Printed soft optical waveguides for delivering light into deep tissue

To implement light-based diagnosis and therapies in the clinic, implantable patient-friendly devices that can deliver light inside the body while being compatible with soft tissues are needed. This Thesis presents the development of optical waveguides for guiding light into tissue, obtained by printing technologies from three different polymer combinations. Firstly, D,L-dithiothreitol (DTT) bridged PEG diacrylate were synthesized and printed into waveguides, which exhibited tunable mechanical properties and degradability, and low optical losses (as low as 0.1 dB cm-1 in visible range). Secondly, degradable waveguides from amorphous poly(D,L-lactide) and derived copolymers were developed by printing, which showed elasticity at body temperature and could guide VIS to NIR light in tissue for tens of centimeters. At last, soft and stretchable optical waveguides consisting of polydimethylsiloxane (PDMS) core and acrylated Pluronic F127 cladding were fabricated by coaxial extrusion printing, which could be stretched to 4 times of their length and showed optical loss values in tissue as low as 0.13 -0.34 dB cm-1 in the range of 405-520 nm. For proof-of-concept, above printed optical waveguides were used to deliver light across 5-8 cm tissue to remotely activate photochemical processes in in vitro cell cultures. The presented work exemplifies how rational study of medically approved biomaterials can lead to useful and cost-effective optical components for light applications.Neue optische Technologien verändern die Zukunft der Medizin und fördern die Entwicklung von Implantaten, die im Körper Licht abgeben. Diese Arbeit beschreibt drei gewebekompatible, optische Wellen¬leiter für medizinische Zwecke, die mit 3D-Extrusionsdruck gefertigt werden. Zum einem wurden Wellen¬leiter mit einstellbaren mechanischen Eigenschaften und kontrollierter Abbaubarkeit im Körper als Funktion des Dithio¬threitol (DTT)-Anteils in DTT-modifizierten Poly¬ethylen¬glykol¬diacrylat-Hydro¬gelen entwickelt. Die bei der Extrusion in-situ-photopolymerisierten Wellen¬leiter haben nur 0,1 dB/cm optischen Verlust im VIS-Bereich und wurden verwendet, um photo¬chemische Prozesse in In-vitro-Zellkulturen zu aktivieren. Zum anderen wurden im Körper abbaubare Wellenleiter aus amorphem Poly(D,L-Lactid) und dessen Copolymeren gedruckt. Diese Wellenleiter sind bei Körpertemperatur elastisch und leiten in mehreren zehn Zentimetern Gewebe Licht vom VIS- bis NIR-Bereich. Schließlich wurden mit koaxialem Extrusions¬druck weiche und dehnbare Wellenleiter hergestellt, die aus einem PDMS-Kern und einer acrylierten Pluronic F127 Hülle bestehen. Diese Wellenleiter sind aufs Vierfache dehnbar und haben in Gewebe nur 0,13 bis 0,34 dB/cm optische Verluste bei 405-520 nm. Die vorgestellte Arbeit zeigt, wie Materialauswahl mit Drucktechnologien kombiniert werden können, um optische Wellenleiter für medizinische Anwendungen mit bemerkenswerter Leistung bei angemessenem Aufwand zu entwickeln

Biomateriaalien valomikrovalmistus soluviljelysovelluksissa – vertaileva tutkimus

The integration of microfluidics, microfabrication technologies and biomaterials has established new means to mimic the natural microenvironment of cells. Using microfluidic culture devices, cells can be stimulated with both mechanical and chemical cues. Light, in the form of UV lamps and lasers, is a powerful microfabrication tool for biomedical applications, offering high resolution and fast production. Special photocurable materials have been developed to meet the needs of this technology. The first part of this thesis is a literature review on this research field, focusing especially on microfabrication using lasers and photosensitive hydrogels in cell-based applications. Recently, photopolymerization by non-linear light absorption has been introduced to microfabrication, breaking the resolution boundaries set by classical optics. This phenomenon is utilized in two-photon polymerization (2PP), a method for the rapid freeform fabrication of 3D micro- and nanostructures. The basic theory of 2PP is provided in the literature review. Unfortunately, 2PP has mainly been studied with common photoresists and investigation of suitable synthetic biomaterials for the biomedical applications of 2PP has remained insufficient. The latter part of this thesis presents an innovative and scientifically original study that aims to widen the selection of 2PP processable biomaterials. In the experiments, 2PP was investigated with a commercial photoinitiator (PI) and two biomaterials: a novel polycaprolactone-based oligomer (PCL-o) and a poly(ethylene glycol) hydrogel (PEGda). PCL-o is a novel photopolymer synthesized for research purposes and has never been used in 2PP; moreover, 2PP of PEGda with the laser type used has not been reported previously. In the study, the two materials were compared in terms of resolution and overall 2PP processability. Using a custom-built fabrication setup based on an affordable Nd:YAG laser, arbitrary microstructures were polymerized on glass substrates and subsequently characterized using SEM imaging. Additionally, the effect of PI concentration on resolution was investigated. Cytotoxicity of the sample materials was tested in order to estimate the applicability of the fabricated microstructures in cell-based applications. The outcome of this study was a success, since 2PP of both PEGda and PCL-o was successfully demonstrated and the Nd:YAG laser proved adequate for the research of novel biomaterial microstructures; resolution in the order of one micrometer was achieved with PCL-o. Based on the cytotoxicity tests, both PEGda and PCL-o were found non-cytotoxic and suitable e.g. for use in guided cell growth. Despite some differences in the fabrication process, the processability of PEGda and PCL-o was found equally well suited for 2PP and research with these materials should definitely be continued in the future. The versatility of the current fabrication system could be improved by experimenting different new photocrosslinkable oligomers, more efficient PIs, optimized equipment, sterilization of the microstructures and cell culturing. /Kir10Biomateriaalien mikrovalmistusmenetelmien kehittymisen myötä voidaan valmistaa yhä paremmin solujen luonnollista mikroympäristöä mimikoivia kasvatusalustoja, joissa soluja stimuloidaan sekä mekaanisin että kemiallisin signaalein mikrofluidistiikan avulla. UV-lamppuja ja lasereita hyödyntävä valoavusteinen mikrovalmistus on yksi kehittyneimmistä menetelmistä tällaisiin sovelluskohteisiin ja menetelmää varten on kehitetty myös uusia valokovettuvia biomateriaaleja. Tämän diplomityön ensimmäinen osa on laaja kirjallisuusselvitys, joka käsittelee mikrofluidistiikan, valoavusteisen mikrovalmistuksen ja biomateriaalien hyödyntämistä soluviljelysovelluksissa. Työssä syvennytään lasermikrovalmistukseen ja valosillottuvien hydrogeelien mikrokuvioitiin. Uusi trendi mikrovalmistuksessa on valon epälineaariseen absorptioon perustuvat menetelmät, joissa päästään nanometriluokan resoluutioon. Yksi niistä on kaksifotonipolymeraatio (2PP), 3D-mikrovalmistukseen sopiva pikamallinnusmenetelmä, jolla voidaan valmistaa mielivaltaisia mikro- ja nanokuvioita. Menetelmän teorian pääpiirteet esitellään kirjallisuusselvityksessä. Biosovelluksissa 2PP:n suurin rajoite on se, että pääsääntöisesti menetelmää on tutkittu perinteisillä fotoresisteillä ja bioyhteensopivien synteettisten materiaalien tutkimus on ollut puutteellista. Tässä työssä tehdyn tieteellisen tutkimuksen tarkoitus oli kokeilla ja esitellä 2PP:oon sopivia biomateriaaleja, joista voidaan valmistaa soluyhteensopivia mikrorakenteita. 2PP-menetelmää tutkittiin polyetyleeniglykolihydrogeelillä (PEGda) sekä uudella polykaprolaktonipohjaisella oligomeerilla (PCL-o) käyttäen kaupallista fotoinitiaattoria. PCL-o on synteettinen biohajoava polymeerimateriaali, jota ei ole ennen testattu 2PP:ssa; myöskään PEGda:n 2PP-valmistusta käytetyllä lasertyypillä ei ole aiemmin raportoitu. Tutkimuksessa vertailtiin PEGda:n ja PCL-o:n sopivuutta 2PP-valmistukseen resoluutio ja prosessoitavuus huomioon ottaen. Itsekoottua Nd:YAG-laseriin perustuvaa tietokoneohjattua laitteistoa käyttäen lasialustalle polymeroitiin mikrokuvioita, jotka karakterisoitiin pyyhkäisyelektronimikroskoopilla. Työssä tutkittiin myös initiaattorikonsentraation vaikutus resoluutioon. Näytemateriaaleille tehtiin sytotoksisuuskokeet, joiden avulla arvioitiin valmistettujen mikrorakenteiden sopivuus biosovelluksiin. Onnistuneeksi osoittautuneen tutkimuksen mukaan sekä PEGda:n että PCL-o:n prosessoitavuus 2PP:ssa oli riittävä ja käytetty laserlaitteisto soveltui tarkoitukseen hyvin; PCL-o:lla saatiin jopa 1 µm:n resoluutio. Sytotoksisuustestien perusteella molemmat materiaalit ovat ei-toksisia ja siten soveltuvat erilaisiin soluviljelysovelluksiin. Vaikka materiaalien välillä ilmeni valmistusprosessissa joitakin eroja, tämän tutkimuksen perusteella molempien materiaalien testaamista 2PP:ssa tulee ehdottomasti jatkaa. Tulevaisuudessa menetelmän käytettävyyttä voisi parantaa mm. testaamalla useampia uusia materiaaleja, tehokkaampia fotoinitiaattoreita, optisesti laadukkaampaa laitteistoa, mik-rorakenteiden sterilointia sekä soluviljelyä polymeroiduilla rakenteilla

Imaging biological water permeability barriers using CARS microscopy

Alterations in the water permeability of the vasculature are attributed to several disease processes including atherosclerosis, reperfusion injury, diabetes mellitus, aging, chronic inflammation, and cancer. While vascular permeability has been extensively studied throughout the years, many of these studies have been limited by their invasiveness, specificity and spatial resolution. Coherent anti-Stokes Raman scattering (CARS) microscopy, offers a means to visualize the water permeability barrier of biological structures at high resolution within intact systems. In this thesis, water CARS imaging was characterized for determining water permeability in both dynamic and steady state conditions in model hydrogel systems. Computational models of water transport were used to validate the accuracy of water-CARS permeability mapping within these systems. This technique was then applied to the first CARS examination of water permeability in the cerebral artery and blood brain barrier. The vessels of mice deficient in the water channel protein aquaporin 1 (AQP1) were examined to evaluate the role of AQP1 in vascular water permeability. A layered hydrogel imaging phantom was constructed from poly(ethylene glycol) diacrylate (PEGDA) to validate ability of water-CARS microscopy to determine the relative permeability differences of adjacent material layers. By imaging the dynamic and steady-state H2O and D2O exchange across the uniform and layered composite hydrogel structures, it was indeed possible to measure the relative permeability differences between individual material layers and to determine the location of material interfaces from analysis of the water-CARS image profile. CARS imaging of intact biological samples poses many significant challenges. Microvessel microfluidic systems have the possibility to facilitate microscopy studies of vascular barrier function due to their flexibility of design, relative ease of preparation, and relative simplicity. In this thesis, the fabrication process of a human umbilical vein endothelial cell (HUVEC) microvessel microfluidic was investigated and optimized for future studies of vascular permeability and physiology. Several factors in the device assembly process were identified and optimized to improve the repeatability and reliability of the microvessel microfluidic fabrication protocol. CARS imaging of the mouse cerebral artery water transport produced higher quality images than what had previously been achieved in the rat mesenteric artery, likely due to their reduced wall thickness. Permeability mapping of these arteries localized the water permeability barrier to the endothelial basolateral membrane, a result consistent with previous measurements performed in the rat mesenteric arteries. Previous work by the Laboratory of Cardiac Energetics has suggested that the exclusively apical expression of the channel protein aquaporin 1 (AQP1) may account for the increase in observed permeability of the apical endothelial cell membrane. To test this hypothesis, water-CARS imaging of H2O/D2O exchange across cerebral arteries of wild type and AQP1 knockout (KO) mice was performed. No significant difference in the location of the water permeability barrier was observed. These findings indicate that AQP1 may not, in fact, be rate limiting for water transport across the apical endothelial membrane, and that it may play some other role in the physiology of the endothelial cell.This project was supported by the NIH Oxford Cambridge Scholars Progra

Advanced hydrogels based on natural macromolecules: chemical routes to achieve mechanical versatility

Advances in synthetic routes to chemically modify natural macromolecules such as polysaccharides and proteins have allowed designing functional hydrogels able to tackle current challenges in the biomedical field. Hydrogels are hydrophilic three-dimensional systems able to absorb or retain a large volume of water, prepared from a low percentage of precursor macromolecules. The typical fragile elastic structure of common hydrogel formulations often limits their usage. Three main fabrication strategies involving several compounds or multimodified materials known as double networks, dual-crosslinked networks, and interpenetrating networks have been explored to impart mechanical strength to hydrogels. Widely investigated for synthetic polymers, these approaches allow obtaining added-value hydrogels with a large spectrum of mechanical properties. Advances in the development of such hydrogels with biomacromolecules as main constituent materials have enabled the fabrication of hydrogels with improved key properties for medical use, including biocompatibility, controlled release of active substances and tailored biodegradability, while exploring sustainable sources. This review describes recent advances in the use of proteins, as well as natural and semi-synthetic polymers for the fabrication of hydrogels for biomedical applications. Structures processed via double network, dual-crosslinked, or interpenetrating network strategies are reviewed, and emphasis is given to the type of chemical modifications and reactions, as well as the covalent and non-covalent interactions/bonds involved in those mechanisms.publishe

PHOTOCURABLE HYDROGELS FOR TISSUE ENGINERING APPLICATIONS

L'abstract è presente nell'allegato / the abstract is in the attachmen

Stimuli-Responsive Microtools for Biomedical and Defense Applications

We live in a 3D world which has embraced ever shrinking technologies, yet the techniques used to create these micro- and nanoscale technologies are inherently 2D. Self-assembly of 2D templates into 3D devices enables the creation of complex tools cheaply, efficiently, and in mass quantity. I utilize this technique to create stimuli-responsive microgrippers, which are shaped like hands with flexible joints and rigid phalanges and range in size from 10 µm to 4 mm. Intrinsic stress within the hinges provides all the energy necessary for gripping, and thus they require no wires or batteries for operation. Here, I demonstrate their use for both biomedical and defense applications. These microgrippers can be used as microsurgical tools, gripping onto tissue in response to body temperature and excising tissue from the gastrointestinal tract in both in vivo and ex vivo porcine models. A Monte Carlo model confirmed that these tiny tools has a higher probability of sampling tissue from a lesion as compared to the traditional biopsy foreceps. These grippers were scaled down to 10 µm and used to capture single cells for in vitro isolation, imaging, and assays. All-polymeric, porous, stimuli-responsive therapeutic grippers or “theragrippers” which swell and de-swell around body temperature were created for drug delivery applications. These theragrippers can be loaded with commercial drugs for biphasic, site-specific controlled release and were successfully demonstrated in an in vitro and an in vivo model. For defense applications, integrating microelectronics like RFID’s onto the microgrippers creates tagging, tracking, and locating (TTL) devices capable of latching onto clothing, hair, and moving animal targets. This integrated design is enabled using high throughput solder-based self-assembly. This defense application, particularly reliant on covert, wireless technology, benefits from our novel photothermal actuation mechanism using low power, handheld lasers. In addition to triggering microgripper closing, this actuation scheme also enables complex sequential folding pathways, a step towards programmable matter

HYDROGEL COMPOSITIONS FOR NONVIRAL GENE DELIVERY

The incorporation of nonviral vectors into biomaterial matrices has been employed to improve localization at the implant site and to protect from loss by clearance or extracellular barriers. However, several limitations such as detrimental crosslinking mechanisms, uncontrolled burst release require improved design of matrix-based gene delivery systems that provides sustained and controlled vector release as well as overcomes extracellular barriers to gene transfer in proximity to target cells. The long-term objective of this dissertation project is to provide the basis for the eventual creation of tissue engineering scaffolds that combine structural and biological activity through the creation of composite materials consisting of polymeric fibers with hydrogel coatings encapsulating nonviral vectors for localized gene delivery. The primary focus of this dissertation was to develop hydrogels with suitable physical/chemical properties for the localized, sustained gene delivery application; including variable degradation rate for controlled release, mild crosslinking conditions compatible with vector encapsulation, and minimal swelling after crosslinking to maintain stability as a fiber coating. The hypothesis was that vector release from hydrogel coatings in intimate proximity with adherent cells will overcome extracellular barriers to gene transfer and increase transfection efficiency relative to conventional bolus delivery methods. In order to concentrate nonviral vectors for efficient hydrogel loading, several approaches to vector lyophilization were investigated. The inclusion of 10% sucrose as a cryoprotectant was shown to preserve DNA structural integrity and biological activity. In addition, incubation of nonviral vectors with sodium tripolyphosphate was shown to effectively release DNA from nonviral gene complexes, which provides a basis for accurate measurement of vector/DNA release. To develop polymer-based gene encapsulation system for gene delivery, hydrolytically degradable hydrogels with various degradation-kinetics were developed and characterized. First, linear poly(ethylene glycol)(PEG) polymers were modified with ester bonds of varying susceptibility to hydrolytic degradation and crosslinkable acrylate groups. Hydrogels were prepared by photopolymerization and shown to variable degradation rates and controlled release of a model macromolecule. Secondly, hydrogels crosslinked by Michael-type addition from various acrylate-terminated four-armed amphiphilic poloxamine (Tetronic®) were evaluated. It was shown that the physical properties of these gels are attributable to both temperature-dependent noncovalent interactions and covalent crosslinking. Hydrogels prepared from Tetronic T904 did not experience significant swelling after crosslinking and were shown to form stable coatings on polymer fibers. Finally, lyophilized nonviral-gene complexes were incorporated within T904-based hydrogels. DNA integrity and sustained release was observed although the kinetics was not considered optimal due to delayed release. Preliminary transfection experiments using an in vitro model with serum-containing medium demonstrated that released vectors could transfect surrounding cells, although the efficiency of transfection was limited and further improvements to the gene delivery system are required

Methods for immobilizing receptors in microfluidic devices: A review

In this review article, we discuss state-of-the-art methods for immobilizing functional receptors in microfluidic devices. Strategies used to immobilize receptors in such devices are essential for the development of specific, sensitive (bio)chemical assays that can be used for a wide range of applications. In the first section, we review the principles and the chemistry of immobilization techniques that are the most commonly used in microfluidics. We afterward describe immobilization methods on static surfaces from microchannel surfaces to electrode surfaces with a particular attention to opportunities offered by hydrogel surfaces. Finally, we discuss immobilization methods on mobile surfaces with an emphasis on both magnetic and non-magnetic microbeads, and finally, we highlight recent developments of new types of mobile supports

Engineering responsive liposome systems for biomedical applications

The design of materials able to undergo changes in response to an applied stimulus (e.g. temperature, pH or magnetic fields) is relevant for biomedical applications. In the context of hydrogels, the design of triggers for hydrogelation has enabled precise control over hydrogelation kinetics and mechanical properties. One trigger that has yet to be explored for hydrogelation is ultrasound; a widely-used biomedical platform that is non-invasive, with tuneable tissue penetration depth and high spatiotemporal control. The use of ultrasound as a remote trigger for enzymatic activity and hydrogelation was explored in this thesis. In particular, the ability of liposomes and microbubble-liposome conjugates to release encapsulated payloads upon ultrasound exposure was leveraged. The designed field-responsive system required that the amount of encapsulated calcium in liposomes was maximised. Hence, cryo-TEM and small-angle neutron scattering were used to investigate the effect of the formulation method and the lipid composition on vesicle lamellarity, which determines the volume of the internal liposomal aqueous compartment. In another study, X-ray and neutron scattering corroborated with all-atom molecular dynamics simulations were used to elucidate the effect of sodium and calcium ions on ethanol-induced lipid membrane interdigitation. The results of this study, which spanned a wide range of length scales, furthered the understanding of ethanol-induced interdigitation of bulk and vesicular lipid formulations, with important implications for the production of interdigitation-fusion vesicles. Calcium-loaded liposomes produced via the interdigitation fusion vesicle method that were able to release their payload upon ultrasound exposure were utilised to trigger the catalytic activity of a calcium-dependent tissue transglutaminase. The ultrasound-activated transglutaminase could then catalyse intermolecular covalent crosslinking between the lysine and glutamine sidechain residues of soluble fibrinogen molecules, yielding fibrinogen hydrogels. Precise control over these processes could be achieved, with the calcium release, catalysis rate and hydrogelation rate all shown to be dependent upon the ultrasound exposure time. Calcium-loaded liposomes were also conjugated to the surface of gaseous microbubbles that are commonly used for in vivo drug delivery. These microbubble-liposome conjugates exhibited enhanced response to the applied acoustic field and could also be used for ultrasound-triggered hydrogelation. Taken together, these results represent an entirely new class of stimuli for enzyme activity and hydrogelation and open up a wide range of opportunities for ultrasound-triggered molecular biology, synthetic biology and material science.Open Acces

Functional surface micropatterns by dewetting of thin polymer films

Patterned polymer surfaces are of great importance with respect to an increasing number of technological and bio-medical applications, due to their great versatility in terms of chemical composition, properties and processing techniques. Surface micro-patterning by spontaneous dewetting of thin polymer films represents a versatile and robust process to fabricate surfaces with controlled topography and chemistry at the micro-scale. In this Thesis, we used polymer dewetting in combination with complementary approaches to engineer both surface chemistry and the ordering of the dewetting patterns. The dewetting of poly(D,L-glycolide-co-lactide) (PLGA) thin films on polystyrene (PS) was combined with the grafting of protein-repellent poly(ethylene glycol) (PEG), in order to form topographical and chemical surface micropatterns consisting in protein-adhesive PS domains surrounded by protein-repellent PEG-grafted PLGA films. The produced micropatterned surfaces were used for site-specific protein adsorption, and represent a promising platform for biological applications, such as proteomics, single-cell studies and tissue engineering. Spatially ordered surface micropatterns were obtained by combining polymer dewetting with microcontact printing and colloidal lithography, respectively. The dewetting of thin PS films was guided within specific regions of the substrate by prestamping of the silicon substrate with self-assembled monolayers of an alkylsilane by microcontact printing. Ordered micropatterns consisting in arrays of holes with tunable size were obtained by exploiting the spontaneous dewetting of poly(4-vinyl pyridine) (P4VP) thin films on PS from the holes produced by colloidal imprinting with two-dimensional colloidal crystals assembled on the polymer bilayer