Chuan Hu,Yujun Song,Yiwei Zhang,Siqin He,Xueying Liu,Xiaotong Yang,Tao Gong,Yuan Huang,Huile Gao
Sequential delivery of PD-1/PD-L1 blockade peptide and IDO inhibitor for immunosuppressive microenvironment remodeling via an MMP-2 responsive dual-targeting liposome
Immunotherapy,Cascade targeting,Liposome,MMP-2 responsive,Breast cancer,ICBs,IDO inhibitors,Immunosuppressive microenvironment
Intelligent responsive drug delivery system opens up new avenues for realizing safer and more effective combination immunotherapy. Herein, a kind of tumor cascade-targeted responsive liposome (NLG919@Lip-pep1) is developed by conjugating polypeptide inhibitor of PD-1 signal pathway (AUNP-12), which is also a targeted peptide that conjugated with liposome carrier through matrix metalloproteinase-2 (MMP-2) cleavable peptide (GPLGVRGD). This targeted liposome is prepared through a mature preparation process, and indoleamine-2,3-dioxygenase (IDO) inhibitor NLG919 was encapsulated into it. Moreover, mediated by the enhanced permeability and retention effect (EPR effect) and AUNP-12, NLG919@Lip-pep1 first targets the cells that highly express PD-L1 in tumor tissues. At the same time, the over-expressed MMP-2 in the tumor site triggers the dissociation of AUNP-12, thus realizing the precise block of PD-1 signal pathway, and restoring the activity of T cells. The exposure of secondary targeting module II VRGDC-NLG919@Lip mediated tumor cells targeting, and further relieved the immunosuppressive microenvironment. Overall, this study offers a potentially appealing paradigm of a high efficiency, low toxicity, and simple intelligent responsive drug delivery system for targeted drug delivery in breast cancer, which can effectively rescue and activate the body's anti-tumor immune response and furthermore achieve effective treatment of metastatic breast cancer.
Sequential delivery of PD-1/PD-L1 blockade peptide and IDO inhibitor for immunosuppressive microenvironment remodeling via an MMP-2 responsive dual-targeting liposome

Intelligent responsive drug delivery system opens up new avenues for realizing safer and more effective combination immunotherapy. Herein, a kind of tumor cascade-targeted responsive liposome (NLG919@Lip-pep1) is developed by conjugating polypeptide inhibitor of PD-1 signal pathway (AUNP-12), which is also a targeted peptide that conjugated with liposome carrier through matrix metalloproteinase-2 (MMP-2) cleavable peptide (GPLGVRGD). This targeted liposome is prepared through a mature preparation process, and indoleamine-2,3-dioxygenase (IDO) inhibitor NLG919 was encapsulated into it. Moreover, mediated by the enhanced permeability and retention effect (EPR effect) and AUNP-12, NLG919@Lip-pep1 first targets the cells that highly express PD-L1 in tumor tissues. At the same time, the over-expressed MMP-2 in the tumor site triggers the dissociation of AUNP-12, thus realizing the precise block of PD-1 signal pathway, and restoring the activity of T cells. The exposure of secondary targeting module II VRGDC-NLG919@Lip mediated tumor cells targeting, and further relieved the immunosuppressive microenvironment. Overall, this study offers a potentially appealing paradigm of a high efficiency, low toxicity, and simple intelligent responsive drug delivery system for targeted drug delivery in breast cancer, which can effectively rescue and activate the body's anti-tumor immune response and furthermore achieve effective treatment of metastatic breast cancer.

Nowadays, with the remarkable therapy outcome achieved by cancer immunotherapy, especially immune checkpoint blockers (ICBs), the field of oncology has been revolutionized. However, the heterogeneous tumor immune microenvironment has a great impact on immunotherapy outcome1, 2, 3, 4. For “altered” tumors, the infiltration of CD3+ and CD8+ T cells is high at the invasive margin but low at the tumor center. Importantly, those CD3+ and CD8+ T cells are mostly exhausted. Therefore, if the functions of these immune cells are restored, it is possible to achieve twice the result that killing tumor cells with half the effort. Due to multiple immune escape mechanisms in tumors, using ICBs alone cannot produce satisfactory results in reactivating exhausted T cells. What's more, there are many limitations while using monoclonal antibody-based ICBs, including high cost, poor tumor penetration, and triggering of immune-related adverse events (irAEs). Peptide-based ICBs and small-molecule-based ICBs have become active research areas. A peptide inhibitor of programmed cell death protein-1 (PD-1) signal pathway, AUNP-12, is designed from the extracellular domains sequences of human PD-1 (hrPD-1), displaying equipotent antagonism effects against PD-L1 expressed on antigen-presenting cells (APC) and many cancer cells, as well as PD-L2 expressed on macrophages, with potent activity in the rescue of lymphocyte proliferation and effector functions, and for better management of irAEs,. Importantly, AUNP-12 has the potential to become a target peptide for PD-1 signal pathway-related cells. Tumor cells overexpress indoleamine 2,3-dioxygenase (IDO-1), which plays an important role in tryptophan (Trp) catabolism. IDO-1 can decrease the Trp and produce a series of toxic kynurenine (Kyn) metabolites, mediating the suppression of T and NK cells, the generation and activation of T regulatory cells and myeloid-derived suppressor cells, promoting the immune toleration and is associated with poor prognosis of cancer,. It has been proved that IDO-1 inhibitors alone cannot achieve effective antitumor efficacy. As an effective IDO-1 inhibitor, navoximod (NLG919) has been applied to decrease the catalytic activity of IDO-1 enzyme and further lessen the number of T regulatory cells for restoring the function of cytotoxic T lymphocytes. But its high hydrophobicity and high dosage limit the clinical application. Therefore, the combination of IDO-1 inhibitors and ICBs may overcome the shortcomings of single treatment and achieve a better immunotherapeutic effect. Nanotechnology has made great success in solving the problems of drug delivery14, 15, 16, 17, 18. In particular, the emergence of stimuli-responsive drug delivery systems (DDSs) has great advantages in overcoming the complex drug delivery barriers facing in vivo through switchable morphological and surface charge, activatable cell-penetrating peptides, and on-demand drug release, etc.19, 20, 21, 22, 23, 24, 25, 26, 27, 28 However, stimuli-responsive DDSs still face many challenges in clinical application, such as complex design, preparation procedure, biocompatibility, and low drug loading capacity. In our previous study, we proposed a simple matrix metalloproteinase 2 (MMP-2) cleavable self-delivery nanoparticles (NPs) for codelivery of hydrophobic photosensitizer chlorin E6 (Ce6) and hydrophilic drugs metformin for synergistic photodynamic-immunotherapy,. Overexpressed MMP-2 in tumor site cleaved GPLGVRGD at a specific site between glycine (G) and valine (V), triggered tumor cell targeting sequence VRGDK-Ce6 re-exposure, thus realizing precise cytotoxicity killing of tumor cells. However, there are still two aspects that can be improved: (1) The tumor accumulation of NPs only depends on the enhanced permeation and retention (EPR) effect alone, which is often much less effective than expected31, 32, 33, 34. (2) Narrow scope of application. In order to design an ideal MMP-2 responsive self-delivery system, drugs need groups that can be linked with the bridging peptide sequence (GPLGVRGD), and the drug activity will not be affected after connection; the synthesized drug-peptide compound should have appropriate hydrophilicity and hydrophobicity. To solve these problems, we utilized liposomes as drug carriers, which are considered inherently biocompatible and biodegradable and are convenient for the modification of targeted peptides. Meanwhile, the hydrophobic lipid layer and the aqueous core can be loaded with hydrophobic and hydrophilic drugs without chemical bond connection. Due to the large amounts of drugs and small molecules liposomes can incorporate, they can be used as combinational agents for drug delivery35, 36, 37, 38, 39. Importantly, the preparation process of liposomes is relatively simple and mature, thus having the potential for clinical use40, 41, 42, 43. In this study, we developed an MMP-2 responsive dual-targeted liposome that co-delivered AUNP-12 and NLG919 to their respective target cells for synergistic immunosuppressive microenvironment remodeling. The GPLGVRGD sequence was used as a bridge, and the N- and C-terminal of GPLGVRGD were connected with two drug modules I and II, respectively, to form a responsive drug-polypeptide compound (I–GPLGVRGD–II). AUNP-12 was used as drug module I and then link to DSPE-PEG to form the lipid part of the liposome. IDO inhibitor NLG919 was encapsulated into the liposome to obtain a tumor cascade-targeted responsive liposome drug delivery system (NLG919@Lip-pep1) (Scheme 1). Mediated by AUNP-12, NLG919@Lip-pep1 first targeted the T cells and tumor cells that were involved in PD-1 signal pathway at the invasive margin tumor tissues. At the same time, overexpressed MMP-2 in the tumor site triggered the dissociation of AUNP-12, thus realizing the precise block of PD-1 signal pathway, and restoring the activity of immune cells. Meanwhile, the exposure of secondary targeting module II, VRGDC-NLG919@Lip-mediated deep tumor cells targeting, and released NLG919 further reverse T-cell exhaustion thus relieving the immunosuppressive microenvironment.

Soybean lecithin (SPC), cholesterol, and DSPE-PEG2000-OMe were purchased from Aiweituo Biotech Co., Ltd. (Shanghai, China). DSPE-PEG2000-Mal was purchased from Avanti Polar Lipids (Birmingham, AL, USA). AUNP-12-GPLGVRGD-Cys (pep1), AUNP-12-GplgvRGD-Cys (pep2), and AUNP-12-GPLGV-Cys (pep3) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). NLG919 was gained from Dalian Meilun Biotech Co., Ltd. (Dalian, China). Anti-mouse PD-L1 antibody (ab213480), antimouse CD34 (ab81289) antibody, and rhMMP-2 (ab125181) were obtained from Abcam Ltd. (Shanghai, China). PE-anti-mouse CD25 antibody (101,904) and APC-anti-mouse CD40 antibody (124,612) were purchased from Biolegend (San Diego, CA, USA). DiD was purchased from Solarbio (Beijing, China). Cy3-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00009-2) was obtained from Proteintech (Rosemont, IL, USA). Murine breast cancer cells line (4T1), mouse mononuclear macrophage leukemia cells (RAW264.7), mouse T lymphocytes (CTLL-2), and mouse dendritic cells (DC2.4) were obtained from the Chinese Academy of Science cells Bank (Shanghai, China). Female BALB/c mice (18–20 g) were obtained from Chengdu ENSIWEIER Biotech Co., Ltd (Chengdu, China). All animal experiments were approved by the Animal Experimentation Ethics Committee of Sichuan University.

Mix DSPE-PEG2000-pep1 (20.0 mg, dissolved in 1 mL of chloroform: methanol = 2: 1, v/v) and DSPE-PEG2000-Mal (50.7 mg, dissolved in 1 mL of chloroform) well, and then add 5 μL of triethylamine to the system. Stirred in the dark for 24 h at room temperature under a nitrogen atmosphere. Then the reaction mixture was condensed and then redissolved in DMSO. After 8 h of dialysis in DMSO, it was changed to dialyze in deionized water for 4 h. DSPE-PEG2000-pep1 was acquired by lyophilization. The molecular weight of the sample was determined by MALDI-TOF-MS (the matrix was sinapic acid). DSPE-PEG2000-pep2 and DSPE-PEG2000-pep3 were synthesized by the same method as above.

Thin film dispersion-ultrasonic method was applied to form liposomes. In detail, SPC, cholesterol, DSPE-PEG2000-OMe, DSPE-PEG2000-pep1 (62:33:4:1, molar ratio), and NLG919 (drug to lipid ratio = 1:30) were weighed and dissolved in chloroform (the total volume of organic solvent was 4 mL). After being mixed by ultrasound, the organic solvent was removed by rotary evaporator to form a uniform lipid film. Dry it overnight in vacuum at 25 °C. Add 4 mL of PBS (pH 7.4), and shake at 25 °C for 30 min at 180 rpm to hydrate (Zhicheng, ZWYR-200D, Shanghai, China). Ultrasonic cell crusher was introduced to emulsify the mixture (80 W, 5 s/5 s a cycle for 3 min). Unencapsulated drug was removed by freezing centrifugation (10,000 rpm, 20 min, Zonkia, KDC-140HR, Hefei, China). When preparing other control liposomes, DSPE-PEG2000-pep1 was replaced with the same equivalent DSPE-PEG2000-MAL and DSPE-PEG2000-pep2/DSPE-PEG2000-pep3.

500 μL of NLG919 (1.2 mg/mL, dissolved in ethanol: PBS = 1:1, v/v), NLG919@Lip-PEG, and NLG919@Lip-pep1 were placed in dialysis bags (molecular weight cut-off was 500 kDa), and dialyzed in 10 mL of release medium at 37 °C 5 mL of medium was transferred out at the designed time points and same volume of fresh medium was added at the same time. The concentration of released compounds was determined by UV at 274 nm.

4T1 cells (5 × 103 cells per well) were added to 96-well plates coated with low melting-point agarose gel. After about 3‒5-day incubation, spherical MCSs with a diameter of about 300 μm were selected and treated with DiD@Lip-PEG, DiD@Lip-pep1, DiD@Lip-pep2 or DiD@Lip-pep3 (2 μg/mL for DiD) for additional 12 h. Then MCSs were washed and fixed before being observed by confocal microscopy.

4T1 cells (3 × 105 cells) were transplanted into the third breast pad on the left side of female BALB/c mice. When the tumor size reached around 200 mm3, mice were randomly divided into four groups (n = 3). Mice were injected with DiD@Lip-PEG, DiD@Lip-pep1, DiD@Lip-pep2 or DiD@Lip-pep3 at an identical DiD dose of 500 μg/kg through tail vein. DiD fluorescence signals were imaged at 2, 6, 12, and 24 h post-injection. After the last imaging, mice were sacrificed and tumors and organs were collected for ex vivo imaging. Then tumors were sliced and stained with anti-CD34 antibody and anti-CD3 antibody. The spleens were sliced and stained with anti-CD3 antibody. The fluorescence signals were monitored using confocal microscope to investigate the deep penetration of DiD in tumor tissue and its co-location with T cells. Tumor cells or spleen cells (1 × 106 cells) were stained with anti-CD3 antibody to investigate the distribution of liposomes in T cells.

4T1 breast cells (3 × 105 cells) were transplanted into the third breast pad on the left side of female BALB/c mice. After 5-day growth, 4T1 cells (1.5 × 105 cells) were intravenously injected to simulate the high lung metastasis of breast cancer. When the tumor size reached around 60 mm3, mice were randomly divided into six groups (1: PBS; 2: NLG919; 3: NLG919@Lip-PEG; 4: NLG919@Lip-pep1; 5: NLG919@Lip-pep2; 6: NLG919@Lip-pep3, n = 7). The mice in each group were injected with indicated formulations (at an identical dose of 6.5 mg/kg NLG919). The treatment was repeated three times at 3-day intervals. Tumor size was measured every two days. The tumor volumes (V) were calculated with the following formula: After 16-day of primary tumor inoculation, parts of tumors and main organs were harvested and fixed in 4% paraformaldehyde and then used for H&E or TUNEL staining. In addition, the total number of lung metastatic lesions was counted to evaluate the anti-metastatic effects of each group (n = 7/group). According to the diameter (d) of the metastases, they can be divided into three grades: Grade I, d < 0.5 mm; Grade II, 0.5 mm < d < 1 mm; Grade III, d > 1 mm. The number of metastases was calculated as: I × 1 + II × 2 + III × 4. Blood and plasma were collected for routine blood indicators detection and blood biochemical indexes.

Fresh spleens and tumors in each group were collected to prepare single-cell suspensions. Flow cytometry was applied to analyze the anti-tumor immunity effects. For the examination of DCs maturation, lymph node cells were stained with anti-CD11c, anti-CD40, anti-CD80, and anti-CD86. For the investigation of the level of T cells, splenocytes were stained with anti-CD3, anti-CD4, anti-CD8, and anti-PD-1; tumor cells were stained with anti-CD3, anti-CD4, anti-CD8, anti-PD-1, anti-PD-L1, and anti-Foxp3. For the evaluation of memory T cell response, splenocytes were stained with anti-CD3, anti-CD4, anti-CD44, and anti-CD62L. To analyze the antigen-specific T cells, splenocytes were re-stimulated with 4T1 tumor cell lysate solution and then stained with anti-CD3, anti-CD8, and anti-IFN-γ. Double-antibody sandwich ELISA method was used to test the expression of inflammatory cytokines (IFN, TNF, IL-2) and inhibitory cytokines (IL-10) in tumor lysates.

The peptides we used have different responsiveness and targeting properties. AUNP-12-GPLGVRGD-Cys (pep1) can be cleaved by MMP-2, and the resulting -VRGDC sequence can further target tumor cells that overexpressed αvβ3; AUNP-12-GplgvRGD-Cys (pep2) cannot be cleaved by MMP-2; AUNP-12-GPLGV-Cys (pep3) can be cleaved by MMP-2, but there is no tumor cells targeting -VRGDC sequence. The three peptides-modified DSPE-PEG2000 lipids were successfully synthesized by MALDI-TOF-MS (Fig. 1A). Negative-charged liposomes were 140.8 ± 1.86 nm in hydrodynamic diameter and revealed spherical under transmission electron microscopy (TEM) imaging (Fig. 1B and C). The size and shape of liposomes were beneficial for tumor accumulation under EPR effects. The hydrodynamic diameter of NLG919@Lip-pep1 in PBS hardly changed within 10 days and PDI was within 0.3, indicating good stability in PBS. While the particle size in PBS containing 10% fetal bovine serum (FBS) increased at 48 h ascribed to protein adsorption. The PDI was stable within 48 h, indicating NLG919@Lip-pep1 is suitable for systemic administration (Supporting Information Fig. S1A and S1B). As shown in Fig. S1C, free NLG919 was released rapidly about 80% within 2 h, while the release amount of NLG919 in NLG919@Lip-PEG and NLG919@Lip-pep1 liposome groups was about 80% within 36 h. These results indicated that liposomes could delay drug release to a certain extent.

To examine the AUNP-12 targeting ability in vitro, we measured the cellular uptake by 4T1 cells, DC2.4 cells, RAW264.7 cells, and CTLL-2 cells. The above cells were first stimulated with IFN-γ to simulate immunosuppressive associated cells in tumor sites. AUNP-12 is a synthesized peptide sequence based on the structure of PD-1, with a high affinity for PD-L1, PD-L2, and CD80. In order to exclude the targeting ability mediated by the RGD sequence, DiD@Lip-pep3 was taken as an example to investigate the targeting of AUNP-12. As shown in Fig. 2A, DiD@Lip-pep3 had a significant increase in the uptake of CTLL-2 cells, 4T1 cells, and RAW264.7 cells compared with DiD@Lip-PEG. However, there is no significant difference in DC2.4 cells uptake between DiD@Lip-PEG and DiD@Lip-pep3 groups, this might be because PD-L1 expressed by DC2.4 cells used in this experiment did not have a binding site for AUNP-12. These results suggest that the modification of AUNP-12 polypeptide can make liposomes accumulated at the tumor site combine with the target cells of AUNP-12 to further improve delivery efficiency. To verify the effect of MMP-2 responsiveness on targeting, we incubated DiD@Lip-pep1, DiD@Lip-pep2, and DiD@Lip-pep3 with MMP-2, respectively, and investigated their uptake in 4T1 cells. The results showed that the uptake of DiD@Lip-pep1 in 4T1 cells after incubation with MMP-2 was significantly higher than the other two groups, indicating that the secondary targeting sequence of 4T1 cells (VRGDC) was responsively exposed after the incubation, which could increase the cellular uptake of 4T1 cells (Fig. 2B). The DiD@Lip-pep2 group did not have MMP-2 responsiveness, while the DiD@Lip-pep3 group was responsive but cannot be exposed to 4T1 cell-targeting sequence. So, there was no significant difference in the uptake between these two groups. Then, the 4T1 multicellular spheroids (MCSs), as a simulated 4T1 tumor model in vitro, were carried out to research the tumor penetration,. After 12-h incubation, the DiD fluorescence intensity of DiD@Lip-pep1 was stronger than the other control groups from 100 to 300 μm (Fig. 2C and D). Taken together, all those data indicated that MMP-2 could trigger the degradation of DiD@Lip-pep1. Thus the exposed secondary targeting molecule -VRGDC- can deliver DiD-loaded liposomes to reach a deeper penetration and stronger retention ability in 4T1 MCSs.

Next, the biodistribution of DiD@Lip-pep1 in 4T1-tumor-bearing mice was investigated. As shown in Fig. 3A, DiD@Lip-pep1 can selectively reach the tumor site after entering the body, and the fluorescence intensity of the tumor site gradually increases with the extension of time. In comparison, DiD@Lip-PEG preferentially distribute to the liver. As for DiD@Lip-pep2 and DiD@Lip-pep3, some of it may initially accumulate at the tumor site, but with time, it gradually distributed to liver and other sites, indicating that AUNP-12 polypeptide could mediate initial tumor-targeting. DiD@Lip-pep1 had the highest accumulation at tumor sites and stronger liver fluorescence signal 24 h after administration, showing that DiD@Lip-pep1 has good tumor targeting and retention ability (Fig. 3B and C). The tumor slices were stained with an endothelial marker, anti-CD34, to evaluate the penetration effects. We found that DiD@Lip-pep1 had the highest fluorescence intensity of DiD at the tumor site, and most of the fluorescence intensity was distributed at the tumor microvessels far away from CD34, indicating that once accumulated, DiD@Lip-pep1 were hard to be pumped out into the systemic circulation (Fig. 3D). Flow cytometry of tumor cells further confirmed that DiD@Lip-pep1 had the highest retention at the tumor site (Fig. 3E). These results jointly demonstrate that DiD@Lip-pep1 can first be efficiently stored at the tumor site via AUNP-12, and then release secondary targeting sequence in response to MMP-2 highly expressed in tumor tissue. DiD@Lip-VRGDC, which can target αVβ3 integrin receptor highly expressed on tumor cells, leading to a high targeting and retention at the tumor site.

We further assessed the tumor growth suppression of NLG919@Lip-pep1 after systemic administration. The tumor growth rates of each preparation group were as follows: PBS >> NLG919@Lip-pep3≈free NLG919 >> NLG919@Lip-pep2≈NLG919@Lip-PEG >> NLG919@Lip-pep1 (Fig. 4A and B). Compared with the PBS group, the free NLG919 group showed a slower tumor growth rate, indicating that NLG919 alone has a certain antitumor effect. Due to the EPR effect and PEG modification, the NLG919@Lip-PEG group showed better tumor growth inhibition compared with the free NLG919 group. But single immunotherapy still can't control tumor growth well. The inhibition rate of NLG919@Lip-pep1 at the tumor in situ reached 73% (consider PBS group as 0%), which was significantly better than NLG919@Lip-pep2 and NLG919@Lip-pep3 group, revealing the advantage of cascade-targeting strategy. To further verify the anti-tumor effect of NLG919@Lip-pep1, paraffin sections of tumor tissues were taken for H&E staining, and the pathological changes were observed intuitively by panoramic scanning images. As shown in Fig. 4C, large areas of nuclear damage loss were observed in tumor tissue sections of the NLG919@Lip-pep1 group. To obverse the cell apoptosis, we also scanned tumor tissue sections after TUNEL staining. As shown in Fig. 4D, the fluorescence signal of FITC in the NLG919@Lip-pep1 group was significantly stronger than in other control groups, demonstrating that a large area of apoptosis was observed. The apoptosis of tumor sites was negatively correlated with the tumor growth curve and tumor quality. In order to investigate the effect of NLG919@Lip-pep1 on inhibiting lung metastasis, the pulmonary metastatic nodules in each group were graded and counted after the treatment. The number of pulmonary metastatic nodules showed a slightly different trend compared with the growth rate of the in-situ tumor (Fig. 5A). The number of nodules in descending order was PBS ≈ free NLG919 >> NLG919@Lip-PEG >> NLG919@Lip-pep3 ≈ NLG919@Lip-pep2 >> NLG919@Lip-pep1. H&E staining showed a consistent trend (Fig. 5B). In addition, the body weight in each group showed a similar trend, and no obvious histological damage in main organs, including heart, liver, spleen, and kidney, were observed in any treatment groups under H&E staining (Supporting Information Figs. S3C and S4A). Then, the blood and serum were collected for blood routine and blood biochemical examination to further evaluate the systemic toxicity (Fig. S4B). Compared with the PBS group, there were no significant differences in red blood cells, platelets, mononuclear cells, and hemoglobin in other treatment groups, indicating that NLG919@Lip-pep1 had good blood compatibility. And there was also no significant difference between the treatment groups and the PBS group in liver function indicators (alanine aminotransferase and aspartate aminotransferase) and renal function indicator (creatinine value), which indicated that NLG919@Lip-pep1 had a high biosafety. Hence, the overall results indicated that NLG919@lip-pep1 is a potential high-efficiency and low-toxic anti-breast cancer strategy.

As an MMP-2 responsive cascade-targeting liposome, NLG919@lip-pep1 can efficiently accumulate to the tumor site first, accurately deliver and release AUNP-12, expose secondary targeting molecules, thus delivering NLG919, which reduces IDO expression in tumor cells, and activates the anti-tumor immune response by synergistically restoring the activity of T cells and reversing the immunosuppression of tumor cells. To confirm the above speculation, we evaluated the ability of in vivo anti-tumor immune response to further verify its anti-cancer mechanism. We first examined the maturation of dendritic cells (DCs) in lymph nodes. NLG919@lip-pep1 group significantly promoted DCs maturation, and the percentages of CD40+CD80+ (Fig. 6A and B) and CD40+CD86+ (Fig. 6C and D) were 1.9- and 2.8-fold higher than PBS group, respectively. The level of cytokines in tumor lysis can directly reflect the immune response of the tumor site, compared with other treatment groups, the pro-inflammatory cytokines IFN-γ (Fig. 7A, Figs. S4C and S4D), TNF-α (Fig. 7B) and IL-2 (Fig. 7C) in NLG919@Lip-pep1 group significantly increased. Furthermore, IL-10 (Fig. 7D), reflecting tumor immunosuppression, was obviously decreased after the treatment. Auxiliary T lymphocytes can promote an anti-tumor immune response by regulating the cellular and humoral immune response. Cytotoxic CD8+ T lymphocytes can directly kill tumor cells by releasing cytokines such as granzyme and perforin. Compared with other treatment groups, the proportion of auxiliary T lymphocytes (CD3+CD4+) in tumor tissue significantly increased in the NLG919@Lip-pep1 group (Fig. 7E) and the absence of significant changes in spleen may indicate that NLG919@Lip-pep1 does not affect the normal immune function of spleen (Fig. 8A and B). The ratio of CD3+CD8+ T cells in tumor tissue and spleen also increased in the NLG919@Lip-pep1 group (Figure 7, Figure 8A and C). Regulatory T lymphocytes (Tregs) negatively regulate the immune response and mediate the immunosuppressive tumor microenvironment. There was no obvious trend between the PBS group and the NLG919@Lip-pep1 group in both tumor and spleen (Figure 7, Figure 8D). The ratio of CD8+ T cells to Tregs can intuitively reflect the level of the anti-tumor immune response at the tumor site. As shown in Fig. 7H, compared with the PBS group, the proportion of CD8+ T cells/Tregs in the NLG919@Lip-pep1 group was increased, indicating a positive anti-tumor immune response. AUNP-12, an inhibitor of PD-1 signal pathway, has been shown to block PD1/PD-L1 and the combination of PD1 and PD-L2. It can regulate the expression of PD-1 through negative feedback. As shown in Fig. 7I, the percentage of PD-1hi CD8+ T cells in tumors was decreased in all drug administration groups. We also found that the effector memory T cells (Tems, CD44+CD62low), which reflects the capacity to call up the adaptive immune responses to suppress tumor recurrence and metastasis after the treatment, was considerably increased in NLG919@Lip-pep1 group (Fig. 8E). In addition, when splenocytes were re-stimulated with 4T1 tumor cell lysate solution, significant enhancement of tumor-specific IFN-γ-secreting CD8+ T cells were detected in the NLG919@Lip-pep1 group (Fig. 8F). All of the above results signified that the combination therapy can effectively stimulate the immune response and long-term memory of the mouse immune system against 4T1 tumor cells, synergistically regulated the immunosuppressive tumor microenvironment, restored T cells function, thus efficiently inhibiting the occurrence of in-situ tumor and distal lung metastasis, which may hold great potential for the treatment of breast cancer.

In summary, we reported a novel MMP-2 responsive cascade target liposome drug delivery system. Cascade targeting increases the infiltration and retention of liposomes in deep tumors and significantly improves the uptake of tumor cells. The simple and biologically safe materials endow the potential for controllable production. Furthermore, this strategy restores the anti-tumor activity of tumor site T cells, synergistically regulates the immunosuppressive tumor microenvironment, induces an effective antitumor immune response, and produces immune memory. Taken together, these results confirm the effectiveness of an MMP-2 responsive cascade target immunotherapy strategy on metastatic breast cancer.