Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX-XXX
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Intelligent “Peptide-Gathering Mechanical Arm” Tames Wild “TrojanHorse” Peptides for the Controlled Delivery of Cancer Nanotherapeutics Nian-Qiu Shi,*,†,⊥ Yan Li,‡ Yong Zhang,# Nan Shen,§ Ling Qi,§ Shu-Ran Wang,∥ and Xian-Rong Qi∇ †
School of Pharmacy, ‡Immunology Department, Laboratory Medical College, §Basic College of Medicine, and ∥School of Public Health, Jilin Medical University, Jilin City 132013, Jilin Province, China ⊥ State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China # College of Life Science, Jilin University, 2699 Qianjin Street, Changchun 130012, Jilin Province, China ∇ Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Beijing 100191, China S Supporting Information *
ABSTRACT: Cell-penetrating peptides (CPPs), also called “Trojan-Horse” peptides, have been used for facilitating intracellular delivery of numerous diverse cargoes and even nanocarriers. However, the lack of targeting specificity (“wildness” or nonselectivity) of CPP-nanocarriers remains an intractable challenge for many in vivo applications. In this work, we used an intelligent “peptide-gathering mechanical arm” (Int PMA) to curb CPPs’ wildness and enhance the selectivity of R9-liposome-based cargo delivery for tumor targeting. The peptide NGR, serving as a cell-targeting peptide for anchoring, and peptide PLGLAG, serving as a substrate peptide for deanchoring, were embedded in the Int PMA motif. The Int PMA construct was designed to be sensitive to tumor microenvironmental stimuli, including aminopeptidase N (CD13) and matrix metalloproteinases (MMP-2/9). Moreover, Int PMA could be specifically recognized by tumor tissues via CD13-mediated anchoring and released for cell entry by MMP-2/9-mediated deanchoring. To test the Int PMA design, a series of experiments were conducted in vitro and in vivo. Functional conjugates Int PMA-R9-poly(ethylene glycol) (PEG)2000-distearoylphosphatidyl-ethanolamine (DSPE) and R9PEG2000-DSPE were synthesized by Michael addition reaction and were characterized by thin-layer chromatography and matrixassisted laser desorption ionization-time-of-flight mass spectrometry. The Int PMA-R9-modified doxorubicin-loaded liposomes (Int PMA-R9-Lip-DOX) exhibited a proper particle diameter (approximately 155 nm) with in vitro sustained release characteristics. Cleavage assay showed that Int PMA-R9 peptide molecules could be cleaved by MMP-2/9 for completion of deanchoring. Flow cytometry and confocal microscopy studies indicated that Int PMA-R9-Lip-DOX can respond to both endogenous and exogenous stimuli in the presence/absence of excess MMP-2/9 and MMP-2/9 inhibitor (GM6001) and effectively function under competitive receptor-binding conditions. Moreover, Int PMA-R9-Lip-DOX generated more significant subcellular dispersions that were especially evident within endoplasmic reticulum (ER) and Golgi apparatus. Notably, Int PMAR9-Lip-DOX could induce enhanced apoptosis, during which caspase 3/7 might be activated. In addition, Int PMA-R9-Lip-DOX displayed enhanced in vitro and in vivo antitumor efficacy versus “wild” R9-Lip-DOX. On the basis of investigations at the molecular level, cellular level, and animals’ level, the control of Int PMA was effective and promoted selective delivery of R9liposome cargo to the target site and reduced nonspecific uptake. This Int PMA-controlled strategy based on aminopeptidaseguided anchoring and protease-triggered deanchoring effectively curbed the wildness of CPPs and bolstered their effectiveness for in vivo delivery of nanotherapeutics. The specific nanocarrier delivery system used here could be adapted using a variety of intelligent designs based on combinations of multifunctional peptides that would specifically and preferentially bind to tumors versus nontumor tissues for tumor-localized accumulation in vivo. Thus, CPPs have a strong advantage for the development of intelligent nanomedicines for targeted tumor therapy. KEYWORDS: “Trojan-Horse” peptides, intelligent “peptide-gathering mechanical arm” (Int PMA) strategy, curbing the wildness, aminopeptidases-guided anchoring, protease-triggered deanchoring
1. INTRODUCTION
more attention due to their abilities to deliver chemotherapeutic
Noticeable progress has been made in the synthesis/fabrication and characterization of engineered nanocarriers for diagnosis and treatment of tumors in recent years. Many conventional nanocarriers, including nanoliposomes, nanoconjugates, nanoparticles, and polymeric nanomicelles, have attracted more and
drugs to specific targets.1−4 Several therapeutic nanoparticle
© XXXX American Chemical Society
Received: October 12, 2017 Accepted: November 10, 2017
A
DOI: 10.1021/acsami.7b15523 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 1. Schematic design of the selective CPP-nanocarrier delivery system based on an intelligent peptide-gathering mechanical arm (Int PMA) and their regulation strategy for selectivity. The multi-intelligent liposomes accumulated in the tumor site due to the EPR effect and the long-circulation effect by the PEG chain. The active targeting effect was achieved by binding of the ligand of the targeting peptide (NGR) with oversecreted CD13 receptor. The unregulated MMP-2/9 in the tumor microenvironment cleaves the MMP-2/9-sensitive substrate peptide (PLGLAG) and removes the targeting motif in the Int PMA, releasing the CPP domain (R9) for cellular internalization. The CPP-nanocarrier (R9-liposome cargo) can cross cellular or subcellular membranes to enter tumor cells for intracellular deep delivery because of the penetration function of CPPs.
dots,19 and small molecules20,21 in vitro or in vivo. Notably, polyarginine peptide conjugation has been demonstrated to improve the cellular uptake of doxorubicin-loaded liposomes15 and other chemotherapeutic drug-based nanocarriers.16,22 However, polyarginine-enabled medication, via the formation of direct links to the carrier surface, can result in a lack of cell selectivity, causing uncontrolled dispersion in vivo.23 Nonselectivity of CPP-nanocarriers toward tumor cells or tissues is a major hurdle for in vivo systemic targeted delivery of anticancer drugs because of low targeting efficiency and indiscriminate nontargeted distributions.24 The “wild” uncontrollable dispersion of CPPs becomes potentially life threatening during delivery of toxic cargoes. Thus, it is urgently necessary to improve selectivity of delivery of CPPs toward tumor cells/ tissues both in vitro and in vivo and effectively harness the penetrating power of CPPs to carry nanocarriers deep into tumors for more effective targeted tumor therapy. Numerous tools have been developed to overcome obstacles of nonspecificity and low recognition that plague nanocarrierbased therapies. Cell-targeting peptides (CTPs), which emerged from library screening or by design, denote a diverse group of amino acid-based molecules that exhibit high specificity and strong affinity for a given target cell line through specific interactions with corresponding receptors exclusively overexpressed by these cells.25,26 Additionally, CTP-medicated nanocarriers can be internalized via receptor-mediated endocytosis and can increase drug accumulation in tumor cells via an active targeting mechanism. Some common CTPs (e.g., RGD or NGR)
platforms (e.g., Doxil, Myocet, and Abraxane) have been approved for cancer therapy by the Food and Drug Administration (FDA). Moreover, many other nanotechnology-enabled therapeutic nanocarriers are currently under clinical investigation.5−7 These nanocarrier delivery systems accumulate at target sites on the basis of a passive targeting mechanism referred to as the enhanced permeability and retention (EPR) effect.8 Despite these advances, the applications of nanodrugs remain limited by their lack of specificity, low target efficacy, poor permeability toward solid tumors, and low penetration of biological barriers.9 Therefore, the development of multiintelligent nanocarrier platforms is still urgently needed for targeted drug-delivery applications. Cell-penetrating peptides (CPPs), also known as “TrojanHorse” peptides, are highly cationic peptides that are usually rich in arginine and lysine amino acids. CPPs have the remarkable ability to cross cellular plasma membranes quickly for entry into almost any live cell.10−12 Frequently involved CPPs are composed of the transactivating (Tat) protein of HIV-1, the homeodomain (penetratin) of Antennapedia, Antennapedia (Antp), VP22, transportan, model amphipathic peptide MAP, signal sequence-based peptide sequence, and synthetic polyarginines.13 Polyarginine peptide is one of the most effective CPPs, which has been reported to present better translocation ability (∼20-fold) than popular TAT49‑57 (RKKRRQRRR) originating from HIV-1 virus.14 Moreover, polyarginine CPP has been shown to deliver a series of cargoes, including liposomes,15 micelles,16 siRNA,17 imaging agents,18 quantum B
DOI: 10.1021/acsami.7b15523 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Ltd. (Darmstadt, Germany). All other chemicals were of analytical or high-performance liquid chromatography (HPLC) grade. The Int PMAR9 peptide (Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Pro-Leu-GlyLeu-Ala-Gly-Asn-Gly-Arg), R9 CPP peptide (Cys-Arg-Arg-Arg-ArgArg-Arg-Arg-Arg-Arg), and NGR peptide (Asn-Gly-Arg) were customsynthesized via a standard Fmoc solid-phase peptide synthesis method by KareBay Biochem, Inc. (Shanghai, China). The purities of the peptides were 98.93% (Int PMA-R9), 98.11% (R9), and 97.79% (NGR). 2.2. Cells and Animals. HT-1080 (human fibrosarcoma) cells were purchased from the Cell Culture Centre, Peking Union Medical College (Beijing, China). The cells were cultured in Minimum Essential Medium with Earle’s salts, L-Glutamine, nonessential amino acids (Macgene), 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Female BALB/c nude mice (22−24 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and kept under specific-pathogen-free conditions for 1 week before the study with free access to standard food and water. All care and handling of animals was performed with approval of the Ethics Committee of Jilin Medical University. 2.3. Synthesis and Characterization of Int PMA-R9-PEG2000DSPE Conjugate. For selective Int PMA-R9-modified liposomes, Int PMA-R9 peptide was conjugated with N-[(3-maleimide-1-oxopropyl) aminopropyl poly(ethylene glycol)-carbamyl] distearoylphosphatidylethanolamine by the Michael addition reaction for the production of a functional compound Int PMA-R9-PEG2000-DSPE. Int PMA-R9 (8.5 mg) and maleimide-PEG2000-DSPE (10 mg) with molar ratio 1:1 were added into 2 mL of HEPES buffer solution (pH 7.2, 20 mM) that had been deoxidized for 30 min in advance. The reaction solution was stirred mildly at 4 °C for 24 h under protection of N2 gas. After 24 h incubation, a dialysis bag (cutoff molecular weight (MW) of 2000 Da) was used to hold the reacted solution. A 48 h dialysis procedure was applied to exclude unreacted raw material. Lyophilization was performed for the final solution, and lyophilized products were preserved under 20 °C. The linkage of Int PMA-R9 with PEG2000-DSPE was affirmed by thinlayer chromatography (TLC) using a developing solvent system of chloroform/methanol mixture (4/1, v/v). The TLC plates were then visualized using Dragendorff’s reagent stain (prepared on-site using a U.S. Pharmacopeia protocol) to detect the PEG chain and sprayed with ninhydrin stain to detect peptides. Molecular weights (MWs) of products were monitored by a matrix-assisted laser desorption ionization-orthogonal time-of-flight mass spectrometry (MALDI-TOF MS). 2.4. Synthesis and Characterization of the R9-PEG2000-DSPE Conjugate. For achieving unselective R9-modified liposomes (control group), R9 CPP peptide was conjugated with maleimide-PEG2000-DSPE by Michael addition to yield the functional conjugate R9-PEG2000-DSPE. R9 (6.5 mg) and maleimide-PEG2000-DSPE (10 mg) with molar ratio 1:1 were added into 2 mL of previously deoxidized HEPES buffer solution. The products were monitored with TLC and MALDI-TOF mass spectrometry and were purified and isolated using dialysis and lyophilization according to the aforementioned procedures (Section 2.3). 2.5. Preparation of Various DOX-Loaded Liposomal Nanocarriers. Liposomes contained soybean phosphatidylcholine (SPC), cholesterol (Chol), or other functional conjugates. Lipids of SPC/Chol (20:10, w/w), SPC/Chol/Int PMA-R9-PEG2000-DSPE (20:10:1, w/w), and SPC/Chol/R9-PEG2000-DSPE (20:10:1, w/w) were the components of common DOX-loaded liposomes (cLip-DOX), Int PMA-R9modified DOX-loaded liposomes (Int PMA-R9-Lip-DOX), and R9modified DOX-loaded liposomes (R9-Lip-DOX), respectively. Liposomes were prepared by thin-lipid-film hydration, followed by sonication. Briefly, lipids above were dissolved in chloroform and dried until a thin lipid film formed on a rotary evaporator (Yarong RE52, Shanghai, China) under reduced pressure. The dried lipid film was hydrated with 300 mM ammonium sulfate and sonicated using a bathtype sonicator. The liposome suspension was eluted using a Sephadex G-50 column preequilibrated with 20 mM HEPES buffer solution containing 150 mM NaCl (HBS, pH 7.4) to form an ammonium sulfate gradient. DOX was remote-loaded via the ammonium sulfate gradient method.34
have been used to specifically bind with targeted receptors (integrin αvβ3 and CD13) that are oversecreted by endothelial cells or human tumors.27 A wide variety of drug-delivery systems have frequently been reported by the decoration of CTPcontaining peptides.28−30 Protease-cleavable substrate peptides (SPs) are another tool, which are amino acid sequences or linkers that are vulnerable to cleavage by upregulated proteases during tumor or other disease progression. Proteolysis is a simple hydrolytic process that separates two adjacent amino acid residues at the level of the amide bond.31 Several drug-delivery systems or molecular probes incorporating protease-sensitive SPs have been crafted to manage diseases (e.g., disease treatment, detection, and imaging) in a controlled manner for biomedical applications.32 Matrix metalloproteinases (MMPs) play critical roles in cancer invasion and metastasis. MMP-2 and MMP-9 are currently the species with the best-established associations with tumor grade/poor prognosis and with relatively specific substrate sequences. Thus, MMP-2 and MMP-9 are two of the most extensively utilized cleavable enzymes in drug-delivery system design.33 Herein, a rational and smart strategy was employed to create a more intelligent delivery system to improve the selectivity of CPP-nanocarriers toward cancer cells by using a combination of both CTP and SP strategies. We designate these multifunctional combinatorial peptides as intelligent “peptide-gathering mechanical arm” (Int PMA) peptides. A nonselectively targeted R9liposome cargo was tethered to Int PMA, generating an Int PMAR9-liposome with higher selectivity. The schematic of an Int PMA-R9-liposome delivery system is shown in Figure 1. The nanocarrier platform includes five units: the cell-penetrating domain (oligoarginine, R9), the MMP-2/9-sensitive cleavable substrate peptide (−PLGLAG−), the cell-targeting peptide (NGR), a circulation-stable protective poly(ethylene glycol) (PEG) chain, and a nanoliposome loaded with antitumor agent doxorubicin. Using this design, Int PMA was anticipated to enhance specific cellular uptake of R9-liposome cargo by selective ligand−receptor binding and substrate−enzyme cleavage biased toward tumor tissues, where MMP-2/9 proteases and CD13 are overexpressed. Engineered nanocarriers with an elaborately designed combination of various functional peptides are endowed with five types of intelligence: traditional EPR effect (derived from nanoparticles), long-circulation properties (from an existing PEG protective layer), responsiveness toward two tumor microenvironmental triggers (CD13 receptor and MMP2/9 enzyme), and ability to penetrate membrane barriers in tumor cells or tissues. This multi-intelligent nanocarrier based on functional peptides holds great promise for the development of nanomedicines for targeted tumor therapy.
2. MATERIALS AND METHODS 2.1. Materials. N-[(3-Maleimide-1-oxopropyl) aminopropyl poly(ethylene glycol)-carbamyl] distearoylphosphatidyl-ethanolamine (maleimide-PEG2000-DSPE) was supplied by NOF Corporation (Tokyo, Japan). Soybean phosphatidylcholine (SPC) was purchased from LIPOID Company (Germany). Doxorubicin hydrochloride (DOX) was provided by Zhejiang Hisun Pharmaceutical Co., Ltd. (Zhejiang, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4-(2-hydroxyethyl)piperazine-1-erhanesulfonic acid (HEPES), cholesterol (Chol), and collagenase IV were purchased from Sigma-Aldrich (St. Louis, MO). GM6001 (MMP-2/9 inhibitor) was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY). Trypsin, penicillin, streptomycin, and fluorescent probe Hoechst 33258 were provided by Macgene Biotech Co., Ltd. (Beijing, China). 4Aminophenylmercuric acetate (APMA) was obtained from Merck Co., C
DOI: 10.1021/acsami.7b15523 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces 2.6. Physicochemical Characteristics of Various DOX-Loaded Liposomal Nanocarriers. 2.6.1. Particle Sizes, Zeta Potentials, and Morphology. We used a Malvern Zetasizer (Malvern, U.K.) to determine the mean diameter of various formulations, including cLipDOX, R9-Lip-DOX, and Int PMA-R9-Lip-DOX based on dynamic light scattering (DLS) at 25 °C. Zeta potentials of the samples were also measured by this apparatus. Each sample was detected for three times (n = 3). Transmission electron microscopy (TEM) was applied to observe the micromorphological structure of samples. 2.6.2. Encapsulation Efficiency. The encapsulation efficiency of all liposomes was measured as described below. Briefly, the final liposomes were passed through a Sephadex G-50 column to remove free DOX, followed by disruption with 10% Triton X-100 (v/v); the DOX in the liposomes was then measured in a spectrofluorometer (RF-5301PC; Shimadzu Corp., Nakagyo-ku, Kyoto, Japan). In addition, the same quality of liposomes was treated as above, except they were passed through a Sephadex G-50 column to obtain the total concentration of DOX. The encapsulation efficiency was calculated by the following formula
were preincubated with excess MMP-2/9 for 4 h to cleave substrate peptide (PLGLAG) within the Int PMA-R9 moiety. After the incubation for 20 h, the medium was removed and the cells were washed with cold PBS. GM6001, a specific MMP-2/9 inhibitor, was present through the process at 250 ng/mL. MMP-2/9 (collagenase IV) was activated with 2.5 mM APMA solution for 1 h at 37 °C. For the competition experiment, the cells were preincubated with excess free NGR peptide (1 mg/mL) for 4 h to saturate the cellular surface receptor (CD13), and then co-incubated with Int PMA-R9-Lip-DOX (DOX, 20 μg/mL) for another 6 h. For MMP-2/9-sensitivity and competition experiments using R9-Lip-DOX, the cells were preincubated with 0.1 mg/mL MMP2/9 or NGR for 4 h and then co-incubated with R9-Lip-DOX (DOX, 20 μg/mL) for another 6 h. Flow cytometry analysis was carried out as described in Section 2.9.1. 2.10. Cellular Location under Various Triggers Detected by Confocal Laser Scanning Microscopy (CLSM). 2.10.1. Cellular Location under Endogenous Triggers. After adherent culturing on a Petri dish for 24 h, HT-1080 cells were treated with free DOX, cLipDOX, R9-Lip-DOX, or Int PMA-R9-Lip-DOX (each containing 2.5 μg/ mL DOX) mixed in culture medium for 24 h at 37 °C. PBS (pH 7.4) was added to wash cells three times. A fixation procedure was carried out for 10 min by adding PBS solution containing 4% paraformaldehyde into cells at room temperature. The fixed cells were imaged using CLSM (Leica, Heidelberg, Germany). 2.10.2. Cellular Location under Exogenous Triggers by CLSM. The sensitivity of Int PMA-R9-Lip-DOX to triggers was analyzed by adding exogenous MMP-2/9, MMP-2/9 inhibitor (GM6001), or free NGR peptide (competitively combined with receptor CD13) using CLSM. For MMP-2/9-sensitivity and competition experiments, the cells were preincubated with 0.1 mg/mL MMP-2/9 (with or without 250 ng/mL GM6001 inhibitor) or 0.1 mg/mL NGR for 4 h, followed by coincubation with various liposomes for 24 h. For MMP-2/9-sensitivity and competition experiments using R9-Lip-DOX, the cells were preincubated with excess MMP-2/9 or NGR for 4 h, followed by coincubation with R9-Lip-DOX for another 24 h. The same CLSM analysis was followed as in Section 2.10.1. 2.11. Subcellular Localization. To determine which organelles are involved in the cytoplasmic distribution of the functional nanoliposomes, we performed triple-labeling experiments of HT-1080 cells, followed by confocal microscopy. The localization of coumarin-6 or various coumarin-6 nanoliposomes in subcellular organelles was visualized by labeling the cells with fluorescent probes specific for each specific subcellular organelle. HT-1080 cells were seeded onto a Petri dish and cultured for 24 h at 37 °C in the presence of 5% CO2, followed by addition of 1.0 μg/mL free coumarin-6 (Cou-6), coumarin-6-loaded liposomes (cLip-Cou-6), coumarin-6-loaded R9-modified liposomes (R9-Lip-Cou-6), or coumarin-6-loaded Int PMA-R9-modified liposomes (Int PMA-R9-Lip-Cou-6). The cells were further incubated for 2 h. The drug-containing medium was removed and the cells were washed with PBS. Next, the cells were stained with organelle-selective dyes (Molecular Probes, Eugene, OR). Lysosomes, mitochondria, endoplasmic reticulum (ER), and Golgi apparatus were visualized by staining cells with 50 nM LysoTracker Red DND-99, 200 nM MitoTracker Red CMXRos, 1 mM ER-Tracker Red (BODIPY TR Glibenclamide), and 5 mM Golgi-Tracker Red (BODIPY TR C5-Ceramide) for 30 min each. The cells loaded with organelle markers were washed with PBS, then cell nuclei were stained with Hoechst 33258 (1 μM) for 3 min, the cells were washed twice with PBS, and observed using CLSM. These procedures were performed gently to avoid detachment of adherent cells. 2.12. Apoptosis Assay. The Annexin V-FITC/propidium iodide (PI) kit was employed to measure apoptotic and necrotic cells induced by DOX formulations. Annexin V-FITC was able to bind with phosphatidyl serine that had transversed from the inner to outer plasma membrane leaflet during the apoptotic stage, whereas necrotic cells were stained with propidium iodide (PI). HT-1080 cells with various DOX formulations at concentrations of 0.5, 5, and 15 μg/mL were incubated for 24 h. Next, the medium was collected and the treated cells were trypsinized to detach them from the bottom of the plate using trypsin solution and suspended in fresh medium. The cells were centrifuged at 4000 rpm for 5 min and then resuspended in 150 μL medium. Annexin
encapsulation efficiency DOX concentration in the filtered liposomes = × 100 DOX concentration in the unfiltered liposomes 2.7. In Vitro Release Measurement. A dialysis approach was adopted to explore the in vitro release profiles of DOX in various formulations. A dialysis bag with MW cutoff of 3500 Da was used to hold 1 mL of liposomal solutions through sealing them at both ends. The bag containing samples was placed into phosphate-buffered saline (PBS) (pH 7.4) and dialyzed with stirring under 37 °C. At predetermined time intervals, aliquots were withdrawn and replaced with an equal volume of fresh medium. The DOX concentrations were calculated based on the fluorescence absorbance intensity of DOX as measured by excitation at 485 nm. The cumulative release of DOX was recorded for 24 h in various groups. 2.8. Cleavage of Int PMA-R9 by an Exogenous MMP-2/9 Trigger. To analyze the enzymatic cleavage of matrix metalloproteinase (MMP-2/9)-sensitive peptide (Int PMA-R9), MMP-2/9-mediated cleavage was studied in the presence of MMP-2/9 in pH 7.4 phosphate-buffered saline (PBS) solution. MMP-2/9 (collagenase IV) was activated with the 2.5 mM 4-aminophenylmercuric acetate (APMA) solution for 1 h at 37 °C. Int PMA-R9 stock solution was mixed with activated MMP-2/9 and incubated at 37 °C. The aliquots were removed after incubation for 1 and 3 h and analyzed by HPLC. The HPLC system employed a Diamonsil ODS C18 column (250 mm × 4.6 mm, 5 mm) on an LC-20A HPLC system (Shimadzu), and chromatograms were detected at 220 nm on the basis of isocratic solvent conditions (solvent A, 0.05% trifluoroacetic acid (TFA) in acetonitrile; solvent B, 0.05% TFA in water; A/B = 20:80, v/v) using a flow rate of 1.0 mL/min at room temperature. 2.9. Cellular Uptake toward Various Triggers Measured by Flow Cytometry. 2.9.1. Cellular Uptake toward Endogenous Triggers. Cellular uptake profiles were analyzed for various DOXrelated formulations (cLip-DOX, R9-Lip-DOX, and Int PMA-R9-LipDOX) using flow cytometry. Typically, six-well plates containing seeded HT-1080 cells (5 × 105 cells/well) were placed and cultured at an incubator (37 °C) for 24 h. PBS (pH 7.4) solution was added in wells, and attached cells were washed two times to exclude growth medium. Then, the cells were treated with serum-free medium predissolved with different samples with a DOX concentration of 5 μg/mL. After the treatment for 20 h, cold PBS solution was added to wash the cells three times. These cells were mixed with 0.5 mL of PBS and resuspended. A flow cytometer (Becton Dickinson) was adopted to determine the fluorescence value of samples. A total of 5000 events were recorded for each flow cytometry readout. 2.9.2. Cellular Uptake toward Exogenous Triggers. The sensitivity of Int PMA-R9-Lip-DOX to triggers was analyzed by adding exogenous MMP-2/9, MMP-2/9 inhibitor (GM6001), or free NGR peptide (competitively combined with receptor CD13) using flow cytometry. For the MMP-2/9-sensitivity testing of Int PMA-R9-Lip-DOX, the cells D
DOI: 10.1021/acsami.7b15523 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 2. Schematic representation of Int PMA-R9 and R9 CPP conjugation with maleimide-PEG2000-DSPE by way of Michael addition (nucleophilic addition) between the cysteine sulfur with maleimide. The reaction was performed in HEPES solution (pH 7.2) deoxidized in advance at 4 °C for 24 h under protection of N2 gas to promote formation of functional conjugates by preventing oxidation of maleimide-PEG2000-DSPE. V-FITC and PI (50 μL) solution were added, and the cells were incubated in the dark for 20 min at room temperature. Finally, the cells were analyzed within 1 h by flow cytometry (MUSE Cell Analyzer, Merck, Germany). 2.13. Caspase 3/7 Activation. Caspase 3/7 activation was evaluated using the MUSE Caspase 3/7 Kit that is specifically designed for use with MUSE Cell Analyzer (Merck). HT-1080 cells with various DOX formulations at dosages of 5 and 15 μg/mL were incubated for 24 h; then, the medium was removed and the treated cells were detached using trypsin solution and suspended in fresh medium. The cells were centrifuged at 4000 rpm for 5 min and resuspended with MUSE Caspase 3/7 working solution. Next, 5 μL of MUSE Caspase 3/7 working solution was added to the 50 μL of cell suspension and then the suspension was incubated at 37 °C for 30 min. 7-AAD working solution (150 μL) was then added and mixed thoroughly. The cell suspension was then analyzed using a MUSE Cell Analyzer. 2.14. Cytotoxicity Assay. In vitro cytotoxicity of different samples was assessed by MTT approach. 96-Well plates containing HT-1080 cells (5000 cells per well) were established and subjected to incubation for 24 h. Next, the cells were incubated by free DOX, cLip-DOX, R9-LipDOX, or Int PMA-R9-Lip-DOX with altered concentrations at 37 °C. In addition, for MMP-2/9-sensitive cytotoxicity testing of Int PMA-R9-LipDOX, MMP-2/9 (0.1 mg/mL) was selectively incubated with the cells in this process. The cells were incubated with treatments for indicated time periods (24 h) and then the viability of the cells was determined by MTT assay. MTT (5 mL, 5 mg/mL) dissolved in PBS (pH 7.4) was added to each well. The plates were incubated for an additional 4 h at 37 °C and then the medium was discarded. Thereafter, 200 mL of dimethyl sulfoxide was added to each well to dissolve the formazan crystals while vigorously agitating the plates using an automated shaker. The absorbance of each well was read on a Bio-Rad 680 microplate reader (Bio-Rad, Hercules, CA) at a test wavelength of 570 nm. In this assay, all of the experiments were done using six replicates. The results were described as ratio (the absorbance of samples vs the absorbance of the
culture medium (control)). The percentage of cell growth inhibition was calculated as follows: survival rate = A570sample/A570control × 100%. 2.15. Transmembrane Mechanisms. Transmembrane transit mechanisms of released R9-liposome cargoes after dual recognition by MMP-2/9 and CD13 were investigated using various treatments. To completely inhibit energy-dependent endocytosis, preincubation was performed at 4 °C for 30 min and the cells were exposed to complete DEME and cultured for another 12 h at 4 °C with R9-Lip-DOX. The cells were then prepared for flow cytometry analysis. To suppress specific endocytic routes, chlorpromazine (20 μM), chloroquine (100 μM), amiloride (50 μM), β-cyclodextrin (100 μM), or heparin (10 μM) was added into the cells. All chemical dilutions were made in serum-free medium and incubated for 30 min, followed by rapid addition of R9-LipDOX at 37 °C for an additional 12 h. Next, the cells were subjected to flow cytometric analysis (as in Section 2.8). All experiments were performed in triplicate. 2.16. Animal Models. For in vivo investigation, an animal model of human fibrosarcoma was established by inoculation of 0.2 mL of HT1080 cell suspension (5 × 106 cells) into the right armpit of female BALB/c nude mice. The HT-1080 tumor line was chosen due to its dual overexpression of both triggers (MMP-2/9 protease35,36 and CD13 receptor37,38) according to published reports.35−38 2.17. In Vivo Antitumor Efficacy. Antitumor efficacies were compared using the HT-1080 tumor animal model. When the tumor reached ∼50 mm3, the animals were administrated with control group (saline), cLip-DOX, R9-Lip-DOX, and Int PMA-R9-Lip-DOX using tail vein injection method (n = 7). Administration dosage was 1.0 mg DOX/ kg every other day for three times, and the animals were observed for 15 days. Tumor volume (V) was determined according to following equation: V = [(a)2 × b]/2 (a denotes the width of the tumor and b denotes the length of the tumor). Relative tumor volume was calculated using the formula V/Vi, where V represents practical tumor volume and Vi represents the initial tumor volume. Body weights were monitored during the period. E
DOI: 10.1021/acsami.7b15523 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces Table 1. Characteristics of cLip-DOX, R9-Lip-DOX, and Int PMA-R9-Lip-DOXa groups (abbreviations) DOX-loaded common liposomes (cLip-DOX) R9-mediated DOX-loaded liposomes (R9-Lip-DOX) Int PMA-R9-mediated DOX-loaded liposomes (Int PMA-R9-Lip-DOX) a
mean size (nm)
polydispersity index (PDI)
zeta potential (mv)
encapsulation efficiency (%)
150.3 ± 6.9 145.3 ± 4.3 158.6 ± 7.2
0.149 ± 0.013 0.168 ± 0.016 0.117 ± 0.020
−2.67 ± 0.14 0.998 ± 0.19 3.49 ± 0.48
96.25 ± 0.26 96.81 ± 0.19 97.01 ± 0.15
Data are represented as mean ± SD (n = 3).
2.18. Histological Analysis. Major organs were fixed in 4% formalin, embedded in paraffin, and then sectioned. Sections of 7 μm thickness were mounted on glass slides and stained with hematoxylin/ eosin (H&E) and examined by light microscopy. 2.19. Data Analysis. Data were expressed as mean ± standard deviation (SD). The difference between any two groups was determined by ANOVA, and p < 0.05 was considered to be statistically significant.
PEG2000-DSPE. In HEPES solution, at pH 7.2, the maleimide group of maleimide-PEG2000-DSPE efficiently reacts with the sulfhydryl group of cysteine-modified peptides. Deoxidization via ultrasonic treatment with protection by N2 gas helps to promote formation of functional polymers by preventing oxidation of maleimide-PEG2000-DSPE. After conjugation, purification, and staining, because of their increased hydrophilicity, Int PMA-R9PEG2000-DSPE (Figure S1A) and R9-PEG2000-DSPE (Figure S1B) remained near the starting point on the TLC plates. Next, MALDI-TOF MS analyses were used to measure molecular weight (MW) and check the correctness of synthesized products. In Figure S1C, maleimide-PEG2000-DSPE shows peaks at 2900− 3100 Da that approximate the calculated MW of 2984 Da. The theoretical molecular weights of R9 (Figure S1D) and Int PMAR9 (Figure S1E) were 1528 and 2364 Da, respectively. After conjugation, the MWs of Int PMA-R9-PEG2000-DSPE (Figure S1F) and R9-PEG2000-DSPE (Figure S1G) were determined to be 5345.66 and 4509.21 Da, respectively. These observed values are close to the calculated MWs of 5347 Da (Int PMA-R9PEG2000-DSPE) and 4512 Da (R9-PEG2000-DSPE). Thus, according to the well-designed synthetic route confirmed by TLC and MALDI-TOF MS results, functional derivatives of Int PMA-R9-PEG2000-DSPE and R9-PEG2000-DSPE were synthesized successfully. 3.2. Preparation and Characterization of Liposomal Formulations. Three types of liposomes were formed by the remote loading method using an ammonium sulfate gradient, including cLip-DOX, Int PMA-R9-Lip-DOX, and R9-Lip-DOX (Table 1). To form Int PMA-R9-Lip-DOX and R9-Lip-DOX, Int PMA-R9-PEG2000-DSPE, and R9-PEG2000-DSPE were individually added into lipid materials. The threshold vesicle size for the extravasation into a tumor’s extracellular space has been shown to be approximately 400 nm,45 and the recommended drugdelivery system (
