Biomaterials 23 (2002) 1485–1494

Nitric oxide releasing silicone rubbers with improved bloodcompatibility: preparation, characterization,

and in vivo evaluation

Huiping Zhanga, Gail M. Annichb, Judiann Miskulinb, Kathryn Osterholzerc,Scott I. Merzc, Robert H. Bartlettb, Mark E. Meyerhoffa,*

aDepartment of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, MI 48109-1055, USAbDepartment of Surgery, University of Michigan, Ann Arbor, MI 48109, USA

cMichigan Critical Care Consultants, Inc., Ann Arbor, MI 48103, USA

Received 25 April 2001; accepted 29 May 2001

Abstract

Nitric oxide (NO) releasing silicone rubbers (SR) are prepared via a three-step reaction scheme. A diamino/triamino-alkyltrimethoxysilane crosslinker is used to vulcanize hydroxyl terminated polydimethylsiloxane (PDMS) in the presence of ambient

moisture and a dibutyltin dilaurate catalyst so that the respective diamine/triamine groups are covalently linked to the cured SRstructure. These amine sites are then diazeniumdiolated, in situ, when the cured SR is reacted with NO at elevated pressure (80 psi).Although nitrite species are also formed during the NO addition reaction, in most cases the diazeniumdiolated polymer is the major

product within the final SR matrix. Temperature appears to be the major driving force for the dissociation of the attacheddiazeniumdiolate moieties, whereas the presence of bulk water bathing the SR materials has only minimal effect on the observed NOrelease rate owing to the low water uptake of the SR matrices. The resulting SR films/coatings release NO at ambient orphysiological temperature for up to 20 d with average fluxes of at least 4� 10�10mol � cm�2 �min�1 (coating thickness X600mm)over first 4 h, comparable to the NO fluxes observed from stimulated human endothelial cells. The NO loading and concomitant NOrelease flux of the SR material are readily adjustable by altering the diamine/triamine loading and film/coating thickness. The newNO releasing SR materials are shown to exhibit improved thromboresistance in vivo, as demonstrated via reduced platelet

activation on the surface of these polymers when used to coat the inner walls of SR tubings employed for extracorporeal circulationin a rabbit model. r 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Nitric oxide; Silicone rubbers; Diazeniumdiolates; Thrombosis; Platelet activation; Extracorporeal circulation; Silanes

1. Introduction

Synthetic polymers, including poly(vinyl chloride)(PVC), polyurethane, and silicone rubber (SR), arewidely used in the medical field with an annualconsumption of over one billion pounds [1]. However,when in direct contact with blood, these polymers arestill prone to initiate the formation of clots, as plateletsand other components of the blood coagulation systemare activated. Systemic anticoagulation is often per-formed clinically to reduce risk of thrombus on the

surfaces of these materials, especially when employed intubing form for extracorporeal circulation. Despitedecades of research, non-thrombogenic surfaces forblood-contacting polymers remain an elusive goal.A potential solution to the problem of thrombogenic

polymers may now be realized by creating polymericmaterials that are capable of releasing low levels of nitricoxide (NO) at the blood/polymer interface. Smith et al.[2], Schoenfisch et al. [3], and Annich, et al. [4] allreported recently that when specific diazeniumdiolatetype NO donors [5], which readily release NO underphysiological conditions, are incorporated within var-ious polymeric matrices (through dispersion or covalentattachment), a dramatic improvement in the observedin vivo blood compatibility results. Indeed, NO is a

*Corresponding author. Tel.: +1-734-763-5916; fax: +1-734-647-

4865.

E-mail address: mmeyerho@umich.edu (M.E. Meyerhoff).

0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.

PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 2 7 4 - 5

potent anti-platelet agent that is naturally generated byhuman endothelial cells to decrease the propensity ofblood to clot [6,7]. Studies have shown that stimulatedhuman endothelial cells continuously generate NO at alevel of ca. 4� 10�10mol cm�2min�1 [8]. Polymers withequal or higher NO fluxes, therefore, may prove effectivein preventing platelet adhesion/activation/aggregationonto their surfaces, thereby lowering the risk ofthrombus formation and other complications associatedwith interactions between blood and foreign materials.Diazeniumdiolate NO donors (also known as NON-

Oates) are typically synthesized by the reaction ofsecondary amines with NO at elevated pressure[5,9,10]. One mole of diazeniumdiolate species readilydissociates into 2mol of NO(g) and 1mol of the donoramine when exposed to water [11] or at relatively hightemperature [12]. When certain structures (e.g., di-methylhexanediamine diazeniumdiolate) were dispersedwithin polymer matrices, however, they were found toleach into aqueous bathing media due to their hydro-philic nature [13]. Furthermore, a measurable level ofpotentially carcinogenic nitrosamine species was alsodetected within the bathing medium as a result of theback reaction of an oxidized NO intermediate specieswith secondary amine sites on the original donormolecule [13]. Covalently linking the diazeniumdiolatemoiety onto polymer structures should avoid suchleaching and possible toxicity issues and yet maintainNO release ability. Recent preliminary studies havedemonstrated that NO releasing silicone materials couldbe prepared with anchored diazeniumdiolate groups[14]. Herein, a more detailed investigation regarding thesynthesis and characterization of these novel SRmaterials is provided. It will also be shown that NOrelease properties of these materials can be varied by thestructure of the diamino/triamino-alkyltrimethoxysilanecrosslinkers used in conjunction with polydimethylsilox-ane (PDMS) to form the rubber films, the amount ofcrosslinker employed in a given formulation, and thethickness of the final polymeric films. It will be furtherdemonstrated that when such NO releasing materials areused to coat the inner walls of SR tubings employed forvenovenous extracorporeal circulation of a rabbit [4], asignificant reduction in the activation of platelets on theinner walls of the tubings is observed vs. control in vivoexperiments using the same polymer coatings butwithout NO release.

2. Experimental section

2.1. Materials

Hydroxyl terminated polydimethylsiloxane (PDMS,viscosity 65, 120, 750, 2000, and 20000 cSt), methyl-trimethoxysilane, and dibutyltin dilaurate, were ob-

tained from Aldrich (Milwaukee, WI). Diamino-alkyltrimethoxysilane crosslinking agents (DACA) in-cluding N-(6-aminohexyl)-3-aminopropyl-trimethoxysi-lane (DACA-6) and N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (DACA-2), and hexamethyldisilazanetreated fumed silica were products of Gelest (Tullytown,PA). Nitric oxide and argon (Ar) gases were obtainedfrom Cryogenic Gases (Detroit, MI). Toluene, xylene,tetrahydrofuran, hexane, and methylene chloride werepurchased from Fisher (Pittsburgh, PA). All chemicalswere used as received.

2.2. Preparation and characterization of NO releasingsilicone rubbers

The NO releasing silicone rubbers were prepared viathe synthetic approach shown in Fig. 1. Briefly, thediaminoalkyltrimethoxylsilane (DACA) species reactswith the terminal hydroxyl groups of PDMS andreleases methanol in the presence of the dibutyltindilaurate catalyst. The resulting aminated PDMSmacromolecules are then crosslinked to each other(condensation-curing) in the presence of ambientmoisture; thereby covalently linking diamine sites tothe SR matrices. The cured DACA-SR films/coatingswere then reacted with NO at 80 psi, and the diaze-niumdiolate species (DACA/N2O2) are formed on thependent amine sites, in situ. For example, PDMS (2000cSt, 1.6 g), DACA-6 (240mg), and dibutyltin dilaurate(1.6mg) were mixed in toluene (8ml) overnight, andthen mixed completely with a fumed silica/toluenesuspension (320mg/8ml), cast into wells (diam. 27mm,depth ca. 5mm) on Teflon plates, and cured underambient moisture for 48 h. The resulting DACA-SRfilms (ca. 250 mm thick) were then peeled off, placedwithin the NO reactor (modified from a Parr hydro-genation system), purged with Ar gas, and charged withNO gas (80 psi) for 24–120 h. A UV absorbance band at230–250 nm that is characteristic of diazeniumdiolateformation [11] was observed when thin films of theresulting NO releasing SR were initially cast on quartzslides. CHN elemental analysis results indicated thatthere was a 40–60mol% increase in nitrogen contentafter the NO addition to the DACA-SR films. Thissuggests that the conversion efficiency from diaminogroups to diazeniumdiolate groups was 40–60% if onlydiazeniumdiolate functional groups were formed uponNO addition, and if for every diazeniumdiolate group,one protonated adjacent amine site serves as therequired counter-cation.The NO release from various DACA/N2O2-SR film

formulations was characterized using either a chemilu-minescence NO analyzer (Sievers, NOATM 280) or aspectrophotometric Griess assay [15]. The former is adirect and selective NO(g) measurement where all theNO generated from samples is swept with N2 into the

H. Zhang et al. / Biomaterials 23 (2002) 1485–14941486

NOA and is then detected based on a gas-phasechemiluminescent reaction between NO and ozone.The latter is an indirect measurement of NO whereNO is first oxidized to nitrite by ambient oxygen inphosphate buffered saline (PBS) solutions and then theresulting nitrite levels are determined via absorbancemeasurement at 540 nm after the nitrite species areconverted to an azo-compound via the Griess reaction[15].

2.3. In vivo blood compatibility evaluation

Approximately one meter lengths of extracorporealSR tubing (i.d. 6.35mm) were coated by adding thePDMS cocktail solution (described above) and allowingthe bolus of solution to flow through the entire length ofthe tubing. The excess solution was discarded, and the

tubing was extended horizontally and rotated slowly toensure even coating during curing. Several coats wereapplied until the coating thickness reached approxi-mately 600 mm. In addition to the tubing, 10-FrenchPVC cannulas and polypropylene tubing connectorswere also coated and connected to the pretreated tubingsegments to create the test circuits. Some of theseDACA-SR coated circuits were used subsequently ascontrols for the extracorporeal experiments. The re-mainder was reacted with NO at 80 psi for 36–64 h priorto carrying out the animal studies. The tubings wereconnected to a rabbit through two cannulas forvenovenous circulation via a roller pump. Blood wascirculated through the tubing for 4 h at a rate of>90ml/min. Platelet counts were recorded each hourthroughout the experiment. At the end of each experi-ment, the tubing was removed, rinsed with normal saline

Fig. 1. Scheme used to prepare DACA/N2O2-SR materials.

H. Zhang et al. / Biomaterials 23 (2002) 1485–1494 1487

(0.9% saline), fixed with 2wt% aqueous glutaraldehydesolution, dehydrated with 30%, 50%, 70%, 90%, 95%,and 100% (v/v) ethanol and hexamethyldisilazane. Onecentimeter segments of each of the circuits were selectedfor examination at the following location: (1) immedi-ately distal to the drainage cannula, (2) region within theroller pump, (3) exit from the roller pump, (4)immediately distal to the infusion cannula. These tubingpieces were cut in half, sputter-coated with gold, andexamined on their inner surfaces for surface plateletadhesion using a scanning electron microscope (SEM,Hitachi S-3200N). The tubing was also examinedvisually for general gross thrombus formation alongthe inner wall of the circuit. Gross necropsy wasperformed on the rabbits at the end of each experimentto determine whether thrombo-embolic injury occurredwithin the lungs.

3. Results and discussion

3.1. DACA-SR preparation

A variety of factors are involved in the curing processfor the DACA-SR films. These generally fall into twocategories: internal and external. The former includesthe molecular weight (MW) of PDMS, types andamounts of DACA, and the amount of tin catalystemployed in the curing reaction. The latter includeshumidity, temperature, and solvents. Numerous curingexperiments were conducted initially to investigate thesefactors so as to obtain suitable conditions for preparingfully cured DACA-SR films. Table 1 summarizes theformulations found to be useful for preparing fullycured DACA-SR films. As suggested by the results, itappears that the MW of PDMS must be greater than18,000 (viscosity X750 cSt) for the successful prepara-tion of DACA-SR films. Indeed, lower MW PDMStends to yield white and brittle films that are difficult topeel off intact from the Teflon plates. The DACA

loading (generally, 3–12 equivalents (3X–12X) com-pared to hydroxyl groups of PDMS) is also important.Lower crosslinker levels (o3X) tend to cause incom-plete curing, while higher loadings (>12X) yield brittle,sticky, and even rippled or cracked films.From Table 1 it is also notable that DACA cross-

linkers require less tin catalyst (typically 0.03–0.3 wt%)to cure PDMS than conventional alkyltrialkoxysilanecrosslinking agents; in fact, several wt% is typicallyrequired for the latter [16]. Thus, it appears that theDACA crosslinkers possess a higher reactivity, probablyowing to the pendant amino groups that can acceleratethe condensation-curing process, similar to aminecatalysis of silylation reactions [17]. The use of largeramounts of the tin catalyst (>0.5wt%), on the otherhand, lead to incomplete curing for DACA-SR films.This is normally not observed for conventional moisturecured SRs. Such failure may be due to the oligomeriza-tion of the DACA crosslinkers at high catalyst levels; asa result, the crosslinking ability of DACA is greatlyreduced, and hence curing of the PDMS is inhibited.All of the formulations reported in Table 1 were

determined for SR films cast at ambient temperatureand humidity (p50%) with a fumed silica (as filler)content in a range of 0–20wt%. It was found that ahigher humidity environment usually yields sticky filmssince the top surface of the films, which is exposed to air,may cure so fast that moisture is probably inhibitedfrom penetrating further into the film [18]; as a result,the back side of the film may only be partially cured.Furthermore, both high boiling point (b.p.), includingtoluene and xylene, and low b.p. solvents, includinghexane, tetrahydrofuran, and methylene chloride, wereexamined to cast DACA-SR films. Smoother, clearer,and less sticky films are generally obtained with thehigher b.p. solvents. In general, the effect of the internaland external variables was found to be nearly the samefor both the DACA-2-SR and DACA-6-SR films.

3.2. NO release characteristics of DACA/N2O2-SR

The formation of diazeniumdiolate structures withinDACA-SR materials reacted with NO under highpressure was confirmed by measuring the release ofNO from the materials by chemiluminescence (NOA)and/or by recording the UV absorbance spectra of thinfilms of the cured polymers after reaction with NO.Figs. 2 and 3 illustrate the typical NO release rates (indry nitrogen atmosphere and in PBS buffer) and theUV-Vis spectra of freshly prepared DACA-6/N2O2-SRfilms. As shown, the NO reacted films release relativelylarge quantities of NO, and the freshly prepared filmsexhibit a strong absorbance band centered at 235 nm,characteristic of the diazeniumdiolate species (230–260 nm) [11]. The diazeniumdiolate content of freshlyNO reacted films was estimated from the CHN

Table 1

Various formulations found to be useful in preparing fully cured

DACA-SR films

PDMS Applicable DACA loadings

mole ratio over

–OH (wt% of PDMS)

Applicable amount

of tin catalyst

(wt% of PDMS)

DACA-6-SR

65c St N/A (brittle) N/A

120 cSt N/A (brittle) N/A

750 cSt 3X–6X (10–20%) 0.05–0.2%

2000 cSt 3X–12X (4.5–18%) 0.03–0.5%

20,000 cSt 3X–18X (2.0–12%) 0.03–0.2%

DACA-2-SR

2000 cSt 3X–12X (3.5–13%) 0.03–0.5%

H. Zhang et al. / Biomaterials 23 (2002) 1485–14941488

elemental analysis. It was found that ca. 0.20 mmol/mgof diazeniumdiolate groups were formed for filmsoriginally containing 0.42 mmol/mg diamine sites (i.e.,

15wt% (10X) of DACA-6 loading), under the assump-tion that the overall nitrogen content increase isprimarily due to the diazeniumdiolate formation.Diazeniumdiolate dissociation to NO from the cross-

linked SR materials is assumed to be the reversepathway to its formation [19], as illustrated in Fig. 4.Such a dissociation pathway should be accelerated whenthe diazeniumdiolate molecules encounter water [11] orwhen the temperature is increased [12]. Indeed, the half-lives of various diazeniumdiolate molecules in PBSbuffer range from minutes to days at ambient tempera-ture [10], but tend to be much longer in the dry state. Inthe presence of water or other proton sources, theprotonation step of the amino nitrogen (linked to thediazeniumdiolate anion) should be favored [20]; thus,the equilibrium shifts to the left (see Fig. 4), anddissociation of the diazeniumdiolate adduct takes place.The NO release profiles of DACA-6/N2O2-SR films in

dry nitrogen atmosphere and in PBS buffer at ambienttemperature (231C) are also shown in Fig. 2. As can beseen, the diazeniumdiolate species within these filmsrelease as much NO in the dry state as when bathed inPBS buffer. Indeed, water uptake by the SR films seemsto barely influence the rate of diazeniumdiolate dis-sociation. A possible explanation for this behaviorrelates to the water uptake of SR, which is reported tobe rather low (typically 0.3–1.5wt% over 197 d [21]).Hence, this amount of water may not be sufficient toaffect the NO release pathway of DACA-6/N2O2.Indeed, thermal decomposition appears to be theprimary driving force for dissociation of DACA-6/N2O2. As shown in Fig. 5, DACA-6/N2O2-SR coatingson inner walls of SR tubing release NO at a much fasterrate at 371C than at 231C in a nitrogen atmosphere (asmeasured by the NOA). As shown in Fig. 3, no obviousloss in the number of diazeniumdiolate groups isobserved for DACA-6/N2O2-SR after storage for morethan 50 d at �201C. Indeed, the characteristic UV-Visband for the diazeniumdiolate species at 235 nm remainsnearly intact upon such storage. However, as also shownin Fig. 3a, the diazeniumdiolate band decreases signifi-

Fig. 4. Hypothesized mechanism of diazeniumdiolate formation and

dissociation (modified from the one proposed by Drago [19]).

Fig. 2. NO release profiles (average of two films) of DACA-6/N2O2-

SR films (diam. 1 cm, thickness ca. 250mm) with 20wt% DACA-6

loading in dry nitrogen atmosphere (a) and in PBS (b), and of the films

with 15wt% DACA-6 loading in dry nitrogen atmosphere (c) and in

PBS (d), as measured by the chemiluminescence NO analyzer (NOA)

at 231C.

Fig. 3. UV-Vis spectra of various SR films on quartz slides; (F)

DACA/N2O2-SR fresh films, (- - -) DACA/N2O2-SR films stored at

various conditions, and (y) DACA-SR control films: (a) a DACA-6/

N2O2-SR film stored at 231C (45 d); (b) a DACA-6/N2O2-SR film

stored at –201C (55 d); and (c) a DACA-2/N2O2-SR film stored at

–201C (55 d).

H. Zhang et al. / Biomaterials 23 (2002) 1485–1494 1489

cantly when films are stored at ambient room tempera-ture. This observation further confirms the resultsshown in Fig. 5 regarding the effect of temperature onthe rate of NO release from the films.Unlike DACA-6/N2O2, DACA-2/N2O2 appears to

dissociate quite readily even at �201C (Fig. 3c),suggesting that DACA-2/N2O2 is much less stable thanDACA-6/N2O2. This observation contradicts what hasbeen reported in the literature for smaller free diaminespecies in aqueous phases [10]. Indeed, Hrabie hasshown that shorter alkyl chains between two secondaryamine nitrogen atoms yield more stable diazeniumdio-late species in aqueous phase experiments [10]. Appar-ently, the much more hydrophobic environment of theSR matrix could be playing a significant role in furtherstabilizing the DACA-6/N2O2 material over that ob-served for DACA-2/N2O2, but additional studies arerequired to determine the precise mechanism of thisenhanced stability.In addition to NO gas release, nitrite release was also

detected from the DACA/N2O2-SR films by determiningthe nitrite level in the PBS buffer used to soak the SRfilms during the chemiluminescence measurements.Based on such measurements (via spectrophotometricGriess assay) it appears that both diazeniumdiolate andnitrite species are present within freshly NO reacted SRmaterials. This is clearly shown in Table 2, whichprovides the mol% of NO release over total moles ofNO plus nitrite released (termed PNO) for variousDACA/N2O2-SR films. In some cases (e.g., early on inthe first 5 h of the 10X DACA-6 sample), more moles ofnitrite are released from the film than moles of NO gas(PNOo50%). The nitrite species may have been formedeither during the NO addition, as NOx species arealways present in the high pressure NO source used forreacting the polymer [22], or during the ambient work-up process following removal of the polymer films from

the NO reactor. In the latter case, diazeniumdiolatespecies may be decomposed to corresponding ammo-nium nitrite species in the presence of ambient oxygenand moisture. However, similar levels of nitrite (e.g.,2.5–4.5� 10�3 mmol/mg) were observed within the first4 h for 10X DACA-6/N2O2-SR films even when oxygenwas excluded during the work-up process (usingnitrogen bag). This result suggests that the observednitrite species within the DACA/N2O2-SR materialsoriginate during the NO addition reaction, likely fromNOx impurities in the NO source.Since ion exchange of any nitrite anions within the

films is relatively fast, the nitrite levels in the PBS bufferused to bath the films during NO chemiluminescencemeasurements should quickly level off with time, whilethe release of NO gas should continue until all of thediazeniumdiolates have decomposed. As shown in Table2, this is exactly what is observed for DACA-6/N2O2-SRfilms. Indeed, DACA-6/N2O2-SR films appear to con-tain primarily diazeniumdiolate species in most cases, asthe amount of NO gas released eventually surpasses thenitrite levels measured by the Griess assay (PNOX66%).The DACA-2/N2O2-SR films, however, contain farmore initial nitrite species than the DACA-6/N2O2-SRfilms, as the former has much lower PNO values (seeTable 2). It is still not clear why the slight difference instructure between DACA-2 and DACA-6 causes such alarge difference in the NO addition product, as well asthe thermal stability of the respective diazeniumdiolatespecies formed within the films (see above).The DACA-6/N2O2-SR films release NO for up to

20 d (Fig. 6), much longer than corresponding smalldiazeniumdiolate molecules made from similar aminestructures and monitored in aqueous phases (minutes)[10]. Such extended NO release may be especially usefulin biomedical areas where relatively long blood contacttimes are required for the polymeric materials used for aparticular application, such as implanted sensors andextracorporeal circulation, specifically extracorporealmembrane oxygenation (ECMO), where blood ofpatients is pumped through an external oxygenator forperiods of 2–3 weeks [23].

Fig. 5. NO flux profiles of SR tubing pieces (i.d. 6.35mm) coated with

DACA-6/N2O2-SR (10X DACA-6 (15wt%), coating thickness ca.

700mm), at 371C (a) and 231C (b) in a nitrogen atmosphere, as

measured by the NOA.

Table 2

Percentage (mol%) of NO(g) released over total moles of NO(g) plus

nitrite released (termed PNO) from various DACA/N2O2-SR films after

given time periods

DACA loading mole ratio over

–OH (wt% of PDMS)

PNO (mol%) (bathing time in PBS)

DACA-6/N2O2-SR

4X (6%) 55% (5.0 h); 66% (11.3 h)

8X (12%) 58% (5.0 h); 70% (10.4 h)

10X (15%) 33% (4.3 h); 63% (11.6 h)

DACA-2/N2O2-SR

10X (11%) 20% (4.1 h); 26% (11.6 h)

H. Zhang et al. / Biomaterials 23 (2002) 1485–14941490

Since the NO flux from the SR surfaces is critical forinhibition of platelet adhesion/activation [24], it isimportant to investigate ways to adjust the totaldiazeniumdiolate loading and/or NO flux from thesurface of DACA-6/N2O2-SR materials. This couldinclude altering DACA-6 content, NO charging time,film/coating thickness, as well as incorporating basesand/or small counter-cations within SR matrices to helpfurther stabilize diazeniumdiolate adduct formation. Ingeneral, it has been found that NO loading and NOfluxes increase with the wt% of DACA-6 loading, asillustrated in Fig. 7. As the DACA-6 loading exceeds8X, however, the amount of NO released changes verylittle; the initial nitrite ion release level, however,appears to increase as the DACA-6 content increases,as can be seen by comparing the PNO values for the 8Xand 10X DACA-6-SR films (see Table 2).The NO release from films of DACA-6/N2O2-SR was

also found to increase with the film thickness (seeFig. 8). Thicker films should contain more total DACA-6 and thus yield more total diazeniumdiolate species perunit surface area of film. Thus, NO generated within theinner bulk of the SR material still diffuses out to thesurface. Even though doubling the film thickness doesnot double the initial NO release rate (Fig. 8), the totalamount of NO released over relatively long time period(>20 d) tend to be linearly proportional to filmthickness (Fig. 6).Increasing the NO charging time also increases the

NO loading via diazeniumdiolate formation (data notshown). However, the observed changes on NO loadingtend to become small (o50%) when the NO additionreaction is continued beyond 48 h.As discussed previously, the diazeniumdiolate forma-

tion can be favored by shifting the equilibrium stepsshown in Fig. 4 towards the product side. Apparently,providing a strong base and/or alternate counter-cationcan force the equilibrium toward diazeniumdiolate

formation, as strong bases would favor the deprotona-tion of the amino nitrogen involved in adduct forma-tion, while added small cations could serve as theseparate counter-cations for the diazeniumdiolate anionthat is formed (in place of a protonated amine on thesame DACA species, or another DACA species in closeproximity). Therefore, a variety of lipophilic bases andlipophilic salts were investigated as potential additives tothe SR formulations to enhance diazeniumdiolateformation. The bases examined included the proton-sponge (N;N;N 0;N 0-tetramethyl-1,8-naphthalenedia-mine), 2,6-di-tert-butyl-4-methyl-pyridine, and tridode-cylamine. The cation salts studied included lipophiliclithium, sodium, and potassium salts; for example,lithium tetrafluoroborate, sodium tetraphenylborate,potassium tetrakis(4-chlorophenyl)borate, and variouscation salts of dinonylnaphthalenesulfonate. In all cases,no improvement on NO release fluxes from the resultingfilms could be observed over that found without any

Fig. 7. NO release profiles (average of two) of DACA-6/N2O2 SR

films (diam. 1 cm, thickness ca. 250mm) in PBS at 231C, with different

DACA-6 loading, 4X (6wt%), 6X (9wt%), 8X (12wt%), and 10X

(15wt%), as measured by the NOA.

Fig. 6. NO release profiles (average of three films) of DACA-6/N2O2-

SR (6X, diam. 1 cm) films in PBS at 371C, film thickness of 175mm (a)

and 450mm (b), measured indirectly by determining the level of the

oxidized NO product (nitrite) using the Griess assay.

Fig. 8. NO release profiles (average of two) of DACA-6/N2O2-SR

films (10X, diam. 1 cm) in PBS at 231C, with different thickness, 130,

250, and 330mm, as measured by the NOA.

H. Zhang et al. / Biomaterials 23 (2002) 1485–1494 1491

such additives. Indeed, in most cases, the presence of theadditives actually enhanced the formation of nitritesalts, either during the NO reaction, or during theambient handling of the materials after removing themfrom the reactor.It should be noted that small amounts of both PDMS

and DACA were found to leach from the cured SR filmsas measured by 1H NMR and weight loss measure-ments. Such leaching is likely due to the presence ofuncrosslinked DACA and PDMS species. The totalweight loss of the DACA-SR films after soaking in PBS(ca. 12 d at ambient or physiological temperature) orSoxhlet extraction (24–48 h, water or methanol) wasdetermined to be 2–5wt%. To eliminate such leaching,the DACA-SR films can be pre-extracted with water ormethanol prior to NO addition. Fortunately, it has beenfound that NO loading of the SR films/coatings aftersuch treatment is essentially the same as that obtainedfor films that are not pre-extracted.A triaminoalkyltrimethoxysilane (namely (3-tri-

methoxysilylpropyl)-diethylenetriamine, TACA-2) wasalso used to crosslink hydroxyl terminated PDMS so asto link triamine sites to the cured SR matrices. Theappended triamine groups can further react with NO toform diazeniumdiolate species (TACA-2/N2O2). Asillustrated in Fig. 9, the resulting TACA-2/N2O2-SRfilms also generate NO over an extended time period(>20 d). Further investigation on this NO releasing SRis currently underway.

3.3. In vivo blood compatibility evaluation

The DACA-6/N2O2-SR with DACA-6 loading of 10X(15wt%) was employed to conduct animal testing, asrelatively high NO fluxes could be achieved with thispolymer formulation (see Fig. 7). The NO flux from SRtubing coated (inner wall) with DACA-6/N2O2-SR(thickness X600 mm) reaches levels of4� 10�10mol � cm�2 �min�1 over the first 4 h at ambienttemperature (see Fig. 5), under the assumption that NOdiffuses out from both the inner and outer surfaces ofthe coated tubing (measured by the NOA). Such NOgeneration rate is comparable to that reported forhuman endothelial cells that line all healthy bloodvessels [8]. It should be noted that the NO fluxes becomeeven higher at 371C (Fig. 5) or for thicker films (Fig. 8),suggesting that the use of films of variable thickness inconjunction with elevated in vivo temperatures can beutilized to develop coatings with the desired NO fluxesfor a given application.Preliminary results using the rabbit extracorporeal

circulation (ECC) model indicate that the NO releasefrom DACA-6/N2O2-SR coated SR loops appears toinhibit platelet consumption and platelet activation oninner surfaces of SR tubing loops coated with thismaterial. Indeed, for 4 experiments with DACA-6/

N2O2-SR coated SR tubings, the ECC circuits did notclot off over the entire 4 h experiment period, and theplatelet counts at the end of experiment were 86724%of the initial values. In contrast, for 4 control experi-ments in which DACA-6 coated SR tubings were used(no NO release), all the circuits clotted off at the cannulasites before the termination of the experiments (o3 h),and the platelet counts dropped to 65710% of theinitial values. Results from gross necropsies of the lungsshowed that for all of the animals in which suchinspections were made, there was no thromobo-embolicinjury for animals on the NO releasing circuits (n ¼ 4),whereas one animal from the control group that wasexamined (n ¼ 3) did suffer such injury. Upon visualinspection, clots were observed for most of the controlcircuits, but the NO releasing circuits appeared toexhibit much less thrombus formation. As can be seen intypical SEM pictures for the different circuits (seeFig. 10), for both uncoated SR and DACA-6-SR coatedcontrol circuits, obvious platelet activation occurs onthese materials, as evidenced by pseudopod extensionsand the spreading of the platelets and platelet aggrega-tion on the surfaces after exposure to flowing blood forat least 2 h. In contrast, nearly all of the plateletsadhered to the NO releasing DACA-6/N2O2-SR coatedsurface are spherical and not activated. This behavior isseen repeatedly and correlates well with previous resultsof reduced platelet consumption and activation withinECC circuits that were coated with PVC containing adispersed diazeniumdiolate species (i.e., dimethylhexa-nediamine diazeniumdiolate) using the same animalmodel [4]. However, the use of polymers containing sucha discrete diazeniumdiolate could have serious toxicityissues since such molecules were found to leach from thepolymer matrix [13]. In contrast, for the DACA/TACA-based materials reported herein, after pre-extraction ofany non-crosslinked materials, only NO gas is releasedfrom the NO loaded polymer when bathed in aqueous

Fig. 9. NO release profiles (average of three films) of TACA-2/N2O2-

SR films (diam. 1 cm, thickness ca. 250mm) in PBS at 371C, with

TACA-2 loading of 6X (a) and 12X (b), as measured using the Griess

assay.

H. Zhang et al. / Biomaterials 23 (2002) 1485–14941492

solutions, making this material potentially very attrac-tive for biomedical applications.

4. Conclusion

New DACA/TACA crosslinked silicone rubbers canbe prepared and subsequently loaded, in situ, with NOgas via formation of diazeniumdiolated adducts on theappended amine sites of the crosslinking reagents. Theresulting SR materials exhibit enhanced thromboresis-tance through local release of NO gas. Nitric oxide fluxproduced from such silicone materials can be adjustedby varying the polymer formulation and/or film thick-ness. These NO releasing SR materials could potentiallybe coated on the inside or outside of tubings/cathetersthat are employed in a variety of biomedical devices thatcontact blood. However, achieving a high NO genera-tion rate and NO loaded SR materials that are stable atroom temperature, remains a challenge. Future studieswill include attempts to improve the efficiency ofdiazeniumdiolate loading within such materials as wellas efforts to stabilize the formation of such adducts bychanging the nature/structure of the secondary aminesites on the trimethoxysilane crosslinkers used tovulcanize PDMS.

Acknowledgements

The authors are grateful for supports from theNational Institutes of Health through grant NIH56991, and Michigan Critical Care Consultants, Inc.

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