Annexin V-Conjugated Mixed Micelles as a ... - ACS Publications

Feb 27, 2017 - ABSTRACT: To alleviate the hemorrhagic side effect of thrombolysis therapy, a thrombus targeted drug delivery system based on the speci...
0 downloads 0 Views 5MB Size
Download PDF
Article pubs.acs.org/Biomac

Annexin V‑Conjugated Mixed Micelles as a Potential Drug Delivery System for Targeted Thrombolysis Yang Pan,† Xiaoting Ren,† Shuang Wang,† Xin Li,† Xianglin Luo,‡ and Zongning Yin*,† †

Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu, 610041, China ‡ College of Polymer Science and Engineering, Sichuan University, Chengdu, 610065, China S Supporting Information *

ABSTRACT: To alleviate the hemorrhagic side effect of thrombolysis therapy, a thrombus targeted drug delivery system based on the specific affinity of Annexin V to phosphatidylserine exposed on the membrane surface of activated platelet was developed. The amphiphilic and biodegradable biomaterial, polycaprolactone-block-poly(2(dimethylamino)ethyl methacrylate)-block-poly(2-hydroxyethyl methacrylate) (PCL-b-PDMAEMA-b-PHEMA (PCDH)) triblock polymer, was synthesized via ring opening polymerization (ROP) and atom transfer radical polymerization (ATRP) to use as the nanocarriers of thrombolytic drug. In order to conjugate Annexin V to the polymer, PCDH was modified by succinic anhydride via ring-opening reaction to introduce the carboxyl group (PCDH-COOH). After preparation of PCDH/PCDH-COOH (9/1, m/m) mixed micelles, Annexin V was coupled with the micelles using carbodiimide chemistry. The blood clot lysis assay in vitro confirmed that lumbrokinase-loaded targeted micelles (LKTM) had stronger thrombolysis potency than free lumbrokinase (LK) and LK-loaded nontargeted micelles (LKM, P < 0.05). In vivo thrombolytic assay, multispectral, optoacoustic tomography (MSOT) was used to assess the target ability of LKTM. The results of MSOT images indicated the fluorescence intensity of the LKTM group located in the blood clot position were significantly stronger than the LKM group. A 5 mm of carotid artery containing blood clot was cut out 24 h later after administration to assess the degree of thrombolysis. The results of thrombolytic assay in vivo were consistent with the assay in vitro, which the differences between LK, LKM, and LKTM groups were both statistically significant. All the results of thrombolysis assays above proved that the capacity of thrombolysis in the LKTM group was optimal. It suggested that Annexin V-conjugated micelles will be a potential drug delivery system for targeted thrombolysis.

1. INTRODUCTION Thrombus, a blood clot formed in a vessel, is a common lifethreatening disease in clinic. Many vascular diseases, including the deep vein thrombosis, pulmonary embolism, stroke, and myocardial infarction, are the result of the formation of thrombus.1−4 To unclog the occlusive vessel, a thrombolytic drug is recommended to use for thrombolysis.5 However, after the administration of thrombolytic agents, normally there is an unavoidable serious side effect: hemorrhage.6 How to alleviate the phenomenon of bleeding is crucial for patients. Recently, the target drug delivery system was widely studied to enhance the therapeutic effect and reduce the side effect, especially in the field of cancer research.7,8 If the thrombolytic drug could be efficiently delivered to the targeted thrombus, both the adverse side effect and dosage of thrombolytic agent will be reduced. Recently, studies on the targeted thrombolysis via novel drug delivery systems have been reported. Among them, the widely applied targeting strategies were based on a developed prodrug delivery system which was seminally done by Yang et al.,9−11 activated platelet targeting mediated by specific single chain antibody,12,13 the magnetic nanoparticles (MN) mediated by magnetic field,14 Arg-Gly-Asp (RGD) containing peptide © XXXX American Chemical Society

conjugated nanoparticles according to the specific recognition and binding ability of RGD to activated platelet membrane glycoprotein GP IIb/IIIa,15 or combining the last two approaches.16 For MN’s application in clinic, magnetic field was needed when MN was administrated. It was often studied as bifunctional agent for detection and therapy, but the application for only thrombolysis in clinic was inconvenient. RGD, a receptor antagonist of glycoprotein GP IIb/IIIa, can competitively inhibit the binding between glycoprotein GP IIb/ IIIa and fibrinogen, von Willebrand factor (vWF), fibronectin in activated platelet.17,18 According to the mechanism of platelet aggregation, the platelet would be activated before the formation of clot.19 Interestingly, the phosphatidylserine (PS) located in the intramembrane begins to expose on the surface of membrane during the early stage of platelet activation.20 This phenomenon also exists at the early stage of apoptosis.21,22 Meanwhile, the superficial PS will further accelerate the thrombus formation by Received: November 28, 2016 Revised: February 11, 2017

A

DOI: 10.1021/acs.biomac.6b01756 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules Scheme 1. Schematic Illustration of Thrombus-Targeted Polymer Micelles

al. prepared PCL-g-PDMAEMA-PHEMA via a combination of ROP and ATRP.47 Monomers of DMAEMA and HEMA were added into the reaction system at the same time, which resulted in random polymerization. Based on the structure of polymers mentioned above, it was difficult to form the micelles possessing the structure of hydrophobic core, the positively charged hydrophilic middle block (the PDMAEMA block) for loading LK and the hydrophilic shell (the PHEMA block) for conjugating with Annexin V and enhancing the hydrophilia. Additionally, the previously synthesized above polymers have not been used as targeted drug delivery carriers. In order to introduce carboxylic group to give polymer (PCDH-COOH) for the conjugation with Annexin V, PCDH was first modified by reaction with succinic anhydride. After the preparation of mixed micelles from PCDH and PCDH-COOH, Annexin V was conjugated to the polymer by EDC/NHS chemistry. The synthesized polymers were characterized by 1H NMR, Fourier Transform Infrared Spectroscopy (FTIR), and Gel Permeation Chromatograph (GPC). The particle size and zeta potential of prepared LK-loaded targeted micelles (LKTM) were characterized by Zetasizer Nano ZS (Malvern instruments). The morphology was observed by transmission electronic microscope (TEM). Critical micelles concentration (CMC) of PCDH was measured to analyze the capacity of antidilution in vitro. A low CMC value can ensure the stability to avoid quick disaggregation before the carriers reach the lesion. Enzyme activity of LK and LKTM was determined to compare the potency before and after a series of treatments in the preparation process. To assess the biocompatibility of synthesized polymer and prepared LKTM, cytotoxicity and hemolysis assay were conducted. In this paper, multispectral optoacoustic tomography (MSOT) technique was first used to evaluate the targeted thrombolytic potency of LKTM in vivo. The thrombolytic effect of different groups in vivo was also assessed by comparing the surplus of blood clot in carotid artery 24 h after therapy. Fibrinogen level of mouse was measured to assess the bleeding risk of different materials.

providing the activity surface to blood coagulation factors in the coagulation process.23 As a result, PS can be used as a signal for the recognition of activated platelet. Annexin V, a 35−36 kDa Ca2+-dependent protein consists of 319 amino acids, can bind PS specifically and inhibit the aggregation of platelets.24−26 It has been widely used in the field of apoptosis detection.27 Annexin V conjugated nanoparticles have also been studied for thrombus imaging or apoptotic cells detection based on its target specificity.28−30 However, a few papers reported the study of Annexin V used for targeted thrombolysis.31−33 Since PS is only located on the surface of membranes of all activated platelets, the Annexin V mediated targeting affinity was highly specific. Based on the property of Annexin V binding PS, Annexin V conjugated micelles were designed for the targeted delivery of thrombolytic enzyme to thrombus. There are many fibrinolytic enzymes used for thrombolysis, such as urokinase (UK), streptokinase (SK), tissue plasminogen activator (tPA), and lumbrokinase (LK).34−37 LK, a group of fibrinolytic enzymes having molecular weights of about 25−40 kDa, is extracted and purified from earthworm.38,39 In consideration of its available source and simplicity, LK was selected as the model of the thrombolytic drug in this study. The isoelectric point of LK is 3−5,38 which means it can be absorbed onto the positive surface of polymer under physiological conditions. Currently, polymer micelles have attracted increasing concern in the field of drug delivery system (DDS). Numerous polymers were synthesized and evaluated as the carriers for small molecule drugs, gene therapeutic agents, and protein drugs.40−44 The aim of this study was to design and develop a delivery system for the targeted thrombolysis. Considering the isoelectric point of LK, an amphiphilic cation polymer was chosen as the delivery carrier for LK using the electrostatic interaction. In this study, polycaprolactone-block-poly(2-(dimethylamino) ethyl methacrylate)-block-poly(2-hydroxyethyl methacrylate) (PCL-PDMAEMA-PHEMA (PCDH)), the amphiphilic and biodegradable triblock polymer, was synthesized via ring opening polymerization (ROP) and atom transfer radical polymerization (ATRP). PDMAEMA has been widely used as gene delivery carrier via the electrostatic interaction.45 Only few polymers composing PCL, PDMAEMA, and PHEMA have been synthesized in the previous studies. Bian et al. synthesized star PCL-b-PHEMA-b-PDMAEMA via reversible addition− fragmentation chain transfer (RAFT) polymerization.46 Han et

2. MATERIALS AND METHODS 2.1. Materials. Caprolactone, dodecanol (>99.0%), stannous octoate (Sn(Oct)2, 95%), N,N,N′,N″,N‴-pentamethyldiethylenetriamine (PMDETA, 99%), 2-bromoisobutyryl bromide (2-BiBB, 98%), 2-(dimethylamino)ethyl methacrylate (DMAEMA, 99%), 2hydroxyethyl methacrylate (HEMA, 95%), and cuprous bromide (99%) were all purchased from Aladdin Coporation (Shanghai, B

DOI: 10.1021/acs.biomac.6b01756 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules Scheme 2. Synthesis of PCL-PDMAEMA-PHEMA and PCL-PDMAEMA-PHEMA-Annexin V

with N2 was immerged in preheated oil bath at 120 °C for 24 h. The reaction was quenched by exposure to air and rapidly cooled to room temperature. After removal of toluene under vacuum in rotary evaporator, the crude product was precipitated in cold methanol and then isolated by filtration. The precipitate was dissolved in THF and then purified using the same protocol three times. The obtained product was dried in vacuum at 40 °C until constant weight was reached and was characterized by 1H NMR (400 MHz, Varian Inc., U.S.A.), FTIR (Perkin Elmer, U.S.A.), and GPC (Tosoh corporation, Japan) using THF as eluent at 40 °C. 1 H NMR (400 MHz, CDCl3) δ (ppm): 0.88 (t, CH3 in dodecanol), 1.26 (s, CH3(CH2)9(CH2)2O in dodecanol), 1.38 (m, -OCOCH2CH2CH2CH2CH2O-), 1.65 (m, -OCOCH2CH2CH2CH2CH2O-), 2.31 (m, -OCOCH2CH2CH2CH2CH2O-), 3.65 (t, -CH2OH and terminal OH group), 4.06 (m, -COCH2CH2CH2CH2CH2OCO- and CH3(CH2)10CH2O in dodecanol). 2.2.2. Synthesis and Characterization of PCL-Br Macromolecule Initiator. R-PCL-OH (Mn(NMR) = 4750 Da, 1.0 g, 0.2 mmol) was dissolved in anhydrous toluene (10 mL) in Schlenk flask. The mixture was degassed using vacuum/nitrogen recharge in an ice bath for 30 min. 2-BiBB (130 μL, 1.05 mmol) and dry triethylamine (150 μL, 1.05 mmol) were added dropwise into the Schlenk flask at 0 °C under N2 atmosphere, then the reaction mixture was stirred at room temperature for 24 h. After removal of the precipitate by filtration, the product was isolated and characterized by the same method as PCL, but without GPC. 1 H NMR (400 MHz, CDCl3) δ (ppm): 0.88 (t, CH3 in dodecanol), 1.26 (s, CH3(CH2)9(CH2)2O in dodecanol), 1.38 (m, -OCOCH2CH2CH2CH2CH2O-), 1.65 (m, -OCOCH2CH2CH2CH2CH2O-), 1.93 (s, -CH2OCO(CH3)2Br), 2.31 (m, -OCOCH2CH2CH2CH2CH2O-), 4.06 (m, -COCH2CH2CH2CH2CH2OCO- and CH3(CH2)10CH2O in dodecanol), 4.18 (t, -CH2OCO(CH3)2Br). 2.2.3. Synthesis and Characterization of PCL-PDMAEMA-Br (PCD) Diblock Polymer as a Macromolecule Initiator. The diblock amphiphilic polymer was synthesized via ATRP. Briefly, PCL-Br (Mn(NMR) = 4981 Da, 0.1 g, 0.02 mmol) was dissolved in 3 mL of anhydrous of DMF/DMSO (1/4, v/v) in a 10 mL Schlenk flask, followed by addition of DMAEMA and PMDETA (8 μL, 0.03 mmol)

China). Caprolactone, N,N- dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and toluene were distilled under reduced pressure after drying with calcium hydride before use. DMAEMA was passed through alkaline alumina to remove the polymerization inhibitor (MEHQ). According to the reported purification protocol of HEMA,48 an aqueous solution (50%) of HEMA was washed by nhexane three times, then salted from an aqueous phase by addition of sodium chloride, dried over magnesium sulfate, and then distilled under reduced pressure. Cuprous bromide was purified by washing with glacial acetic acid, ethyl alcohol, and acetone three times. 1-(3(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Adamas-beta Corporation (Shanghai, China). Annexin V was purchased from AAT Bioquest Inc. (Sunnyvale, CA, U.S.A.). Cy5.5 NHS was purchased from Lumiprobe corporation (Florida, U.S.A.). Lumbrokinase (28000 U/mg) was purchased from Xi’an Realin Biotechnology Co., Ltd. Dulbecco’s modified eagle medium (Hyclone, Utah, U.S.A.), penicillin−streptomycin solution (Hyclone, Utah, U.S.A.), FBS (zqxzbio, Shanghai, China), and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (Biosharp, Hefei, China) were obtained from Chengdu Baoxin biological company (Chengdu, China). Other chemicals used in this project are analytical reagents. L929 cells were used as received from Dr. Qin He (West China School of Pharmacy, Sichuan University). Kunming mice and Sprague−Dawley rats were purchased from Chengdu Dashuo Biological Institute (Chengdu, China). All animal experiments were complied with the guidelines of the Laboratory Protocol of Animal Care and Use Committee, Sichuan University. 2.2. Synthesis and Characterization of PCDH. The synthetic route of PCDH and Annexin V conjugated PCDH are shown as Scheme 2. The distribution of poly(2-hydroxyethyl methacrylate) block and its derivative modified by succinic anhydride was random. 2.2.1. Synthesis and Characterization of PCL. PCL was synthesized via ROP. Briefly, dodecanol (0.1127 g, 0.6 mmol), εcaprolactone (2.0540 g, 18 mmol), and anhydrous toluene (0.5 mL) were added into a 10 mL Schlenk flask, followed by evacuation/ nitrogen recharge three times, then Sn(Oct)2 (0.1% of monomer) as a catalyst was added under a N2 atmosphere. The Schlenk flask filled up C

DOI: 10.1021/acs.biomac.6b01756 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

1 H NMR (400 MHz, DMSO-d6) δ (ppm): 0.76−0.96 (m, terminal CH3 group in dodecanol and -C(CH3)2C(CO)(CH3)C(CO)(CH3)Br), 1.12−1.29 (m, -CH2OCO(CH3)2-, CH3(CH2)9(CH2)2O- and -CH2CH2OCO- in dodecanol, -OCOCH2CH2CH2CH2CH2O- in PCL block), 1.54 (m, -OCOCH2CH2CH2CH2CH2O- in PCL block), 1.78−1.90 (-C(CH3)2CH2C(CO)(CH3)CH2- in PDMAEMA block, -C(CH3)2CH2C(CO)(CH3)- in PHEMA block, -C(CH3)2CH2C(CO)(CH3)Br and -C(CH3)2CH2C(CO)(CH3)Br in PHEMACOOH block), 2.27 (m, -OCOCH2CH2CH2CH2CH2O- in PCL block and -N(CH3)2 in PDMAEMA block), 2.50−2.54 (m, -CH2N(CH3)2, -COCH2CH2COOH), 3.53 (t, COCH2CH2OH), 3.98 (m, -COCH2CH2CH2CH2CH2OCO- in PCL block, CH3(CH2)10CH2O in dodecanol, -COOCH 2CH2 N(CH3 )2 in PDMAEME block, -COCH2CH2OH in PHEMA block and -COCH2CH2OCO- in PHEMA-COOH block), 4.22 (s, -COCH2CH2OCO- in PHEMACOOH block, 5.40 (s, -COCH2CH2OH in PHEMA block). Micelles were prepared by solvent evaporation method. Briefly, 10 mg of PCDH was dissolved in 2 mL of THF, then10 mL of PBS was added while stirring. The mixture was concentrated using a rotary evaporator under vacuum to remove THF and then diluted to 10 mL by distilled water. Micelles prepared by PCDH-COOH with the same concentration were obtained by the same method. Annexin V, the functional protein used in the targeted delivery system, was conjugated to PCDH-COOH by carbodiimide chemistry according to reported method.50 Before conjugation of Annexin V with PCDH-COOH, the mixed micelles consisting of PCDH and PCDH-COOH with a ratio of 9:1 (v/v) in 0.01 M PBS were prepared. 5.76 mL of 1.0 mg/mL PCDH, 0.64 mL of 1.0 mg/mL PCDH-COOH (COOH: 0.53 μmol), 0.42 mL PBS, EDC·HCl (2 mg, 10.4 μmol) and NHS (3 mg, 26 μmol) were mixed and stirred at room temperature for 1 h, then placed in refrigerator at 4 °C for 30 min. Finally, 1.18 mL of 0.2 mg/ mL Annexin V (0.0016 μM) solution was added and stirred in ice bath for 24 h. The products was dialyzed by dialysis bag (MWCO:100 kDa) in 0.01 M PBS for 24 h to remove unreacted Annexin V, EDC and NHS. The purified product was used as the carriers to load LK for targeted thrombolysis. To determine whether the combination of Annexin V and micelles is by chemical binding or physical absorption, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was taken. To ensure the conjugated protein can be tested, Anx-M has been concentrated by ultrafiltration in 5 times. The content of conjugated Annexin V micelles (no LK-loaded) was further measured by micro bicinchoninic acid (BCA) kit following the manufacture’s instruction. The nontargeted micelles without LK were treated by the same protocol as a control for calibration. 2.5. Preparation and Characterization of LK-Loaded Targeted Micelles (LKTM). The LK-loaded targeted micelles were obtained by mixing 1.5 mL of targeted micelles and 0.2 mL of PBS, followed by addition of 0.3 mL of 2.0 mg/mL LK and stirring in an ice bath for 30 min. The size and zeta potential of LKTM were measured by Zetasizer Nano ZS (Malvern instruments) at 25 °C. The PBS buffer solution was replaced by deionized water when zeta potential was measured. The morphological property was observed by transmission electron microscope (TEM). LK-loaded nontargeted micelles (LKM) were also characterized by dynamic light scattering (DLS) to analyze the impact of conjugation on micelles. 2.6. Drug Loading and Release. The LKTM was prepared using the same protocol described in section 2.5. The final concentration of LK in LKTM was 0.30 mg/mL, which corresponds to 8400 U/mL. The free LK in micelles was separated by an ultrafiltration tube (300 kDa) and measured by BCA kit. The drug loading content (DLC) and encapsulation efficient (EE) were calculated using the following formula.

into the reaction mixture. After degassing using freeze−thaw three times, purified CuBr (4.6 mg, 0.03 mmol) was added. The molar ratio of PCL-Br, CuBr, PMDETA, and DMAEMA were 1:1.5:1.5:30. After polymerization at 30 °C for 24 h, the reaction mixtures were diluted using THF and then passed through a short neutral aluminum oxide column. The obtained colorless eluent was concentrated using a rotary evaporator and then dialyzed in pure water for 24 h. After lyophilization, the diblock polymer was obtained and characterized by 1H NMR, FTIR and GPC. 1 H NMR (400 MHz, CDCl3) δ (ppm): 0.88 (s, terminal CH3 group in dodecanol and -C(CH3)2C(CO)(CH3)C(CO)(CH3)Br), 1.03 (m, -CH2OCO(CH3)2), 1.24 (s, CH3(CH2)9(CH2)2O in dodecanol), 1.36 (m, -OCOCH2CH2CH2CH2CH2O-), 1.63 (m, -OCOCH2CH2CH2CH2CH2O-), 1.81−1.89 (m, -C(CH3)2CH2C(CO)CH3-, -C(CO)(CH3)CH2C(CO)(CH3)Br, -CH2C(CO)(CH3)Br), 2.27 (m, -OCOCH2CH2CH2CH2CH2O- in PCL block and -N(CH3)2 in PDMAEMA block), 2.55 (t, -CH2N(CH3)2), 4.04 (m, -COCH2CH2CH2CH2CH2OCO- in PCL block, CH3(CH2)10CH 2O in dodecanol and -COOCH2CH2N(CH3)2 in PDMAEME block). 2.2.4. Synthesis and Characterization of PCL-PDMAEMA-PHEMA (PCDH) Triblock Polymer. PCDH, the triblock amphiphilic copolymer, was also prepared via ATRP using a molar feed ratio of PCD/HEMA/ PMDETA/CuBr as1:30:2:2. Briefly, PCD (0.1 g), HEMA, PMDETA, and 2 mL of DMSO were added to the Schlenk flask and degassed by freeze−thaw three times. After addition of purified CuBr, the mixture was immerged in preheated oil bath at 80 °C for 24 h. The purification and characterization were performed according to PCD. 1 H NMR (400 MHz, CDCl3) δ (ppm): 0.88−0.93 (m, terminal CH3 group in dodecanol and -C(CH3)2C(CO)(CH3)C(CO)(CH3)Br), 1.04 (s, -CH2OCO(CH3)2), 1.25 (s, CH3(CH2)9(CH2)2O- in dodecanol), 1.38 (m, -OCOCH2CH2CH2CH2CH2O- in PCL block and -CH2CH2OCO- in dodecanol), 1.64 (m, -OCOCH2CH2CH2CH2CH2O-), 1.90 (m, -C(CH3)2CH2C(CO)(CH3)CH2- in PDMAEMA block, -CH2C(CO)(CH3)CH2-, -C(CH3)2CH2C(CO)(CH3)Br and -CH2C(CO)(CH3)Br in PHEMA block), 2.31 (m, -OCOCH2CH2CH2CH2CH2O- in PCL block and -N(CH3)2 in PDMAEMA block), 2.60 (t, -CH2N(CH3)2), 3.82 (s, COCH2CH2OH), 4.06 (m, -COCH2CH2CH2CH2CH2OCO- in PCL block, CH 3 (CH 2 ) 10 CH 2 O in dodecanol, -COOCH 2 CH 2 N(CH 3 ) 2 in PDMAEME block and COCH2CH2OH in PHEMA block) . 2.3. Determination of Critical Micelles Concentration (CMC). CMC of PCDH was measured according to the reported method using pyrene as a fluorescence probe.49 The series concentration of PCDH was 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50,100, 500, and 1000 μg/mL, respectively. The concentration of pyrene stock solution was 6 μM in acetone. A total of 0.3 mL of pyrene stock solution was added to a centrifuge tube. After evaporation of acetone at 60 °C, 3 mL of micelles with different concentrations was added to the residue. The concentration of pyrene was 0.6 μM in micelles. The mixture was allowed to reach balance for 24 h, then the excitation spectrum of pyrene was obtained using a RF5301 PC spectrofluorometer (Shimadzu, Japan) scanned from 300−360 nm at an emission wavelength of 393 nm, with a slit width of 5 nm. When increasing the concentration of polymer, the excitation wavelength of 332 nm was shifted to 338 nm. The intensity ratio of I338/I332 was used as an evaluation index against the logarithm of concentration of polymer to determine the CMC of polymer. To confirm whether pyrene as a hydrophobic compound would reduce the CMC value of polymer or not, a lower concentration of pyrene (0.06 μM) in micelles was analyzed by the same protocol. 2.4. Synthesis and Characterization of PCDH-COOH and Its Conjugates. PCDH-COOH was synthesized via ring-opening reaction using PCDH and succinic anhydride in the presence of dimethylaminopyridine (DMAP) as a catalyst. Briefly, PCDH (50 mg, 0.005 mM, -OH: 0.05 mM), succinic anhydride (10 mg, 0.1 mM), and DMAP (12 mg, 0.1 mM) were added to Schleck flask. After N2 replacement three times, 1 mL of anhydrous THF was added to the mixture. After reaction at 30 °C for 24 h, the solution was dialyzed for 24 h to remove the unreacted reagents. The PCDH-COOH was obtained after lyophilization and characterized by 1H NMR and GPC.

DLC(%) =

EE(%) = D

total drug taken − wt of drug in free × 100% wt of micelles

total drug taken − wt of drug in free × 100% total drug of taken DOI: 10.1021/acs.biomac.6b01756 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

of 100 μL of the 2% RBC solution was incubated with 100 μL of LKTM, with the concentration of polymer ranging from 0.12 to 0.30 mg/mL for 1 h at 37 °C. Then the mixture was centrifuged at 1500 rpm for 5 min, and 100 μL of the supernatant was transferred into a 96-well plate for the absorbance measurement at 540 nm by using a microplate reader. The 0.01 M PBS (pH 7.4) and 1% triton × 100 treated RBC were set as the negative and positive control group, respectively. The hemolysis assay of LK was taken using the same protocol. Hemolysis was calculated by the following formula:

Release of LK from LKTM was studied in the 0.01 M PBS (pH 7.4) as release medium with shaking at 100 rpm at 37 ± 0.5 °C. Considering the potential inhibition of dialysis bag to the release of LK, the prepared LKTM was added into PBS directly for the releasing study. As the content of LKTM would be reduced after sampling, each sample containing 1 mL of LKTM and 4 mL of PBS was set in every sampling time point. The free LK content in the released sample was measured by the same method used in the drug loading content measurement. 2.7. In Vitro Colloidal Stability Assay. The colloidal stability of micelles determined whether the drug loaded in carriers could be efficiently delivered to the diseased region. Both the dissociation and aggregation of polymer affected the expected release of drug in vivo. The CMC could determine whether the micelle will be disaggregated easily. Herein, the turbidity assay which was taken to evaluate the colloidal stability of nanoparticles51 was used to assess the phenomenon of aggregation of polymer in the 0.01 M PBS (pH 7.4) containing 10% FBS. Considering the dilution of drug after injection, the LK-loaded targeted micelles had been diluted five times using the medium above. The mixture of 0.3 mL of LKTM, 0.15 mL of FBS, and 1.05 mL of PBS was quickly scanned at the wavelength ranged from 400 to 700 nm by ultraviolet−visible spectrophotometer (Varian Cary 100 Conc, Agilent, U.S.A.) to determine the maximum absorption peak (λmax) of LKTM. After that, the mixture was incubated in a water bath at 37 °C with shaking at 100 rpm. Each time, 0.1 mL of mixture was transferred into a 96-well plate at a predetermined time point (0, 15, 30, 45, 60, 90, 120, 150, 180, and 240 min). Then, the absorbance of pipetted out mixture was measured at λmax by the microplate reader (Thermo Scientific Varioskan Flash, U.S.A.). The turbidity variation of 10% FBS was assessed by the same method. The colloidal stability of LKTM in 10% FBS was also evaluated by DLS according to the reported protocol with modification.52 LKTM was treated with the same protocol mentioned above. 2.8. Enzymatic Activity Assay. The thrombolytic potency of LKloaded targeted micelles was determined by the enzymatic activity (EA) of LK. To ensure enough capacity of thrombolysis, the EA of LK should be maintained the same during the preparation of LK-loaded micelles. Therefore, the EA was measured by a spectrophotometric assay using Nα-p-tosyl-L-arginine methyl ester hydrochloride (TAME, a widely used substrate in the EA measurement for many enzymes53,54) as substrate. The LKTM in centrifuge tube were bathed in water at 37 °C with vibration at 100 rpm. At the sampling time point, 0.2 mL of sample was pipetted out to measure the EA. The free LK was assessed by the same method. Briefly, 0.1 mL of 0.1 M TAME, 0.1 mL of PBS, and 0.1 mL of sample were mixed and then bathed in water at 37 °C for 30 min. During the hydrolysis of methyl ester in TAME by LK, methanol could be formed quantitatively. Thus, 0.2 mL of fresh 15% trichloroacetic acid (TCA) and 0.1 mL of 2% KMnO4 were added with shaking for 1.5 min to oxidized generated methanol to formaldehyde. A 0.1 mL aliquot of 10% sodium sulfite (reducing the surplus KMnO4) and 4.0 mL of chrommotropic acid (to form the complex with formaldehyde) were added to the resulting mixture. After homogeneous mixing was reached, solutions were placed in the boiling water for 25 min. Finally, the absorbance of each sample (0.2 mL) was measured at 574 nm by microplate reader. The calibration curve, prepared by LK, was used for quantification. The blank micelles were set as a control in the assay. Additionally, the EA of LK in LKM was also measured to ensure the same dosage of LKM and LKTM group in thrombolytic assay. 2.9. Biocompatibility Study. 2.9.1. Hemolysis. The occurrence of hemolysis is life threatening, which suggesting the necessity to assess the blood biocompatibility of LK-loaded micelles using hemolysis assay. Hemolysis of materials less than 10% was considered as nontoxic for animal.55,56 Red blood cells (RBC) were separated from whole blood originated from Sprague−Dawley (SD) rat by centrifuge at 1200 rpm in 15 min. After the supernatant was removed, the bottomed RBC was washed by PBS three times. A 2% (v/v) RBC solution was prepared by diluting the obtained RBC with PBS. A total

hemolysis(%) =

A sample − A negative A positive − A negative

× 100

2.9.2. Cytotoxicity. The cytotoxicity of PCDH, Annexin V-polymer micelles (Anx-M), and LKTM were evaluated in L929 cell by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the reported protocol.57 Briefly, 100 μL of L929 cells was seeded into a 96-well plate with a density of 1.0 × 104. After incubation with 5% CO2 for 24 h at 37 °C, the cultural medium was renewed by 140 μL of DMEM supplemented 10% FBS. A total of 60 μL of different concentrations of PCDH, Anx-M, and LKTM were added for incubation in 24 h. Then 20 μL of MTT (5 mg/mL, in PBS) was added to each well, followed by incubation for 4 h. Finally, 200 μL of DMSO was added to dissolve the produced formazan crystal after removal of the medium. The absorbance was measured at 490 nm using a microplate reader. PBS and 1% triton × 100 treated cell under the same condition were set as the negative and positive control group, respectively. cell viability(%) =

A sample − A positive A negative − A positive

× 100

2.10. Blood Clot Lysis In Vitro. The blood clot lysis assay in vitro was prepared using the reported method.9,16 Whole blood was collected from SD rat (250−300 g). The blood was drawn into a 1 mL syringe without anticoagulant to form a clot spontaneously. Blood clots were cut into pieces of 25 mg in weight (measured by electronic balance (BT25S, Sartorius, Germany)). Each cut clot was placed in a weighed 1.5 mL centrifuge tube and incubated with 0.4 mL of the following materials at 37 °C for 2 h: saline, Anx-M, LK, LKTM, and LKM. To avoid the impaction of osmotic pressure on red blood cells in clot, the solvent of different groups except saline was 0.01 M pH 7.4 PBS containing 0.15 M NaCl. The enzymatic activity of LK was 3360 U in LK-loaded group. Then 100 μL of the supernatants was pipetted out to measure the absorbance at 576 nm. The absorbance of the supernatants correlated positively with the content of clot lysis. Meanwhile, after the removal of liquid and evaporation of residual moisture in the tube for overnight at room temperature, the clot with the tube was weighed again to obtain the weight loss of blood clot. The percentage of weight loss (PWL) was calculated using the following formula to assess the thrombolysis ability of different materials.

PWL(%) =

wt of clotbefore lysis − wt of clot after lysis wt of clot

× 100%

2.11. Targeting Potential of Annexin V-Conjugated Micelles in Vivo. Herein, MSOT imaging was taken to assess the thrombus targeting ability of LKTM in vivo. MSOT, a noninvasive in vivo nearinfrared imaging technology combining the high specificity of optical imaging and strong tissue penetration ability with high distinguishability of ultrasonic imaging, has been used in many fields (e.g., tumor imaging, cardiovascular system, brain structure, and pharmacokinetics).58−62 Thrombolytic assay in vivo was studied to further determine the targeted thrombolytic capacity of different materials. 2.11.1. Synthesis of Cy5.5 Labeled LK. Cy5.5-labeled LK, a fluorescent probe used in the photoacoustic imaging system in vivo, was synthesized by the following protocol. Briefly, 50 mg of LK was dissolved in 5 mL of 0.01 M PBS (pH 7.4) and mixed with Cy5.5 NHS (1.5 mg) dissolved in 200 μL of DMSO. After 4 h of stirring in a dark environment at room temperature, the mixture was transferred into a E

DOI: 10.1021/acs.biomac.6b01756 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules dialysis bag (MWCO 3500 Da) and dialyzed in PBS for 24 h to remove the unreacted Cy5.5 NHS. Finally, the target product was obtained by freeze-drying. 2.11.2. FeCl3-Induced Carotid Thrombosis in Mouse. The protocol of carotid thrombosis generation was referenced the reported method with modification.63 Kunming mouse (18−22 g) was anesthetized using 4% (w/v) chloral hydrate (0.1 mL/10 g) by intraperitoneal injection. The hair of the whole neck was shaved off and removed completely by depilatory paste. After the incision of skin in the front of neck, the right carotid was isolated and separated from mucosa by preservative film. A 2 mm × 5 mm piece of the filter paper soaked with 10% FeCl3 was used to wrap the carotid for 3 min to form the thrombus. Finally, the wound was sutured after the clearance of the residual FeCl3 by saline. 2.11.3. MSOT Imaging Assay of Mouse In Vivo. After 18 h of thrombus formation, targeted LKM and nontargeted LKM were injected via tail vein. To avoid the interference of suture line to the image, the stitches were removed. Nontreated normal mouse was used as control to determine the location of vessels in the neck. Kunming mouse was anesthetized by isoflurane and placed in holder with polyethylene membrane and ultrasonic coupling agent. After 30 min of injection, the hairless neck of mouse was scanned in the chamber of MSOT inVision128 (iThera Medical, Germany). The laser excitation wavelengths of 680, 700, 715, 730, 760, 800, and 815 nm were selected. 2.11.4. Image Reconstruction and Spectral Unmixing. The MSOT images were reconstructed using a linear model-based inversion. After reconstruction, images were processed by using spectral unmixing to differ the signals of background, hemoglobin, oxyhemoglobin, and Cy5.5 NHS in the system. To compare the fluorescence intensity in two groups, a 0.72 mm2 region of interested (ROI) in the two-dimensional images was set. Three dimensional images were also reconstructed to observe more accurately the location of carotid artery and fluorescence distribution in vivo. 2.11.5. Thrombolytic Assay In Vivo. Five groups including saline, Anx-M, LK, LKTM, and LKM were used in the thrombolytic assay in vivo. The protocol referenced the previous method.64 Drug was injected via tail veil. The dosage of LK in LK-contained groups was 126000 U/kg, which corresponds to 0.015 mL/g according to the concentration of LK (8400 U/mL). After 24 h of therapy, a 5 mm length of carotid artery containing the developed blood clot was cut out to assess the fibrinolytic ability of different groups. The mouse was anesthetized during surgery. After washing by saline to clean the remaining blood and longitudinal incision by blade, the dissected carotid artery was digested by 200 μL of 2.0 mg/mL proteinase K at room temperature for 2 days. The optical density (OD) of the digested samples were measured at a wavelength of 280 nm to assess the remaining contents of thrombus after therapy by different drugs. The OD280 of 200 μL of proteinase was also measured for the calibration. To observe the thrombolytic effect directly, a same method of thrombolytic assay was taken to obtain the carotid arteries containing thrombi for H&E staining. There were five mice in each group. After fixation in 4% paraformaldehyde and dehydration in 30% sucrose, the excised carotid arteries were sectioned into 20 μm frozen sections. The sections were stained with H&E and examined under a light microscope. 2.12. Evaluation of Bleeding Risk. The fibrinogen level of mouse with carotid artery thrombus was measured by Clauss assay according to the reported protocol and manufacturer’s introductions.65 Design of experiment and collection of sample referenced the previous study.66 After 2 h of treatment by different materials (saline, LK, LKTM, LKM), the carotid artery thrombus model mouse was anesthetized by diethyl ether. The blood was drawn using cardiac puncture and mixed with trisodium citrate (0.106 mol/L) at the ratio of 9/1 (v/v). After centrifuge of anticoagulation blood at 2000 g for 10 min, the plasma was collected and stored at 4 °C for the fibrinogen level assay. The time to clot formation of plasma (not been diluted) was measured by fully automatic blood coagulation analyzer (CA-500, Sysmex, Japan). As the standard plasma of Kunming mouse was not obtained, the

blood clotting time was not converted into the concentration of fibrinogen according to the standard calibration curve. The nonconversion of blood clotting time would not affect the statistic analysis for bleeding risk. 2.13. Statistics. All data in this study were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used to determine the statistical significance by statistical package for social science (SPSS) statistical software. Results were considered statistically significant at P < 0.05 (*P < 0.05, **P < 0.01).

3. RESULTS AND DISCUSSION 3.1. Synthesis of PCDH via ROP and ATRP. PCDH was synthesized via ROP and ATRP in four steps (as shown in

Figure 1. Plot of intensity ratio (I338/I332) vs logarithm of PCDH. The concentrations of pyrene used in assay were (A) 0.6 μM and (B) 0.06 μM, respectively.

Figure 2. DLS profile and TEM image of LKTM.

Scheme 1). 1H NMR and FTIR spectra are shown in Figures S1−S3. First, PCL was synthesized via ROP by using dodecanol as the initiator in the presence of Sn(Oct)2 as catalyst. In the 1 H NMR spectrum of PCL, triplet peak at δ 0.88 and singlet at δ 1.26 were attributed to the methyl and nine methylene adjacent to methyl of dodecanol, respectively. Multiplet at δ 1.38, 1.65, and 2.31 corresponding to the methylene group in the caprolactone unit (-OCOCH2CH2CH2CH2CH2O-, -OCOCH2CH2CH2CH2CH2O-, -OCOCH2CH2CH2CH2CH2O-) confirmed the presence of caprolactone. Triplet at δ 3.65 corresponding to methylene adjacent to the terminal OH group confirmed the successful synthesis of polycaprolactone via ROP. The degree of polymerization was calculated by the ratio of peak areas at δ 2.31 and δ 3.65. The molecular weight and molecular weight distribution measured by GPC were 5.96 kDa and 1.676, as shown in Table S1. In the FTIR spectra of PCL, the nonobvious appearance of the hydroxyl group peak was due to the extremely low ratio to all other groups present in the polymer. F

DOI: 10.1021/acs.biomac.6b01756 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules Table 1. Particle Size, PDI, Zeta Potential, EE, and DLC of LKM and LKTM (n = 3) group

size (nm)

PDI

zeta potential (mV)

EE (%)

DLC (%)

LKM LKTM

117.50 ± 2.63 167.10 ± 2.00

0.143 ± 0.030 0.210 ± 0.010

34.30 ± 0.98 30.47 ± 1.10

89.86 ± 0.58 71.87 ± 0.79

20.74 ± 0.13 18.32 ± 0.2

BiBB attaching to the terminal of PCL confirmed the successful synthesis of PCL-Br. Diblock copolymer was synthesized via ATRP using PCL-Br as the macroinitiator and DMF/DMSO (v/v = 1/4) as the solvent. 1H NMR of PCD showed a new signal at δ 2.55 attributing to the methylene proton next to the tertiary amino of PDMAEMA. Moreover, the signal enhancement at δ 0.88 was due to the augmented numbers of methyl protons in the polymerized DMAEMA, which confirmed the successful polymerization of DMAEMA. The degree of polymerization (DP) of DMAEMA was calculated by comparing the signal integration ratio of peak at 2.55 (CH2CH2N(CH3)2) with the peak at 1.63 (-OCOCH2CH2CH2CH2CH2O- in PCL block (DP of CL was calculated in the first step). The molecular weight of PCD measured by GPC and 1H NMR were listed in the Table S1. Furthermore, triblock amphiphilic copolymer was also synthesized via ATRP using PCD as the macroinitiator and DMSO as the solvent. As shown in Figure S1(D), a new triplet at δ 3.82 corresponding to the methylene proton neighboring to the hydroxyl in HEMA unit confirmed the presence of HEMA unit in synthesized polymer. The DP of HEMA was calculated by analyzing the integration ratio of peak u at δ 3.82 (-CH2CH2OH in PHEMA block) to peak r at δ 1.64 (-OCOCH2CH2CH2CH2CH2O- in PCL block). In the FTIR spectra of PCDH, the broad peak at 3200−3600 cm−1 attributed to the hydroxyl group in the PHEMA block, which further proved the successful polymerization of HEMA. 3.2. Determination of Critical Micelles Concentration. Considering the low ratio of Annexin V-polymer in the mixed micelles and few resources of Annexin V, the CMC value of mixed micelles were not measured herein. The plot of intensity ratio (I338/I332) against logarithm of concentration of PCDH is shown in Figure 1. The determined CMC according to the intersection point were 0.42 and 0.55 mg/mL, respectively, when the concentration of pyrene was 0.6 and 0.06 μM in micelles correspondingly, which indicated the effect of pyrene in 0.6 μM on CMC value was faint. The low CMC suggested the synthesized polymer exert strong resistance to dilution, which ensured the stability in vivo. 3.3. Synthesis and Characterization of PCDH-COOH and Its Conjugates. In order to confirm the structure of PCDH-COOH, 1H NMR spectra of PCDH and PCDHCOOH in DMSO-d6 were also obtained (Figure S2). By comparison, a new peak w at δ 4.221 appeared in product after the ring opening reaction with succinic anhydride. It suggested the ester bond between the hydroxyl group in PCDH and succinic anhydride was formed. Affected by the intermolecular and intramolecular hydrogen bond interaction, the remaining hydromethyl peak was masked by a broad peak of H2O in the 1 H NMR spectrum of PCDH-COOH, which indicated the polarity of product also increased. The chemical shift of diethyl group in the succinic anhydride overlapped with DMSO-d6 and methylene peak adjacent to the dimethylamino group in PCDH. The molecular weight listed in Table S1 increased from 9.37 to 10.81 kDa. Based on the described results above, PCDH-COOH was successful synthesized.

Figure 3. Release curve of LK from LKTM in PBS (n = 3).

Figure 4. Results of colloidal stability assay of LKTM (n = 3). (A) UV scanning curve of LKTM. (B) Absorbance of LKTM in 10% FBS and 10% FBS at different time points.

Figure 5. Results of hemolysis of LK-loaded micelles (n = 3).

Figure 6. Results of cytotoxicity of (A) polymer, Annexin V-mixed micelles, LK-loaded targeted micelles, and (B) LK (n = 3).

Then, macromolecular initiator of PCL-Br was synthesized by nucleophilic substitution reaction between 2-BiBB and the terminal OH group of PCL. As shown in Figure S1(B), the triplet peak at δ 3.65 corresponding to terminal hydroxymethyl group in PCL moved to δ 4.18. Meanwhile, the presence of a new singlet at δ 1.93 corresponding to the methyl group in 2G

DOI: 10.1021/acs.biomac.6b01756 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 7. Blood clot lysis assay in vitro (n = 3). (A) Appearance of different groups after thrombolysis in vitro. (B) OD value of supernatant in different groups after lysis for 2 h. (C) Percentage of weight loss in different groups after lysis for 2 h.

Figure 9. Fluorescence intensity in LKTM group and LKM group (n = 5).

the physical interaction is weak. As shown in Figure S5, Lane D, which loaded the free Annexin V, showed one strip at the location of ∼36 kDa. Lane C loaded the blank micelles showed no strip, which indicated there was no protein in the micelles. Annexin V band was also observed in lane B that loaded the physical complex, which suggested the absorbed protein can be separated in the PAGE gel. As Anx-M was concentrated, the background color in gel was dark. No observed band in lane A loaded Annexin V conjugated micelles showed there was no free or physical absorbed protein in the Annexin V conjugated micelles. As a result, the measured protein in Anx-M by BCA kit was combined by the chemical binding rather than physical absorption. The conjugated content of Annexin V in Anx-M was 24.31 ± 1.15 μg/mg measured by BCA kit. The reaction efficiency was about 80%. 3.4. Size and Morphological Characterize of LKTM and LKM. The size and morphological property of LKTM were characterized by DLS and TEM. As shown in Figure 2, the hydrodynamic diameter and polydispersity index (PDI) of prepared LKTM were about 167 nm and 0.207, respectively. The image obtained by TEM showed that LKTM can be formed as spherical nanoparticle easily with a size of 30−80 nm. The results comparing LKTM and LKM are listed in Table 1. After conjugation with Annexin V, size and PDI of the prepared drug-loaded targeted micelles increased, while zeta potential was decreased. These results suggested the combination process had an adverse effect on the stability of micelles. The coupled Annexin V in the shell of micelles might inhibit the adsorption of micelles onto LK to a certain extent due to the

Figure 8. MSOT images in thrombus targeting study in vivo (n = 5). (A) Background, Hb, and HbO2 signal intensity in normal mice. (B) Background and Cy5.5 signal intensity in normal mice. (C) HbO2 and Cy5.5 signal in LKTM group. (D) Cy5.5 signal in LKTM group. (E) HbO2 and Cy5.5 signal in LKM group. (F) Cy5.5 signal in LKM group. (G) Schematic plot of MSOT imaging.

After conjugation to micelles, Annexin V would be stained at the loading hole as the significant increase of molecular weight. For the physical complex of micelles and Annexin V, the free Annexin V in complex would be separated by electrophoresis if H

DOI: 10.1021/acs.biomac.6b01756 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 10. Results of thrombolysis in vivo (n = 5−6). (A−E) H&E staining images (magnification 100×). (A) Saline group. (B) AnX-M group. (C) LK group. (D) LKM group. (E) LKTM group. (F) Results of thrombolytic assay in vivo evaluated by optical density.

in 4 h indicated that there is no obvious phenomenon of aggregation that occurred in the colloidal system. To further assess the disruption of serum on LKTM under the simulated physiological environment, the variation of relative scattering intensity (I/I0) of LKTM in 10% FBS was studied. As shown in Figure S6, I/I0, difference of scattering intensity between 10% FBS group and LKTM (ΔI) and relative difference of scattering intensity (ΔI/ΔI0) was decreased as time goes on, especially 2 h later. The results mentioned above indicated LKTM has an excellent stability in 0.5 h and a good stability in 2 h. After 2 h, the stability had been extremely disrupted. Considering the location of thrombi, 2 h was enough to meet the circulation time of carriers for delivering drug. Of course, the colloidal stability of carriers used in this project should be improved further for the long circulation. Comparison of the two methods used above indicates the latter manifested a stronger capacity to distinguish difference than the turbidity assay for colloidal stability study. Scattering intensity reflects variation of quantity of particles. However, this variation may not result in optical density change. 3.7. Enzymatic Activity Assay. The standard curve of enzymatic activity measurement is shown in Figure S7(A,B). The measured EA of LK, LKM, and LKTM was almost equivalent within 1 h and varied with similar tendency in the release medium in 24 h, which indicated the preparation process of LKM and LKTM has no effect on EA of LK. After 8 h of release, the EA decreased to a relatively stable level. The decrease of EA demonstrated the instability of LK, which corresponded to the results for release of LK. 3.8. Biocompatibility Study. 3.8.1. Hemolysis. Hemolysis assay was undertaken to predict the hemocompatibility of LKTM. As shown in Figure 5, hemolysis of LKTM showed a concentration-dependent manner. When the concentration of LKTM was no more than 0.18 mg/mL, the hemolysis was more than 10%, a safe value regarded as nontoxic for animal. It was suggested the concentration of LKTM should be confined strictly. A concentration no more than 0.18 mg/mL was advised to ensure an acceptable level of hemolysis (less than 10% of hemolysis). The hemolysis was due to the damage of positively charged polymer to negatively charged membrane of red blood cell. 3.8.2. Cytotoxicity. The cytotoxicity of triblock polymer (PCDH), Anx-M, and LKTM was assessed using MTT assay. The results are shown in Figure 6A; there was a good biocompatibility between the triblock polymer and cell in the

Figure 11. Fibrinogen levels characterized by blood clotting time (n = 5).

steric hindrance and decreased zeta potential. Stirring and dialysis process might also result in the enlarge of size, then the electrostatic force between LK and micelles would be weakened correspondingly. According to the results above, the drug loading capacity of micelles would also be impaired. 3.5. Drug Loading and Release. Before the study of drug loading, the protein content of LK was determined by BCA kit after filtration through a 0.45 μm Millipore membrane. The measured protein content was 60 ± 2% (n = 3). Herein, EE and DLC of both LKM and LKTM were measured for comparative analysis. As shown in Table 1, both EE and DLC of the LKM group were higher than that of the LKTM group. The results met the expectation described in section 3.4. The release behavior of LK from micelles is shown in Figure 3. The cumulative release reached maximum value after 2 h. Then it decreased in 2−4 h, but increased to climax after 8 h again. The decrease may be due to the reabsorption of LK by micelles. After 8 h of release, the content of LK decreased by 5% comparison with the maximum value. The second decline might result from the instability of LK under the release condition. 3.6. Colloidal Stability Assess. Turbidity variation of colloid reflect whether the aggregation of nanoparticles occurred or not. The higher the absorbance of LKTM in PBS containing 10% FBS (10% FBS) measured, the more serious aggregation suggested. The scanning curve showed the λmax of LKTM in 10% FBS was 414 nm at 400−700 nm. The changes of turbidity of 10% FBS were studied under the same conditions as the control. The absorbance of LKTM group and 10% FBS was measured at 414 nm at different time points. As shown in Figure 4B, only a small variation of absorbance was observed in the LKTM group. The relatively stable absorbance I

DOI: 10.1021/acs.biomac.6b01756 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

signal intensity of bilateral carotid artery in the image were similar in normal mice. However, for carotid artery thrombosis mice, as shown in Figure 8D,F, the background signal in the containing thrombus artery (right artery) was stronger than the normal carotid artery (left). It might result from the formed blood clot in vessel. In other words, the difference of signals in bilateral vessel indicated the correct location of carotid artery and thrombogenesis. Confined by the hairless area and depth from carotid to surface, the signal of arterial blood adjacent to heart was weak. After surgery to develop carotid artery thrombosis model, the diameter of neck was elongated since it was affected by wound. Analysis of fluorescence intensity of LKTM and LKM groups from MSOT images, as shown in Figure 8D,F, the intensity of the targeted group was enhanced comparison with nontargeted group. The details of the fluorescence intensity of each group were summarized in Figure 9. As shown, there was a significant difference between the LKTM and LKM groups (P < 0.01). As a result, the Annexin V-conjugated micelles can targeted deliver LK to thrombus. 3.10.3. Thrombolytic Assay In Vivo. To compare the effect of thrombolysis among different administrated drugs, the established model of carotid artery was cut out for the further assay after 24 h of thrombolysis in vivo. After lysis by proteinase K, the OD value was dependent on the amount of dissolved protein. Therefore, the anticipated OD value would be lower in the experimental group with better thrombolysis effect because of lower content of blood clot. As shown in Figure 10F, the saline and Anx-M groups had higher optical density, while the OD value in other groups was lower. By comparing OD280 values in the Anx-M, LK, LKTM, and LKM groups with saline group by student’s test, the obtained P values were 0.105,