Research Article www.acsami.org
Antitumor and Antimetastasis Activities of Heparin-based Micelle Served As Both Carrier and Drug Ling Mei, Yayuan Liu, HuaJin Zhang, Zhirong Zhang, Huile Gao, and Qin He* Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University. No. 17, Block 3, Southern Renmin Road, Chengdu 610041, China S Supporting Information *
ABSTRACT: Effective treatments for tumors are not easy to achieve due to the existence of metastases, which are responsible for most tumor death. Hence, a new drug delivery system is a pressing need, which should be biocompatible, stimuli-responsive, and multifunctional, including antitumor, antimetastasis, and antiangiogenesis effects. However, it is challenging to achieve all of these properties in one drug delivery system. Here, we developed a system of drug DOX and heparin into one self-assemble nanoparticle via pHsensitive hydrazone bond and hydrophobic groups, deoxycholate. In the process, heparin itself was not only as the hydrophilic segments of the carrier, but also processed multiple biological functions such as antiangiogenesis and antimetastasis effect. The micelle nanoparticle HD-DOX processed good stability and acidic pH-triggered drug release property. After systemic administration, heparin-based micelle nanoparticle showed longer half-time and enhanced accumulation of DOX in tumors through the enhanced permeability and retention effect, leading to more efficient antitumor effects. In addition, heparin could hinder platelet-induced tumor cells epithelial−mesenchymal transition (EMT) and partially affect cell actin cytoskeletal arrangement, resulting in the disorganization of the actin cytoskeleton. Therefore, HD-DOX exhibited significant inhibitory effect on the metastasis in melanoma animal model in C57BL/6 mouse. Meanwhile, benefited from the antiangiogenesis effect of heparin, tube formations in endothelial cells were effectively inhibited and tumor vascular density was decreased by HD-DOX. Taken together, our study developed a self-assembly nanoplatform that both the drug and carrier had therapeutic effects with ideal antitumor efficacy. KEYWORDS: metastasis, angiogenesis, micelle, heparin, EMT
1. INTRODUCTION A tumor is a kind of disease with high mortality. Although surgery, chemotherapy, and radiotherapy can effectively control some tumors at the primary site, the survival rate of patients has not been satisfactorily improved. It is mainly due to the metastasis of cancer cells, from primary sites of solid tumors to distant organs,1 and current therapies are focused mainly on targeting primary tumors, while little available therapy to inhibit the spread of metastatic. These findings suggested that developing a new way to kill both the tumor in situ and the metastases is extremely urgent. The treatment of tumor metastasis has been widely studied, and many kinds of drugs are quickly gaining interest such as matrix metalloproteinase (MMP) inhibitors, miR-10b antagonists, kinase inhibitors, and so on. But such use of these drugs in clinic is limited because the adverse effects observed in clinical trials involving MMP inhibitors were not encouraging.2 Thus, the truly available antimetastatic therapeutics should be both effective and safe. Besides, an increasing number of studies have proved that therapeutic effect could be improved if angiogenesis inhibitors were combined with chemotherapeutics during the treatment of solid tumor because antiangiogenesis therapy could cut off the tumor’s oxygen and nutrition supply.3 What’s more, © XXXX American Chemical Society
nanoparticles, as one class of drug delivery systems (DDSs), have become widely used in tumor treatment due to the more accumulation in tumor site.4−6 But the leakage of drugs can bring unexpected adverse effect in the process of circulating. Thus, ideal DDSs should also be capable of releasing the payload in response to stimuli, especially in targeting tissues. Nowadays, nanotoxicity research is gaining more attention. For in vivo application, toxicity is of great importance when evaluating the clinical application potentials of nanoparticles. However, many DDSs led to a certain degree of toxicity because the carriers were made of some materials which were not biodegradable or biocompatibility and only the drugs had therapeutic effect. To overcome all these challenges above, the design of a potential polymer based therapeutic was supposed to achieve following main goals together in one DDS: (1) provided with antimetastasis effect during oncotherapy, (2) specific drug release in tumor tissue, (3) enhanced antitumor efficacy through combining antiangiogenesis therapy and Received: December 18, 2015 Accepted: April 1, 2016
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DOI: 10.1021/acsami.5b12347 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 1. Schematic illustration of heparin-based self-assembled nanoparticles and the multiple functions of which.
cytotoxic agents, and (4) the nanoparticle itself should possess good biocompatibility and biodegradable properties. In addition to the anticoagulant activity of heparin, some independent research has proved that heparin and its derivatives showed many other biological effects.7 Surprisingly, numerous studies showed that heparin could inhibit tumor metastasis in different experimental models.8−10 And a few clinical trials also demonstrated the favorable effects of heparin on human tumors.11−13 Besides, heparin appears to be promising as an angiogenesis inhibitor via intrinsically interacting with various endogenous proteins, including a diverse range of angiogenic factors.14 Meanwhile, heparin belongs to a kind of biodegradable and nontoxic natural product, unlike other toxic inhibitors. These findings indicated that heparin could be used for the treatment of tumors. In this regard, an increasing number of heparin modified nanoparticles have been developed,15,16 and surface modification of heparin has shown the ability to improve the stability of nanoparticles in vivo and prolong the circulation time.17−19 Although these studies have verified a variety of functions of heparin, we were inspired to use heparin itself as a carrier for drug delivery and achieve multiple functions for tumor therapy. In this way, the heparin-based carrier was not just an excipient for drug delivery, but also showed therapeutic effects. To this end, we constructed a novel self-assembled nanoparticle by binding the antitumor drug doxorubicin (DOX) to bioactive material heparin via hydrophobic group deoxycholate and pH sensitive hydrazone bond in this study. We investigated the efficacy of heparin-deoxycholate-doxorubicin (HD-DOX) nanoparticle in malignant melanoma. As shown in Figure 1, our HD-DOX nanoparticles showed multiple functions owing to its unique composition. First, the self-assembled nanoparticle with heparin hydrophilic outer could prolong the circulation time and increase the accumulation in tumor through EPR effect, releasing DOX via the stimulation of lower pH value. Second, heparin itself also showed bioactivities, such as antimetastasis and antiangiogenesis effects, thereby affording better therapeutic effect and
prolonged survival period of tumor-bearing mice. Finally, the formed nanoparticle had good biocompatibility so that simultaneously solving above problems. In this study, we evaluated the characteristics of the nanoparticle to ensure the stability and the pH-sensitivity property. In vitro assays mainly verified the nontoxicity and antimetastasis effect of heparin. Pharmacokinetics and biodistribution of HD-DOX revealed longer circulation time and more accumulation in tumor. Subsequently, HD-DOX served as a powerful drug delivery system to effectively inhibit primary tumor and their metastases in vivo. Additionally, we tried to explore the mechanisms of the inhibition effects on tumor metastasis, and the possible reason might be that heparin could not only disorganize actin stress fibers of tumor cells but also hinder the interactions of platelets and tumor cells so as to hinder platelets induced epithelialmesenchymal transition (EMT), thus inhibiting tumor metastasis.
2. MATERIALS AND METHODS 2.1. Materials. Doxorubicin Hydrochloride was obtained from Beijing Huafeng Technology Co., Ltd. (Beijing, China). Unfractionated heparin and Deoxycholic acid were purchased from Sigma (St. Louis, MO). N-Hydroxy-succinimide (NHS) and 6-aminocaproic acid, 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC) were obtained from Keddia Reagent (Chengdu, China). Matrigel was bought from BD Biosciences (San Jose, CA). ActinRed was obtained from KeyGen Biotech. Co., Ltd. (Nanjing, China). Antibody of anti-CD34 was purchased from Abcam (Cambridge, MA). Both anti-E-cadherin and anti-N-cadherin were purchased from Zen Bioscience (Chengdu, China). Other reagents and chemicals were analytical level. Mouse melanoma cell line (B16F10) and human breast cancer cell line (MDA-MB-231) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10% FBS, 100 μg/mL streptomycin and 100 U/mL penicillin at 37 °C. C57/BL6 mice (5−7 weeks old, 17−21 g) were purchased from the Experimental Animal Center of Sichuan University. All animal experiments were performed according to the rules of Experimental Animals Administrative Committee of Sichuan University. 2.2. Synthesis of HD and HD-DOX. 2.2.1. Compound 1 (Ethyl 6-aminohexanoate Hydrochloride). Thionyl chloride (4.4 mL, 60 B
DOI: 10.1021/acsami.5b12347 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces mmol) was added dropwise to anhydrous ethanol (50 mL) while cooling. Subsequently, 6-aminocaproic acid (6.56 g, 50 mmol) was added and the mixture was stirred for 7 h at 60 °C. After complete reaction (monitored by TLC), excess solvent was evaporated. And the crude product was recrystallized from ethyl acetate to give the hydrochloride that was directly used in the next step. 2.2.2. Compound 2. Deoxycholic acid (414.5 mg, 1 mmol) was mixed with EDC (287.6 mg, 1.5 mmol) and NHS (172.7 mg, 1.5 mmol) in DMSO (15 mL). The mixture was reacted for 3 h at 37 °C under nitrogen atmosphere, and followed by the addition of ethyl 6aminohexanoate hydrochloride (195.7 mg, 1 mmol). The reaction solution was stirred for 12 h and monitored by TLC. Finally, the mixture was extracted with ethyl acetate and water three times. Ethyl acetate extracts were purified via column chromatography on silica gel (dichloromethane: methyl alcohol). 2.2.3. Compound 3 (HD). Heparin (182.4 mg, 0.3 mmol COO−) was dissolved in anhydrous formamide (8 mL) with gentle heating. Then EDC (115.0 mg, 0.6 mmol), NHS (69.1 mg, 1.5 mmol), DMAP (7.33 mg, 0.06 mmol) were sequentially added to the solution and stirred for 3 h, followed by compound 2 (160.0 mg, 0.3 mmol) dissolved in DMF (4 mL). After 24 h, the mixture was precipitated in cold acetone, and the precipitate was washed three times with cold acetone. Then, the dried compound 3 was dissolved in water and freeze-dried. 2.2.4. Compound 4 (HD-DOX). Compound 3 (100 mg) was dissolved in DMF (5 mL) with gentle heating, followed by adding hydrazine hydrate (1 mL). The reaction mixture was stirred for 6 h at room temperature. Then, the mixture was precipitated in cold acetone−diethyl too, and the precipitate was washed three times with diethyl ether. The residue was added to DMF directly (4 mL) and dissolved fully. Doxorubicin hydrochloride (15 mg) and trifluoroacetic acid (10 μL) were added to the reaction mixture with constant stirring in the dark at room temperature for 24 h. The solution was dialyzed exhaustively with ultrapure water using dialysis membrane (molecular weight cutoff = 1000) in the dark. The retentate was a red solid. 2.3. Preparation and Characterization of HD and HD-DOX Nanoparticles. HD or HD-DOX was suspended in PBS buffer (pH = 7.4) with gentle shaking. Then, the solution was sonicated 15 times at 200 W for 15s each. The zeta potential and mean size of conjugates were measured by Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., U.K.). And the morphology of nanoparticles was observed under a transmission electron microscope (TEM; JEM-100CX, JEOL, Japan). It revealed that both HD and HD-DOX were spherical and HD conjugate was taken for analysis to determine the amount of heparin. HD was allowed to react with HCl (0.1 mol/L) for 0.5h and the pH of the solution was adjusted to 6.8. The percent of heparin was determined by toluidine blue spectrophotometry. The content of DOX conjugated to HD was measured by Varioskan Flash Multimode Reader (Thermo, USA) at Ex = 470 nm, Em = 590 nm. 2.4. Cytotoxicity Assays. The cytotoxicity of different formulations was determined by MTT assay. First, 3 × 103 B16F10 cells were seeded into 96-well plates and cultured for 24 h. The culture media was removed and then replaced by 200 μL of DMEM containing heparin, HD, DOX, and HD-DOX for 24h. Then, 20 μL MTT solution (5 mg/mL in PBS) was added into each well and incubated for another 4 h. Finally, the culture media was removed and the residue was dissolved with 150 μL dimethyl sulfoxide (DMSO). The absorbance of each group was measured by Varioskan Flash Multimode Reader (Thermo, USA) at 490 nm. Cell viability (%) was calculated according to Atest/Acontrol × 100. 2.5. Hemolysis Assay. In order to investigate the interactions of nanoparticles (HD and HD-DOX) with blood erythrocytes, we performed the hemolysis assays as shown previously with improvement.20 HD or HD-DOX with different concentrations was incubated with 2% red blood cell suspension from mouse for 1 h at 37 °C. Then, each sample was centrifuged at 3000 rpm for 15 min. The absorbance of supernatant was analyzed at 540 nm by a microplate reader and the precipitated erythrocytes were observed under microscope. The hemolysis (%) was calculated according to the following formula.
hemolysis (%) =
A sample (A ultrapure water − APBS)
× 100
Less than 5% hemolysis was deemed as nontoxic.21 PBS and ultrapure water-treated red blood cell suspension were used as negative and positive controls, respectively. 2.6. Scratch/Wound Healing Assay. To investigate the migration ability of tumor cells, we performed wound healing assay, as previously studied. Briefly, B16F10 cells or MDA-MB-231 cells were seeded in 12-well plates and incubated to near confluence (90%). Then, the cell layer was scratched a straight line using a sterile pipet tip. After incubating with different formulations, images were obtained at 24 and 0 h. 2.7. Confocal Microscopy of Actin Cytoskeleton. Around 1 × 105 B16F10 cells were seeded in 6-well plates and cultured for 24 h. After incubating with heparin, HD, DOX and HD-DOX for 12 h, cells were fixed for with 4% paraformaldehyde 15 min, followed by permeabilized for 5 min using 0.5% Triton X-100. Then, the cells were incubated with 1% FBS in PBS to block nonspecific labeling. Blocked cells were cultured with ActinRed for 20 min, followed by nuclei staining with DAPI for 5 min. After being washed with cold PBS three times, the coverslips were imaged by confocal microscopy. 2.8. Interactions of Tumor Cells with Platelets. Fresh blood was obtained from the orbital and platelets were collected. B16F10 cells were seeded in 6-well plates and cultured for 24 h. After the medium was changed with fresh DMEM, a total of 1 × 105 platelets were added to each well, cells were treated with heparin or HD. The images of cellular morphology were captured at 0 and 12 h. 2.9. Western Blot. Detection of E-cadherin protein and Ncadherin protein levels was performed by Western blot assays. After being cultured for 24 h, B16F10 cells were lysed and centrifuged (14 000 rpm at 4 °C for 15 min). The supernatant proteins were collected. Each protein sample was then separated by 10% SDS-PAGE, and followed by transferring to polyvinylidene difluoride membranes. Finally, the samples were incubated with antibody against E-cadherin or N-cadherin. HRP-labeled goat antirabbit secondary antibody was added and measured using a Bio-Rad ChemiDoc MP System (Bio-Rad Laboratories, Hercules, CA). 2.10. In Vitro Endothelial Tube Formation Assay. Human umbilical vein endothelial cells (HUVECs) were plated onto 24-well plates and coated with matrigel, together with Heparin or HD, and incubated for 6h at 37 °C. The formation of capillary tube was photographed by a microscope. 2.11. In Vivo Biodistribution Study. When the volumes of tumors reached around 100 mm3, biodistribution study was carried out. Mice randomly were divided into two groups. DOX and HDDOX were injected via the tail vein, at the DOX dose of 5 mg/kg. In each treatment group, mice were then anesthetized and the fluorescence imaging was monitored by IVI Spectrum system (Caliper, Hopkington, MA) at 1, 8, and 24 h after injection. After that, mice were sacrificed after heart perfusion with saline and paraformaldehyde. Hearts, livers, spleens, lungs, kidneys, and tumors were collected. First, all the organs were imaged and the other were obtained for qualitative analysis. For analysis of the tissues samples, each tissue was washed and weighted. Then, 200 μL of tissue homogenates were absorbed into tubes. Next, 20 μL of daunorubicin (200 μg/mL) as internal standard solution was added for vortex mixing. Then, 1 mL of extracted solution (chloroform/methanol = 4:1) were added for vortex-ultrasonic extraction many times. After centrifugation for 5 min at 12 000 rpm, organic phase was moved to another centrifuge tube and dried at 37 °C under a stream of nitrogen. The residue was redissolved in 100 μL of mobile phase. After centrifugation for 10 min at 12 000 rpm), the supernatant was obtained for HPLC analysis with a UV detector with 254 nm. For confocal microscopy, tumors were fixed with paraformaldehyde for 24 h and dehydration with 15% sucrose solution, 30% sucrose solution overnight sequentially. Then, the tumors were sectioned at 10 μm via freezing microtome section, followed by staining with Rabbit mAb to CD34 antibody and DAPI. 2.12. In Vivo Antitumor Assay. In vivo study was evaluated in B16F10 cancer model using C57/BL6 mice (5−7 weeks old, 17−21 g) C
DOI: 10.1021/acsami.5b12347 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 2. Schematic of the synthesis route of HD and HD-DOX. which were inoculated subcutaneously and intravenously of 1 × 106 B16F10 cells simultaneously. Treatment groups were divided into 5 groups: saline, heparin, HD, DOX and HD-DOX. When tumor reached approximately 100 mm3, intravenous therapy started with DOX or HD-DOX (DOX-equivalent, 2.5 mg/kg DOX) every 3 days for 5 times. Meanwhile, heparin or HD was intravenously administered with equal amounts of heparin in HD-DOX every 2 days for 8 times. Animal body weights and tumor volumes were monitored. Tumor volumes and animal body weights were monitored. On day 21, mice were sacrificed. Tumor tissues were obtained and immunostained using anti-CD34 antibody to determine blood vessel. For the survival study, each group included 11 mice. After the treatment, one mouse of each group was sacrificed for hematoxylin and eosin (HE) staining and the rest 10 mice were used for the survival study. As for antimetastasis study, 2 × 105 B16F10 cells in 100 μL of PBS were injected into the tail vein of C57BL/6 mice. Groups, doses and times were the same as above. On day 18, the mice were sacrificed, and the lungs were obtained and imaged. Samples were applied for hematoxylin and eosin (HE) staining. 2.13. Statistical Analysis. Statistical comparisons were evaluated with Student’s t test. P value
