Size-variable self-feedback nanomotors for glioblastoma therapy via mitochondrial mineralization

Size-variable self-feedback nanomotors for glioblastoma therapy via mitochondrial mineralization | 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 Article Size-variable self-feedback nanomotors for glioblastoma therapy via mitochondrial mineralization Chun Mao, Tiantian Chen, Yu Duan, Yingjie wang, Tiantian Liang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6451662/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Oct, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Developing targeted treatment strategies for glioblastoma (GBM) is of significant importance but remains highly challenging. Herein, we propose a novel size-variable self-feedback nanorobot system tailored for GBM treatment, leveraging the unique high-calcium microenvironment of GBM. These nanomotors consist of three main components: a self-feedback degradable lipid shell, a nanorobot core with motion ability, and the drug curcumin (inhibiting the efflux of Ca 2+ ). The lipid shell incorporates nitric oxide-releasing lipid (NOR) and NO-responsive degradable lipid (NOD). NOR is catalyzed by inducible nitric oxide synthase (iNOS) to release NO. NOD degrades in response to the self-released NO. The nanorobot core is composed of L-arginine (L-Arg) derivatives and zwitterionic monomers rich in carboxyl groups (facilitating Ca 2+ recruitment) (PAC NMs). Initially, the larger size-variable self-feedback nanomotors (~ 500 nm) can penetrate the blood-brain barrier through chemotaxis, driven by the high expression of iNOS in the GBM microenvironment. During chemotaxis, the self-feedback lipid shell gradually degrades as NO accumulates, releasing smaller PAC NMs (~ 50 nm). These smaller nanomotors target mitochondria, where they recruit Ca²⁺ to induce mitochondrial mineralization in conjunction with curcumin, ultimately leading to tumor cell death and inhibiting GBM progression. This work may provide a new strategy for the development of GBM-specific treatment methods. Biological sciences/Cancer/Cancer therapy/Targeted therapies Biological sciences/Biological techniques/Nanobiotechnology/Nanoparticles glioblastoma mitochondrial mineralization self-feedback nanomotors size-variable Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Glioblastoma (GBM) is one of the most aggressive and deadly brain tumors, classified by the World Health Organization as the most malignant glioma (Grade IV). [ 1 , 2 ] The standard treatment for GBM typically involves a combination of surgical resection followed by radiotherapy and/or chemotherapy. Temozolomide (TMZ) is currently the first-line chemotherapeutic agent for GBM treatment. [ 3 ] However, the infiltrative growth pattern and inherent heterogeneity of GBM, coupled with the side effects of TMZ treatment (including hematologic toxicity and thrombocytopenia associated with long-term and high-dose administration), have significantly limited the efficacy of this therapeutic strategy. Consequently, the median survival for GBM patients remains low at 14.6–20.5 months, with a 5-year survival rate of less than 10%. [ 4 – 6 ] Therefore, developing targeted therapeutic strategies specifically for GBM has emerged as a critical area of research. The unique microenvironment of GBM provides valuable insights for the design of targeted therapeutic strategies ( Table S1 ). GBM is characterized by an immunosuppressive tumor microenvironment, often referred to as a "cold" tumor, which has spurred researchers to actively explore novel immunotherapeutic approaches for GBM. [1a] These include tumor vaccines, [ 2 , 7 , 8 ] immune checkpoint inhibitors (ICIs), [ 9 ] and chimeric antigen receptor T-cell (CAR-T) therapies, [ 10 ] etc. However, the efficacy of immunotherapy is affected by the individual patient variability, leading to insufficient or excessive immune responses that limit its broad application. [ 11 ] In addition to immunotherapy, molecular targeted therapies have been developed to target cellular molecules that promote the proliferation and differentiation of GBM, such as anti-angiogenic therapy [ 12 , 13 ] and tyrosine kinase inhibitors. [ 14 , 15 ] However, their effectiveness is often limited by factors such as drug resistance in tumor cells and high recurrence rates following treatment. [ 16 ] Gene therapy represents another promising approach, targeting specific genes involved in the proliferation, migration, invasion, apoptosis, and angiogenesis of malignant glioma cells including gene editing corrections using CRISPR-Cas9, or suppressing immune-suppressive genes expressed in brain tumors to reprogram the tumor immune microenvironment. [ 4 – 6 ] However, challenges such as the poor stability of nucleic acids in vivo , variable transfection efficiency, and high treatment costs have restricted its widespread application. [ 4 ] Therefore, there is a pressing need to find new therapeutic strategies specifically for GBM. In fact, beyond the aforementioned characteristics, the intracellular Ca 2+ in GBM is significantly higher than that of normal tissues, and this elevated Ca 2+ level is crucial for tumor progression. [ 17 , 18 ] Mitochondria, which contain abundant Ca²⁺ (≈ 570 nM), are key organelles for the storge and regulation of intracellular Ca 2+ . [ 17 , 19 – 21 ] Based on this, we propose a therapeutic strategy that induces mitochondrial mineralization in tumor cells within GBM to inhibit tumor growth, thereby achieving effective treatment of GBM with minimal side effects on normal tissues. Specifically, we developed size-variable self-feedback nanomotors comprising a self-propelled nanorobot core, a degradable lipid shell for self-feedback, and the drug curcumin (Cur), which inhibits Ca²⁺ efflux. Firstly, L-arginine (L-Arg) derivatives and zwitterionic monomer 3-[[2- (methacryloxy) ethyl] dimethylammonium] propionate (CBMA) were used to synthesize PAC nanomotors (PAC NMs) via free radical polymerization reaction. These PAC NMs served as the core of the nanorobot composite system. PAC NMs could target mitochondria via chemotaxis towards the high concentration of inducible nitric oxide synthase (iNOS), and recruit cytoplasmic Ca 2+ through their abundant surface carboxyl groups, thereby increasing the local mitochondrial Ca 2+ concentration and inducing mitochondrial mineralization. To prevent the carboxyl groups on PAC NMs from being occupied by Ca 2+ in the bloodstream and losing their ability to recruit Ca 2+ , self-feedback liposomes were used to encapsulate the PAC NMs along with curcumin (Cur), a compound that inhibits Ca 2+ efflux, to construct the nanorobot composite system NO-Lip@PAC@Cur NMs. The self-feedback liposomes contained nitric oxide (NO)-releasing lipid (NOR) and NO-responsive degradable lipid (NOD). The self-feedback mechanism operates as follows: NO-Lip@PAC@Cur NMs could effectively target GBM via chemotaxis towards highly expressed iNOS in the GBM microenvironment. During this chemotactic process, NO released from NOR reacts with the o-phenylenediamine group in NOD to form an amide-derived benzotriazole group. This compound spontaneously hydrolyzes, leading to the gradual degradation of the liposomes (~ 500 nm). This process releases the encapsulated PAC NMs (~ 50 nm) and Cur. The released PAC NMs recruit Ca 2+ to mitochondria, while Cur inhibits Ca 2+ efflux, thereby enhancing mitochondrial mineralization, inducing mitochondrial dysfunction, and ultimately triggering tumor cell death. Results Preparation of NO-Lip@PAC NMs and their degradation behavior in response to NO NO-Lip@PAC NMs were composed of a nanorobot core (PAC NMs) and a self-feedback degradable lipid shell. Firstly, PAC NMs were prepared. The monomer was prepared according to the method reported in the literature. [ 22 , 23 ] L-Arg was combined with methacrylic anhydride and methacryloyl chloride to form N-methacryloyl-L-arginine (M-Arg) ( Figure S1 ). Both 1 H-NMR and 13 C-NMR confirmed the successful preparation of M-Arg ( Figure S2 and S3 ). [ 22 – 24 ] Next, the zwitterionic monomers CBMA and M-Arg were reacted by radical polymerization to obtain PAC NMs, enriched with carboxyl and guanidine groups ( Figure S4 ). [ 25] Transmission electron microscope (TEM) images demonstrated that the synthesized PAC NMs have a uniform size of approximately 50 nm (Fig. 2 a). According to the dynamic light scattering (DLS) test, the hydration diameter of PAC NMs was 128.7 nm (Fig. 2 b). The structure of PAC NMs was further characterized by Fourier transform infrared spectroscopy (FTIR) spectra. As shown in Figure S5 , there were amino II band of M-Arg (1530 cm -1 ) in PAC, amino I band of BAC (1650 cm -1 ), and C = O (1716 cm -1 ) in CBMA. Meantime, the disappearance of the characteristic peak of C = C double bond that originally belonged to the cross-linker, indicates the reaction of free radical polymerization occurred, confirming the successful synthesis of PAC NMs. [ 26 , 27 ] Subsequently, we used a liposomal shell to protect the PAC NMs, thereby preventing the saturation of the carboxyl groups in the PAC NMs by Ca 2+ during blood circulation. The liposome portion was composed of NOR, NOD, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine- n[methoxy(polyethylene glycol)-2000 (DSPE-PEG2000) and cholesterol. NOR was synthesized through an amide reaction ( Figure S6 ), [ 28 ] using 1,2-distearoyl-sn-glycerin-3-phosphate ethanolamine (DSPE) and N-Boc-N'-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine (Boc-Arg(Pbf)-OH) as raw materials, with 1-Hydroxybenzotriazole (HOBT) serving as the activating agent. This process yielded the intermediate product Boc-Arg(Pbf)-OH-DSPE. Trifluoroacetic acid (TFA) was then used to remove p-toluene sulfonyl (Tos-protecting group) and tert-butoxy carbonyl (Boc-protecting group) from Boc-Arg(Pbf)-OH-DSPE, resulting in the formation of NOR which contains free guanidino groups. The Boc-Arg(Pbf)-OH-DSPE and NOR were characterized using NMR spectrometry ( 1 H-NMR, 13 C-NMR), all characteristic peaks from H and C were labeled in Figure S7-S10 , indicating that the structure of the obtained product aligned with the target structure reported in the literature. [ 17 ] Additionally, peaks corresponding to a molecular weight of 904 Da were observed in the mass spectrometry (MS) analysis ( Figure S11 ), consistent with the theoretical value of 904 Da. NOD was prepared by using tetra decanoic acid and 1,2-diaminobenzene as the reaction materials, 2-(6-Chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) as the active agent, the specific synthetic route was shown in Figure S12 . [ 29 ] The results of 1 H-NMR, 13 C-NMR and MS proved the successful synthesis of NOD ( Figure S13-S15 ). [ 29 ] Subsequently, NO-Lip@PAC NMs were prepared using the thin-film hydration method with NOD, NOR, DSPE-PEG2000, cholesterol and PAC NMs as raw materials. The TEM image showed that the size of the synthesized NO-Lip NMs was about 450.0 nm. After loading PAC NMs in the hydrophilic core, a large number of PAC NMs were found to overlap with the liposomes, suggesting that the liposomes may have encapsulated the PAC NMs (Fig. 2 a ). Further, we used employed cryogenic transmission electron microscopy (Cryo-TEM) to observe the fine structure of NO-Lip@PAC NMs (Figure S16) , the Cryo-TEM image showed that NO-Lip@PAC existed a bilayer membrane structure characteristic of liposomes, with a size of about 400 nm. Notably, some small particles with a size of about 50 nm could be seen inside the liposomes, which might be the PAC NMs encapsulated inside them. The DLS results showed that the hydration diameter of NO-Lip NMs was about 502.2 nm, and the hydration diameter did not change much after loading PAC NMs, which was about 583.3 nm (Fig. 2 b). Subsequently, the Zeta potential of different samples was measured. The potential values of PAC NMs and NO Lip NMs were − 9.3 and − 7.9 mV, respectively. The potential value of NO-Lip@PAC NMs decreased to -19.2 mV ( Fig. 2 c). To further demonstrate the successful preparation of NO-Lip@PAC NMs, the phospholipid bilayer membrane of NO-Lip@PAC NMs was labeled with the lipophilic dye DiO, PAC NMs were labeled with Cy5, and the red fluorescence of PAC NMs overlapped with the green phospholipid bilayer membrane of DiO labeled liposomes (Fig. 2 d). The degradation behavior of NO-Lip@PAC NMs in response to NO was detected. The guanidine group in NOR can react with ROS/iNOS in cancer cells to produce NO. Next, NO released from NOR can react with the o-phenylenediamine group in NOD to form an amide-derived benzotriazole group, [ 29 , 30 ] which spontaneously hydrolyzes and leads to the slow degradation of liposomes and the release of encapsulated PAC NMs ( Figure S17 ). Thus, the performance of NO-Lip@PAC NMs responding to NO degradation was verified by monitoring the changes in DLS of NO-Lip@PAC NMs incubated in PBS or simulated tumor environment with a high concentration of ROS (100 µM H 2 O 2 ) in different time. As shown in Fig. 2 e and 2 f, the DLS of NO-Lip@PAC NMs in PBS remained relatively stable, mostly maintaining 500–600 nm. However, following incubation with H 2 O 2 , NO-Lip@PAC NMs began to degrade gradually, with a peak at around 160.0 nm. This indicated that the smaller PAC NMs were gradually released from the liposomes, and the quantity of degraded liposomal fragments increased with prolonged incubation time. In addition, the TEM images showed the degradation behavior of NO-Lip@PAC NMs more visually (Fig. 2 g and 2 h). The morphology and size of NO-Lip@PAC NMs were basically unchanged in the PBS environment, and the degradation of liposomes started at about 0.5 h after the incubation, and the released PAC NMs can be observed at 1 h (red arrows), and the liposomal structure was completely lost at about 4 h. To better simulate the tumor cell environment, DiO labeled liposomes and Cy5 labeled PAC NMs were used. NO-Lip@PAC NMs were incubated with normal and cancer cellular lysate. As shown in Fig. 2 i, 2 j and Figure S18 , after 4 h of incubation with normal cellular lysates, the red fluorescence of PAC NMs in NO-Lip@PAC NMs still co-localized well with the green phospholipid bilayers of DiO-labeled liposomes. In contrast, in the tumor cellular lysate, the green and red fluorescence began to separate, with the red fluorescence of the PAC NMs appearing independently (purple arrows). The above data demonstrated that NO-Lip@PAC NMs can generate NO in response to the high concentration of ROS/iNOS in the tumor environment and degraded to release the encapsulated PAC NMs. Then, the degradation profiles were evaluated by monitoring turbidity changes. The absorbance values of liposomes at 660 nm were investigated under PBS and simulated tumor high concentration of ROS (100 and 500 µM H 2 O 2 ). As shown in Figure S19 , Lip@PAC maintained stable relative turbidity in both 100 µM and 500 µM H 2 O 2 solutions over 6 h, indicating that it does not degrade at high concentrations of H 2 O 2 . In contrast, the relative turbidity of NO-Lip@PAC in the 100 µM H 2 O 2 solution gradually decreased, with 50% degradation occurring within 2.5 h, progressing to 70% degradation at 6 h. The degradation rate in the 500 µM H 2 O 2 environment was slightly higher than that in the 100 µM H 2 O 2 , with 50% degradation occurring at 2 h and 85% degradation achieved by 6 h. The motion ability of NO-Lip@PAC NMs and their chemotactic behavior in static and dynamic environments It has been proved before that the highly expressed iNOS in the tumor microenvironment can act as chemoattractant to induce the chemotactic effect of the nanorobot, thereby facilitating their effective targeting of tumor tissues. [ 31 – 33 ] In this section, we investigated the motion behavior of PAC NMs, NO-Lip NMs, and NO-Lip@PAC NMs in normal and tumor cellular environment. Firstly, CBMA and N-methacryloyl-L-lysine (M-Lys, without guanidino) were used to prepare PLC NPs as control group, which were structurally similar to PAC NMs but without motion ability ( Figure S20-23 ). TEM image showed that the morphology and size of PLC NPs were similar to those of PAC NMs ( Figure S24 ). As shown in Figure S25 , characteristic peaks such as the amino group of L-Lys (1526 cm -1 ), the amino group of BAC (1630 cm -1 ), and the C = O bond in CBMA (1720 cm -1 ) appeared in FTIR, proving the successful preparation of PLC NPs. [ 26 , 27 ] First, the content of iNOS in bEnd.3 cells and different concentrations of Gl261 cells was detected (Figure S26 ). The concentration of iNOS in bEnd.3 cells (1 × 10 5 ) was about 3.61 µM, whereas Gl261 cells (1 × 10 5 ) was about 12.38 µM. When the density of Gl261 cells increased from 1 × 10 3 to 1 × 10 6 , the amount of intracellular iNOS gradually increased, suggesting that the consistency and stability of iNOS concentration could be maintained by controlling the number of cells. As shown in Figure S27, 28 and Video S1-S4 , the motion displacement of PLC NPs in bEnd.3 and Gl261 cellular environment was relatively small, with an average speed distribution ranging from 0.5 to 1.6 µm s -1 , indicating Brownian motion. PAC NMs showed Brownian motion in bEnd.3 cellular environment, and obvious motion displacement was observed in GL261 cellular environment, with an average speed distribution of 1.7-5.0 µm s -1 , displaying enhanced Brownian motion. Furthermore, the mean square displacement (MSD), which describes the average of the squares of a particle’s displacement from its initial position during motion, reflects the motion state of the particles. [ 34 ] Therefore, we investigated particle motion in different environments by analyzing MSD ( Figure S29 ). The diffusion coefficients of PLC in bEnd.3 and Gl261 cellular environments were 0.33 and 0.29 µm²/s, respectively, indicating Brownian motion. [ 22 ] For PAC NMs, the diffusion coefficient was 0.26 µm²/s in the bEnd.3 cellular environment but increased significantly to 3.8 µm²/s in the Gl261 cellular environment, [ 35 – 37 ] indicating Brownian motion in normal cells and enhanced Brownian motion in cancer cells. To investigate the motion behavior of NO-Lip NMs, non-nanorobot Lip NPs (consisting of DSPE, DSPE-PEG2000, and cholesterol) were constructed as control group. As shown in Figure S30, 31 and Video S5-S8 , the Lip NPs displayed Brownian motion in bEnd.3 and Gl261 cellular environment with an average speed distribution of 0.5–1.5 µm s -1 . The NO-Lip NMs showed Brownian motion in bEnd.3 cells, and had a larger movement displacement in Gl261 cellular environment with an average speed distribution of 2.3–3.8 µm s -1 , indicating enhanced Brownian motion. The MSD results further support this conclusion (Figure S32 ). Based on the above results, the motion behaviors of Lip@PAC NPs and NO-Lip@PAC NMs were further investigated, as shown in Fig. 3 a , 3 b, S33 and Video S9-S10 , the Lip@PAC NPs were showed Brownian motion in bEnd.3 and Gl261 cellular environment, with an average speed distribution of 0.5–1.5 µm s -1 . The observed phenomenon may be attributed to the encapsulation of PAC NMs within liposomes, which lack guanidine groups on their surface to serve as a power source for the movement of nanomotors. As shown in Fig. 3 c, 3 d, S33 and Video S11-S12 , NO-Lip@PAC NMs displayed Brownian motion in bEnd.3 cellular environment, and a significant displacement was observed in Gl261 cellular environment, with an average speed distribution of 1.9–3.7 µm s -1 . As shown in Figure S34 , the diffusion coefficients of Lip@PAC NPs in bEnd.3 and Gl261 cellular environments were 0.24 and 0.32 µm²/s, respectively, indicating Brownian motion. [ 35 – 37 ] For NO-Lip@PAC NMs, the diffusion coefficient was 0.26 µm²/s in the bEnd.3 cellular environment, but it increased significantly to 3.8 µm 2 /s in the Gl261 cellular environment, [ 35 – 37 ] indicating Brownian motion in normal cells and enhanced Brownian motion in cancer cells. Next, we employed a straight single-channel model to evaluate the nanomotors’ chemotactic responses under varying conditions. As shown in Figure S35 , an iNOS gradient was established within the straight channel. DiL-labeled Lip@PAC or NO-Lip@PAC was introduced into reservoir (i), and the motion videos of different samples were recorded at position “ a” to analyze their motion behaviors (Figure S36 ). As shown in Figure S37 and Video S13 , when reservoir (ii) contained bEnd.3 cellular lysate-embedded gel, both Lip@PAC and NO-Lip@PAC exhibited minimal displacement at position “a” , with a speed of 1.0 µm s − 1 , exhibit non-directional Brownian motion. In contrast, when reservoir (ii) was loaded with Gl261 cellular lysate-embedded gel, NO-Lip@PAC displayed significantly enhanced Brownian motion, with a speed of 3.1 µm s − 1 and both moved toward the higher iNOS concentration, exhibiting directional chemotactic movements. To further quantify migration persistence, the chemotaxis index (CI), defined as the ratio of total displacement to path length, was calculated. [ 33 ] Notably, NO-Lip@PAC in the iNOS gradient demonstrated the highest CI value (~ 0.5), surpassing both Lip@PAC and NO-Lip@PAC in the absence of a chemotactic gradient. These results confirmed that the iNOS gradient drives directional migration of NO-Lip@PAC, highlighting its chemotactic specificity. Subsequently, we investigated the chemotactic behavior in static environments with iNOS concentration gradient through Y-shaped channels. As shown in Figure S38 , the iNOS gradient was established in the Y-shaped channels. [ 31 – 33 , 38 ] As shown in Fig. 3 e, regions (i), (ii) and (iii) contained samples, bEnd.3 or Gl261 cellular lysate gels, respectively. The chemotaxis behavior of NO-Lip@PAC NMs was characterized by recording fluorescence images of regions (ii) and (iii) at different times. The fluorescence intensity of the Lip@PAC NMs in regions (ii) and (iii) did not differ significantly, the fluorescence quantification illustrated the same results ( FigureS39 ). As shown in Fig. 3 f and 3 g, the fluorescence intensity in regions (ii) and (iii) gradually increased with time, but the fluorescence intensity in region (iii) was significantly higher than that in region (ii), suggesting that NO-Lip@PAC NMs tended to accumulate more in tumor cells with high concentrations of iNOS. These results suggested that due to the presence of L-Arg in the liposomes, the NO-Lip@PAC NMs could autonomously enrich to sites with higher iNOS concentration and showed good chemotactic performance. In addition, we established a dynamic microfluidic model to investigate the chemotactic ability of nanomotors in a flow state ( Figure S40) . To simulate the capillary blood flow rate, the volume flow rate of the microfluidic syringe pump was controlled to be about 0.6 mL h -1 . [ 39 ] When inlets (i), (ii) were pumped with bEnd.3 cellular lysates, and inlet (iii) was pumped with Lip@PAC NPs or NO-Lip@PAC NMs, there was no fluid shift (Fig. 3 h and 3 i). When inlets (i), (ii) and (iii) were pumped with bEnd.3 cellular lysate, different samples and Gl261 cellular lysate, respectively, the fluorescence signals of the NO-Lip@PAC NMs fluid could be observed to shift towards the Gl261 cellular lysate side (Fig. 3 j). The fluorescence quantification results showed that the NO-Lip@PAC NMs were shifted towards the Gl261 cellular lysate side, indicating that they could diffuse from channel (ii) to the channel with higher iNOS concentration (Fig. 3 k). The ability of NO-Lip@PAC NMs to cross the BBB in vitro and the mitochondrial targeting performance To evaluate the ability of nanomotors crossing the BBB at the cellular level, in vitro BBB model was established using a transwell model containing a porous membrane (Fig. 4 a ) . The upper chamber of the transwell model was inoculated with bEnd.3 cells to simulate a dense BBB layer (which was formed after 10 days of culture), [ 40 , 41 ] and the lower chamber was inoculated with Gl261 cells to simulate GBM, forming an environment with iNOS concentration gradient. Then we added Lip@PAC NPs and NO-Lip@PAC NMs (50 µg mL -1 ) to the upper chamber respectively, and incubated for 6 h. The fluorescence intensity of the samples in the upper and lower chambers of the transwell model were detected by CLSM. Meantime, we collected the liquid and cells in the lower chamber and quantitatively detected the amount of samples in each part. As shown in the CLSM images of bEnd.3 cells in upper chamber (Fig. 4 b and S41), DiL-labelled Lip or NO-Lip colocalized well with Cy5-labelled PAC NMs, indicating minimal of Lip@PAC NPs and NO-Lip@PAC NMs before crossing the BBB. The separation of red and green fluorescence in Gl261 cells treated with NO-Lip@PAC NMs group suggests lipid degradation in the tumor environment, which cannot be found in Lip@PAC NPs treated groups. In addition, the red fluorescence intensity in bEnd.3 cells in the upper chambers treated with NO-Lip@PAC NMs was lower than that treated with Lip@PAC NPs (Fig. 4 c), while Gl261 cells in the lower chambers had stronger red fluorescence signals, and fluorescence quantification showed that their intensity was 8.7 times higher than that of Lip@PAC NPs (Fig. 4 c and 4 d). The calculation of the transport rate in Fig. 4 e showed that the transport efficiency of Lip@PAC NPs was only about 13.7%, while that of NO-Lip@PAC NMs reached about 63.5%. When Gl261 cells in the lower chamber were pretreated with iNOS inhibitor (NG-Monomethyl-L-arginine, L-NMMA) for 24 h. The penetration efficiency of NO-Lip@PAC decreased to 21.7% ( Figure S42 ), which confirmed that iNOS concentration gradient-driven chemotaxis was the NO-Lip@PAC to achieve efficient BBB penetration. Next, we used CLSM to check the structural integrity of BBB after treatment with different samples. As shown in Figure S43 , the cell layer that had formed a dense BBB, there was no significant change in the BBB cellular structure treated with Lip@PAC NPs or NO-Lip@PAC NMs for 24 h. Meanwhile, FITC-dextran (FD-4, 4000 Da) --- a polysaccharide composed of fluorescein isothiocyanate coupled to dextran—was used as a marker to identify BBB leakage, if leakage occurred, we would expect to observe stronger FD-4 fluorescence signals in the lower chamber. [ 42 ] As shown in Figure S44 and Fig. 4 f, compared to the control group, the upper compartment of bEnd.3 cells treated with Lip@PAC NPs and NO-Lip@PAC NMs remained tightly connected, with no significant increase in the intensity of FD-4 fluorescence detected in the lower compartment. This further demonstrated that NO-Lip@PAC NMs penetrated the BBB through chemotactic effects on the iNOS concentration gradient, rather than disrupting the integrity of BBB. Further, we evaluated whether NO-Lip@PAC NMs could degrade and release PAC NMs after crossing the BBB. We used DiL to label Lip or NO-Lip, Cy5 to label the PAC NMs, respectively. Different samples were co-incubated with Gl261 cells for varying durations to observe the release of PAC NMs. As shown in Fig. 4 g, Figure S45 and S46 , when Lip@PAC NPs and NO-Lip@PAC NMs were co-incubated with Gl261 cells for 2 h, the perinuclear showed more green fluorescence representing the outer liposomes, while only minimal red fluorescence representing the PAC NMs. With the extension of the incubation time to 6 h, the red fluorescence representing PAC NPs in the NO-Lip@PAC NMs treated group can be observed, and the intensity of red fluorescence was 1.5 times of that at 2 h. As the incubation time was extended to 12 h, the red fluorescence was gradually enhanced (2.6 times of that at 2 h), while the green fluorescence was gradually reduced, which confirmed that NO-Lip@PAC NMs could gradually degrade and release their contents in response to the self-released NO. This phenomenon was not observed in Lip@PAC NPs treated groups. The ability of PAC NMs to target mitochondria was further assessed. Studies have shown that the concentration of iNOS in cancer cell mitochondria was much higher than that in normal cell mitochondria. [ 43 , 44 ] We hypothesized that PAC NMs could accumulate around mitochondria due to their chemotactic effects on iNOS. As shown in Fig. 4 h and S47, we used Cy5 to label PAC, and the red fluorescence of PAC NMs in NO-Lip@PAC NMs overlapped more with the green of mitochondria, suggesting better mitochondrial targeting. Next, we investigated the iNOS induced chemotaxis kinetics of the nanomotors through Y-shaped channels (Fig. 4 i). The regions (i), (ii) and (iii) comprising different samples, mitochondria lysate, or other organellar lysate gels, respectively. As shown in Fig. 4 j and 4 k, the fluorescence in regions (ii) and (iii) gradually increased over time, regions (ii) and (iii) of the PLC NPs did not differ much, and fluorescence quantification also illustrated the same result. The fluorescence in region (ii) of the PAC NMs group is higher than that in region (iii), suggesting that the PAC NMs tended to aggregate more in mitochondria (Fig. 4 l and 4 m). These results demonstrated that the guanidino group in PAC NMs could drive them to enrich autonomously to the sites with higher iNOS concentration, exhibiting effective chemotactic performance toward mitochondria. Evaluation of the ability to induce mitochondrial mineralization in Gl261 cells by NO-Lip@PAC@Cur NMs The previous results confirmed that NO-Lip@PAC NMs displayed good chemotaxis behavior in the tumor microenvironment, and the NO generated during this process promoted the degradation of liposomes, leading to the release of PAC NMs. The released PAC NMs can be further chemotactically targeted to mitochondria, and the carboxyl groups on their surface can recruit Ca 2+ from the cytoplasm, [ 46 – 48 ] leading to an increase in the local Ca 2+ concentration around the mitochondria, and thus inducing their mineralization. To further enhance the effect of mitochondrial mineralization, we also loaded Cur in the lipid hydrophobic region to inhibit Ca 2+ efflux [ 49 – 52 ] and constructed NO-Lip@PAC@Cur NMs. We prepared NO-Lip@PAC@Cur NMs with different drug-lipid ratios and tested their encapsulation rate and drug loading to explore the optimal incorporation conditions of Cur. As shown in Figure S48 , the encapsulation rate of Cur gradually decreased and the drug loading amount increased with higher drug-lipid ratios. However, its stability decreased and liposomes appeared to agglomerate ( Figure S49 ). Considering this, we chose a drug-lipid ratio of 1:10 for subsequent experiments, achieving an encapsulation rate of 73.8% and a drug loading capacity of 7.6%. The TEM images showed that NO-Lip@PAC@Cur had a diameter of about 500 nm. The hydrated particle size was about 545.0 nm and the zeta potential was − 14.0 mV ( Figure S50). Subsequently, we investigated in detail whether the addition of Cur could play a synergistic role with NO to improve intracellular Ca 2+ levels. Firstly, intracellular NO levels were detected using an NO probe. As shown in Fig. 5 a and Figure S51 , Gl261 cells treated with NO-Lip@PAC and NO-Lip@PAC@Cur exhibited significantly stronger NO fluorescence signals (2.7 and 2.9 times that of the control group, respectively), which were significantly higher than those observed in the other groups. In contrast, the NO fluorescence signals in bEnd.3 cells treated with different samples did not show significant differences, likely due to the lower concentration of iNOS to react with the nanomotors and produce NO. Next, we examined the intracellular Ca 2+ content using inductively coupled plasma-Mass Spectrometry (ICP-MS). Figure 5 b showed that the Ca 2+ content in Gl261 cells treated with NO-Lip@PAC increased to 0.27 µmol, which was 1.6 times higher than that in the control group. This increase is likely due to the high concentration of NO causing endoplasmic reticulum stress and releasing more Ca 2+ into the cytoplasm, decrease in endoplasmic reticulum Ca 2+ leads to Ca2 + influx from the extracellular space. [ 53 – 56 ] Cells treated with NO-Lip@PAC@Cur exhibited an even higher intracellular Ca 2+ content, which was 2.7 times higher than that of the control group, suggesting that the inhibition of Ca 2+ exocytosis by Cur can further increase the intracellular Ca 2+ levels, confirming that Cur and NO can exert a synergistic effect to enhance the mineralization effect. To assess intracellular mitochondrial mineralization, Gl261 cells were lysed after treated with different samples, and mitochondria and other organelles were collected. Then the Ca 2+ concentration was detected using ICP-MS. As shown in Figure S52 , the Ca²⁺ content in mitochondria was 0.17 µmol at 6 h and increased to 0.33 µmol after extending the incubation time to 36 h, which was comparable to 0.36 µmol at 48 h. [ 17 ] In addition, to determine that mitochondrial damage was caused by mineralization rather than Ca 2+ overload. we added a control group–A23187 (a chemical reagent commonly used to induce mitochondrial Ca 2+ overload). [ 57 , 58 ] As shown in Fig. 5 c and S53, the mitochondrial Ca 2+ content was significantly elevated after A23187 treatment, which was 5.3 times higher than that of the control group, resulting in mitochondrial Ca 2+ overload. The mitochondrial Ca 2+ treated with NO-Lip@PAC NMs was about 0.19 µmol, which was 6.3 times that of the control group, and had the similar ability to induce mitochondrial Ca 2+ overload as that of A23187 group. In contrast, the mitochondrial Ca 2+ treated with NO-Lip@PAC@Cur NMs was further increased to 0.36 µmol, which was 12 times higher than that of the control group and about 2 times higher than that of the NO-Lip@PAC and A23187 groups. This result indicates that mitochondrial mineralization requires a higher level of Ca 2+ concentration than Ca 2+ overload, NO-Lip@PAC and A23187 were able to trigger Ca 2+ overload but did not reach the level of mineralization, while NO-Lip@PAC @Cur can recruit more Ca 2+ to gather around mitochondria, which can trigger mitochondrial mineralization. To visually observe mitochondrial calcification, NO-Lip@PAC@Cur-treated Gl261 cells were sectioned and intracellular mitochondrial structure was observed using Bio-TEM. As shown in Fig. 5 d and S54, the cell membrane of Gl261 cells treated with NO-Lip@PAC@Cur exhibited significant disruption compared to the untreated group, and the morphology of the mitochondria was notably altered. In addition, the TEM-mapping images of the mitochondria in the NO-Lip@PAC@Cur group and the control group were compared, and the mitochondria in the NO-Lip@PAC@Cur group had stronger Ca fluorescence signals in the mitochondria (Fig. 5 e and 5 f). Subsequently, Ca 2+ in the cytoplasm was labeled with green fluorescence using Flou-4, Ca 2+ in the mitochondria was labeled with red fluorescence using Rhod-2, and the co-localization was observed by CLSM. As shown in Fig. 5 g and S55, the red fluorescence and green fluorescence of NO-Lip@PAC and NO-Lip@PAC@Cur groups were significantly higher than those of other treatment groups, which proved that the concentration of Ca 2+ in the cytoplasm and mitochondria was increased after treatment. Co-localization analysis showed that the red fluorescence and greenfluorescence in the NO-Lip@PAC@Cur NMs group had a higher degree of overlap, suggesting that the mineralization process occurred in the mitochondria. Further, in order to prove that the Ca 2+ accumulated around mitochondria are insoluble calcium salts, we used Alizarin Red S to stain cells, which can form red complexes by specifically chelating insoluble calcium salts (such as calcium phosphate), which is a classic method to evaluate cellular calcium deposition. [ 59 ] As shown in Figure S56 , cells treated with Lip@PLC, A23187 and NO-Lip@PAC was similar to that of the control group, and the production of red complexes was not observed in the cells, which may be because although they can induce the mitochondrial Ca 2+ overload, the concentration did not reach the formation of insoluble calcium salts, so the signal was weak. And the formation of red complex was obviously observed in the cells treated with NO-Lip@PAC@Cur, which indicated that the NO-Lip@PAC@Cur can induce mitochondria to form insoluble calcium salts, confirming the occurrence of mitochondrial mineralization. Subsequently, the effects of mineralization on mitochondrial function were explored in detail. The stability of mitochondrial membrane potential (MMP) is a prerequisite for mitochondria to maintain normal physiological functions, and the JC-1 dye has been widely used to assess MMP. Under normal conditions, JC-1 accumulates in the mitochondrial stroma and formed aggregates that emit red fluorescence (529 nm); when the mitochondrial transmembrane potential was impaired and depolarized, JC-1 was released from mitochondria at a reduced concentration, and then JC-1 was a monomer and emitted green fluorescence (585 nm). [ 60 , 61 ] As shown in Fig. 5 h, 5 i and S57, JC-1 showed strong red fluorescence and only a small amount of green fluorescence in both control and Lip@PLC NPs group, whereas the red JC-1 aggregates in the NO-Lip@PAC@Cur NMs group were converted in large quantities to green JC-1 monomers. In addition, we assessed the effect of different control groups on the behavior of adenosine triphosphate (ATP) generation in tumor cells. As shown in Fig. 5 j, the intracellular ATP concentration decreased from 8.9 to 2.4 µM after NO-Lip@PAC@Cur NMs treatment compared to the control group. Apparently, NO-Lip@PAC@Cur NMs induced mitochondrial calcification effectively depolarized the cellular mitochondrial membranes, leading to mitochondrial dysfunction and inhibition of intracellular ATP production. The impact of the mitochondrial mineralization process on cellular activity was also explored. As shown in Fig. 5 k, 3 -(4,5-dimethylthiazol-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) results showed that NO-Lip@PAC@Cur NMs had a concentration-dependent cytotoxicity against Gl261 cells, with a progressive decrease in cellular activity as the concentration increased. In addition, the cytotoxicity of different samples on GL261 and bEnd.3 cells was compared (Fig. 5 l), the cell viability of Gl261 cells in the NO-Lip@PAC NMs group was 74.6%, while the viability decreased to 44.5% when Cur was loaded, indicating that mitochondrial mineralization in tumor cells resulted in significant cytotoxicity effects. Meanwhile, the viability of bEnd.3 cells treated with NO-Lip@PAC@Cur NMs was 83.8%, with no significant damage to normal cells, due to the absence of overexpressed iNOS in normal cells, which did not undergo mitochondrial mineralization. Targeting ability of NO-Lip@PAC NMs in GBM model mice To investigate the in vivo targeting ability of NO-Lip@PAC NMs, we established a GBM model in mice through orthotopic implantation of GL261-Luc cells. The establishment of the GBM model was determined by in vivo bioluminescence 10 days after in situ inoculation of Gl261-Luc cells ( Figure S58 ). Subsequently, Lip@PAC NPs and NO-Lip@PAC NMs were injected intravenously and their fluorescence images were captured using an in vivo imaging system (IVIS) various time points post-injection. As shown in Fig. 6 a-c, significant fluorescence aggregation signals could be observed in GBM model mice at 1 h after intravenous injection of NO-Lip@PAC, indicating its ability to rapidly penetrate the BBB. The fluorescence intensity of brain tumors in the NO-Lip@PAC group continued to increase over time, reaching a peak at 12 h after injection, with an intensity 3.5 times greater than that of the Lip@PAC group (Fig. 6 d). The fluorescence signal gradually weakened thereafter. It is noteworthy that the fluorescence signal in the brain tumor region of the Lip@PAC group was weak and basically disappeared after 48 h. In contrast, a significant fluorescence signal remained detectable in the NO-Lip@PAC group at 48 h (Fig. 6 d), confirming its ability to maintain stable retention in the brain microenvironment for at least 48 h. These findings were further validated through ex vivo organ imaging (Fig. 6 e, 6 f and S59). After 12 h of intravenous injection, the Cy5 fluorescence intensity of the brain tissue of GBM mice in the NO-Lip@PAC group was significantly higher than that in 7 h, demonstrating its notable specificity for targeting brain tumors. The fluorescence intensity of the NO-Lip@PAC group was still 3.0 times that of the Lip@PAC group at 24 h after injection, which fully reflected its active targeting characteristics driven by iNOS concentration gradient. In addition, ex vivo organ imaging showed that the both samples were mainly cleared by liver and kidney metabolism ( Figure S60 and S61 ) Further, we collected different organs and examined the proportion of different samples to determine the targeting efficiency in different organs. The results showed that the NO-Lip@PAC NMs group accumulated about 30.0% ID/g in the brain, which was 5.1 times higher than that of the Lip@PAC NPs group ( Fig. 6 g). To further observe the distribution of the nanorobot fluorescence signals in the tumor tissues, whole brain tissues were cryosectioned and stained using immunofluorescence techniques. FITC-CD31 was used to label the vascular endothelium, DAPI was used to label the nucleus, and red fluorescence was derived from Cy5-labelled nanorobot. Equal area region of interest (ROI) was selected perpendicular to the blood vessel and used as the starting point, red fluorescence signals within the ROIs were quantitatively analyzed to evaluate the permeability of different samples from the blood vessel to the GBM. As shown in Fig. 6 h and 6 i, the chemotactic NO-Lip@PAC NMs showed the strongest fluorescence intensity and depth of penetration in the GBM. Obvious red fluorescence was still observed at the distal end of the brain vessels (~ 300 µm), indicating that NO-Lip@PAC NMs could effectively cross the BBB and penetrate deeply into the tumor tissues. Antitumor efficacy of NO-Lip@PAC@Cur NMs in GBM model mice Before evaluating the therapeutic effects in vivo , we quantified the levels of Cur in circulating blood at different time intervals after intravenous injection of free Cur and NO-Lip@PAC@Cur in healthy SD rats. As shown in Figure S62 , NO-Lip@PAC@Cur can prolong the circulation time of Cur in the body. Then, their therapeutic efficacy in vivo was further evaluated. In this section, we chose TMZ, a first-line drug for clinical treatment of GBM, as a control for chemotherapeutic agents. Successful brain tumor construction was confirmed by in vivo bioluminescence imaging on day 11 after Gl261-Luc cell transplantation. Thereafter, mice were randomly divided into 6 groups and sham-operated groups, and different drugs were intravenously every two days (Fig. 7 a). As shown in Fig. 7 b, 7 c and Table S2 , in vivo bioluminescence imaging and quantification of total radiant brightness showed that the bioluminescence signal in the PBS group continued to enhance over time, and was strongest at the end of treatment (60.5 times the fluorescence intensity at the initial moment), suggesting that the tumor was growing rapidly. The fluorescence intensity of the representative tumor in the mouse brain decreased following treatment with free TMZ or NO-Lip@PLC@Cur treatment, suggesting mild inhibition of tumor growth (49.8 and 32.6 times the fluorescence intensity at the initial moment, respectively). In contrast, the anti-GBM growth effect was sequentially enhanced in the PAC@Cur and Lip-PAC@Cur groups, and the bioluminescent signals were significantly decreased (20.8 and 23.8 times of the fluorescence intensity at the initial moment, respectively). Kaplan-Meier survival curves showed that NO-Lip@PAC@Cur significantly prolonged the survival of the GBM model mice, with a median survival time (MST) of 49 days, whereas PAC@Cur and Lip-PAC@Cur groups were both 39 days, which was slightly longer than that of the TMZ and NO-Lip@PAC@Cur groups (both 37 days), while the PBS control group was only 33 days (Fig. 7 d). Further, dissected tumors from different treatments were stained with hematoxylin and eosin (H&E) to determine the destruction of tumor cells. The H&E results showed that the tumor area in the NO-Lip@PAC@Cur group was smaller than that in the other treatment groups (Fig. 7 e and S63). Brain tumors in the PBS group were the largest at the end of treatment, accounting for 41.8% of the whole brain. Brain tumors in the free TMZ-treated mice were reduced and accounted for 31.3% of the whole brain. In contrast, the anti-GBM growth effect was enhanced in the PAC@Cur, Lip@PAC@Cur and NO-Lip@PLC@Cur group, with brain tumors accounting for 22.0%, 24.9% and 25.0% of the whole brain in mice, respectively. NO-Lip@PAC@Cur was able to significantly inhibit the growth of brain tumors, which accounted for 9.5% of the whole brain at the end of treatment in mice. TUNEL and Ki67 immunofluorescence were used to label apoptotic and proliferating cells in the tumor tissue (Fig. 7 e and 7 f). Quantitative analysis showed that mice treated with NO-Lip@PAC@Cur had the highest rate of apoptotic cells in their tumors (approximately 49.9%) and the lowest proportion of positive tumor cells proliferating (approximately 10.0%). These differences demonstrated that NO-Lip@PAC@Cur was able to inhibit the growth of GBM by killing tumor cells through inducing mitochondrial mineralization in brain tumor cells. Safety is another key issue in the treatment of glioblastoma. The biocompatibility and systemic response of the different agents were assessed by hemolysis rate and erythrocyte morphology analysis, weight changes in mice, hematological and histopathological tests. Results showed no significant damage to erythrocytes with the different materials, indicating that the materials have good blood compatibility (Figure S64 and S65) . The mice in the PBS and TMZ groups showed significant weight loss during treatment, which may be attributed to the fact that chemotherapeutic agents inevitably damage normal tissues and their functions during treatment, leading to weight loss. In contrast, the NO-Lip@PAC@Cur NMs constructed in this paper did not observe a significant weight loss trend in mice during treatment ( Figure S66 ). High-dose TMZ treatment leads to bone marrow suppression. [ 62 , 63 ] TMZ treatment group resulted in a significant increase in bone marrow vacuoles ( Figure S65 ) and a decrease in blood cell counts ( Figure S68 ), suggesting that free TMZ may have side effects on bone marrow. Blood biochemistry data showed that alanine aminotransferase (ALT), aspartate aminotransferase (AST) lactate dehydrogenase (LDH) and serum albumin concentration (ALB) indices were increased in the TMZ group, suggesting that the TMZ treatment may cause some damage to the liver function of mice ( Figure S69 ). In contrast, the hematological parameters and H&E staining results of mice treated with NO-Lip@PAC@Cur NMs indicated that the liver and kidney tissues of mice did not show significant pathological damage ( Figure S70 ). In order to observe mitochondrial calcification more visually, the brain tumor tissues of mice in the PBS and NO-Lip@PAC@Cur groups were sectioned, and the structures of cells and mitochondria within the tumor tissues were observed using Bio-TEM. As shown in Fig. 7 g, compared with the tumor cells in PBS group, cell membranes in the NO-Lip@PAC@Cur group were broken, nuclei were fragmented, and mitochondrial structures were lost, indicating cell apoptosis. TEM-Mapping results showed that mitochondria in the NO-Lip@PAC@Cur group had stronger Ca fluorescence signals ( Fig. 7 h and 7 i), further demonstrating that NO-Lip@PAC@Cur could inhibit tumor growth by inducing mitochondrial mineralization in brain tumor cells. In addition, in order to verify whether there is tumor calcification in vivo , von Kossa staining was carried out on the tumor tissues of each group. [ 59 ] As shown in Figure S71 , the brain tumor tissue in NO-Lip@PAC@Cur group showed dark brown areas, indicating that there was calcification in the tumor. The other groups were similar to the PBS group, showing the same dark red color as the normal tumor. These results suggest that the apoptosis of tumor cells is mainly due to mitochondrial mineralization rather than Ca 2+ overload. Conclusion In summary, we successfully developed size-variable self-feedback nanomotors NO-Lip@PAC@Cur NMs, and verified their therapeutic efficacy against GBM. The lipid shell of NO-Lip@PACNMs degraded in response to the NO generated during chemotactic targeting to the GBM microenvironment, thereby releasing the loaded Cur and PAC NMs. The released NO induced endoplasmic reticulum stress and subsequent Ca 2+ release, while Cur inhibited Ca 2+ efflux. This dual action resulted in an increase in the intracellular Ca 2+ concentration (2.7 times higher than that in the control group). Additionally, PAC NMs targeted mitochondria via chemotaxis, where the carboxyl groups recruited Ca 2+ from the cytoplasm, thereby increasing the local mitochondrial Ca 2+ concentration to 6.2 times higher than that in the control group. Fluorescence imaging and TEM-mapping of Ca²⁺ in tumor cell mitochondria revealed that mitochondria in the control group. Fluorescence imaging and TEM-mapping of Ca²⁺ in tumor cell mitochondria revealed that mitochondria in the NO-Lip@PAC@Cur NMs-treated group exhibited stronger fluorescence signals. This indicates that NO-Lip@PAC@Cur NMs effectively induced mitochondrial dysfunction, loss of mitochondrial membrane potential, and a significant decrease in ATP-generating capacity from 8.9 µM to 2.4 µM. In vivo experiments demonstrated that NO-Lip@PAC NMs could recognize the highly expressed iNOS in the tumor microenvironment, cross the BBB, and accumulate in brain tumors. Specifically, the accumulation in brain tissues of GBM model mice reached 30.0% ID/g, which was 5.1 times higher than that of the Lip@PAC NMs group. The treatment results showed that NO-Lip@PAC@Cur NMs effectively inhibited the growth of tumor by inducing mitochondrial mineralization and subsequent tumor cell death (the total radiant brightness of brain tumors in the NO-Lip@PAC@Cur NMs group at the end of the treatment was 15.8% of that in the PBS group). Compared with the chemotherapeutic drug TMZ, NO-Lip@PAC@Cur NMs exibited favorable biocompatibility. At the end of the treatment the weight of the mice remained basically unchanged, and blood biochemistry and hematology indices were within normal ranges. Additionally, histological analysis using H&E staining revealed no significant damage to major organs. Given that tumor cell mitochondria are crucial for their growth and proliferation, the strategy of specifically targeting mitochondrial mineralization in tumor cells is expected to provide valuable ideas for designing therapeutic strategies specifically for GBM. Methods Cell culture The mouse glioma cell line Gl261and Gl261-Luc were purchased from Shanghai Aorui cell Biotechnology CO., Ltd.; the mouse brain microvascular endothelial cells bEnd.3 were purchased from Procell Life Science & Technology CO., Ltd. Gl261 and Gl261-Luc cells were cultured in complete culture medium containing 89% v/v high-sugar Dalberg's modified Eagle's medium (DMEM with 4.5 g L − 1 D-glucose, Jiangsu KeyGEN BioTECH Corp., CO., Ltd.), 10% v/v fetal bovine serum (SKU: SP011010500, Sperikon Life Science & Biotechnology CO., Ltd.), and 1% v/v penicillin-streptomycin mixture in complete culture medium. bEnd.3 cells was cultured in 89% v/v high-sucrose Dalberg's modified Eagle's medium (DMEM, containing 4.5 g L − 1 D-glucose), 10% v/v fetal bovine serum (LONSERA. Suzhou Shuangru Biotechnology Co., Ltd), and 1% v/v penicillin-streptomycin mixture in complete culture medium. All the cells were cultured in a humidified atmosphere that contained 5% CO 2 at 37°C. When not in use, cell cryopreservation with cell saving (PB180438, Pricella Life Science&Technology Co., Ltd.) at -80°C. iNOS content in different cells The bEnd.3 (1.0 mL, 1 × 10 6 cells mL − 1 ) or Gl261 cells (1.0 mL, 10 3 , 10 4 , 10 5 or 10 6 cells mL − 1 ) were broken by sonication, and the supernatant was collected after centrifugation at 3000 rpm for 10 min. The iNOS concentration was detected using an enzyme immunoassay kit (Jiangsu Enzyme Immunoassay, MM-0454M2). Motion behavior of different samples To analyze motion behavior, bEnd.3 or Gl261 cells (1.0 mL, 1 × 10 5 cells mL − 1 ) were inoculated in glass-bottomed confocal petri dishes and incubated overnight. 20.0 µL of Cy5 or DiO-labeled different samples was slowly added to the above petri dishes. The motion behavior of different samples was recorded using the 100 × objective of an inverted fluorescence microscope (Micro-shot MF53-N), and the motion trajectories of the different samples were marked using the tracking plug-in of the Fiji software. The trajectories of 50 particles were randomly selected to calculate the average speed of nanomotors and analyze the speed distribution histogram. iNOS content in straight or Y-shaped channels To determine the iNOS content in the straight or Y-shaped channel, 25.0 µL of agarose solution was mixed with an equal volume of bEnd.3 or Gl261 cellular lysate in the storage chamber (ii) and (iii), respectively. And then the Y-shaped channel was transferred to 4°C to wait for the agarose to form a gel. Subsequently, it was filled with 300.0 µL of PBS and after standing at 4°C for 15 min, samples were collected from different locations of the Y-shaped channel. The iNOS concentration was detected using an enzyme-linked immunoassay kit (Jiangsu Enzyme Free, MM-0454M2). Collective chemotaxis behavior of nanomotors in the Y-shaped channel To observe the collective chemotaxis behavior of nanomotors, a Y-shaped glass substrate microchannel was used. The main channel was 1.0 cm in length, 0.4 cm in width, and the branch channel was 0.7 cm in length and 0.3 cm in width. The concentration gradient of the chemoattractant was created by different types of cell lysates that were placed in reservoirs (II) or (III) in the branched channel. Briefly, 5.0 mg of agarose was completely dissolved in 0.5 mL of PBS at 90°C, and 50.0 µL of Gl261 or bEnd.3 cellular lysate was added when the melted agarose was cooled to room temperature but not solidified. And then, it was transferred to 4°C for gelation. Before assessing the chemotactic motion of the nanomotors, the Y-shaped channel was prefilled with 300.0 µL PBS and quiescence for 10 min. Then 50.0 µL of Cy5-labeled nanomotors was gently dropped into the reservoir (I). The fluorescence microscope of the reservoir (II) and (III) were captured with an inverted fluorescence microscope (Micro-shot MF53-N), equipped with 10 × objective, at specific times. The corresponding fluorescence intensity was quantified using Image J. Dynamic chemotaxis behavior of nanomotors in the microfluidic channel The three-inlet one-outlet glass substrate microfluidic channel with dimensions of 2.2 cm (length) × 1.5 mm (width) × 0.3 mm (height) was used to evaluate the dynamic chemotaxis of nanomotors. Among them, the diluent of Gl261 cellular lysate (lysate: PBS = 1:4, v/v) flowed through inlet (I), the Cy5-labeled nanomotors in PBS was flowed through inlet (II), and the dilution of bEnd.3 cellular lysate (lysate: PBS = 1:4, v/v) was flowed through inlet (III). The flow velocity of each channel was controlled at 0.6 mL h − 1 . The video was captured continuously for 5 min (1 frame per second) at the position near to outlet using an inverted fluorescence microscope (10 × objective). The fluorescence intensity of Cy5 perpendicular to the flow direction was measured using Image J. Moreover, the dilution of bEnd.3 cell lysate (lysate: PBS = 1:4, v/v) flowed through both inlet (I) and (III) as control, while the flow of inlet (II) was not replaced. Detection of intracellular Ca 2+ concentration GL261 cells (1.0 mL, 1 × 10 5 cells mL − 1 ) were inoculated into a 6-well plate and incubated overnight. Then different samples (Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur, 1.0 mL, 200 µg mL − 1 in DMEM) were added and incubated for 48 h. The cells were washed three times with PBS to remove free samples. Cells were collected with cell scraper and placed in a reactor with nitric acid (1.0 mL) and H 2 O 2 (1.5 mL), which was nitrated in vacuum oven at 180°C for 1.5 h. The above system was subsequently volume-determined to 5.0 mL, and the intracellular Ca 2+ concentration was measured using inductively coupled plasma-Mass Spectrometry (ICP-MS, IRIS Intrepid II). Detection of Ca 2+ concentration in mitochondria GL261 cells (1 × 10 8 cells) were inoculated into T175 cell culture flasks and incubated overnight. Then different samples (Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur, 20.0 mL, 200 µg mL − 1 in DMEM or A23187, 20.0 mL, 5 µM) were added and incubated for 48 h. The cells were washed three times with PBS to remove free samples. Cells were then collected and mitochondria were extracted according to the instructions of the mitochondrial isolation kit (C3601, Beyotime Biotechnology). The mitochondria and other organelles were placed in a reactor with nitric acid (1.0 mL) and H 2 O 2 (30%, 1.5 mL), which was nitrated in vacuum oven at 180°C for 1.5 h. The above system was subsequently volume-determined to 5.0 mL, and the Ca 2+ concentration in mitochondria was measured using ICP-MS. Detection of intracellular NO release GL261 cells (1.0 mL, 1 × 10 5 cells mL − 1 ) were inoculated into a 6-well plate and incubated overnight. Then different samples (1.0 mL, 200 µg mL − 1 in DMEM, Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur) were added and incubated for 6 h. 3-Amino, 4-aminomethyl-2’,7’-difluorescein, diacetate (DAF-FM DA, NO fluorescent probe, 5 µM, Beyotime Institute of Biotechnology, China) was incubated with the cells for 30 min and images were taken using inverted fluorescence microscope (Micro-shot MF53-N), their fluorescence being quantified using Image J. Detection of mitochondrial membrane potential Gl261 cells (1.0 mL, 1 × 10 5 cells mL − 1 ) were inoculated in confocal dishes and incubated overnight. Then different samples (1.0 mL, 200 µg mL − 1 in DMEM, Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur) were added and incubation for 24 h. The cells were washed three times with PBS to remove free material. Mitochondria was labeled with JC-1 (Beyotime biotechnology, C2006) and nucleus was labeled with Hoechst 33342 in blue fluorescence. Cells were fixed with 4% paraformaldehyde. Fluorescence images were taken with CLSM, and the fluorescence intensity of the acquired images was analyzed using Image J software. Intracellular ATP concentration assay GL261 cells (1.0 mL, 1 × 10 5 cells mL − 1 ) were inoculated into a 6-well plate and incubated overnight. Then different samples (1.0 mL, 200 µg mL − 1 in DMEM, Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur) were added and incubation for 24 h. The cells were washed three times with PBS to remove free samples. Cells were then collected and intracellular ATP concentration was detected according to the instructions for use of the ATP Assay Kit (S0026, Beyotime Biotechnology). Evaluation of cell viability in vitro GL261 cells or bEnd.3 cells (200.0 µL, 1 × 10 5 cells mL − 1 ) were inoculated in 96-well plates and incubated overnight. The cell cultures were then removed and 200.0 µL of different concentrations in DMEM (0, 50.0, 100.0, 200.0, 400.0, and 800.0 µg mL − 1 ) of NO-Lip@PAC@Cur were added and incubated for 24 h. The cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-dibenzenetetrazolium bromide (MTT, ApexBio Technology Co., Ltd.) colorimetric method to assess cell viability. To compare the in vitro cell viability of different nanomotors, GL261 cells (200.0 µL, 1 × 10 5 cells mL − 1 ) were inoculated in 96-well plates and incubated overnight. Then different samples (1.0 mL, 200 µg mL − 1 in DMEM, Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur) were added and incubated for 24 h. Cell viability was assessed by the MTT colorimetric assay described above. In vitro mitochondrial mineralization detection For Alizarin Red S staining, Gl261 cells (1.0 mL, 1 × 105 cells mL-1) were inoculated in confocal dishes and incubated overnight. After removal of the medium, 1 mL of Lip@PLC, NO-Lip@PAC, NO-Lip@PAC@Cur (200.0 µg mL − 1 ) or A3187 (5 µM) dispersed in fresh medium was added and incubated for 48 h. After removal of the culture medium, and the cells were then washed and fixed with 95% ethanol. Subsequently, the cells were stained with Alizarin Red S (1 mL, Beyotime Biotechnology) for 30 min. After the extra staining solution was washed away by ultrapure water, the stained cell samples were observed under a light microscope. Detection of pharmacokinetics SD rats (purchased from Jiangsu Wukong Biotechnology Co., LTD.) were used for the pharmacokinetic determination of NO-Lip@PAC@Cur NMs. Briefly, Cur (1.0 mL, 15.0 mg mL − 1 ) and NO-Lip@PAC@Cur (1.0 mL, 15.0 mg mL − 1 ) were injected through the tail vein, and 500 µL blood was drawn from the orbits of the rats at different times using capillary glass tubes soaked with sodium heparin. After centrifugation at 2500 r min − 1 for 15 min, the supernatant was obtained and the absorbance of curcumin was measured at 420 nm using a Multi-Function Measuring Instrument (Infinite® E Plex) and the content of the samples was calculated using a standard curve of absorbance of Cur or NO-Lip@PAC@Cur concentration. Establishment of glioblastoma (GBM) model mice. Female C57BL/6 mice (6–8 weeks old) were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. All animal experiments were conducted under the supervision and guidance of the Ethical Review Committee for Laboratory Animal Welfare of Nanjing Normal University (Nanjing, China, approval No. IACUC- 20220901-1 and 20200802). To establish the GBM model mice, GL261-Luc cells (10 6 cells in 8.0 µL Corning® Matrigel® Matrix) were slowly injected into the brain using a brain stereotaxic instrument (RWD Life Science Co., Shenzhen) positioned to (1.8 mm,0.6 mm, 2 mm depth) using the fontanel point of origin, where the craniotomy operation was kept consistent provided that the mice without cell injection were referred to as the sham-operated group as a control. During the operation, the mice were anesthetized by inhalation of 1–5% isoflurane mixed with oxygen. 10 days later, all the mice were intraperitoneally injected D-Luciferin potassium salt (150 mg kg − 1 in PBS, Guangzhou Biolight Biotechnology Co.), 15 min later, bioluminescent imaging was performed immediately using in vivo image system (IVIS Spectrum, Aniview 600 Multi-mode Animal Imaging, Guangzhou Biolight Biotechnology Co.) In vivo targeting and tumor permeability To assess the in vivo targeting ability of different samples, 200.0 µL of Cy5-labled different samples (2.0 mg mL − 1 , Lip@PAC or NO-Lip@PAC) was injected intravenously. Notably, 200.0 µL of PBS was injected as control. Fluorescence imaging (excitation filter: 620 nm, emission filter: 670 nm) was performed at different times by IVIS Spectrum in vivo . Euthanizing mice at different times, and the brain tissue and major organs (heart, liver, spleen, lung, and kidney) were collected for fluorescence imaging using IVIS Spectrum (excitation filter: 620 nm, emission filter: 670 nm). The in vivo targeting ability of different samples was quantified using Living Image software. Subsequently, the above whole brain tissues were subjected to coronal frozen sections. The cell membranes of cerebrovascular endothelial cells were labeled with rabbit anti-mouse CD31 antibody and Aliexa Fluor 488-coupled goat anti-rabbit IgG, and the nucleus were labeled with DAPI, respectively. Tissue sections were blocked and fluorescence imaging was performed using CLSM. Normal brain tissues and GBM tissues were distinguished by cell density, and GBM permeability of different nanomotors was analyzed by Image J software. In vivo delivery efficiency of different samples To investigate the in vivo delivery efficiency and biodistribution of the nanomotors, 200.0 µL of different samples (2.0 mg mL − 1 , Lip@PAC or NO-Lip@PAC) was injected intravenously. and 200.0 µL of PBS was injected as control. After 24 h, the mice were euthanized, and the brain tissue and major organs (heart, liver, spleen, lung, and kidney) were collected and weighed. Then added 1.0 mL RIPA lysis buffer to every 100.0 mg of tissue, and prepare tissue homogenate using a tissue grinder (frequency: 70 Hz, time: 8 min, run times: 8 times). Centrifuge the homogenate (4°C, 3000 rpm, 10 min) and collect the supernatant. Dilute the supernatant and measure the fluorescence intensity using a multifunctional enzyme-linked immunosorbent assay reader. Calculate the injection dose percentage (% ID/g) of each group of samples based on the sample concentration fluorescence intensity standard curve and the initial injection dose. In vivo anti-GBM efficacy and biosafety evaluation of different samples On the 11th, 13th, 15th, 17th, 19th, 21st, and 23rd day after injection of GL261-Luc cells into the skull of mice, 200.0 µL different samples (2.0 mg mL − 1 , PAC@Cur, Lip@PAC@Cur, NO-Lip@PLC @Cur, NO-Lip@PAC@Cur and temozolomide (TMZ, Beyotime Biotechnology)) were injected intravenously. Notably, the sham group received no injections and 200.0 µL PBS was injected as control. The growth of the glioma was monitored by IVIS Spectrum at specific times. The mice were sacrificed after treatment, and the brain tissues were taken for paraffin-embedding and coronal sectioning, which were performed with hematoxylin-eosin (H&E) staining and Ki-67 immunohistochemical staining, respectively. The main tissues including the heart, liver, spleen, lung, kidney and bone marrow were collected and fixed with 4% paraformaldehyde. Then the tissues were embedded in paraffin, and performed with H&E staining to evaluate the histopathological changes after different treatments. The blood samples were obtained from each group for biochemical analysis and routine blood examination. The biochemical analysis was measured by the automatic biochemical analyzer. The routine blood examination was performed by blood cell analyzer. Then, 8 additional mice were used in the survival experiments and survival curves were obtained using the Kaplan-Meier method. Statistical analysis All statistical analyses were finished using SPSS. Statistical tests and P values are detailed in figure legends. Data are presented as the means ± SD. The statistical significance was calculated via one-way ANOVA and LSD posthoc test, *P < 0.05; **P < 0.01; ***P < 0.001. Declarations Competing interests The authors declare no competing interests. Additional information The supplementary figures, supplementary movies are provided in Supplementary information . Acknowledgements The work was supported by National Natural Science Foundation of China (No. 52422306 (the Excellent Young Scholars NSFC), 22275095, 22175096, 22475103), Jiangsu Key Laboratory of Biofunctional Materials, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials. 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Supplementary Files supportinginformation.docx supporting information SupplementaryVideo1PLCNPsinbEnd.3.mp4 Motion behavior of PLC NPs in bEnd.3 cellular environment for 10 s SupplementaryVideo2PLCNPsinGl261.mp4 Motion behavior of PLC NPs in Gl261 cellular environment for 10 s SupplementaryVideo3PACNMsinbEnd.3.mp4 Motion behavior of PAC NRs in bEnd.3 cellular environment for 10 s SupplementaryVideo4PACNMsinGl261.mp4 Motion behavior of PAC NRs in Gl261 cellular environment for 10 s SupplementaryVideo5LipNPsinbEnd.3.mp4 Motion behavior of Lip NPs in bEnd.3 cellular environment for 10 s SupplementaryVideo6LipNPsinGl261.mp4 Motion behavior of Lip NPs in Gl261 cellular environment for 10 s SupplementaryVideo7NOLipNMsinbEnd.3.mp4 Motion behavior of NO-Lip NRs in bEnd.3 cellular environment for 10 s SupplementaryVideo8NOLipNMsinGl261.mp4 Motion behavior of NO-Lip NRs in Gl261 cellular environment for 10 s SupplementaryVideo9LipXPACNPsinbEnd.3.mp4 Motion behavior of Lip@PAC NPs in bEnd.3 cellular environment for 10 s SupplementaryVideo10LipXPACNPsinGl261.mp4 Motion behavior of Lip@PAC NPs in Gl261 cellular environment for 10 s SupplementaryVideo11NOLipXPACNMsinbEnd.3.mp4 Motion behavior of NO-Lip@PAC NRs in bEnd.3 cellular environment for 10 s SupplementaryVideo12NOLipXPACNMsinGl261.mp4 Motion behavior of NO-Lip@PAC NRs in Gl261 cellular environment for 10 s SupplementaryVideo13.mp4 Motion behavior of different samples in straight single-channel mode for 10 s Cite Share Download PDF Status: Published Journal Publication published 09 Oct, 2025 Read the published version in Nature Communications → Version 1 posted 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-6451662","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":449572807,"identity":"a856d5de-e171-4528-8f4f-d8f0cf11881c","order_by":0,"name":"Chun Mao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYJACZiCWY2A4AGMTqcWYdC2JDQg2AaDbfvjg58K2O+nbGc8ek2CosE5sYD97AK8WszNpydIz257l7mw4lybBcCY9sYEnLwG/lgM5Zsy8bYdzNxw4YybB2HY4sUGCxwC/lvNvwFrSDcBa/hGj5QbElgSIlgaitDxLluY5d9gQ6DBji4Rj6cZtPDmEHJZ88DNP2WF5gxtnDG98qLGW7Wc/g18LAkgcYGBIANJsRKoHAv4G4tWOglEwCkbByAIAIclHEEdQfZ0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4085-3414","institution":"Nanjing Normal University","correspondingAuthor":true,"prefix":"","firstName":"Chun","middleName":"","lastName":"Mao","suffix":""},{"id":449572808,"identity":"af5bf66b-76a7-4950-92a3-ff28ae2f4c68","order_by":1,"name":"Tiantian Chen","email":"","orcid":"","institution":"School of Chemistry and Materials Science, Nanjing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Tiantian","middleName":"","lastName":"Chen","suffix":""},{"id":449572809,"identity":"be1d4b47-0f9a-46c2-a0af-5d32738cf77c","order_by":2,"name":"Yu Duan","email":"","orcid":"","institution":"Nanjing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Duan","suffix":""},{"id":449572810,"identity":"251f5f28-be46-4417-a186-fe5d603d5c0d","order_by":3,"name":"Yingjie wang","email":"","orcid":"","institution":"Nanjing Normal University School of Chemistry and Material Science","correspondingAuthor":false,"prefix":"","firstName":"Yingjie","middleName":"","lastName":"wang","suffix":""},{"id":449572811,"identity":"6db91f26-5490-446b-ab9a-3e836dd49e88","order_by":4,"name":"Tiantian Liang","email":"","orcid":"","institution":"Nanjing Normal University School of Chemistry and Material Science","correspondingAuthor":false,"prefix":"","firstName":"Tiantian","middleName":"","lastName":"Liang","suffix":""},{"id":449572812,"identity":"58b3ca03-3329-464c-8270-2cb7f44c020c","order_by":5,"name":"Shiluan Liu","email":"","orcid":"","institution":"Nanjing Normal University School of Chemistry and Material Science","correspondingAuthor":false,"prefix":"","firstName":"Shiluan","middleName":"","lastName":"Liu","suffix":""},{"id":449572813,"identity":"ebe050c2-2994-4b49-a964-d3d256915756","order_by":6,"name":"xue Xia","email":"","orcid":"","institution":"nanjing normaluniversity","correspondingAuthor":false,"prefix":"","firstName":"xue","middleName":"","lastName":"Xia","suffix":""},{"id":449572814,"identity":"3734a2e4-a06f-4297-a589-3b3bd3fabd8b","order_by":7,"name":"Mimi Wan","email":"","orcid":"","institution":"Nanjing Normal University School of Chemistry and Material Science","correspondingAuthor":false,"prefix":"","firstName":"Mimi","middleName":"","lastName":"Wan","suffix":""}],"badges":[],"createdAt":"2025-04-15 06:45:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6451662/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6451662/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-64020-x","type":"published","date":"2025-10-09T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81672577,"identity":"b615995b-b6c8-4e38-b3ac-b2b2be4ef8dd","added_by":"auto","created_at":"2025-04-30 06:25:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":506261,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of (a) preparation of NO-Lip@PAC@Cur and its schematic diagram in response to NO degradation and (b) NO-Lip@PAC@Cur selectively inducing mitochondrial mineralization for the treatment of GBM. By Figdraw.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6451662/v1/4344ad4710c98d7dca854a70.png"},{"id":81672788,"identity":"7d49b4e3-9fbf-40cb-b4a6-e833bc3f0d10","added_by":"auto","created_at":"2025-04-30 06:33:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":635060,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreparation of NO-Lip@PAC NMs and characterization of their degradation behavior in response to NO.\u003c/strong\u003e (a) TEM images (scale bar: 500 nm), (b) DLS and (c) Zeta potential of different samples (I: PAC, II: NO-Lip, III: NO-Lip@PAC); (d) Confocal laser scanning microscope (CLSM) images of NO-Lip@PAC (scale bar: 50 μm, green: DiO labeled NO-Lip, red: Cy5 labeled PAC); DLS changes of NO-Lip@PAC NMs incubated with (e) PBS and (f) 100 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for different times; TEM images of NO-Lip@PAC NMs incubated with (g) PBS and (h) 100 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for different times (scale bar 500 nm; red arrows indicate PAC released by lipid degradation); Representative CLSM images of NO-Lip@PAC incubated with (i) HUVECs and (j) Gl261 cellular lysates incubated for different times (scale bar: 10 μm, green: DiO labeled Lip or NO-Lip; Red: Cy5 labeled PAC; Purple arrows indicate PAC released by lipid degradation). Data in b and c were mean ± s.d (n = 3).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6451662/v1/06c27fef3904b86bb899ce2e.png"},{"id":81672580,"identity":"efd2dc1a-6464-4eed-a0d9-314dd0811b94","added_by":"auto","created_at":"2025-04-30 06:25:24","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1013129,"visible":true,"origin":"","legend":"\u003cp\u003eMovement behavior of different samples and characterization of their chemotaxis in static and dynamic environments. Normalized motion trajectories (n = 20) and motion speed distribution plots (n = 50) of Lip@PAC NPs in (a) bEnd.3 and (b) Gl261 cellular environment (Video S9-S10, Supporting information); Normalized motion trajectories (n = 20) and motion speed distribution plots (n = 50) of NO-Lip@PAC NMs in (c) bEnd.3 and (d) Gl261 cellular environment (Video S11-S12, Supporting information); (e) Schematic of the Y-channel model; (f) Representative fluorescence images (scale bar: 1000 μm) and (g) fluorescence quantification of NO-Lip@PAC NMs in Y-channel regions (ii) and (iii) at different times; Representative fluorescence images of Lip@PAC NPs and NO-Lip@PAC NMs at the exit of the Ψ-shaped microfluidic channel (scale bar: 200 μm) and the corresponding fluorescence quantification in the presence of (h and i) bEnd.3 cellular lysates or (j and k) bEnd.3 cellular lysates and Gl261 cellular lysates. Data in g was mean ± s.d (n = 3).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6451662/v1/10e260dc9c1839d7c2ebddec.jpeg"},{"id":81672809,"identity":"a188722d-6ea3-44f2-8364-beadb58ff5c0","added_by":"auto","created_at":"2025-04-30 06:33:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":992005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of the ability of different samples to cross the BBB \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and the mitochondrial targeting performance.\u003c/strong\u003e (a) Schematic diagram of the transwell model; (b) Representative CLSM images of bEnd.3 cells in the upper chamber and GL261 cells in the lower chamber of the transwell system treated with different samples for 6 h (blue: nucleus, red: Cy5-labelled PACs, green: DiL-labelled Lip or NO-Lip; Scale bar: 50 μm), quantitative analysis of red and green fluorescence in the (c) upper and (d) lower compartments (I: Control, II: Lip@PAC, III: NO-Lip@PAC); (e) BBB transport efficiency of different samples (I: Lip@PAC, II: NO-Lip@PAC I: Lip@PAC, II: NO-Lip@PAC); (f) Paracellular permeability of FD-4 treated with different samples for 24 h (I: Control, II: Lip@PAC, III: NO-Lip@PAC); (g) Representative CLSM images of Gl261 cells treated with different samples for 2 h, 6 h and 12 h (blue: nucleus, green: DiL-labelled Lip or NO-Lip, Red: Cy5-labelled PAC; Scale bar: 50 μm); (h) Representative CLSM images of colocalization of mitochondrial treated with different samples for 2 h and corresponding fluorescence curves (I: PAC, II: PLC, III: Lip@PAC, IV: NO-Lip@PAC; Blue: nucleus, green: mito-tracker-labelled mitochondria, red: Cy5-labelled different samples; Scale bar: 50 μm); (i) Schematic illustration of using the Y-channel model to assess the chemotaxis of different samples towards mitochondria. By Figdraw; Representative fluorescence images of (j) PLC NPs and (l) PAC NMs at different times in Y-channel regions (ii) and (iii) (scale bar: 1000 μm) and fluorescence quantification of (k) PLC and (m) PAC. Data in c, d, e, f, g and i were mean ± s.d (n = 3). Statistical significance was assessed by one-way ANOVA with post hoc LSD tests.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6451662/v1/611ea5289e42ea971e9a08fb.png"},{"id":81672591,"identity":"ceb531eb-f5b8-4961-92e6-455440d065f3","added_by":"auto","created_at":"2025-04-30 06:25:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1232202,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the ability to induce mitochondrial mineralization in Gl261 cells by NO-Lip@PAC@Cur NMs. (a) Fluorescence images of intracellular NO (labelled with NO fluorescent probe, DAF-FM DA) in bEnd.3 cells and Gl261 cells treated with different samples for 6 h (Scale bar: 50 μm); (b) Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration in bEnd.3 cells and Gl261 cells after treated with different samples, (c) Ca\u003csup\u003e2+\u003c/sup\u003e concentration in mitochondria and other organelles treated with different samples for 24 h in Gl261 cells (I: Control, II: Lip@PLC, III: NO-Lip@PAC, IV: NO-Lip@PAC@Cur); (d) Bio-TEM images of Gl261 cells treated with NO-Lip@PAC@Cur for 24 h (I: Control, II: magnification of I, III: NO-Lip@PAC@Cur, IV: magnification of III, Scale bar: 2 μm); TEM-Mapping (O, P and Ca) of Gl261 cellular mitochondria after treated with (e) control and (f) NO-Lip@PAC@Cur; (g) CLSM images of cytoplasmic Ca\u003csup\u003e2+\u003c/sup\u003e and mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e treated with different samples for 24 h and the fluorescence distribution curves along selected lines (indicated by the white lines in the images; Blue: nucleus, green: Fluo-4-labelled cytoplasmic Ca\u003csup\u003e2+\u003c/sup\u003e, red: Rhod-2-labelled mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e; Scale bar: 20 μm); (h) Changes in mitochondrial membrane potential treated with different samples for 24 h (red: JC-aggregates, green: JC-1 monomers; Scale bar: 50 μm) and (i) the corresponding red/green fluorescence ratios; (j) Intracellular ATP concentrations (I: Control, II: Lip@PLC, III: NO-Lip@PAC, IV: NO-Lip@PAC@Cur); (k) Cellular activity of Gl261 cells after treated with different NO-Lip@PAC@Cur concentrations for 24 h, (m) Cellular activity of bEnd.3 and Gl261 cells treated with different samples (200 μg mL\u003csup\u003e-1\u003c/sup\u003e ) for 24 h (I: Control, II: Lip@PLC, III: NO-Lip@PAC, IV: NO-Lip@PAC@Cur). Data in b, c, i, j, k and l were mean ± s.d (n = 3). Statistical significance was assessed by one-way ANOVA with post hoc LSD tests.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6451662/v1/90e800b662eae6b14b41e792.png"},{"id":81673499,"identity":"f4669c79-6d0d-4b3b-92f0-ecf889f6dab8","added_by":"auto","created_at":"2025-04-30 06:41:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":929550,"visible":true,"origin":"","legend":"\u003cp\u003eTargeting ability of NO-Lip@PAC NMs in GBM model mice.\u003cem\u003e In vivo\u003c/em\u003efluorescence imaging of mice injected with (a) PBS, (b) Lip@PAC or (c) NO-Lip@PAC at various time points; (d) Quantification of the mean fluorescence intensities of the brain sites from mice in a-c at various time points (I: PBS, II: Lip@PLC, III: NO-Lip@PAC); (e) Cy5 fluorescence imaging of the brain after intravenous injection of different samples for 24 h (n=3, scale bar: 1 cm) and (f) the corresponding quantitative analysis (I: Control, II: Lip@PLC, III: NO-Lip@PAC); (g) Quantitative analysis of different sample accumulations in major organs, expressed as injected dose per gram of tissue (%ID/g) (H.: heart ; Li.: liver ; S.: spleen; Lu.: lung; K.: kidney; B.: brain); (h) Representative CLSM images of brain tumors in mice (Blue: nucleus, green: FITC-CD31, red: Cy5-labelled different samples; Scale bar: 500 μm) and (i) red fluorescence distribution curves (I: PBS, II: Lip@PLC, III: NO-Lip@PAC) along selected lines (indicated by white lines in the images). Data in b, c and e are mean ± s.d (n = 3). Statistical significance was assessed by one-way ANOVA with post hoc LSD tests.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6451662/v1/681b761acf545d2fab3e130e.png"},{"id":81672790,"identity":"1be87624-a1f5-44c2-9323-1d0f228435f4","added_by":"auto","created_at":"2025-04-30 06:33:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1869295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-tumor efficacy of NO-Lip@PAC@Cur NMs in GBM model mice.\u003c/strong\u003e (a) Treatment protocols for orthotopic brain-GBM-tumor-bearing models. By Figdraw; (b) Representative IVIS spectrum images and (c) quantified signal intensity (n = 3 mice per group) ; (d) Kaplan-Meier survival curves (n = 8) of GBM model mice with different treatments; (e) H\u0026amp;E (scale bar: 4 cm), TUNEL, and Ki67 staining images (scale bar: 500 μm) of dissected brain tissues at the end of treatment; (f) Tunel and Ki67 quantitative fluorescence analysis; (I: sham, II: PBS, III: TMZ, IV: PAC@Cur, V: Lip-PAC@Cur, VI: NO-Lip@PLC@Cur, VII: NO-Lip@ PAC@Cur); (g) Bio-TEM images of brain tumor (I: PBS, II: magnification of I, III: NO-Lip@PAC@Cur, IV: magnification of III, Scale bar of I and III: 5 μm, Scale bar of II and IV: 200 nm); TEM-Mapping (P and Ca) of brain tumor cellular mitochondria after treated with (h) control and (i) NO-Lip@PAC@Cur (Scale bar: 200 nm). Data in c and f are mean ± s.d (n = 3).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6451662/v1/bdb89493a062c8e16bfa87c5.png"},{"id":93201379,"identity":"d3eb38cc-e261-407e-87cf-fefb144d33e1","added_by":"auto","created_at":"2025-10-10 07:06:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8842643,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6451662/v1/2985b383-07d1-452b-a41f-00031e06d041.pdf"},{"id":81672585,"identity":"812e85ac-77b4-4ae3-ba32-578d4d34c858","added_by":"auto","created_at":"2025-04-30 06:25:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13808710,"visible":true,"origin":"","legend":"\u003cp\u003esupporting 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environment for 10 s","description":"","filename":"SupplementaryVideo12NOLipXPACNMsinGl261.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6451662/v1/7964d18f5c0d546a159f4be8.mp4"},{"id":81673506,"identity":"71311fb3-b7e9-4ef2-b722-021014f74135","added_by":"auto","created_at":"2025-04-30 06:41:25","extension":"mp4","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":10348294,"visible":true,"origin":"","legend":"Motion behavior of different samples in straight single-channel mode for 10 s","description":"","filename":"SupplementaryVideo13.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6451662/v1/05b75433e551c7c6476d2f74.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Size-variable self-feedback nanomotors for glioblastoma therapy via mitochondrial mineralization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlioblastoma (GBM) is one of the most aggressive and deadly brain tumors, classified by the World Health Organization as the most malignant glioma (Grade IV).\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e The standard treatment for GBM typically involves a combination of surgical resection followed by radiotherapy and/or chemotherapy. Temozolomide (TMZ) is currently the first-line chemotherapeutic agent for GBM treatment.\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e However, the infiltrative growth pattern and inherent heterogeneity of GBM, coupled with the side effects of TMZ treatment (including hematologic toxicity and thrombocytopenia associated with long-term and high-dose administration), have significantly limited the efficacy of this therapeutic strategy. Consequently, the median survival for GBM patients remains low at 14.6\u0026ndash;20.5 months, with a 5-year survival rate of less than 10%.\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e Therefore, developing targeted therapeutic strategies specifically for GBM has emerged as a critical area of research.\u003c/p\u003e \u003cp\u003eThe unique microenvironment of GBM provides valuable insights for the design of targeted therapeutic strategies (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). GBM is characterized by an immunosuppressive tumor microenvironment, often referred to as a \"cold\" tumor, which has spurred researchers to actively explore novel immunotherapeutic approaches for GBM.\u003csup\u003e[1a]\u003c/sup\u003e These include tumor vaccines, \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e immune checkpoint inhibitors (ICIs),\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e and chimeric antigen receptor T-cell (CAR-T) therapies,\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e etc. However, the efficacy of immunotherapy is affected by the individual patient variability, leading to insufficient or excessive immune responses that limit its broad application.\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e In addition to immunotherapy, molecular targeted therapies have been developed to target cellular molecules that promote the proliferation and differentiation of GBM, such as anti-angiogenic therapy\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e and tyrosine kinase inhibitors.\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e However, their effectiveness is often limited by factors such as drug resistance in tumor cells and high recurrence rates following treatment.\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e Gene therapy represents another promising approach, targeting specific genes involved in the proliferation, migration, invasion, apoptosis, and angiogenesis of malignant glioma cells including gene editing corrections using CRISPR-Cas9, or suppressing immune-suppressive genes expressed in brain tumors to reprogram the tumor immune microenvironment.\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e However, challenges such as the poor stability of nucleic acids \u003cem\u003ein vivo\u003c/em\u003e, variable transfection efficiency, and high treatment costs have restricted its widespread application.\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e Therefore, there is a pressing need to find new therapeutic strategies specifically for GBM.\u003c/p\u003e \u003cp\u003eIn fact, beyond the aforementioned characteristics, the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e in GBM is significantly higher than that of normal tissues, and this elevated Ca\u003csup\u003e2+\u003c/sup\u003e level is crucial for tumor progression.\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e Mitochondria, which contain abundant Ca\u0026sup2;⁺ (\u0026asymp;\u0026thinsp;570 nM), are key organelles for the storge and regulation of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e.\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e Based on this, we propose a therapeutic strategy that induces mitochondrial mineralization in tumor cells within GBM to inhibit tumor growth, thereby achieving effective treatment of GBM with minimal side effects on normal tissues. Specifically, we developed size-variable self-feedback nanomotors comprising a self-propelled nanorobot core, a degradable lipid shell for self-feedback, and the drug curcumin (Cur), which inhibits Ca\u0026sup2;⁺ efflux. Firstly, L-arginine (L-Arg) derivatives and zwitterionic monomer 3-[[2- (methacryloxy) ethyl] dimethylammonium] propionate (CBMA) were used to synthesize PAC nanomotors (PAC NMs) via free radical polymerization reaction. These PAC NMs served as the core of the nanorobot composite system. PAC NMs could target mitochondria via chemotaxis towards the high concentration of inducible nitric oxide synthase (iNOS), and recruit cytoplasmic Ca\u003csup\u003e2+\u003c/sup\u003e through their abundant surface carboxyl groups, thereby increasing the local mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e concentration and inducing mitochondrial mineralization. To prevent the carboxyl groups on PAC NMs from being occupied by Ca\u003csup\u003e2+\u003c/sup\u003e in the bloodstream and losing their ability to recruit Ca\u003csup\u003e2+\u003c/sup\u003e, self-feedback liposomes were used to encapsulate the PAC NMs along with curcumin (Cur), a compound that inhibits Ca\u003csup\u003e2+\u003c/sup\u003e efflux, to construct the nanorobot composite system NO-Lip@PAC@Cur NMs. The self-feedback liposomes contained nitric oxide (NO)-releasing lipid (NOR) and NO-responsive degradable lipid (NOD). The self-feedback mechanism operates as follows: NO-Lip@PAC@Cur NMs could effectively target GBM via chemotaxis towards highly expressed iNOS in the GBM microenvironment. During this chemotactic process, NO released from NOR reacts with the o-phenylenediamine group in NOD to form an amide-derived benzotriazole group. This compound spontaneously hydrolyzes, leading to the gradual degradation of the liposomes (~\u0026thinsp;500 nm). This process releases the encapsulated PAC NMs (~\u0026thinsp;50 nm) and Cur. The released PAC NMs recruit Ca\u003csup\u003e2+\u003c/sup\u003e to mitochondria, while Cur inhibits Ca\u003csup\u003e2+\u003c/sup\u003e efflux, thereby enhancing mitochondrial mineralization, inducing mitochondrial dysfunction, and ultimately triggering tumor cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of NO-Lip@PAC NMs and their degradation behavior in response to NO\u003c/h2\u003e \u003cp\u003eNO-Lip@PAC NMs were composed of a nanorobot core (PAC NMs) and a self-feedback degradable lipid shell. Firstly, PAC NMs were prepared. The monomer was prepared according to the method reported in the literature.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e L-Arg was combined with methacrylic anhydride and methacryloyl chloride to form N-methacryloyl-L-arginine (M-Arg) (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Both \u003csup\u003e1\u003c/sup\u003eH-NMR and \u003csup\u003e13\u003c/sup\u003eC-NMR confirmed the successful preparation of M-Arg (\u003cb\u003eFigure S2 and S3\u003c/b\u003e).\u003csup\u003e[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e Next, the zwitterionic monomers CBMA and M-Arg were reacted by radical polymerization to obtain PAC NMs, enriched with carboxyl and guanidine groups (\u003cb\u003eFigure S4\u003c/b\u003e).\u003csup\u003e[ 25]\u003c/sup\u003e Transmission electron microscope (TEM) images demonstrated that the synthesized PAC NMs have a uniform size of approximately 50 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). According to the dynamic light scattering (DLS) test, the hydration diameter of PAC NMs was 128.7 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The structure of PAC NMs was further characterized by Fourier transform infrared spectroscopy (FTIR) spectra. As shown in \u003cb\u003eFigure S5\u003c/b\u003e, there were amino II band of M-Arg (1530 cm\u003csup\u003e-1\u003c/sup\u003e) in PAC, amino I band of BAC (1650 cm\u003csup\u003e-1\u003c/sup\u003e), and C\u0026thinsp;=\u0026thinsp;O (1716 cm\u003csup\u003e-1\u003c/sup\u003e) in CBMA. Meantime, the disappearance of the characteristic peak of C\u0026thinsp;=\u0026thinsp;C double bond that originally belonged to the cross-linker, indicates the reaction of free radical polymerization occurred, confirming the successful synthesis of PAC NMs.\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSubsequently, we used a liposomal shell to protect the PAC NMs, thereby preventing the saturation of the carboxyl groups in the PAC NMs by Ca\u003csup\u003e2+\u003c/sup\u003e during blood circulation. The liposome portion was composed of NOR, NOD, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine- n[methoxy(polyethylene glycol)-2000 (DSPE-PEG2000) and cholesterol. NOR was synthesized through an amide reaction (\u003cb\u003eFigure S6\u003c/b\u003e),\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e using 1,2-distearoyl-sn-glycerin-3-phosphate ethanolamine (DSPE) and N-Boc-N'-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine (Boc-Arg(Pbf)-OH) as raw materials, with 1-Hydroxybenzotriazole (HOBT) serving as the activating agent. This process yielded the intermediate product Boc-Arg(Pbf)-OH-DSPE. Trifluoroacetic acid (TFA) was then used to remove p-toluene sulfonyl (Tos-protecting group) and tert-butoxy carbonyl (Boc-protecting group) from Boc-Arg(Pbf)-OH-DSPE, resulting in the formation of NOR which contains free guanidino groups. The Boc-Arg(Pbf)-OH-DSPE and NOR were characterized using NMR spectrometry (\u003csup\u003e1\u003c/sup\u003eH-NMR, \u003csup\u003e13\u003c/sup\u003eC-NMR), all characteristic peaks from H and C were labeled in \u003cb\u003eFigure S7-S10\u003c/b\u003e, indicating that the structure of the obtained product aligned with the target structure reported in the literature.\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e Additionally, peaks corresponding to a molecular weight of 904 Da were observed in the mass spectrometry (MS) analysis (\u003cb\u003eFigure S11\u003c/b\u003e), consistent with the theoretical value of 904 Da. NOD was prepared by using tetra decanoic acid and 1,2-diaminobenzene as the reaction materials, 2-(6-Chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) as the active agent, the specific synthetic route was shown in \u003cb\u003eFigure S12\u003c/b\u003e.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e The results of \u003csup\u003e1\u003c/sup\u003eH-NMR, \u003csup\u003e13\u003c/sup\u003eC-NMR and MS proved the successful synthesis of NOD (\u003cb\u003eFigure S13-S15\u003c/b\u003e).\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSubsequently, NO-Lip@PAC NMs were prepared using the thin-film hydration method with NOD, NOR, DSPE-PEG2000, cholesterol and PAC NMs as raw materials. The TEM image showed that the size of the synthesized NO-Lip NMs was about 450.0 nm. After loading PAC NMs in the hydrophilic core, a large number of PAC NMs were found to overlap with the liposomes, suggesting that the liposomes may have encapsulated the PAC NMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e).\u003c/b\u003e Further, we used employed cryogenic transmission electron microscopy (Cryo-TEM) to observe the fine structure of NO-Lip@PAC NMs \u003cb\u003e(Figure S16)\u003c/b\u003e, the Cryo-TEM image showed that NO-Lip@PAC existed a bilayer membrane structure characteristic of liposomes, with a size of about 400 nm. Notably, some small particles with a size of about 50 nm could be seen inside the liposomes, which might be the PAC NMs encapsulated inside them. The DLS results showed that the hydration diameter of NO-Lip NMs was about 502.2 nm, and the hydration diameter did not change much after loading PAC NMs, which was about 583.3 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Subsequently, the Zeta potential of different samples was measured. The potential values of PAC NMs and NO Lip NMs were \u0026minus;\u0026thinsp;9.3 and \u0026minus;\u0026thinsp;7.9 mV, respectively. The potential value of NO-Lip@PAC NMs decreased to -19.2 mV \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). To further demonstrate the successful preparation of NO-Lip@PAC NMs, the phospholipid bilayer membrane of NO-Lip@PAC NMs was labeled with the lipophilic dye DiO, PAC NMs were labeled with Cy5, and the red fluorescence of PAC NMs overlapped with the green phospholipid bilayer membrane of DiO labeled liposomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eThe degradation behavior of NO-Lip@PAC NMs in response to NO was detected. The guanidine group in NOR can react with ROS/iNOS in cancer cells to produce NO. Next, NO released from NOR can react with the o-phenylenediamine group in NOD to form an amide-derived benzotriazole group, \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e which spontaneously hydrolyzes and leads to the slow degradation of liposomes and the release of encapsulated PAC NMs (\u003cb\u003eFigure S17\u003c/b\u003e). Thus, the performance of NO-Lip@PAC NMs responding to NO degradation was verified by monitoring the changes in DLS of NO-Lip@PAC NMs incubated in PBS or simulated tumor environment with a high concentration of ROS (100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) in different time. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, the DLS of NO-Lip@PAC NMs in PBS remained relatively stable, mostly maintaining 500\u0026ndash;600 nm. However, following incubation with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, NO-Lip@PAC NMs began to degrade gradually, with a peak at around 160.0 nm. This indicated that the smaller PAC NMs were gradually released from the liposomes, and the quantity of degraded liposomal fragments increased with prolonged incubation time. In addition, the TEM images showed the degradation behavior of NO-Lip@PAC NMs more visually (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). The morphology and size of NO-Lip@PAC NMs were basically unchanged in the PBS environment, and the degradation of liposomes started at about 0.5 h after the incubation, and the released PAC NMs can be observed at 1 h (red arrows), and the liposomal structure was completely lost at about 4 h. To better simulate the tumor cell environment, DiO labeled liposomes and Cy5 labeled PAC NMs were used. NO-Lip@PAC NMs were incubated with normal and cancer cellular lysate. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej \u003cb\u003eand Figure S18\u003c/b\u003e, after 4 h of incubation with normal cellular lysates, the red fluorescence of PAC NMs in NO-Lip@PAC NMs still co-localized well with the green phospholipid bilayers of DiO-labeled liposomes. In contrast, in the tumor cellular lysate, the green and red fluorescence began to separate, with the red fluorescence of the PAC NMs appearing independently (purple arrows). The above data demonstrated that NO-Lip@PAC NMs can generate NO in response to\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ethe high concentration of ROS/iNOS in the tumor environment and degraded to release the encapsulated PAC NMs.\u003c/p\u003e \u003cp\u003eThen, the degradation profiles were evaluated by monitoring turbidity changes. The absorbance values of liposomes at 660 nm were investigated under PBS and simulated tumor high concentration of ROS (100 and 500 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). As shown in \u003cb\u003eFigure S19\u003c/b\u003e, Lip@PAC maintained stable relative turbidity in both 100 \u0026micro;M and 500 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solutions over 6 h, indicating that it does not degrade at high concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. In contrast, the relative turbidity of NO-Lip@PAC in the 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution gradually decreased, with 50% degradation occurring within 2.5 h, progressing to 70% degradation at 6 h. The degradation rate in the 500 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e environment was slightly higher than that in the 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, with 50% degradation occurring at 2 h and 85% degradation achieved by 6 h.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe motion ability of NO-Lip@PAC NMs and their chemotactic behavior in static and dynamic environments\u003c/h3\u003e\n\u003cp\u003eIt has been proved before that the highly expressed iNOS in the tumor microenvironment can act as chemoattractant to induce the chemotactic effect of the nanorobot, thereby facilitating their effective targeting of tumor tissues.\u003csup\u003e[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e In this section, we investigated the motion behavior of PAC NMs, NO-Lip NMs, and NO-Lip@PAC NMs in normal and tumor cellular environment. Firstly, CBMA and N-methacryloyl-L-lysine (M-Lys, without guanidino) were used to prepare PLC NPs as control group, which were structurally similar to PAC NMs but without motion ability (\u003cb\u003eFigure S20-23\u003c/b\u003e). TEM image showed that the morphology and size of PLC NPs were similar to those of PAC NMs (\u003cb\u003eFigure S24\u003c/b\u003e). As shown in \u003cb\u003eFigure S25\u003c/b\u003e, characteristic peaks such as the amino group of L-Lys (1526 cm\u003csup\u003e-1\u003c/sup\u003e), the amino group of BAC (1630 cm\u003csup\u003e-1\u003c/sup\u003e), and the C\u0026thinsp;=\u0026thinsp;O bond in CBMA (1720 cm\u003csup\u003e-1\u003c/sup\u003e) appeared in FTIR, proving the successful preparation of PLC NPs.\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFirst, the content of iNOS in bEnd.3 cells and different concentrations of Gl261 cells was detected \u003cb\u003e(Figure S26\u003c/b\u003e). The concentration of iNOS in bEnd.3 cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) was about 3.61 \u0026micro;M, whereas Gl261 cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) was about 12.38 \u0026micro;M. When the density of Gl261 cells increased from 1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e to 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e, the amount of intracellular iNOS gradually increased, suggesting that the consistency and stability of iNOS concentration could be maintained by controlling the number of cells. As shown in \u003cb\u003eFigure S27, 28 and Video S1-S4\u003c/b\u003e, the motion displacement of PLC NPs in bEnd.3 and Gl261 cellular environment was relatively small, with an average speed distribution ranging from 0.5 to 1.6 \u0026micro;m s\u003csup\u003e-1\u003c/sup\u003e, indicating Brownian motion. PAC NMs showed Brownian motion in bEnd.3 cellular environment, and obvious motion displacement was observed in GL261 cellular environment, with an average speed distribution of 1.7-5.0 \u0026micro;m s\u003csup\u003e-1\u003c/sup\u003e, displaying enhanced Brownian motion. Furthermore, the mean square displacement (MSD), which\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003edescribes the average of the squares of a particle\u0026rsquo;s displacement from its initial position during motion, reflects the motion state of the particles.\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e Therefore, we investigated particle motion in different environments by analyzing MSD (\u003cb\u003eFigure S29\u003c/b\u003e). The diffusion coefficients of PLC in bEnd.3 and Gl261 cellular environments were 0.33 and 0.29 \u0026micro;m\u0026sup2;/s, respectively, indicating Brownian motion.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e For PAC NMs, the diffusion coefficient was 0.26 \u0026micro;m\u0026sup2;/s in the bEnd.3 cellular environment but increased significantly to 3.8 \u0026micro;m\u0026sup2;/s in the Gl261 cellular environment,\u003csup\u003e[\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e indicating Brownian motion in normal cells and enhanced Brownian motion in cancer cells. To investigate the motion behavior of NO-Lip NMs, non-nanorobot Lip NPs (consisting of DSPE, DSPE-PEG2000, and cholesterol) were constructed as control group. As shown in \u003cb\u003eFigure S30, 31 and Video S5-S8\u003c/b\u003e, the Lip NPs displayed Brownian motion in bEnd.3 and Gl261 cellular environment with an average speed distribution of 0.5\u0026ndash;1.5 \u0026micro;m s\u003csup\u003e-1\u003c/sup\u003e. The NO-Lip NMs showed Brownian motion in bEnd.3 cells, and had a larger movement displacement in Gl261 cellular environment with an average speed distribution of 2.3\u0026ndash;3.8 \u0026micro;m s\u003csup\u003e-1\u003c/sup\u003e, indicating enhanced Brownian motion. The MSD results further support this conclusion \u003cb\u003e(Figure S32\u003c/b\u003e). Based on the above results, the motion behaviors of Lip@PAC NPs and NO-Lip@PAC NMs were further investigated, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea ,\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, S33 \u003cb\u003eand Video S9-S10\u003c/b\u003e, the Lip@PAC NPs were showed Brownian motion in bEnd.3 and Gl261 cellular environment, with an average speed distribution of 0.5\u0026ndash;1.5 \u0026micro;m s\u003csup\u003e-1\u003c/sup\u003e. The observed phenomenon may be attributed to the encapsulation of PAC NMs within liposomes, which lack guanidine groups on their surface to serve as a power source for the movement of nanomotors. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, S33 \u003cb\u003eand Video S11-S12\u003c/b\u003e, NO-Lip@PAC NMs displayed Brownian motion in bEnd.3 cellular environment, and a significant displacement was observed in Gl261 cellular environment, with an average speed distribution of 1.9\u0026ndash;3.7 \u0026micro;m s\u003csup\u003e-1\u003c/sup\u003e. As shown in \u003cb\u003eFigure S34\u003c/b\u003e, the diffusion coefficients of Lip@PAC NPs in bEnd.3 and Gl261 cellular environments were 0.24 and 0.32 \u0026micro;m\u0026sup2;/s, respectively, indicating Brownian motion.\u003csup\u003e[\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e For NO-Lip@PAC NMs, the diffusion coefficient was 0.26 \u0026micro;m\u0026sup2;/s in the bEnd.3\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ecellular environment, but it increased significantly to 3.8 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e/s in the Gl261 cellular environment,\u003csup\u003e[\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e indicating Brownian motion in normal cells and enhanced Brownian motion in cancer cells.\u003c/p\u003e \u003cp\u003eNext, we employed a straight single-channel model to evaluate the nanomotors\u0026rsquo; chemotactic responses under varying conditions. As shown in \u003cb\u003eFigure S35\u003c/b\u003e, an iNOS gradient was established within the straight channel. DiL-labeled Lip@PAC or NO-Lip@PAC was introduced into reservoir (i), and the motion videos of different samples were recorded at position \u0026ldquo;\u003cb\u003ea\u0026rdquo;\u003c/b\u003e to analyze their motion behaviors \u003cb\u003e(Figure S36\u003c/b\u003e). As shown in \u003cb\u003eFigure S37 and Video S13\u003c/b\u003e, when reservoir (ii) contained bEnd.3 cellular lysate-embedded gel, both Lip@PAC and NO-Lip@PAC exhibited minimal displacement at position \u003cb\u003e\u0026ldquo;a\u0026rdquo;\u003c/b\u003e, with a speed of 1.0 \u0026micro;m s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, exhibit non-directional Brownian motion. In contrast, when reservoir (ii) was loaded with Gl261 cellular lysate-embedded gel, NO-Lip@PAC displayed significantly enhanced Brownian motion, with a speed of 3.1 \u0026micro;m s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and both moved toward the higher iNOS concentration, exhibiting directional chemotactic movements. To further quantify migration persistence, the chemotaxis index (CI), defined as the ratio of total displacement to path length, was calculated.\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e Notably, NO-Lip@PAC in the iNOS gradient demonstrated the highest CI value (~\u0026thinsp;0.5), surpassing both Lip@PAC and NO-Lip@PAC in the absence of a chemotactic gradient. These results confirmed that the iNOS gradient drives directional migration of NO-Lip@PAC, highlighting its chemotactic specificity.\u003c/p\u003e \u003cp\u003eSubsequently, we investigated the chemotactic behavior in static environments with iNOS concentration gradient through Y-shaped channels. As shown in \u003cb\u003eFigure S38\u003c/b\u003e, the iNOS gradient was established in the Y-shaped channels.\u003csup\u003e[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, regions (i), (ii) and (iii) contained samples, bEnd.3 or Gl261 cellular lysate gels, respectively. The chemotaxis behavior of NO-Lip@PAC NMs was characterized by recording fluorescence images of regions (ii) and (iii) at different times. The fluorescence intensity of the Lip@PAC NMs in regions (ii) and (iii) did not differ significantly, the fluorescence quantification illustrated the same results (\u003cb\u003eFigureS39\u003c/b\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, the fluorescence intensity in regions (ii) and (iii) gradually increased with time, but the fluorescence intensity in region (iii) was significantly higher than that in region (ii), suggesting that NO-Lip@PAC NMs tended to accumulate more in tumor cells with high concentrations of iNOS. These results suggested that due to the presence of L-Arg in the liposomes, the NO-Lip@PAC NMs could autonomously enrich to sites with higher iNOS concentration and showed good chemotactic performance.\u003c/p\u003e \u003cp\u003eIn addition, we established a dynamic microfluidic model to investigate the chemotactic ability of nanomotors in a flow state (\u003cb\u003eFigure S40)\u003c/b\u003e. To simulate the capillary blood flow rate, the volume flow rate of the microfluidic syringe pump was controlled to be about 0.6 mL h\u003csup\u003e-1\u003c/sup\u003e.\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e When inlets (i), (ii) were pumped with bEnd.3 cellular lysates, and inlet (iii) was pumped with Lip@PAC NPs or NO-Lip@PAC NMs, there was no fluid shift (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). When inlets (i), (ii) and (iii) were pumped with bEnd.3 cellular lysate, different samples and Gl261 cellular lysate, respectively, the fluorescence signals of the NO-Lip@PAC NMs fluid could be observed to shift towards the Gl261 cellular lysate side (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). The fluorescence quantification results showed that the NO-Lip@PAC NMs were shifted towards the Gl261 cellular lysate side, indicating that they could diffuse from channel (ii) to the channel with higher iNOS concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe ability of NO-Lip@PAC NMs to cross the BBB\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand the mitochondrial targeting performance\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the ability of nanomotors crossing the BBB at the cellular level, \u003cem\u003ein vitro\u003c/em\u003e BBB model was established using a transwell model containing a porous membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. The upper chamber of the transwell model was inoculated with bEnd.3 cells to simulate a dense BBB layer (which was formed after 10 days of culture),\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e and the lower chamber was inoculated with Gl261 cells to simulate GBM, forming an environment with iNOS concentration gradient. Then we added Lip@PAC NPs and NO-Lip@PAC NMs (50 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e) to the upper chamber respectively, and incubated for 6 h. The fluorescence intensity of the samples in the upper and lower chambers of the transwell model were detected by CLSM. Meantime, we collected the liquid and cells in the lower chamber and quantitatively detected the amount of samples in each part. As shown in the CLSM images of bEnd.3 cells in upper chamber (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and S41), DiL-labelled Lip or NO-Lip colocalized well with Cy5-labelled PAC NMs, indicating minimal of Lip@PAC NPs and NO-Lip@PAC NMs before crossing the BBB. The separation of red and green fluorescence in Gl261 cells treated with NO-Lip@PAC NMs group suggests lipid degradation in the tumor environment, which cannot be found in Lip@PAC NPs treated groups. In addition, the red fluorescence intensity in bEnd.3 cells in the upper chambers treated with NO-Lip@PAC NMs was lower than that treated with Lip@PAC NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), while Gl261 cells in the lower chambers had stronger red fluorescence signals, and fluorescence quantification showed that their intensity was 8.7 times higher than that of Lip@PAC NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The calculation of the transport rate in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee showed that the transport efficiency of Lip@PAC NPs was only about 13.7%, while that of NO-Lip@PAC NMs reached about 63.5%. When Gl261 cells in the lower chamber were pretreated with iNOS inhibitor (NG-Monomethyl-L-arginine, L-NMMA) for 24 h. The penetration efficiency of NO-Lip@PAC decreased to 21.7% (\u003cb\u003eFigure S42\u003c/b\u003e), which confirmed that iNOS concentration gradient-driven chemotaxis was the NO-Lip@PAC to achieve efficient BBB penetration. Next, we used CLSM to check the structural integrity of BBB after treatment with different samples. As shown in \u003cb\u003eFigure S43\u003c/b\u003e, the cell layer that had formed a dense BBB, there was no significant change in the BBB cellular structure treated with Lip@PAC NPs or NO-Lip@PAC NMs for 24 h. Meanwhile, FITC-dextran (FD-4, 4000 Da) --- a polysaccharide composed of fluorescein isothiocyanate coupled to dextran\u0026mdash;was used as a marker to identify BBB leakage, if leakage occurred, we would expect to observe stronger FD-4 fluorescence signals in the lower chamber.\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e As shown in \u003cb\u003eFigure S44 and\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, compared to the control group, the upper compartment of bEnd.3 cells treated with Lip@PAC NPs and NO-Lip@PAC NMs remained tightly connected, with no significant increase in the intensity of FD-4 fluorescence detected in the lower compartment. This further demonstrated that NO-Lip@PAC NMs penetrated the BBB through chemotactic effects on the iNOS concentration gradient, rather than disrupting the integrity of BBB.\u003c/p\u003e \u003cp\u003eFurther, we evaluated whether NO-Lip@PAC NMs could degrade and release PAC NMs after crossing the BBB. We used DiL to label Lip or NO-Lip, Cy5 to label the PAC NMs, respectively. Different samples were co-incubated with Gl261 cells for varying durations to observe the release of PAC NMs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, \u003cb\u003eFigure S45 and S46\u003c/b\u003e, when Lip@PAC NPs and NO-Lip@PAC NMs were co-incubated with Gl261 cells for 2 h, the perinuclear showed more green fluorescence representing the outer liposomes, while only minimal red fluorescence representing the PAC NMs. With the extension of the incubation time to 6 h, the red fluorescence representing PAC NPs in the NO-Lip@PAC NMs treated group can be observed, and the intensity of red fluorescence was 1.5 times of that at 2 h. As the incubation time was extended to 12 h, the red fluorescence was gradually enhanced (2.6 times of that at 2 h), while the green fluorescence was gradually reduced, which confirmed that NO-Lip@PAC NMs could gradually degrade and release their contents in response to the self-released NO. This phenomenon was not observed in Lip@PAC NPs treated groups. The ability of PAC NMs to target mitochondria was further assessed. Studies have shown that the concentration of iNOS in cancer cell mitochondria was much higher than that in normal cell mitochondria.\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e We hypothesized that PAC NMs could accumulate around mitochondria due to their chemotactic effects on iNOS. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh and S47, we used Cy5 to label PAC, and the red fluorescence of PAC NMs in NO-Lip@PAC NMs overlapped more with the green\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eof mitochondria, suggesting better mitochondrial targeting. Next, we investigated the iNOS induced chemotaxis kinetics of the nanomotors through Y-shaped channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). The regions (i), (ii) and (iii) comprising different samples, mitochondria lysate, or other organellar lysate gels, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek, the fluorescence in regions (ii) and (iii) gradually increased over time, regions (ii) and (iii) of the PLC NPs did not differ much, and fluorescence quantification also illustrated the same result. The fluorescence in region (ii) of the PAC NMs group is higher than that in region (iii), suggesting that the PAC NMs tended to aggregate more in mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em). These results demonstrated that the guanidino group in PAC NMs could drive them to enrich autonomously to the sites with higher iNOS concentration, exhibiting effective chemotactic performance toward mitochondria.\u003c/p\u003e\n\u003ch3\u003eEvaluation of the ability to induce mitochondrial mineralization in Gl261 cells by NO-Lip@PAC@Cur NMs\u003c/h3\u003e\n\u003cp\u003eThe previous results confirmed that NO-Lip@PAC NMs displayed good chemotaxis behavior in the tumor microenvironment, and the NO generated during this process promoted the degradation of liposomes, leading to the release of PAC NMs. The released PAC NMs can be further chemotactically targeted to mitochondria, and the carboxyl groups on their surface can recruit Ca\u003csup\u003e2+\u003c/sup\u003e from the cytoplasm,\u003csup\u003e[\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e leading to an increase in the local Ca\u003csup\u003e2+\u003c/sup\u003e concentration around the mitochondria, and thus inducing their mineralization. To further enhance the effect of mitochondrial mineralization, we also loaded Cur in the lipid hydrophobic region to inhibit Ca\u003csup\u003e2+\u003c/sup\u003e efflux \u003csup\u003e[\u003cspan additionalcitationids=\"CR50 CR51\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e and constructed NO-Lip@PAC@Cur NMs. We prepared NO-Lip@PAC@Cur NMs with different drug-lipid ratios and tested their encapsulation rate and drug loading to explore the optimal incorporation conditions of Cur. As shown in \u003cb\u003eFigure S48\u003c/b\u003e, the encapsulation rate of Cur gradually decreased and the drug loading amount increased with higher drug-lipid ratios. However, its stability decreased and liposomes appeared to agglomerate (\u003cb\u003eFigure S49\u003c/b\u003e). Considering this, we chose a drug-lipid ratio of 1:10 for subsequent experiments, achieving an encapsulation rate of 73.8% and a drug loading capacity of 7.6%. The TEM images showed that NO-Lip@PAC@Cur had a diameter of about 500 nm. The hydrated particle size was about 545.0 nm and the zeta potential was \u0026minus;\u0026thinsp;14.0 mV (\u003cb\u003eFigure S50).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSubsequently, we investigated in detail whether the addition of Cur could play a synergistic role with NO to improve intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels. Firstly, intracellular NO levels were detected using an NO probe. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea \u003cb\u003eand Figure S51\u003c/b\u003e, Gl261 cells treated with NO-Lip@PAC and NO-Lip@PAC@Cur exhibited significantly stronger NO fluorescence signals (2.7 and 2.9 times that of the control group, respectively), which were significantly higher than those observed in the other groups. In contrast, the NO fluorescence signals in bEnd.3 cells treated with different samples did not show significant differences, likely due to the lower concentration of iNOS to react with the nanomotors and produce NO. Next, we examined the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e content using inductively coupled plasma-Mass Spectrometry (ICP-MS). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb showed that the Ca\u003csup\u003e2+\u003c/sup\u003e content in Gl261 cells treated with NO-Lip@PAC increased to 0.27 \u0026micro;mol, which was 1.6 times higher than that in the control group. This increase is likely due to the high concentration of NO causing endoplasmic reticulum stress and releasing more Ca\u003csup\u003e2+\u003c/sup\u003e into the cytoplasm, decrease in endoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e leads to Ca2\u0026thinsp;+\u0026thinsp;influx from the extracellular space.\u003csup\u003e[\u003cspan additionalcitationids=\"CR54 CR55\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e Cells treated with NO-Lip@PAC@Cur exhibited an even higher intracellular Ca\u003csup\u003e2+\u003c/sup\u003e content, which was 2.7 times higher than that of the control group, suggesting that the inhibition of Ca\u003csup\u003e2+\u003c/sup\u003e exocytosis by Cur can further increase the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels, confirming that Cur and NO can exert a synergistic effect to enhance the mineralization effect.\u003c/p\u003e \u003cp\u003eTo assess intracellular mitochondrial mineralization, Gl261 cells were lysed after treated with different samples, and mitochondria and other organelles were collected. Then the Ca\u003csup\u003e2+\u003c/sup\u003e concentration was detected using ICP-MS. As shown in \u003cb\u003eFigure S52\u003c/b\u003e, the Ca\u0026sup2;⁺ content in mitochondria was 0.17 \u0026micro;mol at 6 h and increased to 0.33 \u0026micro;mol after extending the incubation time to 36 h, which was comparable to 0.36 \u0026micro;mol at 48 h.\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e In addition, to determine that mitochondrial damage was caused by mineralization rather than Ca\u003csup\u003e2+\u003c/sup\u003e overload. we added a control group\u0026ndash;A23187 (a chemical reagent commonly used to induce mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e overload).\u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and S53, the mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e content was significantly elevated after A23187 treatment, which was 5.3 times higher than that of the control group, resulting in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e overload. The mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e treated with NO-Lip@PAC NMs was about 0.19 \u0026micro;mol, which was 6.3 times that of the control group, and had the similar ability to induce mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e overload as that of A23187 group. In contrast, the mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e treated with NO-Lip@PAC@Cur NMs was further increased to 0.36 \u0026micro;mol, which was 12 times higher than that of the control group and about 2 times higher than that of the NO-Lip@PAC and A23187 groups. This result indicates that mitochondrial mineralization requires a higher level of Ca\u003csup\u003e2+\u003c/sup\u003e concentration than Ca\u003csup\u003e2+\u003c/sup\u003e overload, NO-Lip@PAC and A23187 were able to trigger Ca\u003csup\u003e2+\u003c/sup\u003e overload but did not reach the level of mineralization, while NO-Lip@PAC @Cur can recruit more Ca\u003csup\u003e2+\u003c/sup\u003e to gather around mitochondria, which can trigger mitochondrial mineralization.\u003c/p\u003e \u003cp\u003eTo visually observe mitochondrial calcification, NO-Lip@PAC@Cur-treated Gl261 cells were sectioned and intracellular mitochondrial structure was observed using Bio-TEM. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and S54, the cell membrane of Gl261 cells treated with NO-Lip@PAC@Cur exhibited significant disruption compared to the untreated group, and the morphology of the mitochondria was notably altered. In addition, the TEM-mapping images of the mitochondria in the NO-Lip@PAC@Cur group and the control group were compared, and the mitochondria in the NO-Lip@PAC@Cur group had stronger Ca fluorescence signals in the mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Subsequently, Ca\u003csup\u003e2+\u003c/sup\u003e in the cytoplasm was labeled with green fluorescence using Flou-4, Ca\u003csup\u003e2+\u003c/sup\u003e in the mitochondria was labeled with red fluorescence using Rhod-2, and the co-localization was observed by CLSM. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg and S55, the red fluorescence and green fluorescence of NO-Lip@PAC and NO-Lip@PAC@Cur groups were significantly higher than those of other treatment groups, which proved that the concentration of Ca\u003csup\u003e2+\u003c/sup\u003e in the cytoplasm and mitochondria was increased after treatment. Co-localization analysis showed that the red fluorescence and greenfluorescence in the NO-Lip@PAC@Cur NMs group had a higher degree of overlap, suggesting that the mineralization process occurred in the mitochondria. Further, in order to prove that the Ca\u003csup\u003e2+\u003c/sup\u003e accumulated around mitochondria are insoluble calcium salts, we used Alizarin Red S to stain cells, which can form red complexes by specifically chelating insoluble calcium salts (such as calcium phosphate), which is a classic method to evaluate cellular calcium deposition.\u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e As shown in \u003cb\u003eFigure S56\u003c/b\u003e, cells treated with Lip@PLC, A23187 and NO-Lip@PAC was similar to that of the control group, and the production of red complexes was not observed in the cells, which may be because although they can induce the mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e overload, the concentration did not reach the formation of insoluble calcium salts, so the signal was weak. And the formation of red complex was obviously observed in the cells treated with NO-Lip@PAC@Cur, which indicated that the NO-Lip@PAC@Cur can induce mitochondria to form insoluble calcium salts, confirming the occurrence of mitochondrial mineralization.\u003c/p\u003e \u003cp\u003eSubsequently, the effects of mineralization on mitochondrial function were explored in detail. The stability of mitochondrial membrane potential (MMP) is a prerequisite for mitochondria to maintain normal physiological functions, and the JC-1 dye has been widely used to assess MMP. Under normal conditions, JC-1 accumulates in the mitochondrial stroma and formed aggregates that emit red fluorescence (529 nm); when the mitochondrial transmembrane potential was impaired and depolarized, JC-1 was released from mitochondria at a reduced concentration, and then JC-1 was a monomer and emitted green fluorescence (585 nm).\u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei and S57, JC-1 showed strong red fluorescence and only a small amount of green fluorescence in both control and Lip@PLC NPs group, whereas the red JC-1 aggregates in the NO-Lip@PAC@Cur NMs group were converted in large quantities to green JC-1 monomers. In addition, we assessed the effect of different control groups on the behavior of adenosine triphosphate (ATP) generation in tumor cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej, the intracellular ATP concentration decreased from 8.9 to 2.4 \u0026micro;M after NO-Lip@PAC@Cur NMs treatment compared to the control group. Apparently, NO-Lip@PAC@Cur NMs induced mitochondrial calcification effectively depolarized the cellular mitochondrial membranes, leading to mitochondrial dysfunction and inhibition of intracellular ATP production.\u003c/p\u003e \u003cp\u003eThe impact of the mitochondrial mineralization process on cellular activity was also explored. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-(4,5-dimethylthiazol-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) results showed that NO-Lip@PAC@Cur NMs had a concentration-dependent cytotoxicity against Gl261 cells, with a progressive decrease in cellular activity as the concentration increased. In addition, the cytotoxicity of different samples on GL261 and bEnd.3 cells was compared (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003el), the cell viability of Gl261 cells in the NO-Lip@PAC NMs group was 74.6%, while the viability decreased to 44.5% when Cur was loaded, indicating that mitochondrial mineralization in tumor cells resulted in significant cytotoxicity effects. Meanwhile, the viability of bEnd.3 cells treated with NO-Lip@PAC@Cur NMs was 83.8%, with no significant damage to normal cells, due to the absence of overexpressed iNOS in normal cells, which did not undergo mitochondrial mineralization.\u003c/p\u003e\n\u003ch3\u003eTargeting ability of NO-Lip@PAC NMs in GBM model mice\u003c/h3\u003e\n\u003cp\u003eTo investigate the \u003cem\u003ein vivo\u003c/em\u003e targeting ability of NO-Lip@PAC NMs, we established a GBM model in mice through orthotopic implantation of GL261-Luc cells. The establishment of the GBM model was determined by \u003cem\u003ein vivo\u003c/em\u003e bioluminescence 10 days after in situ inoculation of Gl261-Luc cells (\u003cb\u003eFigure S58\u003c/b\u003e). Subsequently, Lip@PAC NPs and NO-Lip@PAC NMs were injected intravenously and their fluorescence images were captured using an \u003cem\u003ein vivo\u003c/em\u003e imaging system (IVIS) various time points post-injection. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-c, significant fluorescence aggregation signals could be observed in GBM model mice at 1 h after intravenous injection of NO-Lip@PAC, indicating its ability to rapidly penetrate the BBB. The fluorescence intensity of brain tumors in the NO-Lip@PAC group continued to increase over time, reaching a peak at 12 h after injection, with an intensity 3.5 times greater than that of the Lip@PAC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The fluorescence signal gradually weakened thereafter. It is noteworthy that the fluorescence signal in the brain tumor region of the Lip@PAC group was weak and basically disappeared after 48 h. In contrast, a significant fluorescence signal remained detectable in the NO-Lip@PAC group at 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), confirming its ability to maintain stable retention in the brain microenvironment for at least 48 h. These findings were further validated through ex vivo organ imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef and S59). After 12 h of intravenous injection, the Cy5 fluorescence intensity of the brain tissue of GBM mice in the NO-Lip@PAC group was significantly higher than that in 7 h, demonstrating its notable specificity for targeting brain tumors. The fluorescence intensity of the NO-Lip@PAC group was still 3.0 times that of the Lip@PAC group at 24 h after injection, which fully reflected its active targeting characteristics driven by iNOS concentration gradient. In addition, \u003cem\u003eex vivo\u003c/em\u003e organ imaging showed that the both samples were mainly cleared by liver and kidney metabolism (\u003cb\u003eFigure S60 and S61\u003c/b\u003e) Further, we collected different organs and examined the proportion of different samples to determine the targeting efficiency in different organs. The results showed that the NO-Lip@PAC NMs group accumulated about 30.0% ID/g in the brain, which was 5.1 times higher than that of the Lip@PAC NPs group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). To further observe the distribution of the nanorobot fluorescence signals in the tumor tissues, whole brain tissues were cryosectioned and stained using immunofluorescence techniques. FITC-CD31 was used to label the vascular endothelium, DAPI was used to label the nucleus, and red fluorescence was derived from Cy5-labelled nanorobot. Equal area region of interest (ROI) was selected perpendicular to the blood vessel and used as the starting point, red fluorescence signals within the ROIs were quantitatively analyzed to evaluate the permeability of different samples from the blood vessel to the GBM. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei, the chemotactic NO-Lip@PAC NMs showed the strongest fluorescence intensity and depth of penetration in the GBM. Obvious red fluorescence was still observed at the distal end of the brain vessels (~\u0026thinsp;300 \u0026micro;m), indicating that NO-Lip@PAC NMs could effectively cross the BBB and penetrate deeply into the tumor tissues.\u003c/p\u003e\n\u003ch3\u003eAntitumor efficacy of NO-Lip@PAC@Cur NMs in GBM model mice\u003c/h3\u003e\n\u003cp\u003eBefore evaluating the therapeutic effects \u003cem\u003ein vivo\u003c/em\u003e, we quantified the levels of Cur in circulating blood at different time intervals after intravenous injection of free Cur and NO-Lip@PAC@Cur in healthy SD rats. As shown in \u003cb\u003eFigure S62\u003c/b\u003e, NO-Lip@PAC@Cur can prolong the circulation time of Cur in the body. Then, their therapeutic efficacy \u003cem\u003ein vivo\u003c/em\u003e was further evaluated. In this section, we chose TMZ, a first-line drug for clinical treatment of GBM, as a control for chemotherapeutic agents. Successful brain tumor construction was confirmed by \u003cem\u003ein vivo\u003c/em\u003e bioluminescence imaging on day 11 after Gl261-Luc cell transplantation. Thereafter, mice were randomly divided into 6 groups and sham-operated groups, and different drugs were intravenously every two days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb,\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec \u003cb\u003eand Table S2\u003c/b\u003e, \u003cem\u003ein vivo\u003c/em\u003e bioluminescence imaging and quantification of total radiant brightness showed that the bioluminescence signal in the PBS group continued to enhance over time, and was strongest at the end of treatment (60.5 times the fluorescence intensity at the initial moment), suggesting that the tumor was growing rapidly. The fluorescence intensity of the representative tumor in the mouse brain decreased following treatment with free TMZ or NO-Lip@PLC@Cur treatment, suggesting mild inhibition of tumor growth (49.8 and 32.6 times the fluorescence intensity at the initial moment, respectively). In contrast, the anti-GBM growth effect was sequentially enhanced in the PAC@Cur and Lip-PAC@Cur groups, and the bioluminescent signals were significantly decreased (20.8 and 23.8 times of the fluorescence intensity at the initial moment, respectively). Kaplan-Meier survival curves showed that NO-Lip@PAC@Cur significantly prolonged the survival of the GBM model mice, with a median survival time (MST) of 49 days, whereas PAC@Cur and Lip-PAC@Cur groups were both 39 days, which was slightly longer than that of the TMZ and NO-Lip@PAC@Cur groups (both 37 days), while the PBS control group was only\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e33 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Further, dissected tumors from different treatments were stained with hematoxylin and eosin (H\u0026amp;E) to determine the destruction of tumor cells. The H\u0026amp;E results showed that the tumor area in the NO-Lip@PAC@Cur group was smaller than that in the other treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee and S63). Brain tumors in the PBS group were the largest at the end of treatment, accounting for 41.8% of the whole brain. Brain tumors in the free TMZ-treated mice were reduced and accounted for 31.3% of the whole brain. In contrast, the anti-GBM growth effect was enhanced in the PAC@Cur, Lip@PAC@Cur and NO-Lip@PLC@Cur group, with brain tumors accounting for 22.0%, 24.9% and 25.0% of the whole brain in mice, respectively. NO-Lip@PAC@Cur was able to significantly inhibit the growth of brain tumors, which accounted for 9.5% of the whole brain at the end of treatment in mice. TUNEL and Ki67 immunofluorescence were used to label apoptotic and proliferating cells in the tumor tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef). Quantitative analysis showed that mice treated with NO-Lip@PAC@Cur had the highest rate of apoptotic cells in their tumors (approximately 49.9%) and the lowest proportion of positive tumor cells proliferating (approximately 10.0%). These differences demonstrated that NO-Lip@PAC@Cur was able to inhibit the growth of GBM by killing tumor cells through inducing mitochondrial mineralization in brain tumor cells.\u003c/p\u003e \u003cp\u003eSafety is another key issue in the treatment of glioblastoma. The biocompatibility and systemic response of the different agents were assessed by hemolysis rate and erythrocyte morphology analysis, weight changes in mice, hematological and histopathological tests. Results showed no significant damage to erythrocytes with the different materials, indicating that the materials have good blood compatibility \u003cb\u003e(Figure S64 and S65)\u003c/b\u003e. The mice in the PBS and TMZ groups showed significant weight loss during treatment, which may be attributed to the fact that chemotherapeutic agents inevitably damage normal tissues and their functions during treatment, leading to weight loss. In contrast, the NO-Lip@PAC@Cur NMs constructed in this paper did not observe a significant weight loss trend in mice during treatment (\u003cb\u003eFigure S66\u003c/b\u003e). High-dose TMZ treatment leads to bone marrow suppression. \u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e TMZ treatment group resulted in a significant increase in bone marrow vacuoles (\u003cb\u003eFigure S65\u003c/b\u003e) and a decrease in blood cell counts (\u003cb\u003eFigure S68\u003c/b\u003e), suggesting that free TMZ may have side effects on bone marrow. Blood biochemistry data showed that alanine aminotransferase (ALT), aspartate aminotransferase (AST) lactate dehydrogenase (LDH) and serum albumin concentration (ALB) indices were increased in the TMZ group, suggesting that the TMZ treatment may cause some damage to the liver function of mice (\u003cb\u003eFigure S69\u003c/b\u003e). In contrast, the hematological parameters and H\u0026amp;E staining results of mice treated with NO-Lip@PAC@Cur NMs indicated that the liver and kidney tissues of mice did not show significant pathological damage (\u003cb\u003eFigure S70\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to observe mitochondrial calcification more visually, the brain tumor tissues of mice in the PBS and NO-Lip@PAC@Cur groups were sectioned, and the structures of cells and mitochondria within the tumor tissues were observed using Bio-TEM. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg, compared with the tumor cells in PBS group, cell membranes in the NO-Lip@PAC@Cur group were broken, nuclei were fragmented, and mitochondrial structures were lost, indicating cell apoptosis. TEM-Mapping results showed that mitochondria in the NO-Lip@PAC@Cur group had stronger Ca fluorescence signals \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei), further demonstrating that NO-Lip@PAC@Cur could inhibit tumor growth by inducing mitochondrial mineralization in brain tumor cells.\u003c/p\u003e \u003cp\u003eIn addition, in order to verify whether there is tumor calcification \u003cem\u003ein vivo\u003c/em\u003e, von Kossa staining was carried out on the tumor tissues of each group.\u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e As shown in \u003cb\u003eFigure S71\u003c/b\u003e, the brain tumor tissue in NO-Lip@PAC@Cur group showed dark brown areas, indicating that there was calcification in the tumor. The other groups were similar to the PBS group, showing the same dark red color as the normal tumor. These results suggest that the apoptosis of tumor cells is mainly due to mitochondrial mineralization rather than Ca\u003csup\u003e2+\u003c/sup\u003e overload.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we successfully developed size-variable self-feedback nanomotors NO-Lip@PAC@Cur NMs, and verified their therapeutic efficacy against GBM. The lipid shell of NO-Lip@PACNMs degraded in response to the NO generated during chemotactic targeting to the GBM microenvironment, thereby releasing the loaded Cur and PAC NMs. The released NO induced endoplasmic reticulum stress and subsequent Ca\u003csup\u003e2+\u003c/sup\u003e release, while Cur inhibited Ca\u003csup\u003e2+\u003c/sup\u003e efflux. This dual action resulted in an increase in the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration (2.7 times higher than that in the control group). Additionally, PAC NMs targeted mitochondria via chemotaxis, where the carboxyl groups recruited Ca\u003csup\u003e2+\u003c/sup\u003e from the cytoplasm, thereby increasing the local mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e concentration to 6.2 times higher than that in the control group. Fluorescence imaging and TEM-mapping of Ca\u0026sup2;⁺ in tumor cell mitochondria revealed that mitochondria in the control group. Fluorescence imaging and TEM-mapping of Ca\u0026sup2;⁺ in tumor cell mitochondria revealed that mitochondria in the NO-Lip@PAC@Cur NMs-treated group exhibited stronger fluorescence signals. This indicates that NO-Lip@PAC@Cur NMs effectively induced mitochondrial dysfunction, loss of mitochondrial membrane potential, and a significant decrease in ATP-generating capacity from 8.9 \u0026micro;M to 2.4 \u0026micro;M. In vivo experiments demonstrated that NO-Lip@PAC NMs could recognize the highly expressed iNOS in the tumor microenvironment, cross the BBB, and accumulate in brain tumors. Specifically, the accumulation in brain tissues of GBM model mice reached 30.0% ID/g, which was 5.1 times higher than that of the Lip@PAC NMs group. The treatment results showed that NO-Lip@PAC@Cur NMs effectively inhibited the growth of tumor by inducing mitochondrial mineralization and subsequent tumor cell death (the total radiant brightness of brain tumors in the NO-Lip@PAC@Cur NMs group at the end of the treatment was 15.8% of that in the PBS group). Compared with the chemotherapeutic drug TMZ, NO-Lip@PAC@Cur NMs exibited favorable biocompatibility. At the end of the treatment the weight of the mice remained basically unchanged, and blood biochemistry and hematology indices were within normal ranges. Additionally, histological analysis using H\u0026amp;E staining revealed no significant damage to major organs. Given that tumor cell mitochondria are crucial for their growth and proliferation, the strategy of specifically targeting mitochondrial mineralization in tumor cells is expected to provide valuable ideas for designing therapeutic strategies specifically for GBM.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe mouse glioma cell line Gl261and Gl261-Luc were purchased from Shanghai Aorui cell Biotechnology CO., Ltd.; the mouse brain microvascular endothelial cells bEnd.3 were purchased from Procell Life Science \u0026amp; Technology CO., Ltd. Gl261 and Gl261-Luc cells were cultured in complete culture medium containing 89% v/v high-sugar Dalberg's modified Eagle's medium (DMEM with 4.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e D-glucose, Jiangsu KeyGEN BioTECH Corp., CO., Ltd.), 10% v/v fetal bovine serum (SKU: SP011010500, Sperikon Life Science \u0026amp; Biotechnology CO., Ltd.), and 1% v/v penicillin-streptomycin mixture in complete culture medium. bEnd.3 cells was cultured in 89% v/v high-sucrose Dalberg's modified Eagle's medium (DMEM, containing 4.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e D-glucose), 10% v/v fetal bovine serum (LONSERA. Suzhou Shuangru Biotechnology Co., Ltd), and 1% v/v penicillin-streptomycin mixture in complete culture medium. All the cells were cultured in a humidified atmosphere that contained 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. When not in use, cell cryopreservation with cell saving (PB180438, Pricella Life Science\u0026amp;Technology Co., Ltd.) at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eiNOS content in different cells\u003c/h2\u003e \u003cp\u003eThe bEnd.3 (1.0 mL, 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) or Gl261 cells (1.0 mL, 10\u003csup\u003e3\u003c/sup\u003e, 10\u003csup\u003e4\u003c/sup\u003e, 10\u003csup\u003e5\u003c/sup\u003e or 10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were broken by sonication, and the supernatant was collected after centrifugation at 3000 rpm for 10 min. The iNOS concentration was detected using an enzyme immunoassay kit (Jiangsu Enzyme Immunoassay, MM-0454M2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMotion behavior of different samples\u003c/h2\u003e \u003cp\u003eTo analyze motion behavior, bEnd.3 or Gl261 cells (1.0 mL, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were inoculated in glass-bottomed confocal petri dishes and incubated overnight. 20.0 \u0026micro;L of Cy5 or DiO-labeled different samples was slowly added to the above petri dishes. The motion behavior of different samples was recorded using the 100 \u0026times; objective of an inverted fluorescence microscope (Micro-shot MF53-N), and the motion trajectories of the different samples were marked using the tracking plug-in of the Fiji software. The trajectories of 50 particles were randomly selected to calculate the average speed of nanomotors and analyze the speed distribution histogram.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eiNOS content in straight or Y-shaped channels\u003c/h2\u003e \u003cp\u003eTo determine the iNOS content in the straight or Y-shaped channel, 25.0 \u0026micro;L of agarose solution was mixed with an equal volume of bEnd.3 or Gl261 cellular lysate in the storage chamber (ii) and (iii), respectively. And then the Y-shaped channel was transferred to 4\u0026deg;C to wait for the agarose to form a gel. Subsequently, it was filled with 300.0 \u0026micro;L of PBS and after standing at 4\u0026deg;C for 15 min, samples were collected from different locations of the Y-shaped channel. The iNOS concentration was detected using an enzyme-linked immunoassay kit (Jiangsu Enzyme Free, MM-0454M2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCollective chemotaxis behavior of nanomotors in the Y-shaped channel\u003c/h2\u003e \u003cp\u003eTo observe the collective chemotaxis behavior of nanomotors, a Y-shaped glass substrate microchannel was used. The main channel was 1.0 cm in length, 0.4 cm in width, and the branch channel was 0.7 cm in length and 0.3 cm in width. The concentration gradient of the chemoattractant was created by different types of cell lysates that were placed in reservoirs (II) or (III) in the branched channel. Briefly, 5.0 mg of agarose was completely dissolved in 0.5 mL of PBS at 90\u0026deg;C, and 50.0 \u0026micro;L of Gl261 or bEnd.3 cellular lysate was added when the melted agarose was cooled to room temperature but not solidified. And then, it was transferred to 4\u0026deg;C for gelation. Before assessing the chemotactic motion of the nanomotors, the Y-shaped channel was prefilled with 300.0 \u0026micro;L PBS and quiescence for 10 min. Then 50.0 \u0026micro;L of Cy5-labeled nanomotors was gently dropped into the reservoir (I). The fluorescence microscope of the reservoir (II) and (III) were captured with an inverted fluorescence microscope (Micro-shot MF53-N), equipped with 10 \u0026times; objective, at specific times. The corresponding fluorescence intensity was quantified using Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDynamic chemotaxis behavior of nanomotors in the microfluidic channel\u003c/h2\u003e \u003cp\u003eThe three-inlet one-outlet glass substrate microfluidic channel with dimensions of 2.2 cm (length) \u0026times; 1.5 mm (width) \u0026times; 0.3 mm (height) was used to evaluate the dynamic chemotaxis of nanomotors. Among them, the diluent of Gl261 cellular lysate (lysate: PBS\u0026thinsp;=\u0026thinsp;1:4, v/v) flowed through inlet (I), the Cy5-labeled nanomotors in PBS was flowed through inlet (II), and the dilution of bEnd.3 cellular lysate (lysate: PBS\u0026thinsp;=\u0026thinsp;1:4, v/v) was flowed through inlet (III). The flow velocity of each channel was controlled at 0.6 mL h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The video was captured continuously for 5 min (1 frame per second) at the position near to outlet using an inverted fluorescence microscope (10 \u0026times; objective). The fluorescence intensity of Cy5 perpendicular to the flow direction was measured using Image J. Moreover, the dilution of bEnd.3 cell lysate (lysate: PBS\u0026thinsp;=\u0026thinsp;1:4, v/v) flowed through both inlet (I) and (III) as control, while the flow of inlet (II) was not replaced.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDetection of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration\u003c/h2\u003e \u003cp\u003eGL261 cells (1.0 mL, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were inoculated into a 6-well plate and incubated overnight. Then different samples (Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur, 1.0 mL, 200 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in DMEM) were added and incubated for 48 h. The cells were washed three times with PBS to remove free samples. Cells were collected with cell scraper and placed in a reactor with nitric acid (1.0 mL) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (1.5 mL), which was nitrated in vacuum oven at 180\u0026deg;C for 1.5 h. The above system was subsequently volume-determined to 5.0 mL, and the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration was measured using inductively coupled plasma-Mass Spectrometry (ICP-MS, IRIS Intrepid II).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDetection of Ca\u003csup\u003e2+\u003c/sup\u003e concentration in mitochondria\u003c/h2\u003e \u003cp\u003eGL261 cells (1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells) were inoculated into T175 cell culture flasks and incubated overnight. Then different samples (Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur, 20.0 mL, 200 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in DMEM or A23187, 20.0 mL, 5 \u0026micro;M) were added and incubated for 48 h. The cells were washed three times with PBS to remove free samples. Cells were then collected and mitochondria were extracted according to the instructions of the mitochondrial isolation kit (C3601, Beyotime Biotechnology). The mitochondria and other organelles were placed in a reactor with nitric acid (1.0 mL) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (30%, 1.5 mL), which was nitrated in vacuum oven at 180\u0026deg;C for 1.5 h. The above system was subsequently volume-determined to 5.0 mL, and the Ca\u003csup\u003e2+\u003c/sup\u003e concentration in mitochondria was measured using ICP-MS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDetection of intracellular NO release\u003c/h2\u003e \u003cp\u003eGL261 cells (1.0 mL, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were inoculated into a 6-well plate and incubated overnight. Then different samples (1.0 mL, 200 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in DMEM, Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur) were added and incubated for 6 h. 3-Amino, 4-aminomethyl-2\u0026rsquo;,7\u0026rsquo;-difluorescein,\u003c/p\u003e \u003cp\u003ediacetate (DAF-FM DA, NO fluorescent probe, 5 \u0026micro;M, Beyotime Institute of Biotechnology, China) was incubated with the cells for 30 min and images were taken using inverted fluorescence microscope (Micro-shot MF53-N), their fluorescence being quantified using Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDetection of mitochondrial membrane potential\u003c/h2\u003e \u003cp\u003eGl261 cells (1.0 mL, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were inoculated in confocal dishes and incubated overnight. Then different samples (1.0 mL, 200 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in DMEM, Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur) were added and incubation for 24 h. The cells were washed three times with PBS to remove free material. Mitochondria was labeled with JC-1 (Beyotime biotechnology, C2006) and nucleus was labeled with Hoechst 33342 in blue fluorescence. Cells were fixed with 4% paraformaldehyde. Fluorescence images were taken with CLSM, and the fluorescence intensity of the acquired images was analyzed using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular ATP concentration assay\u003c/h2\u003e \u003cp\u003eGL261 cells (1.0 mL, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were inoculated into a 6-well plate and incubated overnight. Then different samples (1.0 mL, 200 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in DMEM, Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur) were added and incubation for 24 h. The cells were washed three times with PBS to remove free samples. Cells were then collected and intracellular ATP concentration was detected according to the instructions for use of the ATP Assay Kit (S0026, Beyotime Biotechnology).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of cell viability\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGL261 cells or bEnd.3 cells (200.0 \u0026micro;L, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were inoculated in 96-well plates and incubated overnight. The cell cultures were then removed and 200.0 \u0026micro;L of different concentrations in DMEM (0, 50.0, 100.0, 200.0, 400.0, and 800.0 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of NO-Lip@PAC@Cur were added and incubated for 24 h. The cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-dibenzenetetrazolium bromide (MTT, ApexBio Technology Co., Ltd.) colorimetric method to assess cell viability.\u003c/p\u003e \u003cp\u003eTo compare the \u003cem\u003ein vitro\u003c/em\u003e cell viability of different nanomotors, GL261 cells (200.0 \u0026micro;L, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were inoculated in 96-well plates and incubated overnight. Then different samples (1.0 mL, 200 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in DMEM, Lip@PLC, NO-Lip@PAC, or NO-Lip@PAC@Cur) were added and incubated for 24 h. Cell viability was assessed by the MTT colorimetric assay described above.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003emitochondrial mineralization detection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor Alizarin Red S staining, Gl261 cells (1.0 mL, 1 \u0026times; 105 cells mL-1) were inoculated in confocal dishes and incubated overnight. After removal of the medium, 1 mL of Lip@PLC, NO-Lip@PAC, NO-Lip@PAC@Cur (200.0 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) or A3187 (5 \u0026micro;M) dispersed in fresh medium was added and incubated for 48 h. After removal of the culture medium, and the cells were then washed and fixed with 95% ethanol. Subsequently, the cells were stained with Alizarin Red S (1 mL, Beyotime Biotechnology) for 30 min. After the extra staining solution was washed away by ultrapure water, the stained cell samples were observed under a light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDetection of pharmacokinetics\u003c/h2\u003e \u003cp\u003eSD rats (purchased from Jiangsu Wukong Biotechnology Co., LTD.) were used for the pharmacokinetic determination of NO-Lip@PAC@Cur NMs. Briefly, Cur (1.0 mL, 15.0 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and NO-Lip@PAC@Cur (1.0 mL, 15.0 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were injected through the tail vein, and 500 \u0026micro;L blood was drawn from the orbits of the rats at different times using capillary glass tubes soaked with sodium heparin. After centrifugation at 2500 r min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 15 min, the supernatant was obtained and the absorbance of curcumin was measured at 420 nm using a Multi-Function Measuring Instrument (Infinite\u0026reg; E Plex) and the content of the samples was calculated using a standard curve of absorbance of Cur or NO-Lip@PAC@Cur concentration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEstablishment of glioblastoma (GBM) model mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e Female C57BL/6 mice (6\u0026ndash;8 weeks old) were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. All animal experiments were conducted under the supervision and guidance of the Ethical Review Committee for Laboratory Animal Welfare of Nanjing Normal University (Nanjing, China, approval No. IACUC- 20220901-1 and 20200802). To establish the GBM model mice, GL261-Luc cells (10\u003csup\u003e6\u003c/sup\u003e cells in 8.0 \u0026micro;L Corning\u0026reg; Matrigel\u0026reg; Matrix) were slowly injected into the brain using a brain stereotaxic instrument (RWD Life Science Co., Shenzhen) positioned to (1.8 mm,0.6 mm, 2 mm depth) using the fontanel point of origin, where the craniotomy operation was kept consistent provided that the mice without cell injection were referred to as the sham-operated group as a control. During the operation, the mice were anesthetized by inhalation of 1\u0026ndash;5% isoflurane mixed with oxygen. 10 days later, all the mice were intraperitoneally injected D-Luciferin potassium salt (150 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in PBS, Guangzhou Biolight Biotechnology Co.), 15 min later, bioluminescent imaging was performed immediately using \u003cem\u003ein vivo\u003c/em\u003e image system (IVIS Spectrum, Aniview 600 Multi-mode Animal Imaging, Guangzhou Biolight Biotechnology Co.)\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003etargeting and tumor permeability\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the \u003cem\u003ein vivo\u003c/em\u003e targeting ability of different samples, 200.0 \u0026micro;L of Cy5-labled different samples (2.0 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Lip@PAC or NO-Lip@PAC) was injected intravenously. Notably, 200.0 \u0026micro;L of PBS was injected as control. Fluorescence imaging (excitation filter: 620 nm, emission filter: 670 nm) was performed at different times by IVIS Spectrum \u003cem\u003ein vivo\u003c/em\u003e. Euthanizing mice at different times, and the brain tissue and major organs (heart, liver, spleen, lung, and kidney) were collected for fluorescence imaging using IVIS Spectrum (excitation filter: 620 nm, emission filter: 670 nm). The \u003cem\u003ein vivo\u003c/em\u003e targeting ability of different samples was quantified using Living Image software. Subsequently, the above whole brain tissues were subjected to coronal frozen sections. The cell membranes of cerebrovascular endothelial cells were labeled with rabbit anti-mouse CD31 antibody and Aliexa Fluor 488-coupled goat anti-rabbit IgG, and the nucleus were labeled with DAPI, respectively. Tissue sections were blocked and fluorescence imaging was performed using CLSM. Normal brain tissues and GBM tissues were distinguished by cell density, and GBM permeability of different nanomotors was analyzed by Image J software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003edelivery efficiency of different samples\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the \u003cem\u003ein vivo\u003c/em\u003e delivery efficiency and biodistribution of the nanomotors, 200.0 \u0026micro;L of different samples (2.0 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Lip@PAC or NO-Lip@PAC) was injected intravenously. and 200.0 \u0026micro;L of PBS was injected as control. After 24 h, the mice were euthanized, and the brain tissue and major organs (heart, liver, spleen, lung, and kidney) were collected and weighed. Then added 1.0 mL RIPA lysis buffer to every 100.0 mg of tissue, and prepare tissue homogenate using a tissue grinder (frequency: 70 Hz, time: 8 min, run times: 8 times). Centrifuge the homogenate (4\u0026deg;C, 3000 rpm, 10 min) and collect the supernatant. Dilute the supernatant and measure the fluorescence intensity using a multifunctional enzyme-linked immunosorbent assay reader. Calculate the injection dose percentage (% ID/g) of each group of samples based on the sample concentration fluorescence intensity standard curve and the initial injection dose.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eanti-GBM efficacy and biosafety evaluation of different samples\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOn the 11th, 13th, 15th, 17th, 19th, 21st, and 23rd day after injection of GL261-Luc cells into the skull of mice, 200.0 \u0026micro;L different samples (2.0 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, PAC@Cur, Lip@PAC@Cur, NO-Lip@PLC @Cur, NO-Lip@PAC@Cur and temozolomide (TMZ, Beyotime Biotechnology)) were injected intravenously. Notably, the sham group received no injections and 200.0 \u0026micro;L PBS was injected as control. The growth of the glioma was monitored by IVIS Spectrum at specific times. The mice were sacrificed after treatment, and the brain tissues were taken for paraffin-embedding and coronal sectioning, which were performed with hematoxylin-eosin (H\u0026amp;E) staining and Ki-67 immunohistochemical staining, respectively. The main tissues including the heart, liver, spleen, lung, kidney and bone marrow were collected and fixed with 4% paraformaldehyde. Then the tissues were embedded in paraffin, and performed with H\u0026amp;E staining to evaluate the histopathological changes after different treatments. The blood samples were obtained from each group for biochemical analysis and routine blood examination. The biochemical analysis was measured by the automatic biochemical analyzer. The routine blood examination was performed by blood cell analyzer. Then, 8 additional mice were used in the survival experiments and survival curves were obtained using the Kaplan-Meier method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were finished using SPSS. Statistical tests and P values are detailed in figure legends. Data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The statistical significance was calculated via one-way ANOVA and LSD posthoc test, *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAdditional information\u003c/h2\u003e \u003cp\u003eThe supplementary figures, supplementary movies are provided in \u003cb\u003eSupplementary information\u003c/b\u003e.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe work was supported by National Natural Science Foundation of China (No. 52422306 (the Excellent Young Scholars NSFC), 22275095, 22175096, 22475103), Jiangsu Key Laboratory of Biofunctional Materials, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHameedat F, Mendes BB, Conniot J, Di Filippo LD, Chorilli M, Schroeder A, Conde J, Sousa F (2024) Nat Rev Mater 9:628\u0026ndash;642\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamad A, Yusubalieva GM, Baklaushev VP, Chumakov PM, Lipatova AV (2023) Viruses-Basel 15:547\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYalamarty SSK, Filipczak N, Li X, Subhan MA, Parveen F, Ataide JA, Rajmalani BA, Torchilin VP (2023) Cancers 15:2116\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarlsson J, Luly KM, Tzeng SY, Green JJ (2021) Adv Drug Deliv Rev 179:113999\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou Y, Sun XH, Yang QS, Zheng M, Shimoni O, Ruan WM, Wang YB, Zhang DY, Yin JL, Huang XG, Tao W, Park JB, Liang XJ, Leong KW, Shi BY (2022) Sci Adv 8:eabm8011\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Li M, Zheng M, Zou Y, Shi B (2024) Nano Today 56:102310\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa R, Li Z, Chiocca EA, Caligiuri MA, Yu J (2023) Trends Cancer 9:122\u0026ndash;139\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu B, Tian L, Chen J, Wang J, Ma R, Dong W, Li A, Zhang J, Chiocca EA, Kaur B, Feng M, Caligiuri MA, Yu J (2021) Nat Commun 12:5908\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Yang L, Han M, Han Y, Jiang Z, Zheng Q, Dong J, Wang T, Li Z (2024) ACS Nano 18:23001\u0026ndash;23013\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim GB, Aragon-Sanabria V, Randolph L, Jiang H, Reynolds JA, Webb BS, Madhankumar A, Lian X, Connor JR, Yang J, Dong C (2020) Bioact Mater 5:624\u0026ndash;635\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuijkerbuijk KPM, van Eijs MJM, van Wijk F, Eggermont AMM (2024) Nat Cancer 5:557\u0026ndash;571\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Xu T, Jiang Y, Xu H, Yan Y, Fu D, Chen J (2015) Neoplasia 17:239\u0026ndash;255\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeunen O, Johansson M, Oudin A, Sanzey M, Rahim SAA, Fack F, Thorsen F, Taxt T, Bartos M, Jirik R, Miletic H, Wang J, Stieber D, Stuhr L, Moen I, Rygh CB, Bjerkvig R, Niclou SP (2011) \u003cem\u003eProc. 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Herein, we propose a novel size-variable self-feedback nanorobot system tailored for GBM treatment, leveraging the unique high-calcium microenvironment of GBM. These nanomotors consist of three main components: a self-feedback degradable lipid shell, a nanorobot core with motion ability, and the drug curcumin (inhibiting the efflux of Ca\u003csup\u003e2+\u003c/sup\u003e). The lipid shell incorporates nitric oxide-releasing lipid (NOR) and NO-responsive degradable lipid (NOD). NOR is catalyzed by inducible nitric oxide synthase (iNOS) to release NO. NOD degrades in response to the self-released NO. The nanorobot core is composed of L-arginine (L-Arg) derivatives and zwitterionic monomers rich in carboxyl groups (facilitating Ca\u003csup\u003e2+\u003c/sup\u003e recruitment) (PAC NMs). Initially, the larger size-variable self-feedback nanomotors (~\u0026thinsp;500 nm) can penetrate the blood-brain barrier through chemotaxis, driven by the high expression of iNOS in the GBM microenvironment. During chemotaxis, the self-feedback lipid shell gradually degrades as NO accumulates, releasing smaller PAC NMs (~\u0026thinsp;50 nm). These smaller nanomotors target mitochondria, where they recruit Ca\u0026sup2;⁺ to induce mitochondrial mineralization in conjunction with curcumin, ultimately leading to tumor cell death and inhibiting GBM progression. This work may provide a new strategy for the development of GBM-specific treatment methods.\u003c/p\u003e","manuscriptTitle":"Size-variable self-feedback nanomotors for glioblastoma therapy via mitochondrial mineralization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-30 06:25:19","doi":"10.21203/rs.3.rs-6451662/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0d5cf8c9-5a2b-449b-a188-7e289573ac24","owner":[],"postedDate":"April 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47838756,"name":"Biological sciences/Cancer/Cancer therapy/Targeted therapies"},{"id":47838757,"name":"Biological sciences/Biological techniques/Nanobiotechnology/Nanoparticles"}],"tags":[],"updatedAt":"2025-10-10T07:06:19+00:00","versionOfRecord":{"articleIdentity":"rs-6451662","link":"https://doi.org/10.1038/s41467-025-64020-x","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-10-09 04:00:00","publishedOnDateReadable":"October 9th, 2025"},"versionCreatedAt":"2025-04-30 06:25:19","video":"","vorDoi":"10.1038/s41467-025-64020-x","vorDoiUrl":"https://doi.org/10.1038/s41467-025-64020-x","workflowStages":[]},"version":"v1","identity":"rs-6451662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6451662","identity":"rs-6451662","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}