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Preparation of Water-soluble Hyperbranched Polyester Nanoparticles with Sulfonic Acid Functional Groups and Their Micelles Behavior, Anticoagulant Effect and Cytotoxicity Qiaorong Han, Xiaohan Chen, Yanlian Niu, Bo Zhao, Bingxiang Wang, Chun Mao, Libin Chen, and Jian Shen Langmuir, Just Accepted Manuscript • DOI: 10.1021/la400836y • Publication Date (Web): 29 May 2013 Downloaded from http://pubs.acs.org on June 2, 2013
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Preparation of Water-soluble Hyperbranched Polyester Nanoparticles with Sulfonic Acid Functional Groups and Their Micelles Behavior, Anticoagulant Effect and Cytotoxicity Qiaorong Han,†,║ Xiaohan Chen,†,║ Yanlian Niu,† Bo Zhao,† Bingxiang Wang,† Chun Mao,*,† Libin Chen,† and Jian Shen,*,†,‡
†
Jiangsu Key Laboratory of Biofunctional Materials, College of Chemistry and
Materials Science, Nanjing Normal University, Nanjing 210023, P.R. China ‡
School of Chemistry and Chemical Engineering, Nanjing University, Nanjing
210093, P.R. China
TOC
The aliphatic water-soluble hyper branched polyester nanoparticles with sulfonic acid functional groups (HBPE-SO3 NPs) were synthesized and their micelles behavior, anticoagulant effect and cytotoxicity were investigated.
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ABSTRACT: Biocompatibility of nanoparticles has been attracting great interest in the development of nanoscience and nanotechnology. Herein, the aliphatic water-soluble hyperbranched polyester nanoparticles with sulfonic acid functional groups (HBPE-SO3 NPs) were synthesized and characterized. They are amphiphilic polymeric nanoparticles with hydrophobic hyperbranched polyester (HBPE) core and hydrophilic sulfonic acid terminal groups. Based on our observations, we believe there are two forms of HBPE-SO3 NPs in water under different conditions: unimolecular micelles and large multimolecular micelles. The biocompatibility and anticoagulant effect of the HBPE-SO3 NPs were investigated using coagulation tests, hemolysis assay, morphological changes of red blood cells (RBCs), complement and platelet activation detection, and cytotoxicity (MTT). The results confirmed that the sulfonic acid terminal groups can substantially enhance the anticoagulant property of HBPE, and the HBPE-SO3 NPs have the potential to be used in nanomedicine due to their good bioproperties.
Keywords: Hyperbranched; Nanoparticles; Sulfonic Acid; Micelles; Anticoagulant Effect; Cytotoxicity
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■ INTRODUCTION Hyperbranched polymers have attracted significant interests because of their unique architecture and novel properties that include good solubility, special viscosity behavior, and high density of their functional groups.1-6 Owing to the multifunctionality in hyperbranched polymers, the physical properties can be adjusted to a large extent by the chemical modification of the end-groups.7,8 The use of hyperbranched polymers by the chemical modification has attracted increasing attention in recent years.9-12 These features of hyperbranched polymers have been used extensively in diverse fields, such as coatings, additives, blends, nonlinear optics, composites, and copolymers.13-16 Especially, hyperbranched polymers hold great potential as drug delivery agents because of their three-dimensional shapes and availability of a large number of surface functional groups amenable to various modification chemistries for drug conjugation and targeting purposes.17-21 Much attention has been paid to the synthesis methods and the drug delivery efficiency of hyperbranched polymers, but there is not enough research works that focus on the blood compatibility of these materials when they used in the blood circulation system.22-25 As is well known, when in contact with blood, most of conventional and currently used polymers are still prone to induce clot formation, as platelets and other components of the blood coagulation system are activated.26 The blood compatibility of biomaterials continues need to be improved and evaluated for further biomedical applications. Biocompatibility of polymers is directly related to their architecture, molecular
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weight and surface chemistry.27-30 At present, the original material of hyperbranched polyester (HBPE) we used only dissolve in organic solvent (e.g. dimethyl sulphoxide (DMSO), tetrahydrofuran (THF)) but not in water. However, it cannot meet the requirement of biological systems. In this paper, we synthesized water-soluble nanoparticles by the chemical modification of aliphatic HBPE with sulfonic acid functional groups (HBPE-SO3 NPs). The micelles behavior of HBPE-SO3 NPs in aqueous solution was investigated by transmission electron microscopy (TEM) and calculator simulation method. Moreover, a series of specialized experiments were used to assess their blood compatibility and cytotoxicity.
■ EXPERIMENTAL SECTION Materials. 2,2-Bis(hydroxymethyl)propionic acid (DMPA) was purchased from Sigma-Aldrich Co. Ltd. and used as received. Trimethylol propane (TMP), sodium hydride and 1,3-Propanesultone were obtained from Energy Chemical Co. Ltd., China. All solvents are AR grade and purchased from Sinopharm Chemical Reagent Co. Ltd., China. THF was dried by refluxing over sodium and distilled just prior to use. HUVECs were purchased from Bogoo Biotechnology Company, China. Human embryonic kidney (HEK 293) cell lines were purchased from Goybio Biotechnology Company, China. Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM) and trypsin/EDTA 0.25% were obtained from Invitrogen (USA). All the other reagents used in the experiments are AR grade. Doubly distilled deionized (DI) water was obtained from a Milli-Q water purification system and used throughout the study.
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Synthesis of HBPE. All synthetic procedures were carried out under a dry nitrogen atmosphere. HBPE with TMP as a core was prepared by a procedure described in the previous literatures.31-34 The schematic diagram illustrating the synthesis route was presented in Scheme 1. Briefly, esterification reaction was carried out at 140 ºC with p-Toluenesulfonic acid (p-TSA) as an acid catalyst. The chosen molar ratio of TMP to DMPA was 1:9 corresponding to the theoretical molecular weight of 1179 g/mol and a HBPE with 12 terminal hydroxyl groups. The crude polymer was precipitated from acetone in n-hexane and dried under vacuum. Synthesis of HBPE-SO3 NPs. The HBPE (1.00 g, with 9.6 mM of -OH groups) was dissolved in THF in a three-necked round-bottomed flask equipped with a magnetic stirrer and a reflux condenser with a drying tube. Excess sodium hydride (2.0 equiv to -OH groups), corresponding to the theoretical quantity of hydroxy groups of HBPE, was then dissolved in THF and added to the polymer solution. The reaction mixtures were reacted at 70 ºC for 12 hours with stirring. Then 1,3-propane sulfone was added at 70 ºC and the mixture was allowed to react for more than 12 hours . The resulting product was filtered and dissolved in DMSO, then precipitated in THF and dried in vacuum oven. The crude product was purified by dialysis through dialysis bag (MWCO 500) for at least five days. During the dialyzing process, the fresh DI water was exchanged at appropriate intervals. After dialysis, the solution was dried under vacuum to give a product HBPE-SO3 NPs that ranged in colour from off-white to tan (esterifications conducted in THF tended to give more coloured products).35,36 Characterization. The FTIR spectra were obtained with KBr pellets on a Bruker
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Tensor 27 (Bruker, Germany). 1H NMR and 3C NMR (400MHz) spectra were recorded on a Bruker Avance 400 spectrometers (Bruker, Germany) at room temperature. The Electro-spray Ionization Mass Spectrometry (ESI-MS) was obtain from mass spectrometer (LCQ/M/Z=50～1850, Finnigan, USA). The compositions of the sample were determined using energy dispersive spectrometer (EDS) with Vantage Digital Acquisition Engine (Thermo Noran, USA). The morphology and structure of the samples were characterized by TEM which was carried out by HITACHI H-7650 (Hitachi, Japan) and JEOL-2000F (JEOL, Japan). Specimens for inspection were prepared on a 200 mesh copper grid by slowly evaporating a drop of prepared solutions covered by a carbon-supported film at room temperature. The Zeta Potential (ζ) of HBPE-SO3 NPs was detected using a Nano ZS90 Zetasizer (Malvern Instruments, UK). The measurements were made in automatic mode, and the data was analysed using the software supplied by the manufacturer.
The Self-assemble Behavior of the HBPE-SO3 NPs Investigated by Calculator Simulation Method. Molecular docking simulations were performed using the ZDOCK program37,38 integrated into Discovery Studio 2.1 software package,39 The ZDOCK program is an docking algorithm that provides near-native structure predictions, where scoring function includes a combination of shape complementarity. In this study, one HBPE-SO3 unimolecular micelle was defined as a receptor and the other was defined as a ligand for their docking. 100 model complexes were generated and the values of ZDOCK Score were used to choose the optimal complex. The complex was then subjected to energy minimization for 10000 steps by the steepest descents and 10000 steps by conjugation gradient.
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Coagulation Tests. The coagulation assays were performed and measured by using a Semi automated Coagulometer (RT-2204C, Rayto, USA). The antithrombogenicity of the samples were evaluated by in vitro coagulative time tests, activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) tests. Blood was drawn from healthy New Zealand white rabbits containing sodium citrate. The platelet-poor plasma (PPP) was obtained by centrifuging blood at 3000 rpm for 20 min. The final concentrations of the test samples mixed with PPP were 0.1, 1, 10 and 20 mg/mL. HBPE-SO3 NPs were dissolved in phosphate buffered saline (PBS), while HBPE was dissolved in PBS and DMSO mixture solution (HBPE is not hydrosoluble), and the final DMSO concentration was 1% v/v. PBS was used as a control. Haemolysis Assay. Red blood cells (RBCs) were separated from the blood by centrifugation (1500 rpm, 10 min) and washed five times with PBS. They were used immediately after isolation. 2 mL of diluted 2% RBC suspensions were added to 2 mL sample solutions at systematically varied concentrations. The final concentrations were 0.1, 1, 10 and 20 mg/mL, HBPE-SO3 NPs were dissolved in PBS, while HBPE was dissolved in PBS and DMSO mixture solution, and the final DMSO concentration was 1% v/v). PBS was used as a negative control whereas DI was used as a positive control. The mixtures were incubated at 37 ºC for 3h then centrifuged at 1500 r/min for 10 min. The absorbance of the supernatant was measured for release of haemoglobin at 545 nm. The percent haemolysis of RBCs was calculated using the following formula: % haemolysis = ((sample absorbance - negative control absorbance)/(positive control absorbance - negative control absorbance)) × 100%
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Morphological Changes of RBCs. For observing morphological changes of treated RBCs at the early stages of hemolysis, the HBPE-SO3 NPs were diluted to the required concentrations in RBC suspensions. The cell pellets obtained after 1.5h by centrifugation, were diluted in PBS, and mounted on clean glass slides covered with cover slips and observed under an Olympus BX41 microscope with a camera (Olympus E-620, Olympus Ltd., Japan). Complement Activation. The complement activation of the samples was determined by turbidimetry method assessing the depletion of complement C3. Activation studies were performed on PPP isolated by centrifugation from human whole blood donations. The sample solutions were incubated for 1 h at 37 ºC with PPP, the final concentration was 1 mg/mL (the final DMSO concentration in HBPE solution was 1% v/v). The assays were done as per the protocol provided by a commercial C3a enzyme immunoassay kit (BD Biosciences, USA). All the complement activation experiments were done in triplicates. Platelet Activation Assay. To measure the platelet activation, the platelet rich plasma (PRP) was incubated at 37 °C with the sample solution where the final samples concentration was 1 mg/mL (the final DMSO concentration in HBPE solution was 1% v/v). The incubation mixture was removed at 30 min to assess the activation state of the platelets using fluorescence flow cytometry. Expression of the fluorescently labeled platelet activation marker anti-CD62P and the platelet pan-marker anti-CD42a was detected using a BD FACSCalibur (BD Biosciences, USA). All the platelet activation experiments were done in triplicates. MTT Assay. The cytotoxicity of HBPE-SO3 NPs as well as HBPE was assessed by
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MTT assay that carried out according to the methods described previously.40-42 Two kinds of cells (HUVECs and HEK 293 cells) were used as the research objects. The cells were cultured in DMEM medium supplemented with 10% FBS in 96-well culture plates. The culture was kept in a 5% CO2 atmosphere for 48 hours at 37 ºC. Then they were detached using 0.25% trypsin-EDTA. Subsequently, the cells were subcultured once again. The media was changed by fresh ones, and the HBPE (the final DMSO concentration in HBPE solution was 1% v/v) and HBPE-SO3 NPs samples with different concentrations were added to the wells at a density of 2 x 104 cells/well (HUVECs) or 1 x 104 cells/well (HEK 293 cells). The cells of positive control were only incubated with equal Dulbecco's modified Eagle's medium (DMEM) with 10% FBS. All of the cells were allowed to grow for 72 h before 10 mL MTT (5 mg/mL) was added to each well. Then, the cells were incubated at 37 ºC for an additional 4 h until purple precipitates were visible. The medium was replaced by 100 mL DMSO and the cell plate was vibrated for 15 min at room temperature to dissolve the crystals formed by the living cells. Finally, the absorption at 540 nm of each well was measured by an ELISA reader (Behring ELISA Processor, Germany). All of the samples were assayed in triplicate, and the mean value for each experiment was calculated. The obtained results are expressed as a percentage of the control, which is considered to be 100%.
■ RESULTS AND DISCUSSION Characterization of HBPE-SO3 NPs. In this case, the hydroxy-terminated aliphatic HBPE was first activated by NaH to form HBPE-O-(oxygen anion) and then reacted with 1,3-propane sultone to obtain the sulfonic acid functionalized aliphatic HBPE. The presence of HBPE-SO3 NPs was confirmed by the FTIR as shown in
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Figure. As exhibited in the FTIR spectrum of HBPE, the absorptions at 3400 cm−1 and 1735 cm−1 were attributed to the hydroxy stretching and ester carbonyl asymmetrical.43 Comparing with HBPE, the −OH stretching vibration bands of HBPE-SO3 NPs at 3400 cm−1 appeared wider and shifted to higher wave number, suggesting that the original intermolecular hydrogen bonding in HBPE was broken during sulfation.44 Similarly, the intensity of the band at 2900 cm−1, attributed to the stretching and/or deformation vibration of C–O–H bonds, was decreased in the spectrum of HBPE-SO3 NPs. The new vibration bands at 1195 cm−1 and 1045 cm−1 appeared in HBPE-SO3 NPs, which were identified as O=S=O symmetric stretching and SO3− stretching modes in sulfonic acid groups, respectively.45 The results indicates that HBPE-SO3 NPs were synthesized based on a HBPE with terminal hydroxyl groups by modified with sulfonic acid groups. The elemental composition of the HBPE-SO3 NPs was determined using energy dispersive spectrometer (EDS) with Vantage Digital Acquisition Engine (Thermo Noran, USA). It was observed from Figure 2 that the new product HBPE-SO3 NPs are composed of the elements C, O, Na, and S. Appearance of the new element sulfur indicates that the modification is successful. The result is consistent with FTIR. The 1H NMR spectrum of HBPE-SO3 NPs with (-SO3)arm= 6 was shown in Figure 3. Comparing the 1H NMR spectrum of HBPE-SO3 NPs with that of HBPE, new proton signals appeared at 3.14-3.43, 2.46-2.53, 1.68-1.89 ppm (protons h, i and j), which confirmed that 1,3-propane sultone was grafted successfully through the formation of ester bondings. The conversion ratio from hydroxyl groups to sulfonic acid groups was calculated by comparing the integral of peaks f and g with the integral of peaks a and c
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(1-5Sf,
g
/2Sa, c) was about 50%. (HBPE: 1H NMR (DMSO-d6, ppm): 0.81(t, 3H,
CH3CH2–), 1.08(t, 27H, CH3–CR3), 1.33(q, 2H, CH3CH2–), 3.42(t, 24H, CH2OH), 4.10 (m, 18H, R3C –CH2–OOC), 4.62 (6H, CH2OH), 4.94 (6H, CH2OH). HBPE-SO3 NPs: 1H NMR (DMSO-d6, ppm): 0.97(CH3CH2–), 1.08(t, CH3–CR3), 1.21(CH3CH2–), 1.68-1.89 (m, –OCH2–CH2–CH2–SO3Na), 2.46-2.53 (t, –OCH2–CH2–CH2–SO3Na), 3.14-3.43 (m, R3C –CH2–OH, –OCH2–CH2–CH2–SO3Na), 4.03-4.09 (m, R3C –CH2–OOC), 4.50 (CH2OH), 4.71 (CH2OH)). 13
C NMR (in DMSO-d6) spectrum and the ESI-MS of the HBPE-SO3 NPs were also
used to analyze the structure of samples and precisely calculate the functionalization efficiency (Figure 1S and 2S). Micelles Behavior of HBPE-SO3 NPs in Aqueous Solution: TEM images were performed to estimate the size and morphology of the HBPE-SO3 NPs. As shown in Figure 4, the HBPE-SO3 NPs have two forms that include unimolecular micelles (around 5 nm, Figure 4A) and large multimolecular micelles (around 50 nm, Figure 4B) in water under different conditions.46 The amphiphilic architecture of the unimolecular micelles were formed by hydrophobic HBPE core and hydrophilic sulfonic acid outer shell as shown in Figure 4A.47,48 Typical TEM photos in Figure 4B exhibit the fine structures in every large multimolecular micelles. The unimolecular micelles of HBPE-SO3 NPs aggregated into approximate spherical large multimolecular micelles in water. It was driven by the intermolecular interactions.49 The amplified large micelles (arrows) in Figure 4B, clearly indicate that the large micelles were aggregated and composed of small spherical building units (unimolecular micelles).50 The self-assemble behavior and mechanism of the
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HBPE-SO3 NPs were investigated by calculator simulation method (Figure 3S, 4S and Table 1S). The size of micelles is a very important parameter for intracellular drug delivery because the small size (
