MMP2-Sensitive PEG–Lipid Copolymers: A New ... - ACS Publications

Research Article www.acsami.org

MMP2-Sensitive PEG−Lipid Copolymers: A New Type of TumorTargeted P‑Glycoprotein Inhibitor Zhi Dai,† Qing Yao,†,‡ and Lin Zhu*,† †

Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M University Health Science Center, Kingsville, Texas 78363, United States ‡ Department of Pharmaceutics, College of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, People’s Republic of China S Supporting Information *

ABSTRACT: Low tumor targetability and multidrug resistance (MDR) are two major impediments to the success of cancer treatments. Nanomaterials which possess high tumor targetability and the ability to reverse the MDR are rare. This report describes a new type of self-assembling polyethylene glycol-phosphoethanolamine-based copolymers (PEG-pp-PE) which showed both the matrix metalloproteinase 2 (MMP2)-sensitive tumor-targeted drug delivery and ability to inhibit the Pglycoprotein (P-gp)-mediated drug efflux. In this study, we synthesized a series of the homologous analogues of PEG-pp-PE copolymers and investigated the influence of their structures, including PEG lengths and peptide linkers, on the drug efflux, and identified the underlying mechanisms. We found that the whole structure (PEG-peptide-lipid) rather than any parts of the copolymers was key for the P-gp inhibition and a delicate balance between the hydrophilic and lipophilic segments of the PEGpp-PE copolymers was needed for better modulating the P-gp-mediated drug efflux. The best copolymer, PEG2k-pp-PE, showed even higher P-gp inhibition effect than the D-α-tocopherol polyethylene glycol 1000 succinate (TPGS1k). We also found that the P-gp inhibition capability of PEG-pp-PE copolymers was highly associated with the P-gp down-regulation, the increase in the plasma membrane fluidity, and the inhibition of the P-gp ATPase activity. Besides, the excellent physicochemical properties, high drug loading, MMP2-dependent drug release, and improved drug efficacy in the MDR cancer cells suggested that the PEG-pp-PE copolymers might have great potential for building tumor-targeted drug delivery systems for treating drug-resistant cancers. KEYWORDS: polymeric P-gp inhibitor, matrix metalloproteinase, stimuli-sensitive, tumor targeting, multidrug resistance

1. INTRODUCTION Poor water solubility, low tumor specificity, drug resistance, and side effects lead to poor clinical outcomes of many anticancer drugs. Unsuccessful drug delivery is one of the major reasons. Although the current drug delivery technologies, including cell surface receptor-medicated tumor targeting, e.g., monoclonal antibody and antibody drug conjugate (ADC), and nanotechnology-based therapeutics, e.g., Doxil and Abraxane, could improve the drug bioavailability and tumor targetability, they fail to address the drug resistance. A drug delivery system which can address all of the aforementioned issues is needed. The mechanisms of drug resistance described in cancer fall into two categories, pharmacokinetic resistance, e.g., overexpression of drug efflux pumps, and pharmacodynamic © 2016 American Chemical Society

resistance, e.g., apoptosis resistance or altered survival pathways, where the drug reaches the therapeutic concentration in the tumor site but fails to propagate an appropriate cell death response.1 P-glycoprotein (P-gp), one of the major efflux pumps, belongs to the ATP-binding cassette (ABC) transporter superfamily.2,3 It is usually located on the apical membrane of normal epithelial cells of the liver, placenta, kidney, blood brain barrier (BBB), and intestine, as a membrane detoxification system,4 as well as on cancer cells, responsible for the cancer multidrug resistance (MDR).5,6 These ATP-dependent transReceived: March 12, 2016 Accepted: May 4, 2016 Published: May 4, 2016 12661

DOI: 10.1021/acsami.6b03064 ACS Appl. Mater. Interfaces 2016, 8, 12661−12673

Research Article

ACS Applied Materials & Interfaces

Figure 1. Drug delivery and P-gp inhibition by the PEG-pp-PE micelles.

prognosis as well as a therapeutic target for cancer treatments.22 Several MMP inhibitors were developed over the past decade; however, none of their clinical trials was successful, due to the drug resistance and severe toxicity.23 Most recently, MMP2 has been used as a robust stimulus for tumor-targeted drug delivery.24−27 Using the MMP2-sensitive peptide (pp, GPLGIAGQ), we have prepared several MMP2-sensitive drug conjugates26,27 and nanoparticles25,26,28 and found that the MMP2-sensitive moiety in the drug delivery systems significantly improved the tumor targetability and drug efficacy. In the most recent study,29 we have demonstrated that the multifunctional micellar nanocarriers containing the polyethylene glycol 2k−MMP2-sensitive peptide−1,2-dioleoyl-snglycero-3-phosphoethanolamine (PEG2k-pp-PE, an MMP2labile self-assembling block copolymer), TAT-PEG1k-PE (a cell penetrating moiety, in which the TAT refers to the transactivating transcriptional activator peptide), and PEG1kPE could improve the drug uptake and cytotoxicity in not only drug-sensitive but also -resistant cancer cells. However, the mechanisms remained unknown. Here, the possible drug delivery and drug efflux inhibition mechanisms by the PEGpp-PE micelles were proposed in Figure 1. To test our hypothesis, in the current study, a serial of homologous analogues of PEG-pp-PE copolymers with various PEG chain lengths and peptide sequences were synthesized, and the structure−activity relationships and the underlying mechanisms of the antidrug resistance were identified. Furthermore, the physicochemical properties, MMP2-sensitivity, drug release, and cytotoxicity of the drug-loaded PEG-pp-PE micelles were investigated.

membrane transporter proteins can extrude a wide range of structurally diverse compounds out of the cells, including anticancer agents,7 steroid hormones,8 immunosuppressants,9 cardiac glycosides,10 and calcium channel blockers.11 The development of the MDR is highly associated with the P-gp overexpression on the cancer cells that pose a great challenge to anticancer treatments and also give impetus to inhibit P-gp functions for reducing drug resistance and improving drug efficacy. To overcome the P-gp-mediated drug efflux, the use of the small molecule inhibitors (SMI) of the P-gp was the most common way. Currently, three generations of SMIs are investigated in the preclinical and clinical studies, including the first generation SMIs, e.g., quinine or verapamil,12 second generation SMIs, e.g., the D-isomer of verapamil (dexverapamil),13 and third generation SMIs, e.g., tariquidar.14 Although a wide variety of P-gp SMIs have been discovered, research efforts are underway to identify the most appropriate one in terms of higher tumor specificity and lower toxicity. On the other hand, from a drug delivery point of view, the co-administration/co-delivery of the anticancer drug and its SMI which have different physicochemical and pharmacokinetic properties, is complicated and may cause undesired side effects.15,16 Recently, it has been found that some pharmaceutical polymers used in the drug formulation, including N-(2hydroxypropyl) methacrylamide (HPMA), poly(ethylene oxide)-poly(propylene oxide) block copolymers (Pluronic), and D-α-tocopherol polyethylene glycol 1000 succinate (TPGS1k), could sensitize MDR tumors to some extent.17 For example, TPGS has been extensively used as the polymeric P-gp inhibitor in various nanoparticles, e.g., liposome,18 porous PLGA nanoparticles,19 and other TPGS-based nanomedicine.20 Depending on the types of the polymer and its formed nanoparticles, the behind mechanisms are various, including bypassing the P-gp pathway, interference with ATPase activity, depletion of intracellular ATP, and increase of the membrane fluidity, etc.21 Compared to the SMIs, the polymeric P-gp inhibitors are much safer and the formed polymeric nanoparticles usually show the “passive” tumor specificity due to the EPR effect. So, the use of the polymeric P-gp inhibitors-formed nanoparticles for overcoming drug resistance would be a simple and effective way. The matrix metalloproteinase 2 (MMP2) is an essential protein involved in tissue repair, morphogenesis, and angiogenesis. In most cancer tissues, the MMP2 is up-regulated and has been used as a biomarker for cancer diagnosis and

2. EXPERIMENTAL SECTION 2.1. Materials. Polyethylene glycol 1000−succinimidyl propionate (PEG1k-SPA), polyethylene glycol 2000−succinimidyl valerate (PEG2k-SVA), polyethylene glycol 5000− succinimidyl valerate (PEG5k-SVA), 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000] (PEG1k-PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (PEG2k-PE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (PEG5k-PE) were purchased from Laysan Bio, Inc. (Arab, AL, USA). 1,2-Dioleoyl-sn-glycero-3phosphoethanolamine (DOPE) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Oregon Green 488 Paclitaxel Conjugate (PTX-FL), chloroform, dichloromethane (DCM), and methanol were purchased from Thermo Fisher 12662

DOI: 10.1021/acsami.6b03064 ACS Appl. Mater. Interfaces 2016, 8, 12661−12673

Research Article

ACS Applied Materials & Interfaces

Figure 2. Synthesis of the PEG-pp-PE copolymers.

the copolymers were characterized by 1H NMR spectroscopy using deuterated chloroform as the solvent. 2.3. Determination of Critical Micelle Concentration. The critical micelle concentation (CMC) was determined by the fluorescence spectroscopy using pyrene as a hydrophobic fluorescent probe.28 Briefly, the pyrene chloroform solution was added to the testing tube at the final concentration of 8 × 10−5 M and dried overnight under vacuum. Then, the polymers in HBSS were added to the tubes at a 5-fold serial dilution (from 10−1 to 10−8 mg/mL) and incubated with shaking at room temperature for 24 h before measurement. The fluorescence intensity was measured on a Tecan Infinite M1000 Pro microplate reader with the excitation wavelengths (λex) of 338 and 334 nm and an emission wavelength (λem) of 390 nm. The intensity ratio (I338/I334) was calculated and plotted against the logarithm of the polymer concentration. The CMC value was obtained as the crossover point of the two tangents of the curves.26 2.4. Preparation of PTX-Loaded Polymeric Micelles. To prepare PTX-loaded polymeric micelles, PTX (200 μg) and PEG-pp-PE (2 mg) were dissolved in 1 mL of chloroform and dried to form the drug−polymer film, followed by hydration with 1 mL of HBSS using the vortex. The unentrapped PTX was removed by filtration through a 0.45 μm filter (GE Healthcare). The PTX in the filtrate was measured on a reversed-phase C18 column (250 mm × 4.6 mm) using an isocratic mobile phase of acetonitrile and water (60:40, v/v) at a flow rate of 1.0 mL/min and detected at 227 nm on a Waters HPLC system. 2.5. In Vitro Drug Release. The PTX release rate from the polymeric micelles was studied by a dialysis method.26 Briefly, the PTX-loaded polymeric micelles were dialyzed (MWCO 12000−14000 Da) against 30 mL of PBS containing 0.5% Tween 80 to maintain the sink condition at 37 °C. The released drug was determined by RP-HPLC over 48 h. To study the influence of the cleavage on the drug release, the PTX-loaded polymeric micelles were incubated with the 50 μg/mL collagenase Type IV or BSA at 37 °C for 2 h before the drug release study. 2.6. Particle Size Measurement. The particle size and size distribution of the polymeric micelles were measured by dynamic light scattering (DLS) on the NanoBrook 90Plus PALS particle size and zeta potential analyzer (Brookhaven Instruments). Briefly, 0.5 mg of polymers or PTX-loaded micelles in 1 mL of PBS, or the samples after incubation with the collagenase Type IV or BSA, were measured at 25 °C. 2.7. Fluorescence-Activated Cell Sorting. For fluorescence-activated cell sorting (FACS) analysis, the treated cells were trypsinized and collected by centrifugation at 2000 rpm for 4 min. After washing with ice-cold PBS, the cells were resuspended in 200 μL of PBS and immediately applied on a

Scientific (Rockford, IL, USA). Molybdenum Blue Spray reagent, N,N′-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole hydrate (HOBT), and collagenase Type IV were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). TLC plate (silica gel 60 F254) and bovine serum albumin (BSA) were from EMD Biosciences (La Jolla, CA, USA). Dialysis tubings (MWCO 2000 and 12000−14000 Da) were purchased from Spectrum Laboratories, Inc. (Houston, TX, USA). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute medium (RPMI)-1640, penicillin streptomycin solution (PS), Hoechst 33258, and trypsin-EDTA were from Invitrogen Corp. (Carlsbad, CA, USA). Fetal bovine serum (FBS) and Hank’s balanced salt solution (HBSS) were from Mediatech (Manassas, VA, USA). The MMP2-cleavable peptides, pp (GPLGIAGQ) and pp6 (PLGIAG), and uncleavable peptide, unpp (GGGPALIQ), were synthesized by the Sigma BioSciences Inc. (St. Louis, MO, USA). The Alexa Fluor 488 conjugated mouse anti-human CD243 (UIC2) antibody (anti-P-gp antibody) was from BioRad. 1-Pyrenedodecanoic acid was from Marker Gene Technologies Inc. Luminescent ATP detection assay kit was from Abcam. The Pgp-Glo assay system was from Promega. The multidrug-resistant ovarian cancer (NCI/ADR-RES) and breast cancer cells (MDA-MB-231) were grown in complete growth media (DMEM supplemented with 50 U/ mL penicillin, 50 mg/mL streptomycin, and 10% FBS) at 37 °C in a 5% CO2. The non-small cell lung cancer cells (A549) were grown in complete growth media (RPMI1640 supplemented with 50 U/mL penicillin, 50 mg/mL streptomycin, and 10% FBS) at 37 °C in a 5% CO2. 2.2. Synthesis, Purification, And Characterization of PEG-pp-PE Copolymers. The similar synthesis, purification, and characterization protocols were used for all of the PEG-ppPE copolymers (PEG1k-pp-PE, PEG2k-pp-PE, PEG5k-pp-PE, PEG2k-pp6-PE, and PEG2k-unpp-PE; Figure 2). Here, only the synthesis process of PEG5k-pp-PE was described as an example. First, the MMP2-cleavable peptide, pp (4 mg) was reacted with the PEG derivative, PEG5k-SVA (25.5 mg) at the molar ratio of around 1.1:1 in the DMF in the presence of a trace amount of triethylamine at room temperature overnight. The crude was purified by the dialysis (MWCO 2000 Da) against water for 48 h, followed by the freeze-dry, affording 21.2 mg of PEG5k-pp in ∼73% yield as a white powder. Then, the PEG5k-pp (11.8 mg) was activated by the excess amount of the coupling reagents (DCC/HOBT) and conjugated with DOPE (1.8 mg) at the molar ratio of around 1:1.2 in the chloroform in the presence of a trace amount of triethylamine at room temperature overnight. The product PEG5k-pp-PE was purified by preparative TLC (chloroform/methanol, 4:1, v/v), affording 6.3 mg of PEG5k-pp in ∼47% yield as a white powder. All of 12663

DOI: 10.1021/acsami.6b03064 ACS Appl. Mater. Interfaces 2016, 8, 12661−12673

Research Article

ACS Applied Materials & Interfaces BD Accuri C6 flow cytometer (BD Biosciences). The cells were gated upon acquisition using forward vs side scatter to exclude debris and dead cells. The data were collected (2.5 × 104 cell counts) and analyzed with CFlow Plus software. 2.8. Calcein AM Extrusion Assay. The calcein AM extrusion assay was performed on the NCI/ADR-RES cells according to the manufacturer’s instruction and determined by FACS. Briefly, the NCI/ADR-RES cells were seeded at 1 × 105 cells/well in 24-well plates 24 h before treatments. The cells were first incubated with the PBS, empty polymeric micelles at various concentrations or Verapamil (2.5 μM) at 37 °C for 30 min, and then incubated with 0.5 μM calcein AM at 37 °C for an additional 30 min. The PBS-treated cells were the negative control. The cells were washed with ice-cold PBS for three times, followed by the FACS analysis. 2.9. Fluorescence Microscopy. Briefly, the cells were fixed by 4% paraformaldehyde for 5 min at room temperature. To visualize the cell nuclei, the cells were incubated with 5 μM Hoechst 33258 for 1 min in the dark at room temperature. The photographs were taken with a Nikon eclipse 80i fluorescence microscope system at 400 × magnification and analyzed using the NIS-Elements BR software. 2.10. Drug Uptake and Intracellular Accumulation. The drug uptake and intracellular accumulation of doxorubicin (DOX) and Oregon Green 488 Paclitaxel Conjugate (PTX-FL) in NCI/ADR-RES cells were determined by both the FACS and fluorescence microscopy. Briefly, the NCI/ADR-RES cells were seeded at 2 × 105 cells/well in 12-well plates 24 h before treatments. The cells were first incubated with the PBS or empty polymeric micelles at 37 °C for 0.5 h, and then incubated with 45 μM DOX or 1.5 nM PTX-FL at 37 °C for additional 1.5 h. The PBS-treated cells were the negative control. The cells were washed with ice-cold PBS for three times, followed by the FACS and microscopy. 2.11. Determination of the Cell Surface P-gp Level. The cell surface P-gp levels were labeled using fluorescent antiP-gp monoclonal antibody and analyzed by the FACS.30−32 Briefly, the A549 and NCI/ADR-RES cells were seeded at 1 × 105 cells/well in 24-well plates 24 h before treatments. The cells were first incubated with the PBS or empty polymeric micelles at 37 °C for 1 h. The cells were washed, trypsinized, and resuspended in the PBS containing 0.5% BSA. The cell suspensions were incubated with the Alexa Fluor 488conjugated mouse anti-human (CD243) monoclonal antibody at 4 °C for 40 min. Then, the cells were washed with 0.5% BSA and analyzed by the FACS. 2.12. Measurement of the Membrane Fluidity. The plasma membrane fluidity after the polymer incubation was determined using the 1-pyrenedodecanoic acid as a fluorescent probe.33 Briefly, the NCI/ADR-RES cells were seeded at 1 × 105 cells/well in 24-well plates 24 h before treatments. The cells were first incubated with the PBS or polymers at 37 °C for 0.5 h. At the end of the incubation, cells were washed twice and suspended in PBS. The 1-pyrenedodecanoic acid was added to the cell suspension at a final concentration of 2 μM and incubated for 5 min in the dark. Then, the fluorescence intensity was scanned in a microplate reader at an excitation wavelength of 340 nm and the emission wavelengths from 380 to 580 nm. After scanning, the ratio of the maximum fluorescent intensities of excimer to pyrene monomer was calculated at 475 and 407 nm, respectively. 2.13. Determination of the Intracellular ATP Level. The intracellular ATP levels were analyzed by luminescent ATP

detection assay kit (Abcam). The NCI/ADR-RES cells were preincubated with the assay buffer for 30 min, and then incubated with 28 μM of copolymers in assay buffer for an additional 2 h. After treatments, the cells were washed twice with ice-cold PBS, lysed by Triton X-100 (1.0%), and frozen immediately for the subsequent ATP quantification. The ATP was determined using a luciferin/luciferase assay. Briefly, a 100 μL aliquot of cell lysate was mixed with a 100 μL of ATP assay mix. The luminescence was measured with a microplate reader. The ATP concentrations were obtained according to a standard calibration curve established using the ATP standards and normalized by the total protein content which was quantitated by the BCA assay according to the manufacturer’s instructions. 2.14. P-gp ATPase Activity Assay. The effects of the copolymers on the P-gp ATPase activity were evaluated by the Pgp-Glo assay (Promega). Briefly, 25 μg of human P-gp membranes in 20 μL of assay buffer, 10 μL of 25 mM MgATP, and the copolymers in 20 μL of assay buffer were mixed. After incubation at 37 °C for 40 min, 50 μL of ATP detection reagent was added and incubated for an additional 20 min at room temperature, followed by the luminescence measurement on a microplate reader. In order to investigate the effects of the substrate-induced P-gp ATPase activity, 50 μM verapamil, a Pgp substrate, was added to both control and test groups. All the readings were normalized by the subtraction of the non-P-gp ATPase activity which was determined in the presence of 50 μM sodium orthovanadate. 2.15. Cytotoxicity Study. To study the cytotoxicity of the PTX-loaded polymeric micelles, the A549, NCI/ADR-RES, and MDA-MB-231 cells were seeded at 2 × 103 cells/well in 96-well plates 24 h before treatments. The free PTX or PTX formulations containing the same ratio of polymer/drug were incubated with the cells for 72 h in complete growth media, followed by the cell titer-blue cell viability assay (Promega). Briefly, 10 μL of cell titer-blue reagent was diluted with 90 μL of complete growth media per well and incubated with the cells at 37 °C for 2 h. Thereafter, the fluorescence intensity was recorded at λex 560 nm and λ em 590 nm on a microplate reader. 2.16. Statistical Analysis. Data were presented as mean ± standard deviation (SD). The difference between the groups was analyzed using a one-way ANOVA analysis. P < 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of PEG-pp-PE Copolymers. One of the major aims of the current study was to study the structure vs P-gp inhibition relationship of the PEG-pp-PE copolymers and identify the underlying mechanism. So, a series of the homologous analogues of PEG-pp-PE copolymers with various PEG chain lengths (1k, 2k, and 5k Da) and peptide sequences (pp, pp6, and unpp) are prepared. The PEG2k-pp-PTX26 and PEG2k-pp-PE29 have been successfully synthesized in our previous work. Here, the similar two-step synthesis was used for preparation of all these PEG-pp-PE copolymers (Table 1). The chemical structures of PEG1k-ppPE, PEG5k-pp-PE, PEG2k-pp6-PE, and PEG2k-unpp-PE are confirmed by 1H NMR spectroscopy (solvent, CCl3D) (Supporting Information Figure S-1). The -CH2−CH2−O- in PEG was characterized by the peaks at around 3.65 ppm. The peaks at around 1.30 ppm belonged to the -CH2- protons of PE. The spectra were consistent with the previously reported one of PEG2k-pp-PE,29 indicating that these copolymers were 12664

DOI: 10.1021/acsami.6b03064 ACS Appl. Mater. Interfaces 2016, 8, 12661−12673

Research Article

ACS Applied Materials & Interfaces

could be easily loaded into the lipid core of the PEG-pp-PE micelles via self-assembly. No much difference in the CMC values was observed between the PEG-pp-PE copolymers and the commercial PEG-PE polymers at the same PEG length, indicating their great self-assembly property. The increase in the PEG length significantly decreased the CMC values (PEG1k vs PEG 2k and 5k), in agreement with the previous reports;34 while the PEG2k had a slightly lower CMC value than PEG5k in all PEG-pp-PE or PEG-PE copolymers, probably due to their proper HLB values. For PEG-PE diblock copolymers, the increase in the PEG length increased the particle size.34 For PEG-pp-PE triblock copolymers, interestingly, the increase in the PEG length decreased the particle sizes, suggesting that, unlike the PEG-PE polymers, the PEGpp-PE polymers might undergo the different self-assembly processes and formed different nanoarchitectures in the aqueous environment.35 The peptide linker containing more hydrophobic amino acids than hydrophilic ones might increase the hydrophobicity of the micelle core, resulting in higher drug loading (DL) and encapsulation efficiency (EE): 6.2% DL and 66% EE of PEG2k-pp-PE micelles vs 4.4% DL and 46% EE of PEG2k-PE micelles. 3.3. MMP2-Dependent Drug Release and Particle Size. In previous studies, we proved that the pp could be completely cleaved into two parts by the human MMP2, when used as an MMP2-sensitive linker in various PEG-pp containing polymers25,28,29 or drug conjugates.26,27 In this study, using PEG2k-pp-PE as an example, we prepared the PTX-loaded micelles and studied the influence of the MMP2-mediated cleavage on the micelles’ particle size and drug release. The in vitro drug release from the polymeric micelles was investigated in the simulated physiological environment (PBS, pH 7.4, 37 °C). The collagenase IV derived from the fermentation of Clostridium histolyticum was used to pretreat the PEG2k-ppPE micelles, instead of the MMP2 due to the cost issue. After 2 h incubation with 50 μg/mL collagenases IV or BSA, the drug release from the PTX-loaded PEG2k-pp-PE micelles or PTXloaded PEG2k-PE micelles were determined. As shown in Figure 3A, the 2 h incubation with collagenases (b) significantly increased the drug release rate and extent compared to the incubation with BSA (a) (55% vs 30% at 4 h and 90% vs 65% at 48 h). In contrast, the drug release from PEG2k-PE micelles did not show any changes after incubation with the collagenases compared to BSA (c vs d). We also found that the hydrophobicity of the peptide was lower than that of the PE,

Table 1. PEG-pp-PE and Other Polymers Used in the Study MMP2-cleavable copolymers

MMP2-uncleavable copolymers

PEG1k-pp-PE PEG2k-pp-PE PEG5k-pp-PE PEG2k-pp6-PE

PEG2k-unpp-PE PEG1k-PE PEG2k-PE PEG5k-PE TPGS1k

successfully synthesized. After integration of the characteristic peaks, the ratio of PEG/PE was approximately 1:1. 3.2. Micelle Formation and Drug Loading. We have shown that the conjugation of PEG2k to the hydrophobic moiety, including lipids25 and hydrophobic drugs,26,27 imparted the amphiphilicity to the formed conjugates. Here, to study the influence of the PEG length and peptide linker on the micelle formation, the CMC (Figure S-2) and particle size of the copolymers were determined. The theoretical hydrophilic− lipophilic balance (HLB) was calculated by the Griffin equation. The values of the CMC, particle size, and HLB of the PEG-pp-PE and PEG-PE were compared in Table 2. The ζ potentials of the PEG-PE or PEG-pp-PE micelles was close to zero, in agreement with ref 29. Table 2. CMC, Particle Size, and HLB of PEG-pp-PE and PEG−PE Based Copolymersa copolymers

CMC (μM)

HLB

PEG1k-pp-PE PEG2k-pp-PE PEG5k-pp-PE PEG2k-pp6-PE PEG2k-unpp-PE PEG1k-PE PEG2k-PE PEG5k-PE

4.2 2.6 2.9 3.4 3.2 6.3 2.1 3.1

9.35 12.44 15.96 12.23 12.44 11.46 14.58 17.41

particle size (nm) 50.4 33.0 23.6 63.2 67.4 44.3 55.8 59.0

± ± ± ± ± ± ± ±

3.7 1.2 0.8 2.5 3.7 2.0 2.1 2.3

a

HLB, hydrophilic−lipophilic balance, which was calculated based on the Griffin equation. HLB = 20[Mhydrophilic/(Mhydrophilic + Mhydrophobic)], wherein Mhydrophilic = molecular weight of PEG and hydrophilic amino acid and Mhydrophobic = molecular weight of PE and hydrophobic amino acid.

All of the PEG-pp-PE copolymers showed excellent micelle formation properties, as evidenced by the small CMC values (