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ELSEVIER

Biom ateria ls 17 (1996) 359-3640 1996 Elsevier Science Limited

Printed in Great Britain. All rights reserved0142-9612/96/$15.00

Characterization of the formation ofint er facially photopolymer ized th inhydrogels in con tact with a r teria ltissueMichelle D. Lyman, David Melanson and Amarpreet S. SawhneyFocal Inc., 4 Maguire Road, Lexington, MA 02173, USAThe aim of this study was to examine the conditions under which an interfacial photopolymerizationprocess results in hydrogel barriers. Visible light initiated interfacial photopolymerization of apolyoxyethylene glycol (PEG)-co-poly(n-hydroxy acid) copolymer based on PEG 8000 macromonomerwas performed on porcine aortic tissue, resulting in conformal hydrogel barriers. The processconditions were optimized in vitro for the formation of a 5-100 pm thick barrier.Keywords: Hydrogels, interfacial photopolymerization, poly(ethylene glycol)Received 4 November 1994; accepted 13 February 1995

Hydrogels are an important class of biomaterials.The permselective nature of hydrogels makes themsuited for diverse applications ranging fromcontrolled drug delivery matrices to microencapsu-lating media for cellular and tissue transplantation.Several of these applications also depend on thehigh water content and low interfacial tension ofhydrogels to govern the interfacial biocompatibilityof an implant. Because hydrogels do not offersignificant structural support, it is often desirablethat they be deposited as thin films to achievefunction and durability. Most of the conventionallyused hydrogel films are pre-coated on devices anddo not lend themselves well to precise applicationat the local site to form conformally adherent andlow profile barriers in situ.

previously, including microencapsulation for hybridbioartificial organs*, use as barriers for prevention ofthe formation of post-surgical adhesions, use as anintravascular coating for inhibition of thrombosis andvascular intimal thickeningI.

Free radical photopolymerization initiated by EosinY, a xanthene dye and member of the fluoresceinfamily, using triethanolamine as a co-initiator has beendescribed in the literature3p7. Visible laser lightinduced polymerization of polyoxyethylene glycol(PEG) diacrylate and PEG tetraacrylate macromono-mers as well as of polyoxyethylene glycol-co-poly(cc-hydroxy acid) diacrylate macromonomers have beenreported previously5,8. When multifunctional acrylatedspecies are used as macromonomers, polymerizationresults in the formation of cross-linked hydrogels.Photopolymerized hydrogels formed from speciallysynthesized macromonomers can be either bioabsorb-able or non-absorbable depending on the structure ofthe precursor molecule. Some applications of thesephotopolymerized hydrogels have been described

An interfacial approach to photopolymerization thatenables the local formation of hydrogels has beendescribed by Sawhney et c11.~~.Here, the chromo-phore (Eosin Y) is deposited on a substrate (such as amicrocapsule or tissue) and can initiate polymeriza-tion from the surface of the substrate outward intothe macromonomer solution, upon illumination withappropriate light. Eosin Y is anionic due to thepresence of two carboxyl groups which are chargedat physiological pH. The exact mechanism by whichit is immobilized on tissue has not been explored,but is possibly due to a combination of hydrophobicinteractions and ionic interactions with cellularglycosaminoglycans and proteins. This approach hasbeen used in the formation of an immunoprotectivehydrogel barrier on the surface of islets of Langerhansto allow the transplantation of xenogeneic islets totreat type-1 diabetes. The photopolymerizationprocess was seen to be non-toxic to sensitive tissuessuch as islets of Langerhans, which continued tosecrete insulin in response to glucose challenge.Hill-West et al. have reported on interfacialphotopolymerization to form thin hydrogel barriersintravascularly. These gels were shown to be highlythromboprotective and efficacious in the reduction ofvascular intimal thickening in a rabbit modelinvolving balloon angioplasty injury to a rabbitcarotid artery.Correspondence to Dr A.S. Sawhney. The structure and properties of barriers formed by

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360 Hydrogel characterization: M.D. Lyman et a/.interfacial photopolymerization are affected by thephotopolymerization conditions. This study examinesthe effect of the conditions of the photopolymerizationprocess on the nature of the resulting hydrogelbarriers. The variables explored were macromonomerconcentration, illumination intensity (or irradiance),illumination duration, chromophore (Eosin Y)concentration and the concentration of a species thataccelerates the photopolymerization process (here, N-vinyl pyrrolidinone (VP)). The process conditions wereoptimized in vitro for the formation of a 5-100-Ltm-thick barrier on arterial tissue.

summary of the photopolymerization conditionsemployed appears in Table 1.Interfacial gel on porcine aortic tissue: thicknessanalysis

MATERIALS AND METHODSMaterialsA PEG-co-poly(cc-hydroxy acid) copolymer, based onPEG 8000, extended with an average number ofrepeating oligomer units of lactic acid ester of three tofive on either side and end-capped with acrylategroups to form a photopolymerizable macromonomer,was synthesized and purified as describedelsewheres,l. This material was stored under an argonatmosphere at 4C until use. Porcine aortas wereobtained from thawed porcine hearts (BlackstoneButchery, Boston, MA, USA) which had been freshlyharvested and frozen. The aortas were stored in 0.05%sodium azide containing phosphate-buffered saline(PBS: pH 7.4, 8.5 gl-l NaCl, 2.57 81-l Na2HP04(anhydrous), 0.138 g 1-l NaH,PO,) until use.

Sections, less than 0.5 mm thick, of fully hydrated,interfacially formed hydrogel-porcine aortic tissuelaminates prepared as described above were cut with arazor blade applied roughly to the centre of the sampleand moving from the hydrogel side downward to thetissue side. The cross-section was placed on a glassmicroscope slide, flooded with PBS and observedunder an inverted microscope (Nikon TMS, Avon, MA,LJSA) equipped with a mechanical sizer with aresolution of 1 /lrn (Boeckeler Instruments, Tuscan,AZ, USA). Five thickness measurements were takenalong the length of each section using the mechanicalsizer.

RESULTS

Macromonomer solution preparationAll chemicals were purchased from Aldrich(Milwaukee, WI, USA) unless otherwise mentioned. To50 ml of PBS, 0.67 g vacuum distilled triethanolamine(TEOA) was added, mixed well and pH was adjustedto 7.4 using 0.6 M hydrochloric acid. The photopoly-merizable macromonomer was dissolved in the PBS-triethanolamine solution at the concentration desiredfor the studies described, in the range of 5-25% (w/v).N-Vinyl pyrolidinone (VP), distilled over argon, wasthen added at the concentration desired for the studiesdescribed, in the range of O-Z ~1 ml I, to form themacromonomer solution.Interfacial photopolymerization on porcineaortic tissueThe luminal tissue surface of a 1 cm* piece of porcineaortic tissue was patted dry with a paper towel. EosinY was applied at the concentration desired for thestudies described, in the range of 0.0025-0.10 mgEosin Y ml- of PBS, to the entire luminal surface andthen copiously rinsed with PBS. The Eosin Y stainedtissue was submerged in 0.5 ml of macromonomersolution and illuminated with an argon ion laseroperated at multiline, multimode through a ZOO /Irncore optical fibre (Spectra-Physics) at an illuminationintensity desired for the studies described, in the rangeof 25-300 mW cm . The interfacially photopolymer-ized piece of tissue was placed in PBS for at least 1hour to allow the hydrogel to completely hydrate. A

Thickness analysis of a fully hydrated hydrogel onporcine tissue (aortic or artery) was utilized toinvestigate the variables that affect the process ofinterfacial photopolymerization. Table 1 lists all of thevariables and the ranges that were investigated. EosinY concentration was one of these variables. Anapproximately linear relationship between hydrogelthickness and illumination duration was observed inthe initial 15 s, after which a plateau was reached(Figure ZA). This trend was consistent for three EosinY concentrations in the range of 0.02-0.10 mg ml . At10% (w/v) macromonomer concentration, the plateaubarrier thickness observed for 0.02 mgml Eosin Ywas 81 f 13 /lrn, for 0.05 mgml Eosin Y was107 5 12 Llrn and for 0.10 mgml-1 Eosin Y was191* 24 /Irn. The same relationship between hydrogelbarrier thickness and illumination duration wasobserved for both 10% (w/v) and 23% (w/v)macromonomer concentrations. The plateauthicknesses were greater for 23% (w/v)macromonomer than 10% (w/v) macromonomer forthe same Eosin Y concentration (Figure 1B). At 23%(w/v) macromonomer, the plateau hydrogel barrierthickness observed for 0.02 mgml- Eosin Y was140*27Alm, for 0.05 mgml Eosin Y was181 * 12 ALrn and for 0.10 mgml- Eosin Y was257 + 24 km. Eosin Y concentrations lower than0.02 mgml were investigated to determine theminimum amount of Eosin Y necessary to initiatephotopolymerization. Figure 1 C illustrates the linearrelationship observed between hydrogel barrierTable 1 Variables of the interfacial polymerization processand the ranges examinedVariable Range examinedMacromonomer concentrationVP concentrationTEOA concentrationEosin Y concentrationIllumination concentration

5-25% (w/y)O-2 /II ml90.4 mM only0.0025-0.100 mg ml- 25-300 mW cm

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0 10 20 30 40 50 60 70Illumination Time (s)

I* 0.10 mg/ml Eosin-Y0 10 20 30 40 50 60 70

Illumination Time (s)

Eosin Y Concentration (mg/ml)Figure 1 Change in hydrogel barrier thickness as a function of illumination duration for: (A) 10% (w/v) macromonomerconcentration; (B) 23% (w/v) macromonomer concentration, for different chromophore (Eosin Y) concentration and illumina-tion durations. VP = 2 ~1 ml- and TEOA = 90.4 mM. (C) Change in hydrogel barrier thickness with Eosin Y concentrationusing a 10% (w/v) macromonomer concentration for a 30 s exposure. Illumination intensity = 100 mW cm-, VP = 2 ~1 ml-and TEOA = 90.4 mM. The error bars designate averages f s.d. with three samples per group.

thickness and Eosin Y concentration for a 10% (w/v)macromonomer concentration at a 30 s illuminationtime (plateau region). Table 2 summarizes plateauthickness results for a variety of polymerizationconditions.Macromonomer concentration and VP concentration

were also investigated for their effect on thickness ofinterfacially polymerized hydrogels. Hydrogelthickness was observed to be directly proportional toVP concentration at any given illumination time for a23% (w/v) macromonomer concentration (Figure ZA).Hydrogel barrier thickness was also directly

Table 2 In vitro plateau thickness values for a variety of polymerization conditionsFormulation Component concentration Illumination Plateau

Eosin Y TEOA VP Macromonomer intensity (mW cm-) thickness (pm)(mg ml-) (mN (~1 ml-) (%, w/v)1 0.02 90.4 2 10 100 81 1t132 0.05 90.4 2 10 100 107 Zt 123 0.10 90.4 2 IO 100 191 at 244 0.02 90.4 2 23 100 140 f 275 0.05 90.4 2 23 100 181 + 126 0.10 90.4 2 23 100 257 * 247 0.02 90.4 2 23 100 151

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362 Hydrogel characterization: M.D. Lyman et a/.

-n Oul/ml VPq .25 Fl/ml VPq .5 PI/ml VPq 1.0 ullml VP0 2.0 plfml VP

l- A

30Illumination Time (s)

0 IO 20 30Macromonomer Concentration (w/v %)

Figure 2 (A) Change in hydrogel barrier thickness withillumination duration for a 23% (w/v) macromonomerconcentration for different VP concentrations. (B) Changein hydrogel barrier thickness with macromonomer concen-tration for different VP concentrations. Illumination intensity= 100 mW cm- for 30 s, Eosin Y = 0.02 mg ml- andTEOA = 90.4 mM. The error bars designate averages * sd.with three samples per group.

proportional to illumination duration for any given VPconcentration. A plateau barrier thickness was againachieved in 15 s. A linear relationship betweenhydrogel barrier thickness and macromonomerconcentration using a 30 s illumination time (plateauregion) was observed, as illustrated in Figure 2B. Thistrend was consistent for 3 VP concentrations in therange of O-2 plmll. The plateau thickness for a 23%(w/v) macromonomer concentration, 0.02 mg ml-Eosin Y concentration and 2 plmll VP concentrationcalculated from Figure ZB was 151 ,um, as seenpreviously for a 23% (w/v) macromonomer concentra-tion treated under similar conditions.

The local presence at the tissue surface of allcomponents required for photopolymerization allowsinterfacial polymerization to proceed from thesubstrate outward into the surrounding medium. It ispossible to confine the initial event of polymerizationto the interface by immobilizing an anionicchromophore (Eosin Y) on the tissue surface. Thischromophore absorbs visible light from an argon ionlaser and is excited to a triplet state, where it canaccept an electron from the tertiary amine (hereTEOA) to form a bleached product. The TEOA, inturn, forms a radical that can initiate polymerizationof the multifunctional acrylate macromonomer. Thepresence of N-vinyl pyrrolidinone accelerates thegrowth of the hydrogel thickness, possibly due to theability of the small, water-soluble unsaturatedmolecule to migrate freely through the micellesformed by the amphiphilic macromonomer, thusacting to further enhance the rate of propagation overthat of the rate of termination of actively growingradical chain ends. The exact mechanism of its actionis unclear, however, and warrants further study.The components of the macromonomer solution andlight are not confined to the interface, but do notundergo polymerization in the absence of thechromophore. Thus, on illumination, a conformallyadherent hydrogel barrier, whose final thickness is afunction of the components of the photopolymeriza-tion system, is formed at the tissue surface. The rate ofgrowth of this hydrogel was seen to increase linearlywith increase in macromonomer concentration. This isin accordance with the classical rate equationdescribing photopolymerization and its dependence onmonomer concentration:

Illumination intensity (or irradiance) was I?, = k,/(2kJ0 5 [I,,(1 - e- 3A)0,]0 5 [M]

investigated as a process variable. The plateauhydrogel barrier thickness was the same for eachillumination intensity; however, the time in which itwas achieved was reduced with increased illuminationintensity. This trend was consistent for both the 10and 23% (w/v) macromonomer concentrations: theplateau barrier thickness for the 23% (w/v)macromonomer concentration was greater than for the10% (w/v) macromonomer concentration, as seenpreviously (Figure 3A and B). Table 3 summarizes thetime it takes to reach the plateau thickness for eachillumination intensity. A wider range of illuminationintensity was investigated with the 10% (w/v)macromonomer concentration to determine theminimum illumination intensity necessary forinterfacial polymerization. Figure 3C illustrates thatthe hydrogel thickness is directly proportional toillumination intensity at an illumination duration priorto the plateau region (10 s). However, in the plateauregion, illumination intensity had no significant effecton barrier thickness. The plateau thickness rangedfrom 68 to 81 ;

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0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70Illumination Time (s) Illumination Time (s)

q OOmW/cm2q 200 mW/cm2 Cq 00 mWicm2

post-plateau

Illumination Time (s)Figure 3 Change in hydrogel barrier thickness with illumination duration for (A) 10%; (B) 23% (w/v) macromonomer concen-tration for different illumination intensities; and (C) 10% (w/v) macromonomer concentration at illumination durations of 10 and30 s for different illumination intensities. Eosin Y = 0.02 mg ml-, VP = 2 ~1 ml- and TEOA = 90.4 mM. The error barsdesignate averages f s.d. with three samples per group

Table 3 Time to reach plateau thickness as a function ofillumination intensityIllumination intensity(mW cme2)

Time to reach plateauthickness (s)

100 15200 10300 5

where A is the absorbance of the sample, I, is thephoton flux, 8i is the initiation quantum yield and [M]is the macromonomer concentration.This equation was used by Decker and Moussa13 toanalyse photocross-linking reactions. However, DavisIhas pointed out that the classical equation can be usedto analyse photocross-linking reactions only for verylow conversions and is invalidated for higherconversions due to chain mobility restrictions caused

by cross-linking, which affect the rate of termination.The immobilized Eosin Y is bleached and exhaustedover a short period of time and acts to limit thegeneration of free radicals for a given TEOA concentra-tion. The plateau region seen to form as a characteristicof interfacial photopolymerizations is presumably dueto the exhaustion of the source of free radicals due tothe bleaching of all available Eosin Y. The thickness ofthe barrier formed was seen to have a lineardependence on the concentration or reservoir of theimmobilized Eosin Y. The accelerating species, VP,appears to enhance the rate of polymerization as awhole by contributing an increased concentration ofunsaturated reactive moieties. Its effect was seen to besimilar to that of the macromonomer concentration onthe thickness of hydrogel formed. Increasing theillumination intensity resulted in a quicker attainmentof the plateau thickness. Our methods of assessing theextent of photopolymerization were not sensitive

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enough to determine whether the dependence onillumination intensity was in accordance with classicalkinetics.In conclusion, we have demonstrated that it ispossible to apply hydrogel barriers to tissue surfaceswith precision. The interfacial approach to formingconformal hydrogels allows a great measure offlexibility and control in forming hydrogel barriers forvarious biomedical applications. We have previouslyshown that the porosity of these barriers can beregulated to form temporary or permanent barriers.The regulation of the thickness and porosity of thesehydrogels may be used to control the permeation andtransport of bioactive species across the barrier. In theevent that these hydrogels are used as local drugdelivery matrices, control of the thickness of barrierscan be used to regulate the dose and residence time ofthe active agent being released. Preliminary studieshave been performed in freshly harvested ratabdominal aorta arteries as well as rat carotid arteriesin viva. These studies show comparable hydrogelthicknesses under the same process conditions.Comparable hydrogel thicknesses were also achievedin preliminary studies in freshly harvested porcinecarotid and femoral arteries, as well as in viva.Future work will explore the type of gel formed byway of perhaps the extent of polymerization, swellingcharacteristics, degradation characteristics, gel-tissueinteractions and gel adherence to tissue.

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