Bommel, K.J.C. van

Esch, J. van

Feringa, B.L.

Friggeri, A.

Stuart, M.

English

An enzymatically cleavable low molecular weight gelator (LMWG)-drug conjugate is described that is capable of gelating water at concentrations as low as 0.45 mM (=0.03 wt.%). By comparing the enzymatic cleavage kinetics of the LMWG-drug conjugate with those of a nongelating substrate, it was shown that although the enzyme (α-chymotrypsin) is still functional in the gel, molecules present within the gel fibers are protected from enzymatic cleavage

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   University of GroningenProtecting Substrates from Enzymatic CleavageBommel, K.J.C. van; Friggeri, A.; Stuart, M.; Feringa, B.L.; Esch, J. vanPublished in:ChemistryIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2005Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bommel, K. J. C. V., Friggeri, A., Stuart, M., Feringa, B. L., & Esch, J. V. (2005). Protecting Substrates fromEnzymatic Cleavage: Hydrogels of Low Molecular Weight Gelators Do The Trick. Chemistry, 11(18).CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-2022Poster AbstractsI. Polymeric Materials for Drug DeliveryPROTECTING SUBSTRATES FROM ENZYMATIC CLEAV AGE: HYDROGELS OF LOW MOLECULARWEIGHT GELATORSDO THE TRICKK.J.C. van Bommel1, A. Friggeri1 , M. Stuart2, B.L. Feringa2, J. van Esch21BiOMaDe Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands2Department of Organic and Molecular Inorganic Chemistry , Stratingh Institute, University of Groningen, Nijenborgh 4, 9747 AGGroningen, The NetherlandsSummaryAn enzymatically cleavable low molecular weight gelator (LMWG)–drug conjugate is described that is capable of gelating water atconcentrations as low as 0.45 mM (=0.03 wt.%). By comparing the enzymatic cleavage kinetics of the LMWG–drug conjugate with those of anongelating substrate, it was shown that although the enzyme (a-chymotrypsin) is still functional in the gel, molecules present within the gelfibers are protected from enzymatic cleavage.Introd uctionHydrogels of low molecular weight gelators (LMWGs) [1] are an attractive complement or even alternative for pharmaceutically interestingpolymeric gel systems [2] as they possess properties unattainable by polymeric gelators [3]. The most important properties being a veryrapid response to external stimuli, an inherent thermoreversibility owing to the noncovalent nature of the aggregation process, and a lowmolecular weight of the gelator, facilitating a fast clearance from the body after triggering the gel-to-sol transition. As we want to useLMWG systems in pharmaceutical applications, we require responsive and biocompatible systems, especially when envisioning their use forthe triggered release of a pharmaceutical. Enzymatically cleavable systems are especially attractive triggerable systems, as they allow releaseof a pharmaceutical in very specific locations (e.g., tumors or designated areas of the GI tract). Enzymatically cleavable LMWG–drugconjugates (Fig. 1) could result in gels of which the molecules are protected against enzymatic cleavage as a result of their unavailability tothe enzyme because they are incorporated in the fibrous aggregates. Upon triggering the gel-to-sol transition (e.g., via a pH or temperaturechange: D) the fibrous aggregates fall apart and the individual molecules can be cleaved by the enzyme. Here, we present the firstenzymatically cleavable LMWG–drug conjugate system and prove that incorporation of a substrate into the gel fibers does indeed protect itfrom enzymatic cleavage.doi:10.1016/j.jconrel.2004.09.006Journal of Controlled Release 101 (2005) 287 –408www.elsevier.com/locate/jconrelResults and discussionWe designed and synthesized compound 1, containing like many of our other LMWGs a cyclohexane core to which different moieties arelinked via amide bonds [4]. The l-phenylalanylamidoquinoline [5] moiety was introduced to allow enzymatic cleavage to take place,using a-chymotrypsin capable of cleaving amide bonds at the CjO terminus of aromatic amino acids. The thus released fluorogenicQmodel drugQ 6-aminoquinoline (6-AQ) allows easy monitoring of the cleavage kinetics [5]. Although gels of LMWG 1 can be easilyswitched into solutions and back into gels, either by temperature or pH changes, neither of these methods is very suitable for theinclusion of a-chymotrypsin in gels of 1, as a-chymotrypsin does not respond well to large changes in either temperature or pH. Asolution was found by using a mixed solvent system. Gelator 1 was dissolved in a small amount of DMSO, whereas a-chymotrypsin wasdissolved in a buffer solution (Tris–HCl, 0.1 M, pH 7.75). The rapid addition of the aqueous a-chymotrypsin solution to the DMSOsolution of 1 resulted in the instantaneous formation of a clear, homogeneous gel that could be used for fluorescence experiments (cryo-TEM measurements showed no differences in the gel fiber structure in this mixed solvent system when compared to the pure aqueoussystem). Compound 2 was synthesized in order to have a nongelating substrate, allowing us to evaluate the effect of gelation onenzymatic cleavage.In order to investigate the kinetics of enzymatically catalyzed cleavage of LMWG 1 as well as model substrate 2, experiments werecarried out using different concentrations of substrates (i.e., 1 or 2). The initial velocity of hydrolysis of each experiment was then plottedas a function of the substrate concentration (S), resulting in the points plotted in graphic 1. From the points belonging to each substrate theVmax (limiting or maximum enzyme velocity) and Km (Michaelis constant) can be determined, using either an Eadie–Hofstee orLineweaver–Burke plot, both plots giving identical results for Vmax and Km with R2 values of N0.995. Using the values thus obtained forsubstrate 2 (Vmax=22.3 Amol/min, Km=4.9 mM), it was possible to plot the theoretical curve, which accurately followed the experimentallydetermined points. For LMWG 1, however, the experimentally determined points (circles in graphic 1) clearly did not follow the trendcommon for enzyme substrates. Although the values for Vmax did increase with increasing substrate concentrations, they abruptly leveledChart 1. Molecular structures of LMWG 1 and nongelator 2.Fig. 1. Schematical representation of the enzymatic cleavage of a gelator–drug conjugate and the influence of aggregation on enzymaticcleavage.Poster Abstracts288off at a concentration of ca. 1.5 mM (see also inset of Graphic 1). Using only the experimentally obtained points at concentrations below1.5 mM, values were calculated for Vmax and Km (4.1 Amol/min and 1.8 mM, respectively), which again allowed a theoretical curve to bedrawn. It is clear that the experimentally determined points for 1 deviate from the theoretical curve at concentrations above 1.5 mM. As aresult of its gelating properties LMWG 1 has an imposed Vmax of 1.8 Amol/min, rather than the expected 4.1 Amol/min. Interestingly, theconcentration of 1.5 mM corresponds to the critical gelation concentration (CGC) of 1 in this particular solvent mixture. In gels ofLMWGs, the concentration of gelator in solution no longer increases once the total concentration of gelator is above the CGC (i.e., allgelator in excess of the CGC will be in the gel fibers).As the value for Vmax no longer increases once the concentration of LMWG 1 has increased above the CGC, this suggests that onlythe molecules in solution and not those in the gel fibers can be cleaved by the enzyme. By performing an enzymatic cleavage experimentin which both LMWG 1 as well as nongelating substrate 2 were present, it was shown that the incorporation into a gel did not influencethe functioning of the enzyme a-chymotrypsin.ConclusionsIn conclusion, we designed and synthesized the first example of an enzymatically cleavable LMWG–(model) drug conjugate capable ofgelating water at very low concentrations. Furthermore, we proved that although the LMWG–drug conjugate can be cleaved by a-chymotrypsin, the molecules that are incorporated into the gel fibers are protected from enzymatic cleavage. We plan to use similarsystems for the controlled release of pharmaceuticals.References[1] a) P. Terech, R.G. Weiss, Chem. Rev. 97 (1997) 3133–3159; b) D.J. Abdallah, R.G. Weiss, Adv. Mater. 12 (2000) 1237–1247; c) J.H. vanEsch, B.L. Feringa, Angew. Chem. 112 (2000) 2351–2354; Angew. Chem. Int. Ed. 39 2000 2263–2266; d) O. Gronwald, S. Shinkai, Chem.Eur. J. 7 (2001) 4328–4334; e) G. Mieden–Gundert, L. Klein, M. Fischer, F. Vfgtle, K. Heuzé, J.L. Pozzo, M. Vallier, F. Fages, Angew.Chem. 113 (2001) 3266–3267; Angew. Chem., Int. Ed. 40 2001 3164–3166.[2] a) S. Deo, E. Moschou, S. Peteu, P. Eisenhardt, L. Bachas, M. Madou, S. Daunert, Anal. Chem. 75 (2003) 207A–213A; b) K.T. Nguyen, J.L.West, Biomaterials 23 (2002) 4307–4314; c) P. Gupta, K. Vermani, S. Garg, Drug Discov. Today 7 (2002) 569–579; d) N.A. Peppas, P. Bures,W. Leobandung, H. Ichikawa, Eur. J. Pharm. Biopharm. 50 (2000) 27–46.Graphic 1. Initial velocity of hydrolysis of model substrate 2 (squares) and LMWG 1 (circles). Conditions: 25 8C, a-chymotrypsin: 40 AM,buffer solution: Tris–HCl, 0.1 M, pH 7.75. Solid lines: theoretical curves based on the calculated Vmax and Km values. Dashed line: experimentalcurve. Inset: enlarged view of data points and theoretical curve for LMWG 1.Poster Abstracts 289[3] J.C. Tiller, Angew. Chem. 115 (2003) 3180–3183; Angew. Chem., Int. Ed. 42 2003 3072–3075.[4] a) A. Heeres, C. van der Pol, M. Stuart, A. Friggeri, B.L. Feringa, J. van Esch, J. Am. Chem. Soc. 125 (2003) 14252–14253. b) K.J.C. vanBommel, C. van der Pol, I. Muizebelt, A. Friggeri, A. Heeres, A. Meetsma, B.L. Feringa, J. van Esch, Angew. Chem., Int. Ed. accepted.[5] P.J. Brynes, P. Bevilacqua, A. Green, Anal. Biochem. 116 (1981) 408–413.STUDY OF THE SURFACE MOBILITY OF PLGA MICROSPHERES USING HIGH-RESOLUTIONTOPOGRAPHY MEASUREMENTS WITH THE ATOMIC FORCE MICROSCOPEC. Bouissou1, P. Begat1, C. van der Walle2, R. Price11Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY , United Kingdom2Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, G4 0NR, United KingdomSummaryAn atomic force microscope (AFM) has been used to obtain high-resolution topography measurements of surface properties of poly(lactide-co-glycolide) (PLGA) microspheres. The effect of the tapping AFM mode on a low Tg polymer (Tg=37.5F0.2) was investigated and wasshown to have no discernible effect on the surface stability. Real-time AFM measurements provides a novel means of observing moistureinduced transformations to the porous structures of microspheres under controlled environmental conditions.IntroductionBiodegradable polymeric microspheres have received much attention for the sustained release of bioactive macromolecules, such as peptides orproteins. The various microencapsulation techniques lead to a wide variety of microcapsule behaviours [1,2]. The understanding of the differentfeatures is necessary in order to control and tomanufacture specific pattern releases. The scanning electronmicroscope (SEM) is commonly used toobserve the external morphology of the microspheres. However, conventional SEM requires coating of the sample with a conductive layer andoperates in vacuo, thus limiting the mesoscopic resolution and study of the dynamic stabilities of microspheres particles. In contrast, the AFMoffers the possibility of assessing accurately the conformational aspect and stability of anymicrospheres without the need for any significant samplepreparation. Surprisingly, only very limited amount of work has been undertaken to study the surface conformational arrangement [3,4]. The aim ofthis study was to use high-resolution topographical AFM to investigate the surface properties of PLGA microspheres and to measure in situmoisture-induced transformations of porous microspheres.Experimental methodsPreparation of microspheresThe encapsulation procedure used in this study was the water-in-oil-in-water double emulsion evaporation technique [5]. The inner aqueous phasecontained 90 Al of phosphate-buffered saline (PBS, pH 7.5) with 5 Al of poly-vinyl alcohol (PVA, 5%w/v) in water, which was injected into 950 Alof oil phase consisting of dichloromethane (DCM) with 5% w/v PLGA. This primary emulsion was homogenised at 22000 rpm for 15 s (with anUltra TURRAX IKAT 18 basic), and transferred into 40ml of water containing poly-vinyl alcohol (PVA, 0.5%w/v), then stirred at 500 rpm for 2 hat room temperature (lab-egg IKA RW11 basic). The solvent was allowed to evaporate for the capsides to harden. The microspheres were thenharvested by centrifugation at 41000 rpm for 1 min, washed, snap-frozen in liquid nitrogen and finally lyophilised overnight.Scanning Electron Microscope (SEM)Lyophilised microspheres were sprinkled onto a carbon adhesive disc, whichmounted on an aluminium stub. Samples were coated with a thin layerof gold (Edwards Sputter Coater S150B). The SEM was operated at 10 kV (JEOL JSM6310).Topography measurement with the Atomic Force Microscope (AFM)All AFM surface topography images were recorded in TappingModek operation (TM-AFM). The effect of RH on surface topography wasinvestigated using a custom-built perfusion unit. The partial vapour pressure of water within the imaging chamber of AFM was controlled byvarying a mixture of dry nitrogen gas with the same gas humidified to 100% under constant temperature 25 8C (F0.2 8C).Poster Abstracts290

Protecting Substrates from Enzymatic Cleavage:Hydrogels of Low Molecular Weight Gelators Do The Trick

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Protecting Substrates from Enzymatic Cleavage:Hydrogels of Low Molecular Weight Gelators Do The Trick

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