Using low - molecular - weight ligands for targeting in integrated chemodynamic/starvation therapy and chemotherapy for prostate cancer

Using low - molecular - weight ligands for targeting in integrated chemodynamic/starvation therapy and chemotherapy for prostate cancer | 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 Research Article Using low - molecular - weight ligands for targeting in integrated chemodynamic/starvation therapy and chemotherapy for prostate cancer Xiaoli Zhang, Jie He, Yu An, Kehua Jiang, Qing Wang, Wenrui Deng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6045405/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jun, 2025 Read the published version in Cancer Nanotechnology → Version 1 posted 8 You are reading this latest preprint version Abstract Targeted therapy enhances tumor elimination while reducing adverse effects by integrating multiple tumoricidal mechanisms. Low molecular weight (LMW) ligands, offering faster pharmacokinetics and improved tumor permeability, present a viable alternative to antibodies. This study presents a novel nanomedicine for prostate cancer therapy, leveraging mesoporous silica nanoparticles (MSN) as the nanocarrier to encapsulate manganese dioxide (MnO 2 ) and doxorubicin (DOX). The resultant nanoparticles are further coated with a polydopamine (PDA) layer and covalently conjugated with glucose oxidase (GOx), forming the MSN@Mn@PDA-GOx/DOX hybrid system (hereafter termed SMPG/DOX NPs). LMW ligands (small molecule inhibitor DCL and nanobody VHH) targeting prostate-specific membrane antigen (PSMA) were conjugated to create DCL-SMPG/DOX and VHH-SMPG/DOX. Mn 2+ -mediated Fenton-like reactions converted H 2 O 2 into toxic hydroxyl radicals (·OH) under acidic conditions, enabling chemodynamic therapy (CDT). GOx-generated H 2 O 2 and gluconic acid disrupted nutrient supply, inducing tumor starvation therapy (ST). The increased H 2 O 2 and acidity amplified the Fenton-like reaction, creating a "ROS storm" that synergistically enhanced chemotherapy. LMW targeting improved tumor specificity, efficacy, and reduced side effects. In vitro, DCL-SMPG/DOX showed superior tumor cell internalization and cytotoxicity compared to VHH-SMPG/DOX. In vitro, the cellular internalization rates of VHH-SMPG/DOX and DCL-SMPG/DOX were 34.1% and 44.5%, respectively, significantly higher than that of free DOX uptake (10.3%). Moreover, DCL-SMPG/DOX-induced stronger cytotoxicity compared to VHH-SMPG/DOX. In vivo studies further demonstrated the strong anti-tumor activity of the DCL-SMPG/DOX nanomedicine, underscoring its potential as a prostate cancer treatment. Further research is needed to elucidate its antitumor mechanisms. PSMA low molecular weight ligands prostate cancer targeted therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Prostate cancer (PCa) is widely regarded as the second most prevalent malignant neoplasm in men and is a significant contributor to cancer-related mortality in this population. Notably, PCa has the highest incidence among all newly diagnosed male tumors, and its global prevalence has increased considerably over the past few years [ 1 , 2 ] . The absence of distinctive early symptoms in PCa leads to a high incidence of advanced-stage disease at initial diagnosis, ultimately resulting in an overall unfavorable prognosis [ 3 ] . At present, pharmacotherapy is the primary therapy modality for advanced PCa. Unfortunately, the clinical application of high-dose chemotherapeutic agents is significantly restricted by their dose-limiting toxicity, which arises from the unintended systemic exposure of healthy tissues to these drugs [ 4 ] . Furthermore, patients often fail to complete the prescribed chemotherapy regimen due to cumulative drug toxicity, drug resistance, or disease progression. Hence, the side effects and other limitations of chemotherapy pose a significant challenge and remain to be addressed. Nanomedicines have gradually emerged as potential candidates for PCa treatment. Several nanomedicines — such as Abraxane, Onyvide, Marqibo, and Doxil — have demonstrated clinical advantages in reducing chemotherapy-induced drug toxicity and improving overall patient survival [ 5 , 6 ] . However, achieving ideal therapeutic outcomes through a single therapeutic regimen is challenging. Chemodynamic therapy (CDT) induces apoptosis in tumor cells by utilizing endogenous cellular chemical energy to promote reactive oxygen species (ROS) production, eliminating the need for external energy [ 7 – 9 ] . Upon uptake by tumor cells, MnO 2 depletes the intracellular antioxidant glutathione (GSH) and generates Mn 2+ , disrupting the cellular antioxidant defense system. Mn 2+ exhibits remarkable Fenton-like activity, converting endogenous hydrogen peroxide (H 2 O 2 ) into highly toxic hydroxyl radicals (•OH) and thereby enhancing the tumor damage induced by GSH-depleting cancer therapy (i.e., CDT) [ 10 – 12 ] . Glucose oxidase (GOx) can catalytically disrupt glucose metabolism and produce H 2 O 2 in the tumor microenvironment (TME), enabling tumor starvation therapy (ST) [ 13 , 14 ] . This process of GOx-mediated glucose consumption increases the levels of gluconic acid and H 2 O 2 in the TME, thereby improving the effectiveness of CDT. Drugs administered into the bloodstream face several biological barriers before reaching their target sites. The drugs must cross the vascular network, diffuse into tumor tissues, and get internalized by tumor cells. These barriers hamper the delivery efficiency of anti-cancer nanomedicines, decreasing their overall therapeutic effect [ 15 ] . Despite decades of advances, the median delivery efficiency of systemically administered nanomedicines remains critically low (typically < 1%, with some studies reporting values as low as 0.0014% [ 16 , 17 ] ), underscoring the urgent need for more effective tumor-targeted delivery strategies. Thus, to enhance the transmembrane transport of nanomedicines and reduce their phagocytic uptake by non-tumor cells, researchers have modified the surfaces of nanomedicines with specific targeting molecules to achieve selective tumor cell targeting [ 18 ] . These strategies have proven effective at reducing treatment side effects and improving therapeutic efficacy. For instance, Wang et al. utilized anti-PSMA antibodies to modify arsenic nanosheets, achieving higher intratumoral drug concentrations and fewer adverse effects [ 19 ] .Compared to monoclonal antibodies, low - molecular - weight (LMW) ligands possess distinct advantages such as smaller molecular size, improved tumor penetration, faster systemic clearance, and reduced immunogenicity [ 20 , 21 ] . These features allow for more efficient and selective delivery of therapeutic payloads to PCa cells. In particular, PSMA-targeting LMW ligands, including small-molecule inhibitors (DCL) and nanobodies (VHH), have demonstrated strong binding affinity and rapid internalization into PCa cells, enabling enhanced intratumoral accumulation and minimizing off-target toxicity [ 22 – 24 ] . Despite their potential, few studies have directly compared the targeting performance and therapeutic efficacy of multiple LMW ligands within the same nanoplatform. Therefore, our study addresses by systematically evaluating and comparing the tumor-targeting capabilities and therapeutic outcomes of VHH- and DCL-functionalized nanomedicines for PCa treatment. In this study, we rationally designed a hybrid multifunctional nanomedicine(Scheme 1 ) capable of specifically targeting PCa. In this system, mesoporous silica (MSN) served as nanocarriers for the therapeutic agents MnO 2 and doxorubicin (DOX) and was subsequently coated with a polydopamine (PDA) nano-shell to form a core-shell structure. Then, GOx was grafted onto the surface of these nanoparticles via electrostatic interactions. Finally, the nanoparticles were surface-functionalized with LMW (VHH and DCL) to fabricate VHH-SMPG/DOX and DCL-SMPG/DOX nanomedicine. Subsequently, the differences in the therapeutic efficacy of VHH-SMPG/DOX and DCL-SMPG/DOX nanomedicine were examined. In the TME, GOx, MnO 2 increased ROS production by disrupting the nutrient supply of the tumor and initiating a cascade of catalytic reactions, synergistically inducing apoptosis along with chemotherapy. Moreover, functionalization with the LMW specifically enhanced the accumulation of the nanomedicine at tumor sites, reducing off-target toxicity and improving therapeutic efficacy, thereby providing an effective strategy for PCa therapy. 2. Materials and methods 2.1. Materials All the chemicals used in this study were of analytical reactive grade and most of them were from Aladdin Chemical Co., Ltd. 2.2. Synthesis of SMPG NPs MSN nanoparticles (NPs) were synthesized following established protocols, with slight modifications [ 25 ] . 50 mg MSN were dispersed in water, and 20 mL of a KMnO 4 solution (0.5 mM) was added dropwise under dark conditions and stirred for 4 h. 50 mg of MSN@Mn NPs were dispersed in a Tris-HCl buffer solution (10 mM, pH 8.5) containing 25 mg of dopamine hydrochloride and reacted for 3h to obtain MSN@Mn@PDA. 53 mg NHS, 35 mg EDC, 10 mg GOx were dissolved in 20 mL water before adding 500 µL APTES. Subsequently, 10 mg of MSN@Mn@PDA NPs added to stirring for an additional 24h. The final product was GOx-grafted SMPG NPs. 2.3. Synthesis of DCL-SMPG and VHH-SMPG NPs 10 mg of SMPG NPs and 10 mg of DCL-PEG2k-COOH in a 15 mL aqueous solution. The pH was adjusted to 8.5 using an ammonia solution and the mixture was stirred for 24h to obtain DCL-SMPG NPs. 1mg of SMPG NPs were functionalized using HOOC-PEG 2k -NHS. 0.2 mg/mL VHH solution (500 µL) was added to this dispersion. The mixture was incubated overnight at 4°C. Unbound VHH was removed via centrifugation and thorough washing to obtain the final product VHH-SMPG NPs. VHH loading was quantified via BCA assay. Briefly, free VHH in the supernatant was measured against a BSA standard curve. VHH Loading (μ g/mg SMPG) = \(\:\frac{\text{W}\:\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{V}\text{H}\text{H}-\text{W}\:\text{f}\text{r}\text{e}\text{e}\:\text{V}\text{H}\text{H}}{\text{W}\:\text{S}\text{M}\text{P}\text{G}\:\text{N}\text{P}\text{s}\:}\) (W: weight) 2.4. Drug loading and stimulus-triggered drug release assay The resulting product MSN@Mn NPs (5 mg/mL) were dispersed in a 20 mL DOX solution (0.5 mg/mL) and stirred for 24 h. The MSN@Mn/DOX NPs was obtained by centrifugation for further use. The drug loading amounts were determined by analysing the supernatant DOX solution after centrifugation with a UV-Vis test (480 nm). The release of DOX from SMPG/DOX NPs was examined in various media. Specifically, SMPG/DOX NPs (1 mg/mL; total volume, 1 mL) were dispersed in PBS solutions of different pH values (7.4 and 5.2) with or without 10.0 mM GSH. These solutions were placed inside dialysis bags (2000 KDa), which were immersed in 9 mL of the corresponding PBS solutions and incubated at 37°C with gentle shaking. After predetermined intervals, 1 mL of the external solution was sampled and replaced with fresh PBS (same pH). The DOX concentration in the collected samples was determined using a microplate reader (Bio-Rad Model680, USA). DLC (Drug Loading Capacity, %)= \(\:\frac{\text{W}\:\text{l}\text{o}\text{a}\text{d}\text{e}\text{d}\:\text{D}\text{O}\text{X}\:}{\text{W}\:\text{N}\text{P}\text{s}\:+\text{W}\:\text{l}\text{o}\text{a}\text{d}\text{e}\text{d}\:\text{D}\text{O}\text{X}\:}\:ⅹ\:100\%\) 2.5. Nanozyme activity assay 1 mL of SMPG NPs (25 µg/mL) was incubated with 1 mL of GSH (10 mM) for different durations (0, 1, 2, 4, 6 h). The mixtures were reacted with a 1 mL DTNB (1 mM) for 30 min and their absorbance at 412 nm was recorded. To examine TMB oxidation, different concentrations of the SMPG NPs were mixed with an equal volume of H 2 O 2 (20 mM). Equal volumes of TMB (10 mM) were added to the mixture. After 30 min the fluorescence spectrum of the supernatant was measured. Similarly, various concentrations of SMPG NPs were mixed with equal volumes of an MB solution for 30 min. The fluorescence spectrum of MB was then measured. SMPG NPs (0.5 mg/mL) were uniformly mixed with different concentrations of glucose solutions at room temperature. A pH meter was employed to determine the pH value of the mixture. To examine H 2 O 2 generation, different concentrations of SMPG NPs were mixed with glucose solutions (10 mM) at room temperature. Then, different samples were collected and the H 2 O 2 concentration was measured by a H 2 O 2 assay kit. Briefly, 50 µL of samples was mixed with 100 µL of H 2 O 2 detection reagent. After 30 min incubation at room temperature in the dark. Absorbance was measured at 560 nm using a microplate reader. H₂O₂ concentrations were calculated against a standard curve. 2.6. Cellular uptake assay 22RV1 cells were seeded in 24-well plates at a density of 1 × 10 5 cells per well. When the cells reached confluency, the culture medium was replaced with a fresh medium containing equivalent concentrations of DOX in the forms of free DOX, SMPG/DOX, VHH-SMPG/DOX, and DCL-SMPG/DOX NPs. After a 1, 2, and 4h incubation period, the medium was aspirated. The cells were washed thrice with PBS, fixed, and stained with DAPI. Subsequently, cellular uptake was visualized using CLSM (ZEISS, Germany). Additionally, flow cytometry (BDFA Celesta Flow Cytometer, Novocyte) was employed to quantitatively measure intracellular fluorescence intensity. 2.7. In vitro cytotoxicity study 22RV1 cells were seeded into 96-well plates. After the cells adhered sufficiently to the plates, the medium was removed. Subsequently, 100 µL of fresh medium containing different concentrations of free DOX, SMPG, SMPG/DOX, VHH-SMPG/DOX, DCL-SMPG/DOX NPs (containing an equivalent amount of DOX) was added to the wells and cell viability was assessed using the CCK-8 assay. SMPG/DOX nanoparticle concentrations were adjusted to deliver 5 µg/mL of DOX, based on a drug loading capacity DLC of 42.9%. Accordingly, a final SMPG/DOX and SMPG NPs concentration of 12 µg/mL was used. Furthermore, the cells were stained with 5 µL of Calcein-AM and 10 µL of PI for 30 min and then observed under a fluorescence microscope to examine cell viability. Similarly, cells were stained with Annexin V-FITC/7-AAD using apoptosis detection kits, and apoptosis was measured using flow cytometry. The forward scatter (FSC) and side scatter (SSC) voltages were set to 300 and 225, respectively, to accurately gate single, viable cells while excluding debris. Fluorescence signals were detected using a FITC voltage of 168 (Annexin V-FITC) and a 7-AAD voltage of 158. 2.8. ROS detection and the mitochondrial membrane potential of live cells 22RV1 cells were seeded into 24-well plates. Once the cells adhered sufficiently, they were treated with 50 µg/mL of MSN@Mn@PDA, SMPG, VHH-SMPG, and DCL-SMPG NPs treatment 6h. Intracellular ROS levels were quantified using the DCFH-DA probe. Cells were incubated with 10 µM DCFH-DA for 30 min at 37°C, washed with PBS, and analyzed immediately. Fluorescence signals were measured at excitation 488 nm and emission 525 nm using a flow cytometer and fluorescence microscope. Similarly, the changes in the mitochondrial membrane potential were observed by staining the cells with the JC-1 probe. Cells were stained with 5 µg/mL JC-1 for 20 min at 37°C. Fluorescence was detected at two emission wavelengths: aggregates (excitation 585 nm, emission 590 nm) and monomer (excitation 514 nm, emission 529 nm). 2.9. Animal Tumor Models Male BALB/C nude mice (~ 4 weeks old) were procured from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and housed in the Experimental Animal Center of The Affiliated Hospital of Guizhou Medical University, China. Animal procedures were conducted following the guidelines of the Regional Ethics Committee and approved care regulations of The First Affiliated Hospital of Guizhou Medical University. 22RV1 cells were first washed with PBS, collected, and suspended in PBS. Tumor models were established by subcutaneously injecting 5 ×10 6 22RV1 cells (suspended in 100 µL PBS) into the right flank of the mice. 2.10. In vivo tissue distribution To investigate the in vivo tissue distribution of the nanoparticles, tumor-bearing BALB/c nude mice were treated with DCL-SMPG/R6G nanoparticles, with SMPG/R6G-treated mice serving as the control group. Rhodamine 6G (R6G) was loaded onto the nanoparticles to enable subsequent fluorescence imaging. Specifically, MSN@Mn nanoparticles (5 mg/mL) were dispersed in 20 mL of an R6G solution (0.5 mg/mL) and stirred continuously for 24 hours. The resulting MSN@Mn/R6G nanoparticles were collected by centrifugation for further use. The synthesis of DCL-SMPG/R6G nanoparticles was consistent with that of DCL-SMPG/DOX. When the tumor volume reached approximately 100 mm³, R6G-labeled nanoparticles were intravenously administered via the tail vein at a dose of 5 mg/kg. After 24 hours, major organs including the liver, spleen, heart, lungs, kidneys, and the tumors were harvested. Fluorescence imaging and quantitative region-of-interest (ROI) analysis were performed using the Lumina IVIS Spectrum imaging system (Caliper Life Sciences Inc., USA). 2.11. In vivo tumor therapy To assess the in vivo anti-tumor efficacy of the NPs, mice were randomly assigned to five groups(Ten per group) once their average tumor volume reached 100 mm 3 . The mice received intravenous injections of PBS, free DOX, SMPG, SMPG/DOX, and DCL-SMPG/DOX NPs via the tail vein(5mg/kg DOX). During treatment, the changes in their body weight and tumor size were monitored every 2 d over a duration of 21 d post-treatment. At the end of the 21-d period, the mice were euthanized, and their major organs and tumor tissues were collected for hematoxylin and eosin (H&E) staining analysis. Furthermore, immunohistochemistry (IHC) was performed to analyze Ki-67 and caspase-3 expression in the tumor tissues. 2.12. Statistical analysis All data are presented as the mean ± standard deviation (SD). Statistical analysis was conducted using Origin 2021 and GraphPad Prism 8.0 software based on the student’s t-test or one-way analysis of variance (ANOVA). Statistical significance was determined at ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. 3. Results 3.1. Characterization of VHH-OSMPG/DOX and DCL-OSMPG/DOX NPs The synthesis of MSN with disulfide bonds was initially accomplished via the hydrolysis and co-condensation of silane precursors in a mixture of water and ethanol under alkaline conditions. TEM images revealed that the resulting organic silica had an average diameter of approximately 122 nm (Fig. 1 A). Subsequently, the introduction of KMnO 4 triggered an in-situ oxidation-reduction reaction, leading to the confined growth of MnO 2 NPs within the mesoporous channels of the organic silica [ 26 ] . Figure 1 B shows the TEM images of MSN@Mn NPs. Furthermore, the composition of MSN@Mn NPs was confirmed via elemental mapping, which revealed a homogeneous distribution of Si, C, S, O, N and Mn in these particles (Fig. 1 C). The MSN@Mn NPs shows the typical characteristic of N 2 adsorption-desorption behavior with the surface area of 60.46 m 2 /g, average pore size of 6.3 nm, which guarantees the further storage and sustained release of chemotherapeutic drug (Fig. S1 , Table S1 ). Figure 1 D shows the Mn 2p XPS spectra exhibited two distinct peaks at 642 eV and 653 eV, which represented the binding energies of Mn 2p3/2 and Mn 2p1/2 in MnO 2 respectively. To confirm the successful loading of the chemotherapeutic drug DOX into the MSN/Mn NPs, the UV-vis absorption spectra of the different NPs preparations were examined. Following DOX loading into MSN@Mn NPs, a prominent absorption band at approximately 480 nm was observed in MSN@Mn/DOX NPs (Fig. 1 E). These findings demonstrated the successful encapsulation of DOX within the MSN@Mn NPs. The drug loading capacity of the nanocarriers was calculated to be 42.9%. Using DLS analysis, the hydrodynamic diameter of MSN@Mn@PDA NPs was determined to be approximately 220 nm (Fig. 1 F), while the surface zeta potential became − 27 mV (Fig. 1 G). SMPG NPs were generated via electrostatic interactions. The introduction of GOx augmented the hydrodynamic diameter and increased the zeta potential to -19 mV. Furthermore, the successful integration of SMPG NPs was successively confirmed through FTIR spectroscopy (Fig. 1 H). SMPG NPs showed absorption peaks at 1625 cm − 1 and 1030 cm − 1 , corresponding to the stretching vibrations of -CO-NH2 and C = O, respectively [ 27 ] . Figure 1 F, G shows the introduction of the targeting agent PEG-DCL onto the surface of SMPG NPs further altering their hydrodynamic diameter and zeta potential. DCL-SMPG NPs had a hydrodynamic diameter of 342 nm and a zeta potential of -8 mV. Moreover, the presence of the -CH 2 groups of PEG was reflected by a vibrational peak at 2840 cm − 1 in DCL-SMPG NPs (Fig. 1 H). Only negligible changes in the hydrodynamic diameter of DCL-SMPG NPs in PBS, ang FBS were observed after 7 days, suggesting that DCL-SMPG NPs also possessed good colloidal stability in biological media (Fig. S2). Anti-VHH antibodies (Alexa Fluor 488) were incubated with SMPG, VHH-SMPG NPs, the samples were observed using fluorescence microscopy after the excess anti-VHH antibodies were completely removed (Fig. 1 I). The observations confirmed the successful synthesis of VHH-SMPG NPs. BCA quantification confirmed a VHH loading of 73 µg/mg SMPG, demonstrating efficient conjugation. This value aligns with reported ligand densities for targeted nanomedicines and ensures sufficient binding sites for PSMA-specific uptake [ 28 ] . 3.2. Stimulus-responsive DOX release and enzyme activity of SMPG NPs First, we examined the release of DOX from SMPG/DOX NPs. At pH 7.4, DOX release increased gradually for 48h, yielding a cumulative drug release rate of less than 15% (Fig. 2 A). However, the rate of drug release was higher at a pH of 5.2 and increased even further at a pH of 7.4 in the presence of 10 mM GSH. Even after the 48-hour, the drug release rate remained below 22%. In contrast, in the presence of 10 mM GSH under acidic conditions (pH 5.2), approximately 33% of the DOX diffused out of SMPG/DOX NPs. Similarly, we examined DOX release from DCL-SMPG/DOX and VHH-SMPG/DOX NPs (Fig. S3). Within 24 h, the release profiles from both DCL-SMPG/DOX and VHH-SMPG/DOX NPs were essentially consistent with that of SMPG/DOX NPs. MnO 2 can convert reduced GSH to its oxidized form and release Mn 2+ , further catalyzing the generation of cytotoxic •OH from H 2 O 2 . After prolonged incubation with SMPG NPs, the content of GSH gradually decreased (Fig. 2 B) and the absorbance value of GSH decreased with increasing concentrations of SMPG NPs (Fig. S4). Subsequently, •OH production was observed under different conditions using TMB and MB as indicators. TMB readily captures highly reactive •OH species to form a radical cation that generates a blue color and exhibits an absorption peak at 650 nm [ 29 ] . When the presence of SMPG NPs, the absorbance increased with an increase in the SMPG NP concentration (Fig. 2 C). Similarly, when MB was incubated with SMPG NPs, the absorbance of the mixture decreased with an increasing concentration of SMPG NPs (Fig. 2 D). Remarkably, the MB degradation kinetics exhibited a non-monotonic dependence on GSH concentration (Fig. S5). At 1–2 mM GSH, enhanced Mn²⁺ release from SMPG amplified ·OH generation, minimizing MB absorbance. Conversely, ≥ 5 mM GSH scavenged ·OH via thiol-radical interactions, attenuating MB degradation. This biphasic behavior aligns with Mn²⁺-mediated Fenton catalysis and glutathione’s dual redox roles [ 30 , 31 ] We investigated the oxidative activity of SMPG NPs toward glucose by monitoring the changes in the H 2 O 2 concentration and pH value in mixed solutions. At a glucose concentration of 10 mM, the pH value decreased from 6.8 to 3.5 within a 2h period (Fig. 2 E). Additionally, the H 2 O 2 concentration in the solution increased with an increase in the concentration of SMPG NPs (Fig. 2 F). Subsequently, the •OH-producing capacity of SMPG NPs was investigated in the presence of glucose (Fig. S6). When SMPG NPs were incubated with glucose, the absorbance of MB decreased due to the gluconic acid-induced activation of SMPG nanozyme activity, which enhanced •OH generation [ 32 ] . 3.3. Cellular uptake During the incubation period, both VHH-SMPG/DOX and DCL-SMPG/DOX NPs demonstrated rapid internalization in 22RV1 cells, unlike free DOX and SMPG/DOX (Fig. 3 A). This phenomenon could be attributed to the interactions between PSMA- ligand and 22RV1 cells [ 19 , 33 ] . Notably, the cellular fluorescence intensity of DCL-SMPG/DOX NPs was higher than that of VHH-SMPG/DOX NPs at the same incubation time points (Fig. 3 B, C). Moreover, DCL-SMPG/DOX consistently exhibited higher intracellular fluorescence intensity than VHH-SMPG/DOX at all time points, indicating more rapid and efficient internalization(Fig. S7). This enhanced uptake is likely attributable to the high-affinity interaction between DCL and PSMA on the cell surface. We further compared the internalization patterns of NPs between 22RV1 (PSMA-positive) and PC3 (PSMA-negative) cells (Fig. 3 D). The targeting efficiency was notably enhanced in 22RV1 cells incubated with VHH-SMPG/DOX and DCL-SMPG/DOX NPs, resulting in uptake efficiencies of 34.1% and 45.5%, respectively. Meanwhile, the uptake of SMPG/DOX NPs in these cells was only 25.4%. This targeted enhancement (Fig. 3 D) signified the increased interaction between 22RV1 cells and NPs. However, the uptake efficiencies for each type of NPs significantly lower in PC3 cells than in 22RV1 cells. Passive diffusion serves as the primary mechanism for the intracellular uptake of small-molecule drugs. Meanwhile, the intracellular accumulation of NPs is often due to enhanced endocytosis [ 34 ] . Consequently, we investigated the effects of temperature (energy-dependent pathways) and different phagocytosis inhibitors on cellular uptake to identify the internalization mechanism of NPs [ 35 ] . As depicted in Fig. 3 E, cellular uptake rates were significantly poorer at 4°C (only 4.58%) than at 37°C. When chlorpromazine and nystatin were used to block clathrin-mediated endocytosis (CME) and caveolin-mediated endocytosis(CavME), respectively, the internalization rates of all NPs decreased. Specifically, inhibition with chlorpromazine reduced the internalization rate to 15%, while inhibition with nystatin reduced this rate to 35%. When M-β-CD blocked both clathrin and caveolin-mediated pathways, the uptake rates decreased by approximately 40%. However, when amiloride was used to block macropinocytosis, the internalization rates of all NPs remained similar to those observed at 37°C. This indicated that 22RV1 cells primarily internalize all NPs through energy-dependent clathrin- and caveolin-mediated pathways. 3.4. In vitro anti-tumor performance Following the treatment of 22RV1 cells with free DOX, SMPG, SPMG/DOX, VHH-SMPG/DOX, and DCL-SMPG/DOX, cell viability and apoptosis were assessed. As illustrated in Fig. 4 A, dose-dependent cytotoxicity was observed in 22RV1 cells following treatment with free DOX and different NPs. At a DOX concentration of 5 µg/mL, the cell viability after DCL-SMPG/DOX treatment was only 8.4%. This value was markedly lower than the values observed after SPMG/DOX (42.4%) and VHH-SMPG/DOX (24.5%) treatment. The cytotoxicity of DCL-SMPG/DOX was higher than that of free DOX, SMPG, and SPMG/DOX underscoring the potential of combination therapy in exerting synergistic anti-tumor effects [ 36 ] . 22RV1 cells subjected to different treatments were stained using calcein-AM and propidium iodide for live/dead staining. After treatment with DCL-SMPG/DOX, the red fluorescence was the highest and there were virtually no viable cells in this group(Fig. 4 B). After treatment with free DOX, approximately 45.8% of cells were found to be in the early and late stages of apoptosis. Meanwhile, the rate of apoptosis after treatment with SMPG NPs was 42.93%. The synergistic effects of CDT/ST and chemotherapy after SMPG/DOX treatment increased the rate of apoptosis to 56.38%. After DCL-SMPG/DOX treatment, the rate of apoptosis reached 85.57%(Fig. 4 C). This demonstrated that DCL-SMPG/DOX NPs induce pronounced cancer cell apoptosis. The cytotoxicity induced by VHH-SMPG NPs was significantly lower than that induced by DCL-SMPG NPs (Fig. S8). To further validate the PSMA-targeting specificity of our nanoplatforms, we performed comparative in vitro cytotoxicity assays using both 22RV1 and PC3 prostate cancer cell lines (Fig. S9). In PC3 cells, which lack PSMA expression, both targeted (VHH-SMPG/DOX52.1% cell viability and DCL-SMPG/DOX 50.4% cell viability)formulations exhibited no significant increase in cytotoxicity compared to the non-targeted SMPG/DOX (50.0% cell viability). This indicates minimal off-target cytotoxic effects in cells lacking PSMA expression. In contrast, in PSMA-positive 22RV1 cells, both targeted nanoparticles༈VHH-SMPG/DOX 24.5% cell viability and DCL-SMPG/DOX 8.4% cell viability༉ induced significantly higher cytotoxicity relative to the non-targeted formulation(SMPG/DOX 43.0% cell viability). The enhanced cytotoxic effects observed for VHH-SMPG/DOX and DCL-SMPG/DOX were statistically significant, confirming that the functionalization with PSMA-targeting ligands effectively improved therapeutic efficacy via receptor-mediated uptake. Together, these findings demonstrate that the antitumor activity of the targeted nanocarriers is PSMA-dependent, and support the specificity and safety profile of our delivery strategy. Moreover, the cytotoxicity of 200 µg/mL MSN@PDA NPs with SVUHC, PC3, 22RV1 cells remained around 10%(Fig. S10). Similarly, the hemolytic rate induced by SMPG treatment was below 5% (Fig. S11). 3.5. In vitro ROS production DCFH-DA was employed as a fluorescent probe to assess intracellular ROS generation. As shown in Fig. 5 A, all treatment groups—including MSN@Mn@PDA, SMPG, VHH-SMPG, and DCL-SMPG NPs—exhibited markedly enhanced green fluorescence compared to the untreated control group, indicating elevated ROS levels. Among these, the DCL-SMPG group displayed the highest fluorescence intensity. To further confirm intracellular ROS production, quantitative analysis was performed using flow cytometry (Fig. 5 B and Fig.S12). DCL-SMPG nanoparticles significantly promoted ROS generation within cells. Notably, there was no statistically significant difference in ROS induction between the DCL-SMPG and VHH-SMPG groups. ROS has been found to decrease the mitochondrial membrane potential, thereby inducing cell apoptosis [ 37 ] . In this study, the effect of ROS on mitochondrial membrane potential was assessed using the JC-1 probe (Fig. 5 B). After treatment with MSN@Mn@PDA, the mitochondrial membrane potential of 22RV1 cells decreased. Following SMPG treatment, more pronounced mitochondrial damage was observed in 22RV1 cells, suggesting that the synergistic effect between CDT and ST increased ROS levels and thereby induced mitochondrial damage. In 22RV1 cells, VHH-SMPG and DCL-SMPG treatment resulted in predominantly green fluorescence, with DCL-SMPG inducing stronger green fluorescence. During apoptosis, the decline in adenosine triphosphate (ATP) levels typically coincides with the reduction in the mitochondrial membrane potential. GOx effectively catalyzes the breakdown of glucose, leading to a decrease in cellular energy levels [ 38 – 40 ] . Therefore, we further investigated the intracellular ATP levels in cells treated with different NPs. As shown in Fig. 5 D, intracellular ATP levels decreased progressively upon treatment with MSN@Mn@PDA, SMPG, VHH-SMPG, and DCL-SMPG NPs. This trend is consistent with the corresponding changes in mitochondrial membrane potential, indicating mitochondrial dysfunction induced by oxidative stress. The introduction of GOx in SMPG NPs enhanced glucose oxidation and hydrogen peroxide production, further impairing mitochondrial function and reducing ATP synthesis. Importantly, the addition of PSMA-targeting ligands significantly amplified this effect due to enhanced cellular uptake, with DCL-SMPG NPs showing the most pronounced ATP depletion. Furthermore, treatment with these NPs also led to a substantial reduction in the intracellular GSH: oxidized glutathione (GSSG) ratio (Fig. S13), reflecting elevated oxidative stress and diminished antioxidant defenses. This was primarily due to the redox reaction between MnO 2 and GSH, which oxidizes GSH into GSSG and disrupts the cellular redox balance. The DCL-SMPG group exhibited the greatest decrease in GSH:GSSG, consistent with more efficient intracellular MnO 2 delivery and redox activation. 3.6. In vivo biodistribution In vitro experiments demonstrated that DCL-SMPG/DOX NPs exhibit enhanced internalization in 22RV1 prostate cancer cells and induce more potent tumor cell cytotoxicity compared to VHH-SMPG/DOX NPs. Based on these promising results, we further evaluated the in vivo antitumor efficacy of DCL-SMPG/DOX NPs. To investigate the tissue biodistribution, BALB/C nude mice tumor were intravenously administered with Rhodamine 6G (R6G) -labeled SMPG/R6G and DCL-SMPG/R6G NPs. At predetermined time points (24 h), fluorescence images were taken by using a small animal imaging system. Fluorescence imaging revealed that DCL-SMPG/R6G NPs exhibited enhanced accumulation at tumor tissues, as shown by significantly higher R6G fluorescence signals compared to non-targeted SMPG/R6G (Fig. 6 A-B). This selectivity aligns with the PSMA-targeting mechanism of DCL ligands, which bind to tumor vasculature and cell surfaces to promote receptor-mediated endocytosis. Fluorescence signals in major organs were similar between groups, except for slightly elevated kidney accumulation in the DCL group. 3.7. In vivo anti-tumor activity The in vivo anticancer effects of DCL-SMPG/DOX were evaluated based on parameters such as tumor volume, weight, and pathology. Figure 7 A shows the control group, free DOX group, and SMPG group, the mice in these groups experienced rapid tumor growth. In contrast, the SMPG/DOX group experienced a relative delay in tumor growth, accompanied by a considerable reduction in tumor volume. However, the tumor inhibition effects were even more pronounced in the DCL-SMPG/DOX group. Following the 21-day treatment period, all mice were euthanized, and their tumors were excised and weighed(Fig. 7 B, Fig. S14). DCL-SMPG/DOX provided the most potent tumor inhibition after 21 days, demonstrating its efficacy in eliminating tumor cells. During the treatment period, there were no significant changes in body weight among the mice in the different treatment groups (Fig. 7 C). Excised mouse tissues were examined using H&E staining to assess the histocompatibility of DCL-SMPG/DOX NPs. Comparisons with non-tumor tissues from PBS-treated mice revealed that the non-tumor tissues from mice treated with the different NPs retained normal physiological morphologies, devoid of inflammation or pathological changes (Fig. S15). Figure 7 D shows the free DOX and SMPG treatment groups showed a significantly higher degree of tumor cell necrosis than the PBS treatment group. SMPG/DOX treatment, which combined chemotherapy with CDT/ST, resulted in significant tumor cell destruction and extensive necrosis. Notably, targeted treatment with DCL-SMPG/DOX resulted in even more pronounced structural damage to tumor tissues.The expression of Ki-67 and caspase-3 was examined in tumor tissues using immunohistochemical staining. Compared with the PBS-treated group, the group treated with DCL-SMPG/DOX exhibited significantly lower cell proliferation and a higher rate of cell apoptosis, validating the superior efficacy of synergistic CDT/ST and chemotherapy. These findings collectively suggested that DCL-SMPG/DOX, which possesses exceptional biocompatibility and anti-tumor activity, holds significant promise as an effective anticancer agent in vivo . 4. Discussion Traditional prostate cancer therapy are often associated with significant side effects, poor efficacy, and low bioavailability. Effectively delivering drugs to target cells has become a key focus of current research. Targeted nanotherapies can more precisely localize to tumor cells, reducing off-target effects and increasing drug internalization within tumors. The team led by Michael Mitchell developed a class of antibody-conjugated lipid nanoparticles (Ab-LNPs) that exhibit extrahepatic tropism and target T cell markers. This innovation enables the precise delivery of mRNA, minimizing off-target effects and adverse reactions [ 41 ] . The targeted accumulation of nanotherapeutics in prostate cancer tissues or cells is the crucial first step to enhance therapeutic efficacy, followed by the release of therapeutic molecules from the delivery system. Subsequently, the therapeutic effectiveness of nanomedicine in treating prostate cancer is further improved by harnessing synergistic therapy mechanisms [ 4 ] . In this study, we utilized low molecular weight ligands targeting PSMA to construct a multifunctional nanotherapeutic platform. This platform integrates CDT, ST and chemotherapy for the targeted therapy of prostate cancer. CDT and ST are often considered together in antitumor research due to their synergistic effects in enhancing antitumor efficacy. Redox reactions enable the uniform distribution of Mn within the mesoporous channels of MSN, preventing aggregation on the surface of the nanoparticles, thereby enhancing their reactivity and acid sensitivity [ 42 ] . XPS confirmed the binding energy of Mn2p, ensuring the Mn 2+ -mediated Fenton-like reaction selectively converts overexpressed H 2 O 2 into highly toxic ·OH under low pH conditions. Additionally, GOx produces H 2 O 2 and gluconic acid, effectively disrupting nutrient supply and triggering tumor starvation therapy. More importantly, the elevated levels of H 2 O 2 and increased acidity significantly enhance the Fenton-like reactivity, generating a pronounced "ROS (·OH) storm," thus achieving Mn 2+ -mediated cascade amplification of CDT. In vitro, SMPG NPs consumed GSH within a short period (6h), which is half the time compared to the 12h required for Ce6/GOx@ZIF-8/PDA@MnO 2 nanoparticles synthesized via a one-pot method by Zhang et al [ 11 ] . Furthermore, TMB and MB assays were employed to detect the ability of SMPG NPs to catalyze the production of ·OH from H 2 O 2 , confirming that SMPG NPs catalyzed the generation of ·OH under acidic conditions (Fig. 2 D-F). After catalyzing H 2 O 2 and gluconic acid from glucose, SMPG NPs further generated ·OH through a cascade reaction, ensuring the in vivo amplification of CDT, leading to a ROS storm and tumor ablation. DCFH-DA staining revealed intracellular ROS in 22RV1 cells after therapy with SMPG NPs, showing that SMPG NPs induced higher ROS production compared to CDT catalysis mediated by MSN@Mn@PDA NPs alone. ROS caused mitochondrial damage and induced apoptosis. JC-1 staining indicated changes in mitochondrial membrane potential, demonstrating that mitochondrial damage was proportional to the ROS levels. Additionally, intracellular ATP and GSH levels were found to correspond to the changes in mitochondrial membrane potential, indicating that SMPG NPs synergistically enhanced CDT and ST to produce significant ROS. The chemotherapeutic agent DOX was encapsulated within SMPG/DOX nanoparticles, synergistically contributing to the antitumor effects of CDT and ST. Initially, DOX was loaded into MSN@Mn nanoparticles with a loading efficiency of 42.9%, and the surface was coated with PDA to prevent drug leakage during circulation. In vitro drug release studies typically elucidate how encapsulated drugs are released in vivo [ 43 ] . In our study, drug release was investigated in PBS (pH 7.4) with or without 10 mM GSH. Rapid drug release occurred within the first 0–10 hours in both media, followed by a slower release profile. In the medium containing GSH, the release of DOX was enhanced, with even greater and faster drug release observed in the medium at pH 5.2 containing GSH. Given that the TME is often characterized by low pH and high GSH concentration (approximately 10 mM), this suggests that the nanomedicine is responsive to the TME for targeted drug release. Additionally, drug release continued at a sustained, slow pace even after 24 hours in the pH 5.2 GSH-containing medium. This prolonged drug release pattern contributes to maintaining therapeutic drug concentrations within tumor cells over an extended period, enhancing cytotoxicity and accelerating cancer cell death [ 43 ] . In vivo, targeted nanomedicine typically achieve less than 1% accumulation at the tumor site [ 44 ] . However, compared with non-targeted nanomedicine, targeted nanomedicine demonstrate significantly enhanced accumulation within tumor tissues. This indicates that the targeting mechanism is the driving force behind the active uptake of nanomedicine by tumor cells [ 45 ] . Most targeting ligands are traditionally monoclonal antibodies; however, some low-molecular-weight ligands offer additional advantages due to their smaller size, higher tissue penetration, and remarkably low immunogenicity. In this study, two low-molecular-weight ligands, a PSMA small molecule inhibitor (DCL) and anti-PSMA nanobody (VHH), were chosen as targeting moieties. These ligands specifically bind to the overexpressed PSMA on prostate cancer cells, facilitating the rapid attachment of the nanomedicine, enhancing their anti-tumor activity, and reducing off-target toxicity. The conjugation of DCL with the nanomedicine was examined through changes in the hydrodynamic diameter and zeta potential of the nanoparticles, and the successful conjugation of DCL was further confirmed via FTIR analysis. Additionally, fluorescence microscopy revealed green fluorescence after incubating the VHH-conjugated nanomedicine with anti-VHH fluorescent antibodies, indicating successful conjugation. Experimental results demonstrated that ligand surface conjugation led to increased uptake of nanomedicine by prostate cancer 22RV1 cells (Fig. 3 A), as further confirmed by flow cytometry. The uptake of VHH-SMPG/DOX nanoparticles and DCL-SMPG/DOX nanoparticles by 22RV1 cells was 1.3-fold and 1.8-fold higher, respectively, compared to SMPG/DOX nanoparticles. Notably, the uptake of DCL-SMPG/DOX nanoparticles by 22RV1 cells was 1.3 times higher than that of VHH-SMPG/DOX nanoparticles. At the same dosage, DCL-SMPG/DOX nanoparticles exhibited higher cytotoxicity against 22RV1 cells compared to VHH-SMPG/DOX nanoparticles (Fig. 4 A). These findings indicate the nanomedicine conjugated with the PSMA small molecule inhibitor DCL are internalized more rapidly by 22RV1 cells, thus resulting in greater toxicity than those conjugated with VHH. The observed lower internalization efficiency of VHH-SMPG/DOX compared to DCL-SMPG/DOX can be attributed to the monovalent nature of VHH ligands, higher steric hindrance, and less effective induction of receptor clustering on the cell membrane. In contrast, the multivalent architecture of DCL enables stronger PSMA binding, promotes receptor-mediated endocytosis via CME and CavME, and enhances nanoparticle uptake in prostate cancer cells. These findings are consistent with recent studies emphasizing the importance of ligand valency, orientation, and receptor dynamics in optimizing nanoparticle internalization and therapeutic performance [ 46 , 47 ] . Furthermore, the therapeutic efficacy of a drug at its site of action is contingent upon the ability of the nanomedicine to enter the target cells. The primary mechanism by which nanomedicine was internalized into cells is through endocytosis. Depending on the cell type and the proteins, lipids and other molecules involved in the process, endocytosis can be categorized into several types, including phagocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, clathrin/caveolin-independent endocytosis, and micropinocytosis [ 48 ] . In our study, we found that the uptake of nanomedicine (including DOX) by 22RV1 cells primarily occurred through energy-dependent CEM and CaME. In the research conducted by Anna Salvati et al., it was observed that the uptake of silica nanoparticles was independent of clathrin [ 49 ] . The endocytic pathways of different types of nanomedicine vary due to differences in their chemical properties, including size, surface charge, shape and rigidity [ 50 ] . Although DOX is classically considered to enter cells via passive diffusion due to its small molecular size, recent studies have suggested a partial contribution of energy-dependent and endocytic pathways to its intracellular accumulation, particularly in cancer cells with altered membrane composition. In our study, the observation that endocytosis inhibitors moderately reduced free DOX uptake indicates a non-exclusive reliance on passive diffusion. This may result from transient CEM and CaME -mediated internalization of DOX under physiological conditions, as previously observed in other malignancies [ 51 ] . Furthermore, commonly used inhibitors such as chlorpromazine and M-β-CD may alter membrane integrity or metabolic status, indirectly influencing passive permeability and transporter-mediated DOX influx/efflux [ 52 ] . Targeting PSMA receptors with low molecular weight ligands ensured that the nanomedicine was specifically delivered to prostate cancer cells and exhibited efficient cellular internalization. Further studies were conducted using the CCK-8 assay, live/dead cell staining, and apoptosis detection to assess the anticancer effects of the nanomedicine on prostate cancer cells. Figures 4 B-D show that the cytotoxicity of nanomedicine combining chemotherapy, CDT/ST (SMPG/DOX NPs) was significantly higher than that of standalone chemotherapy and nanomedicine utilizing CDT/ST (SMPG NPs). Nanomedicine with low molecular weight ligand targeting (VHH-SMPG/DOX and DCL-SMPG/DOX) exhibited even greater cytotoxicity, far exceeding that of SMPG NPs alone. This increased cytotoxicity is attributed to the ligand-targeting effect, which enhances the internalization of nanomedicine in 22RV1 cells, leading to higher cytotoxicity. Additionally, since the internalization of DCL-SMPG/DOX in 22RV1 cells was greater than that of VHH-SMPG/DOX, the cytotoxicity induced by DCL-SMPG/DOX was higher than that induced by VHH-SMPG/DOX. While the therapeutic efficacy of DCL-SMPG/DOX is supported by significant tumor suppression and enhanced ROS generation, we acknowledge that this study did not include a formal quantitative synergy analysis (e.g., Chou–Talalay method). This limitation arises primarily due to the co-loading design of our nanoplatform, in which MnO 2 , GOx, and DOX are integrated at a fixed ratio. As a result, it was not feasible to independently modulate the concentrations of each component to generate a comprehensive dose–effect matrix required for synergy quantification. Moreover, the cooperative interactions among these therapeutic modalities are highly dependent on tumor-specific biochemical cues—such as acidic pH and endogenous H₂O₂—which dynamically regulate the catalytic performance of MnO 2 and GOx. Therefore, decoupling their individual effects in an artificial in vitro setting may not accurately reflect the in vivo therapeutic synergy. Based on the in vitro anticancer effects of nanomedicine against prostate cancer cells, the in vivo antitumor efficacy of DCL-SMPG/DOX NPs was further studied. In our study, the in vivo biodistribution analysis revealed that DCL-SMPG/R6G nanoparticles exhibited significantly enhanced accumulation in PSMA-expressing prostate tumors compared to non-targeted SMPG/R6G nanoparticles. This targeted delivery is attributed to the DCL ligands affinity for PSMA, facilitating receptor-mediated endocytosis and selective tumor uptake. Furthermore, the observed slight increase in kidney accumulation of DCL-SMPG/R6G nanoparticles aligns with findings from other studies, where PSMA-targeted agents exhibited renal uptake due to endogenous PSMA expression in renal tissues. These results underscore the importance of considering off-target effects in the design of PSMA-targeted therapies [ 53 ] . Tumor volume and body weight changes were analyzed in a 22RV1 mouse model after therapy with DOX, SMPG, SMPG/DOX, and DCL-SMPG/DOX. The results demonstrated that tumor size and volume significantly decreased following therapy with DCL-SMPG/DOX, with H&E staining showing a markedly higher degree of tumor necrosis compared to the other therapy groups. Ki-67 is an important indicator used to evaluate the proliferative activity of tumor cells. The expression level of Ki-67 reflects the extent of cellular proliferation, with higher Ki-67 levels indicating an increased number of cells in the mitotic phase and more active cell division [ 54 ] . Caspase-3 is a biomarker of apoptosis that, upon receiving apoptotic signals, becomes activated and cleaves and modifies various intracellular proteins. It can cleave cytoskeletal proteins, leading to morphological changes, and act on DNA repair-related proteins, rendering the cell incapable of repairing damage [ 55 ] . These actions collectively push the cell toward an irreversible apoptotic process. Immunohistochemistry results showed that the expression of Ki-67 in tumor tissues from the DCL-SMPG/DOX therapy group was lower than in other therapy groups, while caspase-3 expression was higher than in other groups. These findings indicate that DCL-SMPG/DOX therapy can effectively ablate tumors by significantly inhibiting tumor proliferation and promoting apoptosis. In conclusion, we have successfully created a synergistic chemotherapy-CDT/ST nanomedicine platform for the targeted therapy of prostate cancer. This system effectively targets and is internalized by prostate cancer cells, and it responds to the tumor microenvironment with sustained drug release. It induces a ROS storm within tumor cells, promoting chemotherapy-induced apoptosis of prostate cancer. The examination of the in vivo and in vitro efficacy of DCL-SMPG/DOX in inhibiting prostate cancer in animal models suggests that DCL-SMPG/DOX has significant potential for clinical application. 5. Conclusions A targeted nanomedicine delivery system capable of single or multiple reactions was designed. Using MSN and PDA as drug delivery platforms, chemotherapeutic agents doxorubicin, CDT agent MnO 2 , and ST agent GOX were encapsulated to construct a nanomedicine (SMPG/DOX) that synergistically targets prostate cancer. The nanomedicine is coupled with low molecular weight ligands targeting PSMA (DCL-SMPG/DOX or VHH-SMPG/DOX). Notably, DCL-SMPG/DOX exhibits superior cellular internalization capabilities and demonstrates positive antitumor effects in combination with chemotherapy-CDT/ST. Overall, this multi-modal targeted nanomedicine shows significant translational potential for the therapy of prostate cancer. However, further investigation into its underlying antitumor mechanisms is necessary. Abbreviations PCa Prostate cancer PSMA Prostate-specific membrane antigen LMW Low molecular weight CDT Chemodynamic therapy ST Starvation therapy ROS Reactive oxygen species DOX Doxorubicin hydrochloride MnO 2 Manganese dioxide GSH Glutathione H 2 O 2 Hydrogen peroxide •OH Hydroxyl radicals GOx Glucose oxidase TME Tumor microenvironment NPs Nanoparticles MSN Mesoporous silica PDA Polydopamine DCL Small-molecule inhibitor VHH Anti-PSMA nanobody KMnO 4 Potassium permanganate DTNB 5,5′ -dithiobis (2-nitrobenzoic acid) TMB 4,4′-diamino- 3,3′,5,5′-tetramethylbiphenyl DCFH-DA 2,7′ -dichlorodihydrofluorescein diacetate MB Methylene blue TEOS Tetraethyl orthosilicate CTAB Cetyltrimethylammonium bromide DAPI 4′,6-diamidino-2-phenylindole H&E Hematoxylin and Eosin IHC Immunohistochemistry NPs Nanoparticles GSSG Oxidized glutathione ATP Adenosine Triphosphate R6G Rhodamine 6G Declarations Funding This work was supported by the National Natural Science Foundation of China [grant numbers 82060462, 82360558]; the Science and Technology Foundation of Guizhou Province [grant number ZK[2023] 211]; the Foundation of Health Commission of Guizhou Province [grant number gzwkj2021-534]; and the Guizhou Province Youth Science and Technology Talents Growth Project [grant number Foundation- [2024] Youth 296 ]. Author contributions Xiaoli Zhang: Investigation, Methodology, Data curation, Writing – original draft. Jie He: Investigation, Methodology, Data curation. Yu An: Data curation, Writing – original draft. Kehua Jiang: Investigation, Methodology. Qiang Wang: Investigation, Methodology. Wenrui Deng: Investigation, Validation. Qiqi Yang: Investigation, Validation. Fa Sun: Funding acquisition, Project administration. Kun Chen: Conceptualization, Writing – review & editing, Funding acquisition, Project administration. Declarations of ethics approval The animal experiments were approved by the Ethics Committee of Guizhou Medical University. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. References Siegel RL, Miller KD, Wagle NS, Jemal A (2023) Cancer statistics, 2023.[J]. 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Pharmaceutics, 12(4) Cong VT, Tilley RD, Sharbeen G, Phillips PA, Gaus K, Gooding JJ (2021) How to exploit different endocytosis pathways to allow selective delivery of anticancer drugs to cancer cells over healthy cells.[J]. Chem Sci 12(46):15407–15417 Martins PT, Velazquez-Campoy A, Vaz WLC, Cardoso RMS, Valério J, Moreno MJ (2012) Kinetics and thermodynamics of chlorpromazine interaction with lipid bilayers: effect of charge and cholesterol.[J]. J Am Chem Soc 134(9):4184–4195 Dhull A, Wei J, Pulukuri AJ, Rani A, Sharma R, Mesbahi N, Yoon H, Savoy EA, Xaivong Vi S, Goody KJ, Berkman CE, Wu BJ, Sharma A (2024) PSMA-targeted dendrimer as an efficient anticancer drug delivery vehicle for prostate cancer.[J]. Nanoscale 16(11):5634–5652 Yang C, Su H, Liao X, Han C, Yu T, Zhu G, Wang X, Winkler CA, O’Brien SJ, Peng T (2018) Marker of proliferation Ki-67 expression is associated with transforming growth factor beta 1 and can predict the prognosis of patients with hepatic B virus-related hepatocellular carcinoma.[J]. Cancer Manage Res 10:679–696 Asadi M, Taghizadeh S, Kaviani E, Vakili O, Taheri-Anganeh M, Tahamtan M, Savardashtaki A (2022) Caspase-3: Structure, function, and biotechnological aspects.[J]. Biotechnol Appl Chem 69(4):1633–1645 Schemes Scheme 1 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterialReviseclenaversion.docx image1.png Scheme 1 Schematic depicting the synthesis of VHH-SMPG/DOX and DCL-SMPG/DOX nanoparticles and TME-responsive synergistic therapy. Cite Share Download PDF Status: Published Journal Publication published 03 Jun, 2025 Read the published version in Cancer Nanotechnology → Version 1 posted Editorial decision: Accepted 30 Apr, 2025 Reviews received at journal 27 Apr, 2025 Reviewers agreed at journal 17 Apr, 2025 Reviews received at journal 14 Apr, 2025 Reviewers agreed at journal 13 Apr, 2025 Reviewers invited by journal 12 Apr, 2025 Submission checks completed at journal 11 Apr, 2025 First submitted to journal 11 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-6045405","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":442825500,"identity":"0b64369e-7aac-41c4-bb2f-b38dcf454a8e","order_by":0,"name":"Xiaoli Zhang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Zhang","suffix":""},{"id":442825501,"identity":"e3e513ec-5dfe-43d6-9fb6-f2bc2f5eb6ec","order_by":1,"name":"Jie He","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"He","suffix":""},{"id":442825502,"identity":"5b0da7b8-e9a5-4119-be90-961d9734ef07","order_by":2,"name":"Yu An","email":"","orcid":"","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"An","suffix":""},{"id":442825503,"identity":"63a05b7d-14e4-4b78-9032-08c28cf9a3ee","order_by":3,"name":"Kehua Jiang","email":"","orcid":"","institution":"Guizhou Provincial People’ Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kehua","middleName":"","lastName":"Jiang","suffix":""},{"id":442825504,"identity":"f7cc11e4-ef32-4c56-8a77-57039fef8df8","order_by":4,"name":"Qing Wang","email":"","orcid":"","institution":"Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Wang","suffix":""},{"id":442825505,"identity":"880623a2-2957-4a17-a436-854f8e82bc64","order_by":5,"name":"Wenrui Deng","email":"","orcid":"","institution":"Guizhou Provincial People’ Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenrui","middleName":"","lastName":"Deng","suffix":""},{"id":442825506,"identity":"2ec8ceb5-fb3c-4506-9dcb-3e20ed348c95","order_by":6,"name":"Qiqi Yang","email":"","orcid":"","institution":"Guizhou Provincial People’ Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qiqi","middleName":"","lastName":"Yang","suffix":""},{"id":442825507,"identity":"f413dc40-ccc9-4102-b0e3-1c66f89417cc","order_by":7,"name":"Fa Sun","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Fa","middleName":"","lastName":"Sun","suffix":""},{"id":442825508,"identity":"a679a803-4c4e-49ae-99bb-27c9e429d74b","order_by":8,"name":"Kun Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYBACxobDBwwkKv7J8ROthbnxWEKBxZkDxpINxGphbz5j8KGy7UCiwQFitfC2HUvccLPtToLx8eQNDD8qthHWItlz+LDhjHPP8szOPCtg7Dlzm7AWwxnH0owlypiLzW7kGDAzthGhxf7+G/Pff9iYEzfPIFYLY8MZAwOJtsOJGySI13IswUDiDNBxQL8cJMov0Ki0keNvT9744EcFEVqQQALxUYPQQqqOUTAKRsEoGCEAAHCdR7DPMEaIAAAAAElFTkSuQmCC","orcid":"","institution":"Guizhou Provincial People’ Hospital","correspondingAuthor":true,"prefix":"","firstName":"Kun","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-02-17 07:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6045405/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6045405/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12645-025-00325-2","type":"published","date":"2025-06-03T15:57:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80626907,"identity":"a2265b26-7fc6-4310-9f06-f892fbf1886e","added_by":"auto","created_at":"2025-04-15 10:55:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":235810,"visible":true,"origin":"","legend":"\u003cp\u003eThe synthesis and characterization of VHH-SMPG and DCL-SMPG NPs.\u003c/p\u003e\n\u003cp\u003e(A) TEM images of MSNNPs;\u003cstrong\u003e \u003c/strong\u003e(B) TEM images of MSN@Mn NPs; (C) Elemental mapping images of MSN@MnNPs; (D) Mn2p XPS spectrum of MSN@Mn NPs; (E)UV-Vis absorbance spectra of DOX, MSN NPs, and MSN@Mn/DOX NPs;\u003cstrong\u003e \u003c/strong\u003e(F) DLS analysis; (G) Zeta potential; (H)FTIR spectra of MSN, MSN@Mn, MSN@Mn@DOX, VHH-SMPG/DOX, and DCL-SMPG NPs; (I) Fluorescence images obtained after the incubation of SMPG and VHH-SMPG with anti-VHH antibodies (Alexa Fluor 488).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6045405/v1/af4a41c6a3537e3b2d4a8cc9.png"},{"id":80626908,"identity":"25546e28-fc30-4a18-91ee-5abbf4d15ea3","added_by":"auto","created_at":"2025-04-15 10:55:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":205875,"visible":true,"origin":"","legend":"\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e TME-responsive drug release and nanozyme activities of SMPG NPs. (A) The cumulative release of DOX from SMPG/DOX NPs under different conditions; (B) GSH concentrations measured after incubation with SMPG NPs for varying time; (C) UV/vis absorption spectra of TMB solutions after incubation with different concentrations of SMPG NPs; (D) UV/vis absorption spectra of MB after incubation with different concentrations of SMPG NPs. The NPs1, NPs2, and NPs3 are 50, 100, and 200 μ g mL\u003csup\u003e-1\u003c/sup\u003e. (E) The pH changes observed after the incubation of SMPG NPs with different concentrations of glucose; (F) The changes in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels after the incubation of glucose with different concentrations of SMPG NPs.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6045405/v1/8733442cc926a017d28c1f0e.png"},{"id":80626906,"identity":"46d94e4e-2e9e-44be-baab-7d7ea1461f75","added_by":"auto","created_at":"2025-04-15 10:55:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":223992,"visible":true,"origin":"","legend":"\u003cp\u003eThe selective cellular uptake activity of the prepared nanoparticles. (A) CLSM images of 22RV1 cells after co-incubation with free DOX, SMPG/DOX, VHH-SMPG/DOX, and DCL-SMPG/DOX NPs; (B-C) Representative images of cellular uptake analysis using flow cytometry (B) and corresponding mean fluorescence intensity (C); (D) Uptake in 22RV1 and PC3 cells after incubation with free DOX, SMPG/DOX, VHH-SMPG/DOX, and DCL-SMPG/DOX NPs; (E) Uptake rates in 22RV1 cells following various treatments, including NP incubation at 37°C and 4°C, as well as cellular pretreatment with nystatin, M-β-CD, chlorpromazine, and amiloride. ∗p \u0026lt; 0.05, ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001, and ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6045405/v1/64d0a1231b335fa617841ccc.png"},{"id":80627384,"identity":"c99a1d0d-0f27-401f-983f-f86677c91357","added_by":"auto","created_at":"2025-04-15 11:03:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":179615,"visible":true,"origin":"","legend":"\u003cp\u003eSynergistic anti-tumor activity of the prepared NPs. (A) Viability of 22RV1 cells assessed with free DOX, SMPG/DOX, VHH-SMPG/DOX, DCL-SMPG/DOX NPs treatments; (B) and the fluorescence images of 22RV1 cells stained with calcein-AM and propidium iodide; (C) Apoptosis assay of 22RV1 cells. ∗p \u0026lt; 0.05, ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001, and ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6045405/v1/b8825c7d24cad8a1c262475d.png"},{"id":80626911,"identity":"7574e959-c485-4b9b-a6a5-232a34e6a475","added_by":"auto","created_at":"2025-04-15 10:55:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":302790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro \u003c/em\u003eROS production. (A) Intracellular DCF fluorescence in 22RV1 cells after treatment with PBS, MSN@Mn@PDA, SMPG, VHH-SMPG, and DCL-SMPG NPs;(B) Mean fluorescence intensity of ROS generation in 22RV1 cells analyzed by flow cytometry after various treatments. (C) Mitochondrial membrane potential of 22RV1 cells after different treatments;(D) ATP levels in 22RV1 cells after different treatment. ns, not significant, ∗p \u0026lt; 0.05, ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001, and ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6045405/v1/edd35cf815ca5ed38a41d836.png"},{"id":80626910,"identity":"273d7493-98d5-4bf5-932e-1ba15851ec99","added_by":"auto","created_at":"2025-04-15 10:55:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":212891,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo tissue distribution after DCL-SMPG/R6G NPs treatment. (A) Fluorescence images of the retrieved livers, spleens, hearts, lungs, kidneys, and tumors after intravenous treatment with SMPG/R6G and DCL-SMPG/R6GNPs for 24 h. (B) FL quantitative data of tumors and organs. Statistical analysis was performed using one-way ANOVA, ∗p \u0026lt; 0.05, ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001, and ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6045405/v1/291747bc1647f5f6aaabd5bd.png"},{"id":80628196,"identity":"3dba331a-7e46-4e2c-8e65-9bc53e22457a","added_by":"auto","created_at":"2025-04-15 11:11:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":538149,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic efficacy of DCL-SMPG/DOX in 22RV1 tumor-bearing mice. (A-B) Changes in tumor volume (A) and tumor weight (B) in tumor-bearing mice following treatment with free DOX, SMPG, SMPG/DOX, DCL-SMPG/DOX;(C) Body weight changes; (D) H\u0026amp;E stainingand IHC for Ki-67 and caspase-3 in tumor tissues. ∗p \u0026lt; 0.05, ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001, and ∗∗∗∗p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6045405/v1/78e6d9eb59c81a5e505c6463.png"},{"id":84242585,"identity":"e1aed1bc-38bf-43e2-b011-24fba0be903b","added_by":"auto","created_at":"2025-06-09 16:09:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2768219,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6045405/v1/c3e93eaf-e548-4a72-a4db-abab50a86d16.pdf"},{"id":80626916,"identity":"982e04b5-3953-4c5d-b328-54ab2b91df7f","added_by":"auto","created_at":"2025-04-15 10:55:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4346480,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialReviseclenaversion.docx","url":"https://assets-eu.researchsquare.com/files/rs-6045405/v1/5a501cc993a9fe0a48e61a5c.docx"},{"id":80628199,"identity":"d6708601-6296-463b-a23a-e6862b429e54","added_by":"auto","created_at":"2025-04-15 11:11:28","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3473631,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1 \u003c/strong\u003eSchematic depicting the synthesis of VHH-SMPG/DOX and DCL-SMPG/DOX nanoparticles and TME-responsive synergistic therapy.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6045405/v1/424d6949dff3a82ea020e2b2.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Using low - molecular - weight ligands for targeting in integrated chemodynamic/starvation therapy and chemotherapy for prostate cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eProstate cancer (PCa) is widely regarded as the second most prevalent malignant neoplasm in men and is a significant contributor to cancer-related mortality in this population. Notably, PCa has the highest incidence among all newly diagnosed male tumors, and its global prevalence has increased considerably over the past few years \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The absence of distinctive early symptoms in PCa leads to a high incidence of advanced-stage disease at initial diagnosis, ultimately resulting in an overall unfavorable prognosis \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. At present, pharmacotherapy is the primary therapy modality for advanced PCa. Unfortunately, the clinical application of high-dose chemotherapeutic agents is significantly restricted by their dose-limiting toxicity, which arises from the unintended systemic exposure of healthy tissues to these drugs \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Furthermore, patients often fail to complete the prescribed chemotherapy regimen due to cumulative drug toxicity, drug resistance, or disease progression. Hence, the side effects and other limitations of chemotherapy pose a significant challenge and remain to be addressed.\u003c/p\u003e \u003cp\u003eNanomedicines have gradually emerged as potential candidates for PCa treatment. Several nanomedicines \u0026mdash; such as Abraxane, Onyvide, Marqibo, and Doxil \u0026mdash; have demonstrated clinical advantages in reducing chemotherapy-induced drug toxicity and improving overall patient survival \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, achieving ideal therapeutic outcomes through a single therapeutic regimen is challenging. Chemodynamic therapy (CDT) induces apoptosis in tumor cells by utilizing endogenous cellular chemical energy to promote reactive oxygen species (ROS) production, eliminating the need for external energy \u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Upon uptake by tumor cells, MnO\u003csub\u003e2\u003c/sub\u003e depletes the intracellular antioxidant glutathione (GSH) and generates Mn\u003csup\u003e2+\u003c/sup\u003e, disrupting the cellular antioxidant defense system. Mn\u003csup\u003e2+\u003c/sup\u003e exhibits remarkable Fenton-like activity, converting endogenous hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) into highly toxic hydroxyl radicals (\u0026bull;OH) and thereby enhancing the tumor damage induced by GSH-depleting cancer therapy (i.e., CDT) \u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Glucose oxidase (GOx) can catalytically disrupt glucose metabolism and produce H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the tumor microenvironment (TME), enabling tumor starvation therapy (ST) \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. This process of GOx-mediated glucose consumption increases the levels of gluconic acid and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the TME, thereby improving the effectiveness of CDT.\u003c/p\u003e \u003cp\u003eDrugs administered into the bloodstream face several biological barriers before reaching their target sites. The drugs must cross the vascular network, diffuse into tumor tissues, and get internalized by tumor cells. These barriers hamper the delivery efficiency of anti-cancer nanomedicines, decreasing their overall therapeutic effect \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Despite decades of advances, the median delivery efficiency of systemically administered nanomedicines remains critically low (typically\u0026thinsp;\u0026lt;\u0026thinsp;1%, with some studies reporting values as low as 0.0014% \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e ), underscoring the urgent need for more effective tumor-targeted delivery strategies. Thus, to enhance the transmembrane transport of nanomedicines and reduce their phagocytic uptake by non-tumor cells, researchers have modified the surfaces of nanomedicines with specific targeting molecules to achieve selective tumor cell targeting \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. These strategies have proven effective at reducing treatment side effects and improving therapeutic efficacy. For instance, Wang et al. utilized anti-PSMA antibodies to modify arsenic nanosheets, achieving higher intratumoral drug concentrations and fewer adverse effects \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.Compared to monoclonal antibodies, low - molecular - weight (LMW) ligands possess distinct advantages such as smaller molecular size, improved tumor penetration, faster systemic clearance, and reduced immunogenicity \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. These features allow for more efficient and selective delivery of therapeutic payloads to PCa cells. In particular, PSMA-targeting LMW ligands, including small-molecule inhibitors (DCL) and nanobodies (VHH), have demonstrated strong binding affinity and rapid internalization into PCa cells, enabling enhanced intratumoral accumulation and minimizing off-target toxicity \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. Despite their potential, few studies have directly compared the targeting performance and therapeutic efficacy of multiple LMW ligands within the same nanoplatform. Therefore, our study addresses by systematically evaluating and comparing the tumor-targeting capabilities and therapeutic outcomes of VHH- and DCL-functionalized nanomedicines for PCa treatment.\u003c/p\u003e \u003cp\u003eIn this study, we rationally designed a hybrid multifunctional nanomedicine(Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) capable of specifically targeting PCa. In this system, mesoporous silica (MSN) served as nanocarriers for the therapeutic agents MnO\u003csub\u003e2\u003c/sub\u003e and doxorubicin (DOX) and was subsequently coated with a polydopamine (PDA) nano-shell to form a core-shell structure. Then, GOx was grafted onto the surface of these nanoparticles via electrostatic interactions. Finally, the nanoparticles were surface-functionalized with LMW (VHH and DCL) to fabricate VHH-SMPG/DOX and DCL-SMPG/DOX nanomedicine. Subsequently, the differences in the therapeutic efficacy of VHH-SMPG/DOX and DCL-SMPG/DOX nanomedicine were examined. In the TME, GOx, MnO\u003csub\u003e2\u003c/sub\u003e increased ROS production by disrupting the nutrient supply of the tumor and initiating a cascade of catalytic reactions, synergistically inducing apoptosis along with chemotherapy. Moreover, functionalization with the LMW specifically enhanced the accumulation of the nanomedicine at tumor sites, reducing off-target toxicity and improving therapeutic efficacy, thereby providing an effective strategy for PCa therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eAll the chemicals used in this study were of analytical reactive grade and most of them were from Aladdin Chemical Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Synthesis of SMPG NPs\u003c/h2\u003e \u003cp\u003eMSN nanoparticles (NPs) were synthesized following established protocols, with slight modifications \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. 50 mg MSN were dispersed in water, and 20 mL of a KMnO\u003csub\u003e4\u003c/sub\u003e solution (0.5 mM) was added dropwise under dark conditions and stirred for 4 h. 50 mg of MSN@Mn NPs were dispersed in a Tris-HCl buffer solution (10 mM, pH 8.5) containing 25 mg of dopamine hydrochloride and reacted for 3h to obtain MSN@Mn@PDA. 53 mg NHS, 35 mg EDC, 10 mg GOx were dissolved in 20 mL water before adding 500 \u0026micro;L APTES. Subsequently, 10 mg of MSN@Mn@PDA NPs added to stirring for an additional 24h. The final product was GOx-grafted SMPG NPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Synthesis of DCL-SMPG and VHH-SMPG NPs\u003c/h2\u003e \u003cp\u003e10 mg of SMPG NPs and 10 mg of DCL-PEG2k-COOH in a 15 mL aqueous solution. The pH was adjusted to 8.5 using an ammonia solution and the mixture was stirred for 24h to obtain DCL-SMPG NPs. 1mg of SMPG NPs were functionalized using HOOC-PEG\u003csub\u003e2k\u003c/sub\u003e-NHS. 0.2 mg/mL VHH solution (500 \u0026micro;L) was added to this dispersion. The mixture was incubated overnight at 4\u0026deg;C. Unbound VHH was removed via centrifugation and thorough washing to obtain the final product VHH-SMPG NPs. VHH loading was quantified via BCA assay. Briefly, free VHH in the supernatant was measured against a BSA standard curve.\u003c/p\u003e \u003cp\u003eVHH Loading (μ g/mg SMPG) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{W}\\:\\text{i}\\text{n}\\text{i}\\text{t}\\text{i}\\text{a}\\text{l}\\:\\text{V}\\text{H}\\text{H}-\\text{W}\\:\\text{f}\\text{r}\\text{e}\\text{e}\\:\\text{V}\\text{H}\\text{H}}{\\text{W}\\:\\text{S}\\text{M}\\text{P}\\text{G}\\:\\text{N}\\text{P}\\text{s}\\:}\\)\u003c/span\u003e\u003c/span\u003e(W: weight)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Drug loading and stimulus-triggered drug release assay\u003c/h2\u003e \u003cp\u003eThe resulting product MSN@Mn NPs (5 mg/mL) were dispersed in a 20 mL DOX solution (0.5 mg/mL) and stirred for 24 h. The MSN@Mn/DOX NPs was obtained by centrifugation for further use. The drug loading amounts were determined by analysing the supernatant DOX solution after centrifugation with a UV-Vis test (480 nm). The release of DOX from SMPG/DOX NPs was examined in various media. Specifically, SMPG/DOX NPs (1 mg/mL; total volume, 1 mL) were dispersed in PBS solutions of different pH values (7.4 and 5.2) with or without 10.0 mM GSH. These solutions were placed inside dialysis bags (2000 KDa), which were immersed in 9 mL of the corresponding PBS solutions and incubated at 37\u0026deg;C with gentle shaking. After predetermined intervals, 1 mL of the external solution was sampled and replaced with fresh PBS (same pH). The DOX concentration in the collected samples was determined using a microplate reader (Bio-Rad Model680, USA).\u003c/p\u003e \u003cp\u003eDLC (Drug Loading Capacity, %)=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{W}\\:\\text{l}\\text{o}\\text{a}\\text{d}\\text{e}\\text{d}\\:\\text{D}\\text{O}\\text{X}\\:}{\\text{W}\\:\\text{N}\\text{P}\\text{s}\\:+\\text{W}\\:\\text{l}\\text{o}\\text{a}\\text{d}\\text{e}\\text{d}\\:\\text{D}\\text{O}\\text{X}\\:}\\:ⅹ\\:100\\%\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Nanozyme activity assay\u003c/h2\u003e \u003cp\u003e1 mL of SMPG NPs (25 \u0026micro;g/mL) was incubated with 1 mL of GSH (10 mM) for different durations (0, 1, 2, 4, 6 h). The mixtures were reacted with a 1 mL DTNB (1 mM) for 30 min and their absorbance at 412 nm was recorded. To examine TMB oxidation, different concentrations of the SMPG NPs were mixed with an equal volume of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (20 mM). Equal volumes of TMB (10 mM) were added to the mixture. After 30 min the fluorescence spectrum of the supernatant was measured. Similarly, various concentrations of SMPG NPs were mixed with equal volumes of an MB solution for 30 min. The fluorescence spectrum of MB was then measured.\u003c/p\u003e \u003cp\u003eSMPG NPs (0.5 mg/mL) were uniformly mixed with different concentrations of glucose solutions at room temperature. A pH meter was employed to determine the pH value of the mixture. To examine H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e generation, different concentrations of SMPG NPs were mixed with glucose solutions (10 mM) at room temperature. Then, different samples were collected and the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration was measured by a H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e assay kit. Briefly, 50 \u0026micro;L of samples was mixed with 100 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e detection reagent. After 30 min incubation at room temperature in the dark. Absorbance was measured at 560 nm using a microplate reader. H₂O₂ concentrations were calculated against a standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cellular uptake assay\u003c/h2\u003e \u003cp\u003e22RV1 cells were seeded in 24-well plates at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. When the cells reached confluency, the culture medium was replaced with a fresh medium containing equivalent concentrations of DOX in the forms of free DOX, SMPG/DOX, VHH-SMPG/DOX, and DCL-SMPG/DOX NPs. After a 1, 2, and 4h incubation period, the medium was aspirated. The cells were washed thrice with PBS, fixed, and stained with DAPI. Subsequently, cellular uptake was visualized using CLSM (ZEISS, Germany). Additionally, flow cytometry (BDFA Celesta Flow Cytometer, Novocyte) was employed to quantitatively measure intracellular fluorescence intensity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. In vitro cytotoxicity study\u003c/h2\u003e \u003cp\u003e22RV1 cells were seeded into 96-well plates. After the cells adhered sufficiently to the plates, the medium was removed. Subsequently, 100 \u0026micro;L of fresh medium containing different concentrations of free DOX, SMPG, SMPG/DOX, VHH-SMPG/DOX, DCL-SMPG/DOX NPs (containing an equivalent amount of DOX) was added to the wells and cell viability was assessed using the CCK-8 assay. SMPG/DOX nanoparticle concentrations were adjusted to deliver 5 \u0026micro;g/mL of DOX, based on a drug loading capacity DLC of 42.9%. Accordingly, a final SMPG/DOX and SMPG NPs concentration of 12 \u0026micro;g/mL was used. Furthermore, the cells were stained with 5 \u0026micro;L of Calcein-AM and 10 \u0026micro;L of PI for 30 min and then observed under a fluorescence microscope to examine cell viability. Similarly, cells were stained with Annexin V-FITC/7-AAD using apoptosis detection kits, and apoptosis was measured using flow cytometry. The forward scatter (FSC) and side scatter (SSC) voltages were set to 300 and 225, respectively, to accurately gate single, viable cells while excluding debris. Fluorescence signals were detected using a FITC voltage of 168 (Annexin V-FITC) and a 7-AAD voltage of 158.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. ROS detection and the mitochondrial membrane potential of live cells\u003c/h2\u003e \u003cp\u003e22RV1 cells were seeded into 24-well plates. Once the cells adhered sufficiently, they were treated with 50 \u0026micro;g/mL of MSN@Mn@PDA, SMPG, VHH-SMPG, and DCL-SMPG NPs treatment 6h. Intracellular ROS levels were quantified using the DCFH-DA probe. Cells were incubated with 10 \u0026micro;M DCFH-DA for 30 min at 37\u0026deg;C, washed with PBS, and analyzed immediately. Fluorescence signals were measured at excitation 488 nm and emission 525 nm using a flow cytometer and fluorescence microscope. Similarly, the changes in the mitochondrial membrane potential were observed by staining the cells with the JC-1 probe. Cells were stained with 5 \u0026micro;g/mL JC-1 for 20 min at 37\u0026deg;C. Fluorescence was detected at two emission wavelengths: aggregates (excitation 585 nm, emission 590 nm) and monomer (excitation 514 nm, emission 529 nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Animal Tumor Models\u003c/h2\u003e \u003cp\u003eMale BALB/C nude mice (~\u0026thinsp;4 weeks old) were procured from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and housed in the Experimental Animal Center of The Affiliated Hospital of Guizhou Medical University, China. Animal procedures were conducted following the guidelines of the Regional Ethics Committee and approved care regulations of The First Affiliated Hospital of Guizhou Medical University.\u003c/p\u003e \u003cp\u003e22RV1 cells were first washed with PBS, collected, and suspended in PBS. Tumor models were established by subcutaneously injecting 5 \u0026times;10\u003csup\u003e6\u003c/sup\u003e 22RV1 cells (suspended in 100 \u0026micro;L PBS) into the right flank of the mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. In vivo tissue distribution\u003c/h2\u003e \u003cp\u003eTo investigate the in vivo tissue distribution of the nanoparticles, tumor-bearing BALB/c nude mice were treated with DCL-SMPG/R6G nanoparticles, with SMPG/R6G-treated mice serving as the control group. Rhodamine 6G (R6G) was loaded onto the nanoparticles to enable subsequent fluorescence imaging. Specifically, MSN@Mn nanoparticles (5 mg/mL) were dispersed in 20 mL of an R6G solution (0.5 mg/mL) and stirred continuously for 24 hours. The resulting MSN@Mn/R6G nanoparticles were collected by centrifugation for further use. The synthesis of DCL-SMPG/R6G nanoparticles was consistent with that of DCL-SMPG/DOX. When the tumor volume reached approximately 100 mm\u0026sup3;, R6G-labeled nanoparticles were intravenously administered via the tail vein at a dose of 5 mg/kg. After 24 hours, major organs including the liver, spleen, heart, lungs, kidneys, and the tumors were harvested. Fluorescence imaging and quantitative region-of-interest (ROI) analysis were performed using the Lumina IVIS Spectrum imaging system (Caliper Life Sciences Inc., USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. In vivo tumor therapy\u003c/h2\u003e \u003cp\u003eTo assess the \u003cem\u003ein vivo\u003c/em\u003e anti-tumor efficacy of the NPs, mice were randomly assigned to five groups(Ten per group) once their average tumor volume reached 100 mm\u003csup\u003e3\u003c/sup\u003e. The mice received intravenous injections of PBS, free DOX, SMPG, SMPG/DOX, and DCL-SMPG/DOX NPs via the tail vein(5mg/kg DOX). During treatment, the changes in their body weight and tumor size were monitored every 2 d over a duration of 21 d post-treatment. At the end of the 21-d period, the mice were euthanized, and their major organs and tumor tissues were collected for hematoxylin and eosin (H\u0026amp;E) staining analysis. Furthermore, immunohistochemistry (IHC) was performed to analyze Ki-67 and caspase-3 expression in the tumor tissues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was conducted using Origin 2021 and GraphPad Prism 8.0 software based on the student\u0026rsquo;s t-test or one-way analysis of variance (ANOVA). Statistical significance was determined at ns, not significant, \u0026lowast;p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u0026lowast;\u0026lowast;p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u0026lowast;\u0026lowast;\u0026lowast;p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and \u0026lowast;\u0026lowast;\u0026lowast;\u0026lowast;p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of VHH-OSMPG/DOX and DCL-OSMPG/DOX NPs\u003c/h2\u003e \u003cp\u003eThe synthesis of MSN with disulfide bonds was initially accomplished via the hydrolysis and co-condensation of silane precursors in a mixture of water and ethanol under alkaline conditions. TEM images revealed that the resulting organic silica had an average diameter of approximately 122 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Subsequently, the introduction of KMnO\u003csub\u003e4\u003c/sub\u003e triggered an \u003cem\u003ein-situ\u003c/em\u003e oxidation-reduction reaction, leading to the confined growth of MnO\u003csub\u003e2\u003c/sub\u003e NPs within the mesoporous channels of the organic silica \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows the TEM images of MSN@Mn NPs. Furthermore, the composition of MSN@Mn NPs was confirmed via elemental mapping, which revealed a homogeneous distribution of Si, C, S, O, N and Mn in these particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The MSN@Mn NPs shows the typical characteristic of N\u003csub\u003e2\u003c/sub\u003e adsorption-desorption behavior with the surface area of 60.46 m\u003csup\u003e2\u003c/sup\u003e/g, average pore size of 6.3 nm, which guarantees the further storage and sustained release of chemotherapeutic drug (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD shows the Mn 2p XPS spectra exhibited two distinct peaks at 642 eV and 653 eV, which represented the binding energies of Mn 2p3/2 and Mn 2p1/2 in MnO\u003csub\u003e2\u003c/sub\u003e respectively.\u003c/p\u003e \u003cp\u003eTo confirm the successful loading of the chemotherapeutic drug DOX into the MSN/Mn NPs, the UV-vis absorption spectra of the different NPs preparations were examined. Following DOX loading into MSN@Mn NPs, a prominent absorption band at approximately 480 nm was observed in MSN@Mn/DOX NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These findings demonstrated the successful encapsulation of DOX within the MSN@Mn NPs. The drug loading capacity of the nanocarriers was calculated to be 42.9%.\u003c/p\u003e \u003cp\u003eUsing DLS analysis, the hydrodynamic diameter of MSN@Mn@PDA NPs was determined to be approximately 220 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), while the surface zeta potential became \u0026minus;\u0026thinsp;27 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). SMPG NPs were generated via electrostatic interactions. The introduction of GOx augmented the hydrodynamic diameter and increased the zeta potential to -19 mV. Furthermore, the successful integration of SMPG NPs was successively confirmed through FTIR spectroscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). SMPG NPs showed absorption peaks at 1625 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1030 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to the stretching vibrations of -CO-NH2 and C\u0026thinsp;=\u0026thinsp;O, respectively\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G shows the introduction of the targeting agent PEG-DCL onto the surface of SMPG NPs further altering their hydrodynamic diameter and zeta potential. DCL-SMPG NPs had a hydrodynamic diameter of 342 nm and a zeta potential of -8 mV. Moreover, the presence of the -CH\u003csub\u003e2\u003c/sub\u003e groups of PEG was reflected by a vibrational peak at 2840 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in DCL-SMPG NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Only negligible changes in the hydrodynamic diameter of DCL-SMPG NPs in PBS, ang FBS were observed after 7 days, suggesting that DCL-SMPG NPs also possessed good colloidal stability in biological media (Fig. S2).\u003c/p\u003e \u003cp\u003eAnti-VHH antibodies (Alexa Fluor 488) were incubated with SMPG, VHH-SMPG NPs, the samples were observed using fluorescence microscopy after the excess anti-VHH antibodies were completely removed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). The observations confirmed the successful synthesis of VHH-SMPG NPs. BCA quantification confirmed a VHH loading of 73 \u0026micro;g/mg SMPG, demonstrating efficient conjugation. This value aligns with reported ligand densities for targeted nanomedicines and ensures sufficient binding sites for PSMA-specific uptake \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Stimulus-responsive DOX release and enzyme activity of SMPG NPs\u003c/h2\u003e \u003cp\u003eFirst, we examined the release of DOX from SMPG/DOX NPs. At pH 7.4, DOX release increased gradually for 48h, yielding a cumulative drug release rate of less than 15% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, the rate of drug release was higher at a pH of 5.2 and increased even further at a pH of 7.4 in the presence of 10 mM GSH. Even after the 48-hour, the drug release rate remained below 22%. In contrast, in the presence of 10 mM GSH under acidic conditions (pH 5.2), approximately 33% of the DOX diffused out of SMPG/DOX NPs. Similarly, we examined DOX release from DCL-SMPG/DOX and VHH-SMPG/DOX NPs (Fig. S3). Within 24 h, the release profiles from both DCL-SMPG/DOX and VHH-SMPG/DOX NPs were essentially consistent with that of SMPG/DOX NPs.\u003c/p\u003e \u003cp\u003eMnO\u003csub\u003e2\u003c/sub\u003e can convert reduced GSH to its oxidized form and release Mn\u003csup\u003e2+\u003c/sup\u003e, further catalyzing the generation of cytotoxic \u0026bull;OH from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. After prolonged incubation with SMPG NPs, the content of GSH gradually decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and the absorbance value of GSH decreased with increasing concentrations of SMPG NPs (Fig. S4). Subsequently, \u0026bull;OH production was observed under different conditions using TMB and MB as indicators. TMB readily captures highly reactive \u0026bull;OH species to form a radical cation that generates a blue color and exhibits an absorption peak at 650 nm \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. When the presence of SMPG NPs, the absorbance increased with an increase in the SMPG NP concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Similarly, when MB was incubated with SMPG NPs, the absorbance of the mixture decreased with an increasing concentration of SMPG NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Remarkably, the MB degradation kinetics exhibited a non-monotonic dependence on GSH concentration (Fig. S5). At 1\u0026ndash;2 mM GSH, enhanced Mn\u0026sup2;⁺ release from SMPG amplified \u0026middot;OH generation, minimizing MB absorbance. Conversely, \u0026ge;\u0026thinsp;5 mM GSH scavenged \u0026middot;OH via thiol-radical interactions, attenuating MB degradation. This biphasic behavior aligns with Mn\u0026sup2;⁺-mediated Fenton catalysis and glutathione\u0026rsquo;s dual redox roles \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWe investigated the oxidative activity of SMPG NPs toward glucose by monitoring the changes in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration and pH value in mixed solutions. At a glucose concentration of 10 mM, the pH value decreased from 6.8 to 3.5 within a 2h period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Additionally, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration in the solution increased with an increase in the concentration of SMPG NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Subsequently, the \u0026bull;OH-producing capacity of SMPG NPs was investigated in the presence of glucose (Fig. S6). When SMPG NPs were incubated with glucose, the absorbance of MB decreased due to the gluconic acid-induced activation of SMPG nanozyme activity, which enhanced \u0026bull;OH generation \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Cellular uptake\u003c/h2\u003e \u003cp\u003eDuring the incubation period, both VHH-SMPG/DOX and DCL-SMPG/DOX NPs demonstrated rapid internalization in 22RV1 cells, unlike free DOX and SMPG/DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This phenomenon could be attributed to the interactions between PSMA- ligand and 22RV1 cells \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Notably, the cellular fluorescence intensity of DCL-SMPG/DOX NPs was higher than that of VHH-SMPG/DOX NPs at the same incubation time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Moreover, DCL-SMPG/DOX consistently exhibited higher intracellular fluorescence intensity than VHH-SMPG/DOX at all time points, indicating more rapid and efficient internalization(Fig. S7). This enhanced uptake is likely attributable to the high-affinity interaction between DCL and PSMA on the cell surface.\u003c/p\u003e \u003cp\u003eWe further compared the internalization patterns of NPs between 22RV1 (PSMA-positive) and PC3 (PSMA-negative) cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The targeting efficiency was notably enhanced in 22RV1 cells incubated with VHH-SMPG/DOX and DCL-SMPG/DOX NPs, resulting in uptake efficiencies of 34.1% and 45.5%, respectively. Meanwhile, the uptake of SMPG/DOX NPs in these cells was only 25.4%. This targeted enhancement (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) signified the increased interaction between 22RV1 cells and NPs. However, the uptake efficiencies for each type of NPs significantly lower in PC3 cells than in 22RV1 cells.\u003c/p\u003e \u003cp\u003ePassive diffusion serves as the primary mechanism for the intracellular uptake of small-molecule drugs. Meanwhile, the intracellular accumulation of NPs is often due to enhanced endocytosis \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Consequently, we investigated the effects of temperature (energy-dependent pathways) and different phagocytosis inhibitors on cellular uptake to identify the internalization mechanism of NPs \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, cellular uptake rates were significantly poorer at 4\u0026deg;C (only 4.58%) than at 37\u0026deg;C. When chlorpromazine and nystatin were used to block clathrin-mediated endocytosis (CME) and caveolin-mediated endocytosis(CavME), respectively, the internalization rates of all NPs decreased. Specifically, inhibition with chlorpromazine reduced the internalization rate to 15%, while inhibition with nystatin reduced this rate to 35%. When M-β-CD blocked both clathrin and caveolin-mediated pathways, the uptake rates decreased by approximately 40%. However, when amiloride was used to block macropinocytosis, the internalization rates of all NPs remained similar to those observed at 37\u0026deg;C. This indicated that 22RV1 cells primarily internalize all NPs through energy-dependent clathrin- and caveolin-mediated pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4. In vitro anti-tumor performance\u003c/h2\u003e \u003cp\u003eFollowing the treatment of 22RV1 cells with free DOX, SMPG, SPMG/DOX, VHH-SMPG/DOX, and DCL-SMPG/DOX, cell viability and apoptosis were assessed. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, dose-dependent cytotoxicity was observed in 22RV1 cells following treatment with free DOX and different NPs. At a DOX concentration of 5 \u0026micro;g/mL, the cell viability after DCL-SMPG/DOX treatment was only 8.4%. This value was markedly lower than the values observed after SPMG/DOX (42.4%) and VHH-SMPG/DOX (24.5%) treatment. The cytotoxicity of DCL-SMPG/DOX was higher than that of free DOX, SMPG, and SPMG/DOX underscoring the potential of combination therapy in exerting synergistic anti-tumor effects \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e22RV1 cells subjected to different treatments were stained using calcein-AM and propidium iodide for live/dead staining. After treatment with DCL-SMPG/DOX, the red fluorescence was the highest and there were virtually no viable cells in this group(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). After treatment with free DOX, approximately 45.8% of cells were found to be in the early and late stages of apoptosis. Meanwhile, the rate of apoptosis after treatment with SMPG NPs was 42.93%. The synergistic effects of CDT/ST and chemotherapy after SMPG/DOX treatment increased the rate of apoptosis to 56.38%. After DCL-SMPG/DOX treatment, the rate of apoptosis reached 85.57%(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This demonstrated that DCL-SMPG/DOX NPs induce pronounced cancer cell apoptosis.\u003c/p\u003e \u003cp\u003eThe cytotoxicity induced by VHH-SMPG NPs was significantly lower than that induced by DCL-SMPG NPs (Fig. S8). To further validate the PSMA-targeting specificity of our nanoplatforms, we performed comparative in vitro cytotoxicity assays using both 22RV1 and PC3 prostate cancer cell lines (Fig. S9). In PC3 cells, which lack PSMA expression, both targeted (VHH-SMPG/DOX52.1% cell viability and DCL-SMPG/DOX 50.4% cell viability)formulations exhibited no significant increase in cytotoxicity compared to the non-targeted SMPG/DOX (50.0% cell viability). This indicates minimal off-target cytotoxic effects in cells lacking PSMA expression. In contrast, in PSMA-positive 22RV1 cells, both targeted nanoparticles༈VHH-SMPG/DOX 24.5% cell viability and DCL-SMPG/DOX 8.4% cell viability༉ induced significantly higher cytotoxicity relative to the non-targeted formulation(SMPG/DOX 43.0% cell viability). The enhanced cytotoxic effects observed for VHH-SMPG/DOX and DCL-SMPG/DOX were statistically significant, confirming that the functionalization with PSMA-targeting ligands effectively improved therapeutic efficacy via receptor-mediated uptake. Together, these findings demonstrate that the antitumor activity of the targeted nanocarriers is PSMA-dependent, and support the specificity and safety profile of our delivery strategy. Moreover, the cytotoxicity of 200 \u0026micro;g/mL MSN@PDA NPs with SVUHC, PC3, 22RV1 cells remained around 10%(Fig. S10). Similarly, the hemolytic rate induced by SMPG treatment was below 5% (Fig. S11).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5. In vitro ROS production\u003c/h2\u003e \u003cp\u003eDCFH-DA was employed as a fluorescent probe to assess intracellular ROS generation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, all treatment groups\u0026mdash;including MSN@Mn@PDA, SMPG, VHH-SMPG, and DCL-SMPG NPs\u0026mdash;exhibited markedly enhanced green fluorescence compared to the untreated control group, indicating elevated ROS levels. Among these, the DCL-SMPG group displayed the highest fluorescence intensity. To further confirm intracellular ROS production, quantitative analysis was performed using flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and Fig.S12). DCL-SMPG nanoparticles significantly promoted ROS generation within cells. Notably, there was no statistically significant difference in ROS induction between the DCL-SMPG and VHH-SMPG groups.\u003c/p\u003e \u003cp\u003eROS has been found to decrease the mitochondrial membrane potential, thereby inducing cell apoptosis \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. In this study, the effect of ROS on mitochondrial membrane potential was assessed using the JC-1 probe (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). After treatment with MSN@Mn@PDA, the mitochondrial membrane potential of 22RV1 cells decreased. Following SMPG treatment, more pronounced mitochondrial damage was observed in 22RV1 cells, suggesting that the synergistic effect between CDT and ST increased ROS levels and thereby induced mitochondrial damage. In 22RV1 cells, VHH-SMPG and DCL-SMPG treatment resulted in predominantly green fluorescence, with DCL-SMPG inducing stronger green fluorescence.\u003c/p\u003e \u003cp\u003eDuring apoptosis, the decline in adenosine triphosphate (ATP) levels typically coincides with the reduction in the mitochondrial membrane potential. GOx effectively catalyzes the breakdown of glucose, leading to a decrease in cellular energy levels \u003csup\u003e[\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Therefore, we further investigated the intracellular ATP levels in cells treated with different NPs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, intracellular ATP levels decreased progressively upon treatment with MSN@Mn@PDA, SMPG, VHH-SMPG, and DCL-SMPG NPs. This trend is consistent with the corresponding changes in mitochondrial membrane potential, indicating mitochondrial dysfunction induced by oxidative stress. The introduction of GOx in SMPG NPs enhanced glucose oxidation and hydrogen peroxide production, further impairing mitochondrial function and reducing ATP synthesis. Importantly, the addition of PSMA-targeting ligands significantly amplified this effect due to enhanced cellular uptake, with DCL-SMPG NPs showing the most pronounced ATP depletion. Furthermore, treatment with these NPs also led to a substantial reduction in the intracellular GSH: oxidized glutathione (GSSG) ratio (Fig. S13), reflecting elevated oxidative stress and diminished antioxidant defenses. This was primarily due to the redox reaction between MnO\u003csub\u003e2\u003c/sub\u003e and GSH, which oxidizes GSH into GSSG and disrupts the cellular redox balance. The DCL-SMPG group exhibited the greatest decrease in GSH:GSSG, consistent with more efficient intracellular MnO\u003csub\u003e2\u003c/sub\u003e delivery and redox activation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6. In vivo biodistribution\u003c/h2\u003e \u003cp\u003eIn vitro experiments demonstrated that DCL-SMPG/DOX NPs exhibit enhanced internalization in 22RV1 prostate cancer cells and induce more potent tumor cell cytotoxicity compared to VHH-SMPG/DOX NPs. Based on these promising results, we further evaluated the in vivo antitumor efficacy of DCL-SMPG/DOX NPs. To investigate the tissue biodistribution, BALB/C nude mice tumor were intravenously administered with Rhodamine 6G (R6G) -labeled SMPG/R6G and DCL-SMPG/R6G NPs. At predetermined time points (24 h), fluorescence images were taken by using a small animal imaging system. Fluorescence imaging revealed that DCL-SMPG/R6G NPs exhibited enhanced accumulation at tumor tissues, as shown by significantly higher R6G fluorescence signals compared to non-targeted SMPG/R6G (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). This selectivity aligns with the PSMA-targeting mechanism of DCL ligands, which bind to tumor vasculature and cell surfaces to promote receptor-mediated endocytosis. Fluorescence signals in major organs were similar between groups, except for slightly elevated kidney accumulation in the DCL group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.7. In vivo anti-tumor activity\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e anticancer effects of DCL-SMPG/DOX were evaluated based on parameters such as tumor volume, weight, and pathology. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA shows the control group, free DOX group, and SMPG group, the mice in these groups experienced rapid tumor growth. In contrast, the SMPG/DOX group experienced a relative delay in tumor growth, accompanied by a considerable reduction in tumor volume. However, the tumor inhibition effects were even more pronounced in the DCL-SMPG/DOX group. Following the 21-day treatment period, all mice were euthanized, and their tumors were excised and weighed(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, Fig. S14). DCL-SMPG/DOX provided the most potent tumor inhibition after 21 days, demonstrating its efficacy in eliminating tumor cells. During the treatment period, there were no significant changes in body weight among the mice in the different treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eExcised mouse tissues were examined using H\u0026amp;E staining to assess the histocompatibility of DCL-SMPG/DOX NPs. Comparisons with non-tumor tissues from PBS-treated mice revealed that the non-tumor tissues from mice treated with the different NPs retained normal physiological morphologies, devoid of inflammation or pathological changes (Fig. S15). Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD shows the free DOX and SMPG treatment groups showed a significantly higher degree of tumor cell necrosis than the PBS treatment group. SMPG/DOX treatment, which combined chemotherapy with CDT/ST, resulted in significant tumor cell destruction and extensive necrosis. Notably, targeted treatment with DCL-SMPG/DOX resulted in even more pronounced structural damage to tumor tissues.The expression of Ki-67 and caspase-3 was examined in tumor tissues using immunohistochemical staining. Compared with the PBS-treated group, the group treated with DCL-SMPG/DOX exhibited significantly lower cell proliferation and a higher rate of cell apoptosis, validating the superior efficacy of synergistic CDT/ST and chemotherapy. These findings collectively suggested that DCL-SMPG/DOX, which possesses exceptional biocompatibility and anti-tumor activity, holds significant promise as an effective anticancer agent \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTraditional prostate cancer therapy are often associated with significant side effects, poor efficacy, and low bioavailability. Effectively delivering drugs to target cells has become a key focus of current research. Targeted nanotherapies can more precisely localize to tumor cells, reducing off-target effects and increasing drug internalization within tumors. The team led by Michael Mitchell developed a class of antibody-conjugated lipid nanoparticles (Ab-LNPs) that exhibit extrahepatic tropism and target T cell markers. This innovation enables the precise delivery of mRNA, minimizing off-target effects and adverse reactions \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. The targeted accumulation of nanotherapeutics in prostate cancer tissues or cells is the crucial first step to enhance therapeutic efficacy, followed by the release of therapeutic molecules from the delivery system. Subsequently, the therapeutic effectiveness of nanomedicine in treating prostate cancer is further improved by harnessing synergistic therapy mechanisms \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. In this study, we utilized low molecular weight ligands targeting PSMA to construct a multifunctional nanotherapeutic platform. This platform integrates CDT, ST and chemotherapy for the targeted therapy of prostate cancer.\u003c/p\u003e \u003cp\u003eCDT and ST are often considered together in antitumor research due to their synergistic effects in enhancing antitumor efficacy. Redox reactions enable the uniform distribution of Mn within the mesoporous channels of MSN, preventing aggregation on the surface of the nanoparticles, thereby enhancing their reactivity and acid sensitivity \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. XPS confirmed the binding energy of Mn2p, ensuring the Mn\u003csup\u003e2+\u003c/sup\u003e-mediated Fenton-like reaction selectively converts overexpressed H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into highly toxic \u0026middot;OH under low pH conditions. Additionally, GOx produces H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and gluconic acid, effectively disrupting nutrient supply and triggering tumor starvation therapy. More importantly, the elevated levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and increased acidity significantly enhance the Fenton-like reactivity, generating a pronounced \"ROS (\u0026middot;OH) storm,\" thus achieving Mn\u003csup\u003e2+\u003c/sup\u003e-mediated cascade amplification of CDT.\u003c/p\u003e \u003cp\u003eIn vitro, SMPG NPs consumed GSH within a short period (6h), which is half the time compared to the 12h required for Ce6/GOx@ZIF-8/PDA@MnO\u003csub\u003e2\u003c/sub\u003e nanoparticles synthesized via a one-pot method by Zhang et al \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Furthermore, TMB and MB assays were employed to detect the ability of SMPG NPs to catalyze the production of \u0026middot;OH from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, confirming that SMPG NPs catalyzed the generation of \u0026middot;OH under acidic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). After catalyzing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and gluconic acid from glucose, SMPG NPs further generated \u0026middot;OH through a cascade reaction, ensuring the in vivo amplification of CDT, leading to a ROS storm and tumor ablation.\u003c/p\u003e \u003cp\u003eDCFH-DA staining revealed intracellular ROS in 22RV1 cells after therapy with SMPG NPs, showing that SMPG NPs induced higher ROS production compared to CDT catalysis mediated by MSN@Mn@PDA NPs alone. ROS caused mitochondrial damage and induced apoptosis. JC-1 staining indicated changes in mitochondrial membrane potential, demonstrating that mitochondrial damage was proportional to the ROS levels. Additionally, intracellular ATP and GSH levels were found to correspond to the changes in mitochondrial membrane potential, indicating that SMPG NPs synergistically enhanced CDT and ST to produce significant ROS.\u003c/p\u003e \u003cp\u003eThe chemotherapeutic agent DOX was encapsulated within SMPG/DOX nanoparticles, synergistically contributing to the antitumor effects of CDT and ST. Initially, DOX was loaded into MSN@Mn nanoparticles with a loading efficiency of 42.9%, and the surface was coated with PDA to prevent drug leakage during circulation. In vitro drug release studies typically elucidate how encapsulated drugs are released in vivo \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. In our study, drug release was investigated in PBS (pH 7.4) with or without 10 mM GSH. Rapid drug release occurred within the first 0\u0026ndash;10 hours in both media, followed by a slower release profile. In the medium containing GSH, the release of DOX was enhanced, with even greater and faster drug release observed in the medium at pH 5.2 containing GSH. Given that the TME is often characterized by low pH and high GSH concentration (approximately 10 mM), this suggests that the nanomedicine is responsive to the TME for targeted drug release. Additionally, drug release continued at a sustained, slow pace even after 24 hours in the pH 5.2 GSH-containing medium. This prolonged drug release pattern contributes to maintaining therapeutic drug concentrations within tumor cells over an extended period, enhancing cytotoxicity and accelerating cancer cell death\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn vivo, targeted nanomedicine typically achieve less than 1% accumulation at the tumor site \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. However, compared with non-targeted nanomedicine, targeted nanomedicine demonstrate significantly enhanced accumulation within tumor tissues. This indicates that the targeting mechanism is the driving force behind the active uptake of nanomedicine by tumor cells \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Most targeting ligands are traditionally monoclonal antibodies; however, some low-molecular-weight ligands offer additional advantages due to their smaller size, higher tissue penetration, and remarkably low immunogenicity.\u003c/p\u003e \u003cp\u003eIn this study, two low-molecular-weight ligands, a PSMA small molecule inhibitor (DCL) and anti-PSMA nanobody (VHH), were chosen as targeting moieties. These ligands specifically bind to the overexpressed PSMA on prostate cancer cells, facilitating the rapid attachment of the nanomedicine, enhancing their anti-tumor activity, and reducing off-target toxicity. The conjugation of DCL with the nanomedicine was examined through changes in the hydrodynamic diameter and zeta potential of the nanoparticles, and the successful conjugation of DCL was further confirmed via FTIR analysis. Additionally, fluorescence microscopy revealed green fluorescence after incubating the VHH-conjugated nanomedicine with anti-VHH fluorescent antibodies, indicating successful conjugation. Experimental results demonstrated that ligand surface conjugation led to increased uptake of nanomedicine by prostate cancer 22RV1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), as further confirmed by flow cytometry. The uptake of VHH-SMPG/DOX nanoparticles and DCL-SMPG/DOX nanoparticles by 22RV1 cells was 1.3-fold and 1.8-fold higher, respectively, compared to SMPG/DOX nanoparticles. Notably, the uptake of DCL-SMPG/DOX nanoparticles by 22RV1 cells was 1.3 times higher than that of VHH-SMPG/DOX nanoparticles. At the same dosage, DCL-SMPG/DOX nanoparticles exhibited higher cytotoxicity against 22RV1 cells compared to VHH-SMPG/DOX nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These findings indicate the nanomedicine conjugated with the PSMA small molecule inhibitor DCL are internalized more rapidly by 22RV1 cells, thus resulting in greater toxicity than those conjugated with VHH. The observed lower internalization efficiency of VHH-SMPG/DOX compared to DCL-SMPG/DOX can be attributed to the monovalent nature of VHH ligands, higher steric hindrance, and less effective induction of receptor clustering on the cell membrane. In contrast, the multivalent architecture of DCL enables stronger PSMA binding, promotes receptor-mediated endocytosis via CME and CavME, and enhances nanoparticle uptake in prostate cancer cells. These findings are consistent with recent studies emphasizing the importance of ligand valency, orientation, and receptor dynamics in optimizing nanoparticle internalization and therapeutic performance\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, the therapeutic efficacy of a drug at its site of action is contingent upon the ability of the nanomedicine to enter the target cells. The primary mechanism by which nanomedicine was internalized into cells is through endocytosis. Depending on the cell type and the proteins, lipids and other molecules involved in the process, endocytosis can be categorized into several types, including phagocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, clathrin/caveolin-independent endocytosis, and micropinocytosis \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. In our study, we found that the uptake of nanomedicine (including DOX) by 22RV1 cells primarily occurred through energy-dependent CEM and CaME. In the research conducted by Anna Salvati et al., it was observed that the uptake of silica nanoparticles was independent of clathrin \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. The endocytic pathways of different types of nanomedicine vary due to differences in their chemical properties, including size, surface charge, shape and rigidity \u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. Although DOX is classically considered to enter cells via passive diffusion due to its small molecular size, recent studies have suggested a partial contribution of energy-dependent and endocytic pathways to its intracellular accumulation, particularly in cancer cells with altered membrane composition. In our study, the observation that endocytosis inhibitors moderately reduced free DOX uptake indicates a non-exclusive reliance on passive diffusion. This may result from transient CEM and CaME -mediated internalization of DOX under physiological conditions, as previously observed in other malignancies\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Furthermore, commonly used inhibitors such as chlorpromazine and M-β-CD may alter membrane integrity or metabolic status, indirectly influencing passive permeability and transporter-mediated DOX influx/efflux\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTargeting PSMA receptors with low molecular weight ligands ensured that the nanomedicine was specifically delivered to prostate cancer cells and exhibited efficient cellular internalization. Further studies were conducted using the CCK-8 assay, live/dead cell staining, and apoptosis detection to assess the anticancer effects of the nanomedicine on prostate cancer cells. Figures\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D show that the cytotoxicity of nanomedicine combining chemotherapy, CDT/ST (SMPG/DOX NPs) was significantly higher than that of standalone chemotherapy and nanomedicine utilizing CDT/ST (SMPG NPs). Nanomedicine with low molecular weight ligand targeting (VHH-SMPG/DOX and DCL-SMPG/DOX) exhibited even greater cytotoxicity, far exceeding that of SMPG NPs alone. This increased cytotoxicity is attributed to the ligand-targeting effect, which enhances the internalization of nanomedicine in 22RV1 cells, leading to higher cytotoxicity. Additionally, since the internalization of DCL-SMPG/DOX in 22RV1 cells was greater than that of VHH-SMPG/DOX, the cytotoxicity induced by DCL-SMPG/DOX was higher than that induced by VHH-SMPG/DOX. While the therapeutic efficacy of DCL-SMPG/DOX is supported by significant tumor suppression and enhanced ROS generation, we acknowledge that this study did not include a formal quantitative synergy analysis (e.g., Chou\u0026ndash;Talalay method). This limitation arises primarily due to the co-loading design of our nanoplatform, in which MnO\u003csub\u003e2\u003c/sub\u003e, GOx, and DOX are integrated at a fixed ratio. As a result, it was not feasible to independently modulate the concentrations of each component to generate a comprehensive dose\u0026ndash;effect matrix required for synergy quantification. Moreover, the cooperative interactions among these therapeutic modalities are highly dependent on tumor-specific biochemical cues\u0026mdash;such as acidic pH and endogenous H₂O₂\u0026mdash;which dynamically regulate the catalytic performance of MnO\u003csub\u003e2\u003c/sub\u003e and GOx. Therefore, decoupling their individual effects in an artificial in vitro setting may not accurately reflect the in vivo therapeutic synergy.\u003c/p\u003e \u003cp\u003eBased on the in vitro anticancer effects of nanomedicine against prostate cancer cells, the in vivo antitumor efficacy of DCL-SMPG/DOX NPs was further studied. In our study, the in vivo biodistribution analysis revealed that DCL-SMPG/R6G nanoparticles exhibited significantly enhanced accumulation in PSMA-expressing prostate tumors compared to non-targeted SMPG/R6G nanoparticles. This targeted delivery is attributed to the DCL ligands affinity for PSMA, facilitating receptor-mediated endocytosis and selective tumor uptake. Furthermore, the observed slight increase in kidney accumulation of DCL-SMPG/R6G nanoparticles aligns with findings from other studies, where PSMA-targeted agents exhibited renal uptake due to endogenous PSMA expression in renal tissues. These results underscore the importance of considering off-target effects in the design of PSMA-targeted therapies \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. Tumor volume and body weight changes were analyzed in a 22RV1 mouse model after therapy with DOX, SMPG, SMPG/DOX, and DCL-SMPG/DOX. The results demonstrated that tumor size and volume significantly decreased following therapy with DCL-SMPG/DOX, with H\u0026amp;E staining showing a markedly higher degree of tumor necrosis compared to the other therapy groups. Ki-67 is an important indicator used to evaluate the proliferative activity of tumor cells. The expression level of Ki-67 reflects the extent of cellular proliferation, with higher Ki-67 levels indicating an increased number of cells in the mitotic phase and more active cell division \u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. Caspase-3 is a biomarker of apoptosis that, upon receiving apoptotic signals, becomes activated and cleaves and modifies various intracellular proteins. It can cleave cytoskeletal proteins, leading to morphological changes, and act on DNA repair-related proteins, rendering the cell incapable of repairing damage \u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. These actions collectively push the cell toward an irreversible apoptotic process. Immunohistochemistry results showed that the expression of Ki-67 in tumor tissues from the DCL-SMPG/DOX therapy group was lower than in other therapy groups, while caspase-3 expression was higher than in other groups. These findings indicate that DCL-SMPG/DOX therapy can effectively ablate tumors by significantly inhibiting tumor proliferation and promoting apoptosis.\u003c/p\u003e \u003cp\u003eIn conclusion, we have successfully created a synergistic chemotherapy-CDT/ST nanomedicine platform for the targeted therapy of prostate cancer. This system effectively targets and is internalized by prostate cancer cells, and it responds to the tumor microenvironment with sustained drug release. It induces a ROS storm within tumor cells, promoting chemotherapy-induced apoptosis of prostate cancer. The examination of the in vivo and in vitro efficacy of DCL-SMPG/DOX in inhibiting prostate cancer in animal models suggests that DCL-SMPG/DOX has significant potential for clinical application.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eA targeted nanomedicine delivery system capable of single or multiple reactions was designed. Using MSN and PDA as drug delivery platforms, chemotherapeutic agents doxorubicin, CDT agent MnO\u003csub\u003e2\u003c/sub\u003e, and ST agent GOX were encapsulated to construct a nanomedicine (SMPG/DOX) that synergistically targets prostate cancer. The nanomedicine is coupled with low molecular weight ligands targeting PSMA (DCL-SMPG/DOX or VHH-SMPG/DOX). Notably, DCL-SMPG/DOX exhibits superior cellular internalization capabilities and demonstrates positive antitumor effects in combination with chemotherapy-CDT/ST. Overall, this multi-modal targeted nanomedicine shows significant translational potential for the therapy of prostate cancer. However, further investigation into its underlying antitumor mechanisms is necessary.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePCa Prostate cancer\u003c/p\u003e \u003cp\u003ePSMA Prostate-specific membrane antigen\u003c/p\u003e \u003cp\u003eLMW Low molecular weight\u003c/p\u003e \u003cp\u003eCDT Chemodynamic therapy\u003c/p\u003e \u003cp\u003eST Starvation therapy\u003c/p\u003e \u003cp\u003eROS Reactive oxygen species\u003c/p\u003e \u003cp\u003eDOX Doxorubicin hydrochloride\u003c/p\u003e \u003cp\u003eMnO\u003csub\u003e2\u003c/sub\u003e Manganese dioxide\u003c/p\u003e \u003cp\u003eGSH Glutathione\u003c/p\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Hydrogen peroxide\u003c/p\u003e \u003cp\u003e\u0026bull;OH Hydroxyl radicals\u003c/p\u003e \u003cp\u003eGOx Glucose oxidase\u003c/p\u003e \u003cp\u003eTME Tumor microenvironment\u003c/p\u003e \u003cp\u003eNPs Nanoparticles\u003c/p\u003e \u003cp\u003eMSN Mesoporous silica\u003c/p\u003e \u003cp\u003ePDA Polydopamine\u003c/p\u003e \u003cp\u003eDCL Small-molecule inhibitor\u003c/p\u003e \u003cp\u003eVHH Anti-PSMA nanobody\u003c/p\u003e \u003cp\u003eKMnO\u003csub\u003e4\u003c/sub\u003e Potassium permanganate\u003c/p\u003e \u003cp\u003eDTNB 5,5\u0026prime; -dithiobis (2-nitrobenzoic acid)\u003c/p\u003e \u003cp\u003eTMB 4,4\u0026prime;-diamino- 3,3\u0026prime;,5,5\u0026prime;-tetramethylbiphenyl\u003c/p\u003e \u003cp\u003eDCFH-DA 2,7\u0026prime; -dichlorodihydrofluorescein diacetate\u003c/p\u003e \u003cp\u003eMB Methylene blue\u003c/p\u003e \u003cp\u003eTEOS Tetraethyl orthosilicate\u003c/p\u003e \u003cp\u003eCTAB Cetyltrimethylammonium bromide\u003c/p\u003e \u003cp\u003eDAPI 4\u0026prime;,6-diamidino-2-phenylindole\u003c/p\u003e \u003cp\u003eH\u0026amp;E Hematoxylin and Eosin\u003c/p\u003e \u003cp\u003eIHC Immunohistochemistry\u003c/p\u003e \u003cp\u003eNPs Nanoparticles\u003c/p\u003e \u003cp\u003eGSSG Oxidized glutathione\u003c/p\u003e \u003cp\u003eATP Adenosine Triphosphate\u003c/p\u003e \u003cp\u003eR6G Rhodamine 6G\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China [grant numbers 82060462, 82360558]; the Science and Technology Foundation of Guizhou Province [grant number ZK[2023] 211]; the Foundation of Health Commission of Guizhou Province [grant number gzwkj2021-534]; and the Guizhou Province \u0026nbsp;Youth Science and Technology Talents Growth Project [grant number Foundation- [2024] Youth 296 ].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXiaoli Zhang:\u0026nbsp;\u003c/strong\u003eInvestigation, Methodology, Data curation, Writing \u0026ndash; original draft.\u003cstrong\u003e\u0026nbsp;Jie He:\u0026nbsp;\u003c/strong\u003eInvestigation, Methodology, Data curation. \u003cstrong\u003eYu An:\u0026nbsp;\u003c/strong\u003eData curation, Writing \u0026ndash; original draft. \u003cstrong\u003eKehua Jiang:\u0026nbsp;\u003c/strong\u003eInvestigation, Methodology. \u003cstrong\u003eQiang Wang:\u0026nbsp;\u003c/strong\u003eInvestigation, Methodology. \u003cstrong\u003eWenrui Deng:\u0026nbsp;\u003c/strong\u003eInvestigation, Validation. \u003cstrong\u003eQiqi Yang:\u0026nbsp;\u003c/strong\u003eInvestigation, Validation. \u003cstrong\u003eFa Sun:\u0026nbsp;\u003c/strong\u003eFunding acquisition, Project administration. \u003cstrong\u003eKun Chen:\u0026nbsp;\u003c/strong\u003eConceptualization, Writing \u0026ndash; review \u0026amp; editing, Funding acquisition, Project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations of ethics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments were approved by the Ethics Committee of Guizhou Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Wagle NS, Jemal A (2023) Cancer statistics, 2023.[J]. 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Biotechnol Appl Chem 69(4):1633\u0026ndash;1645\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-nanotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cano","sideBox":"Learn more about [Cancer Nanotechnology](https://cancer-nano.biomedcentral.com/)","snPcode":"12645","submissionUrl":"https://submission.nature.com/new-submission/12645/3","title":"Cancer Nanotechnology","twitterHandle":"@CancerNanotech","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PSMA, low molecular weight ligands, prostate cancer, targeted therapy","lastPublishedDoi":"10.21203/rs.3.rs-6045405/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6045405/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTargeted therapy enhances tumor elimination while reducing adverse effects by integrating multiple tumoricidal mechanisms. Low molecular weight (LMW) ligands, offering faster pharmacokinetics and improved tumor permeability, present a viable alternative to antibodies. This study presents a novel nanomedicine for prostate cancer therapy, leveraging mesoporous silica nanoparticles (MSN) as the nanocarrier to encapsulate manganese dioxide (MnO\u003csub\u003e2\u003c/sub\u003e) and doxorubicin (DOX). The resultant nanoparticles are further coated with a polydopamine (PDA) layer and covalently conjugated with glucose oxidase (GOx), forming the MSN@Mn@PDA-GOx/DOX hybrid system (hereafter termed SMPG/DOX NPs). LMW ligands (small molecule inhibitor DCL and nanobody VHH) targeting prostate-specific membrane antigen (PSMA) were conjugated to create DCL-SMPG/DOX and VHH-SMPG/DOX. Mn\u003csup\u003e2+\u003c/sup\u003e-mediated Fenton-like reactions converted H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into toxic hydroxyl radicals (\u0026middot;OH) under acidic conditions, enabling chemodynamic therapy (CDT). GOx-generated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and gluconic acid disrupted nutrient supply, inducing tumor starvation therapy (ST). The increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and acidity amplified the Fenton-like reaction, creating a \"ROS storm\" that synergistically enhanced chemotherapy. LMW targeting improved tumor specificity, efficacy, and reduced side effects. In vitro, DCL-SMPG/DOX showed superior tumor cell internalization and cytotoxicity compared to VHH-SMPG/DOX. In vitro, the cellular internalization rates of VHH-SMPG/DOX and DCL-SMPG/DOX were 34.1% and 44.5%, respectively, significantly higher than that of free DOX uptake (10.3%). Moreover, DCL-SMPG/DOX-induced stronger cytotoxicity compared to VHH-SMPG/DOX. \u003cem\u003eIn vivo\u003c/em\u003e studies further demonstrated the strong anti-tumor activity of the DCL-SMPG/DOX nanomedicine, underscoring its potential as a prostate cancer treatment. Further research is needed to elucidate its antitumor mechanisms.\u003c/p\u003e","manuscriptTitle":"Using low - molecular - weight ligands for targeting in integrated chemodynamic/starvation therapy and chemotherapy for prostate cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-15 10:55:22","doi":"10.21203/rs.3.rs-6045405/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-04-30T11:54:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-27T11:39:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165351110907194962258880440020927688639","date":"2025-04-17T09:12:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-14T16:03:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175271661042376964345308089090090124149","date":"2025-04-14T03:09:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-12T07:48:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-11T11:05:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Nanotechnology","date":"2025-04-11T10:04:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-nanotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cano","sideBox":"Learn more about [Cancer Nanotechnology](https://cancer-nano.biomedcentral.com/)","snPcode":"12645","submissionUrl":"https://submission.nature.com/new-submission/12645/3","title":"Cancer Nanotechnology","twitterHandle":"@CancerNanotech","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"68742b19-ea0e-4d1e-b456-6dfb3b2ef632","owner":[],"postedDate":"April 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:02:39+00:00","versionOfRecord":{"articleIdentity":"rs-6045405","link":"https://doi.org/10.1186/s12645-025-00325-2","journal":{"identity":"cancer-nanotechnology","isVorOnly":false,"title":"Cancer Nanotechnology"},"publishedOn":"2025-06-03 15:57:34","publishedOnDateReadable":"June 3rd, 2025"},"versionCreatedAt":"2025-04-15 10:55:22","video":"","vorDoi":"10.1186/s12645-025-00325-2","vorDoiUrl":"https://doi.org/10.1186/s12645-025-00325-2","workflowStages":[]},"version":"v1","identity":"rs-6045405","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6045405","identity":"rs-6045405","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}