Building Stable MMP2-Responsive Multifunctional Polymeric Micelles

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Building stable MMP2-responsive multifunctional polymeric micelles by an all-in-one polymer-lipid conjugate for tumor-targeted intracellular drug delivery Qing Yao, Zhi Dai, Jong Hoon Choi, Dongin Kim, and Lin Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09511 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 5, 2017

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ACS Applied Materials & Interfaces

Building stable MMP2-responsive multifunctional polymeric micelles by an all-in-one polymer-lipid conjugate for tumor-targeted intracellular drug delivery Qing Yao1, 2, †, Zhi Dai1, †, Jong Hoon Choi1, Dongin Kim1, and Lin Zhu1,* 1

Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M University Health Science Center, Kingsville, Texas 78363, United States 2

Department of Pharmaceutics, College of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, China † These authors contributed equally to this work.

* Corresponding author: Lin Zhu, Ph.D. Assistant Professor, Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M University Health Science Center 1010 West Ave. B, MSC 131, Kingsville, Texas 78363, USA Phone: (361)221-0757 Fax: (361)221-0793 Email: [email protected]

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Abstract In this study, we described an “all-in-one” polymer-lipid conjugate (PEG2k-ppTAT-PEG1k-PE) that could self-assemble to matrix metalloproteinase 2 (MMP2)-sensitive multifunctional micelles. The assembled micelles had several key features, including a protective long chain polyethylene glycol (PEG2k) (the outer shell), an MMP2-sensitive peptide linker (pp) (the tumor-targeting middle layer), a trans-activating transcriptional activator (TAT) peptide (the cell penetrating middle layer), and a stable PEG1k-PE micelle for drug loading (the inner core). In the absence of MMP2, the PEG2k-ppTAT-PEG1k-PE micelles were intact and showed low bioactivity due to the surface-anchored PEG2k, while in the presence of MMP2, the pp was cleaved, resulting in the PEG2k deshielding and exposure of the previously hidden TAT for enhanced intracellular drug delivery. Even if completely cleaved by MMP2, the remaining PEG1k-PE micelles were stable and the micelle’s particle size and drug release were not significantly influenced. The paclitaxel (PTX)-loaded PEG2k-ppTAT-PEG1k-PE micelles showed significant MMP2-depedent cellular uptake, tumor penetration and anticancer activity in various cancer cells and 3D multicellular spheroids. Due to the enhanced intracellular drug accumulation, these multifunctional micelles were able to sensitize the drug-resistant cancer cells and their spheroids to PTX treatments. Furthermore, in vivo tumor uptake and retention data indicated that the PEG2k-ppTAT-PEG1k-PE micelles could dramatically increase the residence time of their payloads in the tumor.

Keywords: multifunctional nanocarrier, polymeric micelles, stimuli-responsive, matrix metalloproteinase, cell-penetrating peptide.


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1.

INTRODUCTION

Undesirable physicochemical properties, low tumor targeting, insufficient cell internalization, and multidrug resistance (MDR) are the unmet needs of most anticancer drugs. Nanotechnology-based drug delivery systems have drawn a lot of attention due to their ability to improve tumor specificity, enhance drug efficacy, and decrease side effects 1, such as Doxil® (liposomal doxorubicin) and Abraxane® (protein-bound paclitaxel) 2. However, their intrinsic nature, such as “passive” tumor targeting and low cellular uptake, unavoidably compromise their anticancer activity. Although further modification of nanoparticles by monoclonal antibodies and small molecules can improve drugs’ tumor targetability and efficacy to some extent, their overall clinical outcomes remain unsatisfactory. More and more drug delivery scientists have realized that the traditional delivery strategies dealing with only one or two aspects of anticancer drugs, such as bioavailability and specificity, might not satisfy the overall clinical needs. The multifunctional drug delivery systems which can simultaneously tackle the aforementioned issues become one of the major current research focuses. However, most multifunctional systems contain multiple components and have complex architecture which make quality control and characterization difficult. A multifunctional but simple drug delivery system will be a critical need. The idea of stimuli-responsive drug delivery comes from the abnormalities of the diseased tissues and cells 3. In the tumor microenvironment, the matrix metalloproteinases (MMPs), including MMP2 and MMP9, are usually up-regulated 4. MMPs are zinc and calcium-dependent proteolytic enzymes and responsible for degrading most extracellular matrix (ECM) proteins and regulating the activity of other proteinases, growth factors, and cell receptors as well. They have been extensively explored as cancer biomarkers for diagnosis and prognosis as well as the therapeutic targets for development of the MMP inhibitors 5. Most recently, they have been used as the robust tumor environmental stimuli for the stimuli-responsive delivery of drugs and imaging agents to the tumor 6-13. For most anticancer drugs, intracellular drug delivery is required because they must reach their intracellular targets to exert pharmacological actions, rather than be released and stuck in the extracellular space. Cell penetrating peptides (CPPs), such as the trans-activating transcriptional activator (TAT) peptide, which are short peptides of less than 30 amino acids with the ability to translocate across the plasma membrane and deliver a wide range of cargoes, including macromolecules and nanoparticles 14. However, the CPP-decorated nanoparticles usually show low disease specificity 15. To decrease the CPPs’ positive charge-induced nonspecific biodistribution 16, the activatable CPP (ACPP) was proposed, in which the polyarginine (positive segment) was coupled with a polyanion via an enzyme-sensitive peptide linker 11-13. The intramolecular electrostatic interaction could block the non-specific cell internalization. The positive charge plays an important role in CPPs’ membrane traslocation, but it is not the only one 17 . Therefore, the efficiency of the ACPP strategy may be compromised when used to decrease the nonspecific distribution of the CPPs, such as TAT peptides, who enter cells via various mechanisms 18.



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Polyethylene glycol (PEG) a flexible hydrophilic polymer, is well-known to improve drug solubility and stability, prolong drug’s blood circulation, and prevent drug’s nonspecific biodistribution, via the “steric hindrance” 19. But, many studies, including our previous ones, indicated that the PEG corona might interfere drug uptake and subcellular events. Therefore, it’s better to remove the protective PEG shell before cell internalization 6, 9-10. We have designed several MMP2-sensitive TAT-modified PEGylated multicomponent nanoparticles including liposomes 10 and micelles 6, 8. Among them, the micelles composed of an MMP2-sensitive polymer-drug conjugate (PEG2k-pp-PTX) 6 or polymer-lipid conjugate (PEG2k-pp-PE) 8, a cell penetrating polymer (TAT-PEG1k-PE), and a micelle building block (PEG1k-PE), showed the decreased nonspecific biodistribution and improved tumor specificity and anticancer activity. To “Keep it simple, Stupid”, in this study, we developed a novel self-assembling “all-in-one” polymer-lipid conjugate (PEG2k-ppTAT-PEG1k-PE) for tumor-targeted intracellular drug delivery. By itself, the multifunctional micelles could be formed. Like the aforementioned multifunctional multicomponent micelles 6, 8, the PEG2k-ppTAT-PEG1k-PE-assembled micelles possessed several key features, including the protective long chain PEGs (PEG2k) (the outer shell), MMP2-sensitive peptides (the tumor-targeting middle layer), TAT peptides (the cell penetrating middle layer), and a stable micelle (PEG1k-PE) (the inner core) (Figure 1A).In this study, the conjugates’ synthesis and characterization were described. Using the poorly water-soluble drug, paclitaxel (PTX), as a model drug, the physicochemical properties, stability/sensitivity, and drug release of the PEG2k-ppTAT-PEG1k-PE-assembled micelles were studied. The cellular uptake and anticancer activity of the PEG2k-ppTAT-PEG1k-PE micelles were determined in human cancer cells with various aspects (MMP2+ vs. MMP2-; drug sensitive vs. resistant). Then, the tumor penetration and anticancer activity of the micelles were evaluated in the 3D cancer cell spheroids. Furthermore, the tumor uptake and retention of the micelles were studied in tumor-bearing mice.


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Figure 1. (A) Drug delivery mechanisms of the PEG2k-ppTAT-PEG1k-PE micelles (stable multifunctional micelles) and PEG2k-ppTAT-PE micelles (unstable micelles). a, self-assembly; b, MMP2-induced cleavage and PEG2k deshielding; c, TAT exposure on the stable micelles; d, extracellular micelle dissociation; e, TAT-mediated intracellular uptake; f, passive drug diffusion; and g, endosomal escape and intracellular drug release. (B) Synthesis schemes of PEG2k-ppTAT-PEG1k-PE and PEG2k-ppTAT-PE. 2. MATERIALS AND METHODS 2.1. Materials Polyethylene glycol 2000-succinimidyl valerate (PEG2k-SVA, average MW 1900Da) and maleimide-PEG1k-DSPE (MAL-PEG1k-PE, average MW of PEG 900Da) were from Laysan Bio, Inc. (Arab, AL). 1,2-dioleoylsn- glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rh-PE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 4-(N-Maleimidomethyl) cyclohexane carboxylic acid N-hydroxysuccinimide ester (SMCC), chloroform, dichloromethane (DCM), dimethylformamide (DMF), methanol, and acetonitrile (ACN) were obtained from Thermo Fisher Scientific (Rockford, IL). Collagenase Type IV was from Sigma-Aldrich Chemicals (St. Louis, MO). Human active MMP2 protein (MW 66000 Da), Bovine serum albumin (BSA) and TLC plate (silica gel 60 F254) were from EMD Biosciences (La Jolla, CA). 1,1'-dioctadecyl-3,3,3',3'- tetramethylindotricarbocyanine iodide (DiR) was from Marker Gene Technologies, Inc (Eugene, OR). Dialysis tubings (MWCO 8000 and 12000-14000 Da) were from Spectrum Laboratories, Inc. (Houston, TX). Fetal bovine serum (FBS) was purchased from VWR International (Radnor, PA). Hank’s Balanced Salt Solution (HBSS) was obtained from Mediatech (Manassas,VA). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute medium (RPMI)-1640, penicillin-streptomycin solution, trypsin-EDTA and Hoechst 33258 were purchased from Invitrogen Corporation (Carlsbad, CA). The MMP2-cleavable peptide, ppTAT (GPLGIAGQYGRKKRRQRRRC) was synthesized by the NeoScientific, Inc. (Cambridge, MA). The CellTiter-Blue® Cell Viability Assay kit was from Promega (Fitchburg, WI). The human fibrosarcoma (HT1080), breast cancer (MDA-MB-231 and MCF-7) and MDR ovarian cancer (NCI/ADR-RES) cells were grown in the DMEM with 10% FBS and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) at 37 ºC and 5% CO2. The human non-small cell lung cancer (A549) and cervical cancer (HeLa) cells were grown in the RPMI1640 with 10% FBS and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) at 37 ºC and 5% CO2 2.2. Synthesis of PEG2k-ppTAT-PEG1k-PE and PEG2k-ppTAT-PE PEG2k-ppTAT-PEG1k-PE was synthesized according to the method reported in the ref. 7 with some modifications. Two steps were involved (Figure 1B). First, the cysteine-modified ppTAT (ppTAT-Cys) was reacted with the maleimide (MAL) group of MAL-PEG1k-PE in DMF with a trace amount of trimethylamine (TEA) at room temperature for 12h. The reaction mixture was dialyzed (MWCO 8000 Da) in water for 48 h to remove the unreacted ppTAT. The ppTAT-PEG1k-PE was lyophilized. Then, ppTAT-PEG1k-PE was reacted with the PEG2k-SVA, in DMF in the presence of TEA overnight, followed by dialysis (MWCO 8000 Da) against water



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and lyophilization. The final product PEG2k-ppTAT-PEG1k-PE was characterized by the thin layer chromatography (TLC) and 1H NMR using CDCl3 as solvent. Three steps were involved in the synthesis of PEG2k-ppTAT-PE (Figure 1B). First, DOPE and SMCC were reacted in the DMF and TEA at room temperature for 12h. The obtained MAL-PE was purified using the preparative TLC with the solvent system composed of chloroform and methanol (5:1, v/v). Second, the ppTAT-Cys was reacted with MAL-PE in DMF in the presence of TEA at room temperature for 12 h. The product ppTAT-PE was dialyzed (MWCO 8000 Da) in water for 48 h to remove the unreacted ppTAT. Third, the ppTAT-PE was reacted with PEG2k-SVA in DMF in the presence of TEA at room temperature overnight. The final product PEG2k-ppTAT-PE was dialyzed (MWCO 8000 Da) in water for 48 h and characterized by the TLC and 1H NMR using CDCl3 as solvent. 2.3. Measurement of critical micelle concentration (CMC) The fluorescence spectroscopy was used to measure the CMC 20. First, the pyrene chloroform solution was added to the test tubes and dried overnight and then the polymer HBSS solution was added. The final concentration of pyrene was 8×10-5M. The polymer concentration was in the range of 10-9 to10-4 M. The mixture was incubated in a shaker at room temperature. After 24 h, the fluorescence was measured on a microplate reader (Tecan Inﬁnite® M1000 PRO) at the excitation wavelengths of 338 nm (I338) and 334 nm (I334) and the emission wavelength of 390 nm. The fluorescence intensity ratio (1338/I334) was plotted as a function of logarithm of polymer concentration. The crossover point of two tangents is considered as the CMC. To study the influence of the serum on the CMC, the nanopreparation was hydrated by HBSS containing 50% mouse serum. The fluorescence intensity was measured on an F-2000 fluorescence spectrometer (Hitachi) with the excitation wavelengths (λex) of 338 nm (I3) and 334 nm (I1) and an emission wavelength (λem) of 390 nm. The intensity ratio (I338 / I334) was calculated and plotted against the logarithm of the micelle concentration. The CMC value was obtained as the crossover point of the two tangents of the curves. 2.4. Preparation of PTX-loaded polymeric micelles The PTX-loaded micelles were prepared by our previously reported film-hydration methods 8, 20. First, PTX (100 µg) and conjugates (2 mg) were co-dissolved in chloroform (1 mL). After removal of solvent, the formed drug-polymer film was hydrated in 1 mL HBSS by vortexing. The micelles were filtered through a 0.45 μm syringe filter (Agela Technologies Inc.) to remove the unentrapped drug. The encapsulated PTX was measured by the same HPLC method in ref 20. The drug loading (DL) and encapsulation efficiency (EE) were calculated by the following equations. DL (%) = EE (%) =

×100 % ×100 %

2.5. Particle size and zeta potential


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The polymeric micelles were diluted by PBS and their particle size was measured by dynamic light scattering (DLS) on a NanoBrook 90Plus PALS (Brookhaven Instruments) at 25 ºC. The zeta potential of the micelles was measured by the same instrument. 2.6. Morphological observations The morphology of the micelles was examined using transmission electron microscopy (TEM). Briefly, one drop of the micelle solution was placed on a 400-mesh carbon coated copper grid (Ted Pella, Inc., USA). The micrographs were recorded on a JEOL JEM-2010 TEM (JEOL, Japan). 2.7. MMP2-mediated cleavage of the PEG2k-ppTAT-PEG1k-PE micelles Two mg/mL of the polymers were incubated with active human MMP2 (10 ng/mL) in pH 7.4 HBS (HEPES buffered saline) containing 10 mM CaCl2 at 37 ºC for 12 h. The MMP2-mediated cleavage and the resultant PEG2k deshielding were determined by the TLC. The samples were run in the solvent system of chloroform and methanol (8:2, v/v), and then stained with the Dragendorff’s reagent6, 9. 2.8. In vitro drug release The in vitro PTX release profile from the polymeric micelles was carried out according to the dialysis method 20. One milliliter of the PTX-loaded micelles was placed in a dialysis bag (MWCO 12000-14000 Da). The bag was stirred in 30 mL PBS containing Tween 80 (0.5%, w/v) at 37 ºC to achieve the “sink” condition. The released PTX was quantitated by HPLC. 20 To investigate the effect of the MMP2-mediated cleavage on the drug release rate, the micelles were preincubated with Collagenase Type IV (50 µg/mL) or BSA at 37 ºC for 2h prior to the drug release study. 2.9. Cellular uptake of the polymeric micelles The Rh-PE was used as a fluorescent indicator to prepare the micelles at a concentration of 2.5 μg/mL. To study the effect of MMP2, the Rh-PE-labeled PEG2k-ppTAT-PEG1k-PE micelles (2 mg/mL) were pretreated with human MMP2 (10 ng/µL) in pH 7.4 HBS containing 10 mM CaCl2 at 37 ºC overnight. Before experiment, the A549 cells were seeded at a density of 1×105 cells/well in 24-well plates for 24 h. After washed with PBS, the cells were incubated with Rh-PE-labeled micelles in serum-free medium for 2 h. Then, the cells were washed three times by serum-free medium. For the fluorescence-activated cell sorting (FACS) analysis, the treated cells were harvested by trypsinization and centrifugation (600 g for 4 min). The collected cells were washed with PBS and resuspended in 200μL PBS, followed by analysis on a BD Accuri C6 flow cytometer. The dead cells and cell debris were excluded from viable cells using the forward scatter (FSC) and side scatter (SSC). For the fluorescence microscopy, the cells were fixed with 4% paraformaldehyde for 10 min. The cell nuclei were stained by Hoechst 33258 (2 μM) for 1 min. The cells were observed on a Nikon ECLIPSE 80i fluorescence microscope system at a 400× magnification.



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2.10. Cellular uptake of the micellar PTX The A549 and MCF-7 cells were seeded in 6-well plates at 4×105 cells/well 24 h before experiments. The PTX-loaded micelles were incubated with the cells at 37°C for 4 h. The cells were trypsinized and collected by centrifugation at 600 g for 4 min. The collected cells were washed with ice-cold PBS for four times to remove any uninternalized PTX. Then, the cells were lysed by 1% Triton X-100 in PBS and 150 μL of cell lysate was mixed with 300 μL of acetonitrile by ultrasonication for drug extraction and protein precipitation. The mixture was centrifuged at 8000 g for 10 minutes, and the supernatant was analyzed by HPLC. 2.11. Cytotoxicity study The cytotoxicity of the formulations on A549, HeLa, HT1080， MCF-7, NCI/ADR-RES and MDA-MB-231 cells was performed by CellTiter-Blue® Cell Viability Assay (Promega) 20. The cells were first seeded at 2×103 cells/well in 96-well plates for 24 h. The cells were incubated with the micelles in the complete growth medium for 72 h. Then, the medium was replaced by 100 µL fresh complete growth medium containing 10 µL of CellTiter-Blue® Reagent and incubated at 37 ºC for 2 h. Finally, the fluorescence intensity was recorded on a microplate reader at λex 560 nm and λ em 590 nm. 2.12. Penetration study on tumor cell spheroids The NCI/ADR-RES multicellular spheroids were established by a liquid overlay method according to [25]. Briefly, the 96-well plates were pre-coated with 1.5% agarose (w/v) in the culture medium. Then, the cell suspensions (9×104cells / 200μL per well) were transferred into the agarose-coated plates and maintained at 37 °C for spheroid formation. The spheroids were identified by their size and morphology. The 4-5 day spheroids with a diameter of 400–600 μm were selected for the penetration study. To study the impact of the MMP2-mediated cleavage on tumor penetration, the Rh-PE (10 μg/mL) labeled micelles with/without the collagenase IV (50 µg/mL) pretreatment were incubated with the spheroids for 2h. Then, the medium was removed and the spheroids were gently washed with PBS for 3 times, followed by observation on a Nikon Eclipse Ti confocal microscope at a 100× magnification. The Z-stack images were obtained at a fixed interval of 25μm. The fluorescence intensity of each image was analyzed by ImageJ software. The curves of the normalized fluorescence intensity vs. the distance from the spheroid periphery were plotted. To study the effect of the endogenous (cell-secreted) MMP2 on micelles’ behavior on the spheroids, the Rh-PE (1μg/mL) labeled micelles were incubated with the spheroids for 24 h, followed by imaging with confocal microscopy. 2.13. Anticancer activity study on tumor cell spheroids The anticancer effects including growth inhibition and cytotoxicity of the formulations on the spheroids were investigated. Here, low cell number (6×104cells / 200μL per well) was used to generate the NCI/ADR-RES spheroids. On day 3 after seeding cells, the PTX formulations were


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added to the spheroids and incubated for additional 12 days. The untreated spheroid was used as a control. The morphology and size of the spheroids were recorded on a confocal microscope a 100× magnification every day. To study the cytotoxicity on the cell spheroids, the formulations were incubated with the 4-5 day spheroids for 72 h, followed by CellTiter-Blue® Cell Viability Assay. 2.14. In vivo tumor uptake and retention study For the in vivo study, 6-8-week athymic nude (BALB/c nu/nu) mice (Charles River Laboratories, Wilmington, MA) were housed at 5 mice/cage and maintained according to an approved protocol from the Texas A&M University Institutional Animal Care and Use Committee. The mice were randomly grouped at 3 mice per group. The tumor xenografts were initiated by subcutaneously implanting 100 µL of NCI/ADR-RES (5×106) cell suspensions in the right rear flank of the mice. The tumor was measured by a caliper. The tumor volume was calculated by the equation, V = (w2×l)/2, where w and l are the width and length of the tumor. When the tumor volume reached 100~300 mm3, each mouse was treated with 100 µL of DiR dye (25 µg)-encapsulated micelles by intratumor injection. A 100 µL of DiR dye (25 µg) itself served as a control. Using an in vivo molecular imaging instrument (Kodak Imaging Station In-vivo F/FX, MA), the mice were scanned to measure the fluorescence intensity of DiR in the tumor at different time points after injection. The DiR intensity was determined in the region encompassing each tumor area at each time point. 2.15. Statistical analysis All experiments were carried out in triplicate and the obtained data were processed using GraphPad Prism 6 software (GraphPad Software, Inc.). The difference between two groups was analyzed using unpaired t-test in this software. P value < 0.05 was considered statistically significant. 3.

RESULTS AND DISCUSSION

The biological membrane, such as cell membrane, is a barrier for most anticancer drugs to exert their therapeutic effects. The TAT peptide has been widely used as an intracellular penetrating enhancer for a variety of cargos, including molecules and particles, while its effectiveness is compromised by its interaction with various biomolecules nonspecifically 15. To utilize TAT’s cell penetrating capability but minimize its downsides, we have designed several multifunctional multicomponent nanoparticles, by which the TAT-mediated cancer cell uptake of nanoparticles could be controlled by the up-regulated MMP2 6, 8, 10. Based on these work, in our recent study, we have designed a new peptide sequence, ppTAT, possessing both the MMP2 sensitivity and cell penetrating capability 7. This peptide has been well studied as the linker in a doxorubicin conjugate (PEG-ppTAT-DOX) 7. However, the formation of the drug conjugate might decrease the drug efficacy and limit the applicability of the idea to other drugs. Here, we developed an “all-in-one” polymer-lipid conjugate, PEG2k-ppTAT-PEG1k-PE, that was able to self-assemble to stable multifunctional polymeric micelles for physical loading of hydrophobic drugs without compromising their drug efficacy. The commercial PTX formulation (Taxol®), non-MMP2 sensitive micelles (PEG5k-PE, with a similar MW as PEG2k-ppTAT-PEG1k-PE) and unstable MMP2-sensitive micelles (PEG2k-ppTAT-PE) were used as the controls.



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3.1. Synthesis of PEG2k-ppTAT-PEG1k-PE and PEG2k-ppTAT- PE In the previous work, we successfully synthesized TAT-PEG1k-PE 6, 8, 10 and PEG2k-ppTAT-Dox 7 . Here, the similar methods were used for the synthesis of PEG2k-ppTAT-PEG1k-PE. The cysteine of ppTAT (GPLGIAGQYGRKKRRQRRRC) was first reacted with maleimide (MAL) of MAL-PEG1k-PE, and then the amine of ppTAT-PEG1k-PE was reacted with PEG2k-SVA. After reactions, the uncoupled ppTAT and PEG2k were removed by dialysis. To synthesize the PEG2k-ppTAT-PE, the synthesis method of the PEG-pp-PE polymers 20 was used. The DOPE was first maleimidylated and reacted with the cysteine of ppTAT. Then, the amine of the ppTAT-PE was reacted with PEG2k-SVA. After conjugation and purification, these conjugates were identified by the thin layer chromatography (TLC) (Figure S1). The PEG of the conjugates could be stained by the Dragendorff's reagent, while the PE could be stained by the molybdenum blue reagent 6, 10. The data indicated that the conjugation reactions of PEG2k-ppTAT-PEG1k-PE (Figure S1A) and PEG2k-ppTAT-PE (Figure S1B) were successful. All the conjugates were also characterized by the 1H NMR in CDCl3 as shown in Figure 2. The -CH2-CH2-O- in PEG was identified by the peak at 3.6 ppm 7. The peaks at 1.3 ppm were assigned to the -CH2- protons of PE 20. The characteristic peaks of ppTAT were presented at around 0.8, 2.0 and 3.0 ppm 7. The results were consistent with the reported NMR results of the PEG-pp-PE and TAT-PEG1k-PE conjugates 7-8, 20 . After integration, the areas of the PEG peak in ppTAT-PEG1k-PE, PEG2k-ppTAT-PEG1k-PE, and PEG2k-ppTAT-PE were 68.8, 246.3, and 161.0, respectively, when the area of the PE peak was assigned as 70. Based on the integration, the actual PEG1k/PE ratios in these polymers were 0.98:1, 3.58:1, and 2.34:1, respectively. Based on the vendor-provided MW of the PEG2k (~ 1900Da) and PEG1k (~ 900Da), the theoretical PEG1k/PE ratios were around 1:1 (ppTAT-PEG1k-PE), 3.11:1 (PEG2k-ppTAT-PEG1k-PE), and 2.11:1 (PEG2k-ppTAT-PE). The characteristic peaks and integration data indicated that all conjugates were successfully synthesized.


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Figure 2. 1H NMR of ppTAT-PEG1k-PE (a), PEG2k-ppTAT-PEG1k-PE (b), ppTAT-PE (c) and PEG2k-ppTAT-PE (d). 3.2. Micelle formation and drug loading It was well known that the PEG-PE polymers had excellent self-assembly capability and could form the nanomicelles for loading hydrophobic drugs 2. To study the influence of the incorporation of ppTAT on the polymer’s amphiphilicity and confirm the micelle formation, the critical micelle concentration (CMC) of the polymers was measured (Figure 3A). The synthesized polymers, ppTAT-PEG1k-PE, PEG2k-ppTAT-PEG1k-PE, and PEG2k-ppTAT-PE, could solubilize the hydrophobic pyrene and showed the significant inflection points in the curves of the intensity ratio (I338/I334) vs. the logarithm of polymer concentrations, indicating the existence of CMC. The CMC values of both PEG2k-ppTAT-PEG1k-PE (2.0×10−7 M) and ppTAT-PEG1k-PE (8.4×10-7 M) were lower than the reported CMC of the popularly used commercial PEG-PE polymers (10-6 ~ 10-5 M) 21, such as the PEG5k-PE (3.1×10-6 M) (Table 1), indicating their excellent self-assembly and micelle formation capability. The PEG2k-ppTAT-PE polymer had a CMC value of 2.2×10−6 M, which was close to the CMC of the PEG-pp-PE polymers in our recent work 20, conforming that the insert of the peptide (ppTAT) into PEG-PE didn’t significantly influence the polymers’ self-assembly. Here, lower CMC values of PEG2k-ppTAT-PEG1k-PE and ppTAT-PEG1k-PE than PEG2k-ppTAT-PE suggested that the modification of the terminus of the PEG-PE polymers might benefit the micelle formation compared to the modification in the middle of the PEG-PE polymers. The data also indicated that the PEG deshielding from the



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PEG2k-ppTAT-PEG1k-PE might not dramatically change the self-assembly and micelle formation capability of the rest part of the polymer (ppTAT-PEG1k-PE). Due to excellent micelle formation, the poorly water-soluble drug, PTX, was able to be loaded to the micelles’ lipid core by hydrophobic interaction. All synthesized polymers could efficiently carry PTX with a more than 3.0 % drug loading (Table 1). For the PEG2k-ppTAT-PEG1k-PE-assembled micelles, the drug loading was about 4.0 % and drug encapsulation efficiency was about 80% according to the HPLC analysis, which was pretty similar to the drug loading capacity of the PEG5k-PE micelles (4.2%). We also noted that the ppTAT-PEG1k-PE micelles had slightly lower drug loading (3.2%) compared to the PEG2k-ppTAT-PEG1k-PE micelles, probably due to their positive charge-caused repulsion. Because the ppTAT was a positively charged peptide at the normal physiological pH 7, the ppTAT-PEG1k-PE-formed micelles showed the positive zeta potential of around 14.9±1.5 mV. Further modification of ppTAT-PEG1k-PE with PEG2k shielded its charge and the resultant PEG2k-ppTAT-PEG1k-PE micelles had a near neutral zeta potential (1.9±0.2 mV), which was similar to that of the PEG5k-PE micelles (1.4±0.1mV). The data confirmed our previous findings that the charged molecules, including TAT 7 and PEI 9, could be shielded via the PEGylation. Similarly, the zeta potential of the PEG2k-ppTAT-PE micelles was also near neutral (2.4±0.3 mV). The data suggested that the TAT’s charge-induced membrane translocation might be inhibited at least in part via the PEGylation. Like most of the amphiphilic polymers, all the synthesized PEG-PE derivatives could self-assemble to nanoscale particles. Based on the particle size (Table 1), the PEG5k-PE and PEG2k-ppTAT-PE micelles had the similar size of 50-60 nm, while the PEG2k-ppTAT-PEG1k-PE micelles were about 90 nm. The large size of PEG2k-ppTAT-PEG1k-PE micelles might be due to the high PEG intensity / thick PEG shell (PEG2k and PEG1k), the micelle’s architecture, and the equilibrium between PE’s hydrophobic force/attraction and positively charged TAT-induced repulsion 22. The PEG2k-ppTAT-PEG1k-PE micelles had a near spherical shape and narrow size distribution, as evidenced by the TEM micrographs and DLS-based size distribution data (Figure 3B). It was also worth noting that ppTAT-PEG1k-PE could self-assemble into a micellar nanoparticle with a relatively larger particle size (142.6±2.1 nm), probably due to the increased repulsion between the exposed positively charged TAT, consistent with the drug loading data. Although having a slightly large size, ppTAT-PEG1k-PE’s micelle formation capability would not be influenced, as evidenced by its small CMC. The appropriate particle size, morphology, zeta potential, and drug loading implied that the PEG-ppTAT-PEG1k-PE micelles would be a great nanocarrier for in vivo tumor-targeted drug delivery 23.


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Figure 3. (A) Critical micelle concentrations (CMC) of PEG2k-ppTAT-PEG1k-PE (a), PEG2k-ppTAT-PE (b), and ppTAT-PEG1k-PE (c), measured by fluorescence spectroscopy. (B) Particle size (DLS) and morphology (TEM) of the PEG2k-ppTAT-PEG1k-PE (a) and PEG2k-ppTAT-PE (b) micelles. Table 1 Summary of polymers’ properties CMC

Zeta potential

Particle size

Drug loading

(μM)

(mV)

(nm)

(%)

PEG2k-ppTAT-PEG1k-PE

0.2

1.9±0.2

90.0±0.4

4.0±0.3

PEG2k-ppTAT-PE

2.2

2.4±0.3

51.2±0.9

3.8±0.2

ppTAT-PEG1k-PE

0.8

14.9±1.5

142.6±2.1

3.2±0.1

PEG5k-PE

3.1

1.4±0.1

59.0±2.3

4.2±0.1

Polymers

3.3. MMP2-dependent micelle stability and drug release We have approved that the MMP2-sensitive peptide sequence (GPLGIAGQ) could be cleaved at the site between G (glycine) and I (isoleucine) by the MMP2 10 when used as a linker in a synthetic peptide in the nanoparticles 8-10 and drug conjugates 6-7. Here, PEG2k-ppTAT-PEG1k-PE could be also cleaved by the active human MMP2 and its PEG2k was deshielded thereafter. Consistent with previous studies, 6b, 6d the released PEG moiety (PEG-GPLG) was visualized as a spot with an Rf value higher than that of the original conjugate on the TLC plate (Figure 4A)7, 9. The drug release from the polymeric micelles was investigated using the dialysis method under the simulated “sink” condition (0.5% Tween 80 in pH 7.4 PBS at 37 °C). For better understanding of the drug release behaviors, the micelle stability (particle size as an indicator) was studied. The collagenase IV, which was obtained from Clostridium histolyticum, was employed to study the impact of the cleavage on the micelle stability and cargo release, instead of MMP2 due to the



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cost 20. The release profiles and particle sizes of the PTX-loaded micelles after incubation with collagenase IV or BSA were shown in Figures 4 B and C. The PEG2k-ppTAT-PEG1k-PE micelles showed a sustained drug release and the incubation of the micelles with either BSA or collagenase IV didn’t significantly influence their drug release pattern (b vs. c) (Figure 4B). Based on the particle size data, the collagenase incubation only slightly increased the size of the PEG2k-ppTAT-PEG1k-PE micelles from 93.4±0.4 to 102.4±6.2 nm (Figure 4C). These data indicated that the micellar structure of the PEG2k-ppTAT-PEG1k-PE micelles was remained even after the MMP2-mediated cleavage and the remaining (IAGQ-TAT-PEG1k-PE) micelles could efficiently hold the hydrophobic drugs. Here, after cleavage, the size of the micelles (IAGQ-TAT-PEG1k-PE) was a little bit smaller than the ppTAT-PEG1k-PE micelles (102.4±6.2 vs. 142.6±2.1 nm) (Table 1), probably due to the shortened peptide length after cleavage. We also noticed that the ppTAT-PEG1k-PE micelles had a relatively lower drug loading than PEG2k-ppTAT-PEG1k-PE micelles (3.2±0.1% vs 4.0±0.3%) (Table 1). However, it didn’t have the remarkable influence on the micelle stability and drug release within 24h (Figure 4B). In contrast, the PEG2k-ppTAT-PE micelles showed a significant initial burst release (~20% with BSA incubation and ~35% with collagenase incubation) compared to the PEG2k-ppTAT-PEG1k-PE micelles (200ng/400000cells), indicating that the PEG2k-ppTAT-PEG1k-PE micelles could not benefit drug uptake without MMP2, while the PEG2k-ppTAT-PE micelles had lower drug uptake (~170 ng), probably due to their initial burst release (Figure 4B). In the A549 cells, the PEG2k-ppTAT-PEG1k-PE micelles had significantly higher drug uptake (>270 ng) compared to the PEG5k-PE micelles (~190 ng) and Taxol® (~170 ng), consistent with the micelle uptake data (Figure 5A-B). The data suggested that the cell-secreted MMP2 was able to induce the pp cleavage, PEG2k deshielding and TAT exposure, resulting in the enhanced drug uptake. High uptake of the PEG2k-ppTAT-PEG1k-PE micelles was most likely secured by their high micelle stability and low drug leakage (Figure 4B-C). In contrast, the drug uptake of the PEG2k-ppTAT-PE micelles was much lower (~100 ng) than other groups (Figure 5C) although they contained both the MMP2-sensitive and cell penetrating moieties. Due to low stability especially in the presence of MMP2, the PEG2k-ppTAT-PE micelles underwent rapid micelle dissociation and drug release (Figure 4B-C) before they could be internalized by the A549 cells. All cellular uptake data indicated that the endogenous MMP2 in the MMP2-expessing cancer cells would be sufficient to trigger the PEG deshielding and TAT-mediated cellular uptake and the PEG2k-ppTAT-PEG1k-PE micelles was stable enough to resist the conformational or architectural change during the cell internalization process. We might speculate that the effective nanoparticles must “survive” not only in the bloodstream but also during the subsequent cellular events, particularly the internalization process, to ensure the intracellular drug delivery rather than extracellular drug leakage.

Figure 5. Cellular uptake of the Rh-PE-labeled polymer micelles after 2h incubation with the A549 cells, determined by FACS (A) and fluorescence microscopy (B). a PEG2k-ppTAT-PEG1k-PE; b, PEG2k-ppTAT-PEG1k-PE with MMP2 preincubation; c, PEG5k-PE; and e, untreated. (C) Cellular uptake of the micellar drug (paclitaxel) after 4h incubation with A549 or MCF-7 cells, determined by HPLC. Data were expressed as mean ± SD. *p < 0.05; **p < 0.01; n.s., non-significant. 3.5. MMP2-dependent cytotoxicity of the polymeric micelles The anticancer activity of the polymeric micelles were tested on several cancer cell lines expressing various levels of MMP2, including the MMP2+ cells (A549, HeLa and HT1080), and


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MMP2- cells (MCF-7). In the MMP2+ cells (a-c, Figure 6), the PEG2k-ppTAT-PEG1k-PE micelles had a high drug response, similar to Taxol®. Based on the 50% inhibitory concentrations (IC50) listed in Table 2, the PEG2k-ppTAT-PEG1k-PE micelles could bring down the IC50 of PTX for more than one-fold compared with the PEG5k-PE and PEG2k-ppTAT-PE micelles. Here, Taxol® showed high cytotoxicity in the long time (72h) cytotoxicity study (Figure 6) in agreement with 32 although its cellular uptake was lower than that of the PEG2k-ppTAT-PEG1k-PE micelles in the short time (4h) uptake study (Figure 5C). The PEG5k-PE micelles had the similar drug uptake as Taxol® but showed much lower drug response, most likely because of their delayed drug release 20. The PEG2k-ppTAT-PEG1k-PE micelles also had a slower drug release (Figure 4B), however, their high cellular uptake especially in the presence of MMP2 (Figure 5) increased drug response. In contrast, the MMP2 sensitivity and cell penetrating capability of the PEG2k-ppTAT-PE micelles were significantly compromised by their low stability and substantial drug leakage (Figure 4B-C) in the presence of MMP2, resulting in low drug uptake (Figure 5C) and cytotoxicity (a-c, Figure 5). In the MMP2- cells, however, all polymeric micelles were structurally stable and showed relatively lower drug uptake (Figure 5C) and resultant lower cytotoxicity compared to Taxol® (d, Figure 6). The different drug responses in these cells were mainly due to their different levels of extracellular MMP2, in agreement with previous observations 7. The data confirmed that the MMP2-responsive TAT exposure played a key role in cell internalization and cytotoxicity of the PEG2k-ppTAT-PEG1k-PE micelles and the cell-secreted MMP2 was sufficient to control these functions. Here, we would like to point out that the long-term incubation reduced the difference between the PEG2k-ppTAT-PEG1k-PE micelles and Taxol® and the quick uptake of the PEG2k-ppTAT-PEG1k-PE micelles would be more efficient for in vivo drug delivery which is a “dynamic” process rather than “static” cell incubation. 3.6. Overcoming drug resistance by PEG2k-ppTAT-PEG1k-PE micelles Insufficient intracellular drug concentration due to the reduced drug uptake and/or increased drug efflux is one of the major reasons of MDR 33. Cell penetrating peptides, e.g. TAT, have been widely used in the nanoparticles for intracellular delivery of drugs 34, while some polymeric micelles, including PEG-PE micelles 8, 20, exhibited the capability of reversing the efflux-induced drug resistance. The PEG2k-ppTAT-PEG1k-PE micelles possessed both characteristics and would be effective when treating the drug-resistant cancers. To investigate if the polymeric micelles are able to overcome the drug resistance, the MDR ovarian (NCI/ADR-RES) and PTX-resistant breast (MDA-MB-231) cancer cells were used as cell models (e-f, Figure 6). The previous studies indicated that they were MMP2+ 7-8, 20. Unlike high drug response in the PTX-sensitive cells, Taxol® showed the lowest cytotoxicity in these resistant cells. The retrospective comparison with free PTX 8, 20 confirmed that Taxol® could not overcome the drug resistance. All the PEG-PE-based polymeric micelles showed the improved cytotoxicity compared to Taxol®, mainly due to their capability to interfere the drug uptake and efflux. Among them, the unstable micelles (PEG2k-ppTAT-PE) had lower cytotoxicity than the stable micelles (PEG5k-PE and PEG2k-ppTAT-PEG1k-PE), indicating the importance of the micelle stability in anticancer treatment. In contrast, the PEG2k-ppTAT-PEG1k-PE micelles showing high stability and low drug leakage even after the MMP2-mediated cleavage (Figure 4B-C), secured their high drug uptake (Figure 5). Moreover, their drug holding and sustained drug release (Figure 4B) were



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able to bypass the drug efflux 35. As a result, the internalized PTX was dramatically accumulated in the intracellular compartment, resulting in the highest cytotoxicity in the drug-resistant cells, although the drug resistance mechanisms in the NCI/ADR-RES and MDA-MB-231 cells were not exactly identical36-37.

Figure 6. Cytotoxicity of the micellar PTX in the drug-sensitive cancer cells: A549 (a), HeLa (b), HT1080 (c), and MCF-7 (d) cells, and in the PTX-resistant cancer cells: NCI/ADR-RES (e) and MDA-MB-231 (f) cells. The CellTiter-Blue® Cell Viability Assay was performed after 72 h incubation. Table 2. The IC50 of the PTX-loaded micelles in the tested cancer cells (nM).


Page 18 of 31

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Micelles

A549

HeLa

HT1080

MCF-7

NCI/ADR-RES

MDA-MB-231

84.2±1.2

44.4±0.7

39.0±0.5

51.7±0.8

20354±287

37315±593

91.4±1.3

29.8±0.5

49.3±0.8

87.4±1.2

6177±87

10134±161

PEG2k-ppTAT-PE

249. 0±3.0

96.24±1.4

102.1±1.4

68.3±0.9

13969±166

22724±315

PEG5k-PE

209.8±2.6

79.66±1.2

92.2±1.3

88.8±1.2

10560±131

17198±258

Taxol

®

PEG2k-ppTAT-PEG1k-PE

3.7. Penetration of the polymeric micelles through cancer cell spheroids The epigenetic and genetic alterations of cancer cells and the tumor heterogeneous microenvironment pose significant challenges to clinical applications of anticancer therapeutics. The data obtained on 2D cell cultures may not tell all behaviors of therapeutics, especially the tumor/tissue penetration and response to the cell-cell/cell-matrix interaction in the tumor microenvironment 38. In this regard, the in vitro 3D cell model, e.g. multicellular spheroids, is developed to mimic the characteristics of solid tumors including poor drug penetration 6, drug resistance 38, pH and hypoxic gradients 39-40, etc.. Here, the multicellular spheroids generated from the MDR NCI/ADR-RES cancer cells were spherical dense cell aggregates with a diameter of around 400 - 600 μm, which were appropriate for studying the drug or nanoparticle penetration and drug response 6, 39. After 2h incubation, the PEG5k-PE micelles were observed only at the periphery of the cell spheroids, as evidenced by the dark spheroid core and weak surrounding red fluorescence (Figure 7A), indicating their limited penetrating ability, although their size was the smallest among the tested micelles (Table 1). Both the PEG2k-ppTAT-PEG1k-PE micelles and PEG2k-ppTAT-PE micelles could penetrate the spheroids and be taken up by cancer cells to a significant extent. The exogenous MMP2 preincubation substantially improved the penetration and cellular uptake of the PEG2k-ppTAT-PEG1k-PE micelles in the spheroids, as evidenced by stronger fluorescence even in the core of the spheroids (Figure 7A), because of their exposed TAT and stable micellar structure, while the MMP2 preincubation couldn’t improve the penetration of the PEG2k-ppTAT-PE micelles significantly, mainly due to the micelle dissociation. The fluorescence intensity vs. penetration distance curves (Figure 7B) also showed that both the overall cellular uptake (the area under curve) and penetration capability and depth (the height of the peak and the distance from the periphery) of the MMP2-preincubated PEG2k-ppTAT-PEG1k-PE micelles were greater than those of other groups. In the cell monolayers, the cell-secreted (endogenous) MMP2 could increase the drug uptake of the PEG2k-ppTAT-PEG1k-PE micelles (Figure 5C). Here, this effect was also investigated in the NCI/ADR-RES spheroids. The cell spheroids were treated with the Rh-PE labeled micelles without MMP2 preincubation for 24 h. Prolongation of incubation time allowed for deeper penetration and enhanced spheroid uptake of all micelles, in agreement with 41. Among them, the PEG2k-ppTAT-PEG1k-PE micelles showed the deepest penetration and highest uptake in the spheroids (Figure 7 C-D), suggesting that the cell-secreted MMP2 in the spheroids could also cleave the peptide, deshield the PEG2k, and expose the TAT, leading to the enhanced micelle penetration and uptake in the 3D cell structure.



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Figure 7. Penetration of the Rh-PE labeled micelles through the NCI/ADR-RES spheroids. (A) Z-stack images of the spheroids and (B) the fluorescence intensity vs. penetration distance curves after 2h incubation with the micelles or MMP2-preincubated micelles; (C) Z-stack images and (D) the fluorescence intensity vs. penetration distance curves after 24h incubation with the micelles without MMP2-preincubation. The scale bar represents 200 μm. ***,

Building Stable MMP2-Responsive Multifunctional Polymeric Micelles

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