Pros and Cons in The Delivery of Doxorubicin Using Renal-clearable Gold Nanoparticles

Pros and Cons in The Delivery of Doxorubicin Using Renal-clearable Gold Nanoparticles | 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 Pros and Cons in The Delivery of Doxorubicin Using Renal-clearable Gold Nanoparticles Lang Liu, Shanshan Qiao, Meiyu Sun, Yusheng Mao, Hai Huang, Yemei Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3940105/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Renal-clearable drug delivery systems (DDSs) offer significant advantages compared to conventional non-renal-clearable DDSs due to their reduced toxicity and enhanced therapeutic efficacy. However, despite the development of renal-clearable DDSs in the past decade, deeper understanding of how the biological barriers, especially the intracellular barriers affect their therapeutic efficiency remain poorly explored. Herein, the antitumor efficiency and the intracellular behavior of renal-clearable Au-DOX which use renal-clearable gold nanoparticles (AuNPs) as delivery vectors for doxorubicin (DOX) were systematically investigated. The results revealed that although the toxicity of Au-DOX was significantly lower than that of free DOX due to efficient elimination of off-target DOX through renal clearance, the altered cellular uptake pathway compromised the antitumor efficacy of Au-DOX. Most Au-DOX was endocytosed and sequestered within lysosomes, preventing it from diffusing into nucleus to elicit therapeutic effect. Our results indicate that the lysosomal barrier induced ineffective intracellular delivery would counteract the therapeutic efficacy of renal-clearable DDSs and highlight the role of overcoming intracellular barriers when designing DDSs. Renal-clearable delivery vector intracellular barrier antitumor efficacy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Understanding the biological barriers in preventing DDSs from eliciting their therapeutic efficacy was critical to the clinical translation of DDSs. Currently identified biological barriers include the mononuclear phagocytic system barrier [1], the tumor blood vessel extravasation barrier [2], the intratumoral penetration barrier and the intracellular barrier [3, 4]. Among them, the intracellular barrier has been drawn little attention in the past few decades despite its critical role in governing the therapeutic efficacy of organelle-targeting chemo drugs such as doxorubicin (DOX) [5], which functions through intercalation into nuclear DNA and disruption of topoisomerase mediated DNA repair, and genetic drugs such as siRNA, mRNA, and gene-editing systems, which have to function in cytosol and nucleus [6, 7]. Nanomaterials were generally taken up by cells through endocytosis [8], phagocytosis [9], and pinocytosis [10] pathways, leading to the internalization of nanomaterials into intracellular vesicles such as endosomes and lysosomes [11]. The confinement of the loaded drugs in these intracellular vesicles would dramatically decrease their therapeutic efficacy when they were unable to escape from the vesicles [12]. Therefore, it was of great significance to understand the intracellular barrier of DDSs when they were used to deliver organelle-specific drugs and genetic drugs. Renal-clearable DDSs deliver therapeutic agents for cancer treatment by employing renal-clearable nanomaterials as delivery vectors [13]. The ultrasmall size of renal-clearable DDSs below the kidney filtration threshold (~5.5 nm) [14], enables enhanced and homogeneous intratumoral penetration, thereby facilitating the effective delivery of therapeutic agents to cancer cells [15]. Furthermore, the efficient renal clearance through the kidneys and bladder allows for the rapid elimination of off-target therapeutic agents, leading to a significant reduction in off-target toxicity [16]. Over the past decade, several renal-clearable DDSs have been developed, demonstrating promising potential for the treatment of various cancers with low toxicity [17]. For instance, Hak Soo Choi and co-workers developed cyclodextrin-based renal-clearable DDSs (“H-Dots”) to deliver imatinib for efficient treatment of unresectable gastrointestinal stromal tumors [18, 19]. Michelle Bradbury team developed renal-clearable silica nanoparticles (“Cornell-dots”), which have been used to deliver different types of anticancer therapeutic agents such as chemo drugs [20], therapeutic proteins [21], and immune drugs [22] with efficient therapeutic efficacy. Moreover, Peng et al. demonstrated the superior efficacy of AuNPs-based renal-clearable DDSs over free drug and the non-renal-clearable counterpart in treating triple-negative breast cancer and lung metastasis due to significantly enhanced intratumoral penetration [23, 24]. However, despite the development of renal-clearable DDSs in the past decade, deeper understanding of how the biological barriers, especially the intracellular barrier, affect their therapeutic efficacy, remains poorly explored at this stage. Herein, by using DOX as the model drug, we developed dual pH/glutathione (GSH)-responsive renal-clearable Au-DOX to investigate the antitumor efficacy and toxicity in treating triple-negative breast cancer 4T1 cancer. Initially, DOX was conjugated with a thiol ligand through an acid-labile linker and subsequently coated onto renal-clearable PEGylated AuNPs via Au-S bond. The loaded DOX exhibits prompt release under acidic pH and in the presence of GSH. However, despite the Au-DOX achieved enhanced tumor delivery efficiency and efficient release of DOX, the therapeutic efficacy of Au-DOX only reached comparable or even lower levels compared to free DOX. Further in vitro studies revealed that, unlike the passive diffusion pathway of free DOX into cells and subsequent nuclear penetration, DOX loaded on AuNPs was internalized into cells alongside the AuNPs via endocytosis. Once internalized, DOX molecules became confined within lysosomes, significantly hindering them from reaching the nucleus and thus limiting their therapeutic functionality. Consequently, this fundamentally diminishes the cellular toxicity and antitumor efficacy of DOX. On the other side, the off-target Au-DOX was efficiently eliminated from the body through renal clearance pathway, which significantly reduced the toxicity of Au-DOX compared to free DOX. Our findings suggest that despite the high tumor targeting efficiency and reduction in off-target toxicity of renal clearable nanoparticles, the role of the lysosomal barrier in mediating the therapeutic efficacy should be carefully considered when design renal clearable DDSs. Material and methods Synthesis of Au-PEG Au-PEG was synthesized through thermal reduction of chloroauric acid (HAuCl 4 ). Briefly, a solution comprising HAuCl 4 ·4H 2 O (0.04 g, 0.1 mmol), mPEG-SH (MW : 5 kDa, 1.5 g, 0.3 mmol), and NaBH 4 (0.075 g, 2 mmol) was dissolved in a methanol/acetic acid mixed solvent (6:1 v/v, 5 mL) and stirred vigorously at room temperature for 2 hours. Subsequently, the resulting mixture was evaporated to dryness under vacuum, and the resulting residue was dissolved in water. The pH value was adjusted to 7.4 using NaOH, and the solution was then subjected to centrifugation at 9000 rpm for 10 minutes. The resulting supernatant was further purified using dialysis and freeze-drying techniques. Synthesis of DOX-PDPH DOX-HCl (22.7 mg, 0.034 mmol) and PDPH-linker (20.7 mg, 0.045 mmol) were dissolved in methanol (14 mL) and stirred in the dark at room temperature for 6 days. The solvent was then evaporated, and acetonitrile was added for purification via centrifugation. The resulting residues were dissolved in dimethyl sulfoxide (DMSO) and subsequently stored at 4°C. Synthesis of Au-DOX DOX-PDPH was conjugated to Au-PEG via an Au-S bond. A solution of Au-PEG (0.5 mL, 0.03 g/mL) and DOX-PDPH (0.2 mL, 0.01 M) was combined and incubated with agitation at room temperature for 48 hours. Once the reaction was completed, the solution was purified by passing it through a Sephadex LH-20 column to remove free DOX-PDPH, and subsequently lyophilized, resulting in a dark red Au-DOX powder. Agarose gel electrophoresis A solution (10 µL) of each sample was introduced into the prepared agarose gel wells, followed by analysis through 2% agarose gel electrophoresis utilizing the DYY-8C Gel electrophoresis system (Beijing Liuyi Biotechnology Co., LTD.) and 0.1X Britton-Robinson buffer solution (pH 7.4) at a constant current of 60 mA for 20 minutes. In vitro drug release In the pH-responsive release assay, a 20 µL aliquot of the Au-DOX solution (10 m M) was added to 180 µL of PBS buffer, and the buffer pH was pH 5.5, 6.5, 7.4, respectively. For the GSH-responsive release experiment, the Au-DOX solution was mixed with a GSH solution to achieve a final concentration of 5 mM. The resulting solution was incubated on a shaker at 37°C and 100 rpm. The fluorescence intensity of the samples was measured and analysed at predetermined time intervals using a fluorescence spectrometer. It was noteworthy that the stability of Au-DOX was evaluated under both acidic (pH 5.5) and neutral (pH 7.4) conditions. Using the static method for a period of 2 hours, with fluorescence measurements taken every ten minutes to assess stability. Cellular uptake Hela and 4T1 cells were purchased from Shanghai Xuanya Biotechnology Co., Ltd, culturing under standard conditions (37°C, 5% CO 2 ). Cells were cultured in 35 mm culture dishes and subjected to individual treatments using either Au-DOX or free DOX, both at equal DOX concentrations (5 µg/mL). After incubation for different time, washing with PBS (pH = 7.4) for three times, lysosomes and nuclei were stained with LysoTracker Green DNA-26 (50 nM) and Hoechst 33342 (1 µg/mL) for 30 min, the cells underwent PBS wash and were subsequently visualized using a confocal fluorescence microscope (OLYMPUS IX73, Olympus Trading (Shanghai) Co., Ltd). In vitro cytotoxicity For the cytotoxicity investigation, 4T1 cells were seeded in a 96-well plate and incubated at 37°C with 5% CO 2 for 24 hours. Following this period, the supernatant was aspirated, and RPMI 1640 medium supplemented with various concentrations of Au-DOX and DOX (ranging from 0.1 µg/mL to 100 µg/mL) were added (n = 3). Following an additional 24 hours of incubation, the culture media in the wells were substituted with 100 µL of fresh medium supplemented with 10 µL of the CCK-8 reagent. This was succeeded by an additional 2 hours incubation period. Finally, the absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using the formula: cell viability (%) = (OD sample – OD blank )/( OD control – OD blank ) × 100%, where OD represents the absorbance of the sample at 450 nm. The experiment was conducted in triplicate. Mouse tumor model In our experimental procedures, we obtained female Balb/c mice from Changzhou Cavens Experimental Animal Co., Ltd. The animal experiments were conducted in accordance with the approved protocols by Soochow University Laboratory Animal Center. To establish the tumor model, 1*10 6 4T1 cells suspended in 50 µL of PBS were subcutaneously injected into the chest of each mouse. Antitumor efficacy and toxicity studies Thirty female Balb/c mice bearing 4T1 tumors (with an initial tumor volume of approximately 60 mm 3 ) were randomly assigned to four treatment groups administered via the tail vein: (1) 125 µL of PBS (administered once every 3 days); (2) 125 µL of Au-PEG (at a dose of AuNPs same with Au-DOX of 50 mg/kg, administered once every 3 days); (3) 125 µL of Au-DOX (at a DOX dose of 5 mg/kg, administered once every 3 days); (4) 125 µL of Au-DOX (at a DOX dose of 5 mg/kg, administered once every 2 days); and (5) 125 µL of DOX (at a dose of 5 mg/kg, administered once every 3 days). The tumor volume (V = L × W 2 /2) was measured, and the relative volume (V/V 0 ) was calculated, where V 0 represents the tumor volume before treatment. Mice were weighed periodically to monitor their weight throughout the treatment. To ensure ethical considerations, tumor volumes exceeding 1500 mm 3 were not permitted during the study. At the end of the study, organs and tumor tissues were dissected, photographed, and weighed. Subsequently, they were embedded in paraffin and sectioned for hematoxylin and eosin (H&E) staining. Blood was procured via cardiac puncture for the purpose of blood analysis. The collected blood samples were kept on ice for 30 minutes to allow coagulation. Subsequently, serum was obtained by centrifuging the samples at 2,000 g for 15 minutes to remove coagulated blood. Biodistribution and renal clearance Female Balb/c mice bearing 4T1 tumors (with an initial tumor volume of approximately 200 mm 3 ) were randomly assigned to two treatment groups: the DOX group and the Au-DOX group. Each group received intravenous injection of DOX at a dose of 5mg/kg through the tail vein. After completing administration therapy, the mice were euthanized, and their heart, liver, spleen, lungs, kidneys, and tumor tissues were dissected. The biodistribution of DOX was quantified by using fluorescence imaging. For AuNPs biodistribution, the main organs were dissected, weighed, dissolved in aqua regia, and diluted appropriately for ICP-OES measurements using the AVIO 200 instrument from Shanghai Platinum Elmer Instrument Co., Ltd. To quantify renal clearance, mice were intravenously injected with Au-DOX and free DOX, and urine samples were collected and measured for volume at various time intervals within 24 hours. To quantify DOX in the urine, the suspension was centrifuged at 21000 rpm for 10 minutes, and the supernatant was collected for DOX concentration analysis based on UV absorption. To quantify AuNPs in the urine, the urine was dissolved in aqua regia and diluted properly for ICP-OES measurement. Results and discussion Figure 2. (a) Chemical structure of DOX-hydrazone linker conjugate (DOX-PDPH). Scheme of renal-clearable Au-DOX, which consists of the gold core, surface coating with poly (ethylene glycol) thiol (mPEG-SH, MW: 5000 Da) and acid sensitive linker (PDPH), and loaded DOX molecules. (b) HR-TEM image and size distribution of Au-DOX. The scale bar is 10 nm. (c) UV-Vis absorbance spectra of Au-PEG, DOX, DOX-PDPH and Au-DOX in aqueous solution. (d) Fluorescence emission spectra of Au-DOX and DOX at the same concentrations. Preparation and characterization of Au-DOX The Au-PEG nanoparticles (AuNPs) were synthesized following a reported method [ 25 ]. DOX was connected with a 3-[2-Pyridyldithio]propionyl hydrazide (PDPH crosslinker) [ 26 ] (Figure S1 c), which not only can form a pH-responsive hydrazone bond with the carbonyl group of DOX, but also can introduce a thiol functional group to conjugate DOX with AuNPs (Fig. 2a) [ 27 ]. The uniform size distribution of Au-DOX nanoparticles was confirmed by high-resolution transmission electron microscopy (HR-TEM) imaging, with a particle size of 1.8 ± 0.2 nm (Fig. 2b). The absorption spectrum of Au-DOX exhibited a characteristic absorbance of DOX at 488 nm, indicating the successful conjugation of DOX onto AuNPs [ 28 ]. Notably, the loaded DOX showed an 11 nm redshift (from 488 to 499 nm) compared to free DOX, suggesting the formation of J-aggregates of DOX after conjugation onto AuNPs, which was resulted from dipole-dipole interactions among the loaded DOX molecules (Fig. 2c). Moreover, the fluorescence intensity of DOX decreased about 9 times due to the fluorescence resonance energy transfer (FRET) process between DOX and AuNPs (Fig. 2d) [ 29 ]. The successful conjugation of DOX onto AuNPs was also confirmed by agarose gel electrophoresis. The gel mobility of Au-DOX was in between that of pure DOX and Au-PEG (Figure S1 a), and the fluorescence imaging of the agarose gel showed a similar mobility behavior (Figure S1 b). Figure 3. (a) The illustration of the acidic pH response of Au-DOX. (b) The fluorescence intensity changes of Au-DOX at pH 5.5, 6.5, and 7.4 in 31 h. (c) Time-dependent DOX release from Au-DOX at different pH values. (d) The illustration of the GSH response of Au-DOX. (e) The change of fluorescence intensity of Au-DOX in 31h after adding 5 mM GSH. (f) Time-dependent DOX release from Au-DOX in 5 mM GSH. Drug release characteristics triggered by pH and GSH in vitro Cell cytosol contains high concentrations of GSH (1–10 mM) [ 30 , 31 ], and the cell endocytic vesicles were acidic with pH as low as 4.5 [ 32 ]. To evaluate the pH sensitivity of Au-DOX, the fluorescence intensity of the NPs at different pH values were tested. Three pH values were selected to monitor the pH of blood (pH 7.4), the tumor microenvironment (pH 6.5), and lysosomes (pH 5.5). As expected, the cleavage of hydrazone bond at acidic environment resulted in the release of DOX from Au-DOX, leading to an increase in fluorescence intensity (Fig. 3a). Within 31 h, the fluorescence intensity increased by factors of 3.9, 4.9, and 7 at pH 7.4, 6.5, and 5.5, respectively (Fig. 3b, c, Figure S2a-c). The result indicated that the pH responsive property would facilitate the release of DOX in the tumor acidic environment [ 33 ]. Since GSH can displace the ligands of gold nanoparticle through Au-S bond [ 34 , 35 ], DOX can effectively release from Au nanoparticle surface in the presence of GSH (Fig. 3d). To assess the thiol activation property of Au-DOX, the NPs were incubated in PBS solution containing 5 mM GSH. As the incubation time elapsed, the fluorescence of Au-DOX increased, as revealed by Figure S2d. The fluorescence intensity at the maximum emission wavelength (594 nm) increased by a factor of 8.9 times gradually after 31h incubation with GSH (Fig. 3e, f). Compromised antitumor efficacy of the drug delivery system The antitumor efficacy of Au-DOX was evaluated by Female Balb/c mice with 4T1 tumors. Following six treatments of DOX and Au-DOX, respectively, the organs (heart, spleen, lungs, kidneys, liver) and tumors were collected from the Balb/c mice. The fluorescence images showed that (Fig. 4 a) the liver fluorescence was much stronger than the other organs for the mice injected with DOX, indicating that DOX were accumulated in liver after intravenous injection. While for the mice injected with Au-DOX, the fluorescence of the tumor area was strong, indicating that the drug accumulated in the tumor through EPR effect after intravenous injection. Quantitative analysis of the fluorescence intensity of the organs and tumors (Fig. 4 b) showed that the liver fluorescence intensity of the mice injected with DOX was 5 times higher than the mice injected with Au-DOX, and lung was 1.5 times higher, while the tumor fluorescence intensity of the mice injected with Au-DOX was 2 times higher than the mice injected with DOX, indicating the renal clearable DDSs can enhance the clearance of DOX from the reticuloendothelial system (RES). H&E images of tumor tissue (Figure S3) revealed extensive necrosis with unstructured eosinophilic material and numerous necrotic cell debris (red arrows) surrounding the necrotic area in both the DOX and Au-DOX groups, confirming the effective therapeutic efficacy of Au-DOX. Interestingly, the fluorescence intensity of the liver collected from the mice 6 times treatment of Au-DOX was 12.26 times lower than the liver collected 24 h after treatment of Au-DOX (Figure S4), further confirmed the efficient clearance ability of the renal clearable DDSs [ 36 , 37 ]. Analysis of the tumor growth curve (Fig. 4 c and S5) revealed a significant inhibition of tumor growth in the Au-DOX treatment group compared to the PBS group when administered once every three days, with the tumor volume and weight approximately half that of the PBS treatment group. However, even though Au-DOX showed higher accumulation at the tumor site than free DOX, it did not exhibit superior antitumor effects, the therapeutic effect of the Au-DOX was not as good as free DOX at the same dosage (5 mg/kg body weight), and the tumor volume was nearly twice that of the DOX group. Interestingly, when the Au-DOX injection frequency was increased to once every two days, a similar therapeutic effect was achieved as with free DOX group administered once every three days, the size and weight of these two groups were comparable (Figs. 4 d and 4 e). To evaluate the possible influence of the antitumor effect of Au-PEG, the Au-PEG and PBS (comparable AuNPs dosage to Au-DOX treatment) were continuously injected into 4T1 tumor-bearing mice for 22 days. As shown in Figure S6a, S6b, and S6c, no significant differences were found in tumor volume growth rate, size, and weight between these two groups, and the Au-PEG treatment group did not exhibit significant tumor injury compared to the PBS group. These findings indicated that the vector Au-PEG did not interfere with the tumor suppression study. Figure 5. (a) Schematic diagram of lysosome escape. (b) Cellular uptake of DOX and Au-DOX within 2 hours. (c) Fluorescence intensity of DOX and Au-DOX in nucleus and lysosomes. (d) Fluorescence images of Hela cells for intracellular tracking of Au-DOX. scale bar = 5 µm. (e) The cell viability of Au-DOX and DOX towards Hela cells for 24 h. Data points represent mean ± SD (n = 3). Lysosomal barrier to the drug delivery system To unveil the origin of the reduced therapeutic efficiency of Au-DOX, intracellular tracking and drug release studies were conducted. After 2h incubation, the small molecule DOX diffused rapidly within the cell, primarily accumulated in the nucleus due to the concentration gradient driving force (Fig. 5b and Figure S7). While for the cells incubated with Au-DOX, a significant fluorescence overlap was observed between the red fluorescence of DOX and the green fluorescence of LysoTracker, indicating the endocytosis and lysosomal localization of the NPs. The fluorescence intensity of Au-DOX and DOX in the lysosome and nucleus were further analysed. As demonstrated in Fig. 5c, the fluorescence intensity of DOX group in the nucleus was 1.6 times higher than that in the lysosome, whereas the fluorescence intensity of Au-DOX group in the lysosome was 3.4 times higher than that in the nucleus. To study whether Au-DOX can evade lysosomal entrapment, the incubation period was extended to 48 hours (Fig. 5d, figure S7) with Hela cells and 4T1 cell, respectively. The result showed that Au-DOX was still remained in the lysosomes. Since Au-DOX being trapped in the lysosomes, the cytotoxicity of Au-DOX within 24 hours was slightly lower compared to an equivalent amount of free DOX (Fig. 5e) [ 38 ]. These results indicated that although renal-clearable gold nanoparticles can effectively release drugs in the presence of acid and GSH, it was worth noting that AuNPs without DOX loading exhibited minimal toxicity to Hela cancer cells (Figure S8), but they face challenges in evading lysosomal entrapment in vivo, as illustrated in diagram 5a, thereby limiting their therapeutic efficacy [ 39 ]. Reduced toxicity of the drug delivery system One advantage of Renal clearable DDSs was reduced toxicity [ 40 ]. After six injections over a 15 days period (administered every three days) (Fig. 6 a), the mice in the DOX group exhibited a weight reduction of 10%, whereas no significant weight loss was observed in the Au-DOX group, no matter whether the drug was administered every two or three days. This observation highlights the biocompatibility and low toxicity of Au-DOX (Figure S6d). Further study showed that Au-DOX exhibited improved renal clearance efficiency compared to DOX (Figure S9a, b, c, d). As depicted in Fig. 6 b, the renal clearance rate of Au-DOX within 24 hours was 47%, whereas that of DOX was only 11%. The utilization of renal clearable AuNPs considerably enhanced the renal clearance efficiency while concurrently reducing kidney injury [ 41 ]. Au-DOX treatment blood chemistry tests showed that the blood urea nitrogen (BUN) and creatinine (CREA) levels were all within the normal range (Fig. 6 c and Figure S9e), indicating the favourable biosafety profile of the Au-DOX. H&E images revealed that the free DOX group induced severe kidney damage, whereas no obvious damage was observed in the Au-DOX group (Fig. 6 d). It was worth noting that the primary elimination pathway for free DOX was the liver, which usually leads to liver injury in approximately 40% of patients. Fluorescence imaging of organs demonstrated a higher accumulation of free DOX in the liver compared to Au-DOX. Further examination via H&E staining revealed that the DOX group caused substantial liver injury, as well as damage to other reticuloendothelial system organs such as the spleen and lungs (Fig. 6 d, Figure S3). Conversely, no evident damage was observed in the Au-DOX group. Conclusion In summary, pH/GSH dual responsive renal-clearable Au-DOX nanoparticles were developed to study the tumor targeting efficiency and intracellular confinement of renal-clearable drug delivery vectors. The delivery vectors successfully enhanced the accumulation of DOX at the tumor area, however, the antitumor efficacy of Au-DOX was lower than that of free DOX. Further cellular study indicated that the intracellular confinement hindered Au-DOX from reaching the nucleus and thus limiting their therapeutic efficiency. On the other hand, the renal clearance property of Au-DOX enhanced the renal clearance of DOX and significantly reduced their toxicity. Overall, our work confirmed the significancy of renal-clearable drug delivery vectors in reducing drug toxicity and emphasized the role of the lysosomal barrier in mediating the therapeutic efficacy of renal-clearable DDSs. Declarations Acknowledgements The authors would like to thank the students of Dr. Aihua Gong’s group for their excellent technical assistance. Author contributions L.L. and S.Q. contributed equally to this work. L.L. collected, analyzed and interpreted the data. S.Q. collected and analyzed the data. Y.M. prepared samples and collected the data. M.S. prepared samples. H.H did the animal study. Y. W. supervised the research. A.G. supervised the research and revised the manuscript. S.T.: conceived and designed the experiments. X.C. supervised the research and revised the manuscript. S.S. supervised the research, designed the experiments and revised the manuscript. Funding This work was financially supported by the National Natural Science Foundation of China (21807050, 22308134), the Natural Science Foundation of Jiangsu Province (BK20220644, BK20210876), Research and Practice Innovation Plan of Postgraduate Training Innovation Project in Jiangsu Province (KYCX22_3752), the start-up fund from Jiangsu University of Science and Technology, the postdoc fund from Jiangsu Cancer Hospital& Jiangsu Institute of Cancer Research. Availability of data and materials All data are available from the corresponding author upon reasonable request. Ethics approval and consent to participate The animal experimental protocols were approved by the ethics committee of Jiangsu University of Science and Technology, and followed the Guidelines for Care and Use of Laboratory Animals from National Institutes of Health. Consent for publication Not applicable. Competing interests The authors declare no conflict of interest. Author details 1 School of Environmental and Chemical Engineering, Jiangsu University of Science and Technology, Zhenjiang, China 2 School of Medicine, Jiangsu University, Zhenjiang, China 3 Department of Medical Oncology, Jiangsu Cancer Hospital& Jiangsu Institute of Cancer Research& The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, China References Lu J, Gao X, Wang S, He Y, Ma X, Zhang T, Liu X. Advanced strategies to evade the mononuclear phagocyte system clearance of nanomaterials. Exploration. 2023;3:20220045. Zhou Q, Dong C, Fan W, Jiang H, Xiang J, Qiu N, Piao Y, Xie T, Luo Y, Li Z, Liu F, Shen Y. Tumor extravasation and infiltration as barriers of nanomedicine for high efficacy: The current status and transcytosis strategy. Biomaterials. 2020;240:119902. Blanco E, Shen H, Ferrari M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 2015;33: 941-951. Poon W, Kingston BR, Ouyang B, Ngo W, Chan WCW. A framework for designing delivery systems. Nat. Nanotechnol. 2020;15:819-829. Thorn CF, Oshiro C, Marsh S, Hernandez-Boussard T, McLeod H, Klein TE, Altman RB. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genom. 2011;21:440-446. Rao C, Sharma S, Garg R, Anjum F, Kaushik K, Nandi CK. Mapping the time dependent DNA fragmentation caused by doxorubicin loaded on PEGylated carbogenic nanodots using fluorescence lifetime imaging and superresolution microscopy. Biomaters Science. 2022;10:4525-4537. Yang F, Teves SS, Kemp CJ, Henikoff S. Doxorubicin, DNA torsion, and chromatin dynamics. Biochim et Biophys Acta. 2014;1845:84-89. Makvandi P, Chen M, Sartorius R, Zarrabi A, Ashrafizadeh M, Dabbagh Moghaddam F, Ma J, Mattoli V, Tay F.R. Endocytosis of abiotic nanomaterials and nanobiovectors: Inhibition of membrane trafficking. Nano Today. 2021;40:101279. Zhou X, Liu X, Huang L. Macrophage-Mediated Tumor Cell Phagocytosis: Opportunity for Nanomedicine Intervention. Adv. Funct. Mater. 2021;31: 2006220. Jayashankar V, Edinger AL. Macropinocytosis confers resistance to therapies targeting cancer anabolism. Nat. Commun. 2020;11:1121. Francia V, Montizaan D, Salvati A. Interactions at the cell membrane and pathways of internalization of nano-sized materials for nanomedicine. Beilstein J. Nanotechnol. 2020;11:338-353. Jiang L, Liang X, Liu G, Zhou Y, Ye X, Chen X, Miao Q, Gao L, Zhang X, Mei L. The mechanism of lauric acid-modified protein nanocapsules escape from intercellular trafficking vesicles and its implication for drug delivery. Drug Deliv. 2018;25:985-994. Gong L, Wang Y, Liu J. Bioapplications of renal-clearable luminescent metal nanoparticles. Biomater. Sci. 2017;5:1393-1406. Soo Choi H, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, Bawendi MG, Frangioni JV. Renal clearance of quantum dots. Nat. Biotechnol. 2007;25:1165-1170. He B, Sui X, Yu B, Wang S, Shen Y, Cong H. Recent advances in drug delivery systems for enhancing drug penetration into tumors. Drug Deliv. 2020;27:1474-1490. Yu M, Zheng J. Clearance Pathways and Tumor Targeting of Imaging Nanoparticles. ACS Nano. 2015;9:6655-6674. Peng C, Huang Y, Zheng J. Renal clearable nanocarriers: Overcoming the physiological barriers for precise drug delivery and clearance. J. Controlled Release. 2020;322:64-80. Kang H, Gravier J, Bao K, Wada H, Lee JH, Baek Y, Fakhri GE, Gioux S, Rubin BP, Coll JL, Choi HS. Renal Clearable Organic Nanocarriers for Bioimaging and Drug Delivery. Adv. Mater. 2016;28:8162-8168. Kang H, Stiles WR, Baek Y, Nomura S, Bao K, Hu S, Park GK, Jo MJ, Hoseok I, Coll JL, Rubin BP, Choi HS. Renal Clearable Theranostic Nanoplatforms for Gastrointestinal Stromal Tumors. Adv. Mater. 2020;32:1905899. Aragon-Sanabria V, Aditya A, Zhang L, Chen F, Yoo B, Cao T, Madajewski B, Lee R, Turker MZ, Ma K, Monette S, Chen P, Wu J, Ruan S, Overholtzer M, Zanzonico P, Rudin CM, Brennan C, Wiesner U, Bradbury MS. Ultrasmall Nanoparticle Delivery of Doxorubicin Improves Therapeutic Index for High-Grade Glioma. Clin. Cancer Res. 2022;28:2938-2952. Madajewski B, Chen F, Yoo B, Turker MZ, Ma K, Zhang L, Chen PM, Juthani R, Aragon-Sanabria V, Gonen M, Rudin CM, Wiesner U, Bradbury MS, Brennan C. Molecular Engineering of Ultrasmall Silica Nanoparticle–Drug Conjugates as Lung Cancer Therapeutics. Clin. Cancer Res. 2020;26:5424-5437. Zhang L, Aragon-Sanabria V, Aditya A, Marelli M, Cao T, Chen F, Yoo B, Ma K, Zhuang L, Cailleau T, Masterson L, Turker MZ, Lee R, DeLeon G, Monette S, Colombo R, Christie RJ, Zanzonico P, Wiesner U, Subramony JA, Bradbury MS. Engineered Ultrasmall Nanoparticle Drug-Immune Conjugates with “Hit and Run” Tumor Delivery to Eradicate Gastric Cancer. Adv. Therap. 2023;6:2200209. Peng C, Xu J, Yu M, Ning X, Huang Y, Du B, Hernandez E, Kapur P, Hsieh JT, Zheng J. Tuning the in vivo transport of anticancer drugs using renal-clearable gold nanoparticles. Angew. Chem. Int. Ed. 2019;58:8479-8483. Peng C, Yu M, Hsieh JT, Kapur P, Zheng J. Correlating Anticancer Drug Delivery Efficiency with Vascular Permeability of Renal Clearable Versus Non-renal Clearable Nanocarriers. Angew. Chem. Int. Ed. 2019;131:12204-12208. Liu J, Yu M, Ning X, Zhou C, Yang S, Zheng J. PEGylation and Zwitterionization: Pros and Cons in the Renal Clearance and Tumor Targeting of Near-IR-Emitting Gold Nanoparticles. Angew. Chem. Int. Ed. 2013;52:12572-12576. Lelle M, Hameed A, Ackermann LM, Kaloyanova S, Wagner M, Berisha F, Nikolaev VO, Peneva K. Functional Non‐Nucleoside Adenylyl Cyclase Inhibitors. Chem. Biol. Drug Des. 2014;85:633-637. Zhang Y, Ang CY, Li M, Tan SY, Qu Q, Zhao Y. Polymeric Prodrug Grafted Hollow Mesoporous Silica Nanoparticles Encapsulating Near-Infrared Absorbing Dye for Potent Combined Photothermal-Chemotherapy. ACS Appl. Mater. Interfaces. 2016;8:6869-6879. Yang X, Zhang X, Liu Z, Ma Y, Huang Y, Chen Y. High-Efficiency Loading and Controlled Release of Doxorubicin Hydrochloride on Graphene Oxide. J. Phys. Chem. C. 2008;112:17554-17558. Chen B, Mei L, Fan R, Wang Y, Nie C, Tong A, Guo G. Facile construction of targeted pH-responsive DNA-conjugated gold nanoparticles for synergistic photothermal-chemotherapy. Chin. Chem. Lett. 2021;32: 1775-1779. Xing J, Gong Q, Zou R, Yao J, Xiang L, Wu A. GSH responsive traditional clinical drugs probe for cancer cell fluorescence imaging and therapy. Chin. Chem. Lett. 2023;34:107786. Zhao H, Ruan H, Li H. Progress in the research of GSH in cells. Chin. Sci. Bull. 2011;56:3057-3063. Wang Z, Luo M, Mao C, Wei Q, Zhao T, Li Y, Huang G, Gao J. A Redox-Activatable Fluorescent Sensor for the High-Throughput Quantification of Cytosolic Delivery of Macromolecules. Angew. Chem. Int. Ed. Engl. 2017;56:1319-1323. Mu J, Zhong H, Zou H, Liu T, Yu N, Zhang X, Xu Z, Chen Z, Guo S. Acid-sensitive PEGylated paclitaxel prodrug nanoparticles for cancer therapy: Effect of PEG length on antitumor efficacy. J. Controlled Release. 2020;326:265-275. Awotunde O, Okyem S, Chikoti R, Driskell JD. Role of Free Thiol on Protein Adsorption to Gold Nanoparticles. Langmuir. 2020;36:9241-9249. Meng J, Hu Z, He M, Wang J, Chen X. Gold nanocluster surface ligand exchange: An oxidative stress amplifier for combating multidrug resistance bacterial infection. J. Colloid Interface Sci. 2021;602:846-858. Ding D, Yang C, Lv C, Li J, Tan W. Improving Tumor Accumulation of Aptamers by Prolonged Blood Circulation. Anal. Chem. 2020;92:4108-4114. Singh B, Mitragotri S. Harnessing cells to deliver nanoparticle drugs to treat cancer. Biotechnol Advances. 2020;42:107339. Tian X, Shi A, Yin H, Wang Y, Liu Q, Chen W, Wu J. Nanomaterials Respond to Lysosomal Function for Tumor Treatment. Cells. 2022;11:3348. Zhai X, Hiani YE. Getting Lost in the Cell-Lysosomal Entrapment of Chemotherapeutics. Cancers. 2020;12:12123669. Zhang XD, Wu D, Shen X, Liu PX, Fan FY, Fan SJ. In vivo renal clearance, biodistribution, toxicity of gold nanoclusters. Biomaterials. 2012;33:4628-4638. Peng C, Gao X, Xu J, Du B, Ning X, Tang S, Bachoo RM, Yu M, Ge WP, Zheng J. Targeting orthotopic gliomas with renal-clearable luminescent gold nanoparticles. Nano Res. 2017;10:1366-1376. Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3940105","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273237133,"identity":"aa333db5-5e8f-4c1f-bcfe-53178c680c91","order_by":0,"name":"Lang Liu","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Lang","middleName":"","lastName":"Liu","suffix":""},{"id":273237134,"identity":"6c2452ba-cf2d-4bf8-867e-11110514ae22","order_by":1,"name":"Shanshan Qiao","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shanshan","middleName":"","lastName":"Qiao","suffix":""},{"id":273237135,"identity":"c2659825-5296-45ee-b65d-499bd83bc0e7","order_by":2,"name":"Meiyu Sun","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Meiyu","middleName":"","lastName":"Sun","suffix":""},{"id":273237136,"identity":"08c6d849-1be1-4aa2-8fe8-a630bf012af2","order_by":3,"name":"Yusheng Mao","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yusheng","middleName":"","lastName":"Mao","suffix":""},{"id":273237137,"identity":"4c5321dd-d1c9-4a6d-ad59-998d2b7a4faf","order_by":4,"name":"Hai Huang","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hai","middleName":"","lastName":"Huang","suffix":""},{"id":273237138,"identity":"f983b7bd-3d19-44a6-bbcc-0dfc6eb7a12d","order_by":5,"name":"Yemei Wang","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yemei","middleName":"","lastName":"Wang","suffix":""},{"id":273237139,"identity":"f0a471f3-cdfe-4e95-8161-ab321427ce65","order_by":6,"name":"Shasha Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYJACZiBOYGBvSIBwDxCthedAAogiRYtEAgNxWuRn5B7+XFBTl8c/88EzyZ8/GOT4biQwfi7Ao8XgRl6a9Ixjh4slbiekSfMkMBhL3khglp6BT4tEjhkzD9uBxAaQFqDDEjfcSGBj5sHrsBzjzzz/6hLn3zyQJvkjgaGeoBaGGzkG0rxtzEDDGdIkgA5LMCCkxeDMGzNp3r7DiRvPJCRb86RJGM4887BZGq/D2kEO+1aXOO/4mcSbP2xs5PmOJx/8jNdhCAB0FQODBBAzNhCngYGB/QCxKkfBKBgFo2CEAQALpUxNOV2elAAAAABJRU5ErkJggg==","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Shasha","middleName":"","lastName":"Sun","suffix":""},{"id":273237140,"identity":"394ab9f8-3421-4eb2-aebe-2938ab802696","order_by":7,"name":"Xianzhi Chai","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xianzhi","middleName":"","lastName":"Chai","suffix":""},{"id":273237141,"identity":"b7ccd4c2-068b-4255-8ab7-24c528cb1850","order_by":8,"name":"Shaoheng Tang","email":"","orcid":"","institution":"Jiangsu University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shaoheng","middleName":"","lastName":"Tang","suffix":""},{"id":273237142,"identity":"e54b1ff5-49a8-4dc3-938c-9d965f610cbe","order_by":9,"name":"Aihua Gong","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Aihua","middleName":"","lastName":"Gong","suffix":""}],"badges":[],"createdAt":"2024-02-08 13:51:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3940105/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3940105/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51384087,"identity":"bb1bcc67-7248-444f-bf35-95431dd54a9d","added_by":"auto","created_at":"2024-02-20 16:56:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":164278,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illumination of the tumor targeting and intracellular behaviour of renal clearable AuNPs drug delivery system. (a) Au-DOX accumulated at the tumor area through EPR effect, and exhibited efficient renal clearance. (b) Free Dox passively diffused into cells and got into nucleus, while the DOX loaded on AuNPs were internalized into cells via endocytosis and confined within lysosome, significantly hindering them from reaching the nucleus.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3940105/v1/4392ba8c6aefc4da565aa1ff.png"},{"id":51384088,"identity":"8de5be45-2bb1-4f8f-883a-9cdaff001723","added_by":"auto","created_at":"2024-02-20 16:56:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":249958,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Chemical structure of DOX-hydrazone linker conjugate (DOX-PDPH). Scheme of renal-clearable Au-DOX, which consists of the gold core, surface coating with poly (ethylene glycol) thiol (mPEG-SH, MW: 5000 Da) and acid sensitive linker (PDPH), and loaded DOX molecules. (b) HR-TEM image and size distribution of Au-DOX. The scale bar is 10 nm. (c) UV-Vis absorbance spectra of Au-PEG, DOX, DOX-PDPH and Au-DOX in aqueous solution. (d) Fluorescence emission spectra of Au-DOX and DOX at the same concentrations.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3940105/v1/61efa5ed4e2917ceace29ee8.png"},{"id":51384089,"identity":"2b3f4a28-0a92-44f4-8499-af5ad5fed2a5","added_by":"auto","created_at":"2024-02-20 16:56:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":162658,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The illustration of the acidic pH response of Au-DOX. (b) The fluorescence intensity changes of Au-DOX at pH 5.5, 6.5, and 7.4 in 31 h. (c) Time-dependent DOX release from Au-DOX at different pH values. (d) The illustration of the GSH response of Au-DOX. (e) The change of fluorescence intensity of Au-DOX in 31h after adding 5 mM GSH. (f) Time-dependent DOX release from Au-DOX in 5 mM GSH.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3940105/v1/475588cbdaf393d9af8d28ff.png"},{"id":51384090,"identity":"387d28a4-dcb8-4c52-93ff-8eb14ed0dc65","added_by":"auto","created_at":"2024-02-20 16:56:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":436180,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Organ fluorescence imaging of Balb/c mice after DOX and Au-DOX treatment. (b) Comparison of fluorescence intensity in different organs after DOX and Au-DOX treatment. \u0026nbsp;(c) Tumor volume growth curves of 4T1 tumor-bearing mice. (d) Tumor size and (e) tumor weight after treatment.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3940105/v1/f4e3a2493633c4d829f1cc88.png"},{"id":51384093,"identity":"f8ed6452-01a1-46da-b3c8-cb8f5449d507","added_by":"auto","created_at":"2024-02-20 16:56:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1051007,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic diagram of lysosome escape. (b) Cellular uptake of DOX and Au-DOX within 2 hours. (c) Fluorescence intensity of DOX and Au-DOX in nucleus and lysosomes. (d) Fluorescence images of Hela cells for intracellular tracking of Au-DOX. scale bar = 5 μm. (e) The cell viability of Au-DOX and DOX towards Hela cells for 24 h. Data points represent mean ± SD (n=3).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3940105/v1/bec8a485ed45743215f73f90.png"},{"id":51384092,"identity":"9a6c866f-3222-47ef-853e-9a12b86ad45e","added_by":"auto","created_at":"2024-02-20 16:56:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":790834,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Body weight changes curve of 4T1 tumor-bearing mice. (b) Renal clearance of DOX after intravenous injection of Au-DOX and free DOX. (c) Blood tests for renal function BUN. (d) H\u0026amp;E images of organs in mice after PBS, Au-DOX (2 days) and DOX (3 days) treatment. (e) After 24 h of administration, organ distribution of carrier AuNPs.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3940105/v1/9264b524179e168badc88de8.png"},{"id":68735037,"identity":"a67fa8a4-563a-4e1b-883f-0aebc1db45cc","added_by":"auto","created_at":"2024-11-11 13:17:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3382735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3940105/v1/f244892e-1041-4f97-b71e-d29f217f39e9.pdf"},{"id":51384094,"identity":"e2817569-412e-4d83-9c33-7b0d82a63aeb","added_by":"auto","created_at":"2024-02-20 16:56:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10018903,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3940105/v1/cb6499a1d3e7a8c3a3863357.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pros and Cons in The Delivery of Doxorubicin Using Renal-clearable Gold Nanoparticles","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUnderstanding the biological barriers in preventing DDSs from eliciting their therapeutic efficacy was critical to the clinical translation of DDSs. Currently identified biological barriers include the mononuclear phagocytic system barrier\u0026nbsp;[1], the tumor blood vessel extravasation barrier\u0026nbsp;[2], the intratumoral penetration barrier and the intracellular barrier\u0026nbsp;[3, 4]. Among them, the intracellular barrier has been drawn little attention in the past few decades despite its critical role in governing the therapeutic efficacy of organelle-targeting chemo drugs such as doxorubicin (DOX)\u0026nbsp;[5], which functions through intercalation into nuclear DNA and disruption of topoisomerase mediated DNA repair, and genetic drugs such as siRNA, mRNA, and gene-editing systems, which have to function in cytosol and nucleus\u0026nbsp;[6, 7]. Nanomaterials were generally taken up by cells through endocytosis\u0026nbsp;[8], phagocytosis\u0026nbsp;[9], and pinocytosis\u0026nbsp;[10]\u0026nbsp;pathways, leading to the internalization of nanomaterials into intracellular vesicles such as endosomes and lysosomes\u0026nbsp;[11]. The confinement of the loaded drugs in these intracellular vesicles would dramatically decrease their therapeutic efficacy when they were unable to escape from the vesicles\u0026nbsp;[12]. Therefore, it was of great significance to understand the intracellular barrier of DDSs when they were used to deliver organelle-specific drugs and genetic drugs.\u003c/p\u003e\n\u003cp\u003eRenal-clearable DDSs deliver therapeutic agents for cancer treatment by employing renal-clearable nanomaterials as delivery vectors\u0026nbsp;[13]. The ultrasmall size of renal-clearable DDSs below the kidney filtration threshold (~5.5 nm)\u0026nbsp;[14], enables enhanced and homogeneous intratumoral penetration, thereby facilitating the effective delivery of therapeutic agents to cancer cells\u0026nbsp;[15]. Furthermore, the efficient renal clearance through the kidneys and bladder allows for the rapid elimination of off-target therapeutic agents, leading to a significant reduction in off-target toxicity\u0026nbsp;[16]. Over the past decade, several renal-clearable DDSs have been developed, demonstrating promising potential for the treatment of various cancers with low toxicity\u0026nbsp;[17].\u0026nbsp;For instance, Hak Soo Choi and co-workers developed cyclodextrin-based renal-clearable DDSs (\u0026ldquo;H-Dots\u0026rdquo;) to deliver imatinib for efficient treatment of unresectable gastrointestinal stromal tumors\u0026nbsp;[18, 19]. Michelle Bradbury team developed renal-clearable silica nanoparticles (\u0026ldquo;Cornell-dots\u0026rdquo;), which have been used to deliver different types of anticancer therapeutic agents such as chemo drugs\u0026nbsp;[20], therapeutic proteins\u0026nbsp;[21], and immune drugs\u0026nbsp;[22]\u0026nbsp;with efficient therapeutic efficacy. Moreover, Peng et al. demonstrated the superior efficacy of AuNPs-based renal-clearable DDSs over free drug and the non-renal-clearable counterpart in treating triple-negative breast cancer and lung metastasis due to significantly enhanced intratumoral penetration\u0026nbsp;[23, 24]. However, despite the development of renal-clearable DDSs in the past decade, deeper understanding of how the biological barriers, especially the intracellular barrier, affect their therapeutic efficacy, remains poorly explored at this stage.\u003c/p\u003e\n\u003cp\u003eHerein, by using DOX as the model drug, we developed dual pH/glutathione (GSH)-responsive renal-clearable Au-DOX to investigate the antitumor efficacy and toxicity in treating triple-negative breast cancer 4T1 cancer. Initially, DOX was conjugated with a thiol ligand through an acid-labile linker and subsequently coated onto renal-clearable PEGylated AuNPs via Au-S bond. The loaded DOX exhibits prompt release under acidic pH and in the presence of GSH. However, despite the Au-DOX achieved enhanced tumor delivery efficiency and efficient release of DOX, the therapeutic efficacy of Au-DOX only reached comparable or even lower levels compared to free DOX. Further in vitro studies revealed that, unlike the passive diffusion pathway of free DOX into cells and subsequent nuclear penetration, DOX loaded on AuNPs was internalized into cells alongside the AuNPs via endocytosis. Once internalized, DOX molecules became confined within lysosomes, significantly hindering them from reaching the nucleus and thus limiting their therapeutic functionality. Consequently, this fundamentally diminishes the cellular toxicity and antitumor efficacy of DOX. On the other side, the off-target Au-DOX was efficiently eliminated from the body through renal clearance pathway, which significantly reduced the toxicity of Au-DOX compared to free DOX. \u0026nbsp;Our findings suggest that despite the high tumor targeting efficiency and reduction in off-target toxicity of renal clearable nanoparticles, the role of the lysosomal barrier in mediating the therapeutic efficacy should be carefully considered when design renal clearable DDSs.\u003c/p\u003e\n"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of Au-PEG\u003c/h2\u003e \u003cp\u003eAu-PEG was synthesized through thermal reduction of chloroauric acid (HAuCl\u003csub\u003e4\u003c/sub\u003e). Briefly, a solution comprising HAuCl\u003csub\u003e4\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO (0.04 g, 0.1 mmol), mPEG-SH (MW : 5 kDa, 1.5 g, 0.3 mmol), and NaBH\u003csub\u003e4\u003c/sub\u003e (0.075 g, 2 mmol) was dissolved in a methanol/acetic acid mixed solvent (6:1 v/v, 5 mL) and stirred vigorously at room temperature for 2 hours. Subsequently, the resulting mixture was evaporated to dryness under vacuum, and the resulting residue was dissolved in water. The pH value was adjusted to 7.4 using NaOH, and the solution was then subjected to centrifugation at 9000 rpm for 10 minutes. The resulting supernatant was further purified using dialysis and freeze-drying techniques.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of DOX-PDPH\u003c/h2\u003e \u003cp\u003eDOX-HCl (22.7 mg, 0.034 mmol) and PDPH-linker (20.7 mg, 0.045 mmol) were dissolved in methanol (14 mL) and stirred in the dark at room temperature for 6 days. The solvent was then evaporated, and acetonitrile was added for purification via centrifugation. The resulting residues were dissolved in dimethyl sulfoxide (DMSO) and subsequently stored at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSynthesis of Au-DOX\u003c/h3\u003e\n\u003cp\u003eDOX-PDPH was conjugated to Au-PEG via an Au-S bond. A solution of Au-PEG (0.5 mL, 0.03 g/mL) and DOX-PDPH (0.2 mL, 0.01 M) was combined and incubated with agitation at room temperature for 48 hours. Once the reaction was completed, the solution was purified by passing it through a Sephadex LH-20 column to remove free DOX-PDPH, and subsequently lyophilized, resulting in a dark red Au-DOX powder.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAgarose gel electrophoresis\u003c/h2\u003e \u003cp\u003eA solution (10 \u0026micro;L) of each sample was introduced into the prepared agarose gel wells, followed by analysis through 2% agarose gel electrophoresis utilizing the DYY-8C Gel electrophoresis system (Beijing Liuyi Biotechnology Co., LTD.) and 0.1X Britton-Robinson buffer solution (pH 7.4) at a constant current of 60 mA for 20 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro drug release\u003c/h2\u003e \u003cp\u003eIn the pH-responsive release assay, a 20 \u0026micro;L aliquot of the Au-DOX solution (10 m M) was added to 180 \u0026micro;L of PBS buffer, and the buffer pH was pH 5.5, 6.5, 7.4, respectively. For the GSH-responsive release experiment, the Au-DOX solution was mixed with a GSH solution to achieve a final concentration of 5 mM. The resulting solution was incubated on a shaker at 37\u0026deg;C and 100 rpm. The fluorescence intensity of the samples was measured and analysed at predetermined time intervals using a fluorescence spectrometer. It was noteworthy that the stability of Au-DOX was evaluated under both acidic (pH 5.5) and neutral (pH 7.4) conditions. Using the static method for a period of 2 hours, with fluorescence measurements taken every ten minutes to assess stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCellular uptake\u003c/h2\u003e \u003cp\u003eHela and 4T1 cells were purchased from Shanghai Xuanya Biotechnology Co., Ltd, culturing under standard conditions (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e). Cells were cultured in 35 mm culture dishes and subjected to individual treatments using either Au-DOX or free DOX, both at equal DOX concentrations (5 \u0026micro;g/mL). After incubation for different time, washing with PBS (pH\u0026thinsp;=\u0026thinsp;7.4) for three times, lysosomes and nuclei were stained with LysoTracker Green DNA-26 (50 nM) and Hoechst 33342 (1 \u0026micro;g/mL) for 30 min, the cells underwent PBS wash and were subsequently visualized using a confocal fluorescence microscope (OLYMPUS IX73, Olympus Trading (Shanghai) Co., Ltd).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vitro cytotoxicity\u003c/h3\u003e\n\u003cp\u003eFor the cytotoxicity investigation, 4T1 cells were seeded in a 96-well plate and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours. Following this period, the supernatant was aspirated, and RPMI 1640 medium supplemented with various concentrations of Au-DOX and DOX (ranging from 0.1 \u0026micro;g/mL to 100 \u0026micro;g/mL) were added (n\u0026thinsp;=\u0026thinsp;3). Following an additional 24 hours of incubation, the culture media in the wells were substituted with 100 \u0026micro;L of fresh medium supplemented with 10 \u0026micro;L of the CCK-8 reagent. This was succeeded by an additional 2 hours incubation period. Finally, the absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using the formula: cell viability (%) = (OD\u003csub\u003esample\u003c/sub\u003e \u0026ndash; OD\u003csub\u003eblank\u003c/sub\u003e)/( OD\u003csub\u003econtrol\u003c/sub\u003e \u0026ndash; OD\u003csub\u003eblank\u003c/sub\u003e) \u0026times; 100%, where OD represents the absorbance of the sample at 450 nm. The experiment was conducted in triplicate.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMouse tumor model\u003c/h2\u003e \u003cp\u003eIn our experimental procedures, we obtained female Balb/c mice from Changzhou Cavens Experimental Animal Co., Ltd. The animal experiments were conducted in accordance with the approved protocols by Soochow University Laboratory Animal Center. To establish the tumor model, 1*10\u003csup\u003e6\u003c/sup\u003e 4T1 cells suspended in 50 \u0026micro;L of PBS were subcutaneously injected into the chest of each mouse.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntitumor efficacy and toxicity studies\u003c/h3\u003e\n\u003cp\u003eThirty female Balb/c mice bearing 4T1 tumors (with an initial tumor volume of approximately 60 mm\u003csup\u003e3\u003c/sup\u003e) were randomly assigned to four treatment groups administered via the tail vein: (1) 125 µL of PBS (administered once every 3 days); (2) 125 µL of Au-PEG (at a dose of AuNPs same with Au-DOX of 50 mg/kg, administered once every 3 days); (3) 125 µL of Au-DOX (at a DOX dose of 5 mg/kg, administered once every 3 days); (4) 125 µL of Au-DOX (at a DOX dose of 5 mg/kg, administered once every 2 days); and (5) 125 µL of DOX (at a dose of 5 mg/kg, administered once every 3 days). The tumor volume (V = L × W\u003csup\u003e2\u003c/sup\u003e/2) was measured, and the relative volume (V/V\u003csub\u003e0\u003c/sub\u003e) was calculated, where V\u003csub\u003e0\u003c/sub\u003e represents the tumor volume before treatment. Mice were weighed periodically to monitor their weight throughout the treatment. To ensure ethical considerations, tumor volumes exceeding 1500 mm\u003csup\u003e3\u003c/sup\u003e were not permitted during the study. At the end of the study, organs and tumor tissues were dissected, photographed, and weighed. Subsequently, they were embedded in paraffin and sectioned for hematoxylin and eosin (H\u0026amp;E) staining. Blood was procured via cardiac puncture for the purpose of blood analysis. The collected blood samples were kept on ice for 30 minutes to allow coagulation. Subsequently, serum was obtained by centrifuging the samples at 2,000 g for 15 minutes to remove coagulated blood.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBiodistribution and renal clearance\u003c/h2\u003e \u003cp\u003eFemale Balb/c mice bearing 4T1 tumors (with an initial tumor volume of approximately 200 mm\u003csup\u003e3\u003c/sup\u003e) were randomly assigned to two treatment groups: the DOX group and the Au-DOX group. Each group received intravenous injection of DOX at a dose of 5mg/kg through the tail vein. After completing administration therapy, the mice were euthanized, and their heart, liver, spleen, lungs, kidneys, and tumor tissues were dissected. The biodistribution of DOX was quantified by using fluorescence imaging. For AuNPs biodistribution, the main organs were dissected, weighed, dissolved in aqua regia, and diluted appropriately for ICP-OES measurements using the AVIO 200 instrument from Shanghai Platinum Elmer Instrument Co., Ltd. To quantify renal clearance, mice were intravenously injected with Au-DOX and free DOX, and urine samples were collected and measured for volume at various time intervals within 24 hours. To quantify DOX in the urine, the suspension was centrifuged at 21000 rpm for 10 minutes, and the supernatant was collected for DOX concentration analysis based on UV absorption. To quantify AuNPs in the urine, the urine was dissolved in aqua regia and diluted properly for ICP-OES measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eFigure 2.\u003c/b\u003e (a) Chemical structure of DOX-hydrazone linker conjugate (DOX-PDPH). Scheme of renal-clearable Au-DOX, which consists of the gold core, surface coating with poly (ethylene glycol) thiol (mPEG-SH, MW: 5000 Da) and acid sensitive linker (PDPH), and loaded DOX molecules. (b) HR-TEM image and size distribution of Au-DOX. The scale bar is 10 nm. (c) UV-Vis absorbance spectra of Au-PEG, DOX, DOX-PDPH and Au-DOX in aqueous solution. (d) Fluorescence emission spectra of Au-DOX and DOX at the same concentrations.\u003c/p\u003e\u003ch2\u003ePreparation and characterization of Au-DOX\u003c/h2\u003e\u003cp\u003eThe Au-PEG nanoparticles (AuNPs) were synthesized following a reported method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. DOX was connected with a 3-[2-Pyridyldithio]propionyl hydrazide (PDPH crosslinker) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec), which not only can form a pH-responsive hydrazone bond with the carbonyl group of DOX, but also can introduce a thiol functional group to conjugate DOX with AuNPs (Fig.\u0026nbsp;2a) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The uniform size distribution of Au-DOX nanoparticles was confirmed by high-resolution transmission electron microscopy (HR-TEM) imaging, with a particle size of 1.8 ± 0.2 nm (Fig.\u0026nbsp;2b). The absorption spectrum of Au-DOX exhibited a characteristic absorbance of DOX at 488 nm, indicating the successful conjugation of DOX onto AuNPs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Notably, the loaded DOX showed an 11 nm redshift (from 488 to 499 nm) compared to free DOX, suggesting the formation of J-aggregates of DOX after conjugation onto AuNPs, which was resulted from dipole-dipole interactions among the loaded DOX molecules (Fig.\u0026nbsp;2c). Moreover, the fluorescence intensity of DOX decreased about 9 times due to the fluorescence resonance energy transfer (FRET) process between DOX and AuNPs (Fig.\u0026nbsp;2d) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The successful conjugation of DOX onto AuNPs was also confirmed by agarose gel electrophoresis. The gel mobility of Au-DOX was in between that of pure DOX and Au-PEG (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea), and the fluorescence imaging of the agarose gel showed a similar mobility behavior (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e \u003cb\u003eFigure 3.\u003c/b\u003e (a) The illustration of the acidic pH response of Au-DOX. (b) The fluorescence intensity changes of Au-DOX at pH 5.5, 6.5, and 7.4 in 31 h. (c) Time-dependent DOX release from Au-DOX at different pH values. (d) The illustration of the GSH response of Au-DOX. (e) The change of fluorescence intensity of Au-DOX in 31h after adding 5 mM GSH. (f) Time-dependent DOX release from Au-DOX in 5 mM GSH.\u003c/p\u003e\u003ch2\u003eDrug release characteristics triggered by pH and GSH in vitro\u003c/h2\u003e\u003cp\u003eCell cytosol contains high concentrations of GSH (1–10 mM) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and the cell endocytic vesicles were acidic with pH as low as 4.5 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To evaluate the pH sensitivity of Au-DOX, the fluorescence intensity of the NPs at different pH values were tested. Three pH values were selected to monitor the pH of blood (pH 7.4), the tumor microenvironment (pH 6.5), and lysosomes (pH 5.5). As expected, the cleavage of hydrazone bond at acidic environment resulted in the release of DOX from Au-DOX, leading to an increase in fluorescence intensity (Fig.\u0026nbsp;3a). Within 31 h, the fluorescence intensity increased by factors of 3.9, 4.9, and 7 at pH 7.4, 6.5, and 5.5, respectively (Fig.\u0026nbsp;3b, c, Figure S2a-c). The result indicated that the pH responsive property would facilitate the release of DOX in the tumor acidic environment [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Since GSH can displace the ligands of gold nanoparticle through Au-S bond [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], DOX can effectively release from Au nanoparticle surface in the presence of GSH (Fig.\u0026nbsp;3d). To assess the thiol activation property of Au-DOX, the NPs were incubated in PBS solution containing 5 mM GSH. As the incubation time elapsed, the fluorescence of Au-DOX increased, as revealed by Figure S2d. The fluorescence intensity at the maximum emission wavelength (594 nm) increased by a factor of 8.9 times gradually after 31h incubation with GSH (Fig.\u0026nbsp;3e, f).\u003c/p\u003e\u003ch2\u003eCompromised antitumor efficacy of the drug delivery system\u003c/h2\u003e\u003cp\u003eThe antitumor efficacy of Au-DOX was evaluated by Female Balb/c mice with 4T1 tumors. Following six treatments of DOX and Au-DOX, respectively, the organs (heart, spleen, lungs, kidneys, liver) and tumors were collected from the Balb/c mice. The fluorescence images showed that (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) the liver fluorescence was much stronger than the other organs for the mice injected with DOX, indicating that DOX were accumulated in liver after intravenous injection. While for the mice injected with Au-DOX, the fluorescence of the tumor area was strong, indicating that the drug accumulated in the tumor through EPR effect after intravenous injection. Quantitative analysis of the fluorescence intensity of the organs and tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) showed that the liver fluorescence intensity of the mice injected with DOX was 5 times higher than the mice injected with Au-DOX, and lung was 1.5 times higher, while the tumor fluorescence intensity of the mice injected with Au-DOX was 2 times higher than the mice injected with DOX, indicating the renal clearable DDSs can enhance the clearance of DOX from the reticuloendothelial system (RES). H\u0026amp;E images of tumor tissue (Figure S3) revealed extensive necrosis with unstructured eosinophilic material and numerous necrotic cell debris (red arrows) surrounding the necrotic area in both the DOX and Au-DOX groups, confirming the effective therapeutic efficacy of Au-DOX. Interestingly, the fluorescence intensity of the liver collected from the mice 6 times treatment of Au-DOX was 12.26 times lower than the liver collected 24 h after treatment of Au-DOX (Figure S4), further confirmed the efficient clearance ability of the renal clearable DDSs [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Analysis of the tumor growth curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and S5) revealed a significant inhibition of tumor growth in the Au-DOX treatment group compared to the PBS group when administered once every three days, with the tumor volume and weight approximately half that of the PBS treatment group. However, even though Au-DOX showed higher accumulation at the tumor site than free DOX, it did not exhibit superior antitumor effects, the therapeutic effect of the Au-DOX was not as good as free DOX at the same dosage (5 mg/kg body weight), and the tumor volume was nearly twice that of the DOX group. Interestingly, when the Au-DOX injection frequency was increased to once every two days, a similar therapeutic effect was achieved as with free DOX group administered once every three days, the size and weight of these two groups were comparable (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). To evaluate the possible influence of the antitumor effect of Au-PEG, the Au-PEG and PBS (comparable AuNPs dosage to Au-DOX treatment) were continuously injected into 4T1 tumor-bearing mice for 22 days. As shown in Figure S6a, S6b, and S6c, no significant differences were found in tumor volume growth rate, size, and weight between these two groups, and the Au-PEG treatment group did not exhibit significant tumor injury compared to the PBS group. These findings indicated that the vector Au-PEG did not interfere with the tumor suppression study.\u003c/p\u003e\u003cp\u003e \u003cb\u003eFigure 5.\u003c/b\u003e (a) Schematic diagram of lysosome escape. (b) Cellular uptake of DOX and Au-DOX within 2 hours. (c) Fluorescence intensity of DOX and Au-DOX in nucleus and lysosomes. (d) Fluorescence images of Hela cells for intracellular tracking of Au-DOX. scale bar = 5 µm. (e) The cell viability of Au-DOX and DOX towards Hela cells for 24 h. Data points represent mean ± SD (n = 3).\u003c/p\u003e\u003ch2\u003eLysosomal barrier to the drug delivery system\u003c/h2\u003e\u003cp\u003eTo unveil the origin of the reduced therapeutic efficiency of Au-DOX, intracellular tracking and drug release studies were conducted. After 2h incubation, the small molecule DOX diffused rapidly within the cell, primarily accumulated in the nucleus due to the concentration gradient driving force (Fig.\u0026nbsp;5b and Figure S7). While for the cells incubated with Au-DOX, a significant fluorescence overlap was observed between the red fluorescence of DOX and the green fluorescence of LysoTracker, indicating the endocytosis and lysosomal localization of the NPs. The fluorescence intensity of Au-DOX and DOX in the lysosome and nucleus were further analysed. As demonstrated in Fig.\u0026nbsp;5c, the fluorescence intensity of DOX group in the nucleus was 1.6 times higher than that in the lysosome, whereas the fluorescence intensity of Au-DOX group in the lysosome was 3.4 times higher than that in the nucleus. To study whether Au-DOX can evade lysosomal entrapment, the incubation period was extended to 48 hours (Fig.\u0026nbsp;5d, figure S7) with Hela cells and 4T1 cell, respectively. The result showed that Au-DOX was still remained in the lysosomes. Since Au-DOX being trapped in the lysosomes, the cytotoxicity of Au-DOX within 24 hours was slightly lower compared to an equivalent amount of free DOX (Fig.\u0026nbsp;5e) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. These results indicated that although renal-clearable gold nanoparticles can effectively release drugs in the presence of acid and GSH, it was worth noting that AuNPs without DOX loading exhibited minimal toxicity to Hela cancer cells (Figure S8), but they face challenges in evading lysosomal entrapment in vivo, as illustrated in diagram 5a, thereby limiting their therapeutic efficacy [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eReduced toxicity of the drug delivery system\u003c/h2\u003e\u003cp\u003eOne advantage of Renal clearable DDSs was reduced toxicity [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. After six injections over a 15 days period (administered every three days) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), the mice in the DOX group exhibited a weight reduction of 10%, whereas no significant weight loss was observed in the Au-DOX group, no matter whether the drug was administered every two or three days. This observation highlights the biocompatibility and low toxicity of Au-DOX (Figure S6d). Further study showed that Au-DOX exhibited improved renal clearance efficiency compared to DOX (Figure S9a, b, c, d). As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, the renal clearance rate of Au-DOX within 24 hours was 47%, whereas that of DOX was only 11%. The utilization of renal clearable AuNPs considerably enhanced the renal clearance efficiency while concurrently reducing kidney injury [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Au-DOX treatment blood chemistry tests showed that the blood urea nitrogen (BUN) and creatinine (CREA) levels were all within the normal range (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and Figure S9e), indicating the favourable biosafety profile of the Au-DOX. H\u0026amp;E images revealed that the free DOX group induced severe kidney damage, whereas no obvious damage was observed in the Au-DOX group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). It was worth noting that the primary elimination pathway for free DOX was the liver, which usually leads to liver injury in approximately 40% of patients. Fluorescence imaging of organs demonstrated a higher accumulation of free DOX in the liver compared to Au-DOX. Further examination via H\u0026amp;E staining revealed that the DOX group caused substantial liver injury, as well as damage to other reticuloendothelial system organs such as the spleen and lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, Figure S3). Conversely, no evident damage was observed in the Au-DOX group.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, pH/GSH dual responsive renal-clearable Au-DOX nanoparticles were developed to study the tumor targeting efficiency and intracellular confinement of renal-clearable drug delivery vectors. The delivery vectors successfully enhanced the accumulation of DOX at the tumor area, however, the antitumor efficacy of Au-DOX was lower than that of free DOX. Further cellular study indicated that the intracellular confinement hindered Au-DOX from reaching the nucleus and thus limiting their therapeutic efficiency. On the other hand, the renal clearance property of Au-DOX enhanced the renal clearance of DOX and significantly reduced their toxicity. Overall, our work confirmed the significancy of renal-clearable drug delivery vectors in reducing drug toxicity and emphasized the role of the lysosomal barrier in mediating the therapeutic efficacy of renal-clearable DDSs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the students of Dr. Aihua Gong\u0026rsquo;s group for their excellent technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.L. and S.Q. contributed equally to this work. L.L. collected, analyzed and interpreted the data. S.Q. collected and analyzed the data. \u0026nbsp;Y.M. prepared samples and collected the data. M.S. prepared samples. \u0026nbsp;H.H did the animal study. Y. W. supervised the research. A.G. supervised the research and revised the manuscript. S.T.: conceived and designed the experiments. X.C. supervised the research and revised the manuscript. S.S. supervised the research, designed the experiments and revised the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (21807050,\u0026nbsp;22308134), the Natural Science Foundation of Jiangsu Province (BK20220644, BK20210876), Research and Practice Innovation Plan of Postgraduate Training Innovation Project in Jiangsu Province (KYCX22_3752), the start-up fund from Jiangsu University of Science and Technology, the postdoc fund from\u0026nbsp;Jiangsu Cancer Hospital\u0026amp; Jiangsu Institute of Cancer Research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experimental protocols were approved by the ethics committee of Jiangsu University of Science and Technology, and followed the Guidelines for Care and Use of Laboratory Animals from National Institutes of Health.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e School of Environmental and Chemical Engineering, Jiangsu University of Science and Technology, Zhenjiang, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e School of Medicine, Jiangsu University, Zhenjiang, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e Department of Medical Oncology, Jiangsu Cancer Hospital\u0026amp; Jiangsu Institute of Cancer Research\u0026amp; The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLu J, Gao X, Wang S, He Y, Ma X, Zhang T, Liu X. Advanced strategies to evade the mononuclear phagocyte system clearance of nanomaterials. Exploration. 2023;3:20220045. \u003c/li\u003e\n\u003cli\u003eZhou Q, Dong C, Fan W, Jiang H, Xiang J, Qiu N, Piao Y, Xie T, Luo Y, Li Z, Liu F, Shen Y. Tumor extravasation and infiltration as barriers of nanomedicine for high efficacy: The current status and transcytosis strategy. Biomaterials. 2020;240:119902. \u003c/li\u003e\n\u003cli\u003eBlanco E, Shen H, Ferrari M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 2015;33: 941-951. \u003c/li\u003e\n\u003cli\u003ePoon W, Kingston BR, Ouyang B, Ngo W, Chan WCW. A framework for designing delivery systems. Nat. Nanotechnol. 2020;15:819-829. \u003c/li\u003e\n\u003cli\u003eThorn CF, Oshiro C, Marsh S, Hernandez-Boussard T, McLeod H, Klein TE, Altman RB. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genom. 2011;21:440-446. \u003c/li\u003e\n\u003cli\u003eRao C, Sharma S, Garg R, Anjum F, Kaushik K, Nandi CK. Mapping the time dependent DNA fragmentation caused by doxorubicin loaded on PEGylated carbogenic nanodots using fluorescence lifetime imaging and superresolution microscopy. Biomaters Science. 2022;10:4525-4537. \u003c/li\u003e\n\u003cli\u003eYang F, Teves SS, Kemp CJ, Henikoff S. Doxorubicin, DNA torsion, and chromatin dynamics. Biochim et Biophys Acta. 2014;1845:84-89. \u003c/li\u003e\n\u003cli\u003eMakvandi P, Chen M, Sartorius R, Zarrabi A, Ashrafizadeh M, Dabbagh Moghaddam F, Ma J, Mattoli V, Tay F.R. Endocytosis of abiotic nanomaterials and nanobiovectors: Inhibition of membrane trafficking. Nano Today. 2021;40:101279. \u003c/li\u003e\n\u003cli\u003eZhou X, Liu X, Huang L. Macrophage-Mediated Tumor Cell Phagocytosis: Opportunity for Nanomedicine Intervention. Adv. Funct. Mater. 2021;31: 2006220. \u003c/li\u003e\n\u003cli\u003eJayashankar V, Edinger AL. Macropinocytosis confers resistance to therapies targeting cancer anabolism. Nat. Commun. 2020;11:1121. \u003c/li\u003e\n\u003cli\u003eFrancia V, Montizaan D, Salvati A. Interactions at the cell membrane and pathways of internalization of nano-sized materials for nanomedicine. Beilstein J. Nanotechnol. 2020;11:338-353. \u003c/li\u003e\n\u003cli\u003eJiang L, Liang X, Liu G, Zhou Y, Ye X, Chen X, Miao Q, Gao L, Zhang X, Mei L. The mechanism of lauric acid-modified protein nanocapsules escape from intercellular trafficking vesicles and its implication for drug delivery. Drug Deliv. 2018;25:985-994. \u003c/li\u003e\n\u003cli\u003eGong L, Wang Y, Liu J. Bioapplications of renal-clearable luminescent metal nanoparticles. Biomater. Sci. 2017;5:1393-1406. \u003c/li\u003e\n\u003cli\u003eSoo Choi H, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, Bawendi MG, Frangioni JV. Renal clearance of quantum dots. Nat. Biotechnol. 2007;25:1165-1170. \u003c/li\u003e\n\u003cli\u003eHe B, Sui X, Yu B, Wang S, Shen Y, Cong H. Recent advances in drug delivery systems for enhancing drug penetration into tumors. Drug Deliv. 2020;27:1474-1490. \u003c/li\u003e\n\u003cli\u003eYu M, Zheng J. Clearance Pathways and Tumor Targeting of Imaging Nanoparticles. ACS Nano. 2015;9:6655-6674. \u003c/li\u003e\n\u003cli\u003ePeng C, Huang Y, Zheng J. Renal clearable nanocarriers: Overcoming the physiological barriers for precise drug delivery and clearance. J. Controlled Release. 2020;322:64-80. \u003c/li\u003e\n\u003cli\u003eKang H, Gravier J, Bao K, Wada H, Lee JH, Baek Y, Fakhri GE, Gioux S, Rubin BP, Coll JL, Choi HS. Renal Clearable Organic Nanocarriers for Bioimaging and Drug Delivery. Adv. Mater. 2016;28:8162-8168. \u003c/li\u003e\n\u003cli\u003eKang H, Stiles WR, Baek Y, Nomura S, Bao K, Hu S, Park GK, Jo MJ, Hoseok I, Coll JL, Rubin BP, Choi HS. Renal Clearable Theranostic Nanoplatforms for Gastrointestinal Stromal Tumors. Adv. Mater. 2020;32:1905899. \u003c/li\u003e\n\u003cli\u003eAragon-Sanabria V, Aditya A, Zhang L, Chen F, Yoo B, Cao T, Madajewski B, Lee R, Turker MZ, Ma K, Monette S, Chen P, Wu J, Ruan S, Overholtzer M, Zanzonico P, Rudin CM, Brennan C, Wiesner U, Bradbury MS. Ultrasmall Nanoparticle Delivery of Doxorubicin Improves Therapeutic Index for High-Grade Glioma. Clin. Cancer Res. 2022;28:2938-2952. \u003c/li\u003e\n\u003cli\u003eMadajewski B, Chen F, Yoo B, Turker MZ, Ma K, Zhang L, Chen PM, Juthani R, Aragon-Sanabria V, Gonen M, Rudin CM, Wiesner U, Bradbury MS, Brennan C. Molecular Engineering of Ultrasmall Silica Nanoparticle\u0026ndash;Drug Conjugates as Lung Cancer Therapeutics. Clin. Cancer Res. 2020;26:5424-5437. \u003c/li\u003e\n\u003cli\u003eZhang L, Aragon-Sanabria V, Aditya A, Marelli M, Cao T, Chen F, Yoo B, Ma K, Zhuang L, Cailleau T, Masterson L, Turker MZ, Lee R, DeLeon G, Monette S, Colombo R, Christie RJ, Zanzonico P, Wiesner U, Subramony JA, Bradbury MS. Engineered Ultrasmall Nanoparticle Drug-Immune Conjugates with \u0026ldquo;Hit and Run\u0026rdquo; Tumor Delivery to Eradicate Gastric Cancer. Adv. Therap. 2023;6:2200209. \u003c/li\u003e\n\u003cli\u003ePeng C, Xu J, Yu M, Ning X, Huang Y, Du B, Hernandez E, Kapur P, Hsieh JT, Zheng J. Tuning the in vivo transport of anticancer drugs using renal-clearable gold nanoparticles. Angew. Chem. Int. Ed. 2019;58:8479-8483. \u003c/li\u003e\n\u003cli\u003ePeng C, Yu M, Hsieh JT, Kapur P, Zheng J. Correlating Anticancer Drug Delivery Efficiency with Vascular Permeability of Renal Clearable Versus Non-renal Clearable Nanocarriers. Angew. Chem. Int. Ed. 2019;131:12204-12208. \u003c/li\u003e\n\u003cli\u003eLiu J, Yu M, Ning X, Zhou C, Yang S, Zheng J. PEGylation and Zwitterionization: Pros and Cons in the Renal Clearance and Tumor Targeting of Near-IR-Emitting Gold Nanoparticles. Angew. Chem. Int. Ed. 2013;52:12572-12576. \u003c/li\u003e\n\u003cli\u003eLelle M, Hameed A, Ackermann LM, Kaloyanova S, Wagner M, Berisha F, Nikolaev VO, Peneva K. Functional Non‐Nucleoside Adenylyl Cyclase Inhibitors. Chem. Biol. Drug Des. 2014;85:633-637.\u003c/li\u003e\n\u003cli\u003eZhang Y, Ang CY, Li M, Tan SY, Qu Q, Zhao Y. Polymeric Prodrug Grafted Hollow Mesoporous Silica Nanoparticles Encapsulating Near-Infrared Absorbing Dye for Potent Combined Photothermal-Chemotherapy. ACS Appl. Mater. Interfaces. 2016;8:6869-6879. \u003c/li\u003e\n\u003cli\u003eYang X, Zhang X, Liu Z, Ma Y, Huang Y, Chen Y. High-Efficiency Loading and Controlled Release of Doxorubicin Hydrochloride on Graphene Oxide. J. Phys. Chem. C. 2008;112:17554-17558. \u003c/li\u003e\n\u003cli\u003eChen B, Mei L, Fan R, Wang Y, Nie C, Tong A, Guo G. Facile construction of targeted pH-responsive DNA-conjugated gold nanoparticles for synergistic photothermal-chemotherapy. Chin. Chem. Lett. 2021;32: 1775-1779. \u003c/li\u003e\n\u003cli\u003eXing J, Gong Q, Zou R, Yao J, Xiang L, Wu A. GSH responsive traditional clinical drugs probe for cancer cell fluorescence imaging and therapy. Chin. Chem. Lett. 2023;34:107786. \u003c/li\u003e\n\u003cli\u003eZhao H, Ruan H, Li H. Progress in the research of GSH in cells. Chin. Sci. Bull. 2011;56:3057-3063. \u003c/li\u003e\n\u003cli\u003eWang Z, Luo M, Mao C, Wei Q, Zhao T, Li Y, Huang G, Gao J. A Redox-Activatable Fluorescent Sensor for the High-Throughput Quantification of Cytosolic Delivery of Macromolecules. Angew. Chem. Int. Ed. Engl. 2017;56:1319-1323. \u003c/li\u003e\n\u003cli\u003eMu J, Zhong H, Zou H, Liu T, Yu N, Zhang X, Xu Z, Chen Z, Guo S. Acid-sensitive PEGylated paclitaxel prodrug nanoparticles for cancer therapy: Effect of PEG length on antitumor efficacy. J. Controlled Release. 2020;326:265-275. \u003c/li\u003e\n\u003cli\u003eAwotunde O, Okyem S, Chikoti R, Driskell JD. Role of Free Thiol on Protein Adsorption to Gold Nanoparticles. Langmuir. 2020;36:9241-9249. \u003c/li\u003e\n\u003cli\u003eMeng J, Hu Z, He M, Wang J, Chen X. Gold nanocluster surface ligand exchange: An oxidative stress amplifier for combating multidrug resistance bacterial infection. J. Colloid Interface Sci. 2021;602:846-858. \u003c/li\u003e\n\u003cli\u003eDing D, Yang C, Lv C, Li J, Tan W. Improving Tumor Accumulation of Aptamers by Prolonged Blood Circulation. Anal. Chem. 2020;92:4108-4114. \u003c/li\u003e\n\u003cli\u003eSingh B, Mitragotri S. Harnessing cells to deliver nanoparticle drugs to treat cancer. Biotechnol Advances. 2020;42:107339. \u003c/li\u003e\n\u003cli\u003eTian X, Shi A, Yin H, Wang Y, Liu Q, Chen W, Wu J. Nanomaterials Respond to Lysosomal Function for Tumor Treatment. Cells. 2022;11:3348. \u003c/li\u003e\n\u003cli\u003eZhai X, Hiani YE. Getting Lost in the Cell-Lysosomal Entrapment of Chemotherapeutics. Cancers. 2020;12:12123669. \u003c/li\u003e\n\u003cli\u003eZhang XD, Wu D, Shen X, Liu PX, Fan FY, Fan SJ. In vivo renal clearance, biodistribution, toxicity of gold nanoclusters. Biomaterials. 2012;33:4628-4638. \u003c/li\u003e\n\u003cli\u003ePeng C, Gao X, Xu J, Du B, Ning X, Tang S, Bachoo RM, Yu M, Ge WP, Zheng J. Targeting orthotopic gliomas with renal-clearable luminescent gold nanoparticles. Nano Res. 2017;10:1366-1376.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Renal-clearable, delivery vector, intracellular barrier, antitumor efficacy","lastPublishedDoi":"10.21203/rs.3.rs-3940105/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3940105/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRenal-clearable drug delivery systems (DDSs) offer significant advantages compared to conventional non-renal-clearable DDSs due to their reduced toxicity and enhanced therapeutic efficacy. However, despite the development of renal-clearable DDSs in the past decade, deeper understanding of how the biological barriers, especially the intracellular barriers affect their therapeutic efficiency remain poorly explored. Herein, the antitumor efficiency and the intracellular behavior of renal-clearable Au-DOX which use renal-clearable gold nanoparticles (AuNPs) as delivery vectors for doxorubicin (DOX) were systematically investigated. The results revealed that although the toxicity of Au-DOX was significantly lower than that of free DOX due to efficient elimination of off-target DOX through renal clearance, the altered cellular uptake pathway compromised the antitumor efficacy of Au-DOX. Most Au-DOX was endocytosed and sequestered within lysosomes, preventing it from diffusing into nucleus to elicit therapeutic effect. Our results indicate that the lysosomal barrier induced ineffective intracellular delivery would counteract the therapeutic efficacy of renal-clearable DDSs and highlight the role of overcoming intracellular barriers when designing DDSs.\u003c/p\u003e","manuscriptTitle":"Pros and Cons in The Delivery of Doxorubicin Using Renal-clearable Gold Nanoparticles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-20 16:56:05","doi":"10.21203/rs.3.rs-3940105/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aaf20061-d950-4592-8ee7-4ba642897862","owner":[],"postedDate":"February 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-11T13:08:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-20 16:56:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3940105","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3940105","identity":"rs-3940105","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}