Dual Stable Nanomedicines Prepared by Cisplatin-Crosslinked

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Dual Stable Nanomedicines Constructed by Cisplatin Crosslinked Camptothecin Prodrug Micelles for Effective Drug Delivery Yinwen Li, Hongzhi Lu, Shiming Liang, and Shoufang Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 22, 2019

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ACS Applied Materials & Interfaces

Dual Stable Nanomedicines Constructed by Cisplatin Crosslinked Camptothecin Prodrug Micelles for Effective Drug Delivery Yinwen Li a, *, Hongzhi Lu a, Shiming Liang a, Shoufang Xu a, * a School of Materials Science & Engineering, Linyi University, Linyi 276000, People’s Republic of China

*

Corresponding Author: Prof. Y. W. Li; S. F. Xu

*E-mail: [email protected]; [email protected]. Address: Shuangling Road, Linyi 276000, China. Tel: +86-539-7258660. Fax: +86-539-7258660.

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ABSTRACT: Polymer micelles based drug delivery system has a bravely faced deficiency for lack of stability especially after diluted in blood and resulted in premature release. Herein, we developed a camptothecin (CPT)-conjugated prodrug micelles (CPTP) in which CPT was grafted to the poly (ethylene glycol)-poly (glutamic acid) block copolymer via disulfide bond linker for a redox-triggered drug release. Then the cisplatin (CDDP) crosslinked CPT-prodrug micelles (CPTP/CDDP) with hybrid complex as stable structure were successfully established via CDDP (Pt)-carboxyl (COOH) chelate interaction. The resultant dual CPTP/CDDP had average hydrodynamic radius about 50 nm with narrow distribution, which was conducive to the promotion of solid tumor accumulation. Importantly, CPT chemical bonding to the polymer backbone obviously stabilizes the CPT-prodrug micelles and prolongs its circulation time. Moreover, both CPT and CDDP are clinically used antitumor drugs, CDDP not only behaves ancillary anticarcinogen but also serves as a crosslinker to restraint the untimely and burst release of CPT and to achieve synergistic antitumor efficacy. In addition, the CPTP/CDDP also exhibited a sustained reductive responsive release of CPT accompanied with the dissociative of CDDP-COOH complex. This design ingeniously solved the contradiction between stability and release of polymer micelles based nanomedicines. Both in vitro and in vivo tests demonstrated an amazing antineoplastic efficacy compared with free drugs (CPT or CDDP) and their just physical mixing, and indicating its great promising for cancer treatments. Key Words: Cisplatin; CPT-prodrug micelles; Coordination; Stability; Nanomedicines


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1. INTRODUCTION Drugs loaded in nanocarriers, referred to as drug delivery system or nanomedicines are formed by the combination of chemotherapeutic drugs through electrostatic adsorption1, 2, complexation3-7 and covalent bonding8-10 with nanocarriers to improve their water solubility, extend their blood circulation time, target cancerous tissues by enhanced permeability and retention (EPR) effect and/or using targeting groups11, and finally improved antitumor efficacy, which make it being a mainstay in clinical cancer treatment12-14. Among kinds of nanocarriers15, polymer micelles are regarded as ideal drug carriers because of its significantly merits, such as nano-size, core-shell structure, and relatively high stability, etc16-20. And so far, many polymer micelles based nanomedicines (NK91121, NC-600422, and Genexol-PM23, etc.) have entered the clinical trial stages24, 25. However, it is indeed reduced the toxic and side effects of the original drugs, but its antitumor effect does not meet the expectations. This perplexing puzzle is reckoned to the premature drugs release before reaching tumor cells because of the imperfect stability against dilution and scouring in plasma26-28. Therefore, stability is the key prerequisite which polymer micelles based nanomedicines are facing at present. Currently, diverse chemical crosslinking approaches have been successfully used to make micelles stable by strongly holding the assembled nanostructures29-31. In addition, more and more elaborately crosslinked methods and structures for enhancing antitumor efficacy were designed32-34. However, most of the above designs have more elaborate structures and components and require abundantly time-consuming and laborious organic and polymer synthesis. Moreover, the degree and density of chemical crosslinking are inevitably involved and thus increase the complexity and uncertainty of clinical translation. Recently, metal-organic complexes have received significant attention in numerous realms especially in biomedical and diagnostic fields35-39. In particular, using metal-organic complexes as drug delivery platform is of particular interest to researchers40-42. For instance, Kataoka et al.43 prepared a cisplatin-incorporated PEG-polyglutamate (Glu) block copolymer micelle via carboxylate complexation with Pt(II) ions (NC-6004). Although NC-6004 did not significantly improved the antitumor effect compared with free CDDP, it significantly reduced the drug toxicity. Lee et al.44 reported a pH-responsive core-shell polymer micelle



with catechol-ferri ion coordinated core crosslinks, which provided robustness to drug-loaded polymer micelles and also allowed the intracellular release of loaded anticancer drugs. Chen et al.45 fabricated a cisplatin (CDDP)-crosslinked hyaluronic acid nanogel for effective delivery of doxorubicin (DOX), in which CDDP acted as a crosslinker and anticarcinogen to prevent the premature release of DOX and to achieve synergistic therapeutic performance. On the other hand, compared to simple encapsulation by the nanoassemblies through physical interactions, the drug molecules direct or via linkers chemically bonded to polymer chains referred to as polymer-drug conjugates or prodrugs have been extensively proposed and studied46-50. As a consequence, polymer-drug conjugates can self-assemble into prodrug micelles with lipophilic drugs as the hydrophobic inside cavity. The obtained prodrug micelles have all advantages of traditional micelles, and the deadly shortcomings such as dynamic instability, untimely and burst release can be eliminated easily. Moreover, the prodrugs also could be used as drug carriers and result in co-existence of the encapsulated drug and the conjugated drug jointly, which increase the drug content and improve the drug release kinetics51-55. Herein, we describe a facile strategy to introduce chelate crosslinking with prodrug micelles to heighten the stability and antitumor efficacy of polymer micelles based nanomedicines jointly. To be specific, camptothecin (CPT)-prodrug with poly (ethylene glycol)-poly (glutamic acid) block copolymer as reactive scaffold and with the attachment of CPT via the disulfide bond was designed, CPT-prodrug micelles (CPTP) were formed firstly, and then the dual stable cisplatin (CDDP) crosslinked CPT-prodrug micelles (CPTP/CDDP) with hybrid CDDP-COOH complex as crosslinked structures were constructed using the carboxyl (COOH) coordinated with CDDP. The resultant CPTP/CDDP showed a remarkably prolonged blood circulation, controlled CPT release, and high antitumor efficacy. The excellent antitumor efficacy make the CPTP/CDDP as promising nanomedicines for cancer therapy (Figure 1), and this study might offer a new formulation by combination of chemotherapeutic drugs itself as chelate crosslinking agents and prodrugs for jointly improving the stability of polymer micelles based nanomedicines. 2. EXPERIMENTAL SECTION 2.1 Materials. γ-benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) was prepared


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referring to previously reported procedure56. Methoxy poly (ethylene glycol) amine (mPEG-NH2, Mn=5000 Da) was bought from Yare Biotech Co., Ltd. (Shanghai, China). Camptothecin (CPT), 2-hydroxyethyl disulfide (DTE), dithiothreitol (DTT), triphosgene, trifluoroacetic acid (TFA), and hydrogen bromide (HBr, 33 wt.% in acetic acid) were all bought from Energy Chemical Co., Ltd. (Shanghai, China). Cisplatin (CDDP) was bought from Boyuan Chemical Co., Ltd. (Jinan, China). Other reagents and solvents were all bought from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Dichloromethane (DCM), tetrahydrofuran (THF), N, N-dimetylformamide (DMF) were all dried over calcium hydride (CaH2) and distilled before use. Dechlorination of CDDP were react with AgNO3, respectively, and the detailed procedure of CDDP was as follows: CDDP (100 mg) and AgNO3 (112 mg) were all dissolved into 5 mL H2O, then shaken in darkness at room temperature for 24 h and filtrated with a 0.22 μm filter, finally obtained the dechlorinated CDDP solution (20 mg mL-1). 2.2. Instruments. The 1H NMR spectra were recorded on a Bruker ECX 400 spectrometer operating at 400 MHz using CDCl3 and DMSO-d6 as solvents. The gel permeation chromatography (GPC/SEC) was determined by a Wyatt GPC/SEC-MALS (Wyatt Technology Corp., USA) system using DMF as mobile phase with a flow rate of 0.8 mL min-1. The sizes and distribution were determined by dynamic laser light scattering (DLS, Nano-ZS, Malvern Instrument Ltd., UK). The transmission electron microscope (TEM, JEM-1200EX, JEOL, Japan) was used to characterize the micelle morphology and the samples was dropped onto a copper grid, and stained with 1% (w/v) phosphotungstic acid solutions for 30 s. The UV-Vis and fluorescence spectra were recorded on microplate reader (SpectraMax® M2/M2e; Molecular devices, USA). The MTT assay was evaluated by measuring the absorbance at 562 and 620 nm on a SpectraMax M2e (Molecular Devices, USA). The CPT amounts were determined by reverse-phase HPLC (Agilent Technologies, USA) using a C18 column at 35 oC, and the mobile phase was a mixture of triethylamine acetate (TEAA) buffer (pH 5.5-5.9) and acetonitrile with a ratio of 7:3 (v/v), and the UV detector was performed at 368 nm. The CDDP concentrations (Pt content, μg mL-1) were determined by inductively coupled plasma mass spectrometry (ICP-MS) (Perkin Elmer Optima 3100XL, USA).



2.3. Synthesis of Methoxypolyethylene Glycols-Poly (glutamic acid) (PEG-b-PGA). PEG-b-PGA was synthesized via the ring-opening polymerization of BLG-NCA and subsequent deprotection. Taking PEG-b-PGA40 for example, mPEG-NH2 (1 mmol) and BLG-NCA (40 mmol) was all dissolved in 30 mL dehydrated DMF with a nitrogen protected atmosphere. The polymerization was performed at 35 oC for 96 h and precipitated by excess absolute diethyl ether. After dried under vacuum a white solid (PEG-b-PGAp) was obtained. 1H

NMR (400 MHz, CDCl3): δ (ppm) 7.27 (5H, C6H6CH2-), 5.06-5.01 (2H, C6H6CH2-), 3.93

(1H, -COCH-), 3.39-3.80 (4nH, -CH2CH2-), 3.34 (3H, CH3O-), 2.21 (2H, -CH2COO-), 2.05 (2H, -CHCH2-). The deprotection of PEG-b-PGAp is described as follows: firstly dissolved the PEG-b-PGAp in 20 mL TFA, then added 15 mL of HBr and stirred at room temperature for 5 h before precipitated into excess absolute diethyl ether. The precipitate was solved in appropriate amount of DMF and dialyzed (MWCO 3500) with deionized water for 24 h, and finally freeze-dried to obtain target product. 2.4. Synthesis of Camptothecin Derivatives (CPT-DTE). CPT (0.38 g) and DMAP (0.37 g) were suspended in anhydrous DCM. After stirring for 30 min turned into bright yellow clear liquid, added triphosgene (0.11 g) and stirred for 4 h, then added 2, 2-dithiodiethanol (0.25 g) was and stirred for 48 h under ambient condition. The obtained mixture was filtrated, washed with HCl aqueous solution, brine and water for three times, respectively. The organic phase was separated, collected and dried over anhydrous MgSO4, then concentrated by rotary evaporation and the residue was recrystallized from chloroform/methanol (3:10/v: v), finally purified by column chromatography (dichloromethane/methanol, 20:1/v: v). 1H NMR (400 MHz, CDCl3): δ (ppm) 8.45 (s, 1H), 8.27 (d, J=8.5 Hz, 1H), 7.97 (d, J=8.0 Hz, 1H), 7.88 (t, J=7.7 Hz, 1H), 7.71 (t, J=7.4 Hz, 1H), 7.51 (d, J=9.5 Hz, 1H), 5.74 (t, J=16.8 Hz, 1H), 5.35 (dd, J=41.1, 18.6 Hz, 3H), 4.56-4.27 (m, 2H), 4.04-3.84 (m, 2H), 3.13-2.75 (m, 4H), 2.44-2.18 (m, 2H), 1.13-0.95 (m, 3H). 2.5. Synthesis of Camptothecin Grafting Methoxypolyethylene Glycols-Poly (glutamic acid) (PEG-b-PGA-CPT). Taking PEG-b-PGA40 for example, PEG-b-PGA40 (1 mmol, GA repeating unit: 40 mmol) and DTE-CPT (7 mmol) was all dissolved in 20 mL DMSO containing DMAP (1mmol) and DCC (7 mmol) and stirred for 2 h at 0 °C, and followed with stir at 30 oC for 48 h, the reaction process is tracked by TLC. After 48 h no residual CPT


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existed then precipitated into excess ether, and the precipitate was further purified via dialysis (MWCO 3500) against DMSO followed by lyophilisation. 2.6. Preparation of CDDP Crosslinked CPT-prodrug Micelles. (1) Preparation of CPT-prodrug micelles (short for CPTP). PEG-b-PGA-CPT (10 mg) was dissolved in DMSO (1 mL) under vigorous stirring, and then added slowly into 10 mL deionized water at a flow rate of about 0.2 mL min-1. After addition finished, the dispersion solution was stirred for another 1 h. The CPTP (1 mg mL-1) was obtained by dialysis against deionized water. (2) Preparation of CDDP crosslinked CPT-prodrug micelles (short for CPTP/CDDP). Taken 5 mL CPTP, added dechlorination CDDP with different molar ratios (MCOOH:MCDDP =4:1, 2:1, 1:1 and 1:2, separately), then shaken at 37 oC for 72 h away from light, finally CPTP/CDDP was obtained by dialysis (MWCO 3500) away from light to remove free CDDP. The CPT encapsulation efficiency (EE, %) and the CPT loading content (DLC, %) were obtained by the HPLC measurement. 2.7. Stability of CPTP/CDDP. (1) Stability was evaluated by diluting the CPTP and CPTP/CDDP with fetal bovine serum (FBS) from 1.0 mg mL-1 to 1.0×10-3 mg mL-1, respectively, and the particles size and distribution were determined by DLS. (2) Stability was further determined by fluorescence method. Briefly, Nile red (1.0×10-4 mol L-1, 20 μL) in CH2Cl2 was put into each vial away from light, after CH2Cl2 was evaporated absolutely followed by adding micelles (4 mL, 1.0 mg mL-1). All vials were stirred at 37 °C for different times in darkness. The fluorescence emission intensity over time was measured and calculated at the wavelength of 620 nm (exited at 560 nm). (3) Both CPTP and CPTP/CDDP were all treated with DTT (10 mM) at 37 oC in PBS (pH 5.0, 7.4) buffer, the particle size and distribution of CPTP and CPTP/CDDP were monitored by DLS at determined time intervals. 2.8. In Vitro Release and Blood Circulation. (1) The CPT release was determined at 37 oC in different media: PBS buffer (0.01 M, pH 7.4), PBS buffer (0.01 M, pH 7.4) with DTT, and PBS buffer (0.01 M, pH 5.0) with DTT. 2.0 mL CPTP or CPTP/CDDP were added into a dialysis bag (MWCO 3500), and dialyzed against 40 mL mediums in a 37 °C shaker with a speed of 200 r min-1, respectively. 100 μL solutions were taken out and equal volume mediums were added meanwhile at predetermined time intervals. The CPT concentrations were determined by reverse-phase HPLC. (2) The ICR mice were randomly divided into



three groups and injected intravenously with Irinotecan, CPTP and CPTP/CDDP, respectively. At predetermined time point (5 min, 1 h, 2 h, 6 h, 12 h, 24 h, and 48 h), blood samples (50 μL) were taken out from eyes retro-orbital plexus, heparinized, and centrifuged (12000 rpm, 5 min) to obtain the plasma. The obtained plasma (20 μL) was extracted with acetonitrile, centrifuged, and then subject to reverse-phase HPLC analysis 2.9. In Vitro Cytotoxicity Assays. The cytotoxicity of CPTP and CPTP/CDDP was determined using 4T1 cells by MTT assay. Briefly, 4T1 cells were evenly seeded in 96-well culture plates with a density about 5.0×103 cells per well in 100 μL DMEM and cultured at 37 °C in CO2 incubator for 24 h. Then the cells were incubated with 200 μL DMEM with serial dilutions of Irinotecan, CDDP, CPTP and CPTP/CDDP, and CDDP mixed with CPTP freshly (short for CPTP+CDDP) for another 48 h, 72 h, respectively. After above treatment, the 96-well culture plates were centrifuged for 5 min at 3000 rmp, and the cells were incubated with fresh 200 μL DMEM containing 20 μL (0.75 mg mL-1) of MTT for 4 h. Finally, the culture medium was replaced by 200 μL DMSO to dissolve the formazan crystals. The cell viability was determined in a BioRad 680 microplate reader under the wavelength of 560 nm and 612 nm. 2.10. Biodistribution of polymers. Balb/C mice were subcutaneously inoculated with 4T1 tumors. After the tumors grown up to about 100 mm3, 200 μL Cy5 labeled CPTP and CPTP/CDDP (5 mg kg-1 on the basis of CDDP) were injected intravenously. Each group with 4 mice was imaged using a non-invasive NIRF imaging system (PerkinElmer IVIS Lumina XRMS Series Ⅲ imaging system) at predetermined time point (1 h, 2 h, 6 h, 12 h, and 24 h). Then sacrificed the mice as soon as the experiment was finished, major organs (heart, liver, spleen, lung, kidney and intestine) and tumors were collected, washed with 0.9% saline rapidly for imaging and analyzing via fluorescence quantization. 2.11. In Vivo Antitumor Efficiency and Histological Analysis. Female Balb/C mice (6 weeks old, 20 g body weight) were bought from SLRC Laboratory Animal Company (Shanghai, China). All mice received full care in according with the guidelines in the Guide for the Care and Use of Laboratory Animals and all procedures were approved by the Animal Care and Use Committee of Linyi University. A mouse orthotopical breast adenocarcinoma implantation model was established by subcutaneous injection of 4T1 cells (0.15 mL, 1.5 ×


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106 cells). When the tumor grown up to about 50 mm3, six mice were randomly assigned as a group, and the mice were injected via tail vein with PBS control, Irinotecan (5 mg kg-1), CPTP (at an equivalent CPT dose of 5 mg kg-1), CPTP/CDDP (at an equivalent CPT and CDDP dose of 5 mg kg-1 and 2 mg kg-1, respectively), and CPTP+CDDP (at an equivalent CPT and CDDP dose of 5 mg kg-1 and 2 mg kg-1, respectively) on every other day. The sizes of tumor and body weights were measured every two days during the treatment process. The tumor volume (V; mm3) was evaluated by the following equation: V(mm3) =

𝑡h𝑒 𝑙𝑜𝑛𝑔𝑒𝑠𝑡 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 (𝑚𝑚) × 𝑡h𝑒 𝑠h𝑜𝑟𝑡𝑒𝑠𝑡 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟2 (𝑚𝑚) 2

Finally, sacrificed the mice, taken out and weighted the tumor. The tumor inhibition rate (IRT; %) was calculated using the following formula: IRT (%) =

𝑉𝑐𝑜𝑛𝑡𝑟𝑜𝑙 –𝑉𝑠𝑎𝑚𝑝𝑙𝑒 𝑉𝑐𝑜𝑛𝑡𝑟𝑜𝑙

× 100%

Where, Vsample and Vcontrol represented the tumor volumes of experiment groups and control group, respectively. The histological examination was summarized as follows: the collected tumors and major organs were fixed with 4% paraformaldehyde for 24 h followed by paraffin fixation and cut into 5 μm thickness slices. At last, stained with haematoxylin/eosin (H&E) for histological assessment under the optical microscopy.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of PEG-b-PGA-CPT. As illustrated in Figure 2, PEG-b-PGA-CPT was prepared as follows: firstly, block copolymer mPEG-PGAp with different glutamic acid (GA) repeating unit (20, 40, and 60) were synthesized by ring-opening polymerization of BLU-NCA initiated by active methoxy polyethylene glycol amine (mPEG-NH2). The intermediates and target products were characterized through the NMR and GPC (Figure S1, S2, S3). The polymerization degree of glutamic acid (GA) were obtained by using the peak integral ratios of CH3O- (3.34 ppm) in mPEG to C6H5- (5H, 7.27 ppm) or C6H5CH2- (2H, 5.01~5.06 ppm) in PGAp. Then reacted with trifluoroacetic acid the graft benzyl groups converted to carboxyl groups and PEG-b-PGA was finally obtained. Lastly, the CPT-prodrug, PEG-b-PGA-CPT was prepared by esterification between the graft carboxyl groups of PEG-b-PGA and the hydroxyl group of camptothecin derivative (CPT-DTE) which was synthesized from camptothecin (CPT) and 2-hydroxyethyl disulfide



(DTE) according to previous report with small modification57. The molecular weight (Mn, Mw, and PDI), polymerization degree of glutamic acid (GA) and CPT grafting amount are shown in Table S1. Taking the PEG-b-PGA40-CPT7 for example, in order to ensure adequate carboxyl residue, the feed ratio (MCOOH:MCPT=40:7) of CPT grafting esterification was strictly controlled and the reaction process was tracked by TLC. Moreover, the following CPT release amount measured by HPLC further confirmed the CPT grafting amount (w/w, %) was about 20.6%, which was basically consistent with the theoretical feeding quantity. 3.2. Preparation and Characterization of CPTP/CDDP. The CPTP/CDDP was easily obtained in two steps. The first is to prepare the stable CPT-prodrug micelles (CPTP), then further stabilized with dechlorinated CDDP through CDDP (Pt)-carboxyl (COOH) chelate interaction and got the CPTP/CDDP. CPTP1, CPTP2, and CPTP3 were easily prepared from PEG-b-PGA20-CPT3, PEG-b-PGA40-CPT7 and PEG-b-PGA60-CPT10, respectively; and the DLS results were shown in Figure S4. However, when used for further crosslinking with CDDP under the theoretical chelate molar ratios (MCOOH:MCDDP =2:1), We found that the CPTP1, CPTP2 and CPTP3 indeed crosslinked with CDDP, but further increase the PGA repeating unit, CPTP3 would induce precipitation of crosslinked micelles. It is probably that the chelate ability of CDDP (Pt)-COOH pair is too strong, and results in the intermolecular coordination and precipitation. A similar result was obtained by previous report45. Therefore, PEG-b-PGA40-CPT7 with proper PGA units and considerable CPT grafting amounts (20.6%, w/w) was chosen to prepare the CPTP and CPTP/CDDP in the following discussion. As shown in Figure 3, when the MCDDP:MCOOH=1:1, the volume average hydrodynamic diameters of CPTP/CDDP were kept in 50 nm with narrow size distribution (PDI ≤ 0.14). Meanwhile, the TEM images results showed that both the CPTP and CPTP/CDDP presented regular spherical shape with average size of about 30 nm, and which were slightly smaller than that of corresponding DLS results. As known, the dechlorinated CDDP contains two coordination holes, 1 mol CDDP theoretically needs 2 mol COOHs to reach the coordination saturation. Series of CPTP/CDDP with different feed molar ratios (MCOOH:MCDDP =4:1, 2:1, 1:1 and 1:2) were prepared, and the residual CDDP amounts was measure by ICP-MS. Although the particle size of CPTP/CDDP remained basically unchanged (Figure S5), the DLC (%) of CDDP changed significantly;


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when the MCOOH:MCDDP =4:1 and 2:1, the DLC (%) of CDDP were 18.2% and 36.4% which were almost the same as the theoretical feed values (18.6% and 37.2%), and further indicated that almost all the CDDP was just act as crosslinkers. While as the molar ratio increasing to 1:1, the DLC (%) of CDDP were 40.2%, which were much lower than the feed value (74.0%) and further demonstrated that only few part of the excess CDDP was locked in the hydrophobic core of CPTP/CDDP. This result was different from the cisplatin-incorporated PEG-polyglutamate (Glu) block copolymer micelle via carboxylate complexation with CDDP which reported by Kataoka43, and this probably results from the hydrophobic inner core of CPTP/CDDP rejects the hydrophilic dechlorinated CDDP. Therefore, for overall considering the particle size, CPT grafting amount, and CDDP crosslink content and efficiency, the CPTP/CDDP were prepared with the MCDDP: MCOOH=1:1 for the following in vitro and in vivo study. 3.3. Stability of CPTP/CDDP. As known, stability is one of the most crucial properties for polymer micelles based nanomedicines, which can effectively help to avoid the untimely drug release dilemma during the delivery process. Firstly, both the CPTP and CPTP/CDDP were diluted from 1.0 mg mL-1 to 1.0×10-3 mg mL-1 with 10% FBS at 37 °C, separately. As shown in Figure 4, after diluted with FBS and incubated for 24 h, the hydrodynamic sizes of CPTP with different concentrations changed significantly. While for CPTP/CDDP, there was hardly changes of the hydrodynamic sizes, indicated that the chelate interaction between the CDDP (Pt) and carboxyl (COOH) was indeed improved the stability of CPT-prodrug micelles. Fluorescent probe technology is often used to track the formation of micelles, In order to further prove that the chelate crosslinking did take place between the CDDP (Pt) and carboxyl (COOH), Nile red served as an effective probe of characterization the change of hydrophilicity and hydrophobicity in microenvironment was chosen as the fluorescent probe to monitor this coordination process. As shown in Figure 4(c), the fluorescence intensity increased obviously with time increasing for the CPTP, while for the CPTP/CDDP, the fluorescence intensity increased very slowly and far below the uncrosslinked CPTP. In addition, the fluorescence intensity of the micelles prepared from CDDP directly coordinated with PEG-b-PGA40 was almost unchanged. These results clearly indicated that the



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coordination between the CDDP (Pt) and carboxyl (COOH) indeed created a tight crosslinking layer or structure, which prevented the migration of Nile red into hydrophobic nuclei composed of hydrophobic CPT segments. 3.3. In Vitro Release. In order to solve the contradiction between stability and controlled release of prodrugs, In this manuscript, among the polymer backbone and drug, a disulfide bond was introduced as a linker to make PEG-b-PGA-CPT reductively breakable with response to tumor microenvironment. In order to demonstrate the responsivity, the CPTP before and after CDDP crosslinked were blended with 10 mM DTT thoroughly, and the size and distribution were measured at predetermined time point (0 h ， 1 h, 6 h, and 12 h). As illustrated in Figure 5(a), (b), the average diameters of DTT treated CPTP were chaotically changed, resulting in disordered distribution of microsized and nanosized particles after 12 h. While for DTT treated CPTP/CDDP, the hydrodynamic sizes were still remain unchanged for 12 h, further indicated that the CDDP coordinated with residual carboxyl groups and formed stable crosslinked structures. In addition, we further adjust the pH to 7.4 and 5.0 of the DTT treated CPTP/CDDP. As shown in Figure 5 (c), (d), at pH 5.0 the average volume diameters of DTT treated CPTP/CDDP were chaotically changed, resulting in microsized particles and larger size distribution aggregations after 12 h; while at pH 7.4 the average volume diameters of DTT treated CPTP/CDDP were hardly changed. The in vitro drug release from CPTP before and after CDDP crosslinked was carried out in PBS (pH 7.4) at 37 oC. Since the CPT was chemical connected to the polymer via reductive disulfide linker, it is believed that the CPT could release under reductive condition50. Just as shown in Figure 6(a), both the drug release behaviors of the CPTP and CPTP/CDDP in the absence of DTT behaved gradually accumulative profiles, and nearly about 17.6% and 12.3% of CPT was released in 48 h resulted from the gradually hydrolysis of ester linker bond. While in the presence of 10 mM DTT, the CPT release rate accelerated apparently. For example, ca. nearly 90% CPT was released in 48 h for CPTP. It was thought that the structure of PEG-b-PGA-CPT was destroyed due to the cleavage of disulfide linker in a reductive condition, which resulted in fast CPT release. This was in line with the results of the CPT-prodrug micelles were destabilized in response to DTT. Moreover, only nearly about 20% CPT release within 48 h for CPTP/CDDP treatment with DTT. It is evident that the CDDP


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coordinated crosslinking indeed improved the stability of CPTP. In addition, for CPTP/CDDP treatment with DTT, when adjusting the pH to 5.0, at the beginning the CPT release behaved a low release rate; after 12 h the release rate becoming fast and reach to nearly about 70% CPT release at 48 h. 3.4. In Vitro Cytotoxicity. In order to investigate the antitumor efficacy of the CPTP/CDDP, the MTT assay was used to evaluate its cytotoxicity, and the in vitro cytotoxicity assays of Irinotecan, CDDP, CPTP, CPTP/CDDP, and CDDP mixed with CPTP freshly (short for CPTP+CDDP) were evaluated on 4T1 cancer cells. As shown in Figure 7(a), (b), both the CPTP and CPTP/CDDP showed dose- and time-dependent inhibiting efficacy which was comparable to that of free CDDP and Irinotecan. But the CPTP/CDDP was consistently lower toxic than that of free CDDP, Irinotecan, and even the CPTP+CDDP. The cytotoxicity of the CPTP with before and after CDDP crosslinking were evaluated in the presence and absence of 10 mM DTT using the 4T1 cell model to further confirm the CPT release under reductive tumor microenvironment in vitro. As shown in Figure7(c), (d), as expected, the CPTP with 10 mM DTT behaved a much stronger antitumor activity against 4T1 cells as the concentration increasing than that without DTT. While for CPTP/CDDP, as expected, both the CPTP/CDDP with before and after adding DTT showed relatively low cytotoxicity; moreover, unlike the CPTP with DTT, the change of cell cytotoxicity was not obvious for CPTP/CDDP in the presence of DTT, which further indicated that the formed stable crosslinked structure prevented DTT from contacting the disulfide linker, and thus exhibited low cytotoxicity. 3.5. Pharmacokinetics and Biodistribution. Subsequently, the in vivo pharmacokinetics of the CPT-prodrug micelles with before and after CDDP crosslinked was investigated. Blood samples were taken out at predetermined time point after a single intravenous injection. As illustrated in Figure 6(b), both CPTP and CPTP/CDDP showed remarkably extended blood circulation time, whereas Irinotecan was subject to a fast from blood circulation. The CPTP and CPTP/CDDP exhibited prolonged blood circulation with 8.6 % and 33.4 % of injected dose still remained in the blood even after 24 h. The relatively long circulation time of CPT-prodrug micelles especially for CDDP crosslinked CPT-prodrug micelles was conducive to increase accumulation of CPTP/CDDP in tumors via the EPR effect, and thus enhance the antitumor efficacy.



In addition, the biodistribution was also evaluated by determining the real-time fluorescence intensity in tumors and different organs. Firstly, PEG-b-PGA-CPT was covalently labeled with Cy5, then Cy5 labeled CPTP micelles with and without CDDP crosslinked were intravenously injected into 4T1-xenografted BALB/c mice, the time-related accumulation and distribution were monitored by using a non-invasive NIRF imaging system. As illustrated in Figure S6 and Figure 8, the NIRF signals in the whole bodies were obviously observed due to the rapid circulation of Cy5 labeled CPTP and CPTP/CDDP in the blood. Although there was a relatively high Cy5 labeled CPTP/CDDP level in the tumors and abundance of CPTP/CDDP were mainly accumulated in reticuloendothelial of main organs (heart, liver, spleen, lung, kidneys, and intestine), the NIRF signals in the tumor sites were almost as strong as that in the liver, indicating that the CPTP/CDDP had predominantly accumulated in the tumor tissue. 3.6. In Vivo Antitumor Efficacy. Finally, an orthotropic breast cancer mouse model was introduced to evaluate the antitumor efficacy of the CDDP crosslinked CPT-prodrug micelles. The tumor volume, growth situation and body weight were all monitored and recorded after intravenous injection of PBS control, Irinotecan, CPTP, CPTP/CDDP and CPTP+CDDP at a CPT equivalent dose of 5.0 mg kg-1 every two days from 7 days after the tumor volume reach to 50 mm3, respectively. As illustrated in Figure 9(a)-(d), in contrast to the PBS control groups and the other groups (Irinotecan, CPTP, and CPTP+CDDP), the CPTP/CDDP group could significantly inhibit and slow the 4T1 tumor growth; moreover, during the whole treatment process the body weights of mice behaved steady without significant weight loss. The CPTP/CDDP group exhibited the highest IRT of 80.4%, which indicated that the CPTP/CDDP not only had excellent antitumor efficacy, but also could reduce its toxicity and side effect. Meanwhile, the histological staining measurements of tumors and major organs (heart, liver, spleen, lung, and kidneys) were used to evaluate the therapeutic effects. As illustrated in Figure 9(e) and Figure S7, compared with the tightly and regularly dispersed tumor cells in the PBS control group, the tumor cells treated with the CPTP/CDDP group exhibited obvious vacuolization and apoptosis. In addition, the H&E staining results showed that there were no obvious pathological changes in these major organs in the CPTP/CDDP treated group and PBS groups, and further indicated the low in vivo toxicity. Therefore, the


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CDDP crosslinked CPT-prodrug micelles achieved high antitumor efficacy and low toxicity. 4. CONCLUSIONS In summary, we established CDDP crosslinked CPT-prodrug micelles (CPTP/CDDP) via CDDP (Pt)-carboxyl (COOH) coordination to effectively overcome multiple barriers for polymer micelles based nanomedicines. Besides the stability of the CPT-prodrug micelles (CPTP), after CDDP in situ crosslinked with COOH, the obtained CPTP/CDDP behaved a remarkably enhancement of stability and tolerability. Moreover, the CPTP/CDDP also exhibited a sustained reductive responsive release of CPT accompanied with the dissociative of CDDP-COOH complex. In addition, CDDP also as antineoplastic drug, and the CPTP/CDDP would exhibit synergistic antitumor effect. Based on these fabrications, those designs jointly led to the reduced multiorgan toxicity, optimized biodistribution, and enhanced antitumor potency of the drug in vivo. The results confirmed that the CPTP/CDDP presented remarkable performances in delivering CPT into tumor cells and suppressing the growth of 4T1 breast cancer xenografts with the IRT as high as 80.4% in vivo. In view of the above-mentioned merits the CPTP/CDDP had great potentials as antitumor nanomedicines for clinical application. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Molecular weight, polymerization degree and CPT grafting amount of PEG-b-PGA-CPT. GPC spectra of PEG-b-PGAp with different polymerization degree. 1H NMR spectra of CPT-DTE and PEG-b-PGA-CPT.

13C

NMR spectra of PEG-b-PGAp40. Sizes of CPTP and

CPTP/CDDP with feed molar ratios (MCOOH:MCDDP= 2:1). Sizes of CPTP/CDDP with different feed molar ratios (MCOOH:MCDDP= 4:1, 2:1, 1:1 and 1:2). Images of in vivo real-time biodistribution of Balb/C mice bearing 4T1 tumors injected with Cy5 labeled CPTP and CPTP/CDDP at predetermined time intervals. Representative histological images of major organs (liver, lung, spleen, kidney and heart) after stained with haematoxylin/eosin (H&E).

AUTHOR INFORMATION



Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors appreciate the Chinese Postdoctoral Science Foundation (2017M621908) and the Shandong Provincial Natural Science Foundation, China (ZR2017BB036), and the Natural Science Foundation of China (21777065).

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Graphical abstract


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Figure 1 Schematic illustration of dual stable nanomedicines constructed by cisplatin crosslinked camptothecin prodrug micelles for effective drug delivery.



Figure 2 Synthetic routes PEG-b-PGA-CPT and CPT-DTE.


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Figure 3 The sizes (a) and images of CPTP (b) and CPTP/CDDP with feed molar ratio (MCDDP: MCPTP =1:1) (c) measured by DLS and TEM.



Figure 4 Stability of CPTP and CPTP/CDDP. The CPTP (a) and CPTP/CDDP (b) diluted with FBS from 1.0 mg mL-1 to 1.0 × 10-3 mg mL-1 and kept for 24 h before the DLS measurement. The fluorescence intensity of CPTP and CPTP/CDDP with Nile red (1.0×10-4 mol L-1, 20 μL) as fluorescence probe and kept for 24 h before the measurement (c).


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Figure 5 Micelles sizes measured at different intervals by DLS. (a) CPTP; (b) CPTP /CDDP; (c) CPTP /CDDP with DTT at pH 7.4; (d) CPTP /CDDP with DTT at pH 5.0.



Figure 6 Drug release profiles (a) of CPTP and CPTP/CDDP with and without DTT in mediums of PBS (at pH 7.4 and 5.0, respectively). Blood circulation curves (b) of Irinotecan, CPTP, and CPTP/CDDP in ICR mice by measuring the concentration of CPT in blood at different time points post injection.


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Figure 7 Cytotoxicity assays of the CDDP, Iriontecan, CPTP, CPTP/CDDP and CPTP+CDDP incubated with 4T1 for 48 h (a) and 72 h (b), respectively; Cytotoxicity assays of CPTP (c) and CPTP/CDDP (d) with and without DTT incubated with 4T1 for 48 h, respectively. The concentration of CDDP is the same in all the groups.



Figure 8 In vivo real-time biodistribution of Balb/C mice bearing 4T1 tumors injected with Cy5 labeled CPTP (a) and CPTP/CDDP (b), and ex vivo NIRF imaging of isolated normal organs (liver, lung, spleen, kidney, heart and intestine) and tumors taken out at 24 h post-injection. A quantification of in vivo biodistribution was recorded as total photon counts per centimeter squared per steradian (p/sec/cm2/sr) per each excised organs at 24 h post-injection (c).


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Figure 9 The antitumor effects of Balb/C mice with 4T1 tumors received PBS, Irinotecan, CPTP, CPTP/CDDP and CPTP+CDDP. (a) Images of resected tumors; (b) Changes of tumor volumes during the whole treatment; (c) Body weight changes during the whole treatment; (d) the average tumor weights of each group after sacrificed and taken out from the mice. (e) Representative histological images of 4T1 tumor slices treated with PBS, Irinotecan, CPTP, CPTP/CDDP and CPTP+CDDP after stained with H&E (magnification×20), and the scale bar is 100 μm.


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