Transforming “cold” tumors into “hot” ones via tumor-microenvironment-responsive siRNA micelleplexes for enhanced immunotherapy

•The siRNA micelleplexes were designed for turning “cold” tumors into “hot” ones•Tumor-microenvironment-responsive nanomaterial precise drug delivery•Normalize the tumor vasculature to enhance oxygen-dependent photoimmunotherapy It is highly desirable turn ones improve efficacy of antitumor immunotherapy. Photoimmunotherapy can induce immunogenic cell death (ICD), providing a new path pursue this goal. But its therapeutic effect significantly weakened by hypoxic microenvironment (TME). Herein, we type chimeric matrix metalloproteinase-2 (MMP-2)-responsive, peptide-based micelleplex achieve vascular-normalization-mediated reversion immunosuppressive TME and synergistically enhanced photoimmunotherapy. develop small interfering RNA (siRNA) (PA7R@siPD-L1) normalizing vascular-immune crosstalk establish positive feedback loop in potentiating These are equipped with tumor-microenvironment-responsive property delivery release. The prepared PA7R@siPD-L1-mediated photodynamic therapy could eliminate solid trigger generate systematic immune responses. Meanwhile, antiangiogenic peptide A7R normalizes converting chaotic vascular systems matured organized ones, thus alleviating hypoxia promoting intratumoral infiltration cells. Antitumor immunogenicity would be further strengthened aid siPD-L1 muting resistance cells against effector This study provides unique strategy enabled nanomaterial. Angiogenesis inevitable tumors, as they need abundant nutrients rapid expansion.1Folkman J. Tumor angiogenesis: implications.N. Engl. Med. 1971; 285: 1182-1186https://doi.org/10.1056/nejm197111182852108Crossref PubMed Google Scholar,2Kuczynski E.A. Vermeulen P.B. Pezzella F. Kerbel R.S. Reynolds A.R. Vessel co-option cancer.Nat. Rev. Clin. Oncol. 2019; 16: 469-493https://doi.org/10.1038/s41571-019-0181-9Crossref Scopus (214) Scholar Such abnormal growth drives formation aberrant structure, including excessive vessel leakiness uneven distribution.3Jain R.K. Normalizing treat cancer: bench bedside biomarkers.J. 2013; 31: 2205-2218https://doi.org/10.1200/jco.2012.46.3653Crossref Pathological angiogenesis compromises oxygen alters metabolic pathways, resulting hypoxia, acidity, high interstitial fluid pressure (TME), which favors progression, metastasis, treatment resistance.4Carmeliet P. Jain Principles mechanisms normalization cancer other angiogenic diseases.Nat. Drug Discov. 2011; 10: 417-427https://doi.org/10.1038/nrd3455Crossref (1219) Scholar,5Jain Normalization vasculature: an emerging concept therapy.Science. 2005; 307: 58-62https://doi.org/10.1126/science.1104819Crossref (4404) Specifically, induces continuous production proangiogenic factors, particularly endothelial factor (VEGF), neovessels within tumor, creating vicious cycle.6Sun Y. Chen W. Torphy R.J. Yao S. Zhu G. Lin R. Lugano Miller E.N. Fujiwara Bian L. et al.Blockade CD93 pathway facilitate immunotherapy.Sci. Transl. 2021; 13: eabc8922https://doi.org/10.1126/scitranslmed.abc8922Crossref (29) In addition, contributes tolerance secretion cytokines such interleukin-10 (IL-10) transforming β (TGF-β), well upregulating expression checkpoint molecules, PD-L1, on cells.7Barsoum I.B. Smallwood C.A. Siemens D.R. Graham C.H. A mechanism hypoxia-mediated escape from adaptive immunity cells.Cancer Res. 2014; 74: 665-674https://doi.org/10.1158/0008-5472.can-13-0992Crossref (0) Scholar, 8Jain Rakesh K. Antiangiogenesis strategies revisited: starving hypoxia.Cancer Cell. 26: 605-622https://doi.org/10.1016/j.ccell.2014.10.006Abstract Full Text PDF (993) 9Mpekris Panagi M. Voutouri C. Martin J.D. Samuel Takahashi Gotohda N. Suzuki T. Papageorgis Demetriou al.Normalizing overcomes compression nano-immunotherapy breast lung metastasis.Adv. Sci. 2020; 8: 2001917https://doi.org/10.1002/advs.202001917Crossref (35) Furthermore, leaky vessels characterized loosely attached adjacent downregulate cell-adhesion producing anergy decreasing translocation tumor.10Huang Kim B.Y.S. Chan C.K. Hahn S.M. Weissman I.L. Jiang Improving immune-vascular immunotherapy.Nat. Immunol. 2018; 18: 195-203https://doi.org/10.1038/nri.2017.145Crossref (253) intense not only fosters but also antagonizes effectiveness treatment.11Kabir A.U. Subramanian Lee D.H. Wang X. Krchma Wu Naismith Halabi C.M. J.Y. Pulous F.E. al.Dual role Myct1 immunity.Sci. eabb6731https://doi.org/10.1126/scitranslmed.abb6731Crossref (18) Conventional that aims block starvation easily trapped cycle hypoxia-angiogenesis, aggravation immunotherapy resistance.12Liu Z. Huang Shan H. D. vasculatures: target immunotherapy.Trends Pharmacol. 40: 613-623https://doi.org/10.1016/j.tips.2019.07.001Abstract (61) 13Potente Gerhardt Carmeliet Basic aspects angiogenesis.Cell. 146: 873-887https://doi.org/10.1016/j.cell.2011.08.039Abstract (1958) 14Shigeta Datta Hato Kitahara I.X. Matsui A. Kikuchi Mamessier E. Aoki Ramjiawan R.R. programmed receptor-1 receptor-2 blockade promotes enhances responses hepatocellular carcinoma.Hepatology. 71: 1247-1261https://doi.org/10.1002/hep.30889Crossref (174) Comparatively, adopts judicious acquires access reprogram immunostimulatory phenotype repairing structure biological function network.15Schmittnaegel Rigamonti Kadioglu Cassará Wyser Rmili Kiialainen Kienast Mueller H.-J. Ooi C.-H. Laoui De Palma Dual angiopoietin-2 VEGFA inhibition elicits PD-1 blockade.Sci. 2017; 9: eaak9670https://doi.org/10.1126/scitranslmed.aak9670Crossref (361) Immunotherapy, especially (ICB) therapy, has revolutionized traditional elicited remarkable clinical few patients.16Chen D.S. Mellman I. Elements cancer–immune set point.Nature. 541: 321-330https://doi.org/10.1038/nature21349Crossref (2746) However, patients (non-immunogenic) have poor ICB owing immune-desert (which lacks cells) or immune-excluded blocks out periphery stroma).17Nam Son Park K.S. Zou Shea L.D. Moon J.J. Cancer nanomedicine combination Mater. 4: 398-414https://doi.org/10.1038/s41578-019-0108-1Crossref (494) Thus, it (immunogenic increased T infiltration) response therapy.18Galon Bruni Approaches hot, altered cold tumours immunotherapies.Nat. 197-218https://doi.org/10.1038/s41573-018-0007-yCrossref (1438) Scholar,19Martin Cabral Stylianopoulos using nanomedicines: progress, opportunities challenges.Nat. 17: 251-266https://doi.org/10.1038/s41571-019-0308-zCrossref (285) recent discovery (ICD) offers succumbing ICD release tumor-associated antigens (TAAs) danger-associated molecular patterns (DAMPs), act “in situ” vaccines promote recruitment maturation dendritic (DCs) priming cells.20Krysko D.V. Garg A.D. Kaczmarek Krysko O. Agostinis Vandenabeele Immunogenic DAMPs therapy.Nat. Cancer. 2012; 12: 860-875https://doi.org/10.1038/nrc3380Crossref (1483) 21Kono Rock K.L. How dying alert system danger.Nat. 2008; 279-289https://doi.org/10.1038/nri2215Crossref (1313) 22Galluzzi Buqué Kepp Zitvogel Kroemer infectious disease.Nat. 97-111https://doi.org/10.1038/nri.2016.107Crossref (1582) Studies proven (PDT) able ICD, TME.23Duan Nanoparticle-mediated enables potentiates immunotherapy.Angew. Chem. Int. Ed. 58: 670-680https://doi.org/10.1002/anie.201804882Crossref (482) Scholar,24Zhu Duo Suo Zhao Xia Zheng Li Tang B.Z. Tumor-exocytosed exosome/aggregation-induced emission luminogen hybrid nanovesicles efficient penetration therapy.Angew. 59: 13836-13843https://doi.org/10.1002/anie.202003672Crossref (87) To reinforce PDT, self-delivering intratumor-generating been extensively exploited, utilizing transport carriers like hemoglobin25Chen Liu Liang Luo He Tian Ma Cai Bioinspired protein nanocarrier amplified eliciting anti-tumor abscopal effect.ACS Nano. 8633-8645https://doi.org/10.1021/acsnano.8b04371Crossref (237) Scholar,26Li Yang Qin B. Yin Yuan Zhang al.Targeting photothermal endoplasmic reticulum death.Nat. Commun. 3349https://doi.org/10.1038/s41467-019-11269-8Crossref (478) introducing substances catalyze decomposition H2O2 produce O2.27Gao Q. Oxygen-generating nanoparticles fluorescent/photoacoustic/ultrasound imaging guided therapy.Biomaterials. 112: 324-335https://doi.org/10.1016/j.biomaterials.2016.10.030Crossref (213) Scholar,28Zeng Deng Bao Guo Ji Zeng Mei Dual-response oxygen-generating MnO2 polydopamine modification combined photothermal-photodynamic therapy.Chem. Eng. 389: 124494https://doi.org/10.1016/j.cej.2020.124494Crossref (131) these methods cannot fundamentally solve problems PDT limited distribution TME, angiogenesis, desmoplasia. By system, established ameliorate potentiate delivery. holds great promise breaking limitation improving Firstly, polyethylene glycol (PEG)-R9 conjugated chlorin e6 (Ce6) was linked via MMP-2 substrate spacer (GPLGVRG). Then, obtained assembled lecithin29Zhang Pan Feng Ou [email protected]2-SP nanosheet-based intelligent nanoplatform second near-infrared light-mediated theranostics.Adv. Funct. 29: 1903791https://doi.org/10.1002/adfm.201903791Crossref (67) through hydrophobic interaction acquire PA7R micelleplexes, bound PD-L1 electrostatic adsorption R9 (designated PA7R@siPD-L1; Figure 1A ). Upon reaching MMP-2-rich broken down hydrolyzation peptide. Subsequently, dissociate VEGF receptor 2 (VEGFR-2) neuropilin-1 (NRP-1) process contribute blood-vessel alleviation immunosuppression TME. PEG-R9K(Ce6)-LLGPLG released simultaneously enter cell-penetrating segment R9. laser irradiation, photosensitizer reactive species (ROS) ICD; meantime, lysosome disrupted cytotoxic ROS, facilitating diffusion cytoplasm silencing (Figure 1B). sum up, work superior photoimmunotherapeutic triggering normalization. PA7R@siPD-L1 exert strong durable inhibit recurrence metastasis. Moreover, newly developed our kind promising universal platform might load siRNAs targeting different variety molecules (e.g., mRNA, microRNA [miRNA], single guide [sgRNA], etc.), treating myriad malignancies. PEG-R9K(Ce6)-LLGPLGVRG-A7R first Fmoc solid-phase synthesis (SPPS) S1) electrospray ionization mass spectrometry (ESI-MS) S2) high-performance liquid chromatography (HPLC) S3), verifying weight purity. produced assembly lecithin at molar ratio 1:2. After incubating 30 min room temperature, uniform well-defined spherical morphology confirmed transmission electron microscopy (TEM) 2A ), average hydrodynamic diameter 53.42 ± 4.63 nm determined dynamic light scattering (DLS) 2C). low polydispersity index (PDI) (0.140) indicated good uniformity dispersion micelleplexes. No obvious size changes DMEM containing 10% fetal bovine serum (FBS) PBS observed over period 3 days S4). critical micelle concentration (CMC) 6.04 μM measured pyrene solubilization method stability nanostructures 2D). incubation 37°C h, collapsed excellent MMP-2-responsive disassembly 2B). We explored pH-dependent degradability pH 48 h. As S5A shows, 7.4 maintained turned larger irregularly 6.5 S5B), ascribed proton under weak acidity (pH 6.5), because guanidine groups easy capture H+. ultraviolet-visible (UV-vis) absorption peaks located 402 660 nm, indicating successful conjunction Ce6 2E). investigate singlet (1O2)-generating capability irradiation after encapsulation PA7R@siPD-L1, Singlet Oxygen Sensor Green (SOSG) fluorescent probe employed. shown 2F, pretreated displayed more increase fluorescence intensity than without pretreatment, although both them acquired similar amount 1O2 25 min. disassembled presence MMP-2, synchronously exposed accelerate generation enhancing accessibility O2. explore possibility complexes, gel retardation assay performed 2G). revealed binding affinity when PA7R-to-siPD-L1 N/P exceeded 10. zeta potential decreased 23.06 1.60 mV 9.33 2.27 mV, confirming preparation stable complexes 2H). assess being complexed PA7R, RNase protection carried out. seen 2I, free completely degraded physiological conditions (lanes 1 4). protected enzymolysis 2, 3, 5, 6). hemolysis experiment initially evaluate their biomedical applications. result S6) showed activity still stayed therapeutical (5 μg/mL). cellular uptake labeled dye carboxyfluorescein (FAM). S7, effectively internalized 4T1 time-dependent manner. Interestingly, slightly higher efficiency attributed cleavage MMP-2. far transfection commercial reagent Lipofectamine 2000 3A challenge better did 3A), consistent 2I). photo-induced ROS level investigated employing 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assessing confocal scanning (CLSM) flow cytometry (Figures 3B S8). Under relative exhibited highest production, MMP-2-mediated contributed exposure 1O2. inquire whether intracellular endo- lysosomal colocalization (red) lysosomes (green) assessed CLSM S9). Without overlapping red green entrapment lysosomes. most separated illustrating destroyed integrated membrane facilitated siPD-L1-FAM cytoplasm. cytotoxicities formulations normal evaluated typical counting kit-8 (CCK-8) assay. Figures 3C S10, Ce6, + M negligible cytotoxicity murine mammary carcinoma line (4T1 (HC11 epithelium), supporting biosafety PA7R@siPD-L1. pretreatment phototoxicity upon concentration-dependent greater generation. Because essential driving occurrence, development, before tube 3D S11) transwell migration S12). results human umbilical vein (HUVECs) treated absence less tubule compared control group. worth noting activity, separation VEGFR-2 NRP-1 enzyme pretreatment. Afterward, knockdown verified western blotting analysis 3E). PA7R@ siR.C (scrambled siRNA) expression, whereas suppressed demonstrating effective inhibitory form associated DAMPs, signals response.30Zhou Yu microenvironment-activatable prodrug vesicles nanoenabled chemoimmunotherapy combining induction CD47 blockade.Adv. 1805888https://doi.org/10.1002/adma.201805888Crossref (295) Surface-exposed calreticulin (CRT) (“eat” me signal), extracellular mobility group box (HMGB1) (“danger” adenosine triphosphate (ATP) (“find me” signal) pivotal well-known recruit immature DCs stimulate help cells.31Sun Saeed Xu Zhong Song Huai Xie al.Regulating glucose metabolism pancreatic cancer.Adv. 2002746https://doi.org/10.1002/advs.202002746Crossref (65) vitro mentioned above detected incubated [email protected] had almost no CRT HMGB1 ATP release, elevated application underlining significant impact 3F–3I). DC collecting bone marrow (BM) BALB/c mice differentiating (BMDCs). then co-incubated BMDCs percentage CD11c, CD80, CD86 3J S13). Compared group, level, while much receiving played major activating immunity. certain extent. L 12.5% rate lipopolysaccharide (LPS) activation condition. Before carrying vivo studies, biochemistry complete blood panel PA7R@siP-L1, investigated. differences liver kidney indicators count among S14 S15), PA7R@siP-L1 suitable Next, biodistribution behavior bearing investigated, since accumulation retention site prerequisites efficacy. intravenously injected identical dose 5 mg/kg tracked time 4A S16A). Unlike S16B), accumulated gradually reached maximum 24 h post-injection 4B). long circulation PEG layer inherent long-term circulating reported previous studies.32Jeong W.-J. Bu Kubiatowicz L.J. S.S. Hong Peptide-nanoparticle conjugates: next diagnostic platforms?.Nano Converg. 5: 38https://doi.org/10.1186/s40580-018-0170-1Crossref (111) Scholar,33Taleb Atabakhshi-Kashi Rezavani Alanagh Farhadi Sabet Cheng Qi Nie Ying Bifunctional exerting improved activities inhibition.Adv. Healthc. 2100051https://doi.org/10.1002/adhm.202100051Crossref (12) Ex images possessed ability tissue even injection 4C 4D), mainly distributed S16C S16D). unilateral model 4E). A7R, another amphiphilic Pcon synthesized replaced meaningless (characterized ESI-MS HPLC; S17 S18). Pcon@siPD-L1 55.30 3.64 DLS S19). Mice randomly divided eight (n = per group) volume 90 mm3 either PBS, Pcon@siPD-L1, [email protected], Four underwent intravenous alone, four received additional post-injection, 2.5 mg/kg. 4F–4H, volumes shar