Design of nanoparticle systems with antimicrobial properties

Infektiöse Komplikationen im Zusammenhang mit Implantaten stellen einen Großteil aller Krankenhausinfektionen dar und treiben die Gesundheitskosten signifikant in die Höhe. Die bakterielle Kolonisation von Implantatoberflächen zieht schwerwiegende medizinische Konsequenzen nach sich, die unter Umständen tödlich verlaufen können. Trotz umfassender Forschungsaktivitäten auf dem Gebiet der antibakteriellen Oberflächenbeschichtungen ist das Spektrum an wirksamen Substanzen aufgrund der Anpassungsfähigkeit und Ausbildung von Resistenzen verschiedener Mikroorganismen eingeschränkt. Die Erforschung und Entwicklung neuer antibakterieller Materialien ist daher von fundamentaler Bedeutung.rnIn der vorliegenden Arbeit wurden auf der Basis von Polymernanopartikeln und anorganischen/polymeren Verbundmaterialien verschiedene Systeme als Alternative zu bestehenden antibakteriellen Oberflächenbeschichtungen entwickelt. Polymerpartikel finden Anwendung in vielen verschiedenen Bereichen, da sowohl Größe als auch Zusammensetzung und Morphologie vielseitig gestaltet werden können. Mit Hilfe der Miniemulsionstechnik lassen sich u. A. funktionelle Polymernanopartikel im Größenbereich von 50-500 nm herstellen. Diese wurde im ersten System angewendet, um PEGylierte Poly(styrol)nanopartikel zu synthetisieren, deren anti-adhesives Potential in Bezug auf P. aeruginosa evaluiert wurde. Im zweiten System wurden sog. kontakt-aktive kolloide Dispersionen entwickelt, welche bakteriostatische Eigenschaften gegenüber S. aureus zeigten. In Analogie zum ersten System, wurden Poly(styrol)nanopartikel in Copolymerisation in Miniemulsion mit quaternären Ammoniumgruppen funktionalisiert. Als Costabilisator diente das zuvor quaternisierte, oberflächenaktive Monomer (2-Dimethylamino)ethylmethacrylat (qDMAEMA). Die Optimierung der antibakteriellen Eigenschaften wurde im nachfolgenden System realisiert. Hierbei wurde das oberflächenaktive Monomer qDMAEMA zu einem oberflächenaktiven Polyelektrolyt polymerisiert, welcher unter Anwendung von kombinierter Miniemulsions- und Lösemittelverdampfungstechnik, in entsprechende Polyelektrolytnanopartikel umgesetzt wurde. Infolge seiner oberflächenaktiven Eigenschaften, ließen sich aus dem Polyelektrolyt stabile Partikeldispersionen ohne Zusatz weiterer Tenside ausbilden. Die selektive Toxizität der Polyelektrolytnanopartikel gegenüber S. aureus im Unterschied zu Körperzellen, untermauert ihr vielversprechendes Potential als bakterizides, kontakt-aktives Reagenz. rnAufgrund ihrer antibakteriellen Eigenschaften wurden ZnO Nanopartikel ausgewählt und in verschiedene Freisetzungssysteme integriert. Hochdefinierte eckige ZnO Nanokristalle mit einem mittleren Durchmesser von 23 nm wurden durch thermische Zersetzung des Precursormaterials synthetisiert. Durch die nachfolgende Einkapselung in Poly(L-laktid) Latexpartikel wurden neue, antibakterielle und UV-responsive Hybridnanopartikel entwickelt. Durch die photokatalytische Aktivierung von ZnO mittels UV-Strahlung wurde der Abbau der ZnO/PLLA Hybridnanopartikel signifikant von mehreren Monaten auf mehrere Wochen verkürzt. Die Photoaktivierung von ZnO eröffnet somit die Möglichkeit einer gesteuerten Freisetzung von ZnO. Im nachfolgenden System wurden dünne Verbundfilme aus Poly(N-isopropylacrylamid)-Hydrogelschichten mit eingebetteten ZnO Nanopartikeln hergestellt, die als bakterizide Oberflächenbeschichtungen gegen E. coli zum Einsatz kamen. Mit minimalem Gehalt an ZnO zeigten die Filme eine vergleichbare antibakterielle Aktivität zu Silber-basierten Beschichtungen. Hierbei lässt sich der Gehalt an ZnO relativ einfach über die Filmdicke einstellen. Weiterhin erwiesen sich die Filme mit bakteriziden Konzentrationen an ZnO als nichtzytotoxisch gegenüber Körperzellen. Zusammenfassend wurden mehrere vielversprechende antibakterielle Prototypen entwickelt, die als potentielle Implantatbeschichtungen auf die jeweilige Anwendung weiterhin zugeschnitten und optimiert werden können.

Formation of DNA complexes in aqueous and organic solvents

From a physical-chemical point of view, it is challenging to form complexes with polyelectrolytes, consisting of only molecule of the largest component, i.e. the component with the highest number of charges. In this study, complexes are formed with DNA because of its potential applications as an artificial vector for gene delivery. The aim of this work is to prepare complexes in aqueous solutions as well as in organic solvents containing only one DNA molecule. For this purpose, the topology, equilibrium and conformation of complexes between a supercoiled DNA pUC19 (2686 base pairs) and spermine containing hydrophilic and/or hydrophobic moieties or a polylysine with a hydrophilic block are determined by means of dynamic (DLS) and static light scattering (SLS), atomic force microscopy (AFM), and circular dichroism (CD) spectroscopy. It is demonstrated that all of these complexes consisted of only one molecule of the polyanion. Only the polylysine-b-polyethylene glycol copolymer satisfied the conditions: 1) 100% neutralization of DNA charges and with a small excess of the cation (lower than 30%) and 2) form stable complexes at every charge ratio. rnDNA complex formation is also investigated in organic solvents. Precipitation is induced by neutralizing the charge of the supercoiled DNA pUC19 with the surfactants dodecyltrimethylammonium bromide (DTAB) and tetradecyltrimethylammonium bromide (TTAB). After isolation and drying of the solids, the complexes are dissolved in organic solvents. DNA-TTA complexes are only soluble in methanol and DNA-DTA in DMF. The complexes again consisted of only one DNA molecule. The final topology of the complexes is different in methanol than in DMF. In the former case, DNA seems to be compacted whereas in the latter case, the DNA-DTA complexes seem to have an expanded conformation. Upon complex formation with polycations in organic solvents (with polyvilylpyridine brush (b-PVP) in methanol and with a protected polylysine in DMF), DNA aggregates and precipitates. rnDNA is linearized with an enzyme (SmaI) to investigate the influence of the initial topology of the polyanion on the final conformation of the complexes in organic solvents. Two main differences are evidenced: 1. Complexes in organic solvents formed with linear DNA have in general a more expanded conformation and a higher tendency to aggregate. 2. If a polycation, i.e. the b-PVP, is added to the linear DNA-TTA complexes in methanol, complexes with the polycation are formed at a higher charge ratio. In DMF, the addition of the same b-PVP and of b-PLL did not lead to the formation of complexes.rn

Selective oxidation of 5-hydroxymethylfurfural using monometallic and bimetallic supported nanoparticles

This work deals with the oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA) using metal supported catalysts. Catalysts were prepared from the immobilisation of preformed monometallic (Au, Pd) and bimetallic (AuCu, AuPd) nanoparticles on commercial oxides (TiO2, CeO2). Au-TiO2 catalyst was found to be very active for HMF oxidation; however, this system deactivated very fast. For this reason, we prepared bimetallic gold-copper nanoparticles and an increase in the catalytic activity was observed together with an increase in catalyst stability. In order to optimise the interaction of the metal active phase with the support, Au and AuCu nanoparticles were supported onto CeO2. Au-CeO2 catalyst was found to be more active than the bimetallic one, leading to the conclusion that in this case the most important feature is the interaction between gold and the support. Catalyst pre-treatments (calcination and washing) were carried out to maximise the contact between the metal and the oxide and an increase in the FDCA production could be observed. The presence of ceria defective sites was crucial for FDCA formation. Mesoporous cerium oxide was synthesised with the hard template method and was used as support for Au nanoparticles to promote the catalytic activity. In order to study the role of active phase in HMF oxidation, PdAu nanoparticles were supported onto TiO2. Au and Pd monometallic catalysts were very active in the formation of HMFCA (5-hydroxymethyl-2-furan carboxylic acid), but Pd was not able to convert it, leading to a low FDCA yield. The calcination of PdAu catalysts led to Pd segregation on the particles surface, which changed the reaction pathway and included an important contribution of the Cannizzaro reaction. PVP protected PdAu nanoparticles, synthesised with different morphologies (core-shell and alloyed structure), confirmed the presence of a different reaction mechanism when the metal surface composition changes.

Polykation-DNA-Komplexe : Eigenschaften und Anwendung in der Gentransfektion

Polykationen bilden mit DNA spontan Komplexe. Triebkraft ist der Entropiegewinn durch Freisetzung der Gegenionen auf den Polyelektrolyten. Solche Komplexe können in der Gentechnik verwendet werden, um fremde DNA in eine Zelle einzuschleusen. Dies bezeichnet man als Gentransfektion. In dieser Arbeit werden erstmals bürstenförmige Polykationen mit wurmförmiger Topologie zur Gentransfektion verwendet. Dazu wurde die Komplexierung von DNA mit Bürstenpolymeren mit Poly-L-Lysin- und Polyvinylpyridinium-Seitenketten und linearen Polykationen untersucht. Die Komplexbildung verläuft in allen Fällen kinetisch kontrolliert, alle Polykationen bilden sphärische Komplexe, die Topologie hat keinen Einfluss auf die Komplexgröße. Komplexe aus Bürstenpolymeren transfizieren mehr als 25% der gesamten Zellpopulation bei Schweinehirnendothelzellen. Gegenüber dem kommerziellen Transfektionsmittel Lipofektamin konnte eine deutliche Steigerung um bis zu 400% erreicht werden. Komplexe, die mit linearen Analoga gebildet wurden, zeigten bei gleicher Komplexgröße Transfektionsraten unter 5%. Freisetzungsversuche zeigen, dass die Komplexe, die gut transfizieren, recht labil sind, also die DNA unter Kompetitoreinfluss freisetzen können. Stabile Komplexe haben geringe Transfektionseffizienzen. Ebenso wichtig ist der Schutz der DNA vor Abbau durch DNase. Die PVP-Bürste bietet als einziges der untersuchten Polykationen diesen Schutz und zeigt auch die besten Transfektionsraten. Zusätzlich zu der medizinischen Anwendung wurde die Kinetik der Komplexbildung untersucht. Dazu wurde ein spezieller Aufbau entwickelt, der es ermöglicht die Streuintensität der Komplexlösung bei kleinen Streuwinkeln zeitaufgelöst im Millisekundenbereich zu detektieren. Die Komplexbildung verläuft diffusionskontrolliert, im Bereich von Ladungsverhältnissen (positive zu negativen Ladungen) von 1.8 bis 4.0 schließt sich ein fraktales Wachstum an.

Determination of effective charge of flexible polyelectrolytes by light scattering

The most important property controlling the physicochemical behaviour of polyelectrolytes and their applicability in different fields is the charge density on the macromolecular chain. A polyelectrolyte molecule in solution may have an effective charge density which is smaller than the actual charge density determined from its chemical structure. In the present work an attempt has been made to quantitatively determine this effective charge density of a model polyelectrolyte by using light scattering techniques. Flexible linear polyelectrolytes with a Poly(2-Vinylpyridine) (2-PVP) backbone are used in the present study. The polyelectrolytes are synthesized by quaternizing the pyridine groups of 2-PVP by ethyl bromide to different quaternization degrees. The effect of the molar mass, degree of quaternization and solvent polarity on the effective charge is studied. The results show that the effective charge does not vary much with the polymer molar mass or the degree of quaternization. But a significant increase in the effective charge is observed when the solvent polarity is increased. The results do not obey the counterion condensation theory proposed by Manning. Based on the very low effective charges determined in this study, a new mechanism for the counterion condensation phenomena from a specific polyelectrolyte-counterion interaction is proposed

Selbstorganisation von Amphiphilen auf Basis von Hexaphenylbenzol und Hexa-peri-hexabenzocoronen

Die Selbstorganisation von amphiphilen Molekülen wird genutzt, um in Lösung, auf der Oberfläche, in der festen Phase und an der Flüssig/Fest-Grenzfläche nanoskopisch strukturierte Materialien zu erhalten. Ziel hierbei ist es, die Dynamik der niedermolekularen Amphiphile mit der Stabilität der hochmolekularen Amphiphile zu vereinigen, um damit die Selbstorganisation der Moleküle zu kontrollieren. Drei Konzepte zur Strukturierung von Kohlenstoff durch Selbstorganisation werden vorgestellt. Im ersten Konzept werden aus Hexaphenylbenzol-Polyethylenglykol- (HPB-PEG) und Hexa-peri-hexabenzocoronen- (HBC-PEG) Derivaten wurmähnliche bzw. faserförmige Strukturen in wässriger Lösung erhalten. Der Wassergehalt in den Hydrogelfasern aus den HPB-PEG-Derivaten kann durch das Substitutionsmuster der Amphiphile und die Länge der PEG-Ketten eingestellt werden. Die Hydrogelfasern ähneln anders als die bisherigen Verfahren, die zur Faserherstellung verwendet werden (Extrudieren, Mikrofluid-Verarbeitung oder Elektrospinning), Systemen in der Natur. Der Beweis für die Bildung von Hydrogelfasern wird mittels spezieller Methoden der polarisierten und depolarisierten dynamischen Lichtstreuung erbracht. Im zweiten Konzept werden durch Elektronenbestrahlung und Pyrolyse von 3',4',5',6'-Tetraphenyl-[1,1':2',1''-terphenyl]-4,4''-dithiol homogene Kohlenstoffmembranen mit Poren erzeugt, die Anwendung in der Filtration finden können und im dritten Konzept wird die Selbstorganisation von einem ortho-verknüpften HPB-Trimer an der Flüssig/Fest-Grenzfläche untersucht. Auf diese Weise werden hochgeordnete lamellare Strukturen erhalten. In allen drei Konzepten sind die Geometrie und die Größe der Moleküle die entscheidenden Parameter zur Erzeugung definierter Strukturen.

Hydrogels for DNA isolation from blood

This dissertation describes the synthesis of surface attached hydrogel biomaterials, characterization of their properties, evaluation of structuring concepts and the investigation of these materials in the isolation of DNA from human whole blood. Photosensitive hydrogel precursor materials on the basis of hydroxyethylmethacrylate (HEMA) were synthesized by free radical polymerization. In order to obtain surface bound hydrogel films, the precursors were deposited on a suitable substrate and subsequently irradatiated with UV - light to accomplish the formation of crosslinks in the film and create surface attachment. The composition of the polymerization precursor materials was determined by comprehensive NMR and GPC studies, revealing the copolymerizationrnbehaviour of the used monomers - HEMA derivatives and the photocrosslinkerrnMABP - and their respective distribution in the hydrogel precursors. The degree of crosslinking of the hydrogels was characterized with UV/vis spectroscopy. Stress-strain measurements were conducted in order to investigate the mechanical properties of the biomaterials. Moreover, the swelling process and biomolecule adsorption properties of the hydrogels were investigated with SPR/OW spectroscopy. For this, the deposition and binding of the hydrogels on gold or SiO2 surfaces was facilitated with photocrosslinkable adhesion promotors. The produced hydrogels were mechanically rigid and stablernunder the conditions of PCR and blood lysis. Furthermore, strategies towards the increase of hydrogel surface structure and porosity with porosigens, 2D laser interference lithography and photocleavable blockcopolymers were investigated. At last, a combinatorial strategy was used for the determination of the usefulness of hydrogels for the isolation from DNA from blood. A series of functionalized hydrogel precursors were synthesized, transferred to the surface inside a PCR tube and subsequently screened in regard to DNA adsorption properties with Taqman quantitative PCR. This approach yielded a promising candidate for a functional PCR tube coating that would allow the entire DNA isolation procedure being carried out in a single reaction container.rnThereforce, the practical application of such macromolecular architectures can be envisioned to improve industrial DNA diagnostic processes.

Improving chondrogenesis: potential and limitations of SOX9 gene transfer and mechanical stimulation for cartilage tissue engineering

Articular cartilage injuries and degeneration affect a large proportion of the population in developed countries world wide. Stem cells can be differentiated into chondrocytes by adding transforming growth factor-beta1 and dexamethasone to a pellet culture, which are unfeasible for tissue engineering purposes. We attempted to achieve stable chondrogenesis without any requirement for exogenous growth factors. Human mesenchymal stem cells were transduced with an adenoviral vector containing the SRY-related HMG-box gene 9 (SOX9), and were cultured in a three-dimensional (3D) hydrogel scaffold composite. As an additional treatment, mechanical stimulation was applied in a custom-made bioreactor. SOX9 increased the expression level of its known target genes, as well as its cofactors: the long form of SOX5 and SOX6. However, it was unable to increase the synthesis of sulfated glycosaminoglycans (GAGs). Mechanical stimulation slightly enhanced collagen type X and increased lubricin expression. The combination of SOX9 and mechanical load boosted GAG synthesis as shown by (35)S incorporation. GAG production rate corresponded well with the amount of (endogenous) transforming growth factor-beta1. Finally, cartilage oligomeric matrix protein expression was increased by both treatments. These findings provide insight into the mechanotransduction of mesenchymal stem cells and demonstrate the potential of a transcription factor in stem cell therapy.

Effect of enamel matrix derivative and parathyroid hormone on bone formation in standardized osseous defects: an experimental study in minipigs

Previous experimental studies have indicated that locally administered enamel matrix derivative (EMD) and parathyroid hormone (PTH) may have a stimulatory effect on bone formation. However, it is not clear if the positive effect of EMD is related to its effect on the periodontium as a whole or directly on the bone-forming cells. In addition, it is not known if the presentation of PTH by adding the amino acid sequence Arg-Gly-Asp (RGD) is essential for its osteopromotive effect. Local delivery of a bioactive substance at the right time and in the right concentration often constitutes a major challenge. Polyethylene glycol-based hydrogel (PEG) is a degradable vehicle developed for delivery of bioactive proteins. To enhance the mechanical stability of the PEG-bioactive substance complex, an osteoconductive bone substitute material is often needed.

Glycan-binding specificities of Streptococcus mutans and Streptococcus sobrinus lectin-like adhesins

Since the adhesion of bacteria to the tooth surface is a prerequisite for dental plaque and subsequent caries development, a promising caries preventive strategy could be to block the lectin-glycan-mediated adherence of cariogenic bacteria. The aim of the study was to evaluate potential differences in glycan-binding specificities of two Streptococcus mutans strains (DSM 20523 and DSM 6178) and Streptococcus sobrinus (DSM 20381). A competitive enzyme-linked lectin-binding assay was used to identify the binding specificities of isolated bacterial surface lectins. Blotting of the microbial proteins on neoglycoprotein-coated PVP membranes enabled a qualitative protein analysis of all specific bacterial lectins. Different glycan-binding sites could be identified for the S. mutans strains in comparison to S. sobrinus. An earlier reported glycan-binding specificity for terminal galactose residues could be confirmed for the S. mutans strains. For the S. sobrinus strain, more than one glycan-binding specificity could be found (oligomannose and terminal sialyl residues). Each of the tested strains showed more than one surface lectin responsible for the specific lectin-binding with varying molecular weight (S. mutans, 90/155 kDa and S. sobrinus, 35/45 kDa). The established experimental setup could be used as future standard procedure for the identification of bacterial lectin-derived binding specificities. The findings from this study might serve as basis for the design of an individual 'glycan cocktail' for the competitive inhibition of lectin-mediated adhesion of mutans streptococci to oral surfaces.

Continuous Formation and Separation of Calcium-Alginate Capsules Containing Viable Mammalian Cells in Microfluidic Devices

Microfluidic devices can be used for many applications, including the formation of well-controlled emulsions. In this study, the capability to continuously create monodisperse droplets in a microfluidic device was used to form calcium-alginate capsules.Calcium-alginate capsules have many potential uses, such as immunoisolation of cells and microencapsulation of active drug ingredients or bitter agents in food or beverage products. The gelation of calcium-alginate capsules is achieved by crosslinking sodiumalginate with calcium ions. Calcium ions dissociated from calcium carbonate due to diffusion of acetic acid from a sunflower oil phase into an aqueous droplet containing sodium-alginate and calcium carbonate. After gelation, the capsules were separated from the continuous oil phase into an aqueous solution for use in biological applications. Typically, capsules are separated bycentrifugation, which can damage both the capsules and the encapsulated material. A passive method achieves separation without exposing the encapsulated material or the capsules to large mechanical forces, thereby preventing damage. To achieve passiveseparation, the use of a microfluidic device with opposing channel wa hydrophobicity was used to stabilize co-laminar flow of im of hydrophobicity is accomplished by defining one length of the channel with a hydrogel. The chosen hydrogel was poly (ethylene glycol) diacrylate, which adheres to the glass surface through the use of self-assembled monolayer of 3-(trichlorosilyl)-propyl methacrylate. Due to the difference in surface energy within the channel, the aqueous stream is stabilized near a hydrogel and the oil stream is stabilized near the thiolene based optical adhesive defining the opposing length of the channel. Passive separation with co-laminar flow has shown success in continuously separating calcium-alginatecapsules from an oil phase into an aqueous phase. In addition to successful formation and separation of calcium alginate capsules,encapsulation of Latex micro-beads and viable mammalian cells has been achieved. The viability of encapsulated mammalian cells was determined using a live/dead stain. The co-laminar flow device has also been demonstrated as a means of separating liquid-liquidemulsions.

Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel™) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.

A MICROFLUIDIC METHOD TO MEASURE DIFFUSION IN HYDROGELS

A novel microfluidic method is proposed for studying diffusion of small molecules in a hydrogel. Microfluidic devices were prepared with semi-permeable microchannels defined by crosslinked poly(ethylene glycol) (PEG). Uptake of dye molecules from aqueous solutions flowing through the microchannels was observedoptically and diffusion of the dye into the hydrogel was quantified. To complement the diffusion measurements from the microfluidic studies, nuclear magnetic resonance(NMR) characterization of the diffusion of dye in the PEG hydrogels was performed. The diffusion of small molecules in a hydrogel is relevant to applications such asdrug delivery and modeling transport for tissue-engineering applications. The diffusion of small molecules in a hydrogel is dependent on the extent of crosslinking within the gel, gel structure, and interactions between the diffusive species and the hydrogel network. These effects were studied in a model environment (semi-infinite slab) at the hydrogelfluid boundary in a microfluidic device. The microfluidic devices containing PEG microchannels were fabricated using photolithography. The unsteady diffusion of small molecules (dyes) within the microfluidic device was monitored and recorded using a digital microscope. The information was analyzed with techniques drawn from digital microscopy and image analysis to obtain concentration profiles with time. Using a diffusion model to fit this concentration vs. position data, a diffusion coefficient was obtained. This diffusion coefficient was compared to those from complementary NMR analysis. A pulsed field gradient (PFG) method was used to investigate and quantify small molecule diffusion in gradient (PFG) method was used to investigate and quantify small molecule diffusion in hydrogels. There is good agreement between the diffusion coefficients obtained from the microfluidic methods and those found from the NMR studies. The microfluidic approachused in this research enables the study of diffusion at length scales that approach those of vasculature, facilitating models for studying drug elution from hydrogels in blood-contacting applications.

Quantifying The Diffusion And Erosion Of Degradable Hydrogels Using A Microfluidic Technique

Hydrogels are composed of cross-linked networks of hydrophilic polymers that are biocompatible due to their high water content. Mass transfer through hydrogels has been suggested as an effective method of drug delivery, specifically in degradable polymers to minimize lasting effects within the body. Diffusion of small molecules in poly (ethylene glycol) diacrylate (PEG-DA) and dextran methacrylate (dex-MA) hydrogels was characterized in a microfluidic device and by complementary techniques. Microfluidic devices were prepared by crosslinking a formulation of hydrogel and photo-initiator, with and without visible dye, using photolithography to define a central microchannel. Channel sizes within the devices were approximately 600 ¿m to simulate vessels within the body. The microfluidic technique allows for both image and effluent analyses. To visualize the diffusive behavior within the dextran hydrogel, methylene blue and sulforhodamine 101 dyes were used in both elution and uptake experiments. Three analysis techniques for measuring diffusion coefficients were used to quantify the diffusion of solute in the hydrogel, including optical microscopy, characterization of device effluent, and NMR analyses. The optical microscopy technique analyzes images of the dye diffusion captured by a stereomicroscope to generate dye concentration v. position profiles. The data was fit to a diffusion model to determine diffusion coefficients and the dye release profile. In a typical elution experiment, aqueous solution is pumped through the microchannel and dye diffuses out of the hydrogel and into the aqueous phase. During elution, images are taken at regular time intervals and the effluent was collected. Analysis of the device effluent was performed using ultraviolet-visible (UV/Vis) spectroscopy to determine the effluent dye concentration and thus a short-time diffusion coefficient. Nuclear magnetic resonance (NMR) was used to determine a free diffusion coefficient of molecules in hydrogel without the effect of a concentration gradient. Diffusion coefficients for methylene blue and sulforhodamine 101 dyes in dex-MA hydrogel calculated using the three analysis methods all agree well. It was determined that utilizing a combination of the three techniques offers greater insight into molecular diffusion in hydrogels than employing each technique individually. The use of the same microfluidic devices used to measure diffusion is explored in the use of studying the degradation of dex-MA hydrogels. By combining what is known about the degradation rate in regards to the effect of pH and crosslinking and the ability to use a dye solution in contrast to establish the hydrogel boundaries could be a novel approach to studying hydrogel degradation.

A Microfluidic Method to Measure Small Molecule Diffusion in Hydrogels

Drug release from a fluid-contacting biomaterial is simulated using a microfluidic device with a channel defined by solute-loaded hydrogel; as water is pumped through the channel, solute transfers from the hydrogel into the water. Optical analysis of in-situ hydrogels, characterization of the microfluidic device effluent, and NMR methods were used to find diffusion coefficients of several dyes (model drugs) in poly( ethylene glycol) diacrylate (PEG-DA) hydrogels. Diffusion coefficients for methylene blue and sulforhodamine 101 in PEG-DA calculated using the three methods are in good agreement; both dyes are mobile in the hydrogel and elute from the hydrogel at the aqueous channel interface. However, the dye acid blue 22 deviates from typical diffusion behavior and does not release as expected from the hydrogel. Importantly, only the microfluidic method is capable of detecting this behavior. Characterizing solute diffusion with a combination of NMR, optical and effluent methods offer greater insight into molecular diffusion in hydrogels than employing each technique individually. The NMR method made precise measurements for solute diffusion in all cases. The microfluidic optical method was effective for visualizing diffusion of the optically active solutes. The optical and effluent methods show potential to be used to screen solutes to determine if they elute from a hydrogel in contact with flowing fluid. Our data suggest that when designing a drug delivery device, analyzing the diffusion from the molecular level to the device level is important to establish a complete picture of drug elution, and microfluidic methods to study such diffusion can play a key role. (C) 2013 Elsevier B.V. All rights reserved.