Perfluorocarbons enhance oxygen transport inalginate-based hydrogels†

Joseph C. Whitea‡ Megan E. Godseya§ Surita R. Bhatiaa,b,c*

A major limitation of current soft biomaterials for tissue engineering and cell encapsulation is inadequate transportof oxygen to cells and tissues. Oxygen transport is a challenge in nearly all aqueous hydrogel biomaterials. Here, wereport the effective diffusivity of oxygen in alginate-based hydrogels containing stable perfluorocarbon (PFC)emulsions. Incorporation of 7% perfluorooctyl bromide into the alginate gels was found to increase oxygenpermeability by a factor of three. Our work also demonstrates that the increase in oxygen transport is largely dueto improved oxygen solubility in PFC-containing gels. Although promising, this improved oxygen transport comeswith a trade-off in terms of mechanical robustness. This must be carefully considered in future development ofPFC-containing hydrogels for biomedical devices. Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: oxygen; hydrogels; perfluorocarbon; biomaterials; tissue engineering

INTRODUCTION

One of the major challenges in synthetic biomaterial fabricationis insufficient oxygen supply throughout the device. Without theuse of an oxygen delivery system, such as vascular growth intothe device[1,2] or a network of synthetic perfusion channels,[3]

hypoxia within hydrogel-based devices limits the efficacy ofcell-laden, macroscopic constructs for three-dimensional (3D)artificial tissue implants.[4] Another strategy for overcoming pooroxygen is to incorporate compounds with larger capacity todissolve oxygen, such as perfluorocarbons (PFC), in order toincrease the overall oxygen solubility in the material.[5]

PFCs are hydrophobic compounds that are capable of dissolv-ing large amounts of oxygen (about 20 times more than water)[6]

due to their weak intermolecular forces.[7] PFC emulsions havebeen investigated for their oxygen delivery capabilities in manyaqueous biomedical applications, most notably in the creation of ar-tificial blood substitutes (e.g. Oxygent™). Recently, research has fo-cused on methods to incorporate these emulsions into cell-basedtissue engineering applications.[8–10] Our collaborative studies withRoberts[11–13] as well as research by other groups[14–16] have ex-plored the addition of PFC emulsions to alginate-based hydrogelsystems to enhance encapsulated cell viability and functionality.

Our laboratory has previously reported protocols for formingstable alginate hydrogels that incorporate perfluorooctyl bro-mide (PFOB) as emulsion droplets within the hydrogel, stabilizedby poly(ethylene oxide)-poly(propylene oxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymers, specifically commer-cially available PluronicW F68 (F68) (Fig. 1).[13] We have describedthe mechanical properties and transport of biomolecules inthese systems,[13] as well as in vitro studies of encapsulated cellsin alginate-PFOB gels.[11,12] These latter studies showed improve-ments in cell viability and cell functionality related to oxygen-dependent metabolism, presumably due to enhanced oxygentransport. However, direct measurement of oxygen transportcharacteristics within these gels, including dependence ofoxygen transport characteristics on formulations characteristics(e.g. PFOB and F68 concentration), has not yet been performed.

Here, we report the impact of F68/PFOB emulsions on overall ox-ygen transport within alginate hydrogels based on permeabilityexperiments. Although current research has shown mixed resultsfor how beneficial PFCs are to tissue-engineered constructdesign,[8,10,12,17] a complete understanding of oxygen transportin aqueous gels containing PFC emulsion is crucial for thecontinued development and optimization of these materials,and eventual translation into clinical settings.

EXPERIMENTAL

Materials

Cell culture-grade sodium alginate (lot#1353824, %G ≥60, Mv

~240kDa, determined from intrinsic viscosity in 100mM sodiumchloride[19]) was obtained from Sigma Aldrich (St. Louis, MO).

* Correspondence to: Surita R. Bhatia, Department of Chemistry, Stony BrookUniversity, Stony Brook, NY 11794, USA.E-mail: surita.bhatia@stonybrook.edu

† This article is published in Journal of Polymers for Advanced Technologies as aspecial issue on 12th PAT Conference in Berlin, 2013, edited by Prof. AndreasLendlein and Prof. Marc Behl, Institute of Biomaterial Science, Helmholtz-ZentrumGeesthacht GmbH, Centre for Materials and Coastal Research, Kantstr. 55, 14513Teltow, Germany.

‡ Current address: University of Maryland, College Park, MD, USA

§ Current address: Department of Biomedical Engineering, Duke University,Durham, NC, USA

a J. C. White, M. E. Godsey, S. R. BhatiaDepartment of Chemical Engineering, University of Massachusetts Amherst,Amherst, MA, 01003, USA

b S. R. BhatiaDepartment of Chemistry, Stony Brook University, Stony Brook, NY, 11794, USA

c S. R. BhatiaCenter for Functional Nanomaterials, Brookhaven National Laboratory, Upton,NY, 11793, USA

Special issue: Research article

Received: 29 January 2014, Accepted: 27 February 2014, Published online in Wiley Online Library: 1 April 2014

(wileyonlinelibrary.com) DOI: 10.1002/pat.3296

Polym. Adv. Technol. 2014, 25 1242–1246 Copyright © 2014 John Wiley & Sons, Ltd.

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Polymer solutions were made with water purified using a ThermoScientific Barnstead NANOPureW Infinity system (nanopure water)purified to 18MΩ-cm. Cell culture-grade PluronicW F68, HEPESbuffer, and D(+)-glucose were obtained from Sigma Aldrich.

Sample preparation

Sodium alginate solutions were prepared in nanopure waterwith final concentrations of glucose and HEPES buffer of 0.1%w/v and 10mM, respectively, and stirred for 24 h. F68, PFOBand nanopure water were emulsified for 30–60min using aThermo Fisher AquasonicW 75HT sonicator (Waltham, MA) untilno visible phase separation was observed. The emulsion wasadded to a concentrated alginate solution to create a finalalginate concentration of 1% w/v. Solutions with 1 or 2% F68and 0–7% PFOB were stable for more than 30min.Alginate hydrogels were prepared using in situ release of

calcium ions, using a modified method to that previouslyreported.[13,18] Briefly, CaEDTA was added to an alginate oralginate/F68/PFOB solution at a final concentration of 50mMCaEDTA. Glucono-δ-lactone (GDL) was added to initiate calciumrelease, and samples were stored in a humidified chamber(90% humidity, 25°C) to gel and equilibrate. All hydrogel formu-lations were then soaked in 100mM calcium chloride (CaCl2)containing the equivalent F68 concentration for 24–48 h. Therewas some PFOB loss due to syneresis, and gels remainedopaque. Care was required to prevent fracture of the alginate/F68/PFOB hydrogels. The final alginate concentration for all solu-tion formulations in this study is 1% w/v, and all concentrationsreported refer to the pre-gel solution during sample preparation.

Oxygen transport

All oxygen diffusion measurements were performed using aside-by-side two-chamber diffusion cell system (LabECX, SantaClarita, CA), as depicted in Fig. 2, and measurements were takenin a method adapted from previously published work.[19] Briefly,the donor chamber was filled with 75ml of air-saturatednanopure water maintained, and the receiver chamber was

initially filled with 75ml of nitrogen-saturated nanopure water.The donor chamber was continuously circulated with air-saturated water to maintain a constant oxygen concentration.A gel membrane was fit into a custom-built cartridge (schematicin Fig. 1), which included surrounding the membrane betweentwo strips of 6–8 kDa dialysis tubing to minimize the amount ofF68 and PFOB leaching into either chambers and interfering withdiffusion measurements. The system was allowed to reachpseudo-steady state for about 1 h, and measurements wererecording from a YSI 5300 oxygen meter (Yellow Springs, OH).The output of the meter was in arbitrary units which werecalibrated to initial and final concentrations of the receiverchamber determined using a Winkler test titration kit (LaMotte,Chestertown, MD). All diffusion experiments were run at 37°C.All samples were run, at a minimum, in triplicate (n≥ 3). Statisti-cal significance was determined using Student’s t-test, p≤ 0.05.All error bars reported represent one standard deviation.

Effective diffusivity was determined from the evolution ofoxygen in the receiver chamber after pseudo-steady state wasreached. For pseudo-steady diffusion through a membrane,[20]

VRdcRdt

¼ AM jM þ QB (1)

jM ¼ Deff

LcD � cRð Þ (2)

QB ¼ DB c� � cRð Þ (3)

where VR is the volume of the receiver chamber (cm3), cR is theconcentration of oxygen in the receiver chamber (mg/cm3), AMis the effective area of diffusion (cm2), jM is the flux throughthe membrane per unit area (mg/cm2/s), and t is time (s). Theparameter QB is the experimental baseline input of oxygen into

Figure 2. Schematic of the temperature-controlled oxygen diffusionsetup. (above) A bath of continuously air-saturated water exchangesinto the donor chamber. The receiver chamber is initially filled withnitrogen-saturated water. (below) Hydrogel was encased in a custom-made polystyrene cartridge and sandwiched between dialysismembrane to minimize PluronicW leaching into either chamber.

Figure 1. Schematic of hydrogel system investigated in this work,comprising an alginate hydrogel which contains a perfluorocarbon(perfluorooctyl bromide (PFOB)) and a non-ionic surfactant (PluronicWF68) at varying concentrations.[13] Hydrogels are formed by the additionof 50mM CaEDTA which is hydrolyzed by 50mM GDL. This figure is avail-able in colour online at wileyonlinelibrary.com/journal/pat

PERFLUOROCARBONS ENHANCE OXYGEN TRANSPORT IN ALGINATE-BASED HYDROGELS

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the receiver chamber (mg/s) and can be determined experimen-tally by performing the procedure using a polystyrene cartridgewith no holes. In eqn (2), Deff is the effective diffusion coefficient(cm2/s), L is the diffusion path length (cm; gel thickness), and cDis the concentration of oxygen in the donor chamber (μg/cm3).In eqn (3), c* is the maximum concentration of oxygen solublein the solution, which in this case was taken as cD since the con-centration was held constant at saturation, and DB is the baselinediffusivity (ml/s). Combining eqns (1–3) and rearranging yields:

dcRcD � cRð Þ ¼ D′dt (4)

D′ ¼ β′Deff þ DB

VR(5)

β′ ¼ AM

LVR(6)

Solving eqn (4) using the initial condition at t= 0, cR= cRo,yields:

lncD � cRocD � cR

� �¼ D′t (7)

As discussed above, the parameter DB is determined from abaseline experiment performed with a polystyrene barrier. Forthis experiment, Deff can be taken as zero, and then eqn (7) canbe written as

lncD � cRocD � cR

� �¼ DB

VRt (8)

With DB experimentally determined, Deff remains the onlyunknown to solve for. D′ was taken as the slope of the linear fitfor the log of the dimensionless plot of concentration versustime. An example plot is shown Fig. 3. Deff is the effective diffu-sivity, or permeability, which is the product of diffusivity, D,and the partition coefficient k (ml oxygen in PFC/ml in water).

RESULTS

Prior to investigation of the impact of F68/PFOB emulsions, theeffect F68 on oxygen transport was determined as a control. Bycomparing the Deff of neat alginate hydrogels to alginate/1%F68 and alginate/2% F68 formulations, it was possible determineto whether oxygen transport is affected by the surfactant. Forneat alginate, experimental results yielded an average Deff valueof 6.2 ± 3 × 10�5 cm2/s, and for alginate/1% F68 and alginate/2%F68, 4.7 ± 1× 10�5 cm2/s and 4.0 ± 1 × 10�5 cm2/s, respectively.Based on these results, it was determined that (i) the presenceof F68 does not significantly impact oxygen transport of alginatehydrogels and (ii) Deff was independent of the studied F68concentrations. For the former, each Deff measured was foundto be statistically similar to the value reported by Li et al.,7.5 × 10�5 cm2/s (this value was extrapolated from the reporteddata).[21] As for the latter, this was expected since, to the bestof our knowledge, neither dilute PEO, PPO, nor PluronicsW hasbeen reported to have an effect on oxygen solubility in aqueousmedia. Thus, in further discussion and analysis, we treat the twoalginate/F68 concentrations as being from similar formulations:alginate + F68.Increasing oxygen permeability can be achieved by increasing

the diffusivity through a medium as well as increasing the overalloxygen solubility. Figure 3 shows the measured Deff for alginate,alginate + F68 and alginate + F68 + PFOB hydrogel formulations.Previous work from our laboratory on the mechanical propertiesof PFOB-containing alginate gel demonstrated that PFOBconcentrations higher than 10% (w/v) significantly decreasedthe fracture stress of the composite.[13] Thus, for this work, wedid not investigate PFOB concentrations higher than 7%.From Fig. 4, the incorporation of 7% PFOB into the hydrogel

formulation significantly increased oxygen permeability by a fac-tor of about three as compared to formulations without PFOB.However, we found that 5% PFOB incorporation was not highenough to significantly impact the overall oxygen transport.Although we have primarily focused on PFOB concentrations

<10% in this study, other groups have incorporated higher

Figure 3. Representative plot of evolution of oxygen concentration inthe receiver chamber. The natural logarithm of the dimensionlessconcentration is plotted versus time, and the slope is used to calculatethe effective diffusion coefficient, Deff, from eqn (5). Solid line is a linearregression analysis.

Figure 4. Effective diffusivity, or permeability, for alginate hydrogelscontaining PFOB emulsion. Permeability of alginate and alginate + F68was statistically similar to the reported value in the literature.[21] Sevenpercent PFOB incorporation significantly increased permeabilitycompared to alginate (*) and alginate+ F68 (+) formulations (p≤ 0.05).

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concentrations of PFCs in their alginate constructs for 3D cellculture.[22] To accurately assess and predict the effect of PFCsof varying concentrations in devices with varying thicknessesand geometries, it is advantageous to understand not only thepermeability, Deff, but also to extract the impact of F68 and PFOBconcentration on oxygen diffusivity and solubility. This can bedetermined from the definition of permeability, eqn (9), and asimple estimate of the partition coefficient, k, based on reportedoxygen solubility values for PFOB and water from the literature,as shown in eqn (10):

Deff ¼ Dk (9)

k ¼ 1� φPFOBð ÞSwater þ φPFOBSPFOB (10)

where φPFOB is the volume fraction of PFOB, SPFOB is oxygensolubility in PFOB, 98.56ml O2/ml PFOB,[6] and Swater is oxygensolubility in water, 4.93ml O2/ml water.[6]

Table 1 contains the estimated solubility, k, and calculated dif-fusivity, D (determined from eqn (9)), for alginate + F68+ PFOBhydrogels. The partition coefficient k was calculated based onthe volumetric concentration of PFOB incorporated (%v/v). Forthese materials, the increase in oxygen solubility was thegreatest contribution to the observed increase in oxygen perme-ability. Theoretical solubility of at 7% (3.6% v/v) PFOB incorpora-tion is about 8.45, compared to 4.93 for water. Diffusivity of atwo-phase system can be determined from many models,including parallel, series, and Maxwellian models,[23] most ofwhich are volume based. Additionally, we have previouslyreported a different model that uses the ratio of fluxes todetermine diffusivity of a two-phase medium.[11] Regardless ofmodel choice, the calculated contribution to Deff of PFOB fromD is negligible compared to k, where a change in k is an orderof magnitude larger than in D. Thus, the dominant factor thatimpacts oxygen transport for alginate hydrogels containingF68/PFOB emulsion is the increase in overall oxygen solubilityprovided by the incorporation of PFOB.

DISCUSSION

Our transport studies show a significant improvement in oxygendiffusivity in alginate hydrogels with PFOB concentrationsgreater than 5%. However, in vitro experiments with a varietyof cell lines has shown mixed results in terms of biologicalbenefits of added PFCs.[8,10,12,17] Thus, although incorporationof PFCs at an appropriate concentration does lead to increasedoxygen transport, other factors may be contributing to limitedutility of PFC-containing alginate gels in biomedical devices.

Our previous studies[13] showed that although the smallstrain mechanical properties, i.e. storage modulus, G′, were notaffected by a 10% PFOB incorporation, the large strain properties(e.g. fracture stress) were negatively impacted at high PFOBconcentrations. Additionally, higher concentrations of PFOBmay also impede diffusivity of larger biomolecules throughoutalginate gels,[13] which may also have a negative impact on bio-logical function.

Particularly for applications where high mechanical stress is en-countered, such as replacement of joint cartilage or ligaments, theuse of PFC emulsions as oxygen vectors in hydrogel-basedmaterialsmay be of concern. A trade-off between increased oxygen transportand mechanical integrity is evident and must be considered duringengineered-construct design. By contrast, for applications that donot require biomaterials with high strength, such as ocular implantsor artificial extracellular matrix, this trade-off may not be as impor-tant. However, even in these cases, the impact of PFCs on transportof relevant large biomolecules must be evaluated.

One way to combat the decrease in fracture stress arising fromaddition of PFOBs may be to incorporate the oxygen vector directlyinto the polymer that comprises the hydrogel network.[15]

Improvements to mechanical robustness of alginate gels mayalso be achieved through modification or addition of a secondcomponent that provides either additional soft crosslinking sites orsome “stretchiness” into the ionically crosslinked alginate backbone.To this end, we have pursued hydrophobic modification of thealginate backbone,[24] incorporation of alginate as a component ininterpenetrating or semi-interpenetrating network systems,[25] andaddition of an additional gel-forming component into the alginategel.[26] However, combining these strategies with addition of PFCsremains a challenge.

The measured increase in oxygen permeability in PFOB-containing hydrogels includes the effects of both oxygen solubil-ity and oxygen diffusivity in the composite gels. Our estimatesindicate that the most important factor impacting oxygentransport in our gels is the overall oxygen solubility providedby the incorporation of PFOB. Therefore, when modeling oxygentransport of these and related systems for device design, asimple estimation of diffusivity is likely to suffice; however, anaccurate value of the overall oxygen solubility is vital.

CONCLUSIONS

We have shown that PFC emulsions can be used to increase therate of oxygen transport in alginate-based hydrogels. However,this is heavily dependent upon the amount of PFC that is ableto be successfully incorporated into the formulation. As shown

Table 1. Calculated diffusivity (D) and solubility (k) for oxygen in a two-phase system

Parallel model[23] Maxwell model[23] Flux model[11]

%w/vPFOB

%v/vPFOB

D (cm2/s)× 105

%change*

D (cm2/s)× 105

%change*

D (cm2/s)× 105

%change*

Calculatedk (ml O2/ml alg/PFOB sol)

%change*

0 0 1.50 1.50 1.50 4.935 2.59 1.60 7% 1.55 4% 1.55 4% 7.35 49%7 3.63 1.64 10% 1.57 5% 1.58 5% 8.32 69%10 5.18 1.70 13% 1.60 7% 1.62 8% 9.78 98%

Value of k calculated from eqn (10). Values marked with * are compared to the 0% PFOB value.

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in previous studies,[13] if the concentration of PFOB is too high,this will significantly decrease the fracture strength of the com-posite. This limitation guided the design of the oxygen transportexperiments presented herein, in which we showed the incorpo-ration of 5% PFOB into an alginate formulation was insufficientto impact oxygen transport, but 7% increases oxygen permeabil-ity by a factor of approximately three. Analysis of oxygen perme-ability made it clear that the increased oxygen transport was dueto the increase in oxygen solubility provided by the PFOB phase.A trade-off between mechanical stability and required oxygensupply must be heavily weighed for the efficacious design ofaqueous biomaterials containing PFC emulsions.

Acknowledgements

The authors acknowledge support from the NSF-funded Institutefor Cellular Engineering IGERT Program (DGE-0654128), the NSF-funded Center for Hierarchical Manufacturing (CMMI-0531171),and a UMass Graduate School Dissertation Fellowship for J.C.W., and support from an NSF REU program (EEC-1005083) forM.E.G. The sponsors had no role in the experimental design, datainterpretation, or presentation of results. Finally, we thank theorganizers of the PAT 2013 conference for invitation and oppor-tunity to present this work.

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