Biocompatibility and biofunctionalization of mesoporous silicon particles

Several of the newly developed drug molecules experience poor biopharmaceutical behavior, which hinders their effective delivery at the proper site of action. Among the several strategies employed in order to overcome this obstacle, mesoporous silicon-based materials have emerged as promising drug carriers due to their ability to improve the dissolution behavior of several poorly water-soluble drugs compounds confined within their pores. In addition to improve the dissolution behavior of the drugs, we report that porous silicon (PSi) nanoparticles have a higher degree of biocompatibility than PSi microparticles in several cell lines studied. In addition, the degradation of the nanoparticles showed its potential to fast clearance in the body. After oral delivery, the PSi particles were also found to transit the intestines without being absorbed. These results constituted the first quantitative analysis of the behavior of orally administered PSi nanoparticles compared with other delivery routes in rats. The self-assemble of a hydrophobin class II (HFBII) protein at the surface of hydrophobic PSi particles endowed the particles with greater biocompatibility in different cell lines, was found to reverse their hydrophobicity and also protected a drug loaded within its pores against premature release at low pH while enabling subsequent drug release as the pH increased. These results highlight the potential of HFBII-coating for PSi-based drug carriers in improving their hydrophilicity, biocompatibility and pH responsiveness in drug delivery applications. In conclusion, mesoporous silicon particles have been shown to be a versatile platform for improving the dissolution behavior of poorly water-soluble drugs with high biocompatibility and easy surface modification. The results of this study also provide information regarding the biofunctionalization of the THCPSi particles with a fungal protein, leading to an improvement in their biocompatibility and endowing them with pH responsive and mucoadhesive properties.Heikot biofarmaseuttiset ominaisuudet vaikeuttavat uusien lääkemolekyylien keksintää ja kehittämistä, mikä estää molekyylien tehokasta annostelua vaikutuspaikkaansa elimistössä. Tämän ongelman ratkaisemiseksi on tutkittu useita menetelmiä ja materiaaleja, joista yksi lupaavimmista perustuu mesohuokoisten silikonipohjaisten (PSi) materiaalien käyttöön lääkeannostelussa. PSi-pohjaiset lääkekantajat parantavat niukkaliukoisten lääkeaineiden liukenemisnopeutta, mikä perustuu mesohuokosten pieneen kokoon ja suureen pinta-alaan. Useissa solulinjoissa tehdyissä kokeissa havaittiin, että huokoisesta piistä valmistetut nanopartikkelit ovat biologiselta yhteensopivuudeltaan parempia kuin vastaavat PSi-mikropartikkelit. PSi-nanopartikkelien etuna on lisäksi nopea hajoaminen ja sitä kautta nopea poistuminen elimistöstä. Väitöskirjatyössä annosteltiin radio-leimattuja PSi-nanopartikkeleita rotan laskimoverenkiertoon, jolloin ne kohdentuivat nopeasti koe-eläimen maksaan ja pernaan ilman näkyviä toksisia vaikutuksia. Suun kautta annosteltuina PSi-nanopartikkelit kulkeutuivat suoliston läpi. PSi-partikkelien biologista yhteensopivuutta tutkituissa solulinjoissa parannettiin päällystämällä ne itsejärjestäytyvillä hydrofobiini-proteiineilla (hydrofobiini-luokka II, HFBII), mikä muutti hydrofobiset PSi-partikkelit hydrofiilisemmiksi. Päällystäminen myös suojasi PSi-partikkeleita ennenaikaiselta lääkeaineen vapautumiselta matalassa pH:ssa; kun ympäristön pH nousi, myös lääkevapautuminen nopeutui. Tulosten perusteella HFBII-päällystetyt PSi-pohjaiset lääkekantajat paransivat materiaalin hydrofiilisyyttä, biologista yhteensopivuutta ja pH-herkkyyttä, mitä voidaan käyttää hyväksi erilaisissa lääkeannosteluun liittyvissä sovellutuksissa. Yhteenvetona väitöskirjan tuloksista voidaan todeta, että biologisesti hyvin yhteensopivat mesohuokoiset silikonipohjaiset mikro- ja nanopartikkelit soveltuvat erinomaisesti niukkaliukoisten lääkemolekyylien liukoisuusnopeuden parantamiseen. Lääkeannostelua ja biologista yhteensopivuutta voidaan edelleen helposti parantaa ja säädellä partikkelien pintaa muokkaamalla. Hydrofobiini-proteiinien kanssa suoritetut PSi-partikkelien biofunktionalisoinnit mahdollistavat lääkekantajan pH-herkkyyteen ja mukoadhesiivisuuteen perustuvan säädellyn lääkeannostelun

Biopolymeric Scaffold obtained by Electrospinning for Intima Vessel regeneration

Atherosclerosis is the main cause of death in Western countries for coronary heart disease or stroke; the disease process is progressive and typically starts at an early age and is expressed clinically during the middle and old age. It's a multifactorial disease that, to become clinically manifest, requires the formation of a plaque fibro-lipid within the wall of an artery that reduces blood flow. The formation of advanced atherosclerotic lesions is the consequence of three processes: 1) the accumulation of lipids, mainly cholesteryl esters and free cholesterol, in the space subendothelium of the arteries; 2) the establishment of an inflammatory infiltration of lymphocytes and macrophages, engulfing lipids accumulated, they become foam cells (foam cells); 3) migration and proliferation of smooth muscle cells (smooth muscle cells – SMC) Despite the term "atherosclerosis" derives from the greek ãthere, "mush" and sclerosis,"hardening",it is important to emphasize that the injury could be a great variability in tissue formed because of the prevalence of each of the three processes.Consequently, some atherosclerotic lesions appear predominantly dense and fibrous, others can contain large amounts of lipid and necrotic debris, while most have combinations and variations of each of these characteristics. The distribution of lipids and connective tissue within the lesions determines the stability, the ease to rupture and thrombosis, with consequent clinical effects. The purpose of the following doctoral work is part of the European project "THE GRAIL" ( Tissue in Host Engineering Guided Regeneration of Arterial Intimal Layer Grant nr 278557) whose purpose is to regenerate diseased arteries by removing the inner Atherosclerotic plaque and substituting it with a temporary scaffold referred to as Synthetic Intimal Layer(SIL),that will lead to the regeneration of an intimal layer. In particular, the project will be focused on the realization of a selectively porous, resorbable and preshaped scaffold made by Electrospinning, based on recombinant Elastin Like Polymers and a copolymer made by conjugation of ELP with silk. This two types of scaffold will be deployed once in contact with healthy medial layer of arterie as a new internal elastic lamina

Towards rational design of peptides for selective interaction with inorganic materials

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.MIT Science Library copy: printed in leaves.Also issued printed in leaves.Includes bibliographical references (p. 127-141).Utilizing molecular recognition and self-assembly, material-specific biomolecules have shown great promise for engineering and ordering materials at the nanoscale. These molecules, inspired from natural biomineralization systems, are now commonly selected against non-natural inorganic materials through biopanning random combinatorial peptide libraries. Unfortunately, the challenge of studying the biological inorganic interface has slowed the understanding of interactions principles, and hence limited the number of downstream applications. This work focuses on the facile study of the peptide-inorganic interface using Yeast Surface Display. The general approach is to use combinatorial selection to suggest interaction principles followed by rational design to refine understanding. In this pursuit, two material groups-II-VI semiconductors and synthetic sapphire (metal oxides)-are chosen as inorganic targets due to their technological relevance and ease of study. First, yeast surface display (YSD) was established as a broadly applicable method for studying peptide-material interactions by screening a human scFv YSD library against cadmium sulfide (CdS), a II-VI semiconductor. The presence of multiple histidine residues was found to be necessary for mediating cell binding to CdS. As a follow-up, a systematic screen with yeast displayed rationally designed peptides was performed on a panel of II-VI semiconductors and gold. Cell binding results indicated that peptide interaction was mediated by a limited number of amino acids that were influenced by locally situated residues. Interpretation of the results facilitated design of new peptides with desired material specificities. Next, the nature of peptide/metal oxide binding interface was interrogated using sapphire crystalline faces as model surfaces.(cont.) Biopanning a random peptide YSD library and subsequent characterization of the identified binding partners revealed the importance of multiple basic amino acids in the binding event. Study of rationally designed basic peptides revealed a preference for those amino acids to be spaced in such a manner that maximized simultaneous interaction with the surface. Fusing peptides to maltose binding protein (MBP) allowed for quantitative affinity measurement with the best peptides having low nanomolar equilibrium dissociation constants. Finally, peptides were demonstrated as facile affinity tags for protein immobilization in micro-patterning and biosensor assays.by Eric Mark Krauland.Ph.D

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

Thermal-AFM under aqueous environment

The aim of this thesis is to describe the work developing and demonstrating the use of Scanning Thermal Microscopy (SThM) in an aqueous electrically conductive environment for the first time. This has been achieved by using new instrumentation to allow conventional SThM probes to measure and manipulate the temperature of non-biological and biological samples. For the latter, the aqueous environment is crucial to allow in-vitro experimentation, which is important for the future use of SThM in the life sciences. SThM is known to be a powerful technique able to acquire simultaneous topographic and thermal images of samples. It is able to measure the microscopic thermal properties of a surface with nanoscale spatial resolution. However, SThM has traditionally been limited to use in vacuum, air and electrically inert liquids. The aqueous Scanning Thermal Microscopy (a-SThM) described in this thesis is an entirely novel technique that opens up a new field for thermal-AFM. The first challenge addressed in this work was the adaptation of a commercial Multimode Nanoscope IIIa AFM to permit electrical access to a SThM probe completely immersed in aqueous solutions. By employing a newly designed probe holder and electronic instrumentation, the probe could then be electrically biased without inducing electrochemical reactions. This approach permitted conventional microfabricated thermal probes to be operated whilst fully immersed in water. This innovation allowed SThM measurements under deionized (DI) water to be performed on a simple solid sample (Pt on Si3N4) and the results compared with in-air scans and accurate 3D Finite Element (FE) simulations. Once the validity of the technique was proven, its performance was investigated, including crucially the limit of its thermal-spatial resolution; this was investigated using nanofabricated solid samples (Au on Si3N4) with well-defined features. These results were compared to the FE model, allowing an understanding of the mechanisms limiting resolution to be developed. In order to demonstrate the advantages granted by the water’s superior thermal conductivity compared to air or other liquids, non-contact thermal images were also acquired using the same samples. The final part of this thesis was focused on extending SThM into the biological area; a completely new field for this technique. New results are presented for soft 4 samples: I-collagen gel and collagen fibrils, which were thermally manipulated using a self-heated SThM probe. This successfully demonstrated the possibility of using heat to alter a biological sample within a very well localised area while being operated for long time in an aqueous environment. The difference in force response originated from the AFM scans with different levels of self-heating further proved the robustness of the technique. Finally, the technique was employed to study MG-63 living cells: The SThM probe was left in contact with each cell for a pre-determined period of time, with and without self heating. The results demonstrated that only the heated cells, directly beneath the probe tip died, tallying with the highly localised temperature gradient predicted by FE analysis

Prostate Cancer Molecular Aspects to Direct Visualization Utilizing a Bioreactor

Prostate cancer is the most common cancer in males, and the second leading cause of cancer deaths in American men. Most of the mortality associated with this disease is a result of widespread dissemination of tumors cells from the primary tumor mass. In order for metastasis to occur, the cancer cell must overcome multiple barrier which include development, neovascularization, intravastion, adherence or attachment, extravasation, and ectopic growth. As dissemination from the primary tumor mass is a rate-limiting step during metastasis, tumor cells undergo an epithelial to mesenchymal transition (EMT) to acquire enhanced invasiveness and increased motility. A key step within EMT is a loss of cadherin mediated cell-cell adhesion. Unfortunately, current understanding of the regulatory mechanism of this decreased cell-cell adhesion is poorly understood. Herein this work utilizes the LHRH antagonist Cetrorelix to investigate the regulation of E-cadherin expression in invasive prostate cancer cells. We provide direct evidence that E-cadherin expression can be reinstated upon abrogation of EGFR signaling via LHRH antagonist Cetrorelix or specific inhibitors of EGFR signaling thereby limiting the invasiveness of these cells. In concert, we developed a microscale liver perfusion culture system that provides a tissue-relevant environment to assess metastasis behavior of human prostate cancer cell line DU-145 in the liver capillary bed as a model system. This system offers the currently unavailable features of real time observation of in vivo microenvironment with the manipulation of in vitro cultures. Within this system we were able to observe three dimensional growth and invasion of prostate cancer cells juxtaposed to hepatic tissue, revealing an exceptionally defined cell border at the interface of prostate cancer cells and hepatic tissue. Although not completed defined within this system, we hypothesize that exists heterotypic cell-communication between prostate cancer cells and hepatocytes.The very distinct cell border observed within our liver microreactor, coupled with our previous findings of reexpression of E-cadherin expression lead us to investigate the involvement of E-cadherin in this heterotypic communication. Consequently, prostate cancer cells utilize E-cadherin at the point of initial adherence to parenchymal hepatocytes (heterotypic interaction) and throughout the development of the metastatic tumor mass (homotypic interaction). Our observed expression pattern of E-cadherin has not been reported before. These findings constitute a new paradigm in the adhesiveness or lack there of in cancer cells during tumor invasion. The differentiation or redifferentiation (EMT) of the cancer cell during the pathophysiological events of metastasis is likely a characteristic of adaptability to the microenvironment. The term epithelial mesenchymal transition (EMT) only summates the dedifferentiation of epithelial cells to escape the primary tumor, although we have provided evidence of phenotypic reversion. Therefore we provide the impetus that Epithelial Mesenchymal Transition (EMT) should be renamed "Meschenymal Epithelial reverting Transition (MErT)" to underscore the dynamics of the cancer cell progression

SURFACE ENABLED LAB-ON-A-CHIP (LOC) DEVICE FOR PROTEIN DETECTION AND SEPARATION

Sensitive and selective chemical/biological detection/analysis for proteins is essential for applications such as disease diagnosis, species phenotype identification, product quality control, and sample examination. Lab-on-a-chip (LOC) device provides advantages of fast analysis, reduced amount of sample requirements, and low cost, to magnificently facilitate protein detection research. Isoelectric focusing (IEF) is a strong and reliable electrophoretic technique capable of discerning proteins from complex mixtures based on the isoelectric point (pI) differences. It has experienced plenty of fruitful developments during previous decades which has given it the capability of performing with highly robust and reproducible analysis. This progress has made IEF devices an excellent tool for chemical/biological detection/analysis purposes. In recent years, the trends of simple instrument setting, rapid analysis, small sample requirement, and light labor intensity have inspired the LOC concept to be combined with IEF to evolve it into an “easily-handled chip with hours of analysis” from the earlier method of “working with big and heavy machines in a few days.” Although IEF is already a mature technique being applied, further LOC-IEF developments are still experiencing challenges related to its limitations such as miniaturizing the device scale without harming the resolving/discerning ability. With the facilitation of newly technologically advanced/improved fabrication tools, it is completely possible to address challenges and approach new limits of LOC-IEF. In this dissertation, a surface enabled printing technique, which can transfer liquid to a surface with prescribed patterns, was firstly introduced to IEF device fabrication. By employing surface enabled printing, a surface enabled IEF (sIEF) device running at a scale of 100 times smaller than those previously reported was designed and fabricated. Commercial carrier ampholytes (PharmalyteTM) with different pH range were engaged to generate a continuous pH gradient on sIEF device. Device design and optimized fabrication conditions were practically investigated; establishment of pH gradient was verified by fluorescent dyes; dependencies of electric field strength and carrier ampholytes concentration were systematically examined. To further optimize the sIEF system, dependencies of surface treatment and additive chemicals were explored. Fluorescent proteins and peptides were tested for the separation capability of sIEF. Finally, the well optimized sIEF system was used as a tool for real protein (hemoglobin variants and monoclonal antibody isoforms) separations. Hemoglobin variants test results revealed that sIEF is capable of separating amphoteric species with pI difference as small as 0.2. Monoclonal protein tests demonstrated the capability of sIEF to be a ready-to-use tool for protein structural change monitoring. In conclusion, this new sIEF approach has lower applied voltages, smaller sample requirements, a relatively quick fabrication process, and reusability, making it more attractive as a portable, user-friendly platform for qualitative protein detection and separation

Soft and flexible material-based affinity sensors

Recent advances in biosensors and point-of-care (PoC) devices are poised to change and expand the delivery of diagnostics from conventional lateral-flow assays and test strips that dominate the market currently, to newly emerging wearable and implantable devices that can provide continuous monitoring. Soft and flexible materials are playing a key role in propelling these trends towards real-time and remote health monitoring. Affinity biosensors have the capability to provide for diagnosis and monitoring of cancerous, cardiovascular, infectious and genetic diseases by the detection of biomarkers using affinity interactions. This review tracks the evolution of affinity sensors from conventional lateral-flow test strips to wearable/implantable devices enabled by soft and flexible materials. Initially, we highlight conventional affinity sensors exploiting membrane and paper materials which have been so successfully applied in point-of-care tests, such as lateral-flow immunoassay strips and emerging microfluidic paper-based devices. We then turn our attention to the multifarious polymer designs that provide both the base materials for sensor designs, such as PDMS, and more advanced functionalised materials that are capable of both recognition and transduction, such as conducting and molecularly imprinted polymers. The subsequent content discusses wearable soft and flexible material-based affinity sensors, classified as flexible and skin-mountable, textile materials-based and contact lens-based affinity sensors. In the final sections, we explore the possibilities for implantable/injectable soft and flexible material-based affinity sensors, including hydrogels, microencapsulated sensors and optical fibers. This area is truly a work in progress and we trust that this review will help pull together the many technological streams that are contributing to the field

Design of Engineered Biomaterial Architectures Through Natural Silk Proteins

Silk proteins have provided a source of unique and versatile building blocks in the fabrication of biomedical devices for addressing a range of applications. Critical to advancing this field is the ability to establish an understanding of these proteins in their native and engineered states as well as in developing scalable processing strategies, which can fully exploit or enhance the stability, structure, and functionality of the two constituent proteins, silk fibroin and sericin. The research outlined in this dissertation focuses on the evolution in architecture and capability of silks, to effectively position a functionally-diverse, renewable class of silk materials within the rapidly expanding field of smart biomaterials. Study of the process of building macroscopic silk fibers provides insight into the initial steps in the broader picture of silk assembly, yielding biomaterials with greatly improved attributes in the assembled state over those of protein precursors alone. Self-organization processes in silk proteins enable their aggregation into highly organized architectures through simple, physical association processes. In this work, a model is developed for the process of aqueous behavior and aggregation, and subsequent two-dimensional behavior of natural silk sericin, to enable formation of a range of distinct, complex architectures. This model is then translated to an engineered system of fibroin microparticles, demonstrating the role of similar phenomena in creating autonomously-organized structures, providing key insight into future “bottom up” assembly strategies. The aqueous behavior of the water-soluble silk sericin protein was then exploited to create biocomposites capable of enhanced response and biocompatibility, through a novel protein-template strategy. In this work, sericin was added to the biocompatible and biodegradable poly(amino acid), poly(aspartic acid), to improve its pH-dependent swelling response. This work demonstrated the production of a range of porous scaffolds capable providing meaningful response to environmental stimuli, with application in tissue engineering scaffolds and biosensing technologies. Finally, to expand the capabilities of silk proteins beyond process-driven parameters to directly fabricate engineered architectures, a method for silk photopatterning was explored, enabling the direct fabrication of biologically-relevant structures at the micro and nanoscales. Using a facile bioconjugation strategy, native silk proteins could be transformed into proteins with a photoactive capacity. The well-established platform of photolithography could then be incorporated into fabrication strategies to produce a range of architectures capable of addressing spatially-directed material requirements in cell culture and further applications in the use of non-toxic, renewable biological materials