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Complex Hydrogel Systems Composed of Polymers, Liposomes, and Cyclodextrins: Implications of Composition on Rheological Properties and Aging

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Complex Hydrogel Systems Composed of Polymers, Liposomes, and Cyclodextrins: Implications of Composition on Rheological Properties and Aging
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  8480  DOI:  10.1021/la804305z  Langmuir  2009,  25(15),  8480–8488Published on Web 06/04/2009 pubs.acs.org/Langmuir © 2009 American Chemical Society Complex Hydrogel Systems Composed of Polymers, Liposomes, andCyclodextrins: Implications of Composition on Rheological Propertiesand Aging † Spyridon Mourtas, ‡ Christos A. Aggelopoulos, § Pavlos Klepetsanis, ‡,§ Christos D. Tsakiroglou, § and Sophia G. Antimisiaris* ,‡,§ ‡ LaboratoryofPharmaceutical Technology,DepartmentofPharmacy,SchoolofHealthSciences,Universityof Patras, 26510 Rio, Greece, and   § Foundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes, 26504 Rio, GreeceReceived December 30, 2008. Revised Manuscript Received April 24, 2009 Rheological properties of complex hydrogels containing different amounts of liposomes and/or cyclodextrin (CD)were evaluated. Sonicated unilamellar vesicles (SUV) were loaded in a hydrogel composed of Carbopol 974 NF andhydroxyethylcellulose (Natrosol 250 HX). Phosphatidylcholine (PC) and hydrogenated-PC (HPC) liposomes, bothmixed with cholesterol in a 2:1 lipid/chol mol ratio, were used. In some cases, hydroxypropyl-  β -cyclodextrin was alsoadded (100 or 400 mg/mL). Gels were incubated at 40   C/75% humidity for 7 days or 1 month to evaluate the effect of aging on their rheological properties. FTIR and DSC studies were performed to investigate possible interactionsbetween the polymers and CD molecules at different CD concentrations. Static and dynamic rheological measurementswere carried out. All gels had shear-thinning behavior (fitted well by the Cross model) with the exception of gelscontaining high concentrations ofCD that weretransformed into nonflowing elastic stickysolids, especially after aging.The more pronounced elastic behavior of gels containing 400 mg/mL CD is reflected by the higher values of relaxationstrengths over all relaxation times. Complete interaction between polymers and CD, in the high-CD-content gels, asproven by FTIR and DSC studies, explains the dominating contribution of CD on gel characteristics. The addition of liposomestosuchCD-containinggelshasasubstantialeffectontheirrheologicalproperties,whicharedependentontheliposome type (HPC/chol liposomes > PC/chol) and the lipid/CD ratio. This is explained by the “neutralization” of some CD molecules that prefer to interact with chol molecules that they extract from the lipid membranes. Gels with ahigh CD concentration (400 mg/mL) are almost insensitive to aging, whereas all other gels become slightly more elasticand less viscous as aging proceeds. 1. Introduction When mucosal or topical (especially vaginal) delivery of liposomal formulations is considered, the rheological and/ormucoadhesive properties of liposomes should be adjusted. 1 Thiscan bemanagedbyaddinggelling agentstoliposomedispersions,in which case complex drug-in-liposome-in-gel formulations areformed. 2 - 5 Such gelling agents can be polymer blends consistingof Carbopol 974 and hydroxyethylcellulose (Natrosol). 6 Carbo-pol974,anacrylicacid-basedpolymer,andhydroxyethylcellulose(HEC), a cellulose-based polymer, the structures of which arepresentedinFigure1,arethemaincomponentsofmanysemisolidformulations(commercially available orunderpreclinicalevalua-tion). It was recently proposed that mixtures of the above twopolymer types have improved rheological properties for thevaginal administration of drugs. 6 Indeed, such mixture gels werefound to be more stable toward temperature and pH changescompared to gels composed of each polymer alone when usedindividually. 7 For the above stated reasons, we chose to use thisspecific type of mixture gel in our study.It was recently demonstrated that when liposomes are dis-persedinsuchgelstheyareprotectedfromthedisruptiveeffectsof specific excipients because of the higher viscosity of the geldispersion compared to that of aqueous dispersions, whichprevents (or delays) contact between the various components of the formulation. 8 Nevertheless, the rigidity of liposomal mem-branes determines their integrity, 8 and the release of liposomaldrugs from drug-in-liposome-in-gel complex systems is deter-mined by different factors according to the physicochemicalproperties of each drug. 9 Indeed, hydrophilic drug release isretarded when rigid membrane liposomes are used, but releaseis not affected by the amount of lipid loaded into the gels.Oppositely, in the case of amphiphilic/lipophilic drugs (that havethe ability to permeate the lipid membrane), the drug release rateisprimarilydeterminedbytheamountoflipidloadedintothegel.Whenalargeamountofdrug(comparedtoitsaqueoussolubility)is loaded into the gel, the drug isreleased at a constant rate that isnot affected by the liposome type and is primarily determined bythe solubility of the drug in the aqueous environment. In the later † Part of the Molecular and Polymer Gels; Materials with Self-AssembledFibrillar Networks special issue. *Corresponding author. Tel: 0030-2610-969332. Fax: 0030-2610-996302.E-mail: santimis@upatras.gr. (1) Kieweg, S. L.; Katz, D. F.  J. Pharm. Sci.  2007 ,  96 , 835–85.(2) Moldovan, M.; Leucuta, S. E.; Bakri,A.  J.DrugDeliverySci.Technol. 2006 , 16 , 127–132.(3) Mishra, V.; Mahor, S.; Rawat, A.; Dubey, P.; Gupta, P. N.; Singh, P.; Vyas,S. P.  Vaccine  2006 ,  24 , 5559–5570.(4) Boulmedarat, L.; Grossiord, J. L.; Fattal, E.; Bochot, A. Int.J.Pharm. 2003 , 254 , 59–64.(5) Pavelic,Z.;Skalko-Basnet,N.;Filipovic-Grcic,J.;Martinac,A.;Jalsenjak,I. J. Controlled Release  2005 ,  106 , 34–43.(6) Wang, Y.; Lee, C. H.  Contraception  2002 ,  66 , 281–287.(7) Owen, D. H.;Peters, J.J.; Lavine, M. L.;Katz, D. F. Contraception 2003 , 67  ,57–64.(8) Mourtas, S.; Fotopoulou, S.; Duraj, S.; Sfika, V.; Tsakiroglou, C.;Antimisiaris, S. G.  Colloids Surf., B  2007 ,  55 , 212–221.(9) Mourtas, S.; Duraj, S.; Fotopoulou, S.; Antimisiaris, S. G.  Colloids Surf., B 2008 ,  61 , 270–276.  DOI:  10.1021/la804305z  8481 Langmuir  2009,  25(15),  8480–8488 Mourtas et al. Article case, another important factor is the degree of dilution of theliposome dispersion. Depending on the specific therapeutic need,itmayberequiredtocontrolthedrugreleasekinetics.Becausethedilution factor of liposome dispersions is connected with thephysiology of the drug administration site and therefore rangesbetween specific values that cannot be considerably modified, thesolubility of the drug in the aqueous environment of the site ispossiblythemostimportant,andperhapstheeasiesttomodulate,factor. Cyclodextrins (CDs) 10 are cone-shaped oligosaccharides(Figure 1) that are known to increase the solubility of amphiphi-lic/lipophilicdrugsbyformingsolublecomplexesthatincorporatethe drug within their lipophilic “cave”, mainly by hydrophobicand van der Waals interactions. 11 - 13 Therefore, the addition of CDs to such formulations may increase the release rate of thedrugs from the dispersed liposomes (which act as lipid-phasereservoirs). 14 - 16 Furthermore, it may be possible to control therelease rate by adding different amounts and different types of drug-bindingCDstotheaqueousphaseofsuchformulations. 15,16 It was recently demonstrated that the presence of liposomes insuch polymer blend gels modifies their rheological properties,which are greatly affected by the lipid composition of theliposomes used. 17 Thereby,itisimportant toknow ifthe additionof another component to the gels, such as CDs, will causeadditional changes to their rheological profile.Furthermore, it is important to evaluate the effect of agingon the rheology of such complex systems, especially if theyare intended for commercial use. In particular, aging mayhave important implications on the characteristics of suchgels because the occurrence of interactions between theirmain components (polymers, lipids, and CDs) has been pre-viously reported. 18 - 22 Herein, we investigate the effect on a hydrogel’s rheologicalpropertiesofaddingincreasingamountsofCDsand/orliposomesto a polymer blend hydrogel. Two different concentrations (100and 400 mg/mL) of hydroxypropyl-  β -cyclodextrin (HP-  β -CD)were added to the gel in order to study the effect of CDconcentration.ThisspecificCDwasselectedbecauseofitsknownability to complex many drug molecules and produce complexeswith very high aqueous solubility (compared to other CDs).Additionally, two different types of small unilamellar vesicles(SUV) consistingofphosphatidylcholine PC orhydrogenatedPC(HPC) were used.Because of the complexity of the system and to understand theimplications of the various components on the properties of thefinal system, in a systematic way, various control gels containing(i)onlyliposomesor(ii)onlyCDswereconstructedandevaluatedunder identical experimental conditions.Furthermore, the effect of aging on the rheological propertiesof all gels was studied by incubating the gels under constanttemperature and humidity conditions (ICH accelerated stabilitytest) for 1 week or 1 month. 2. Material and Methods Phosphatidylcholine (PC, egg lecithin) and hydrogenated-PC(egg) (HPC) were purchased from Lipoid Gmbh (Ludwigshafen,Germany). The chemical purity of the phospholipids was verifiedby thin layer chromatography, as described before. 23 In brief,the lipids were developed on silicic acid-coated plates (Merck,Darmstandt, Germany) using chloroform/methanol/water(65:25:4 v/v/v) as the solvent system, and both lipids gavesingle spots. Cholesterol (99%) (chol) was purchased fromSigma-Aldrich Hellas (Chemilab, Athens, Greece). Hydroxy-ethylcellulose (HEC), as Natrosol 250 HX (Hercules Inc.)was kindly provided by Unipharma (Athens, Greece). Carbopol974 P NF (CRB) was kindly provided by Chemix S.A. (Athens,Greece). Hydroxypropyl-  β -cyclodextrin was purchased fromTCI Europe N.V.All solvents used were of analytical or HPLC grade and werepurchased from Merck (Darmstandt, Germany). All other mate-rials were of analytical grade and were purchased from Sigma-Aldrich (Chemilab, Athens, Greece). Figure 1.  Structure sof (A) hydroxyethyl cellulose, (B) hydroxy-propyl-  β -CD, and (C) polyacrylic acid. (10) Loftsson, T.; Duchene, D.  Int. J. Pharm.  2007 ,  329 , 1–11.(11) Harries, D.; Rau, D. C.; Parsegian, V. A.  J. Am. Chem. Soc.  2005 ,  127  ,2184–2190.(12) Lui, L.; Guo, Q. X.  J.InclusionPhenom.MacrocyclicChem. 2002 ,  42 , 1–14.(13) Rekharsky, M. V.; Inoue, Y.  Chem. Rev.  1998 ,  98 , 1875–1917.(14) Boulmedarat, L.; Piel, G.; Bochot, A.; Lesieur, S.; Delattre, L.; Fattal, E. Pharm. Res.  2005 ,  22 , 962–971.(15) Joguparthi, V.; Anderson, B. D.  Pharm. Res.  2008 ,  25 , 2505–2515.(16) Cal, K.; Centkowska, K.  Eur. J. Pharm. Biopharm.  2008 ,  68 , 467–478.(17) Mourtas, S.; Haikou, M.; Theodoropoulou, M.; Tsakiroglou, C.;Antimisiaris, S. G.  J Colloid Interface Sci.  2008 ,  317  , 611–619.(18) Hatzi, P.; Mourtas, S. G.; Klepetsanis, P.; Antimisiaris, S. G.  Int.J.Pharm. 2007 ,  333 , 167–176.(19) Piel, G.; Piette, M.; Barillaro, V.; Castagne, D.; Evrard, B.; Delattre, L.  J.Inclusion Phenom. Macrocyclic Chem.  2007 ,  57  , 309–311.(20) Alexanian, C.; Papademou, H.; Vertzoni, M.; Archontaki, H.; Valsami, G. J. Pharm. Pharmacol.  2008 ,  60 , 1433–1439.(21) Zhang, L.; Hsieh, Y.-L.  J. Nanosci. Nanotechnol.  2008 ,  8 , 4461–4469.(22) Melzak, K. A.; Bender, F.; Tsortos, A.; Gizeli, E.  Langmuir 2008 ,  24 ,9172– 9180.(23) New, R. R. C., Ed.  Liposomes: A Practical Approach ; IRL Press:New York, 1990; Chapter 2.  8482  DOI:  10.1021/la804305z  Langmuir  2009,  25(15),  8480–8488 Article Mourtas et al. A Shimatzu UV-1205 spectrophotometer was utilized for themeasurement of liposomal lipid. Rheological measurements wereperformed on a stress rheometer (Rheometrics SR-200). 2.1. Preparation of Liposomes.  Multilamellar vesicles(MLV) were prepared by the thin film hydration method. 24 Inbrief,the appropriate weights oflipid and cholwere dissolved in achloroform/methanol (2:1 v/v) mixture and subsequently evapo-rated ina round-bottomedflask connected toa rotoryevaporatoruntilathinlipidfilmwasformedonthesidesoftheflask.Thelipidfilm was hydrated with the appropriate volume of citrate buffer(pH 5.0) at 40   C in the case of PC/chol liposomes and at 60   C inthe case of HPC/chol. After complete lipid hydration and theformation of liposomes, the vesicle dispersion was placed in aprobe sonicator (Sonics, Vibra Cell, U.K.) for the reduction of vesicle size. Sonicated unilamellar (SUV) liposomes were pre-pared by subjecting the MLV dispersions to probe sonication forone or two 10 min cycles or until the dispersions became com-pletely clear. After this, the SUV dispersions were centrifuged at10000rpm (HeraeusBiofuge28RS,Germany)for10mininorderto precipitate any titanium fragments released from the probeduring sonication.Finally, all of the liposome dispersions were incubated at thetemperature of preparation for 1 to 2 h in order to annealstructural defects. 2.2. Characterization of Liposome Preparations.  The li-pid concentration of liposomes was measured by the Stewartcolorimetric assay. In this assay, phospholipids form a coloredcomplex with ammonium ferrothiocyanate (OD 485 nm) that issubsequently extracted in chloroform. 25 An appropriate calibra-tion curve using known concentrations of lipid was constructed.After measurement, the lipid concentrations of the liposomedispersions were adjusted to the desired value in order to preparethe liposome-containing gelswith 5 or20mg oflipid/mL ofgel,asdescribed below.The size distribution (mean diameter and polydispersity index)and  ζ  potential of liposomes were measured by dynamic lightscattering (DLS) and laser Doppler electrophoresis (LDE), re-spectively, on a Nano-ZS nanosizer Nanoseries (Malvern Instru-ments, U.K.), which enables the mass distribution of particle sizeas well as the electrophoretic mobility to be obtained. Measure-mentsweremadeat25  C atafixedangleof173  .Sizesquotedarethe  z  average means (d z ) for the liposomal hydrodynamic dia-meter (nm). The zeta potential (mV) was calculated by theinstrument (from electrophoretic mobility). 2.3. PreparationofGels. The different types of gel formula-tions used in this study are presented in Table 1. For theirpreparation, appropriate amounts of Carbopol 974 NF andNatrosol 250-HX were weighted and added slowly to a citratebuffer solution (pH 5.0) for the control gel preparation or to theappropriate liposomal dispersion for complex gel preparations,under constant stirring by a paddle stirrer (100 - 150 rpm). In thecase of CD-containing gels, the appropriate amount of HP-  β -CDwas weighed and dissolved in the liposomal dispersion (if lipo-somes were also added) or in a small volume of buffer. After theaddition of the full amount of solid materials, the gels wereallowed to swell under moderate stirring (50 rpm) for at least 2h.Inallformulations,sodium benzoate(0.02% w/v)wasincludedin the buffer used, as a preservative, and also 1.0% (v/v) glycerinwas added to all of the gels at the end of their preparation for theprevention of dehydration. 2.4. Measurement of Rheological Properties and Rheo-logical Models Applied.  All measurements were performed oncone-and-plate geometry (diameter = 4 cm, slope = 2  ) atconstant temperature ( T   = 37 ( 0.5   C). In steady stress-sweeptests,arangeofshearstressesatconstantamplitude(0.1 - 1000Pa)was applied to the sample, the shear rate was recorded, and theshear viscosity was calculated as the ratio of shear stress to shearrate. In dynamic stress-sweep tests, a range of sinusoidal stressesat constant frequency (0.005 - 20 rad/s) were applied to thesample, and the storage and loss moduli,  G 0 and  G 00 , respectively,were measured. The storage modulus (or elasticity modulus)  G 0 isa measure of the elastic behavior of a gel, which is associated withenergy storage, and the loss modulus (or viscosity modulus)  G 00 isameasureoftheviscousbehaviorofagel,whichisassociatedwithviscous energy dissipation. 26,27 The shear viscosity values of the gels were fitted with the Crossmodel, 26 given by eq 1  μ  ¼  μ ¥  þ ð  μ 0 -  μ ¥ Þ½ 1  þ ð γ = γ 0 Þ 2  ð n - 1 Þ = 2 ð 1 Þ where  μ ¥  is the infinite shear rate viscosity,  μ 0  is the zero shearrate viscosity,  γ 0  is the critical shear rate where the slope of the relationship  μ ( γ ) drops (namely, the fluid transitionsfrom Newtonian to power law behavior), and  n  is the powerlaw index (at high shear rates,  n  -  1 tends asymptotically tothe slope of the regression line when  μ  is plotted vs  γ  on alogarithmic scale).To estimate parameters  μ 0 ,  γ 0 , and  n  (  μ ¥  was set equal to theviscosity of water) of the Cross model, nonlinear regressionanalysis was done using the Bayesian estimator of Athena soft-ware package (Stewart and Associates). For an estimation of the elastic modulus (relaxation strength) and relaxationtimes, previously reported equations were used 26 - 29 (SupportingInformation). 2.4.1. Gel Aging Studies.  All rheological measurementsperformed on the various gel types were repeated after 7 and30 days of aging. For this, the gels were placed in sealed contai-ners and incubated in a homemade constant temperature/humidity oven at 40  (  2   C/75  (  5% (ICH accelerated testingconditions). 2.5. Differential Scanning Calorimetry Experiments. DSC experiments were carried out, in duplicate, in order toinvestigate the interactions between HP-  β -CD molecules andpolymers. Three aqueous solutions of cyclodextrin in buffercitrate at pH 5.0 were prepared (10, 50, and 100 mg/mL). Tothesesolutions,appropriate amountsofCarbopol(4mg/mL)andNatrosol (15 mg/mL) were added in order to have the same ratioof the two polymers as that in the gels. The mixtures were stirreduntil the polymers were completely dispersed. Each solution wasfreeze dried.A Star DSC1 (Mettler-Toledo) system with a refrigeratedcooling accessory was used. Nitrogen was used as the purge gasat a flow rate of 20 mL min - 1 . The calorimeter was calibrated forthe baseline using empty pans and for the cell constant andtemperature using indium (melting point 156.61   C, enthalpy of fusion 28.71 J g - 1 ). The samples were heated from 50 to 250   C ata rate of 10   C min - 1 . 2.6. Fourier Transform Infrared Spectroscopy (FTIR). FTIRwasusedto investigatepossibleinteractionsbetweenHP-  β -CD molecules and polymers. The same samples that were pre-pared for DSC experiments were also evaluated by FTIR. Solidsof cyclodextrin, Carbopol, and Natrosol were also used asreferences.Spectra were recorded with an FTIR spectrometer (DigilabExcalibur, Randolph, MA) at a resolution of 2 cm - 1 . Scans wererun over the range of 400 - 4000 cm - 1 using the KBr pellettechnique. To obtain good-quality spectra, a minimum of 20scans were accumulated. (24) Bangham, A D.; Standish, M M.; Watkins, J C.  J.Mol.Biol. 1965 ,  13 , 238– 252.(25) Stewart, J. C. M.  Anal. Biochem.  1980 ,  104 , 10–14.(26) Macosco, C. W.  Rheology: Principles, Measurements, and Applications ;Wiley-VCH: New York, 1994.(27) Bird, R. B., Armstrong, R. A., Hassager, P., Eds.  Dynamics of PolymerLiquids ; John Wiley & Sons: New York, 1977; Vol.  1 .(28) Robb, I. D.; Smeulders, J. B. A. F.  Polymer  1997 ,  38 , 2165–2169.(29) Tan, H.; Tam, K. C.; Jenkins, R. D.  J. Colloid Interface Sci.  2000 ,  231 , 52– 58.  DOI:  10.1021/la804305z  8483 Langmuir  2009,  25(15),  8480–8488 Mourtas et al. Article 3. Results 3.1. Liposome Physicochemical Properties.  The size dis-tribution (mean diameter and polydispersity index) and  ζ  poten-tial of some of the liposome dispersions are presented in Table 2.As anticipated, SUV liposomes are small with mean diametersranging between 101 and 110 nm, slightly larger when comparedto SUV liposomes without chol in their lipid membranes, aspreviously reported. 17 The  ζ -potential values show that thesevesicles have no surface charge. This was expected because thelipids used for their preparation are not charged. 3.2. Gel Rheological Properties.  3.2.1. Shear Viscos-ity versus Shear Rate.  3.2.1.1. Blank Gel: Effect of Tem-perature .  The shear viscosity as a function of shear rate for theblank gel is shown in Figure 2. Measurements were performed atdifferent temperatures ranging from 20 to 37   C. It is evident thatthere is practically no temperature effect on the viscosity of thisgel, as anticipated by previous studies. 6,7 Furthermore, the blankgel demonstrates shear-thinningbehavior that isfitted well by theCross model (the fitting of the experimental data (points) ispresented as lines in the graphs). 3.2.1.2. Complex Gels.  In Figures 3 and 4, the viscosity/rategraphs are shown for some of the HPC/chol- and the PC/chol-containinggels,respectively.Themeasurementsperformedonthedifferent types of gels are presented as symbols, and the curvespredicted by the Cross model are presented as lines. All graphs inFigures 3 and 4 are plotted on identical  x -axis/  y -axis scales inorder to permit direct comparison between the different cases. Inall gels, the fitting of the experimental data (points) to the Crossmodel (lines) was very good, especially over the shear-thinningflow regime. In Table 3, the parameters estimated by the Crossmodel, zero shear rate viscosity  μ 0 , and power law index  n  aregiven for all of the gel types evaluated. The zero shear rateviscosity is a measure of the fluidity of gels under stress-freeconditions. The power law index,  n,  of the Cross model is ameasureoftheslopeofthe  μ ( γ )curveathighshearrateswherethegeltendstobehaveasapowerlawfluid.Actually,theslopeof   μ ( γ )athigh γ valuestendstobecomeequalto n - 1,andasthevalueof  n  decreases, the drop in viscosity with  γ  becomes sharper .When observing the modulation of the shear viscosity of thegels as a function of shear rate in Figures 3 and 4, we realize thatalmostallofthegelsareclearlyshear-thinningfluidsandhavethetendencytobecomeNewtonianatasymptoticallylowshearrates.ThegelthatcontainsPC/cholliposomes(5mg/mL)togetherwith400 mg/mLCD (plotted astrianglesinFigure4A)has a veryhighzero-rate viscosity, and no shear-thinning behavior is observedover the range of shear stresses applied by the rheometer.However, gel CD2, which contains the same concentration of HP-  β -CDbutnolipid(plottedastrianglesinFigure3A),isshear-thinning, indicating that the addition of PC/chol liposomes maybe responsible for the reduction of the fluidity of the previous gel(CG-2-P). Nevertheless, when a higher number of liposomes isaddedtothesamegel,inwhichcasegelCG-4-Pisformed,itagainbecomes shear-thinning (triangles in Figure 4B). This indicatesthat the ratio of CD to lipid in the gel may have an effect on therheological behavior of the final product. Additionally, bycomparing the viscosity graphs of similar gels (that contain thesameamountsofCDandlipidbutdifferenttypesofliposomes,asinFigures3Band4A;formoredataseeSupportingInformation),onecaneasilyconcludethattheflowbehaviorofthefinalproductisalsoaffected bythe lipid compositionofthe liposomesaddedtothe complex gels, in agreement with earlier observations. 17 3.2.1.3. Effect of Aging.  The rheological data measured forthe gels after 1 week (7 days) and 1 month (30 days) of aging arepresented in the Supporting Information. For the blank gel, theviscosity increases with aging. Indeed, as seen in Table 3, thecalculatedzero-rateviscosity  μ 0 isincreasedbyfactorsof2and16after 1 week and 1 month of aging, respectively. However, the gelremains shear-thinning with no significant modification of theshear rate value  γ 0  at which the viscosity of the gel starts todecrease.Ingeneral,theeffectofCDmoleculesontherheologicalprofileof the complex gels during aging seems to be influenced by thepresence of liposomes and also by the number and type of liposomes added to the gels. Indeed, gel CD2, which contains400 mg/mL CD and no liposomes, has a very high zero-rateviscosity after 1 month of aging (see Supporting Information fordata)andabolishesitsshear-thinningbehavior(overthefullshearstress range of the rheometer). Nevertheless, when HPC/chol Table 1. Compositions of the Gels Studied a gel name polymers lipid comp b HP-  β -CDconc (mg/mL)lipid conc(mg/mL) c BL (blank) no lipid 0 0HPC1 all gels: 0, 40%(w/v) Carbopol(974 NF) and1,5% (w/v)Natrosol(250-HX)HPC 0 5HPC2 HPC 0 20CD1 no lipid 100 0CD2 no lipid 400 0CG-1-H HPC 100 5CG-2-H HPC 400 5CG-3-H HPC 100 20CG-4-H HPC 400 20PC1 PC 0 5PC2 PC 0 20CG-1-P PC 100 5CG-2-P PC 400 5CG-3-P PC 100 20CG-4-P PC 400 20 a All gels contain Carbopol 974 NF and hydroxyethyl cellulose(Natrosol) at the concentrations reported as well as citrate buffer pH5.00 (0.10 M), glycerin (1% w/v), and sodium benzoate (0.20% w/v). b All liposomes contain chol at lipid/chol 2:1 (mol/mol).  c Lipid concen-trations were remeasured after gel preparation and were found to be(5.013 ( 0.022) - (20.045 ( 0.050). Table 2. Mean Diameter and Zeta Potential Values of the LiposomesUsed in Gels a lipid comp mean diameter (nm) PI b ζ  potential (mV)PC/chol (2:1) 100.9 ( 7.5 0.201  - 2.1 ( 4.1HPC/chol (2:1) 109.6 ( 5.2 0.163 2.05 ( 2.75 a Each value is the mean, calculated from at least three separatepreparations,andthestandarddeviationofthemeanispresented. b PIisthe polydispersity index for the measurements (mean ( SD). Figure 2.  Viscosity versus shear rate graph of the blank gel (BL),measured at various temperatures ranging between 20 and 37   C.The symbol key is included in the graph inset.  8484  DOI:  10.1021/la804305z  Langmuir  2009,  25(15),  8480–8488 Article Mourtas et al. liposomes are added to that same gel at5 or 20 mg/mL, the initialshear-thinning behavior of the gels is maintained even after the 1month aging period (Figure 1 in Supporting Information). Incontrast, the addition of 5 mg/mL PC/chol liposomes to the gelseemstoaffectthegelnegativelybecauseitlosesitsshear-thinningability even at time 0 (Figure 4A) and the same behavior is alsoobserved after aging. However, when PC/chol liposomes areadded at a higher concentration (20 mg/mL), the gel regains itshear-thinning behavior initially, but after 1 week of aging, itstops flowing and becomes very sticky again (Figure 2 inSupporting Information). This behavior indicates that when highamounts of PC/chol liposomes are added to the gel that containshigh amount of HP-  β -CD the interactions that occur between geland liposome components are slow and time is needed for thesystem to equilibrate.As shown in Table 3, the effect of adding HPC/chol liposomesto the blank gel on the zero shear rate viscosity  μ 0  and power lawindex  n  is similar to that observed previously for HPC lipo-somes; 17  μ 0  values increase by an order of magnitude when 5 mg/mL liposomes is included (comparison between BL and HPC1,gels) and furthermore when the lipid content is increased to 20mg/mL (comparison between BL and HPC2, gels), whereas the  n index decreases in both cases. However, the addition of PC/cholliposomestotheblankgel(PC1,PC2)alsoresultsinanincreasein  μ 0  and a slight decrease in  n,  although this increase/decrease islower than that caused by equivalent amounts of HPC/cholliposomes. This was not the case for PC liposomes that werestudied previously in the same blank gel system 17 and wereobserved to cause a decrease in  μ 0  values and a slight increasein n values. Thereby,itisevident thatthe rigidityofthe liposomalmembraneisindeedoneoftheparametersthatinfluencetheeffectof liposome addition to polymeric gels on the gel properties;PC/chol liposomes used herein behave differently compared tothe PC liposomes used before 17 as a result of the addition of cholesteroltotheirlipidmembrane,whichresultsintheformationof a more rigid lipid membrane. 3.2.2. Dynamic Frequency Sweep Tests.  From the resultsof the oscillatory measurements performed (DFS  -  dynamicfrequency sweep), one can extract information about the net-work structure of gels. For each formulation, the elasticity(storage) modulus ( G 0 ) and the viscosity (loss) modulus ( G 00 )were measured as a function of the oscillatory frequency (hertz).The gel structure was examined over the frequency range of 0.005 - 20 rad/s. 3.2.2.1. Blank Gel.  In Figure 5, the dynamic measurementsof the blank gel are compared with corresponding predictions of the multimodal Maxwell model. (See Supporting Informationfor more details about the model.) As seen, the Maxwellmodel satisfactorily predicts the experimental frequency re-sponses (predictions are presented as lines and experimentalresponses as symbols), although some discrepancy is observedover the very low frequencies measured. In general, the storagemodulus(plottedassolidsymbols)ishigherthanthelossmodulus(plotted as hollow symbols), particularly with reference to the Figure 4.  Viscosity versusshear rategraphof5 mg/mL PC/chol - liposome-containing complex gels measured immediately afterpreparation. (For the results of gels with higher liposome concen-tration and the results obtained after gel aging, see SupportingInformation.) The symbol key is included in the graph inset. Figure 3.  Viscosity versus shear rate graph of various types of controlandcomplexgels,measuredimmediatelyafterpreparation.Graph A is for the gels without lipid; B, for 5 mg/mL HPC/chol - liposome-containing gels. (For the results of gels with higherliposome concentration and the results obtained after gel aging,see Supporting Information.) The symbol key is included in thegraph inset.

Orb of Quietus

May 6, 2018

04721627

May 6, 2018
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