with CD11c+ DCs to facilitate immune synapse formation. Notably, despite rapid lymphatic drainage mediated by cationic surfaces and sub-20 nm hydrodynamic sizes, short-term LN retention appeared to be governed primarily by charge and size, with chiral topology exerting a negligible influence on migration kinetics. These findings were consistent with recent studies highlighting the need for engineering strategies to prolong LN retention without compromising initial delivery [24].
To investigate the immunomodulatory effects of chiral nanovaccines on DC activation and antigen presentation under conditions of equivalent LN migration efficiency, a single-dose subcutaneous injection model was established. Draining LNs were harvested three days post-injection for flow cytometry (FCM) analysis (Fig. 3C). Experimental data indicated that the G0-PD-Lys50-OVA nanovaccine significantly enhanced immune activation. The proportions of CD11c+CD40+ and CD11c+CD80+ cells were 2.5- and 1.3-fold higher than in the OVA group (Figs. 3D and E). CD11c+SIINFEKL-H-2Kb+ cells, indicative of the ability of antigen cross-presentation, increased 1.8-fold compared to the OVA group (Fig. 3F). Compared to the G0-PL-Lys50-OVA, the G0-PD-Lys50-OVA nanovaccine consistently outperformed across all three markers by 50%, 40%, and 30%, respectively (Figs. 3D–F). Upregulation of CD40 and CD80 suggests the maturation of DCs and the ability to deliver co-stimulatory signals to T cells. In parallel, elevated SIINFEKL-H-2Kb expression indicates enhanced antigen processing and presentation, supporting T cell priming.
To evaluate the capacity of nanovaccines in eliciting antigen-specific T cell immunity in vivo, a triple-dose prime-boost immunization protocol with subcutaneous injections was implemented (Fig. 3C). On day 21, splenocytes were isolated and stimulated ex vivo stimulation with OVA257–264 peptide. FCM analysis revealed that the G0-PD-Lys50-OVA nanovaccine significantly enhanced the frequency of IFN-γ+ in CD8+ T cells, achieving a 2.5-fold elevation compared to the OVA group and a 1.9-fold increase over the structural control G0-PL-Lys50-OVA group (Fig. 3G). These results demonstrate that chiral nanovaccine enhances DC-mediated antigen cross-presentation, thereby promoting the differentiation of CD8+ T cells into effector subsets. This supports the establishment of robust vaccine-induced adaptive immune response and provides insight for studies on anti-tumor efficacy.
The therapeutic efficacy of tumor vaccines is a critical metric for assessing their translational potential. To evaluate this, a LLC—OVA tumor model was establised. As shown in Fig. 4A, a six-dose prime-boost immunization regimen was administered subcutaneously post-tumor implemented across four experimental groups: PBS, OVA, G0-PL-Lys50-OVA, and G0-PD-Lys50-OVA. Longitudinal monitoring up to day 23 revealed that tumor growth in the OVA and G0-PL-Lys50-OVA groups was statistically indistinguishable from that in the PBS controls. In contrast, G0-PD-Lys50-OVA treatment resulted in a 50% tumor growth inhibition relative to G0-PL-Lys50-OVA, demonstrating superior anti-tumor efficacy (Fig. 4B). Notably, all groups maintained body weight within 5% variation (Fig. 4C). To evaluate the biocompatibility of nanovaccine, critical organs, and serum samples were collected from mice at the experimental endpoint. The hematoxylin-eosin staining of major organs revealed no signs of inflammation, necrosis, or fibrosis (Figs. S13–S17 in Supporting information). Moreover, the analysis of serum biomarkers, including aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and creatinine, revealed no statistically significant differences among all treatment groups, underscoring the nanovaccine's excellent biocompatibility (Fig. S18 in Supporting information). Collectively, these results validate the potent and precise immunomodulatory capacity of the chirally engineered G0-PD-Lys50-OVA nanovaccine. This platform achieves robust anti-tumor immunity through optimized antigen delivery while avoiding systemic toxicity commonly associated with conventional vaccines.
To explore the mechanisms underlying anti-tumor immunity, comprehensive immune profiling of tumor tissues was performed at the end of treatment using FCM analysis. Within the tumor microenvironment, G0-PD-Lys50-OVA markedly elevated the proportions of CD11c+CD40+ (1.9-fold) and CD11c+MHC II+ (1.8-fold) among CD45+ cells compared to G0-PL-Lys50-OVA, concomitant with a 1.5-fold increase in CTLs. The proportion of myeloid-derived suppressor cells (MDSCs) decreased by 50% compared to control (Figs. 4D–G). These findings demonstrate that the chirally engineered vaccine orchestrates a mechanism by reprogramming the tumor microenvironment through enhanced immune activation and reduced immunosuppression, thereby enabling sustained tumor surveillance. This mechanism is essential for anti-tumor immunotherapy [25–27].
Building upon prior findings that D-configured poly(amino acid)s exhibit potent immunoadjuvant properties, this study introduces a unimolecular chiral polymer library using PAMAM dendrimers as molecular scaffolds [13,24]. Through comprehensive screening, G0-PD-Lys50 polymer emerged as a promising candidate nanoadjuvant. To confirm chiral-specific immunological effects, we synthesized its L-enantiomer counterpart, G0-PL-Lys50, and prepared nanovaccine formulations, G0-PL-Lys50-OVA and G0-PD-Lys50-OVA, via complexation with OVA, ensuring comparable morphology, particle size, and surface charge. Regarding immune activation, G0-PD-Lys50-OVA significantly enhanced the cell uptake, activation, and antigen presentation of DCs and effectively induced antigen-specific T responses. These immune responses translated into superior tumor suppression in the LLC—OVA model. Compared with G0-PL-Lys50-OVA, G0-PD-Lys50-OVA demonstrated more potent anti-tumor activity in the LLC—OVA model. The increased levels of mature DCs and CTLs, along with the decreased levels of MDSCs in the tumor tissues, indicate a significantly improved tumor immune microenvironment. This research provides a crucial theoretical basis for designing chiral nanovaccines and supports their potential as a versatile strategy in cancer immunotherapy.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Qi Wei: Writing – original draft, Validation, Investigation, Conceptualization. Hua Xin: Supervision, Funding acquisition, Conceptualization. Xiaolong Wang: Writing – review & editing. Changjuan Qin: Writing – review & editing. Yuanzhen Su: Writing – review & editing, Conceptualization. Di Li: Writing – review & editing, Supervision, Investigation. Jianxun Ding: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.
The study was financially supported by the National Natural Science Foundation of China (Nos. U23A20591, 82472144, 52273158, 52273159, and W2421115), the Science and Technology Department Project of Jilin Province (No. 20220204018YY), the Industrial Technology Research and Development Project of Jilin Province (No. 2023C040-8), the Health Research Talent Special Project of Jilin Province (Nos. 2023SCZ70 and 2024SCZ46), the Youth Innovation Promotion Association of Chinese Academy of Sciences (No. Y2023066), and the Bethune Project of Jilin University (No. 2023B01).
Supplementary material associated with this article can be found, in the online version, at doi:
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Scheme 1 Mechanism of unimolecular chiral poly(amino acid)s as adjuvants in nanovaccines for cancer immunotherapy. G0-PD-Lys50, a unimolecular chiral poly(amino acid), significantly enhances the endocytosis, activation, and cross-presentation of antigens by DCs. This, in turn, promotes the activation of CTLs, ultimately leading to tumor reduction.
Figure 1 Synthesis, characterizations, and screen of dendritic PAMAM poly(D-lysine). (A) Synthesis process of dendritic PAMAM poly(D-lysine). (B) 1H NMR spectra of G0-PD-Lys50 (up) and G0-P(Cbz-D-Lys)50 (down). (C) FT-IR of G0-PD-Lys50 (up) and G0-P(Cbz-D-Lys)50 (down). (D) GPC of dendritic PAMAM poly(D-lysine) with different branching. The letters a-i represent as: G0-P(Cbz-D-Lys)10, G0-P(Cbz-D-Lys)25, G0-P(Cbz-D-Lys)50, G1-P(Cbz-D-Lys)10, G1-P(Cbz-D-Lys)25, G1-P(Cbz-D-Lys)50, G2-P(Cbz-D-Lys)10, G2-P(Cbz-D-Lys)25, and G2-P(Cbz-D-Lys)50, respectively. (E) Quantification of IFN-γ secretion levels in DC2.4 cell culture supernatants across incubation with dendritic poly(D-lysine) of different branching after 24 h. (F) OVA loading efficiency of OVA-compound dendritic poly(D-lysine) nanovaccines. (G) Size distribution of OVA-compound dendritic poly(D-lysine) nanovaccines. The statistical data are represented as mean ± standard deviation (SD) (n = 3). P < 0.05. NS: no significance.
Figure 2 Preparation of G0-PL-Lys50-OVA and G0-PD-Lys50-OVA nanovaccines and their effects on lysosome escape and maturation on DCs. (A) Dh and (B) transmission electron microscope (TEM) images of G0-PL-Lys50-OVA. (C) Dh and (D) TEM images of G0-PD-Lys50-OVA. Scale bar: 20 nm. (E) Fluorescence imaging and (F) co-localization analysis of DC2.4 cells incubation with different vaccine formations for 8 and 12 h. Scale bar: 20 µm. Hoechst (blue), lysosome (green), and OVA-Cy5.5 (red). (G) CD11c+CD80+ cells, (H) CD11c+CD40+ cells, and (I) CD11c+MHC Ⅱ+ cells on BMDCs incubated with different vaccine formations for 24 h. The statistical data are represented as mean ± SD (n = 3; P < 0.05, ***P < 0.001).
Figure 3 In vivo biodistribution and immune response of nanovaccines. (A) Fluorescence images and semi-quantitative analysis of inguinal LNs at 12 and 24 h after injection with different vaccine formations. (B) Fluorescence images of biodistribution and intensity curves of different vaccine formations in LNs at 12 h. DAPI (blue), CD11c (green), and OVA-Cy5.5 (red). Scale bar: 500 µm. Right curve quantifies OVA-Cy5.5 along white dot line edge-to-center. (C) Scheme of the study of immune response in vivo. (D) CD11c+CD40+ cells, (E) CD11c+CD80+ cells, and (F) CD11c+ SIINFEKL-H-2kb+ cells in LNs. (G) IFN-γ+ in CD8+ T cells of restimulated splenocytes. The statistical data are represented as mean ± SD (n = 5 for D–F, n = 6 for G; P < 0.05, **P < 0.01, ***P < 0.001).
Figure 4 Elevating the treatment efficiency and anti-tumor immunomodulatory mechanism of nanovaccines. (A) Schematic illustrations of LLC—OVA treatment model. (B) Tumor volume and (C) body weight of mice injected with different vaccine formations from day 0 to 23. (D) CD11c+CD40+ in CD45+, (E) CD11c+MHC II+ in CD45+ cells, (F) CD8+ in CD3+ cells, and (G) CD11b+Gr-1+ in CD45+ cells in tumor tissues. The statistical data are represented as mean ± SD (n = 8 for B and C, n = 5 for D–G). P < 0.05, **P < 0.01, ***P < 0.001.
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