Polymethacrylate-Based Nitric Oxide Donors with Pendant N

Biomacromolecules 2005, 6, 780-789

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Polymethacrylate-Based Nitric Oxide Donors with Pendant N-Diazeniumdiolated Alkyldiamine Moieties: Synthesis, Characterization, and Preparation of Nitric Oxide Releasing Polymeric Coatings Zhengrong Zhou and Mark E. Meyerhoff* Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055 Received September 1, 2004; Revised Manuscript Received November 3, 2004

A series of new nitric oxide (NO) releasing copolymers have been prepared by covalently anchoring alkyldiamine side chains onto a polymethacrylate-based polymer backbone, followed by NO addition to form the desired pendant diazeniumdiolate structures. The resulting diazeniumdiolated copolymers were characterized via UV spectroscopy, and their proton-driven decomposition to release NO was also examined by UV and FTIR as well as chemiluminescence. Polymers with up to 22.1 mol % of incorporated amine sites that can be converted to corresponding diazeniumdiolates could be prepared, and such polymers release up to 0.94 µmol/mg of NO. Further, novel NO releasing polymeric coatings were formulated by doping one of the new polymethacrylate-based NO donors within inert polymeric matrixes. Biodegradable poly(lactideco-glycolide) was employed as a film additive to greatly prolong the NO release of such coatings by continuously generating protons within the organic phase of the polymeric films, thereby driving decomposition of the diazeniumdiolates. Introduction There has been considerable interest in creating truly blood compatible polymeric materials for use in fabricating or coating a wide variety of medical devices (e.g., catheters, vascular grafts, extracorporeal circuits, oxygenators, implantable chemical sensors, etc.).1-4 To date, however, achieving a fully nonthrombogenic blood-contacting polymeric surface remains an elusive goal. A persistent problem with current synthetic polymers is their propensity to induce a thrombogenic response when in contact with blood. This can cause serious complications (e.g., platelet activation, thrombosis, etc.) in patients and ultimately functional device failure.5-11 As a result, systemic or localized anticoagulation treatments (e.g., use of heparin or heparin coatings) or use of antiplatelet agents (e.g., Plavix) are often required to reduce the risk of clot formation during certain clinical procedures and/ or for more long-term implants.12,13 One approach that can potentially solve this problem is to create materials that mimic the endothelium that lines all healthy blood vessels and that continuously releases a low level of nitric oxide (NO) locally (ca. 0.5-4 × 10-10 mol cm-2 min-1),14,15 at the vessel’s inner wall/blood interface. Indeed, it is well-known that NO is a potent inhibitor of platelet function,16 and recent research in this laboratory17-24 and elsewhere25-29 has demonstrated that polymers capable of releasing physiological levels of NO at the polymer/blood * To whom correspondence should be addressed. Address: 930 N. University Ave., The University of Michigan, Ann Arbor, MI 48109-1055. Tel: 734-763-5916. Fax: 734-647-4865. E-mail: [email protected].

interface can effectively decrease the platelet adhesion and thrombus formation on the surfaces of such materials. Previous research has also shown that N-diazeniumdiolates are a very useful class of NO donors, since they readily release NO when exposed to water (Figure 1).30-36 During the past decade, substantive efforts have been reported on the preparation of NO releasing materials based on Ndiazeniumdiolate chemistry. The initial focus of these efforts was to dope/disperse small N-diazeniumdiolate molecules (e.g., N-methyl-N-[6-(N-methylammoniohexyl)-amino]diazen-1-ium-1,2-diolate (MAHMA/NO)) into various hydrophobic polymers to enhance blood compatibility via NO release.5,18 Although considerable success was initially achieved, such hydrophilic NO donors were found to leach from the polymers, creating the risk of contaminating the blood with carcinogenic N-nitrosamine species.19 Thus, more recent work has emphasized avoiding the toxicity of the NO release precursor materials while maintaining controlled NO fluxes. This can be accomplished by either increasing the lipophilicity of the discrete diazeniumdiolate molecules24 or covalently attaching such moieties directly to polymers (e.g., piperazine modified PVC,19 silicone rubbers,20 methacrylatebased polymers,22 sol-gel materials,28,29 etc.19,25,26,37) or to the surface of reinforcing filler particles used in polymeric materials (e.g., fumed silica23). It has been shown that the thromboresistivity of such polymers is dramatically improved, with a significant decrease in platelet adhesion and activation on surface of such materials when tested in vivo.5,20,22-24 Parzachowski et al. have previously described polymethacrylate-based NO donors with N-diazeniumdiolated

10.1021/bm049462l CCC: $30.25 © 2005 American Chemical Society Published on Web 01/08/2005

Polymethacrylate-Based Nitric Oxide Donors

Figure 1. Schematic representation of NO release chemistry that occurs with newly developed polymethacrylate-based NO donors in the presence of water.

alkymonoamine side chains and demonstrated enhanced thromboresistance of silicone rubber films that were doped with these polymers.22 Herein, we report efforts to further enhance/alter the NO release capabilities of polymethacylate polymers by replacing the alkylmonoamines with various diamine moieties (Figure 1). This new polymer design allows us to explore the possibility of converting the pendant alkyldiamine moieties to zwitterionic diazeniumdiolate structures (typically formed in some of the discrete aklydiamine analogues24,34) on the methacrylate backbone without the need for an exogenous base (e.g., sodium methoxide). Additionally, we describe the use of these novel polymeric NO donors as agents to create NO releasing hydrophobic polymeric films (e.g., in PVC and silicone rubber). Such coatings consist of the newly prepared diazeniumdiolated polymethacrylate polymers as the NO donors and a biodegradable poly(lactide-co-glycolide) (PLGA) polymer as an additive. It will be shown that the PLGA can greatly help in maintaining a desirable NO release flux for an extended period of time (usually at or above 1 × 10-10 mol cm-2 min-1 for 24 h). The mechanism by which PLGA additive impacts the NO release profiles of such films is discussed in detail. Results and Discussion Synthesis of Monomers. Monomers 6a-d were designed and synthesized based on the desired copolymer structures (Scheme 1). These structures contain a polymethacrylate backbone with pendant alkyldiamine side chains. The syntheses were accomplished by unsymmetrically modifying diamine 1a or 1c via stepwise amidation reactions with esters possessing various chain lengths, yielding amides 3a-c. These molecules were further reduced to hydroxyl functionalized unsymmetric diamines 4a-c using lithium aluminum hydride (LAH) reduction (4d is a commercially available compound). Before incorporation of the polymerizable methacrylate group onto the terminal hydroxyl, the two primary or secondary amine sites were protected using ditert-butyl dicarbonate (Boc2O). The resulting N-Bocprotected alcohols 5a-d were then reacted with methacryloyl chloride to form the target monomers 6a-d. These monomers contain different lengths of methylene spacers between the two amine sites or different spacers between the methacrylate group and the diamine segments of the monomers.

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Polymerization of Monomers. Appropriate amounts of the resulting monomers 6a-d were reacted with methyl methacrylate (MMA) or a mixture of MMA and isodecyl acrylate (IDA) to form a series of N-Boc-protected diamine copolymers 7a-d′′ using 2,2′-azo-bis-isobutyrylnitrile (AIBN) initiated radical copolymerization (Scheme 2). Table 1 summarizes the series of copolymers prepared using various monomer species at varying ratios. High yields (above 90%) were achieved for all of the polymerization reactions. However, only in the case of copolymer 7d′ did the mol % of diamine monomer (Ma) match the amount added to the reaction mixture. Indeed, the actual compositions of Ma in the other copolymers are lower than expected. This trend indicates that most of the N-Boc-protected diamine monomers may have somewhat lower reactivity than MMA during the free radical polymerization reaction. The polymerization reactions were monitored by FTIR and 1 H NMR spectroscopies (Figure 2). In the FTIR spectrum (Figure 2, I-A), the methacrylate group of the N-Bocprotected diamine monomer exhibits CdC and CdO stretching frequencies at 1638 and 1718 cm-1, respectively. Upon polymerization, the CdC stretch disappears, and the CdO stretch is blue shifted to 1730 cm-1 (Figure 2, I-B) due to a loss of conjugation. These observations, in combination with the disappearance of 1H NMR signals a associated with the methacrylate double bond (Figure 2, II-A and B), indicate a transformation of the methacrylate group. The success of incorporating the MMA was also confirmed by the appearance of its characteristic 1H NMR signals e from the methoxyl group (Figure 2, II-B). Molecular weights of all of the resulting copolymers were determined by gel permeation chromatography (GPC) and tabulated in Table 2. The data indicates that small diamine monomer 6d when polymerized with MMA tends to form copolymers 7d and 7d′ with relatively higher molecular weights and higher polydispersities than the other reaction products. Deprotection of N-Boc-Protected Diamine Copolymers. Trifluoroacetic acid (TFA), a commonly used N-Boc deprotection reagent,38 was employed to remove the Boc-group of copolymers 7a-d′′ yielding the free alkyldiamines as pendant side chains (Scheme 2). The reactions were carried out in a dichloromethane solution containing TFA (10:1 volume ratio) at room temperature in high yields (average above 90%). These reaction conditions were applied previously in the polymethacrylate system prepared with monoamine side chains, and no backbone and amide bond cleavages were observed.22 We further confirmed this by measuring the nitrogen content increase of the copolymer 7d before and after the deprotection step. It was found that the nitrogen weight percentage increased from 3.17% to 4.01%, close to the expected 1.01 wt % increase (given the instrumental error of (0.1 wt %). It was also observed that as the deprotection proceeds, the initially clear solution becomes cloudy due to the decreased solubility of the diamine copolymers in the dichloromethane solvent. The resulting copolymers were dissolved in appropriate amounts of chloroform and washed thoroughly with sodium bicarbonate solution to completely remove the excess TFA. This is essential for the subsequent NO addition reaction since any

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Scheme 1. Synthesis of N-Boc-Protected Methacrylate Monomers 6a-d

a Glycolic acid ethylester (n ) 2) and 12-hydroxydodecanoic acid ethylester (n ) 12) were synthesized from their corresponding acids via esterification. Only compound 3b was acetylated on the terminal hydroxyl group to form soluble 3b′ before reduction with LAH. c Compound 4d is commercially available.

b

Scheme 2. Three-step Synthesis of NO Releasing Copolymersa Including Polymerization, Deprotection and NO Addition

a Copolymers 7d′ and 7d′′ and their deprotected and diazeniumdiolated products were also synthesized using this method.

remaining acid sites will dramatically decrease the amount of diazeniumdiolate formed. The deprotection reaction was also examined using FTIR and 1H NMR spectroscopies. After addition of TFA, the FTIR signal at 1698 cm-1 (CdO stretch of Boc) as well as the 1H NMR Boc-peak disappear completely indicating the complete removal of the Boc-groups (Figure 2, I and II-C). In addition, due to the loss of such an electron-withdrawing group, the proton signals c associated with two NCH2 groups shift upfield by approximately 0.6 ppm. NO Addition to the Diamine Copolymers 8a-d′′. THF solutions or suspensions of the diamine copolymers were exposed to 80 psi NO(g) at room temperature for 3 d in the presence or absence of NaOMe. Due to various potential side reactions (e.g., decomposition, nitrosamine/nitrite forma-

tion), it is important to maintain extremely anhydrous and anaerobic reaction conditions. Moreover, to prevent decomposition, final compounds require timely workup and must be stored under dry N2/Ar at -20 °C. Table 3 summarizes the characteristic UV absorbances and total NO release capabilities (measured by chemiluminescence) of all of the NO addition products as a function of amount of base added. In general, exogenous base is clearly required for diazeniumdiolate formation to occur. Indeed, this data indicates that spontaneous formation of intramolecular zwitterionic diazeniumdiolates of the alkyldiamine side chains in the absence of exogenous base is not favored (see below). However, presence of the exogenous base allows conversion of at least 4.1-22.1 mol % of the amine sites within the copolymers to stable diazeniumdiolates with sodium as the countercation. The total released NO (µmol/ mg) appears to be related to the base amount used during the reaction as well as the exact structure of the alkyldiamine pendant chains, with the highest percentage conversion occurring with the use of monomer 6b. The UV spectra of all of the diazeniumdiolated copolymers in methanol exhibit the characteristic absorption band for N-diazeniumdiolates in the range of 244-256 nm.34,35 For the four copolymers, 9a and 9b that have a six-carbon spacer between the two amine sites exhibit λmax values at approximately 246 nm. In contrast, 9c and 9d that have a two-carbon spacer between the two amine sites have λmax values at approximately 255 nm. No significant amounts of NO were released and only a weak absorbance band at ∼360 nm was observed for the products of the NO addition reactions that were carried out in the absence of exogenous base (Table 3). This absorbance band suggests the formation of N-nitrosamines,39 a competing side product of diazeniumdiolate formation. This was further confirmed by 1H NMR spectroscopy since protons alpha to the nitrosamines usually show two clusters of typical peaks at around 3.5 and 4.1 ppm. For example, Figure 3 compares the 1H NMR signals a of the methylene groups adjacent to nitrogens in the side chains of copolymer 8c and relative signals b of its nitrosamine product when reacted with NO



Table 1. Compositions and Yields of Various Methacrylate Copolymers with Pendant Diamine Side Chains copolymer

Ma

7a 7b 7c 7d 7d′ 7d′′

6a 6b 6c 6d 6d 6d

Mb MMA MMA MMA MMA MMA MMA-IDAd

CT a (mol % of Ma)

CA b (mol % of Ma)

XT c

XA c

yield (%)

20 20 20 20 10 20

12.7 16.1 17.9 17.2 10.2 N/Ae

4 4 4 4 9 4

6.9 5.2 4.6 4.8 8.8 N/Ae

90 93 97 97 99 92

a Theoretical composition (C ) is based on the amount of monomer (M ) and monomer (M ) used in reaction. b Actual composition (C ) is determined T a a b b A by comparing the 1H NMR integration of the respective incorporated monomers. c XT and XA are the theoretical and the actual molar ratios of Mb/Ma, respectively. d Theoretical composition of MMA and IDA is 40 mol % each. e CA and XA of 7d′′ are unavailable because its 1H NMR signals overlap, thereby preventing accurate compositional analysis.

Figure 2. Typical IR (I) and 1H NMR (II) spectra of monomer 6d (spectra A), N-Boc-protected copolymer 7d (spectra B), and deprotected copolymer 8d (spectra C) (1H NMR solvent ) CDCl3). Table 2. Molecular Weights of the N-Boc-Protected Copolymers copolymera

Mw

Mn

polydispersity

7a 7b 7c 7d 7d′ 7d′′

10 267 10 842 28 723 64 560 48 589 79 581

7189 7651 15 713 21 971 22 883 52 328

1.43 1.42 1.83 2.93 2.12 1.52

a Molecular weights were measured by GPC using refractive index detector. The concentration of the injected sample solution was 2 mg/mL in THF.

in the absence of NaOMe. Due to the electron-withdrawing character and resonance structures of the nitroso group (s NsNdO), the proton signals exhibit a downfield shift and a complicated splitting pattern. Without NaOMe to push the equilibrium toward formation of the diazeniumdiolate species, trace amounts of O2 or NOx that are always present in

the reaction system appear to lead to formation of the N-nitrosamine structures that are much more stable than N-diazeniumdiolates.39-41 Although intramolecular hydrogen bond stabilized zwitterionic diazeniumdiolate structures are formed in the case of small diamine molecules (i.e., MAHMA/NO34 and DBHD/ NO24), no significant quantities were observed in this polymer system regardless of the exact structure of the alkyldiamine monomer employed in the polymerization reaction. For the zwitterionic diazeniumdiolate structure, the lack of stability with an increase of either alkyl side chain length of the diamine (e.g., didodecylhexamethylenediamine, a small molecule analogue of the diamine polymers if one considers the polymer backbone as an infinitely long side chain) has already been reported,24 though the precise reason for this instability is not yet known. In the case of the new polymers reported here, destabilization is hypothesized to occur because the intramolecular hydrogen-bond formation between the organic ammonium NH and the oxygen on the diazeniumdiolate is weakened, probably by polymer chain entanglement. Based on the current understanding of this copolymer system, sodium methoxide is therefore an essential driving force for the deprotonation of the amine site that becomes diazeniumdiolated. Moreover, the sodium ion serves as the countercation that can stabilize the final diazeniumdiolated polymeric structure. To further demonstrate that an increase in base used can shift the reaction toward diazeniumdiolate product formation, the amount of NaOMe was varied from 1 equiv (same as the amount of all the free amine sites as determined by 1H NMR) to 2 equiv when reacting 8d with NO. It was found that this increase enhances the reaction efficiency more than 1.5-fold based on the amount of total NO released from the final products (0.30 µmol/mg vs 0.46 µmol/mg). Since further increases in the base content of the reaction mixture may cause the structural cleavage of the polymer, 2 equiv of NaOMe was chosen as the optimal quantity of base used for all subsequent NO addition reactions. Beyond varying the concentration of base, modifying the composition of the copolymer components can also lead to variation in the NO loading potentials for the final copolymers. Indeed, as expected, increasing the content of alkyldiamine units within the polymer resulted in a greater number of diazeniumdiolate moieties in the final copolymer products. For example, NO released from the diazeniumdiolated copolymers increased 2-fold from 0.23 µmol/mg (9d′) to 0.46 µmol/mg (9d) due to the increase of the diamine unit

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Table 3. UV and Chemiluminescence (CL) Characterization of the NO Addition Copolymers copolymer 9a 9b 9c 9d 9d 9d′ 9d′′ 9a 9b 9c 9d 9d′′

NaOMe useda 2 equiv 2 equiv 2 equiv 2 equiv 1 equiv 2 equiv 2 equiv 0 0 0 0 0

UV absorbance λmax (nm)

total NO releasedb (µmol/mg)

amine sitesc (µmol/mg)

D%d

247 244 253 256 256 256 252 N/A 363 360 360 N/A

0.31 ( 0.94 0.48 0.46 ( 0.05e 0.30 ( 0.04e 0.23 ( 0.04e 0.17 0.001 0.003 0.005 0.005 0.002

1.60 2.13 2.07 2.45 2.45 1.64 2.08 1.60 2.13 2.07 2.45 2.08

9.7 ( 0.3 22.1 11.6 9.4 ( 1.0 6.1 ( 0.8 7.0 ( 1.2 4.1 0.03 0.07 0.1 0.1 ∼0

0.01e

a The amount of NaOMe used for NO addition is with respect to the total free amine sites within the copolymers (1eq of NaOMe equals to the total amine sites). b Total NO release was measured without acid addition. c Amine sites were calculated based on the actual composition of the copolymers obtained from the 1H NMR data (theoretical composition was used for 9d′′). d D% represents the percentage of total amine sites that yield diazeniumdiolate groups. The calculation is based on the total NO release at the physiological pH (pH ) 7.4). e Average of 2 to 3 measurements.

Figure 3. Example of the 1H NMR spectra showing the methylene groups adjacent to nitrogens in the side chains of diamine copolymer 8c and its nitrosamine product that forms when the copolymer is reacted with NO in the absence of NaOMe. *One cluster of the nitrosamine signals b at 3.5-3.6 ppm is overlapped by the signal of the methoxyl groups on the polymer backbone (1H NMR solvent ) CDCl3).

composition from 10.2% (8d′) to 17.2% (8d) in the corresponding precursor copolymers (Table 3). In contrast, substitution of 40 mol % methyl methacrylate with same mole amount of long alkyl chain component (isodecyl acrylate) greatly decreased the diazeniumdiolate yield of the resulting copolymer from 0.46 µmol/mg (9d) to 0.17 µmol/ mg (9d′′). Decomposition and NO Release of Diazeniumdiolated Copolymers. N-Diazeniumdiolates undergo complicated dissociations via different mechanisms. When exposed to proton sources, such as when soaked in PBS buffer or water, they decompose into NO and parent amines predominantly.33 When exposed to ambient conditions, moisture and oxygen can accelerate their decomposition mainly to nitrosamines.19,20 Efforts have been made to utilize UV and IR spectroscopies as well as NO-selective chemiluminescence measurements to monitor the decomposition of the new diazeniumdiolated polymethacrylates with time in protic solvents, as well as in room air (containing moisture). The decreases in the absorbance in the range of 244-256 nm can be used to follow the decomposition of the diazeniumdiolates within copolymers 9a-d′′. As shown in Figure 4 for 9b in deoxygenated methanol, the intensity of absorp-

Figure 4. Example of decomposition of diazeniumdiolates within copolymer 9b with time as determined by UV spectroscopy at room temperature (in deoxygenated methanol).

tion at the characteristic wavelength decreases with time, illustrating the proton-driven decomposition of the diazeniumdiolates in the methanol solution. Accurate kinetic constants cannot be obtained from these data, since the copolymers are not completely soluble in the methanol solutions. Hence, it is not possible to know the solution phase concentration of soluble diazeniumdiolate in the starting solution. However, from measurements of the half-life in such solutions, the pseudo-first-order22,33 rate constant can be estimated to be 6.19 × 10-4 s-1, assuming that the reaction obeys first-order behavior for the soluble fraction of species in the methanol solution that contains trace amount of water and therefore a given proton activity. Infrared spectroscopy was also employed to monitor the decomposition of the diazeniumdiolated copolymers but under ambient conditions, which is primarily proton-driven as well. It was found that, in the presence of oxygen, the diazeniumdiolate moieties in the polymethacrylate structures could exhibit auxiliary decomposition to nitrosamines, which is consistent with what has been reported elsewhere for diazeniumdiolates examined by 1H NMR.22 Of course, nitrosamines can also form when diazeniumdioates are releasing NO in solution phase, provided oxygen is present. The mechanism of such nitrosamine formation is very similar to that reported previously,19,20 resulting from the initial reaction between air moisture and the diazeniumdiolates, followed by the back reaction of an oxidized NO intermediate



Figure 5. Example of decomposition of diazeniumdiolates in terms of nitrosamine formation within copolymer 9d with time under ambient conditions as determined by IR spectroscopy. (A) diamine copolymer 8d (starting material); (B) diazeniumdiolated copolymer 9d (immediately after NO loading); (C) 9d (after workup); (D) exposed to ambient conditions for few hours; (E) exposed to the ambient conditions for few days.

species with amine sites on the original donor molecule. For example, Figure 5 illustrates the decomposition of 9d under ambient conditions during workup and over several days thereafter. Two signals, likely from the NdO stretching of N-nitroso species, increase during this period. Nitrosamines could initially result when the polymer is reacted with NO in the presence of a trace level of residual oxygen that is inevitably present no matter how much care is taken to purge the reactor with argon. During the workup and later during storage, ambient oxygen and moisture could play a role to disassociate the diazeniumdiolates at room temperature. Under such conditions, diazeniumdiolates decompose to free amines as well as nitrosamine species.22 It was found that this decomposition can be eliminated by storage of the diazeniumdiolated copolymers under argon at -20 °C for extended time periods. N-diazeniumdiolates have been shown to decompose and release NO by two mechanisms, proton-driven33 and thermal20 dissociation. Given the fact that the NO donors studied herein undergo very little decomposition in dry N2 at physiological temperature (only thermal dissociation), we assume that their dissociation in deoxygenated aqueous solution, which prevents the possible nitrosamine formation, to release NO is primarily proton-mediated. A chemiluminescence NO analyzer (NOA) was therefore used to examine the decomposition of the various diazeniumdiolated copolymers in terms of total NO release capability. The typical NO release profiles for 9d and 9d′ (obtained from different NO loading conditions) suspended in PBS buffer at 37 °C as determined by the NOA are shown in Figure 6 (I). For each of the diazeniumdiolated copolymers studied, water diffusion into the insoluble particles of the copolymer initiates NO(g) release that reaches a maximum rate during the first half hour. As shown in Figure 6 (I)A-C, NO release slows significantly after the first 1 h period, but detectable levels of NO (at > 20 ppb) can still be observed after 24 h for most of the materials examined. After being soaked in PBS buffer for a more extended period of time, the NO donors release very low levels of NO (e.g., below 5 ppb); however, once an acid is added to the soaking solution, at this point,

Figure 6. NO release from different diazeniumdiolated copolymers at 37 °C (I), and the same copolymer at different temperatures (II). Five mg of each material as measured by chemiluminescence NO analyzer (NOA) in 4 mL of deoxygenated 0.1 M PBS buffer. (A) 9d made with 2eq NaOMe; (B) 9d with 1 equiv NaOMe; (C) 9d′ with 2 equiv NaOMe; (D) 9d with no NaOMe (** showing no significant NO release).

a sharp increase in the amount of NO release can be observed. It is very likely that this sudden increase in NO release results from two sources: acidification of the nitrite side product from the NO loading reaction and intact diazeniumdiolates whose activity are inhibited by the basic (high pH) microdomain environment within the donor polymer structures (see discussion below).24 It should also be noted that proton-driven NO release from the same material appears to be temperature dependent, but this effect is not very significant. Figure 6 (II) shows that the highest loaded 9d has a slightly faster initial NO release rate and a higher (ca. 15% in 3 h) accumulated NO release amount when soaked in the PBS buffer at a higher temperature. Polymeric Coatings for Potential Biomedical Applications. To demonstrate that the newly developed NO donors can be used to prepare polymeric coating materials that release NO for extended time periods, the copolymer 9d was doped into poly(vinyl chloride) (PVC) and silicone rubber (SR) matrixes (two commonly used polymers employed for extracorporeal tubing systems and intravascular catheters). The corresponding NO fluxes from a 7-mm circular PVC film and a SR coated catheter containing NO donors were studied, respectively. Additives were used to enhance the NO release from such polymeric coatings doped with 9d since it was found recently that continued release of NO from hydrophobic polymers containing diazeniumdiolate species requires regulation of organic phase pH within the polymeric matrix.24 Indeed, without control of pH, after an initial bolus of NO is released from the 7-mm polymeric film, the NO flux quickly decreases with time (see Figure 7C), only

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Figure 7. NO surface fluxes and total NO release curves for polymethacrylate-based diazeniumdiolate 9d (8 wt %) embedded in 2:1 PVC/DOS matrix (circular disks with a diameter of 7 mm and a thickness of approximately 150 µm, top coated with PVC) as measured by CL in PBS buffer (pH 7.4) at 37 °C. (A) PLGA as additive (8 wt % of lactide:glycolide (50:50), average Mw ) 50 000-75 000); (B) KTpClPB as additive (8 wt %); (C) no additive. Scheme 3. Hydrolysis of Poly(lactide-co-glycolide) in the Aqueous Environment

achieving 24% of the expected NO release capacity (measured via the acid injected condition for the doped NO donor, pH ) 1-2). This inhibition of NO release with time is due to an increase in pH within the organic phase, resulting from the formation of sodium hydroxide as well as free highly basic amine sites after diazeniumdiolate decomposition. Since loss of NO is primarily proton driven, the increase in pH greatly slows the NO release rate. In previous studies with discrete lipophilic diazeniumdiolates, the use of lipophilic anionic salts (e.g., potassium tetrakis(4-chlorophenylborate) (KTpClPB)) was employed to help drive protons into the organic film, as organic amine sites were generated.24 As shown in Figure 7B, when KTpClPB is added to the PVC film containing 9d, an approximately 100% enhancement in the total amount of NO that can be released is observed, compared to the polymer film that did not contain this additive. An even greater enhancement in both rate and total moles of NO (approximately 90% of its NO release capacity) released from such films could be achieved by adding the biodegradable poly(lactide-co-glycolide) (PLGA) polymer to the plasticized PVC films containing 9d (Figure 7A). Unlike KTpClPB’s proton exchange mechanism,24 it is hypothesized that the ester linkages of the biodegradable polymer (PLGA) are hydrolyzed as small amounts of water penetrate the polymer film from the surrounding aqueous environment to generate acid (lactic acid and glycolic acid) within the polymer matrix (e.g., pH < 4 in PLGA microspheres) as show in Scheme 3.42,43 Although the proton concentration in the matrix may not be as high as from the exogenous acid (pH ) 1-2) to achieve 100% conversion into NO, the presence of this continuous reaction compensates for the increase in pH from generation of sodium hydroxide and the free amines from NO release reaction, thereby maintain-

Figure 8. NO surface fluxes (I) and total NO release curves (II) for 9d (8 wt %) embedded in SR matrix (with a thickness of 150-200 µm) as measured by CL in PBS buffer (pH 7.4) at 37 °C. (A) PLGA as additive (8 wt %); (B) KTpClPB as additive (8 wt %).

ing a greater rate of NO release for longer periods of time. Moreover, this biodegradable additive is potentially harmless to the body since the final hydrolytic products may enter the tricarboxylic acid cycle and may be eliminated from the body as carbon dioxide and water. To further demonstrate PLGA’s capability of maintaining desirable NO surface flux for polymeric coatings, this polymer was also added to a SR matrix that contained 8 wt % of polymethacrylate NO donor 9d. NO release from catheters containing such outer-coating was compared to that from another SR control coating with KTpClPB added in the same manner. Figure 8 shows that with PLGA added NO surface flux from the coating was maintained above 10 × 10-10 mol cm-2 min-1 for at least 4 h, which is 10-fold more than the NO flux level of endothelial cells (EC) (ca. 1 × 10-10 mol cm-2 min-1). The same coating using KTpClPB as an additive releases NO at a greatly reduced rate. It should be noted that in Figure 8, NO release is shown for only the first 4 h in terms of NO surface flux. Actually, both coatings can release potentially thromboresistant levels of NO (approximately 1.5 × 10-10 mol cm-2 min-1) even after 24 h. Conclusions A series of new NO releasing methacrylate copolymers were designed and synthesized by covalently anchoring alkyldiamine side chains with various spacers onto a polymethacrylate-based copolymer backbone, followed by NO addition to form the desired pendant diazeniumdiolate structures. Based on the current study of this copolymer


system, sodium methoxide is an essential driving force for the deprotonation of the pendant amine sites that are diazeniumdiolated. UV and IR spectroscopies as well as chemiluminescence were used to investigate the formation and decomposition of the diazeniumdiolated copolymers. It was found that the release of NO from the final diazeniumdiolated products is proton mediated; therefore, exposure of this class of NO donors to physiological conditions stimulates NO release predominantly, and surely some formation of nitrosamine products on the polymer backbone due to the presence of oxygen. The fact that the parent amine sites are anchored to the polymer makes such nitrosamine formation less of a concern. Under the ambient condition, the products can slowly dissociate to release NO as well, with oxidized NO intermediate species (e.g., N2O3) also back-reacting with original amine sites to form nitrosamines. However, such dissociation can be greatly eliminated by storage of the diazeniumdiolated products under argon in the freezer. The NO release properties of these donors were studied, by themselves and after being doped into hydrophobic PVC and SR polymeric coatings with/without additives. The use of PLGA as a novel polymeric additive was shown to provide a means to enhance the release rate of NO from the diazeniumdiolated methacrylate copolymers incorporated these matrixes, by providing organic phase protons to maintain a proton activity required for diazeniumdiolate decomposition. Polymer films with a desired NO surface flux at or above 1 × 10-10 mol cm-2 min-1 for at least 24 h could be prepared using this concept. With such behavior, it is expected that such polymeric coatings can potentially be used to enhance the blood compatibility of various biomedical devices without significant concern about loss of the NO precursors (large polymethacrylate-based NO donors) from the organic phase of such coatings. Experimental Section Materials. Methyl methacrylate (MMA) and isodecyl acrylate (IDA) were freshly distilled under N2 before use and stored in a refrigerator. Triethylamine (TEA) was distilled over calcium hydride, and tetrahydrofuran (THF) was distilled over sodium and potassium benzophenone ketyl prior to use. Details on the synthesis of the diamino-alcohols (4a-c) are provided in the Supporting Information. Compound 2-(2-aminoethylamino)-ethanol (4d) was used as purchased from Aldrich. Additive poly(lactide-co-glycolide) (PLGA, 50:50, average Mw ) 50 000-75 000) was purchased from Aldrich and potassium tetrakis(4-chlorophenylbrorate) (KTpClPB) from Fluka. Measurements. 1H and 13C NMR spectra of all of the intermediates and the final products were obtained on a Varian 300 MHz or a 400 MHz spectrometer. FTIR spectra were collected on a Perkin-Elmer spectrum BX FT-IR system. High-resolution (HR) and low-resolution (LR) mass spectra were measured by a MicroMass LCT or a VG 70250-S mass spectrometer using either electrospray or chemical ionization. UV spectra were recorded as methanol solutions using a Beckman DU 640B or a Perkin-Elmer Lambda 35 spectrometer. Polymer molecular weights were


determined by gel permeation chromatography (GPC) using THF as the eluent and polystyrene standards on Waters Styragel HT 3 columns (500-30 000 molecular weight range). The instrument is equipped with a Waters UV detector (254 nm) and a Wyatt Optilab refractive index detector. All NO measurements were performed using a Sievers Nitric Oxide Analyzer (NOA), model 280. The instrument was calibrated before each experiment using an internal two-point calibration (zero gas and 45 ppm). The flow rate was set to 200 mL/min with a cell pressure of 5.4 Torr and an oxygen pressure of 6.0 psi. Elemental analyses were performed by the University of Michigan Microanalysis Laboratory. Synthesis. Monomer Precursors, N-Boc-Protected Diamino Alcohols (5a-d). A solution of 2.6 g (11.7 mmol) of di-tert-butyl dicarbonate in 15 mL of dry THF was added dropwise to a vigorously stirred solution of 1 g (5.3 mmol) of diamine-alcohol 4b and 2.7 g (26.5 mmol) of triethylamine in 100 mL of dry THF under argon at 0 °C. The reaction mixture was allowed to gradually warm to room temperature and stirred for 3 d. Upon completion of the reaction monitored by TLC, the solvent was removed under reduced pressure, and the residue was dissolved in ethyl ether and washed with water and brine. The organic layer was dried with sodium sulfate and the ethyl ether was removed in vacuo. The crude product 5b was purified by column chromatography on silica gel with a hexane/ethyl acetate mixture. Similar syntheses were carried out to obtain 5a, 5c, and 5d. 5a: Yield 64%, light oil. IR νmax (neat): 3388, 2923, 1692, 1415, 1162 cm-1; 1H NMR (400 MHz, CDCl3, δ): 3.59 (t, 2H, J ) 6.6 Hz, CH2OH), 3.24-3.00 (bs, 8H, 4NCH2), 1.55-1.43 (m, 8H, 4CH2), 1.41 (d, 18H, 2But), 1.38-1.15 (bs, 20H, 10CH2), 1.04 (t, 3H, J ) 7.0 Hz, CH3CH2); 13C NMR (100 MHz, CDCl3, δ): 155.6, 78.8, 62.9, 47.0, 46.9, 46.5, 41.7, 32.7, 29.5, 29.4, 29.3, 28.6, 28.4, 26.8, 26.7, 25.7, 13.5; MS (HR-ESI) m/z: [M+Na]+ calcd for C30H60N2O5, 551.4400; found 551.4396. 5b: Yield 43%, light oil. IR νmax (neat): 3416, 2929, 1693, 1416, 1165 cm-1; 1H NMR (400 MHz, CDCl3, δ): 3.70 (d, J ) 4.0 Hz, 2H, CH2OH), 3.34 (bs, 2H, NCH2), 3.44-3.06 (m, 6H, 3NCH2), 1.52-1.44 (m, 4H, 2CH2), 1.43 (s, 9H, But), 1.42 (s, 9H, But), 1.30-1.20 (m, 4H, 2CH2), 1.06 (t, 3H, J ) 7.0 Hz, CH3CH2); 13C NMR (100 MHz, CDCl3, δ): 155.4, 79.9, 78.9, 62.7, 50.2, 48.7, 46.5, 41.8, 28.4, 28.3, 26.5, 13.8; MS (HR-ESI) m/z: [M+Na]+ calcd for C20H40N2O5, 411.2835; found 411.2837. 5c: Yield 43%, light oil. IR νmax (neat): 3465, 2926, 1694, 1416, 1165 cm-1; 1H NMR (400 MHz, CDCl3, δ): 3.50 (t, 2H, J ) 6.6 Hz, CH2OH), 3.22-3.00 (bs, 8H, 4NCH2), 1.48-1.38 (m, 4H, 2CH2), 1.35 (s, 18H, 2But), 1.30-1.08 (bs, 18H, 9CH2), 0.99 (bs, 3H, CH3CH2); 13C NMR (100 MHz, CDCl3, δ): 155.1, 79.1, 62.5, 47.9, 47.5, 45.5, 45.1, 44.7, 42.8, 42.2, 32.6, 29.4, 29.3, 29.2, 29.1, 28.5, 28.3, 26.6, 25.6, 13.7, 13.3; MS (HR-ESI) m/z: [M+Na]+ calcd for C26H52N2O5, 495.3774; found 495.3775. 5d: Yield 68%, light oil. IR νmax (neat): 3358, 2975, 1692, 1411, 1170 cm-1; 1H NMR (400 MHz, CDCl3, δ): 5.104.75 (bs, 1H, NH), 3.71 (bs, 2H, CH2OH), 3.50-3.00 (bs,

788


6H, 3NCH2), 1.45 (s, 9H, 1But), 1.41 (s, 9H, 1But); 13C NMR (100 MHz, CDCl3, δ): 156.5, 80.2, 79.2, 61.5, 61.2, 51.5, 51.1, 48.4, 48.1, 40.7, 39.6, 28.2; MS (HR-ESI) m/z: [M+Na]+ calcd for C14H28N2O5, 327.1896; found 327.1892. Introduction of Polymerizable Group to Form Monomers (6a-d). A solution containing 0.38 g (0.97 mmol) of the protected diamine alcohol 5b and 0.98 g (9.7 mmol) of triethylamine was prepared in dry THF at 0 °C under argon. The solution was then stirred vigorously, and 0.3 g (2.91 mmol) of methacryloyl chloride was added dropwise maintaining the temperature below 10 °C. The reaction mixture was then stirred at room temperature for 3 d, monitoring by TLC. A white precipitate of triethylamonium chloride was filtered off and washed with THF. The combined organic phases were evaporated under reduced pressure, and the resulting residue was dissolved in ethyl ether. This solution was washed with saturated sodium bicarbonate solution, water and brine, and then dried over sodium sulfate. After removal of the solvent, the product 6b was purified by column chromatography on silica gel with a hexane/ethyl acetate mixture. Compounds 6a, 6c, and 6d were prepared in the similar way. 6a: Yield 64%, light oil. IR νmax (neat): 2926, 1717, 1694, 1638, 1416, 1163 cm-1; 1H NMR (400 MHz, CDCl3, δ): 6.07 (s, 1H, CHH)), 5.52 (s, 1H, CHH)), 4.11 (t, 2H, J ) 6.6 Hz, CH2CH2O), 3.26-3.02 (bs, 8H, 4NCH2), 1.92 (s, 3H, CH3C)), 1.70-1.58 (m, 2H, CH2), 1.52-1.40 (bs, 6H, 3CH2), 1.42 (s, 18H, 2But), 1.40-1.16 (bs, 20H, 10CH2), 1.06 (t, 3H, J ) 7.2 Hz, CH3CH2); 13C NMR (100 MHz, CDCl3, δ): 167.4, 155.5, 136.5, 125.0, 78.8, 64.7, 47.0, 46.8, 46.5, 41.7, 29.5, 29.4, 29.3, 29.1, 28.5, 28.4, 26.8, 26.6, 25.9, 18.2, 13.5; MS (HR-ESI) m/z: [M+Na]+ calcd for C34H64N2O6, 619.4662; found 619.4664. 6b: Yield 32%, light oil. IR νmax (neat): 2929, 1721, 1692, 1638, 1415, 1157 cm-1; 1H NMR (400 MHz, CDCl3, δ): 6.04 (s, 1H, CHH)), 5.51 (s, 1H, CHH)), 4.17 (d, 2H, J ) 3.6 Hz, CH2CH2O), 3.45-3.25 (m, 2H, NCH2), 3.28-2.90 (bs, 6H, 3NCH2), 1.88 (s, 3H, CH3C)), 1.56-1.30 (bs, 4H, 2CH2), 1.38 (s, 18H, 2But), 1.21 (bs, 4H, 2CH2), 1.01 (t, 3H, J ) 7.2 Hz, CH3CH2); 13C NMR (100 MHz, CDCl3, δ): 167.1, 155.3, 136.2, 125.7, 79.4, 78.8, 62.8, 62.7, 47.9, 47.7, 46.5, 45.8, 45.7, 41.6, 28.6, 28.4, 28.3, 28.1, 26.6, 26.5, 18.2, 13.7; MS (HR-ESI) m/z: [M+Na]+ calcd for C24H44N2O6, 479.3097; found 479.3083. 6c: Yield 77%, light oil. IR νmax (neat): 2926, 1718, 1694, 1638, 1414, 1164 cm-1; 1H NMR (400 MHz, CDCl3, δ): 6.04 (s, 1H, CHH)), 5.48 (s, 1H, CHH)), 4.08 (t, 2H, J ) 6.6 Hz, CH2CH2O), 3.24-3.00 (bs, 8H, 4NCH2), 1.88 (s, 3H, CH3C)), 1.64-52 (m, 2H, CH2), 1.40 (s, 18H, 2But), 1.38-1.10 (bs, 18H, 9CH2), 1.04 (bs, 3H, CH3CH2); 13C NMR (100 MHz, CDCl3, δ): 167.4, 155.2, 136.4, 125.0, 79.2, 64.7, 48.0, 47.6, 45.6, 45.2, 44.8, 42.9, 42.3, 29.5, 29.4, 29.3, 29.1, 28.7, 28.5, 28.4, 26.7, 25.9, 13.8, 13.4; MS (HRESI) m/z: [M+Na]+ calcd for C30H56N2O6, 563.4036; found 563.4039. 6d: Yield 70%, light oil. IR νmax (neat): 3368, 2975, 1718, 1698, 1638, 1411, 1164 cm-1; 1H NMR (400 MHz, CDCl3, δ): 6.09 (s, 1H, CHH)), 5.56 (s, 1H, CHH)), 5.04-4.68 (bs, 1H, NH), 4.22 (bs, 2H, CH2CH2O), 3.48 (bs, 2H, NCH2),

Zhou and Meyerhoff

3.35 (bs, 2H, NCH2), 3.25 (bs, 2H, NCH2), 1.92 (s, 3H, CH3C)), 1.44 (s, 9H, But), 1.41 (s, 9H, But); 13C NMR (100 MHz, CDCl3, δ): 166.8, 155.7, 135.8, 125.7, 79.9, 78.8, 62.6, 62.4, 47.3, 46.9, 46.3, 46.1, 40.2, 39.1, 28.1, 18.0; MS (HR-ESI) m/z: [M+Na]+ calcd for C18H32N2O6, 395.2158; found 395.2158. Polymerization to Form 7a-d′′. Individual methacrylate monomer 6a-d and MMA or MMA-IDA were mixed in appropriate mole ratio aiming to achieve copolymers 7ad′′ (where the composition of each monomer 6a-d is 1020 mol %, Table 1). A 0.22 mmol of monomer mixture was dissolved in 1 mL of dry THF and placed in a 5 mL reaction vial equipped with a small stir bar and Teflon seal. The initiator AIBN (0.5 mol %) was added. Before heating, the vial was flushed with argon for 10 min and the reactor was sealed and then placed in an oil bath at 65-70 °C. The mixture was stirred for 48 h at this temperature. The solution was then concentrated to around 0.3 mL, and the polymer was precipitated with 5-10 mL hexane. This dissolution and precipitation procedure was repeated three times for purification. The polymer was dried under vacuum overnight. 1H NMR spectra were collected to confirm the structures and actual compositions of the copolymers (Figure 1s). GPC measurements were performed to determine the molecular weights of the copolymers (Table 2). Deprotection to Form 8a-d′′. A total of 40∼50 mg of each copolymer 7a-d′′ was dissolved in 2 mL of dichloromethane, and then 200 µL of TFA was added dropwise. The solution was stirred at room temperature for 3 h. The reaction mixture was diluted with 20 mL of dichloromethane, and the organic phase was then washed with sodium bicarbonate, water and brine, and finally dried with sodium sulfate. After the solvent was evaporated, the resulting copolymer was dissolved and precipitated with THF/hexane, and then dried under vacuum overnight. 1H NMR spectra were collected to confirm the structures of the copolymers (Figure 2s). NO addition to Form 9a-d′′. A total of 20∼30 mg of each deprotected copolymer 8a-d′′ was dissolved in 3 mL of dry THF and placed in a high-pressure reactor with a stir bar and flushed with argon for 10 min. Sodium methoxide (0 or 1 or 2 equiv with respect to the total amine sites) was then added, and the reactor was closed. The reactor was purged with argon several times and charged with NO at 80 psi. The reaction mixture was stirred at room temperature and after 72 h each copolymer was precipitated with dry hexane under argon. After quick removal of the remaining solvent using vacuum, the NO addition copolymers 9a-d′′ were then examined by UV and NO release was assessed using the NOA (Table 3). The resulting diazeniumdiolated copolymers were stored in sealed vials charged with argon in the freezer. Preparation of Polymeric Coatings Containing NO Donor (9d). A PVC film containing the polymer-based NO donor 9d was prepared by dissolving 133 mg of poly(vinyl chloride) (PVC), 66 mg of dioctyl sebacate (DOS), and 20 mg of poly(lactide-co-glycolide) (50:50) (PLGA 50:50) in 3-4 mL of THF. A total of 20 mg of polymer-base NO donor 9d was then dispersed within the polymer cocktail


via sonication for 10 min to obtain a slightly cloudy dispersion of the diazeniumdiolate within the polymer solution. A trilayer film configuration was employed to fabricate films. A straight PVC solution (44 mg/mL in THF) was first cast into a 3.7 cm diameter Teflon ring with a Teflon base. Four hours later, the polymer cocktail was then cast on top of the PVC layer. After another 4 h, the same straight PVC solution was cast again as a top layer. The membrane was allowed to cure overnight. Polymer films containing KTpClPB and no additives were prepared in a similar manner. Small circular disks with a diameter of 7 mm and a thickness of ∼150 µm were cut from the parent films the next morning and measured for their NO release via chemiluminescence. A SR coating containing the polymer-based NO donor 9d was prepared by dissolving 200 mg SR and 20 mg PLGA in 3-4 mL of THF. A total of 20 mg of polymer-base NO donor 9d was dispersed within the polymer cocktail. SR catheter sleeves were dip coated with such cocktail at a 20 min interval 8 times and then dip coated with a top PVC layer using a PVC solution (44 mg/mL in THF). The catheter sleeves were covered and allowed to cure overnight, and NO release was measured subsequently. Acknowledgment. We gratefully acknowledge the NIH (EB-00783) and Michigan Critical Care Consultants, Inc., for supporting this work. We also thank Dr. Pawel G. Parzachowski and Dr. Jeremy T. Mitchell-Koch for helpful discussions. Supporting Information Available. Details on the synthesis of the diamino-alcohols (4a-c) and the typical 1H NMR spectra for copolymers (7a-d) and (8a-d). This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) (2) (3) (4) (5) (6) (7)

(8) (9) (10) (11)

Keefer, L. K. Nat. Mater. 2003, 2, 357-358. Brash, J. L. J. Biomater. Sci. Polym. Ed. 2000, 11 (11), 1135-1146. Kim, S. W.; Jacobs, H. Blood Purif. 1996, 14, 357-372. Peppas, N. A.; Langer, R. Science 1994, 263, 1715-1720. Annich, G. M.; Meinhardt, J. P.; Mowery, K. A.; Ashton, B. A.; Merz, S. I.; Hirschl, R. B.; Meyerhoff, M. E.; Bartlett, R. H. Crit. Care Med. 2000, 28 (4), 915-920. Ratner, B. D. J. Biomed. Mater. Res. 1993, 27, 283-287. Wise, D. L.; Gresser, J. D.; Trantolo, D. J.; Cattaneo, M. V.; Lewandrowski, K. U.; Yaszemski, M. J.; Eds. Biomaterials Engineering and DeVices: Human Applications; Humana Press: Totowa, NJ, 2000; Vol. 1. Van Der Kamp, K. W. H. J. The Interactions of Blood with Polymeric Materials; Groningen: Rijksuniversiteit Groningen, 1995. Szycher, M. Medical/Pharmaceutical Markets for Medical Plastics. In High Performance Biomaterials; Szycher, M., Ed.; Technomic: Lancaster, PA, 1991; pp 3-40. Greco, R. S., Ed.; Implantation Biology: The Host Response and Biomedical DeVices; CRC Press: Boca Raton, FL, 1994. Tanzawa, H. Biomedical Polymers: Current Status and Overview. In Biomedical Applications of Polymeric Materials; Tsuruta, T., Hayashi, T., Kataoka, K., Ishihara, K., Kimura, Y., Eds.; CRC Press: Boca Raton, FL, 1993; pp 1-15.

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BM049462L

Polymethacrylate-Based Nitric Oxide Donors with Pendant N

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