ARTICLE pubs.acs.org/bc
NONOates Polyethylenimine Hydrogel for Controlled Nitric Oxide Release and Cell Proliferation Modulation Jihoon Kim,† Yanggy Lee,‡ Kaushik Singha,† Hyun Woo Kim,† Jae Ho Shin,§ Seongbong Jo,^ Dong-Keun Han,‡ and Won Jong Kim*,† †
Department of Chemistry, Pohang University of Science and Technology, Pohang 790-784, Korea Biomaterials Research Center, Korea Institute of Science and Technology, P.O. Box 131, Seoul 130-650, Korea § Department of Chemistry, Kwangwoon University, Seoul 139-701, Korea ^ Department of Pharmaceutics, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States ‡
bS Supporting Information ABSTRACT: In recent years, numerous research activities have been devoted to the controlled release of nitric oxide (NO) due to its potential as a restenosis inhibitor which inhibits the proliferation of vascular smooth muscle cells, the apoptosis of vascular endothelial cells, and aggregation of platelets. This work has demonstrated the development of a novel NO-conjugated gel system comprising of thermosensitive Pluronic F127, branched polyethylenimine (BPEI), and diazeniumdiolates (NONOates). Synthesis of conjugated Pluronic-BPEI-NONOates involved coupling of activated F127 to BPEI followed by the installation of NONOates at the secondary amine sites of branched PEI backbone under high pressure. NO-conjugated gel system, F127-BPEI-NONOates, reduced the initial burst of NO release and prolonged NO release. Furthermore, F127-BPEI-NONOates polymer coated on cell culture dish displayed much higher increase of endothelial cell proliferation and reduction of smooth muscle cell proliferation than that exhibited by non-NO releasing control. Such an NO-releasing device can operate locally and has a great potential in several biomedical applications due to high biocompatibility imparted by the conjugated F127.
’ INTRODUCTION Nitric oxide (NO) is a small reactive molecule that plays a major role in proliferation and apoptosis of cells, neurotransmitters, and regulation of vasomotor tone.1 3 Especially in vascular vessels, NO has been known to affect relaxation, inhibition of proliferation, and induction of apoptosis of vascular smooth muscle cells. In contrast to these effects on vascular smooth muscle cells, NO is also known to induce the growth of endothelial cells.4,5 In addition, NO exerts an inhibitory effect on platelet aggregation, which can cause thrombogenicity.6 8 Despite the various and selective functions of NO, as well as in vivo generation of NO by three types of nitric oxide synthase, it is very difficult to apply NO to biomedical and therapeutic fields because NO could easily react with other components to produce a variety of nitrate/nitrite and could be released very rapidly from NO donors. Thus, there has been enormous interest in developing biomaterials which could facilitate the controlled and prolonged release of NO for modulating its concentrationdependent functions.9 Several types of NO donors have been developed and evaluated in vitro and in vivo. Nitroglycerin,10 sodium nitroprusside,11 S-nitrosothiol,12 15 NO aspirin,16 18 and 1-substituted diazen-1-ium-1,2-diolates (NONOates)19 28 and its derivatives16,29 are typical examples of NO donors. r 2011 American Chemical Society
Among these types of NO donors, NONOates, containing the [N(O)NO] functional group, are considered as the most attractive candidates for practical application due to their spontaneous dissociation under physiological conditions to yield 2 mol of NO per mole of [N(O)NO] functional group and structural flexibility of synthesis.26 Recently, NO donors were incorporated into stents covalently and noncovalently and thus used as restenosis inhibitors.12,14,20 22,25,27 There are mainly two distinct structural systems which are utilized as NO donors in coating materials for drug-eluting stent (DES). The majority of the NO-releasing polymers incorporate NO through covalent conjugation as found in diaminoalkyltrimethoxylsilane silicone rubber-NONOates (DACA/NONO-SR), 27 polypropylenimine dendrimersNONOates,22 and polyethyleneimine-NONOates.19 Using this system, it is possible to incorporate NO in abundance into polymers; however, a supporting material which could bind NO donors into the substrate is essential. Unless NONOates polymers remain bound to the supporting material, the detached Received: September 9, 2010 Revised: March 10, 2011 Published: April 29, 2011 1031
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Bioconjugate Chemistry polymer could be dispersed into the body through the bloodstream and may induce toxicity. Another type of NO donor includes a mixture of small NO donor noncovalently mixed with polymers, for example, the mixture of diethylamine-NONOates (DEANONO) with poly(ethylene glycol) (PEG),28 diethylenetriamine-NONOates (DETA-NONO) with polycaprolactone,28 S-nitrosoglutathione with poly(vinyl alcohol)/poly(vinyl pyrrolidone) film,15 and S-nitrosoglutathione or S-nitroso-N-acetylcysteine with Pluronic F127 gel.14 Especially, it has been reported that NO release from S-nitrosoglutathione or S-nitroso-N-acetylcysteine in thermosensitive Pluronic F127 hydrogel is slower than that in H2O. However, these mixed systems may induce leaching out of small molecules, which can cause cytotoxicity. In this study, we developed a conjugated gel system by integrating advantageous features of two distinct systems in a single system. We used Pluronic F127, a commercially available thermosensitive triblock copolymer which was approved by FDA, to prolong the NO release by a cage recombination mechanism.14 In addition, we have conjugated BPEI which has a large amount of secondary amines, designed for conjugation of NONOates to Pluronic F127. We have also investigated the kinetics of NO release from F127-BPEI-NONOates hydrogel and their effects on endothelial as well as smooth muscle cell proliferation.
’ EXPERIMENTAL PROCEDURES Materials. Branched PEIs (BPEI, MW 600 Da, 10 kDa,) were obtained from Polyscience, Inc., (Warrington, PA) and high molecular weight BPEI (MW 25 kDa), sodium methoxide (NaOMe), Pluronic F127, and p-nitrophenyl chloroformate (p-NPC) were purchased from Sigma Aldrich (St. Louis, MO). Tetrahydrofuran (THF) and diethyl ether were purchased from Dae Jung Chemical Co. (Seoul, Korea). Dichloromethane (DCM) was purchased from Samchun Chemical Co. (Korea). Methyl alcohol (MeOH) was purchased from Mallinckrodt (St. Louis, MO). A Griess assay system was purchased from Promega (Madison, WI). Nitric oxide (NO) and argon (Ar) gases were obtained from HANA gas (Gimhae, Korea). Human umbilical vein endothelial cells (HUVEC), coronary artery smooth muscle cells (SMC), endothelial basal medium-2 (EBM-2) with full supplements (EGM-2 siglequots), and smooth muscle basal medium (SmBM) with full supplements were obtained from Lonza (Walkersville, MD). Dulbecco’s phosphate buffered saline (DPBS) was purchased from Invitrogen-Gibco (Carlsbad, CA). Cell viability was estimated by Cell Counting Kit-8 obtained from Dojindo Molecular Technologies, Inc. (Gaithersburg, MD). Synthesis and Charaterization of BPEI-NONOates. BPEINONOates was prepared as previously reported.19 Briefly, a solution of BPEI (0.5 g each for Mw: 600 Da, 10 kDa, and 25 kDa) in dry MeOH (10 mL) and dry THF (20 mL) was prepared in 100 mL beaker. After the solution was stirred, NaOMe (1 mol equiv with respect to the total amine sites) in MeOH (10 mL) was added to the BPEI solution and placed in an in-house high-pressure reactor. The high-pressure reactor was flushed with 20 psi Ar (g) twice and then subsequently charged with NO (g) at 80 psi. After 3 days, NO was vented and the reactor was flushed with 20 psi Ar (g) twice before each product was precipitated with cold dry ether. The solvent was quickly removed by filtration, and the product was washed with cold dry ether and then vacuum-dried to yield a light yellow product. The products were stored at 20 C. The conjugation ratios of
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BPEI-NONOates were characterized by 1H NMR in D2O using 300 MHz NMR spectrometer (Bruker, Germany). The characteristic absorption bands for NONOates and N-nitrosoamine were 250 260 nm and 330 360 nm, respectively, as measured by UV/vis spectrophotometry (UV 2550, Shimadzu, Japan). Measurement of NO Release from BPEI-NONOates by Griess Assay. NO release profile was monitored using the Griess assay (Promega), which quantifies the amount of nitrites, the primary degradation byproduct of NO. In brief, each 250 mg aliquot of BPEI-NONOates was incubated in DPBS (50 mL) at 37 C. After sampling 60 μL in predetermined time intervals, fresh DPBS (60 μL) was added to the vial to maintain the overall volume. After mixing the acquired samples with DPBS, the samples (50 μL) were added to a sulfanilamide solution (50 μL) and incubated in the dark at room temperature for 5 min. Naphthylethylenediamine (50 μL) was added to the mixture to stimulate a colorimetric change; intensity was measured at 548 nm by microplate spectrofluorometer (VICTOR3 V Multilabel Counter, Perkin-Elmer, Wellesley, MA) and compared to known sodium nitrite standards. Synthesis and Characterization of F127-BPEI. F127-BPEI was prepared using the modified method as reported previously.30,31 To prepare the hydroxyl-activated Pluronic F127, Pluronic F127 (10 g, 0.79 mmol) was dissolved in anhydrous DCM (70 mL). The solution was added dropwise to a stirred solution of p-NPC (1.29 g, 6.35 mmol) in anhydrous DCM (70 mL). The reaction was carried out for 24 h at room temperature with gentle stirring under nitrogen atmosphere. The activated Pluronic F127 was precipitated by addition of ice-cold ether and dried under vacuum. To synthesize the F127BPEI, a DCM solution (20 mL) containing activated Pluronic F127 (0.6 g) was added dropwise to a solution of BPEI600 (1.5, 3, and 10 mol equiv with respect to activated F127) and triethylamine (1 mL) in DCM (20 mL) at ice-cold conditions. The reaction was carried out for 24 h at room temperature with gentle stirring under nitrogen atmosphere. After evaporating the DCM and dissolving with water, each product was dialyzed by a Spectra/Por dialysis membrane Mw cutoff of 10 000 against water. The conjugation ratios of F127-BPEI were determined by 1H NMR taken in D2O using 300 MHz NMR spectrometer (Bruker, Germany). Temperature-responsive sol gel transition behaviors were monitored using a modified vial tilting method.32 Synthesis and Characterization of F127-BPEI-NONOates. To a solution of F127-BPEI (0.5 g) in dry THF (2.3 mL) was added a solution of NaOMe in dry MeOH (0.5 M, 2.3 mL). The solution was stirred for 30 min and was then placed in a highpressure reactor. The high-pressure reactor was flushed with 20 psi Ar (g) twice and subsequently charged with NO (g) at 80 psi. After 3 days, NO was vented and the reactor was flushed with 20 psi Ar (g) twice before each product was precipitated with cold dry ether. The solvent was quickly removed by filtration, and the product was washed with cold dry ether and then vacuum-dried. The products were stored at 20 C. The conjugation ratios of BPEI-NONOates were determined by 1H NMR in D2O using 300 MHz NMR spectrometer (Bruker, Germany). Measurement of NO Release from F127-BPEI-NONOates by Griess Assay. Each of F127-BPEI-NONOates (150 mg) was dissolved in deionized water at 4 C to prepare 20 wt % solutions and incubated in 600 μL DPBS at 37 C. Release of NO was monitored using the Griess assay (Promega). In brief, after sampling 600 μL at predetermined time intervals, fresh DPBS (600 μL) was added to the vial. A sample (50 μL) was added to a 1032
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Bioconjugate Chemistry sulfanilamide solution (50 μL) and incubated at room temperature for 5 min, protected from light. Then, naphthylethylenediamine (50 μL) was added to the mixture to stimulate a colorimetric change; intensity was measured at 548 nm by microplate spectrofluorometer (VICTOR3 V Multilabel Counter, Perkin-Elmer, Wellesley, MA) and compared to known sodium nitrite standards. In Vitro Cell Proliferation Test. All materials such as F127, F127-BPEI, F127-BPEI-NONOates, deionized water, DPBS, and media were sterilized under UV for 30 min prior to carrying out the in vitro experiments. F127 (100 mg), a mixture of F127 (50 mg) and F127-BPEI (50 mg) or a mixture of F127 (50 mg) and F127-BPEI-NONOates (50 mg) were dissolved separately in deionized water at 4 C to yield 25 wt % solutions and kept at 4 C for 1 h, and then gelation was performed on the cell culture dish at 37 C for 2 min. HUVEC and SMC were seeded on the coated cell culture plates (24 well) at a density of 1 104 cells/ well and further incubated at 37 C for 24 h. After 24 h incubation, cell proliferation was investigated by cell counting kit-8 (CCK-8) assay.33 This CCK-8 uses the water-soluble tetrazolium salts (WST-8) (2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) which is a derivative of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).
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Scheme 1. Synthetic Route for (a) BPEI-NONOates and (b) F127-BPEI-NONOates
’ RESULTS AND DISCUSSION Synthesis and Characterization of BPEI-NONOates. Three types of BPEI-NONOates having BPEI with different MW were synthesized by the method described above, and the structural characteristics of each type of BPEI-NONOates were confirmed by 1H NMR and UV/vis spectroscopy (Scheme 1, and Figures 1 and 2). In 1H NMR, the proton signals of methylene groups adjacent to amine sites of BPEI at 2.7 ppm were shifted downfield to about 3.1 ppm in BPEI-NONOates due to the electron-withdrawing effect of the NONOates group. The integration of the shifted proton signals compared to the total proton integration of each type of BPEI-NONOates revealed that the conjugation ratios of NONOates to BPEI600, BPEI10K, and BPEI25K were 15.3%, 13.8%, and 16.0%, respectively (Figure 1 and Table 1). The UV/vis absorption spectra also showed the characteristic absorption peaks for NONOates at about 250 nm19 as measured in DPBS buffer (Figure 2). NO Release Profile of BPEI-NONOates. NO release from three types of BPEI-NONOates was measured in DPBS solution at physiological temperature using a Griess assay (Figure 3) method for nitrite measurement based on the oxidation of highly reactive NO to nitrite. As shown in NO release profile in Figure 3, at initial stages NO was released rapidly from BPEI25K-NONOates and release was completed within 10 h. Likewise, BPEI10K-NONOates showed a similar initial NO release profile, but the NO release was completed within 22 h. However, BPEI600-NONOates having low MW of BPEI showed a different NO release profile. NO was released rapidly at the initial few hours, but showed a sustained NO release pattern. Moreover, NO was released from BPEI600-NONOates for an extended time over 50 h. Longevity can be defined as the duration of NO release, while sustainability can be defined as the degree of sustained release of NO at the initial state, and quantitative evaluation of longevity and sustainability of NO release was summarized in Table 1. These results could be rationalized by considering the protonation of the primary amine group. The protonated primary amine groups (pKa = 9.73 11.02) are
expected to confer stability to NONOates derivative through the hydrogen bonding formation between protonated primary 1033
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Bioconjugate Chemistry
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Figure 2. UV vis spectra of BPEIs and the corresponding BPEINONOates at a concentration of 0.14 mg/mL.
Table 1. NO Release Properties of BPEI-NONOates and F127-BPEI-NONOatesa t[NO] polymer
% convb
t1/2c
(μmol/mg)d longevity (h)
BPEI600-NONOates
15.3
