Rational Design of an Amphiphilic Chlorambucil ... - ACS Publications

Feb 7, 2018 - School of Pharmaceutical Science, Shandong University, 44 West Wenhua Road ... Key Laboratory of Colloid & Interface Chemistry, Shandong...
0 downloads 0 Views 5MB Size
Download PDF
Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Rational Design of an Amphiphilic Chlorambucil Prodrug Realizing Self-Assembled Micelles for Efficient Anticancer Therapy Xu Hu,† Ruiling Liu,† Di Zhang,† Jing Zhang,† Zhonghao Li,‡ and Yuxia Luan*,† †

School of Pharmaceutical Science, Shandong University, 44 West Wenhua Road, Jinan, Shandong Province 250012, P. R. China Key Laboratory of Colloid & Interface Chemistry, Shandong University, Ministry of Education, Jinan, Shandong Province 250100, P. R. China

‡

S Supporting Information *

ABSTRACT: The application of anticancer drug chlorambucil (CLB) in chemotherapy is severely restricted by its insolubility, lability, and toxic side effects; therefore, it is challenging to realize a highly efficient anticancer therapy of chlorambucil. To solve the above drawbacks encountered by chlorambucil, herein we proposed an amphiphilic chlorambucil prodrug-based self-assembled micelle strategy to realize the highly efficient anticancer therapy of chlorambucil. 1,6-Hexanediamine hydrochloride (HDH) serving as the hydrophilic segment was covalently bound to hydrophobic CLB to prepare an amphiphilic prodrug CLB-HDH which could selfassemble into micelles in aqueous solution. These micelles can passively target tumor tissues via the enhanced permeability and retention (EPR) effect, leading to enhanced cellular internalization. Both the cytotoxicity assay in vitro and anticancer study in vivo confirmed the excellent therapeutic activity of CLB-HDH micelles in comparison with free chlorambucil. Moreover, the hemolysis examination and histological analysis demonstrated the designed CLB-HDH micelles are safe in drug delivery. Therefore, our designed amphiphilic prodrug CLB-HDH micelles bring new opportunity for chlorambucil clinical application to combat cancers. KEYWORDS: amphiphilic molecule, chlorambucil, prodrug, micelle, cancer therapy

■

INTRODUCTION In the field of cancer therapy, the efficient delivery of chemotherapeutic agents is confronted with multiple barriers. Central obstacles include low water-solubility,1 poor stability, rapid clearance from the bloodstream,2,3 severe multidrug resistance,4,5 lack of site specificity, and adverse side effects for healthy tissues.6,7 In order to address these issues, nanosized vehicles for drug delivery such as polymeric micelles,8−11 polymeric nanoparticles,12,13 liposomes,14−17 and inorganic materials18,19 have been developed. Unfortunately, numerous carriers are characterized with low/unstable drug loading,20−22 and usually bring about serious side effects during the course of degradation, metabolism, and excretion.23,24 Therefore, it is of great interest to develop a drug-delivery system which can solve the central obstacles described above and realize a high/stable drug loading for achieving highly efficient anticancer therapy. Chlorambucil (CLB), a commercially available nitrogen mustard drug,25 has been widely used against a variety of cancers including chronic lymphocytic leukemia,26 ovarian cancer,27 breast cancer,28 lymphomas,29 and some other malignancies.30−32 Chlorambucil produces its cytotoxic effects by the two reactive chloroethyl side chains which bind preferably to the N7 atoms of guanine bases,33 causing interstrand and intrastrand linking of DNA,34,35 and thus leading to halted DNA replication and DNA damage. However, chlorambucil has low solubility and easily suffers from rapid © XXXX American Chemical Society

degradation in an aqueous environment, resulting in a short half-life in vivo.36 In addition, its toxic side effects,37 such as myelotoxicity and neurotoxicity,38 are factors limiting its application and development in chemotherapy. Improving the solubility and stability of chlorambucil and increasing its accumulation at the action sites to reduce side effects are highly demanded for improving its anticancer therapy effect. Micelles based on chlorambucil conjugates with high drug loading are effective drug carriers to overcome the shortcomings of CLB. Chlorambucil conjugates need the hydrophobic drug molecule and a hydrophilic group or segment to endow them with amphipathy to self-assemble into micelles at a relatively low concentration in aqueous solution. With the advantages of simple structure with cation and low toxicity, 1, 6-hexanediamine hydrochloride was chosen to act as the hydrophilic part. Herein, we rationally designed and synthesized a new amphiphilic prodrug CLB-HDH prodrug, whereby the hydrophobic CLB molecule was covalently linked to a HDH molecule working as a hydrophilic part. The synthesized amphiphilic CLB-HDH prodrug is capable of self-assembling into micelles when dispersed in aqueous solution. The asprepared micelles can be utilized directly as a bioactive drug Received: November 19, 2017 Accepted: February 7, 2018 Published: February 7, 2018 A

DOI: 10.1021/acsbiomaterials.7b00892 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

solution. The resulting mixture was stirred in the dark. After 48 h, rotary evaporation and vacuum were employed to remove solvent. The crude product was further purified by column chromatograph using petroleum ether and ethyl acetate (from 5:1 to 1.5:1, v/v) as the eluent. The final product appeared to be amorphous white solid (0.37 g, 90%). The product was confirmed by nuclear magnetic resonance spectroscopy (NMR, Bruker Avance 400 spectrometer). 1H NMR (400 MHz, CDCl3) δ: 7.26 (solvent residual peak of CDCl3), 7.08 (d, J = 8.55 Hz, H13 and H13′), 6.67 (d, J = 8.56 Hz, H14 and H14′), 5.55 (broad s, 1H, H9), 4.71 (solvent residual peak of water), 4.54 (broad s, 1H, H2), 3.71 (t, 4H, H15 and H15′), 3.62 (t, 4H, H16 and H16′), 3.23 (t, 2H, H8), 3.10 (t, 2H, H3), 2.56 (t, J = 7.51 Hz, H12), 2.17 (t, J = 7.53 Hz, H10), 1.96−1.87 (m, 2H, H11), 1.52−1.21 (17H, H1, H4, H5, H6 and H7). Synthesis of CLB-HDH. CLB-HD-BOC (0.37 g, 0.74 mmol) was directly dissolved into 3 mL of ethyl acetate with saturated HCl. The resulting mixture was stirred for 3 h in the dark. Then the reaction solution was filtered and the obtained product was then washed with ether and dried in a vacuum oven to give CLB-HDH. The product was white solid (0.27 g, 83%). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.91 (s, 3H, H1), 7.79 (s, 1H, H8), 7.01 (d, J = 8.48 Hz, H12 and H12′), 6.66 (d, J = 8.60 Hz, H13 and H13′), 3.69 (8H, H14, H14′, H15 and H15′), 3.02 (t, 2H, H7), 2.74 (t, 2H, H2), 2.42 (t, 2H, H11), 2.04 (t, 2H, H9), 1.76−1.66 (m, 2H, H10), 1.55−1.49 (m, 2H, H6), 1.39−1.34 (m, 2H, H3), 1.30−1.25 (4H, H4 and H5).13C NMR (DMSO-d6) δ (ppm): 172.23, 144.81, 130.47, 129.76 (2C), 112.40 (2C), 52.70 (2C), 41.62 (2C), 39.09, 38.67, 35.39, 34.12, 29.44, 27.90, 27.36, 26.35, 25.98. Preparation and Characterization of Micelles Formed by CLB-HDH. The CLB-HDH micelles were prepared by the direct dissolution method. To research the self-assembly behavior of amphiphilic CLB-HDH in aqueous solution, the critical micelle concentration (CMC) was determined via electrical conductivity experiments making use of a DDS-11A conductivity meter, according to the previous study.43,44 CLB-HDH aqueous solutions of different concentrations were prepared from 0.0025 to 2.25 mmol L−1. All the samples were measured at room temperature. The average size and size distribution of CLB-HDH micelles were obtained from dynamic light scattering (DLS, BIC·Brook-Haven). Zeta potential (Malvern, Zetasizer NanoZS) was also measured. The morphology was observed using transmission electron microscopy (TEM, JEM-200CX). In Vitro Drug Release Study. To measure the release profile, the dialysis method was used. For comparison, the release profile of free CLB solution was also studied. The prepared sample solution was loaded into a dialysis bag (MWCO = 1000 Da). The dialysis bag was then put into 30 mL of phosphate buffer saline (PBS, pH = 7.4) containing 0.5% (w/w) Tween-80, stirred slightly with the rate of 100 rpm at 37 °C in the dark. At the selected time intervals, 3 mL of the released solution was collected for drug quantification, and then 3 mL of fresh release medium was added immediately to maintain the sinking condition. The amounts of released CLB-HDH and CLB were investigated with UV−vis spectrophotometry. Each measurement was carried out in triplicate. Cell Culture. Human leukemic cells (K562), human breast cancer cells (MCF-7), and mouse melanoma cells (B16F10) were kindly provided by Shandong Analysis and Test Center. The three cell lines were cultivated in RPMI-1640 culture media. The culture medium contained 10% fetal bovine serum (FBS) at the temperature of 37 °C under a humidified atmosphere containing 5% CO2. In Vitro Cellular Uptake Assay. The cellular uptake behavior of CLB-HDH micelles was studied qualitatively in MCF-7 cells. Because CLB molecules possess no inherent fluorescent property, 5dodecanoylaminofluorescein (DAF) acting as a fluorescent marker was chosen to replace a small portion of CLB-HDH in the micelles. MCF-7 cells were seeded in six-well microplates at 2.0 × 105 cells per well in culture medium and incubated for 24 h at the temperature of 37 °C. Afterward, the culture medium was substituted with medium containing CLB-HDH micelles (DAF content: 2%) and free DAF molecules, respectively. After the incubation of 2 h, the culture medium was removed, and cells were then washed three times with

delivery system for cancer therapy because they are formed by a CLB-based prodrug (Scheme 1). The major advantages of the Scheme 1. Schematic Illustration of CLB-HDH Micelles as Drug-Delivery System: Circulation in the Bloodstream, Accumulation via EPR Effect, Internalization by Endocytosis, and Intracellular Drug Release

present newly developed prodrug-formed micelles as drug delivery system are as follows: (i) CLB drug solubility is significantly improved, and extremely high drug loading is obtained as the amphiphilic prodrug itself contains CLB segment; (ii) the CLB segment lies in the interior of the micelle based on the molecular structure of the amphiphilic CLB-HDH prodrug, shielding the bioactive CLB segment from degradation and expanding its lifetime in the bloodstream; (iii) the asprepared micelles can accumulate at tumor tissues via the enhanced permeability and retention effect (EPR),39 and then they are effectively internalized by cells via the endosomal pathway,40 which is speculated to be a major reason for micelles to bypass the P-glycoprotein (P-gp) mediated drug efflux associated with multidrug resistance (MDR);41 (iV) the micelles equipped with positive charges could show increased cellular binding and uptake in a nonspecific manner, owing to the electrostatic interactions between cationic micelles and cell membranes equipped with negative charges.42

■

EXPERIMENTAL SECTION

Materials. Chlorambucil was obtained from Dalian Meilun Biology Technology Co., Ltd. N-Boc-1,6-Hexanediamine (HD-BOC) was purchased from Aladdin Industrial Corporation. Ethyl chloroformate (C3H5ClO2) was provided by Chengdu Beisite Reagent Co., Ltd. Triethylamine (TEA, C6H15N), dichloromethane (CH2Cl2), petroleum ether, and ethyl acetate (EA, C4H8O2) were purchased from Tianjin Fuyu Fine Chemical Co., Ltd. TEA was used as received without further purication. Dichloromethane was refluxed with calcium hydride and distilled before use. Water used in all experiments was deionized. Synthesis of CLB-HD-BOC. Chlorambucil (0.25 g, 0.82 mmol) was directly dissolved in anhydrous CH2Cl2 (8 mL), and then the solution was stirred in ice-bath under nitrogen atmosphere. Then, triethylamine (142 μL, 0.96 mmol) and ethyl chloroformate (90 μL, 0.91 mmol) were sequentially added to the stirring solution. After 20 min, a solution of N-Boc-1,6-hexanediamine (0.19 g, 0.88 mmol) in anhydrous CH2Cl2 (8 mL) was added dropwise to the reaction B

DOI: 10.1021/acsbiomaterials.7b00892 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

where V is the tumor volume, L is the longest diameter, and W is the shortest diameter of the tumor.47 Every treatment group consisted of five mice (n = 5). In Vivo Anticancer Efficacy. When the tumor volumes reached 50−100 mm3 (10 days after implantation), the tumor-bearing mice were then randomly divided into three groups. Mice of different treatment groups were all intravenously injected via the tail vein with saline and CLB related formulations (the equivalent CLB dose was kept at 4.5 mg kg−1) every other day for 12 days. As indications of the antitumor activity and safety of our formulations, tumor volume and body weight were measured before every injection.48 Relative body weight was calculated according to W/W0 (W0 was the body weight when the treatment was initiated).49 The tumor inhibition ratio (IR) was calculated using the following equation:50

fresh PBS for the sake of removing the sample that had not been ingested. Cells were imaged with the help of a fluorescent inverted microscope (ECLIPSE-Ti, Nikon) with identical setting parameters. In Vitro Cytotoxicity Assay. The in vitro cytotoxicity of CLBHDH micelles against K562 cells, MCF-7 cells, and B16F10 cells were estimated by virtue of MTT assay.45 Exponentially growing cells were seeded in 96-well plates, reaching a cell density of 5 × 103 for every well in culture media and then incubated for 24 h. After the discard of cell growth medium, free CLB and CLB-HDH micelle solutions with various CLB concentrations (0.5, 1, 10, 50, 150 μmol L−1) were added into the culture medium, respectively. The cells were grown for 24 and 48 h. After scheduled time intervals, 10 μL of PBS-containing MTT (5 mg mL−1) was added to each cell well, and cells were afterward incubated for another 4 h at the temperature of 37 °C. Afterward, the supernatant was cleared away and 150 μL of DMSO was added to each well to make the obtained blue formazan crystals dissolved. The absorbance at 490 nm of each well was measured by a microplate reader (ELIASA of PerkinElmer) to work out the cell inhibition rates and IC50 values according to the following formula: cell inhibition rate (%) =

A positive − A sample A positive − A blank

tumor inhibition rate (%) =

A sample − A negative A positive − A negative

× 100% (1)

■

RESULTS AND DISCUSSION The synthetic processes of CLB-HDH were depicted in Figure 1A. The chemical structures of compound CLB-HD-BOC and CLB-HDH were confirmed by 1H NMR and mass spectrometry (MS). In Figure S1 (Supporting Information), the peak at 5.55 ppm belonged to the -CO-NH- of CLB-HD-BOC. The absence of the carboxyl group peak of CLB and the appearance of characteristic peaks from HD-BOC demonstrated the successful linkage between CLB and HD-BOC. As shown in Figure S2 (Supporting Information), a specific [M + H+] peak of CLB-HD-BOC was observed at 502.4, which is consistent with theoretically calculated value. In Figure 1B, compared with the 1H NMR spectroscopy of compound CLB-HD-BOC, the characteristic peak related to the BOC group disappeared completely and the new peak at 7.91 ppm corresponded to the terminal -NH3+ of CLB-HDH, confirming the successful preparation of compound CLB-HDH. The MS data (Figure S3, Supporting Information) showed that the molecular weight of CLB-HDH (m/z, M + H+) was 402.6 due to the loss of HCl, which was in accordance with the calculated value. In the 13C NMR spectrum (Figure S4) of the CLB-HDH conjugate, an obvious peak appeared at 172.23 ppm belonging to the -CONH- group. The molecular structure of CLB-HDH is similar to the surfactant, which endows it with the ability to self-assemble into micelles upon dispersion in aqueous solution. The conductivity method was used to measure the CMC of the CLB-HDH. According to the curve (Figure 2A), there was a growing tendency in the conductivity along with the increase of concentration. Notably, as the concentration grew beyond a certain value, the conductivity started to increase remarkably with the increasing concentration. Based on the inflection of the curve, the CMC value of the CLB-HDH micelles is about 142 μmol L−1. DLS was further applied to characterize the size and size distribution of the micelles. In Figure S5, the micelles show an average hydrodynamic diameter of 49 nm with a polydispersity index (PDI) of 0.271. From the TEM image revealed in Figure 2B, it is demonstrated that the CLB-HDH

× 100% (2)

where Asample, Anegative, and Apositive refer to the absorbance of sample, negative control, and positive control at 540 nm, respectively. Animal Melanoma Models. Healthy male Kunming mice (18−22 g) were obtained from the Shandong University laboratory animal center (Jinan, China) and housed under optimum care. All animalrelated experimental procedures were performed under the requirements of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Animal Experiment Ethics Review of Shandong University. B16F10 cells were provided by the Laboratory of Pharmacology, School of Pharmaceutical Science, Shandong University. The animal tumor models were built by subcutaneous injection with 0.1 mL B16F10 cell suspension at a density of 1 × 107 cells mL−1 into the right anterior armpit region of each mouse. The tumor volume was calculated according to the following formula: V=

L × W2 2

(4)

where Wt and Wc refer to the average tumor weight of treatment group and control group injected by normal saline, respectively. Histological Examination. After the continuous treatments for 12 days, all the mice were sacrificed. Major organs including heart, liver, spleen, lung, and kidney were dissected, and tumors were harvested, weighed, and photographed. Tumors as well as major organs were fixed in 4% formaldehyde, embedded in paraffin, and sliced routinely. The prepared sections were used for histological analysis with hematoxylin and eosin (HE) staining.51

Cells without treatment were used as the positive group and medium without drugs or cells was considered as the blank group. The IC50 values referring to half maximal inhibitory concentrations were further calculated. For all samples, the cytotoxicity experiment was repeated three times. Hemolytic Evaluation. Hemolytic study was performed to evaluate the blood compatibility of CLB-HDH micelles with regard to the feasibility for intravenous administration. The procedures were based on the previous literature with minor modifications.46 Derived from the New Zealand white rabbit, all the plasma samples were centrifuged and resuspended in normal saline to obtain the red blood cells (RBC, 2%, v/v). 1.25 mL RBC suspension was dispersed in 1.25 mL normal saline solution to work as the negative control and 1.25 mL distilled water to serve as the positive control, respectively. A series of 0.15 mL micelles solutions with different concentrations ranging from 0.01 mg mL−1 to 0.2 mg mL−1 were mixed with normal saline solution (1.10 mL) and incubated with the prepared RBC suspension (1.25 mL) under a temperature of 37.0 ± 1.0 °C for 2 h, which was followed by centrifugation at 1500 rpm for 15 min. Subsequently, an ultraviolet spectrophotometer was employed to measure the absorbance of the supernatant at 540 nm which was the typical absorbance of hemoglobin released from RBCs. All groups were conducted in triplicate, and the degree of hemolysis was calculated on the basis of the following equation:

hemolysis (%) =

Wc − Wt × 100% Wc

(3) C

DOI: 10.1021/acsbiomaterials.7b00892 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

The accumulative release curves were provided in Figure 3. As it could be seen, free CLB molecules underwent a rapid release

Figure 3. In vitro drug release curves of CLB solution and CLB-HDH micelles.

due to its relatively small molecular size. For example, the cumulative release amount almost reached 100% in the initial 3 h. In contrast, a sustained drug release profile was obtained for CLB-HDH micelles. For example, the release amount of CLBHDH was only 62% at the same time point of 3 h. After being released from micelles, the CLB-HDH molecules could be cleaved to active CLB and HDH molecules through the cleavage of amide bonds in the tumor.52−54 CLB tends to be released in the cell on the grounds that the amide linkage deriving from the COOH group of CLB and the amino group of each linker can be easily cleaved through lysosomal proteases.55 It could be speculated from the experimental results that the formation of micelles delayed the drug release to a large extent, indicating the promising applications of CLBHDH micelles in drug delivery for CLB. To determine whether the CLB-HDH micelles could effectively deliver drugs into cells, the cellular uptake by MCF-7 cells was investigated using a fluorescent inverted microscope. Because CLB molecules possess no inherent fluorescent property, 5-dodecanoylaminofluorescein (DAF) acting as a fluorescent marker was chosen as a model molecule to replace the CLB molecules or small portion of CLB-HDH in the micelles. Fluorescent dye DAF is a widely used lipophilic fluorescein probe. The DAF could bind with the hydrophobic moiety of CLB-HDH molecules and thus the alkyl tail of DAF protrudes into the hydrophobic interior of micelles. Therefore, the DAF together with CLB-HDH could form the mixed micelles. In the experiments, the cellular uptake efficiency of the DAF-carrying CLB-HDH micelles was assessed, which was compared with free DAF molecules. As demonstrated by the fluorescence presented in Figure 4, both free DAF molecules and micelles could be effectively taken up by the cells after incubation for 2 h. Notably, cells treated with CLB-HDH micelles exhibited obviously higher fluorescence intensity than the cells treated with free DAF, which indicated the higher cell internalization efficiency of micelles. The results could be attributed to the distinct ways free DAF and micelles enter into cells. The free DAF molecules entered into cells through diffusion, which would be affected by the functions of P-gp efflux pumps. Nevertheless, the DAF-carried CLB-HDH micelles were internalized via endocytosis which provided an opportunity for the micelles to avoid the P-gp-mediated drug efflux efficiently. Furthermore, the positively charged micelles could work well in cell-membrane penetration and cellular

Figure 1. Synthetic route of CLB-HDH (A) and 1H NMR spectrum of CLB-HDH (B).

Figure 2. Electrical conductivity (A) at different concentrations of CLB-HDH. TEM image (B) of CLB-HDH micelles.

could self-assemble into well-dispersed micelles with an average diameter of approximate 40 nm, which was slightly smaller than that determined by DLS as a result of the drying state of micelles during the process of TEM sample preparation. The zeta potential of the micelles was measured to be 19.5 ± 7.8 mV, indicating the positive charge characteristic of the micelles which was consistent with the molecular structure of CLBHDH. When the compound dissociates in water, chlorine ion is in a free state, leading to a -NH3+ group endowed with a positive charge at the terminal of CLB-HDH. Therefore, the micelles show positive charge characteristic. The in vitro release study was carried out via a dialysis method under simulated physiological conditions to understand the drug release property of our prepared micelles. For comparison, the release profile of free CLB was also studied. D

DOI: 10.1021/acsbiomaterials.7b00892 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

Figure 5. Cell inhibition rates of different samples to K562, MCF-7 and B16F10 cells after 24 and 48 h incubation, *p < 0.05, **p < 0.01, *** p < 0.001, n = 3.

Figure 4. Cellular uptake images of free DAF (A) and DAF-carried CLB-HDH micelles (B) in MCF-7 cells after incubation for 2 h.

Table 1. IC50 (μmol L−1) of Different Samples against K562, MCF-7, and B16F10 Cells after 24 and 48 h Incubation

internalization owing to their effective binding to the cell surface equipped with negatively charged groups.56 The in vitro cytotoxic activity of CLB-HDH micelles was investigated by standard MTT assay toward K562, MCF-7, and B16F10 cell lines. For comparison, the in vitro cytotoxic activity of free CLB was also studied. The results were illustrated in Figure 5. The cytotoxicity of both CLB and CLB-HDH micelles showed a concentration-dependent and time-dependent manner. It was obvious that there was an increase in the cell inhibition rates along with the increase of drug concentrations and incubation time. However, CLB-HDH micelles exhibited obvious higher cytotoxic effects against all of the cell lines than that of an equivalent dose of free CLB after 48 h incubation. For example, the inhibition rates reached up to 94%, 98%, and 86% for K562, MCF-7, and B16F10 cell lines, respectively, for CLB-HDH micelles at 150 μmol L−1. The corresponding IC50 values of free CLB and CLB-HDH micelles at 24 and 48 h incubation time were calculated to quantitatively evaluate the proliferation inhibition, and the data was shown in Table 1. As displayed in the table, CLB-HDH micelles possessed a much lower IC50 value than free CLB after incubation of 24 or 48 h for all the cell lines. Especially for K562 and MCF-7 cells, the IC50 values of CLB-HDH micelles were lower by orders of magnitude than that of free CLB after 48 h. In a word, despite containing the same drug dosage, CLB-HDH micelles exhibited significantly improved inhibition to the cell proliferation. It is reported that cancer cells contain more negative charge than healthy cells on the surface.57 When the concentration was below the CMC value, the CLB-HDB molecules were present as monomers. The monomer had not only a hydrophobic part to facilitate cell membrane binding and penetration but also a

IC50 cells

formulation

24 h

48 h

K562

free CLB CLB-HDH micelles free CLB CLB-HDH micelles free CLB CLB-HDH micelles

119.8 55.3 77.2 19.7 122.7 104.4

71.0 7.0 54.5 4.7 29.1 11.4

MCF-7 B16F10

cationic domain which would contribute electrostatic cellular adhesion,58 leading to superior uptake and cytotoxicity than CLB. When the concentration was above CMC value, the higher in vitro cytotoxic activity of CLB-HDH micelles could be mainly attributed to the higher cellular uptake by endocytosis mechanism in comparison with the diffusion mechanism of free CLB. In this case, CLB-HDH micelles were expected to be effectively internalized by the cells, evading the P-gp efflux pump. Moreover, the positively charged micelles tended to bind to negatively charged cancer cells, increasing tumor uptake. Hemolytic study was carried out to assess the feasibility of intravenous administration of CLB-HDH micelles. The study is based on the fact that hemoglobin will be released from red blood cells when cell membranes are destroyed. The results in Figure 6 demonstrated that the hemolytic rates of all the prepared CLB-HDH micelle solutions were less than 2% within the studied concentration range. Positive charges on the surface of CLB-HDH micelles should be responsible for the negligible hemolytic toxicity to red blood cells with negatively charged E

DOI: 10.1021/acsbiomaterials.7b00892 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

to tumor weight. From Figure 7C, the TIR of CLB-HDH micelles is determined to be 88%, which is remarkably higher than that of free CLB (50%). As an indication of systemic toxicity, the body weight of mice was monitored during the whole administration process. The changes of relative body weight were showed in Figure 7D. No significant body weight loss was observed in CLB-HDH micelle group, implying no noticeable systemic toxicity. Figure 7E showed the images of the dissected tumors, which were in accordance with the tumor growth curves. The predominant antitumor activity of CLBHDH micelles could be ascribed to the following reasons. On one hand, the bioactive CLB segment lies in the interior of the micelle structure, and therefore, the degradation of CLB segment in the blood could be considerably reduced, leading to prolonged stability. On the other hand, the improved accumulation in tumor tissues via the EPR effect and efficient cellular uptake could be achieved owing to the micelle structure of appropriate nanoscale. All the results thus suggested a bright prospect of our as-prepared CLB-HDH micelles as highly efficient drug-delivery system for cancer therapy. Histology analysis was further performed on the major organs (heart, liver, spleen, lung, and kidney) and tumors with various treatments to confirm the safety and anticancer effectiveness of our CLB-HDH micelles. The results were depicted in Figure 8. It could be observed that major organs

Figure 6. Hemolytic rates of CLB-HDH micelles at different concentrations.

surface. All the hemolytic rates were evidently below the reported requirement of 5%, implying the biocompatibility and security of CLB-HDH micelles for intravenous injection.59 The in vivo anticancer efficacy of our CLB-HDH micelles was estimated against melanoma-bearing Kunming mice by tail vein injection. For comparison, the mice were treated with normal saline, CLB, and CLB-HDH micelles, respectively. As exhibited in Figure 7A, the tumors of normal saline group

Figure 8. HE-stained images of major organs (heart, liver, spleen, lung, and kidney) and tumors after treatments with normal saline, CLB and CLB-HDH micelles.

including heart, liver, spleen, lung, and kidney did not exhibit any histological changes after the administration of CLB-HDH micelles. Tumor tissue morphology showed palpable differences between normal-saline and drug-treated (CLB and CLBHDH micelles) groups. The group treated with normal saline was observed with large nuclei in the tumor cells, signifying a rapid growth tendency. In addition, with a degree of reduction of cell nuclei, moderate inhibition of tumor growth was obtained in the CLB group. Interestingly, significant tumor tissue damage in large regions and severe nuclear shrinkage were observed in CLB-HDH micelles group, indicating that the CLB-HDH micelle group has the best antitumor effect. Therefore, CLB-HDH micelles could be used as a promising drug-delivery system for generating an excellent antitumor effect.

Figure 7. (A) Changes of tumor volumes after treatment with normal saline, CLB and CLB-HDH micelles. (B) Mean weights of tumors separated from mice. (C) Average tumor inhibition ratios of each group. (D) Relative body weight changes of mice in each group. (E) Image of separated tumors after different treatments.

rapidly grew up to about 1500 mm3 after 12 days. The tumor growth was inhibited in the group treated with free CLB during initial 4 days; however, the tumors exhibited a further rapid growth afterward. In contrast, the tumor volumes of mice treated with CLB-HDH micelles were significantly smaller than that of mice treated with normal saline and free CLB, which is indicative of the superior tumor growth inhibitory efficacy of the micelles. To further determine the therapeutic efficacy, tumors were dissected, imaged, and weighed after the completion of the experiments. As illustrated in Figure 7B, CLB-HDH micelles group possessed the smallest mean tumor weight value, compared with normal saline and CLB groups. Then, the tumor inhibitory rate (TIR) was calculated according

■

CONCLUSIONS In summary, a new amphiphilic CLB-HDH prodrug was rationally designed and synthesized to solve the drawbacks such as low solubility, high lability, and high toxic side effects encountered by CLB for cancer therapy. The as-synthesized F

DOI: 10.1021/acsbiomaterials.7b00892 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

(7) Gou, P.; Liu, W.; Mao, W.; Tang, J.; Shen, Y.; Sui, M. Selfassembling doxorubicin prodrug forming nanoparticles for cancer chemotherapy: synthesis and anticancer study in vitro and in vivo. J. Mater. Chem. B 2013, 1, 284−292. (8) Zhang, H.; Wang, K.; Zhang, P.; He, W.; Song, A.; Luan, Y. Redox-sensitive micelles assembled from amphiphilic mPEG-PCL-SSDTX conjugates for the delivery of docetaxel. Colloids Surf., B 2016, 142, 89−97. (9) Zhang, P.; Zhang, H.; He, W.; Zhao, D.; Song, A.; Luan, Y. Disulfide-linked amphiphilic polymer-docetaxel conjugates assembled redox-sensitive micelles for efficient antitumor drug delivery. Biomacromolecules 2016, 17, 1621−1632. (10) Zhao, D.; Zhang, H.; Yang, S.; He, W.; Luan, Y. Redox-sensitive mPEG-SS-PTX/TPGS mixed micelles: An efficient drug delivery system for overcoming multidrug resistance. Int. J. Pharm. 2016, 515, 281−292. (11) Guo, Y.; He, W.; Yang, S.; Zhao, D.; Li, Z.; Luan, Y. Co-delivery of docetaxel and verapamil by reduction-sensitive PEG-PLGA-SS-DTX conjugate micelles to reverse the multi-drug resistance of breast cancer. Colloids Surf., B 2017, 151, 119−127. (12) Griset, A. P.; Walpole, J.; Liu, R.; Gaffey, A.; Colson, Y. L.; Grinstaff, M. W. Expansile nanoparticles: synthesis, characterization, and in vivo efficacy of an acid-responsive polymeric drug delivery system. J. Am. Chem. Soc. 2009, 131, 2469−2471. (13) Nicolas, J.; Mura, S.; Brambilla, D.; Mackiewicz, N.; Couvreur, P. Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery. Chem. Soc. Rev. 2013, 42, 1147−1235. (14) Gao, J.-Q.; Lv, Q.; Li, L.-M.; Tang, X.-J.; Li, F.-Z.; Hu, Y.-L.; Han, M. Glioma targeting and blood−brain barrier penetration by dual-targeting doxorubincin liposomes. Biomaterials 2013, 34, 5628− 5639. (15) Zhang, Q.; Deng, C.; Fu, Y.; Sun, X.; Gong, T.; Zhang, Z. Repeated administration of hyaluronic acid coated liposomes with improved pharmacokinetics and reduced immune response. Mol. Pharmaceutics 2016, 13, 1800−1808. (16) Zhang, X.; Guo, S.; Fan, R.; Yu, M.; Li, F.; Zhu, C.; Gan, Y. Dual-functional liposome for tumor targeting and overcoming multidrug resistance in hepatocellular carcinoma cells. Biomaterials 2012, 33, 7103−7114. (17) Pedersen, P. J.; Christensen, M. S.; Ruysschaert, T.; Linderoth, L.; Andresen, T. L.; Melander, F.; Mouritsen, O. G.; Madsen, R.; Clausen, M. H. Synthesis and biophysical characterization of chlorambucil anticancer ether lipid prodrugs. J. Med. Chem. 2009, 52, 3408−3415. (18) Wang, Y.; Zhao, Q.; Han, N.; Bai, L.; Li, J.; Liu, J.; Che, E.; Hu, L.; Zhang, Q.; Jiang, T.; Wang, S. Mesoporous silica nanoparticles in drug delivery and biomedical applications. Nanomedicine 2015, 11, 313−327. (19) Tang, F.; Li, L.; Chen, D. Mesoporous silica nanoparticles: synthesis, biocompatibility and drug delivery. Adv. Mater. 2012, 24, 1504−1534. (20) Lee, E. S.; Na, K.; Bae, Y. H. Doxorubicin loaded pH-sensitive polymeric micelles for reversal of resistant MCF-7 tumor. J. Controlled Release 2005, 103, 405−418. (21) Tong, R.; Cheng, J. Paclitaxel-initiated, controlled polymerization of lactide for the formulation of polymeric nanoparticulate delivery vehicles. Angew. Chem., Int. Ed. 2008, 47, 4830−4834. (22) Fang, S.; Niu, Y.; Zhang, W.; Zhang, Y.; Yu, L.; Zhang, Y.; Li, X. Liposome-like nanocapsules of dual drug-tailed betaine for cancer therapy. Int. J. Pharm. 2015, 493, 460−465. (23) Luo, C.; Sun, J.; Sun, B.; He, Z. Prodrug-based nanoparticulate drug delivery strategies for cancer therapy. Trends Pharmacol. Sci. 2014, 35, 556−566. (24) Versluis, F.; van Esch, J. H.; Eelkema, R. Synthetic SelfAssembled Materials in Biological Environments. Adv. Mater. 2016, 28, 4576−4592. (25) Chen, H.; Tham, H. P.; Ang, C. Y.; Qu, Q.; Zhao, L.; Xing, P.; Bai, L.; Tan, S. Y.; Zhao, Y. Responsive prodrug self-assembled vesicles

amphiphilic prodrug can self-assemble into micelles. Specifically, high drug loading (70 wt %) is obtained as the amphiphilic CLB-HDH prodrug itself contains CLB segment. The CLB segment lies in the interior of the micelles, which protects the bioactive CLB segment from degradation and expands its lifetime in the bloodstream. It was demonstrated that the CLB-HDH micelles showed significant higher cellular uptake than that of free CLB. Moreover, compared with free CLB, the CLB-HDH micelles exhibited significantly improved cytotoxicity in vitro and remarkably enhanced inhibition to tumor growth without obvious side effects in vivo. As a result, our newly designed CLB-HDH micelles successfully solve the drawbacks of free CLB as anticancer drug, which offers a new and efficient platform for cancer therapy.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00892. 1 H NMR spectrum of CLB-HD-BOC; MS spectrum of CLB-HD-BOC; MS spectrum of CLB-HDH; 13C NMR spectrum of CLB-HDH; The size and size distribution of CLB-HDH micelles; UV spectra of CLB and CLB-HDH in PBS (pH = 7.4) (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (86) 531-88382007. Fax: (86) 531-88382548. ORCID

Zhonghao Li: 0000-0003-0699-300X Yuxia Luan: 0000-0002-7480-2642 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (NSFC, No. 21373126 and No. 21673128).

■

REFERENCES

(1) Liang, L.; Lin, S. W.; Dai, W.; Lu, J. K.; Yang, T. Y.; Xiang, Y.; Zhang, Y.; Li, R. T.; Zhang, Q. Novel cathepsin B-sensitive paclitaxel conjugate: Higher water solubility, better efficacy and lower toxicity. J. Controlled Release 2012, 160, 618−629. (2) Maksimenko, A.; Mougin, J.; Mura, S.; Sliwinski, E.; Lepeltier, E.; Bourgaux, C.; Lepêtre, S.; Zouhiri, F.; Desmaële, D.; Couvreur, P. Polyisoprenoyl gemcitabine conjugates self- assemble as nanoparticles, useful for cancer therapy. Cancer Lett. 2013, 334, 346−353. (3) Liu, J.; Liu, J.; Zhao, D.; Ma, N.; Luan, Y. Highly enhanced leukemia therapy and oral bioavailability from a novel amphiphilic prodrug of cytarabine. RSC Adv. 2016, 6, 35991−35999. (4) Li, N.; Huang, C.; Luan, Y.; Song, A.; Song, Y.; Garg, S. Active targeting co-delivery system based on pH-sensitive methoxy-poly (ethylene glycol)2K-poly(e-caprolactone)4K-poly(glutamic acid)1K for enhanced cancer therapy. J. Colloid Interface Sci. 2016, 472, 90−98. (5) Huang, P.; Wang, D.; Su, Y.; Huang, W.; Zhou, Y.; Cui, D.; Zhu, X.; Yan, D. Combination of small molecule prodrug and nanodrug delivery: amphiphilic drug−drug conjugate for cancer therapy. J. Am. Chem. Soc. 2014, 136, 11748−11756. (6) Jain, R. K. Delivery of molecular and cellular medicine to solid tumors. Adv. Drug Delivery Rev. 1997, 26, 71−90. G

DOI: 10.1021/acsbiomaterials.7b00892 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering for targeted chemotherapy in combination with intracellular imaging. ACS Appl. Mater. Interfaces 2016, 8, 24319−24324. (26) Rai, K. R.; Peterson, B. L.; Appelbaum, F. R.; Kolitz, J.; Elias, L.; Shepherd, L.; Hines, J.; Threatte, G. A.; Larson, R. A.; Cheson, B. D.; Schiffer, C. A. Fludarabine compared with chlorambucil as primary therapy for chronic lymphocytic leukemia. N. Engl. J. Med. 2000, 343, 1750−1757. (27) Horton, J. K.; Roy, G.; Piper, J. T.; Van Houten, B. V.; Awasthi, Y. C.; Mitra, S.; Alaoui-Jamali, M. A.; Boldogh, I.; Singhal, S. S. Characterization of a chlorambucil-resistant human ovarian carcinoma cell line overexpressing glutathione S-transferase μ. Biochem. Pharmacol. 1999, 58, 693−702. (28) Millard, M.; Gallagher, J. D.; Olenyuk, B. Z.; Neamati, N. A selective mitochondrial-targeted chlorambucil with remarkable cytotoxicity in breast and pancreatic cancers. J. Med. Chem. 2013, 56, 9170−9179. (29) Nicolle, A.; Proctor, S. J.; Summerfield, G. P. High dose chlorambucil in the treatment of lymphoid malignancies. Leuk. Lymphoma 2004, 45, 271−275. (30) Qin, X.; Fang, L.; Chen, F.; Gou, S. Conjugation of platinum (IV) complexes with chlorambucil to overcome cisplatin resistance via a “joint action” mode toward DNA. Eur. J. Med. Chem. 2017, 137, 167−175. (31) Lin, J.; Qiu, L.; Lv, G.; Li, K.; Wang, W.; Liu, G.; Zhao, X.; Wang, S. Synthesis and preliminary biological evaluation of a 99mTcchlorambucil derivative as a potential tumor imaging agent. J. Labelled Compd. Radiopharm. 2017, 60, 116−123. (32) Mukherjee, S.; Sarma, J. D.; Shunmugam, R. pH-sensitive nanoaggregates for site-specific drug-delivery as well as cancer cell imaging. ACS Omega 2016, 1, 755−764. (33) Kohn, K. W.; Hartley, J. A.; Mattes, W. B. Mechanisms of DNA sequence selective alkylation of guanine-N7 positions by nitrogen mustards. Nucleic Acids Res. 1987, 15, 10531−10549. (34) Verwilst, P.; Han, J.; Lee, J.; Mun, S.; Kang, H.-G.; Kim, J. S. Reconsidering azobenzene as a component of small-molecule hypoxiamediated cancer drugs: A theranostic case study. Biomaterials 2017, 115, 104−114. (35) Shin, W. S.; Park, S. K.; Verwilst, P.; Koo, S.; Lee, J. H.; Chi, S.G.; Kim, J. S. Targeted combinational therapy inducing mitochondrial dysfunction. Chem. Commun. 2017, 53, 1281−1284. (36) Tadros, M. I.; Al-mahallawi, A. M. Long-circulating lipoproteinmimic nanoparticles for smart intravenous delivery of a practicallyinsoluble antineoplastic drug: development, preliminary safety evaluations and preclinical pharmacokinetic studies. Int. J. Pharm. 2015, 493, 439−450. (37) Fan, M.; Liang, X.; Li, Z.; Wang, H.; Yang, D.; Shi, B. Chlorambucil gemcitabine conjugate nanomedicine for cancer therapy. Eur. J. Pharm. Sci. 2015, 79, 20−26. (38) Ganta, S.; Paxton, J. W.; Baguley, B. C.; Garg, S. Pharmacokinetics and pharmacodynamics of chlorambucil delivered in parenteral emulsion. Int. J. Pharm. 2008, 360, 115−121. (39) Fang, J.; Nakamura, H.; Maeda, H. The EPR effect: unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv. Drug Delivery Rev. 2011, 63, 136−151. (40) Iversen, T.-G.; Skotland, T.; Sandvig, K. Endocytosis and intracellular transport of nanoparticles: present knowledge and need for future studies. Nano Today 2011, 6, 176−185. (41) Pavan, B.; Paganetto, G.; Rossi, D.; Dalpiaz, A. Multidrug resistance in cancer or inefficacy of neuroactive agents: innovative strategies to inhibit or circumvent the active efflux transporters selectively. Drug Discovery Today 2014, 19, 1563−1571. (42) Bertrand, N.; Wu, J.; Xu, X.; Kamaly, N.; Farokhzad, O. C. Cancer nanotechnology: the impact of passive and active targeting in the era of modern cancer biology. Adv. Drug Delivery Rev. 2014, 66, 2− 25. (43) Šegota, S.; Težak, D. Spontaneous formation of vesicles. Adv. Colloid Interface Sci. 2006, 121, 51−75.

(44) Rao, J. P.; Geckeler, K. E. Polymer nanoparticles: preparation techniques and size-control parameters. Prog. Polym. Sci. 2011, 36, 887−913. (45) Wu, L.; Zou, Y.; Deng, C.; Cheng, R.; Meng, F.; Zhong, Z. Intracellular release of doxorubicin from core-crosslinked polypeptide micelles triggered by both pH and reduction conditions. Biomaterials 2013, 34, 5262−5272. (46) Li, M.; Song, W.; Tang, Z.; Lv, S.; Lin, L.; Sun, H.; Li, Q.; Yang, Y.; Hong, H.; Chen, X. Nanoscaled poly (L-glutamic acid)/ doxorubicin-amphiphile complex as pH-responsive drug delivery system for effective treatment of nonsmall cell lung cancer. ACS Appl. Mater. Interfaces 2013, 5, 1781−1792. (47) Mou, Q.; Ma, Y.; Zhu, X.; Yan, D. A small molecule nanodrug consisting of amphiphilic targeting ligand−chemotherapy drug conjugate for targeted cancer therapy. J. Controlled Release 2016, 230, 34−44. (48) Lu, J.; Liu, C.; Wang, P.; Ghazwani, M.; Xu, J.; Huang, Y.; Ma, X.; Zhang, P.; Li, S. The self-assembling camptothecin-tocopherol prodrug: an effective approach for formulating camptothecin. Biomaterials 2015, 62, 176−187. (49) Shen, J.-M.; Gao, F.-Y.; Yin, T.; Zhang, H.-X.; Ma, M.; Yang, Y.J.; Yue, F. cRGD-functionalized polymeric magnetic nanoparticles as a dual-drug delivery system for safe targeted cancer therapy. Pharmacol. Res. 2013, 70, 102−115. (50) Zhong, Y.; Goltsche, K.; Cheng, L.; Xie, F.; Meng, F.; Deng, C.; Zhong, Z.; Haag, R. Hyaluronic acid-shelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast tumor xenografts in vivo. Biomaterials 2016, 84, 250− 261. (51) Dai, L.; Yu, Y.; Luo, Z.; Li, M.; Chen, W.; Shen, X.; Chen, F.; Sun, Q.; Zhang, Q.; Gu, H.; Cai, K. Photosensitizer enhanced disassembly of amphiphilic micelle for ROS-response targeted tumor therapy in vivo. Biomaterials 2016, 104, 1−17. (52) Bui, D. T.; Nicolas, J.; Maksimenko, A.; Desmaele, D.; Couvreur, P. Multifunctional squalene-based prodrug nanoparticles for targeted cancer therapy. Chem. Commun. 2014, 50, 5336−5338. (53) Arias, J. L.; Reddy, L. H.; Othman, M.; Gillet, B.; Desmaele, D.; Zouhiri, F.; Dosio, F.; Gref, R.; Couvreur, P. Squalene based nanocomposites: a new platform for the design of multifunctional pharmaceutical theragnostics. ACS Nano 2011, 5, 1513−1521. (54) Zhang, D.; Li, D.; Shang, L.; He, Z.; Sun, J. Transporter-targeted cholic acid-cytarabine conjugates for improved oral absorption. Int. J. Pharm. 2016, 511, 161−169. (55) Guaragna, A.; Chiaviello, A.; Paolella, C.; D’Alonzo, D.; Palumbo, G.; Palumbo, G. Synthesis and evaluation of folate-based chlorambucil delivery systems for tumor-targeted chemotherapy. Bioconjugate Chem. 2012, 23, 84−96. (56) Zhao, F.; Zhao, Y.; Liu, Y.; Chang, X.; Chen, C.; Zhao, Y. Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials. Small 2011, 7, 1322−1337. (57) Toft, D. J.; Moyer, T. J.; Standley, S. M.; Ruff, Y.; Ugolkov, A.; Stupp, S. I.; Cryns, V. L. Coassembled cytotoxic and pegylated peptide amphiphiles form filamentous nanostructures with potent antitumor activity in models of breast cancer. ACS Nano 2012, 6, 7956−7965. (58) Koppelhus, U.; Shiraishi, T.; Zachar, V.; Pankratova, S.; Nielsen, P. E. Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (catLip) domain. Bioconjugate Chem. 2008, 19, 1526−1534. (59) Rajagopal, K.; Mahmud, A.; Christian, D. A.; Pajerowski, J. D.; Brown, A. E. X.; Loverde, S. M.; Discher, D. E. Curvature-coupled hydration of semicrystalline polymer amphiphiles yields flexible worm micelles but favors rigid vesicles: polycaprolactone-based block copolymers. Macromolecules 2010, 43, 9736−9746.

H

DOI: 10.1021/acsbiomaterials.7b00892 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX