Aptamer-modified GSH-sensitive honokiol polyprodrug nanoparticles for ovarian cancer-specific targeting therapy

Aptamer-modified GSH-sensitive honokiol polyprodrug nanoparticles for ovarian cancer-specific targeting therapy | 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 Aptamer-modified GSH-sensitive honokiol polyprodrug nanoparticles for ovarian cancer-specific targeting therapy Chunhua Guo, Xiaowei Cheng, Yuxing Yang, Lijuan Wang, Wenfang Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4783145/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 Background Ovarian cancer is a major threat to women's lives. Chinese medicine honokiol (HK) is a polyphenol isolated from Magnolia, which can effectively suppress the growth of ovarian cancer. However, low water solubility and lack of tumor-targeting ability have greatly hindered the clinical application of HK. Results Herein, a glutathione (GSH) sensitive HK polyprodrug was prepared by using HK as the backbone. Then, an EpCAM-specific aptamer and poly(ethylene glycol) (PEG) were conjugated to HK polyprodrug, and the obtained polyprodrug was assembled into nanoparticles in water. The HK polyprodrug-formed nanoparticles achieved high drug loading and GSH-responsiveness drug release. Moreover, after optimization, HK polyprodrug nanoparticles (A/P-PHK NP40) formed by aptamer-modified and PEG-modified prodrug at feed molar ratio at 2: 3 had the highest ability to target EpCAM overexpression ovarian cancer cells. A/P-PHK NP40 also exhibited a higher cell growth inhibition effect in ovarian cancer cells than free HK and control HK nanoparticles. Conclusion All in all, this work reported a novel strategy for HK delivery based on microenvironment responsiveness polyprodrug, which provided a potential method for ovarian cancer targeting therapy. Polyprodrug GSH-responsive cancer aptamer targeting honokiol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Ovarian cancer (OC) is one of the major malignancies in women (Pathak et al., 2020 ). It is reported that over three hundred thousand women were diagnosed with OC, and over two hundred thousand women have died from OC in the USA in 2023 (Siegel et al., 2024 ). Currently, chemotherapy remains the major method for OC therapy, which has significantly improved the survival rate of patients with localized OC, however, patients with advanced, metastatic, and relapsed OC remain a challenge in the clinic (Akter et al., 2022 ; Barani et al., 2021 ; Li et al., 2020 ). Moreover, conventional chemotherapy with many drawbacks including side effects and drug resistance has greatly decreased the quality of life of patients (Liu et al., 2021 ; Sharma et al., 2022 ). Hence, the identification of superior and more efficacious therapy approaches for OC is an urgent need. Honokiol (HK) is an extractive polyphenol of the genus Magnolia species, which exhibited various biological activities, such as anticancer and anti-inflammatory activities (Ju et al., 2018 ; Rauf et al., 2021 ; Rauf et al., 2018 ). Currently, researchers are focused on the anticancer potential of honokiol. Various preclinical studies showed that HK can effectively suppress the growth of numerous cancer cells (e.g., OC, breast cancer, and colon cancer) and inhibit tumor growth and metastasis in mice models (Godugu et al., 2017 ; Li et al., 2008 ; Ponnurangam et al., 2012 ). However, the low aqueous solubility and lack of tumor-targeting ability of HK has greatly hindered its application in the clinical and prompted the development of delivery systems (such as liposomes and polymeric micelles) to improve the physicochemical properties of HK (Pan et al., 2024 ; Yang et al., 2017 ; Zhang et al., 2015 ). Among various HK-based delivery systems, the polymeric prodrug has drawn more and more attention. The polymeric prodrug is prepared by conjugating the drug to polymer, which enables prolonged therapeutics drug blood circulation time, enhances the drug’s stability and water solubility, and reduces immunogenicity (Zhou et al., 2020 ). For instance, Wang’s group developed an oxidation and reduction dual-responsive HK polymeric prodrug by grafting HK to the side of dextran via diselenium linker for efficient laryngeal carcinoma combination therapy (Zhou et al., 2022 ). However, polymers typically have many functional groups, which makes it difficult to precisely control conjugation sites, resulting in heterogeneous products and inconsistent batch-to-batch reproducibility (Cheng et al., 2022 ; Xu et al., 2017 ). Moreover, in these strategies, the polymer carrier remains after the cleavage of the linker between the drug and polymer, which could hinder drug release (Yu et al., 2021 ). To address these issues, the polyprodrug concept has emerged by polymerizing stimuli-responsive linkers with therapeutic drugs (Yang et al., 2022 ). The obtained polyprodrugs are stable and inactive under normal environments but can release drugs triggered by endogenous and exogenous stimuli including pH and redox (Seidi et al., 2022 ). Thus, developed HK-based polyprodrugs not only can achieve constant and high drug-loading levels but also realize synchronous drug activation and polymer backbone degradation. Until now, there have been no reports about polyprodrugs with HK as the skeleton. On the other hand, effective delivery of drugs to tumor tissues also requires overcoming the tumor microenvironment, such as tumor cell active targeting (Adityan et al., 2020 ). Aptamers, small single-stranded oligonucleotide segments with unique three-dimensional (3D) structures, can specifically and high affinity bind to their protein targets (Li et al., 2021 ). Aptamers have been widely employed as tumor-targeting ligands for cancer-specific drug delivery (Sanjanwala & Patravale, 2023 ). Moreover, studies have shown that aptamers have low immunogenicity and are less likely to develop resistance compared with antibodies (He et al., 2023 ). Besides, excellent stability and easily targeted cell penetration of aptamers are also desired advantages (Ning et al., 2020 ). The success of AS1411 in phase II trial (NCT00740441) for treating metastatic renal cell carcinoma further demonstrates the superiority of aptamers (Vandghanooni et al., 2020 ). Moreover, epithelial cell adhesion molecules (EpCAM), an epithelial cell adhesion molecule, are overexpressed in over 70% of OC specimens, and their levels correspond to chemoresistance, malignant ascites, and reduced survival rates of OC patients (Zheng et al., 2017 ). While EpCAM expression is normal in epithelial tissues, it appears to be tumor-specific in the peritoneal cavity since EpCAM is negative in abdominal mesothelial cells (Reichert & Valge-Archer, 2007 ). Hence, the EpCAM-specific aptamer will be a promising tumor-targeting ligand for developing HK-based polyprodrugs. Herein, we designed and prepared a reduction-responsive honokiol polyprodrug NPs, A/P-PHK NPs, for OC efficiency therapy (Scheme 1 ). A/P-PHK NPs were prepared by EpCAM-specific aptamer (Ap) modified HK polyprodrug Ap-PHK and poly(ethylene glycol) (PEG) modified HK polyprodrug PEG-PHK at a molar ratio of 2: 3. Ap-PHK and PEG-PHK were synthesized by copolymerizing HK with reduction linker 3,3’-dithiodipropionic acid, and finally modified with Ap or PEG, respectively. The hydrophilic Ap and PEG act at the outer shell, which protects the HK in body fluid. Ap leads to A/P-PHK NPs actively targeted to OC cancer cells. In cancer cells glutathione (GSH) rich environment, A/P-PHK NPs were degraded and immediately released HK to kill cancer cells. This work not only provides a promising method for OC therapy but also offers a strategy to develop tumor-specific targeting polyprodrugs. Methods and agents Materials The sulfhydryl-modified EpCAM aptamer, Ap (5’-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG -SH-3’, Mw: ~15000 Da), was purchased from Sangon Biotech Co., Ltd (China). Methoxy poly(ethylene glycol) thiol (PEG-SH, Mw: 5 kDa) was purchased from Beijing Jenkem Technology Co., Ltd (China). N-(2-Aminopropyl)maleimide (Mal-NH 2 ) was obtained from BioChemPartner Biotechnology Co. LTD (China). HK, dimethylsulfoxide (DMSO), N,N -dimethylformamide (DMF), and 3,3’-dithiodipropionic acid were purchased from Shanghai Aladdin Biotech Co., Ltd (China). The EpCAM overexpression OC SKOV3 and OVCAR3 cells and the EpCAM negative expression OC A2780 cells were purchased from Nanjing Cobioer Biosciences Co., LTD (China) and incubated in RPMI 1640 medium containing 10% FBS and antibiotics (100 U/mL penicillin and 100 U/mL streptomycin) at 37°C with 5% CO 2 . Synthesis of maleimide-modified HK polyprodrug Mal-PHK The Mal-PHK was synthesized by two-step reactions in one pot according to the previous report (Vandghanooni et al., 2020 ). First, 3,3′-dithiodipropionyl chloride was synthesized. 1.0 equiv of 3,3′-dithiodipropionic acid was added to a 250 mL round bottom flash and purged with nitrogen at room temperature, then 6.0 equiv of thionyl chloride was added dropwise. After heating to 90°C for 24 h, the reaction changed color to yellow, and the reaction was stopped. The residual product was dried under reduced pressure after excess thionyl chloride was removed with vacuum transfer. The product without further purification was mixed with an equal ratio of HK in DMF for reaction with 24 h at 50°C. Then, the Mal-NH 2 is added and the polymerization is continued for another 6 h at 50°C under nitrogen protection. At last, the mixture is dialyzed (MWCO: 3500 Da) for 48 h against DMSO and then distilled water, and the Mal-PHK is obtained after lyophilization. Synthesis of Ap-PHK and PEG-PHK Ap-PHK and PEG-PHK were synthesized by conjugating Ap or PEG to Mal-PHK through the click reaction, respectively. In brief, sulfydryl-modified Ap (15 mg, 1 µmol) or PEG-SH (5 mg, 1 µmol) dissolved in 200 µL water was added to the solution of Mal-PHK (4 mg, 0.4 µmol) in 200 µL DMSO in a 1.5 mL centrifuge tube. The reaction solution was mixed at 37°C for 6 h, then it was immediately purified by high-performance liquid chromatography (HPLC) and finally lyophilized to obtain Ap-PHK and PEG-PHK. A/P-PHK NPs preparation Ap-PHK (4 mg) and PEG-PHK (6 mg) were dissolved in 1 mL of DMSO with the aid of ultrasonication and subsequently added dropwise to 1 mL of water with vigorous stirring (1000 rpm). The solution was continuously stirred at 1000 rpm for 2 h at room temperature. Subsequently, the solution was subjected to dialysis in water for one day using a 5 kDa MWCO dialysis bag to eliminate the DMSO, followed by lyophilization to generate nanoparticles. Characterization The proton nuclear magnetic resonance ( 1 H NMR) spectra of all drugs were carried on a Bruker AVANCE III 300 MHz NMR spectrometer in DMSO- d6 . Nano-ZS 90 (Malvern, UK) is used to measure the size distribution of NPs. Transmission electron microscopy (TEM, JEM-1400plus, Japan) is conducted to observe the morphology of A/P-PHK NPs. The concentration of HK in the NPs was quantified using HPLC on a SHIMADU LC-20 system (SHIMADU, Japan). The mobile phase consisted of water, acetonitrile, and methanol at a volume ratio of 25: 20: 55, with the detection wavelength set at the maximum UV absorption of HK at 294 nm. The polymer dispersity index (PDI), number–average molecular weight, and weight-average molecular weight of prodrugs were determined by gel permeation chromatography (GPC) measurements on a Waters GPC system (Waters, Milford, MA). The system was equipped with a Waters 2414 refractive index detector and a Waters 1515 HPLC solvent pump. Polyacrylamide gel electrophoresis (PAGE) analysis PAGE analysis was used to characterize the Ap-PHK. DNA samples (250 nM) were combined with 6 × loading buffer and loaded into a 12% gel using 20 µL. After electrophoresis for 1.5 h at 80 V, the gels were imaged by a molecular imager. In vitro drug release PHK release from the A/P-PHK NPs was studied by dialysis method by using PBS (0.01M, pH 7.4) with different GSH concentrations of 0, 0.01, 1, or 10 mM as the release medium. First, the A/P-PHK NPs (3 mg/mL) were added into a dialysis tube (MWCO: 3.5 kDa), and the tube was immersed and incubated in 50 mL of release medium at 37°C. At predetermined time points, 3 mL of release solution was collected and another 3 mL of fresh-released medium was added. The concentration of HK in the release medium was measured by HPLC. As a result of carrying out the experiments in triplicate, the average value is presented. Cytotoxicity experiment CCK-8 assay was conducted to evaluate the cytotoxicity of prepared NPs. Cells were grown in 96-well plates (6000 cells per well) for 24 h with 150 µL cell culture medium. Thereafter, the cells were treated with HK or NPs, at various concentrations for 48 h. Following the addition of 10 µL of CCK-8 solutions, the wells were further incubated for 2 h. Cell proliferation was monitored using a microplate reader at 450 nm. Cell viability is determined by the equation provided as follows: Cell viability (%) = (As-An)/(Ac-An) × 100 Where As is the sample group; Ac is the control group, in which, the cells were treated with complete DMEM, and An is the adsorption of complete RPMI 1640. Cellular uptake The cells were first cultured in a six-well plate for 24 h. Then, they were treated for 4 h with coumarin-6 loaded NPs (500ng/mL coumarin-6). Immediately following treatment, cells were washed three times with PBS and imaged by a fluorescence microscope (Nikon-112, Nikon, Japan). Statical analysis In analyzing the differences between the groups, one-way ANOVA with Tukey multiple comparisons tests (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001) was carried out. Results and discussion HK polyprodrug preparation and characterization As shown in Fig. 1 , Ap-PHK and PEG-PHK were synthesized in two steps: (i) HK was polymerized with 3,3′-dithiodipropionyl chloride, and then modified with Mal-NH 2 to obtain Mal-PHK; (ii) Ap or PEG-SH were conjugated to Mal-PHK via click reaction to obtain Ap-PHK and PEG-PHK, respectively. The 1 H NMR and GPC were used to characterize the structure of Mal-PHK. In the 1 H NMR spectrum, all the peaks were well assigned (Fig. 2 ). The peaks at 4.9–5.1 ppm, 5.9-6.0 ppm, and 6.8 ~ 7.5 ppm is attributed to the double bond and benzene ring of HK, respectively. The peaks at 2.7 and 2.8 ppm are derived from 3,3′-dithiodipropionic acid. The peaks at 2.0, 4.3–4.6 ppm were belonged to Mal. Moreover, the GPC results showed that the M w of Mal-PHK was around 10900 Da, and its M w distribution (PDI) was close to 1 (Table 1 ). These results confirmed the successfully prepared Mal-PHK. Subsequently, Ap or PEG-SH was conjugated to Mal-PHK to obtain Ap-PHK and PEG-PHK, respectively. The 1 H NMR and GPC results demonstrated that the PEG-PHK was also successfully synthesized. The GPC results showed that the M w of Ap-PHK and PEG-PHK was 17540 Da and 17157 Da, respectively, with both PDIs close to 1. Additionally, the successful preparation of Ap-PHK was confirmed by PGAE and GPC (Table 1 ). In the PAGE image, the migration of Ap-PHK was significantly lower than free Ap, suggesting that the molecular weight increased after the reaction of Ap and Mal-PHK (Fig. 3 ). These results also demonstrated the successful preparation of Ap-PHK. Table 1 GPC results. Polymers M n (Da) a M w (Da) b PDI c Mal-PHK 10257 10912 1.06 Ap-PHK 16325 17540 1.07 PEG-PHK 16035 17157 1.07 Note : a: number–average molecular weight. b: weight-average molecular weight. c: polymer dispersity index (Mw/Mn). A/P-PHK NPs preparation and optimization A/P-PHK NPs were prepared by dialysis method according to the previous report with several modifications. According to the previous reports, the target segments on the surface of NPs are not “more is better”. NPs with a high density of target segments may result in low binding ability to their target. Thereby, a series of A/P-PHK NPs with different molar ratios between Ap-PHK and PEG-PHK were prepared, and these NPs were denoted as PEG-PHK NP, A/P-PHK NP20 to A/P-PHK NP80, and Ap-PHK NPs, respectively, with the increase of the molar ratio of Ap-PHK (Table 2 ). The particle size, zeta potential, and HK content of NPs are shown in Fig. 4 A to 4 D. It can be found that the particle size of NPs and size distribution index (polymer dispersity index, PDI) with the different molar ratio of Ap-PHK and PEG-PHK has no significant difference (Fig. 4 A and 4 B), while the zeta potential is increased with the increase of the molar ratio of Ap-PHK, because of the high negative charge characteristic of nucleic acid (Fig. 4 C). Moreover, the HK content decreases with the increase of the molar ratio of Ap-PHK, because of the high molecular weight of Ap than that of PEG (Fig. 4 D). Subsequently, the cancer cells targeting capability of these NPs were investigated in EpCAM overexpression human ovarian cancer SKOV3 cells and OVCAR3 cells by fluorescence microscope and flow cytometry (Fig. 5 A to 5 C). As shown in Fig. 5 A, after a 4 h treatment, the fluorescence image results in SKOV3 cells showed that the NPs contained 40% of Ap-PHK (A/P-PHK NP40) had the highest fluorescence intensity compared with no Ap-modified NPs (PEG-PHK NPs) and other Ap-modified NPs, suggesting that the A/P-PHK NP40 has the highest targeting ability. This result was also verified by the quantitative results of flow cytometry in two EpCAM overexpression cell lines (Fig. 5 B and 5 C). Thus, A/P-PHK NP40 was used in the following studies. Moreover, to further confirm the increased uptake of A/P-PHK NP40 by cells is attributed to the specifical targeting of Ap, the EpCAM overexpression cells (OVCAR3 and SKOV3) and EpCAM negative cells (A2780) were pretreated with EpCAM antibody, and then treated with coumarin-6 loaded A/P-PHK NP40. As presented in Fig. 5 D, in the EpCAM overexpression OVCAR3 and SKOV3 cells, antibody pretreated significantly inhibited the cell uptake of A/P-PHK NP40, while in EpCAM negative A2780 cells, the cell uptake has no significant difference with or without antibody pretreatment. These results demonstrated that Ap modification significantly improves the OC targeting of NPs. Table 2 Component of different NPs (molar content %). PEG-PHK NP A/P-PHK NP20 A/P-PHK NP40 A/P-PHK NP60 A/P-PHK NP80 Ap-PHK NP Ap-PHK 0 20 40 60 80 100 PEG-PHK 100 80 60 40 20 0 GSH-responsiveness investigation In the design, the disulfide bond within A/P-PHK NPs could cleave and release active HK in a rich environment. To confirm this conception, the degradation of A/P-PHK NPs after incubation in 10 mM GSH for 12 h was observed by TEM. As shown in Fig. 6 A, A/P-PHK NPs have spheroid morphology and uniform distribution in water. On the contrary, after being treated with GSH, the A/P-PHK NPs were degraded, and cannot observe obvious particles. Dynamic light scattering also demonstrated the structural degradation of A/P-PHK NPs in response to GSH (Fig. 6 B). Subsequently, the dialysis method was employed to evaluate the release of HK in various conditions. As presented in Fig. 6 C, the HK release rate is positively associated with the GSH concentration. In detail, in the absence of GSH, almost no HK was released from A/P-PHK NPs, as supported by the cumulative release of HK was lower than 10% at 48 h. In the low GSH condition (0.01 mM GSH), the release of HK was increased compared with the GSH absence condition, while only 8% of HK was released from NPs at 48 h. When the concentration of GSH increased to 10 mM, the release rate of HK was significant, and around 87.3% of HK was released within 48 h. These results demonstrated the good GSH-responsiveness of A/P-PHK NPs and can avoid premature leakage in low GSH conditions. In vitro anti-OC efficiency After a step-by-step evaluation, the in vitro anti-OC efficiency of optimized A/P-PHK NPs was further measured in SKOV3, OVCAR3, and A2780 cells via CCK-8 detection. As shown in Fig. 7 A to 6 D, after incubation for 48 h, the growth inhibition efficiency of NPs groups was all higher than that of the free HK group, suggesting that the polyprodrug strategy can enhance the therapeutics efficiency of HK. Moreover, in the EpCAM negative expression A2780 cells, the growth inhibition efficiency of PEG-PHK NPs, A/P-PHK NP40, and Ap-PHK NPs is not significantly different. On the contrary, in the EpCAM overexpression SKOV3 and OVCAR3 cells, the growth inhibition efficiency of A/P-PHK NP40 was significantly higher than other NPs, as evidenced by the IC 50 value of A/P-PHK NP40 was 1.5/1.5 and 1.4/1.3-fold lower than that of PEG-PHK NPs and Ap-PHK NPs in SKOV3 and OVCAR3 cells, respectively. These results, further demonstrated that the aptamer-modified GSH-sensitive polyprodrug strategy can significantly enhance the therapeutics efficiency of HK. Conclusion In summary, we created a novel aptamer-modified GSH-sensitive HK-polyprodrug Ap-PHK, which provided a potential method for OC therapy. Ap-PHK can self-assemble into NPs in an aqueous solution with PEG-modified HK-polyprodrug PEG-PHK. The obtained NPs can specifically target EpCAM overexpression OC cells and release HK in cell-high GSH conditions. This designed strategy not only improves the anti-OC efficiency of HK but also provides a delivery strategy for other drugs with two modifiable groups similar to HK, such as curcumin, mitoxantrone, etc. All in all, this approach offers a potential new strategy for the treatment of OC and expands the application of traditional Chinese medicine. Abbreviations 1 H NMR proton nuclear magnetic resonance spectra Ap EpCAM-specific aptamer A/P-PHK NPs reduction-responsive honokiol polyprodrug NPs Ap-PHK Ap-modified HK polyprodrug DMF N,N -dimethylformamide DMSO dimethylsulfoxide EpCAM epithelial cell adhesion molecules GPC gel permeation chromatography GSH glutathione HK Honokiol HPLC high-performance liquid chromatography NPs nanoparticles OC Ovarian cancer PAGE polyacrylamide gel electrophoresis PDI the polymer dispersity index PEG poly(ethylene glycol) PEG-PHK PEG-modified HK polyprodrug TEM Transmission electron microscopy Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article. Competing interests The authors declare that they have no competing interests. Funding Not applicable. Authors' contributions C.H.G and L.P.S designed the study. 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Theranostics 7(5):1373–1388. https://doi.org/10.7150/thno.17826 Zhou L, Hou B, Wang D, Sun F, Song R, Shao Q, Wang H, Yu H, Li Y (2020) Engineering Polymeric Prodrug Nanoplatform for Vaccination Immunotherapy of Cancer. Nano Lett 20(6):4393–4402. https://doi.org/10.1021/acs.nanolett.0c01140 Zhou L, Wu J, Sun Z, Wang W (2022) Oxidation and Reduction Dual-Responsive Polymeric Prodrug Micelles Co-delivery Precisely Prescribed Paclitaxel and Honokiol for Laryngeal Carcinoma Combination Therapy. Front Pharmacol 13:934632. https://doi.org/10.3389/fphar.2022.934632 Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files PAGEimage.tif Scheme1.png 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-4783145","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":337275469,"identity":"ca73bdd1-754c-4afa-82fb-557aa159558c","order_by":0,"name":"Chunhua Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3PsYoCMRCA4QkDqdbbdkTZe4WAIHa+SsKCnYdyTQo5F5RsoWKrb2FpuSJslWNbS32DszrBRq0Vs3YW+er5mRkAz3tDPJz/n0+ChiHiZi/1wJ18UCZqoFuymvJY7G3uTiKQ18RqKYqgWT2MscRhkMlG39AXbKGpVcIhTCfyeYJJFi8NfbMRdHZqXQeyvyvHlk2SVQyxEUK+U5aDoK4riVlySwwy01MGyyQdxMCSmiJyKJeQ5WyhqUHIkaTNA+cvn/Mphz/xE7WL4ng86UEUprPnyZ3gtXHP8zzvoQu8GkThdn0M9wAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Chunhua","middleName":"","lastName":"Guo","suffix":""},{"id":337275470,"identity":"77ba0be2-b1f8-4d7f-850b-3ea636ddaac2","order_by":1,"name":"Xiaowei Cheng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiaowei","middleName":"","lastName":"Cheng","suffix":""},{"id":337275472,"identity":"7c927034-133f-43de-b161-948cbdcbcf5e","order_by":2,"name":"Yuxing Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yuxing","middleName":"","lastName":"Yang","suffix":""},{"id":337275474,"identity":"265b1a21-8df6-4034-8926-82f8fcbd3203","order_by":3,"name":"Lijuan Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lijuan","middleName":"","lastName":"Wang","suffix":""},{"id":337275476,"identity":"5accc684-fa1e-4df5-863a-87b47082a374","order_by":4,"name":"Wenfang Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wenfang","middleName":"","lastName":"Wang","suffix":""},{"id":337275478,"identity":"aee932a7-7b09-4d18-9b29-589f62709c02","order_by":5,"name":"Liping Shao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"Shao","suffix":""}],"badges":[],"createdAt":"2024-07-22 16:06:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4783145/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4783145/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63358928,"identity":"6e308cc5-0bd2-4103-899f-0b687885fb98","added_by":"auto","created_at":"2024-08-27 09:51:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70833,"visible":true,"origin":"","legend":"\u003cp\u003eThe synthesis route of Ap-PHK and PEG-PHK. (i): DMF, 50 °C, 24 h; (ii) DMF, 50 °C, 6 h; (iii) water/DMSO = 1/1 (v/v), 37 °C, 6 h; (iv) water/DMSO = 1/1 (v/v), 37 °C, 6 h.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4783145/v1/a19fc681ceed81cab793a194.png"},{"id":63359681,"identity":"d3c04a2a-5c6d-44c7-b0e4-909af5567153","added_by":"auto","created_at":"2024-08-27 09:59:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectrum of HK, Mal-PHK, and PEG-PHK, respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4783145/v1/9699b92742be499e6513f27e.png"},{"id":63358941,"identity":"0df7fe82-7bbe-4bd2-a824-0e22540ee5f2","added_by":"auto","created_at":"2024-08-27 09:51:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":114415,"visible":true,"origin":"","legend":"\u003cp\u003ePAGE images of Ap and Ap-PHK.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4783145/v1/33ac65e5cb9100ffd63ae670.png"},{"id":63358961,"identity":"e2d5599c-5ed8-48a3-8fbd-8ebb7cee1942","added_by":"auto","created_at":"2024-08-27 09:51:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":107335,"visible":true,"origin":"","legend":"\u003cp\u003eNPs characterization. (A-B) The particle size (A) and size distribution (B) of different NPs detected by dynamic light scattering. (C) Zeta potential of different NPs. (D) HK content in different NPs. Data is shown as mean ± SD, \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4783145/v1/b02297f1901f2e58de9873f6.png"},{"id":63358965,"identity":"ac94a928-4dac-44e9-a188-e8fefd9ea26d","added_by":"auto","created_at":"2024-08-27 09:51:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":184785,"visible":true,"origin":"","legend":"\u003cp\u003eSpecifical targeting ability of NPs characterization. (A) The fluorescence images of SKOV3 cells after being treated with coumarin-6 loaded NPs with or without EpCAM antibody pretreated. (B-C) Flow cytometry results of SKOV3 and OVCAR3 cells after being treated with coumarin-6 loaded NPs. (D) Flow cytometry results of SKOV3, OVCAR3, and A2780 cells after being treated with coumarin-6 loaded A/P-PHK NP40 with or without EpCAM antibody pretreated. Data is shown as mean ± SD, \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4783145/v1/391f277ea271f84b3ba05d34.png"},{"id":63358943,"identity":"711aa0e6-6b0d-4604-b412-78a13761b73f","added_by":"auto","created_at":"2024-08-27 09:51:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":99407,"visible":true,"origin":"","legend":"\u003cp\u003eGSH-responsiveness of A/P-PHK NPs characterization. (A-B) TEM images (A) and size distribution (B) of A/P-PHK NPs with or without GSH treatment. (C) Cumulative release of HK in PBS containing 0, 0.01, 1, and 10 mM GSH, respectively. Data is shown as mean ± SD, \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4783145/v1/de99f5acd8ff896d21d3e62a.png"},{"id":63358955,"identity":"dd16c823-f3fc-43ef-945d-915c69342de8","added_by":"auto","created_at":"2024-08-27 09:51:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":129722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn\u003c/em\u003e \u003cem\u003evitro\u003c/em\u003e anti-OC efficiency of different drug formulations. Cell viability of A2780 (A), SKOV3 (B), OVCAR3 (C), and IC\u003csub\u003e50\u003c/sub\u003e (D) after being treated with different drug formulations. Data is shown as mean ± SD, \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4783145/v1/b5cd65aa9e24a8bba52929bc.png"},{"id":67211956,"identity":"a5261b4b-df46-4df8-a142-dc6259330901","added_by":"auto","created_at":"2024-10-22 12:32:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1201276,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4783145/v1/5e9a3f39-591b-499d-b160-a4926cdc6802.pdf"},{"id":63358966,"identity":"7b10d960-d530-4d01-aa4a-ce0132dd8669","added_by":"auto","created_at":"2024-08-27 09:51:20","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1701692,"visible":true,"origin":"","legend":"","description":"","filename":"PAGEimage.tif","url":"https://assets-eu.researchsquare.com/files/rs-4783145/v1/cef6e2438e47e77626da3971.tif"},{"id":63358967,"identity":"bfed771a-0de6-4a89-85e5-eea62217d617","added_by":"auto","created_at":"2024-08-27 09:51:20","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":187607,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-4783145/v1/611867cfb5cc07eef736f72d.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Aptamer-modified GSH-sensitive honokiol polyprodrug nanoparticles for ovarian cancer-specific targeting therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer (OC) is one of the major malignancies in women (Pathak et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is reported that over three hundred thousand women were diagnosed with OC, and over two hundred thousand women have died from OC in the USA in 2023 (Siegel et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Currently, chemotherapy remains the major method for OC therapy, which has significantly improved the survival rate of patients with localized OC, however, patients with advanced, metastatic, and relapsed OC remain a challenge in the clinic (Akter et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Barani et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, conventional chemotherapy with many drawbacks including side effects and drug resistance has greatly decreased the quality of life of patients (Liu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sharma et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Hence, the identification of superior and more efficacious therapy approaches for OC is an urgent need.\u003c/p\u003e \u003cp\u003eHonokiol (HK) is an extractive polyphenol of the genus Magnolia species, which exhibited various biological activities, such as anticancer and anti-inflammatory activities (Ju et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rauf et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rauf et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Currently, researchers are focused on the anticancer potential of honokiol. Various preclinical studies showed that HK can effectively suppress the growth of numerous cancer cells (e.g., OC, breast cancer, and colon cancer) and inhibit tumor growth and metastasis in mice models (Godugu et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ponnurangam et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, the low aqueous solubility and lack of tumor-targeting ability of HK has greatly hindered its application in the clinical and prompted the development of delivery systems (such as liposomes and polymeric micelles) to improve the physicochemical properties of HK (Pan et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Among various HK-based delivery systems, the polymeric prodrug has drawn more and more attention.\u003c/p\u003e \u003cp\u003eThe polymeric prodrug is prepared by conjugating the drug to polymer, which enables prolonged therapeutics drug blood circulation time, enhances the drug\u0026rsquo;s stability and water solubility, and reduces immunogenicity (Zhou et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For instance, Wang\u0026rsquo;s group developed an oxidation and reduction dual-responsive HK polymeric prodrug by grafting HK to the side of dextran via diselenium linker for efficient laryngeal carcinoma combination therapy (Zhou et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, polymers typically have many functional groups, which makes it difficult to precisely control conjugation sites, resulting in heterogeneous products and inconsistent batch-to-batch reproducibility (Cheng et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, in these strategies, the polymer carrier remains after the cleavage of the linker between the drug and polymer, which could hinder drug release (Yu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To address these issues, the polyprodrug concept has emerged by polymerizing stimuli-responsive linkers with therapeutic drugs (Yang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The obtained polyprodrugs are stable and inactive under normal environments but can release drugs triggered by endogenous and exogenous stimuli including pH and redox (Seidi et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, developed HK-based polyprodrugs not only can achieve constant and high drug-loading levels but also realize synchronous drug activation and polymer backbone degradation. Until now, there have been no reports about polyprodrugs with HK as the skeleton.\u003c/p\u003e \u003cp\u003eOn the other hand, effective delivery of drugs to tumor tissues also requires overcoming the tumor microenvironment, such as tumor cell active targeting (Adityan et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Aptamers, small single-stranded oligonucleotide segments with unique three-dimensional (3D) structures, can specifically and high affinity bind to their protein targets (Li et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Aptamers have been widely employed as tumor-targeting ligands for cancer-specific drug delivery (Sanjanwala \u0026amp; Patravale, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, studies have shown that aptamers have low immunogenicity and are less likely to develop resistance compared with antibodies (He et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Besides, excellent stability and easily targeted cell penetration of aptamers are also desired advantages (Ning et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The success of AS1411 in phase II trial (NCT00740441) for treating metastatic renal cell carcinoma further demonstrates the superiority of aptamers (Vandghanooni et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, epithelial cell adhesion molecules (EpCAM), an epithelial cell adhesion molecule, are overexpressed in over 70% of OC specimens, and their levels correspond to chemoresistance, malignant ascites, and reduced survival rates of OC patients (Zheng et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). While EpCAM expression is normal in epithelial tissues, it appears to be tumor-specific in the peritoneal cavity since EpCAM is negative in abdominal mesothelial cells (Reichert \u0026amp; Valge-Archer, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Hence, the EpCAM-specific aptamer will be a promising tumor-targeting ligand for developing HK-based polyprodrugs.\u003c/p\u003e \u003cp\u003eHerein, we designed and prepared a reduction-responsive honokiol polyprodrug NPs, A/P-PHK NPs, for OC efficiency therapy (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A/P-PHK NPs were prepared by EpCAM-specific aptamer (Ap) modified HK polyprodrug Ap-PHK and poly(ethylene glycol) (PEG) modified HK polyprodrug PEG-PHK at a molar ratio of 2: 3. Ap-PHK and PEG-PHK were synthesized by copolymerizing HK with reduction linker 3,3\u0026rsquo;-dithiodipropionic acid, and finally modified with Ap or PEG, respectively. The hydrophilic Ap and PEG act at the outer shell, which protects the HK in body fluid. Ap leads to A/P-PHK NPs actively targeted to OC cancer cells. In cancer cells glutathione (GSH) rich environment, A/P-PHK NPs were degraded and immediately released HK to kill cancer cells. This work not only provides a promising method for OC therapy but also offers a strategy to develop tumor-specific targeting polyprodrugs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methods and agents","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe sulfhydryl-modified EpCAM aptamer, Ap (5\u0026rsquo;-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG -SH-3\u0026rsquo;, Mw: ~15000 Da), was purchased from Sangon Biotech Co., Ltd (China). Methoxy poly(ethylene glycol) thiol (PEG-SH, Mw: 5 kDa) was purchased from Beijing Jenkem Technology Co., Ltd (China). N-(2-Aminopropyl)maleimide (Mal-NH\u003csub\u003e2\u003c/sub\u003e) was obtained from BioChemPartner Biotechnology Co. LTD (China). HK, dimethylsulfoxide (DMSO), \u003cem\u003eN,N\u003c/em\u003e-dimethylformamide (DMF), and 3,3\u0026rsquo;-dithiodipropionic acid were purchased from Shanghai Aladdin Biotech Co., Ltd (China).\u003c/p\u003e \u003cp\u003eThe EpCAM overexpression OC SKOV3 and OVCAR3 cells and the EpCAM negative expression OC A2780 cells were purchased from Nanjing Cobioer Biosciences Co., LTD (China) and incubated in RPMI 1640 medium containing 10% FBS and antibiotics (100 U/mL penicillin and 100 U/mL streptomycin) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of maleimide-modified HK polyprodrug Mal-PHK\u003c/h2\u003e \u003cp\u003eThe Mal-PHK was synthesized by two-step reactions in one pot according to the previous report (Vandghanooni et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). First, 3,3\u0026prime;-dithiodipropionyl chloride was synthesized. 1.0 equiv of 3,3\u0026prime;-dithiodipropionic acid was added to a 250 mL round bottom flash and purged with nitrogen at room temperature, then 6.0 equiv of thionyl chloride was added dropwise. After heating to 90\u0026deg;C for 24 h, the reaction changed color to yellow, and the reaction was stopped. The residual product was dried under reduced pressure after excess thionyl chloride was removed with vacuum transfer. The product without further purification was mixed with an equal ratio of HK in DMF for reaction with 24 h at 50\u0026deg;C. Then, the Mal-NH\u003csub\u003e2\u003c/sub\u003e is added and the polymerization is continued for another 6 h at 50\u0026deg;C under nitrogen protection. At last, the mixture is dialyzed (MWCO: 3500 Da) for 48 h against DMSO and then distilled water, and the Mal-PHK is obtained after lyophilization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of Ap-PHK and PEG-PHK\u003c/h2\u003e \u003cp\u003eAp-PHK and PEG-PHK were synthesized by conjugating Ap or PEG to Mal-PHK through the click reaction, respectively. In brief, sulfydryl-modified Ap (15 mg, 1 \u0026micro;mol) or PEG-SH (5 mg, 1 \u0026micro;mol) dissolved in 200 \u0026micro;L water was added to the solution of Mal-PHK (4 mg, 0.4 \u0026micro;mol) in 200 \u0026micro;L DMSO in a 1.5 mL centrifuge tube. The reaction solution was mixed at 37\u0026deg;C for 6 h, then it was immediately purified by high-performance liquid chromatography (HPLC) and finally lyophilized to obtain Ap-PHK and PEG-PHK.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eA/P-PHK NPs preparation\u003c/h2\u003e \u003cp\u003eAp-PHK (4 mg) and PEG-PHK (6 mg) were dissolved in 1 mL of DMSO with the aid of ultrasonication and subsequently added dropwise to 1 mL of water with vigorous stirring (1000 rpm). The solution was continuously stirred at 1000 rpm for 2 h at room temperature. Subsequently, the solution was subjected to dialysis in water for one day using a 5 kDa MWCO dialysis bag to eliminate the DMSO, followed by lyophilization to generate nanoparticles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization\u003c/h2\u003e \u003cp\u003eThe proton nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH NMR) spectra of all drugs were carried on a Bruker AVANCE III 300 MHz NMR spectrometer in DMSO-\u003cem\u003ed6\u003c/em\u003e. Nano-ZS 90 (Malvern, UK) is used to measure the size distribution of NPs. Transmission electron microscopy (TEM, JEM-1400plus, Japan) is conducted to observe the morphology of A/P-PHK NPs. The concentration of HK in the NPs was quantified using HPLC on a SHIMADU LC-20 system (SHIMADU, Japan). The mobile phase consisted of water, acetonitrile, and methanol at a volume ratio of 25: 20: 55, with the detection wavelength set at the maximum UV absorption of HK at 294 nm. The polymer dispersity index (PDI), number\u0026ndash;average molecular weight, and weight-average molecular weight of prodrugs were determined by gel permeation chromatography (GPC) measurements on a Waters GPC system (Waters, Milford, MA). The system was equipped with a Waters 2414 refractive index detector and a Waters 1515 HPLC solvent pump.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePolyacrylamide gel electrophoresis (PAGE) analysis\u003c/h2\u003e \u003cp\u003ePAGE analysis was used to characterize the Ap-PHK. DNA samples (250 nM) were combined with 6 \u0026times; loading buffer and loaded into a 12% gel using 20 \u0026micro;L. After electrophoresis for 1.5 h at 80 V, the gels were imaged by a molecular imager.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003edrug release\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePHK release from the A/P-PHK NPs was studied by dialysis method by using PBS (0.01M, pH 7.4) with different GSH concentrations of 0, 0.01, 1, or 10 mM as the release medium. First, the A/P-PHK NPs (3 mg/mL) were added into a dialysis tube (MWCO: 3.5 kDa), and the tube was immersed and incubated in 50 mL of release medium at 37\u0026deg;C. At predetermined time points, 3 mL of release solution was collected and another 3 mL of fresh-released medium was added. The concentration of HK in the release medium was measured by HPLC. As a result of carrying out the experiments in triplicate, the average value is presented.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity experiment\u003c/h2\u003e \u003cp\u003eCCK-8 assay was conducted to evaluate the cytotoxicity of prepared NPs. Cells were grown in 96-well plates (6000 cells per well) for 24 h with 150 \u0026micro;L cell culture medium. Thereafter, the cells were treated with HK or NPs, at various concentrations for 48 h. Following the addition of 10 \u0026micro;L of CCK-8 solutions, the wells were further incubated for 2 h. Cell proliferation was monitored using a microplate reader at 450 nm. Cell viability is determined by the equation provided as follows:\u003c/p\u003e \u003cp\u003eCell viability (%) = (As-An)/(Ac-An) \u0026times; 100\u003c/p\u003e \u003cp\u003eWhere \u003cb\u003eAs\u003c/b\u003e is the sample group; \u003cb\u003eAc\u003c/b\u003e is the control group, in which, the cells were treated with complete DMEM, and \u003cb\u003eAn\u003c/b\u003e is the adsorption of complete RPMI 1640.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCellular uptake\u003c/h2\u003e \u003cp\u003eThe cells were first cultured in a six-well plate for 24 h. Then, they were treated for 4 h with coumarin-6 loaded NPs (500ng/mL coumarin-6). Immediately following treatment, cells were washed three times with PBS and imaged by a fluorescence microscope (Nikon-112, Nikon, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatical analysis\u003c/h2\u003e \u003cp\u003eIn analyzing the differences between the groups, one-way ANOVA with Tukey multiple comparisons tests (\u0026lowast; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u0026lowast;\u0026lowast; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and \u0026lowast;\u0026lowast;\u0026lowast; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was carried out.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHK polyprodrug preparation and characterization\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Ap-PHK and PEG-PHK were synthesized in two steps: (i) HK was polymerized with 3,3\u0026prime;-dithiodipropionyl chloride, and then modified with Mal-NH\u003csub\u003e2\u003c/sub\u003e to obtain Mal-PHK; (ii) Ap or PEG-SH were conjugated to Mal-PHK \u003cem\u003evia\u003c/em\u003e click reaction to obtain Ap-PHK and PEG-PHK, respectively. The \u003csup\u003e1\u003c/sup\u003eH NMR and GPC were used to characterize the structure of Mal-PHK. In the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum, all the peaks were well assigned (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The peaks at 4.9\u0026ndash;5.1 ppm, 5.9-6.0 ppm, and 6.8\u0026thinsp;~\u0026thinsp;7.5 ppm is attributed to the double bond and benzene ring of HK, respectively. The peaks at 2.7 and 2.8 ppm are derived from 3,3\u0026prime;-dithiodipropionic acid. The peaks at 2.0, 4.3\u0026ndash;4.6 ppm were belonged to Mal. Moreover, the GPC results showed that the \u003cem\u003eM\u003c/em\u003ew of Mal-PHK was around 10900 Da, and its \u003cem\u003eM\u003c/em\u003ew distribution (PDI) was close to 1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results confirmed the successfully prepared Mal-PHK.\u003c/p\u003e \u003cp\u003eSubsequently, Ap or PEG-SH was conjugated to Mal-PHK to obtain Ap-PHK and PEG-PHK, respectively. The \u003csup\u003e1\u003c/sup\u003eH NMR and GPC results demonstrated that the PEG-PHK was also successfully synthesized. The GPC results showed that the \u003cem\u003eM\u003c/em\u003ew of Ap-PHK and PEG-PHK was 17540 Da and 17157 Da, respectively, with both PDIs close to 1. Additionally, the successful preparation of Ap-PHK was confirmed by PGAE and GPC (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the PAGE image, the migration of Ap-PHK was significantly lower than free Ap, suggesting that the molecular weight increased after the reaction of Ap and Mal-PHK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results also demonstrated the successful preparation of Ap-PHK.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGPC results.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolymers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM\u003c/em\u003en (Da)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eM\u003c/em\u003ew (Da)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePDI\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMal-PHK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10912\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAp-PHK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17540\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG-PHK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNote\u003c/b\u003e: \u003cem\u003ea: number\u0026ndash;average molecular weight. b: weight-average molecular weight. c: polymer dispersity index (Mw/Mn).\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eA/P-PHK NPs preparation and optimization\u003c/h2\u003e \u003cp\u003eA/P-PHK NPs were prepared by dialysis method according to the previous report with several modifications. According to the previous reports, the target segments on the surface of NPs are not \u0026ldquo;more is better\u0026rdquo;. NPs with a high density of target segments may result in low binding ability to their target. Thereby, a series of A/P-PHK NPs with different molar ratios between Ap-PHK and PEG-PHK were prepared, and these NPs were denoted as PEG-PHK NP, A/P-PHK NP20 to A/P-PHK NP80, and Ap-PHK NPs, respectively, with the increase of the molar ratio of Ap-PHK (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The particle size, zeta potential, and HK content of NPs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA to \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD. It can be found that the particle size of NPs and size distribution index (polymer dispersity index, PDI) with the different molar ratio of Ap-PHK and PEG-PHK has no significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), while the zeta potential is increased with the increase of the molar ratio of Ap-PHK, because of the high negative charge characteristic of nucleic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Moreover, the HK content decreases with the increase of the molar ratio of Ap-PHK, because of the high molecular weight of Ap than that of PEG (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eSubsequently, the cancer cells targeting capability of these NPs were investigated in EpCAM overexpression human ovarian cancer SKOV3 cells and OVCAR3 cells by fluorescence microscope and flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, after a 4 h treatment, the fluorescence image results in SKOV3 cells showed that the NPs contained 40% of Ap-PHK (A/P-PHK NP40) had the highest fluorescence intensity compared with no Ap-modified NPs (PEG-PHK NPs) and other Ap-modified NPs, suggesting that the A/P-PHK NP40 has the highest targeting ability. This result was also verified by the quantitative results of flow cytometry in two EpCAM overexpression cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Thus, A/P-PHK NP40 was used in the following studies.\u003c/p\u003e \u003cp\u003eMoreover, to further confirm the increased uptake of A/P-PHK NP40 by cells is attributed to the specifical targeting of Ap, the EpCAM overexpression cells (OVCAR3 and SKOV3) and EpCAM negative cells (A2780) were pretreated with EpCAM antibody, and then treated with coumarin-6 loaded A/P-PHK NP40. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, in the EpCAM overexpression OVCAR3 and SKOV3 cells, antibody pretreated significantly inhibited the cell uptake of A/P-PHK NP40, while in EpCAM negative A2780 cells, the cell uptake has no significant difference with or without antibody pretreatment. These results demonstrated that Ap modification significantly improves the OC targeting of NPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComponent of different NPs (molar content %).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePEG-PHK NP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA/P-PHK NP20\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA/P-PHK NP40\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA/P-PHK NP60\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA/P-PHK NP80\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAp-PHK NP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAp-PHK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG-PHK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGSH-responsiveness investigation\u003c/h2\u003e \u003cp\u003eIn the design, the disulfide bond within A/P-PHK NPs could cleave and release active HK in a rich environment. To confirm this conception, the degradation of A/P-PHK NPs after incubation in 10 mM GSH for 12 h was observed by TEM. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, A/P-PHK NPs have spheroid morphology and uniform distribution in water. On the contrary, after being treated with GSH, the A/P-PHK NPs were degraded, and cannot observe obvious particles. Dynamic light scattering also demonstrated the structural degradation of A/P-PHK NPs in response to GSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eSubsequently, the dialysis method was employed to evaluate the release of HK in various conditions. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, the HK release rate is positively associated with the GSH concentration. In detail, in the absence of GSH, almost no HK was released from A/P-PHK NPs, as supported by the cumulative release of HK was lower than 10% at 48 h. In the low GSH condition (0.01 mM GSH), the release of HK was increased compared with the GSH absence condition, while only 8% of HK was released from NPs at 48 h. When the concentration of GSH increased to 10 mM, the release rate of HK was significant, and around 87.3% of HK was released within 48 h. These results demonstrated the good GSH-responsiveness of A/P-PHK NPs and can avoid premature leakage in low GSH conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eanti-OC efficiency\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter a step-by-step evaluation, the \u003cem\u003ein vitro\u003c/em\u003e anti-OC efficiency of optimized A/P-PHK NPs was further measured in SKOV3, OVCAR3, and A2780 cells \u003cem\u003evia\u003c/em\u003e CCK-8 detection. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA to \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, after incubation for 48 h, the growth inhibition efficiency of NPs groups was all higher than that of the free HK group, suggesting that the polyprodrug strategy can enhance the therapeutics efficiency of HK. Moreover, in the EpCAM negative expression A2780 cells, the growth inhibition efficiency of PEG-PHK NPs, A/P-PHK NP40, and Ap-PHK NPs is not significantly different. On the contrary, in the EpCAM overexpression SKOV3 and OVCAR3 cells, the growth inhibition efficiency of A/P-PHK NP40 was significantly higher than other NPs, as evidenced by the IC\u003csub\u003e50\u003c/sub\u003e value of A/P-PHK NP40 was 1.5/1.5 and 1.4/1.3-fold lower than that of PEG-PHK NPs and Ap-PHK NPs in SKOV3 and OVCAR3 cells, respectively. These results, further demonstrated that the aptamer-modified GSH-sensitive polyprodrug strategy can significantly enhance the therapeutics efficiency of HK.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we created a novel aptamer-modified GSH-sensitive HK-polyprodrug Ap-PHK, which provided a potential method for OC therapy. Ap-PHK can self-assemble into NPs in an aqueous solution with PEG-modified HK-polyprodrug PEG-PHK. The obtained NPs can specifically target EpCAM overexpression OC cells and release HK in cell-high GSH conditions. This designed strategy not only improves the anti-OC efficiency of HK but also provides a delivery strategy for other drugs with two modifiable groups similar to HK, such as curcumin, mitoxantrone, etc. All in all, this approach offers a potential new strategy for the treatment of OC and expands the application of traditional Chinese medicine.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003csup\u003e1\u003c/sup\u003eH NMR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eproton nuclear magnetic resonance spectra\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAp\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpCAM-specific aptamer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eA/P-PHK NPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereduction-responsive honokiol polyprodrug NPs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAp-PHK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAp-modified HK polyprodrug\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eN,N\u003c/em\u003e-dimethylformamide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edimethylsulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEpCAM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eepithelial cell adhesion molecules\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGPC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egel permeation chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglutathione\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHonokiol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-performance liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enanoparticles\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOvarian cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePAGE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolyacrylamide gel electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethe polymer dispersity index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePEG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epoly(ethylene glycol)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePEG-PHK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePEG-modified HK polyprodrug\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransmission electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\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\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.H.G and\u0026nbsp;L.P.S designed the study. C.H.G, Y.X.Y, L.J.W, and W.F.W collected and analyzed the data. C.H.G and L.P.S wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdityan S, Tran M, Bhavsar C, Wu SY (2020) Nano-therapeutics for modulating the tumour microenvironment: Design, development, and clinical translation. 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Front Pharmacol 13:934632. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2022.934632\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2022.934632\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":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":"Polyprodrug, GSH-responsive, cancer, aptamer targeting, honokiol","lastPublishedDoi":"10.21203/rs.3.rs-4783145/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4783145/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eOvarian cancer is a major threat to women's lives. Chinese medicine honokiol (HK) is a polyphenol isolated from Magnolia, which can effectively suppress the growth of ovarian cancer. However, low water solubility and lack of tumor-targeting ability have greatly hindered the clinical application of HK.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHerein, a glutathione (GSH) sensitive HK polyprodrug was prepared by using HK as the backbone. Then, an EpCAM-specific aptamer and poly(ethylene glycol) (PEG) were conjugated to HK polyprodrug, and the obtained polyprodrug was assembled into nanoparticles in water. The HK polyprodrug-formed nanoparticles achieved high drug loading and GSH-responsiveness drug release. Moreover, after optimization, HK polyprodrug nanoparticles (A/P-PHK NP40) formed by aptamer-modified and PEG-modified prodrug at feed molar ratio at 2: 3 had the highest ability to target EpCAM overexpression ovarian cancer cells. A/P-PHK NP40 also exhibited a higher cell growth inhibition effect in ovarian cancer cells than free HK and control HK nanoparticles.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eAll in all, this work reported a novel strategy for HK delivery based on microenvironment responsiveness polyprodrug, which provided a potential method for ovarian cancer targeting therapy.\u003c/p\u003e","manuscriptTitle":"Aptamer-modified GSH-sensitive honokiol polyprodrug nanoparticles for ovarian cancer-specific targeting therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-27 09:51:07","doi":"10.21203/rs.3.rs-4783145/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":"f5fc4fe2-18f6-4e78-a817-928c17cca882","owner":[],"postedDate":"August 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-22T12:23:55+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-27 09:51:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4783145","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4783145","identity":"rs-4783145","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}