A Redox-Responsive Dihydroartemisinin Dimeric Nanoprodrug For Enhanced Antitumor Activity

A Redox-Responsive Dihydroartemisinin Dimeric Nanoprodrug For Enhanced Antitumor Activity | 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 A Redox-Responsive Dihydroartemisinin Dimeric Nanoprodrug For Enhanced Antitumor Activity Yawei Li, Qing Pei, Baiji Cui, Hongmei Zhang, Liu Han, Wenqing Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-981484/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Dec, 2021 Read the published version in Journal of Nanobiotechnology → Version 1 posted 15 You are reading this latest preprint version Abstract Redox-responsive drug delivery system emerges as a hopeful platform for tumor treatment. Dihydroartemisinin (DHA) has been investigated as an innovative tumor therapeutic agent. Herein, a DHA dimeric prodrug bridged with disulfide bond as linker (DHA 2 -SS) has been designed and synthesized. The prepared prodrugs could self-assemble into nanoparticles (SS NPs) with high DHA content (>90%) and robust stability. These SS NPs display sensitive redox responsive capability and can release DHA under the tumor heterogeneity microenvironment. SS NPs possess preferable antitumor therapeutic activity in contrast with free DHA. Moreover, the possible anti-cancer mechanism of these nanoparticles was investigated through RNA-seq analysis, bioinformatics and molecular biological method. SS NPs could induce apoptosis via mitochondrial apoptosis pathway, as well as glycolysis inhibition associate with the regulation of PI3K/AKT/HIF-1α signal path, which may offer an underlying therapeutic target for liver cancer. Our study highlights the potential of using redox responsive prodrug nanoparticles to treat cancer, meanwhile provides insights into the anti-cancer mechanism of DHA prodrug. Nanoscience Dimeric nanoprodrug dihydroartemisinin redox-responsive antitumor activity PI3K/AKT/HIF-1α signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Currently, chemotherapy remains the predominant therapy in cancer treatment due to its versatility and high efficiency [ 1 – 4 ]. For the past few years, traditional herbal medicines have generated many chemotherapy drugs which could inhibit a variety of tumor entities [ 5 ]. For instance, dihydroartemisinin (DHA), one derivative of artemisinin, has been proved to possess a potent and broad anti-tumor effect in addition to anti-malarial [ 6 – 9 ]. However, some existing problems, for example strong hydrophobicity, nonspecific distribution, and rapid elimination from the body, impede the application of DHA in cancer treatment [ 10 – 12 ]. To overcome these limitations, nano drug-delivery systems have been employed to increase the solubility, prolong systemic circulation, and promote passive tumor targeting owing to EPR effect [ 13 – 17 ]. However, low drug content and uncontrolled drug release of these nanoparticle formulations are still far from satisfactory [ 18 – 20 ]. Hence, developing effective nanoplatforms with the tumor microenvironment-responsive drug release and high drug content is highly desirable for various chemotherapeutic drugs. In recent years, growing evidences demonstrate that dimerization of drug molecules has emerged as a powerful tool for developing new prodrugs [ 16 , 21 – 25 ], and organic dimers are liable to self-assemble into NPs in aqueous solution [ 18 , 25 – 30 ]. In addition, the drug-delivery system should also possess the responsive and controllable drug release, which can realize specific selectivity towards tumor cells and the low side effects [ 31 – 36 ]. As we known, in contrast with normal cells, tumor cells usually exhibit higher levels of intracellular glutathione (GSH) and reactive oxygen species (ROS) [ 37 – 40 ]. A variety of redox-responsive chemical bonds have been developed to fabricate the tumor microenvironment-responsive nanomaterials [ 41 – 46 ]. Therefore, it is of great significance to design DHA nanoprodrug with redox-responsive linker, which can effectively resolve the existing problems of DHA and further improve its therapeutic effects. In our present work, a DHA dimer containing disulfide bond linker, which has been proven to possess dual-responsiveness [ 47 – 49 ], was designed and successfully synthesized, named as DHA 2 -SS. And this DHA dimer could self-assembly into nanoparticles (SS NPs) in aqueous media by nanoprecipitation method. The formed SS NPs possess nanoscale size, robust stability, and ultra-high drug content. The redox response, cellular uptake and antitumor efficacy of SS NPs have been studied, and the gene expression of tumor cells after SS NPs treatment was analyzed by RNA-seq analysis (Scheme 1 ). Results And Discussion Preparation and characterization of DHA dimeric NPs Firstly, the DHA 2 -SS was synthesized through the esterification reaction of DHA with dicarboxylic acid (Fig. S1) [ 23 ]. After purification by silica gel column chromatography, DHA 2 -SS was obtained in high yields (>90%) and the chemical construction has been characterized via proton nuclear magnetic resonance ( 1 H NMR) spectroscopy and a linear ion trap mass spectrometer (LTQ-MS). In 1 H NMR spectra, the characteristic peak of 10-hydroxyl group at 2.85 ppm corresponding to DHA disappeared, validating the success of esterification reaction and the reaction site was at the 10-hydroxyl group of DHA (Fig. S2). The peak value corresponding to DHA 2 -SS at around 737 in mass spectrometry was consistent with the theoretical calculated value (Fig. S3), further confirming the structure of DHA dimer. In order to compare the redox responsiveness of DHA 2 -SS, we also synthesized another DHA dimer with the same length of carbon chain (DHA 2 -C6) as control (Fig. S4). It is reported that the organic dimers could self-assemble into NPs in aqueous solution [ 25 – 30 ]. As anticipated, both kinds of resulting dimers formed spherical nanoparticles (abbreviated as SS NPs and C6 NPs, respectively) through nanoprecipitation method as observed via transmission electron microscopy (TEM) (Fig. 1 A and 1 B). And SS NPs had an average hydrodynamic diameter of approximately 167.2 nm as determined by dynamic light scattering (DLS), which was similar to those of C6 NPs (181.4 nm) (Fig. 1 C). These two kinds of NPs were found to be negative, and the zeta potential values were around -20 mV (Fig. 1 D). The drug content of SS and C6 NPs was 90.6% and 91.7%, respectively. In addition, SS and C6 NPs both had robust stability with negligible changes in size and size distribution in one week (Fig. 1 E). And these NPs also kept stable in PBS (pH 7.4) containing 10% FBS after 24 h (Fig. 1 F), and a slight size increase in the first two hours was largely ascribed to the protein absorption on the surface of NPs. DTT and GSH triggered release of DHA As mentioned above, disulfide bond possess distinct redox response capability [ 47 – 49 ]. Therefore, we investigated the responsiveness of DHA 2 -C6 and DHA 2 -SS by using dithiothreitol (DTT) and H 2 O 2 as reducing and oxidizing agents, respectively. As shown in Fig. 2 A, after incubation with 10 mM DTT, the HPLC peak of DHA 2 -SS at 10.6 min declined significantly and entirely disappeared in 24 h, while the new peak for DHA at 6.4 min emerged and enhanced gradually, indicating the dissociation of disulfide linkage and the release of DHA. In comparison, drug release from DHA 2 -C6 dimer was quite slow under the same condition, and only approximately 10% of DHA released even after 48 h treatment (Fig. 2 B). In addition, we further investigated the oxidation responsiveness of these dimers. Similar to reduction responsiveness, DHA 2 -SS also exhibited sensitive oxidation responsiveness. As presented in Fig. 2 D, DHA 2 -SS could release about 60% DHA after 48 h of H 2 O 2 treatment (10 mM), while negligible degradation was detected for DHA 2 -C6 at the same time (Fig. 2 E). The release curves of DHA over time in the presence of DTT and H 2 O 2 are revealed in Fig. 2 C and 2 F, respectively. These results validate the redox responsiveness of disulfide bond linker and the controlled release of DHA. Cellular uptake and in vitro cytotoxicity of DHA dimeric NPs Human hepatoma HepG2 cells were used to study the cellular internalization of these NPs via confocal laser scanning microscopy (CLSM). The fluorescent dye nile red (NR) was utilized as a marker and encapsulated into the NPs by coassembly with DHA dimer, and the blue fluorescence of Hoechst 33258 was employed to localize the cell nucleus. As exhibited in Fig. 3 A, the red fluorescence principally distributed in the cytoplasm of tumor cells in a time-dependent manner (Fig. S5), implying the efficient and sustained cellular uptake. Furthermore, the effect of temperature on cellular uptake has also been carried out. It can be observed that a strong fluorescence signal at 37°C and significantly reduced cellular uptake at 4°C from Fig. S6, suggesting an energy-dependent endocytosis. Next, the cytotoxicity of DHA dimeric NPs was evaluated against human HepG2 and HeLa cells through a standard MTT assay. As shown in Fig. 3 B, both free DHA and SS NPs showed efficient suppression of HepG2 viability in concentration dependent manner for 48 h, and SS NPs exhibited higher cytotoxicity than free DHA. The cell viability of SS NPs was less than 30%, while it was approximately 40% for free DHA at the equivalent DHA concentration. This result may be attributed to the enhanced cellular uptake of NPs into tumor cells and the rapid release of DHA. And SS NPs could quickly release active DHA once in living cells because of the responsiveness of disulfide bond in redox environment. However, C6 NPs exhibited apparent lower cytotoxicity compared with SS NPs (Fig. S7). Similarly, SS NPs still possessed the strongest cellular toxicity towards HeLa cells (Fig. S8). In addition, we evaluated the cytotoxicity of SS NPs toward normal hepatocytes (HL-7702), and also compared its toxicity on HepG2, HeLa and HL-7702 cells. As revealed in Fig. 3 C, SS NPs exhibited no significant cellular toxicity against HL-7702 cells, and enhanced cytotoxicity against these two kinds of tumor cells (Fig. S9), indicating the selectivity of DHA 2 -SS prodrug towards tumor cells. This result is ascribed to the different redox conditions in normal cells and tumor cells. To further understand the contribution of SS NPs on apoptosis, we stained HepG2 cells with Annexin V-FITC and PI, and analyzed them by flow cytometry. As presented in Fig. 3 D, the ratio of early apoptotic cells was 9.40%, 15.00%, and 24.56%, respectively, as the drug concentration increases (20, 40 and 60 µM), validating the cell apoptosis in a concentration-dependent manner. Additionally, we also examined the nuclear morphological changes by CLSM. The cell nucleus emerged as a homogeneous blue chromatin with an organized structure in normal cells, whereas the cells incubated with SS NPs displayed representative morphological changes (Fig. S10), including intense fluorescent spots, nuclear pyknosis, and extensive blebbing, further verifying the apoptosis of tumor cells. Antitumor mechanism of SS NPs To elucidate the mechanism of SS NPs in inhibiting tumor cell proliferation and inducing apoptosis, RNA sequencing (RNA-seq) technology was applied to collect the gene expression [ 50 ]. The total RNA extracted from SS NPs treatment group (SS) and the control group (C) have been analyzed for quality and integrity by utilizing formaldehyde agarose gel electrophoresis. The results verified that the obtained RNA was intact, undegraded, and suitable for RNA-seq analysis. Meanwhile, principal component analysis (PCA) of samples was performed on the complete dataset [ 51 ], which can display changes of overall gene expression. As shown in the PCA results (Fig. S11), there were two clusters, verifying a distinct directionality between SS NPs treatment and the control groups based on the similarity of gene expression. Subsequently, the differentially expressed genes (DEGs) induced by SS NPs have been identified and described by volcano plots and heatmaps. After comparing with the untreated control group (log 2 fold-change ≥ 2.0 and adjusted P value < 0.05), we distinguished 6546 DEGs, including 3288 up-regulated and 3258 down-regulated expression genes (Fig. 4 A and 4 B). The above results imply that SS NPs play an important role on gene expression of the treated tumor cells. According to the RNA-seq data, we performed the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for 6546 differentially expressed genes. Three domains were involved: cellular components (CC), biological processes (BP) and molecular function (MF). In the BP domain, a large proportion of genes were associated with cellular process, metabolic process, biological regulation (Fig. 5 A). For the CC domain, the enriched genes covered cell, cell part, organelle, membrane and so forth (Fig. 5 B). Binding, catalytic activity, transcription regulator activity and molecular function regulator were primarily influenced for the MF domain (Fig. 5 C). On the basis of the DEGs results, we also executed KEGG functional enrichment analysis and pathway classification. The KEGG functional enrichment analysis result (Fig. 5 D) demonstrated that multiple pathways have been affected by DHA 2 -SS NPs, and the signaling pathways of carbon metabolism, biosynthesis of amino acids, fatty acid metabolism, HIF-1 signaling pathway and pathways in cancer were among the top 20 pathways. On the other hand, KEGG pathway classification results (Fig. S12) displayed that there were six branches for KEGG pathways, including cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems. And the DEGs in HepG2 cells after SS NPs treatment were mainly enriched in PI3K-Akt signaling pathway, MAPK signaling pathway and pathways in cancer. From GO and pathway analysis results, we discovered that the metabolic process was involved in the inhibition of tumor cells proliferation, thus we carried out network statistical analysis on the protein-protein interaction (PPI) of these genes correlated with metabolism. As shown in Fig. 6 , the metabolism of carbohydrates, amino acids and lipids have been remarkably regulated in the cells after SS NPs treatment, indicating that SS NPs could induce metabolic reprogramming in tumor cells. Therefore, we concentrated on the metabolism reprogramming of tumor cells after treated with SS NPs. Apoptosis is a highly regulated process of cell death, which is important to maintain the inherent stability of multicellular organisms, and involves a variety of signal pathways [ 52 – 54 ]. The Bcl-2 protein family contains pro-apoptotic and anti-apoptotic regulators of programmed cell death/apoptosis, and plays a dominant role in regulating cell apoptosis. In this family, Bax gene, a pro-apoptotic member, can form heterodimers with Bcl-2 protein, and the ratio of Bax/Bcl-2 could determine the sensitivity of cells to apoptosis. The activation of Bax can release cytochrome c (Cyt C), and Cyt C activates caspase-9 and downstream caspase-3 through a cascade reaction to promote cell apoptosis, whereas the anti-apoptotic Bcl-2 operates in the opposite way. Therefore, we detected expression of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9 and Cyt C by western blotting. As displayed in Fig. 7 , the expression levels of Bax, caspase-3, caspase-9, and Cyt C were obviously increased, and the expression levels of Bcl-2 was reduced compared to the control group. The above results demonstrate that mitochondrial apoptosis pathway is involved in the apoptosis of HepG2 cells induced by SS NPs. As we known, tumor cells generally reveal aberrant metabolism due to metabolic reprogramming [ 55 ]. As a hallmark of tumor cells, the Warburg effect means that tumor cells rely heavily on glycolysis for energy, rather than oxygen [ 56 – 58 ]. To investigate whether SS NPs treatment could suppress the glycolysis of tumor cells, the glucose uptake, contents of lactic acid and ATP products of HepG2 cells treated with SS NPs were detected. As shown in Fig. 8 , the glucose uptake was decreased, and the contents of intracellular ATP and extracellular lactic acid were also declined, indicating that SS NPs may inhibit glycolysis of tumor cells. Cancer metabolic reprogramming is regulated by multiple pathways, which includes the PI3K-AKT signaling pathway [ 59 ]. The activated PI3K-AKT can promote the transition to aerobic glycolysis, and AKT could result in the phosphorylation of some important downstream targets, such as Bcl-2 apoptosis-related family, and mammalian target of rapamycin (mTOR), to protect cells from apoptosis. Meanwhile, this pathway also could regulate HIF-1α through mTOR, and activated HIF-1α is related to the up-regulation of glucose transporters (Gluts) and glycolytic enzymes. To understand whether the activation of PI3K/AKT and HIF-1α were involved in metabolic reprogramming of tumor cells treated by SS NPs, we detected the related protein expression. As displayed in Fig. 9 , SS NPs treatment could decrease the expression of glycolytic enzymes, such as PFKP, HK2, LDH, Glut1. The ratio of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR and HIF-1α were also reduced accordingly. Collectively, these findings confirm that SS NPs could induce apoptosis and suppress glycolysis by regulating the PI3K/AKT/HIF-1α signaling pathway. In vitro antitumor efficacy of DHA dimeric NPs We further evaluated the anti-cancer activity of SS NPs on H22 tumor-bearing Kunming mice. Mice bearing the tumors were randomly divided into 4 groups with different treatments: PBS, free DHA, C6 NPs, and SS NPs, and injected intravenously at equivalent DHA doses every second day. As illustrated in Fig. 10 A, DHA treatment exhibited a moderate inhibitory effect on tumor growth compared with the control group, which is mainly owing to its intrinsic toxicity. Notably, SS NPs exhibited evident antitumor activity, which is more potent than free DHA group. The improved therapeutic efficacy of SS NPs should be ascribed to the multiple advantages of nanoparticle formulations, including enhanced tumor accumulation, effective endocytosis, and rapid drug release in tumor sites. Unsurprisingly, C6 NPs group displayed the weakest tumor growth inhibition effect in these treatment groups on account of the insensitivity of C6 linker to redox microenvironment. What’s more, the tumor weight (Fig. 10 B) and the photographs of resected tumors (Fig. 10 C) visually demonstrated the greatest tumor inhibition efficacy obtained by SS NPs, further validating the enhanced antitumor effect of disulfide-bond bridged prodrug nanoparticles. In addition, all mice had no significant weight fluctuation during the whole treatment period (Fig. 10 D), and there was also no detectable histological damage observed after SS NPs treatment from the hematoxylin and eosin (H&E) stained tissue sections of major organs (heart, liver, spleen, lung, and kidney) (Fig. S13). The above results validate SS NPs at current doses possess favorable biosafety and ignorable systemic toxicity. The H&E staining of tumor slices revealed that SS NPs group exhibited the most severe cellular damage compared with the control group. The tumor cells after SS NPs treatment shrank largely and the tumor tissue significantly decreased. (Fig. 10 E). All of the results substantiate that DHA 2 -SS NPs could be safely used for the in vivo treatment, and possess better treatment effects in contrast with free DHA. Conclusions In conclusion, a DHA dimeric nanoprodrug using disulfide bond as linkage (DHA 2 -SS) was obtained. The SS NPs not only possess favorable stability and high drug content of 90.6 wt%, but also can respond to the tumor redox microenvironment, thus resulting in the effective release of DHA for chemotherapy. Importantly, both in vitro and in vivo therapy experiments indicate these obtained SS NPs have efficient endocytosis, potent cytotoxicity and enhanced antitumor efficacy in contrast with free DHA. RNA sequencing and bioinformatics analysis demonstrate that SS NPs could induce apoptosis via the intrinsic mitochondrial apoptosis pathway, as well as inhibit glycolysis through PI3K/AKT/HIF-1α signaling pathway. Our finding provides a reference for the rational design of responsive prodrug nanoparticles, and could potentially spur the similar study of Chinese medicine and related natural active ingredients. Declarations Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s129 51 -021-#####-#. Authors’ contributions The manuscript was written through contributions of all authors. All authors read and approved the fnal manuscript Funding This work was supported by the Science and Technology Development Project of Jilin Province (No.YDZJ202101ZYTS094), Scientific Research Project of Education Department of Jilin Province (No.JJKH20210493KJ), and Traditional Chinese Medicine Science and Technology Project of Jilin Province (No.2021090). Availability of data and materials All data generated or analysed during this study are included in this article and its additional file. Ethics approval and consent to participate Not applicable. Consent for publication All authors gave their consent for publication. Competing interests The authors declare that they have no competing interests. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A. F. Bray. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA-Cancer J Clin. 2021;71(3):209–49. Bocci G, Kerbel RS. Pharmacokinetics of metronomic chemotherapy: a neglected but crucial aspect. Nat Rev Clin Oncol. 2016;13(11):659–73. Lee SM, O'Halloran TV, Nguyen ST. Polymer-Caged Nanobins for Synergistic Cisplatin -Doxorubicin Combination Chemotherapy. J Am Chem Soc. 2010;132(48):17130–8. Li HH, Zhang P, Luo JW, Hu DR, Huang Y, Zhang ZR, Fu Y. T. Gong. 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Supplementary Files GraphicalAbstract.docx SupportingInformation.docx Cite Share Download PDF Status: Published Journal Publication published 01 Dec, 2021 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Major revision 02 Nov, 2021 Review # 1 received at journal 01 Nov, 2021 Review # 2 received at journal 31 Oct, 2021 Review # 4 received at journal 28 Oct, 2021 Review # 3 received at journal 26 Oct, 2021 Reviewer # 4 agreed at journal 20 Oct, 2021 Reviewer # 3 agreed at journal 19 Oct, 2021 Reviews received at journal 19 Oct, 2021 Reviewers invited by journal 19 Oct, 2021 Editor invited by journal 18 Oct, 2021 Editor assigned by journal 18 Oct, 2021 Reviewer # 2 agreed at journal 18 Oct, 2021 Reviewer # 1 agreed at journal 18 Oct, 2021 Submission checks completed at journal 17 Oct, 2021 First submitted to journal 15 Oct, 2021 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-981484","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":57640449,"identity":"3893b7cb-4e3b-4d8d-b149-3cd5274e7103","order_by":0,"name":"Yawei Li","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yawei","middleName":"","lastName":"Li","suffix":""},{"id":57640450,"identity":"fb222c1e-8f2c-4a6a-a797-3f124c030adb","order_by":1,"name":"Qing Pei","email":"","orcid":"","institution":"Chang Chun Institute of Applied Chemistry: Chang Chun Institute of Applied Chemistry Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Pei","suffix":""},{"id":57640451,"identity":"c09c8b57-eb93-4bcc-8141-fa7b0ffde0a4","order_by":2,"name":"Baiji Cui","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Baiji","middleName":"","lastName":"Cui","suffix":""},{"id":57640452,"identity":"abd07a11-bbd8-4cbb-b504-8b09c1be17b5","order_by":3,"name":"Hongmei Zhang","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongmei","middleName":"","lastName":"Zhang","suffix":""},{"id":57640453,"identity":"3aa49c22-255e-413f-ab28-fbf51351dc68","order_by":4,"name":"Liu Han","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"Han","suffix":""},{"id":57640454,"identity":"e1d282d2-f6b1-440a-8172-506fa17db663","order_by":5,"name":"Wenqing Li","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenqing","middleName":"","lastName":"Li","suffix":""},{"id":57640455,"identity":"3dadabf7-5776-4025-92cc-f8b7463cec47","order_by":6,"name":"Wenhe Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYBACAwYGxgMJDAwJ/MzMhx8Qq4UBrEWynS3NgHgtQJxgcJ5HQYIoLeYSyQcOPPhzOM/4MA9Qf41NNEEtljPSEg4k8BwuNjvMe+ABw7G03AaCDruRY3AgQeJ24rbDfAkGjA2HidVicDtxczOPgQQJWhJuJ25gJlrLmWdAvxz4nzjjMDCQE4jyy/Hkgw9//ElL7O8/fPjBhxobwlpQQQJpykfBKBgFo2AU4AIAhylHKmngFTIAAAAASUVORK5CYII=","orcid":"","institution":"Jilin Medical Univesity","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wenhe","middleName":"","lastName":"Zhu","suffix":""},{"id":57640456,"identity":"38969e29-569e-417b-887b-09ae1015f1cf","order_by":7,"name":"Xianmin Feng","email":"","orcid":"","institution":"Jilin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xianmin","middleName":"","lastName":"Feng","suffix":""},{"id":57640457,"identity":"bdd46704-b317-4160-8702-e53b59ff37f4","order_by":8,"name":"Zhigang Xie","email":"","orcid":"","institution":"Changchun Institute of Applied Chemistry Chinese Academy of Sciences: Chang Chun Institute of Applied Chemistry Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhigang","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2021-10-16 09:23:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-981484/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-981484/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-021-01200-z","type":"published","date":"2021-12-01T12:55:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":14801159,"identity":"d51277a6-70e3-4bb8-b619-0b28f1b59f1d","added_by":"auto","created_at":"2021-10-22 14:45:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":222406,"visible":true,"origin":"","legend":"Basic characterization of DHA dimeric NPs. TEM images of (A) SS NPs and (B) C6 NPs. (C) Size distribution and (D) zeta potential of prepared SS and C6 NPs. Changes of hydrodynamic diameter and PDI of two kinds of NPs (E) in water and (F) in PBS with FBS (10%) over different times measured by DLS. Data are expressed as mean ± SD (n = 3).","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/835be6082cebf2cf1d6e22ff.png"},{"id":14801148,"identity":"702fa5ba-605b-4286-8319-2ff8a3662c99","added_by":"auto","created_at":"2021-10-22 14:45:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":143330,"visible":true,"origin":"","legend":"Reduction responsiveness of (A) DHA2-SS and (B) DHA2-C6 dimers degradation and (C) the rate of DHA released from DHA dimers in the presence of 10 mM DTT at 37 °C. Oxidation responsiveness of (D) DHA2-SS and (E) DHA2-C6 dimers degradation and (F) the rate of DHA released from DHA dimers in the presence of 10 mM H2O2 at 37 °C. ","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/59722cfc7c10c8f11e24f73c.png"},{"id":14801149,"identity":"5020db03-9d52-4f32-bd44-1424b740f066","added_by":"auto","created_at":"2021-10-22 14:45:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":491615,"visible":true,"origin":"","legend":"(A) CLSM images of HepG2 cells incubated with SS and C6 NPs at 37 °C for 4 h. In vitro cytotoxicity against (B) HepG2 cells and (C) HL-7702 cells of free DHA and SS NPs at different concentrations after incubation for 48 h. (D) Flow cytometry data for apoptosis in HepG2 cells treated with different concentrations of SS NPs. ","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/d91a3b3702e994b09699413c.png"},{"id":14801151,"identity":"f000b964-a207-41a0-8412-fcf7afb61504","added_by":"auto","created_at":"2021-10-22 14:45:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103079,"visible":true,"origin":"","legend":"(A) Volcano plots to determine DEGs of SS NPs treatment group and control group. The x-axis represents the log 2.0-fold changes (FCs) of genes and the y-axis represents the -log10 of the p-values for the various condition pairs. Each dot represents a gene. The gray points represent a non-statistically significant difference in gene expression. The red field represents the upregulated genes and the blue field represents the downregulated genes. (B) Heat map displaying the overview of the differentially expressed genes induced by SS NPs treatment.","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/9563568243f9b2d05bba95fa.png"},{"id":14801799,"identity":"55df4f33-29b1-482c-af66-c7f715916d19","added_by":"auto","created_at":"2021-10-22 14:48:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":872571,"visible":true,"origin":"","legend":"GO and KEGG pathway functional enrichment analysis of DEGs in SS NPs treated HepG2 cells. (A) Biochemical processes. (B) Cellular components. (C) Molecular function. (D) KEGG pathway functional enrichment of DEGs. ","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/4d9770c9f341a778ebd001c6.png"},{"id":14801801,"identity":"27565075-876a-4735-83f8-9aad1ee51471","added_by":"auto","created_at":"2021-10-22 14:48:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":357331,"visible":true,"origin":"","legend":"PPI network of the DEGs correlation with (A) carbohydrates, (B) amino acids and (C) lipids metabolism.","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/3c8954e9bcd1916f4328dad8.png"},{"id":14801153,"identity":"ff8e009b-9661-4312-8308-a7203052c329","added_by":"auto","created_at":"2021-10-22 14:45:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":119134,"visible":true,"origin":"","legend":"Effects of SS NPs on expression of mitochondrial apoptosis pathway relative proteins. (A) Protein expression was analyzed using western blotting and (B) quantified in relation to β-actin. Densitometric values were normalized by β-actin and expressed as mean ± SD, n = 3. Statistical significance: *p \u003c 0.05, and **p \u003c 0.01.","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/3fea10ceeb8a11035a02d601.png"},{"id":14801152,"identity":"22b163b7-391a-48ba-a9f6-be562a30bb87","added_by":"auto","created_at":"2021-10-22 14:45:42","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":49987,"visible":true,"origin":"","legend":"SS NPs suppress glycolysis level in HepG2 cells. (A) After HepG2 cells were treated with SS NPs, glucose content in the culture media was immediately tested, and glucose uptake was calculated. (B) Lactic acid product was detected. (C) ATP content was detected through bioluminescence assay. Data arepresented as mean ± SD, n = 3. Statistical significance: *p \u003c 0.05, and **p \u003c 0.01.","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/018126c92831b00ca0999e65.png"},{"id":14801158,"identity":"7e869321-f40d-4242-a658-0753ed7956c9","added_by":"auto","created_at":"2021-10-22 14:45:42","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":228139,"visible":true,"origin":"","legend":"Effects of SS NPs on expression of PI3K/AKT/HIF-1α signaling pathway relative proteins. (A) Protein expression of glycolytic enzymes were analyzed using western blotting and (B) quantified in relation to β-actin. (C) Protein expression of PI3K/AKT pathway were analyzed and (D) quantified in relation to β-actin. Densitometric values were normalized by β-actin and expressed as mean ± SD, n = 3. Statistical significance: *p \u003c 0.05, and **p \u003c 0.01. ","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/f3c44b25086f8b2aa2a92312.png"},{"id":14801154,"identity":"8f1b71b9-8218-43b7-b4c1-4e49465c2921","added_by":"auto","created_at":"2021-10-22 14:45:42","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":585124,"visible":true,"origin":"","legend":"In vivo antitumor efficacy evaluation. (A) Tumor growth curves and (B) tumor weights of H22 tumor-bearing mice after different treatments. (C) Photographs of excised tumors after the last treatment. (D) Body weight curves of tumor-bearing mice in each group during treatments. (E) H\u0026E-stained images of tumor slices collected from mice after different treatments. Scale bars, 100 μm. Data are expressed as mean ± SD (n = 3). Statistical significance: **p \u003c 0.01, and ***p \u003c 0.001.","description":"","filename":"fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/2aa9824a71ecbf5b171571b7.png"},{"id":16609548,"identity":"9c87857c-1387-4875-b0a8-45e69cf9ffdc","added_by":"auto","created_at":"2021-12-20 12:55:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2879594,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/9d370bf6-f028-45fa-8932-fe99a2285163.pdf"},{"id":14801157,"identity":"23c57fca-1760-43cb-88cf-de7ee7b0ef90","added_by":"auto","created_at":"2021-10-22 14:45:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":647744,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/20d9eb15b7a52102b23e3b37.docx"},{"id":14801800,"identity":"55f15328-607c-4a54-9a28-0a3ffa7491e0","added_by":"auto","created_at":"2021-10-22 14:48:42","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4578255,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-981484/v1/2b370ec42b8b6dd4a1a2fabf.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eA Redox-Responsive Dihydroartemisinin Dimeric Nanoprodrug For Enhanced Antitumor Activity\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCurrently, chemotherapy remains the predominant therapy in cancer treatment due to its versatility and high efficiency [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For the past few years, traditional herbal medicines have generated many chemotherapy drugs which could inhibit a variety of tumor entities [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For instance, dihydroartemisinin (DHA), one derivative of artemisinin, has been proved to possess a potent and broad anti-tumor effect in addition to anti-malarial [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, some existing problems, for example strong hydrophobicity, nonspecific distribution, and rapid elimination from the body, impede the application of DHA in cancer treatment [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To overcome these limitations, nano drug-delivery systems have been employed to increase the solubility, prolong systemic circulation, and promote passive tumor targeting owing to EPR effect [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, low drug content and uncontrolled drug release of these nanoparticle formulations are still far from satisfactory [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Hence, developing effective nanoplatforms with the tumor microenvironment-responsive drug release and high drug content is highly desirable for various chemotherapeutic drugs.\u003c/p\u003e \u003cp\u003eIn recent years, growing evidences demonstrate that dimerization of drug molecules has emerged as a powerful tool for developing new prodrugs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and organic dimers are liable to self-assemble into NPs in aqueous solution [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26 CR27 CR28 CR29\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, the drug-delivery system should also possess the responsive and controllable drug release, which can realize specific selectivity towards tumor cells and the low side effects [\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. As we known, in contrast with normal cells, tumor cells usually exhibit higher levels of intracellular glutathione (GSH) and reactive oxygen species (ROS) [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. A variety of redox-responsive chemical bonds have been developed to fabricate the tumor microenvironment-responsive nanomaterials [\u003cspan additionalcitationids=\"CR42 CR43 CR44 CR45\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Therefore, it is of great significance to design DHA nanoprodrug with redox-responsive linker, which can effectively resolve the existing problems of DHA and further improve its therapeutic effects.\u003c/p\u003e \u003cp\u003eIn our present work, a DHA dimer containing disulfide bond linker, which has been proven to possess dual-responsiveness [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], was designed and successfully synthesized, named as DHA\u003csub\u003e2\u003c/sub\u003e-SS. And this DHA dimer could self-assembly into nanoparticles (SS NPs) in aqueous media by nanoprecipitation method. The formed SS NPs possess nanoscale size, robust stability, and ultra-high drug content. The redox response, cellular uptake and antitumor efficacy of SS NPs have been studied, and the gene expression of tumor cells after SS NPs treatment was analyzed by RNA-seq analysis (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003ePreparation and characterization of DHA dimeric NPs\u003c/h2\u003e\n \u003cp\u003eFirstly, the DHA\u003csub\u003e2\u003c/sub\u003e-SS was synthesized through the esterification reaction of DHA with dicarboxylic acid (Fig. S1) [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. After purification by silica gel column chromatography, DHA\u003csub\u003e2\u003c/sub\u003e-SS was obtained in high yields (\u0026gt;90%) and the chemical construction has been characterized \u003cem\u003evia\u003c/em\u003e proton nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH NMR) spectroscopy and a linear ion trap mass spectrometer (LTQ-MS). In \u003csup\u003e1\u003c/sup\u003eH NMR spectra, the characteristic peak of 10-hydroxyl group at 2.85 ppm corresponding to DHA disappeared, validating the success of esterification reaction and the reaction site was at the 10-hydroxyl group of DHA (Fig. S2). The peak value corresponding to DHA\u003csub\u003e2\u003c/sub\u003e-SS at around 737 in mass spectrometry was consistent with the theoretical calculated value (Fig. S3), further confirming the structure of DHA dimer. In order to compare the redox responsiveness of DHA\u003csub\u003e2\u003c/sub\u003e-SS, we also synthesized another DHA dimer with the same length of carbon chain (DHA\u003csub\u003e2\u003c/sub\u003e-C6) as control (Fig. S4).\u003c/p\u003e\n \u003cp\u003eIt is reported that the organic dimers could self-assemble into NPs in aqueous solution [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. As anticipated, both kinds of resulting dimers formed spherical nanoparticles (abbreviated as SS NPs and C6 NPs, respectively) through nanoprecipitation method as observed \u003cem\u003evia\u003c/em\u003e transmission electron microscopy (TEM) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). And SS NPs had an average hydrodynamic diameter of approximately 167.2 nm as determined by dynamic light scattering (DLS), which was similar to those of C6 NPs (181.4 nm) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). These two kinds of NPs were found to be negative, and the zeta potential values were around -20 mV (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). The drug content of SS and C6 NPs was 90.6% and 91.7%, respectively. In addition, SS and C6 NPs both had robust stability with negligible changes in size and size distribution in one week (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). And these NPs also kept stable in PBS (pH 7.4) containing 10% FBS after 24 h (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF), and a slight size increase in the first two hours was largely ascribed to the protein absorption on the surface of NPs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003eDTT and GSH triggered release of DHA\u003c/h2\u003e\n \u003cp\u003eAs mentioned above, disulfide bond possess distinct redox response capability [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. Therefore, we investigated the responsiveness of DHA\u003csub\u003e2\u003c/sub\u003e-C6 and DHA\u003csub\u003e2\u003c/sub\u003e-SS by using dithiothreitol (DTT) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as reducing and oxidizing agents, respectively. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, after incubation with 10 mM DTT, the HPLC peak of DHA\u003csub\u003e2\u003c/sub\u003e-SS at 10.6 min declined significantly and entirely disappeared in 24 h, while the new peak for DHA at 6.4 min emerged and enhanced gradually, indicating the dissociation of disulfide linkage and the release of DHA. In comparison, drug release from DHA\u003csub\u003e2\u003c/sub\u003e-C6 dimer was quite slow under the same condition, and only approximately 10% of DHA released even after 48 h treatment (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). In addition, we further investigated the oxidation responsiveness of these dimers. Similar to reduction responsiveness, DHA\u003csub\u003e2\u003c/sub\u003e-SS also exhibited sensitive oxidation responsiveness. As presented in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD, DHA\u003csub\u003e2\u003c/sub\u003e-SS could release about 60% DHA after 48 h of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment (10 mM), while negligible degradation was detected for DHA\u003csub\u003e2\u003c/sub\u003e-C6 at the same time (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). The release curves of DHA over time in the presence of DTT and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e are revealed in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF, respectively. These results validate the redox responsiveness of disulfide bond linker and the controlled release of DHA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eCellular uptake and in vitro cytotoxicity of DHA dimeric NPs\u003c/h2\u003e\n \u003cp\u003eHuman hepatoma HepG2 cells were used to study the cellular internalization of these NPs \u003cem\u003evia\u003c/em\u003e confocal laser scanning microscopy (CLSM). The fluorescent dye nile red (NR) was utilized as a marker and encapsulated into the NPs by coassembly with DHA dimer, and the blue fluorescence of Hoechst 33258 was employed to localize the cell nucleus. As exhibited in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, the red fluorescence principally distributed in the cytoplasm of tumor cells in a time-dependent manner (Fig. S5), implying the efficient and sustained cellular uptake. Furthermore, the effect of temperature on cellular uptake has also been carried out. It can be observed that a strong fluorescence signal at 37\u0026deg;C and significantly reduced cellular uptake at 4\u0026deg;C from Fig. S6, suggesting an energy-dependent endocytosis.\u003c/p\u003e\n \u003cp\u003eNext, the cytotoxicity of DHA dimeric NPs was evaluated against human HepG2 and HeLa cells through a standard MTT assay. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, both free DHA and SS NPs showed efficient suppression of HepG2 viability in concentration dependent manner for 48 h, and SS NPs exhibited higher cytotoxicity than free DHA. The cell viability of SS NPs was less than 30%, while it was approximately 40% for free DHA at the equivalent DHA concentration. This result may be attributed to the enhanced cellular uptake of NPs into tumor cells and the rapid release of DHA. And SS NPs could quickly release active DHA once in living cells because of the responsiveness of disulfide bond in redox environment. However, C6 NPs exhibited apparent lower cytotoxicity compared with SS NPs (Fig. S7). Similarly, SS NPs still possessed the strongest cellular toxicity towards HeLa cells (Fig. S8). In addition, we evaluated the cytotoxicity of SS NPs toward normal hepatocytes (HL-7702), and also compared its toxicity on HepG2, HeLa and HL-7702 cells. As revealed in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, SS NPs exhibited no significant cellular toxicity against HL-7702 cells, and enhanced cytotoxicity against these two kinds of tumor cells (Fig. S9), indicating the selectivity of DHA\u003csub\u003e2\u003c/sub\u003e-SS prodrug towards tumor cells. This result is ascribed to the different redox conditions in normal cells and tumor cells.\u003c/p\u003e\n \u003cp\u003eTo further understand the contribution of SS NPs on apoptosis, we stained HepG2 cells with Annexin V-FITC and PI, and analyzed them by flow cytometry. As presented in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD, the ratio of early apoptotic cells was 9.40%, 15.00%, and 24.56%, respectively, as the drug concentration increases (20, 40 and 60 \u0026micro;M), validating the cell apoptosis in a concentration-dependent manner. Additionally, we also examined the nuclear morphological changes by CLSM. The cell nucleus emerged as a homogeneous blue chromatin with an organized structure in normal cells, whereas the cells incubated with SS NPs displayed representative morphological changes (Fig. S10), including intense fluorescent spots, nuclear pyknosis, and extensive blebbing, further verifying the apoptosis of tumor cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003eAntitumor mechanism of SS NPs\u003c/h2\u003e\n \u003cp\u003eTo elucidate the mechanism of SS NPs in inhibiting tumor cell proliferation and inducing apoptosis, RNA sequencing (RNA-seq) technology was applied to collect the gene expression [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]. The total RNA extracted from SS NPs treatment group (SS) and the control group (C) have been analyzed for quality and integrity by utilizing formaldehyde agarose gel electrophoresis. The results verified that the obtained RNA was intact, undegraded, and suitable for RNA-seq analysis. Meanwhile, principal component analysis (PCA) of samples was performed on the complete dataset [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e], which can display changes of overall gene expression. As shown in the PCA results (Fig. S11), there were two clusters, verifying a distinct directionality between SS NPs treatment and the control groups based on the similarity of gene expression. Subsequently, the differentially expressed genes (DEGs) induced by SS NPs have been identified and described by volcano plots and heatmaps. After comparing with the untreated control group (log\u003csub\u003e2\u003c/sub\u003e fold-change \u0026ge; 2.0 and adjusted \u003cem\u003eP\u003c/em\u003e value \u003cem\u003e\u0026lt;\u003c/em\u003e 0.05), we distinguished 6546 DEGs, including 3288 up-regulated and 3258 down-regulated expression genes (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). The above results imply that SS NPs play an important role on gene expression of the treated tumor cells.\u003c/p\u003e\n \u003cp\u003eAccording to the RNA-seq data, we performed the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for 6546 differentially expressed genes. Three domains were involved: cellular components (CC), biological processes (BP) and molecular function (MF). In the BP domain, a large proportion of genes were associated with cellular process, metabolic process, biological regulation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). For the CC domain, the enriched genes covered cell, cell part, organelle, membrane and so forth (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Binding, catalytic activity, transcription regulator activity and molecular function regulator were primarily influenced for the MF domain (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eOn the basis of the DEGs results, we also executed KEGG functional enrichment analysis and pathway classification. The KEGG functional enrichment analysis result (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD) demonstrated that multiple pathways have been affected by DHA\u003csub\u003e2\u003c/sub\u003e-SS NPs, and the signaling pathways of carbon metabolism, biosynthesis of amino acids, fatty acid metabolism, HIF-1 signaling pathway and pathways in cancer were among the top 20 pathways. On the other hand, KEGG pathway classification results (Fig. S12) displayed that there were six branches for KEGG pathways, including cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems. And the DEGs in HepG2 cells after SS NPs treatment were mainly enriched in PI3K-Akt signaling pathway, MAPK signaling pathway and pathways in cancer.\u003c/p\u003e\n \u003cp\u003eFrom GO and pathway analysis results, we discovered that the metabolic process was involved in the inhibition of tumor cells proliferation, thus we carried out network statistical analysis on the protein-protein interaction (PPI) of these genes correlated with metabolism. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, the metabolism of carbohydrates, amino acids and lipids have been remarkably regulated in the cells after SS NPs treatment, indicating that SS NPs could induce metabolic reprogramming in tumor cells. Therefore, we concentrated on the metabolism reprogramming of tumor cells after treated with SS NPs.\u003c/p\u003e\n \u003cp\u003eApoptosis is a highly regulated process of cell death, which is important to maintain the inherent stability of multicellular organisms, and involves a variety of signal pathways [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. The Bcl-2 protein family contains pro-apoptotic and anti-apoptotic regulators of programmed cell death/apoptosis, and plays a dominant role in regulating cell apoptosis. In this family, Bax gene, a pro-apoptotic member, can form heterodimers with Bcl-2 protein, and the ratio of Bax/Bcl-2 could determine the sensitivity of cells to apoptosis. The activation of Bax can release cytochrome c (Cyt C), and Cyt C activates caspase-9 and downstream caspase-3 through a cascade reaction to promote cell apoptosis, whereas the anti-apoptotic Bcl-2 operates in the opposite way. Therefore, we detected expression of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9 and Cyt C by western blotting. As displayed in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, the expression levels of Bax, caspase-3, caspase-9, and Cyt C were obviously increased, and the expression levels of Bcl-2 was reduced compared to the control group. The above results demonstrate that mitochondrial apoptosis pathway is involved in the apoptosis of HepG2 cells induced by SS NPs.\u003c/p\u003e\n \u003cp\u003eAs we known, tumor cells generally reveal aberrant metabolism due to metabolic reprogramming [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. As a hallmark of tumor cells, the Warburg effect means that tumor cells rely heavily on glycolysis for energy, rather than oxygen [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]. To investigate whether SS NPs treatment could suppress the glycolysis of tumor cells, the glucose uptake, contents of lactic acid and ATP products of HepG2 cells treated with SS NPs were detected. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e, the glucose uptake was decreased, and the contents of intracellular ATP and extracellular lactic acid were also declined, indicating that SS NPs may inhibit glycolysis of tumor cells.\u003c/p\u003e\n \u003cp\u003eCancer metabolic reprogramming is regulated by multiple pathways, which includes the PI3K-AKT signaling pathway [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e]. The activated PI3K-AKT can promote the transition to aerobic glycolysis, and AKT could result in the phosphorylation of some important downstream targets, such as Bcl-2 apoptosis-related family, and mammalian target of rapamycin (mTOR), to protect cells from apoptosis. Meanwhile, this pathway also could regulate HIF-1\u0026alpha; through mTOR, and activated HIF-1\u0026alpha; is related to the up-regulation of glucose transporters (Gluts) and glycolytic enzymes. To understand whether the activation of PI3K/AKT and HIF-1\u0026alpha; were involved in metabolic reprogramming of tumor cells treated by SS NPs, we detected the related protein expression. As displayed in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, SS NPs treatment could decrease the expression of glycolytic enzymes, such as PFKP, HK2, LDH, Glut1. The ratio of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR and HIF-1\u0026alpha; were also reduced accordingly. Collectively, these findings confirm that SS NPs could induce apoptosis and suppress glycolysis by regulating the PI3K/AKT/HIF-1\u0026alpha; signaling pathway.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003eIn vitro antitumor efficacy of DHA dimeric NPs\u003c/h2\u003e\n \u003cp\u003eWe further evaluated the anti-cancer activity of SS NPs on H22 tumor-bearing Kunming mice. Mice bearing the tumors were randomly divided into 4 groups with different treatments: PBS, free DHA, C6 NPs, and SS NPs, and injected intravenously at equivalent DHA doses every second day. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA, DHA treatment exhibited a moderate inhibitory effect on tumor growth compared with the control group, which is mainly owing to its intrinsic toxicity. Notably, SS NPs exhibited evident antitumor activity, which is more potent than free DHA group. The improved therapeutic efficacy of SS NPs should be ascribed to the multiple advantages of nanoparticle formulations, including enhanced tumor accumulation, effective endocytosis, and rapid drug release in tumor sites. Unsurprisingly, C6 NPs group displayed the weakest tumor growth inhibition effect in these treatment groups on account of the insensitivity of C6 linker to redox microenvironment. What\u0026rsquo;s more, the tumor weight (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eB) and the photographs of resected tumors (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eC) visually demonstrated the greatest tumor inhibition efficacy obtained by SS NPs, further validating the enhanced antitumor effect of disulfide-bond bridged prodrug nanoparticles. In addition, all mice had no significant weight fluctuation during the whole treatment period (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eD), and there was also no detectable histological damage observed after SS NPs treatment from the hematoxylin and eosin (H\u0026amp;E) stained tissue sections of major organs (heart, liver, spleen, lung, and kidney) (Fig. S13). The above results validate SS NPs at current doses possess favorable biosafety and ignorable systemic toxicity. The H\u0026amp;E staining of tumor slices revealed that SS NPs group exhibited the most severe cellular damage compared with the control group. The tumor cells after SS NPs treatment shrank largely and the tumor tissue significantly decreased. (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eE). All of the results substantiate that DHA\u003csub\u003e2\u003c/sub\u003e-SS NPs could be safely used for the in vivo treatment, and possess better treatment effects in contrast with free DHA.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, a DHA dimeric nanoprodrug using disulfide bond as linkage (DHA\u003csub\u003e2\u003c/sub\u003e-SS) was obtained. The SS NPs not only possess favorable stability and high drug content of 90.6 wt%, but also can respond to the tumor redox microenvironment, thus resulting in the effective release of DHA for chemotherapy. Importantly, both in vitro and in vivo therapy experiments indicate these obtained SS NPs have efficient endocytosis, potent cytotoxicity and enhanced antitumor efficacy in contrast with free DHA. RNA sequencing and bioinformatics analysis demonstrate that SS NPs could induce apoptosis \u003cem\u003evia\u003c/em\u003e the intrinsic mitochondrial apoptosis pathway, as well as inhibit glycolysis through PI3K/AKT/HIF-1α signaling pathway. Our finding provides a reference for the rational design of responsive prodrug nanoparticles, and could potentially spur the similar study of Chinese medicine and related natural active ingredients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e accompanies this paper at https://doi.org/10. 1186/s129 51 -021-#####-#. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript was written through contributions of all authors. All authors read and\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eapproved the fnal manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Development Project of Jilin Province (No.YDZJ202101ZYTS094), Scientific Research Project of Education Department of Jilin Province (No.JJKH20210493KJ), and Traditional Chinese Medicine Science and Technology Project of Jilin Province (No.2021090).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article and its\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eadditional file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors gave their consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A. 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Cells. 2021;10: 1056.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dimeric nanoprodrug, dihydroartemisinin, redox-responsive, antitumor activity, PI3K/AKT/HIF-1α signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-981484/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-981484/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRedox-responsive drug delivery system emerges as a hopeful platform for tumor treatment. Dihydroartemisinin (DHA) has been investigated as an innovative tumor therapeutic agent. Herein, a DHA dimeric prodrug bridged with disulfide bond as linker (DHA\u003csub\u003e2\u003c/sub\u003e-SS) has been designed and synthesized. The prepared prodrugs could self-assemble into nanoparticles (SS NPs) with high DHA content (\u0026gt;90%) and robust stability. These SS NPs display sensitive redox responsive capability and can release DHA under the tumor heterogeneity microenvironment. SS NPs possess preferable antitumor therapeutic activity in contrast with free DHA. Moreover, the possible anti-cancer mechanism of these nanoparticles was investigated through RNA-seq analysis, bioinformatics and molecular biological method. SS NPs could induce apoptosis\u003cem\u003e via\u003c/em\u003e mitochondrial apoptosis pathway, as well as glycolysis inhibition associate with the regulation of PI3K/AKT/HIF-1α signal path, which may offer an underlying therapeutic target for liver cancer. Our study highlights the potential of using redox responsive prodrug nanoparticles to treat cancer, meanwhile provides insights into the anti-cancer mechanism of DHA prodrug.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"A Redox-Responsive Dihydroartemisinin Dimeric Nanoprodrug For Enhanced Antitumor Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-10-22 14:45:40","doi":"10.21203/rs.3.rs-981484/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-11-02T10:12:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-11-02T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-11-01T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-10-29T00:00:00+00:00","index":4,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-10-27T00:00:00+00:00","index":3,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-10-21T00:00:00+00:00","index":4,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-10-20T00:00:00+00:00","index":3,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-10-19T13:18:07+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-10-19T13:12:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Nanobiotechnology","date":"2021-10-19T02:00:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-10-19T01:42:17+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-10-19T01:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-10-19T00:00:00+00:00","index":1,"fulltext":""},{"type":"checksComplete","content":"","date":"2021-10-17T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2021-10-16T02:37:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"210f3594-7f1a-4234-8c15-b7b5f60a62cb","owner":[],"postedDate":"October 22nd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":8023042,"name":"Nanoscience"}],"tags":[],"updatedAt":"2021-12-20T12:55:32+00:00","versionOfRecord":{"articleIdentity":"rs-981484","link":"https://doi.org/10.1186/s12951-021-01200-z","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2021-12-01 12:55:32","publishedOnDateReadable":"December 1st, 2021"},"versionCreatedAt":"2021-10-22 14:45:40","video":"","vorDoi":"10.1186/s12951-021-01200-z","vorDoiUrl":"https://doi.org/10.1186/s12951-021-01200-z","workflowStages":[]},"version":"v1","identity":"rs-981484","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-981484","identity":"rs-981484","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}