ISSN 1998-0124 CN 11-5974/O4

2020, 13(6): 1509–1518 https://doi.org/10.1007/s12274-020-2737-5

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Reverse immune suppressive microenvironment in tumor draininglymph nodes to enhance anti-PD1 immunotherapy via nanovaccinecomplexed microneedle Zhongzheng Zhou1, Jianhui Pang1, Xuanjin Wu1, Wei Wu1, Xiguang Chen1,2, and Ming Kong1 ()

1 College of Marine Life Science, Ocean University of China, 5 Yushan Road, Qingdao 266003, China 2 Qingdao National Laboratory for Marine Science and Technology, Wenhai Road, Qingdao 266237, China © Tsinghua University Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020 Received: 23 December 2019 / Revised: 26 February 2020 / Accepted: 28 February 2020

ABSTRACT The maturation of dendritic cells (DCs) and infiltration effector T cells in tumor-draining lymph node (tdLN) and tumor tissue are crucial for immunotherapy. Despite constructive progresses have been made with anti-programmed death-1 (anti-PD1) checkpoint blockade for immunotherapy, the efficacy of PD1/PD-L1 therapy deserves to be improved. Here, we constructed a novel transfersomes based nanovaccine complexed microneedles to enhance anti-PD1 immunotherapy via transdermal immunization for skin tumor therapy. Transfersomes were functionalized with DCs targeting moiety CD40, co-encapsulated with antigens and adjuvant poly I:C. Moreover, transdermal administration promoted accumulation in tumor-draining lymph nodes (tdLN), which could facilitate cellular uptake, activate DCs maturation and enhance Th1 immune responses. Using a mouse melanoma model, combined therapy of such nanovaccine complexed microneedles with pembrolizumab (PD1) was able to enhance cytotoxic T lymphocytes activation, promote infiltration and reduce regulatory T cells frequency in tdLN and tumor tissues, which achieved reversion of the immunosuppressive microenvironment into immune activation. This study highlighted the potential of transfersomes based nanovaccines complexed microneedles as an attractive platform for tumor immunotherapy.

KEYWORDS transfersomes, microneedles, immunosuppressive microenvironment, tumor draining lymph node, PD1

1 Introduction As cancer is a leading cause of death around the world, a number of strategies have been described to discover new anti- cancer drugs or technologies that could replace or supplement classical cancer therapies [1]. For skin cancer, immunotherapy is a promising manner in treatment, which aims to activate or enhance the patient’s cellular immune responses to increase the frequency or potency of antitumor T cells, and has the advantage of high specificity and low toxicity by acting on the immune system [2, 3]. In clinical trials for advanced melanoma, programmed death-1 (PD1) plays a pivotal role in regulation of tumor-infiltrating lymphocytes [4, 5]. Blocking antibodies (PD1) are utilized to disable the interaction between PD1 and its ligand programmed death-ligand 1 (PD-L1), stopping exhaustion of effector T cells and stimulate the immune system to eliminate cancer cells [6]. However, only 30% to 40% of patients respond to PD1, such as ipilimumab, demonstrating the efficacy of PD1 deserves to be improved [7, 8]. Combined therapy of PD1 with extra immune activation might exert synergetic efficacies.

Lymph nodes (LNs) play important roles in initiating adaptive immune response and could be a strategic target for vaccine delivery [9]. Furthermore, tumor-draining lymph node (tdLN) often acts as a mediator to induce anti-tumor response and involves in the malignant metastases of cancer cells [10, 11].

However, due to the continuous excessive stimulation of tumor secretion and accumulation of regulatory T cells, the maturation of dendritic cells (DCs) and the activation of effector T cells are inhibited in tdLN [9, 12]. The resulted immune compressive microenvironment of tdLN allow tumor cells to evade immune surveillance [13]. For immunotherapy, it is therefore crucial to reverse the local immunosuppression of tdLN, activate specific effector T cells, and remodel the anti-tumor immune microenvironment finally.

DCs are dedicated to antigen presentation, which is essential for regulating adaptive immunity and inducing cytotoxic T lymphocytes (CTL) responses [14–16]. Efficient DCs maturation is the key to inhibit the formation of immune tolerance and stimulate immune response in tdLN thereby. CD40 is a cell- surface receptor highly expressed on DCs and also a key signal for activating CD4+ T helper cells through binding to its ligand (CD40L) [17]. Targeting nanovaccines to DCs via CD40 antibody (CD40) facilitated vaccines entering into early-endosomes, promoted antigen cross-presentation and induced tumor-specific T cell responses [18, 19]. In addition, polyino-sinic:polycytidylic acid (polyI:C) is a synthetic analog of double-stranded RNA and can trigger both apoptosis and necroptosis, enhance type I inflammatory cytokines and provoke natural killer or CD8+ T cells to activate anti-tumor immunity [20, 21]. And it has been applied as an adjuvant to stimulate adaptive immune responses targeted to human DCs via activating TLR3 signaling

Address correspondence to kongming@ouc.edu.cn

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pathway [15, 22]. Skin is rich in antigen presenting cells (APCs) and distributed

with subcutaneous diffuse lymphatic vessels. We previously utilized transfersomes (T), an ultra-elastic or deformable vesicle, to enhance lymphatic absorption through transdermal route [23]. Microneedles (MNs) as an efficient transdermal vehicle have significantly evolved for four decades, and recently were applied in cancer immunotherapy [24–26]. MNs can overcome the challenges of the skin barrier for macromolecular drugs delivery [27]. We prepared dissolvable MNs based on hyaluronic acid (HA) enabled retention and sustained release of the laden active ingredients within skin [28]. We also constructed a transfersome based nanovaccines complexed microneedle platform for transdermal immunization and elicited enhanced Th1 immune responses [29].

In this study, transfersomes functionalized with DCs targeting capacity by assembling HA-GMS-CD40 on the surface. The CD40 functionalized transfersomes co-encapsulated with ovalbumin (OVA) or tumor associated antigen (TAA), PD1 and polyI:C were formulated and complexed with microneedle to facilitate delivery towards tdLN through transdermal administration. Targeting capacity to DCs, DCs maturation, effector T cells (CD4+ T and CD8+ T) activation and regulatory T cells frequency were evaluated. Through the adjuvant efficacies of the complex vaccine, the immunosuppressive microenvironment of tdLN was hypothesized to be reversed into immune activation, which was expected to enhance anti-PD1 immunotherapy (Fig. 1).

2 Materials and methods

2.1 Materials

Hyaluronic acid (HA, molecular weight 10 kDa and 2,000 kDa) was purchased from Bloomage Freda Biopharm Co., Ltd., China. Egg yolk phosphocholine (EPC), ovalbumin (OVA), CD40 antibody (CD40) and Monostearin (GMS) were obtained from Sigma-Aldrich, St. Louis. The polyI:C, TAA (TRP-2),

pembrolizumab (PD1) and the mouse cytokine Elisa kit were purchased from Solebo Biotechnology Co., Ltd. Bone Marrow-Derived Dendritic Cells (BMDC) were obtained through primary culture. Dendritic cells (DC2.4) were supplied by Microbiology Laboratory of Ocean University of China. The polyquaternium-7 (PQ-7) and the other chemical reagents used in the study were all analytical grade and acquired from Huasheng Chemical Reagent Company (Qingdao, China). All animal experiments in this study were conducted in accordance with the guidelines of Ocean University of China Laboratory Animal Ethical Committee.

2.2 Synthesis of functionalized transfersomes

2.2.1 Synthesis and characterization of amphiphilic HA

GMS was conjugated to HA by the formation of ester linkages through an EDC-mediated reaction. And the preparation details of HA-GMS (H) were described previously [30]. Furthermore, HA-GMS-CD40 (H-CD40) was prepared by linked CD40 to the side chain of HA-GMS, which could enhance the targeting to DC2.4. Briefly, 2.0 g HA-GMS was dissolved into phosphate buffer saline (PBS) solution and stirred for 5 min at room temperature. Then, EDC/NHS (50 mmol/L) solution was mixed with HA-GMS solution for 60 min. 20 μL CD40 was added into mixture solution and stirred for 12 h. The residual reactants were removed under dialyzing for three days, and the final mass productions were obtained by freeze-drying.

The structure of HA-GMS and H-CD40 was confirmed by

1H NMR on a Bruker ARX 400 MHz spectrometer (Germany).

2.2.2 Antigen-loaded transfersomes with or without HA-CD40

coatings

The transfersomes were prepared by the lipid film method [23]. 70 mg EPC was dissolved in chloroform/ethanol (1:1, v/v) and evaporated to dry to form lipid film under reduced pressure for 30 min. Then the lipid film was hydrated by PQ-7 solution (0.1 mg/mL, 5 mL) to obtain the transfersomes vesicles. Besides, OVA or TAA and polyI:C (1 mg/mL) were encapsulated by

 Figure 1 Schematics of functionalized transfersomes induced immune responses in tdLNs through microneedles assisted transdermal immunization.Transdermal administration enhanced accumulation in tdLN, αCD40 functionalized promoted cellular uptake by DCs, activated DCs maturation andnaïve T-cells differentiate into effector T lymphocyte, reduced regulatory T lymphocytes to achieve reversion of immnosuppressive microenvironment in tdLN into immune activation.

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dissolving in PQ-7 solution during the hydration process. HA-GMS or H-CD40 (1 mg/mL) was added to self-assembled on surface of transfersomes vesicles which were subjected to size reduction via sonication. Different components of transfersomes were shown in Table S1 in the ESM. The blank transfersomes (T) and HA-GMS modified transfersomes (H-T) were used as controls.

2.3 Characterization of transfersomes

The size analysis and zeta potential of transfersomes were determined by a Zetasizer ZEN 3600 Nano Series apparatus (ZEN, UK). The morphology images of transfersomes vesicles were obtained on transmission electron microscopy (TEM, 100 CXII JEOL Ltd., Japan).

To measure the polyI:C release profile from the transfersomes vesicles, 5 mL polyI:C loaded transfersomes suspensions were transferred into a dialysis bag. And the dialysis bag was placed into test tubes with 45 mL PBS solution (pH 7.2–7.4) in a shaking bed at a rate of 100 rpm and 37 °C. At predetermined time, 2 mL medium was withdrawn for analysis and replaced with fresh PBS solution. The absorbance was measured at 260 nm to evaluate the amounts of polyI:C that released from the transfersomes. Besides, the release profiles of OVA from transfersomes vesicles were measured with similar methods. 5 mL OVA loaded transfersomes suspensions were mixed into 45 mL PBS solution directly. 2 mL medium was removed and fresh release media was then added. The amount of OVA was also determined by BCA kit.

2.4 Cellular uptake and intracellular localization in

DC2.4

DC2.4 cells were used to evaluate cellular uptake efficiency. The cells were incubated with T(OVA), H-T(OVA), H-T(OVA+ polyI:C) and H-CD40-T(OVA+polyI:C) for 2 h, where HA- GMS or H-CD40 was labeled with FITC and transfersomes were labeled with DiI. Then, the cells were rinsed three times with PBS and fixed with 4% paraformaldehyde for 30 min, which were then stained by 20 μL DAPI for 5 min under room temperature. Then cells were observed by confocal laser scanning microscopy (CLSM) after washed with PBS three times.

For flow cytometry, the cells were treated with the above four groups formulations containing stained by DiI for 0.5 or 2 h. The harvested cells were washed with PBS and detected by flow cytometry.

For intracellular localization assay, DC 2.4 cells were incubated with free OVA and H-CD40-T(OVA+polyI:C) for 1 or 4 h, where the OVA was labeled by FITC. Subsequently, the cells were stained by Cell Navigator TM Lysosomal Staining Kit *Red Fluorescence* for 30 min at 37 °C. Furthermore, the cells were stained with DAPI for 10 min after fixing with 4% paraformaldehyde and then observed using confocal laser scanning microscope.

2.5 Activation of bone marrow-derived dendritic cells

(BMDCs)

BMDCs from Balb/c mice (female, 6–8 weeks old, Qingdao Daren Fortune Animal Technology Co. Ltd, China) were used to evaluate DCs maturation with functionalized transfersomes. In brief, for BMDCs primary culture, the cells were completely washed out of the marrow cavity and separated from bone marrow precursors with 1640 medium containing GM-CSF (20 ng/mL) and IL-4 (10 ng/mL). The prepared BMDCs were then incubated with T(OVA), T(OVA+polyI:C), H-T(OVA+polyI:C) and H-CD40-T(OVA+polyI:C) with an OVA concentration

of 20 μg/mL before incubation for 28 h. The cells culture supernatants were harvested to assess maturation of BMDCs through measuring the cytokines TNF-a and IL-12p70 with ELISA kits. Lipopolysaccharides (LPS) stimulated BMDCs and blank BMDCs were taken as a positive or negative control, respectively.

2.6 Fabrication and characterization of microneedles

MNs were fabricated using silicone molds with two steps as reported in previous study [28]. In brief, the needles of MNs were composed of 40% HA solution (Mw ~ 10 kDa) and the substrates were 2% HA solution (Mw ~ 2,000 kDa). The HA solution was evenly distributed into molds (20 mm × 20 mm, 35 × 35 array, pyramid geometry, 500 μm spacing between needle tips) and bubbles were excluded by centrifugation. Besides, to obtain transfersomes loaded microneedles, the needles of MNs were prepared with 40% HA solution, where the HA powders were dissolved in transfersomes suspension. The microneedles were peeled off from molds after drying under room temperature. Finally, 15 × 15 arrays with needle heights of 400 μm, base width of 300 μm and tips spacing of 500 μm were obtained by cutting the MN into pieces. The morphology of MNs were observed with optical microscope and SEM.

The SD rat (male, 4 weeks age, average weight: 200 g, Qingdao Daren Fortune Animal Technology Co., Ltd., China) cadaver dorsal skin was used to evaluate skin insertion capacity of microneedles. The skin was inserted by 15 × 15 array MNs with 15 N strength for 1 min, and stained by 0.2% trypan blue for 2 h after removing the MNs array. The extra dye was cleaned by ethanol swabs. The skin was imaged to identify the perforation. The insertion ratio was calculated by dividing the number of blue spots on the skin after insertion by the number of array needles. In addition, MNs fabricated with DiI-labeled transfersomes or FITC-labeled OVA, were applied on rat skin and observed by CLSM to observe the distribution of transfersomes or OVA in skin.

2.7 ELISA analysis of OVA-specific IgG and spleen

cytokines

15 Balb/c mice (female, 6–8 weeks old, Qingdao Daren Fortune Animal Technology Co., Ltd., China) weighting ~ 200 g were used for this study to measure immunity serum titer of OVA and isotypes by the routine ELISA. The dorsal skin of the mice was carefully shaved 24 h before administration and fasted overnight. The mice randomized into three groups: (1) the blank microneedles as control group; (2) the mice treated with H-CD40-T(OVA+polyI:C) loaded MNs (H-CD40-T(OVA+ polyI:C)/MNs); (3) the mice treated with free OVA-loaded microneedles. After hair removal and anesthesia, 4 pieces of 15 × 15 microneedle patches were inserted into rat skin and maintained for 15 min. This process was repeated 3 times every seven days. On day 21, the blood was collected and centrifuged to obtain serum for IgG/IgG1/IgG2a titer analysis using ELISA. Besides, the spleen cells of three group mice were collected to assess the secretion of cytokines IFN-γ, IL-2 and IL-4 for analyzing the immune induction effects with ELISA kits.

2.8 Mice and in vivo tumor models

The anti-tumor activity of functionalized transfersomes was evaluated with 60 female C57B6 mice (~ 20 g) as animal models. The B16F10 tumor cells (~ 106) were transplanted into left forelimb axilla of mice and the tumor growth was observed until the volume of tumor reached 50 mm3. The 60 tumor-bearing

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mice were randomly divided into 6 groups with different treatment: (I) The blank MNs group; (II) H-T(PolyI:C)/MN group; (III) H-CD40-T(PolyI:C)/MN group; (IV) H-CD40- T(PolyI:C+TAA)/MN group; (V) H-CD40-T(PolyI:C+TAA+ PD1)/MN group and (VI) H-CD40-T(PolyI:C+TAA+PD1)/ i.v. group. On the days 5, 7, and 9, microneedles therapy or intravenous injection was intervened every other day. It was emphasized that the tumor-associated antigen (tyrosinase- related protein-2 (TRP-2), H-Ser-Val-Tyr-Asp-Phe-Phe-Val- Trp-Leu-OH) was loaded in transfersomes instead of OVA, and the pembrolizumab (2 mg) was also encapsulated into transfersomes. The tumor volumes (mm3) of 6 mice out of 10 were measured by vernier caliper and calculated as (long diameter × short diameter2)/2.

On the day 13, the tdLN and tumor of 4 mice were excised for lymphocyte examination after the animals were sacrificed under anesthesia. The lymph node (0.02 mg) and tumor (0.1 mg) were grinded with 1 mL PBS and the obtained cells were stained with FITC-labeled anti-mouse antibodies against CD4, CD8 and PE-labeled CD25. The quantity and types of lymphocyte were detected by flow cytometry after washing with PBS. Furthermore, the immunohistochemistry of TDLN and immunofluorescence of tumor were equally observed to estimate the infiltration of immunocytes.

2.9 Statistical analysis

All experiments were performed at least in triplicate indepen-dently. All data were expressed as means ± standard deviation. Difference analysis among groups were calculated by One-Way ANOVA test. All tests were analyzed by SPSS and a p-values of p < 0.05 suggested the presence of significant difference.

3 Results and discussion

3.1 Synthesis and characterizations of functionalized

transfersomes

Functionalized transfersomes were designed to enhance anti- PD1 immunotherapy, which were loaded in microneedles to faciliate lymphatic delivery via transdermal administration. In order to evaluate the immune responses of transfersomes

based nanovaccine, OVA was used as model exogenous antigen. Instead, for anti-tumor assay, TRP-2 was encapsulated in nanovaccine as endogenous melanoma TAA. Moreover, pembrolizumab (PD1) was utilized to block the reaction between PD-1 and PD-L1 for combining therapy [31].

The synthesis of HA-GMS and HA-GMS-CD40 was con-firmed by amidation specific peaks on 1H NMR spectrum [32] (Fig. S1 in the ESM). The morphology and size of transfersomes were respectively characterized by TEM and dynamic light scattering (DLS). As shown in TEM images (Fig. 2(a)), transfersomes vesicles were approximately spherical with homogeneous dispersion. Compared with T(OVA), H-T(OVA) owned smaller mean size of 116.4 ± 9.5 nm and lower zeta potential of −13.3 ± 1.1 mV (Table S1 in the ESM). With PolyI:C encapsulation (encapsulation rates: 89.03%), the size of transfersomes increased obviously to 137 nm.

The release profiles of OVA (encapsulation rates: 65.24%) and PolyI:C from H-CD40-T(OVA+ PolyI:C) were assessed in vitro (Figs. 2(b) and 2(c)). A high initial burst release of more than 50% during 20 h was observed for OVA, followed by a sustained release till 80 h to achieve a cumulative OVA release of 70%. Some OVA molecular were adsorbed on the surface of transfersomes via electrostatic nonspecific interaction, leading to easier detachment from the transfersomes by centrifugation. In contrast, the smaller molecule PolyI:C remained slow release within 20 h. The results showed that the transfersomes had excellent protection of PolyI:C, which could prevent its premature release and degradation so as to achieve sustained drug effect. In addition, the biocompatibility of functionalized transfersomes was investigated by hemolysis and cytotoxicity assay. The hemolysis rate (HR%) of transfersomes was around 4% (Fig. S2 in the ESM), less than 5% of the upper limit for hemocompatibility material assessment [33, 34]. Furthermore, it showed no toxicity toward DC2.4 with relative cells growth rate above 75% (Fig. S3 in the ESM).

3.2 Cellular uptake and intracellular localization

inDC 2.4

Antigens cross-presentation is mainly carried out by DCs, and the cellular uptake efficiency and intracellular localization thereinto dramatically influence DCs maturation and subsequent

 Figure 2 Characterization of functionalized transfersomes. (a) Representative TEM images of transfersomes vesicles (I, T(OVA); II, H-T(OVA);III, H-T(OVA+PolyI:C); IV, H-αCD40-T(OVA+PolyI:C) ). Scale bar: 50 nm. OVA (b) and PolyI:C (c) release profiles from transfersomes, respectively. Error bars represent standard deviation (n = 3 independent experiments).

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immune responses [35]. The ability of functionalized trans-fersomes to deliver OVA and PolyI:C into DC2.4 was evaluated by CLSM. As shown in Fig. 3(a), for T(OVA) group, few red fluorescence appeared around nucleus. The cellular uptake efficiency was enhanced with HA-GMS (green fluorescence) assembled on the surface of transfersomes, which might be attributed to HA-CD44 receptor-mediated endocytosis [36]. Comparing group II with group III, the efficiency of cellular uptake had no significant difference regardless of PolyI:C loading. However, in the presence of CD40 (Group IV), the relative fluorescence intensity was much higher than the other groups, suggesting CD40 could enhance the targeting capacity of transfersomes to DC2.4 and further promote cellular uptake efficiency. The flow cytometry data of endocytosis were consistent with CLSM images (Fig. S4 in the ESM). The cellular uptake rate of H-CD40-T(OVA+PolyI:C) group was almost 100% due to CD40 mediated targeting effect after co-incubation for 2 h (p < 0.05).

The complete or partial escape of the transfersomes from the lysosomes into the cell plasma is critical for stimulating cellular immune response. The location of FITC-OVA-loaded transfersomes in DC2.4 was tracked after incubation for 1 or 4 h

(Fig. S5 in the ESM). After 1 h, the green fluorescence was colocalized with red fluorescence for both free OVA and functionalized transfersomes, indicating they mainly resided in lysosomes. In contrast, for the H-CD40-T(OVA+PolyI:C) group, the co-localization of two fluorescent colors was not observed and showed that H-CD40-T(OVA+PolyI:C) had escaped from lysosomes or endosomes, probably due to that functionalized transfersomes broke osmotic pressure balance of lysosomes through proton sponge effect [37]. For free OVA, without the protection of outer covering, it was quickly hydrolyzed by proteinase causing failure escape from lysosomes.

3.3 Efficient activation of BMDCs

The ability of DCs to regulate adaptive immunity is decided by their degree of maturation [38]. TNF- and IL-12p70 are representative stimulating factors of DCs maturation and important to elicit protective cellular immune responses [39]. The levels of both TNF- and IL-12p70 induced by PolyI:C loaded transfersomes showed significantly higher than T(OVA) without PolyI:C, indicating that the transfersomes with PolyI:C had stronger efficacy in BMDCs activation (Figs. 3(b) and 3(c)). The highest efficiency of DCs activation was found

 Figure 3 (a) Representative CLSM images of DC2.4 cells treated with various formulations (I, T(OVA); II, H-T(OVA); III, H-T(OVA+PolyI:C); IV, H-αCD40-T(OVA+PolyI:C)). Nucleus: blue fluorescence, transfersomes: red fluorescence, HA-GMS or HA-GMS-αCD40: green fluorescence, scale bar: 20 μm. TNF-α (b) and IL-12 p70 (c) secretion by BMDCs. Data depict mean ± standard deviation. * p < 0.05 compared with T(OVA) (n = 4).

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in H-CD40-T(OVA+PolyI:C) group, due to cellular uptake increased by introduction of CD40 antibody, which was equivalent to increasing the polyI:C content in cells.

3.4 Characterization of microneedles with or without

transfersomes loaded

The HA and entrapped functionalized transfersomes served as the matrix material for the structure of MNs to achieve transdermal vaccine delivery with ease administration. The optical microscope and SEM images showed that transfersomes complexed microneedles were uniformly arrayed with rectangular pyramid morphology (Figs. 4(a) and 4(b)). The insertion ratio (~ 84.4%) was calculated on the basis of trypan blue spots on the skin after MNs insertion (Fig. 4(c)). For histological examination, a clear triangular perforation was observed in stratum corneum, which coincided with the shape of micro-needles (Fig. 4(d) and Fig. S6(a) in the ESM). In addition, arrayed holes were found on the surface of skin after MNs insertion (Fig. 4(h)). Overall, the results revealed that the MNs could pierce into the skin homogeneously. For safety of the MNs, there was no significant erythema or edema observed 24 h after injection compared to the surrounding tissue (Fig. S7 in the ESM), indicating that HA based microneedles owned desirable potential for transdermal drug delivery with good biocompatibility.

To comfirm whether the transfersomes were encapsulated in the MNs, the distribution of transfersomes was detected by DiI fluorescence labeling. The tips of MNs were covered by red fluorescence, suggesting the transfersomes were effectively loaded in MNs (Figs. 4(e) and 4(f)). With DiI labeled transfersomes and FITC labeled OVA, CLSM images of skin cryostat section showed that transfersomes could be transited into skin by MNs and achieved drug release (Figs. 4(g) and 4(i), Figs. S6(b) and S8 in the ESM). Besides, the solubility in skin was an important property for dissolvable microneedles. For HA based microneedles, the tips appeared to dissolve

during 1 min and completely dissolved in rat skin within 15 min (Fig. 4(j)). Accordingly, the insertion time was set at 15 min in subsequent animal experiments.

3.5 In vivo lymphatic targeting and immune responses

of nanovaccine complexed microneedles

tdLN acts as a mediator to support adaptive immune cell priming responses, has increasingly explored as target for immunotherapy [40]. The intradermal fluorescence intensity was observed to assess the distribution of transfersomes (DiI labeled) in vivo. Four bright fluorescent regions appeared in mice dorsum, indicating that the tips of the microneedles retained in the skin (Fig. S9 in the ESM). The fluorescence intensity decreased gradually with time and eventually dis-appeared at 120 h. It could be concluded that transfersomes were transited continuously with time delivering loaded immune adjuvants or antigens to stimulate the body's immune responses constantly. To ascertain tdLNs targeting capacity, the fluorescences distribution in lymph nodes, hearts, livers, spleens and kidneys were observed (Fig. 5(a)). Compared with blank microneedles, the DiI fluorescence appeared at the lymph node 48 h after H-CD40-T(OVA+PolyI:C)/MNs transdermal administration, which reached the maximum at 96 h, indicating that the transfersomes were accumulated in the lymph nodes. The fluorescence intensity increased gradually in livers after 96 h, and there was no significant variations in heart and spleen. Fluorescence quantification results were consistent with the above analysis (Fig. S10 in the ESM). The photon quantum intensity was highest in lymph nodes (~ 25 × 104/g) and twice higher than that in livers at 96 h (p < 0.05).

To investigate the effect of H-CD40-T(OVA+PolyI:C)/MNs on antibody responses, serum IgG/IgG1/IgG2a titers were analyzed using ELISA. As shown in Fig. 5(b), the OVA-loaded H-CD40-T(OVA+PolyI:C)/MNs induced significantly higher antigen-specific IgG titers than the free OVA/MNs group (p < 0.05). Besides, the IgG2a and IgG1 levels were also measured to

 Figure 4 Characterization of microneedles with or without transfersomes loaded. (a) Photograph of a representative MN patch (scale bar: 500 μm). (b) SEMimage of the MN patch (scale bar: 500 μm). (c) Image of the trypan blue stained mouse skin after pricked by microneedles. (d) H&E-stained of rat skin after insertion of microneedles (scale bar: 200 μm). (e) and (f) Images of DiI-labeled transfersomes distribution in microneedles tips (scale bar: 200 μm). (g) The cryo-section of rat skin pricked by DiI-labeled transfersomes loaded microneedles (scale bar: 200 μm). (h) SEM images of perforations left on mouse skin after microneedles insertion. (i) Representative CLSM image showing transfersomes retention in mouse skin (red fluorescence: DiI-labeled transfersomes, green fluorescence: FITC labeled OVA, scale bar: 100 μm). (j) Dissolution of microneedles in rat skin along with time.

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determine the types of T helper cell immune responses. Compared with free OVA/MNs group, H-CD40-T(OVA+PolyI:C)/MNs could induce higher IgG2a/IgG1 ratios, demonstrating trans-fersomes based vaccines preferred to enahnceTh1 immune response [39].

In order to further determine the type of Th immune response induced by transfersomes, hallmark cytokines secreted by spleen cells were evaluated (Fig. 5(c)). IFN-γ and IL-2 were important Th1 type cytokine and their high expressions were an indicator for T cells activation. On the other hand, IL-4, produced by Th2 cells, effected on B cells and T cells which involved in humoral responses [41, 42]. Compared with OVA/ MNs, the expression levels of IFN-γ and IL-2 in H-CD40-T (OVA+PolyI:C)/MNs group were significantly increased. Although the expression of IL-4 was also increased, the relative increment was not so significant as IFN-γ and IL-2. It could be concluded that the transfersomes based nanovaccines were preferable to stimulate Th1 immune response, which was consistent with the results of the above serum antibody level.

3.6 Anticancer efficiency of nanovaccine complexed

microneedles

To evaluate anti-tumor efficiency of nanovaccine complexed microneedles, melanoma (B16F10) mouse model was established. TRP-2 was taken as endogenous melanoma TAA and loaded into nanovaccines instead of OVA. As shown in Fig. 6(c) and Fig. S11 in the ESM, the number of CD4+ T or CD8+ T cells, especially in tumors, was the lowest with blank microneedles administration. With CD40 conjugation and poly (I:C) loading in transfersomes, the content of lymphocytes increased significantly compared to blank MNs group, suggesting that effector T lymphocytes could be activated by DCs mediated cascade reactions. Compared with H-CD40- T(PolyI:C)/MN group, the lymphocytes infiltration was not obviously improved in the presence of TAA addition in H-CD40-T(PolyI:C+TAA)/ MN. It probably attributed to the fact that the tdLNs and tumors were infiltrated with TAA continuously and formed immunosuppressive microenvironment, and resulted in tolerant native T cells and inhibited DC maturation. Furthermore, higher

accumulations of lymphocytes infiltrated in lymph nodes and tumors treated with PD1 combined therapy (H-CD40- T(PolyI:C+TAA+PD1)/MN), especially for the CD8+ T cells in tumor (~ 9%), which was over two times higher than any other groups (~ 1%–3%) and exhibited the strongest anti- tumor efficacy. The results were consistent with the tumor volume variations (Figs. 6(b) and 6(d)). For intravenous injection group (H-CD40-T(PolyI:C+TAA+PD1)/i.v.), dramatical anti- tumor effect was found at the beginning of treatment. However, the tumor volume significantly accelerated in the following 4 days, suggesting the intravenously injected nanovaccines might be lack of targeting capacity and were quickly metabolized in vivo, thereby could not maintain long-term effect. In addition, 60% of mice survived 25 days treated with H-CD40-T(PolyI: C+TAA+PD1)/MN, while no mice survived in the control group (Fig. 6(e)).

Continuous excessive stimulation of tumor secretions and accumulation of regulatory T cells (TRegs) in tdLNs could induce immune tolerance, inhibit the activation and expansion of effector T cells, alter the expression of costimulatory molecules in dendritic cells and thus fail to produce effective anti-tumor effects [43]. As shown in Fig. S12 in the ESM, 12.3% of CD4+ CD25+ and 14.6% of CD8+ CD25+ TRegs appeared for mice treated with blank MNs, which related with the immunosuppressive microenvironment of lymph nodes. As treated with H- CD40-T(PolyI:C)/MN, the number of both TRegs isotypes reduced but no significant difference was found. The largest reductions of TRegs shown in H-CD40-T(PolyI:C+TAA+PD1)/ MN group, descended to 5.07% or 5.33% for CD4+ or CD8+ TRegs, respectively. The expansions of TRegs were inhibited in both tdLN and tumor tissues, demonstrating the immunosuppressive microenvironment ascribing from negative regulations of regulatory T lymphocytes might be alleviated to promote immune activation.

The infiltration of T lymphocytes into tdLNs and tumor tissues were also assessed. The tumor-infiltrating T lymphocytes from tdLNs and tumor were harvested and analyzed by the immunohistochemistry and immunofluorescence, respec-tively. For tdLNs (Fig. 7(a)), limited amount of T lymphocytes

 Figure 5 Transfersomes induced immune responses and OVA-specific antibody production. (a) Representative fluorescence images showing retention ofDiI labeled transfersomes in different tissues in vivo at different time after insertion of transfersomes complexed microneedles. (b) OVA-specific serum IgG, IgG1, IgG2a and IgG2a/IgG1 antibodies titer in mice sera measured by ELISA assay after treatment of OVA/MNs or H-αCD40-T(OVA+PolyI:C)/MNs.(c) Secretion of representative cytokines (IL-2, IFN-γ and IL-4) in supernatant of cultured spleen cells treated with different formulations. Data depict mean ± standard deviation (n = 4, * p < 0.05, ** p < 0.01).

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 Figure 7 The in vivo T lymphocytes infiltration in tdLNs and tumor tissue from with different groups administration: (I) blank MN; (II) H-T(PolyI:C)/ MN; (III) H-CD40-T(PolyI:C)/MN; (IV) H-CD40-T(PolyI:C+TAA)/ MN; (V) H-CD40-T(PolyI:C+TAA+PD1)/MN; (VI) H-CD40-T(PolyI: C+TAA+PD1)/i.v. (a) Representative immunohistochemical analysis of tdLNs showing CD4+ T and CD8+ T cells infiltration (scale bar: 50 μm). (b) Representative immunofluorescence analysis of T lymphocytes in tumor (pink: CD25+ TRegs cells; green: CD8+ T cells; red: CD4+ T cells; blue: nucleus, size bar: 100 μm).

(brown-labeled) could be seen with blank MNs group (I). In contrast, CD8+ and CD4+ T cells remarkably infiltrated both in tdLNs and tumors (Fig. 7(b)) with combined therapy, especially for group V. As noted, CD25 labeled TRegs (Pink fluorescence) remarkably decreased, consistent with the results of Fig. S12 in the ESM, confirming that the immunosuppressive microenvironment in lymph nodes was alleviated and enable activation of the individual autoimmune response to exert stronger anti-tumor efficacy.

4 Conclusions In conclusion, we described novel transfersomes based nanova-ccines complexed microneedles to enhance anti-PD1 immu-notherapy for skin cancer. The functionalized transfersomes based nanovaccine could achieve targeted delivery of antigen and adjuvants to tdLNs, promote DCs maturation and enhance Th1 immune responses. Combined therapy with the nanovaccines complexed microneedle and PD1 through transdermal immunization significantly promoted infiltration of CD8+ T and CD4+ T cells in tdLN and tumor tissues, and reducd regulatory T cells frequency to reverse immunosuppressive microenviron-ment into immune activation. Such nanovaccine complexed microneedles exhibited great potential in the research and clinical application for tumor immunotherapy.

Acknowledgements This work was supported by the National Natural Science

 Figure 6 In vivo anti-tumor effects on B16F10 melanoma mice models treated with different formulations. (a) Experimental protocol, B16F10 cells wereimplanted into each mouse on day 0, and the mice were treated every two days from day 5 to day 9. (b) Tumor volume on 11th day after treatment with various formulations: (I) blank MN; (II) H-T(PolyI:C)/MN; (III) H-αCD40-T(PolyI:C)/MN; (IV) H-αCD40-T(PolyI:C+TAA)/MN; (V) H-αCD40-T(PolyI:C+TAA+αPD1)/MN; (VI) H-αCD40-T(PolyI:C+TAA+αPD1)/i.v. (c) The percentages of CD4+ T or CD8+ T cells among all lymph nodes or tumors tissue cells measured by flow cytometry (n = 4). (d) Tumor volume variations in mice treated with different formulations (n = 6). (e) Survival rates of mice treated with different formulations (n = 6). Data depict mean ± standard deviation. *p < 0.05, compared with MN.

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Foundation of China (No. 31670972), and the Taishan Scholar Program, China.

Electronic Supplementary Material: Supplementary material (1H NMR spectra, hemolysis rates pattern, DC2.4 viability images, cellular uptake images, intracellular localization pattern, LCSM images, fluorescence intensity variations of DiI labeled transfersomes, flow cytometry images, and Table S1) is available in the online version of this article at https://doi.org/10.1007/s12274-020-2737-5.

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