Abstract

Background

   The efficacy of current therapies for ovarian cancer is limited due to
   the multilevel and complex tumor microenvironment (TME), which induces
   drug resistance and tumor progression in a single treatment regimen.
   Additionally, poor targeting and insufficient tissue penetration are
   important constraints in ovarian cancer treatment.

Result

   We constructed PH20-overexpressing cancer-associated fibroblast
   (CAF)-cancer hybrid-cell membrane vesicles (PH20/CCM) for the
   dual-targeted delivery of carboplatin (CBP) and siRNA targeting p65
   (sip65) loaded on the poly (dimethyl diallyl ammonium chloride)
   (PDDA)-modified MXene (PMXene), named PMXene@CBP-sip65 (PMCS). The
   nanoparticle PH20/CCM@PMCS could penetrate the extracellular matrix of
   tumor tissues and target both cancer cells and CAFs. After tumor cell
   internalization, these nanoparticles significantly inhibited cancer
   cell proliferation, generated reactive oxygen species, induced
   endoplasmic reticulum stress, and triggered immunogenic cell death.
   After CAF internalization, they inhibited pro-tumor factor release and
   activated immune effects, promoting immune system infiltration. In an
   experiment with ID8 homograft-carrying mice, PH20/CCM@PMCS
   significantly improved tumor inhibition and enhanced immune
   infiltration in tumor tissues.

Conclusion

   These new therapeutic nanoparticles can simultaneously target tumor
   cells, CAFs, immune cells, and the extracellular matrix, thereby
   increasing treatment sensitivity and improving the TME. Therefore,
   these TME-regulating nanoparticles, combining specificity, efficiency,
   and effectiveness, provide new insights into ovarian cancer treatment.

Graphical Abstract

   [44]graphic file with name 12951_2025_3165_Figa_HTML.jpg

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12951-025-03165-9.

   Keywords: Cancer-associated fibroblasts, Tumor microenvironment,
   Membrane-coated nanoparticles, Targeted therapy

Introduction

   Epithelial ovarian cancer (EOC) is the deadliest gynecological
   malignancy globally. In 2022, an estimated 324,603 new cases of EOC
   were reported worldwide, with 206,956 deaths [[45]1]. Most patients are
   already in the advanced stages at diagnosis [[46]2]. Currently, the
   most widely used treatment methods in clinical practice include
   surgery, platinum-based drugs, and paclitaxel chemotherapy. Although
   most patients achieve good therapeutic outcomes in the early treatment
   stages, nearly half of them experience multiple relapses and tumor
   metastasis [[47]3]. Some patients may develop resistance to platinum
   drugs, leading to a decline in chemotherapy effectiveness.

   Eliminating tumor cells is crucial for cancer treatment. The ability of
   the treatment to eradicate tumor cells and whether residual tumor cells
   can be eliminated in the microenvironmental “soil” are key factors in
   preventing tumor recurrence. The tumor microenvironment (TME) plays a
   key role in promoting immune suppression and chemotherapy resistance in
   tumors [[48]4]. The TME comprises components such as tumor cells,
   stromal cells, immune cells, the factors secreted by these cells, and
   the extracellular matrix (ECM ) [[49]5]. Cancer-associated fibroblasts
   (CAFs) are a major component of the TME that interact with other TME
   components and play an important role in tumor development. CAFs
   secrete various cytokines, growth factors, and ECM components to
   stimulate tumor cell growth, metastasis, and angiogenesis [[50]6,
   [51]7]. Additionally, they interact with tumor cells and suppress
   immune effects by inhibiting immune cell activity [[52]8, [53]9]. CAFs
   also contribute to remodeling the ECM of tumor tissues, fostering a
   supportive environment for tumor cell survival [[54]10, [55]11].
   Following in-depth research and understanding of how CAFs drive the
   pro-cancer effects of the TME, they are emerging as one of the most
   promising targets for cancer intervention, offering hope for patients
   with refractory and easily relapsing tumors.

   Owing to the multiple levels and complexity of the TME, a single
   treatment regimen may induce dynamic tumor evolution through
   compensatory feedback mechanisms, leading to drug resistance and
   promoting tumor progression, thus resulting in treatment outcomes that
   fall short of expectations [[56]12–[57]16]. Drug combination therapy is
   an effective strategy to improve anti-cancer efficacy and overcome drug
   resistance [[58]17]. However, the use of two or more drugs remains
   controversial due to different pharmacokinetic characteristics and
   increased patient burden. Accordingly, constructing a platform that can
   multi-target tumor cells, CAFs, immune cells, and the ECM to promote
   the TME toward an antitumor state represents an appealing strategy for
   developing new therapeutic approaches.

   Cell membrane-coated nanoparticles (CNPs) are a promising new type of
   nanocarrier. CNPs consist of a core of nanoparticles and an outer shell
   containing a natural cell membrane used for membrane camouflage
   purposes, typically composed of natural or engineered red blood cells,
   neutrophils, macrophages, and cancer cells [[59]18–[60]21]. The
   adhesion proteins, antigens, and membrane structures of the source cell
   can be retained on the surface of the nanoparticles after cell membrane
   encapsulation; therefore, the resulting nanoparticles exhibit various
   characteristics of the source cell, such as good biocompatibility,
   immune evasion, homologous targeting, and tumor targeting, thereby
   improving the effectiveness of drug delivery and tumor treatment
   [[61]22]. Hybrid membrane technology involves mixing and hybridizing
   two or more types of cell membranes before coating the nanoparticles to
   achieve the inheritance of different cell membrane functions
   [[62]23–[63]25]. The abovementioned studies have primarily focused on
   the delivery to tumor cells or immune evasion using cell membrane
   coating methods. Although biomimetic nanoparticles offer a promising
   strategy to enhance the efficacy of chemotherapy, these cell
   membrane-camouflaged nanomedicines neglect the overall control of the
   TME, and the effective transport of nanoparticles in the TME is
   hindered. Therefore, we developed a “multi-pronged” therapeutic
   program. Notably, CAFs are pivotal in regulating the TME and pose a
   barrier to nanomedicine penetration. Consequently, we addressed the
   above challenges by modifying the nanoparticles using hybrid CAF-cancer
   cell membrane vesicles. Compared with previous membrane-camouflage
   technologies, these hybrid CAF-cancer cell membrane vesicles
   demonstrated dual-targeting capability to CAFs and cancer cells,
   simultaneously directing nanoparticles to kill tumor cells while
   inhibiting the pro-tumor effects of CAFs. This novel strategy offers
   multiple advantages, such as reducing tumor burden, enhancing
   penetration, activating immunity, and reshaping the TME. To the best of
   our knowledge, no reports on the hybridization of CAFs obtained from
   ovarian cancer tissues as cell membrane vesicles with cancer cells to
   disguise nanoparticles for cancer treatment exist. In addition, the
   increase in interstitial fluid pressure and the dense tumor stroma in
   tumor tissues hinder the transport of nanoparticles across cell layers.
   In previous studies, hyaluronoglucosaminidase PH-20 (PH20) has been
   used to assist drug delivery in overcoming this barrier [[64]26]. As a
   cell membrane surface protein, PH20 can maintain its activity in both
   neutral and acidic environments, hydrolyzing hyaluronic acid in the TME
   to reduce tumor tissue density and achieve deep penetration and
   accumulation of drugs in tumor tissue [[65]27, [66]28]. Plasmid
   transfection technology can be used to achieve high expression on the
   cell membrane in a relatively simple and efficient manner, making it a
   desirable choice for engineered modification of cell membrane vesicles.

   The nuclear factor kappa B (NF-κB) pathway is a critical regulator in
   shaping TME, playing a crucial role in tumor cell and CAF dynamics. In
   tumor cells, the NF-κB pathway contributes to infiltration and
   metastasis during chemotherapy [[67]29, [68]30]. In CAFs, the NF-κB
   pathway promotes the formation of an immunosuppressive TME and
   therapeutic resistance by regulating inflammatory factors, chemokines,
   and cytokines in the TME [[69]31–[70]33]. Therefore, the NF-κB pathway
   can serve as a common therapeutic target for CAFs and tumor cells. The
   p50/p65 heterodimer is the best-characterized form of the NF-κB complex
   [[71]34]. The NF-κB complex functions by translocating to the nucleus
   and regulating target gene expression [[72]35]. Based on this, we
   attempted to combine cytotoxic chemotherapy drugs with p65 siRNA to
   eliminate tumor cells and regulate the TME. MXene, a 2D
   bio-nanoparticle with ultrathin planar nanostructures, which provides
   abundant attachment sites for drugs owing to its large surface area and
   characteristics [[73]36, [74]37], enhanced the delivery efficiency of
   drugs and siRNAs, ultimately improving drug adsorption. Moreover, MXene
   exhibits negligible cytotoxicity and good biodegradability [[75]38,
   [76]39]. These advantages endow MXene with enormous potential in
   biomedical research fields, such as drug delivery and cancer therapy.
   To optimize the loading capacity negatively of charged drugs and siRNAs
   on MXene, we modified Ti3C2 MXene with PDDA, resulting in a positively
   charged surface, which facilitates the enrichment of drugs and nucleic
   acids [[77]40, [78]41].

   In the present study, we constructed PH20-overexpressing CAF-cancer
   hybrid-cell membrane vesicles (PH20/CCM) for dual-targeted delivery of
   carboplatin (CBP) and siRNA targeting p65 (sip65), which were loaded
   onto PDDA-modified MXene (PMXene), referred to as PMXene@CBP-sip65
   (PMCS). The biomimetic nanovesicle PH20/CCM@PMCS exhibited tumor
   penetration capability and dual targeting of tumor cells and CAFs.
   After their internalization by tumor cells and CAFs, the loaded drugs
   and siRNAs were released, leading to a significantly reduced
   proliferation of cancer cells and CAF inhibition. PH20/CCM@PMCS
   regulated TME by reducing the secretion of immunosuppressive,
   angiogenic, and matrix-remodeling factors in CAFs, thereby enhancing
   the drug sensitivity of cancer cells and immune cell infiltration. In
   summary, we believe that PH20/CCM@PMCS has effective antitumor effects
   and satisfactory biosafety and is expected to serve as an efficient,
   specific, targeted, and integrated new strategy for the treatment of
   ovarian cancer.

Results

Enrichment of the NF-κB Pathway in Both Tumor Epithelial and CAFs and
Positive Correlation with the Disease Progression

   We integrated two single-cell RNA sequencing datasets of high-grade
   serous ovarian cancer from the GEO database ([79]GSE235329 and
   [80]GSE184880). A total of 80,850 cells were clustered for further
   bioinformatics analysis after low-quality cells were removed,
   normalized, dimensionality reduced, and integrated. We identified
   20,455 fibroblasts, which clustered into five subclusters, comprising
   one of the normal fibroblasts (NFs) and four of CAFs (Figure [81]S1A,
   B). Initially, we compared genes between the NF and CAFs from the four
   clusters to explore their functions. PROGENy enrichment analysis
   revealed significant enrichment of JAK-STAT, PI3K, VEGF, hypoxia
   signaling, NF-κB, TNFa, EGFR, and MAPK pathways for each CAF cluster
   (Figure [82]S1C). Similarly, we performed PROGENy enrichment analysis
   of differential genes between normal and malignant ovarian epithelium.
   VEGF, NF-κB, and TNFa pathways were significantly enriched (Figure
   [83]S1D). The NF-κB pathway plays a vital role in the TME, promoting
   tumor cell progression, influencing CAFs to regulate matrix
   reorganization and cytokine secretion, and regulating the expression of
   immunosuppressive molecules [[84]31, [85]42]. Activation of the NF-κB
   pathway triggers tumors to generate downstream responses, such as
   apoptotic escape and TME remodeling, leading to tumor insensitivity to
   platinum-based chemotherapeutic agents [[86]43]. p65 plays a key role
   in the NF-κB pathway and is a major transcription factor in the NF-κB
   pathway, regulating the transcription of a wide range of genes. The EOC
   survival curves plotted using the Kaplan–Meier plotter revealed a
   negative correlation of high p65 expression with overall survival
   (Figure [87]S1E). Collectively, our findings suggest that the NF-κB
   pathway is significantly upregulated in tumor epithelial cells and CAFs
   and plays an important role in TME regulation, making it a potential
   therapeutic target.

Characterization of PH20/CCM@PMCS

   Figure [88]1A illustrates the preparation of PH20/CCM@PMCS in the
   following steps: (a) extraction of PH20-overexpressing tumor cell
   membranes and CAF membranes to form PH20/CCM; (b) MXene modification
   using PDDA to obtain the PMXene and enriching CBP and siRNA on the
   surface of PMXene to construct PMCS; (c) fusing PH20/CCM on the surface
   of PMCS via extrusion.

Fig. 1.

   [89]Fig. 1
   [90]Open in a new tab

   Characterization of hybrid membrane-coated nanoparticles. (A) The
   preparation procedure of PH20/CCM@PMCS. (B) TEM images of PH20/SKOV3
   membrane vesicle (PH20/SKOV3-M), PH20/ID8 membrane vesicle
   (PH20/ID8-M), h_CAFs membranes (h_CAF-M), m_CAFs membranes (m_CAF-M),
   h_PH20/CCM, and m_PH20/CCM (Scale bar: 100 nm). (C) SDS-PAGE protein
   analysis of cancer cell membrane, cancer-associated fibroblast
   membrane, and hybrid cell membrane proteins. (D) Western Blot analysis
   of characteristic proteins from cancer cell membrane, cancer-associated
   fibroblast membrane, and hybrid cell membrane proteins. (E) Images of
   mixed membranes and hybrid membranes (Scale bar: 20 μm). (F) (a) SEM
   images of stacked block shape MXene (Scale bar: 300 nm). (b) TEM images
   of stacked block shape MXene (Scale bar: 100 nm) (c) TEM images of
   dispersed MXene (Scale bar: 100 nm) (G) UV-visible absorption spectra
   of PMXene and PMCS. (H) AFM image of PMCS. (I) TEM images of PMXene,
   hybrid cell membranes and hybrid cell membranes coated PMXene (Scale
   bar: 100 nm). (J) The Zeta potential of PMXene, PMCS, h_PH20/CCM,
   h_PH20/CCM@PMCS, m_PH20/CCM, m_ PH20/CCM@PMCS. Data are expressed as
   mean ± standard deviation (S.D.)

   The PH20-overexpressing SKOV3 (PH20/SKOV3) membrane vesicles,
   human-derived CAF (h_CAF) membrane vesicles and human-derived PH20/CCM
   membrane vesicles (h_PH20/CCM) were obtained by extruding the extracted
   PH20/SKOV3 membranes, h_CAF membranes, and PH20/SKOV3 and h_CAF
   membranes, respectively. Furthermore, the PH20-overexpressing ID8
   (PH20/ID8) membrane vesicles, mouse-derived CAF (m_CAF) membrane
   vesicles and mouse-derived PH20/CCM membrane vesicles (m_PH20/CCM) were
   obtained by extruding the extracted PH20/ID8 membranes, m_CAF
   membranes, and PH20/ID8 and m_CAF membranes, respectively. Transmission
   electron microscopy (TEM) images revealed a dark–bright–dark cell
   membrane structure of the vesicles with 10–20 nm thickness
   (Fig. [91]1B), consistent with a previous study [[92]44]. The cell
   membrane vesicles exhibited a negative zeta potential (Figure [93]S2),
   with no significant differences in appearance from different cell
   sources. We further evaluated the protein features of the CAF membrane
   vesicles, PH20-overexpressing cancer cell membrane vesicles, and
   PH20/CCM using SDS–PAGE. The proteins on the hybrid membrane vesicles
   shared common characteristics with the PH20-overexpressing cancer cell
   membrane vesicles and CAF membrane vesicles (Fig. [94]1C). Western
   blotting revealed the presence of specific proteins on the
   PH20-overexpressing cancer cell membrane vesicles, CAF membrane
   vesicles, and PH20/CCM, including E-cadherin, fibroblast activation
   protein alpha (FAP), and PH20 (Fig. [95]1D). This indicated that the
   functionalized membrane proteins were retained in the membrane vesicles
   and co-expressed on the hybrid membrane vesicles after membrane
   hybridization. To verify that the two cell membranes were fused through
   co-extrusion and not simple mixing, the tumor cell membranes were
   labeled with DIO (green), and the CAF membranes were labeled with DID
   (red). The tumor cell (green) and CAF membranes (red) did not overlap
   in the mixing group, but overlapped in the co-extrusion group
   (Fig. [96]1E), indicating a successful membrane hybridization after
   co-extrusion.

   A high degree of dispersibility and nanoscale planar size are essential
   for MXene nanosheets to meet the stringent biomedical application
   requirements. MXene is shown in stacked blocks in scanning electron
   microscope (SEM) and TEM images (Fig. [97]1F(a) and [98]1F(b)), whereas
   further sonication reveals highly dispersed MXene in TEM images
   (Fig. [99]1F(c)). Subsequently, PMXene, PMXene@CBP (PMC), PMXene@sip65
   (PMS), and PMCS were successfully synthesized. CBP and siRNA adhered to
   the surface of PMXene through electrostatic adsorption, and their
   loading efficiency and loading contents were calculated (Figure
   [100]S3). Considering the entrapment efficiency and drug loading
   contents, we ultimately chose to synthesize PMCS in a ratio of CBP:
   PMXene: siRNA = 30:10:0.25. Among them, the entrapment efficiency of
   CBP was 82.98%, the drug loading content was 71.34%; the entrapment
   efficiency of siRNA was 71.79%, and the drug loading content was 1.75%.
   The ultraviolet-visible absorption spectra were used to analyze the
   main components of the PMCS. As shown in Fig. [101]1G, PMCS exhibited a
   strong absorption peak near 229 and 260 nm, corresponding to the
   binding of CBP and siRNA to PMXene, respectively. For drug release
   (Figure [102]S4), CBP initially showed explosive release within the
   first 16 h and reached a plateau after 24 h. CBP exhibited a higher
   release rate at pH 5.0 than at pH 7.4, with approximately 70% drug
   release. The release of siRNA also followed this pattern, with a drug
   release rate of approximately 50% at pH 5.0. These findings indicated
   the excellent performance of PMXene as a nanocarrier for loading and
   delivering CBP and siRNA. Images of the nanoparticles were obtained
   using an atomic force microscope (AFM). The AFM image revealed a
   layered structure of PMCS with a maximum diameter of approximately
   100 nm and a thickness of 4–5 nm (Fig. [103]1H). PMCS was then
   encapsulated using hybrid membrane vesicles. TEM images showed a
   layered morphology of PMCS with excellent monodispersity. PH20/CCM@PMCS
   exhibited a core-shell structure with a dark–bright–dark cell membrane
   structure observed on the surface, indicating the successful
   modification using the hybrid membrane (Fig. [104]1I). The overall size
   distribution of PH20/CCM@PMCS detected using dynamic light scattering
   (DLS) was approximately 110 nm, indicating uniform particle size of
   PH20/CCM@PMCS (Figure [105]S5). The zeta potentials of PMCS,
   m_PH20/CCM@PMCS, and h_PH20/CCM@PMCS were (23.1 ± 4.1) mV,
   (− 12.1 ± 4.5) mV, and (− 13.3 ± 4.3) mV, respectively (Fig. [106]1J).
   The surface of the PH20/CCM@PMCS was negatively charged, which was
   beneficial for the blood circulation time of the nanoparticles. After
   incubation with PBS, DMEM/F12, and DMEM/F12 with 10% FBS at 37 °C for
   24 h, the diameter of the PH20/CCM@PMCS particles remained
   approximately 110 nm (Figure [107]S6), indicating satisfactory
   dispersibility and stability. After incubation of PH20/CCM@PMCS for
   36 h, the cell membranes were ruptured and nanoparticles were rapidly
   released (Figure [108]S7).

Function of PH20/CCM@PMCS

   We validated the dual-targeting ability of PH20/CCM@PMCS toward cancer
   cells and CAFs. Fluorescein isothiocyanate (FITC, green) was used to
   label PMXene. PMCS encapsulated by the SKOV3 cell membrane can be
   internalized by SKOV3 cells but not by h_CAFs. In contrast, PMCS
   encapsulated by h_CAF cell membranes can be internalized by h_CAFs, but
   not by SKOV3. SKOV3 and h_CAFs can simultaneously encapsulate PMCS in
   the hybrid membrane. PMCS encapsulated by mouse-derived membranes also
   exhibited similar properties (Fig. [109]2A). Specifically, the hybrid
   membrane exhibited a dual-targeting ability. In addition, PMCS
   encapsulated by h_PH20/CCM and m_PH20/CCM were minimally internalized
   by other cancer cells or NFs (Fig. [110]2B), indicating the high
   targeting effect of PH20/CCM@PMCS. Escape from the endothelial
   reticular system is necessary for all nanomedicines injected into the
   bloodstream. PMCS and PH20/CCM@PMCS were subsequently co-cultured with
   RAW264.7 cells. The cell membrane shell effectively reduced the
   internalization of nanoparticles by macrophages (Fig. [111]2C). This
   may be attributed to the immune evasion function of cancer cell
   membranes [[112]45]. To verify the biocompatibility of PH20/CCM@PMCS in
   vitro, we performed CCK8 experiments to observe the activity of six
   cell types, SKOV3, ID8, h_CAFs, m_CAFs, 293T, and HUVECs, after
   co-incubation with PH20/CCM, PMXene, PH20/CCM@PMXene, and
   PH20/CCM@PMCS. During co-incubation with cancer cells, CAFs, 293T
   cells, and HUVECs, PH20/CCM, PMXene, and PH20/CCM@PMXene did not
   inhibit cell viability, indicating that they are not significantly
   toxic. PH20/CCM@PMCS inhibited the viability of cancer cells and CAFs,
   but had no significant effect on 293T cells or HUVECs (Fig. [113]2D).
   The effects of different concentrations of PH20/CCM@PMCS on cancer
   cells and CAFs are demonstrated in Figure [114]S8. The above results
   demonstrate that the cell membranes and nanocarrier PMXene themselves
   do not have inhibitory effects on cells. PH20/CCM@PMXene only exerts
   inhibitory effects on its target cells, indicating the good biosafety
   of PH20/CCM@PMXene.

Fig. 2.

   [115]Fig. 2
   [116]Open in a new tab

   Dual-targeting, penetrating capability, and biocompatibility of
   PH20/CCM@PMCS. (A) Images of SKOV3 and h_CAF treated with SKOV3
   membrane-coated PMCS, h_CAFs membrane-coated PMCS, and
   h_PH20/CCM-coated PMCS for 7 h. Images of ID8 and m_CAF treated with
   ID8 membrane-coated PMCS, m_CAFs membrane-coated PMCS, and m_
   PH20/CCM-coated PMCS for 7 h. The nucleus is stained with DAPI (blue),
   and PMCS is labeled with FITC (green) (Scale bar: 12.5 μm). (B) Images
   of HEK-293T and h_NF treated with h_ PH20/CCM@PMCS for 7 h. Images of
   B16 and m_NF treated with m_PH20/CCM@PMCS for 7 h (Scale bar: 12.5 μm).
   (C) Images of RAW264.7 treated with PMCS, h_PH20/CCM@PMCS, and
   m_PH20/CCM@PMCS for 7 h (Scale bar: 12.5 μm). (D) CCK-8 assay indicated
   PH20/CCM, PMxene, and PH20/mPMxene had no significant inhibitory effect
   on either cancer cells or normal cells (N = 3). (E) The penetrating
   ability of PH20/CCM@PMCS was investigated by the orthotopic homograft
   in vivo (N = 3). The fluorescence images of harvested major organs and
   tumor at 24 h post-injection. (F) Percentage of red blood cell
   hemolysis at different concentrations of PH20/CCM@PMCS (N = 3). Data
   are expressed as mean ± S.D. and analyzed by ordinary one-way ANOVA. (*
   P < 0.05, ** P ＜ 0.01, *** P ＜ 0.001, n.s. no significance)

   The targeting and penetrating abilities of PH20/CCM@PMCS were evaluated
   in vivo. PMXene was labeled with CY7 and encapsulated in PH20/CCM
   before injection into orthotopic tumor-bearing C57BL/6 mice through the
   tail vein. Using an in vivo fluorescence imaging system (Fig. [117]2E),
   significant aggregation of CCM@PMCS and PH20/CCM@PMCS at the tumor site
   was observed in mice 24 h after intravenous injection, which was not
   observed in the PMCS group. PH20/CCM@PMCS was more concentrated within
   the tumor tissue than CCM@PMCS, indicating that PH20/CCM@PMCS could
   effectively penetrate the physical barrier in the tumor tissue,
   achieving deep treatment. Neither the heart nor the spleen showed
   significant fluorescence accumulation; however, in the CCM@PMCS and
   PH20/CCM@PMCS groups, the liver, lungs, and kidneys exhibited slight
   fluorescence after 24 h following intravenous injection, indicating the
   tumor-targeting ability of cell membrane-encapsulated nanoparticles.

   A red blood cell hemolysis assay was used to evaluate the blood
   compatibility of PH20/CCM@PMCS at different concentrations (PMXene
   concentration: 10–10,000 µg mL^-1). The percentage of hemolysis was
   calculated based on the absorbance of the supernatant of the
   PH20/CCM@PMCS and the red blood cell solution mixture (Fig. [118]2F).
   Under the working concentration (10 µg mL^-1), negligible hemolysis
   (< 5%) was observed, confirming the good blood compatibility of the
   nanoparticles.

Effect of PH20/CCM@PMCS on ovarian tumors in Vitro

   To select the most effective siRNA targeting p65 in human cells, we
   verified three siRNAs (sip65 #1, sip65 #2, and sip65 #3) targeting
   human p65 RNA in SKOV3 cells (Fig. [119]3A). Another three siRNAs
   targeting p65 in mouse cells (sip65 #4, sip65 #5, and sip65 #6) were
   assessed in ID8 for knockdown efficiency (Fig. [120]3B). The two siRNA
   strands with the highest knockdown efficiency (sip65 #3 and sip65 #4)
   were selected to construct the nanoparticles. Subsequently, we
   evaluated the mRNA and protein expression of p65 in the cells treated
   with PMC, PMS, PMCS, CCM@PMCS, and PH20/CCM@PMCS. The RNA and protein
   levels of p65 decreased significantly following PMS treatment,
   indicating that the nanoparticles successfully introduced siRNA into
   the cells and knocked down the target RNA. The RNA and protein levels
   of p65 increased following PMC treatment, but those in the PMCS,
   CCM@PMCS, and PH20/CCM@PMCS groups treated with sip65 were lower than
   those in the NC group (Fig. [121]3C, D, E). Next, we explored the
   antitumor efficacy of PH20/CCM@PMCSs in vitro. The cloning formation
   assay showed that PH20/CCM@PMCS significantly inhibited ovarian cancer
   cell growth (Fig. [122]3F). In addition, compared with that in the NC
   and PMCS groups, a significant increase in apoptosis was observed in
   the tumor cells treated with PH20/CCM@PMCS (Fig. [123]3G, H). We
   further investigated the antitumor efficacy of the engineered cell
   membrane-coated nanoparticles PH20/CCM@PMCS under complex 3D culture
   conditions by incubating PH20/CCM@PMCS with 3D cultured tumorspheres. A
   comparison of the changes in the volume of the tumorspheres after 7
   days revealed that the volume of the tumorspheres decreased most after
   treatment with PH20/CCM@PMCS (Fig. [124]3I, J). This may be attributed
   to the stronger penetration of the encapsulation membrane after the
   engineered PH20 modification, indicating high efficacy of PH20/CCM@PMCS
   in antitumor therapy in vitro.

Fig. 3.

   [125]Fig. 3
   [126]Open in a new tab

   Anti-tumor effect of PH20/CCM@PMCS in vitro. (A) Knockdown efficiency
   of sip65 in SKOV3 (N = 3). (B) Knockdown efficiency of sip65 in ID8
   (N = 3). (C) Relative RNA expression of p65 to β-actin in SKOV3 in each
   group (N = 3). (D) Relative RNA expression of p65 to β-actin in ID8 in
   each group (N = 3). (E) Relative protein expression of p65 in SKOV3 and
   ID8 in each group. (F) Colony formation assay images of tumor cells
   treated with PMC, PMS, PMCS, CCM@PMCS, PH20/CCM@PMCS. (G, H) Detection
   and quantitative analysis of apoptosis in SKOV3 and ID8 treated in each
   group by flow cytometry (N = 3). (I, J) 3D growth of tumors image and
   quantitative analysis of tumor sphere in NC, PMC, PMS, PMCS, CCM/PMCS,
   and PH20/CCM@PMCS group (Scale bar: 100 μm, N = 3). Data are expressed
   as mean ± S.D. and analyzed by ordinary one-way ANOVA. (* P ＜ 0.05,  **
   P < 0.01, *** P < 0.001, n.s. no significance)

PH20/CCM@PMCS Induced Immunogenic Cell Death in Tumor cells

   Considering that NF-κB is an important regulatory pathway for reactive
   oxygen species (ROS) production in tissue cells, the promotion of cell
   apoptosis by PH20/CCM@PMCS may depend on increased intracellular
   oxidative stress [[127]46]. The generation of ROS was detected via
   DCFH-DA probes (green). The PMC group exhibited few ROS, whereas the
   PH20/CCM@PMCS group exhibited an intense increase in ROS levels
   (Fig. [128]4A, B). Moreover, the mitochondrial membrane potential was
   assessed using the JC-1 probe. After PH20/CCM@PMCS treatment, the
   mitochondrial membrane potential decreased (Fig. [129]4C, D),
   indicating an increase in cellular oxidative stress. High intracellular
   ROS can result in endoplasmic reticulum (ER) dysfunction [[130]47,
   [131]48]. Therefore, we performed protein blotting analysis to
   investigate whether treatments of each group would affect ER stress.
   The identification of ER stress-related proteins indicated that
   PH20/CCM@PMCS remarkably induced ER dysfunction compared with other
   tested groups (Fig. [132]4E). ER stress and ROS are important
   intracellular pathways that induce immunogenic cell death (ICD)
   [[133]49]. The translocation of calcium reticulum protein (CRT) to the
   cell membrane, extracellular release of adenosine 5ʹ-triphosphate
   (ATP), and secretion of high mobility group protein cassette 1 (HMGB1)
   are classic features of ICD [[134]50–[135]52]. Therefore, we explored
   the above three indicators in each group to detect whether cells
   underwent ICD. We collected the cell culture supernatant from the NC,
   PMC, PMS, PMCS, CCM@PMCS, and PH20/CCM@PMCS groups. The ELISA results
   show that the supernatant of the PH20/CCM@PMCS group exhibits a
   significant increase in the level of HMGB1 (Fig. [136]4F). Significant
   ATP secretion was observed in the PH20/CCM@PMCS group using the
   luciferase assay (Fig. [137]4G). To verify the membrane transfer of
   calcium reticulum proteins, cell membrane immunofluorescence staining
   and western blot analyses were performed. The PH20/CCM@PMCS group
   showed a significant increase in the membrane transfer of calcium
   reticulum protein (Fig. [138]4H, I,J). In short, these results
   demonstrate that PH20/CCM@PMCS effectively increased intracellular ROS
   and led to ER stress to induce the production of damage-associated
   molecular patterns, thus triggering ICD in tumor cells.

Fig. 4.

   [139]Fig. 4
   [140]Open in a new tab

   PH20/CCM@PMCS induced immunogenic cell death in tumor cells. (A, B)
   Fluorescence images with semiquantitative analyses of intracellular ROS
   production in SKOV3 and ID8 treated in each group (Scale bar: 50 μm).
   (C, D) Fluorescence images with semiquantitative analyses of SKOV3 and
   ID8 mitochondrial membrane potential (JC-1 probe) (Scale bar: 25 μm).
   (E) Levels of CHOP, elF2α, ATF6, and BIP in SKOV3 and ID8 in each
   group. (F) The concentration of HGBM1 released by SKOV3 and ID8 treated
   in each group (N = 3). (G) The concentration of ATP released by SKOV3
   and ID8 treated in each group (N = 3). (H, I) Fluorescence images with
   semiquantitative analyses of CRT expression on the cell membrane of
   SKOV3 and ID8 (Scale bar: 12.5 μm). (J) Protein expression of CRT on
   the cell membrane of SKOV3 and ID8. Na+, K+-ATPase serves as an
   internal reference for cell membrane proteins. β-actin, a cytoplasmic
   protein was not observed to demonstrate cytoplasmic removal. Data are
   expressed as mean ± S.D. and analyzed by ordinary one-way ANOVA. (*
   P < 0.05, ** P ＜ 0.01,  *** P < 0.001)

PH20/CCM@PMCS regulate cytokine secretion in CAFs to reverse tumor-promoting
TME

   CAFs foster a supportive environment for tumor growth [[141]53,
   [142]54]. Furthermore, CAFs promote tumor formation and progression by
   producing various cytokines, growth factors, and ECM proteins, which
   contribute to blood and lymphatic vessel formation, ECM remodeling,
   immune system suppression, drug resistance, and tumor invasion
   [[143]55–[144]58]. Hence, suppressing the release of cancer-promoting
   substances from CAFs might alter the TME, making it beneficial for
   inhibiting tumor growth. To further investigate the effect of
   PH20/CCM@PMCS on CAFs, the cytokine levels in the CAF supernatants
   treated with PH20/CCM@PMCS were measured using the Cytokine Proteome
   Profiler Array (Fig. [145]5A, B). Among them, 8 cytokines were
   downregulated, and 15 were upregulated. KOBAS3.0 was used for pathway
   enrichment analysis of the differential cytokines, and significant
   enrichment in angiogenesis, inflammatory immunity, and cell migration
   and adhesion was observed (Fig. [146]5C). The 23 differentially
   expressed cytokines were categorized into three groups based on their
   functions and the results of KEGG enrichment analysis (Fig. [147]5D),
   and their functions are listed in Table [148]S1. The top six
   differentially expressed cytokines included GDF-15, GROα, Dkk-1, MMP-9,
   CD14, and osteopontin. Among them, osteopontin, Dkk-1, and MMP-9
   participate in ECM remodeling and tumor cell invasive migration.
   Specifically, osteopontin induces HIF-1α to promote ovarian cancer
   progression and metastasis by activating the PI3-K/Akt survival pathway
   [[149]59]. The DKK-1 protein inhibits tumor cell survival and migration
   by regulating β-catenin/E-cadherin signaling [[150]60]. MMP-9 allows
   migration and invasion of ovarian cancer cells by degrading basement
   membrane components, ultimately leading to metastasis [[151]61]. CD14,
   DPPIV, and GDF-15 are involved in inflammatory cell recruitment and
   immune cell infiltration and activation. Specifically, CD14^high
   CD16^low monocytes induce M2-like tumor-associated macrophages
   phenotype and suppress dendritic cell-activated CD4 + T-cell responses
   in ovarian cancer [[152]62]. DPPIV is involved in the
   signal-transducing process. Cross-linking of DPPIV and CD3 with
   immobilized mAbs can induce T-cell activation and IL-2 production
   [[153]63]. GDF15 promotes the production of inducible Treg cells of
   peripheral origin in hepatocellular carcinoma by interacting with CD48
   [[154]64]. Moreover, platinum-based chemotherapy has been reported to
   increase the levels of circulating GDF-15 in patients with cancer
   [[155]65, [156]66]. Additionally, cytokines that play a key role in
   angiogenesis—angiogenin and VEGF—were notably reduced. In conclusion,
   changes in cytokines secreted by CAFs related to invasion,
   angiogenesis, and immune infiltration were observed following treatment
   with PH20/CCM@PMCS, and their real effects on TME required verification
   through further experiments.

Fig. 5.

   [157]Fig. 5
   [158]Open in a new tab

   PH20/CCM@PMCS alters cytokine levels secreted by CAFs. (A, B) Cytokine
   Proteome Profiler Array images and semiquantitative analysis of the
   supernatant of h-CAF treated with PH20/CCM@PMCS. (C) Pathway enrichment
   analysis of differentially secreted cytokines. (D) Schematic diagram of
   the effects of PH20/CCM@PMCS on CAF-mediated TME remodeling from three
   aspects: inhibition of angiogenesis, suppression of cancer cell
   invasion, and activation of immunity. Data are expressed as mean ± S.D

   Next, we co-cultured cancer cells with conditioned medium (CM). The
   invasion capability of tumor cells was investigated using Transwell
   assay (Fig. [159]6A). Compared with tumor cells incubated with CM from
   other groups, a significant reduction in the migration rate was
   observed in the cells treated with PH20/CCM@PMCS CM. Thereafter, we
   explored the anti-angiogenic characteristics of CAFs treated in each
   group. The CM from the PH20/CCM@PMCS group hindered the migration and
   tube formation abilities of vascular endothelial cells in vitro
   compared with those from other groups (Fig. [160]6B, C,D).

Fig. 6.

   [161]Fig. 6
   [162]Open in a new tab

   PH20/CCM@PMCS remodels TME by influencing cytokine secretion of CAFs.
   (A) Images of transwell assay and the relative migratory number of
   ovarian cancer cells treated with NC CM, PMC CM, PMS CM, PMCS CM,
   CCM@PMCS CM, and PH20/CCM@PMCS (Scale bar: 100 μm, N = 3). (B) Tube
   formation assays of HUVEC treated with different CMs (Scale bar:
   100 μm). (C) Wound healing assays of HUVEC treated with different CMs
   (Scale bar: 200 μm). (D) The statistical results of tube formation
   assays and wound healing assays (N = 3). Data are expressed as
   mean ± S.D. and analyzed by ordinary one-way ANOVA. (* P < 0.05, **
   P < 0.05, *** P < 0.001)

   Subsequently, we investigated the effects of CAFs on immune function
   after PH20/CCM@PMCS treatment. Compared with NC, the CM from
   h_PH20/CCM@PMCS increased the average percentage of
   CD86 + CD11b + macrophages from 14.2 to 19.3%; whereas, the CM from
   m_PH20/CCM@PMCS increased the average percentage of
   CD86 + F4/80 + macrophages from 15.4 to 55.2% (Fig. [163]7A, B,C, D).
   This may be attributed to the inhibition of the NF-κB pathway, which is
   involved in the tumor inflammatory response. The cytokines secreted by
   CAFs treated with PH20/CCM@PMCS significantly stimulated macrophage
   polarization toward the M1 type compared to the PMCS treatment. This
   effect may likely occur due to the increased internalization of
   nanoparticles by CAFs and the enhanced internalization of hybrid-cell
   membranes by macrophages as tumor antigens, further promoting their
   polarization toward the M1 type. These results indicated that
   PH20/CCM@PMCS may contribute to macrophage polarization and reshape the
   TME. CD8 + cytotoxic T cells (CTL) are one of the key immune
   surveillance cells in the TME, which are a positive indicator of
   prognosis. Increasing the proportion of CTL in the tumor tissues of the
   patients can help inhibit tumor progression. To test the effect of CAFs
   on CTL activation following PH20/CCM@PMCS treatment, we cultured T
   cells using CMs from different treatment groups. The number of
   activated T cells that differentiated into CD8 + T cells in the
   PH20/CCM@PMCS group was almost 50% higher than that in the NC group
   (Fig. [164]7E, F,G, H). Further, we assessed IFN-γ and TNF-α secreted
   from T cells in each treatment group through ELISA. The highest IFN-γ
   and TNF-α levels were observed in the PH20/CCM@PMCS group, indicating
   functional activation of CTL (Fig. [165]7I). Collectively,
   PH20/CCM@PMCS regulates cytokine secretion in CAFs to reverse the
   tumor-promoting TME.

Fig. 7.

   [166]Fig. 7
   [167]Open in a new tab

   PH20/CCM@PMCS remodels the immune environment by influencing cytokine
   secretion of CAFs. (A) Proportion of M1 macrophages (CD86 + CD11b+) in
   THP-1 after treatment with different CMs (N = 3). (B) Proportion of M1
   macrophages (CD86 + F4/80+) in RAW264.7 after treatment with different
   CMs. (C) The statistical results of M1 type THP-1 proportion. (D) The
   statistical results of M1 type RAW264.7 proportion. (E) Proportion of
   human CD8 + T cells after treatment with different CMs (N = 3). (F)
   Proportion of mouse CD8 + T cells after treatment with different CMs
   (N = 3). (G) The statistical results of human CD8 + T cell proportion.
   (H) The statistical results of mouse CD8 + T cell proportion. (I) IFN γ
   and TNF α were quantified by ELISA, in the culture supernatants of T
   cells stimulated with different groups (N = 3). Data are expressed as
   mean ± S.D. and analyzed by ordinary one-way ANOVA. (* P < 0.05, **
   P < 0.05, *** P < 0.001)

Inhibitory effect of PH20/CCM@PMCS in orthotopic homograft

   To confirm the role of PH20/CCM@PMCS in EOC growth in vivo, we selected
   healthy C57BL/6 mice to construct orthotopic homografts and designed in
   vivo experiments (Fig. [168]8A). Tumor-bearing mice were randomly
   divided into six groups and injected with physiological saline, PMC,
   PMS, PMCS, CCM@PMCS, or PH20/CCM@PMCS through the tail vein on Day 0,
   4, 8, 12, 16, and 20. During the treatment period, no significant
   differences in body weight were observed among different groups of mice
   (Figure [169]S9). After treatment, we collected the tumors from each
   group and measured their volumes. Treatment of PMC and PMS showed a
   slight inhibitory effect on tumors compared with the NC group. In
   contrast, tumor growth was significantly inhibited in the PH20/CCM@PMCS
   group. Compared with the tumor volume of the PMCS group without hybrid
   membrane coverage, the tumor volume of the CCM@PMCS and PH20/CCM@PMCS
   groups with hybrid membrane coverage was reduced by 63.37% and 85.30%,
   respectively. This suggests a stronger anti-tumor effect of hybrid
   membrane-coated nanoparticles, which may be related to the greater
   amounts of hybrid membrane-coated nanoparticles being ingested.
   Furthermore, the strong penetration of PH20/CCM@PMCS increases the
   delivery of nanoparticles, making the efficacy even more significant.
   In conclusion, PH20/CCM@PMCS significantly reversed the growth of the
   homograft tumors (Fig. [170]8B, C). Compared with other groups, the
   number of Ki67-positive cells stained via immunohistochemistry (IHC)
   was significantly reduced after treatment with PH20/CCM@PMCS or
   CCM@PMCS (Fig. [171]8D, E), indicating a stronger inhibition of tumor
   proliferation. The tumor tissue was stained using Masson’s staining to
   observe its histopathology. Notably, in the PH20/CCM@PMCS treatment
   group, the tumor tissue was sparse, and the cell nucleus size
   decreased, whereas in the control group, the tumor tissue was dense,
   and the cell nucleus size showed strong heterogeneity (Figure
   [172]S10).

Fig. 8.

   [173]Fig. 8
   [174]Open in a new tab

   Anti-tumor effect of PH20/CCM@PMCS in orthotopic homograft. (A)
   Experimental procedure in vivo. (B) Tumor volume after 21-day treatment
   (N = 5). (C) Excised ovaries and uterus from NC, PMC, PMS, PMCS,
   CCM@PMCS, PH20/CCM@PMCS after 21-day treatment. (D, E)
   Immunohistochemical staining of Ki67 with semiquantitative analyses of
   excised tumor slices in each group (Scale bar: 10X: 100 μm, 40X: 25 μm,
   N = 3). (F, G) Immunohistochemical staining of CD31 with
   semiquantitative analyses of excised tumor slices in each group (Scale
   bar: 10X: 100 μm, 40X: 25 μm, N = 3). Data are expressed as mean ± S.D.
   and analyzed by ordinary one-way ANOVA. (* P < 0.05, ** P < 0.05, ***
   P < 0.001)

   Furthermore, we investigated the antitumor mechanisms of these
   nanoparticles. We evaluated the formation of blood vessels in tumor
   tissues by measuring CD31 expression. Interestingly, PMC promoted tumor
   angiogenesis, which was similar to a previous clinical study and may be
   associated with chemotherapeutic drug-induced hypoxia [[175]67].
   Compared with NC, the CD31 levels in the PMS and PMCS groups did not
   change significantly, whereas the CD31 levels in the PH20/CCM@PMCS
   group went down nearly 75%, suggesting that PH20/CCM@PMCS had an
   inhibitory effect on tumor angiogenesis (Fig. [176]8F, G).

   To verify the effect of PH20/CCM@PMCS on immune activation in vivo, we
   measured the number of macrophages in the tumor tissues of each mouse
   group. Compared with other treatments, PH20/CCM@PMCS significantly
   increased the aggregation of F4/80 (green) positive macrophages in the
   tumor tissue, and the macrophages expressed CD86 (red), indicating
   polarization toward the M1 type (Fig. [177]9A, B). To further assess
   TME transformation, we measured the levels of dendritic cell maturation
   at the tumor site. The levels of CD11c+(green) CD86+(red) dendritic
   cells were elevated in the PH20/CCM@PMCS group compared with the other
   groups (Fig. [178]9C, D), indicating that PH20/CCM@PMCS may contribute
   to dendritic cell maturation. Subsequently, we evaluated the T cells
   expressing CD8 and granzyme B in the tumor tissues using IHC and
   observed increased cytotoxic T-cell aggregation in the
   PH20/CCM@PMCS-treated group. The staining of T cells with
   immunosuppressive function (FOXP3-positive) revealed decreased Treg
   aggregation in the PH20/CCM@PMCS-treated group (Fig. [179]9E, F). These
   results indicate that PH20/CCM@PMCS relieved the TME by promoting
   immune infiltration of the tumor tissue.

Fig. 9.

   [180]Fig. 9
   [181]Open in a new tab

   Effects of PH20/CCM@PMCS on remodeling immune infiltration in TME. (A,
   B) Immunofluorescence staining for F4/80 (green) and CD86 (red) with
   semiquantitative analyses of excised tumor slices in each group (Scale
   bar: 64X: 20 μm, X: 180X: 10 μm). (C, D) Immunofluorescence staining
   for CD11c (green) and CD86 (red) with semiquantitative analyses of
   excised tumor slices in each group (Scale bar: 64X: 20 μm, 180X:
   10 μm). (E, F) Immunohistochemical staining of CD8, Foxp3, and Granzyme
   B with semiquantitative analyses of excised tumor slices in each group
   (Scale bar: 10X: 100 μm, 40X: 25 μm, N = 3). Data are expressed as
   mean ± S.D. and analyzed by ordinary one-way ANOVA. (* P < 0.05, **
   P < 0.05, *** P < 0.001)

   Apart from these, varying degrees of cancer lung metastasis were
   observed in mouse lungs. Hematoxylin and eosin (H&E)-stained
   pathological sections of metastatic lesions were observed (Figure
   [182]S11), and compared with the NC group, both the PH20/CCM@PMCS and
   CCM@PMCS groups exhibited a significant reduction in metastatic
   lesions, indicating the inhibitory effect of PH20/CCM@PMCS on distant
   ovarian cancer metastasis.

   After 21 days of treatment, H&E staining of important organs of the
   mice, including the heart, liver, spleen, lungs, and kidneys, revealed
   no significant damage to these organs (Figure [183]S12A). The venous
   blood of the mice was collected for biochemical analysis. Indicators
   such as the UA, CR, ALT, AST, albumin, and total protein levels were
   all within the normal ranges (Figure [184]S12B). The above results
   demonstrated excellent targeting and penetration abilities and good
   biosafety of PH20/CCM@PMCS in mice in vivo.

   Assessment of metal nanodrug metabolism is essential for further in
   vivo application. To investigate the systemic circulation time of the
   nanoparticles, 10 µL of blood was collected for measurement at
   different time points after injection into healthy C57BL/6 mice. The
   plasma concentration of the nanoparticles was negligible at 48 h after
   injection (Figure [185]S13). Next, we investigated the excretion and
   tissue deposition of PH20/CCM@PMCS in tumor-bearing mice. PH20/CCM@PMCS
   was injected intravenously into C57BL/6 mice loaded with orthotopic
   homografts. Ti levels were measured in urine and feces at 12, 24, and
   48 h to monitor excretion. Ti excretion in the urine and feces of mice
   after 48 h reached 20.09% and 16.80%, respectively (Figure [186]S14).
   The heart, liver, spleen, lungs, and kidneys of mice were collected 21
   days after treatment to detect the biodistribution of nanoparticles.
   Intravenous PH20/CCM@PMCS was mainly deposited in the liver and spleen
   due to nanoparticle capture by the endothelial reticular system.
   Nanoparticles were deposited in the kidneys, likely due to the
   excretion of nanoparticles through the kidneys (Figure [187]S15).
   Overall, nanoparticle deposition in main organs was low, similar to the
   results from in vivo fluorescence imaging in small animals. High
   excretion and low off-target effects of nanoparticles ensure the safety
   of their clinical translation for cancer therapy.

PH20/CCM@PMCS potentiates Anti-PD-L1 blockade therapy

   PD-L1 monoclonal antibody is a widely used immune checkpoint inhibitor
   for cancer treatment, but its response rate is low in ovarian cancer
   [[188]68]. As PH20/CCM@PMCS induced ICD and enhanced immune
   infiltration in the TME, we evaluated its potential to improve the
   efficacy of tumor immune checkpoint therapy. We administered
   PH20/CCM@PMCS separately, anti-PD-L1, or a combination of both in ID8
   tumor-bearing mice (Figure [189]S16A), whereas CBP was used as a
   standard treatment for control. Efficacy assessment of ovarian in situ
   tumor growth showed that anti-PD-L1 alone did not significantly affect
   tumor growth, suggesting that this tumor subtype exhibited a limited
   response to immune checkpoint inhibition. In contrast, PH20/CCM@PMCS
   alone significantly inhibited tumor growth and was more effective than
   CBP alone. Moreover, tumor inhibition by PH20/CCM@PMCS was
   substantially augmented in combination with anti-PD-L1 (Figure
   [190]S16B, C). The therapeutic interventions did not adversely affect
   physiological parameters such as body weight (Figure [191]S16D) and
   liver and kidney function (Figure [192]S16E). Tumor growth inhibition
   achieved using PH20/CCM@PMCS was further enhanced in combination with
   anti-PD-L1.

Discussion

   Owing to the high degree of malignancy and the difficulty of early
   diagnosis, EOC patients are often diagnosed at an advanced stage, with
   frequent relapse following initial treatment, resulting in a five-year
   survival rate of approximately 50% [[193]69]. Although immunotherapy
   has been proposed as a novel therapeutic option, EOC tumors, classified
   as “cold tumors”, are generally less sensitive to immunotherapy.
   Furthermore, patients suffer from severe adverse effects during
   treatment, in addition to poor survival rates. Hence, targeted
   therapies to enhance patient sensitivity to platinum-based chemotherapy
   and stimulate tumor immune infiltration must be developed.

   Tumor cells grow in a complex TME ecosystem, and focusing on tumor cell
   growth alone is not sufficient to inhibit tumor development. The TME
   constructs a complex and changeable tumor growth “soil” through
   crosstalk and communication of various physical and biochemical factors
   [[194]70]. Chemotherapy resistance and immune silencing induced by the
   TME are remarkable challenges in cancer treatment. Cancer cells
   previously exposed to chemotherapy drugs undergo oncogene activation
   and tumor suppressor gene dysregulation following interaction with the
   TME, resulting in resistance to subsequent treatments [[195]71].
   Meanwhile, the TME also helps tumor tissue achieve immune evasion by
   recruiting immunosuppressive cells and secreting immunosuppressive
   factors [[196]72]. Therefore, TME regulation is a favorable choice for
   reversing tumor drug resistance and immune evasion to improve treatment
   efficacy. As the main component of the TME, CAFs produce numerous
   factors that facilitate the establishment of a pro-tumorigenic TME.
   First, CAFs produce a dense ECM that reduces drug absorption and immune
   cell invasion. CAFs synthesize large amounts of several collagen types,
   hyaluronic acid, and fibronectin, constituting the ECM and basement
   membrane. Furthermore, CAFs can recruit endothelial progenitor cells to
   the tumor site, resulting in hyperpermeability of the neovascularized
   blood and increasing interstitial pressure at the tumor site [[197]73].
   Subsequently, the CAF-produced MMP provides pre-metastatic ecological
   niches for tumor cells and directs tumor cells to invade collectively
   by degrading the basement membrane [[198]74]. Third, CAFs secrete
   several immunosuppressive factors, including TGF-β, IL-6, and CCL2, to
   promote the organization and recruitment of immunosuppressive cells,
   including regulatory T cells and M2-type macrophages [[199]75].
   Therefore, we propose a strategy to reprogram the TME by targeting CAFs
   to disrupt the microenvironment that supports tumor growth and
   recurrence. Wound healing assay, angiogenesis assay, invasion assay,
   and immune cell co-culture demonstrated that PH20/CCM@PMCS effectively
   inhibited the tumor-promoting TME. The primary mechanism by which
   PH20/CCM@PMCS specifically disrupts the tumor-promoting activity of
   CAFs was attributed to its highly permeable delivery to tumors and
   specific targeting of CAFs, which disrupted the dense stromal barrier
   and remodeled the TME. PH20/CCM@PMCS reduced the secretion of
   pro-angiogenic factors, such as VEGF and angiogenin, by CAFs,
   inhibiting tumor angiogenesis, and regulated the secretion of stromal
   remodeling factors, such as osteopontin and Dkk-1 to prevent tumor cell
   invasion and metastasis. In addition, PH20/CCM@PMCS increased the
   number of immunoreactive cells, such as CD8T cells and M1-type
   macrophages in TME.

   Platinum-based drugs are susceptible to resistance mechanisms such as
   reduced drug accumulation, activation of survival signaling pathways,
   and the regulation of TME. The accumulation of drugs in Pt-resistant
   tumor cells is reduced. Clinically relevant resistance to cisplatin or
   carboplatin is associated with reduced drug uptake due to the loss of
   subunits LRRC8A and LRRC8D of volume-regulated anion channel (VRAC)
   heterodimeric LRRC8 VRAC [[200]76]. Antitumor drug resistance is also
   associated with the activation of pro-survival signaling. Yang et al.
   recently reported that NF-κB p65 binds to the mortalin promoter,
   stimulating the proliferation and migration of ovarian cancer cells
   [[201]77]. Inhibition of mortalin enhances platinum-drug effectiveness
   against ovarian cancer by translocating p53 to the nucleus [[202]43].
   CAF leads to drug resistance by providing signals that favor tumor
   progression. For example, activation of NF-κB signaling in response to
   oxidative stress mediates the upregulation of MCP-1 secretion and
   promotes tumor growth in oral cancer [[203]78]. In our study,
   PH20/CCM@PMCS likely enhanced tumor sensitivity to platinum-based drugs
   in several ways. First, PH20/CCM@PMCS actively targeted the tumor site,
   and the PH20 on its surface effectively improved the drug delivery
   efficiency and enhanced drug accumulation. Second, PH20/CCM@PMCS
   inhibited the anti-apoptotic and immunosuppressive effects induced by
   the NF-κB pathway. Finally, PH20/CCM@PMCS remodeled TME and enhanced
   immune infiltration by modulating CAFs.

   In this study, we achieved multi-dimensional remodeling of the TME by
   constructing engineered hybrid membrane-coated nanoparticles
   PH20/CCM@PMCS with dual targeting of tumor cells and CAFs.
   PH20/CCM@PMCS were suitable for intravenous injection in terms of size,
   biocompatibility, cytotoxicity, and targeting. Furthermore, in terms of
   function, the dual-targeting mechanism ablated tumor cells while
   significantly inhibiting the tumor-promoting function of CAFs, thereby
   improving TME in three aspects: immune infiltration, angiogenesis, and
   matrix remodeling.

   Nevertheless, challenges and opportunities lie ahead regarding the
   translation of PH20/CCM@PMCS nanoparticles for clinical drug delivery.
   Although no significant toxicity was observed in mice injected
   intravenously with MXene for up to 50 days and MXene can be biodegraded
   in the presence of human myeloperoxidase [[204]79], the long-term role
   of nanoparticles in the body remains unexplored. In particular, the
   biodegradation mechanism and long-term toxicity of PH20/CCM@PMCS in
   vivo have not been sufficiently explored. In-depth studies on the
   biological fate and safety of nanoparticles are essential to ensure
   their safe application. On the other hand, considering the
   heterogeneity and complexity of CAF origins, TME contains several CAF
   subtypes. The specific mechanisms by which different CAF subtypes
   regulate TME remain to be further determined. For targeting CAFs, the
   limitations and challenges associated with each subtype must be
   considered. A deeper understanding of CAFs and TME communication is
   required for future studies, whether they are directly targeting a
   specific subtype of CAFs or targeting molecules and pathways common to
   different subtypes of CAFs. Additionally, deciphering the interactions
   between nanoparticles and various cell types in the TME can help
   advance the design of nanoparticles to enhance therapeutic efficacy and
   safety. For instance, nanoparticle delivery may be affected by cellular
   heterogeneity and their stereochemistry through single-cell sequencing
   analysis [[205]80, [206]81]. High-resolution techniques can help
   characterize the complexity and heterogeneity of cell–nanoparticle
   interactions. Delogu et al. and Tae et al. revealed the interaction
   between graphene and immune cells and between Ag nanoparticles and
   different subpopulations of immune cells, respectively, using
   single-cell mass spectrometry flow cytometry [[207]82, [208]83]. Our
   group is exploring cellular communication in the TME using
   high-resolution methods such as single-cell sequencing and proteomics
   to support the clinical translation of the dual-targeting strategy to
   remodel the TME in this study.

Conclusion

   In summary, we successfully designed and synthesized the engineered
   cell-membrane-camouflaged nanoparticles, PH20/CCM@PMCS. PH20/CCM@PMCS
   nanoparticles could efficiently break through the physical barrier of
   the TME to achieve intratumoral delivery and exhibited dual-targeting
   ability toward tumor cells and CAFs. After internalization by tumor
   cells, these nanoparticles disrupted the redox homeostasis within tumor
   cells, induced ICD, and exerted significant antitumor effects.
   Simultaneously, after internalization by CAFs, these nanoparticles
   inhibited the adverse effects of CAFs on the ECM, tumor blood vessels,
   and immune infiltration, thereby reshaping the TME. Therefore,
   PH20/CCM@PMCS achieved a multi-dimensional and multilevel response for
   each component in the TME, unifying specificity, efficiency, and
   effectiveness for ovarian tumor treatment.

Experimental section

   Available in Supporting Information.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [209]Supplementary Material 1^ (32.9MB, docx)

Acknowledgements