Dissertations / Theses on the topic 'Bioresponsive'

Polyethyleneglycol (PEG) conjugates of peptides, proteins and an aptamer are in routine clinical use as first generation nanomedicines. Here a new family of polymer therapeutics based on PEG conjugates containing a coiled-coil peptide motif as a molecular switch are proposed. The coiled-coil motif is adopted by many naturally occurring proteins/peptides, including transcription factors key to cancer progression (E2F1/AP-1) and Ebola virus proteins (VP35/GP2). These were chosen as the first targets, however there is potentially a much wider role for this novel family of therapeutics. First studies selected coiled-coil motif peptide sequences (using computational prediction software and published literature) that were then synthesised using a solid phase approach, purified and characterised. To facilitate subsequent PEGylation, peptides were engineered to include an N-terminal cysteine residue. mPEG-maleimide (-5,500 g mol 1) was then conjugated site-specifically via the cysteine thiol. A purification method optimised using cation-exchange chromatography enabled the removal of both unreacted mPEG-maleimide and free peptide purity was > 95 % for each conjugate. Proof of concept was obtained with mPEG-FosWc, which was designed to inhibit coiled-coil heterodimerisation of native c-Jun and c-Fos proteins (AP-1). 1H, 15N HSQC spectroscopy confirmed target hybridisation of heterodimeric coiled-coils FosWc : c-Jun and mPEG-FosWc : c-Jun. In addition, both NMR and CD spectroscopy showed that both heterodimers adopted very similar structures under physiological conditions, irrespective of the presence or absence of PEG. Further studies using fluorescently labelled conjugates investigated cellular uptake in MCF-7 cells, and biological activity was assessed using the MTT assay with and without the use of a cationic transfection reagent. These studies demonstrate the potential of mPEG-coiled-coil motifs as therapeutic agents. However, demonstrating reproducible biological activity was not possible with the intracellular targets. Investigating the biological activity of the conjugates designed to target the extracellular Ebola virus fusion proteins remains an exciting prospect.

Bioresponsive hydrogels are emerging with technological significance in targeted drug delivery, biosensors and regenerative medicine. The design challenge is to effectively link the conferred biospecificity with an engineered response tailored to the needs of a particular application. Moreover, the fundamental phenomena governing the response must support an appropriate dynamic range, limit of detection and the potential for feedback control. The design of these systems is inherently complicated due to the high interdependency of the governing phenomena that guide sensing, transduction and actuation of the hydrogel. The objective of the dissertation is to review the current state of bioresponsive hydrogel technology and introduce a method of extending the technology through integrated control loops; explore fundamental phenomena which affect ion transport within biomimetic hydrogels; and investigate, via in silico studies, the fundamental design parameters for the implementation of a feedback control loop within a bioresponsive hydrogel.

In one study, effects of valence number, temperature and polymer swelling on release profiles of monovalent potassium and divalent calcium ions elucidates mechanistic characteristics of polymer interactions with charged species. For comparison, ions were loaded during hydrogel formulation or loaded by partitioning following construct synthesis. Using the Korsmeyer-Peppas release model, the diffusional exponents were found to be Fickian for pre- and post-loaded potassium ions while preloaded calcium ions followed an anomalous behavior and postloaded calcium ions followed Case II behavior. Results indicate divalent cations interact through cation-polyelectrolyte anion complexation while monovalent ions do not interact with the polymer. Temperature dependence of potassium ion release was shown to follow an Arrhenius relation and calcium ion release was temperature independent.

In another study, data generated from the previous Chymotrypsin system is used to build and validate a finite element model. The model provides insight into key engineering parameters for the design of an enzymatically actuated, feedback controlled release. A drug delivery platform comprising a biocompatible, bioresponsive hydrogel and possessing a covalently tethered peptide-inhibitor conjugate was engineered to achieve stasis, via a closed control loop, of the external biochemical activity of the actuating enzyme. The FEM model was used to investigate the release of a competitive protease inhibitor, MAG283, via cleavage of Acetyl-Pro-Leu-Gly|Leu-MAG-283 by MMP-9 in order to achieve targeted homeostasis of MMP-9 activity, a goal for the treatment of chronic wound pathophysiology. It was found the key engineering parameters for the delivery device are the radii of the hydrogel microspheres and the concentration of the peptide-inhibitor conjugate loaded into the hydrogel.

Homeostatic drug delivery, where the focus turns away from the drug release rate and turns towards achieving targeted control of biochemical activity within a biochemical pathway, is an emerging approach in drug delivery methodologies for which the potential has not yet been fully realized. By understanding mechanistic phenomena and key engineering parameters for design, advancements in bioresponsive hydrogels will continue to produce novel technologies in biomedical applications.

The long-term stability and brittle nature of ceramic bone replacements in physiological conditions makes them prone to mechanical failure. These problems have led to the development of bioresorbable bone replacement materials. Bioresorbable biomaterials are expected to degrade at a rate which is proportional to the rate of formation of new bone tissue. In the majority of cases, however, resorption is driven by simple dissolution and so it is difficult to ensure an appropriate degradation rate for all patients. This thesis seeks to develop a material that can degrade in response to the bone formation process, thus linking implant resorption to tissue formation. We have shown that this can be achieved by linking implant resorption to a biological stimulus, such as the enzyme alkaline phosphatase (ALP), which is found on the surface of bone forming cells (osteoblasts). ALP causes bone mineralisation by removing the pyrophosphate (P\(_2\)O\(_7\)\(^4\)\(^-\)) ion, a known inhibitor to calcium phosphate formation. By removing the P\(_2\)O\(_7\)\(^4\)\(^-\) P2O74- ions from solution the dissolution of calcium pyrophosphate ((Ca\(_2\)P\(_2\)O\(_7\)\(^4\)\(^-\)) crystals were accelerated in accordance with Le Chetalier's principle. We demonstrated that for this accelerated dissolution to occur, the ALP did not require access to the crystal surface. This is contrary to previous work which suggested that CPPD dissolution occurred as a result of ALP cleaving the crystal surface. Bulk (Ca\(_2\)P\(_2\)O\(_7\)\ ceramics were successfully produced by sintering brushite cement at temperatures ≥ 400°C, the dissolution of which could accelerated in the presence of ALP but was heavily dependent on material specific surface area. The process of sintering limits the possibility of producing biomaterials of complex morphology; therefore the final part of this thesis involved the fabrication of ((Ca\(_2\)P\(_2\)O\(_7\) ceramic using stereolithography.

Tuberculosis is one of the most prevalent infectious diseases globally due to the successful survival mechanisms displayed by Mycobacterium tuberculosis (Mtb). Mtb primarily infects alveolar macrophages (AMs) and is able to live intracellularly for extended periods of time due to a number of virulence factors which inhibit the antibacterial mechanisms of the AMs. This aspect of the Mtb life cycle means TB treatments suffer from poor bioavailability and efficacy. Additionally, the rise in resistant strains of Mtb means the use of higher doses and the use of alternative second and third line drugs which increase the risk of systemic toxicity. Drug encapsulation is a novel approach that can provide more favourable drug pharmacokinetics and pharmacodynamics. The aim of this project was to develop a liposomal drug delivery system to target Mtb infected alveolar macrophages. The system involved the encapsulation of two drugs; the antibiotic gatifloxacin (GFLX) and Mtb virulence factor inhibitor CV7. The hypothesis was that the two different antibacterial mechanisms would work in synergy and increase the efficacy of the treatment. AM targeting and receptor-mediated endocytic uptake was encouraged by the presence of a ligand attached to the surface of the liposome. Furthermore a pH-sensitive release mechanism was to be incorporated into the liposome to encourage the release of the encapsulated drugs in the vicinity of the intracellular bacteria. The intention was to produce a drug delivery system to enable a TB therapy regime of fewer, lower doses to increase compliance and reduce systemic toxicity by increasing efficacy through improved bioavailability. GFLX was successfully encapsulated using a weak base active loading method. To establish encapsulation efficiency, a homogeneous fluorescence assay able to quantify intra- and extra-liposomal gatifloxacin simultaneously was developed. pH-sensitive release of the payload could be achieved using a pH-sensitive peptide with a novel design based on chimeric structure, namely P3. CV7 was successfully encapsulated using a weak acid active loading method. CV7 liposomes were able to be functionalised by the incorporation of a mannose ligand on the surface of the liposome. An inhibition assay using the target enzyme of CV7, MptpB, was optimised to assess efficacy of liposomally encapsulated and released CV7. Flow cytometry and confocal microscopy studies confirmed that the liposomal formulations were internalised by the target macrophage cell line, J774a.1. Mannose liposomes conveyed superior uptake kinetics. Further confocal microscopy showed that after internalisation the liposomes entered the endolysosomal pathway and colocalised with BCG. A BCG-macrophage infection model was used to determine the intracellular efficacy of the liposomal formulations. Encapsulated CV7 displayed increased efficacy over free CV7, while encapsulation in functionalised liposomes showed better efficacy still. The encapsulation of GFLX did not increase the efficacy of GFLX and synergy between the two drugs was not achieved. In conclusion, the liposomal encapsulation of CV7 increased uptake of the drug by the target cell line and facilitated colocalisation of the drug with the target pathogen thereby increasing efficacy. Such a formulation could potentially increase bioavailability and efficacy in vivo for a more tolerable TB therapy.

Drug eluting beads (DEB) are employed in the treatment of solid hypervascularised malignant tumours by a method called trans-arterial chemoembolisation (TACE). When the microcirculation to a tumour is blocked, oxygen levels decrease to critically low levels causing the tumour to become hypoxic. Hypoxic tumours are known to be chemoresistant and send out growth factor signals leading to angiogenesis and metastasis of tumour cells to other parts of the body. Commercially available DEB are unable to respond to the conditions of hypoxia and will continue to release drug at a constant rate via ionic exchange through the hydrogel. It is therefore recognised that an avenue for improvement would be the development of novel bioresponsive DEB that are able to react to the conditions of hypoxia to overcome chemoresistance associated with the tumour cells.

PEGylation has become very popular for the generation of nanomedicines with improved protein delivery properties, despite its lack of biodegradability. Researchers usually try to maximise retained protein activity during PEGylation. However, this proof of principle study aimed to create an inactive peptide or enzyme product, using a biodegradable polymer, that would elicit minimal activity/non-specific toxicity on administration. Following triggered site-specific degradation of the polymer, the hypothesis was that protein activity could be slowly regenerated in the general circulation or localised to a specific target site. Model conjugates were synthesised by coupling dextrin degraded by amylase to trypsin and melanocyte stimulating hormone MSH, to test this concept and targeted delivery for both an enzyme and a receptor-binding ligand. Hyaluronic acid HA degraded by hyaluronidase conjugates of trypsin and ribonuclease A were also synthesised. The latter was intended to develop the possibility of designing novel anti cancer conjugates. A higher molecular weight dextrin 47,200 g/mol, 26 mol succinoylation was shown to best mask 34 trypsin activity and reinstate 58 of the activity by addition of amylase. When a HA fraction molecular weight 130,000 g/mol was prepared by acid hydrolysis and conjugated to trypsin 4 w/w, trypsin activity was masked to 6 and immediately re-instated to 24 on addition of hyaluronidase. Similarly, the dextrin-MSH conjugate reduced melanin production to 11 of the control and only restored to 33 on addition of amylase. RNase A alone was not cytotoxic up to 1 mg/mL, whereas, the HA-RNase A conjugate 0.1 mg/mL RNase A equivalent was cytotoxic in B16F10 and CV-1 cells 72 h. This work provides proof of principle for the concept of using biodegradable polymers to mask and reinstate conjugated protein activity in the presence of the appropriate enzyme 'unmasking' trigger.

Increasingly sophisticated new treatments such as trastuzumab (Herceptin ) and Bevacizumab (Avastin ) have contributed to reduced mortality from breast cancer over recent years, nevertheless 40--60 % of those affected still die from metastatic disease. Thus there remains an urgent need for novel therapies for breast cancer. As PLA2 (crotoxin) has proven anticancer activity but its use is limited by non-specific toxicity, and polymer-drug and polymer-protein conjugates are finding growing use as anticancer agents, the aim of this thesis was to explore the potential of polymer-PLA2 conjugates as a new treatment for breast cancer. Polymer conjugation has previously been shown to reduce systemic toxicity of proteins, prolong their plasma half-life and promote tumour-specific targeting by the enhanced permeability and retention (EPR) effect. First, the synthesis and characterisation methods were optimised using trypsin as a model. After these studies highlighted dextrin as the best polymer for conjugation, dextrin-PLA2 (Apis mellifera venom) conjugates were prepared. Dextrin was chosen for conjugation as it can be used to mask protein activity in the protein masked-unmasked polymer therapy (PUMPT) concept. Such conjugates retained 36 % enzyme activity compared to free PLA2, and moreover, unmasking by a-amylase degradation of dextrin regenerated full enzyme activity. However, while free PLA2 was found to be very haemolytic, dextrin-PLA2 displayed no haemolytic activity, and unmasking by a-amylase degradation of dextrin did not reinstate this activity. The conjugate displayed significant toxicity towards several tumour cell lines, including human breast cancer. Indirect evidence that epidermal growth factor receptor (EGFR) status and tyrosine kinase activity of the receptor influences PLA2-induced anti-proliferative activity were shown. Uptake studies have revealed that conjugation of dextrin to PLA2 reduces non-specific binding to breast cancer cells. In a further study, dextrin-PLA2's ability to burst DaunoXome using the polymer-enzyme liposome therapy (PELT) concept was assessed. Here, it was seen that the conjugate released liposomally encapsulated drug and the combination caused enhanced cytotoxicity in MCF-7 cells. These studies confirm the potential of dextrin-PLA2 as a novel anticancer agent and/or as trigger for liposomal drug release and highlight the feasibility of developing a candidate for further in vivo pharmacokinetic and activity studies.

Thesis (Ph. D.)--Harvard-MIT Program in Health Sciences and Technology, 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references.
The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have been held back by immunogenicity and toxicity concerns, but synthetic vectors have lagged in transfection efficiency. This thesis describes the rational design and systematic study of three classes of bioresponsive polymers for nucleic acid delivery. A central theme of the study was understanding how the structure of the polymers impacted each of the intracellular steps of delivery, rather than solely the end result. A powerful tool for efficiently quantifying endosomal escape was developed and applied to each of the material systems described. First, a linear-dendritic poly(amido amine) -poly(ethylene glycol) (PAMAM-PEG) block copolymer system previously developed in our lab was evaluated and its ability to overcome the sequential barriers of uptake, endosomal escape, and nuclear import were characterized. Next, a class of crosslinked linear polyethyleimine (xLPEI) hyperbranched polymers, which can contain disulfideresponsive linkages, were synthesized and investigated. It was demonstrated that free polymer in solution, not the presence of a functional bioresponsive domain, was responsible for the highly efficient and relatively nontoxic DNA delivery of this promising class of crosslinked polyamines. Finally, this analysis was applied to siRNA delivery by a library of amine-functionalized synthetic polypeptides. The pH-responsive secondary structure, micelle formation, and ester hydrolysis were studied prior to the discrete barrier-oriented analysis of the siRNA delivery potential of this library. It is hoped that the tools, materials, and systemic analysis of structure-function relationships in this thesis will enhance the process of discovery and development of clinically relevant gene carriers.
by Daniel Kenneth Bonner.
Ph.D.

This dissertation is aimed towards using stimuli-responsive pNIPAm-co-AAc microgels synthesized via free-radical precipitation polymerization to prepare stimuli-responsive hydrogel microlenses. Chapter 1 gives a detailed background of hydrogels, and their applications using responsive hydrogels. Chapter 2 describes the use of colloidal hydrogel microparticles as microlens elements and the fabrication method to form the hydrogel microlens arrays via Coulombic interactions. Chapter 3 shows the demonstration of tunable microlenses prepared by the method used in Chapter 2. In this chapter the microlenses are subjected to various pH and temperature in aqueous solutions. Chapter 4 describes that the microlens arrays constructed on Au nanoparticle-functionalized glass substrates by self-assembly display dramatic changes in lensing power in response to an impingent frequency-doubled Nd:YAG laser. The microlens photoswitching is highly reversible, with sub-millisecond lens switching times. Chapter 5 describes the development of bioresponsive hydrogel microlenses as a new protein detection technology. The microlens method is shown to be very specific for the target protein, with no detectable interference from nonspecific protein binding. Chapter 6 describes the use of bioresponsive hydrogel microlenses as a label-free biosensing scaffolding. These microstructures simultaneously act as the biosensors scaffolding/immobilization architecture, transducer, amplifier, and also allow for broad tunability of the analyte concentration to which the microlens is sensitive.

Multidrug-resistant Gram-negative infection is an important cause of mortality and morbidity. Management of these infections is often dependent upon “treatment of last resort” "small molecule" antibiotics which suffer from significant toxicity and an indiscriminate volume of distribution. The aim of this study was to develop a prototype polymer-antibiotic conjugate that may be customised by polymer modification and binding chemistry to afford selective, controlled release at an infected site. These studies employed the biodegradable, naturally-occurring polymer, dextrin, and a polymyxin antibiotic, colistin, as the first model combination. Physicochemical characterisation of a library of succinoylated dextrins and dextrin-colistin conjugates demonstrated that conjugation of dextrin to colistin was feasible and reproducible, resulting in masking of colistin's amino groups through incorporation in peptide bonds. Exposure to physiological �-amylase activity resulted in controlled degradation of the dextrin component, leading to sustained colistin release. Following exposure of the conjugates to physiological concentrations of �-amylase, minimally-modified, low molecular weight dextrin, conjugated to colistin, demonstrated significantly earlier, maximal release of colistin and subsequent reinstatement of antimicrobial activity. At maximum unmasking, the lead conjugate reported equivalent antimicrobial activity to the current clinical formulation of colistin (Colimycin®)against a range of MDR organisms including: A. baumannii, K. pneumoniae and E. coli. A static two-compartment dialysis bag model was developed under infinite sink conditions, which demonstrated that the conjugates were able to suppress bacterial growth over a significantly greater duration than colistin sulfate. Ex vivo studies of infected human wound fluid samples confirmed that colistin could be readily liberated from conjugate in infected sites. Significantly higher amylase activity in these wound fluid samples supported the notion of locally-triggered, enzymatically-mediated unmasking. An in vivo intravenous, pharmacokinetic model in rats demonstrated the increased half-life associated with conjugation and succinoylation. Moreover,the dextrin-colistin conjugates were better tolerated than colistin sulfate at higher concentrations. These studies have demonstrated the feasibility of developing this new class of “nanoantibiotics” and highlighted their potential usefulness as bioresponsive nanomedicines for the treatment of MDR Gram-negative infection.

Anisotropic metal nanoparticles have been successful used in a wide range of biomedical applications, such as diagnostics and therapy, because of their unique optical and electronic properties. Even though there is a wide range of morphologies synthetically available, the understanding of the mechanism behind the anisotropic growth of the nanoparticles is still incomplete. Regarding their application in diagnostics, metallic nanoparticle-based biosensors are facing new challenges, such as the discovery of novel circulating cancer biomarkers (e.g. cell-free DNA), which require sensitivities that cannot be achieved by traditional approaches. The research of this thesis covers current challenges in three specific areas found in the interface between bio- and nanoscience. (1) Colloidal synthesis, where a novel synthesis of gold nanorods (AuNRs) has been developed by the addition of Hofmeister salts into the growth solution. The thorough characterization of the surfactant micelles in the growth solution provided a better understanding of the role of the surfactant as symmetry breaking component in the anisotropic growth. (2) Diagnostics and disease prevention, where two new metal nanoparticle-based biosensors have been developed. The first one exploits the control of a photoresponsive fluid over the dimensions of anisotropic gold nanoparticles for UV exposure sensing and erythema prediction, where the nanoparticles are synthesized and used for sensing purpose at the same time. The second one is a AuNR-based biosensor for circulating cell-free DNA with inverse sensitivity, i.e. the lower the analyte concentration, the higher the response intensity. (3) Bio-inspired materials, where a hybrid system made of AuNR-DNA has been designed to study the sequence-specific binding between transcription factors and DNA. This system has been further expanded to build a versatile multi-logic gate platform, capable of performing six different logic operations. Finally, the use of alternative plasmonic nanomaterials for sensing and bio-inspired materials has also been explored.

Chronic wounds are a major financial and clinical burden causing the deaths of millions per year. Over expression of elastase is well documented as the main culprit that delays the normal wound repair process within chronic wounds. The aim of this thesis is to design a responsive chronic wound dressing based on the hydrogel polymer, PEGA (polyethylene glycol acrylamide) in the form of particles to mop-up excess elastase by exploiting polymer collapse in response to elastase hydrolytic activity within sample fluids mimicking the environment of chronic wounds. PEGA particles were functionalised with enzyme cleavable peptides (ECPs) containing charged residues. Upon cleavage the charge balance changes, causing polymer swelling and consequent elastase entrapment. The pH range of chronic wounds is reported in the range of 5.45 - 8.65. Due to its pI which is around 8.3, within this range elastase exist both in its cationic and anionic forms. To accommodate a hydrogel dressing that could selectively entrap excess elastase both in its cationic and anionic, oppositely charged ECPs were designed. In its cationic form, elastase was found to have a high preference of cleaving ECPs and penetrating into PEGA particles bearing negative charges. In contrast, in its anionic form the opposite effect was observed, wherein elastase preferred to cleave ECPs and penetrate PEGA particles bearing positive charges. The diffusion, accessibility and entrapment of elastase into functionalised PEGA particles was explored using various fluorescence microscopy techniques. Removal of the charged residue by elastase showed a reduction in particle swelling causing the pores of PEGA particles to become restricted. In this manner, cleaved PEGA particles prevented the accessibility of molecules with a molecular weight as low as 20 kDa into the cleaved PEGA particles. Since elastase has a molecular weight of 25.9 kDa the collapsing of the pores within PEGA particles entrapped elastase inside the interior of cleaved PEGA particles. In its cationic form (at pH 7.4) elastase was found to penetrate and become trapped more into both negative and positive PEGA particles compared to neutral particles. The negative particles were shown to trapped cationic elastase within 2 minutes compared to the positive particles. In contrast, the neutral particles failed to retain and encapsulate elastase as the fluorescence inside the neutral particles was found to decrease. Coinciding with these observations, after sample fluids containing elastase were treated with functionalised PEGA particles, the residual elastase activity in sample fluids was reduced more by the charged PEGA particles compared to neutral particles. The cell culture studies demonstrated that the elastase activity observed in human dermal fibroblasts (HDF) was also reduced more by the charged particles compared to the neutral particles. However, the positive particles were found to significantly reduced HDF-elastase activity compared to both the negative and neutral PEGA particles. Overall, this thesis exemplifies that on the basis of charge selective cleaving of ECPs coupled to PEGA particles can be exploited to selectively remove excess proteases such as elastase from sample fluids mimicking the environment of chronic wounds.

Effective treatment of ocular diseases presents a formidable task, dually attributed to their nature, and the presence of the ocular barriers. This was exemplified in the presented review of developments in ocular drug delivery systems. Conceptualization of novel polymeric systems with intelligent (e.g. stimulus-responsive) mechanisms and advances in nanotechnology are at the forefront of achieving directed and controlled delivery for treating vision-threatening diseases. It was thus the pertinent goal of this investigation to assimilate these observations in the design of an intelligent ocular drug delivery system. The design of an autofeedback polymeric platform, employing biodegradable polymers is exemplified through the implementation of two-way communication systems between our bodies and the delivery platform to create innovative drug delivery systems that recognize a biochemical process that is characteristic of a disease, and then responding via drug release. To achieve this intelligence in design, the delivery platform was based on novel stimulus-responsive polymeric materials. The concept of an autofeedback polymeric platform was intrinsically implemented in the design of an intelligent intraocular implant - called the I3 - employing inflammation-responsive polymers, having application as a smart release system capable of delivering controlled therapeutic levels of anti-inflammatory and/or antibiotic drug/s for posterior segment disorders of the eye in response to inflammation and infection. Inner and outer bioresponsive polymeric matrices (BPMs) were designed that released the incorporated anti-inflammatory and antibiotic in a fashion responsive to a stimulus, such as the highly reactive intermediates including hydroxyl radicals (OH.) that are released from activated leukocytes both in vitro and during acute and chronic intraocular inflammatory reactions in vivo (Hawkins and Davies, 1996). The first step in developing the intelligent device implicated design of an anti-inflammatory nanosystem (NS) with satisfactory size and surface properties, adequate permeation potential, uptake by inflamed cells, low cellular toxicity, and enhanced anti-inflammatory effect availed to the incorporated drug. A composite lipoidal-polymeric NS was developed (Lipo-Chit-PCL NS) and compared to a purely polymeric NS. The NS was ultimately enclatherated as a NS-polymer superlattice, forming the inner BPM of the I3. The designed composite lipoidal-polymeric NS attested its significant potential for selective drug delivery to inflamed tissues, demonstrating significantly enhanced tissue permeation, cell uptake, and anti-inflammatory activity compared to an indomethacin suspension. Subsequent molecular modeling revealed that the composite NS displayed an enhanced lipophilicity and superior cellular internalization efficiency. The preferred NS was subsequently selected for optimization via a Plackett-Burman Statistical Design Method. Design of the NS was proceeded by development and optimization of the inner and outer BPMs, being the stimulus-responsive component of the device, via implementation of a novel methodology for simultaneous design of the two intimately crosslinked matrices. Inflammation-responsive polymers such as hyaluronic acid, alginate, poly(acrylic) acid, and chitosan, were ultimately selected for design of the I3drug delivery system. Intensive device optimization was then undertaken, first employing a Response Surface Methodology, embodied by the Box-Behnken Design, ensued by Artificial Neural Networks. Characterization of the drug release kinetics from the optimized I3 is pivotally provided, as well as molecular modeling. The reagent (N-hydroxysuccimide, NHS) and catalyst employed (aluminium chloride, AlCl3) had a significant or notable effect on the mean dissolution time of indomethacin under normal and pathological conditions, respectively (p=0.048; p=0.058). The interaction between the inflammation-responsive hyaluronic acid and carbodiimide crosslinker emanated in a significant effect on the change in mean dissolution time of indomethacin from normal to inflammatory conditions (p=0.050). Subsequent execution of ANN with further training of the data confirmed the adequacy of the design. Analysis of the drug release kinetics from the optimum I3 under both normal and pathological conditions was in coherence with the anticipated behavior of an inherently bioresponsive device. Molecular simulations generated provided clear evidence for the catalytic effect of the hydroxyl radicals, specifically in hyaluronic acid hydrolysis. It was imperative to elucidate the intricate modus operandi of the optimized I3. The intricately crosslinked polymeric system comprising the I3 responds at an innate level predicted by its molecular make-up to inflammatory conditions as indicated by the results of the rheological analysis, MRI and SEM imaging. FTIR explicated the formation of pivotal intra- and intermolecular bonds within and between the inflammation-responsive polymers of the I3, while TMDSC confirmed the extent to which the composite polymeric system had altered from its native consituents to form a device of the desired functionality. Tensile analysis provided an indication of the overall mechanical performance of the implant. The porosity ascertained for both the inner and outer BPMs was a predictor of the overall internal architecture and potential drug release characteristics of the BPMs comprising the I3, as were critical morphological changes, visualized via SEM and interpreted via image analysis displayed during device erosion. Furthermore, molecular mechanics simulations were carried out to model the interaction between the polymeric components of the inner and outer BPM and confirmed the formation of a ‘secure-fit’ dual polymeric matrix system by highlighting the anticipated interconnectivity between the inner and outer BPM of the I3. The in vivo performance of the device was assessed to further provide a convincing argument as to the ocular suitability and overall contrasting performance under normal and inflammatory conditions. Histological assessment was key to predicting significant inflammatory changes, as well as reductions in inflammation initiated by the I3. Analysis of ocular drug levels under normal and inflammatory conditions was undertaken for correlation with in vitro results, for ultimate establishment of an in-vitro-in-vivo correlation (IVIVC). The device was well-tolerated following implantation in the rabbit eye. Investigations of drug concentrations attained and device erosion were a good indication that the I3 expresses bioresponsive capabilities in vivo. There was enhanced release of both drugs in the inflamed rabbit eye even after 7 days (the maximum period in which the induced inflammation was permitted to ensue), with indomethacin levels of 0.749±0.126μg/mL and 1.168±0.186μg/mL, and ciprofloxacin levels of 1.181±0.150μg/mL and 6.653±0.605μg/mL being attained in the normal and inflamed eye, respectively. At 28 days in the normal eye, concentrations of indomethacin detected were only 0.564±0.111μg/mL and those of ciprofloxacin were 1.226±0.209μg/mL. Furthermore, the enhanced erosion of the I3 in the inflamed eye is also exemplified, with the I3 eroding 1.504±0.505% in the normal eye and 22.609±2.421% in the inflamed eye after 7 days; and only reaching 13.830±1.010% erosion after 28 days in the normal rabbit eye. Elaboration of the IVIVC undertaken for both indomethacin and ciprofloxacin approached or attained a Level A correlation, respectively, and provided further evidence for the feasibility of the I3 and for advancement of this concept toward application in a clinical setting. Establishment of such a correlation for the inflamed rabbit eye would be the main consideration in prospective investigations.

Progressive loss of skeletal muscle mass, strength and function poses a major threat to independence and quality of life, particularly in the elderly. To date, sarcopenia therapy consists of resistance exercise training in combination with protein supplementation due to the limited efficacy of available pharmacological options in counteracting the effects of muscle wasting. Therapeutic intervention with growth factors including insulin-like growth factor I (IGF-I) or inhibitors of myostatin  a potent suppressor of myogenesis  hold potential to rebalance the altered activity of anabolic and catabolic cytokines. However, dosing limitations due to acute side effects and disruptions of the homeostasis have so far precluded clinical application. Intending to provide a therapy with a superior safety and efficacy profile by directing drug release to inflamed tissue and minimizing off-target activity, we designed bioresponsive delivery systems for an anti-catabolic peptide and anabolic IGF-I responding to local flares of muscle wasting. In Chapter I, current concepts for bioorthogonal conjugation methods are discussed and evaluated based on various drug delivery applications. With a focus on protein delivery, challenges and potential pitfalls of each chemical and enzymatic conjugation strategy are analyzed and opportunities regarding their use for coupling of biomolecules are given. Based on various studies conjugating proteins to polymers, particles and biomaterials using different site-directed approaches, the chapter summarizes available strategies and highlights certain aspects requiring particular consideration when applied to biomolecules. Finally, a decision process for selection of an optimum conjugation strategy is exemplarily presented. Three of these bioorthogonal coupling reactions are applied in Chapter II detailing the potential of site-directed conjugation in the development of novel, homogenous drug delivery systems. The chapter describes the design of a delivery system of a myostatin inhibitor (MI) for controlled and local release counteracting myositis flares. MI release from the carrier is driven by increased matrix metalloproteinase (MMP) levels in compromised muscle tissues cleaving the interposed linker, thereby releasing the peptide inhibitor from the particulate carrier. Release experiments were performed to assess the response towards various MMP isoforms (MMP-1, -8, -9 and -13) – as upregulated during skeletal muscle myopathies – and the release pattern of the MI in case of disease progression was analyzed. By selection of the protease-sensitive linker (PSL) showing variable susceptibilities to proteases, release rates of the MI can be controlled and adapted. Immobilized MI as well as released MI as response to MMP upregulation was able to antagonize the effects of myostatin on cell signalling and myoblast differentiation. The approach of designing bioresponsive protein delivery systems was also applied to the anabolic growth factor IGF-I, as described in Chapter III. Numerous studies of PEGylated proteins or peptides reveal, that successful therapy is challenged by safety and efficacy issues, as polymer attachment considerably alters the properties of the biologic, thereby jeopardizing clinical efficacy. To this end, a novel promising approach is presented, intending to exploit beneficial effects of PEGylation on pharmacokinetics, but addressing the pharmacodynamic challenges by releasing the protein upon entering the target tissue. This was realized by integration of a PSL between the PEG moiety and the protein. The soluble polymer conjugate was produced by site-directed, enzymatic conjugation of IGF-I to the PSL, followed by attachment of a 30 kDa-PEG using Strain-promoted azide-alkyne cycloaddition (SPAAC). This strategy illustrates the potential of bioorthogonal conjugation (as described in Chapter I) for generation of homogenous protein-polymer conjugates with reproducible outcome, but also emphasizes the altered protein properties resulting from permanent polymer conjugation. As compared to wild type IGF-I, the PEGylated protein showed considerable changes in pharmacologic effects – such as impaired insulin-like growth factor binding protein (IGFBPs) interactions, submaximal proliferative activity and altered endocytosis patterns. In contrast, IGF-I characteristics were fully restored upon local disintegration of the conjugate triggered by MMP upregulation and release of the natural growth factor. For successful formulation development for the proteins and conjugates, the careful selection of suitable excipients is crucial for a safe and reliable therapy. Chapter IV addresses one aspect by highlighting the chemical heterogeneity of excipients and associated differences in performance. Polysorbate 80 (PS80) is a surfactant frequently used in protein formulations to prevent aggregation and surface adsorption. Despite being widely deployed as a standard excipient, heterogeneous composition and performance entails the risk of eliciting degradation and adverse effects on protein stability. Based on a comprehensive study using different batches of various suppliers, the PS80 products were characterized regarding chemical composition and physicochemical properties, facilitating the assessment of excipient performance in a formulation. Noticeable deviations were recorded between different suppliers as well as between batches of the same suppliers. Correlation of all parameters revealed, that functionality related characteristics (FRCs) could be reliably predicted based on chemical composition alone or by a combination of chemical and physicochemical properties, respectively. In summary, this thesis describes and evaluates novel strategies for the targeted delivery and controlled release of biologics intended to counteract the imbalance of anabolic and catabolic proteins observed during aging and musculoskeletal diseases. Two delivery platforms were developed and characterized in vitro – (i) using anti-catabolic peptides immobilized on a carrier for local delivery and (ii) using soluble IGF-I polymer conjugates for systemic application. Both approaches were implemented by bioorthogonal coupling strategies, which were carefully selected in consideration of limitations, side reactions and efficiency aspects. Bioresponsive release of the active biomolecules following increased protease activity could be successfully realized. The therapeutic potential of these approaches was demonstrated using various cell-based potency assays. The systems allow targeted and controlled release of the growth factor IGF-I and anti-catabolic peptides thereby overcoming safety concerns of current growth factor therapy and thus positively impacting the benefit-risk profile of potent therapeutics. Taking potential heterogeneity and by-product concerns into account, comprehensive excipient characterization was performed and a predictive algorithm for FRCs developed, in order to facilitate formulation design and guarantee a safe and efficient therapy from start to finish
Der zunehmende Verlust an Skelettmuskelmasse, Kraft und Funktion stellt insbesondere bei Älteren eine wesentliche Gefährdung der Unabhängigkeit und Lebensqualität dar. Bislang besteht die Sarkopenie-Therapie infolge der eingeschränkten Wirksamkeit verfügbarer pharmakologischer Möglichkeiten, den Auswirkungen des Muskelschwunds entgegenzuwirken, aus einer Kombination von Krafttraining und erhöhter Proteinzufuhr. Therapeutische Intervention mit Wachstumsfaktoren wie Insulin-like growth factor (IGF-I) oder Inhibitoren von Myostatin – eines wirkungsvollen Hemmstoffes der Myogenese – bietet das Potenzial, die veränderte Aktivität der anabolen und katabolen Zytokine wieder ins Gleichgewicht zu bringen. Allerding haben Dosiseinschränkungen aufgrund akuter Nebenwirkungen und Beeinträchtigungen der Homöostase bislang eine klinische Anwendung ausgeschlossen. Mit der Absicht, eine Therapie mit besserem Sicherheits- und Wirksamkeitsprofil zu bieten, indem die Freisetzung des Wirkstoffs auf entzündetes Gewebe gelenkt wird und Aktivitäten außerhalb des Zielgewebes minimiert werden, entwickelten wir bioresponsive Freisetzungssysteme für ein antikataboles Peptid und das anabole IGF-I, die auf lokalen Ausbruch von Muskelschwund reagieren. In Kapitel I werden aktuelle Konzepte bioorthogonaler Konjugationsmethoden diskutiert und auf Basis einer Vielzahl von Drug Delivery Anwendungen beurteilt. Mit besonderem Fokus auf die Verabreichung von Proteinen werden Herausforderungen und Schwierigkeiten jeder chemischen und enzymatischen Konjugationsstrategie analysiert und Möglichkeiten im Hinblick auf ihre Verwendung für die Kopplung von Biomolekülen aufgezeigt. Auf Grundlage diverser Studien zur Verknüpfung von Proteinen mit Polymeren, Partikeln und Biomaterialien unter Verwendung verschiedener ortsspezifischer Ansätze, fasst das Kapitel vorhandene Strategien zusammen und hebt gewisse Aspekte hervor, die bei Anwendung auf Biomoleküle besondere Beachtung erfordern. Abschließend wird ein Entscheidungsprozess zur Auswahl einer optimalen Verknüpfungsstrategie exemplarisch dargestellt. Drei dieser bioorthogonalen Kopplungsreaktionen werden in Kapitel II angewendet, wodurch das Potenzial der ortsgerichteten Konjugation für die Entwicklung neuer, homogener Drug Delivery Systeme detailliert aufgezeigt wird. Dieses Kapitel beschreibt die Gestaltung eines Delivery Systems für einen Myostatin- Inhibitor (MI) für kontrollierte und lokale Freisetzung, um Myositis-Ausbrüchen entgegenzuwirken. Die Freisetzung des MI vom Träger wird durch erhöhte Konzentration an Matrix-Metalloproteinasen (MMPs) in betroffenem Muskelgewebe vorangetrieben, die durch Spaltung des dazwischen positionierten Linkers das Peptid vom Partikelträger freisetzen. Es wurden Freisetzungsexperimente durchgeführt, um die Reaktion gegenüber mehreren MMP-Isoformen (MMP-1, -8, -9 und -13), die im Verlauf von Skelettmuskelmyopathien hochreguliert sind, festzustellen, und es wurde das Freisetzungsmuster des MI im Falle einer Krankheitsprogression analysiert. Durch Auswahl der Protease-sensitiven Linker (PSL), die unterschiedliche Empfindlichkeit gegenüber Proteasen zeigen, können die Freisetzungsraten des MI kontrolliert und angepasst werden. Sowohl der immobilisierte MI, als auch der auf MMP-Hochregulation hin freigesetzte MI, waren dazu in der Lage, die Wirkungen von Myostatin auf Signaltransduktion von Zellen und Myoblastendifferenzierung aufzuheben. Das Konzept, bioresponsive Delivery Systeme für Proteine zu designen, wurde auch auf den anabolen Wachstumsfaktor IGF-I angewendet, wie in Kapitel III beschrieben wird. Zahlreiche Studien zu PEGylierten Proteinen oder Peptiden offenbaren, dass eine erfolgreiche Therapie durch Sicherheits- und Wirksamkeitsprobleme herausgefordert wird, da der Polymeranhang die Eigenschaften des biologischen Wirkstoffs beachtlich verändern und dadurch die klinische Wirksamkeit gefährden kann. Zu diesem Zweck wird ein neuer, vielversprechender Ansatz vorgestellt, mit der Absicht, die vorteilhaften Auswirkungen der PEGylierung auf die Pharmakokinetik zu nutzen, aber auch die pharmakodynamischen Herausforderungen dadurch zu adressieren, dass das Protein bei Eintritt ins Zielgewebe freigesetzt wird. Das wurde durch Einfügen eines PSL zwischen den PEG-Teil und das Protein erreicht. Das lösliche Polymerkonjugat wurde durch ortsspezifische, enzymatische Konjugation von IGF-I an den PSL hergestellt, gefolgt von Verknüpfung mit einem 30k Da-PEG unter Verwendung von kupferfreier Azid-Alkin Cycloaddition (SPAAC). Diese Strategie veranschaulicht das Potenzial der bioorthogonalen Konjugation (wie in Kapitel I beschrieben) zur Erzeugung homogener Protein-Polymer-Konjugate mit reproduzierbarem Ergebnis, aber betont auch die veränderten Proteineigenschaften, die sich aus der dauerhaften Polymerkonjugation ergeben. Verglichen mit dem Wildtyp-IGF-I zeigte das PEGylierte Protein beachtliche Veränderungen der pharmakologischen Eigenschaften, wie verminderte Interaktionen mit Insulin-like growth factor Bindungsproteinen (IGFBPs), eine submaximale proliferative Aktivität und ein verändertes Endozytosemuster. Im Gegensatz dazu wurden die Eigenschaften von IGF-I bei lokaler Spaltung des Konjugates durch MMP-Hochregulation und Freisetzung des natürlichen Wachstumsfaktors vollständig wiederhergestellt. Für eine erfolgreiche Formulierungsentwicklung der Proteine und Konjugate ist eine sorgfältige Auswahl geeigneter Hilfsstoffe für eine sichere und zuverlässige Therapie essenziell. Kapitel IV befasst sich mit einem Aspekt davon, indem die chemische Heterogenität von Hilfsstoffen und damit verbundene Unterschiede in der Leistung hervorgehoben werden. Polysorbat 80 (PS80) ist ein in Proteinformulierungen häufig verwendeter Hilfsstoff, der Aggregation und Oberflächenadsorption verhindern soll. Trotz dieser breiten Anwendung als Standardhilfsstoff birgt die heterogene Zusammensetzung und Performance Risiken, wie eine begünstigte Zersetzung und nachteilige Auswirkungen auf die Proteinstabilität. Auf Basis einer umfassenden Studie mit verschiedenen Chargen diverser Anbieter wurden die PS80 Produkte hinsichtlich ihrer chemischen Zusammensetzung und ihrer physikochemischen Eigenschaften charakterisiert, um eine Beurteilung der Hilfsstoffperformance in einer Formulierung zu ermöglichen. Auffällige Abweichungen sowohl zwischen unterschiedlichen Anbietern, also auch zwischen Chargen des gleichen Anbieters konnten verzeichnet werden. Die Korrelation aller Parameter ergab, dass funktionalitätsbezogene Eigenschaften (FRCs) auf Basis der chemischen Zusammensetzung alleine bzw. durch eine Kombination aus chemischen und physikochemischen Eigenschaften zuverlässig prognostiziert werden konnten. Zusammenfassend beschreibt und bewertet diese Dissertation neue Strategien für eine zielgerichtete und kontrollierte Freisetzung von biologischen Wirkstoffen mit der Absicht, dem Ungleichgewicht zwischen anabolen und katabolen Proteinen, welches im Laufe der Alterung und im Zuge muskuloskelettaler Erkrankungen beobachtet wird, entgegenzuwirken. Zwei Wirkstoff-Verabreichungsplattformen wurden entwickelt und in vitro charakterisiert: (i) unter Verwendung antikataboler Peptide, die für eine lokale Applikation auf einem Träger immobilisiert werden, und (ii) unter Verwendung löslicher IGF-I-Polymer Konjugate für die systemische Anwendung. Beide Ansätze wurden mittels bioorthogonaler Kopplungsstrategien, die unter Berücksichtigung von Einschränkungen, Nebenreaktionen und Effizienzaspekten sorgfältig ausgewählt wurden, durchgeführt. Die bioresponsive Freisetzung der aktiven Biomoleküle als Folge einer erhöhten Proteaseaktivität konnte erfolgreich umgesetzt werden. Das therapeutische Potenzial dieser Ansätze wurde anhand mehrerer zellbasierter Wirksamkeitsassays gezeigt. Die Systeme ermöglichen eine zielgerichtete und kontrollierte Freisetzung des Wachstumsfaktors IGF-I und antikataboler Peptide, wobei sie die Sicherheitsbedenken aktueller Wachstumsfaktortherapie bewältigen und somit das Nutzen-Risiko-Profil hochwirksamer Therapeutika positiv beeinflussen. Unter Berücksichtigung der potenziellen Bedenken bezüglich Heterogenität und Nebenprodukten wurde eine umfassende Hilfsstoffcharakterisierung durchgeführt und ein prognostischer Algorithmus für FRCs entwickelt, um die Formulierungsentwicklung zu erleichtern und eine sichere und effiziente Therapie von Anfang bis zum Ende zu garantieren

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, South Africa Johannesburg 2016
The perception of wound healing within the current decade goes beyond the straightforward assertion of the three phases assembling the wound healing cascade. Healing of wounds is a complex process that involves a dynamic series of interactions and reactions and requires a collaboration of the many cell pedigrees, mediators and different tissues. The skin is the largest organ of the body and serves as a protective barrier against foreign objects therefore a loss in its veracity may lead to a decrease quality of life or even death. The primary goal for wound care and treatment is an aesthetically pleasing scar with close to complete functionality at the wound site and rapid wound closure. Attainment of these features requires incorporation of various characteristics such as a moisture retention, absorption and debridgement amongst others. A huge variety of wound dressings are available however not all of these meet the specific requirements of an ideal wound healing device to cover every aspect within the wound healing cascade. Highlighted within this thesis is the design and development of a Bioresponsive transdermal delivery system (BTDS) for wound healing that aims at the incorporation of the significant characteristics for optimal wound management and treatment. Nanobiotechnology is an interdisciplinary field that combines many avenues to revolutionise the development of drug delivery systems specific to wound healing. Delivery systems produced on the nanoscale can encourage the promotion of biologically active new molecular entities that were previously considered underdeveloped by the enhancement of the therapeutic efficacy of wound healing materials. Recent research interest has focused on the development of smart biomaterials. Combining biomaterials that are crucial for wound healing will provide opportunities to synthesize matrices that are inductive to cells and that stimulate and trigger target cell responses crucial to the wound healing process. Stimuli responsive systems provides an attractive, novel and alternate approach to the process of healing by offering an advanced alternative to simple wound dressings as they have the ability to adapt to the surrounding wound environment and regulate the healing process by thermal, chemical, biochemical, electrical and mechanical means on exposure to an external stimulus that triggers the effect. The research focused on the development and characteristic analysis of a complete prototyped device for wound healing incorporating a nanofibrous mat as well as a bioresponsive component to inflammation which could be the first novel prototype developed as an inflammation bioresponsive device for superior wound healing incorporating a nanofibrous mat. The BTDS was synthesized by the attainment of a statistically derived Box- Behnken Design Template, whereby 15 formulations were generated to fabricate a wound healing nanofibrous mats as well as a lyophilized inflammatory dependent matrix. The technique entailed the process of electrospinning for nanofiber formation as well as blending and lyophilization for the inflammatory responsive component. Elucidation of the various polymeric and crosslinker concentrations greatly influenced the properties and characteristics of the system. An endorsement in intensity and conjugation is noted by the FTIR spectra whereby greater shifts in wavelengths from 3260.11cm-1 to 3278.79cm-1 is noted when enhancements in crosslinking bridges is undertaken. Structural morphological analysis revealed the synthesis of smooth, cylindrical, uniformly aligned nanofibres without the presence of nanobeads as well as the formation of a lyophilized matrix having a tough backbone structure at higher concentrations. Upon nanotensile mapping, variation in Young‟s Modulus was observed at 4.25MPa providing flexibility whereas a higher Young‟s Modulus provides rigidity and stiffness to the structure. Determination of the bioresponsive nature was carried out in a stimulated inflammatory environment by utilisation of the Fentons reaction: Fe2+ + H2O2 → Fe3+ + OH∙ + OH- . Results amongst the experimentally derived formulations revealed the reliance of bioactive release on the hyaluronic acid concentration and degradation by hydroxyl radicals present. MDT results obtained depicted a value at 42.39 at a higher hyaluronic concentration and degree of crosslinkage whereas at lower concentrations, MDT values at 33.21 and 35.76 were depicted. In vivo histological examination revealed the healing progression whereby the presence of the nanofibrous mat illucidated a close to complete re-epithelisation and remodelling of the wound site represented by thick, vascular granulation tissue dominated by fibroblasts and extensive collagen deposition. The approach of introducing a topical device for wound management containing both nanotechnology and stimuli responsive techniques provide an innovative and encouraging proposal for wound care to the pharmaceutical industry.
MT2016

abstract: Minimally invasive endovascular embolization procedures decrease surgery time, speed up recovery, and provide the possibility for more comprehensive treatment of aneurysms, arteriovenous malformations (AVMs), and hypervascular tumors. Liquid embolic agents (LEAs) are preferred over mechanical embolic agents, such as coils, because they achieve homogeneous filling of aneurysms and more complex angioarchitectures. The gold standard of commercially available LEAs is dissolved in dimethyl sulfoxide (DMSO), which has been associated with vasospasm and angiotoxicity. The aim of this study was to investigate amino acid substitution in an enzyme-degradable side group of an N-isopropylacrylamide (NIPAAm) copolymer for the development of a LEA that would be delivered in water and degrade at the rate that tissue is regenerated. NIPAAm copolymers have a lower critical solution temperature (LCST) due to their amphiphilic nature. This property enables them to be delivered as liquids through a microcatheter below their LCST and to solidify in situ above the LCST, which would result in the successful selective occlusion of blood vessels. Therefore, in this work, a series of poly(NIPAAm-co-peptide) copolymers with hydrophobic side groups containing the Ala-Pro-Gly-Leu collagenase substrate peptide sequence were synthesized as in situ forming, injectable copolymers.. The Gly-Leu peptide bond in these polypeptides is cleaved by collagenase, converting the side group into the more hydrophilic Gly-Ala-Pro-Gly-COOH (GAPG-COOH), thus increasing the LCST of the hydrogel after enzyme degradation. Enzyme degradation property and moderate mechanical stability convinces the use of these copolymers as liquid embolic agents.
Dissertation/Thesis
Masters Thesis Biomedical Engineering 2019

博士
國立陽明大學
臨床醫學研究所
88
Current efforts of new stent technology have been aimed largely at the improvement of intravascular stent biocompatibility. Among the chemical characteristics of metallic stents, surface oxide corrosion properties are paramount. Using our unique technique, the currently marketed 316 L stainless steel and nitinol stent wires covered with polycrystalline oxide were chemically etched and then passivated to form amorphous oxide. Excellent metallic stent corrosion resistance with an amorphous oxide surface was demonstrated in our previous in vitro study. For in vivo validation, we compared the corrosion behavior of different oxide surfaces on various forms of test wires in the abdominal aorta of mongrel dogs using open-circuit potential and cyclic anodic polarization measurements. After conduction, the retrieved test wires were observed under scanning electron microscope (SEM). No passivity breakdown was found for wires covered with amorphous oxide, while wires with polycrystalline oxide showed breakdown at potentials between +0.2 to + 0.6 V. SEM showed that severe pitting or crevice corrosion occurred on the surface of polycrystalline oxide, while the surface of amorphous oxide was free of degradations in our experiment. We have demonstrated that this amorphous oxide coating on metallic material provides better corrosion resistance, but whether the corrosion products released from the wires with polycrystalline oxide are harmful to the surrounding tissue or not? This issue needs further be clarified. Although both 316 L stainless steel and nitinol are most popular materials of intravascular stents, there are still few confirmative biocompatibility data available, especially in vascular smooth muscle cells. The released nickel ions have been proven to be toxic to cultured fibroblasts but the potential cytotoxicity of stent corrosion products on vascular smooth muscle cells has still not been highlighted. In this doctoral thesis research, the 316 L stainless steel and nitinol wires were corroded in Dulbecco''s modified Eagle''s medium applied with constant electrochemical breakdown voltage, and the supernatant and precipitates of corrosion products were prepared as culture media. The dose and time effects of different concentrations of corrosion products on the growth and morphology of smooth muscle cells were evaluated with [3H]-thymidine uptake ratio and cell cycle sorter. Both the supernatant and precipitates of the corrosion products were toxic to the primary cultured rat aortic smooth muscle cells. The growth inhibition was correlated well with the increased concentrations of the corrosion products. For nitinol wire, small growth stimulation was found with released nickel concentration of 0.95±0.23 ppm, but this stimulation effect was not observed for 316 L stainless steel wires with mild leaching. The growth inhibition became significant when the nickel concentration was above 9 ppm for nitinol wires and above 11.7 ppm for 316 L stainless steel wires. The corrosion products also altered cell morphology, induced cell necrosis and decreased cell numbers. The cell growth inhibition occurred at the G0/G1 to S transition phase. This was the first study to demonstrate the cytotoxicity of corrosion products of current nitinol and 316 L stainless steel stent wires on smooth muscle cells, which might affect the post- stenting neointimal hyperplasia and the patency rate of cardiovascular stents. In conclusion, this thesis study demonstrated that in comparison with polycrystalline oxide, amorphous oxide coating on metallic material provides better corrosion resistance, not only in vitro but also in vivo, and it is superior not only in strength safety but also in medical device biocompatibility.