Precise Ratiometric Control of Dual Drugs through a Single

Jun 2, 2015 - Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, P. R. Chin...
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Precise Ratiometric Control of Dual Drugs through a Single Macromolecule for Combination Therapy Shiying Luo,†,‡ Ying Gu,‡,§ Yuannian Zhang,†,‡ Pei Guo,† Jean Felix Mukerabigwi,† Min Liu,† Shaojun Lei,† Yu Cao,*,† Hongxuan He,∥ and Xueying Huang*,† †

Key Laboratory of Pesticide and Chemical Biology (Ministry of Education), College of Chemistry, Central China Normal University, Wuhan 430079, P. R. China § Prenatal Diagnosis Center, Lianyungang Maternal and Child Hospital, Lianyungang, 222002, P. R. China ∥ Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, P. R. China S Supporting Information *

ABSTRACT: A major challenge of combinatorial therapy is the unification of the pharmacokinetics and cellular uptake of various drug molecules with precise control of the dosage thereby maximizing the combined effects. To realize ratiometric delivery and synchronized release of different drugs from a single carrier, a novel approach was designed in this study to load dual drugs onto the macromolecular carrier with different molar ratio by covalently preconjugating dual drugs through peptide linkers to form drug conjugates. In contrast to loading individual types of drugs separately, these drug conjugates enable the loading of dual drugs onto the same carrier in a precisely controllable manner to reverse multidrug resistance (MDR) of human hepatoma (HepG2) cells. As a proof of concept, the synthesis and characterization of xyloglucan−mitomycin C/doxorubicin (XG−MMC/DOX) conjugates were demonstrated. This approach enabled MMC and DOX to be conjugated to the same polymeric carrier with precise control of drug dosage. The cytotoxicity and combinatorial effects were significantly improved compared to the cocktail mixtures of XG−MMC and XG−DOX as well as the individual conjugate of the mixture. Moreover, the results also showed that there was an optimum ratio of dual drugs showing the best cytotoxicity effect and greatest synergy among other tested polymeric conjugate formulations. KEYWORDS: drug delivery, combinational therapy, polymeric conjugates, ratiometric drug loading, reversion of multidrug resistance

1. INTRODUCTION Carcinoma affects millions of individuals and is responsible for many million deaths annually worldwide.1 It is difficult to treat with current approaches and often displays multidrug resistance, which may help to explain why so many of the clinical trials using standard cytotoxic drug therapy have been disappointing. Multidrug resistance (MDR) is a major obstacle for chemotherapy.2,3 Combined therapy could provide a promising strategy to suppress cancer-drug resistance because different drugs may damage or kill cancer cells at different stages of their growth cycles through distinct mechanisms of action.4 Several drug delivery systems, such as liposomes and nanoparticles, have displayed the ability to codeliver multiple drugs, but precisely controlling the loading ratio and release kinetics of the multiple-drug payloads remains an unmet expectation. Polymer−drug conjugates have become increasingly attractive in cancer drug delivery systems because of their decreased toxicity, great accumulation at the tumors through enhanced permeability and retention (EPR) effect, and the enhanced bioavailability and prolonged plasma half-life.5−8 In © 2015 American Chemical Society

recent years, several studies have been conducted which highlight the fitness of polymer−drug conjugates to deliver drug combinations.9,10 This conjugation approach is different from other drug encapsulation methods due to its ability to precisely control the molar ratios of different drugs.1,11 It has been reported in clinics that the polymeric conjugation for drug combinations can elicit synergisms among them and block disease recurrence.12,13 However, the unification of pharmacokinetics and cellular uptake of various drug molecules to precisely control the dosage and scheduling of multiple drugs remains a major challenge, due to the variability in drug-to-drug and drug-to-polymer interactions as well as steric hindrance between different drug molecules.14,15 Despite the success in maintaining the ratio between multiple drugs with different properties, ratiometric delivery and synchronized release of Received: Revised: Accepted: Published: 2318

December 29, 2014 May 13, 2015 June 2, 2015 June 2, 2015 DOI: 10.1021/mp500867g Mol. Pharmaceutics 2015, 12, 2318−2327

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Molecular Pharmaceutics

insoluble protein fraction was removed by centrifugation. After centrifugation, anhydrous ethanol (twice volume of the solution) was added, which was followed by suction filtration with a sand core funnel (G4, pore size, 5−15 μm). The filtrate solution was lyophilized to obtain pure XG for further experiments. The molecular weight of XG was about 700 kDa measured by GPC (Prominence GPC, Shimadzu Co., Japan). 2.3. Preparation of Peptide-DOX/Peptide-MMC Derivatives. 1 g of DOX (1.84 mmol) and 0.75 g of Boc-GlyLeu-Gly-OSu (1.70 mmol) were dissolved in dry DMF, and 0.4 g (0.25 mmol) of diethylphosphoryl cyanide (DEPC) was added under stirring. 0.3 mL of triethylamine (TEA) was added at 0 °C under stirring for 0.5 h. After overnight reaction in the dark at room temperature, the solvent was evaporated in vacuum and ethyl acetate was added to dissolve the dry residue. The reaction mixture was extracted with a 10% citric acid solution (3 × 5 mL) and saturated sodium bicarbonate (3 × 5 mL). The organic part was isolated and the water layer was extracted with ethyl acetate (2 × 5 mL). Ethyl acetate extracts were evaporated to dryness in vacuum, and the residue was purified by column chromatography on silica. The selected fraction was dried over MgSO4. After removal of the solvent, the Boc-Gly-Leu-Gly-DOX derivative was finally obtained. 0.1 g of Boc-Gly-Leu-Gly-DOX was dissolved in 2 mL of DMF, and 0.2 mL of trifluoroacetic acid (TFA) was added. The reaction was conducted at room temperature for 1 h under stirring. The solvent was evaporated in vacuo. The residue was dissolved in 5 mL of methanol, and the solution was filtered. The Gly-LeuGly−DOX conjugate was finally obtained after evaporation of the solvent. The preparation of Gly-Leu-Gly−MMC was similar to that of Gly-Leu-Gly−DOX. 0.44 g of Boc-Gly-Leu-Gly-OSu (1 mmol) and 0.37 g of MMC (1.1 mmol) were dissolved in 20 mL of dry DMF, and 0.22 g of diethylphosphoryl cyanide (DEPC) was added with stirring. 0.15 mL of triethylamine (TEA) was added at 0 °C and stirred for 0.5 h. The next steps were almost in the same way according to our previous preparation.25 2.4. Preparation of the XG−DOX/MMC Conjugates. XG (2 g, 12.3 mmol) and 4-dimethylaminopyridine (DMAP) (0.15 g, 1.2 mmol) were dissolved in 20 mL of DMSO/pyridine solution (volume ratio 1/1). 4-Nitrophenyl chloroformate (0.9 g, 4.4 mmol) was added at 0 °C. The reaction mixture was stirred at 0 °C for 4 h and then precipitated with anhydrous ethanol. A white precipitate was gained and washed repetitively with the same solvent. The XG-COO(C6H4)NO2 was finally dried in vacuum. The carbonate content was determined by UV analysis after activated XG hydrolysis in NaOH. 2 g of XG-COO(C6H4)NO2 (1.3 mmol reactive groups) and 2 g of Gly-Leu-Gly−DOX and 2 g of Gly-Leu-Gly−MMC (1.2 mmol) were dissolved in dry DMSO, and then TEA (0.1 mL) was added. After 48 h of reaction in the dark, the conjugate was separated by precipitation in anhydrous ethanol. The product was washed and dried. Finally, the conjugate was purified by preparative GPC (Sephadex G25) with water as eluent and freeze-drying. The content of DOX and MMC in the conjugates was determined by UV analysis in water. A series of XG−MMC/DOX conjugates loading dual drugs in a precisely controllable ratio were prepared using peptideDOX/peptide-MMC derivatives in different molar ratios (0.5:1; 0.75:1; 1:1; 2.5:1).

different drugs from a single carrier remain powerful challenges.15−18 In this study, a new approach to address these issues was to load dual drugs with different molar ratios onto a single macromolecule. Doxorubicin (DOX) and mitomycin C (MMC) were chosen as model drugs. DOX and MMC are among the most common chemotherapeutics in clinical use, but the two drugs can cause cardiac toxicity and other severe toxicities, which limited the administration of these free drugs.6,19 They are anthracycline-based agents with the same mechanism of action against DNA. The synergistic effect of DOX and MMC involves an interaction of cross-link-activated DNA repair machinery and drug−DNA adducts.20 The common intracellular destination makes it possible for them to generate an improved synergistic effect after being conjugated to the same deliverer. In recent decades, several clinical studies of the combination of MMC and DOX against cancer have been conducted by using particulate carriers including microspheres and nanoparticles.21−23 However, these systems have not realized control of the intracellular ratios of dual drugs as well as transportation of the two drugs into the cell simultaneously. It was reported that tripeptide Gly-Leu-Gly possesses the essential requisite that it can be degraded by lysosome enzymes,24,25 and in this study, it was used as the spacer for drug binding. These ideal polymer−drug spacers could offer more stability in the bloodstream and degrade in cancer cells to release drug. The polymer conjugates were transferred into the cell and then directed into a secondary lysosomal compartment which contains a variety of lysosomal enzymes. The release rates were significantly controlled by the oligo peptide spacer in polymer conjugates. Xyloglucan (XG), a kind of polysaccharide, was chosen as the drug carrier.26 XG consists of a (1→4)-β-D-glucan main chain and (1→6)-α-Dxylose branches which are partially substituted by (1→2)-β-Dgalactoxylose.27 It has been widely utilized in the pharmaceutical industry because it is a natural, water-soluble, biodegradable, and nontoxic polysaccharide. Particularly, the hydroxyl groups of xyloglucan are anchoring points for conjugation of some kinds of antitumor drugs or the targeting unit.28,29 Herein, we present a polymer−drug conjugate strategy to meet the aforementioned need in which two drugs were linked to the polymeric carrier by covalently conjugating through cleavable linkers. The therapeutic effect on drug resistant variants of hepatoma cells (HepG2/DR) was evaluated in vitro and in vivo. This approach allowed the precise control of the dosage of the therapeutic agents, thereby maximizing the combinatorial effects.

2. MATERIALS AND METHODS 2.1. Materials. XG was prepared from tamarind seed powder (TCI, Shanghai, China). DOX and MMC were purchased from Chuangcheng Pharmaceutical (Wuhan) Ltd., China. N-t-Boc-glycyl-L-leucyl-glycine N-hydroxysuccinimide ester (Boc-Gly-Leu-Gly-OSu) was purchased from GL Biochem (Shanghai) Ltd., China. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and collagenase IV were purchased from the Sigma-Aldrich Co., USA. All other chemicals used were analytical grade unless otherwise stated. 2.2. Preparation and Purification of XG. XG was purified from tamarind seed powder. Tamarind solution (2 wt %) was prepared by dissolving the tamarind kernel powder in distilled water. The solution was stirred in a boiling water bath for 2 h. The clear solution was kept overnight, and then the water2319

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Molecular Pharmaceutics 2.5. In Vitro Study. 2.5.1. In Vitro Release of MMC and DOX from the Conjugates. The study of drug release was carried out in serum, phosphate buffered saline (PBS, pH 7.4), and buffer incubated with collagenase IV (0.3 mg/mL) at 37 °C with mild stirring. The XG−MMC/DOX conjugate was diverted in 10 mL of PBS to a dialysis tube. The samples were analyzed by HPLC with a Shimadzu HPLC system composed of two pumps (LC-10Avp and LC-10AS) and a SPD-10Avp ultraviolet detector (Shimadzu Corporation, Japan) in reverse phase mode at different points of time. An Extend-C18 column (4.6 × 250 mm i.d., 5 μm) was used, the mobile phase used for the analysis was methanol−acetonitrile− phosphate buffer (pH 5.0, 0.2 M) (50:20:30, v/v/v), and the flow rate was 0.5 mL/min. The amount of DOX in the solution was determined by UV spectroscopy at 245 nm, and the amount of MMC was determined by UV spectroscopy at 360 nm. 2.5.2. In Vitro Cytotoxicity Assay. The cytotoxicity of the conjugates was investigated against human hepatoma cell line (HepG2) with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium) assay. HepG2 cells were cultured in a flask in RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum (FCS, Sigma), penicillin (100 μg/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere containing 5% CO2. In a few months, the drug resistant HepG2 cell line (HepG2/DOX) was developed from HepG2 cells incubated with DOX in a stepwise increasing concentration, from 0.01 to 2 μg/mL. The resistant cells were selected by removing the dead nonresistant cells. The drug resistance was maintained by culturing the cells at 1 μg/mL DOX. Data was expressed by cell survival. The reversal of MDR was measured by the 50% inhibitory concentrations (IC50). Combination index (CI), one of the simplest formalisms to describe synergy, is defined as CI =

IC50(A)pair IC50(A)

+

previously.32 XG−drug and released drug were determined by HPLC as described previously.33 2.6. In Vivo Study. All work implemented on animals was in accordance with the “Guidelines for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). This study was approved by the Ethics Committee of Central China Normal University. 2.6.1. Safety Assessment of the Conjugates in Normal Mice. Male BALB/c nude mice at the age of 4 weeks were injected at different doses of 25.0, 50.0, and 75.0 μmol/kg every week for four doses (days 0, 7, 14, and 21), separately (n = 10 mice/group). Toxicity and mortality were recorded afterward for the next 2 weeks. At the end of the fifth week, a blood sample was collected from each mouse and liver samples were obtained and weighed. Serum was collected and tested for alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH) with enzymatic reagent kits. 2.6.2. Body Distribution of Drugs (MMC and DOX) in Tumor-Bearing Mice. Every male BALB/c nude mouse (provided by the Experimental animal center of Wuhan Institute of Biological Products) was injected with 0.2 mL of HepG2/DOX cell suspension (5 × 106 cells/mL) in the right axillary region. Three weeks later, solid tumor growth was obviously established in nude mice. 108 tumor-bearing mice were randomly divided into 3 groups with 36 mice in each group and separately injected with free MMC/DOX and XG−MMC/DOX at a single dose (equivalent dose of drug = 25 μmol/kg). At a certain time interval (48 h), a 0.5 mL blood sample was withdrawn by retroorbital venous plexus puncture from tumor-bearing mice. The livers, hearts, tumors, spleens, and kidneys of all the mice were immediately separated and washed with 10 mM Na2HPO4 buffer, followed by homogenization with ethyl acetate solvent. The drug was extracted by incubation with acidic isopropanol at 4 °C for 4 h. The mixture was treated with a vortex mixer for 1 min, followed by centrifugation. The MMC and DOX concentration in the supernatant solution was detected by HPLC quantitatively. Free drug or released drug was extracted and determined without incubation by HPLC as described previously.24,32,33 To determine the distribution of drugs in tumor tissue, the tissue was homogenized after being cut into small pieces. The liquid supernatant was extracted for test of drug concentration. Then the tissue was added with 10-fold 0.5 wt % pancreatic enzyme with mild stirring at a constant temperature of 37 °C, pH 7.4 for 20 to 30 min. The digestive juice was extracted and was passed through a filter screen (100 mesh). Then the filtrate was centrifuged with 1000 rpm for 5 min. The tumor cells were produced, and then drug concentration was tested. The MMC and DOX concentration in the supernatant solution was detected by HPLC as described previously.24,32,33 2.6.3. In Vivo Cytotoxicity of XG−MMC/DOX Conjugates against Drug Resistant HepG2 Cells in BALB/c Nude Mice. Male BALB/c nude mice (Shanghai Institute of Materia Medica, Chinese Academy of Sciences; 4 weeks old, 20−24 g) were injected subcutaneously with drug resistant HepG2 cells. Three weeks later, the growth of solid tumor was distinctly established in most mice, and XG−MMC/DOX conjugates or free DOX and MMC (equivalent dose of MMC and DOX = 25 μmol/kg) suspended in PBS were injected into tail veins of mice every week for four doses (days 0, 7, 14, and

IC50(B)pair IC50(B)

where IC50(A)pair and IC50(B)pair are the half inhibitory concentrations when drug is given as an A−B pair; IC50(A) and IC50(B) are the half inhibitory concentrations when drug A or B acts singly. The CI values lower than, equal to, and higher than 1 indicate synergism, additivity, and antagonism, respectively.30,31 2.5.3. In Vitro Cellular Uptake Studies. HepG2/DOX cells were preincubated with the different formulations for 4 h at a dose equivalent to free MMC/DOX (mixture of free MMC and DOX). 1 mL of cell suspension (107 HepG2/DOX cells) was combined with 300 mL of TM-2 buffer solution (10 mM TrisHCl, pH 7.4, 2 mM MgCl2, 0.5 mM PMSF). The mixture was kept in an ice bath for 5 min and combined with 300 μL of 1.0% Triton X-100. Then the mixture was passed through a filtration membrane (0.22 μm) 6 times. The contents of MMC and DOX were detected by HPLC. Free MMC or released MMC was extracted and determined without incubation by HPLC as described previously.24 Free DOX or liberated DOX was determined after protein denaturation. Tissue homogenate or plasma was combined with AgNO3 (3.0 M), and then the suspension was mixed for 10 min at room temperature. The excess of silver ions was precipitated with NaCl (3.0 M). Then DOX was extracted and determined by HPLC as described 2320

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Figure 1. FTIR spectra of xyloglucan (XG), XG−MMC, XG−DOX, and XG−MMC/DOX conjugate.

Figure 2. 1H NMR spectroscopy of xyloglucan (XG), XG−MMC, XG−DOX, and XG−MMC/DOX conjugate.

2321

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Molecular Pharmaceutics 21). A major axis and a minor axis of tumors were measured with calipers. The volume of tumor was then determined. The number of long-term survivors and the survival times were determined. 2.7. Statistical Analysis. Data was expressed as means ± standard deviations of multireplicated determinations. We used one-way analysis of variance (ANOVA) with the Student− Newman−Keuls multiple comparisons or t test to compare the differences between the means of two groups at the same time point. Differences were considered to be statistically significant if p < 0.05.

3. RESULTS 3.1. Preparation and Characterization of the XG− MMC/DOX Conjugate. In this study, the activation of xyloglucan was conducted by mixing polysaccharide with the 4-nitrophenyl chloroformate, in order to introduce the prepared peptide derivatives into the carrier. The carbonyl and amino groups of MMC and DOX were suitable for grafting the drugs to polymeric carriers by amide bond and hydrazone bond, respectively. The efficiency of grafting drugs to the XG carrier was 70.65% for DOX and 84.62% for MMC. The amount of carbonate in glucan, calculated with Beer’s law, was about 19.2 mol %. The content of drugs, determined by detection and calculation via spectra, was about 4.7 mol %. Through UV analysis, the content of DOX and MMC in the conjugates was the same as what we designed. In Figure 1, the strong absorptions of the methyl and methylene groups of the conjugate between 2800 and 2960 cm−1 are different from the absorption peaks of xyloglucan because of the tripeptide spacer residue. The peaks about 1550 and 1505 cm−1 might be the absorption of the amide groups in the conjugated DOX and MMC. In Figure 2, the peaks about 7.21 ppm might be the absorption of the hydrogen of the amine group in the conjugated DOX. The peaks about 2.12 ppm might be the absorption of the hydrogen in the conjugated DOX. The peaks about 7.38 ppm might be the absorption of the hydrogen of the amine group in the conjugated MMC. The peaks about 2.22 and 2.42 ppm might be the absorption of the hydrogen in the conjugated MMC. 3.2. Drug Released from the XG−MMC/DOX Conjugate. The in vitro release activities of DOX and MMC from the XG−MMC/DOX conjugate were tested by culturing the conjugate with collagenase IV, serum, buffer at pH 7.4 (extracellular pH), and buffer at pH 5.0 (lysosomal pH) at 37 °C. As shown in Figure 3, the release of drugs (DOX and MMC) was almost negligible in serum and buffer at pH 7.4. Namely, the XG−MMC/DOX conjugates held stability in serum and buffer at extracellular pH. The stability of the conjugates in serum indicated that the conjugates were stable during plasma circulation. When treated with collagenase IV and buffer at pH 5.0, DOX and MMC were released as time proceeded. The amount of drug released was approximately 50% at 8 h under processing with collagenase IV, and the total release was 80% after 48 h. Compared with the drug released from the conjugate incubated with collagenase IV, the drug released from the conjugate treated with buffer at pH 5.0 was much lower and the apparent release did not exceed 30% after 24 h (p < 0.05). The total release from the conjugate treated with buffer at pH 5.0 was even less than half the amount of the release from the conjugate treated with collagenase IV after 48 h. It has been reported that highly aggressive human tumors all exhibit elevated levels of type IV collagenase activity.34,35 Hence

Figure 3. Release profiles of XG−MMC/DOX conjugate (incubating with collagenase IV (■), pH 5.0 buffer (▲), serum (●), pH 7.4 buffer (◆)). Data are given as mean ± SD (**p < 0.05).

drugs were released from the conjugates by the specific hydrolysis of collagenase IV but were not released in pH 7.4 buffer and serum.25 3.3. In Vitro Cytotoxicity of XG−MMC/DOX Conjugate against Drug Resistant Hepatoma Cells. The cytotoxicity of XG−MMC/DOX conjugate was determined by MTT assay against HepG2/DOX cells. HepG2/DOX cells were cultured for 72 h under exposure to the samples. As shown in Table 1 Table 1. IC50 of Different Formulations in HepG2 Cell formulation

IC50 μM

DOX MMC MMC/DOX(1:1)

0.159 ± 0.009 0.389 ± 0.033 0.265 ± 0.022

and Table 2, the IC50 value of DOX in HepG2/DOX cells was 45.86 ± 0.95 μM, which was 288 times higher than that of normal HepG2 cells (0.159 ± 0.009 μM). The IC50 value of MMC in HepG2/DOX cells was 4.533 ± 0.221 μM, which was 11 times larger than that of HepG2 cells (0.389 ± 0.033 μM). The combination of MMC and DOX (molar ratio = 1:1) resulted in an IC50 of 4.746 ± 0.164 μmol/L in HepG2/DOX cells, which was 17 times larger than that of HepG2 cells (0.265 ± 0.022 μM). Table 2 shows the IC50 and combination indexes of the therapeutics of different polymeric conjugates. The IC50 value and CI value of all XG−MMC/DOX conjugates with different molar ratios were lower than those of the XG−DOX + XG−MMC groups (p < 0.01). The lowest IC50 value of 0.48 ± 0.019 μM was detected in the XG−MMC/DOX (0.75:1) group, showing significant difference with other tested drug delivery systems (p < 0.01). The lowest CI value of 0.514 was detected in the XG−MMC/DOX (0.75:1) group, showing greater effect of synergy (p < 0.01), although the difference of the CI value between the XG−MMC/DOX (0.75:1), (0.5:1), and (1:1) groups was not significant. It was found that the XG−MMC/DOX (0.75:1) group displayed the best cytotoxicity effect and the greatest synergy in all conjugates. 3.4. In Vivo Pharmacokinetic Investigation of Free MMC/DOX and XG−MMC/DOX Conjugate in TumorBearing Mice. As shown in Figure 4, in plasma, the total concentration of DOX and MMC produced by the administration of XG−MMC/DOX was much higher than that of free DOX and MMC. It was also displayed that free MMC/DOX (combination of free MMC and DOX), free 2322

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Molecular Pharmaceutics Table 2. IC50 Values of Different Formulations in Drug Resistant HepG2 Cella

Data are given as mean ± SD (p < 0.05; for XG−MMC + XG−DOX groups and XG−MMC/DOX groups, **p < 0.01; for XG−MMC/DOX (0.75:1) and other XG−MMC/DOX groups, ***p < 0.01).

a

extended by formulating DOX and MMC in the XG−MMC/ DOX conjugates. As for the drug biodistribution in tumor-bearing mice (Figure 5), in comparison with free MMC/DOX, the

Figure 5. Drug biodistribution in tumor-bearing mice (n = 10 per group, equivalent dose of drug = 25 μmol/kg). Data are given as mean ± SD (p < 0.05). *The intracellular concentration of dual drugs was 9.697 ± 0.619 μM, including released MMC/DOX, which was 4.072 ± 0.367 μM, and the molar ratio of MMC/DOX was 0.75:1.

conjugation in the XG−MMC/DOX lowered the uptake of released drug significantly in heart, spleen, lung, and kidney (p < 0.05). In spite of the subequal uptake of free MMC/DOX and XG−MMC/DOX in kidney, spleen, and liver, the drug released from the conjugates was much lower. The XG− MMC/DOX uptake in heart and lung was less than that of free MMC/DOX, and the uptake of released drug was also much lower. On the contrary, the XG−MMC/DOX uptake in tumor was 7-fold higher than that of free MMC/DOX, and MMC and DOX released from the conjugate was 3-fold higher than free MMC/DOX (p < 0.05). The intracellular concentration of dual drugs was 9.697 ± 0.619 μM, including released MMC/DOX, which was 4.072 ± 0.367 μM. The molar ratio of intracellular drug concentration of MMC/DOX was 0.75:1, which was in keeping with the initial molar ratios.

Figure 4. (a) Concentration of MMC/DOX in plasma of free MMC/ DOX (▲) in tumor-bearing mice; concentration of MMC/DOX in plasma of XG−MMC/DOX conjugate (■) in tumor-bearing mice. Data are given as mean ± SD (**p < 0.05). (b) Concentration in plasma of MMC (▲) and DOX (●) of XG−MMC/DOX conjugate in tumor-bearing mice; concentration in plasma of free MMC (◆) and free DOX (■) in tumor-bearing mice. Data are given as mean ± SD (**p < 0.05).

MMC, and free DOX were all eliminated quickly from the circulation. The drug retention in circulation was substantially 2323

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Table 3. Serum Biochemical Parameters and Relative Liver Weight at 2 Weeks after Administration of Different Doses of the Conjugates to Micea dose (μmol/kg) control (0) 25 50 75 a

ALT (U/L) 26.8 28.1 26.9 27.4

± ± ± ±

4.6 4.9 3.9 4.8

AST (U/L)

AST/ALT

± ± ± ±

1.20 1.18 1.22 1.25

32.2 33.1 32.7 31.9

5.5 4.2 5.6 4.9

LDH (U/L) 486.2 468.1 478.6 478.2

± ± ± ±

58.8 63.8 59.9 56.4

CK (U/mL) 0.25 0.23 0.32 0.33

± ± ± ±

0.17 0.13 0.15 0.12

liver/body (wt %) 6.29 6.32 6.25 6.39

± ± ± ±

0.52 0.34 0.29 0.46

ALT alanine aminotransferase, AST aspartate aminotransferase, LDH lactate dehydrogenase, CK creatine kinase.

3.5. Toxicological Study. The median lethal dose (LD50) of XG−MMC/DOX (0.75:1) was 142.9 μmol/kg (drug equiv). In comparison with DOX (LD50 of DOX: 32.94 μmol/kg) or MMC (LD50 of MMC: 39.84 μmol/kg), the security of XG− MMC/DOX conjugate was enhanced. Using a method of monitoring the body weights of mice treated with the conjugates, no significant change was observed, indicating that there was nonspecific toxicity under treatment with these conjugates. ALT (alanine aminotransferase) is abundant in liver cells, but liver tissue will release the ALT into bloodstream when lesions occur and led to the increase of ALT levels in serum. AST (aspartate aminotransferase) and LDH (lactate dehydrogenase) are mainly in heart and liver, and both of them are important indicators of the degree of heart and liver disease. CK (creatine kinase) is mainly in heart issue, and when heart is diseased, the CK levels in serum will increase. In Table 3, the mitigative effect of the conjugates on heart was further sustained due to there being no obvious increase of LDH, AST, or CK enzyme levels at all doses (p < 0.05). The evaluation of hepatotoxicity of the conjugates was based on the serum biochemical parameters and relative liver weight (Table 3). No significant change in ALT, AST, LDH, and liver weight was produced by the conjugate, even though the doses used were equivalent to 75 μmol/kg for continuous four times. 3.6. Reversion of Multidrug Resistance of XG−MMC/ DOX Conjugate. In order to determine the effect of free MMC/DOX and XG−MMC/DOX conjugate against MDR, in vivo antitumor activities were examined in BALB/c nude mice implanted with drug resistant HepG2 cells. As shown in Figure 6, compared with free MMC/DOX in animal models, the conjugates proved an aggressive therapeutic profile in the field of tumor growth inhibition. When mice were treated with saline alone or free MMC/DOX, tumor volumes increased rapidly and exponentially, with little difference between these two treatment groups. When treated with XG−DOX + XG−MMC or the XG−MMC/DOX conjugate, the tumor growth was inhibited. In terms of inhibiting the growth of the drug resistant HepG2 cells xenografts, the XG−MMC/DOX conjugate was found to be more effective than XG−DOX + XG−MMC at a similar dose due to the fact that the tumor volume administered with XG−DOX + XG−MMC was 1.3-fold higher than the tumor volume treated with the XG−MMC/DOX conjugate after 21 days (p < 0.05). The survival data also reflected these results (Figure 7). Administration with XG−MMC/DOX conjugate showed an impressive treatment efficacy of up to 30% long-term survivors, resulting in obviously prolonged survival (46.9 days) in comparison with the administration with saline (17.9 days), free MMC/DOX (21.6 days), or XG−DOX + XG−MMC (41.5 days) respectively (Figure 7). Administration with free MMC/DOX produced observable side effects such as lessened activities and reduction in weight of mice. On the contrary, mice treated with the conjugates did not

Figure 6. Tumor size changes of the treated xenograft nude mice bearing the DOX resistant HepG2 tumors [saline (●), free MMC/ DOX (◆), XG−DOX + XG−MMC conjugates (■), and XG−MMC/ DOX conjugate (▲)]. After injection of HepG2/DR cells for 3 weeks, these DOX resistant HepG2 tumor-bearing mice were treated with drugs (25 μmol/kg) by tail vein injection every week for four doses (days 1, 7, 14, and 21). Data are given as mean ± SD (**p < 0.05).

Figure 7. Survival curve of the treated xenograft nude mice bearing the DOX resistant HepG2 tumors [saline (●), free MMC/DOX (◆), XG−DOX + XG−MMC conjugates (■), and XG−MMC/DOX conjugate (▲)]. After injection of HepG2/DR cells for 3 weeks, these DOX resistant HepG2 tumor-bearing mice were treated with drugs (25 μmol/kg) by tail vein injection every week for four doses (days 1, 7, 14, and 21). The survival time and number of long-term survivors (LTS) until day 50 were monitored (**p < 0.05).

show obvious side effects and XG−MMC/DOX conjugate may be used to achieve a higher therapeutic efficacy.

4. DISCUSSION A novel polymer−drug conjugate formulation of coloaded DOX and MMC with high efficiency has been devised in this study. This conjugate with precise ratiometric control of two drugs can reverse MDR of HepG2/DOX cells achieving passive 2324

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conjugates was investigated by the biodistribution of the drug delivery systems in human tumor xenograft nude mice. The concentration of XG−MMC/DOX conjugate, including drug released from the conjugates, was much higher than that of free MMC/DOX. The drug distribution in kidney, lung, spleen, and heart was significantly reduced when the drug was delivered via the conjugates (Figure 5). Meanwhile, the conjugates reduced the cardiotoxicity and hepatotoxity of DOX and MMC (Table 3). These were the indication of the improvement in the safety and targeting of the conjugates to reverse MDR. Taking together the methods of tumor response to treatment (Figures 6 and 7), tumor volumes and survival treatments with free MMC/DOX were not much different than no treatment. The responses to treatment with XG−MMC/DOX conjugate and XG−DOX + XG−MMC had evident improvement over free MMC/DOX or no treatment. Compared to treatment with free MMC/DOX, the polymer−drug systems slowed down tumor progression with concomitant smaller tumors and increased life span. In addition, the XG−MMC/DOX conjugate showed higher therapeutic efficiency than XG−DOX + XG−MMC. The results indicated that single polymeric carrier (XG) carrying a combination of DOX and MMC displayed higher cytotoxicity and synergy than the combination of XG−DOX + XG−MMC, which was corresponding to the results of the in vitro cytotoxicity investigation. The enhanced synergy effect of passive accumulating and intracellular delivery should be responsible for the improved therapeutic efficacy of XG− MMC/DOX over XG−DOX + XG−MMC.

targeting by the EPR effect. Xyloglucan represented one of the most promising carriers for macromolecular drug delivery.25,26 The attachment of DOX and MMC to the xyloglucan was achieved using the tripeptide glycyl-L-leucyl-glycine as linkers. The polymer−drug linkers are designed to be effectively hydrolyzed by the lysosomal enzymes and to be resistant to attack in the serum. In this study, we aimed to design a variety of formulations of polymeric conjugates with different molar ratios of MMC and DOX to determine the cytotoxicity (in vitro) of these conjugates. When the molar ratio of MMC/DOX was 1:1, the XG−MMC/DOX conjugate showed higher cytotoxicity against HepG2/DOX cells than XG−DOX + XG−MMC (Table 2). Then a series of XG−MMC/DOX conjugates with different molar ratios of MMC and DOX (0.5:1, 0.75:1, 1:1, 2.5:1) were devised. The mechanism of endocytic uptake was favorable to the combinatorial drug delivery system presented in the current study. The drug-loaded polymeric conjugates could enter tumor cells by endocytosis and be internalized.34−37 Once engulfed by the plasma membrane, conjugates were transported by endosomal vesicles before unloading their effective drug loads.15 Interestingly, in the combination of free MMC/DOX (molar ratio, 1:1), the molar ratio of intracellular drug concentration of MMC/DOX in HepG2/DOX cells was 0.75:1. The main reason was that the structures and properties of MMC and DOX were different so that the action of endocytosis was different between dual drugs, which led to the difference between the intracellular drug concentration and the initial molar ratios. However, in our polymer−drug conjugation strategy using XG−MMC/DOX (0.75:1), the concentration of dual drugs in tumor issue was 9.945 ± 0.568 μM, including released MMC/DOX, which was 4.096 ± 0.476 μM. The intracellular concentration of dual drugs was 9.697 ± 0.619 μM, including released MMC/DOX, which was 4.072 ± 0.367 μM (Figure 5). It is shown that the drugs were mainly released intracellularly. In addition, the molar ratio of intracellular drug concentration of MMC/DOX was 0.75:1, which was in accordance with the designed molar ratio. The main reason was that MMC and DOX were chemically conjugated to xyloglucan by the tripeptide linker. When the XG−MMC/ DOX conjugates ultimately arrived in the lysosomal compartment of the cell following their pinocytic capture, drug− polymer linkers were hydrolyzed by lysosomal enzymes simultaneously. There are abundant enzymes including proteases in the lysosomes which play a role in the degradation of drug−polymer linker to achieve efficient intracellular drug release.38,39 These results recognized that this approach enabled different types of drugs to be conjugated to the same polymeric carrier with ratiometric control over drug loading and hydrolyzed by lysosomal enzymes achieving better effect of synergy. Many drug delivery systems have not achieved the ratiometric delivery and synchronized release of different drugs from a single carrier.9,14,36,40 Since our results showed that the XG−MMC/DOX (0.75:1) group displayed the best cytotoxicity effect and the greatest synergy in all conjugates (Table 2), in vivo investigation was administered using the XG−MMC/DOX (0.75:1) group. In this study, the XG−MMC/DOX conjugate (0.75:1) enhanced the retention of DOX and MMC in the circulation due to the many hydroxyl residues in xyloglucan, which equipped the conjugates with the hydrophilic coat to keep the long-term retention in the circulation (Figure 4). The efficiency of the

5. CONCLUSION This work provides a novel approach to load different drugs into the same polymeric conjugates in a precisely optimum control to reverse MDR. The combinational therapy proposed here showed superior therapeutic effect and synergetic effect compared to the cocktail mixtures of XG−MMC and XG− DOX. Especially there was an optimum ratio of dual drugs showing better cytotoxicity effect and greater synergy than other tested drug delivery systems. Our study offers an approach to solving the aforementioned limitations in multidrug encapsulation into the same drug-delivery vehicle. Since this polymer−drug conjugation strategy can be generalized to various therapeutic agents, this combinatorial drug delivery system will hold promise in nanomedicine for different drug combinations.

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures, including dynamic laser scattering (DLS) results of DOX, MMC, XG, and XG−MMC/DOX. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/mp500867g.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-27-61311087. Author Contributions ‡

S.L., Y.G., and Y.Z. were equal contributors for the work.

Notes

The authors declare no competing financial interest. 2325

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ACKNOWLEDGMENTS This project was sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars (State Education Ministry, 2010-1174) and the project of Hubei Key Laboratory of Genetic Regulation and Integrative Biology (201002). This work was also supported by selfdetermined research funds of Central China Normal University (CCNU) from the colleges’ basic research and operation of MOE (CCNU11A02013).

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NOTE ADDED AFTER ASAP PUBLICATION This paper published ASAP on June 16, 2015. The Abstract graphic was replaced and the revised version was reposted on July 6, 2015.

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