A Novel Doxorubicin-Loaded in Situ Forming Gel Based High

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A Novel Doxorubicin-Loaded in Situ Forming Gel Based High Concentration of Phospholipid for Intratumoral Drug Delivery Wenqi Wu,† Hui Chen,† Fengying Shan,† Jing Zhou,† Xun Sun,† Ling Zhang,*,‡ and Tao Gong*,† †

Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, Sichuan University, 29 Wangjiang Rd, Chengdu, Sichuan 610041, People’s Republic of China ‡ Faculty of Life Sciences, The University of Manchester, Oxford Rd, Manchester M13 9PT, United Kingdom ABSTRACT: The purpose of this study was to develop a safe and effective drug delivery system for local chemotherapy. A novel injectable in-situ-forming gel system was prepared using small molecule materials, including phospholipids, medium chain triglycerides (MCTs), and ethanol. Thus, this new sustained release system was named PME (first letter of phospholipids, MCT, and ethanol). PME has a well-defined molecule structure, a high degree of safety, and better biocompatible characteristics. It was in sol state with low viscosity in vitro and turned into a solid or semisolid gel in situ after injection. When loaded with doxorubicin (Dox), PME-D (doxorubicin-loaded PME) exhibited notably antitumor efficiency in S180 sarcoma tumors bearing mice after a single intratumoral injection. In vitro, PME-D had remarkable antiproliferative efficacies against MCF-7 breast cancer cells for over 5 days. Moreover, the initial burst effect can hardly be observed from PME system, which was different from many other in-situforming gels. The in vivo biodistribution study showed the high Dox concentration in tumors compared with other major organs after PME-D intratumoral administration. The strong signal in tumors was retained for more than 14 days after one single injection. The high concentration of Dox in tumor and long-term retention may explain the superior therapeutic efficacy and reduced side effects. The PME-D in-situ-forming gel system is a promising drug delivery system for local chemotherapy. KEYWORDS: doxorubicin, in-situ-forming gel, phospholipid, solid tumors, intratumoral injection

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INTRODUCTION Cancer is the leading cause of death worldwide.1 According to the World Health Organization (WHO), there were 7.6 million people who died of cancer (around 13% of all deaths) in 2008, and the number is projected to be 13.1 million in 2030.2 Surgery, radiotherapy, and chemotherapy are regular methodologies for treating malignant solid tumors, and systemic chemotherapy is the primary treatment for cancer patients.3 Severe side effects are generally associated with the systemic administration of chemotherapy agents due to the nonselectivity on normal and cancer cells, the rapid clearance of anticancer drugs from blood circulation, and low accumulation in tumor tissue.4 Regional chemotherapy directly delivers chemotherapeutics to the tumor site via local administration.5 Compared with systemic administration, it could significantly increase the drug concentration in tumor sites and alleviate dose-dependent toxicities due to the low drug distribution in other normal organs and tissues.6,7 Among the local delivery systems, intratumoral injectable in-situ-forming gel system has attracted increasing attention. It has many advantages such as site specificity, prolonged action, and improved patient compliance.8−10 Most importantly, it can be injected into tumors in a liquid state and then transforms into gel in situ to act as a drug reservoir. In conclusion, this intratumoral © XXXX American Chemical Society

injectable in-situ-forming gel is a promising approach for the regional chemotherapy delivery system. In the past years, many in-situ-forming gel systems have been developed.11−18 The transitions from sol to gel are obtained by different stimulus, such as solvent exchange, UV or visible light irradiation, chemical reactions, and temperature change. Though these systems have advantages as mentioned above, there are many limitations. For example, in solvent exchange gels, polymers were precipitated from solution with the organic solvent diffused. Those organic solvents may include harmful dimethyl sulfoxide (DMSO) and N-methyl-2-pyrrolidone (NMP).19 Kranz et al. already confirmed that the intramuscular injection of DMSO and NMP into Sprague−Dawley rats may cause myotoxic and muscle damage.20 Moreover, other concerns are drug burst release and safety.6,21 In addition to the complex instrumentation requirement, the application of Special Issue: Recent Molecular Pharmaceutical Development in China Received: January 9, 2014 Revised: March 25, 2014 Accepted: April 15, 2014

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used. The MCF-7 cells were cultured in a 5% CO2 incubator at 37 °C. Kunming mice (male, 22 ± 2 g) were purchased from Experimental Animal Center of Sichuan University. All of the animal procedures were approved by Institute Laboratory Animal guidelines of Sichuan University. PME-D Preparation and Viscosity Measurements. Doxloaded PME (PME-D) was prepared by mixing E80, MCT, and ethanol (72:17:11, mass ratio) and stirred for 30 min. Dialysis bags were used to simulate the transition process. PME-D was added into dialysis bag and immersed in prewarmed PBS (pH 6.8). The transition from sol to gel finished in a few minutes. The viscosities of PME-D before (sol) and after transition (gel) were measured by a Brookfield DV-C rotational viscometer at room temperature. Each measurement was repeated three times. Data were recorded as the mean ± standard deviation (SD). In Vitro Dox Release. The in vitro Dox releases were performed according to the following processes. Dox solution (200 μL, 3 mg/mL) or PME-D (200 μL, equivalent to 3 mg/ mL Dox) was added into dialysis bags and then incubated in 5 mL of different releasing media containing 0.1 M PBS (pH 6.8) and ethanol (0, 5, 15, or 40% v/v) at 37 °C with mild shaking at 100 rpm. At predetermined time points, all releasing medium was removed and replaced with 5 mL of prewarmed fresh medium. The concentration of Dox in the medium was measured by spectrofluorophotometer (RF-5301PC, Shimadzu Corporation) with Ex = 467 nm and Em = 556 nm. The amount of Dox was calculated according to a calibration curve. All of the procedures were finished in conditions devoid of light. In Vitro Antitumor Activity. MCF-7 cells were used to evaluate the in vitro antitumor activity. MCF-7 cells at logarithm phase were plated in Transwell plates (Corning Co. Ltd., USA) and treated with different doses of Dox solution (0.5, 1 mg/mL) or PME-D (equivalent to 0.5, 1 mg/mL Dox). At 1, 2, 3, and 5 days after administration, the survival rate of MCF-7 cells was evaluated using the WST-1 assay (Nanjing KeyGEN Biotechnology Co. Ltd., Nanjing, China). Briefly, 100 μL of WST-1 solution was added to each well. The plate was shaken for 30 s and then incubated at 37 °C. After 4 h incubation, 330 μL of solution from each well was transferred to a 96-well plate using 110 μL for each well. Then the absorbance was measured using a Microplate reader (Varioskan Flash, Thermo Scientific, USA) at 450 nm. Each experiment was repeated three times. All of the procedures were finished in conditions devoid of light. In Vivo Antitumor Activity. The in vivo antitumor activities were evaluated in Kunming mice (male, 22 ± 2 g) with xenograft S180 sarcoma tumors. The S180 cell suspension (5.0 × 107 cells in 200 μL of saline) was subcutaneously injected into the left flank of the mice. The treatments started when the tumor volume reached 500 mm3 (approximately 10 mm in diameter). Mice were randomly divided into four groups with nine mice in each group: group 1, intratumoral injected with 200 μL of saline, one injection; group 2, intratumoral injected with 200 μL of free Dox solutions (30 mg/kg), one injection; group 3, intravenous injected with free Dox solution (3 mg/kg) every other day; group 4, intratumoral injected with 200 μL of PME-D (30 mg/kg of Dox), one injection. Tumor sizes and body weight were monitored daily. Tumor volumes (V) were calculated based on the length and width of tumor {V = [length × (width)2]/2}. The relative change (%) of body weight was calculated according to the following equation:

photopolymerization gel systems is limited in subcutaneous administration for the light penetration through tissue.22 In situ gel systems based on chemical reactions were also explored, but side reactions with the drugs can occur, and the highly selective reaction pairs are not many.23 A thermosensitive gel system, which is one of the most common in-situ-forming gels, mainly includes positive thermosensitive gels and negative thermosensitive gels.24 Positive thermosensitive gels have an upper critical solution temperature (UCST). This system is injected in a melted state and forms a solid or semisolid gel upon cooling to body temperature. The high injection temperature may be harmful to the tissues.25 Negative thermosensitive gels have a lower critical solution temperature (LCST) and have been studied most extensively; poly(N-isopropylacrylamide), polycaprolactone, poly(ethylene glycol)-b-polycaprolactone diblock copolymer, MPEG-b-(PCL-ran-PLLA) diblock copolymers, MPEG−PCL copolymer, and MPEG−PLCPPA were also explored recently.11,12,15,26−28 However, most of these materials are polymers and may present biological incompatibility and an initial burst effect.10,26,29,30 Recently, our group developed an injectable in-situ-forming gel (PME) in order to improve biological compatibility and reduce the initial burst effect of in-situ-forming gels. Different from the in-situ-forming gels that have been reported before, the novel PME system is composed of biocompatible small molecular materials, including phospholipids (E80), medium chain triglycerides (MCT), and ethanol. E80 is an amphiphilic molecule with poor water solubility under physiological conditions but very high solubility in ethanol (even up to 85 wt %/v). After intratumoral injection of PME, ethanol rapidly diffused into the surrounding tissue. The precipitation of E80 was accompanied by a sharp system viscosity increase. Through this way, PME in the sol state with a low viscosity at the time of injection turned into a solid or semisolid gel in situ after injection and may act as a drug depot. The purpose of the current study was to investigate the application of the PME system for intratumoral anticancer drug delivery, trying to increase the antitumor efficacy and decrease the toxicities associated with systemic administration. Doxorubicin (Dox), an anticancer drug found 30 years ago31 and widely used in clinic, was researched as a model drug. In this study, Dox-loaded PME (PME-D) was prepared. The releasing profiles were investigated. The in vitro antitumor activities were tested in human breast cancer cell line MCF-7. The in vivo therapeutic efficacies, blood Dox concentration, and biodistribution were evaluated in S180 solid tumors xenograft mice. To our best knowledge, this is the first time reporting the intratumor in-situ-forming gel system based on biocompatible small molecules and their application as a drug delivery system.

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MATERIALS AND METHODS Materials. The phospholipid (E80) was purchased from Lipoid (Germany, 510300-1124016/940). The medium chain triglyceride (MCT) was provided by Beiya Medical Oil Co. Ltd. (Tieling, China, y110501-3-01). Ethanol (HPLC grade) was obtained from Kemiou (Tianjin, China). All of the other agents were of analytical grade. Cells and Animals. The human breast cancer cell line MCF-7 was purchased from Shanghai Institutes for Biological Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, USA) supplemented with 10% fetal bovine serum (Fumeng Gene Co., Ltd., Shanghai, China), penicillin (100 IU/mL), and streptomycin (100 μg/mL) was B

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change rate (%) = [(Wi − W0)/W0] × 100; Wi indicated the weight of the mouse on the day of measurement; W0 was the initial weight of the mouse before treatment. All of the measurements were repeated three times at each time points. The survival rate was evaluated using another batch of mice with nine animals in each group. In Vivo Dox Release and Biodistribution. PME-D (200 μL, 30 mg/kg Dox) was intratumorally injected to mice bearing S180 tumor. Animals were sacrificed at different time points. The blood, major organs (heart, liver, spleen, lung, and kidney), and tumors were harvested. The Dox biodistribution was qualitatively analyzed using a Whole Body Imaging System (Lightools Research, USA). Blood samples were stored at −80 °C before analysis. A portion of 800 μL of acetonitrile was added to 200 μL of plasma. The mixture was vortexed for 15 min, followed by centrifuging at 13 500 rpm for 15 min. Then 1 μL of supernatant was analyzed by liquid chromatography/ mass spectrometry (LC-MS/MS) (Agilent Technologies Co. Ltd., USA). The amount of Dox was calculated based on a standard calibration curve. Data were represented as mean ± SD (n = 5). The LC-MS/MS system consisted of an Agilent 1200 series rapid resolution liquid chromatograph (RRLC), including a SL binary pump, degasser, SL autosampler, and an Agilent triplequadrupole MS. A Diamonsil ODS column (50 × 4.6 mm, 1.8 μm) with a corresponding guard column (ODS, 5 μm) was employed for separation and maintained at 30 °C. The mobile phase consisted of 72% deionized water (0.1% formic acid) and 28% acetonitrile (v/v) at a flow rate of 0.4 mL/min. The triple-quadrupole mass spectroscopy operated under positive electrospray ionization (ESI). The quantification analysis was performed using multiple reaction monitoring (MRM). The transitions of m/z 544.2 → 397.2 were adopted to quantify Dox. Voltages of fragmentor potential and collision energy were 112 and 6 eV, respectively. Nitrogen was used as a nebulizer gas, with a temperature of 350 °C and a gas flow of 10 mL/min. The nebulizer was 35 psi, and the capillary was 4000 V. Histological Analysis. Mice in saline and PME-D groups were sacrificed 14 days after treatment. Mice in free Dox solution group were sacrifice on the eighth day due to the low survival rate. The tumors were collected, fixed with 4% formalin, paraffin embedded, and sectioned. The tumor slides were stained with hematoxylin and eosin (H&E). Apoptotic cells were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; Roche Diagnostics GmbH, Germany) according to the manufacturer’s protocol. Briefly, slides were incubated with proteinase K (20 μg/mL) at 37 °C for 20 min. After rinsing, they were stained with TUNEL solution at 37 °C for 1 h in dark followed by 5 min 4′,6diamidino-2-phenylindol (DAPI) staining. Then the slides were examined using fluorescence microscopy (LEICADM4000B, Germany).

Figure 1. Viscosity of PME-D in sol and gel states at 25 °C. Data are represented as the mean ± standard deviation (SD) (n = 3).

Figure 2. In vitro release profiles of Dox from PME-D or Dox solution in different release media. 0%, 5%, 15%, and 40% ethanol represented 0%, 5%, 15%, and 40% ethanol (v/v) of PBS (pH 6.8) solution. Data are represented as the mean ± standard deviation (SD) (n = 3).

day. In contrast, the releasing profile of Dox from PME-D showed obvious delayed trends in all media. It was found that the faster Dox release was accompanied by a higher amount of ethanol in the solution. Less than 30% Dox was released from PME-D in 20 days when the ethanol content was 0 or 5% in 0.1 M PBS (pH 6.8). However, nearly 55% Dox was released when the release medium containing 15% (v/v) ethanol, and there was more than 80% Dox release when the ethanol content reached 40% (v/v). In Vitro Antitumor Activity. The in vitro antitumor activities were evaluated in MCF-7 cell line. As showed in Figure 3, nearly all of the MCF-7 cells treated with Dox solution (both low and high doses) were killed in 1 day. In contrast, the survival rate of MCF-7 cells gradually decreased during a 5-day period in PME-D treated groups. The PME-D high dose showed a stronger antitumor efficacy when compared with the low dose group. The burst release of Dox from Dox solution and the sustained release of Dox from PME-D may explain the differences in the survival rates. In Vivo Antitumor Activity. As shown in Figure 4A, the tumor volume was approximately 500 mm3 before treatment for all of the groups. The tumor size increased for almost 28 times after 14 days in saline-treated mice. In contrast, there were only very slight increases in free Dox solution and PME-D treated groups, indicating the significant antitumor effects. However, all mice were dead within 4 days in free Dox solution intratumoral injection group due to the high systemic toxicity

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RESULTS PME-D Preparation and Viscosity Measurements. The appearances and viscosities of PME-D are shown in Figure 1. The red color came from Dox. PME-D was a sol with a low viscosity at 25 °C. After transition, PME-D gel was formed. There was a significant increase in viscosity. In Vitro Dox Release. Figure 2 showed the in vitro release profile of Dox. In free Dox solution groups, the release of Dox was very fast in all media with nearly 100% Dox release in 1 C

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received PME-D treatment showed gradually weight gain, which was similar to the growth curve of saline control. The difference in weight changes also indicated the reduced toxicities by PME-D. Figure 4C shows the survival rate of the animals. As mentioned above, mice that received an intratumoral injection of free Dox solution died within 4 days, indicating the high systemic toxicity. Mice were dead within 8 days in the Dox solution intravenous injection group. However, mice survived in PME-D treated group during the 2-week experiment period, indicating the reduced systemic toxicity. In Vivo Dox Release and Biodistribution. As shown in Figure 5A, the Dox concentration increased quickly in the Figure 3. In vitro antitumor activities of PME-D and free Dox solutions in human breast cancer cell line MCF-7. Data were represented as means ± standard deviation (SD) (n = 3).

Figure 5. Dox concentration in blood plasma from mice bearing S180 sarcoma cancer cell xenografts treated with PME-D (A), intratumoral injection of free Dox solution (B), and intravenous injection of free Dox solution (C). Data points are represented as the mean ± standard deviation (SD) (n = 5). Figure 4. In vivo antitumor activity in mice bearing S180 sarcoma cancer cell xenografts. The changes of tumor volume (A), relative body weight (B), and survival rate (C) (p < 0.001) were monitored to evaluate the antitumor activity. Data are represented as the mean ± standard deviation (SD) (n = 9).

bloodstream in the first 4 h with the highest concentration of 47 ng/mL. Then it gradually decreased, followed by maintaining at a relatively stable concentration of 20−25 ng/ mL for several days. Figure 5B shows a significantly higher blood plasma concentration of Dox in the Dox solution intratumoral injected group. The Dox concentration reached 2849.49 ng/mL within 10 min, which was 59.79 times that of the peak concentration in the PME-D group. Then it decreased very quickly in the following 20 min and reached 165.22 ng/mL after 2 days. As shown in Figure 5C, after administrating at a dose of 3 mg/kg, the Dox concentration in the intravenous

of free Dox. Mice in PME-D treated group survived during the 14-day experiment period, suggesting the significantly less toxicity. The body weight changes of the mice are shown in Figure 4B. There were obviously body weight decreases for mice treated with free Dox solution, especially by intratumoral injection (20% weight changes within 4 days). The mice D

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Figure 6. Biodistribution of Dox in different organs, including tumor (a), heart (b), liver (c), kidney (d), lung (e), and spleen (f) after intratumoral injection of PME-D.

Figure 7. H&E stained tumor tissues of saline, free Dox solution, and PME-D groups. The arrow indicates a blood vessel.

Figure 8 shows the results from TUNEL staining. The bright blue fluorescence indicated the nuclei of live cells by DAPI staining. The green fluorescence by TUNEL staining represented apoptotic cells. For the saline treated group, almost no green fluorescence was observed, indicating the absence of apoptotic cells. The free Dox solution group exhibited much brighter green fluorescence than the saline group. However, there were less bright blue and more green fluorescence regions in PME-D treated group when compared with free Dox solution treated mice, suggesting the superior antitumor efficacy of PME-D. In addition, the red fluorescence from Dox in PME-D treated tumor tissue even 14 days after administration further proved the sustained release of Dox from PME-D.

injection group reached 282.52 ng/mL in 10 min and decreased to 161.72 ng/mL in the following 2 days. The biodistribution of Dox in different tissues including heart, liver, spleen, lung, kidney, and tumor are shown in Figure 6. There was a strong signal of Dox in tumor tissue, with very weak fluorescence in other organs, indicating the high concentration of Dox in the tumors. After one single injection, the strong signal in tumor remained for more than 14 days, suggesting the sustained release of Dox from PME-D. Histological Analysis. At predetermined time points, the tumors were harvested for histological analysis. As shown in Figure 7, for tumors injected with saline, blood vessels were observed (as the arrow indicated), and there was no evidence of necrosis in the H&E staining slides. Some necrosis regions were observed in the free Dox solution treated group, whereas there were significantly more necrosis regions in tumors treated with PME-D. Meanwhile, no blood vessel was observed in the PMED group.

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DISCUSSION In this study, a novel in-situ-forming gel system composed of small molecule injectable materials was developed to improve E

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Figure 8. DAPI and TUNEL stained tumor sections of saline, free Dox solution, and PME-D groups. DAPI stained bright blue, indicating the nuclei in live cells. The bright green fluorescence from TUNEL staining is a sign of apoptotic cells. The red fluorescence came from Dox as the arrow indicates.

erosion and release of Dox from PME-D in vivo were partially simulated. The in vitro antitumor activities were evaluated in MCF-7 cells. In this study, the survival rate of MCF-7 cells should be evaluated several times at different time points. A very sensitive WST-1 assay was used for evaluation. It can be performed in the same microtiter plate without extra steps such as cell solubilization. Compared with the sharp decrease of cell viability in 1 day for free Dox solution treated group, the approximately 95% of cell viability in the first day after administration and the gradual cell death in 5 days by PME-D treatment indicated a sustained release of Dox for an extended time period. In addition, the in vitro Dox release profile and the in vivo Dox concentration in blood data suggested a slight initial burst release of Dox from PME-D. The peak concentration of Dox in plasma after PME-D administration approximately doubled the Dox concentration in the stable stage, while the highest Dox concentration was approximately 7 μg/mL and the relatively stable concentration was approximately 1 μg/mL for mPEG−PCL diblock copolymer gel;40 the Cmax was 290 ng/ mL, and the relatively stable concentration was approximately 30 ng/mL for poly(organophosphazene) hydrogel.14 After the slight initial burst release, the sustained released of Dox from PME-D guaranteed the long-term tumor inhibition effects. All of the mice in the free Dox group died within 4 days after the intratumoral injection, indicating the high toxicity of Dox. However, animals survived well in the PME-D treated group with the same administration route and Dox dosage. The significantly decreased toxicity of PME-D could be explained by the sustained Dox release from PME-D gel depot after phase transition. In Dox solution intratumoral injected group, Dox was released very fast and cleared out quickly. Compared with the PME-D group, the highest concentration of Dox in free Dox solution group was 59.79 times that in the PME-D group, suggesting the negligible burst release profile of PME-D. In addition, a significant tumor inhibition effect was observed after a single intratumoral injection of PME-D. Repeated intravenous injections of free Dox solution showed a comparable antitumor efficacy with PME-D but a significantly higher mortality (mice were dead within 8 days). Considering the rapid clearance of

biologically compatibility and optimize the initial burst effect of in-situ-forming gels. E80 and MCT, the main components of PME, have good biocompatibilities.32,33 The ethanol content in PME gel is very low, which would not cause severe toxicity.34−36 The presence of MCT and ethanol, especially the amphiphilic feature of E80, allows PME to encapsulate both hydrophobic and hydrophilic chemotherapeutic agents. Considering the viscosity of the formulation and the sustained release time, the following formulation: E80−MCT−ethanol = 72:17:11 (mass ratio) were selected as the prescription. Doxloaded PME could be easily prepared by simply stirring. PME remained in a sol state in vitro and rapidly turned into gel after intratumoral injection. The mechanism for this sol−gel transition is different from usual stimuli, such as temperature, pH, and ionic strength. It was solvent exchange that lead to this sol−gel transition of the PME system. The solubility of E80 in water was very low. After intratumoral injection, E80 quickly precipitates due to the rapid diffusion of ethanol from injection site to adjacent tissues and therefore leads to the significant viscosity increase. Further studies are needed for the detailed transition mechanism. This solvent exchange mechanism for the sol−gel transition has two main advantages when compared to other mechanisms. First, it does not require rigorous physical and chemical conditions, such as suitable temperature, pH, and ionic strength, which are needed for other in-situ-forming gels.37−39 Second, it does not need complex components; the materials used are commercially available. However, it also has some disadvantages. For example, ethanol, the solvent of PME system, may cause the denaturation of some drugs, such as proteins and peptides. Therefore, the PME system may not be applicable for these drugs. The in vitro study showed fast release of Dox from Dox solution in all media. In contrast, Dox release from PME-D changed with the releasing medium. The more ethanol content in the releasing medium, the faster Dox release was observed. It could be explained by the high solubility of E80 and Dox in ethanol. There was a quicker erosion of PME gel in the releasing medium containing a higher amount of ethanol. By using media containing different percentages of ethanol, the F

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Dox in vivo, the plasma concentration of Dox would be of great change with repeated intravenous injections, which may explain the higher mortality of the repeated intravenous injection group. The superior therapeutic effect and significantly decreased toxicity of PME-D compared with free Dox intravenous injection could be explained by site-specific administration, high Dox concentration maintaining at the tumor site and low Dox level in bloodstream and other major organs. In addition, compared with the repeated administration, a single intratumoral injection of PME-D is predicted to improve patience compliance. The in vivo Dox biodistribution data can be evaluated by several techniques, such as computed tomography (CT) and magnetic resonance imaging (MRI).41 In this study, a whole body imaging system was employed. The results showed very limited Dox distribution in normal tissues, indicating the potential of decreased systemic toxicity in other organs by intratumoral injection. In addition, the Dox signal remained high in tumor tissues after single injection over 14 days, suggesting the retention of Dox in the tumor site. Fluorescence from Dox gradually decreased over time, which was another evidence of sustained release of Dox in PME-D gel. Histology evaluation by H&E and TUNEL stainings showed the cells apoptosis in the tumor tissue. Very few apoptotic cells were found in the saline group. Larger apoptotic cell regions were observed in the PME-D treated group when compared with the free Dox solution group, indicating the superior therapeutic efficacy of PME-D. In addition, a small amount of Dox was observed in the tumor tissue slides from PME-D treated group, suggesting the long-term retention of Dox even at the end of 14 days after a single intratumoral injection of PME-D.

2013CB932504), and the National Natural Science Foundation of China (No. 81273443).

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ABBREVIATIONS: MCT, medium chain triglycerides; PME, the novel in-situforming gel system, first letter of phospholipid, MCT, and ethanol; PME-D, doxorubicin-loaded PME

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CONCLUSION A novel injectable in-situ-forming gel based on biocompatible small molecule materials was developed. The gel was in the low viscosity sol state at the time of injection and then rapidly turned into gel state in vivo. The gel served as a drug reservoir in the tumor site, sustaining the release Dox for more than 14 days after a single injection. Moreover, the initial burst effect can hardly be observed from the PME system, which was different from many other in-situ-forming gels. PME-D displayed significant inhibition of tumor growth during the 14 day treatment period after a single intratumoral injection. In summary, the novel injectable in-situ-forming gel has superior therapeutic efficacy and an improved patient compliance. It is a promising drug delivery system for local chemotherapy.

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REFERENCES

(1) Siegel, R.; Naishadham, D.; Jemal, A. Cancer statistics, 2012. CA: Cancer J. Clin. 2012, 62 (1), 10−29. (2) WHO. 10 facts on cancer; World Health Organization: Geneva, 2006. (3) WHO. Cancer control; World Health Organization: Geneva, 2006. (4) Chun, C.; Lee, S. M.; Kim, C. W.; Hong, K.-Y.; Kim, S. Y.; Yang, H. K.; Song, S.-C. Doxorubicin−polyphosphazene conjugate hydrogels for locally controlled delivery of cancer therapeutics. Biomaterials 2009, 30 (27), 4752−4762. (5) Nsereko, S.; Amiji, M. Localized delivery of paclitaxel in solid tumors from biodegradable chitin microparticle formulations. Biomaterials 2002, 23 (13), 2723−2731. (6) Hatefi, A.; Amsden, B. Biodegradable injectable in situ forming drug delivery systems. J. Controlled Release 2002, 80 (1), 9−28. (7) Anseth, K. S.; Metters, A. T.; Bryant, S. J.; Martens, P. J.; Elisseeff, J. H.; Bowman, C. N. In situ forming degradable networks and their application in tissue engineering and drug delivery. J. Controlled Release 2002, 78 (1), 199−209. (8) Liu, F.; Urban, M. W. Recent advances and challenges in designing stimuli-responsive polymers. Prog. Polym. Sci. 2010, 35 (1), 3−23. (9) Jeong, B.; Kim, S. W.; Bae, Y. H. Thermosensitive sol−gel reversible hydrogels. Adv. Drug Delivery Rev. 2002, 54 (1), 37−51. (10) He, C.; Kim, S. W.; Lee, D. S. In situ gelling stimuli-sensitive block copolymer hydrogels for drug delivery. J. Controlled Release 2008, 127 (3), 189−207. (11) Kang, Y. M.; Kim, G. H.; Kim, J. I.; Kim, D. Y.; Lee, B. N.; Yoon, S. M.; Kim, J. H.; Kim, M. S. In vivo efficacy of an intratumorally injected in situ-forming doxorubicin/poly (ethylene glycol)-b-polycaprolactone diblock copolymer. Biomaterials 2011, 32 (20), 4556− 4564. (12) Kang, Y. M.; Lee, S. H.; Lee, J. Y.; Son, J. S.; Kim, B. S.; Lee, B.; Chun, H. J.; Min, B. H.; Kim, J. H.; Kim, M. S. A biodegradable, injectable, gel system based on MPEG-b-(PCL-ran-PLLA) diblock copolymers with an adjustable therapeutic window. Biomaterials 2010, 31 (9), 2453−2460. (13) Kakinoki, S.; Taguchi, T.; Saito, H.; Tanaka, J.; Tateishi, T. Injectable in situ forming drug delivery system for cancer chemotherapy using a novel tissue adhesive: characterization and in vitro evaluation. Eur. J. Pharm. Biopharm. 2007, 66 (3), 383−390. (14) Al-Abd, A. M.; Hong, K.-Y.; Song, S.-C.; Kuh, H.-J. Pharmacokinetics of doxorubicin after intratumoral injection using a thermosensitive hydrogel in tumor-bearing mice. J. Controlled Release 2010, 142 (1), 101−107. (15) Lee, J. Y.; Kim, K. S.; Kang, Y. M.; Kim, E.; Hwang, S.-J.; Lee, H. B.; Min, B. H.; Kim, J. H.; Kim, M. S. In vivo efficacy of paclitaxelloaded injectable in situ-forming gel against subcutaneous tumor growth. Int. J. Pharmaceutics 2010, 392 (1), 51−56. (16) Liu, W.; MacKay, J. A.; Dreher, M. R.; Chen, M.; McDaniel, J. R.; Simnick, A. J.; Callahan, D. J.; Zalutsky, M. R.; Chilkoti, A. Injectable intratumoral depot of thermally responsive polypeptide− radionuclide conjugates delays tumor progression in a mouse model. J. Controlled Release 2010, 144 (1), 2−9. (17) Shikanov, A.; Shikanov, S.; Vaisman, B.; Golenser, J.; Domb, A. J. Paclitaxel tumor biodistribution and efficacy after intratumoral injection of a biodegradable extended release implant. Int. J. Pharmaceutics 2008, 358 (1), 114−120.

AUTHOR INFORMATION

Corresponding Authors

*T.G.: Tel./Fax: +86-28-85501615. E-mail: gongtaoy@126. com. *L.Z.: Tel.: +44 7565 120516. E-mail: ling.zhang@manchester. ac.uk. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by grants from the National S&T Major Project of China (Grant No.: 2012ZX09304004), the National Basic Research Program of China (973 program, No.: G

dx.doi.org/10.1021/mp500019p | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

(18) Le Renard, P.-E.; Jordan, O.; Faes, A.; Petri-Fink, A.; Hofmann, H.; Rüfenacht, D.; Bosman, F.; Buchegger, F.; Doelker, E. The in vivo performance of magnetic particle-loaded injectable, in situ gelling, carriers for the delivery of local hyperthermia. Biomaterials 2010, 31 (4), 691−705. (19) Royals, M. A.; Fujita, S. M.; Yewey, G. L.; Rodriguez, J.; Schultheiss, P. C.; Dunn, R. L. Biocompatibility of a biodegradable in situ forming implant system in rhesus monkeys. J. Biomed. Mater. Res. 1999, 45 (3), 231−239. (20) Kranz, H.; Brazeau, G.; Napaporn, J.; Martin, R.; Millard, W.; Bodmeier, R. Myotoxicity studies of injectable biodegradable in-situ forming drug delivery systems. Int. J. Pharmaceutics 2001, 212 (1), 11− 18. (21) Parent, M.; Nouvel, C.; Koerber, M.; Sapin, A.; Maincent, P.; Boudier, A. PLGA in situ implants formed by phase inversion: Critical physicochemical parameters to modulate drug release. J. Controlled Release 2013, 172 (1), 292−304. (22) Packhaeuser, C.; Schnieders, J.; Oster, C.; Kissel, T. In situ forming parenteral drug delivery systems: an overview. Eur. J. Pharm. Biopharm. 2004, 58 (2), 445−455. (23) Elbert, D. L.; Pratt, A. B.; Lutolf, M. P.; Halstenberg, S.; Hubbell, J. A. Protein delivery from materials formed by self-selective conjugate addition reactions. J. Controlled Release 2001, 76 (1), 11−25. (24) Kumbhar, A. B.; Rakde, A. K.; Chaudhari, P. In situ gel forming injectable drug delivery system. Int. J. Pharm. Sci. Res. 2013, 4 (2), 597−609. (25) Tae, G.; Kornfield, J. A.; Hubbell, J. A. Sustained release of human growth hormone from in situ forming hydrogels using selfassembly of fluoroalkyl-ended poly (ethylene glycol). Biomaterials 2005, 26 (25), 5259−5266. (26) Bajpai, A.; Shukla, S. K.; Bhanu, S.; Kankane, S. Responsive polymers in controlled drug delivery. Prog. Polym. Sci. 2008, 33 (11), 1088−1118. (27) Hong, D.-W.; Lai, P.-L.; Ku, K.-L.; Lai, Z.-T.; Chu, I. Biodegradable in situ gel-forming controlled vancomycin delivery system based on a thermosensitive mPEG-PLCPPA hydrogel. Polym. Degrad. Stab. 2013, 98 (9), 1578−1585. (28) Ding, H.; Wu, F.; Huang, Y.; Zhang, Z.-r.; Nie, Y. Synthesis and characterization of temperature-responsive copolymer of PELGA modified poly(N-isopropylacrylamide). Polymer 2006, 47 (5), 1575− 1583. (29) Ruel-Gariépy, E.; Leroux, J.-C. In situ-forming hydrogels review of temperature-sensitive systems. Eur. J. Pharm. Biopharm. 2004, 58 (2), 409−426. (30) Camargo, J.; Sapin, A.; Nouvel, C.; Daloz, D.; Leonard, M.; Bonneaux, F.; Six, J.-L.; Maincent, P. Injectable PLA-based in situ forming implants for controlled release of Ivermectin a BCS Class II drug: solvent selection based on physico-chemical characterization. Drug. Dev. Ind. Pharm. 2013, 39 (1), 146−155. (31) Young, R. C.; Ozols, R. F.; Myers, C. E. The anthracycline antineoplastic drugs. N. Engl. J. Med. 1981, 305 (3), 139. (32) Kimura, M.; Takai, M.; Ishihara, K. Biocompatibility and drug release behavior of spontaneously formed phospholipid polymer hydrogels. J. Biomed. Mater. Res. A 2007, 80 (1), 45−54. (33) Schwab, M.; Kessler, B.; Wolf, E.; Jordan, G.; Mohl, S.; Winter, G. Correlation of in vivo and in vitro release data for rh-INFα lipid implants. Eur. J. Pharm. Biopharm. 2008, 70 (2), 690−694. (34) Montaguti, P.; Melloni, E.; Cavalletti, E. Acute intravenous toxicity of dimethylsulfoxide, polyethylene glycol 400, dimethylformamide, absolute ethanol, and benzyl alcohol in inbred mouse strains. Arzneim.-Forsch. 1994, 44, 566−570. (35) Alstota, R. L.; Tarrania, M. E.; Forney, R. B. The acute toxicities of 1-methylxanthine, ethanol and 1-methylxanthine/ethanol combinations in the mouse. Toxicol. Appl. Pharmacol. 1973, 24 (3), 393−404. (36) Malcolm, R. D.; Alkana, R. L. Temperature dependence of ethanol lethality in mice. J. Pharm. Pharmcol 1983, 35 (5), 306−311. (37) Liu, F.; Urban, M. W. Recent advances and challenges in designing stimuli-responsive polymers. Prog. Polym. Sci. 2010, 35 (1− 2), 3−23.

(38) Packhaeuser, C. B.; Schnieders, J.; Oster, C. G.; Kissel, T. In situ forming parenteral drug delivery systems: an overview. Eur. J. Pharm. Biopharm 2004, 58 (2), 445−455. (39) Kempe, S.; Mäder, K. In situ forming implantsan attractive formulation principle for parenteral depot formulations. J. Controlled Release 2012, 161 (2), 668−679. (40) Hyun, H.; Kim, Y. H.; Song, I. B.; Lee, J. W.; Kim, M. S.; Khang, G.; Park, K.; Lee, H. B. In vitro and in vivo release of albumin using a biodegradable MPEG-PCL diblock copolymer as an in situ gel-forming carrier. Biomacromolecules 2007, 8 (4), 1093−1100. (41) Ding, H.; Wu, F. Image guided biodistribution and pharmacokinetic studies of Theranostics. Theranostics 2012, 2 (11), 1040.

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dx.doi.org/10.1021/mp500019p | Mol. Pharmaceutics XXXX, XXX, XXX−XXX