Biomimetic Human Serum Albumin Nanoparticle for Efficiently

Feb 2, 2017 - Triple-negative breast cancers (TNBCs), devoid of hormone receptors and ... human serum albumin (HSA), via an innovative disulfide reduc...
2 downloads 0 Views 6MB Size
Download PDF
Research Article www.acsami.org

Biomimetic Human Serum Albumin Nanoparticle for Efficiently Targeting Therapy to Metastatic Breast Cancers Lisha Liu,†,‡,§ Yunke Bi,†,‡,∥ Muru Zhou,§ Xinli Chen,§ Xi He,†,‡ Yujie Zhang,†,‡ Tao Sun,†,‡ Chunhui Ruan,†,‡ Qingjun Chen,†,‡ Hao Wang,*,§ and Chen Jiang*,†,‡ †

Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China ‡ State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China § National Pharmaceutical Engineering and Research Center, China State Institute of Pharmaceutical Industry, Shanghai 201203, China ∥ Department of Neurosurgery, Shanghai First People’s Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 201620, China S Supporting Information *

ABSTRACT: Triple-negative breast cancers (TNBCs), devoid of hormone receptors and human epidermal growth-factor receptor-2/Neu expression, bring about poor prognosis and induce a high rate of systematic metastases. The ineffectiveness of current therapies on TNBCs could be attributed to the lack of efficient targeted therapy. Paclitaxel (PTX) is considered one of first-line chemotherapeutics for TNBC treatment but, due to its low aqueous solubility and nonspecific accumulation, results in poor antitumor efficacy. The present study is aimed at enhancing the chemotherapeutic potency of PTX by improving the stability and targeting efficiency of PTX-loaded nanoparticulate drug carriers. Here, PTX was incorporated in nontoxic and endogenous material, human serum albumin (HSA), via an innovative disulfide reduction method to construct HSA-based PTX nanoparticle (HSA-PTX NP) to not only realize redox-responsive drug release but also improve in vivo stability. Besides, W peptide was selected as a target ligand to be conjugated with HSA-PTX NP for endowing active targeting ability. The resulting Wpep-HSA-PTX NP possessed a spherical structure (118 nm), 9.87% drug-loading content, and 86.3% entrapment efficiency. An in vitro drug release test showed that PTX release from Wpep-HSA-PTX NP was of a redox-responsive manner. Furthermore, cellular uptake of Wpep-HSA-PTX NP was significantly enhanced, exhibiting the improved antiproliferation and antitube formation effects of PTX in vitro. In comparison with those commercial formulations and conventional HSA NP, WpepHSA-PTX NP exhibited better pharmacokinetic behaviors and tumor homing characteristics. The antitumor efficacy of WpepHSA-PTX NP was further confirmed by the strong pro-apoptotic effect and reduced tumor burden. In a word, this evidence highlighted the proof of concept for Wpep-HSA NP as a promising conqueror to the ineffectiveness of TNBC therapy. KEYWORDS: triple-negative breast cancer, human serum albumin, disulfide reduction, W peptide, drug delivery

1. INTRODUCTION Triple-negative breast cancers (TNBCs) is well-considered as short of expression of human epidermal growth factor 2, estrogen, and progesterone receptors and thus impedes development of target therapies at the moment. Current treatment for TNBC mainly focuses on surgery, traditional chemotherapies, and radiotherapy. Among them, chemotherapy is still regarded as the most frequently used regimen for TNBCs treatment because it could obviously induce apoptosis of cancer cells and inhibits the metastasis by cytotoxic agents.1 Paclitaxel (PTX), one of the most-effective chemotherapeutic agents, has always been considered as the only recommended guideline of clinical therapy for TNBCs, while the efficacy of paclitaxel is © 2017 American Chemical Society

significantly restricted with poor pharmacokinetics from poor water solubility and nonselective distribution from rapid systemic clearance.2 To resolve the dilemma, Taxol, a classical market formulation of PTX containing ethanol and Cremphor EL, was approved by FDA for cancer therapy. Unfortunately, regardless of the prominent clinical potency, Taxol has widely demonstrated that the demand for solvents or surfactants (i.e., ethanol and Cremphor EL) contributed to hypersensitivity, neurotoxicity, and nephrotoxicity. Meanwhile, the rapid blood Received: November 14, 2016 Accepted: February 2, 2017 Published: February 2, 2017 7424

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic illustration presenting the preparation of Wpep-HSA-PTX NP and the in vivo performance through FPR receptors.

leaking out due to the complex in vivo environment abundant in proteins or enzymes, which could dissemble those exotic nanocarriers.10,11 Considering that drugs were basically adsorbed or encapsulated in those drug carriers, the poor stability of biomimetic carriers resulted in the early drug release and further reduced the drug efficacy. Given those drawbacks, appropriate modification on these carriers was utilized to improve the bioinspired design of new drug-delivery systems. A hyaluronic acid modified high-density lipoproteins have been developed for longer half-life and fewer plasma clearance for efficient delivery to atherosclerosis.12 Poly(lactide-co-glycolide) (PLGA) was utilized as the core of protein particles to allow the controlled release of drug.13 Because albumin-based nanoparticle systems represent an attractive strategy among the available potential colloidal drug carrier systems, attractive macromolecule human serum albumin (HSA) was employed to incorporate PTX in the present study.14−16 However, similar to other natural materials, such as high-density lipoprotein17 and apo-ferritin,18 Abraxane, the commercially available PTXloaded albumin nanoparticle, was also found unstable in circulation because of their native characteristics, leading to instable drug loading, poor pharmacokinetic behaviors, and offtarget effect post-administration.19,20 Thus, to improve physical adsorption between drug and protein and increase the in vivo stability of protein-based carrier, cross-linkers frequently applied on polymeric materials would be utilized in this study.21,22 Due to the ample amino acid residues in albumin molecules, a nontoxic disulfide bond reduction method could

clearance and nonspecific accumulation in normal tissues directly post-administration still exist and bring about the low therapeutic index.3,4 Consequently, the development of alternative strategy for delivering PTX into tumors is pivotal for improving therapeutic outcomes of TNBCs while reducing systemic toxicity. Over the past few decades, nanoparticulate-based formulations have been extensively studied for offering an intriguing solution to overcome the severe adverse effects of chemotherapy. Nanocarriers have been generally represented as one of the most-promising drug-delivery systems especially for targeting delivery into the tumor site, improved pharmacokinetics, and reduced systemic toxicities of anticancer drugs. Many efforts have been attempted for developing formulations such as liposomes, nanostructured lipid nanoparticles, polymeric micelles, and drug conjugates.5 However, most of the developed materials fabricating drug carriers synthesized through tedious procedures might not be fully biocompatible or nontoxic and were still in the phase of laboratory research.6,7 As an increasingly emerging trend of manufacturing naoncarriers, biomimetic materials not only have demonstrated that they could replicate features from the biological system but also could incorporate drugs for efficient drug delivery.8,9 Thus, constructing therapeutics loaded nanocarriers with much-more biocompatible materials via simple approaches are of great significance to both research and clinical settings. Despite biomimetic materials possessing good biocompatibility and degradability, encapsulated drugs were still prone to 7425

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435

Research Article

ACS Applied Materials & Interfaces

ultracentrifugal filter device (MWCO = 10 kDa). After that, PEGylated HSA was reacted with WKYMVm peptide via copper (I)-catalyzed azide−alkyne click chemistry. PEGylated HSA (1 equiv) and W peptide (2.5 equiv) were mixed in PBS (pH 7.4) under nitrogen. A freshly prepared catalytic solution of CuSO4 (0.5 equiv), sodium ascorbate (2.5 equiv), and DIPEA (2.5 equiv) was added to the mixture, and the reaction was performed overnight at 35 °C. The final product, denoted as Wpep-HSA, was purified by dialysis against 10 mM EDTA (pH 7.4) for 24 h and then deionized water for 24 h and was lyophilized for storage. 2.2.2. Preparation of Wpep-HSA-PTX NP. Wpep-HSA-PTX NP was prepared through the disulfide reduction method reported from other groups with minor modification.32,33 At first, 20 mg Wpep-HSA was first dissolved in 1 mL of deionized water with 25 mM GSH at 37 °C for 1 h to reduce the intramolecular disulfide bonds to free sulfhydryl groups. Next, the solution was dialyzed (MWCO: 12−14 kDa) against PBS (pH 8.0) at 4 °C so as to remove excess GSH. Then, 2 mg of PTX dissolved in 2 mL of ethanol was put drop-wise into Wpep-HSA solution, exploiting ethanol as an antisolvent. The suspension was kept under stirring at room temperature for 30 min to form intermolecular disulfide bonds. After that, the suspension was sonicated for 5 min and dialyzed (MWCO: 12−14 kDa) to remove residual free PTX and ethanol. 2.3. Physicochemical Characterization of HSA NPs. Synthesized Wpep-HSA was characterized by 1H NMR spectra measured by NMR spectrometer (Bruker, Billerica, MA) using deuterium oxide (D2O) as solvent and tetramethylsilane (TMS) as internal reference. Mean size and ζ potential of HSA NPs were measured by dynamic light scattering (DLS) (Zetasizer Nano-ZS, Malvern, U.K.). Measurements after appropriate dilution were carried out in triplicate at 25 °C. Atom force microscopy (AFM) (Bruker, Billerica, MA) was used to visualize the morphology of Wpep-HSA-PTX NP. To determine drug loading (DL) and entrapment efficiency (EE), HSA-PTX NP and Wpep-HSA-PTX NP were diluted in 2 mL of methanol and sonicated for 30 min to completely extract PTX. The above extraction method was based on organic solvent reported by Wang’s group with minor modification.33 All of the measurements were performed in triplicate. PTX amount were determined by a highperformance liquid chromatography (HPLC) method using an Agilent 1260 series device (Palo Alto, CA) coupled with an UV detector set at 227 nm and a C18 column (250 × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile and water (60:40, v/v) and the flow rate was maintained at 1.0 mL/min. In addition, to investigate the stability of HSA-PTX NP and WpepHSA-PTX NP, we employed pH 7.4 PBS buffer to simulate the in vivo environment. Briefly, nanoparticles were reconstituted in pH 7.4 PBS buffer, respectively. Within a week, mean particle diameters were investigated by DLS. 2.4. In Vitro Redox-Responsive Behaviors of HSA NPs. First, the transition from intermolecular to intramolecular disulfide bonds was determined by Ellman’s test (see the Supporting Information). To track the size changes under the treatment of GSH, different HSA NPs were incubated in pH 7.4 PBS buffer with or without the addition of 10 mM GSH, respectively. At predetermined time points, mean particle diameters and polydispersity index were recorded by DLS. To determine the PTX release behavior, different HSA NP suspensions were performed using dialysis method (n = 3). In brief, 0.15 mL of solution of different preparations were sealed into a dialysis bag (MWCO: 2000 Da) under the same drug amount of NPs. Next, the bag was immersed into 15 mL of PBS buffer (pH 7.4) with or without 10 mM GSH and shaken at 37 °C for 48 h. At each predetermined time point, an aliquot of release medium (150 μL each) was withdrawn for HPLC assay, and the same volume of fresh medium was added. 2.5. Cell Culture. MDA-MB-231/luci cells were grown in DMEM (Gibco, Tulsa, OK) supplemented with 10% fetal bovine serum (FBS) and 1% v/v penicillin−streptomycin solution and cultured at 37 °C 2.6. Cytotoxicity of HSA-NP in MDA-MB-231 Cells. The cytotoxicity of each HSA NP preparation to MDA-MB-231 human metastatic breast tumor cells (PerkinElmer) was evaluated using MTT

assemble albumin particle via intermolecular disulfide as well as encapsulate drug within hydrophobic core of albumin to realize controlled release with a redox-responsive property.23 A promising drug-delivery system should not only possess excellent in vivo stability but also realize effective drug delivery into the target. The enhanced uptake of albumin-based nanoparticle in solid tumors is primarily mediated by gp60 and SPARC receptor, which are expressed on endothelial cells.24 Functionalizing serum albumin nanoparticles with different targeting moieties, such as Arg-Gly-Asp (RGD) peptide25 or Cetuximab26 modified HSA nanoparticles, could significantly improve target transport via receptor-mediated endocytosis into tumor cells and further reinforce the tumor cell-specific target efficiency without affecting albumin’s bioactivity. To attain target therapy for TNBCs, formyl peptide receptors (FPR), a novel family of G-protein coupled receptors, are found to be selectively overexpressed in several malignant tumors such as gliomas and TNBCs.27,28 Trp-Lys-Tyr-Met-ValD-Met (W peptide, Wpep), one of the FPR-binding ligands, could be probably utilized as a targeting moiety to actively deliver therapeutics to FPR-overexpressing cancerous cells.29,30 In this study, our main purpose was to improve the in vivo serum stability of HSA-based nanoparticle as well as actively increase the drug accumulation in tumor site. To be specific, we first constructed a stable HSA nanoparticle-loading PTX (HSAPTX NP) via a sensitive disulfide bond reducing method based on glutathione and then selected the Wpep as the target moiety to achieve the active targeted tumor accumulation of WpepHSA-PTX NPs. Moreover, the physicochemical properties of Wpep-HSA-PTX NPs, including physicochemical characterizations, morphology, and redox-responsive behavior were investigated. Furthermore, the pharmacokinetics behaviors of Wpep-HSA-PTX NP were determined. The in vitro and in vivo antitumor efficacies of those NP formulations were evaluated in MDA-MB-231 human triple-negative breast cancer cell based systems at length. The main scheme of our present study was presented in Figure 1.

2. MATERIALS AND METHODS 2.1. Materials. Human serum albumin was purchased from Jiangxi Boya Biological Technology Co.Ltd., (China). WKYMVm peptide was synthesized by China Petides Co., Ltd. (Suzhou, China). Azidepolyethylene glycol-ω-succinimidyl carbonate (Azide-PEG2000-NHS) was purchased from JenKem technology Co., Ltd. (Beijing, China). Paclitaxel (PTX) was purchased from Meilune Biological Technology Co., Ltd. (Dalian, China). Sodium ascorbate and N,N-diisopropylethylamine (DIPEA) were purchased from J & K Chemical Co., Ltd. (Shanghai, China). [4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI), coumarin-6 (Cou-6), and Hoechst 33342 were all purchased from Sigma-Aldrich (St. Louis, MO). The fluorescent probe BODIPY was synthesized as described previously. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Carlsbad, CA). A DNA fragmentation detection kit and a cell cycle detection kit were purchased from KeyGEN BioTECH (Nanjing, China). D-Luciferin was purchased from Pierce (Rockford, IL). All other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) unless mentioned otherwise. 2.2. Synthesis and Preparation of HSA NPs. 2.2.1. Synthesis of Wpep-HSA. W peptide modified HSA (Wpep-HSA) was synthesized in two steps. First, PEGylated human serum albumin was prepared by using a previously described procedure with slight modification.31 HSA (20 mg, 3.01 × 10−8 mol) was reacted with azide-PEG2000-NHS (60.2 mg, 3.0 × 10−6 mol) in phosphate-buffered saline (PBS) (pH 8.0) for 4 h. The residual PEG was then removed using the 7426

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435

Research Article

ACS Applied Materials & Interfaces

2.11. Pharmacokinetics in SD Rats. Healthy female Sprague− Dawley (SD) rats (200−250 g) were purchased from Department of Experimental Animal center (Fudan University) and maintained under standard laboratory conditions. To quantitatively evaluate the in vivo biodistribution of different formulations, SD rats were randomly divided into the next three groups (n = 5): (1) Taxol; (2) HSA-PTX NP; (3) Wpep-HSA-PTX NP. Next, these three formulations were intravenously administered via the tail vein at a PTX dose of 10 mg/ kg. An aliquot of 500 μL of blood was collected from the tail vein into heparinized Eppendorf tubes at predeterimined time points. After 3000 rpm centrifugation, 200 μL of plasma containing diazepam as the internal standard was extracted by 2 mL of anhydrous ether, followed by vortexing for 2 min. After centrifugation (1200 rpm, 5 min), the organic layer was transferred to another tube and was dried under vacuum. Finally, samples were redissolved with acetonitrile and centrifuged at 12 000 rpm for 5 min. Each sample was injected into HPLC system for PTX quantification, which was mentioned above in the section concerning drug-loading content assay.36 In addition, pharmacokinetic parameters were further analyzed using the DAS 2.0 software. 2.12. Triple-Negative Breast-Cancer Models Establishment. Female BALB/C nude mice of ∼20 g body weight were purchased from Department of Experimental Animal (Fudan University) and maintained under standard laboratory conditions. The xenograft tumor model were established by subcutaneous injection of 1 × 106 MDAMB-231/luci cells, which were suspended in 100 μL of 5 mg/mL Matrigel in PBS into the second right mammary fat pad of nude mice. 2.13. In Vivo Biodistribution of Wpep-HSA-BODIPY NPs. TNBC-bearing nude mice were subjected to in vivo imaging. According to the previously published report,37,38 a class of BF2chelated tetraarylazadipyrromethene fluorophore exhibited excellent absorption and fluorescence properties in the 650−750 nm spectral region. The tetraaryl analogue 1 (BODIPY) has an excitation λmax at 700 nm and emission at 729 nm in water. To trace the biodistribution of HSA NPs, BODIPY was encapsulated in the HSA NPs, referring to the same preparation method described above. The concentration of BODIPY was determined by UV absorption at 705 nm. HSA-BODIPY and Wpep-HSA-BODYPY were injected intravenously via the tail vein into the TNBC bearing mice at a BODIPY dose of 0.5 mg/kg. After 24 h, all mice were placed onto the chamber maintaining anesthesia by 2% isoflurane/oxygen mixture and imaged by Xenogen IVIS Spectrum with Living Image Software 4.2 (Caliper Life Science). After in vivo imaging, mice were anesthetized with diethyl ether and killed by decapitation. Tumors and organs were dissected and photographed. 2.14. Antitumor Efficacy. Antitumor efficacy was evaluated in MDA-MB-231/luci-bearing BALB/C nude mice. Mice were randomly divided into four groups (n = 6), treated with HSA-PTX, Wpep-HSAPTX, Taxol (at an equal dose of 10 mg/kg PTX), and saline (negative control), respectively. All the formulations were intravenously injected to mice via tail vein every 4 days for four times. The body weight of each animal was measured every 2 days. In addition, on days 5, 10, and 15, D-luciferin (3 mg per mouse) was intraperitoneally preinjected, respectively. After luciferin injection, bioluminescence signal was scanned and imaged by the in vivo real-time bioluminescence system. At last, all of the mice were sacrificed on day 16. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed on 5 μm frozen tumor slices using a DNA fragmentation detection kit according to the manufacturer’s instructions and observed by fluorescent microscope. DNA fragment in apoptotic cells was stained with fluorescein-conjugated deoxynucleotides (green), and the nucleus was stained with DAPI. 2.15. Data Analysis. Comparisons of parameters between two groups were performed with an unpaired Student t test, and comparisons of parameter among multiple groups were made with a one-way analysis of variance. All of the data were presented as mean ± standard derivation (SD) unless otherwise indicated. A value of p < 0.05 was considered statistically significant.

assay. MDA-MB-231 cells were seeded on 96 well plates at a density of 5 × 103 cells per well and incubated for 24 h at 37 °C. Afterward, spent media were discarded and replaced with fresh media containing free PTX and different HSA-PTX formulations ranging from various concentrations of PTX (0.01 to 200 μg/mL), and cells were cultured for 48 h. A control group was set up to represent cells without any treatment. Next, 100 μL of MTT solution was added to each well and incubated for an additional 4 h. Subsequently, the medium was replaced with 150 μL of DMSO per well and shaken for 10 min. Absorbance was read at 570 nm and corrected at 630 nm by dualwavelength detection using microplate reader (Thermo Scientific.). 2.7. Cellular Uptake. Qualitative analysis of cellular uptake of HSA NPs was visualized under a fluorescent microscope (Leica, Wetzlar, Germany), using Cou-6 as the fluorescent probe. MDA-MB231/luci cells were seeded in 24 well plates at a density of 5 × 104 cells per well and incubated at 37 °C for 24 h. When 80−90% confluence was achieved, the medium was treated with a series of HSA NPs containing 200 μg/mL HSA/Cou-6 NP and Wpep-HSA/Cou6 NP at different concentrations (50, 100, and 200 μg/mL) for 0.5 h. As for the fabrication of HSA/Cou-6 NP and Wpep-HSA/Cou6 NP, similar to the PTX incorporation, a solution of 0.5 mg Cou-6 dissolved in 2 mL of ethanol was put drop-wise into Wpep-HSA or HSA solution to form different Cou-6 formulations. After the incubation, cells were washed three times with D-Hank’s solution and photographed by fluorescence microscopy (Leica Wetzlar). Quantitative study of cellular uptake of HSA NPs was assessed by fluorescence-activated cell sorting (FACS, BD Biosciences, Bedford, MA). MDA-MB-231/luci cells were seeded in 6 well plates at a density of 5 × 105 cells per well and incubated at 37 °C for 24 h. After 80− 90% confluence was reached, the medium was replaced with different HSA NPs depicted above and incubated for 0.5 h. After the incubation, cells were washed three times with D-Hank’s solution and collected after digestion and centrifugation. The cellular uptake was processed with Flowjo 7.2. The data were collected from a minimum of 10 000 events per sample. 2.8. Mechanism of Cellular Uptake of Wpep-HSA NPs. To investigate the mechanism of internalization, MDA-MB-231/luci cells were first preincubated with different inhibitors at 37 °C for 15 min, including 10 μM free W peptide as FPR receptor competitor and 0.5 μg/mL filipin, 1 μg/mL colchicine, and 0.5 μg/mL phenylarine oxide (PhAsO) as different kinds of endocytic inhibitors.21,34 Then, 200 μg/ mL Wpep-HSA/Cou6 NP was introduced to each well. After 30 min of incubation, the supernatants were discarded and washed three times with D-Hank’s solution. Both qualitative and quantitative analysis were performed as described above. 2.9. Intracellular Tracking of Wpep-HSA/cou6 NPs. MDAMB-231 cells were seeded in confocal cell culture dish (GBS-35-20, NEST Biotech) at a density of 2 × 104 cells per dish and incubated at 37 °C for 24 h in a humidified 5% CO2 incubator. Next, media were replaced with Wpep-HSA/Cou-6 NP for 15, 30, and 60 min, respectively. At 10 min before the predetermined time point, each dish was added with 10 μL Hoechst 33342 (1 mM) into the media. After incubation, the cells were washed twice with D-Hank’s solution (pH 7.4) and then observed by confocal laser scanning microscopy (Leica TCS SP2, Germany). Fluorescence of both Cou-6 and Hoechst 33342 were excited with the 466 or 350 nm wavelength of an argon laser, and the emission was detected around 504 nm (Cou-6) or 461 nm (Hoechst 33342). 2.10. Cell-Cycle Analysis. To investigate cell cycle distribution, MDA-MB-231/luci cells were grown in 6 well plates at 37° C for 24 h. After 80−90% confluence was reached, cells in each well were replaced with fresh media containing free PTX, HSA-PTX NP, and Wpep-HSAPTX NP (equivalent to 5 μM of PTX) at 37 °C for 12 h. Next, the cell pellets were washed with PBS (pH 7.4) and fixed with 500 μL of cold ethanol (70%) for 2 h. Cells were further washed with PBS and treated with RNase A for 30 min and then with 200 uL of propidium iodide (PI) for 30 min. Fluorescent intensity was determined by FACS, and histograms of cell events versus PI intensity were used as a nonviable cell indicator to analyze the distribution in the cell cycle.35 7427

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435

Research Article

ACS Applied Materials & Interfaces

Figure 2. Synthesis route and 1H NMR characterization of Wpep-PEG-HSA.

3. RESULTS 3.1. Preparation and Characterization of HSA NP. PEG-HSA was first synthesized successfully via the reaction between the NHS group on PEG and the amino group on HSA. W peptide was then conjugated on another functionalend azide group on PEG through the alkyne group of the peptide to construct the targeting material. As shown in Figure 2, the triazole proton appeared in Wpep-PEG-HSA, indicating that the success of click chemistry. HSA-NPs encapsulating PTX were prepared by a desolvation method. Table 1 concluded the physicochemical characteristics

solution (Figure S1). During the whole week, the mean particle size of Wpep-HSA-PTX NP remained constant. 3.2. In Vitro Redox-Responsive Behaviors of HSA NPs. Intermolecular disulfide bonds forming HSA nanoparticles were supposed to act as redox-responsive cross-linkers under the intracellular GSH concentration. First, intermolecular disulfide bonds were successfully formed in HSA nanoparticles (Figure S2). For tracking the size change under a GSH environment, relative results showed that the particle size of each Wpep-HSA-PTX NP decreased approximately from 110 to 39 nm, and the PDI enlarged, respectively (Figure S3). Subsequently, the release behaviors were observed under the PBS buffer at pH 7.4 with and without the reductant GSH. Figure 4 presented that as the GSH concentration reached to intracellular level (10 mM), PTX release from Wpep-HSA-PTX NP was very quickly within 2h (∼40%), which was little slower than free PTX (∼60%). While in the environment, PTX release from Wpep-HSA-PTX NP was relatively slow under the protection of intermolecular disulfide bond. 3.3. Cytotoxicity of HSA-NP in MDA-MB-231 Cells. In vitro cytotoxicities of free PTX, HSA-PTX NP, and WpepHSA-PTX NP against MDA-MB-231 cells were evaluated by MTT assay to determine IC50 values (Figure 5 and Table S1). Figure 5 displayed the viabilities of MDA-MB-231 cells after 48 h of incubation with different PTX formulations. Different formulations have various inhibitory effect on cancer cells. Given the data collected from the Figure 5, the IC50 value could be fitted by GraphPad Prsim5 so as to reflect the cytotoxicity of different PTX formulations indirectly. It was shown that the IC50 of HSA-PTX NP and Wpep-HSA-PTX NP were significantly lower than that of free PTX (Table S1), demonstrating that the redox-responsive release behavior due to the disulfide cross-linker within HSA-PTX and Wpep-HSAPTX NP promoted the intracellular PTX release. 3.4. Cellular Uptake and Internalization Mechanism. The cellular uptake of nanoparticles was investigated in MDA-

Table 1. Physical−Chemical Characterization of HSA NPa

a

nanoparticles

HSA-PTX NP

Wpep-HSA-PTX NP

size (nm) PDI ζ potential (mV) DL (%) EE (%)

103.2 ± 2.1 0.209 ± 0.021 −28.2 ± 3.5 10.31 ± 1.42 88.3 ± 0.8

118.8 ± 1.9 0.221 ± 0.031 −24.3 ± 1.9 9.87 ± 0.98 86.3 ± 1.3

Data represented as mean ± SD (n = 3).

of HSA-PTX NP and Wpep-HSA-PTX NP. The mean size of Wpep-HSA-PTX NP was larger than that of HSA-PTX NP due to the conjugation with W peptide slightly increasing the nanoparticle size. Meanwhile, the ζ potential of HSA-PTX NP and Wpep-HSA-PTX NP were −28.2 ± 3.5 and −24.3 ± 1.9 mV, respectively. The drug loading content of two formulations were higher than 9%, with satisfactory entrapment efficiencies. As shown in Figure 3, the average hydrodynamic diameter of Wpep-HSA-PTX was 118.8 nm measured by DLS (Figure 3A), while the AFM image indicated that Wpep-HSA-PTX NP revealed spherical morphology (Figure 3B). To evaluate the stability of Wpep-HSA-PTX NPs, the changes of particle size were recorded in the pH 7.4 PBS 7428

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435

Research Article

ACS Applied Materials & Interfaces

Figure 3. Size distribution of Wpep-HSA-PTX NP (A) and AFM images of Wpep-HSA-PTX NP (B).

concentration-dependent. Consistent with the qualitative results, FACS data demonstrated the same concentrationdependent cellular internalization behavior (Figure 6c). In specific, the mean fluorescent intensity of coumarin-6 among different groups were quantitatively analyzed from FACS results. The fluorescent intensities of Wpep-HSA/Cou-6 NP exhibited the concentration-dependent tendency, which were higher than HSA/Cou-6 NP. To investigate the possible internalization mechanism of Wpep-HSA/Cou-6 NP, various endocytic inhibitors were used to evaluate cellular uptake. As shown in Figure 6b, treatment with free W peptide could significantly down-regulate the internalization of Wpep-HSA NP. At the meantime, different endocytic inhibitors further confirmed the receptor-mediated endocytosis pathway. Among the three endocytic inhibitors, cells treated with PhAsO and colchicine exhibited the higher inhibitory effects. Consistent with the qualitative analysis, the quantitative analysis also proved the similar tendency (Figure 6c). Specifically speaking, the fluorescent intensity was lower in the W peptide competition group than that in the filipin inhibition group. Compared with the filipin inhibition group, the fluorescent intensity were higher in groups inhibited by PhAsO and colchicine. 3.5. Intracellular Tracking of Wpep-HSA/Cou-6 NPs. To discover the intracellular manner of Wpep-HSA nanoparticles, MDA-MB-231 cells were treated with Wpep-HSA/ Cou6 NP for different time intervals. As presented in Figure 7, the green fluorescent signal represented the intracellular WpepHSA/Cou6 NP, and the blue signal represented the nucleus. The fluorescence intensity of green signal possessed a timedependent trend. 3.6. Cell-Cycle Distribution. The formation of microtubules was proved highly specific in the G2/M phase of cell cycle and was a pivotal step in cell proliferation. PTX is wellknown to damage microtubules and arrest the cell cycle in the G2/M phase so that PTX could inhibit uncontrollable proliferation of cancer cells.39 The higher cell distribution in the G2-M phase, the stronger inhibition effect on the microtubules formation paclitaxel performs. Figure 8 concluded the FACS results of cell cycle distribution after cells exposed to free PTX, HSA-PTX NP, and Wpep-HSA-PTX NP, respectively. The percentage of different phases reflected the various cell distribution. PTX encapsulated in HSA nanoparticles exhibited a significantly higher cell distribution in the G2-M phase (23.05% from HSA-PTX NP and 30.39% from Wpep-HSA-PTX NP) than free PTX (19.99% G2-M phase).

Figure 4. In vitro drug release behaviors from Wpep-HSA-PTX NP triggered by GSH or not in PBS (pH 7.4) at 37 °C with free PTX as control group. Data are represented as mean ± SD (n = 3).

Figure 5. In vitro cytotoxicity of different PTX formulations at various concentrations against MDA-MB-231 tumor cells 48 h after incubation. Data are represented as mean ± SD (n = 6).

MB-231 cells. Photographs captured by fluorescence microscope indicated that cells treated with Wpep-HSA/Cou-6 NP exhibited a higher level of internalization compared with HSA/ Cou-6 NP under the same concentration of HSA, suggesting that Wpep could improve the intracellular uptake of nanoparticles into MDA-MB-231 cells (Figure 6a). Meanwhile, the level of internalization of Wpep-HSA/Cou-6 NP was 7429

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435

Research Article

ACS Applied Materials & Interfaces

Figure 6. (a) Cellular uptake of HSA/cou6 NP and Wpep-HSA/Cou6 NPs with different concentrations in MDA-MB- 231 tumor cells 30 min after incubation; (b) possible uptake mechanism of Wpep-HSA/Cou6 NP internalization into MDA-MB-231 tumor cells. The cells were blocked by different inhibitors. (c) Flow cytometry analysis of cellular uptake and uptake mechanism experiment.

(50:50, v/v). As shown in Figure 9, the elimination behaviors of different PTX formulations were almost the same rate within 2 h post-administration. However, the concentration of PTX in blood plasma decreased more rapidly following treatment by Taxol after 12 h post-administration compared with the other two groups. Meanwhile, the concentration of PTX in blood plasma was maintained for a longer circulation time, even 24 h after administration, when PTX was encapsulated in albumin nanoparticle due to the stable structure forming by intramolecular disulfide bonds. The corresponding pharmacokinetic parameters of PTX in different formulations were summarized in Table.2. It was pretty clear that there were significant differences of the pharmacokinetic parameters between Taxol and PTX-HSA nanoparticles, in which the area under the plasma-concentration curves (AUC0 → ∞) of PTX-HSA and Wpep-PTX-HSA were 1.2-fold and 2.5-fold higher than that of Taxol, respectively; correspondingly, the mean residence time (MRT) of PTX-HSA and Wpep-PTX-HSA were 2.3-fold and 7.5-fold longer than that of Taxol, and the clearance (CL) of PTX-HSA and Wpep-PTX-HSA was 1.1-fold and 2.3-fold shorter than that of Taxol, respectively. 3.8. In Vivo Biodistribution. In vivo imaging was employed to evaluate the targeting efficiency of HSA-BODIPY NP and Wpep-HSA-BODIPY NP in tumor bearing nude mice. As displayed in Figure 10 A, mice administered with WpepHSA-BODIPY NPs had a stronger signal of NIR dye in the tumor area 24 h after injection compared to results with HSABODIPY NPs. The bottom portion of Figure 10 A shows the signal amplification version of the upper portion by the increase of the exposure time for both groups, exhibiting a much stronger signal for the Wpep-HSA-BODIPY NP group. Moreover, tumors and major organs were excised for ex vivo imaging to observe the tissue distribution (Figure 10C). NIR fluorescence intensity of Wpep-HSA-BODIPY NP had much stronger accumulation in the tumor section than that of HSABODIPY (Figure 10 B).

Figure 7. Confocal microscopy of Wpep-HSA/Cou6 NP incubation with MDA-MB-231 tumor cells at different time points. The nuclei were stained by Hoechest (blue), and green is the signal of coumarin6.

3.7. Pharmacokinetics Studies. To investigate whether the disulfide cross-link barrier could improve stability of albumin nanoparticles during blood circulation, we studied the pharmacokinetic behaviors of various PTX formulations. The mean plasma concentration−time profiles of PTX after intravenous administration of different PTX formulations were plotted in Figure 9, and the corresponding pharmacokinetic parameters were listed in Table.2, respectively. The Taxol was prepared as the clinical parenteral Taxol, in which PTX was dissolved in a mixture solvent of Cremophor EL and ethanol 7430

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435

Research Article

ACS Applied Materials & Interfaces

Figure 8. Representative cell cycle distribution in MDA-MB-231 after incubation-free PTX (b), HSA-PTX NP (c), and Wpep-HSA-PTX NP (d) at the equivalent drug concentration with analysis was performed by flow cytometry and (a) the negative control group. Each figure individually represents the population present in the G1-S and G2-M phases.

Figure 11, control group (saline) exhibited a sharp growth in body weight ranging from 20 to 25 g, while all of the PTX formulation groups possessed a slight change of body weight compared to the control group. There is no obvious difference among those groups mainly due to the individual difference of animals. Meanwhile, luciferase expression was visualized and imaged using the IVIS Spectrum, which was indicated in Figure 12. Hence, the luminescence intensity represented the progression of MDA-MB-231 breast cancer. To be specific, on Day 0, the luminescence intensity among different groups remained the same. However, along with the treatment, luminescence intensity of mice treated by Wpep-HSA-PTX NP was gradually reduced, and intensities of other groups were on the increase. After the whole regimen, all mice were sacrificed to further compare the tumor apoptosis among different groups using TUNEL assay, both Wpep-HSA-PTXand HSA-PTX-treated groups showed comparable proapoptosis ability compared with the Taxol group (Figure 13).

Figure 9. Pharmacokinetic profiles of PTX in SD rats after injection with different PTX formulations at a dose of 10 mg/kg. Data are represented as mean ± SD (n = 5).

Table 2. Pharmacokinetic Parameters of PTX Formulations after Intravenous Administration at the PTX Dose of 5 mg/ kg in SD Rats (n = 5) formulations Wpep-HSAPTX HSA-PTX Taxol

AUC0∼∞ (mg/L/h)

MRT0∼∞ (h)

24.025 ± 8.657

18.846 ± 3.239

11.174 ± 5.829 9.831 ± 3.718b a

4. DISCUSSION Currently, chemotherapy is still the major regimen for treatment to most TNBC patients, of which PTX is represented as first-line chemotherapeutic drugs for TNBC. However, its clinical applications of PTX were seriously limited by its high water insolubility. Along with the progress of novel nanoparticulate formulations, Abraxane, utilizing HSA as drug carrier, is one of the most-promising and the only clinically approved PTX-loading nanocarrier that exhibits greater antitumor efficacy than Taxol (Cremophor formulation of PTX). Thus, constructing drug carriers with biomimetic

CL (L/h/kg) 0.396 ± 0.014

5.862 ± 1.423 2.510 ± 0.767b b

0.813 ± 0.162a 0.898 ± 0.082a

a

p < 0.05. bp < 0.01, significantly different from that of Wpep-HSAPTX.

3.9. Antitumor Efficacy. The antitumor efficacy was evaluated in TNBC bearing mice after the treatment of different formulations every 4 days for four times. As shown in 7431

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435

Research Article

ACS Applied Materials & Interfaces

only verified by 1H NMR and following self-assembly with PTX into nanoparticle by innovative nontoxic disulfide bond reducing method, in which the number of sulfhydryl group was determined by the Ellman’s method. As HSA was incubated with GSH, the number of free sulfhydryl groups in each molecule increased from 0.35 to 3.5; after forming into HSA NP, the number of the sulfhydryl group decreased back to 0.47, which proved the transition from intermolecular disulfide bonds into intramolecular disulfide bonds (Figure S1). In vitro physicochemical characterization results showed the larger mean size of Wpep-HSA-PTX NP, suggesting the successful surficial conjugation with W peptide, the negatively charged potential of HSA-PTX NP and Wpep-HSA-PTX NP, satisfactory drug loading content, and the entrapment efficiencies of both formulations. Additionally, photographs captured by AFM indicated that Wpep-HSA-PTX NP presented a spherical shape (Figure 3B) that was similar to the average hydrodynamic diameter (118.8 nm) of Wpep-HSAPTX (Figure 3A). To investigate the in vitro redox-responsive characteristics of Wpep-HSA-PTX NP, the size change of Wpep-HSA-PTX NP with or without GSH treatment was carried out first. The size gradually decreased within 24 h, demonstrating the breakdown of the disulfide bond of the nanoparticles. Unlike the micelle’s structure, albumin nanoparticles were cut into individual albumin particles under the reduction effect of intramolecular sulfide bonds. Correspondingly, PDI became larger after incubation time extended. To investigate the redox-responsive drug release behavior of Wpep-HSA-PTX NP, PBS buffers at pH 7.4 with and without the GSH as reductant were used to mimic the intracellular environment. As presented in Figure 4, Wpep-HSA-PTX NP was quickly released within 2 h (∼40%) as the GSH concentration reached to intracellular level (10 mM), which was as fast as free PTX.21 At the meantime, WpepHSA-PTX NP was barely released within 2 h in the nonreductant environment, which was ascribed to the intact of most intermolecular disulfide bond assuring the stability of drug carrier. Thus, relative results demonstrated that drug release behavior of Wpep-HSA-PTX NP was redox-responsive to the reductants’ glutathione. Furthermore, the significantly lower IC50 of HSA-PTX NP and Wpep-HSA-PTX NP than that of free PTX, which was due to the higher intracellular PTX release, indirectly proved the redox-responsive intracellular release mechanism. Next, in vitro drug efficacy was evaluated on TNBC cells. Specifically, the cellular uptake of nanoparticles by MDA-MB-231 cells indicated that cells treated with WpepHSA/Cou-6 NP exhibited a higher efficiency of internalization compared with HSA/Cou-6 NP under the same concentration of HSA, suggesting that Wpep increased the cellular uptake of nanoparticles into MDA-MB-231 cells through FPR-mediated active endocytosis (Figure 6a). Meanwhile, the extent of WpepHSA/Cou-6 NP internalization was concentration-dependent. Moreover, investigation on endocytosis mechanism was carried out with the help of different pathway inhibitors. As shown in Figure 6b,c, the internalization of Wpep-HSA NP was greatly inhibited by free W peptide due to the competitive inhibition and receptor saturation effect, which indicated that Wpep was responsible for actively binding to MDA-MB-231 cell through FPR-mediated endocytosis.29 In addition, two of three endocytic inhibitors (PhAsO and colchicine) exhibited obvious inhibitory effects, suggesting that the internalization endocytosis was mainly through clarthrin-mediated and micropinocytosis-mediated pathways.34 Furthermore, efficient time-

Figure 10. Biodistribution and targeting effects of different PTX formulation in tumor-bearing mice. (A) In vivo 2D imaging 24 h after intravenous (iv) injection of HSA/BODIPY (nontargeting group) and Wpep-HSA/BODIPY (targeting group) at a dose of 0.5 mg BODIPY/ kg in MDA-MB-231-bearing mice. The red dotted circle was indicated as the location of tumor implantation (B) via 3D imaging 24 h after iv injection of Wpep-HSA/BODIPY in tumor-bearing mice. (C) Representative ex vivo optical images of tumors and organs of MDA-MB-231-bearing mice sacrificed at 24 h.

Figure 11. Body-weight change after iv injections of Taxol (10 mg/ kg), HSA-PTX NP (10 mg/kg), and Wpep-HSA- PTX NP (10 mg/ kg) on days 0, 4, 8, and 12 (saline serving as control). At day 20, mice were sacrificed. Date are presented as mean ± SD (n = 6).

materials (e.g., HDL, albumin, and ferritin) could incorporate drugs via simple procedures. However, most of the drugs were physically adsorbed on those materials, resulting in low drug loading and poor plasma stability.40 In the present study, considering the pathological features of TNBC and limitations of bioinspired nanocarriers, a novel albumin-based drugdelivery system was developed through the cross-linking of disulfide bond and the modification with target group, thus leading to greater efficacy in management of this prevalent and challenging disease. Wpep-HSA-PTX NP was prepared by the initial synthesis of Wpep-modified HSA by click chemistry. The conjugation efficiency is a tough issue for biomacromolecule conjugation due to the heterogeneous characteristic of protein and could 7432

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435

Research Article

ACS Applied Materials & Interfaces

Figure 12. Real-time bioluminescence photographs of MDA-MB-231 luci cell bearing nude mice injected with saline, Taxol, HSA-PTX NP, and Wpep-modified HSA-PTX NP on days 5, 10, and 15, respectively.

Figure 13. Representative histological images of MDA-MB-231 tumor xenografts excised from those nude mice used in the experiment of the in vivo antitumor efficacy of micelles. Blue, DAPI-stained cell nuclei; green, apoptosis cells. Original magnification: 100 ×.

dependent intracellular delivery of Wpep-HSA/Cou-6 nanoparticles into MDA-MB-231 cells indicated the feasibility and utility of Wpep as targeting ligand-mediating nanoparticle endocytosis. Considering that the antitumor mechanism of PTX is the disruption of microtubule dynamics, a cell-cycle distribution test was performed to quantify the population of MDA-MB-231 cells in different stages upon treatment with different PTX formulations. The population in each phase of the cell cycle was

indicated as histograms. Relative results exhibited that both HSA nanoparticles had significantly higher cell distribution in the G2-M phase (23.05% from HSA-PTX NP and 30.39% from Wpep-HSA-PTX NP) than free PTX (19.99%); the most potent efficacy of Wpep-HSA-PTX might be attributed to the synergistic role of disulfide-introduced redox-responsive behavior and Wpep-mediated target effects. As mentioned above, the in vivo stability of Wpep-HSA-PTX NP might be improved due to controlled-release behavior and 7433

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435

Research Article

ACS Applied Materials & Interfaces

antitumor efficacy. Collectively, in this research, we successfully developed a new platform for improving the stability of commercially available human serum albumin-based nanocarriers and first exploited the W peptide as a target moiety for the realization of targeted TNBC therapy.

demonstrated by pharmacokinetics study. At present, the lower AUC and Cmax of commercial albumin-based PTX formulation (Abraxane) was reported compared with those of Taxol,41 mainly due to instability of albumin in circulation, resulting in rapid drug metabolism and clearance out of body. As for the nanoparticulate carriers, the major hurdles of poor deposition in circulation may be attributed to nanoparticle aggregation and disassembly and following the rapid clearance of the leaked drug. To resolve these hurdles, the PK parameters of nanocarriers could be advanced by PEGylation, size adjustment, and surface modification. As shown in Figure 9, the similar elimination behaviors among three groups treated with different PTX formulations after 2 h post-administration was probably due to unencapsulated PTX exposed on the albumin surface. However, compared with rapid elimination of free PTX administrated in Taxol, the higher concentration of PTX in blood plasma was still maintained, even 24 h after administration, when PTX was loaded in HSA NP, relying on strong binding with albumin and cross-linking protection by intramolecular disulfide bonds. Those improved pharmacokinetics parameters of Wpep-HSA-PTX, including an increased AUC, MRT, and lowered CL, laid the groundwork for the in vivo anticancer efficacy. In short, the PK behaviors of WpepHSA-PTX has been remarkably improved by the W peptide modification and intramolecular disulfide bonds introduction. After the establishment of TNBCs-bearing nude mice, the targeting effect of Wpep was confirmed by the in vivo imaging method post-administration with HSA-BODIPY NP and Wpep-HSA-BODIPY NP, respectively. The accumulation of BODIPY signal was presented in HSA-BODIPY NP group 24 h after administration based on the enhanced permeability and retention (EPR) effect, in addition to the gp60 or SPARC receptor-mediated targeted pathway. In contrast, the fluorescent signal of Wpep-HSA-BODIPY NP was significantly augmented in cancerous tissue, demonstrating that the specific binding effect between W peptide and FPR, which might play a pivotal role in tumor targeting. The ex vivo analysis also confirmed the tumor homing ability of Wpep-HSA-BODIPY NP. At last, the in vivo drug efficacy of the different PTX formulation, the favored targeting efficiency and the redoxresponsive property of Wpep-HSA were ultimately investigated and demonstrated. In detail, Wpep-HSA-PTX showed much more potent in vivo antitumor activity compared with that of Taxol and HSA-PTX NPs, showing a gradually decreased signal of bioluminescence in tumor foci and stable body weight changes. Besides, TUNEL assay indicated that the Wpep-HSAPTX NP group exerted a strong pro-apoptotic effect on cancerous tissue.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b14390. Sulfhydryl groups determined by Ellman’s method, in vitro stability of Wpep-HSA-PTX NP, in vitro redoxresponsive behaviors of Wpep-HSA-PTX NP, and IC50 values of different PTX formulations. (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*H.W. tel: +86-21-51320211; fax: +86-21-51320728; e-mail: [email protected]. *C.J. tel and fax: +86-021-51980079; e-mail: jiangchen@shmu. edu.cn. ORCID

Chen Jiang: 0000-0002-4833-9121 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the grants from National Basic Research Program of China (973 Program, grant no. 2013CB932500), National Science Fund for Distinguished Young Scholars (grant no. 81425023), and the National Natural Science Foundation of China (grant no. 81373355).

■

REFERENCES

(1) Foulkes, W. D.; Smith, I. E.; Reis-Filho, J. S. Triple-negative Breast Cancer. N. Engl. J. Med. 2010, 363 (20), 1938−1948. (2) Mustacchi, G.; De Laurentiis, M. The Role of Taxanes in Triplenegative Breast Cancer: Literature Review. Drug Des., Dev. Ther. 2015, 9, 4303−4318. (3) Perez-Herrero, E.; Fernandez-Medarde, A. Advanced Targeted Therapies in Cancer: Drug Nanocarriers, the Future of Chemotherapy. Eur. J. Pharm. Biopharm. 2015, 93, 52−79. (4) Xiao, K.; Luo, J.; Fowler, W. L.; Li, Y.; Lee, J. S.; Xing, L.; Cheng, R. H.; Wang, L.; Lam, K. S. A Self-assembling Nanoparticle for Paclitaxel Delivery in Ovarian Cancer. Biomaterials 2009, 30 (30), 6006−6016. (5) Anselmo, A. C.; Mitragotri, S. An Overview of Clinical and Commercial Impact of Drug Delivery Systems. J. Controlled Release 2014, 190, 15−28. (6) Zhang, Y.; Chan, H. F.; Leong, K. W. Advanced Materials and Processing for Drug Delivery: the Past and the Future. Adv. Drug Delivery Rev. 2013, 65 (1), 104−120. (7) Yokoyama, M. Polymeric Micelles as Drug Carriers: Their Lights and Shadows. J. Drug Target. 2014, 22 (7), 576−583. (8) Yoo, J. W.; Irvine, D. J.; Discher, D. E.; Mitragotri, S. Bioinspired, Bioengineered and Biomimetic Drug Delivery Carriers. Nat. Rev. Drug Discovery 2011, 10 (7), 521−535. (9) Zeugolis, D. I.; Pandit, A. Biofunctional Biomaterials–The Next Frontier. Bioconjugate Chem. 2015, 26 (7), 1157. (10) Liu, W.; Rose, J.; Plantevin, S.; Auffan, M.; Bottero, J. Y.; Vidaud, C. Protein Corona Formation for Nanomaterials and Proteins of a Similar Size: Hard or Soft Corona? Nanoscale 2013, 5 (4), 1658− 1668.

5. CONCLUSIONS In summary, the present work reported the successful application of W-peptide-conjugated HSA NP as a delivery vehicle for targeting PTX accumulation into TNBC cells and enhancing antitumor efficacy. Related results showed that Wpep-HSA NP augmented both cell apoptosis extent and cellular uptake via efficient FPR-mediated endocytosis. Meanwhile, pharmacokinetic behaviors were dramatically improved through the protection of intramolecular disulfide bonds on HSA and surface modification by PEG and W peptides. As a result, following intravenous administration, significant drug accumulation at the tumor site was discovered in the WpepHSA-PTX NP-treated group, which thus exhibited stronger 7434

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435

Research Article

ACS Applied Materials & Interfaces

Formyl Peptide Receptors (FPRs) 1 and 2. FASEB J. 2011, 25 (2), 483−496. (28) Xu, C. P.; Zhang, H. R.; Chen, F. L.; Yao, X. H.; Liang, Z. Q.; Zhang, R.; Cui, Y.; Qian, C.; Bian, X. W. Human Malignant Glioma Cells Expressing Functional Formyl Peptide Receptor Recruit Endothelial Progenitor Cells for Neovascularization. Int. Immunopharmacol. 2010, 10 (12), 1602−1607. (29) Wong, D. Y.; Yeo, C. H.; Ang, W. H. Immuno-Chemotherapeutic Platinum(IV) Prodrugs of Cisplatin as Multimodal Anticancer Agents. Angew. Chem., Int. Ed. 2014, 53 (26), 6752−6756. (30) Kim, S. D.; Lee, H. Y.; Shim, J. W.; Kim, H. J.; Baek, S. H.; Zabel, B. A.; Bae, Y. S. A WKYMVm-Containing Combination Elicits Potent Anti-Tumor Activity in Heterotopic Cancer Animal Model. PLoS One 2012, 7 (1), e30522. (31) Xu, R.; Fisher, M.; Juliano, R. L. Targeted Albumin-based Nanoparticles for Delivery of Amphipathic Drugs. Bioconjugate Chem. 2011, 22 (5), 870−878. (32) Wang, S.; Gong, G.; Su, H.; Liu, W.; Wang, Z.; Li, L. SelfAssembly of Plasma Protein through Disulfide Bond Breaking and its Use as a Nanocarrier for Lipophilic Drugs. Polym. Chem. 2014, 5 (17), 4871−4874. (33) Wang, W.; Huang, Y.; Zhao, S.; Shao, T.; Cheng, Y. Human Serum Albumin (HSA) Nanoparticles Stabilized with Intermolecular Disulfide Bonds. Chem. Commun. (Cambridge, U. K.) 2013, 49 (22), 2234−2236. (34) He, X.; Li, J.; An, S.; Jiang, C. pH-Sensitive Drug-Delivery Systems for Tumor Targeting. Ther. Delivery 2013, 4 (12), 1499− 1510. (35) Pawar, V. K.; Panchal, S. B.; Singh, Y.; Meher, J. G.; Sharma, K.; Singh, P.; Bora, H. K.; Singh, A.; Datta, D.; Chourasia, M. K. Immunotherapeutic Vitamin E Nanoemulsion Synergies the Antiproliferative Activity of Paclitaxel in Breast Cancer Cells via Modulating Th1 and Th2 Immune Response. J. Controlled Release 2014, 196, 295−306. (36) Rezazadeh, M.; Emami, J.; Mostafavi, A.; Rostami, M.; Hassanzadeh, F.; Sadeghi, H.; Minaiyan, M.; Lavasanifar, A. A Rapid and Sensitive HPLC Method for Quantitation of Paclitaxel in Biological Samples using Liquid-Liquid Extraction and UV Detection: Application to Pharmacokinetics and Tissues Distribution Study of Paclitaxel Loaded Targeted Polymeric Micelles in Tumor Bearing Mice. J. Pharm. Pharm. Sci. 2015, 18 (5), 647−660. (37) Shao, K.; Zhang, Y.; Ding, N.; Huang, S.; Wu, J.; Li, J.; Yang, C.; Leng, Q.; Ye, L.; Lou, J.; Zhu, L.; Jiang, C. Functionalized Nanoscale Micelles with Brain Targeting Ability and Intercellular Microenvironment Biosensitivity for Anti-Intracranial Infection Applications. Adv. Healthcare Mater. 2015, 4 (2), 291−300. (38) Han, L.; Liu, M.; Ye, D.; Zhang, N.; Lim, E.; Lu, J.; Jiang, C. Tumor Cell Membrane-Targeting pH-Dependent Electron DonorAcceptor Fluorescence Systems with Low Background Signals. Biomaterials 2014, 35 (9), 2952−2960. (39) Bhattacharyya, J.; Bellucci, J. J.; Weitzhandler, I.; McDaniel, J. R.; Spasojevic, I.; Li, X.; Lin, C. C.; Chi, J. T.; Chilkoti, A. A paclitaxelLoaded Recombinant Polypeptide Nanoparticle Outperforms Abraxane in Multiple Murine Cancer Models. Nat. Commun. 2015, 6, 7939. (40) De Jong, W. H.; Borm, P. J. Drug Delivery and Nanoparticles: Applications and Hazards. Int. J. Nanomed. 2008, 3 (2), 133−149. (41) Sparreboom, A.; Scripture, C. D.; Trieu, V.; Williams, P. J.; De, T.; Yang, A.; Beals, B.; Figg, W. D.; Hawkins, M.; Desai, N. Comparative Preclinical and Clinical Pharmacokinetics of a Cremophor-Free, Nanoparticle Albumin-Bound Paclitaxel (ABI-007) and Paclitaxel Formulated in Cremophor (Taxol). Clin. Cancer Res. 2005, 11 (11), 4136−4143.

(11) Pearson, R. M.; Juettner, V. V.; Hong, S. Biomolecular Corona on Nanoparticles: a Survey of Recent Literature and Its Implications in Targeted Drug Delivery. Front. Chem. 2014, 2, 108. (12) Liu, L.; He, H.; Zhang, M.; Zhang, S.; Zhang, W.; Liu, J. Hyaluronic Acid-Decorated Reconstituted High Density Lipoprotein Targeting Atherosclerotic Lesions. Biomaterials 2014, 35 (27), 8002− 8014. (13) Sanchez-Gaytan, B. L.; Fay, F.; Lobatto, M. E.; Tang, J.; Ouimet, M.; Kim, Y.; van der Staay, S. E.; van Rijs, S. M.; Priem, B.; Zhang, L.; Fisher, E. A.; Moore, K. J.; Langer, R.; Fayad, Z. A.; Mulder, W. J. HDL-mimetic PLGA Nanoparticle to Target Atherosclerosis Plaque Macrophages. Bioconjugate Chem. 2015, 26 (3), 443−451. (14) Kundranda, M. N.; Niu, J. Albumin-bound Paclitaxel in Solid Tumors: Clinical Development and Future Directions. Drug Des., Dev. Ther. 2015, 9, 3767−3777. (15) Wang, Y.; Yang, T.; Ke, H.; Zhu, A.; Wang, Y.; Wang, J.; Shen, J.; Liu, G.; Chen, C.; Zhao, Y.; Chen, H. Smart AlbuminBiomineralized Nanocomposites for Multimodal Imaging and Photothermal Tumor Ablation. Adv. Mater. 2015, 27 (26), 3874−3882. (16) Chen, Q.; Liang, C.; Wang, C.; Liu, Z. An Imagable and Photothermal ″Abraxane-like″ Nanodrug for Combination Cancer Therapy to Treat Subcutaneous and Metastatic Breast Tumors. Adv. Mater. 2015, 27 (5), 903−910. (17) Zhang, W.; He, H.; Liu, J.; Wang, J.; Zhang, S.; Zhang, S.; Wu, Z. Pharmacokinetics and Atherosclerotic Lesions Targeting Effects of Tanshinone IIA Discoidal and Spherical Biomimetic High Density Lipoproteins. Biomaterials 2013, 34 (1), 306−319. (18) Liang, M.; Fan, K.; Zhou, M.; Duan, D.; Zheng, J.; Yang, D.; Feng, J.; Yan, X. H-ferritin-Nanocaged Doxorubicin Nanoparticles Specifically Target and Kill Tumors with a Single-dose Injection. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (41), 14900−14905. (19) Li, C.; Li, Y.; Gao, Y.; Wei, N.; Zhao, X.; Wang, C.; Li, Y.; Xiu, X.; Cui, J. Direct Comparison of Two Albumin-based Paclitaxel-loaded Nanoparticle Formulations: is the Crosslinked Version More Advantageous? Int. J. Pharm. 2014, 468 (1−2), 15−25. (20) Zhao, M.; Lei, C.; Yang, Y.; Bu, X.; Ma, H.; Gong, H.; Liu, J.; Fang, X.; Hu, Z.; Fang, Q. Abraxane, the Nanoparticle Formulation of Paclitaxel Can Induce Drug Resistance by Up-Regulation of P-gp. PLoS One 2015, 10 (7), e0131429. (21) Guo, Y.; Zhang, Y.; Li, J.; Zhang, Y.; Lu, Y.; Jiang, X.; He, X.; Ma, H.; An, S.; Jiang, C. Cell Microenvironment-controlled Antitumor Drug Releasing-nanomicelles for GLUT1-targeting Hepatocellular Carcinoma Therapy. ACS Appl. Mater. Interfaces 2015, 7 (9), 5444− 5453. (22) An, S.; Jiang, X.; Shi, J.; He, X.; Li, J.; Guo, Y.; Zhang, Y.; Ma, H.; Lu, Y.; Jiang, C. Single-component Self-assembled RNAi Nanoparticles Functionalized with Tumor-targeting iNGR Delivering Abundant siRNA for Efficient Glioma Therapy. Biomaterials 2015, 53, 330−340. (23) Jiang, L.; Xu, Y.; Liu, Q.; Tang, Y.; Ge, L.; Zheng, C.; Zhu, J.; Liu, J. A Nontoxic Disulfide Bond Reducing Method for Lipophilic Drug-loaded Albumin Nanoparticle Preparation: Formation Dynamics, Influencing Factors and Formation Mechanisms Investigation. Int. J. Pharm. 2013, 443 (1−2), 80−86. (24) Chen, Q.; Liu, Z. Albumin Carriers for Cancer Theranostics: A Conventional Platform with New Promise. Adv. Mater. 2016, 28, 10557. (25) Look, J.; Wilhelm, N.; von Briesen, H.; Noske, N.; Gunther, C.; Langer, K.; Gorjup, E. Ligand-Modified Human Serum Albumin Nanoparticles for Enhanced Gene Delivery. Mol. Pharmaceutics 2015, 12 (9), 3202−3213. (26) Low, K.; Wacker, M.; Wagner, S.; Langer, K.; von Briesen, H. Targeted Human Serum Albumin Nanoparticles for Specific Uptake in EGFR-Expressing Colon Carcinoma Cells. Nanomedicine 2011, 7 (4), 454−463. (27) Khau, T.; Langenbach, S. Y.; Schuliga, M.; Harris, T.; Johnstone, C. N.; Anderson, R. L.; Stewart, A. G. Annexin-1 Signals MitogenStimulated Breast Tumor Cell Proliferation by Activation of the 7435

DOI: 10.1021/acsami.6b14390 ACS Appl. Mater. Interfaces 2017, 9, 7424−7435