Complete regression of xenograft tumors using biodegradable mPEG-PLA-SN38 block copolymer micelles

Colloids and Surfaces B: Biointerfaces 142 (2016) 417–423

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Complete regression of xenograft tumors using biodegradable mPEG-PLA-SN38 block copolymer micelles Lu Lu a,b,1 , Zheng Yan a,b,1 , Weng Shuqiang c,1 , Zhu Wenwei a,b,1 , Chen Jinhong a,b,1 , Zhang Xiaomin d , Robert J. Lee e , Bo Yu d , Jia Huliang a,b,∗ , Qin Lunxiu a,b,∗ a

Department of General Surgery, Huashan Hospital, Fudan University, & Institutes of Biomedical Science, Fudan University, Shanghai 200040, China Institutes of Cancer Metastasis, Fudan University, Shanghai 200040, China c Department of Digestion, Zhongshan Hospital, Fudan University, Shanghai, China d Hangzhou PushKang Biotechnology Co., Ltd., Zhejiang, China e Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA b

a r t i c l e

i n f o

Article history: Received 2 September 2015 Received in revised form 27 November 2015 Accepted 16 February 2016 Available online 18 February 2016 Keywords: Block co-polymers SN38 Micelles Cancer Regression

a b s t r a c t 7-Ethyl-10-hydroxy-comptothecin (SN38) is an active metabolite of irinotecan (CPT-11) and the clinical application of SN38 is limited by its hydrophobicity and instability. To address these issues, a series of novel amphiphilic mPEG-PLA-SN38-conjugates were synthesized by linking SN38 to mPEG-PLA-SA, and they could form micelles by self-assembly. The effects of mPEG-PLA composition were studied in vitro and in vivo. The mean diameters of mPEG2K -PLA-SN38 micelles and mPEG4K -PLA-SN38 micelles were 10–20 nm and 120 nm, respectively, and mPEG2K -PLA-SN38 micelles showed greater antitumor efficacy than mPEG4K -PLA-SN38 micelles both in vitro and in vivo. These data suggest that the lengths of mPEG and PLA chains had a major impact on the physicochemical characteristics and antitumor activity of SN38-conjugate micelles. © 2016 Elsevier B.V. All rights reserved.

1. Introduction 7-Ethyl-10-hydroxy-camptothecin (SN38) belongs to the 20(S)camptothecin (CPT) group of compounds that act as a potent topoisomerase I inhibitor. It interferes with replication and transcription processes in the cell cycle [1–3] and exhibits significant antineoplastic activity against various human cancers, including colorectal, ovarian and non-small-cell lung cancers [4–6]. SN38 is an active metabolite of irinotecan (CPT-11), which is up to 1000fold more potent than CPT-11 against several tumor cell lines [7]. The hydroxyl lactone ring moiety of SN-38 is the essential structure for its antitumor potency. However, the clinical application of SN38 is limited by its poor aqueous solubility and instability of lactone ring at physiological pH [8]. In order to overcome these problems, several approaches have been proposed, such as incorporation into liposomes [7], nanoparticles [9,10], polymeric micelles [11–13], or conjugation to water-soluble polymers as polyethylene glycol [14]. Among drug carriers, polymeric micelles have mani-

∗ Corresponding authors at: No. 12 Middle Urumqi Road, Shanghai 200040, China. E-mail addresses: [email protected] (H. Jia), qin [email protected] (L. Qin). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2016.02.035 0927-7765/© 2016 Elsevier B.V. All rights reserved.

fested several attractive features and advantages over others, for example, their core-shell structure with drug in the core, which can effectively protect the drug from decomposition and prolong circulation in the blood [15]. In addition, the passive accumulation of polymeric micelles in solid tumor is achieved by the enhanced permeability and retention (EPR) effect in the tumor [16,17]. However, burst release of the drug from micelles is still a major problem that can not be avoided because of their dynamic instability. To overcome this problem, a drug molecule was chemically linked to an amphiphilic copolymer chain to form a polymer-drug conjugate. These polymer-drug conjugates have demonstrated several advantages over corresponding original drugs, including enhanced therapeutic efficacy, reduced side effects, decreased drug administration and improved patient compliance. The polymerdrug conjugates not only retained the desirable properties of the micelles, but also enhanced control release property [18–22]. Methoxypoly(ethylene glycol)-b-poly(lactide) (mPEG-PLA) is a typical amphiphilic block copolymer and behaves like a surfactant. It can self-assemble into micelles in aqueous medium with hydrophobic PLA as inner core and mPEG as shell [23–25]. The PLA core can incorporate a hydrophobic drug and facilitate its controlled release. The mPEG shell acts as a hydrophilic shield against plasma

418

L. Lua et al. / Colloids and Surfaces B: Biointerfaces 142 (2016) 417–423

Fig. 1. Schematic illustration of formation of self-assembled SN38-conjugate micelles.

proteins and reduces clearance by the mononuclear phagocyte system. A conjugate of copolymer mPEG-PLLA with docetaxel (DTX) was reported in 2009 [26], which had similar antitumor activity to unconjugated DTX. In theory, mPEG-PLA conjugates of SN38 may be also an effective prodrug for the delivery of SN38. The purpose of this study was to construct a series of mPEGPLA-SN38 conjugates micelles to increase its tumor targeting ability and antitumor activity. These micelles were then evaluated in vitro and in vivo to determine the effects of polymer composition on therapeutic efficacy.

2.3. Preparation of SN38-conjugate micelles SN38-conjugate micelles were prepared by a solvent diffusion method. Briefly, 40 mg PEG-PLA-SN38 was dissolved in 5 mL acetone solvent and dispersed using an ultrasonic water bath. The organic phase was added into 6 mL deionized water and the organic phase was evaporated, resulted in formation of SN38-conjugate micelles.

2.4. Characterization of SN38-conjugate micelles 2. Material and methods 2.1. Materials 7-Ethyl-10-hydroxy camptothecin (SN38) was obtained from Dalian Meilun Biotechnology Inc. ( > 95%; Dalian, China). Its purity was determined by 1 H NMR and HPLC. N-Ethyl-N -(3dimethylaminopropyl) carbodiimide (EDC), 4-(dimethylamino) pyridine (DMAP) and 3-(4, 5-dimethyl-thiazol-2-yl)-2,5diphenyl-tetrazolium bromide(MTT) were purchased from China), penicillin-streptomycin, Sigma-Aldrich(Shanghai, RPMI1640 media, fetal bovine serum (FBS), and 0.25% (w/v) trypsin, and 0.03% (w/v) EDTA solution were purchased fromHyclone.Acetonitrile (HPLC grade) was obtained from Nanjing Xinhuayuan Chemical Agents Co. (Nanjing, China). Succinic anhydride terminated block copolymer mPEG- PLA (mPEG-PLA-SA) was purchased from Advanced Polymer Materials Inc (Canada). DiR was purchasedfrom Caliper Life Sciences (Hopkinton, MA).

2.2. Synthesis of mPEG-PLA-SN38-conjugates mPEG-PLA-SN38-conjugates were synthesized by linking SN38 to mPEG-PLA-SA. Briefly, SN-38, mPEG-PLA-SA, EDC and DMAP were dissolved in dry DMF. Then, 20 ␮L dry pyridine was added. The reaction continued for 24 h at room temperature. The solvent was removed under vacuum. The crude product obtained was purified by silica gel column chromatography (DCM: MeOH = 30: 1). The obtained oily yellow liquid dried in vacuo over night to give the final product PEG-PLA-SN38 (88.0% yield), as characterized by 1 H NMR spectroscopy and Fourier Transform Infrared Spectroscopy (FT-IR).

SN38-conjugate micelles were analyzed for particle size on a Malvern ZetaSizer (Malvern, UK). The analysis was performed in triplicate and the results were expressed as mean ± SD. The sample solutions were diluted in filtered double-distilled water prior to analysis. The morphology of the micelles was investigated by transmission electron microscopy (TEM) (JEOL JEM-1010). A drop of micelle solution was deposited onto a TEM grid covered with carbon film and the solvent was evaporated completely at room temperature.

2.5. Critical micelle concentration (CMC) determination To determine the CMC of SN38-conjugate polymers in deionized water, an Ultraviolet–visible (UV–vis) spectroscopy method was used, using iodine as a hydrophobic probe [27]. A KI/I2 standard solution was prepared by dissolving 0.5 g of potassium iodide and 0.25 g of iodine in 25 mL deionized water. polymers solutions with concentrations ranging from 1 × 10−7 mg/ml to 5 × 10−1 mg/ml were prepared. To each 2.5 mL of SN38-conjugate polymers solutions, 12.5 ␮Lof KI/I2 standard solution was added. The mixtures were incubated for 24 h in the dark at 37 ◦ C in incubator shakers before measurement. Ultraviolet absorbance values of varying solutions with polymer concentrations were measured at 366 nm using a UV–vis spectrometer (UV-2450; Shimadzu). Experiments were performed in triplicate. The absorbance was plotted against the logarithm of polymer concentration. The CMC values corresponded to concentrations of the polymer at which a sharp increase in absorbance was observed.

L. Lua et al. / Colloids and Surfaces B: Biointerfaces 142 (2016) 417–423

419

Fig. 2. Particle size and size distribution of SN38-conjugate micelles (A) were obtained from dynamic light scattering (DLS). Representative TEM images, mPEG2K-PLA8.9KSN38 micelles (B) and mPEG4K-PLA1K-SN38 micelles (C).

2.6. In vitro drug release In vitro drug release of SN-38 from SN38-conjugate micelles was monitored in phosphate-buffered saline (PBS) (pH 7.4) with 0.2% Tween 80 or PBS with 20% human serum albumins (HSA)(v/v). Briefly, a 2 mL aqueous solution of SN38-conjugate micelles (0.1 mg/mL) was introduced into a dialysis membrane bag (MWCO 14000 Da), which was sealed and incubated in 20 mL of release media into an orbital shaker. At predetermined time intervals, aliquot samples (0.5 mL) were collected and the external media was replaced with fresh release media. The collected samples were combined with equal volume of acetonitrile and then analyzedby HPLC (LC-20A, Shimadzu) at a flow rate of 1.0 mL/min, using a gradient of 20–80% acetonitrile in water as mobile phase for 30 min, UV detection at a wavelength of 377 nm. AHypersil ODS2 column (4.6 mm × 250 mm, 5 ␮m) was utilized. 2.7. Cell viability assay Cytotoxicity of SN38-conjugate micelles on humancolon cancercell lines HCT116 and human liver cancer cell lines BEL-7402 were evaluated by MTT assay and compared with the cytotoxicity of CPT-11 [28]. Cells were cultured in the RPMI1640 growth medium supplemented by 10% (v/v) FBS, 1% (w/v) penicillin-streptomycin at 37 ◦ C in a humidified atmosphere of 5% CO2 . Briefly,5 × 103 cells per well were plated in a96-well pate. After 24 h of incubation, cells were treated withdifferent concentrations (SN-38 equivalent from 0.01 to 100 ␮g/mL) of SN38-conjugate micelles or CPT-11 and incubated for 48 h. After 48 h incubation, 30 ␮L of MTT (5 mg/mL)

solution was added to each well. This was followed by 4 h of incubation. Then, culture medium was removed and 100 ␮L of DMSO was added to dissolve the formazan crystals, and the optical density was measured using a microplate reader at 492 nm. The resultsare expressed as the percentage of cell viability relative to untreated control. 2.8. In vivo biodistribution study by live imaging investigations Nude mice bearing subcutaneous HCT116 tumors were intravenously injected with 200 ␮L of DiR loaded micelles (100 ␮g/mL). The preparation method for DiR loaded micelles was the same as that for SN38-conjugate micelles, except the addition of DiR in acetone. At different time point (2, 6 and 24 h), The mice were anesthetized using isoflurane in oxygen and placed in the imaging chamber.Fluorescent images were collected using the Clairvivo OPT (SHIMADZU Corporation, Kyoto, Japan) with a 735 nm single laser. The exposure time was 5 s for each image. After in vivo imaging, the mice were sacrificed by cervical dislocation, and major organs including hearts, livers, spleens, lungs, kidneys and tumors were excised. The fluorescent images of tissues were photographed and the near-infrared fluorescence signal intensity in different tissues was measured. 2.9. In vivoantitumor efficacy The therapeutic effect of SN38-conjugate micelles was evaluated using female BALB/c nude mice model (5–6 weeks, 18–22 g), inoculated subcutaneously with 5 × 106 HCT116 human colon can-

420

L. Lua et al. / Colloids and Surfaces B: Biointerfaces 142 (2016) 417–423

Fig. 3. Plot of ultraviolet absorbance of I2 versus concentration of SN38-conjugate micelles in water. CMC value was calculated by determining the polymer concentration at which a sharp increase in absorbance was observed.

Table 1 Physicochemical characterization of SN38-conjugate micelles (n = 3). Composition

Zeta potential (mV)

Mean diameter(nm)

PDI

mPEG2K -PLA4·2K -SN38 mPEG2K -PLA8·9K -SN38 mPEG4K -PLA1K -SN38

5.64 6.91 9.99

14.2 13.4 120

0.298 0.275 0.476

cer cells. When the tumor volume reached about 100 mm3 , mice were randomly assigned to six groups (n = 7) and treated with six different formulations: PBS and CPT-11 as control group, the other four groups were treated with SN38-conjugate micelles, respectively. Mice were administrated injection through the tail vein every 3 days for four times. The tumor diameters were measured every other day with a Vernier caliper in two dimensions. Individual tumor volume (V) was calculated using the formula: V = (L × W2 )/2, wherein length (L) is the longest diameter and width (W) is the shortest diameter perpendicular to length. In addition, for safety evaluation of the control and SN38-conjugate micelles, the body weight of each mouse was determined every other day. Weight reduction was used as an indicator of acute toxicity. 3. Result and discussion 3.1. Characterization of SN38-conjugate micelles For the synthesis of SN38-conjugates, mPEG-PLA-SA was reacted with SN38 through esterification reaction between the carboxyl group in mPEG-PLA-SA and the phenolic hydroxyl group in SN38. The product was characterized by 1 HNMR and FT-IR (Fig. S1 and Fig. S2). In 1 H NMR spectrum, the characteristic peak of phenolic hydroxyl (10.33 ppm) disappeared and the multiple peaks (7.38 ppm) of C-9 and C-11 divided appeared at 7.72 ppm and 7.23 ppm. In FT-IR spectrum, O H vibration of phenolic hydroxyl of SN38 at 3596 cm−1 and O H vibration of carboxyl of PEG-PLA-SA at 1762 cm−1 disappeared and C O stretching bands at 1762 cm−1 was observed. These characteristic peaks confirmed the formation of ester bond. Therefore, mPEG-PLA-SN38 was successfully synthesized. The drug loading efficiency of SN38 conjugate micelles was about 20% (mol/mol). The scheme for the preparation of the SN38-conjugate micelles is displayed in Fig. 1. mPEG-PLA-SN38 has two hydrophobic segments, PLA and SN38. When micelles were formed, the two hydrophobic segments are likely to form a double-layer core, with

Table 2 Cytotoxicity of various SN38-conjugate micelles against BEL-7402 and HCT116 cells (means ± SD, n = 3). IC50 (␮g/mL) CPT-11 mPEG2K -PLA4·2K -SN38 mPEG2K -PLA8·9K -SN38 mPEG4K -PLA1K -SN38

BEL-7402 7.51 ± 0.77 5.33 ± 0.84 2.35 ± 0.11 21.38 ± 2.44

HCT116 5.57 ± 0.72 0.93 ± 0.18 0.52 ± 0.05 21.06 ± 1.19

SN38 in inner layer and PLA in outer layer. This double-layer core contributes to the stability of micelles. After SN38-conjugate micelles were injected, the ester bond of PLA is gradually hydrolyzed and the active drug of SN38 is released. Fig. 2A shows the size distribution of SN38-conjugate micelles, the sizes of mPEG2K -PLA4·2K -SN38 and mPEG2K -PLA8·9K -SN38 micelles were about 15 nm, and much smaller than mPEG4K- PLA1K SN38 micelleswith a mean diameter of about 100 nm. The small particle sizes could reduce RES uptake and prolong the circulation time in the blood, and facilitate extravasation from leaky capillaries [29]. Hence, the size of SN38-conjugate micelleswas suitable for tumor specific accumulation via the EPR effect. Fig. 2B and 2C are transmission electron micrographs of mPEG2K -PLA8·9K -SN38 andmPEG4K -PLA1K- SN38 micelles. These two images showed that the SN38-conjugate micelles are discrete spherical particles. The average particle size, polydispersity index (PDI) and zeta (␨) potential of SN38-conjugate micelles are presented in Table 1. All of the SN38-conjugate micelles were of nanoscale sizes with small PDI, and there were negative correlations between PLA chain length and the size of the micelles and positive correlation between PEG chain length and the size of micelles. This result is expected on the basis of theoretical approaches to the structure of polymeric micelles [30,31]. Zeta (␨) potential of SN38-conjugate micelles increased with increasing of the length of PLA and PEG chains.

L. Lua et al. / Colloids and Surfaces B: Biointerfaces 142 (2016) 417–423

421

micelles and accelerated release of SN38 based on the mechanism of disassembly-induced exposure of phenyl ester bonds to aqueous environment [35]. In Fig. 4, addition of HSA was shown to accelerate the release rate of SN38. In the two different dialysis medium, the release of SN38 from mPEG4K -PLA1K -SN38 micelles was faster than from mPEG2K PLA4·2K -SN38 and mPEG2K -PLA8·9K -SN38 micelles.The release profiles indicated that sustained release of SN38 was achieved and the drug release rate could be controlled by modulating the chain length of mPEG and PLA blocks [36]. This phenomenon also can be explained with the differingCMC of micelles. The CMC of mPEG4K PLA1K -SN38 was higher than that of mPEG2K -PLA4.2 K-SN38 and mPEG2K -PLA8·9K -SN38, so the mPEG4K- PLA1K -SN38 micelles had lower stability compared to mPEG2K -PLA4·2K -SN38 and mPEG2K PLA8·9K -SN38 micelles. Compared with traditional micelles, these SN38 conjugated micelles had significantly decreased rate of release of SN38 [37,38]. Fig. 4. In vitro release profile of SN38-conjugate micelles. Drug release study was performed respectively in PBS (pH7.4) with addition of 0.2% Tween 80 and in PBS (pH7.4) with 20% (HSA) dialysis medium. Eg·mPEG2K -PLA4·2K -SN38 indicates the release profile in PBS (pH7.4) with addition of 0.2% Tween 80, and H-mPEG2K PLA4·2K -SN38 indicates the release profile in PBS (pH7.4) with 20% (HSA).

3.2. CMC determination The CMC of the micelles influences their in vitro and in vivo stability. Low CMC values of polymers underlie high stability of micelles in solutions upon dilution [32]. In this study, the formation of micelles was monitored by using iodine as a hydrophobic probe [33]. Solubilized I2 prefers the hydrophobic interior of polymer micelles, causing the conversion of I3 − to I2 from the excess potassium iodide (KI) in the solution. The relationship of absorbance intensity of I2 as a function of the concentration of the SN38conjugate polymers aqueous solution is plotted in Fig. 3. It was found that absorbance of I2 increased sharply when the concentration exceeded a certain value, which corresponds to the concentration of micelle formation. Therefore, the intersection of the two straight lines with a value was determined to be the CMC of SN38-conjugate polymers. The CMC values of SN38-conjugate polymers with the same compositions but with different molecular were measured, i.e. 0.012 mg mL−1 for mPEG2K -PLA4·2K -SN38, 0.011 mg mL−1 for mPEG2K -PLA8·9K -SN38 and 0.023 mg mL−1 for mPEG4K -PLA1K -SN38. Obviously, when hydrophilic segment is constant, the longer the hydrophobic segment, the lower the value of CMC. When hydrophobic segment is constant, the longer the hydrophilic segment, the higher the value of CMC. The variation of CMC values suggested fact that the polymer composition may affect micelle self-assembly [34]. 3.3. In vitro drug release The structural integrity of prodrug micelles circulating in the bloodstream is necessary for tumor targeting via the EPR effect. Therefore, we studied the drug release property of SN38-conjugate micelles in physiological pH (7.4) at 37 ◦ C. As shown in Fig. 4, about 20% of SN38 was released from mPEG2K -PLA4·2K -SN38 and mPEG2K- PLA8·9K -SN38 micelles within the first 12 h, while almost 30% of SN38 was released from mPEG4K -PLA1K -SN38 micelles during the same time period. However, after 12 h, the release rate of SN38 from these three micelles was much slower. The fast release of SN38 maybe due to the presence of free drug embedded in SN38 conjugated micelles. In order to provide a more realistic simulation of bloodstream condition, 20% (v/v) human serum albumin (HSA) was added into PBS (pH 7.4). This should lead to disruption of assembled

3.4. Cytotoxicity of SN38-conjugate micelles The cytotoxicity of SN38-conjugate micelles was evaluated by the MTT cell viability assay in human hepatoma BEL-7402 and colon cancer HCT-116 cell lines. The half-maximal inhibitory concentration (IC50) values are summarized in Table 2. As shown in Fig. 5, the in vitro antitumor efficacy of mPEG2K- PLA-SN38 micelles was higher than that of mPEG4K- PLA1K -SN38 micelles and CPT-11. SN38-conjugate micelles and CPT-11 showed higher cytotoxic to HCT116 cells than to BEL-7402 cells (Fig. 5B). Fixed PEG chain length at 2 K, the in vitro antitumor activity of SN38-conjugate micelles was higher for conjugates with longer PLA chains. This might be attributed to protection of SN38 by PLA core in the micelles. Longer PLA chains form more stable core-shell structure micelles. Thus, the IC50 values of mPEG4K -PLA1K -SN38 micelles were higher than that of mPEG2K -PLA4·2K -SN38 and mPEG2K -PLA8·9K -SN38 micelles. Based on CMC value and in vitro release rate, mPEG4K-PLA1K-SN38 micelles should have the greatest cytotoxicity. However, the result indicated otherwise. It is possible that drugs released from conjugated micelles were not taken up the cells due to the low solubility of SN38. For the mPEG4K -PLA1K -SN38 micelles, a part of SN38 was quickly released before the micelles entered cells, and the overall amount of SN38 entered cells is reduced, so the in vitro antitumor activity ofmPEG4K -PLA1K -SN38 micelles was lower than mPEG2K PLA4·2K -SN38 and mPEG2K -PLA8·9K -SN38 micelles. 3.5. In vivo distribution of SN38-conjugate micelles To estimate direct and real-time distribution for SN38conjugate micelles in HCT116 tumor-bearing mice, non-invasive live animal imaging technology was utilized. This was because the time-dependent clearance profile, in vivo distribution, and tumor targeting efficacy of SN38-conjugate micellescould easily be monitored by imaging DiR loaded SN38-conjugate micelles in tumor-bearing mice. We chose DiR loaded mPEG2K -PLA8·9K -SN38 micelles as representative to study their distribution in tumor-bearing mice. After 2 h, the DiR fluorescence signal of the tumor site was at a low level. But as time goes on, the DiR fluorescence signal grew stronger at 6 h and 24 h (Fig. 6A). In order to more directly and clearly show the distribution of mPEG2K -PLA8·9K -SN38 micelles, mice were sacrificed and major organs were excised at 2 h, 6 h and 24 h time points for ex vivo evaluation (Fig. 6B). Ex vivo images of excised major tissues including heart, liver, spleen, lung, kidneyandtumor indicated that the mPEG2K -PLA8·9K -SN38 micelles were mainly taken up by the tumor tissue, which exhibited strong fluorescence intensity, whereas the uptake levels of mPEG2K -PLA8·9K -SN38 micelles in normal tissues (heart, liver, spleen, lung and kidney) were moder-

422

L. Lua et al. / Colloids and Surfaces B: Biointerfaces 142 (2016) 417–423

Fig. 5. In vitro cytotoxicity of SN38-conjugate micelles against human colon cancer cell line HCT116 (A) and human liver cancer cell line BEL-7402 (B).

Fig. 6. In vivo fluorescent images of HCT116 bearing mice at different time point after i.v. injection of DiR loaded micelles. The fluorescent images of time-dependent whole body imaging of HCT116 tumor bearing mice at 2 h, 6 h and 24 h after intravenous injected DiR loaded PEG2K-PLA8.9K-SN38 micelles (A). Tissue distribution of DiR loaded micelles was determiend 2 h, 6 h and 24 h after intravenous administration. Tissues were harvested and then DiR fluorescence signals were measured (B).

ate, indicatingpassive targeting ability of SN38-conjugate micelles. However, the liver and kidney showed strong fluorescence intensity after 6 h and 24 h post-injection, which was indicative of probe excretion by liver and renal clearance, presumably due to the degradation of the polymer. It has been reported that small nanoparticles (<20 nm) are excreted through kidneys, medium sized nanoparticles (30–150 nm) accumulate in the bone marrow, heart, kidney and stomach, while large nanoparticles (150–300 nm) are found in the spleen and the liver [39,40]. However, the tissue distribution of SN38-conjugate micelles of about 15 nm did not seem to follow this pattern.

3.6. Antitumor effect of SN38-conjugate micelles in HCT116-bearing mice To demonstrate the antitumor efficacy of SN38-conjugate micelles, CPT-11 and SN38-conjugate micelles were injected every three days in HCT116-bearing mice via lateral tail vein. The dose of SN38 equivalent was 10 mg/kg. Fig. 7Ashows the tumor growth observed for 28 days in the mice injected with PBS, CPT-11 and different SN38-conjugate micelles. Compared with PBS and CPT11 groups, it was found that the tumors of mPEG2K -PLA8·9K -SN38 micelles treated group was most inhibited, followed by mPEG2K PLA4·2K -SN38 micelles group. Meanwhile, mPEG4K -PLA1K -SN38 micelles did not show greaterantitumor activity to CPT-11. These data illustrated that the length of PLA chain was critical for the property of SN38-conjugate micelles. Fig. 7B shows the body weight changes of the tumor-bearing mice during the study of antitumor efficacy. After the administra-

Fig. 7. (A) Antitumor effect of control and SN38-conjugate micelles on BALB/c HCT116 tumor-bearing nude mice. (B) Body weight change of BALB/c tumor-bearing nude mice after intravenous injection according to a dose schedule regimen of three injections at 3 days intervals. (n = 7).

L. Lua et al. / Colloids and Surfaces B: Biointerfaces 142 (2016) 417–423

tion of 12 days, the body weight of CPT-11 group appeared falling, but recovered within 4 days. However, the body weight of SN38conjugate micelles was normal. The survival curves were examined and no animal died during the 28 days observation. These phenomenon indicated that SN38-conjugate micelles have reduced toxicity as well as increased antitumor efficacy. 4. Conclusion A series of novel amphiphilic mPEG-PLA-SN38-conjugates were synthesized by linking SN38 to mPEG-PLA-SA. SN38-conjugate micelles were formed spontaneously by self-assembly. Through in vitro and in vivo evaluation, we studied the effect of mPEGPLA composition on the micelle formation and antitumor activity. The result showed that the lengths of mPEG and PLA chains had large impacts. As for the particle sizes of SN38-conjugates micelles, mPEG2K -PLA-SN38 micelles had a mean diameter of 10–20 nm, while mPEG4K -PLA-SN38 micelles had a mean diameter about 120 nm. As for antitumor activity, mPEG2K -PLA-SN38 micelles showed greater efficacy than mPEG4K -PLA-SN38 micelles in vitro and in vivo. Among these series of SN38-conjugates micelles, mPEG2K -PLA8·9K -SN38 micelles were the best candidate for an antitumor agent. SN38-conjugate micelles possess advantages of reducing toxicity of anticancer drugs, simultaneously increasing their anticancer efficacy. Thus, mPEG-PLA-SN38-conjugate micelles may have potential for clinical translation. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81472677 and 81270536). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.02. 035. References [1] Y. Pommier, Chem. Rev. 109 (2009) 2894–2902. [2] H. Ulukan, P.W. Swaan, Drugs 62 (2002) 2039–2057. [3] N. Zeghari-Squalli, E. Raymond, E. Cvitkovic, F. Goldwasser, Clin. cancer. res. 5 (1999) 1189–1196. [4] Y. Kawato, M. Aonuma, Y. Hirota, H. Kuga, K. Sato, Cancer Res. 51 (1991) 4187–4191.

423

[5] Y. Saga, H. Mizukami, M. Suzuki, T. Kohno, M. Urabe, K. Ozawa, I. Sato, Clin. Cancer Res. 8 (2002) 1248–1252. [6] J. van Ark-Otte, M.A. Kedde, W.J. van der Vijgh, A.M. Dingemans, W.J. Jansen, H.M. Pinedo, E. Boven, G. Giaccone, Br. J. Cancer 77 (1998) 2171–2176. [7] J.A. Zhang, T. Xuan, M. Parmar, L. Ma, S. Ugwu, S. Ali, I. Ahmad, Int. J. Pharm. 270 (2004) 93–107. [8] H. Zhao, B. Rubio, P. Sapra, D. Wu, P. Reddy, P. Sai, A. Martinez, Y. Gao, Y. Lozanguiez, C. Longley, L.M. Greenberger, I.D. Horak, Bioconjugate Chem. 19 (2008) 849–859. [9] P. Ebrahimnejad, R. Dinarvand, A. Sajadi, M.R. Jaafari, A.R. Nomani, E. Azizi, M. Rad-Malekshahi, F. Atyabi, Nanomed.-Nanotechnol. 6 (2010) 478–485. [10] K.K. Vangara, J.L. Liu, S. Palakurthi, Anticancer Res. 33 (2013) 2425–2434. [11] Q. Gu, J.Z. Xing, M. Huang, C. He, J. Chen, Nanotechnology 23 (2012) 205101. [12] A. Takahashi, N. Ohkohchi, M. Yasunaga, J.-i. Kuroda, Y. Koga, H. Kenmotsu, T. Kinoshita, Y. Matsumura, Clin. Cancer Res. 16 (2010) 4822–4831. [13] A. Carie, J. Rios-Doria, T. Costich, B. Burke, R. Slama, H. Skaff, K. Sill, J. drug deliv, (2011) 869026–869027. [14] A. Patnaik, K. Papadopoulos, A. Tolcher, M. Beeram, S. Urien, L. Schaaf, S. Tahiri, T. Bekaii-Saab, F. Lokiec, K. Rezaï, A. Buchbinder, Cancer Chemoth. Pharm. 71 (2013) 1499–1506. [15] K. Kataoka, A. Harada, Y. Nagasaki, Adv Drug Deliver Rev. 47 (2001) 113–131. [16] Y. Matsumura, H. Maeda, Cancer Res. 46 (1986) 6387–6392. [17] H. Maeda, Adv. Enzyme Regul. 41 (2001) 189–207. [18] Y. Zhang, M. Gao, C. Chen, Z. Wang, Y. Zhao, RSC Adv. 5 (2015) 34800–34802. [19] X. Hu, X. Jing, Expert Opin Drug Del. 6 (2009) 1079–1090. [20] X. Zhang, Y. Li, X. Chen, X. Wang, X. Xu, Q. Liang, J. Hu, X. Jing, Biomaterials 26 (2005) 2121–2128. [21] L. Cheng, Q. Hu, L. Cheng, W. Hu, M. Xu, Y. Zhu, L. Zhang, D. Chen, Colloids Surf. B 136 (2015) 37–45. [22] J.-P. Nam, J.-K. Park, D.-H. Son, T.-H. Kim, S.-J. Park, S.-C. Park, C. Choi, M.-K. Jang, J.-W. Nah, Colloids Surf. B 120 (2014) 168–175. [23] J.-H. Kim, K. Emoto, M. Iijima, Y. Nagasaki, T. Aoyagi, T. Okano, Y. Sakurai, K. Kataoka, Polym. Advan. Technol. 10 (1999) 647–654. [24] S.C. Kim, D.W. Kim, Y.H. Shim, J.S. Bang, H.S. Oh, S.W. Kim, M.H. Seo, J. Control. Release 72 (2001) 191–202. [25] Y. Dong, S.-S. Feng, Biomaterials 25 (2004) 2843–2849. [26] Z. Xie, T. Lu, X. Chen, Y. Zheng, X. Jing, J. Biomed. Mater. Res. A 88 (2009) 238–245. [27] V. Saxena, M.D. Hussain, In J nanomed. 7 (2012) 713–721. [28] S.Y. Kim, Y.M. Lee, D.J. Baik, J.S. Kang, Biomaterials 24 (2003) 55–63. [29] L. Qiu, Y. Bae, Pharm. Res. 23 (2006) 1–30. [30] S. Förster, M. Zisenis, E. Wenz, M. Antonietti, J. Chem. Phys. 104 (1996) 9956–9970. [31] Z. Tuzar, P. Kratochvíl, Adv Colloid Interface 6 (1976) 201–232. [32] J. Logie, S.C. Owen, C.K. McLaughlin, M.S. Shoichet, Chem. Mater. 26 (2014) 2847–2855. [33] Z. Wei, J. Hao, S. Yuan, Y. Li, W. Juan, X. Sha, X. Fang, Int. J. Pharm. 376 (2009) 176–185. [34] A.N. Lukyanov, V.P. Torchilin, Adv Drug DeliverRev. 56 (2004) 1273–1289. [35] D. Amado Torres, M. Garzoni, A.V. Subrahmanyam, G.M. Pavan, S. Thayumanavan, J. Am. Chem. Soc. 136 (2014) 5385–5399. [36] Y. Zhang, R.-x. Zhuo, Biomaterials 26 (2005) 6736–6742. [37] K. Duan, X. Zhang, X. Tang, J. Yu, S. Liu, D. Wang, Y. Li, J. Huang, Colloids Surf. B 76 (2010) 475–482. [38] Q. Guo, P. Luo, Y. Luo, F. Du, W. Lu, S. Liu, J. Huang, J. Yu, Colloids Surf. B: Biointerfaces 100 (2012) 138–145. ˇ Konák, ˇ ´ L.W. Seymour, K. Ulbrich, J. Control. Release [39] T. Reschel, C.r. D. Oupicky, 81 (2002) 201–217. [40] M.D. Chavanpatil, A. Khdair, J. Panyam, J. Nanosci. Nanotechnol. 6 (2006) 2651–2663.