The novel hydrogen-PT2385-silncARSR nanocomplex impairs tumor angiogenesis and mitochondrial activity in sunitinib-resistant renal cancer

Suxian Hu; Yan Zhu; Yi Duan; Liting Wang; Jian Yu; Zhihua Wu; Yourong Duan; Ying Sun

doi:10.1016/j.mtbio.2025.102450

. 2025 Oct 22;35:102450. doi: 10.1016/j.mtbio.2025.102450

The novel hydrogen-PT2385-silncARSR nanocomplex impairs tumor angiogenesis and mitochondrial activity in sunitinib-resistant renal cancer

Suxian Hu ^1,¹, Yan Zhu ^1,¹, Yi Duan ^1,¹, Liting Wang ¹, Jian Yu ¹, Zhihua Wu ¹, Yourong Duan ^1,^⁎, Ying Sun ^1,^⁎⁎

PMCID: PMC12606021 PMID: 41235366

Abstract

Hydrogen therapy has emerged as a promising agent for cancer treatment. Earlier research demonstrated that hydrogen (H₂) possesses anti-angiogenic effects across multiple tumor types. However, no studies have yet investigated the anti-angiogenic effects of H₂ in sunitinib-resistant clear cell renal cell carcinoma (ccRCC) or elucidated the mechanism involved. Besides, both Hypoxia-inducible factor 2α (HIF-2α) and lncRNA Activated in RCC with sunitinib resistance (lncARSR) play essential roles in mediating ccRCC sunitinib resistance. Nevertheless, traditional multidrug combination strategy fails to achieve precise and effective suppression of drug-resistance related targets in conjunction with gas therapy. Therefore, we engineered a tumor-targeted nanocomplex, enabling localized H₂ generation and efficient PT2385 and small interfering RNA targeting lncARSR (silncARSR) delivery to inhibit molecular targets associated with sunitinib resistance in ccRCC. Mechanistically, in situ generated hydrogen and lncARSR knockdown effectively suppresses tumor angiogenesis by downregulating vascular endothelial growth factor A(VEGFA) secretion from sunitinib-resistant cancer cells and M2-like tumor-associated macrophages (TAMs). Thus, the anti-angiogenic activity of PT2385 (HIF-2α inhibitor) was potentiated by H₂ and silncARSR significantly. Moreover, the combination of H₂, silncARSR and PT2385 exerts significantly potentiated efficacy in modulating apoptosis-related protein expression and ultimately enhancing cancer cell mitochondrial apoptosis. The demonstrated high therapeutic efficacy and great biocompatibility of this Hydrogen-PT2385-silncARSR nanocomplex underscore the clinical translation potential for overcoming ccRCC sunitinib resistance.

Keywords: Tumor-targeted nanocomplex, Hydrogen therapy, Angiogenesis inhibition, Potentiated efficacy, Sunitinib-resistant clear cell renal cell carcinoma

Graphical abstract

1. Introduction

Among all the histological subtypes, clear cell renal cell carcinoma (ccRCC) accounts for 75–80 % of kidney cancer [1,2]. Sunitinib is a tyrosine kinase inhibitor (TKI) used to block tumor angiogenesis and growth and indicated for the first-line therapy of patients with advanced or metastatic ccRCC [3]. However, for patients who initially benefit from sunitinib treatment, resistance inevitably emerges within 1 year, leading to tumor progression, recurrence or metastasis and therefore negatively affect 5-year survival rate [4,5]. Moreover, the current combination therapies provide limited survival benefits for sunitinib-resistant renal cancer patients, owing to elevated toxicity, narrow spectrum of target coverage and limited selectivity towards tumor cells [[6], [7], [8]]. Accordingly, the development of an efficient and systemic strategy to overcome sunitinib resistance is critically urgent.

Sunitinib resistance is mediated via various tumor and environmental changes [9,10]. As an inherently highly vascularized solid tumor, ccRCC exhibits a rebound increase in angiogenesis following the acquiring of resistance to sunitinib [[11], [12], [13]]. Mounting evidence has revealed that evasive resistance to antiangiogenic TKIs arises from multiple adaptive mechanisms, including the upregulation of hypoxia-inducible factor (HIF) signaling, activation of alternative pro-angiogenic pathways and increased pericyte coverage that stabilizes the tumor vasculature [14,15]. In addition, M2-polarized tumor-associated macrophages (M2 TAMs) are significantly enriched in ccRCC, thereby orchestrating a pro-angiogenic and immunosuppressive tumor microenvironment that fosters malignant progression and therapeutic resistance [16,17].

Apart from the aforementioned features of tumor microenvironment (TME), the sunitinib-resistant tumor cells exhibit extensive intrinsic adaptations and reprogramming. The mitochondrion is a sophisticated organelle that regulates energy generation and allocation according to the levels of available nutrients and oxygen, as well as the cellular requirements for maintenance and proliferation [18]. Previous studies have demonstrated that sunitinib-resistant renal cancer tumor cells display elevated mitochondrial membrane potential and content, along with enhanced mitochondrial function and upregulated expression of anti-apoptotic proteins [19,20]. Therefore, we seek to develop a multifunctional platform aiming to efficiently, cooperatively and safely overcome sunitinib resistance issue in ccRCC by harnessing both intracellular and extracellular mechanisms.

Hydrogen (H₂), the lightest molecule with excellent permeability and biocompatibility, is widely considered one of the most promising therapeutic gases [[21], [22], [23]]. According to previous research, the therapeutic action of H₂ was attributed to its selective reduction of ·OH and ONOO⁻ while maintaining essential reactive oxygen species (ROS) for normal cellular signaling [24]. In the field of cancer therapy, emerging studies have revealed that H₂ can inhibit tumor development via acting as a mediator in apoptosis signaling, blocking tumor-induced angiogenesis and promoting the functional repolarization of TAMs toward M1-like phenotype with antitumor activity [[25], [26], [27]]. However, the mechanistic involvement of hydrogen in sunitinib-resistant ccRCC remains insufficiently characterized. In this research, we constructed a nanoplatform for Chlorophyll α (Chlα, a photosensitizer), L-ascorbic acid (AA, an electron donor) and AuNPs (gold nanoparticles, catalyst) to generate hydrogen gas (H₂) under the irradiation of 660 nm NIR (near infrared radiation) [28]. Moreover, to enable in situ generation in tumor tissue and better investigate its effects on drug-resistant ccRCCC, antibodies against overexpressed surface proteins of drug-resistant tumor cells were conjugated to the nanoplatform. And the subsequent findings validated this modification overcame the major drawbacks of traditional hydrogen administration-random diffusion, uncontrolled release and suboptimal efficacy [29,30].

HIF-2α and lncARSR are pivotal regulators of sunitinib resistance in ccRCC, serving as independent predictors for poor prognosis [31,32]. HIF-2α is a transcription factor accumulated in ccRCC, participated in many malignant processes including tumor angiogenesis, cell proliferation and metabolic reprogramming via regulating downstream molecules (vascular endothelial growth factor A(VEGFA), cyclin D1 (CCND1), solute carrier family 2 member 1 (SLC2A1), etc.) [33,34]. As a highly selective small-molecule inhibitor, PT2385 is designed to target and disrupt the functional dimerization of HIF-2α with HIF-1β, thereby inhibiting its transcriptional activity [12]. In addition, both clinical and preclinical investigations have substantiated that PT2385 displayed great therapeutic effect by significantly inhibiting tumor angiogenesis and suppressing proliferation and metabolic adaptation [35,36]. Long non-coding RNA (lncRNA) is defined as the non-protein-coding transcripts with >200 nucleotides and regulates multilevel gene expression via various mechanisms [37,38]. Prior research has confirmed that lncARSR (lncRNA activated in RCC with sunitinib resistance) induces ccRCC sunitinib resistance by binding to miR-34 and miR-449 in a competitive manner and promoting M2-like macrophages polarization through stat-3 pathway [39,40]. Therefore, the simultaneous inhibition of HIF-2α and lncARSR supports multi-targeted regulation of critical sunitinib resistance genes, offering a promising strategy to overcome drug resistance.

Nanotechnology has made tremendous progress in recent years [41]. The amphiphilic monomethoxy (polyethylene glycol)-poly (_{D, L}-lactide-co-glycolide)-poly (_L-lysine) triblock copolymer (mPEG-PLGA-PLL) is of great properties with high biocompatibility and superior drug-loading efficiency [42]. Also, the circulation time of drugs and nucleic acids encapsulated in this material could be effectively prolonged in vivo as the notably inhibition of RES (reticuloendothelial system) recognition [[43], [44], [45], [46]]. Notably, the amidogen in PLL allows for the modification of antibody via condensation reaction to enhance the targeting capability for specific tissues. Building on these insights, we sought to leverage the biomedical potential of the polymeric carrier-PEAL to construct a multifunctional therapeutic platform that enables the precise and cooperative integration of gas therapy, targeted molecular therapy and gene therapy, specifically tailored to overcome sunitinib resistance in renal cell carcinoma.

As depicted in Scheme 1, the PEAL was used as backbone material, encapsulating PT2385 for selectively inhibiting HIF-2α, small interfering RNAs for downregulating lncARSR (silncARSR) and H₂-producing substances (Chlα, AA and AuNPs) to generate hydrogen. Moreover, according to our previous investigations into ccRCC sunitinib resistance, Integrin alpha 1 (ITGA1, a membrane-associated integrin subunit) was found to be markedly upregulated on the surface of drug-resistant tumor cells [47,48]. Thus, the anti-ITGA1 antibody was further conjugated to the surface of this nanocomplex to facilitate tumor-specific targeting, thereby enabling efficient and site-specific co-delivery of H₂, PT2385 and silncARSR within sunitinib-resistant ccRCC (P/S/CVA@NPs-AI). In this work, H₂ and lncARSR knockdown collaboratively suppress tumor angiogenesis by two distinct mechanisms: directly downregulating VEGFA secretion from ccRCC tumor cells and promoting M2-like macrophages repolarization into M1-like macrophages, subsequently attenuating their pro-angiogenic functions. Combined with the action of PT2385, the pathological hypervascularization in drug-resistant ccRCC was robustly suppressed. In addition, the mitochondria integrity of sunitinib-resistant renal cancer tumor cells was also compromised by the combined intervention of H₂-PT2385-silncARSR nanocomplex, thereby enhancing pro-apoptotic signaling cascades and culminating in effective tumor cell eradication. We also established orthotopic sunitinib-resistant ccRCC tumor models to assess the in vivo therapeutic efficacy and biocompatibility of this nanocomplex(P/S/CVA@NPs-AI). In summary, multifunctional nanocomplex co-delivering H₂, PT2385 and silncARSR was developed to integrate gas therapy, molecular targeting and gene silencing, precisely addressing cellular and TME features of sunitinib-resistant ccRCC for efficient and low-toxicity overcoming of drug resistance.

2. Results and discussion

2.1. The construction and characterization of tumor targeting P/S/CVA@NPs-AI

Fig. 1A schematically portrays the construction of P/S/CVA@NPs-AI process. Briefly, after the preparation of P/S/CVA@NPs via double emulsion method, the anti-ITGA1 antibody was coupled to the amino groups of PLL through EDC/NHS-catalyzed aminoacyl reaction. The hydrodynamic diameter and Zeta potential of this nanocomplex were subsequently characterized by dynamic light scattering (DLS): 218 ± 2.7 nm and 1.20 ± 0.13 mV, respectively (Fig. S1). The efficiency of anti-ITGA1 antibody conjugation to the surface of nanoparticles was analyzed by flow cytometry. As shown in Fig. S2, the P/S/CVA@NPs-AI + laser group exhibited the lowest protein expression, suggesting that anti-ITGA1antibody-conjugated nanoparticles occupied most target sites on 786-O-R cells, which confirms the successful antibody conjugation to the nanocomplex surface. Moreover, the nanocomplex showed great storage stability as no obvious changes in particle size and PDI (Polydispersity Index) were observed in 7 days at 4 °C and 25 °C (Fig. S3). Also, the transmission electron microscopy (TEM) revealed the spherical shape and well-dispersed property of P/S/CVA@NPs-AI (Fig. 1B). The elemental mapping images showed the uniform distribution of Au, F, Mg and P elements, confirming the structural integrity of this nanocomplex (Fig. 1C).

The efficient cellular uptake capacity is a prerequisite for exerting optimal anti-tumor effect. Therefore, the uptake ability of nanoparticles was evaluated after the successful construction. As displayed in Fig. 1D–E and Fig. S4, Rhodamine encapsulated in PEAL material were more largely endocytosed by both 786-O and 786-O-R (sunitinib-resistant 786-O) cells than free Rhodamine, with increased red fluorescence intensity in RhB NPs group. Crucially, the anti-ITGA1 antibody modification could further enhance the uptake ability of nanocomplex, as the red fluorescence in RhB@NPs-AI group are stronger than that in RhB@NPs. This result confirms that conjugating anti-ITGA1 antibody to the nanoparticle surface enhances its delivery efficiency and accelerates the internalization process. And the quantitative flow cytometry analysis of intracellular fluorescence intensity further confirms the consistency of the conclusion (Fig. 1F and Fig. S5). Collectively, these findings validate the successful formulation of P/S/CVA@NPs-AI nanocomplex, characterized by superior cell targeting property and desirable storable stability.

Enlightened from the natural photosynthesis, the chlorophyll (Chlα, a photosensitizer), L-ascorbic acid (AA, an electron donor) and AuNPs (a catalyst) could work together to produce H₂, under the irradiation of 660 nm near-infra red light [28]. The mPEG-PLGA-PLL (PEAL) provides a proper reactive environment for these components (Chlα, AA and AuNPs) than in a bulk solution. Subsequently, the Ultraviolet (UV) and MB (methylene blue) -Pt (platinum) methods were utilized to further evaluate the production of H₂ among conditions. The peak UV absorption spectrum of chlorophyll α is 669 nm and Fig. 1G depicts the consumption process of Chlα (encapsulated in NPs), triggered by the NIR irradiation. As the exposure time grows, the absorption strength at 669 nm decreased and remained unchangeable since 11 min, indicating that a certain amount of Chlα was fully consumed at this moment. Besides, the MB-Pt probe was used to further quantify the generation of H₂. The Methylene blue (MB) can be reduced by H₂ into colorless MBH₂ (catalyzed by Pt nanoparticles) and the change in absorbance of MB at 664 nm is linearly related to the amount of H₂ [29]. Therefore, the production of hydrogen gas could be calculated according to the reduction degree of MB absorbance by virtue of the standard MB concentration curve (Fig. S6) [49]. As shown in Fig. 1H, the P/S/CVA@NPs-AI + laser group demonstrated more efficient production of H₂ under the irradiation of 660 nm NIR, in comparison with the group that Chlα, AA and AuNPs in bulk solution (free P/S/CVA+AI). In addition, Fig. 1I visually demonstrated intracellular H₂ reductivity of nanocomplex + laser. In the treatment groups where nanoparticles served as a reaction platform (CVA@NPs + laser, P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser), methylene blue exhibited a significantly lighter color compared to unencapsulated hydrogen-generating substances group (free CVA + laser). Furthermore, the enhanced reduction reaction in P/S/CVA@NPs-AI + laser group relative to P/S/CVA@NPs + laser indicated that anti-ITGA1antibody conjugation improved the sunitinib-resistant tumor cell-targeting capability of this nanocomplex, leading to efficient in situ hydrogen generation.

2.2. P/S/CVA@NPs-AI disrupted mitochondrial activity and subsequently induced apoptosis in ccRCC cells

Accumulating evidence suggests that lncARSR is critical for various cancer initiation and progression and may serve as a biomarker and therapeutic target. In renal cell carcinoma, Qu et al. found that lncARSR drives tumor-initiating cell aggressiveness via a YAP (Yes-associated protein) feed-forward loop that inhibits LATS1 (large tumor suppressor kinase 1)-mediated YAP phosphorylation and facilitates its nuclear translocation [50]. Besides, according to the studies by Liao et al. and Li et al., lncARSR upregulates SOX4 (SRY-box transcription factor 4) and HK1 (hexokinase 1) to promote bladder and colorectal cancer progression by acting as a ceRNA (competing endogenous RNA) for miR-129-5p/miR-34a-5p [51,52]. Furthermore, Tian et al. and Yang et al. reported that lncARSR activates STAT3 signaling to promote tumor growth, metabolic reprogramming and stemness in glioma and hepatocellular carcinoma [53,54]. Meanwhile, molecular hydrogen has recently attracted attention as a novel therapeutic gas in oncology, and previous studies has shown its ability to regulate mitochondrial energy metabolism and trigger cancer cell apoptosis [55].

Therefore, the successful synthesis of P/S/CVA@NPs-AI fostered us to further explore the antitumor effect and the underlying mechanism of this nanocomplex. To start with, the Cell Counting Kit-8 assay (CCK-8) was utilized to assess the possible toxicity of PEAL NPs, 660 nm NIR, Chlα and AuNPs to 786-O and 786-O-R cells. The effects of different concentrations of materials (PEAL NPs, Chlα and AuNPs) on the cellular safety of normal human renal proximal epithelial cells (HK-2 cells) were also evaluated. The results in Figs. S7–11 demonstrated the great biosafety of this nanocomplex. Hence, the CCK-8 assay and flow cytometry analysis were further utilized to evaluate the anticancer performance of nanocomplex + laser in vitro. Fig. 2E and F illustrates that negligible cell lethality was measured in free silncARSR - as the lack of nucleic acid delivery vector; free PT2385 and P@NPs groups - as the high selective inhibition of HIF-2α signaling pathway rather than general cytotoxicity in vitro. However, a significant decrease in cell viability can be noticed in H₂-production groups (free CVA + laser, CVA@NPs + laser), nucleic delivery group (S@NPs) and combinational-effect groups (P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser). What is more, P/S/CVA@NPs-AI + laser group shows higher 786-O and 786-O-R cells mortality than P/S/CVA@NPs + laser group, indicating that NPs with anti-ITGA1 body conjugated on the surface could be endocytosed by tumor cells more efficiently and precisely. The above therapeutic conclusions could be drawn by flow cytometry analysis (Fig. 2A–D), consistent with the above CCK-8 assay. And the P/S/CVA@NPs-AI + laser treated group induced the highest apoptosis rate among all the treatment groups (32.31 ± 2.82 % in total).

Fig. 2 — The antitumor effect of P/S/CVA@NPs-AI + laser *in vitro*. A-D) The flow cytometric analysis of 786-O and 786-O-R cell apoptosis under different treatments. E, F) The cell viability of 786-O and 786-O-R cells after co-cultured with nanomedicine for 24, 48 and 72 h, respectively. (G–J) The mitochondrial membrane of cancer cells in different treatment groups detected by flow cytometry and fluorescence microscope. Scale bar = 100 μm. K-L) The intracellular ATP levels of 786-O and 786-O-R cells in different conditions. Data are presented as means ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001.

Mitochondria are central regulators of the intrinsic apoptotic pathway [28,56]. Therefore, the mitochondrial membrane potential and the intracellular ATP levels of cancer cells were further measured in different treatment groups. As shown in Fig. 2G–I, the flow cytometry and quantitative analysis demonstrates the PBS, PT2385, free silncARSR and P@NPs groups has the highest red/green fluorescence ratio, referring to the healthy state of mitochondria. On the contrary, the ratio was decreased in free CVA + laser, S@NPs, CVA@NPs + laser, P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser groups, pointing to the hydrogen and lncARSR-knockdown could disturb the function of mitochondria. Among them, the lower ratio in CVA@NPs + laser than in Free CVA + laser group revealed the higher H₂-generation efficiency in NPs. In addition, the significant decrease in P/S/CVA@NPs-AI + laser group than P/S/CVA@NPs + laser representing the anti-ITGA1antibody realized the more efficient targeting capabilities to cancer cells. The results are consistent with the representative images taken by fluorescence microscope (Fig. 2J and Fig. S12). Meanwhile, the results of intracellular ATP levels in different conditions portrayed a similar trend (Fig. 2K and L). Briefly, the H₂ production groups (Free CVA + laser and CVA@NPs + laser) and lncARSR knockdown group (S@NPs) demonstrated decreased intracellular ATP level. Moreover, the combinational effect groups (P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser) showed the most significant diminishing effect among all the groups. Apoptosis in tumor cells with or without Bcl-2 overexpression was further evaluated via flow cytometry analysis. As illustrated in Fig. S13, a significant reduction in apoptosis was observed in the Bcl-2 overexpression group, suggesting that this nanocomplex induces cell death through mitochondrial dysfunction.

2.3. P/S/CVA@NPs-AI suppresses the pro-angiogenic capacity of tumor cells and M2-like macrophages

Tumor angiogenesis plays a vital role in the survival, progression and therapy resistance of solid tumors [[57], [58], [59]]. One of the hallmarks of sunitinib-resistant ccRCC is highly aberrant vascularization, and high levels of various proangiogenic factors could be secreted by ccRCC tumor cells [5,13,60]. Therefore, the tube formation assay was used to assess the anti-angiogenesis effect of nanocomplex + laser in vitro. As portrayed in Fig. 3A–C, the tube formation ability of human endothelial vein cells (HUVEC) was broadly inhibited by nanomedicines. It was surprisingly found for the first time that H₂ and lncARSR-knockdown could weaken the pro-angiogenic ability of ccRCC tumor cells. Subsequently, the underlying mechanism was further explored by qPCR analysis and Elisa test. VEGFA (vascular endothelial growth factor A), Angpt2 (angiopoietin-2) and bFGF (basic fibroblast growth factor) are well-established pro-angiogenic factors that play central roles in the initiation and progression of angiogenesis [10,61]. Hence, the impact of various treatment regimens on the secretion of pro-angiogenic factors by 786-O-R cells was systematically investigated. According to Fig. 3E–G, the HIF-2α antagonist (PT2385) groups (PT2385 and P@NPs groups), H₂-generation groups (Free CVA + laser and CVA@ groups + laser) and lncARSR knockdown group (S@NPs group) all present the capability to reduce the VEGFA secretion from 786-O-R cells. Moreover, in the combinational treatment groups with enhanced inhibition effect, the anti-ITGA1 antibody-conjugated nanocomplex (P/S/CVA@NPs-AI + laser) exhibited significantly greater suppression than the non-modified group (P/S/CVA@NPs + laser), demonstrating the effectiveness of delivery. It is also noteworthy that all treatment groups failed to downregulate the expression of Angpt2 and bGFG in ccRCC tumor cells significantly.

M2-like tumor-associated macrophages contributes to malignant progression involved pro-angiogenesis, extracellular transformation, immunosuppression and resistance to multi-therapy [62]. Previous studies confirmed that H₂ and silncARSR promote M2-to-M1 macrophage polarization and lncARSR downregulation markedly impairs macrophage-driven angiogenesis [25,49]. However, the specific pro-angiogenic mediators modulated by H₂ and silncARSR following M2-like macrophage repolarization remain inadequately explored. Accordingly, upon flow cytometric and qRT-PCR confirmation of H₂ and silncARSR-induced M2-to-M1 transition (Fig. 3D and Figs. S14–15), the expression levels of VEGFA, Angpt2 and bFGF in macrophages were subsequently assessed. As shown in Fig. 3H, there were significant lower VEGFA expression in Free CVA + laser, CVA@NPs + laser, S@NPs, P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser groups. The most significant suppression of VEGFA secretion was observed in P/S/CVA@NPs-AI + laser treatment group as the cooperation of silncARSR with H₂ and the enhanced targeting capability facilitated by anti-ITGA1 antibody. For Angpt2 and bFGF expression, the nanocomplex + laser has no statistically significant inhibition effect on M2-like macrophages (Fig. 3I and J).

In addition, the results in Fig. 3K–M demonstrates that nanocomplex + laser has no effect on the secretion of pro-angiogenic factors in HUVEC, indicating that P/S/CVA@NPs-AI + laser shifts the angiogenic ratio in vitro by modulating the VEGFA expression levels secreted by 786-O-R and M2-like macrophages. All the Elisa tests trend are consistent with qPCR analysis in Figs. S16–18.

2.4. P/S/CVA@NPs-AI therapeutic efficacy in drug-resistant ccRCC orthotopic model

Inspired by the above experimental results in vitro, we were motivated to further evaluate the therapeutic efficacy of various formulations in orthotopic mice model bearing sunitinib-resistant ccRCC tumor. Firstly, the tumor-targeting capability was affirmed via the small animal in vivo imaging system. After the successful construction of orthotopic sunitinib-resistant ccRCC tumor model in BALB/c nude mice, free Dir, Dir@NPs and Dir@NPs-AI were injected into the mice through tail vein to study the biodistribution feature of nanoparticles with time and the enrichment in tumor tissues. According to Fig. 4A and B, we found that the fluorescence signal of free Dir group was significantly lower in vivo than the Dir@NPs and Dir@NPs-AI groups after 48 h, indicating the quick metabolism of mice for free Dir. And the fluorescence intensity of Dir@NPs and Dir@NPs-AI were significantly enriched at livers and tumor sites. In addition, the Dir@NPs-AI group demonstrates stronger fluorescence intensity in tumor tissues than the Dir@NPs. In short, the fluorescence results intuitively suggest that anti-ITGA1 antibody-modified nanocomplex had a superior tumor-targeting ability. This feature brings advantages for improving therapeutic efficacy and minimizing treatment toxicity to off-target organs.

Fig. 4 — The antitumor activity of P/S/CVA@NPs-AI + laser in sunitinib-resistant ccRCC orthotopic model. A) Representative *in vivo* fluorescence images of major organs 48 h after the tail vein injection of free Dir, Dir@NPs and Dir@NPs-AI. B) The statistical analysis of the quantitated average radiance *in vivo*. C) The body weight of tumor-bearing mice in 6 weeks among different treatments groups. D, E) The tumor volume and the corresponding inhibition ratio under different treatment groups. F-H) The analysis of immunofluorescence/immunohistochemistry staining for TUNEL, Ki67 and CD31, exerted by Image J software. I) The images of orthotopic xenografts from different groups (n = 5/group). J) Representative images of HE, TUNEL immunofluorescence and Ki67, CD31 immunohistochemistry staining for tumor tissues among different groups. All the data are presented as the means ± SD, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.

Followingly, the tumor-bearing mice were randomly divided into 9 groups and treated with different formulations via tail vein injection (3 times a week, for 6 weeks, Fig. S19). The laser-treated groups (Free CVA, CVA@NPs, P/S/CVA@NPs and P/S/CVA@NPs-AI) were exposed to laser irradiation 24 h post-injection. As depicted in Fig. 4I, the P/S/CVA@NPs-AI + laser group exhibited the smallest tumor site among all the treatment groups. The quantified treatment effect was shown in Fig. 4D and E by calculating the tumor volume and inhibition rate. Notably, the inhibition rate of P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser groups were calculated at 89.96 % and 95.78 %, respectively. This result indicates a more potent therapeutic effect attributed to the augmented tumor-targeting efficiency via anti-ITGA1 antibody modification. Besides, the mice weights were measured throughout the duration of therapy. The data in Fig. 4C demonstrates a relatively stable increase among all the interventions, affirming the superior biosafety of this developed nanocomplex.

The HE stained tumor sites confirmed significantly higher necrotic area in P/S/CVA@NPs-AI + laser group (treatment group) than PBS group (control group), indicating the combination of PT2385, silncARSR and H₂ could induce tumor cell death effectively (Fig. 4J). The results of TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) showed that the P/S/CVA@NPs-AI + laser group presented the strongest green fluorescence intensity, revealing the superior capability of nanocomplex + laser inducing tumor cell apoptosis. Moreover, the immunohistochemical analysis of Ki67 and CD31 demonstrated that tumor cell proliferation and microvessel density (MVD) of ccRCC were significantly surpressed by P/S/CVA@NPs-AI + laser (Fig. 4F, H and J).

2.5. P/S/CVA@NPs-AI regulated apoptosis and angiogenesis-related proteins in vivo

The above results have confirmed that P/S/CVA@NPs-AI + laser could induce tumor cell mitochondrial apoptosis and decrease tumor angiogenesis in vitro and in vivo. We subsequently evaluated the differential expression of critical proteins involved in apoptosis and angiogenesis within tumor tissues subjected to distinct treatment regimens.

Bcl-2, Bax and cleaved-caspase 3 are critical apoptosis-related indicators [63]. The Immunohistochemistry (IHC) staining was used to evaluate the expression of them. As shown in Fig. 5A–D, the P/S/CVA@NPs-AI + laser group has the lowest expression of pro-survival protein (Bcl-2) and highest pro-apoptotic proteins (Bax and cleaved-caspase 3). Notably, the expression of apoptosis-associated proteins in tumor tissues following PT2385 treatment demonstrated marked alterations, in contrast to the relatively unchanged profiles observed in the in vitro apoptosis models. This observation is consistent with previous studies that PT2385 exerts antitumor effect by impairing the function of HIF-2α and therefore downregulating the mRNA expression of HIF-2α target genes, including VEGFA (vascular endothelial growth factor A), GLUT1 (glucose transporter 1), PAI-1 (plasminogen activator inhibitor-1) and CCND1 (cyclin D1), rather than acting as a conventional cytotoxic agent [12,35]. Moreover, the favorable tolerability of PT2385, coupled with the absence of dose-limiting toxicities, supports its potential for long-term use in combination with other therapeutic modalities.

Fig. 5 — The P/S/CVA@NPs-AI regulates the expression of critical proteins related to apoptosis and angiogenesis *in vivo*. A) Immunohistochemistry staining images of Bcl-2 (scale bar = 100 μm), Bax (scale bar = 100 μm), cleaved-caspase3 (scale bar = 50 μm) and VEGFA (scale bar = 100 μm) in tumor tissues. (B–E) The Immunohistochemistry staining scores for Bcl-2, Bax, cleaved-caspase3 and VEGFA from tumor tissues in different groups. All the data are presented as the means ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001.

The VEGFA is a central regulator of angiogenesis [64]. Hence, the IHC assay was utilized to assess the VEGFA expression in tumor sites under different interventions. The results in Fig. 5A and E demonstrated that HIF-2α antagonist groups (free PT2385 and P@NPs groups), H₂-generation groups (Free CVA + laser and CVA@NPs + laser groups), lncARSR knockdown group (S@NPs) and combinational effect groups (P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser groups) all have decreased VEGFA expression. Combining our in vitro data and CD31 immunohistochemistry staining above, the major conclusions can be drawn: the angiogenesis in ccRCC can be effectively reduced via the inhibition of HIF-2α/VEGFA pathway (PT2385, HIF-2α antagonist) [35], directly suppressed VEGFA expression secreted by tumor cells and inhibited pro-angiogenesis ability of macrophages (H₂ and silncARSR).

2.6. Biocompatibility and biosafety assessment of P/S/CVA@NPs-AI in vivo

Biocompatibility refers to the description of interactions between foreign materials and the body, expecting no undesirable side effects on the recipients [65,66]. Accordingly, it is vitally important to examine the biocompatibility and biosafety of nanomedicines for evaluating clinical translation potential. In this research, a three-week intravenous injection of nanomedicines into healthy BALB/c nude mice were conducted, and each group blood serum would be collected for further biosafety evaluation.

As NPs primarily accumulate in the liver, spleen and kidney, and then metabolized by liver and kidney, the detection of liver and kidney functions are quite imperative. The key indicators to evaluate liver and kidney functions including ALT (Alanine Aminotransferase), AST (Aspartate Aminotransferase), ALP (Alkaline phosphatase), BUN (Blood Urea Nitrogen) and Crea (Creatinine). According to Fig. 6A–E, the above indicators in P/S/CVA@NPs-AI + laser group exhibit no significant change comparing to the PBS group, indicating the nanocomplex caused no visible harm to the liver and kidneys’ function of experimental mice. Furthermore, the HE staining of vital organs including the heart, liver, spleen, lung and kidneys displays no obvious damage or pathological changes, indicating that P/S/CVA@NPs-AI + laser has a great tissue biocompatibility.

Fig. 6 — The biosafety of P/S/CVA@NPs-AI + laser *in vivo* (n = 3 independent experiments). A - E) The serum biochemical analysis of ALT (*Alanine Aminotransferase*), AST (*Aspartate Aminotransferase*), ALP (Alkaline phosphatase), BUN (Blood Urea Nitrogen) and Crea (Creatinine) to evaluate liver and kidney functions on day 21 after intravenous administration of certain drugs, three times per week. F) HE staining of important organs with different interventions after 21days of intravenous injection. Scale bar = 100 μm.

These results indicated that P/S/CVA@NPs-AI + laser possess high biosafety and biocompatibility in vivo. Combining with effective targeting capability to tumor sites, cooperatively suppressed tumor angiogenesis and enhanced mitochondrial apoptosis, our well-designed nanocomplex exhibits great potential for clinical translation to effectively overcome ccRCC sunitinib-resistance in the future.

3. Material and methods

3.1. Materials

lncARSR siRNA was purchased from Ribo-Bio (Guangzhou, China). Sunitinib, PT2385, N, N-Dimethylacetamide (DMSO), PEG300 (Polyethylene glycol 300) and Tween 80 were purchased from Selleck Chemicals (Shanghai, China). Chlα, L-ascorbic Acid, AuNPs (5 nm in diameter), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (>99 %), N-Hydroxy succinimide (NHS) (>98 %) and PluronicTM F68 were purchased from Sigma-Aldrich (St. Louis, USA). Trichloromethane was bought from Aladdin Reagent (Shanghai) Co., Ltd. Annexin V-FITC Apoptosis Detection Kit was obtained from BD Bioscience (Franklin Lakes, USA). Enhanced ATP Assay Kit and Enhanced mitochondrial membrane potential assay kit with JC-1 were purchased from Beyotime Biotechnology (Jiangsu, China). Anti-ITGA1 antibody was purchased from Novus (Colorado, USA). All other chemicals and organic solvents were of analytical grade.

3.2. Cell lines

The clear cell renal cell carcinoma cell line 786-O and human monocytic leukemia THP-1 cell line were purchased from the Cell Institute of Chinese Academy of Sciences, and were carefully cultured in PRMI-1640 medium (Gibco, USA) containing 10 % fetal bovine serum (FBS, Sigma-Aldrich, USA) and 1 % penicillin/streptomycin (Gibco, USA). The Human Umbilical Vein Endothelial Cell (HUVEC) was provided by State Key Laboratory of Systems Medicine for Cancer, maintained and expanded in high glucose DMEM medium (Gibco, USA) supplemented with 10 % FBS (Sigma Aldrich, USA) and 1 % penicillin/streptomycin (Gibco, USA).

The sunitinib-resistant 786-O cells (786-O-R) were established by exposing to increasing concentrations of sunitinib, starting at the concentration of 5 μM. It took 48 h for a fraction of 786-O cells to adapt and survive in each concentration. After that, the culture medium was replaced by drug-free medium. The recovered and proliferated 786-O cells were passaged after reaching to approximately 80 % confluence. This process was repeated 6–8 times for each sunitinib concentration until the parental 786-O cells could grow stably at this concentration. Followingly, the concentration of sunitinib was improved to a higher level until the parent 786-O cells survived and adapted to the highest concentration.

3.3. Preparation of P/S/CVA@NPs-AI

The double emulsion method was used to construct P/S/CVA@NPs-AI nanoparticles. Briefly, PEAL (12.5 mg), Chlα (45 μM) and PT2385 (0.06 mg) were dissolved in 500 μL trichloromethane (organic phase). L-ascorbic acid (227 mM), silncARSR (5 nmol) and AuNPs (200 nM) were dissolved in 100 μL DEPC water (inner aqueous phase). Next, the organic phase and inner aqueous phase were mixed and sonicated in ice water bath for 1 min to get colostrum solution (Power: 390w; on/off = 2s/2s). Followingly, the colostrum solution was added into 2.5 mL of 1 % F68 aqueous solution and sonicated again for 3 min under the same conditions. After that, the rotary evaporator was employed to remove the trichloromethane.

The EDC/NHS-mediated amide reaction was utilized to conjugated anti-ITGA1 antibody onto the surface of P/S/CVA@NPs. Firstly, 5 mg EDC・HCL, 5 mg NHS and 20 μL anti-ITGAl antibody were added into 100 μL double distilled water and stirred at temperature for 5 min. After that, the 2.5 mL P/S/CVA@NPs solution were mixed and gently stirred with the above mixture for 2 h.

3.4. Characterization of P/S/CVA@NPs-AI

The Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) was operated to detect the zeta potential, size distribution and PDI of P/S/CVA@NPs-AI. The morphology and structure of P/S/CVA@NPs-AI were measured by transmission electron microscope (TEM, Hitachi, Japan). Besides, the P/S/CVA@NPs-AI were stored at different temperatures (4 and 25 °C) in PBS for a week to evaluate the storage ability.

3.5. The detection of H₂ production

To decide the optimal irradiation time (660 nm NIR) of generating hydrogen gas, the absorption band of chlorophyll α were measured after 0, 1, 3, 5, 7, 9, 11, 13 min exposure, respectively. Also, the MB-Pt (Methylene blue-Pt nanoparticles) was utilized to further quantify the generation of hydrogen, as the blue Methylene blue can be reduced by H₂ into color-less MBH₂ (catalyzed by Pt Nanoparticles) and the change of 664 nm (the most characteristic peak of MB) absorbance of MB is linearly related to the generation of hydrogen gas. Briefly, the detection experiments were divided into two groups: free P/S/CVA+AI and P/S/CVA@NPs-AI. The absorbance of MB solution in the two groups were measured by the UV–Vis at 1, 3, 5, 7, 9, 11, 13 min points.

3.6. Cellular uptake

The qualitative cellular uptake of nanocomplex was observed and evaluated via Fluorescence microscopy and flow cytometry. The 786-O and 786-O-R cells were seeded in 6-well culture plate at the density of 1 × 10⁵ cells per well overnight. Subsequently, free RB solution (10 μg/mL RB), RB NPs (10 μg/mL RB) and RB NPs-AI (10 μg/mL RB) were added into different plates. After 4 h of co-incubation, the phosphate buffered saline was utilized to wash cells for 3 times to remove the reagents. Then, the 4 % paraformaldehyde was added to fix the cells at 37 °C for 10 min. Finally, the 786-O and 786-O-R cells were stained with Hoechst 33342 (5 μg/mL) for 20 min and observed using Fluorescence microscopy (Olympus, Japan, IX71). To quantify the cellular uptake of free RB, RB@NPs and RB@NPs-AI, the cells were collected by 0.25 % trypsin and centrifuged at 1000 rpm for 5 min. Resuspend the harvested cells with 0.2 mL of PBS and assess the uptake condition via flow cytometry analysis (Becton Dickinson, USA).

3.7. Intracellular reductive hydrogen gas release analysis

Firstly, the 786-O-R cells were cultivated in 6-well plate and treated with MB solution (diluted with 1640 PRMI medium). After 1 h of incubation, the supernatant was replaced by free CVA + laser, CVA@NPs + laser, P/S/CVA@NPs + laser and P/S/VA@NPs-AI + laer, respectively (PBS group was treated as control). Subsequently, the color change of MB was monitored by microscope.

3.8. Antitumor efficiency in vitro

The antitumor efficacy of various treatment groups on 786-O and 786-O-R cells were assessed by CCK-8 assay (Cell Counting Kit-8, Dojindo Molecular Technologies, Kyushu, Japan). Briefly, cells were cultured in 96-well plates containing 5 × 10³ cells per well. After one-night incubation, the drugs were added in different wells and co-incubated with tumor cells for 24 h–72 h. Subsequently, the different treated wells would be added CCK-8 reagent and measured the absorbance at 450 nm wavelength using a spectrophotometer, under the manufacturer’s instructions. Also, the tumor cell apoptosis in vitro was detected by Annexin-V/PI assay. Briefly, the 786-O and 786-O-R cells in logarithmic growth phase were co-cultured with PBS, free PT2385, free silncARSR, free CVA + laser, P@NPs, S@NPs, CVA@NPs + laser, P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser for 48 h in six well plate. Afterwards, the 786-O and 786-O-R cells were double stained using the apoptosis kit from BD Bioscience (Franklin Lakes, USA) and examined the apoptosis condition with a BD flow cytometer. Data were computed as the mean ± SD of triplicates.

3.9. Rt-qPCR

The total RNA was extracted from the 786-O and 786-O-R cells by TRIzol Reagent (Invitrogen, USA). Followingly, the Nanodrop (Thermo Fisher, USA) was used to determine the concentration and purity of the RNA. Rt-qPCR was then conducted with the SYBR-Green master kit (Vazyme, Nanjing, China) on the LightCycler 480 II (Roche Dignostics) instrument. The house-keeping gene - GAPDH was used to normalize the mRNA levels in different cells. The primer sequences are listed in Supplementary Table 1. And the expressions of genes were calculated with the ^ΔΔCt method.

3.10. Evaluation of the repolarization of M2 macrophages

Firstly, THP-1 cells were differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate, 100 ng/mL), seeded in 6-well plate. Afterwards, 20 ng/mL of IL-4 (Interleukin-4) were added in order to obtain the M2 polarized macrophages (co-cultured for 48 h). Then, different treatment groups, including PBS, PT2385, free silncARSR, Free CVA + laser, P@NPs, S@NPs, CVA@NPs + laser, P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser were co-incubated with M2-like macrophages. Subsequently, the cells in different groups were collected and labeled by various antibodies to further analyzed via flow cytometry. The anti-human antibodies were as followed: Brilliant Violet 421 anti-mouse/human CD11b Antibody (#101251 Biolegend), FITC anti-human CD80 (#305205, Biolegend) and FITC anti-human CD206 (#321103, Biolegend).

3.11. Enzyme-linked immunosorbent assay (Elisa)

The vascular endothelial growth factor A (VEGFA), angiopoietin-2 (Angpt2) and basic fibroblast growth factor (bFGF) concentrations of 786-O-R, THP-1 and human umbilical vein endothelial cells’ supernatant under different treatment groups were measured using Enzyme-linked immunosorbent assay Kit (mlbio, Shanghai, China). Briefly, PBS, free PT2385, free silncARSR, free CVA + laser, P@NPs, S@NPs, CVA@NPs + laser, P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser were added into different wells of cells for 48 h. The PBS group was treated as a control. The supernatants in different treatment groups were then collected and processed according to the manufacturer’s instructions to detect the expression of VEGFA, ANGPT2 and bFGF, respectively.

3.12. Tube formation assay

The 786-O-R cells were seeded on the upper chamber of transwell plate (0.4 μM pore size polycarbonate membrane filters) and the HUVEC were seeded on the lower plates. After the co-culture under the designated treatments for 72 h, the tube formation assay was further exerted. Briefly, the pre-melted Matrigel gel (#354230, BD Biosciences) was placed in pre-cooled 24-well plates (30μL/well). After 30–40 min of solidification at 37 °C, the digested and counted HUVEC (1.5 × 10⁴) from different treatment groups were added into each well. Then, the results were observed and analyzed under microscope after 6–8 h incubation at 37 °C. The number of tubes were counted in five random microscopic fields and the Image J software was used for quantification.

3.13. Detection of intracellular ATP

To detect the intracellular ATP levels, the logarithmic-growth 786-O and 786-O-R cells were cultured in six-well plates and incubated overnight for the attachment. The PBS, free PT2385, free silncARSR, free CVA + laser, P@NPs, S@NPs, CVA@NPs + laser, P/S/CVA@NPs + laser, P/S/CVA@NPs-AI + laser were then added into different wells for 48 h, respectively. Discard the medium and add 200 μL lysate into each well to fully lyse cells. Afterwards, the conduction of drawing standard curve, preparation of working fluid for ATP detection and final determination of the ATP level in different treatment groups were all performed according to the manufacturer’s instructions (Beyotime, China).

3.14. Detection of mitochondria membrane potential

JC-1 is an ideal fluorescent probe for detecting mitochondrial membrane potential. 786-O and 786-O-R cells were cultured in 6-well plate. After the attachment, PBS, free PT2385, free silncARSR, free CVA + laser, P@NPs, S@NPs, CVA@NPs + laser, P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser were added into the different wells, co-culturing for 48 h. Subsequently, detect the mitochondrial membrane potential of cells under different treatment groups using the BD flow cytometer and fluorescence microscope, according to the guidelines that Mitochondrial Membrane Potential Test Kit (Beyotime, China) provided.

3.15. Sunitinib-resistant mice model construction

Briefly, the sunitinib-resistant 786-O subcutis implantation into renal capsule was used to construct the orthotopic ccRCC animal model. And all experimental procedures were approved by the Ethics Committee of Renji Hospital (Shanghai, China).

Firstly, the 786-O-R cells (5 × 10⁶ cells in PBS) in logarithmic stage were injected subcutaneously into the 6-week-old nude mice. After the successful construction of sunitinib-resistant subcutaneous tumor model, the mice were euthanized and the tumor tissues were stripped under aseptic conditions. PBS buffer was used to remove the connective tissue and blood clot. Followingly, the tumor tissues were cut into 1 mm³ pieces using ophthalmic scissors and surgical blades for further use. After that, the nude mice were anesthetized and placed in the right lateral decubitus position. And the lateral incisions were made in the left flank through the skin and peritoneum to fully expose the left kidney of the nude mice. Finally, the 2–3 mm incision were made on the renal capsule, using ophthalmic tweezers and scalpels. And the treated ccRCC tissue blocks were placed under the renal capsule through the incision and kept away from the incision. The tumor-bearing mice were kept in SPF condition after the suture of wounds.

3.16. Targeting of nanoparticles in vivo

After sunitinib-resistant orthotopic ccRCC mice was successfully constructed, 200 μL of free Dir, Dir@NPs, Dir@NPs-AI was injected into the mice by tail vein injection. Followingly, the tumor-bearing mice were humanly sacrificed and the main organs including kidney (with tumor tissue), heart, liver, spleen and lung were extracted for fluorescence imaging by fluorescence imaging apparatus (Berthold Technologies, Germany). The biodistribution of various forms of Dir were calculated by the fluorescence intensity on unit area of these extracted organs.

3.17. Antitumor efficiency in vivo

The sunitinib-resistant orthotopic ccRCC mice were divided into 9 treatment groups randomly: PBS (treated as control), free PT2385, free silncARSR, free CVA + laser, P@NPs, S@NPs, CVA@NPs + laser, P/S/CVA@NPs + laser and P/S/CVA@NPs-AI + laser. The tumor-bearing mice were treated via tail vein injection three times a week, and the weight of the mice were measured and recorded per week. Also, the mice in free CVA, CVA@NPs, P/S/CVA@NPs and P/S/CVA@NPs-AI groups underwent NIR (660 nm) irradiation 24 h post-injection. After 6 weeks of treatment, the mice were humanly euthanized and the organs were collected, weighed and photographed. Afterwards, the organs were immersed in 4 % paraformaldehyde and cut into slices. The kidney sections (bearing tumor tissues) were stained with HE, Ki67, CD31, TUNEL, Bcl-2, Bax, cleaved-caspase 3 and VEGFA for histological analysis.

3.18. Assessment of systemic toxicity

To evaluate the toxicity nanoparticles in vivo, healthy BALB/c nude mice (6 weeks) were randomly divided into nine groups and intravenously injected with PBS, free PT2385, free silncARSR, free CVA + laser, P@NPs, S@NPs, CVA@NPs + laser, P/S/CVA@NPs + laser, P/S/CVA@NPs-AI + laser, three times a week. After three weeks of injection, the serum samples were collected for the standard biochemistry test of liver functions (ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase) and kidney functions (BUN, blood urea nitrogen; Crea, creatinine). The important organs (heart, liver, spleen, lung and kidney) were collected for tissue sections by HE staining analysis.

3.19. Statistical analysis

All data are presented as the means ± standard deviations (SD) of at least 3 independent experiments and were analyzed by Student’s-test with GraphPad Prism 9.0 software. All methods were carried out according to relevant guidelines and regulations. P < 0.05 was considered statistically significant (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 and NS: no significant difference). Also, the data that support findings of this study are available from the corresponding author upon reasonable request.

4. Conclusion

In summary, the P/S/CVA@NPs-AI + laser realized the superior therapeutic effect for overcoming sunitinib-resistant ccRCC through the following aspects: 1) the anti-ITGA1 antibody modified on the surface of P/S/CVA@NPs increased tumor-targeting capability of nanocomplex, realized the expected effect of generating H₂ in situ and improving the efficiency of overcoming sunitinib-resistant ccRCC. 2) The H₂-PT2385-silncARSR nanocomplex realized the surprising anti-vascular effect through the following mechanism cooperatively: inhibiting the upstream factors of angiogenesis (PT2385), decreasing the secretion of VEGFA from tumor cells and weakening the pro-angiogenic capabilities of macrophages (H₂ and silncARSR). 3) The P/S/CVA@NPs-AI + laser induced tumor cell apoptosis cooperatively via jeopardizing the function of mitochondria, decreasing the generation of ATP and regulating the apoptosis-related protein. 4). The rationally engineered H₂-PT2385-silncARSR nanocomplex achieves a seamless integration of gas therapy, transcription-targeted intervention and gene therapy. Also, the favorable safety profile and minimal systemic toxicity render it highly suitable for sustained therapeutic applications.

Taken together, this research offers a novel insight into overcoming sunitinib-resistant ccRCC in clinic and lays a solid ground from in vitro and in vivo experiments.

Looking forward to future clinical translation, the therapeutic efficacy of this approach could be further enhanced through integration with various innovative strategies, including implantable wireless systems enabling localized irradiation or self-luminescent nanomaterials that eliminate external light dependence [67], which would allow more precise and controllable light-mediated therapeutic responses in clinical tumors. The convergence of these developing technologies is anticipated to not only optimized treatment efficacy but also expand the clinical applicability of light-associated therapeutic systems.

CRediT authorship contribution statement

Suxian Hu: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Formal analysis, Data curation, Conceptualization. Yan Zhu: Writing – original draft, Validation, Software, Methodology, Data curation. Yi Duan: Validation, Software, Methodology, Conceptualization. Liting Wang: Writing – original draft, Methodology, Investigation, Conceptualization. Jian Yu: Project administration, Methodology, Conceptualization. Zhihua Wu: Writing – original draft, Methodology, Data curation. Yourong Duan: Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Funding acquisition. Ying Sun: Writing – review & editing, Writing – original draft, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supposed by the National Natural Science Foundation of China (No. 82172736 and 82272829), the Natural Science Foundation of Shanghai (No. 22ZR1459700) and the State Key Laboratory of Oncogenes and Related Genes (ZZ-RCPY-24-42, ZZ-RCPY-25-37).

Footnotes

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2025.102450.

Contributor Information

Yourong Duan, Email: yrduan@shsci.org.

Ying Sun, Email: ysun@shsci.org.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1

mmc1.docx^{(17.9MB, docx)}

Data availability

Data will be made available on request.

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PERMALINK

The novel hydrogen-PT2385-silncARSR nanocomplex impairs tumor angiogenesis and mitochondrial activity in sunitinib-resistant renal cancer

Suxian Hu

Yan Zhu

Yi Duan

Liting Wang

Jian Yu

Zhihua Wu

Yourong Duan

Ying Sun

Abstract

Graphical abstract

1. Introduction

Scheme 1.

2. Results and discussion

2.1. The construction and characterization of tumor targeting P/S/CVA@NPs-AI

Fig. 1.

2.2. P/S/CVA@NPs-AI disrupted mitochondrial activity and subsequently induced apoptosis in ccRCC cells

Fig. 2.

2.3. P/S/CVA@NPs-AI suppresses the pro-angiogenic capacity of tumor cells and M2-like macrophages

Fig. 3.

2.4. P/S/CVA@NPs-AI therapeutic efficacy in drug-resistant ccRCC orthotopic model

Fig. 4.

2.5. P/S/CVA@NPs-AI regulated apoptosis and angiogenesis-related proteins in vivo

Fig. 5.

2.6. Biocompatibility and biosafety assessment of P/S/CVA@NPs-AI in vivo

Fig. 6.

3. Material and methods

3.1. Materials

3.2. Cell lines

3.3. Preparation of P/S/CVA@NPs-AI

3.4. Characterization of P/S/CVA@NPs-AI

3.5. The detection of H2 production

3.6. Cellular uptake

3.7. Intracellular reductive hydrogen gas release analysis

3.8. Antitumor efficiency in vitro

3.9. Rt-qPCR

3.10. Evaluation of the repolarization of M2 macrophages

3.11. Enzyme-linked immunosorbent assay (Elisa)

3.12. Tube formation assay

3.13. Detection of intracellular ATP

3.14. Detection of mitochondria membrane potential

3.15. Sunitinib-resistant mice model construction

3.16. Targeting of nanoparticles in vivo

3.17. Antitumor efficiency in vivo

3.18. Assessment of systemic toxicity

3.19. Statistical analysis

4. Conclusion

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgements

Footnotes

Contributor Information

Appendix A. Supplementary data

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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Links to NCBI Databases

3.5. The detection of H₂ production