Research Article www.acsami.org
Ultrasound-Responsive Polymeric Micelles for SonoporationAssisted Site-Specific Therapeutic Action Pengying Wu,#,† Yali Jia,#,† Fei Qu,† Yue Sun,† Pan Wang,† Kun Zhang,† Chuanshan Xu,*,‡ Quanhong Liu,† and Xiaobing Wang*,† †
Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi 710119, China ‡ School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China S Supporting Information *
ABSTRACT: Targeting drug delivery remains a challenge in various disease treatment including cancer. The local drug deposit could be greatly enhanced by some external stimuliresponsive systems. Here we develop pluronic P123/F127 polymeric micelles (M) encapsulating curcumin (Cur) that are permeabilized directly by focused ultrasound, in which ultrasound triggers drug release. Tumor preferential accumulation and site-specific sonochemotherapy were then evaluated. Cur-loaded P123/F127 mixed micelles (Cur-M) exhibited longer circulating time and increased cellular uptake compared to free Cur. With the assistance of focused ultrasound treatment, Cur-M showed tumor-targeting deposition in a time-dependent manner following systemic administration. This was due to enhanced permeabilization of tumor regions and increased penetration of Cur-M in irradiated tumor cells by ultrasound sonoporation. Furthermore, Cur-M self-assembly could be regulated by ultrasound irradiation. In vitro Cur release from mixed micelles was greatly dependent on ultrasound intensity but not on duration, suggesting the cavitational threshold was necessary to initiate subsequent sonochemotherapy. In vivo site-specific drug release was demonstrated in dual-tumor models, which showed spatial-temporal release of entrapped drugs following intratumoral injection. The sonoporation-assisted sitespecific chemotherapy significantly inhibited tumor growth and the decrease in tumor weight was approximately 6.5-fold more than without exposure to ultrasound irradiation. In conclusion, the established ultrasound-guided nanomedicine targeting deposit and local release may represent a new strategy to improve chemotherapy efficiency. KEYWORDS: mixed micelles, ultrasound-responsive, targeting accumulation, local release, sonoporation-assisted chemotherapy
1. INTRODUCTION
phototoxicity and simultaneous release of inhibitors, synergistically inhibited tumor regrowth.9 Chen et al. developed a new type of nanoparticle that can be activated by microwaves to produce singlet oxygen for drug release.10 Zhang et al. showed a novel modality for combining radiotherapy and photodynamic therapies based on luminescence-dependent drug release under X-rays exposure.11 In particular, ultrasound represents a unique approach to control the “on and off” switch of micelles and induces biological effects.15 As a noninvasive modality, ultrasound (Us) has shown great potentials in diagnosis, therapeutics, as well as in drug delivery.16 Numerous studies in respect of Us-mediated drug delivery demonstrate that the application of Us could promote molecular drugs’ intracellular transportation.17−19 Hence, Us would be an effective tool to activate drug release in situ with
Nanoparticles have been preferentially used to transport anticancer drugs into tumors site, with their superiority of moderate magnitude.1−3 Up to now, several nanocarriers including micelles have been approved to enhance the biodistribution and anticancer efficacy of chemotherapeutic agents clinically.4 The core−shell structure of polymeric micelles allows the incorporation of poorly soluble drugs, and meanwhile, it provides protection from inactivation in biological environment. Furthermore, their relatively small size (10−100 nm) confers the system many advantages like passive targeting, long circulation, and easy preparation.5,6 However, this delivery system is partially impeded by physiological barriers and the bioavailability of loaded drugs, which are inevitably suboptimal.7,8 In order to improve the efficacy of therapeutic agents in tumor, some strategies of external stimuli such as light, ultrasound, thermo, X-ray, and microwave have been pursued.9−14 Hasan et al. recently reported a photoactivatable multi-inhibitor nanoparticle which could produce © XXXX American Chemical Society
Received: April 19, 2017 Accepted: July 13, 2017
A
DOI: 10.1021/acsami.7b05469 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) assay kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). An Annexin V-FITC Apoptosis Detection Kit and red fluorescent dye (DiR iodide) were obtained from Keygen Technology Co., LTD (Nanjing, China). 2.2. Ultrasound Apparatus. The experimental ultrasonic devices were similar as previously described.41,42 A cell-based therapeutic pulsed ultrasound apparatus was used for the in vitro test. For cell treatment, such Us parameters (0.4 W/cm2, 1.0 MHz, duty cycle of 30%, duration of 1 min) were used. For in vivo experiments, a focused Us transducer was utilized. The load power (LP) indicated as 1 (Us1), 2 (Us2), and 3 W (Us3) with a frequency of 1.90 MHz were adopted in this study. 2.3. Formation of Polymeric Micelles. Cur-M was prepared by thin-film hydration methods.38 Briefly, 15 mg of Cur, 180 mg of P123, and 90 mg of F127 were dissolved in 5 mL of tetrahydrofuran, and then the organic solvent was removed by rotary evaporation at 50 °C for 1 h, until a thin film was formed. The thin film was hydrated for 30 min at 60 °C to obtain a micelle solution, a 0.22 μm syringe filter was used to remove the excess drug and the final micelles solution were store at 4 °C in the dark. 2.4. Characterization of Micelles. The particle size and surface charge of Cur-M in an aqueous medium were measured by DLS at 25 °C using a Delsa Nano C Size/Zeta Potential Analysis Instrument (Beckman Instruments, German). A transmission electron microscope (TEM) operating at 80 kV (HT-7700, Hitachi) was used to observe the micelles morphology. The encapsulation efficiency of Cur within Cur-micelles was determined through a spectrophotometric method. The micelles were disrupted with methanol, and then the concentration of Cur was determined using a UV−vis spectrophotometer (Spectra Max M5, Molecular Device, U.S.A.) at 425 nm. The drug loading (DL) and encapsulation efficiency (EE) were calculated using the formula: DL% = (weight of encapsulated Cur)/(weight of polymer + weight of feeding Cur) × 100 (%), encapsulation efficiency (%) = (weight of encapsulated Cur)/(weight of feeding Cur) × 100 (%). The Cur-M kept at 4 or 37 °C for 2 days (n = 3 batches) after preparation, and the particle size distribution and zeta potential were monitored to reflect the stability of Cur-M. Meanwhile, in order to evaluate the stability of Cur-M in blood circulation, we used saline containing fetal bovine serum (FBS) to simulate the blood environment.43 The time-dependent colloidal stability of Cur-M in PBS containing 10%, 20%, or 40% FBS at 37 °C was monitored as described above. Then we examined the fluorescence changes under different conditions. Briefly, a thin-walled polypropylene tube with 0.5 mL of Cur-M was placed in the focal zone of an Us transducer. Before and after treatment with Us3 for 3 min, the fluorescence of Cur-M at 530 nm was recorded using a fluorescence photometer (LS-55, PE, U.S.A.), and the free Cur was used for comparison. The drug release profiles of Cur-M upon dialysis against PBS were determined to evaluate the influence of Us on Cur-M. The drug release in different pH and physiological conditions in the absence and presence of Us treatment was also monitored. Subsequently, we estimated the ultrasonic cavitation using TA (terephthalic acid) dosimetry. Nonfluorescent TA reacts with hydroxyl radicals that were generated during acoustic cavitation and produces fluorescent 2-hysroxyterephthalate ions (HTA). TA solution (1 mM) was subjected to Us treatment (1.90 MHz) for 1 min when LP indicated 1 (Us1), 2 (Us2), and 3 W (Us3), and then a fluorescence photometer was used to determine the fluorescence of HTA at 426 nm immediately after sonication. 2.5. Cell Experiments. Human breast cancer MDA-MB-231 cells and mouse breast cancer 4T1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, Life Technologies, Carlsbad, CA, U.S.A.) containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% glutamine. Cultures were maintained at 37 °C with humidity and 5% CO2. Cells in the exponential phase were used for this study. For qualitative assay of cellular uptake of Cur and Cur-M in MDA-MB-231 cell, the same final concentration of Cur (10 μM) were added to the cells (2 × 105 cells/ml) and irradiated with Us (1.0 MHz, 0.4 W/cm2, 1 min) after 4 h of coincubation. At 30 min after
increased therapeutic values and decreased side-effects. In multiple types of drug or gene delivery, sonoporation, based on Us cavitation, has received much attention.20 It has been reported that cavitational effects such as jet formation, shock waves, and microstreaming could enhance cell membrane permeabilization and a reversible opening of tight junctions in the vascular endothelium.21,22 At the current time, sonoporation has been proved to be an effective method to overcome the biomembranes in vitro.23−25 In vivo sonoporation are mainly applied in the permeation of vasculature and the bloodbrain barrier.26 Chen et al. have reported that Us sonoporation can open up the blood-brain barrier and improve interleukin12’s delivery into a brain tumor.27 Bekeredjian’s study indicates that sonoporation notably enhanced the transfer of genes into rat myocardium under optimized Us conditions.28 Preliminary study also shows sonoporation plays a role to ameliorate drug delivery into solid tumors.29 These reports suggest Us would be potentially applied in future clinic drug delivery and deserves deep explorations. Compressible objects (e.g., micelles and microbubbles) contract, expand, and explode as exposed to acoustic waves, then facilitate cargo deposit at specific sites.30−33 However, few studies have especially evaluated the multifunctional advantages of Us irradiation on preferential tumor accumulation, deep and homogeneous penetration of particles, controllable drug-release from micelles, and sitespecific sonochemotherapy. Curcumin (Cur), as one of the commonly used anticancer drugs, has been applied for treatment of colorectal, breast, prostate, pancreatic, and other types of carcinomas.34,35 Nevertheless, challenges like low aqueous solubility, biological instability, and rapid metabolism, make the bioavailability of Cur very poor.36 Thus, developing a novel Cur-loading nanocarrier to overcome the above problems is of great importance. Pluronic is the FDA approved PEO−PPO-PEO triblock copolymer, and has been clinically used as a pharmaceutical adjuvant.37,38 Compared to a single system, a binary mixing system with pluronic shows additional advantages that attract investigators to develop pluronic copolymers such as P123/F127 as new drug carriers.39,40 Herein, a new treatment strategy that utilizes Us to first promote local deposition of P123/F127 mixed micelles in irradiated regions is introduced. Following this, the Cur release from micelles was triggered to facilitate targeting sonochemotherapy. In this established system, the Us2 treatment with a load power of 2 W was sonicated for 5 min immediately after intravenous injection of Cur-loaded P123/F27 micelles (CurM), aiming to increase vascular permeability and enhance tumor accumulation. After 12 h of Us2 treatment, Us3 (load power = 3 W, 1.90 MHz, 3 min) was applied to stimulate local drug release and exert site-specific therapeutic action. Our findings represent a method for excellent spatial and temporal control of cargos using Us-responsive nanocarriers, which could be developed as a simple, noninvasive, specific, and effective approach for improving chemotherapeutics.
2. EXPERIMENTAL SECTION 2.1. Materials. Plurconic P123, Plurconic F127, 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT), propidium iodide (PI), and curcumin were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Primary antibody against proliferating cell nuclear antigen (PCNA) was purchased from Abcam (Cambridge, U.K.). Secondary antibodies were obtained from Zhong Shan Golden Bridge Biotechnology (Beijing, China). Glutamic-pyruvic B
DOI: 10.1021/acsami.7b05469 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 1. Characterization of Cur-M. (A) Schematic illustration of the simple synthetic process of Cur-M. (B) TEM image (left) of Cur-M, scale bar in TEM = 100 nm. DLS analysis (right) showing an average size distribution around 23 nm. (C,D) Changes of diameters (C) and zeta potential (D) distributions of Cur-M after different storage times at 4 or 37 °C. Error bars represent the SD for n = 3. treatment, cells were detected by flow cytometer (NovoCyte, ACEA Biosciences Inc., U.S.A.). To observe the subcellular localization of Cur, MDA-MB-231 cells were incubated with 10 μM Cur or Cur-M with/without Us treatment and visualized using a confocal laser scanning microscope (LSM 510 Meta, Zeiss, Germany) SEM was used to observe the cells damage. At 24 h post treatment, cells were fixed in glutaraldehyde, washed by PBS, dehydrated by graded alcohol, displaced, dried at the critical point, gold evaporated, and observed under a SEM microscope (S-3400N, Hitachi, Tokyo, Japan). Cell viability was measured by regular MTT assay at 24 h as previously reported,44 and cell apoptosis was determined through Annexin V-FITC/PI double staining with flow cytometry. 2.6. In Vivo Experiments. Animal experiments were carried out with approval form the university’s Animal Care and Use Committee. The BALB/c mice (female) were subcutaneously injected with 1 × 106 4T1 cells in the flank region. In order to detect Cur circulation in blood and tissues, the BALB/c mice were assigned into two groups and intravenously injected with Cur or Cur-M (50 mg/kg Cur), respectively. At different time points after injection, the blood samples were collected to separate the plasma. At 1, 6, 12, and 24 h after injection, the mice were euthanized, and tissues were excised, followed by homogenization and extraction with methanol. The Cur content in plasma and tissue extraction was measured by fluorescence photometer. To investigate the biodistribution of micelles in vivo and whether Us could promote drug accumulation in tumor tissue, a dye, DiR was used to replace Cur due to its strong red fluorescence in the NIR region. The mice (n = 3) were subcutaneously injected with 4T1 cancer cells (1 × 106) in both flank regions to build dual-xenograft model. After 1 week, DiR-micelles was injection intravenously (all injection doses: 120 μL, 1 mg/mL), after 5 min post injection, the right tumor was treated with exposure system (Us2, LP = 2W, 1.90 MHz, 5 min). By using the strong fluorescence of DiR, an in vivo imaging system was performed at 1, 3, 6, 12, 24, and 48 h after administration. To illustrate the ex vivo biodistribution of micells, the tumor-bearing mice were sacrificed at 12 h post injection, and the main organs were collected and imaged. To further quantify the
concentration of Cur in vivo, the 4T1 xenograft mice (n = 3) were intravenously injected with Cur or Cur-M at an identical dose of 50 mg/kg, after 5 min, the right tumor was treated with Us2 exposure system. At different time intervals, the mice were sacrificed and tissues were collected, extracted, and subjected to fluorescence analysis. The efficiency of Us-triggered drug release in vivo was examined in 4T1 tumor-bearing mice. The mice (n = 3) with dual tumors were intratumorally injected with DiR-micelles (8 μL, 1 mg/mL) in the same depth (3 mm) with the aid of stereotaxic instrument (Stoelting, Zenda Inc., Shanghai, China) when the tumors grew to about 4−6 mm in diameter. Then the left tumor was treated with ultrasonic exposure system (Us3, LP = 3 W, 1.90 MHz, 3 min), 10 min after Us, the mice was placed into NIR fluorescence imaging system to collect the signal of DiR. For morphological observation, the tumors with or without Us treatment were collected, continuously frozen sectioned at 10 μm thickness until down into 500 μm in total and the fluorescence distribution of Cur was observed using Stereo Fluorescence Microscope (Discovery V20, Zeiss, German). To evaluate the antitumor efficacy, the 4T1 xenograft mice were divided into five groups (BALB/c, female, n = 7): (A) Cur alone, (B) Cur+Us2+Us3, (C) Cur-M, (D) Cur-M+Us2, (E) Cur-M+Us2+Us3. The Cur dose of 50 mg/kg were injected into the caudal vein and exposed to Us2 (LP = 2 W, 1.90 MHz, 5 min) for improving the drug accumulation in tumor sites, then after 12 h, the tumors were second irradiated by Us3 (LP = 3 W, 1.90 MHz, 3 min). This procedure were repeated thrice (7 days interval). The body weight and tumor volume were recorded every 2 days since the first treatment procedure. The mice were sacrificed on day 21. The survival period was monitored each day according the requirement of Animal Care and Use Committee within our university. For histological analysis, the tumors and major organs were collected after three procedures and fixed with 10% formalin for 24 h, then paraffin-embedded, sectioned, and stained with H&E or subjected to immunohistochemistry analysis of PCNA according to the manufacturers’ protocol. The biocompatibility of Cur-M was also detected. The whole blood and main organs were harvested after treatment. The GOT and GPT C
DOI: 10.1021/acsami.7b05469 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 2. Cur release from P123/F127 micelles in the absence and presence of Us exposure. (A) Fluorescence changes under different conditions (Free Cur: 50 μg/mL free Cur; Cur-M: 50 μg/mL Cur in P123/F127 micelles; Cur-M+Us3:50 μg/mL Cur in P123/F127 micelles with Us3 treatment). (B) Evaluation of Us cavitation by TA method. (Control: without Us treatment; Us1: Us treatment with LP = 1 W; Us2: Us treatment with LP = 2 W; Us3: Us treatment with LP = 3 W. **p < 0.01 versus control. (C) Drug release with or without Us3 irradiation for 2, 4, and 6 min, respectively. D (a) Drug release with or without Us irradiation (Us1, Us2, Us3) for 3 min, respectively; (b) Drug release at 30 min post Us irradiation (Us1, Us2, Us3) for 3 min. **p < 0.01 versus Us1 and Us2. (E) Drug release curve after twice Us3 stimulation. First Us3 irradiation for 3 min, then incubation until 15 min, a second Us3 irradiation for 3 min, and a final incubation until 30 min. Data shown are mean ± SD of three batches. levels were measured by biochemical analysis. The major organs were fixed in formalin, sectioned, and histopathologically analyzed.
Cur in Cur-M was much lower than free Cur, indicating the aggregation state after being encapsulated in micelles (Figure 2A). However, the fluorescence intensity of Cur in Cur-M was significantly increased after Us3 exposure, suggesting that micelles were disrupted by Us irradiation and the cargos could be freely released. Further, the drug release profiles were examined against PBS at room temperature. The drug leakage trends were lower than 1.1% within 30 min in the absence of Us irradiation (Figure 2C,D). On the other hand, drug release was approximately 19.78%, 28.34%, and 38.64% at 30 min following different intensities of Us irradiation for 3 min when LP indicated 1 (Us1), 2 (Us2) and 3 W (Us3), respectively (Figure 2D). Yet, the sonication time did not result in an obvious impact on the release, which was approximately 35.23%, 38.45%, and 40.43% at 30 min after Us3 irradiation for 2, 4, and 6 min, respectively (Figure 2C). The results suggested that the Us intensity was a determinant factor in initiating cargo release form P123/F123-mixed micelles, which may be related to Us-induced cavitation. Besides, we know that the tumor metabolic profile is different from interstitial matrix, improved levels of lactic acid due to the poor oxygen perfusion within tumor result in a reduction in pH from 7.40 to 6.00,48 and recent studies showed that Cur has a poor chemical stability at physiological pH and a better chemical stability at acidic condition.49 The result in Figure S2 shows that the acidic condition did not cause an obvious impact on the release of Cur-M under different pH conditions with the absence and presence of Us treatment. The measurement of hydroxyl radicals using TA method indirectly demonstrates cavitational events,50 which indeed occurred under the applied Us intensities. A gradual increase in hydroxyl radicals by Us1, Us2, and Us3 treatment was found (Figure 2B). Therefore, the pronounced Cur leakage in the
3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Cur-M. Micelles have been widely investigated as a nanocarrier due to their versatile loading capacities, efficient tumor accumulating ability, and excellent biocompatibility.5 Here, the standard formulation of pluronic mixed micelles was chosen as a drug loading platform for the antitumor inhibitor Cur, which was encapsulated into the hydrophobic core (Figure 1A). The concentration of polymer micelles used in vitro and in vivo assays were 80.47 μg/mL and 135 mg/mL, respectively. The morphology of Cur-M was characterized by TEM confirming their nearly spherical shape with nice monodispersity (Figure 1B, left panel). DLS indicates the average particle size of Cur-M was approximately 23 ± 1.06 nm (Figure 1B, right panel), which are favorable for passive targeting in tumor via the enhanced permeability and retention effect (EPR).45 In this system, the loading efficiency of Cur was 4.56%, and the encapsulation efficiency was 86.67%. No significant change in the particle size and zeta potential could be observed within 48 h (Figure 1C,D), suggesting Cur-M stability. Furthermore, the result in Figure S1 shows that the micelles displayed excellent colloidal stability after 24 h storage in PBS buffer containing 10%, 20%, or 40% FBS at 37 °C in vitro, indirectly supporting the good stability of Cur-M in blood circulation. This may attribute to the surrounding corona of micelles, which produces stable interfaces between aqueous media and the hydrophobic cores, preventing the aggregation of micelles.46 3.2. Ultrasound Triggered Drug Release from Micelles. Previous studies have shown that most fluorescent molecules loaded in micelles lead to high local concentrations with fluorescence quenching.47 The fluorescence intensity of D
DOI: 10.1021/acsami.7b05469 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 3. Cellular uptake of MDA-MB-231 cells in the absence and presence of Us exposure. (A) Quantification of intracellular uptake. (Error bars represent the SD for n = 3). **p < 0.01 versus control; &&p < 0.01 versus Cur, Cur-M, and Cur + Us group. (B) In vitro cellular uptake of Cur and Cur-M with or without Us exposure using CLSM (scale bar = 20 μm). (C) SEM images of MDA-MB-231 cells after distinct treatment. Represent images from three independent experiments are shown. Magnification = 2000×.
cytometry. Cur-M displayed more intracellular uptake than free Cur, and Us treatment (0.4 W/cm2) further enhanced cellular internalization (p < 0.01) (Figure 3A). Confocal images show much brighter fluorescent signal in Cur-M plus Ustreated MDA-MB-231 cells (Figure 3B). The results suggest Cur-M shared more intracellular uptake compared to free Cur by passive diffusion across biomembranes and Us prompted the process. This phenomenon could be derived from the increased permeability of cytoplasmic membrane by sonoporation, which has been previously reported by Yu et al.54 It has been shown that more chemotherapeutic agents are able to get into cells thus improving therapeutic effects by sonoporation.55 Cur-M utilized in the present study plus Us sonoportation would result in increased cytotoxicity and accelerated disassembly of the cytoskeleton and alterations in cell shape. Scanning microscopy (Figure 3C) demonstrated that well-adherent cells in control group displayed typical fusiform morphology. Treatment with either Cur alone or Cur-M produced no obvious changes and cells in the Us alone group presented shrinkage but kept a regular shape. However, following treatment with Cur and Us, cells clearly showed morphological changes with irregular shape. Notably, the cell morphology in Cur-M+Us group were seriously damaged, showing cytoskeletal collapse. These findings suggest that Us treatment was efficient in promoting cellular uptake of therapeutic agents or particles. 3.4. Evaluation of In Vitro Cytotoxicity. To determine whether the increased Cur uptake caused serious cytotoxicity, MTT assay and apoptotic analysis were performed. Cell cytotoxicity revealed does-dependent tendencies against both
present study was mainly attributed to Us-induced cavitation, which is consistent with previous reports on Us-induced cavitation facilitated micelle disassembly and drug release.33 Cur release in response to Us treatment was very rapid, almost completed within 5 min, and the prolonged incubation until 30 min post-treatment caused slight effects on the release profiles (Figure 2C,D). Such a quick response pattern will enable a high-speedy therapeutic drug concentration around tumor, which is superior to other controlled release systems, such as pH-responsive systems that are limited in the tumor microenvironment and light-sensitive systems that are hindered by the penetration of light.51 Us cavitation-induced sonoporation is a reversible process. Us-induced membranes can be timely permeabilized and are able to recover integrity within a few minutes postirradiation.18 In order to test the temporary permeability of micelles by Us irradiation, the continuous Cur release profile during Us “on” and “of” in real time was examined. Cur release from P123/ F123 micelles occurred mostly during Us exposure and ceased within minutes of turning Us off, indicating that the present delivery system could be temporarily controlled for unloading of cargo on demand (Figure 2E). 3.3. In Vitro Cellular Uptake. Therapeutic drugs exert their tumor inhibition effects based on the intracellular accumulation doses. As an extensively investigated anticancer drug, Cur has been reported to inhibit proliferation and induce apoptosis in numerous cancer cell lines.52,53 In order to evaluate its cellular uptake performance, intracellular Cur content was determined in MDA-MB-231 cells by flow E
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Figure 4. Effects of Cur-M on cultured MDA-MB-231 cells. (A) Detection of cell viability by MTT assay at 24 h after distinct treatment. (a) Cell viability curve as a function of Cur dose; (b) Comparison of different treatment on cell viability of MDA-MB-231 cells when Cur was 10 μM. Results are mean ± SD for n = 3. *p < 0.05, **p < 0.01 compared with Cur, &&p < 0.01 compared with the other groups. (B) Apoptosis assessment at 24 h after different treatments.
free Cur and Cur-M (Figure 4Aa). Free Cur caused no obvious decrease in MDA-MB-231cells viability at the selected dose range. The cell viability loss caused by Cur-M was approximately 10.60% at the concentration of 10 μM and the presence of Us (0.4 W/cm2) increased the cell viability loss to 59.57% (p < 0.01) (Figure 4Ab). The calculated cell toxicity of Cur-M+Us was 5.61-fold of Cur-M without Us and 2.15-fold of Cur+Us. Cell apoptosis was determined through Annexin VFITC/PI double staining with flow cytometry, and the result (Figure 4B) demonstrated that Cur-M plus Us irradiation obviously induced apoptosis of MDA-MB-231 cells. 3.5. In Vivo Long Circulating Test. The hydrophobic block of micelle comprises a hydrophobic core incorporating lipophilic drugs and protecting them from inactivation in biological environment, and additionally, hydrophilic segment maintains the dispersion stability of micelles, which can prevent aggregation and recognition by the reticuloendothelial system and ensure long blood circulation. Through this design, Cur embedded within the mixed P123/F123 micelles will avoid premature drug release in the blood.56 To assess whether the micelles have a long circulation lifetime, pharmacokinetic studies were carried out using a BALB/c mouse model. Briefly, Cur and Cur-M were intravenously injected (Cur dose: 50 mg/ kg). After injection, Cur-M exhibited significantly prolonged blood retention time over a span of 48 h compared to free Cur (Figure S3Aa). More than 18.42% of Cur-M was found in blood vessels after 48 h injection, whereas the free Cur was almost eliminated from the blood 12 h after injection. Mixed-
micelles could extend the half-life of free Cur about 3.63-fold (Figure S3Ab), as shown in Figure S3B. Cur-M exhibited significant retention time in the major organs compared to free Cur, and the majority of Cur-M was found in the liver, spleen, and lung. As reported by others,57 Cur concentration decreased rapidly within 6 h after injection, due to the poor bioavailability, low aqueous solubility, instability, and rapid metabolism of free Cur. By 12 h, Cur concentration in these tissues was hardly to be detected. On the contrary, 24 h post Cur-M injection, Cur concentration in liver, spleen, and lung was still highly detected (Figure S3B). These results suggest that the mixed micelles could obviously influence the blood circulating time of Cur. Similar results have also been reported with pH-responsive micelles.58 3.6. Sonoporation-Guided Drug Accumulation and Release in Tumor Sites. The tumor vasculature normally lacks an effective lymphatic drainage system compared to normal tissues, thus permitting the passive targeting of tumor sites by nanoparticles based on the EPR.59 Polymeric micelles, due to their small size and passive targeting ability, have been clinically used as nanocarriers.5 In the above section, the mixed micelles were able to increase the long circulation lifetime. Next, Cur-M retention in tumor tissues and whether Us stimulus could promote drug accumulation in tumor sites were investigated. In this study, DiR with strong NIR fluorescence was used to replace Cur incorporation into P123/F127. 4T1 cancer cells (1 × 106 cells) were injected in both flanks of mice to build a dual-tumor model. After 1 week, the right tumors F
DOI: 10.1021/acsami.7b05469 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 5. Sonoporation promotes drug accumulation and drug release in 4T1 xenograft tumors. (A) In vivo NIR fluorescence imaging at different time points post DiR-loaded micelles injection (left: without Us; right: with Us, LP = 2W, 1.90 MHz, 5 min). (B) Biodistribution of NIR images of DiR-M in major organs/tissues at 12 post injection (**p < 0.01 compared with Us2 -irradiated tumor). (C) Cur content in tumor tissues after injection of free Cur and Cur-M at different time points with or without Us (Us2, LP = 2 W, 5 min). Data show mean ± SD for n = 3. *p < 0.05, **p < 0.01 between groups. (D) NIR fluorescence imaging after intratumoral injection of DiR-M with or without Us (Us3, LP = 3 W, 1.90 MHz, 3 min) (a: without Us; b: with Us on the left site tumor); (Dc) The average fluorescence intensity changes after Us3 in tumor tissues (**p < 0.01 versus group). (E) Fluorescence changes in intratumor distribution of Cur with/without Us3 treatment (scale bar = 0.5 mm). Data shown are mean ± SD of three batches.
were sonicated for 5 min (LP = 2 W, 1.90 MHz) after injection to increase vascular permeability, while the left tumors did not undergo Us irradiation. After which, in vivo real time imaging was performed sequentially. The NIR fluorescence signal in left tumor site of xenograft mice after injection of DiR-loaded micelles increased gradually and reached a maximum at 24 h, suggesting passive EPR-based tumor targeting (Figure 5A). By comparison, the DiR signal in right tumor site reached a maximum at 12 h after intravenous injection followed by immediate Us2 treatment. Furthermore, the fluorescent signals were far higher than the self-left contrast, indicating that selective irradiation by focused Us could promote drug accumulation in the tumor site. Moreover, each excised organ was examined at 12 h post injection. Consistent with results in Figure S4, the micelles retained high levels in liver, spleen, lung
as well as in the tumors. Notably, the levels accumulated in the right tumor treated with Us (U-tumor) were greater than that in the left tumor without Us treatment (N-tumor) (Figure 5B). For further quantitative detection, Cur concentration in tumor tissues was specifically examined in a time-dependent manner with or without Us. As shown in Figure 5C, free Cur showed little distribution in tumor tissues with or without Us treatment. Cur-M accumulation in tumor site was significantly higher than that of free Cur, and the Us treatment further increased its’ tumor accumulation, which was about 2.07-fold of Cur-M without Us at 12 h after administration. The results indicate Us guided the targeting-accumulation of Cue-M following sitespecific irradiation. Previous studies have suggested that Us exposure can increase vascular permeability and enhance tumor accumuG
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Figure 6. Antitumor effects in vivo. (Aa) Tumor growth curves of 4T1 xenograft mice after treatment; (Ab) Tumor volume inhibition ratios at 21 days. **p < 0.01 versus control, &&p < 0.01 Cur-M+Us2+Us3 group versus other groups. (A: Cur alone; B: Cur+Us2+Us3; C: Cur-M; D: Cur-M +Us2; E: Cur-M+Us2+Us3). (B) Tumor weight at 21 days. **p < 0.01 versus control, &&p < 0.01 Cur-M+Us2+Us3 group versus other groups. (A: Cur alone; B: Cur+Us2+Us3; C: Cur-M; D: Cur-M+Us2; E: Cur-M+Us2+Us3). (C) Survival curves of 4T1 xenograft mice. (D) Microscopic observation of tumor sections by H&E staining (upper) and PCNA expression level (lower); scale bar = 20 μm.
lation.60 Furthermore, the efficiency of Us-mediated drug delivery could be improved with targeted factors such as folate and other targeting peptides.61 Husseini et al. reported that Usinduced cavitation promoted micelle disintegration and drug release, and the drug rapid reincorporated after Us removal.33 In this study, Us was utilized to control the “on and off” of micelles, aiming to evaluate the in vivo spatial-temporal Cur release from Cur-M. Most fluorescent molecules including DiR and Cur loaded in micelles result in local fluorescence quenching. DiR-M exhibited little fluorescent signal compared to the same dose of free DiR, however, Us treatment (1.90 MHz, LP = 3 W, 3 min) increased the signal close to that of free DiR, indicating Us-stimulated drug release from micelles in vitro (Figure S5). In order to exclude the Us-guided micelle accumulation to increase DiR fluorescent signal, intratumor injection was utilized to reflect drug release, followed by Us treatment. DiR-M were successfully injected into the dualtumor tissue in situ in the same depth with the help of a stereotaxic instrument and sonication with Us3 for 3 min. Subsequently, the signal of DiR was collected by a fluorescent imaging system with 780 nm/810 nm filter. Fluorescent signals
in bilateral tumors without Us can be seen in Figure 5Da; however, when the left tumor was treated with Us, the fluorescent signals were much stronger than without Us (p < 0.01) (Figure 5Db,c), which indirectly suggests that Us could trigger drug release in the tumor mass. Many solid tumors are characterized by a dense collagen network and tight perivascular cell coverage, thus limiting drug penetration into tumors, especially with uneven distribution in the central tumor site where tumor cells are not exposed to drugs. This is the key cause of tumor recurrence after chemotherapy.62 Therefore, the distribution of Cur in situ under Us stimulus was further analyzed. Tumor-bearing mice were also injected intratumorally with Cur-M in both flank regions. Then, tumor tissues were collected and continuously frozen sectioned and Cur fluorescence was observed by a Stereo Fluorescence Microscope. Interestingly, green fluorescence of Cur diffused throughout the tumor section after Us treatment, while fluorescence only gathered brightly in a small region in the untreated group (Figure 5E), suggesting that Us can promote intratumoral diffusion and penetration of Cur. H
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Figure 7. Evaluation of side effects. (A) Body weight changes of 4T1 xenograft mice after different treatments. (B) H&E staining showing no obvious histopathological changes of major organs post distinct treatment (scale bar = 20 μm). (C) GOT (a) and GPT (b) levels in different groups (A: Cur alone; B: Cur+Us2+Us3; C: Cur-M; D: Cur-M+Us2; E: Cur-M+Us2+Us3; F: Normal).
3.7. In Vivo Antitumor Effects. The above-mentioned Us2 (LP = 2 W) guided tumor targeting accumulation, and Us3 (LP = 3 W) further triggered efficient Cur release from Cur-M. The in vivo antitumor efficacy was subsequently investigated by making use of Us2 and Us3 at different time widows post intravenous injection of Cur-M. Us2 was performed followed after Cur-M administration, and Us3 was introduced at 12 h post-Us2 treatment. To evaluate the therapeutic efficacy of Us combined with Cur-M, 4T1 xenograft mice were divided into five groups with free Cur used as control. Both Cur+Us2+Us3 and Cur-M groups inhibited tumor growth to some extent. Importantly, the combination of Cur-M with Us irradiation greatly suppressed tumor growth during the observation period (Figure 6A,B). The Cur-M+Us2 group inhibited tumor growth to a large extent, which could be due to Us promoting drug accumulation in the tumor. After twice Us treatments, tumor suppression effect was better than single Us treatment. On day 21, the tumor volume inhibition ratios in Cur-M+Us2 and Cur +Us2+Us3 groups were 62.54% and 75.56%, respectively (Figure 6Aa,b). The tumor weight inhibition of Cur-M +Us2+Us3 was approximately 6.5-fold more than that of tumors without Us exposure (Figure 6B), which may be attributed to Us triggering Cur release from micelles and promoting intratumor diffusion and deep penetration. Mice survival indicates that Cur-M+Us2+Us3 obviously extended the survival of 4T1 xenograft mice (Figure 6C). Histological analysis indicates that the tumor tissue damage was more serious in Cur-M+Us2+Us3 group compared to CurM+Us2 and Cur+Us2+Us3 groups (Figure 6D, upper). Moreover, the expression level of PCNA declined significantly in Cur-M+Us2+Us3 groups than others (Figure 6D, lower), suggesting that tumor proliferation was obviously inhibited by the synergy of Cur-M and Us2 + Us3. 3.8. Evaluation of Side Effects. Despite the inspiring therapeutic efficacy of Cur-M combined with Us, the potential
adverse effects should be examined. In this manuscript, various treatments did not produce alterations in body weight (Figure 7A). Histological analysis shows no obvious damage of major organs (Figure 7B). In addition, the biochemical detection of GOT and GPT levels suggest no evident acute hepatic damage caused by the designed treatment procedure (Figure 7C). Together, these data gives a relatively safe evaluation of our platform. From the findings above, the work presented here supports the idea that mixed micelles have a good Us-responsive activity. The established combination system could serve as an ideal platform for cancer therapy and a hydrophobic drug delivery approach. Additionally, the Cur concentration in this study was not particularly high. While the tumor did not completely regress, the therapeutic potential of Cur-M can be further improved by functional modifications, which will be exploited in future research.
4. CONCLUSIONS Novel Cur-loaded polymeric micelles with Us-responsive properties were successfully developed to conduct chemotherapy in cancer. The present study confirmed that the mixed micelles possess superior anticancer activity through sonoporation-assisted chemotherapy. Sonoporation induced by Us could significantly increase intracellular Cur accumulation. Both in vivo and in vitro studies confirmed that Us-triggered drug release from Cur-M was intensity-dependent. Furthermore, Us could guide the targeting accumulation and deep penetration of mixed micelles in situ. Localized Us irradiation following systemic injection of Cur-M effectively suppressed tumor with no adverse effect in xenograft mice. Therefore, the potential therapeutic effects of this platform will facilitate its future clinical applications. I
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b05469. Estimated serum stability, drug release in different pH and physiological conditions with or without ultrasound trigger, in vivo circulation, and micelles distribution in major organs (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Xiaobing Wang: 0000-0002-7589-9618 Notes
The authors declare no competing financial interest. # (P.W., Y.J.) Co-first author.
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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (Grant Nos. 81472846, 81571834), the project funded by China Postdoctoral Science Foundation (Grant Nos. 2016M600684, 2017T100649) and the Fundamental Research Funds for the Central Universities (Grant Nos. GK201502009, GK201504010, 2016CSZ007).
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