Peptide-modified phase-transition nanoparticles co-deliver FTO siRNA and Ce6 for sonodynamic metabolism-immunotherapy of melanoma

Peptide-modified phase-transition nanoparticles co-deliver FTO siRNA and Ce6 for sonodynamic metabolism-immunotherapy of melanoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article Peptide-modified phase-transition nanoparticles co-deliver FTO siRNA and Ce6 for sonodynamic metabolism-immunotherapy of melanoma Xintong Li, Weinan Sun, Weidong Yu, Zhuo Wang, Ping Sun, Jianfeng Chen, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7251971/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted 16 You are reading this latest preprint version Abstract Background Melanoma, the most aggressive skin cancer, remains challenging to treat due to limited therapeutic options. Sonodynamic therapy (SDT) has emerged as a promising strategy for combating malignant tumors. However, the excessive accumulation of lactate in the tumor microenvironment after sonodynamic therapy limits the activation of immune cells, leading to the unsatisfactory therapeutic effect of SDT. FTO inhibition can effectively inhibit glycolysis of melanoma cells and relieve the obstacle of immune cell activation caused by lactic acid. FTO silencing in tumors eliminates can metabolic barriers to T cell activation and further enhances the antitumor effect of CD8 + T cells. Combining FTO inhibition with SDT may enhance tumor cell elimination and remodel the immunosuppressive tumor immune microenvironment. Result In this study, tLyp-1 modified ultrasound phase-transforming nanoparticles loaded with sonosensitizer (Ce6) and FTO siRNA were constructed to achieve FTO inhibition and sonodynamic therapy. The tLyp-1 peptide modification facilitates efficient tumor targeting and enhances deep tissue penetration, therefore improving drug delivery efficacy. Ce6 produces ROS in response to ultrasound to induce ICD in tumor cells. At the same time, ultrasound promoted FTO siRNA transfection to inhibit B16-F10 cells glycolysis, which significantly increased the activation and infiltration of dendritic cells and T lymphocytes in the tumor microenvironment, effectively enhanced the therapeutic effect of SDT and inhibited tumor growth. Conclusion This study demonstrated that si-Ce6@tLyP-1 NPs serve as a platform for targeted the tumor site and efficiently deliver si-FTO and Ce6. The realization of FTO inhibition combined with sonodynamic therapy provides an effective treatment strategy for the treatment of melanoma. melanoma sonodynamic therapy FTO immunotherapy glycolysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Melanoma is the most malignant and aggressive form of skin cancer which accounting for up to 90% of all skin cancer deaths [1–3]. Although melanoma can be treated clinically by surgery, traditional chemotherapy and immunotherapy, it is still plagued by drug resistance and recurrence. Therefore, it is essential to seek an effective treatment for melanoma [4,5]. Photodynamic therapy (PDT) has been clinically recognized for the treatment of melanoma, which has attracted great attention [6,7]. Due to the poor laser penetration and the absorption of light by melanin in melanoma, PDT has limited therapeutic effect [8]. Sonodynamic therapy can overcome the above shortcomings of photodynamic therapy. Under low-intensity ultrasound (US) irradiation, sonosensitizer combineing with oxygen can produce excess ROS which induce apoptosis of tumor cells [9,10]. Besides, ROS can also trigger the immunogenic cell death (ICD) of tumor cells and release damage‐associated molecular patterns such as calreticulin (CRT) and high‐mobility group box 1 (HMGB1) [11,12]. ICD can induce the maturation of DCs and activate T lymphocytes, increasing the infiltration of immune cells in the tumor microenvironment, thereby enhancing the immunogenicity of cancer and improving the therapeutic effect [13,14]. However, current studies have found that the therapeutic effect of SDT is still not ideal, which may be due to the aggravation of hypoxia of tumor tissue during sonodynamic therapy, leading to enhanced glycolysis of tumor cells and further increasing the accumulation of lactate [15,16]. The production and release of lactate adversely affect immune responses by impairing cytotoxic T cell function and inhibiting DC differentiation and maturation [17]. Recent studies have suggested that fat mass and obesity-associated (FTO) can regulate glycolytic metabolism in a variety of tumor cells and increase the expression of glycolytic genes such as HK1, GLUT1, PKM and so on [18]. In the meanwhile, evidence indicates that the absence of FTO can enhence tumor-infliltrting T cells and removes the metabolic barrier for T cells activation. The combination of FTO inhibitors with other immunotherapies are effective strategies to improve adaptive immune responses [19] . Therefore, it is reasonable to assume that the inhibition of FTO is able to greatly enhance the effect of sonodynamic therapy. On the one hand, inhibition of FTO can regulate the glycolysis of tumor cells [19] and thus alleviate the metabolic disorder of immune cell activation after sonodynamic therapy. On the other hand, it can enhance the killing effect of immune cells [19] and finally achieve sonodynamic -immune-metabolism synergistic therapy. RNA interference therapy is in the spotlight in recent years. Small interfering Rnas can be precisely regulated. Small interfering RNA (siRNA) has specific gene silencing ability that can precisely knock out the target gene [20]. However, its short half-life, poor bioavailability and easy clearance in vivo limit the clinical application of siRNA [21]. With the approval of coronavirus disease 2019 (COVID-19) mRNA vaccines, lipid nanoparticles have attracted more and more attention as a promising RNA delivery vector [22–24]. Furthermore, as a drug delivery vehicle, lipid-based nanoparticles can efficiently carry fat-soluble drugs, which improves drug stability and bioavailability in vivo [25,26]. Therefore, we propose that lipid-based nanoparticles carrying FTO siRNA and the fat-soluble sonosensitizer Chlorin e6 (Ce6) can be used for effective co-delivery of therapeutic agents. Despite so-called enhanced permeability and retention (EPR) effect existing can make nanomedicine passively target to accumulate in tumor tissue, the delivery efficiency of this process remains very poor [27]. Currently, the surface of lipid-based nanoparticles can be modified by a variety of biomolecules to target the tumor site actively and specifically [28,29]. TLyp-1 (amino acid sequence CGNKRTR) peptide designed specifically binding to the neuropilin-1 (NRP-1) overexpressed on the surface of B16-F10 cells, possess the capabilities of tumor targeting and tumor penetration activities, so that the lipid-based nanoparticles whose surface modificated with it can active target and accumulate in tumor sites [30–32]. In addition, the local controlled targeted release of drugs at the tumor site is also an urgent application challenge to be solved. Ultrasound-targeted microbubble destruction (UTMD) technology has been increasingly applied to accurately control the release of drugs and genes at tumor sites to improve the efficacy of various therapies [30,33]. Under the irradiation of low-power focused ultrasound (LIFU), the phase-change material as a core of Lipid nanoparticles can produce UTMD effect to promote the targeted delivery and release of drugs/genes [34]. In this study, we proposed and constructed a lipid nanoparticle modified by tLyp-1 peptide with an inner core containing PFP (a phase-change material), sonosensitizer Ce6 and FTO siRNA were encapsulating together for the combination therapy of melanoma (designated as si-Ce6@tLyP-1 NPs). The tLyP-1 peptide enables the nanoparticles to target and penetrate deep into the melanoma, with the PFP underwent a liquve-gas phase transition under the irradiation of ultrasound, releasing siRNA and Ce6. Ce6, as a sonosensitizer, has significant advantages in sonodynamic therapy (SDT). In response to ultrasound, Ce6 can efficiently produces ROS to kill tumor cells and instigates ICD to induce DCs maturation and activate T lymphocytes [6]. FTO siRNA transfects into tumor cells to regulate glycolysis thereby reducing the production of lactate and contacting metabolic inhibition, further promoting the effector states of T cells. As a result, the anti-tumor immunity of nanomedicine was enhanced and synergistic anti-tumor effects were achieved (Scheme1). 2. Results and Discussion 2.1. Preparation and characterization of si-Ce6@tLyP-1 NPs The nanoparticles of Ce6@tLyp-1-NPs were prepared by filming-rehydration method and the acoustic vibration method with encapsulated Ce6. FTO siRNA were loading with the nanoparticles by electrostatic adsorption, forming si-Ce6@tLyP-1-NPs ( Fig. 1 A ) . The structure of si-Ce6@tLyP-1 NPs was characterized by transmission electron microscopy (TEM), showing that the nanoparticles were spherical and dispersed uniformly ( Fig. 1 B ) . Based on DLS measurements, the average size of Ce6@tLyp-1-NPs and si-Ce6@tLyp-1-NPs were 271.00 ± 3.60 nm and 294.93 ± 3.17 nm. The zeta potential of Ce6@tLyp-1-NPs and si-Ce6@tLyp-1-NPs were35.61 ± 0.97 mV and − 31.00 ± 0.29 mV ( Fig. 1 C ) . It was indicated that the particle size of nanoparticles increased slightly and the surface transform from a positive charge to a negative charge after siRNA adsorption, suggesting FTO siRNA loading onto the nanoparticles successfully. Excellent physiological stability is a prerequisite for the in vivo application of nanopreparations, so we verified the stability of si-Ce6@tLyp-1-NPs by observing its particle size and appearance. The results showed that there was no significant change in particle size in PBS and 1640 medium with 10% FBS revealing a good seven-day stability of the nanoparticles ( Fig. 1 D, S1 ) . The UV/Vis spectra showed that si-Ce6@tLyp-1-NPs were aligned with free CE6, with two absorption peaks presenting at 400 and 660nm, which confirmed the successful encapsulation of Ce6 ( Fig. 1 E ) . A standard curve of Ce6 was measure and constructed by UV/Vis spectra, and the encapsulation efficiency (EE%) of Ce6 in si-Ce6@tLyp-1-NPs were 76.20% (Figure S2) . To verify the SDT effect of si-Ce6@tLyp-1-NPs singlet oxygen sensor green (SOSG) probe was used as a probe to examine the ability of si-Ce6@tLyp-1-NPs to generate cytotoxic singlet oxygen (Figure S3) . The results showed that after ultrasonic treatment, an absorption peak at 525nm was detected by the fluorescence spectrophotometer. The absorption intensity gradually increased with the extension of ultrasonic irradiation time, indicating that the singlet oxygen produced gradually increased with the prolongation of irradiation time. To investigate the loading ability of Ce6@tLyp-1-NPs to complex the siRNA, we performed agarose gel experiments. With increasing Ce6@tLyp-1-NPs mass, the fluorescence intensity of the free siRNA bands in the supernatants after centrifugation gradually decreased. When the mass ratio of siRNA to nanoparticles reached 1:8, the siRNA in the supernatant completely disappeared (Figure. 1F) , indicating the FTO siRNA was totally loaded by Ce6@tLyp-1-NPs. Therefore, the ratio between siRNA and Ce6@tLyp-1-NPs was determined to be 1:8. After determining the ratio of nanoparticles to siRNA, to further verify the efficiency of nanoparticles carrying siRNA, FAM-labeled siRNA was incubated with DiI-labeled nanoparticles detected by confocal microscopy and flow cytometry. Confocal laser scanning microscopy (CLSM) images showed an overlap of the fluorescence of siFTO with the luminescence of Ce6@tLyp-1-NPs, confirming the successful encapsulation (Figure. 1G). In addition, the obtained double-labeled nanoparticles were incubated with B16-F10. Confocal images showed that FAM and Dil fluorescence were scattered around the nucleus and co-localized with each other, validating the efficient co-delivery of siRNA and the nanoparticles (Figure. 1H) . Flow cytometry showed the loading rate of nanoparticles to siRNA is 99.4% when the mass ratio of siRNA to nanoparticles reached 1:8 (Figure. 1I). 2.2. In vitro cellular uptake and tumor spheroid penetration efficiency Before validating the in vitro therapeutic effects of the nanoparticles, the biocompatibility of the nanoparticles was tested to ensure their subsequent safe application. After co-culturing B16-F10 cells at different concentrations of si-Ce6@tLyp-1-NPs for 24 hours, cell viability was assessed by CCK-8 assay. As shown in Fig. 2 A, the cell viability remained above 90% at concentrations of 1.0mg/ml, which indicated the low cytotoxicity of the nanoparticles. The ability of si-Ce6@tLyp-1-NPs targeted to B16-F10 cells was next evaluated by confocal laser scanning microscopy and flow cytometry. B16-F10 cells were incubated with DiI labeled si-Ce6@tLyp-1-NPs and si-Ce6@NPs at different times (0.5h, 1h, 2h, and 4h). CLSM revealed that the DiI labeled si-Ce6@tLyp-1-NPs emitting red fluorescence clearly began to accumulate around the cell membrane after 1h, the aggregation increased significantly at 2h, and a large number of nanoparticles had infiltrated the cytoplasm at 4h. However, the above phenomenon was not observed in the B16-F10 cells co-incubated with DiI labeled si-Ce6@NPs, and only less red fluorescence was observed around the cell membrane after 4h ( Fig. 2 B ) . Furthermore, we assessed the efficiency of si-Ce6@tLyp-1-NPs uptake by B16-F10 cells at the same time points by flow cytometry. We discovered that the number of DiI-positive cells in the si-Ce6@tLyp-1-NPs group reached 99% at 4h, which was much higher than that in the si-Ce6@NPs group. The results were consistent with the confocal microscopy observations ( Fig. 2 C, D ) . Therefore, these findings confirmed that tLyp-1 peptide-modified nanoparticles possess high targeting specificity toward B16-F10 cells, which is conducive to efficient gene and drug delivery. The tLyp-1 peptide not only has tumor cell targeting ability, but also has strong tumor tissue penetration. Therefore, we used melanoma cell spheres to verify its ability of penetration. CLSM observation showed that after 4 hours of co-incubation with Dil-labeled si-Ce6@tLyp-1-NPs, red fluorescence was distributed throughout the spheres and deep into the tumor core, while Dil-labeled si-Ce6@NPs nanoparticles only marginally bindind to tumor cell spheres ( Fig. 2 E ) . In summary, si-Ce6@tLyp-1-NPs acquired good tumor-targeted penetration capacity, thereby increasing the deep tissue delivery of drugs and genes. 2.3. si-Ce6@tLyp-1NPs gene transfection efficiency assay In order to prove whether si-Ce6@tLyp-1NPs could cause liquid–gas phase transition under LIFU irradiation, we observed the NPs by optical microscopy showing that the nanoparticles expanded and transitioned into microbubbles after 3min of irradiation (Figure S4) . Additionally, we examined the UTMD ability of the nanoparticles in vitro by using ultrasound (US) mode and contrast-enhanced ultrasound (CEUS) mode. The results showed that the ultrasound signal intensity of nanoparticles gradually increased with the extension of irradiation time and the increase of irradiation power ( Fig. 3 A, B ) . As an important gene regulatory molecule, siRNA can specifically silence the expression of target genes through RNA interference (RNAi). To prove the siRNA transfection ability of si-Ce6@tLyp-1-NPs in vitro , the FAM si-Ce6@NPs and FAM si-Ce6@tLyp-1-NPs were co-cultured with B16-F10 cells for 4h and then irradiated with ultrasound ( Fig. 3 C ) . The nanoparticles carrying FAM-labeled siRNA were used to verify the transfection efficiency of si-Ce6@tLyp-1-NPs combined with ultrasound irradiation. The cytoskeleton was stained with phalloidin to observed the cytoplasmic transfection of FAM siRNA by CLSM. More green fluorescence was observed in the cytoplasm of B16-F10 in the FAM si-Ce6@tLyp-1-NPs (+) group ( Fig. 3 D ) . At 24 h after transfection, the mRNA level of FTO in the cells was detected by qRT-PCR. The results showed that the expression level of FTO protein in B16-F10 cells in the si-Ce6@tLyp-1-NPs (+) group was significantly lower than other groups ( Fig. 3 E ) . Additionally, 48 hours after transfection, we extracted the protein of B16-F10 cells and detected the intracellular FTO level by western blot. Consistent with the qRT-PCR results, it showed that the expression level of FTO protein in B16-F10 cells treated with si-Ce6@tLyp-1-NPs (+) was significantly decreased ( Fig. 3 F, G ) . All the results showed that targeted nanoparticles combined with ultrasound irradiation could significantly promote siRNA transfection into the cytoplasm, and the transfection efficiency of siRNA was significantly higher than that of the non-targeted nanoparticles combined with ultrasound irradiation group and siRNA treatment alone group. si-Ce6@tLyp-1-NPs combined with ultrasound can effectively inhibit the expression of FTO gene machinery protein in B16-F10 cells. 2.4. si-Ce6@tLyp-1NPs mediated regulation of glycolytic metabolism To further investigate the effect of FTO inhibition on cellular glycolysis, we used siFTO to knock down FTO expression in B16-F10 cells and performed RNA-seq. Transcriptome analysis showed that 2562 genes were up-regulated and 2525 genes were down-regulated with FTO knockdown in melanoma cells ( Fig. 3 H ) . Among them, 14 genes related to the glycolytic pathway were down-regulated, and the downregulation of PGAM1 was consistent with the results of Liu et al [18] ( Fig. 3 I ) . Phosphoglycerate mutase 1 (PGAM1) is also a key glycolytic enzyme, which can regulate the rate of glycolysis. Studies have shown that PGAM1 is involved in aerobic glycolysis in melanoma and indicates poor prognosis in melanoma [35,36]. Moreover, study had shown that inhibition of glycolysis by PGAM1 knockdown combined with chemotherapy can synergistically treat non-small cell lung cancer with high glycolysis [37]. In addition, the hexokinase 2 (HK2) gene, which plays an important role in glycolysis, was also significantly down-regulated. The hexokinase (HK) protein family is one of the core regulators of the glycolytic pathway. HK2 is highly expressed in cancer cells and plays an important role in the "Warburg effect" (metabolic reprogramming from oxidative phosphorylation to glycolysis) [38]. HK2 inhibition leads to a significant reduction in glycolysis [39]. For Gene set enrichment analysis (GSEA), we predefined gene ranks and utilized the R package clusterProfiler as well as from Molecular Signatures Database (DOI: 10.1093 / bioinformatics/btr260, http://www.gsea-msigdb.org/gsea/downloads.jsp ) to download the c2. Cp. Kegg. V7.4. Symbols. The GMT Ensemble to evaluate glycolysis related pathways and molecular mechanisms. Results showed that FTO was associated with glycolytic pathway ( Fig. 3 J ). Therefore, inhibition the expression of FTO can decrease the expression of key genes in the glycolytic pathway and down-regulate the level of cell glycolysis. We examined the expression levels of HK2 and PGAM1 in the glycolytic pathway. The mRNA level of HK2 and PGAM1 in B16-F10 cells treated with Ce6@tLyp-1-NPs (+)、si-Ce6@NPs(+) and si-Ce6@tLyp-1-NPs(+) was detected by qRT-PCR. The results showed that the mRNA level of HK2 and PGAM1 in B16-F10 cells treated with si-Ce6@tLyp-1-NPs(+) was significantly lower than that in the control group and Ce6@tLyp-1-NPs(+) ( Fig. 3 K, L ) . And the protein expression of HK2 and PGAM1 in B16-F10 cells treated with Ce6@tLyp-1-NPs (+), si-Ce6@NPs (+) and si-Ce6@tLyp-1-NPs (+) was detected by WB. The results also showed that the expressions of HK2 and PGAM1 protein in B16-F10 cells treated with si-Ce6@tLyp-1-NPs (+) were significantly down-regulated in B16-F10 cells after si-Ce6@tLyp-1-NPs (+) treatment ( Fig. 3 M-O ) . To evaluate the inhibitory effect of FTO knockdown on glycolysis in melanoma cells, we collected and analyzed the supernatants of cell cultures following different treatments. Compared with the other groups, the lactate concentration in si-Ce6@tLyp-1-NPs (+) groups decreased significantly ( Fig. 3 P ) . Thus, the glycolytic pathway is inhibited after effective silencing of the FTO gene, reducing the consequent production of lactate. In conclusion, our results showed that UTMD combined with targeted nanoparticles had a high intracellular transfection efficiency and the expression of FTO was effectively inhibited in B16-F10 cells. FTO inhibition can further lead to the down-regulation of HK2, PGAM1 and other glycolytic pathway genes, thereby inhibiting glycolysis of tumor cells and reducing the production of lactate. 2.5. si-Ce6@tLyp-1-NPs -Elicited ICD in vitro Upon ultrasound irradiation, Ce6 could efficiently produce ROS to trigger immunogenic cell death, which resulted in the release of damp-associated molecular patterns (DAMPs), such as CRT and HMGB [40 ,41]. The ROS generation in B16-F10 cells receiving different treatments was observed by CLSM. Under ultrasound exposure, the green fluorescence intensity of ROS probe 2',7'- Dichlorodihydrofluorescein diacetate (DCFH‐DA) in the group of B16-F10 cells treated with si-Ce6@tLyp-1-NPs increased significantly ( Fig. 4 A ) . It was demonstrated that si-Ce6@tLyp-1-NPs + US could efficiently induce the production and accumulation of ROS in cells, which in turn produced ICD. Increased CRT expression on the surface of dying cells during SDT can act an "eat me" signal, thereby stimulate DCs maturation and activation of specific effector T cells, enhancing the host anti-tumor immune response [42,43]. We examined CRT and HMGB1 expression in different treated tumor cells by CLSM (Fig. 4 B, C). Consistent with the trend of ROS production ( Fig. 4 D ) , CRT exposure on the surface of B16-F10 cells in the si-Ce6@tLyp-1-NPs (+) group was significantly increased compared with PBS and other treatment groups ( Fig. 4 E ) . However, in the si-Ce6@NPs (+) group, the localization of HMGB1 in the nucleus was reduced because a small amount of HMGB1 was transferred from the nucleus to the cytoplasm and the cell membrane, while in the si-Ce6@tLyp-1-NPs (+) group, most of HMGB1 was released to the outside of the cells and only a small amount of green fluorescence was distributed in the cytoplasm and the cell membrane. Meanwhile, the CLSM images showed the green fluorescence of HMGB1 was located inside the nucleus and had not been released to the outside of the cells in the PBS, US (+) and si-Ce6@tLyp-1-NPs groups. Research has confirmed that HMGB1 can be transferred from the nucleus to the cytoplasm and finally released outside the cell when ICD occurs in tumor cells, and host antitumor immunity can be induced [44]. We detected the extracellular release of HMGB1 from B16-F10 cells by ELISA, and the results showed that the extracellular HMGB1 content was significantly increased in the si-Ce6@tLyp-1-NPs group ( Fig. 4 F ) . The results indicated that ICD triggered by SDT could induce massive release of HMGB1 outside the cell. The consistent trend of ROS production and DAMP release confirmed that si-Ce6@tLyp-1-NPs was highly effective in triggering ICD, which in turn enhanced anti-tumor immune response. The reduction of lactate production can relieve the activation barrier of immune cells, enhance the infiltration and killing ability of immune cells. As antigen-presenting cells, dendritic cells (DCs) can activate anti-tumor immune responses and induce T cell activation. We incubated different treatments of B16F10 tumor cells with Bone Marrow-Derived Dendritic Cells (BMDCs) ( Fig. 4 I ) . Flow cytometry was used to analyze the proportion of mature DCs after co-culture with B16F10 cells, showing that after co-cultured with si-Ce6@tLyp-1-NPs(+) pretreated B16F10 cells the proportion of mature DC could reach 30.9%, which was significantly higher than that of the control group, si-Ce6@tLyp-1-NPs, si-Ce6@NPs(+) and Ce6@tLyp-1-NPs(+)groups ( Fig. 4 G, J ) . It was confirmed that FTO inhibition combined with sonodynamic therapy can increase the maturation of DCs cells. At the same time, we co-cultured B16F10 cells after different treatments with spleen single fine suspensions and examined the proportion of CD8 + T cells positive for IFN-γ ( Fig. 4 I ) . Flow cytometry staining analysis showed that CD8 T cells in the group that were co-incubated with si-Ce6@tLyp-1-NPs (+) pretreated B16F10 cells expressed higher levels of activated IFN-γ ( Fig. 4 H, K ) . Therefore, we demonstrated that inhibiting FTO expression in tumor cells combined with sonodynamic therapy could further enhance T cell activation and mediated cytotoxicity. All these findings showed that inhibition of FTO is able to reprogram tumor cell metabolism to relieve the obstacle of immune cell activation caused by lactate accumulation. And then we examined the apoptosis of B16-F10 cells after different treatments. It was shown that si-Ce6@tLyp-1-NPs (+) group induced the highest apoptosis rate, which could reach 85.4%, indicating a strong therapeutic effect ( Fig. 4 L, M ) . The results of live and dead cell staining also fully proved that si-Ce6@tLyp-1-NPs (+) had a good combined anti-tumor effect ( Fig. 4 N ) . The cell killing effect of si@tLyp-1-NPs (+) group was small, indicating that knockdown of FTO alone has a small killing effect on tumor cells. The higher cell survival rate of the si-Ce6@NPs (+) untargeted group verified that si-Ce6@tLyp-1-NPs could enter more tumor cells and produce ROS in response to ultrasound stimulation to kill tumor cells with high efficiency. A large number of dead cells also existed in the Ce6@tLyp-1-NPs (+) group, indicating that the targeted nanoparticles had good SDT efficacy. Finally, the dead cells with red fluorescence in the si-Ce6@tLyp-1-NPs (+) group were the most, which verified that si-Ce6@tLyp-1-NPs had a good combined treatment effect. 2.6. Targeting and penetration capacity detection of si-Ce6@tLyp-1-NPs in vivo In vitro experiments showed that si-Ce6@tLyp-1-NPs could produce ROS to induce ICD in tumor cells under ultrasound irradiation. UTMD could promote the efficient delivery of siFTO, inhibiting glycolysis and reducing lactic acid accumulation, thereby increasing immune cell infiltration and improving immunosuppressive TME. Next, we will discuss its anti-tumor effects in vivo . Nanodrugs for systemic administration can cross the tumor vascular endothelial space and accumulate in solid tumors. Peptide-modified nanoparticle surfaces can further improve pharmacokinetics, enhance tissue targeting, and promote cell and tissue penetration, thereby avoiding off-target effects and improving therapeutic efficacy[45]. Therefore, we evaluated the in vivo targeting and penetration of tLyp-1 peptide-modified NPs in melanoma using an animal imaging system (IVIS Lumina XRMS, PerkinElmer, USA). DiR-labeled si-Ce6@NPs and si-Ce6@tLyp-1-NPs were injected via the tail vein of a melanoma mouse model, and the accumulation of nanoparticles at the melanoma site during different time points (4h,8h,12h and 24h) was observed by small animal in vivo imaging. The in vivo imaging results indicated si-Ce6@tLyp-1-NPs gradually accumulated in the tumor site and reached the peak at 12 hours. In addition, a large number of nanoparticles were still retained in the tumor tissue at 24h. However, the fluorescence intensity of si-Ce6@NPs was significantly lower than that of si-Ce6@tLyp-1-NPs at all time points (Fig. 5A) . Therefore, we performed organ ex vivo imaging of tumors and other major organs 12 h after intravenous injection of nanoparticles to investigate the in vivo distribution of nanoparticles. As shown in Fig. 5B , a significant accumulation of si-Ce6@tLyp-1-NPs at the tumor site was observed, but not in the si-Ce6@NPs group. These results demonstrated that tLyp-1 modification can significantly increase the tumor targeting of the nanoparticles and improve the efficiency of gene and drug delivery to the tumor site. In addition, according to the above results, we can confirm that drug accumulation at the tumor site reaches a peak 12h after injection of nanoparticles. Twelve hours after injection of DiI-labeled si-Ce6@NPs and si-Ce6@tLyp-1-NPs, the mice were sacrificed and tumor tissues were collected for section staining to observe the distribution of si-Ce6@NPs and si-Ce6@tLyp-1-NPs in the tumor site (Fig. 5C) . It can be observed that the red fluorescence in the si-Ce6@tLyp-1-NPs group enters more into the deep part of the tumor tissue, while only a small amount of red fluorescence is observed in the si-Ce6@NPs group at the edge of the tumor (Fig. 5D) . This demonstrated that tLyp-1 also enhanced the tumor tissue penetration of the nanoparticles, allowing the drug to penetrate deeper into the tumor tissue to enhance the sonodynamic therapy and FTO inhibition effect. Since hypoxia in the core of the tumor leads to increased cell glycolysis, the well tumor permeability of si-Ce6@tLyp-1-NPs can further inhibit cell glycolysis in the core of the tumor and reduce the accumulation of lactic acid to improve tumor immunity. 2.7. In vivo anti-tumor activity To investigated the anti-tumor effect of different treatments in vivo , we used the subcutaneous tumor model in C57 mice established by B16-F10 cells. When the tumor volume reached about 100 mm 3 , the mice were randomly divided into 7 groups with 5 mice in each group, and the drugs were administered every other day for a total of 3 times. In addition, some groups were subjected to ultrasound irradiation 12 h after administration, tumor size was monitored every 3 days, and euthanized on the 15th day ( Fig. 6 A ) . To analyze the antitumor effect, all tumor samples were collected and weighed. Treatment with si-Ce6@tLyp-1-NPs (+) significantly inhibited tumor growth compared with the other groups ( Fig. 6 B ) . The results showed that the tumor volume of the si@tLyp-1-NPs (+) group was slightly reduced. Although the tumor weight of the Ce6@tLyp-1-NPs (+) group was significantly reduced, the smallest tumor volume and tumor weight could be observed in the si-Ce6@tLyp-1-NPs (+) group, which proved that FTO inhibition combined with sonodynamic therapy could achieve better anti-tumor treatment effect ( Fig. 6 C, E ) . Hematoxylin-eosin (H&E), FTO, Ki67 immunohistochemical staining and terminal deoxynucleotidyl transferase-mediated dutP-biotin Nick end labeling (TUNEL) staining were performed to evaluate the effects of nanomedicine on tumor necrosis, FTO inhibition, proliferation and apoptosis. H&E stained tumor sections showed extensive karyopyknosis, karyorhexis, and karyolysis in the si-Ce6@tLyp-1-NPs (+) group, confirming that the si-Ce6@tLyp-1-NPs (+) group had the most severe tumor tissue necrosis ( Fig. 6 D ) . The results showed that FTO inhibition combined with SDT treatment had the most significant tumor killing effect. Immunohistochemical staining showed that the expression of FTO was significantly reduced in G4 and G7 groups, indicating that the peptide-modified tumor tissue-targeted nanoparticles had a high FTO inhibition efficiency after ultrasound irradiation in vivo ( Fig. 6 F ) . The results of Ki-67 staining ( Fig. 6 G ) and Tunel staining ( Fig. 6 H ) also confirmed that the tumor cell proliferation was significantly decreased and the level of tumor cell apoptosis was significantly increased in the si-Ce6@t-NPs (+) group. The above results fully demonstrated the excellent anti-tumor efficacy of si-Ce6@tNPs (+). 2.8. In vivo immune response activation. SDT can produce a stronger ICD effect by producing ROS. During ICD, tumor cells produce a series of DAMPs, including surface-exposed calreticulin (CRT) and passively released high mobility group box 1 (HMGB1), which together trigger the phagocytosis of dying tumor cells by dendritic cells (DCs) [46]. Our previous results showed that si-Ce6@tLyp-1-NPs can efficiently induce tumor cell ICD in vitro . We next examined the effect of si-Ce6@tLyp-1-NPs on reshaping the immune microenvironment in vivo ( Fig. 7 A ) . The ability of si-Ce6@tLyp-1-NPs + US treatment to induce ICD in vivo to trigger anti-tumor immune responses was assessed by measuring CRT and HMGB1 expression in tumor tissues. The results of immunofluorescence staining showed that CRT was almost not expressed in the control group and si-Ce6@tLyp-1-NPs, little expressed in the si-Ce6@NPs (+) group, more expressed in the Ce6@tLyp-1-NPs (+) group, and the most expressed in si-Ce6@tLyp-1-NPs. The results of HMGB1 immunofluorescence staining showed that the si-Ce6@tLyp-1-NPs (+) group had a significant release compared with other groups. These discharges indicated that the non-targeted nanoparticles can only enter the tissue in a small amount and cause a small amount of tumor cell ICD. Ce6@tLyp-1-NPs can efficiently enter the tumor tissue and cause ICD, while si-Ce6@tLyp-1-NPs (+) group can induce a stronger ICD effect, increased CRT exposure, and a large release of HMGB1 ( Fig. 7 B, C ) . The occurrence of ICD will improve the immunogenicity of tumor microenvironment. Recruitment of immune cells to the TME is directly related to antitumor immune responses. Therefore, to determine whether sonodynamic therapy combined with FTO inhibition could further enhance the anti-tumor immune response by enhancing immune cell infiltration and activation, we further investigated the proportion of immune cells in the tumor tissue. Firstly, we examined the proportion of DCs (CD11c + CD80 + CD86 +) cells in the tumor tissues. Flow cytometry showed that the percentage of mature dendritic cells infiltrating the tumor in the si-Ce6@tLyp-1-NPs + US group was significantly higher than that in the other experimental groups, which could reach 60.3% ( Fig. 7 D, E ) . In addition, we evaluated the levels of CD4 + T cells and CD8 + T cells in the tumor tissue. The results showed that the ratio of CD4 + T cells and CD8 + T cells in the tumor tissues of the si-Ce6@tLyp-1-NPs (+) group was also increased, with the proportion of CD4 + T cells increasing by 23% and the proportion of CD8 + T cells increasing by 13%, demonstrating that si-Ce6@tLyp-1-NPs (+) could intensely promoted the differentiation of CD8 + T cells in the tumor microenvironment, increasing the ratio of CD8 + T cells / CD4 + T cells ( Fig. 7 F-H ) . Compared with Ce6@tLyp-1NPs (+) group, si-Ce6@tLyp-1-NPs + US group significantly promoted the infiltration and maturation of tumor DCs and T lymphocytes, proving that FTO inhibition further enhanced the anti-tumor immune response after SDT treatment. To test whether FTO inhibition could enhance CD8 + T cell-mediated cytotoxicity, we measured the proportion of CTL (CD3 + CD8 + IFN-γ+) in tumor tissues. The percentage of tumor tissue CTL (CD3 + CD8 + IFN-γ+) in si-Ce6@tLyp-1-NPs (+) group was the highest, which was 6.17 fold higher than control in the tumor ( Fig. 7 I , J) . These results indicate that FTO inhibition combined with SDT treatment significantly increased the number of immune cell infiltrates in the tumor tissue and clearly enhanced the antitumor immune response. In addition, the proportion of DCs and CD8 + T cells in the spleen of the mice was measured. The results showed that the percentages of DCs and CD8 + T cells in spleen were also significantly higher than those in other treatment groups, reaching 51.3% and 42.4%, respectively ( Fig. 7 K-N ) . Therefore, si-Ce6@tLyp-1-NPs (+) can not only improve the immune microenvironment in tumor, but also activated the systemic immune response to enhance the systemic immune response and obtain better anti-tumor immunity. The above results confirmed our hypothesis that compared with SDT treatment alone, FTO combined with SDT stimulated a potent antitumor immune response and significantly increased the rate of immune cell tumor tissue infiltration. The “cold tumor” can be transformed into a “hot tumor” with a better anti-tumor immune response, resulting in a more powerful tumor treatment effect. 2.9. In vivo biosafety evaluation Finally, in order to evaluate the in vivo biosafety of si-Ce6@tLyp-1-NPs. Healthy mice receiving si-Ce6@tLyp-1-NPs intravenous injection were euthanized on the scheduled date, blood samples were collected for biochemical examination and blood routine analysis, and major organs were collected for H&E staining. As shown in Figure S5 , compared with the control group, the biochemical indexes and blood routine analysis showed no abnormalities at 1, 3, 7, 14, and 28 d after the tail vein injection of si-Ce6@tLyp-1-NPs. As shown in Figure S6 , each tissue section showed no significant histopathological changes. These results indicate that si-Ce6@tLyp-1-NPs has good biosafety, and showed potential clinical applications as novel agents for cancer therapy. 3. Conclusion In conclusion, this study successfully constructed si-Ce6@tLyp-1-NPs and demonstrated that FTO inhibition can effectively enhance the therapeutic effect of SDT in melanoma. The tLyp-1 peptide-modified nanoparticles have superior tumor targeting ability and penetration, which could achieve efficient co-delivery of sonosensitizer Ce6 and FTO siRNA. Moreover, PFP could respond to ultrasound irradiation to further enhance the delivery efficiency of siRNA, and Ce6 could produce ROS under ultrasound irradiation, thereby inducing ICD in tumor cells. Notably, downregulation of FTO gene expression by si-Ce6@tLyp-1-NPs effectively inhibited melanoma cell glycolysis and relieved lactic acid-mediated immune cell suppression. Furthermore, the tumor immunosuppressive environment was improved, and the infiltration of DCs and T lymphocytes was significantly increased. FTO inhibition can significantly enhance the cytotoxicity of CD8 + T cells, resulting in a strong cellular immune response and significantly enhanced tumor therapeutic efficacy. In conclusion, FTO inhibition combined with SDT treatment significantly inhibited the growth of melanoma, providing a potential new strategy for sonodynamic immuno-metabolic synergistic therapy. 4. Materials and methods 4.1. Materials 1,2-distearoyl-sn-glycero-3-phosphocho-line (DSPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n (DSPE), 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) and DSPE-PEG-tLyp-1, were purchased from Xi’an Ruixi Biological Technology Co., Ltd. (Xi’an, China). FTO siRNA sense strand, 5′-GUCAGACCUUCCUAAAGCUTT-3′, negative control siRNA, FAM labeled negative control siRNA were purchased from GenePharma (Shanghai, China). The Chlorin e6 (Ce6) and Singlet Oxygen Sensor Green (SOSG) was purchased from Meilunbio (Dalian, China). The Calcein/PI Cell Viability/Cytotoxicity Assay Kit, Annexin V-Alexa Fluor 647/PI apoptosis detection kit, Reactive Oxygen Species Assay Kit and DiI Iodide were obtained from Beyotime (Shanghai, China). The lactic acid assay kit was purchased from Jiancheng Bioengineering Institute (Nanjing, China). HMGB1 antibody and HK2 antibody were purchased from Immunoway (US). CRT antibody, FTO antibody, PGAM1 antibody were obtained from Proteintech (Wuhan, China). FITC anti-mouse CD11c, APC anti-mouse CD80, APC anti-mouse CD80, PE anti-mouse CD86, APC anti-mouse CD3, PE anti-mouse CD4, FITC anti-mouse CD8a for flow cytometry were obtained from Biolegend (USA). 4.2. Cell culture and animal model B16-F10 (RRID:CVCL_0159) tumor cells were procured from Procell Life Science & Technology Co., Ltd. (Wuhan, China), culturing in RPMI-1640 with 10% FBS and 1% penicillin‒streptomycin at 37°C in a 5% CO 2 incubator. All C57BL/6 mice, aged 6–8 weeks, were purchased from the Changsheng biotechnology (Liaoning, China). To establish the melanoma cancer model, B16F10 cells at a density of 1 × 106 cells/mL in 0.1 mL PBS were injected to the dorsal flank of C57BL/6 mice. All animal e ments were approved by the Animal Management and Use Ethics Committee of the Second Affiliated Hospital of Harbin Medical University. 4.3. Preparation of si-Ce6@tLyp-1-NPs We employed traditional rotary evaporation and a one-step emulsion method to prepare [email protected] mass of hybrid lipid (6 mg DSPC, 2 mg DOTAP, 2 mg DSPE-PEG- tLyp-1) and 0.5mg Ce6 was dissolved in 4 mL trichloromethane (CHCl 3). Then transferred the resultant mixture into a round-bottom flask, and homogeneous lipid film were formed through rotary evaporation in a water bath at 40°C. Added 4 mL of DEPC-water to rehydrate the films, and then 200 µL of PFP was added to the mixture. Finally, the mixture was sonicated (60 W, 6 min, 5 s on, 5 s off) in an ice bath, and the nanoparticles were washed by twice centrifugation (8000 rpm, 3 min) to obtain the cationic nanoparticles. The obtained Ce6@tLyp-1-NPs was then mixed with FTO siRNA to form si-Ce6@tLyp-1-NPs by electrostatic adsorption. DiR/DiI was added to the mixed lipid solution to synthesize the fluorescently labeled si-Ce6@tLyp-1-NPs. 4.4. Characterization of si-Ce6@tLyp-1-NPs The structure of the nanoparticles was observed by Transmission electron microscopy (Hitachi H-7600; Japan). The potential of si-Ce6@tLyp-1-NPs and the size of si-Ce6@tLyp-1-NPs at 0, 1, 3, and 7d after preparation were measured using a DLS detector from Malvern Instruments (ZEN3600; Malvern, U.K.). A UV spectrophotometer (UV-2600, Shimadzu, Japan) was used to obtain UV–visible absorption spectra. Ce6 was dissolved in methanol and the absorbance of different concentrations of Ce6 solutions at 400nm was determined by a UV spectrophotometer (UV-2600, Shimadzu, Japan). The drug loading rate of Ce6 was calculated by constructing a linear correlation curve between the concentration of Ce6 and its absorption peak. Encapsulation rate (%) = (weight of drugs in nanoparticles/initial weight of drugs) × 100% Mix siRNA (NCsiRNA) and Ce6@tLyp-1-NPs at ratios of 1:2, 1:4, 1:6, 1:8, 1:10 (w: w) to a total volume of 50 µL. The mixture was incubated for 20 min at room temperature before centrifugation to obtain the supernatant. The mixture was incubated at room temperature for 20 min, followed by centrifugation to obtain the supernatant. The supernatant was mixed with loading buffer and loaded onto a 2% agarose gel for electrophoresis at 120 V for 20 min to observe the change in siRNA bands. FAM siRNA was incubated with DiI-labeled Ce6@tLyp-1-NPs drops at a mass ratio of 1:8 and then centrifuged to obtain si-Ce6@tLyp-1-NPs. Observing the fluorescence co-localization of siRNA and nanoparticles by fluorescence microscopy to determine the successful loading of siRNA was. B16-F10 cells were incubated with double-labeled nanoparticles, and the localization of cell, nanoparticles and siRNA was observed by CLMS. The FAM siRNA was incubated with Ce6@tLyp-1-NPs at a mass ratio of 1:8 and centrifuged to obtain si-Ce6@tLyp-1-NPs, and the ligation efficiency of the siRNA to Ce6@tLyp-1-NPs was determined by flow cytometry. The SOSG probe was added to si-Ce6@tLyp-1-NPs solution and irradiated with LIFU for 15s, 30s, 60s, 90s, 120s, 180s (pulse mode, interval 2s, power 3 W). ROS production was determined by observing the fluorescence spectra. 4.5. Cellular uptake and penetration ability of nanoparticles in vitro B16-F10 cells were seeded in the confocal dishes at a density of 1 × 10 5 cells/ml. DiI labeled si-Ce6@tLyp-1-NPs and si-Ce6@NPs were incubated with the cells for 0.5, 1, 2, and 4h, respectively. The cells were washed twice with PBS and fixed with 4% paraformaldehyde. After staining B16-F10 cells with DAPI (10 µg/mL) for 15 min, the uptake of nanoparticles by B16-F10 cells was observed by CLSM. And B16-F10 cells were seeded in a twelve-well plate at a density of 1 × 10 5 cells/ml. Culturing cells as in the CLSM experiment, the uptake efficiency of the nanoparticles was quantified by FCM after the cells were digested with trypsin. B16-F10 cells were seeded in ultra-low adsorption 6-well plates at a density of 3 × 10 4 cells/well to form 3D tumor spheroids. Spheroids were co-cultured with nanoparticles labeled with DiI, staining the cell nucleus by Hoechst. The penetration of nanoparticles was detected by observing fluorescence within the spheres by CLSM. 4.6. In vitro cell safety B16-F10 cells were seeded in 96-well plates at a density of 3000 cells/well and cultured overnight. Different concentrations of si-Ce6@tLyp-1-NPs were added to each well, and cell viability was assessed 24h later using the CCK-8 assay. Add 100 µl of culture medium containing 10 µL of CCK-8 solution to each well and read the optical absorbance of each well at 450 nm using a microplate reader. 4.7. Intracellular ROS production The DCFH-DA probe was used to determine 1O2 generation. The experiment was divided into five groups: PBS group, si-Ce6@tLyp-1-NPs group, si-Ce6@ NPs (+) group, Ce6@tLyp-1-NPs (+) group and si-Ce6@tLyp-1-NPs (+) group. Briefly, B16-F10 cells were incubated with nanoparticles for 4 h at 37°C. Cells were washed and incubated with DCFH-DA probe for 30 min and then sonicated the cells. Hoechst was added for staining of nucleus. The fluorescence was observed by CLSM and the results were quantified. 4.8. Immunofluorescence staining of ICD markers in vitro B16-F10 cells were seeded on confocal dishes overnight. Grouping and treating the cells as ROS assay experiment, 4% paraformaldehyde, Triton (0.3%) and blocking solution were added successively. After the blocking solution was removed, primary antibodies against calreticulin (CRT) and high mobility group b1 protein (HMGB1) diluted 1:400 were added and incubated overnight at 4°C. After washing three times with PBS, AbFluor 488 goat anti-rabbit IgG diluted in 1:500 dilution of PBS was added and incubated for 1 h at 37 ° C in the dark. Nuclei were stained with DAPI for 5 min, and cells were visualized by CLSM. 4.9. In vitro phase transition ability assay To evaluate the in vitro phase transition ability of si-Ce6@tLyp-1-NPs under ultrasound irradiation, we constructed constructing a 3.5% agarose (w/v) gel model. si-Ce6@tLyp-1-NPs were exposed to different intensities of ultrasound (0, 1, 2, 3W/ cm², duty cycle 50%) for 1, 2, and 3 minutes, respectively. Subsequently, B-mode ultrasound and contrast-enhanced ultrasound (CEUS) imaging were performed to observe the liquid-gas phase transition process of the phased-transition nanoparticles. The ultrasound intensity value was quantitatively analyzed by software. Using ultrasound to irradiate si-Ce6@tLyp-1-NPs (power 3W/cm², time 3min), the morphology of the nanoparticles was observed by optical microscope. 4.10. Detection of siRNA transfection ability in vitro B16-F10 was seeded in confocal dishes overnight. The cells were incubated with free FAM-siRNA, FAM-si-Ce6@NPs and FAM-si-Ce6@tLyp-1-NPs for 4h and then irradiated with ultrasound. Cells were fixed with 4% paraformaldehyde, staining the cytoskeleton with phalloidin ring and staining nucleu with DAPI. Finaly, the transfection efficiency of siRNA was observed by CLMS. 4.11. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was isolated from B16-F10 cells using the SPARKeasy Improved Tissue/Cell RNA Kit (Sparkjade, Shandong, China). First-strand cDNA was synthesized by reverse transcription using the PrimeScript™ RT Reagent Kit (Takara Technologies, Shiga, Japan). For quantitative real-time PCR (qRT-PCR), the cDNA was amplified using TB Green® Premix Ex Taq™ II (Takara Technologies, Shiga, Japan) in a real-time PCR system. 4.12. Western blot analysis To further quantify the transfection efficiency of FTO siRNA, B16-F10 cells were incubated with Ce6@tLyp-1-NPs, si-Ce6@NPs and si-Ce6@tLyp-1-NPs and treated with ultrasound irradiation after 4h. Cells were harvested 48 hours later, and protein extracted from untreated B16-F10 cells served as a control. The expression of FTO was detected by Western blot. Cells were lysed in RIPA lysate. Total protein was quantified using the BCA Protein Assay kit and equilibrated prior to sample loading. Equal amounts of protein extracts were electrophoresed in SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was then blocked with 5% nonfat milk and incubated with FTO for primary antibody followed by secondary antibody. Finally, the band is captured by the Odyssey scanning system. Band intensities were quantified using the Image J program. The expression levels of HK2 and PGAM1 in each group were quantitatively analyzed by WB, and the changes of key enzymes in the glycolytic pathway after treatment with nanoparticles were detected. 4.13. RNA Sequencing B16-F10 cells were seeded in 6-well plates (1 × 10 5 cells/well) overnight. FTO was knocked down in cells by Lipofectamine 2000 combined with FTO siRNA, and 24 h after transfection, total RNA was extracted using TRIzol reagent. A cDNA library was established for data processing. The quality of the library was checked by Agilent 2100 Bioanalyzer, and then the total concentration of the library and the effective concentration of the library were detected. The libraries containing different index sequences were then scaled according to the effective concentration of the library and the amount of data required for the library. The mixed libraries were unified to 2nM and, by alkali denaturation, single-stranded libraries were formed. After RNA extraction, closure and library construction, Next-Generation Sequencing (NGS) technology was used to perform Paired-end (PE) sequencing of these libraries based on Illumina sequencing platform. Clean reads were mapped to the reference genome with HISAT2. The distribution of Reads aligned to the genome was counted, and HTSeq statistics were used to calculate the Read Count value on each gene and the original expression level of the gene. Differentially expressed genes were screened and analyzed by R software. 4.14. Regulation of glycolysis by FTO inhibition Lactic acid detection kit was used to detect the content of lactic acid in the cell supernatant of the experimental group and the control group, and glucose detection kit was used to detect the content of glucose. 4.15. In vitro immune cell stimulation Bone marrow-derived dendritic cells (BMDCs) were generated from 8–10 week-old male C57BL/6 mice. All mice were first euthanized, and their femurs and tibias were dissected under sterile conditions. Adherent muscle tissue was carefully stripped away from the bones, and then rinsed with sterile PBS. The femurs and tibias were bisected with scissors to expose the marrow cavities. Using a syringe, the bone marrow was thoroughly flushed from the cavities with PBS, and the resulting cell suspension was filtered through a 200-mesh nylon mesh to eliminate debris and clumps. Following filtration, the cells were treated with red blood cell (RBC) lysate for 5 minutes, leaving a purified population of bone marrow cells. The isolated bone marrow cells were cultured in RPMI 1640 medium supplemented with 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 10% fetal bovine serum (FBS), and interleukin-10 (IL-10)—cytokines essential for driving BMDC differentiation. The immature BMDCs were harvested for subsequent experiments after 6 days of incubation. To evaluate how tumor-derived factors affect DC maturation, BMDCs were incubated with tumor cell culture supernatants after different treatments for 24 hours. Following this stimulation, flow cytometry was used to analyze the surface expression of CD80 and CD86—key co-stimulatory molecules—on CD11c⁺ cells. The spleens of the mice were aseptically removed after euthanasia and placed in precooled PBS. The spleens were shredded with forceps, ground into a single-cell suspension with a syringe piston, collagenase IV was added, and incubated at 37 ° C for 30 min. The cell suspension was slowly spread on the top layer of lymphocyte separation medium and centrifuged at 2000 rpm for 20 minutes. The lymphocyte layer was aspirated and washed twice with PBS. The obtained cells were incubated with the supernatants of tumor cells after different treated for 24h. Finally, the cells were stained for surface markers (CD3⁺, CD8⁺) and intracellular IFN-γ⁺ using fluorophore-conjugated antibodies, and flow cytometry was used to quantify the proportion of CD3⁺CD8⁺IFN-γ⁺ cells. 4.16. In vitro cell killing ability B16-F10 cells were seeded in six well plate. The experiment was divided into seven groups: control, US (+), si-Ce6@tLyp-1-NPs, si@tLyp-1-NPs (+), si-Ce6@NPs (+), Ce6@tLyp-1-NPs (+), si-Ce6@tLyp-1-NPs (+). The nanoparticles were incubated for 4h, and Ce6 was added at the same concentration (2.5×10 − 4 M). After incubation, cells were exposed to ultrasound and cultured for another 24 h. Double staining was performed using the Annexin V-FITC apoptosis detection kit according to the manufacturer's instructions. Flow cytometry was used to analyze the apoptosis rate. B16-F10 cells were seeded in confocal dishes, and cells were treated in the same manner as in the apoptosis experiments. The cells were stained with calcein acetoxymethyl ester (AM) (2×10 − 6 m) and PI (2×10 − 6 m) and visualized by CLSM. 4.17. In vivo distribution In vivo imaging study of the nanoparticle distribution was performed on melanoma mice intravenously injected with DiR-labeled si-Ce6@NPs and si-Ce6@tLyp-1-NPs. Mice were anesthetized with isoflurane and photographed under the IVIS spectral imaging system at 2, 6, 12, and 24 h after injection. To study tissue distribution, mice were sacrificed 12h after injection, with tumors and major organs collecting for in vitro imaging. The in vivo tissue penetration of the nanoparticles was tested in tumor-bearing mice by intravenous injection of DiI-labeled si-Ce6@NPs and si-Ce6@tLyp-1-NPs. After 12 hours, tumors were harvested and cryosections were prepared with a cryomicrotome. Frozen sections were fixed with 4% paraformaldehyde, counterstained with DAPI and observed under a fluorescence microscope.4.16 In vivo antitumor effects. 4.18. In vivo antitumor study Tumor-bearing male C57BL/6 mice aged 6–8 weeks were randomly divided into 7 groups (n = 5) : control, US (+), si-Ce6@tLyp-1-NPs, si@tLyp-1-NPs (+), si-Ce6@NPs (+), Ce6@tLyp-1-NPs (+), si-Ce6@tLyp-1-NPs (+). The corresponding nanoparticles injected into mice via the tail vein. After 12 h, the tumor was exposed to ultrasound (3 W/cm 2 , 5 min), and the treatment was administered every two days for a total of three times. The tumors were monitored every 3 days. After 15 days, the mice were sacrificed, and the tumor embedded sections were collected for hematoxylin-eosin (H&E) staining, FTO and Ki-67 immunohistochemical staining, and terminal deoxynucleotidyl transferase-mediated dutP Nick end labeling (Tunel). 4.19. Analysis of immune response in vivo To verify the improvement of immune microenvironment in vivo , the tumor-bearing mice were divided into five groups: control; si-Ce6@tLyp-1-NPs; si-Ce6@NPs (+); Ce6@tLyp-1-NPs (+); si-Ce6@tLyp-1-NPs (+) group, treated with the same protocol to evaluate the antitumor effect. Immunofluorescence staining was used to observe the expression of CRT and HMGB1 in tumor tissues. The percentages of CD8 + T cells, CD4 + T cells and DCs cells in tumor tissues were detected by flow cytometry. 4.20. In vivo biosafety assessment To assess the biological safety of si-Ce6@tLyp-1-NPs, 25 healthy C57BL/6 mice were injected intravenously with 200µL si-Ce6@tLyp-1-NPs (1 mg/mL). Blood samples and main organs were collected on days 1, 3, 7, 14 and 28 for serum biochemical, blood routine tests and H&E staining. Five healthy mice were selected as the control group. 4.21. Statistics All data were expressed as mean ± standard deviation (SD). GraphPad Prism9.0 software was used for statistical analysis. t test was used for comparison between two groups, and analysis of variance (ANOVA) was used for comparison between multiple groups. p < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001). Declarations Supp l em e nt a ry Information Supporting Information is available from the Wiley Online Library or from the author. Authors’ Contributions X.P.L., P.L., and Z.Y. conceived and designed the experiments, X.T.L., W.N.S., W.D.Y., J.F.C, H.D.L., Y.P.L., J.T.R., L.W., R.T., Y.P.W. and P.Z. carried out the experiments. X.T.L, Z.W. and P.S. analysed the data. X.T.L., P.S., X.P.L. and P.L. wrote and edited the manuscript. All authors agreed to be responsible for the content of the work. Data Availability All data generated or analyzed during this study are included in this published article. Ethics Approval and Consent to Participate All experimental procedures conformed with the National Institutes of Health Guidelines for the Use of Laboratory Animals and were approved by the The Second Affiliated Hospital of Harbin Medical University Medical Ethics Committee (ethical approval number:YJSDW2024-127). Funding This work was supported by the Natural Science Foundation of China (Grant Nos. U22A20346). References Eddy K, Chen S. Overcoming Immune Evasion in Melanoma. Int J Mol Sci. 2020;21:8984. Hossain SM, Eccles MR. Phenotype Switching and the Melanoma Microenvironment; Impact on Immunotherapy and Drug Resistance. Int J Mol Sci. 2023;24:1601. Giammona A, De Vellis C, Crivaro E, Maresca L, Amoriello R, Ricci F, et al. Tumor-derived GLI1 promotes remodeling of the immune tumor microenvironment in melanoma. J Exp Clin Cancer Res. 2024;43:214. Arozarena I, Wellbrock C. Phenotype plasticity as enabler of melanoma progression and therapy resistance. Nat Rev Cancer. 2019;19:377–91. Naidoo C, Kruger CA, Abrahamse H. Photodynamic Therapy for Metastatic Melanoma Treatment: A Review. Technol Cancer Res Treat. 2018;17:1533033818791795. Yu L, Gao L, Liang B, Zhang L, Wu M, Liu J. Polymer-based nanodrugs enhance sonodynamic therapy through epigenetic reprogramming of the immunosuppressive tumor microenvironment. J Controlled Release. 2025;380:125–37. Geng S, Guo M, Zhan G, Shi D, Shi L, Gan L, et al. NIR-triggered ligand-presenting nanocarriers for enhancing synergistic photothermal-chemotherapy. J Controlled Release. 2023;353:229–40. Feng T, Tang Z, Karges J, Shen J, Jin C, Chen Y, et al. Exosome camouflaged coordination-assembled Iridium(III) photosensitizers for apoptosis-autophagy-ferroptosis induced combination therapy against melanoma. Biomaterials. 2023;301:122212. Liang J, Qiao X, Qiu L, Xu H, Xiang H, Ding H, et al. Engineering Versatile Nanomedicines for Ultrasonic Tumor Immunotherapy. Adv Sci. 2024;11:2305392. Jiang Q, Qiao B, Lin X, Cao J, Zhang N, Guo H, et al. A hydrogen peroxide economizer for on-demand oxygen production-assisted robust sonodynamic immunotherapy. Theranostics. 2022;12:59–75. Guo X, Tu P, Zhu L, Cheng C, Jiang W, Du C, et al. Nanoenabled Tumor Energy Metabolism Disorder via Sonodynamic Therapy for Multidrug Resistance Reversal and Metastasis Inhibition. ACS Appl Mater Interfaces. 2023;15:309–26. Hu X, Zhang M, Quan C, Ren S, Chen W, Wang J. ROS-responsive and triple-synergistic mitochondria-targeted polymer micelles for efficient induction of ICD in tumor therapeutics. Bioact Mater. 2024;36:490–507. Kim J, Shim MK, Moon Y, Kim J, Cho H, Yun WS, et al. Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy. J Nanobiotechnology. 2024;22:109. Li M, Liu Q, Xie S, Weng D, He J, Yang X, et al. Transformable Tumor Microenvironment‐Responsive Oxygen Vacancy‐Rich MnO 2 @Hydroxyapatite Nanospheres for Highly Efficient Cancer Sonodynamic Immunotherapy. Adv Sci. 2025;12:2414162. Qiao K, Luo C, Huang R, Xiang J, Pan Y, Zhang S, et al. Ultrasound Triggered Tumor Metabolism Suppressor Induces Tumor Starvation for Enhanced Sonodynamic Immunotherapy of Breast Cancer. Int J Nanomedicine. 2023;Volume 18:3801–11. Chen Y, Li K, Du H, Yao Y, Xie D, Zhou Z. Breaking Barriers in Oncology: Harnessing Sonodynamic Therapy for Enhanced Tumor Metabolism Regulation. Small. 2025;2502323. Chen Z, Chen L, Ma Y, Liu Y, Zhang Q, Qin H, et al. Peptide‐Appended Nanosonosensitizers Targeting Tumor Glycolysis for Synergistic Sonodynamic–Immunometabolic Therapy of Spinal‐Metastasized Tumors. Adv Mater. 2023;35:2304246. Wang F, Hu Y, Wang H, Hu P, Xiong H, Zeng Z, et al. LncRNA FTO-IT1 promotes glycolysis and progression of hepatocellular carcinoma through modulating FTO-mediated N6-methyladenosine modification on GLUT1 and PKM2. J Exp Clin Cancer Res. 2023;42:267. Liu Y, Liang G, Xu H, Dong W, Dong Z, Qiu Z, et al. Tumors exploit FTO-mediated regulation of glycolytic metabolism to evade immune surveillance. Cell Metab. 2021;33:1221-1233.e11. Sun N, Lei Q, Wu M, Gao S, Yang Z, Lv X, et al. Metal-organic framework-mediated siRNA delivery and sonodynamic therapy for precisely triggering ferroptosis and augmenting ICD in osteosarcoma. Mater Today Bio. 2024;26:101053. Xu C, Liu W, Hu Y, Li W, Di W. Bioinspired tumor-homing nanoplatform for co-delivery of paclitaxel and siRNA-E7 to HPV-related cervical malignancies for synergistic therapy. Theranostics. 2020;10:3325–39. Zong Y, Lin Y, Wei T, Cheng Q. Lipid Nanoparticle (LNP) Enables mRNA Delivery for Cancer Therapy. Adv Mater. 2023;35:2303261. Yan Y, Liu X-Y, Lu A, Wang X-Y, Jiang L-X, Wang J-C. Non-viral vectors for RNA delivery. J Controlled Release. 2022;342:241–79. Charbe NB, Amnerkar ND, Ramesh B, Tambuwala MM, Bakshi HA, Aljabali AAA, et al. Small interfering RNA for cancer treatment: overcoming hurdles in delivery. Acta Pharm Sin B. 2020;10:2075–109. Large DE, Abdelmessih RG, Fink EA, Auguste DT. Liposome composition in drug delivery design, synthesis, characterization, and clinical application. Adv Drug Deliv Rev. 2021;176:113851. Yang M, Wang X, Peng M, Wang F, Hou S, Xing R, et al. Nanomaterials Enhanced Sonodynamic Therapy for Multiple Tumor Treatment. Nano-Micro Lett. 2025;17:157. Sun R, Xiang J, Zhou Q, Piao Y, Tang J, Shao S, et al. The tumor EPR effect for cancer drug delivery: Current status, limitations, and alternatives. Adv Drug Deliv Rev. 2022;191:114614. Izci M, Maksoudian C, Manshian BB, Soenen SJ. The Use of Alternative Strategies for Enhanced Nanoparticle Delivery to Solid Tumors. Chem Rev. 2021;121:1746–803. Aronson MR, Medina SH, Mitchell MJ. Peptide functionalized liposomes for receptor targeted cancer therapy. APL Bioeng. 2021;5:011501. Bai J, Duan J, Liu R, Du Y, Luo Q, Cui Y, et al. Engineered targeting tLyp-1 exosomes as gene therapy vectors for efficient delivery of siRNA into lung cancer cells. Asian J Pharm Sci. 2020;15:461–71. Cheng C, Jiang W, Luo Y, Wan L, Guo X, Xie Z, et al. NIR Activated Multimodal Therapeutics Based on Metal–Phenolic Networks‐Functionalized Nanoplatform for Combating against Multidrug Resistance and Metastasis. Small. 2023;19:2206174. Wang R, Wang X, Li J, Di L, Zhou J, Ding Y. Lipoprotein-biomimetic nanostructure enables tumor-targeted penetration delivery for enhanced photo-gene therapy towards glioma. Bioact Mater. 2022;13:286–99. Yin C, Wang G, Zhang Q, Li Z, Dong T, Li Q, et al. Ultrasound nanodroplets loaded with Siglec-G siRNA and Fe3O4 activate macrophages and enhance phagocytosis for immunotherapy of triple-negative breast cancer. J Nanobiotechnology. 2024;22:773. Wang Z. MSCs biomimetic ultrasonic phase change nanoparticles promotes cardiac functional recovery after acute myocardial infarction. 2025; Wang Q, Lang Z, Yin G, Zheng P, Lv H, Fang H, et al. Analysis on the involvement of phosphoglycerate mutase 1 in the aerobic glycolysis of melanoma cells. Int J Biol Macromol. 2024;283:137898. Yang G-J, Tao F, Zhong H-J, Yang C, Chen J. Targeting PGAM1 in cancer: An emerging therapeutic opportunity. Eur J Med Chem. 2022;244:114798. Zhang W, Gong C, Chen Z, Li M, Li Y, Gao J. Tumor microenvironment-activated cancer cell membrane-liposome hybrid nanoparticle-mediated synergistic metabolic therapy and chemotherapy for non-small cell lung cancer. J Nanobiotechnology. 2021;19:339. He K, Tao F, Lu Y, Fang M, Huang H, Zhou Y. The Role of HK2 in Tumorigenesis and Development: Potential for Targeted Therapy with Natural Products. Int J Med Sci. 2025;22:790–805. Garcia SN, Guedes RC, Marques MM. Unlocking the Potential of HK2 in Cancer Metabolism and Therapeutics. Curr Med Chem. 2020;26:7285–322. Zhu D, Lu Y, Yang S, Hu T, Tan C, Liang R, et al. PAD4 Inhibitor‐Functionalized Layered Double Hydroxide Nanosheets for Synergistic Sonodynamic Therapy/Immunotherapy Of Tumor Metastasis. Adv Sci. 2024;11:2401064. Ji P, Sun W, Zhang S, Xing Y, Wang C, Wei M, et al. Modular Hydrogel Vaccine for Programmable and Coordinate Elicitation of Cancer Immunotherapy. Adv Sci. 2023;10:2301789. Yang Y, Huang J, Liu M, Qiu Y, Chen Q, Zhao T, et al. Emerging Sonodynamic Therapy‐Based Nanomedicines for Cancer Immunotherapy. Adv Sci. 2023;10:2204365. Zheng X, Shi Y, Tang D, Xiao H, Shang K, Zhou X, et al. Near‐Infrared‐II Nanoparticles for Vascular Normalization Combined with Immune Checkpoint Blockade via Photodynamic Immunotherapy Inhibit Uveal Melanoma Growth and Metastasis. Adv Sci. 2023;10:2206932. Wan J, Zhang X, Tang D, Liu T, Xiao H. Biodegradable NIR‐II Pseudo Conjugate Polymeric Nanoparticles Amplify Photodynamic Immunotherapy via Alleviation of Tumor Hypoxia and Tumor‐Associated Macrophage Reprogramming. Adv Mater. 2023;35:2209799. Spicer CD, Jumeaux C, Gupta B, Stevens MM. Peptide and protein nanoparticle conjugates: versatile platforms for biomedical applications. Chem Soc Rev. 2018;47:3574–620. Lu Z, Bai S, Jiang Y, Wu S, Xu D, Zhang J, et al. Amplifying Dendritic Cell Activation by Bioinspired Nanometal Organic Frameworks for Synergistic Sonoimmunotherapy. Small. 2022;18:2203952. Scheme 1 Scheme 1 is available in the Supplementary Files section. Supplementary Figure Supplementary Figure 1 is not available with this version Additional Declarations No competing interests reported. Supplementary Files floatimage1.png Scheme 1 Diagram of the functions of si-Ce6@tLyP-1 NPs. Following systemic administration, si-Ce6@tLyP-1 NPs actively accumulated in melanoma tissue through tLyP-1 peptide-mediated tumor targeting. Upon ultrasound irradiation, the nanoparticles executed a dual therapeutic action. On hand, ultrasound-triggered release of Ce6 generated cytotoxic ROS, inducing immunogenic cell death (ICD) in tumor cells. On the other hand, concurrent ultrasound-enhanced siRNA delivery achieved FTO gene silencing, suppressing tumor glycolysis to alleviate lactate accumulation post SDT. This metabolic intervention synergistically reversed immunosuppression by promoting dendritic cell maturation and cytotoxic T lymphocyte infiltration. Cite Share Download PDF Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 16 Aug, 2025 Reviews received at journal 16 Aug, 2025 Reviews received at journal 15 Aug, 2025 Reviews received at journal 14 Aug, 2025 Reviewers agreed at journal 13 Aug, 2025 Reviews received at journal 08 Aug, 2025 Reviewers agreed at journal 07 Aug, 2025 Reviews received at journal 07 Aug, 2025 Reviewers agreed at journal 07 Aug, 2025 Reviewers agreed at journal 07 Aug, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers agreed at journal 05 Aug, 2025 Reviewers invited by journal 05 Aug, 2025 Editor assigned by journal 01 Aug, 2025 Submission checks completed at journal 01 Aug, 2025 First submitted to journal 30 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7251971","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":496731410,"identity":"d4d51944-8403-4305-ab1c-845bd7f4c1c1","order_by":0,"name":"Xintong Li","email":"","orcid":"","institution":"The Second Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xintong","middleName":"","lastName":"Li","suffix":""},{"id":496731411,"identity":"4d92931f-8843-4b68-a241-b74b98585e4b","order_by":1,"name":"Weinan Sun","email":"","orcid":"","institution":"The Second Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weinan","middleName":"","lastName":"Sun","suffix":""},{"id":496731412,"identity":"2a8d0a0f-9771-4fc2-8a45-a5b0a14344a5","order_by":2,"name":"Weidong Yu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weidong","middleName":"","lastName":"Yu","suffix":""},{"id":496731413,"identity":"8f638333-746c-4834-a297-e67df600407a","order_by":3,"name":"Zhuo Wang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhuo","middleName":"","lastName":"Wang","suffix":""},{"id":496731414,"identity":"d8c0bf95-7478-4b1c-b17c-5f0357938717","order_by":4,"name":"Ping Sun","email":"","orcid":"","institution":"The Second Affiliated Hospital of Harbin Medical 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Leng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYFAC5gYgYQNisTEwNoCFDAhoAStLI13LYRK0GNxubHxc8Ou8nG7/AbbHlTu2JTawN2+TYKi5g1OL5JyDzcYz+24bm91IYDc8e+Z2YgPPsTIJhmPPcGrhl0hsk+btuZ247QYDm2RjG1CLRI6ZBGPDYZxa2CQS23/z9pyr33b+AFSL/Bv8WkC2MPP8OJBgdiABZgsPfi0gv0jzNiQbbruR2CbZeOa2cRtPWrFFwjHcWgxuNx/8zPPHTt7s/OFjko07bsv2sx/eeONDDW4tDBJAzNgGYkEjhQ1EJODWANHC8AefilEwCkbBKBjxAACUxVnCP29NQwAAAABJRU5ErkJggg==","orcid":"","institution":"The Second Affiliated Hospital of Harbin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaoping","middleName":"","lastName":"Leng","suffix":""}],"badges":[],"createdAt":"2025-07-30 10:53:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7251971/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7251971/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-025-03872-3","type":"published","date":"2025-11-26T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88574900,"identity":"1621f12b-7255-4db7-8c3b-adaca9dfa6e9","added_by":"auto","created_at":"2025-08-08 01:16:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":265108,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of si-Ce6@tLyp-1-NPs\u003c/p\u003e\n\u003cp\u003e(A) Appearance of si-Ce6@tLyp-1-NPs (created with BioRender.com.) (B) Transmission electron microscopy (TEM) images of si-Ce6@tLyp-1-NPs Size bar = 600 nm. (C) Graphs of the particle size and zeta potential of Ce6@tLyp-1-NPs and si-Ce6@tLyp-1-NPs. (D) Size variations of Size variations of si-Ce6@tLyp-1-NPs in PBS and DMEM + 10% FBS at 1, 3, 5, and 7 days. (E) UV-Vis absorption spectra of free Ce6,NPs,si-Ce6@tLyp-1-NPs. (F) Agarose gel e resis of the siRNA carrying capacity of si-Ce6@tLyp-1-NPs. (G) Fluorescence microscope image of the FAM siRNA and DiI labaled Ce6@tLyp-1-NPs. Scale bar = 10 µm. (H) Intracellular co-localization of the FAM siRNA and DiI labaled [email protected] bar = 50 µm. (I) FAM siRNA loading efficiency was analyzed by flow cytometry.Data are presented as the mean ± SD, n = 3\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7251971/v1/0ca7d8f5b0d4b42aad309f0b.png"},{"id":88574897,"identity":"d1050f12-84df-4c3a-8ea9-8b7ef3c31808","added_by":"auto","created_at":"2025-08-08 01:16:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":500707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e cell targeting, penetration efficiency of si-Ce6@tLyp-1-NPs\u003c/p\u003e\n\u003cp\u003e(A) Viability of B16-F10 cells treated with different concentrations of si-Ce6@tLyp-NPs. (B) CLSM images of B16-F10 cells treated with si-Ce6@NPs and si-Ce6@tLyp-1-NPs. (C) FCM results of intracellular uptake in B16-F10 cells treated with DiI labeled si-Ce6@NPs and si-Ce6@tLyp-1-NPs. (D) Analyzed the number of Dil-positive cells. (E) CLSM results of the infiltration of tumor cell spheres treated with si-Ce6@NPs and si-Ce6@tLyp-1-NPs\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7251971/v1/43b9663d11faf804146a219f.png"},{"id":88574896,"identity":"37bbee15-491a-4cfe-9a84-0c5db6d3f43e","added_by":"auto","created_at":"2025-08-08 01:16:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":558345,"visible":true,"origin":"","legend":"\u003cp\u003eGene silencing and glycolytic regulation of si-Ce6@tLyp-1-NPs\u003c/p\u003e\n\u003cp\u003e(A) Effect of UTMD \u003cem\u003ein vitro\u003c/em\u003e (B) CEUS mode, n = 3 (C) Schematic illustration of the B16F10 cells treated with si-Ce6@tLyP-1NPs combined with US (D) CLSM observation of the localization of FAM-siRNA in B16-F10 cells after transfection with free siRNA, si-Ce6@NPs+US, si-Ce6@tLyP-1NPs +US. The scale was 10 μm, and Phalloidin was the cytoskeleton. DAPI is the nucleus. (E) The relative mRNA expression of FTO in different group of B16F10 (F, G) The protein expression of FTO in different group of B16F10. (H) Volcano plot of DEGs. (I) Heat map of genes involved in the glycolytic pathway. (J) Representative pathways enriched in the identified genes as determined by GSEA. (K, L) The mRNA expression levels of HK2 and PGAM1 in the control group and B16F10 cells treated with different methods. (M) Representative western blot images of HK2 and PGAM1 expression in control and treated B16F10 cell groups. (N, O) Quantitative analysis of HK2 and PGAM1 protein expression. (P) Lactate levels in cell culture medium were measured.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7251971/v1/178504a1710a16365efe9fa6.png"},{"id":88574908,"identity":"4c438f2a-8ea8-43db-9301-94109eb313c6","added_by":"auto","created_at":"2025-08-08 01:16:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":720931,"visible":true,"origin":"","legend":"\u003cp\u003esi-Ce6@tLyp-1-NPs induce tumor cells ICD and activate immune cells \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(A-C) CLSM images, mean DCFH-DA fluorescence intensity, CRT and HMGB1 expression of B16-F10 cells after different treatments. (D, E) Quantitative analysis of intracellular reactive oxygen species production and CRT expression in tumor cells. (F) ELISA analysis of extracellular HMGB1 content. (G) The maturation ratio of BMDCs co-incubated with different treatments of B16F10 tumor cells. (H) The IFN-γ expression levels in CD8+ T cells after ex vivo coculture assay. (I) Schematic of co-culturing differentially-treated tumor cells with BMDCs and spleen lymphocytes. (J) Flow cytometry analysis of BMDCs co-incubated with different treatments of B16F10 tumor cells. (K) Flow cytometry analysis of IFN-γ expression levels in CD8+ T cells after co-incubated with different treatments of B16F10 tumor cells. (L, M) Annexin V-FITC/PI assay was used to analyze the apoptosis of B16-F10 cells after different treatments. (N) Confocal microscopy was used to observe the Calcein AM/PI staining of B16-F10 cells after various treatments (scale = 50µm).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7251971/v1/a4ef475cdadccfba280eb3f5.png"},{"id":88574901,"identity":"528bd365-4d49-40b7-aad3-89535473caea","added_by":"auto","created_at":"2025-08-08 01:16:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":396038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e targeting and penetration of si-Ce6@tLyp-1-NPs\u003c/p\u003e\n\u003cp\u003e(A) \u003cem\u003eIn vivo\u003c/em\u003e fluorescence images of mice treated at different time points DiR-si-Ce6@NPs and DiR-si-Ce6@tLyp-1-NPs. (B) Ex vivo fluorescence images of major organs and tumors of mice were collected after 12 hours. (C) Quantitative analysis of fluorescence intensity of major organs and tumors \u003cem\u003ein vitro\u003c/em\u003e. (D) Frozen sections of tumor tissues were stained 12h after injection of DiI-si-Ce6@NPs and DiI-si-Ce6@tLyp-1-NPs. (scale = 50µm)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7251971/v1/c9418eac5ae267096f128231.png"},{"id":88575408,"identity":"0aef0bf3-d658-441e-b548-5680617a2a62","added_by":"auto","created_at":"2025-08-08 01:24:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":949503,"visible":true,"origin":"","legend":"\u003cp\u003eThe antitumor effect of si-Ce6@tLyp-1-NPs \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of the \u003cem\u003ein vivo\u003c/em\u003e treatment algorithm (created by BioRender.com). (B) Digital photographs of different groups of tumors (n=5). (C) Curves of tumor volume with time in different groups. (D) H\u0026amp;E staining images, FTO, Ki67 IHC images, and Tunel images of tumor sections from different groups (scale: 50μm). (E) Tumor weight at day 15 in different groups. (F) FTO area (%) in different groups. (G) Ki-67 area (%) in different groups. (H) Quantitative analysis of Tunel positive area in different groups.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7251971/v1/da6282d2a9ac20d5c951e0ff.png"},{"id":88575405,"identity":"21755d97-3e14-425b-8e08-ae1e722a2ea7","added_by":"auto","created_at":"2025-08-08 01:24:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":404484,"visible":true,"origin":"","legend":"\u003cp\u003esi-Ce6@tLyp-1-NPs \u003cem\u003ein vivo\u003c/em\u003e remodeling the Immune Microenvironment\u003c/p\u003e\n\u003cp\u003e(A) Schematic design of the \u003cem\u003ein vivo\u003c/em\u003e flow cytometry experiment (created by BioRender.com). (B)Immunofluorescence staining images of CRT in different groups. (C) Immunofluorescence staining images of HMGB1 in different groups. (D) Flow cytometry analysis of DCs. (E-G) proportion of DCs, CD4/CD8 T cells in tumor tissues. (H) Flow cytometry analysis of CD4/CD8 T cells. (I) Flow cytometry analysis of CD8 + IFN-γ + T cells in tumor tissues. (J-L) Proportions of CD8 + IFN-γ + T cells in tumor tissue, DCs (CD80 +CD86+) and CD8+T in the spleen. (M, N) Flow cytometry analysis of DCs and CD8 +T cells in the spleen.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7251971/v1/29102fd79c72353e00c4aeb2.png"},{"id":97179350,"identity":"68876891-2764-49c0-9e9b-b05a7ee676c3","added_by":"auto","created_at":"2025-12-01 16:14:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5080652,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7251971/v1/5741ea9b-9059-448a-ba90-71a7d67bc7e6.pdf"},{"id":88574895,"identity":"4f296445-aa42-4e9b-bb8b-1ef6c55a747e","added_by":"auto","created_at":"2025-08-08 01:16:23","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":675187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e Diagram of the functions of si-Ce6@tLyP-1 NPs. Following systemic administration, si-Ce6@tLyP-1 NPs actively accumulated in melanoma tissue through tLyP-1 peptide-mediated tumor targeting. Upon ultrasound irradiation, the nanoparticles executed a dual therapeutic action. On hand, ultrasound-triggered release of Ce6 generated cytotoxic ROS, inducing immunogenic cell death (ICD) in tumor cells. On the other hand, concurrent ultrasound-enhanced siRNA delivery achieved FTO gene silencing, suppressing tumor glycolysis to alleviate lactate accumulation post SDT. This metabolic intervention synergistically reversed immunosuppression by promoting dendritic cell maturation and cytotoxic T lymphocyte infiltration.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7251971/v1/5d20f8c0b27572a1389eff2f.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Peptide-modified phase-transition nanoparticles co-deliver FTO siRNA and Ce6 for sonodynamic metabolism-immunotherapy of melanoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMelanoma is the most malignant and aggressive form of skin cancer which accounting for up to 90% of all skin cancer deaths [1\u0026ndash;3]. Although melanoma can be treated clinically by surgery, traditional chemotherapy and immunotherapy, it is still plagued by drug resistance and recurrence. Therefore, it is essential to seek an effective treatment for melanoma [4,5]. Photodynamic therapy (PDT) has been clinically recognized for the treatment of melanoma, which has attracted great attention [6,7]. Due to the poor laser penetration and the absorption of light by melanin in melanoma, PDT has limited therapeutic effect [8]. Sonodynamic therapy can overcome the above shortcomings of photodynamic therapy. Under low-intensity ultrasound (US) irradiation, sonosensitizer combineing with oxygen can produce excess ROS which induce apoptosis of tumor cells [9,10]. Besides, ROS can also trigger the immunogenic cell death (ICD) of tumor cells and release damage‐associated molecular patterns such as calreticulin (CRT) and high‐mobility group box 1 (HMGB1) [11,12]. ICD can induce the maturation of DCs and activate T lymphocytes, increasing the infiltration of immune cells in the tumor microenvironment, thereby enhancing the immunogenicity of cancer and improving the therapeutic effect [13,14].\u003c/p\u003e\u003cp\u003eHowever, current studies have found that the therapeutic effect of SDT is still not ideal, which may be due to the aggravation of hypoxia of tumor tissue during sonodynamic therapy, leading to enhanced glycolysis of tumor cells and further increasing the accumulation of lactate [15,16]. The production and release of lactate adversely affect immune responses by impairing cytotoxic T cell function and inhibiting DC differentiation and maturation [17]. Recent studies have suggested that fat mass and obesity-associated (FTO) can regulate glycolytic metabolism in a variety of tumor cells and increase the expression of glycolytic genes such as HK1, GLUT1, PKM and so on [18]. In the meanwhile, evidence indicates that the absence of FTO can enhence tumor-infliltrting T cells and removes the metabolic barrier for T cells activation. The combination of FTO inhibitors with other immunotherapies are effective strategies to improve adaptive immune responses \u003csup\u003e[19]\u003c/sup\u003e. Therefore, it is reasonable to assume that the inhibition of FTO is able to greatly enhance the effect of sonodynamic therapy. On the one hand, inhibition of FTO can regulate the glycolysis of tumor cells [19] and thus alleviate the metabolic disorder of immune cell activation after sonodynamic therapy. On the other hand, it can enhance the killing effect of immune cells [19] and finally achieve sonodynamic -immune-metabolism synergistic therapy.\u003c/p\u003e\u003cp\u003eRNA interference therapy is in the spotlight in recent years. Small interfering Rnas can be precisely regulated. Small interfering RNA (siRNA) has specific gene silencing ability that can precisely knock out the target gene [20]. However, its short half-life, poor bioavailability and easy clearance \u003cem\u003ein vivo\u003c/em\u003e limit the clinical application of siRNA [21]. With the approval of coronavirus disease 2019 (COVID-19) mRNA vaccines, lipid nanoparticles have attracted more and more attention as a promising RNA delivery vector [22\u0026ndash;24]. Furthermore, as a drug delivery vehicle, lipid-based nanoparticles can efficiently carry fat-soluble drugs, which improves drug stability and bioavailability \u003cem\u003ein vivo\u003c/em\u003e [25,26]. Therefore, we propose that lipid-based nanoparticles carrying FTO siRNA and the fat-soluble sonosensitizer Chlorin e6 (Ce6) can be used for effective co-delivery of therapeutic agents.\u003c/p\u003e\u003cp\u003eDespite so-called enhanced permeability and retention (EPR) effect existing can make nanomedicine passively target to accumulate in tumor tissue, the delivery efficiency of this process remains very poor [27]. Currently, the surface of lipid-based nanoparticles can be modified by a variety of biomolecules to target the tumor site actively and specifically [28,29]. TLyp-1 (amino acid sequence CGNKRTR) peptide designed specifically binding to the neuropilin-1 (NRP-1) overexpressed on the surface of B16-F10 cells, possess the capabilities of tumor targeting and tumor penetration activities, so that the lipid-based nanoparticles whose surface modificated with it can active target and accumulate in tumor sites [30\u0026ndash;32].\u003c/p\u003e\u003cp\u003eIn addition, the local controlled targeted release of drugs at the tumor site is also an urgent application challenge to be solved. Ultrasound-targeted microbubble destruction (UTMD) technology has been increasingly applied to accurately control the release of drugs and genes at tumor sites to improve the efficacy of various therapies [30,33]. Under the irradiation of low-power focused ultrasound (LIFU), the phase-change material as a core of Lipid nanoparticles can produce UTMD effect to promote the targeted delivery and release of drugs/genes [34].\u003c/p\u003e\u003cp\u003eIn this study, we proposed and constructed a lipid nanoparticle modified by tLyp-1 peptide with an inner core containing PFP (a phase-change material), sonosensitizer Ce6 and FTO siRNA were encapsulating together for the combination therapy of melanoma (designated as si-Ce6@tLyP-1 NPs). The tLyP-1 peptide enables the nanoparticles to target and penetrate deep into the melanoma, with the PFP underwent a liquve-gas phase transition under the irradiation of ultrasound, releasing siRNA and Ce6. Ce6, as a sonosensitizer, has significant advantages in sonodynamic therapy (SDT). In response to ultrasound, Ce6 can efficiently produces ROS to kill tumor cells and instigates ICD to induce DCs maturation and activate T lymphocytes [6]. FTO siRNA transfects into tumor cells to regulate glycolysis thereby reducing the production of lactate and contacting metabolic inhibition, further promoting the effector states of T cells. As a result, the anti-tumor immunity of nanomedicine was enhanced and synergistic anti-tumor effects were achieved (Scheme1).\u003c/p\u003e"},{"header":"2. Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Preparation and characterization of si-Ce6@tLyP-1 NPs\u003c/h2\u003e\n \u003cp\u003eThe nanoparticles of Ce6@tLyp-1-NPs were prepared by filming-rehydration method and the acoustic vibration method with encapsulated Ce6. FTO siRNA were loading with the nanoparticles by electrostatic adsorption, forming si-Ce6@tLyP-1-NPs \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. The structure of si-Ce6@tLyP-1 NPs was characterized by transmission electron microscopy (TEM), showing that the nanoparticles were spherical and dispersed uniformly \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e. Based on DLS measurements, the average size of Ce6@tLyp-1-NPs and si-Ce6@tLyp-1-NPs were 271.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60 nm and 294.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.17 nm. The zeta potential of Ce6@tLyp-1-NPs and si-Ce6@tLyp-1-NPs were35.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97 mV and \u0026minus;\u0026thinsp;31.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 mV \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cstrong\u003e)\u003c/strong\u003e. It was indicated that the particle size of nanoparticles increased slightly and the surface transform from a positive charge to a negative charge after siRNA adsorption, suggesting FTO siRNA loading onto the nanoparticles successfully. Excellent physiological stability is a prerequisite for the \u003cem\u003ein vivo\u003c/em\u003e application of nanopreparations, so we verified the stability of si-Ce6@tLyp-1-NPs by observing its particle size and appearance. The results showed that there was no significant change in particle size in PBS and 1640 medium with 10% FBS revealing a good seven-day stability of the nanoparticles \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, S1\u003cstrong\u003e)\u003c/strong\u003e. The UV/Vis spectra showed that si-Ce6@tLyp-1-NPs were aligned with free CE6, with two absorption peaks presenting at 400 and 660nm, which confirmed the successful encapsulation of Ce6 \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cstrong\u003e)\u003c/strong\u003e. A standard curve of Ce6 was measure and constructed by UV/Vis spectra, and the encapsulation efficiency (EE%) of Ce6 in si-Ce6@tLyp-1-NPs were 76.20% \u003cstrong\u003e(Figure S2)\u003c/strong\u003e. To verify the SDT effect of si-Ce6@tLyp-1-NPs singlet oxygen sensor green (SOSG) probe was used as a probe to examine the ability of si-Ce6@tLyp-1-NPs to generate cytotoxic singlet oxygen \u003cstrong\u003e(Figure S3)\u003c/strong\u003e. The results showed that after ultrasonic treatment, an absorption peak at 525nm was detected by the fluorescence spectrophotometer. The absorption intensity gradually increased with the extension of ultrasonic irradiation time, indicating that the singlet oxygen produced gradually increased with the prolongation of irradiation time. To investigate the loading ability of Ce6@tLyp-1-NPs to complex the siRNA, we performed agarose gel experiments. With increasing Ce6@tLyp-1-NPs mass, the fluorescence intensity of the free siRNA bands in the supernatants after centrifugation gradually decreased. When the mass ratio of siRNA to nanoparticles reached 1:8, the siRNA in the supernatant completely disappeared \u003cstrong\u003e(Figure. 1F)\u003c/strong\u003e, indicating the FTO siRNA was totally loaded by Ce6@tLyp-1-NPs. Therefore, the ratio between siRNA and Ce6@tLyp-1-NPs was determined to be 1:8. After determining the ratio of nanoparticles to siRNA, to further verify the efficiency of nanoparticles carrying siRNA, FAM-labeled siRNA was incubated with DiI-labeled nanoparticles detected by confocal microscopy and flow cytometry. Confocal laser scanning microscopy (CLSM) images showed an overlap of the fluorescence of siFTO with the luminescence of Ce6@tLyp-1-NPs, confirming the successful encapsulation (Figure. 1G). In addition, the obtained double-labeled nanoparticles were incubated with B16-F10. Confocal images showed that FAM and Dil fluorescence were scattered around the nucleus and co-localized with each other, validating the efficient co-delivery of siRNA and the nanoparticles \u003cstrong\u003e(Figure. 1H)\u003c/strong\u003e. Flow cytometry showed the loading rate of nanoparticles to siRNA is 99.4% when the mass ratio of siRNA to nanoparticles reached 1:8 (Figure. 1I).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. \u003cem\u003eIn vitro\u003c/em\u003e cellular uptake and tumor spheroid penetration efficiency\u003c/h2\u003e\n \u003cp\u003eBefore validating the \u003cem\u003ein vitro\u003c/em\u003e therapeutic effects of the nanoparticles, the biocompatibility of the nanoparticles was tested to ensure their subsequent safe application. After co-culturing B16-F10 cells at different concentrations of si-Ce6@tLyp-1-NPs for 24 hours, cell viability was assessed by CCK-8 assay. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, the cell viability remained above 90% at concentrations of 1.0mg/ml, which indicated the low cytotoxicity of the nanoparticles.\u003c/p\u003e\n \u003cp\u003eThe ability of si-Ce6@tLyp-1-NPs targeted to B16-F10 cells was next evaluated by confocal laser scanning microscopy and flow cytometry. B16-F10 cells were incubated with DiI labeled si-Ce6@tLyp-1-NPs and si-Ce6@NPs at different times (0.5h, 1h, 2h, and 4h). CLSM revealed that the DiI labeled si-Ce6@tLyp-1-NPs emitting red fluorescence clearly began to accumulate around the cell membrane after 1h, the aggregation increased significantly at 2h, and a large number of nanoparticles had infiltrated the cytoplasm at 4h. However, the above phenomenon was not observed in the B16-F10 cells co-incubated with DiI labeled si-Ce6@NPs, and only less red fluorescence was observed around the cell membrane after 4h \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e. Furthermore, we assessed the efficiency of si-Ce6@tLyp-1-NPs uptake by B16-F10 cells at the same time points by flow cytometry. We discovered that the number of DiI-positive cells in the si-Ce6@tLyp-1-NPs group reached 99% at 4h, which was much higher than that in the si-Ce6@NPs group. The results were consistent with the confocal microscopy observations \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, D\u003cstrong\u003e)\u003c/strong\u003e. Therefore, these findings confirmed that tLyp-1 peptide-modified nanoparticles possess high targeting specificity toward B16-F10 cells, which is conducive to efficient gene and drug delivery. The tLyp-1 peptide not only has tumor cell targeting ability, but also has strong tumor tissue penetration. Therefore, we used melanoma cell spheres to verify its ability of penetration. CLSM observation showed that after 4 hours of co-incubation with Dil-labeled si-Ce6@tLyp-1-NPs, red fluorescence was distributed throughout the spheres and deep into the tumor core, while Dil-labeled si-Ce6@NPs nanoparticles only marginally bindind to tumor cell spheres \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE\u003cstrong\u003e)\u003c/strong\u003e. In summary, si-Ce6@tLyp-1-NPs acquired good tumor-targeted penetration capacity, thereby increasing the deep tissue delivery of drugs and genes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. si-Ce6@tLyp-1NPs gene transfection efficiency assay\u003c/h2\u003e\n \u003cp\u003eIn order to prove whether si-Ce6@tLyp-1NPs could cause liquid\u0026ndash;gas phase transition under LIFU irradiation, we observed the NPs by optical microscopy showing that the nanoparticles expanded and transitioned into microbubbles after 3min of irradiation \u003cstrong\u003e(Figure S4)\u003c/strong\u003e. Additionally, we examined the UTMD ability of the nanoparticles \u003cem\u003ein vitro\u003c/em\u003e by using ultrasound (US) mode and contrast-enhanced ultrasound (CEUS) mode. The results showed that the ultrasound signal intensity of nanoparticles gradually increased with the extension of irradiation time and the increase of irradiation power \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, B\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003eAs an important gene regulatory molecule, siRNA can specifically silence the expression of target genes through RNA interference (RNAi). To prove the siRNA transfection ability of si-Ce6@tLyp-1-NPs \u003cem\u003ein vitro\u003c/em\u003e, the FAM si-Ce6@NPs and FAM si-Ce6@tLyp-1-NPs were co-cultured with B16-F10 cells for 4h and then irradiated with ultrasound \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cstrong\u003e)\u003c/strong\u003e. The nanoparticles carrying FAM-labeled siRNA were used to verify the transfection efficiency of si-Ce6@tLyp-1-NPs combined with ultrasound irradiation. The cytoskeleton was stained with phalloidin to observed the cytoplasmic transfection of FAM siRNA by CLSM. More green fluorescence was observed in the cytoplasm of B16-F10 in the FAM si-Ce6@tLyp-1-NPs (+) group \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cstrong\u003e)\u003c/strong\u003e. At 24 h after transfection, the mRNA level of FTO in the cells was detected by qRT-PCR. The results showed that the expression level of FTO protein in B16-F10 cells in the si-Ce6@tLyp-1-NPs (+) group was significantly lower than other groups \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE\u003cstrong\u003e)\u003c/strong\u003e. Additionally, 48 hours after transfection, we extracted the protein of B16-F10 cells and detected the intracellular FTO level by western blot. Consistent with the qRT-PCR results, it showed that the expression level of FTO protein in B16-F10 cells treated with si-Ce6@tLyp-1-NPs (+) was significantly decreased \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF, G\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003eAll the results showed that targeted nanoparticles combined with ultrasound irradiation could significantly promote siRNA transfection into the cytoplasm, and the transfection efficiency of siRNA was significantly higher than that of the non-targeted nanoparticles combined with ultrasound irradiation group and siRNA treatment alone group. si-Ce6@tLyp-1-NPs combined with ultrasound can effectively inhibit the expression of FTO gene machinery protein in B16-F10 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. si-Ce6@tLyp-1NPs mediated regulation of glycolytic metabolism\u003c/h2\u003e\n \u003cp\u003eTo further investigate the effect of FTO inhibition on cellular glycolysis, we used siFTO to knock down FTO expression in B16-F10 cells and performed RNA-seq. Transcriptome analysis showed that 2562 genes were up-regulated and 2525 genes were down-regulated with FTO knockdown in melanoma cells \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH\u003cstrong\u003e)\u003c/strong\u003e. Among them, 14 genes related to the glycolytic pathway were down-regulated, and the downregulation of PGAM1 was consistent with the results of Liu et al [18] \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI\u003cstrong\u003e)\u003c/strong\u003e. Phosphoglycerate mutase 1 (PGAM1) is also a key glycolytic enzyme, which can regulate the rate of glycolysis. Studies have shown that PGAM1 is involved in aerobic glycolysis in melanoma and indicates poor prognosis in melanoma [35,36]. Moreover, study had shown that inhibition of glycolysis by PGAM1 knockdown combined with chemotherapy can synergistically treat non-small cell lung cancer with high glycolysis [37]. In addition, the hexokinase 2 (HK2) gene, which plays an important role in glycolysis, was also significantly down-regulated. The hexokinase (HK) protein family is one of the core regulators of the glycolytic pathway. HK2 is highly expressed in cancer cells and plays an important role in the \u0026quot;Warburg effect\u0026quot; (metabolic reprogramming from oxidative phosphorylation to glycolysis) [38]. HK2 inhibition leads to a significant reduction in glycolysis [39]. For Gene set enrichment analysis (GSEA), we predefined gene ranks and utilized the R package clusterProfiler as well as from Molecular Signatures Database (DOI: 10.1093 / bioinformatics/btr260, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.gsea-msigdb.org/gsea/downloads.jsp\u003c/span\u003e\u003c/span\u003e) to download the c2. Cp. Kegg. V7.4. Symbols. The GMT Ensemble to evaluate glycolysis related pathways and molecular mechanisms. Results showed that FTO was associated with glycolytic pathway \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eJ\u003cstrong\u003e).\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTherefore, inhibition the expression of FTO can decrease the expression of key genes in the glycolytic pathway and down-regulate the level of cell glycolysis. We examined the expression levels of HK2 and PGAM1 in the glycolytic pathway. The mRNA level of HK2 and PGAM1 in B16-F10 cells treated with Ce6@tLyp-1-NPs (+)、si-Ce6@NPs(+) and si-Ce6@tLyp-1-NPs(+) was detected by qRT-PCR. The results showed that the mRNA level of HK2 and PGAM1 in B16-F10 cells treated with si-Ce6@tLyp-1-NPs(+) was significantly lower than that in the control group and Ce6@tLyp-1-NPs(+) \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eK, L\u003cstrong\u003e)\u003c/strong\u003e. And the protein expression of HK2 and PGAM1 in B16-F10 cells treated with Ce6@tLyp-1-NPs (+), si-Ce6@NPs (+) and si-Ce6@tLyp-1-NPs (+) was detected by WB. The results also showed that the expressions of HK2 and PGAM1 protein in B16-F10 cells treated with si-Ce6@tLyp-1-NPs (+) were significantly down-regulated in B16-F10 cells after si-Ce6@tLyp-1-NPs (+) treatment \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eM-O\u003cstrong\u003e)\u003c/strong\u003e. To evaluate the inhibitory effect of FTO knockdown on glycolysis in melanoma cells, we collected and analyzed the supernatants of cell cultures following different treatments. Compared with the other groups, the lactate concentration in si-Ce6@tLyp-1-NPs (+) groups decreased significantly \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eP\u003cstrong\u003e)\u003c/strong\u003e. Thus, the glycolytic pathway is inhibited after effective silencing of the FTO gene, reducing the consequent production of lactate. In conclusion, our results showed that UTMD combined with targeted nanoparticles had a high intracellular transfection efficiency and the expression of FTO was effectively inhibited in B16-F10 cells. FTO inhibition can further lead to the down-regulation of HK2, PGAM1 and other glycolytic pathway genes, thereby inhibiting glycolysis of tumor cells and reducing the production of lactate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. si-Ce6@tLyp-1-NPs -Elicited ICD \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eUpon ultrasound irradiation, Ce6 could efficiently produce ROS to trigger immunogenic cell death, which resulted in the release of damp-associated molecular patterns (DAMPs), such as CRT and HMGB [40 ,41]. The ROS generation in B16-F10 cells receiving different treatments was observed by CLSM. Under ultrasound exposure, the green fluorescence intensity of ROS probe 2\u0026apos;,7\u0026apos;- Dichlorodihydrofluorescein diacetate (DCFH‐DA) in the group of B16-F10 cells treated with si-Ce6@tLyp-1-NPs increased significantly \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. It was demonstrated that si-Ce6@tLyp-1-NPs\u0026thinsp;+\u0026thinsp;US could efficiently induce the production and accumulation of ROS in cells, which in turn produced ICD. Increased CRT expression on the surface of dying cells during SDT can act an \u0026quot;eat me\u0026quot; signal, thereby stimulate DCs maturation and activation of specific effector T cells, enhancing the host anti-tumor immune response [42,43]. We examined CRT and HMGB1 expression in different treated tumor cells by CLSM (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Consistent with the trend of ROS production \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cstrong\u003e)\u003c/strong\u003e, CRT exposure on the surface of B16-F10 cells in the si-Ce6@tLyp-1-NPs (+) group was significantly increased compared with PBS and other treatment groups \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cstrong\u003e)\u003c/strong\u003e. However, in the si-Ce6@NPs (+) group, the localization of HMGB1 in the nucleus was reduced because a small amount of HMGB1 was transferred from the nucleus to the cytoplasm and the cell membrane, while in the si-Ce6@tLyp-1-NPs (+) group, most of HMGB1 was released to the outside of the cells and only a small amount of green fluorescence was distributed in the cytoplasm and the cell membrane. Meanwhile, the CLSM images showed the green fluorescence of HMGB1 was located inside the nucleus and had not been released to the outside of the cells in the PBS, US (+) and si-Ce6@tLyp-1-NPs groups. Research has confirmed that HMGB1 can be transferred from the nucleus to the cytoplasm and finally released outside the cell when ICD occurs in tumor cells, and host antitumor immunity can be induced [44]. We detected the extracellular release of HMGB1 from B16-F10 cells by ELISA, and the results showed that the extracellular HMGB1 content was significantly increased in the si-Ce6@tLyp-1-NPs group \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF\u003cstrong\u003e)\u003c/strong\u003e. The results indicated that ICD triggered by SDT could induce massive release of HMGB1 outside the cell. The consistent trend of ROS production and DAMP release confirmed that si-Ce6@tLyp-1-NPs was highly effective in triggering ICD, which in turn enhanced anti-tumor immune response.\u003c/p\u003e\n \u003cp\u003eThe reduction of lactate production can relieve the activation barrier of immune cells, enhance the infiltration and killing ability of immune cells. As antigen-presenting cells, dendritic cells (DCs) can activate anti-tumor immune responses and induce T cell activation. We incubated different treatments of B16F10 tumor cells with Bone Marrow-Derived Dendritic Cells (BMDCs) \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI\u003cstrong\u003e)\u003c/strong\u003e. Flow cytometry was used to analyze the proportion of mature DCs after co-culture with B16F10 cells, showing that after co-cultured with si-Ce6@tLyp-1-NPs(+) pretreated B16F10 cells the proportion of mature DC could reach 30.9%, which was significantly higher than that of the control group, si-Ce6@tLyp-1-NPs, si-Ce6@NPs(+) and Ce6@tLyp-1-NPs(+)groups \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG, J\u003cstrong\u003e)\u003c/strong\u003e. It was confirmed that FTO inhibition combined with sonodynamic therapy can increase the maturation of DCs cells. At the same time, we co-cultured B16F10 cells after different treatments with spleen single fine suspensions and examined the proportion of CD8\u0026thinsp;+\u0026thinsp;T cells positive for IFN-\u0026gamma; \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI\u003cstrong\u003e)\u003c/strong\u003e. Flow cytometry staining analysis showed that CD8 T cells in the group that were co-incubated with si-Ce6@tLyp-1-NPs (+) pretreated B16F10 cells expressed higher levels of activated IFN-\u0026gamma; \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH, K\u003cstrong\u003e)\u003c/strong\u003e. Therefore, we demonstrated that inhibiting FTO expression in tumor cells combined with sonodynamic therapy could further enhance T cell activation and mediated cytotoxicity. All these findings showed that inhibition of FTO is able to reprogram tumor cell metabolism to relieve the obstacle of immune cell activation caused by lactate accumulation.\u003c/p\u003e\n \u003cp\u003eAnd then we examined the apoptosis of B16-F10 cells after different treatments. It was shown that si-Ce6@tLyp-1-NPs (+) group induced the highest apoptosis rate, which could reach 85.4%, indicating a strong therapeutic effect \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eL, M\u003cstrong\u003e)\u003c/strong\u003e. The results of live and dead cell staining also fully proved that si-Ce6@tLyp-1-NPs (+) had a good combined anti-tumor effect \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eN\u003cstrong\u003e)\u003c/strong\u003e. The cell killing effect of si@tLyp-1-NPs (+) group was small, indicating that knockdown of FTO alone has a small killing effect on tumor cells. The higher cell survival rate of the si-Ce6@NPs (+) untargeted group verified that si-Ce6@tLyp-1-NPs could enter more tumor cells and produce ROS in response to ultrasound stimulation to kill tumor cells with high efficiency. A large number of dead cells also existed in the Ce6@tLyp-1-NPs (+) group, indicating that the targeted nanoparticles had good SDT efficacy. Finally, the dead cells with red fluorescence in the si-Ce6@tLyp-1-NPs (+) group were the most, which verified that si-Ce6@tLyp-1-NPs had a good combined treatment effect.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Targeting and penetration capacity detection of si-Ce6@tLyp-1-NPs \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e experiments showed that si-Ce6@tLyp-1-NPs could produce ROS to induce ICD in tumor cells under ultrasound irradiation. UTMD could promote the efficient delivery of siFTO, inhibiting glycolysis and reducing lactic acid accumulation, thereby increasing immune cell infiltration and improving immunosuppressive TME. Next, we will discuss its anti-tumor effects \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eNanodrugs for systemic administration can cross the tumor vascular endothelial space and accumulate in solid tumors. Peptide-modified nanoparticle surfaces can further improve pharmacokinetics, enhance tissue targeting, and promote cell and tissue penetration, thereby avoiding off-target effects and improving therapeutic efficacy[45]. Therefore, we evaluated the \u003cem\u003ein vivo\u003c/em\u003e targeting and penetration of tLyp-1 peptide-modified NPs in melanoma using an animal imaging system (IVIS Lumina XRMS, PerkinElmer, USA). DiR-labeled si-Ce6@NPs and si-Ce6@tLyp-1-NPs were injected via the tail vein of a melanoma mouse model, and the accumulation of nanoparticles at the melanoma site during different time points (4h,8h,12h and 24h) was observed by small animal \u003cem\u003ein vivo\u003c/em\u003e imaging. The \u003cem\u003ein vivo\u003c/em\u003e imaging results indicated si-Ce6@tLyp-1-NPs gradually accumulated in the tumor site and reached the peak at 12 hours. In addition, a large number of nanoparticles were still retained in the tumor tissue at 24h. However, the fluorescence intensity of si-Ce6@NPs was significantly lower than that of si-Ce6@tLyp-1-NPs at all time points \u003cstrong\u003e(Fig.\u0026nbsp;5A)\u003c/strong\u003e. Therefore, we performed organ ex vivo imaging of tumors and other major organs 12 h after intravenous injection of nanoparticles to investigate the \u003cem\u003ein vivo\u003c/em\u003e distribution of nanoparticles. As shown in \u003cstrong\u003eFig.\u0026nbsp;5B\u003c/strong\u003e, a significant accumulation of si-Ce6@tLyp-1-NPs at the tumor site was observed, but not in the si-Ce6@NPs group. These results demonstrated that tLyp-1 modification can significantly increase the tumor targeting of the nanoparticles and improve the efficiency of gene and drug delivery to the tumor site. In addition, according to the above results, we can confirm that drug accumulation at the tumor site reaches a peak 12h after injection of nanoparticles.\u003c/p\u003e\n \u003cp\u003eTwelve hours after injection of DiI-labeled si-Ce6@NPs and si-Ce6@tLyp-1-NPs, the mice were sacrificed and tumor tissues were collected for section staining to observe the distribution of si-Ce6@NPs and si-Ce6@tLyp-1-NPs in the tumor site \u003cstrong\u003e(Fig.\u0026nbsp;5C)\u003c/strong\u003e. It can be observed that the red fluorescence in the si-Ce6@tLyp-1-NPs group enters more into the deep part of the tumor tissue, while only a small amount of red fluorescence is observed in the si-Ce6@NPs group at the edge of the tumor \u003cstrong\u003e(Fig.\u0026nbsp;5D)\u003c/strong\u003e. This demonstrated that tLyp-1 also enhanced the tumor tissue penetration of the nanoparticles, allowing the drug to penetrate deeper into the tumor tissue to enhance the sonodynamic therapy and FTO inhibition effect. Since hypoxia in the core of the tumor leads to increased cell glycolysis, the well tumor permeability of si-Ce6@tLyp-1-NPs can further inhibit cell glycolysis in the core of the tumor and reduce the accumulation of lactic acid to improve tumor immunity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. \u003cem\u003eIn vivo\u003c/em\u003e anti-tumor activity\u003c/h2\u003e\n \u003cp\u003eTo investigated the anti-tumor effect of different treatments \u003cem\u003ein vivo\u003c/em\u003e, we used the subcutaneous tumor model in C57 mice established by B16-F10 cells. When the tumor volume reached about 100 mm\u003csup\u003e3\u003c/sup\u003e, the mice were randomly divided into 7 groups with 5 mice in each group, and the drugs were administered every other day for a total of 3 times. In addition, some groups were subjected to ultrasound irradiation 12 h after administration, tumor size was monitored every 3 days, and euthanized on the 15th day \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. To analyze the antitumor effect, all tumor samples were collected and weighed. Treatment with si-Ce6@tLyp-1-NPs (+) significantly inhibited tumor growth compared with the other groups \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e. The results showed that the tumor volume of the si@tLyp-1-NPs (+) group was slightly reduced. Although the tumor weight of the Ce6@tLyp-1-NPs (+) group was significantly reduced, the smallest tumor volume and tumor weight could be observed in the si-Ce6@tLyp-1-NPs (+) group, which proved that FTO inhibition combined with sonodynamic therapy could achieve better anti-tumor treatment effect \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, E\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003eHematoxylin-eosin (H\u0026amp;E), FTO, Ki67 immunohistochemical staining and terminal deoxynucleotidyl transferase-mediated dutP-biotin Nick end labeling (TUNEL) staining were performed to evaluate the effects of nanomedicine on tumor necrosis, FTO inhibition, proliferation and apoptosis. H\u0026amp;E stained tumor sections showed extensive karyopyknosis, karyorhexis, and karyolysis in the si-Ce6@tLyp-1-NPs (+) group, confirming that the si-Ce6@tLyp-1-NPs (+) group had the most severe tumor tissue necrosis \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cstrong\u003e)\u003c/strong\u003e. The results showed that FTO inhibition combined with SDT treatment had the most significant tumor killing effect. Immunohistochemical staining showed that the expression of FTO was significantly reduced in G4 and G7 groups, indicating that the peptide-modified tumor tissue-targeted nanoparticles had a high FTO inhibition efficiency after ultrasound irradiation \u003cem\u003ein vivo\u003c/em\u003e \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF\u003cstrong\u003e)\u003c/strong\u003e. The results of Ki-67 staining \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG\u003cstrong\u003e)\u003c/strong\u003e and Tunel staining \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH\u003cstrong\u003e)\u003c/strong\u003e also confirmed that the tumor cell proliferation was significantly decreased and the level of tumor cell apoptosis was significantly increased in the si-Ce6@t-NPs (+) group. The above results fully demonstrated the excellent anti-tumor efficacy of si-Ce6@tNPs (+).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. \u003cem\u003eIn vivo\u003c/em\u003e immune response activation.\u003c/h2\u003e\n \u003cp\u003eSDT can produce a stronger ICD effect by producing ROS. During ICD, tumor cells produce a series of DAMPs, including surface-exposed calreticulin (CRT) and passively released high mobility group box 1 (HMGB1), which together trigger the phagocytosis of dying tumor cells by dendritic cells (DCs) [46]. Our previous results showed that si-Ce6@tLyp-1-NPs can efficiently induce tumor cell ICD \u003cem\u003ein vitro\u003c/em\u003e. We next examined the effect of si-Ce6@tLyp-1-NPs on reshaping the immune microenvironment \u003cem\u003ein vivo\u003c/em\u003e \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. The ability of si-Ce6@tLyp-1-NPs\u0026thinsp;+\u0026thinsp;US treatment to induce ICD \u003cem\u003ein vivo\u003c/em\u003e to trigger anti-tumor immune responses was assessed by measuring CRT and HMGB1 expression in tumor tissues. The results of immunofluorescence staining showed that CRT was almost not expressed in the control group and si-Ce6@tLyp-1-NPs, little expressed in the si-Ce6@NPs (+) group, more expressed in the Ce6@tLyp-1-NPs (+) group, and the most expressed in si-Ce6@tLyp-1-NPs. The results of HMGB1 immunofluorescence staining showed that the si-Ce6@tLyp-1-NPs (+) group had a significant release compared with other groups. These discharges indicated that the non-targeted nanoparticles can only enter the tissue in a small amount and cause a small amount of tumor cell ICD. Ce6@tLyp-1-NPs can efficiently enter the tumor tissue and cause ICD, while si-Ce6@tLyp-1-NPs (+) group can induce a stronger ICD effect, increased CRT exposure, and a large release of HMGB1 \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB, C\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003eThe occurrence of ICD will improve the immunogenicity of tumor microenvironment. Recruitment of immune cells to the TME is directly related to antitumor immune responses. Therefore, to determine whether sonodynamic therapy combined with FTO inhibition could further enhance the anti-tumor immune response by enhancing immune cell infiltration and activation, we further investigated the proportion of immune cells in the tumor tissue. Firstly, we examined the proportion of DCs (CD11c\u0026thinsp;+\u0026thinsp;CD80\u0026thinsp;+\u0026thinsp;CD86 +) cells in the tumor tissues. Flow cytometry showed that the percentage of mature dendritic cells infiltrating the tumor in the si-Ce6@tLyp-1-NPs\u0026thinsp;+\u0026thinsp;US group was significantly higher than that in the other experimental groups, which could reach 60.3% \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD, E\u003cstrong\u003e)\u003c/strong\u003e. In addition, we evaluated the levels of CD4\u0026thinsp;+\u0026thinsp;T cells and CD8\u0026thinsp;+\u0026thinsp;T cells in the tumor tissue. The results showed that the ratio of CD4\u0026thinsp;+\u0026thinsp;T cells and CD8\u0026thinsp;+\u0026thinsp;T cells in the tumor tissues of the si-Ce6@tLyp-1-NPs (+) group was also increased, with the proportion of CD4\u0026thinsp;+\u0026thinsp;T cells increasing by 23% and the proportion of CD8\u0026thinsp;+\u0026thinsp;T cells increasing by 13%, demonstrating that si-Ce6@tLyp-1-NPs (+) could intensely promoted the differentiation of CD8\u0026thinsp;+\u0026thinsp;T cells in the tumor microenvironment, increasing the ratio of CD8\u0026thinsp;+\u0026thinsp;T cells / CD4\u0026thinsp;+\u0026thinsp;T cells \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF-H\u003cstrong\u003e)\u003c/strong\u003e. Compared with Ce6@tLyp-1NPs (+) group, si-Ce6@tLyp-1-NPs\u0026thinsp;+\u0026thinsp;US group significantly promoted the infiltration and maturation of tumor DCs and T lymphocytes, proving that FTO inhibition further enhanced the anti-tumor immune response after SDT treatment. To test whether FTO inhibition could enhance CD8\u0026thinsp;+\u0026thinsp;T cell-mediated cytotoxicity, we measured the proportion of CTL (CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;IFN-\u0026gamma;+) in tumor tissues. The percentage of tumor tissue CTL (CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;IFN-\u0026gamma;+) in si-Ce6@tLyp-1-NPs (+) group was the highest, which was 6.17 fold higher than control in the tumor \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eI ,\u003cstrong\u003eJ)\u003c/strong\u003e. These results indicate that FTO inhibition combined with SDT treatment significantly increased the number of immune cell infiltrates in the tumor tissue and clearly enhanced the antitumor immune response. In addition, the proportion of DCs and CD8\u0026thinsp;+\u0026thinsp;T cells in the spleen of the mice was measured. The results showed that the percentages of DCs and CD8\u0026thinsp;+\u0026thinsp;T cells in spleen were also significantly higher than those in other treatment groups, reaching 51.3% and 42.4%, respectively \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eK-N\u003cstrong\u003e)\u003c/strong\u003e. Therefore, si-Ce6@tLyp-1-NPs (+) can not only improve the immune microenvironment in tumor, but also activated the systemic immune response to enhance the systemic immune response and obtain better anti-tumor immunity.\u003c/p\u003e\n \u003cp\u003eThe above results confirmed our hypothesis that compared with SDT treatment alone, FTO combined with SDT stimulated a potent antitumor immune response and significantly increased the rate of immune cell tumor tissue infiltration. The \u0026ldquo;cold tumor\u0026rdquo; can be transformed into a \u0026ldquo;hot tumor\u0026rdquo; with a better anti-tumor immune response, resulting in a more powerful tumor treatment effect.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9. \u003cem\u003eIn vivo\u003c/em\u003e biosafety evaluation\u003c/h2\u003e\n \u003cp\u003eFinally, in order to evaluate the \u003cem\u003ein vivo\u003c/em\u003e biosafety of si-Ce6@tLyp-1-NPs. Healthy mice receiving si-Ce6@tLyp-1-NPs intravenous injection were euthanized on the scheduled date, blood samples were collected for biochemical examination and blood routine analysis, and major organs were collected for H\u0026amp;E staining. As shown in \u003cstrong\u003eFigure S5\u003c/strong\u003e, compared with the control group, the biochemical indexes and blood routine analysis showed no abnormalities at 1, 3, 7, 14, and 28 d after the tail vein injection of si-Ce6@tLyp-1-NPs. As shown in \u003cstrong\u003eFigure S6\u003c/strong\u003e, each tissue section showed no significant histopathological changes. These results indicate that si-Ce6@tLyp-1-NPs has good biosafety, and showed potential clinical applications as novel agents for cancer therapy.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eIn conclusion, this study successfully constructed si-Ce6@tLyp-1-NPs and demonstrated that FTO inhibition can effectively enhance the therapeutic effect of SDT in melanoma. The tLyp-1 peptide-modified nanoparticles have superior tumor targeting ability and penetration, which could achieve efficient co-delivery of sonosensitizer Ce6 and FTO siRNA. Moreover, PFP could respond to ultrasound irradiation to further enhance the delivery efficiency of siRNA, and Ce6 could produce ROS under ultrasound irradiation, thereby inducing ICD in tumor cells. Notably, downregulation of FTO gene expression by si-Ce6@tLyp-1-NPs effectively inhibited melanoma cell glycolysis and relieved lactic acid-mediated immune cell suppression. Furthermore, the tumor immunosuppressive environment was improved, and the infiltration of DCs and T lymphocytes was significantly increased. FTO inhibition can significantly enhance the cytotoxicity of CD8\u0026thinsp;+\u0026thinsp;T cells, resulting in a strong cellular immune response and significantly enhanced tumor therapeutic efficacy. In conclusion, FTO inhibition combined with SDT treatment significantly inhibited the growth of melanoma, providing a potential new strategy for sonodynamic immuno-metabolic synergistic therapy.\u003c/p\u003e"},{"header":"4. Materials and methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Materials\u003c/h2\u003e\u003cp\u003e1,2-distearoyl-sn-glycero-3-phosphocho-line (DSPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n (DSPE), 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) and DSPE-PEG-tLyp-1, were purchased from Xi\u0026rsquo;an Ruixi Biological Technology Co., Ltd. (Xi\u0026rsquo;an, China). FTO siRNA sense strand, 5\u0026prime;-GUCAGACCUUCCUAAAGCUTT-3\u0026prime;, negative control siRNA, FAM labeled negative control siRNA were purchased from GenePharma (Shanghai, China). The Chlorin e6 (Ce6) and Singlet Oxygen Sensor Green (SOSG) was purchased from Meilunbio (Dalian, China). The Calcein/PI Cell Viability/Cytotoxicity Assay Kit, Annexin V-Alexa Fluor 647/PI apoptosis detection kit, Reactive Oxygen Species Assay Kit and DiI Iodide were obtained from Beyotime (Shanghai, China). The lactic acid assay kit was purchased from Jiancheng Bioengineering Institute (Nanjing, China). HMGB1 antibody and HK2 antibody were purchased from Immunoway (US). CRT antibody, FTO antibody, PGAM1 antibody were obtained from Proteintech (Wuhan, China). FITC anti-mouse CD11c, APC anti-mouse CD80, APC anti-mouse CD80, PE anti-mouse CD86, APC anti-mouse CD3, PE anti-mouse CD4, FITC anti-mouse CD8a for flow cytometry were obtained from Biolegend (USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Cell culture and animal model\u003c/h2\u003e\u003cp\u003eB16-F10 (RRID:CVCL_0159) tumor cells were procured from Procell Life Science \u0026amp; Technology Co., Ltd. (Wuhan, China), culturing in RPMI-1640 with 10% FBS and 1% penicillin‒streptomycin at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\u003cp\u003eAll C57BL/6 mice, aged 6\u0026ndash;8 weeks, were purchased from the Changsheng biotechnology (Liaoning, China). To establish the melanoma cancer model, B16F10 cells at a density of 1 \u0026times; 106 cells/mL in 0.1 mL PBS were injected to the dorsal flank of C57BL/6 mice. All animal e ments were approved by the Animal Management and Use Ethics Committee of the Second Affiliated Hospital of Harbin Medical University.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Preparation of si-Ce6@tLyp-1-NPs\u003c/h2\u003e\u003cp\u003eWe employed traditional rotary evaporation and a one-step emulsion method to prepare [email protected] mass of hybrid lipid (6 mg DSPC, 2 mg DOTAP, 2 mg DSPE-PEG- tLyp-1) and 0.5mg Ce6 was dissolved in 4 mL trichloromethane (CHCl 3). Then transferred the resultant mixture into a round-bottom flask, and homogeneous lipid film were formed through rotary evaporation in a water bath at 40\u0026deg;C. Added 4 mL of DEPC-water to rehydrate the films, and then 200 \u0026micro;L of PFP was added to the mixture. Finally, the mixture was sonicated (60 W, 6 min, 5 s on, 5 s off) in an ice bath, and the nanoparticles were washed by twice centrifugation (8000 rpm, 3 min) to obtain the cationic nanoparticles. The obtained Ce6@tLyp-1-NPs was then mixed with FTO siRNA to form si-Ce6@tLyp-1-NPs by electrostatic adsorption. DiR/DiI was added to the mixed lipid solution to synthesize the fluorescently labeled si-Ce6@tLyp-1-NPs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.4. Characterization of si-Ce6@tLyp-1-NPs\u003c/h2\u003e\u003cp\u003eThe structure of the nanoparticles was observed by Transmission electron microscopy (Hitachi H-7600; Japan). The potential of si-Ce6@tLyp-1-NPs and the size of si-Ce6@tLyp-1-NPs at 0, 1, 3, and 7d after preparation were measured using a DLS detector from Malvern Instruments (ZEN3600; Malvern, U.K.). A UV spectrophotometer (UV-2600, Shimadzu, Japan) was used to obtain UV\u0026ndash;visible absorption spectra. Ce6 was dissolved in methanol and the absorbance of different concentrations of Ce6 solutions at 400nm was determined by a UV spectrophotometer (UV-2600, Shimadzu, Japan). The drug loading rate of Ce6 was calculated by constructing a linear correlation curve between the concentration of Ce6 and its absorption peak.\u003c/p\u003e\u003cp\u003eEncapsulation rate (%) = (weight of drugs in nanoparticles/initial weight of drugs) \u0026times; 100%\u003c/p\u003e\u003cp\u003eMix siRNA (NCsiRNA) and Ce6@tLyp-1-NPs at ratios of 1:2, 1:4, 1:6, 1:8, 1:10 (w: w) to a total volume of 50 \u0026micro;L. The mixture was incubated for 20 min at room temperature before centrifugation to obtain the supernatant. The mixture was incubated at room temperature for 20 min, followed by centrifugation to obtain the supernatant. The supernatant was mixed with loading buffer and loaded onto a 2% agarose gel for electrophoresis at 120 V for 20 min to observe the change in siRNA bands. FAM siRNA was incubated with DiI-labeled Ce6@tLyp-1-NPs drops at a mass ratio of 1:8 and then centrifuged to obtain si-Ce6@tLyp-1-NPs. Observing the fluorescence co-localization of siRNA and nanoparticles by fluorescence microscopy to determine the successful loading of siRNA was. B16-F10 cells were incubated with double-labeled nanoparticles, and the localization of cell, nanoparticles and siRNA was observed by CLMS. The FAM siRNA was incubated with Ce6@tLyp-1-NPs at a mass ratio of 1:8 and centrifuged to obtain si-Ce6@tLyp-1-NPs, and the ligation efficiency of the siRNA to Ce6@tLyp-1-NPs was determined by flow cytometry. The SOSG probe was added to si-Ce6@tLyp-1-NPs solution and irradiated with LIFU for 15s, 30s, 60s, 90s, 120s, 180s (pulse mode, interval 2s, power 3 W). ROS production was determined by observing the fluorescence spectra.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e4.5. Cellular uptake and penetration ability of nanoparticles \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eB16-F10 cells were seeded in the confocal dishes at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/ml. DiI labeled si-Ce6@tLyp-1-NPs and si-Ce6@NPs were incubated with the cells for 0.5, 1, 2, and 4h, respectively. The cells were washed twice with PBS and fixed with 4% paraformaldehyde. After staining B16-F10 cells with DAPI (10 \u0026micro;g/mL) for 15 min, the uptake of nanoparticles by B16-F10 cells was observed by CLSM. And B16-F10 cells were seeded in a twelve-well plate at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/ml. Culturing cells as in the CLSM experiment, the uptake efficiency of the nanoparticles was quantified by FCM after the cells were digested with trypsin. B16-F10 cells were seeded in ultra-low adsorption 6-well plates at a density of 3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well to form 3D tumor spheroids. Spheroids were co-cultured with nanoparticles labeled with DiI, staining the cell nucleus by Hoechst. The penetration of nanoparticles was detected by observing fluorescence within the spheres by CLSM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e4.6. \u003cem\u003eIn vitro\u003c/em\u003e cell safety\u003c/h2\u003e\u003cp\u003eB16-F10 cells were seeded in 96-well plates at a density of 3000 cells/well and cultured overnight. Different concentrations of si-Ce6@tLyp-1-NPs were added to each well, and cell viability was assessed 24h later using the CCK-8 assay. Add 100 \u0026micro;l of culture medium containing 10 \u0026micro;L of CCK-8 solution to each well and read the optical absorbance of each well at 450 nm using a microplate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e4.7. Intracellular ROS production\u003c/h2\u003e\u003cp\u003eThe DCFH-DA probe was used to determine 1O2 generation. The experiment was divided into five groups: PBS group, si-Ce6@tLyp-1-NPs group, si-Ce6@ NPs (+) group, Ce6@tLyp-1-NPs (+) group and si-Ce6@tLyp-1-NPs (+) group. Briefly, B16-F10 cells were incubated with nanoparticles for 4 h at 37\u0026deg;C. Cells were washed and incubated with DCFH-DA probe for 30 min and then sonicated the cells. Hoechst was added for staining of nucleus. The fluorescence was observed by CLSM and the results were quantified.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e4.8. Immunofluorescence staining of ICD markers \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eB16-F10 cells were seeded on confocal dishes overnight. Grouping and treating the cells as ROS assay experiment, 4% paraformaldehyde, Triton (0.3%) and blocking solution were added successively. After the blocking solution was removed, primary antibodies against calreticulin (CRT) and high mobility group b1 protein (HMGB1) diluted 1:400 were added and incubated overnight at 4\u0026deg;C. After washing three times with PBS, AbFluor 488 goat anti-rabbit IgG diluted in 1:500 dilution of PBS was added and incubated for 1 h at 37 \u0026deg; C in the dark. Nuclei were stained with DAPI for 5 min, and cells were visualized by CLSM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e4.9. \u003cem\u003eIn vitro\u003c/em\u003e phase transition ability assay\u003c/h2\u003e\u003cp\u003eTo evaluate the \u003cem\u003ein vitro\u003c/em\u003e phase transition ability of si-Ce6@tLyp-1-NPs under ultrasound irradiation, we constructed constructing a 3.5% agarose (w/v) gel model. si-Ce6@tLyp-1-NPs were exposed to different intensities of ultrasound (0, 1, 2, 3W/ cm\u0026sup2;, duty cycle 50%) for 1, 2, and 3 minutes, respectively. Subsequently, B-mode ultrasound and contrast-enhanced ultrasound (CEUS) imaging were performed to observe the liquid-gas phase transition process of the phased-transition nanoparticles. The ultrasound intensity value was quantitatively analyzed by software. Using ultrasound to irradiate si-Ce6@tLyp-1-NPs (power 3W/cm\u0026sup2;, time 3min), the morphology of the nanoparticles was observed by optical microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e4.10. Detection of siRNA transfection ability \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eB16-F10 was seeded in confocal dishes overnight. The cells were incubated with free FAM-siRNA, FAM-si-Ce6@NPs and FAM-si-Ce6@tLyp-1-NPs for 4h and then irradiated with ultrasound. Cells were fixed with 4% paraformaldehyde, staining the cytoskeleton with phalloidin ring and staining nucleu with DAPI. Finaly, the transfection efficiency of siRNA was observed by CLMS.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e4.11. RNA extraction and quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated from B16-F10 cells using the SPARKeasy Improved Tissue/Cell RNA Kit (Sparkjade, Shandong, China). First-strand cDNA was synthesized by reverse transcription using the PrimeScript\u0026trade; RT Reagent Kit (Takara Technologies, Shiga, Japan). For quantitative real-time PCR (qRT-PCR), the cDNA was amplified using TB Green\u0026reg; Premix Ex Taq\u0026trade; II (Takara Technologies, Shiga, Japan) in a real-time PCR system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e4.12. Western blot analysis\u003c/h2\u003e\u003cp\u003eTo further quantify the transfection efficiency of FTO siRNA, B16-F10 cells were incubated with Ce6@tLyp-1-NPs, si-Ce6@NPs and si-Ce6@tLyp-1-NPs and treated with ultrasound irradiation after 4h. Cells were harvested 48 hours later, and protein extracted from untreated B16-F10 cells served as a control. The expression of FTO was detected by Western blot. Cells were lysed in RIPA lysate. Total protein was quantified using the BCA Protein Assay kit and equilibrated prior to sample loading. Equal amounts of protein extracts were electrophoresed in SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was then blocked with 5% nonfat milk and incubated with FTO for primary antibody followed by secondary antibody. Finally, the band is captured by the Odyssey scanning system. Band intensities were quantified using the Image J program.\u003c/p\u003e\u003cp\u003eThe expression levels of HK2 and PGAM1 in each group were quantitatively analyzed by WB, and the changes of key enzymes in the glycolytic pathway after treatment with nanoparticles were detected.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e4.13. RNA Sequencing\u003c/h2\u003e\u003cp\u003eB16-F10 cells were seeded in 6-well plates (1 \u0026times; 10 \u003csup\u003e5\u003c/sup\u003e cells/well) overnight. FTO was knocked down in cells by Lipofectamine 2000 combined with FTO siRNA, and 24 h after transfection, total RNA was extracted using TRIzol reagent. A cDNA library was established for data processing. The quality of the library was checked by Agilent 2100 Bioanalyzer, and then the total concentration of the library and the effective concentration of the library were detected. The libraries containing different index sequences were then scaled according to the effective concentration of the library and the amount of data required for the library. The mixed libraries were unified to 2nM and, by alkali denaturation, single-stranded libraries were formed. After RNA extraction, closure and library construction, Next-Generation Sequencing (NGS) technology was used to perform Paired-end (PE) sequencing of these libraries based on Illumina sequencing platform. Clean reads were mapped to the reference genome with HISAT2. The distribution of Reads aligned to the genome was counted, and HTSeq statistics were used to calculate the Read Count value on each gene and the original expression level of the gene. Differentially expressed genes were screened and analyzed by R software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e4.14. Regulation of glycolysis by FTO inhibition\u003c/h2\u003e\u003cp\u003eLactic acid detection kit was used to detect the content of lactic acid in the cell supernatant of the experimental group and the control group, and glucose detection kit was used to detect the content of glucose.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e4.15. \u003cem\u003eIn vitro\u003c/em\u003e immune cell stimulation\u003c/h2\u003e\u003cp\u003eBone marrow-derived dendritic cells (BMDCs) were generated from 8\u0026ndash;10 week-old male C57BL/6 mice. All mice were first euthanized, and their femurs and tibias were dissected under sterile conditions. Adherent muscle tissue was carefully stripped away from the bones, and then rinsed with sterile PBS. The femurs and tibias were bisected with scissors to expose the marrow cavities. Using a syringe, the bone marrow was thoroughly flushed from the cavities with PBS, and the resulting cell suspension was filtered through a 200-mesh nylon mesh to eliminate debris and clumps. Following filtration, the cells were treated with red blood cell (RBC) lysate for 5 minutes, leaving a purified population of bone marrow cells. The isolated bone marrow cells were cultured in RPMI 1640 medium supplemented with 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 10% fetal bovine serum (FBS), and interleukin-10 (IL-10)\u0026mdash;cytokines essential for driving BMDC differentiation. The immature BMDCs were harvested for subsequent experiments after 6 days of incubation. To evaluate how tumor-derived factors affect DC maturation, BMDCs were incubated with tumor cell culture supernatants after different treatments for 24 hours. Following this stimulation, flow cytometry was used to analyze the surface expression of CD80 and CD86\u0026mdash;key co-stimulatory molecules\u0026mdash;on CD11c⁺ cells.\u003c/p\u003e\u003cp\u003eThe spleens of the mice were aseptically removed after euthanasia and placed in precooled PBS. The spleens were shredded with forceps, ground into a single-cell suspension with a syringe piston, collagenase IV was added, and incubated at 37 \u0026deg; C for 30 min. The cell suspension was slowly spread on the top layer of lymphocyte separation medium and centrifuged at 2000 rpm for 20 minutes. The lymphocyte layer was aspirated and washed twice with PBS. The obtained cells were incubated with the supernatants of tumor cells after different treated for 24h. Finally, the cells were stained for surface markers (CD3⁺, CD8⁺) and intracellular IFN-γ⁺ using fluorophore-conjugated antibodies, and flow cytometry was used to quantify the proportion of CD3⁺CD8⁺IFN-γ⁺ cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e4.16. \u003cem\u003eIn vitro\u003c/em\u003e cell killing ability\u003c/h2\u003e\u003cp\u003eB16-F10 cells were seeded in six well plate. The experiment was divided into seven groups: control, US (+), si-Ce6@tLyp-1-NPs, si@tLyp-1-NPs (+), si-Ce6@NPs (+), Ce6@tLyp-1-NPs (+), si-Ce6@tLyp-1-NPs (+). The nanoparticles were incubated for 4h, and Ce6 was added at the same concentration (2.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M). After incubation, cells were exposed to ultrasound and cultured for another 24 h. Double staining was performed using the Annexin V-FITC apoptosis detection kit according to the manufacturer's instructions. Flow cytometry was used to analyze the apoptosis rate.\u003c/p\u003e\u003cp\u003eB16-F10 cells were seeded in confocal dishes, and cells were treated in the same manner as in the apoptosis experiments. The cells were stained with calcein acetoxymethyl ester (AM) (2\u0026times;10 \u0026minus;\u0026thinsp;6 m) and PI (2\u0026times;10 \u0026minus;\u0026thinsp;6 m) and visualized by CLSM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e4.17. \u003cem\u003eIn vivo\u003c/em\u003e distribution\u003c/h2\u003e\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e imaging study of the nanoparticle distribution was performed on melanoma mice intravenously injected with DiR-labeled si-Ce6@NPs and si-Ce6@tLyp-1-NPs. Mice were anesthetized with isoflurane and photographed under the IVIS spectral imaging system at 2, 6, 12, and 24 h after injection. To study tissue distribution, mice were sacrificed 12h after injection, with tumors and major organs collecting for \u003cem\u003ein vitro\u003c/em\u003e imaging.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e tissue penetration of the nanoparticles was tested in tumor-bearing mice by intravenous injection of DiI-labeled si-Ce6@NPs and si-Ce6@tLyp-1-NPs. After 12 hours, tumors were harvested and cryosections were prepared with a cryomicrotome. Frozen sections were fixed with 4% paraformaldehyde, counterstained with DAPI and observed under a fluorescence microscope.4.16 \u003cem\u003eIn vivo\u003c/em\u003e antitumor effects.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e4.18. \u003cem\u003eIn vivo\u003c/em\u003e antitumor study\u003c/h2\u003e\u003cp\u003eTumor-bearing male C57BL/6 mice aged 6\u0026ndash;8 weeks were randomly divided into 7 groups (n\u0026thinsp;=\u0026thinsp;5) : control, US (+), si-Ce6@tLyp-1-NPs, si@tLyp-1-NPs (+), si-Ce6@NPs (+), Ce6@tLyp-1-NPs (+), si-Ce6@tLyp-1-NPs (+). The corresponding nanoparticles injected into mice via the tail vein. After 12 h, the tumor was exposed to ultrasound (3 W/cm\u003csup\u003e2\u003c/sup\u003e, 5 min), and the treatment was administered every two days for a total of three times. The tumors were monitored every 3 days. After 15 days, the mice were sacrificed, and the tumor embedded sections were collected for hematoxylin-eosin (H\u0026amp;E) staining, FTO and Ki-67 immunohistochemical staining, and terminal deoxynucleotidyl transferase-mediated dutP Nick end labeling (Tunel).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e4.19. Analysis of immune response \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo verify the improvement of immune microenvironment \u003cem\u003ein vivo\u003c/em\u003e, the tumor-bearing mice were divided into five groups: control; si-Ce6@tLyp-1-NPs; si-Ce6@NPs (+); Ce6@tLyp-1-NPs (+); si-Ce6@tLyp-1-NPs (+) group, treated with the same protocol to evaluate the antitumor effect. Immunofluorescence staining was used to observe the expression of CRT and HMGB1 in tumor tissues. The percentages of CD8\u0026thinsp;+\u0026thinsp;T cells, CD4\u0026thinsp;+\u0026thinsp;T cells and DCs cells in tumor tissues were detected by flow cytometry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\u003ch2\u003e4.20. \u003cem\u003eIn vivo\u003c/em\u003e biosafety assessment\u003c/h2\u003e\u003cp\u003eTo assess the biological safety of si-Ce6@tLyp-1-NPs, 25 healthy C57BL/6 mice were injected intravenously with 200\u0026micro;L si-Ce6@tLyp-1-NPs (1 mg/mL). Blood samples and main organs were collected on days 1, 3, 7, 14 and 28 for serum biochemical, blood routine tests and H\u0026amp;E staining. Five healthy mice were selected as the control group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\u003ch2\u003e4.21. Statistics\u003c/h2\u003e\u003cp\u003eAll data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). GraphPad Prism9.0 software was used for statistical analysis. t test was used for comparison between two groups, and analysis of variance (ANOVA) was used for comparison between multiple groups. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupp\u003c/strong\u003e\u003cstrong\u003el\u003c/strong\u003e\u003cstrong\u003eem\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003ent\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003ery\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting Information is available from the Wiley Online Library or from the author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.P.L., P.L., and Z.Y. conceived and designed the experiments, X.T.L., W.N.S., W.D.Y., J.F.C, H.D.L., Y.P.L., J.T.R., L.W., R.T., Y.P.W. and P.Z. carried out the experiments. X.T.L, Z.W. and P.S. analysed the data. X.T.L., P.S., X.P.L. and P.L. wrote and edited the manuscript. All authors agreed to be responsible for the content of the work.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics Approval and Consent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures conformed with the National Institutes of Health Guidelines for the Use of Laboratory Animals and were approved by the The Second Affiliated Hospital of Harbin Medical University Medical Ethics Committee (ethical approval number:YJSDW2024-127).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of China (Grant Nos. U22A20346).\u0026nbsp;\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEddy K, Chen S. Overcoming Immune Evasion in Melanoma. Int J Mol Sci. 2020;21:8984. \u003c/li\u003e\n\u003cli\u003eHossain SM, Eccles MR. Phenotype Switching and the Melanoma Microenvironment; Impact on Immunotherapy and Drug Resistance. Int J Mol Sci. 2023;24:1601. \u003c/li\u003e\n\u003cli\u003eGiammona A, De Vellis C, Crivaro E, Maresca L, Amoriello R, Ricci F, et al. Tumor-derived GLI1 promotes remodeling of the immune tumor microenvironment in melanoma. J Exp Clin Cancer Res. 2024;43:214. \u003c/li\u003e\n\u003cli\u003eArozarena I, Wellbrock C. Phenotype plasticity as enabler of melanoma progression and therapy resistance. Nat Rev Cancer. 2019;19:377\u0026ndash;91. \u003c/li\u003e\n\u003cli\u003eNaidoo C, Kruger CA, Abrahamse H. Photodynamic Therapy for Metastatic Melanoma Treatment: A Review. Technol Cancer Res Treat. 2018;17:1533033818791795. \u003c/li\u003e\n\u003cli\u003eYu L, Gao L, Liang B, Zhang L, Wu M, Liu J. Polymer-based nanodrugs enhance sonodynamic therapy through epigenetic reprogramming of the immunosuppressive tumor microenvironment. J Controlled Release. 2025;380:125\u0026ndash;37. \u003c/li\u003e\n\u003cli\u003eGeng S, Guo M, Zhan G, Shi D, Shi L, Gan L, et al. NIR-triggered ligand-presenting nanocarriers for enhancing synergistic photothermal-chemotherapy. J Controlled Release. 2023;353:229\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eFeng T, Tang Z, Karges J, Shen J, Jin C, Chen Y, et al. Exosome camouflaged coordination-assembled Iridium(III) photosensitizers for apoptosis-autophagy-ferroptosis induced combination therapy against melanoma. Biomaterials. 2023;301:122212. \u003c/li\u003e\n\u003cli\u003eLiang J, Qiao X, Qiu L, Xu H, Xiang H, Ding H, et al. Engineering Versatile Nanomedicines for Ultrasonic Tumor Immunotherapy. Adv Sci. 2024;11:2305392. \u003c/li\u003e\n\u003cli\u003eJiang Q, Qiao B, Lin X, Cao J, Zhang N, Guo H, et al. A hydrogen peroxide economizer for on-demand oxygen production-assisted robust sonodynamic immunotherapy. Theranostics. 2022;12:59\u0026ndash;75. \u003c/li\u003e\n\u003cli\u003eGuo X, Tu P, Zhu L, Cheng C, Jiang W, Du C, et al. Nanoenabled Tumor Energy Metabolism Disorder via Sonodynamic Therapy for Multidrug Resistance Reversal and Metastasis Inhibition. ACS Appl Mater Interfaces. 2023;15:309\u0026ndash;26. \u003c/li\u003e\n\u003cli\u003eHu X, Zhang M, Quan C, Ren S, Chen W, Wang J. ROS-responsive and triple-synergistic mitochondria-targeted polymer micelles for efficient induction of ICD in tumor therapeutics. Bioact Mater. 2024;36:490\u0026ndash;507. \u003c/li\u003e\n\u003cli\u003eKim J, Shim MK, Moon Y, Kim J, Cho H, Yun WS, et al. Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy. J Nanobiotechnology. 2024;22:109. \u003c/li\u003e\n\u003cli\u003eLi M, Liu Q, Xie S, Weng D, He J, Yang X, et al. Transformable Tumor Microenvironment‐Responsive Oxygen Vacancy‐Rich MnO\u003csub\u003e2\u003c/sub\u003e @Hydroxyapatite Nanospheres for Highly Efficient Cancer Sonodynamic Immunotherapy. Adv Sci. 2025;12:2414162. \u003c/li\u003e\n\u003cli\u003eQiao K, Luo C, Huang R, Xiang J, Pan Y, Zhang S, et al. Ultrasound Triggered Tumor Metabolism Suppressor Induces Tumor Starvation for Enhanced Sonodynamic Immunotherapy of Breast Cancer. Int J Nanomedicine. 2023;Volume 18:3801\u0026ndash;11. \u003c/li\u003e\n\u003cli\u003eChen Y, Li K, Du H, Yao Y, Xie D, Zhou Z. Breaking Barriers in Oncology: Harnessing Sonodynamic Therapy for Enhanced Tumor Metabolism Regulation. Small. 2025;2502323. \u003c/li\u003e\n\u003cli\u003eChen Z, Chen L, Ma Y, Liu Y, Zhang Q, Qin H, et al. Peptide‐Appended Nanosonosensitizers Targeting Tumor Glycolysis for Synergistic Sonodynamic\u0026ndash;Immunometabolic Therapy of Spinal‐Metastasized Tumors. Adv Mater. 2023;35:2304246. \u003c/li\u003e\n\u003cli\u003eWang F, Hu Y, Wang H, Hu P, Xiong H, Zeng Z, et al. LncRNA FTO-IT1 promotes glycolysis and progression of hepatocellular carcinoma through modulating FTO-mediated N6-methyladenosine modification on GLUT1 and PKM2. J Exp Clin Cancer Res. 2023;42:267. \u003c/li\u003e\n\u003cli\u003eLiu Y, Liang G, Xu H, Dong W, Dong Z, Qiu Z, et al. Tumors exploit FTO-mediated regulation of glycolytic metabolism to evade immune surveillance. Cell Metab. 2021;33:1221-1233.e11. \u003c/li\u003e\n\u003cli\u003eSun N, Lei Q, Wu M, Gao S, Yang Z, Lv X, et al. Metal-organic framework-mediated siRNA delivery and sonodynamic therapy for precisely triggering ferroptosis and augmenting ICD in osteosarcoma. Mater Today Bio. 2024;26:101053. \u003c/li\u003e\n\u003cli\u003eXu C, Liu W, Hu Y, Li W, Di W. Bioinspired tumor-homing nanoplatform for co-delivery of paclitaxel and siRNA-E7 to HPV-related cervical malignancies for synergistic therapy. Theranostics. 2020;10:3325\u0026ndash;39. \u003c/li\u003e\n\u003cli\u003eZong Y, Lin Y, Wei T, Cheng Q. Lipid Nanoparticle (LNP) Enables mRNA Delivery for Cancer Therapy. Adv Mater. 2023;35:2303261. \u003c/li\u003e\n\u003cli\u003eYan Y, Liu X-Y, Lu A, Wang X-Y, Jiang L-X, Wang J-C. Non-viral vectors for RNA delivery. J Controlled Release. 2022;342:241\u0026ndash;79. \u003c/li\u003e\n\u003cli\u003eCharbe NB, Amnerkar ND, Ramesh B, Tambuwala MM, Bakshi HA, Aljabali AAA, et al. Small interfering RNA for cancer treatment: overcoming hurdles in delivery. Acta Pharm Sin B. 2020;10:2075\u0026ndash;109. \u003c/li\u003e\n\u003cli\u003eLarge DE, Abdelmessih RG, Fink EA, Auguste DT. Liposome composition in drug delivery design, synthesis, characterization, and clinical application. Adv Drug Deliv Rev. 2021;176:113851. \u003c/li\u003e\n\u003cli\u003eYang M, Wang X, Peng M, Wang F, Hou S, Xing R, et al. Nanomaterials Enhanced Sonodynamic Therapy for Multiple Tumor Treatment. Nano-Micro Lett. 2025;17:157. \u003c/li\u003e\n\u003cli\u003eSun R, Xiang J, Zhou Q, Piao Y, Tang J, Shao S, et al. The tumor EPR effect for cancer drug delivery: Current status, limitations, and alternatives. Adv Drug Deliv Rev. 2022;191:114614. \u003c/li\u003e\n\u003cli\u003eIzci M, Maksoudian C, Manshian BB, Soenen SJ. The Use of Alternative Strategies for Enhanced Nanoparticle Delivery to Solid Tumors. Chem Rev. 2021;121:1746\u0026ndash;803. \u003c/li\u003e\n\u003cli\u003eAronson MR, Medina SH, Mitchell MJ. Peptide functionalized liposomes for receptor targeted cancer therapy. APL Bioeng. 2021;5:011501. \u003c/li\u003e\n\u003cli\u003eBai J, Duan J, Liu R, Du Y, Luo Q, Cui Y, et al. Engineered targeting tLyp-1 exosomes as gene therapy vectors for efficient delivery of siRNA into lung cancer cells. Asian J Pharm Sci. 2020;15:461\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eCheng C, Jiang W, Luo Y, Wan L, Guo X, Xie Z, et al. NIR Activated Multimodal Therapeutics Based on Metal\u0026ndash;Phenolic Networks‐Functionalized Nanoplatform for Combating against Multidrug Resistance and Metastasis. Small. 2023;19:2206174. \u003c/li\u003e\n\u003cli\u003eWang R, Wang X, Li J, Di L, Zhou J, Ding Y. Lipoprotein-biomimetic nanostructure enables tumor-targeted penetration delivery for enhanced photo-gene therapy towards glioma. Bioact Mater. 2022;13:286\u0026ndash;99. \u003c/li\u003e\n\u003cli\u003eYin C, Wang G, Zhang Q, Li Z, Dong T, Li Q, et al. Ultrasound nanodroplets loaded with Siglec-G siRNA and Fe3O4 activate macrophages and enhance phagocytosis for immunotherapy of triple-negative breast cancer. J Nanobiotechnology. 2024;22:773. \u003c/li\u003e\n\u003cli\u003eWang Z. MSCs biomimetic ultrasonic phase change nanoparticles promotes cardiac functional recovery after acute myocardial infarction. 2025; \u003c/li\u003e\n\u003cli\u003eWang Q, Lang Z, Yin G, Zheng P, Lv H, Fang H, et al. Analysis on the involvement of phosphoglycerate mutase 1 in the aerobic glycolysis of melanoma cells. Int J Biol Macromol. 2024;283:137898. \u003c/li\u003e\n\u003cli\u003eYang G-J, Tao F, Zhong H-J, Yang C, Chen J. Targeting PGAM1 in cancer: An emerging therapeutic opportunity. Eur J Med Chem. 2022;244:114798. \u003c/li\u003e\n\u003cli\u003eZhang W, Gong C, Chen Z, Li M, Li Y, Gao J. Tumor microenvironment-activated cancer cell membrane-liposome hybrid nanoparticle-mediated synergistic metabolic therapy and chemotherapy for non-small cell lung cancer. J Nanobiotechnology. 2021;19:339. \u003c/li\u003e\n\u003cli\u003eHe K, Tao F, Lu Y, Fang M, Huang H, Zhou Y. The Role of HK2 in Tumorigenesis and Development: Potential for Targeted Therapy with Natural Products. Int J Med Sci. 2025;22:790\u0026ndash;805. \u003c/li\u003e\n\u003cli\u003eGarcia SN, Guedes RC, Marques MM. Unlocking the Potential of HK2 in Cancer Metabolism and Therapeutics. Curr Med Chem. 2020;26:7285\u0026ndash;322. \u003c/li\u003e\n\u003cli\u003eZhu D, Lu Y, Yang S, Hu T, Tan C, Liang R, et al. PAD4 Inhibitor‐Functionalized Layered Double Hydroxide Nanosheets for Synergistic Sonodynamic Therapy/Immunotherapy Of Tumor Metastasis. Adv Sci. 2024;11:2401064. \u003c/li\u003e\n\u003cli\u003eJi P, Sun W, Zhang S, Xing Y, Wang C, Wei M, et al. Modular Hydrogel Vaccine for Programmable and Coordinate Elicitation of Cancer Immunotherapy. Adv Sci. 2023;10:2301789. \u003c/li\u003e\n\u003cli\u003eYang Y, Huang J, Liu M, Qiu Y, Chen Q, Zhao T, et al. Emerging Sonodynamic Therapy‐Based Nanomedicines for Cancer Immunotherapy. Adv Sci. 2023;10:2204365. \u003c/li\u003e\n\u003cli\u003eZheng X, Shi Y, Tang D, Xiao H, Shang K, Zhou X, et al. Near‐Infrared‐II Nanoparticles for Vascular Normalization Combined with Immune Checkpoint Blockade via Photodynamic Immunotherapy Inhibit Uveal Melanoma Growth and Metastasis. Adv Sci. 2023;10:2206932. \u003c/li\u003e\n\u003cli\u003eWan J, Zhang X, Tang D, Liu T, Xiao H. Biodegradable NIR‐II Pseudo Conjugate Polymeric Nanoparticles Amplify Photodynamic Immunotherapy via Alleviation of Tumor Hypoxia and Tumor‐Associated Macrophage Reprogramming. Adv Mater. 2023;35:2209799. \u003c/li\u003e\n\u003cli\u003eSpicer CD, Jumeaux C, Gupta B, Stevens MM. Peptide and protein nanoparticle conjugates: versatile platforms for biomedical applications. Chem Soc Rev. 2018;47:3574\u0026ndash;620. \u003c/li\u003e\n\u003cli\u003eLu Z, Bai S, Jiang Y, Wu S, Xu D, Zhang J, et al. Amplifying Dendritic Cell Activation by Bioinspired Nanometal Organic Frameworks for Synergistic Sonoimmunotherapy. Small. 2022;18:2203952. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e\n"},{"header":"Supplementary Figure","content":"Supplementary Figure 1 is not available with this version"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"melanoma, sonodynamic therapy, FTO, immunotherapy, glycolysis","lastPublishedDoi":"10.21203/rs.3.rs-7251971/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7251971/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eMelanoma, the most aggressive skin cancer, remains challenging to treat due to limited therapeutic options. Sonodynamic therapy (SDT) has emerged as a promising strategy for combating malignant tumors. However, the excessive accumulation of lactate in the tumor microenvironment after sonodynamic therapy limits the activation of immune cells, leading to the unsatisfactory therapeutic effect of SDT. FTO inhibition can effectively inhibit glycolysis of melanoma cells and relieve the obstacle of immune cell activation caused by lactic acid. FTO silencing in tumors eliminates can metabolic barriers to T cell activation and further enhances the antitumor effect of CD8\u0026thinsp;+\u0026thinsp;T cells. Combining FTO inhibition with SDT may enhance tumor cell elimination and remodel the immunosuppressive tumor immune microenvironment.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e\u003cp\u003eIn this study, tLyp-1 modified ultrasound phase-transforming nanoparticles loaded with sonosensitizer (Ce6) and FTO siRNA were constructed to achieve FTO inhibition and sonodynamic therapy. The tLyp-1 peptide modification facilitates efficient tumor targeting and enhances deep tissue penetration, therefore improving drug delivery efficacy. Ce6 produces ROS in response to ultrasound to induce ICD in tumor cells. At the same time, ultrasound promoted FTO siRNA transfection to inhibit B16-F10 cells glycolysis, which significantly increased the activation and infiltration of dendritic cells and T lymphocytes in the tumor microenvironment, effectively enhanced the therapeutic effect of SDT and inhibited tumor growth.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study demonstrated that si-Ce6@tLyP-1 NPs serve as a platform for targeted the tumor site and efficiently deliver si-FTO and Ce6. The realization of FTO inhibition combined with sonodynamic therapy provides an effective treatment strategy for the treatment of melanoma.\u003c/p\u003e","manuscriptTitle":"Peptide-modified phase-transition nanoparticles co-deliver FTO siRNA and Ce6 for sonodynamic metabolism-immunotherapy of melanoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 01:16:19","doi":"10.21203/rs.3.rs-7251971/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-17T01:10:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-16T19:03:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-15T17:36:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-14T17:32:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9314976012983013730461409270405947305","date":"2025-08-13T16:14:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-08T05:14:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68520165495857387073902651407708967657","date":"2025-08-08T00:55:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-07T16:05:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256548179302836650564159713580318339832","date":"2025-08-07T16:02:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290834891970835622931518140753039019947","date":"2025-08-07T11:33:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1422626878251634423210235050776225211","date":"2025-08-06T16:44:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"231769890470818682015004617542246440282","date":"2025-08-05T15:01:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-05T05:36:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-01T04:54:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-01T04:52:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-07-30T10:47:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cfb8a382-26cf-4020-adab-fa6bff7ff194","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:09:15+00:00","versionOfRecord":{"articleIdentity":"rs-7251971","link":"https://doi.org/10.1186/s12951-025-03872-3","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2025-11-26 15:57:42","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-08-08 01:16:19","video":"","vorDoi":"10.1186/s12951-025-03872-3","vorDoiUrl":"https://doi.org/10.1186/s12951-025-03872-3","workflowStages":[]},"version":"v1","identity":"rs-7251971","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7251971","identity":"rs-7251971","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}