Dual Stable Nanomedicines Prepared by Cisplatin-Crosslinked

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Cite This: ACS Appl. Mater. Interfaces 2019, 11, 20649−20659

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

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ABSTRACT: A polymer micelle-based drug delivery system has faced many challenges due to the lack of stability especially after being diluted in blood, resulting in a premature release. Herein, we developed camptothecin (CPT)-conjugated prodrug (CPTP) micelles in which CPT was grafted to the poly(ethylene glycol)−poly(glutamic acid) block copolymer via a disulfide bond linker for a redox-triggered drug release. Then, the cisplatin (CDDP)-crosslinked CPT-prodrug micelles (CPTP/ CDDP) with a hybrid complex as a stable structure were successfully established via the CDDP (Pt)−carboxyl (COOH) chelate interaction. The resulting dual CPTP/CDDP had an average hydrodynamic radius of about 50 nm with a 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 their circulation time. Moreover, both CPT and CDDP are clinically used antitumor drugs; CDDP not only behaves as an ancillary anticarcinogen but also serves as a crosslinker to restrain the untimely burst release of CPT and to achieve synergistic antitumor efficacy. In addition, the CPTP/CDDP also exhibited a sustained reduction responsive release of CPT accompanied by the dissociation of the CDDP−COOH complex. This design ingeniously solved the contradiction between the stability and release of polymer micelle-based nanomedicines. Both in vitro and in vivo tests demonstrated an amazing antineoplastic efficacy compared with free drugs (CPT or CDDP) and just their physical mixing, indicating great promise for cancer treatment. KEYWORDS: cisplatin, CPT-prodrug micelles, coordination, stability, nanomedicines holding the assembled nanostructures.25,29,30 In addition, more and more elaborately crosslinked structures and crosslinking methods for enhancing antitumor efficacy were designed.31−33 However, most of the above designs have more elaborate structures and components and require laborious organic and polymer synthesis, which is more time-consuming. 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 fields.34−38 In particular, using metal−organic complexes as a drug delivery platform is of particular interest to researchers.39−41 For instance, Kataoka et al.42 prepared cisplatin (CDDP)-incorporated poly(ethylene glycol) (PEG)polyglutamate (Glu) block copolymer micelles via carboxylate complexation with Pt(II) ions (NC-6004). Although NC-6004 did not significantly improve the antitumor effect compared with free CDDP, it significantly reduced the drug toxicity. Lee et al.43 reported a pH-responsive core−shell polymer micelle

1. INTRODUCTION Drugs loaded in nanocarriers, referred to as drug delivery systems or nanomedicines, are formed by the combination of chemotherapeutic drugs through electrostatic adsorption,1,2 complexation,3−7 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 groups,11 and finally antitumor efficacy, which make it a mainstay in clinical cancer treatment.12−14 Among the different kinds of nanocarriers,15 polymer micelles are regarded as ideal drug carriers because of their significant merits, such as nanosize, core−shell structure, relatively high stability, etc.16−20 So far, many polymer micelle-based nanomedicines (NK911,21 NC-6004,22 Genexol-PM,23 etc.) have entered the clinical trial stages.24,25 Although they indeed reduced the toxic and side effects of the original drugs, their antitumor effect does not meet the expectations. This perplexing puzzle is reckoned to the premature drug release before reaching tumor cells because of the imperfect stability against dilution and scouring in plasma.26−28 Therefore, stability is the key problem that polymer micelle-based nanomedicines are facing at present. Currently, diverse chemical crosslinking approaches have been successfully used to make micelles stable by strongly © 2019 American Chemical Society

Received: March 4, 2019 Accepted: May 22, 2019 Published: May 22, 2019 20649

DOI: 10.1021/acsami.9b03960 ACS Appl. Mater. Interfaces 2019, 11, 20649−20659

Research Article

ACS Applied Materials & Interfaces

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

CPTP/CDDP as promising nanomedicines for cancer therapy (Figure 1), and this study might offer a new formulation by a combination of chemotherapeutic drugs themselves as chelate crosslinking agents and prodrugs for jointly improving the stability of polymer micelle-based nanomedicines.

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.44 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 directly or via linkers chemically bonded to polymer chains, referred to as polymer−drug conjugates or prodrugs, have been extensively proposed and studied.45−49 As a consequence, polymer−drug conjugates can self-assemble into prodrug micelles with lipophilic drugs as the hydrophobic inner cavity. The obtained prodrug micelles have all advantages of traditional micelles, and the deadly shortcomings, such as dynamic instability and untimely burst release, can be eliminated easily. Moreover, the prodrugs could also be used as drug carriers and result in the co-existence of the encapsulated drug and the conjugated drug jointly, which increase the drug content and improve the drug release kinetics.50−54 Herein, we describe a facile strategy to introduce chelate crosslinking with prodrug micelles to increase the stability and antitumor efficacy of polymer micelle-based nanomedicines jointly. To be specific, camptothecin (CPT)-prodrug with the poly(ethylene glycol)−poly(glutamic acid) block copolymer as a reactive scaffold and with the attachment of CPT via the disulfide bond was designed, CPT-prodrug (CPTP) micelles were formed first, and then the dual stable cisplatin (CDDP)crosslinked CPT-prodrug micelles (CPTP/CDDP) with the hybrid CDDP−COOH complex as crosslinked structures were prepared using the carboxyl (COOH) coordinated with CDDP. The resulting CPTP/CDDP showed a remarkably prolonged blood circulation, controlled CPT release, and high antitumor efficacy. The excellent antitumor efficacy makes the

2. EXPERIMENTAL SECTION 2.1. Materials. γ-Benzyl-L-glutamate-N-carboxyanhydride (BLGNCA) was prepared by referring the previously reported procedure.55 Methoxypoly(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, and N,N-dimethylformamide (DMF) were all dried over calcium hydride (CaH2) and distilled before use. Dechlorination of CDDP occurred with AgNO3, and the detailed procedure is as follows: CDDP (100 mg) and AgNO3 (112 mg) were dissolved in 5 mL of H2O, then shaken in the dark at room temperature for 24 h, and filtered with a 0.22 μm filter, and finally the dechlorinated CDDP solution (20 mg mL−1) was obtained. 2.2. Instruments. The 1H NMR spectra were recorded on a Bruker ECX 400 spectrometer operating at 400 MHz using CDCl3 and dimethyl sulfoxide (DMSO)-d6 as solvents. Gel permeation chromatography (GPC/size exclusion chromatography (SEC)) was carried out by a Wyatt GPC/SEC-MALS (Wyatt Technology Corp.) system using DMF as the 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 Instruments Ltd., U.K.). The transmission electron microscope (TEM, JEM-1200EX, JEOL, Japan) was used to characterize the micelle morphology, and the samples were 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 a microplate reader (SpectraMax M2/M2e, Molecular Devices). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was evaluated by measuring the 20650

DOI: 10.1021/acsami.9b03960 ACS Appl. Mater. Interfaces 2019, 11, 20649−20659

Research Article

ACS Applied Materials & Interfaces absorbance at 562 and 620 nm on SpectraMax M2e (Molecular Devices). The CPT amounts were determined by reverse-phase highperformance liquid chromatography (HPLC) (Agilent Technologies) using a C18 column at 35 °C, the mobile phase was a mixture of triethylamine acetate 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) (PerkinElmer Optima 3100XL). 2.3. Synthesis of Methoxypoly(ethylene 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) were dissolved in 30 mL of dehydrated DMF in a nitrogen-protected atmosphere. The polymerization was performed at 35 °C for 96 h followed by precipitation by excess absolute diethyl ether. After being 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 (4H, −CH 2CH 2−), 3.34 (3H, CH3O−), 2.21 (2H, −CH2COO−), 2.05 (2H, −CHCH2−). The deprotection of PEG-b-PGAp is described as follows: first, PEG-bPGAp was dissolved in 20 mL of TFA, then 15 mL of HBr was added and stirred at room temperature for 5 h before being precipitated into excess absolute diethyl ether. The precipitate was dissolved in an appropriate amount of DMF, dialyzed (molecular weight cut off (MWCO) 3500) with deionized water for 24 h, and finally freezedried to obtain the target product. 2.4. Synthesis of Camptothecin Derivatives (CPT-DTE). CPT (0.38 g) and 4-dimethylaminopyridine (DMAP) (0.37 g) were suspended in anhydrous DCM. After stirring for 30 min, the reaction mixture turned into a bright yellow clear liquid, triphosgene (0.11 g) was added and stirred for 4 h, and then 2,2-dithiodiethanol (0.25 g) was added and stirred for 48 h under ambient conditions. The obtained mixture was filtered and washed with HCl aqueous solution, brine, and water three times, respectively. The organic phase was separated, collected, dried over anhydrous MgSO4, and then concentrated by rotary evaporation, and the residue was recrystallized from chloroform/methanol (3:10, v/v) and 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-Grafted Methoxypoly(ethylene glycols)-Poly(glutamic acid) (PEG-b-PGA-CPT). Taking PEG-b-PGA40 for example, PEG-b-PGA40 (1 mmol, glutamic acid (GA) repeating unit: 40 mmol) and DTE-CPT (7 mmol) were dissolved in 20 mL of DMSO containing DMAP (1 mmol) and dicyclohexylcarbodiimide (7 mmol), stirred for 2 h at 0 °C followed by stirring at 30 °C for 48 h, and the reaction process is tracked by thin-layer chromatography (TLC). After 48 h, no residual CPT existed and then precipitated into excess ether, and the precipitate was further purified via dialysis (MWCO 3500) against DMSO, followed by lyophilization. 2.6. Preparation of CDDP-Crosslinked CPT-Prodrug Micelles. (1) Preparation of CPT-prodrug micelles (short form CPTP). PEG-b-PGA-CPT (10 mg) was dissolved in DMSO (1 mL) under vigorous stirring and was then added slowly into 10 mL of deionized water at a flow rate of about 0.2 mL min−1. After the addition was completed, 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 form CPTP/CDDP). In 5 mL of CPTP, dechlorination of CDDP was achieved with different molar ratios (MCOOH/MCDDP = 4:1, 2:1, 1:1 and 1:2, separately); then, the reaction mixture was then shaken at 37 °C for 72 h away from light, and finally CPTP/CDDP was obtained by dialysis (MWCO 3500) away from light to remove free CDDP.

The CPT encapsulation efficiency (%) 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 to 1.0 × 10−3 mg mL−1, respectively, and the particle size and distribution were determined by DLS. (2) Stability was further determined by the fluorescence method. Briefly, Nile red (1.0 × 10−4 mol L−1, 20 μL) in CH2Cl2 was added into each vial away from light and then CH2Cl2 was evaporated absolutely followed by addition of micelles (4 mL, 1.0 mg mL−1). All vials were stirred at 37 °C for different times in the dark. The fluorescence emission intensity over time was measured and calculated at a wavelength of 620 nm (excited at 560 nm). (3) Both CPTP and CPTP/CDDP were treated with DTT (10 mM) at 37 °C in phosphate-buffered saline (PBS) (pH 5.0, 7.4) buffer, and the particle size and distribution of CPTP and CPTP/ CDDP were monitored by DLS at predetermined time intervals. 2.8. In Vitro Release and Blood Circulation. (1) The CPT release was determined at 37 °C 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. Then, 2.0 mL CPTP or CPTP/CDDP was added into a dialysis bag (MWCO 3500) and dialyzed against 40 mL of media in a 37 °C shaker with a speed of 200 rpm−1, respectively. Solutions (100 μL) were taken out, and equal volume media 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 points (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 (12 000 rpm, 5 min) to obtain the plasma. The obtained plasma (20 μL) was extracted with acetonitrile, centrifuged, and then subjected to reverse-phase HPLC analysis. 2.9. In Vitro Cytotoxicity Assays. The cytotoxicity of CPTP and CPTP/CDDP was determined using 4T1 cells by the MTT assay. Briefly, 4T1 cells were evenly seeded in 96-well culture plates with a density of about 5.0 × 103 cells per well in 100 μL of Dulbecco’s modified Eagle’s medium (DMEM) and cultured at 37 °C in a CO2 incubator for 24 h. Then, the cells were incubated with 200 μL of DMEM with serial dilutions of irinotecan, CDDP, CPTP, and CPTP/ CDDP, and CDDP mixed with CPTP freshly (short form CPTP + CDDP) for another 48 and 72 h, respectively. After the above treatment, the 96-well culture plates were centrifuged for 5 min at 3000 rpm, and the cells were incubated with fresh 200 μL of DMEM containing 20 μL (0.75 mg mL−1) of MTT for 4 h. Finally, the culture medium was replaced with 200 μL of DMSO to dissolve the formazan crystals. The cell viability was determined by a BioRad 680 microplate reader under wavelengths of 560 and 612 nm. 2.10. Biodistribution of Polymers. BALB/c mice were subcutaneously inoculated with 4T1 tumors. After the tumors grew up to about 100 mm3, 200 μL of Cy5-labeled CPTP and CPTP/ CDDP (5 mg kg−1 on the basis of CDDP) was injected intravenously. Each group with 4 mice was imaged using a noninvasive near-infrared fluorescence (NIRF) imaging system (PerkinElmer IVIS Lumina XRMS Series III imaging system) at predetermined time points (1, 2, 6, 12, and 24 h). Then, the mice were sacrificed as soon as the experiment was completed. The major organs (heart, liver, spleen, lungs, kidneys, and intestine) and tumors were collected and 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-week-old, 20 g body weight) were bought from the SLRC Laboratory Animal Company (Shanghai, China). All mice received full care according to 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 orthotopic breast adenocarcinoma implantation model was established by subcutaneous injection of 4T1 cells (0.15 mL, 1.5 × 106 cells). When the tumor grew up to about 50 mm3, six mice were randomly assigned as a group, and the mice were injected 20651

DOI: 10.1021/acsami.9b03960 ACS Appl. Mater. Interfaces 2019, 11, 20649−20659

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ACS Applied Materials & Interfaces

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

Figure 3. Sizes (a) and images of CPTP (b) and CPTP/CDDP with the feed molar ratio (MCDDP/MCPTP = 1:1) (c) measured by DLS and TEM. 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 equivalent CPT and CDDP doses of 5 and 2 mg kg−1, respectively), and CPTP + CDDP (at equivalent CPT and CDDP doses of 5 and 2 mg kg−1, respectively) on every other day. The sizes of tumor and body weights were measured every 2 days during the treatment process. The tumor volume (V; mm3) was evaluated by the following equation

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of PEG-b-PGACPT. As illustrated in Figure 2, PEG-b-PGA-CPT was prepared as follows: first, the block copolymer mPEG-PGAp with different glutamic acid (GA) repeating units (20, 40, and 60) was synthesized by ring-opening polymerization of BLU-NCA initiated by active methoxypoly(ethylene glycol) amine (mPEG-NH2). The intermediates and target products were characterized through NMR and GPC (Figures S1−S3). The polymerization degree of glutamic acid (GA) was obtained 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, upon reacting with trifluoroacetic acid, the grafted benzyl groups converted to carboxyl groups and PEG-b-PGA was finally obtained. Finally, the CPT-prodrug PEG-b-PGA-CPT was prepared by esterification between the grafted carboxyl groups of PEG-b-PGA and the hydroxyl group of the camptothecin derivative (CPT-DTE) that was synthesized from camptothecin (CPT) and 2-hydroxyethyl disulfide (DTE) according to the previous report with a small modification.56 The molecular weight (Mn, Mw, and polydispersity index (PDI)), the polymerization degree of glutamic acid (GA), and CPT grafting amount are shown in Table S1. Taking PEG-b-PGA40-CPT7 for example, to ensure adequate carboxyl residue, the feed ratio (MCOOH/MCPT = 40:7) of CPT

V (mm 3) =

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

Finally, the mice were sacrificed and the tumor was taken out and weighed. 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 the experimental group and control group, respectively. The histological examination can be 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 thick slices. Finally, they were stained with hematoxylin/eosin (H&E) for histological assessment under an optical microscope. 20652

DOI: 10.1021/acsami.9b03960 ACS Appl. Mater. Interfaces 2019, 11, 20649−20659

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

with an average size of about 30 nm, which was slightly smaller than that in corresponding DLS results. As known, the dechlorinated CDDP contains two coordination holes, 1 mol of CDDP theoretically needs 2 mol of 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 were measured by ICP-MS. Although the particle size of CPTP/CDDP remained basically unchanged (Figure S5), the DLC (%) of CDDP changed significantly; when the MCOOH/MCDDP = 4:1 and 2:1, the DLCs (%) of CDDP were 18.2 and 36.4%, respectively, which were almost the same as the theoretical feed values (18.6 and 37.2%), and further indicated that almost all CDDP were just acting as crosslinkers. While the molar ratio increased to 1:1, the DLC (%) of CDDPs was 40.2%, which was much lower than the feed value (74.0%), and further demonstrated that only a few parts of the excess CDDP were locked in the hydrophobic core of CPTP/ CDDP. This result was different from the cisplatinincorporated PEG-polyglutamate (Glu) block copolymer micelle via carboxylate complexation with CDDP, which was reported by Kataoka,42 and this probably results from the hydrophobic inner core of CPTP/CDDP that rejects the hydrophilic dechlorinated CDDP. Therefore, for considering the overall particle size, CPT grafting amount, and CDDP crosslink content and efficiency, the CPTP/CDDP was 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 micelle-based nano-

grafting esterification was strictly controlled and the reaction process was tracked by TLC. Moreover, the following CPT release amount measured by HPLC further confirmed that 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. First, stable CPT-prodrug micelles (CPTP) were prepared and further stabilized with dechlorinated CDDP through CDDP (Pt)−carboxyl (COOH) chelate interaction and the CPTP/ CDDP was obtained. 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 are 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 on further increasing the PGA repeating unit, CPTP3 would induce precipitation of crosslinked micelles. It is probably that the chelate ability of the CDDP (Pt)−COOH pair is too strong and results in intermolecular coordination and precipitation. A similar result was obtained in the previous report.44 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 as discussed below. As shown in Figure 3, when MCDDP/MCOOH = 1:1, the volume-average hydrodynamic diameter of CPTP/ CDDP was found to be 50 nm with narrow size distribution (PDI ≤ 0.14). Meanwhile, the TEM results showed that both the CPTP and CPTP/CDDP presented regular spherical shape 20653

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Figure 5. Micelle sizes measured at different intervals by DLS. (a) CPTP; (b) CPTP/CDDP; (c) and 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 media of PBS (at pH values 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 postinjection.

medicines, which can effectively help to avoid the untimely drug release dilemma during the delivery process. First, both CPTP and CPTP/CDDP were diluted from 1.0 to 1.0 × 10−3 mg mL−1 with 10% FBS at 37 °C, separately. As shown in Figure 4, after being diluted with FBS and incubated for 24 h, the hydrodynamic sizes of CPTP with different concentrations changed significantly. However, for CPTP/CDDP, there was hardly any change of the hydrodynamic sizes, which indicated that the chelate interaction between the CDDP (Pt) and carboxyl (COOH) indeed improved the stability of CPTprodrug micelles. Fluorescent probe technology is often used to track the formation of micelles to further prove that the chelate

crosslinking did take place between the CDDP (Pt) and carboxyl (COOH). Nile red served as an effective probe to characterize the change of hydrophilicity and hydrophobicity in the microenvironment and was chosen as the fluorescent probe to monitor this coordination process. As shown in Figure 4c, the fluorescence intensity increased obviously with an increase in time 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 remained almost unchanged. These results clearly indicated that the coordination between the CDDP (Pt) and carboxyl (COOH) indeed created a tight 20654

DOI: 10.1021/acsami.9b03960 ACS Appl. Mater. Interfaces 2019, 11, 20649−20659

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ACS Applied Materials & Interfaces

Figure 7. Cytotoxicity assays of the CDDP, irinotecan, CPTP, CPTP/CDDP, and CPTP + CDDP incubated with 4T1 for 48 h (a) and 72 h (b). 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 of the groups.

crosslinking layer or structure, which prevented the migration of Nile red into hydrophobic nuclei composed of hydrophobic CPT segments. 3.4. In Vitro Release. To solve the contradiction between the stability and controlled release of prodrugs, in this article, between the polymer backbone and drug, a disulfide bond was introduced as a linker to make PEG-b-PGA-CPT reductively breakable in response to the tumor microenvironment. To demonstrate the responsivity, the CPTP before and after crosslinking with CDDP was blended with 10 mM DTT thoroughly, and the size and distribution were measured at predetermined time points (0, 1, 6, and 12 h). As illustrated in Figure 5a,b, the average diameters of DTT-treated CPTP were chaotically changed, resulting in disordered distribution of microsized and nanosized particles after 12 h. However, for DTT-treated CPTP/CDDP, the hydrodynamic sizes still remain unchanged for 12 h, further indicating that the CDDP coordinated with residual carboxyl groups and formed stable crosslinked structures. In addition, we further adjust the pH of the DTT-treated CPTP/CDDP to 7.4 and 5.0. As shown in Figure 5c,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; whereas 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 crosslinking was carried out in PBS (pH 7.4) at 37 °C. Since CPT was chemically connected to the polymer via a reductive disulfide linker, it is believed that CPT could release under reductive conditions.49 Just as shown in Figure 6a, the drug

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, lungs, spleen, kidneys, heart, and intestine) and tumors taken out at 24 h postinjection. Quantification of in vivo biodistribution was recorded as total photon counts per centimeter squared per steradian (p s−1 cm−2 sr−1) for each excised organ at 24 h postinjection (c).

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Figure 9. Antitumor effects of BALB/c mice with 4T1 tumors that 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 being 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 staining with H&E (magnification 20×), and the scale bar is 100 μm.

release behaviors of both CPTP and CPTP/CDDP in the absence of DTT exhibited gradually accumulative profiles, and nearly about 17.6 and 12.3% of CPT was released in 48 h, resulting from the gradual hydrolysis of the ester linker bond. However, in the presence of 10 mM DTT, the CPT release rate accelerated apparently. For example, 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 the disulfide linker in reductive conditions, which resulted in fast CPT release. This was in line with the results of the CPTprodrug micelles, which were destabilized in response to DTT. Moreover, only nearly 20% CPT release was observed within 48 h for CPTP/CDDP treatment with DTT. It is evident that the CDDP coordinated crosslinking indeed improved the stability of CPTP. In addition, for CPTP/CDDP treatment with DTT, when adjusting the pH to 5.0, in the beginning, the CPT release exhibited a low release rate. After 12 h, the CPT release rate became fast and reached nearly 70% at 48 h. 3.5. In Vitro Cytotoxicity. 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 form CPTP + CDDP) were evaluated on 4T1 cancer cells. As shown in Figure 7a,b, both the CPTP and CPTP/CDDP showed dose- and timedependent inhibiting efficacy, which was comparable to that of free CDDP and irinotecan. However, the CPTP/CDDP was consistently less toxic than free CDDP, irinotecan, and even the CPTP + CDDP. The cytotoxicity of the CPTP before and after CDDP crosslinking was evaluated in the presence and absence of 10 mM DTT using the 4T1 cell model to further confirm the CPT release under the reductive tumor microenvironment in vitro. As shown in Figure 7c,d, as expected, the CPTP with 10 mM DTT exhibited much stronger antitumor activity against 4T1 cells with the increasing concentration compared to that without DTT. However, for CPTP/CDDP, as expected, both the CPTP/CDDP 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 20656

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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 micelle-based nanomedicines. Besides the stability of the CPT-prodrug micelles (CPTP), after CDDP in situ crosslinked with COOH, the obtained CPTP/CDDP exhibited a remarkable enhancement of stability and tolerability. Moreover, the CPTP/CDDP also exhibited a sustained reduction-responsive release of CPT accompanied by the dissociation of the CDDP−COOH complex. In addition, CDDP, also as an antineoplastic drug, and the CPTP/CDDP would exhibit synergistic antitumor effects. Based on these fabrications, these 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.

further indicated that the formed stable crosslinked structure prevented DTT from contacting the disulfide linker and thus exhibited low cytotoxicity. 3.6. Pharmacokinetics and Biodistribution. Subsequently, the in vivo pharmacokinetics of the CPT-prodrug micelles before and after CDDP crosslinking was investigated. Blood samples were taken out at predetermined time points after a single intravenous injection. As illustrated in Figure 6b, both CPTP and CPTP/CDDP showed remarkably extended blood circulation time, whereas irinotecan was subjected to a fast blood clearance. The CPTP and CPTP/CDDP exhibited prolonged blood circulation with 8.6 and 33.4% of injected dose still remaining 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 the 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. First, PEG-b-PGA-CPT was covalently labeled with Cy5, then Cy5-labeled CPTP micelles with and without CDDP crosslinking were intravenously injected into 4T1-xenografted BALB/c mice, and the time-related accumulation and distribution were monitored using a noninvasive NIRF imaging system. As illustrated in Figures S6 and 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 Cy5labeled CPTP/CDDP level in the tumors and abundant CPTP/CDDP was mainly accumulated in the main reticuloendothelial organs (heart, liver, spleen, lungs, kidneys, and intestine), the NIRF signals in the tumor sites were almost as strong as those in the liver, indicating that the CPTP/ CDDP had predominantly accumulated in the tumor tissue. 3.7. 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 2 days from 7 days after the tumor volume reaches 50 mm3, respectively. As illustrated in Figure 9a−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 remained 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 effects. Meanwhile, the histological staining measurements of tumors and major organs (heart, liver, spleen, lungs, and kidneys) were used to evaluate the therapeutic effects. As illustrated in Figures 9e and 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 CDDP-crosslinked CPT-prodrug micelles achieved high antitumor efficacy and low toxicity.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b03960. Molecular weight, polymerization degree and CPT grafting amount of PEG-b-PGA-CPT; GPC spectra of PEG-b-PGAp with different polymerization degrees; 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; and representative histological images of major organs (liver, lungs, spleen, kidneys, and heart) after staining with haematoxylin/eosin (H&E) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel/Fax: +86-539-7258660 (Y.L.). *E-mail: [email protected] (S.X.). ORCID

Yinwen Li: 0000-0003-2929-6223 Shoufang Xu: 0000-0002-6410-3258 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

The authors appreciate the Chinese Postdoctoral Science Foundation (2017M621908), the Shandong Provincial Natural Science Foundation, China (ZR2017BB036), and the Natural Science Foundation of China (21777065). 20657

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