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Self-Assembled Polymer/Inorganic Hybrid Nanovesicles for Multiple Drug Delivery To Overcome Drug Resistance in Cancer Chemotherapy Meng-Qing Gong, Jin-Long Wu, Bin Chen, Ren-Xi Zhuo, and Si-Xue Cheng* Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China S Supporting Information *

ABSTRACT: With the aim to develop a facile strategy to prepare functional drug carriers to overcome multidrug resistance (MDR), we prepared heparin/protamine/calcium carbonate (HP/PS/CaCO3) hybrid nanovesicles with enhanced cell internalization, good serum stability, and pH sensitivity for drug delivery. All the functional components including protamine to improve the cell uptake, heparin to enhance the stability, and CaCO3 to improve drug loading and endow the system with pH sensitivity were introduced to the nanovesicles by self-assembly in an aqueous medium. An antitumor drug (doxorubicin, DOX) and a drug resistance inhibitor (tariquidar, TQR) were coloaded in the nanovesicles during self-assembly preparation of the nanovesicles. The drug loaded nanovesicles, which had a mean size less than 200 nm, exhibited a pH-sensitive drug release behavior. In vitro study was carried out in both nonresistant cells (HeLa and MCF-7) and drug-resistant cancer cells (MCF-7/ ADR). Because of the enhanced intracellular and nuclear drug accumulation through effective inhibition of the P-gp efflux transporter, DOX/TQR coloaded nanovesicles showed significantly improved tumor cell inhibitory efficiency, especially for drugresistant cells. These results suggest the self-assembled nanovesicles have promising applications in multidrug delivery to overcome drug resistance in tumor treatments.

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INTRODUCTION Since drug delivery systems offer various advantages as compared with conventional formulations, they are of special importance in tumor treatments. Among numerous drug delivery systems, drug delivery systems with nanosizes have achieved increasing scientific interest.1,2 Because of their small sizes as well as large surface areas, nanosized drug delivery systems have favorable properties including the capabilities to increase drug availability and to achieve passive tumor targeting resulted from enhanced permeability and retention (EPR) effect and active targeting by surface functionalizations. In addition, the small size also endows the delivery systems with increased sensitivity, which can optimize the drug delivery upon particular stimuli.3−6 Over the past decade, there have been intense efforts on the design and fabrication of nanosized drug carriers based on polymers, liposomes, inorganic compounds (mesoporous silica, CaCO3, CaP, etc.), metals (gold nanoparticles, iron oxide nanoparticles, etc.), and other materials (graphene oxide, carbon nanotube, etc.).2,6−8 Although many drug carriers with © XXXX American Chemical Society

diverse functions have good performance to realize efficient drug delivery, their biocompatibility and biodegradability are still a major concern.7,8 To develop nanosized drug carriers with various functions as well as ideal biocompatibility and biodegradability is still a challenge. The purpose of the current study is to construct multifunctionalized nanosized drug delivery systems with ideal biocompatibility and biodegradability by a facile and effective strategy. We prepared heparin/protamine/calcium carbonate (HP/PS/CaCO3) hybrid nanovesicles with enhanced cell internalization, good serum stability, and pH sensitivity for efficient multiple drug delivery. All functional components in the drug carrier including protamine, heparin, and CaCO3 were introduced to the nanovesicles by self-assembly in an aqueous medium under mild conditions, and the whole preparation process did not involve any organic solvent and surfactant. Received: February 10, 2015 Revised: March 23, 2015

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DOI: 10.1021/acs.langmuir.5b00542 Langmuir XXXX, XXX, XXX−XXX

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Langmuir As we know, natural polymers have ideal biocompatibility and biodegradability. Because of electrostatic interactions, oppositely charged natural polymer chains possess strong affinity to each other and can form nanosized complexes.9−11 Most commonly, the self-assembled complexes based on two natural polyelectrolytes are used to deliver protein and peptide drugs.9,10 Because of the hydrophilicity of the natural polyelectrolytes, the highly hydrolyzed polyelectrolyte complexes have difficulty to encapsulate anticancer drugs with relatively low molecular weights since the drugs can diffuse out easily and quickly, leading to low encapsulation efficiency and a fast drug release.12,13 In our investigation, positively charged protamine (PS) was condensed with negatively charged heparin (HP) to form nanovesicles for drug delivery. Inorganic component calcium carbonate (CaCO3) was introduced to the HP/PS complexes to form hybrid complexes and to improve the drug loading and release properties of the natural polymer based drug carriers. In the nanosized drug carriers we prepared, all components have good biocompatibility and biodegradability. Both heparin and protamine are FDA-approved drugs.14 Heparin, a natural glycosaminoglycan, involves in diverse physiological processes through interacting with proteins having heparin-binding domains.15 In addition, heparin also exhibits various anticancer activities in tumor progression and metastasis.16 In the current study, the introduction of negatively charged heparin to the drug carrier could endow the nanosize drug delivery system with good stability, especially in the presence of serum. Protamine, a biocompatible naturally occurring polypeptide with an arginine-rich sequence, is known to show unique membrane translocating and nuclear-localizing activities. In pharmaceutics, protamine is used as a heparin antidote and a vaccine stabilizer.17 The presence of protamine in drug or gene delivery systems is favorable for overcoming delivery barriers.18,19 The inorganic compound in the hybrid system CaCO3 exhibits good biocompatibility and biodegradability since the degradation products of bioresorbable CaCO3 are inorganic ions naturally presented in bodies.20 In the current study, in addition to improve the drug loading and release properties,14 CaCO3 also has favorable pH-sensitive dissolution behavior since its high solubility at a low pH facilitates the intracellular drug release.20 In cancer chemotherapy, multidrug resistance (MDR), which involves particular changes in tumor cells developed drug resistance such as overexpression of drug efflux transporters and antiapoptotic factors, is a major obstacle to the success of chemotherapy.21,22 One of the major mechanisms responsible for the acquired drug resistance is the overexpression of Pglycoprotein (P-gp), a member of the ATP-binding cassette (ABC) family of proteins.21 The overexpressed P-gp results in the reduced accumulation of an anticancer drug inside cancer cells so that increased doses are required to produce an equivalent toxicity. Third-generation P-gp inhibitors, like tariquidar, have shown high efficacy in overcoming MDR. However, an important concern is that these agents may increase the side effects since P-gp plays important roles in the physiological regulation of endogenous and xenobiotic compounds in the body. Therefore, it is important to limit the exposure of normal cells and tissues to the efflux inhibitor. Codelivery of an anticancer drug and an efflux inhibitor can realize the colocalization of the drug and the inhibitor in the same tumor cell population to maximize therapeutic efficacy and minimize the systemic toxicity.23,24

In this study, with the purpose to reverse MDR with a minimized side effect, we codelivered an anticancer drug (doxorubicin) and a P-gp inhibitor (tariquidar) into drugresistant tumor cells using HP/PS/CaCO3 nanovesicles as drug carriers. The dual-drug (DOX/TQR) loaded nanovesicles showed a significantly higher tumor cell inhibitory effect as compared with monodrug (DOX) loaded nanovesicles due to the increased accumulation of doxorubicin in drug-resistant tumor cells. The multifunctional self-assembled nanovesicles have promising applications in reversal of drug resistance in tumor treatments.

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EXPERIMENTAL SECTION

Materials. Heparin (HP) (sodium salt, Mw = 6000−20 000 g/mol, 185 USP units/mg) was supplied by Aladdin Chemistry Co. Ltd. (Shanghai, China). Protamine sulfate (PS) was obtained from SigmaAldrich. Calcium chloride (CaCl2), anhydrous sodium carbonate (Na2CO3), and dimethyl sulfoxide (DMSO) supplied by Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) were of analytical grade and used as received. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Amresco. Doxorubicin hydrochloride (DOX) was from Zhejiang Hisun Pharmaceutical Co. Ltd. (China). Tariquidar (TQR) was obtained from MedChem Express. HeLa cells and MCF-7 cells were obtained from China Center for Typical Culture Collection (Wuhan, China) and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mg/mL NaHCO3, and 100 U/mL antibiotics (penicillin−streptomycin). MCF-7/ADR cells were purchased from KeyGEN Biotech Co. Ltd. (Nanjing, China) and were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS, 2 mg/mL NaHCO3, and 100 U/mL antibiotics (penicillin−streptomycin) at 37 °C in a humidified 5% CO2 atmosphere. To maintain the drug resistance property of MCF-7/ADR cells, DOX was added into the cell culture medium to get a final concentration of 1 μg/mL. Preparation of Self-Assembled Nanovesicles and Drug Loaded Nanovesicles. HP/PS nanovesicles were prepared by the following procedures. 500 μL of protamine sulfate solution (2 mg/ mL) and 500 μL of heparin (sodium salt) solution (4 mg/mL) were mixed together and stirred for 1 h at room temperature to obtain HP/ PS nanovesicles. HP/PS/CaCO3 nanovesicles were prepared by following procedures. 500 μL of protamine sulfate solution (2 mg/mL) and 58 μL of Na2CO3 solution (0.02 M) were mixed and stirred for 1 h to obtain solution A. 500 μL of heparin (sodium salt) solution (4 mg/mL) and 116 μL of CaCl2 solution (0.02 M) were mixed and stirred for 1 h to obtain solution B. Then solution A was added into the solution B dropwise, and the mixture was stirred for 1 h at room temperature to obtain HP/PS/CaCO3 nanovesicles. DOX/TQR dual-drug loaded HP/PS/CaCO3 nanovesicles were prepared by following procedures. 1 mg of TQR was dissolved in 1 mL of DMSO to obtain the TQR solution (1 mg/mL), and then 100 μL of TQR solution (1 mg/mL) was diluted with 100 μL of deionized water to obtain diluted TQR solution (0.5 mg/mL). 500 μL of protamine sulfate solution (2 mg/mL) was mixed with 58 μL of Na2CO3 solution (0.02 M) and stirred for 1 h to obtain solution C. 150 μL of DOX solution (0.5 mg/mL), 15 μL of TQR solution (0.5 mg/mL), and 116 μL of CaCl2 solution (0.02 M) were mixed together and stirred for 1 h, and then 500 μL of heparin (sodium salt) solution (4 mg/mL) was added and stirred for 1 h to obtain solution D. Then solution C was added into solution D dropwise and stirred for 1 h at room temperature to obtain HP/PS/CaCO3/DOX/TQR nanovesicles. For comparison, HP/PS/DOX nanovesicles and HP/PS/CaCO3/ DOX nanovesicles were prepared under similar conditions in the absence of CaCO3/TQR and TQR, respectively. Characterizations of Nanovesicles. The size and zeta potential of nanovesicles in deionized water as well as in DMEM cell culture medium with 10% FBS were determined by a Zetasizer (Nano ZS, B

DOI: 10.1021/acs.langmuir.5b00542 Langmuir XXXX, XXX, XXX−XXX

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Langmuir Malvern Instruments). The data are given as mean ± standard deviation (SD) based on three independent measurements. The morphologies of nanovesicles were visualized by transmission electron microscopy (TEM) (JEOL JEM-2100 HR). Before TEM observation, the nanovesicle contained solution was centrifugated. After removal of the supernatant, the nanovesicles were washed by deionized water twice and redispersed in deionized water. Then a droplet of nanovesicles contained solution was placed on a copper grid with Formvar film, naturally dried in air, and negative stained by phosphotungstic acid. Determination of Drug Loading Content and Encapsulation Efficiency. The concentrations of nonencapsulated DOX were determined by the absorbance at 485 nm, using a UV−vis spectrophotometer (PerkinElmer Lambda Bio 40). The drug loading content and encapsulation efficiency were calculated as follows:

loading content = (WT − WF)/ WNP × 100%

min at 37 °C, and then removed. The cells were washed three times with cell culture medium. The cells were observed by CLSM (Nikon Ni-E C2+) under magnification of 400.

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RESULTS AND DISCUSSION Preparation of Blank Nanovesicles and Drug Loaded Nanovesicles by Self-Assembly. In this investigation, we developed a new method to fabricate nanosized multifunctional drug carriers based on the self-assembly of natural polymers. All functional components, including protamine to enhance the cell uptake and nuclear translocation, heparin to endow the nanosize drug delivery system with good serum stability, and CaCO3 to improve the drug loading and release properties, were introduced to the nanovesicles by self-assembly in an aqueous medium. During the preparation of the nanosized drug carrier, the solution containing protamine and CO32− anions was mixed with the solution containing heparin and Ca2+ cations. In an aqueous solution with a neutral pH, heparin chains were highly negatively charged. As a result, there were electrostatic interactions between positively charged Ca2+ ions and the negatively charged groups of heparin.25 After the solution containing protamine and CO32− anions was added, the electrostatic interaction between protamine with positively charged −NH3+ groups and heparin with negatively charged −COO−, −OSO3−, and NHSO3− groups led to ionotropic gelation.9,14 Since these was a competition between heparin and CO32− anions, free CO32− anions were trendy to deprive the calcium ions interacted with the heparin chains, leading to the precipitation of CaCO3 with the polymer chains surrounded. As an overall result, HP/PS/CaCO3 hybrid nanosized selfassemblies were formed. In HP/PS/CaCO3 hybrid selfassemblies, the presence of bound Ca2+ ions reduced the electrostatic repulsion between the negative charged groups in HP chains. As a result, the HP chains in the Ca2+-rich domains became less hydrophilic, and the HP chains in the Ca2+deficient domains had a higher affinity with water molecules. It should be noted that the total amount of Ca2+ ions used for nanovesicle preparation was higher than that of CO32− ions since Ca2+ ions interacted with both heparin chains and CO32− ions. According to previous studies, the morphology of selfassemblies prepared by natural polymers is dependent on the preparation conditions which affect the hydrophilic/hydrophobic balance of self-assemblies, including the ratio of two oppositely charged polymers and the concentration of ions presented during self-assembly. Nanosized self-assemblies with diverse morphologies such as nanospheres and nanovesicles can be obtained under particular self-assembly conditions.13 In the current study, the HP/PS/CaCO3 nanosized self-assemblies we prepared exhibited vesicular morphologies. In the nanovesicles, the Ca2+-rich parts formed the central parts of the vesicular membranes, and the Ca2+-deficient parts formed the outer and inner layers of the vesicles. The negatively charged heparin chains on the particle surface ensured good dispersibility and high colloidal stability of the nanovesicles in aqueous media. Most commonly, drug delivery systems fabricated by hydrophilic natural polymers have low encapsulation efficiency and cannot effectively sustain the drug release if the molecular weight of the drug is relatively low due to the quick diffusion of the encapsulated drug. According to previous studies, introducing inorganic components like calcium phosphate and calcium carbonate to these drug carriers can enhance the

(1)

where WT is the total weight of DOX fed, WF is the weight of nonencapsulated free DOX, and WNP is the weight of nanovesicles.

encapsulation efficiency = (WT − WF)/ WT × 100%

(2)

where WT is the total weight of DOX fed and WF is the weight of nonencapsulated free DOX. In Vitro Drug Release. Drug loaded nanovesicles were sealed in a dialysis bag (MWCO 8000−10 000) and immersed in 30 mL of PBS (0.02 M, pH 7.4 and 5.3) and shaken in water bath at 37 °C. At predetermined intervals, 4 mL of solution was withdrawn from the release medium and replaced with 4 mL of fresh PBS. The concentration of DOX was determined by fluorescence spectroscopy (Shimadzu RF-5301 PC). The fluorescence spectrum of was recorded at λex = 488 nm and λem = 300−500 nm. The data were given as mean ± standard deviation (SD) based on three independent measurements. Evaluation of in Vitro Cell Inhibition. In vitro cytotoxicity of particular agents was determined by MTT assay. The cells in 100 μL of culture medium containing 10% FBS (DMEM for HeLa cells and MCF-7 cells, and RPMI-1640 for MCF-7/ADR) were seeded directly in the well of a 96-well plate at a density of 6000 cells per well and incubated at 37 °C for 24 h; then the medium was replaced by 100 μL of fresh culture medium containing a particular agent (blank nanovesicles, drug loaded nanovesicles, and free drugs). The cells were co-incubated with the particular agent at 37 °C for 48 h. After that, the culture medium was replaced with 100 μL of fresh culture medium, and 20 μL of MTT (5 mg/mL) was added in each well, followed by incubation at 37 °C for 4 h. Thereafter, the supernatant was carefully removed, and 200 μL of DMSO was added to dissolve the Formosan crystals produced by viable cells. The absorbance of the solution was measured at 570 nm using microplate reader (Bio-Rad 550) to determine the OD value. The cell viability was calculated as follows:

cell viability = ODtreated /ODcontrol × 100%

(3)

where ODtreated was obtained from the cells treated by a particular agent and ODcontrol was obtained from the cells without any treatments. The statistical significance between two sets of data was calculated using Student’s t test. A p value 100 μg/mL). An obviously enhanced cell inhibition was observed in HP/PS/CaCO3/DOX nanovesicles. As we know, the drug efflux pumps on the membrane can easily sense free drug molecules when they cross the cellular membrane, making them spatially vulnerable to P-gp-mediated efflux. Nanocarriers are internalized by nonspecific endocytosis, which is known as “stealth endocytosis”.3 The carriers can prevent the drugs from being recognized by efflux pumps when crossing the cell membrane. As a result, the nanoparticles internalized may release the drug molecules near the perinuclear region, which is away from membrane ATP-binding cassette (ABC) transporters, leading to better therapeutic efficiency. In the current study, the improved cell inhibitory effect in MCF-7/ADR cells with overexpression of P-gp was mainly due to the “stealth endocytosis” of the HP/PS/CaCO3/DOX nanovesicles. The difference between the cells treated by free DOX and the cells treated by monodrug loaded nanovesicles was statistically significant (p < 0.05). As compared with HP/PS/CaCO3/DOX nanovesicles, dual-drug loaded HP/PS/CaCO3/DOX/TQR nanovesicles demonstrated a significantly improved performance in cell inhibition, and the difference was statistically significant (p < 0.05). IC50 of HP/PS/CaCO3/DOX/TQR was decreased to 2.5 μg/mL, which was 40 times lower than that of HP/PS/CaCO3/DOX. The dramatically enhanced cell inhibition could be attributed to the tariquidar mediated inhibition of P-gp-mediated drug efflux in MCF-7/ADR cells with the high level of P-gp expression. F

DOI: 10.1021/acs.langmuir.5b00542 Langmuir XXXX, XXX, XXX−XXX

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Langmuir Observation by Confocal Laser Scanning Microscopy. To study the cellular uptake of drug loaded nanovesicles and the mechanism of reversal of drug resistance, the cells after being treated by different agents with equivalent DOX concentrations for 4 h was visualized by confocal laser scanning microscopy (CLSM). As shown in Figure 6, DOX loaded nanovesicles exhibited enhanced intracellular drug concentration as compared with

existence of negatively charged HP. As compared with free DOX, monodrug loaded HP/PS/CaCO3/DOX nanovesicles exhibited increased cell inhibition due to the “stealth endocytosis” as well as the favorable effect of PS in overcoming delivery barriers. Because of the enhanced intracellular DOX accumulation achieved by inhibition of the P-gp efflux, simultaneous delivery of DOX and TQR into drug resistance tumor cells resulted in significantly enhanced cell growth inhibition through effective reversal of drug resistance; i.e., IC50 of dual-drug loaded HP/PS/CaCO3/DOX/TQR nanovesicles was 40 times lower than that of monodrug loaded HP/PS/ CaCO3/DOX nanovesicles. This study provided a facile strategy to construct a multidrug delivery platform to reverse MDR in cancer treatment, and the HP/PS/CaCO3 hybrid nanovesicles prepared have promising applications in multidrug delivery to overcome tumor drug resistance.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details for Western blot analysis of P-gp expression; cell viability of HeLa cells after treated by free DOX, HP/PS/DOX nanovesicles and HP/PS/CaCO3/DOX nanovesicles; cell viability of MCF-7/ADR cells after treated by HP/PS/CaCO3/DOX/TQR nanovesicles and the mixture of HP/PS/CaCO3/DOX nanovesicles and free TQR; confocal images of HeLa cells after different treatments. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b00542.

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Figure 6. Confocal images of MCF-7/ADR cells after being treated by free DOX (A), mixture of free DOX and free TQR (B), HP/PS/ CaCO3/DOX nanovesicles (C), mixture of HP/PS/CaCO3/DOX nanovesicles and free TQR (D), and HP/PS/CaCO3/DOX/TQR nanovesicles (E) for 4 h. Cell nuclei (blue) were stained with Hoechst 33258. DOX concentration for all treatments was 25 μg/mL.

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected], [email protected]; Tel +86-27-68754061; Fax +86-27-68754509 (S.-X.C.). Notes

The authors declare no competing financial interest.

free DOX in drug resistance MCF-7/ADR cells, and dual-drug loaded nanovesicles exhibited highest DOX intracellular accumulation in both nuclei and perinuclear regions. TQR exhibited a strong capability in increasing DOX concentration in MCF-7/ADR cells with a high P-gp expression. In other words, with the presence of TQR, more significant enhancement in intracellular DOX concentration, especially for the DOX concentration in nuclei, could be achieved in the drugresistant cells. The CLSM observation was consistent with the cytotoxicity determined by MTT assay. These results demonstrated that TQR could effectively enhance drug accumulation in drug resistance cancer cells and facilitate nuclear drug accumulation through effective inhibition of the Pgp efflux transporter. Codelivery of the drug resistance inhibitor (TQR) and the antitumor drug (DOX) could achieve highest cell inhibition efficiency since HP/PS/CaCO3/DOX/TQR nanovesicles facilitated the accumulation of two drugs in the same cell population. Taken together, these results suggest the multifunctional self-assembled nanovesicles have great potential for multidrug delivery to overcome drug resistance in tumor treatments.

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ACKNOWLEDGMENTS Financial support from National Natural Science Foundation of China (21274113) and Ministry of Science and Technology of China (National Basic Research Program of China 2011CB606202) is gratefully acknowledged.

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REFERENCES

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CONCLUSIONS HP/PS/CaCO3/DOX/TQR nanovesicles for codelivery of a chemotherapy drug (DOX) and a P-gp inhibitor (TQR) were prepared by a facile self-assembly method. The drug loaded nanovesicles with a mean size less than 200 nm and a negative zeta potential exhibited good serum stability due to the G

DOI: 10.1021/acs.langmuir.5b00542 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.5b00542 Langmuir XXXX, XXX, XXX−XXX