Dijkstra, Pieter J.

Feijen, Jan

Jin, Rong

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Rapid gelation of injectable hydrogels based on hyaluronic acid and poly(ethylene glycol) via Michael-type addition.

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pestle in a mortar for 30 min. Nanoparticles were then preparedusing an amount of mixture containing 100 mg CyA.NMR spectra of a solution of the CyA/HPβCD mixture in CD3OD(99.8%D) were recorded at 30 °C on a Bruker DRX 400 NMRspectrometer operating at 400 MHz. 64 scans were accumulated.In order to study the release of CyA, 40 mg of freeze-driednanoparticles were accurately weighed and transferred to a glass vialcontaining 10 ml PBS pH 7.4. The vials were oscillated in a water bathat 32 °C. To avoid water evaporation, the vials were covered withrubber caps. Throughout the experiment 4 ml sample was withdrawnat specified time intervals and replaced by an equal volume of freshbuffer solution. The sample was centrifuged for 3 h (4000 rpm, 32 °C)and the CyA concentration of the supernatant was determined by avalidated HPLC method.Result and discussionIn a CyA/HPβCD inclusion complex it is expected that thelipophilic side chains of CyA will enter the hydrophobic cavity ofHPβCD and that shielding of methyl groups will be observed.Therefore the aliphatic region (0.7–1.7 ppm) of the NMR spectrumof the CyA/HβPCD sample was compared in detail to the spectrum ofa CyA solution [5]. No substantial differences (<0.003 ppm) could bedetected hence there is no evidence for complexation. Consideringthe large structure of CyA containing 8 lipophilic side chains, apossible explanation may be that a dynamic equilibrium, whereby alldifferent lipophilic side chains only enter the hydrophobic cavity for arelatively short time (i.e. on the average 1/8 of the total time), leadsto a proportional reduction of the shielding effect.The cumulative amount of CyA released from the nanoparticleswas determined. The results are reported as mean±SD of threeexperiments in Table 1. In Fig. 1 the release curves are presented. Aninitial burst effect followed by a slower release rate was observed. Ateach time interval, the amount of CyA released is about 5% higher inthe presence of HPβCD resulting in a cumulative release of 63% and81% after 27 h from nanoparticles loaded by respectively CyA andCyA/HPβCD mixture.Table 1The cumulative percentage of CyA release from CyA and CyA/HPβCD loadednanoparticles.Time (min) CyA release (%)±SD n=3CyA loaded nanoparticles CyA/HPβCD loaded nanoparticles240 37±1 42±3360 51±1 61±1540 60±0 71±21620 63±1 81±2Taking into account the NMR results, the improvement of CyArelease in the presence of HPβCD should not be due to formation of acomplex with higher aqueous solubility, but to the formation of poresinside the PLGA nanoparticles.Fig. 1. Release curves of CyA from PLGA nanoparticles (mean±SD, n=3).ConclusionNMR spectroscopy of the prepared CyA/HPβCD mixture showed noevidence for complexation of CyA with HPβCD. Nevertheless, animprovement of CyA release from PLGA nanoparticles containing theCyA/HPβCDmixture compared to CyA loaded nanoparticles is obtained.AcknowledgementThe authors are grateful to Prof. Dr. L. Pieters for the performanceof NMR spectroscopy (Department of Pharmaceutical Sciences,University of Antwerp, Belgium).References[1] G. Ismailos, C. Reppas, J.B. Dressman, P. Macheras, Unusual solubility behaviour ofcyclosporine A in aqueous mediaJ. Pharm. Pharmacol. 43 (1990) 287–289.[2] B. Malaekeh-Nikouei, H. Nassirli, N. Davies, Enhancement of cyclosporine aqueoussolubility using α-and hydroxypropyl β-cyclodextrin mixtures, J. Inclusion Phenom.Macrocyclic Chem. 59 (2007) 245–250.[3] Y. Ran, L. Zhao, Q. Xu, S.H. Yalkowsky, Solubilization of Cyclosporine A, Pharm. Sci.Tech. 2 (1) (2001) article 2.[4] J. Jaiswal, S.K. Gupta, J. Kreuter, Preparation of biodegradable cyclosporinenanoparticles by high-pressure emulsification-solvent evaporation process, J. Control.Release 96 (2004) 169–178.[5] H. Kessler, H.R. Loosli, H. Oschkinat, Assignment of the 1H, 13C and 15N-NMR spectraof Cyclosporine A in CDCL3 and C6D6 by a combination of homo-and heteronuclear two-dimensional techniques, Helv. Chim. Acta 68 (1985) 661–681.doi:10.1016/j.jconrel.2010.07.048Rapid gelation of injectable hydrogels based on hyaluronic acidand poly(ethylene glycol) via Michael-type additionRong Jin, Pieter J. Dijkstra, Jan Feijen⁎MIRA Institute for Biomedical Technology and Technical Medicine,Department of Polymer Chemistry and Biomaterials, Faculty of Scienceand Technology, University of Twente, P.O. Box 217, 7500 AE Enschede,The Netherlands⁎Corresponding author.E-mail: j.feijen@utwente.nl.Abstract summaryInjectable hydrogels were formed by a Michael-type additionreaction between thiolated poly(ethylene glycol) (PEG) and malei-mide-functionalized hyaluronic acid (HA). Depending on the polymerconcentration and the degree of substitution (DS) of HAwithmaleimidegroups, the gelation time and storage moduli of the hydrogels can bevaried. The gelation time ranged from less than 5 s to about 0.5 min andstorage moduli ranged from 39 to 375 Pa. Moreover, frequency andstrain sweeps demonstrated the elastic nature of these hydrogels.IntroductionInjectable hydrogels are generally formed bymixing two precursorsolutions that gelate in situ by crosslinking. They are attractive forbiomedical applications because a minimally invasive procedure isemployed and cells and bioactive molecules can be easily andhomogeneously incorporated prior to gelation. Michael-type additionis a commonly used crosslinking method to obtain injectablehydrogels. In this study, maleimide-functionalized hyaluronic acids(HA-Mal) with different degree of substitution (DS) were crosslinkedwith thiolated 4-arm poly(ethylene glycol) (PEG-4SH, MW=10 kg/mol) via Michael-type addition. The influences of the DS and polymerconcentration on the gelation time and modulus were studied.Experimental methodsPolymer synthesisHyaluronic acid (HA) (MW=45 kg/mol, measured by viscosity)was functionalized with maleimide moieties according to previouslyAbstracts / Journal of Controlled Release 148 (2010) e21–e56 e41reported method [1]. Briefly, HA (100 mg) was dissolved in 50 mL of0.1 M MES buffer. To the solution, EDC (144 mg, 0.75 mmol) and NHS(86 mg, 0.75 mmol) were added and the resulting mixture wasstirred for 30 min. Afterwards, N-(2-aminoethyl) maleimide trifluor-oacetate salt (AEM) (63.5 mg, 0.25 mmol) was dissolved in 50 mL of0.1 M MES buffer and added to the mixture. After 4 h, the mixturewas dialyzed first against 50 mM NaCl and then deionized water. Thepolymers were obtained as a white foam after freeze-drying. The DS,defined as the number of maleimide groups per 100 disacchariderings of HA, was determined by 1H NMR.Hydrogel preparation and gelation timeHydrogel samples (~0.25 mL) were prepared in vials by mixingPBS solutions of HA-Mal and PEG-4SH. The polymer concentration(1–3% w/v) was defined as the percentage of total polymer weightper volume of PBS solution. A thiol/maleimide molar ratio of 1.2 wasapplied in the preparation of the hydrogels. The gelation time wasdetermined by the vial tilting method.Rheological analysisRheological experiments were carried out with a MCR 301rheometer (Anton Paar) using a parallel plate (25 mm diameter, 0°)configuration at 37 °C in oscillatory mode. A layer of oil wasintroduced around the sample to avoid water evaporation. Theevolution of the storage (G′) and loss (G″) modulus was recordedas a function of time at a frequency of 1 Hz and a strain of 1%. Afterthe oscillation experiment, a frequency sweep was performed withthe frequency varying from 0.01 to 10 Hz and at 1% applied strain.Finally, a strain sweep was performed varying from 0.1% to 10% at afrequency of 1 Hz.Result and discussionPolymer synthesisMaleimide-functionalized HA (HA-Mal) polymers were preparedby reacting HA with AEM using EDC/NHS activation (Fig. 1). Thereaction was performed in 0.1 MES buffer solutions with differentmolar feed ratios of carboxylic acid groups of HA to amino groups ofAEM. After 4 h reaction, the resulting HA-Mal polymers werepurified by dialysis and isolated by freeze-drying. The 1H NMRspectrum of HA-Mal showed that a signal at δ 7.0 was presentcorresponding to the vinyl protons of the maleimide groups (Mal),compared to the spectrum of unmodified HA (Fig. 2). The degree ofsubstitution (DS) of HA-Mal, defined as the number of maleimidegroups per 100 disaccharide rings of HA, was determined from the1H NMR spectra by comparing the integrals of signals at δ 2.0 and 7.0,attributed to the acetamide methyl protons in HA and vinyl protonsof the maleimide moieties, respectively. The DS of HA-Mal increasedfrom 4 to 7 when the [NH2]/[COOH] feed ratio was increased from 1to 2.Fig. 1. Synthesis of maleimide-functionalized HA (HA-Mal) using EDC/NHSactivation.Fig. 2. 1H NMR spectra of HA and HA-Mal (D2O).Hydrogel formationMixing a solution of HA-Mal in PBS with a solution of PEG-4SH inPBS induced rapid hydrogel formation. In Fig. 3 it is shown that anincrease in the polymer concentration from 1 to 3% (w/v) results in adecrease in gelation time. For example, for the hydrogels preparedfrom HA-Mal with a DS of 4, the gelation time is about 35 s for the 1%(w/v) hydrogels, but only 10 s for the 3% (w/v) hydrogels. Moreover,hydrogels based on HA-Mal with a DS of 7 had shorter gelation timesthan those with a DS of 4.In general, the gelation times for HA-Mal/PEG-4SH hydrogelsranged from less than 5 s to about 0.5 min. Obviously, the highreactivity between maleimide and thiol groups makes this in-situhydrogel formation highly interesting for application in tissueregeneration and as drug delivery systems.Fig. 3. Gelation time of hydrogels prepared from PEG-4SH and HA-Mal with a DS of 4and 7 as a function of total polymer concentration.Rheological analysisRheological analysis was used to determine the storage modulus,loss modulus and damping factor of the HA-Mal/PEG-4SH hydrogelsas a function of time (Fig. 4). In general, the storage moduli increasedwhen the polymer concentration was increased from 1 to 3% (w/v)most likely due to the increased crosslinking density. Hydrogels fromHA-Mal with a DS of 7 showed a higher G′ compared to hydrogelsbased on HA-Mal with a DS of 4. The hydrogels from PEG-4SHcrosslinked with HA-Mal DS 7 at a polymer concentration of 3% (w/v)showed the highest storage modulus of 375 Pa. Besides, the dampingfactor of the hydrogels prepared from HA-Mal DS of 7 ranged from0.01 to 0.04, which is much lower than those prepared from HA DS of4 (0.1–0.3). This indicated that the hydrogels with a higher DS weremore elastic.Abstracts / Journal of Controlled Release 148 (2010) e21–e56e42Fig. 4. Storage modulus (G′), loss modulus (G″) and damping factor of the hydrogelsprepared from PEG-4SH and HA-Mal with a DS of (a) 4 or (b) 7 as a function oftime.Frequency and strain sweeps of HA-Mal/PEG-4SH hydrogels arepresented in Fig. 5. A frequency sweep conducted at a strain of 1%revealed that G′ was relatively independent of frequency within thefrequency range of 0.01−10 Hz for all the hydrogels, confirming thatthese materials behaved as an elastic solid. For all the hydrogels,strain sweep experiments (0.1−10%) were performed at an oscilla-tory frequency of 1 Hz. It shows that a strain ranging from 0.1% to 10%is in the linear viscoelastic range for all tested hydrogels.Fig. 5. Frequency and strain sweep of hydrogels prepared from PEG-4SH and HA-Malwith a DS of 4 or 7 as a function of (a and b) frequency and (c and d) strain.ConclusionWe demonstrated that injectable hydrogels from thiolated PEGand Maleimide-functionalized HA can be prepared by Michael-typeaddition. The hydrogels showed fast gelation ranging from less than5 s to about 0.5 min. Besides, storage moduli of the hydrogels can beeasily adjusted by the polymer concentration and DS of maleimidemoieties. Rheological analysis demonstrated that these gels wereelastic.References[1] T. Nie, A. Baldwin, N. Yamaguchiand, K.L. Kiick, Production of heparin-functionalizedhydrogels for the development of responsive and controlled growth factor deliverysystems, J. Control. Release 122 (3) (2007) 287–296.doi:10.1016/j.jconrel.2010.07.049Mesoporous silicon microparticles as carriers for peptidesM. Kilpeläinen1,3,⁎, J. Mönkäre1, J. Riikonen1, M. Vlasova1, J. Salonen2,V.P. Lehto1, K.H. Herzig3, K. Järvinen11University of Eastern Finland, 70211 Kuopio, Finland2University of Turku, 20014 Turku, Finland3University of Oulu, 90014 Oulu, Finland⁎Corresponding author.E-mail: Miia.Kilpelainen@uef.fi.Abstract summaryPorous silicon is considered to be an interesting and safematerial fordrug delivery. Mesoporous thermally hydrocarbonized silicon (THCPSi)microparticles were examined as peptide carrier material for a modelpeptide, melanotan II (MTII). THCPSi microparticles showed to sustainthe MTII the release in vitro. Moreover, THCPSi microparticles changedtheprofile of pharmacological response curves ofMTII in vivo, indicatingsustained release. In conclusion, THCPSi microparticles are promisingcarrier material for sustained peptide release.IntroductionPeptides are interesting molecules for drug development, becausethey could serve as better medical therapies in various pathologicalconditions. However, peptide delivery interfaces many challenges,such as low oral bioavailability, which typically lead on to use ofparenteral administration routes. In addition, the biological half-lifeof peptides is very short, which generates a need to developcontrolled release systems in order to prolong the duration of actionand to lower the frequency of administration.Particulate drug delivery systems are one possibility to enhancepeptide delivery. Porous silicon (PSi) offers a number of advanta-geous properties as a drug carrier material [1], such as a highadsorbing surface area and easily controllable pore size. In thepresent study THCPSi microparticles were investigated in peptidedelivery in vitro and in vivo. Melanotan II (MTII), of whichpharmacological actions include induction of heart rate and inhibi-tion of water consumption [2,3], was used as model a peptide.Experimental methodsThe unloaded microparticles (38–53 μm) were prepared as describedpreviously [4]. The present peptide was loaded into microparticles frommethanol solution in two batches (the loading degreeswere 14.8–15.1w/w %). In vitro release was examined in sink conditions in pH 7.4 buffer(+37 °C) and MTII was detected by HPLC. Male Wistar rats and CD2F1(Balb/c x DBA2) hybrid male mice were purchased from NationalLaboratory Animal Center (Kuopio, Finland) at the age of 7–8 weeks.They were housed individually in a regulated environment; temperature22±1 °C, relative air humidity 55±15% and 12/12 h light/dark cyclewithlights on at 7 am. Commercial pellets (Lactamin R36, Sweden) and tapwater were available ad libitum, unless otherwise indicated.The Institutional Animal Care and Use Committee of the ProvincialGovernment approved the experiments. Procedures were conductedin accordance with the guidelines set by the European CommunityCouncil Directives 86/609/EEC.For the in vivo experiments, the animals were divided into followingtreatment groups: 1) 0.5% carboxymethylcellulose sodium in 0.9% NaCl(Vehicle), 2) unloaded THCPSi microparticles suspended in the vehicle(THCPSi), 3) peptide loaded THCPSimicroparticles suspended in vehicle(THCPSi+MTII) and 4) the peptide dissolved in vehicle (MTII).Water consumption measurementsMalemice (Balb/c xDBA2)were acclimatized to conditions similar tothe experiment and fasted 16 h before the measurements. All thetreatments were injected subcutaneously with a 3.7 mg/kg MTII dose.Abstracts / Journal of Controlled Release 148 (2010) e21–e56 e43

Rapid gelation of injectable hydrogels based on hyaluronic acid and poly(ethylene glycol) via Michael-type addition

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Rapid gelation of injectable hydrogels based on hyaluronic acid and poly(ethylene glycol) via Michael-type addition

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