Rozprawy doktorskie na temat „Surfactant-Protein System - Drug Delivery”

Skin cancer affects many people worldwide and is life threatening without treatment. People with light skin, genetic diseases, and high exposure to ultraviolet radiation (UVR) are at a high risk of developing skin cancer. Once developed, skin cancer can spread to the rest of the body, including organs inside the body. If the cancer is established, it is difficult to control and treat. Skin cancer is diagnosed by the depth at which it has spread below the skin. Conventional treatments such as creams and lotions treat only the outer skin surface, while cancer below the skin is allowed to thrive. Treating only the outer layer and not the inner layer of skin can make it difficult to diagnose cancer because the severity of it can be hidden. The ideal treatment is to treat cancer from the inside out. Administering common cancer medicines means killing healthy cells as well as cancer cells; therefore, developing a drug delivery system (DDS), which can be injected into the body and release medicine at an engineered rate and location, is needed. In this thesis, a special drug delivery system is proposed—one that lessens the toxicity of a therapeutic agent from an intraperitoneal (IP) injection by reducing diffusion of that injection into sensitive areas of the body. This DDS uses both magnetic forces to hold it at the affected location and also a protein, encapsulated into the DDS, to reduce an immune response. The protein can also encourage uptake of the DDS into the cancer cell where the DDS releases the therapeutic agent. It is shown that this DDS is successful in treating cancer, and no toxic effects were found, which makes this treatment a possible alternative to conventional therapies.
Thesis (Ph.D.)--Wichita State University, College of Engineering, Dept. of Mechanical Engineering

The goal of this thesis was to develop a new drug-delivering material to deliver anti-inflammatory protein for treating OA. Our central hypothesis for this work is that a controlled release/presentation system will more effectively deliver anti-inflammatory protein therapies to the OA joint. The primary goal of this work was to synthesize a block copolymer that could self-assemble into injectable, sub-micron-scale particles and would allow an anti-inflammatory protein, IL-1ra, to be tethered to its surface for efficient protein delivery. The block copolymer incorporated an oligo-ethylene monomer for tissue compatibility and non-fouling behavior, a 4-nitrophenol group for efficient protein tethering, and cyclohexyl methacrylate, a hydrophobic monomer, for particle stability. We engineered the copolymer and tested it in both in vitro culture experiments and an in vivo model to evaluate protein retention in the knee joint. The rationale for this project was that the rational design and synthesis of a new drug- and protein-delivering material can create a modular polymer particle that can deliver multi-faceted therapies to treat OA. This work characterizes the in vitro and in vivo behavior of our polymer particle system. The protein tethering strategy allows IL-1ra protein to be tethered to the surface of these particles. Once tethered, IL-1ra maintains its bioactivity and actively targets synoviocytes, cells crucial to the OA pathology. This binding happens in an IL-1-dependent manner. Furthermore, IL-1ra-tethered particles are able to inhibit IL-1beta-induced NF-kappaB activation. These studies show that this particle system has the potential to deliver IL-1ra to arthritic joints and that it has potential for localizing/targeting drugs to inflammatory cells of interest as a new way to target OA drug treatments.

This thesis deals with the study of preparation and characterization of hyaluronan-micelle aggregates. The theoretical part deals with drug delivery systems, characterization of used materials and methods especially fluorescence spectroscopy, dynamic light scattering and turbidimetry. Methods of determination of measured data are summarized in the experimental section. The result section is divided into two subsections dedicated to different preparation methods of hyaluronan-surfactant complexes. Induced aggregates of hyaluronan with Septonex are characterized in terms of their origin and stability, and the results are compared with previously studied surfactants CTAB. In the second part are discussed so-called decorated micelles, their formation, properties and stability.

The targeted delivery of drugs is one of the most actively pursued goals in anti-HIV and anti-cancer chemotherapy. This project takes a proof-of-concept approach to the development of protein-based drug delivery systems - delivery systems that would package, target, and deliver cytotoxins to diseased cells. Primarily, this project explores the use of the potent anti-HN protein, cyanovirin-N (CV-N), to actively target and deliver cytotoxic natural products to HN-infected cells. This project also investigates the use of human serum albumin (HSA), a 66 kDa protein, as a macromolecular carrier to passively target and deliver cytotoxic natural products to cancerous cells. To facilitate release of the toxin within infected cells, an enzymatically-cleavable tetra peptide was incorporated in the conjugates. Maleimido-activated tetra peptide toxin constructs were prepared in readiness for selective reaction with proteins carrying thiol functionalities. Release of the toxin, norhomohalichondrin B, was demonstrated in vitro. Native CV -N conjugates were prepared by thiolation of the lysine ε-amino groups, and the subsequent reaction with maleimido-activated compounds. Reaction across all lysine residues was demonstrated. A singly-substituted tyrosinamide conjugate of CV-N was prepared. Two recombinantly produced mutant CV-N proteins allowed for the production of selectively modified, double- and single-norhomohalichondrin B conjugates of CV-N. The conjugates retained the anti-HN activity of the parent protein. Homohalichondrin B, doxorubicin, and tyrosinamide conjugates of HSA were prepared. The syntheses exploited the availability of a free thiolmoiety at cysteine-34 of HSA, and the specific and selective reaction of this thiol with the maleimido-activated tetra peptide derivatives. All toxin conjugates demonstrate excellent cell toxicity. Further research to investigate whether this is targeted toxicity is currently underway.

Introduction: Embolic agents are used to block blood flow of hypervascular tumours, ultimately resulting in target tissue necrosis. However, this therapy is limited by the formation of new blood vessels within the tumour, a process known as angiogenesis. Targeting angiogenesis led to the discovery of anti-angiogenic factors, large molecular weight proteins that can block the angiogenic process. The aim of this research is development of poly (vinyl alcohol) (PVA) aqueous solutions that cross-link in situ to form a hydrogel that functions as an embolic agent for delivery of macromolecular drugs. Methods: PVA (14 kDa, 83% hydrolysed), functionalised by 7 acrylamide groups per chain, was used to prepare 10, 15, and 20wt% non-degradable hydrogels, cured by UV or redox initiation. Structural properties were characterised and the release of FITCDextran (20kDa) was quantified. Degradable networks were then prepared by attaching to PVA (83% and 98 % hydrolysed) ester linkages with an acrylate end group. The effect on degradation profiles was assessed by varying parameters such as macromer concentration, cross-linking density, polymer backbone and curing method. To further enhance the technology, radiopaque degradable PVA was synthesised, and degradation profiles were determined. Cell growth inhibition of modified PVA and degradable products were also investigated. Results: Redox initiation resulted in non-degradable PVA networks of well-controlled structural properties. Increasing the solid content from 10 to 20wt% prolonged the release time from few hours to ~ 2 days but had no effect on the percent release, with only a maximum release of 65% achieved. Ester attachment to the PVA allowed flexibility in designing networks of variable swelling behaviors and degradation times allowing ease of tailoring for specific clinical requirements. Synthesis of radiopaque degradable PVA hydrogels was successful without affecting the polymer solubility in water or its ability to polymerize by redox. This suggested that this novel hydrogel is a potential liquid embolic with enhanced X-ray visibility. Degradable products had negligible cytotoxicity. Conclusion: Novel non-degradable and radiopaque degradable PVA hydrogels cured by redox initiation were developed in this research. The developed PVA hydrogels showed characteristics in vitro that are desirable for the in vivo application as release systems for anti-angiogenic factors.

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2004.
"A dissertation in pharmaceutical sciences and chemistry." Advisor: Thomas P. Johnston. Typescript. Vita. Description based on contents viewed Feb. 23, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 202-223). Online version of the print edition.

Thesis (Ph.D.)--University of Mississippi, 2008.
Typescript. Vita. Major advisor: John O' Haver "June 2008." Includes bibliographical references (leaves 120-163). Also available online via ProQuest to authorized users.

Cancer is one of the leading causes of death throughout the world and the number of cases per year is reported to rise to 23.6 million by 2030. Amongst the different types of treatments available, chemotherapy represent the most common one. Despite its proven capability of tumour shrinkage and prevention from coming back after surgery, several factors limit its potential. These include poor bioavailability and biodistribution of the majority of the chemotherapeutic agents commonly used, the high dose required, their numerous adverse side effects, the development of drug resistance and non-specific targeting. To overcome these limitations, I propose to develop novel protein-based drug delivery system capable of selectively transporting large quantities of chemotherapeutics at the tumour-site thus conferring greater therapeutic indices and efficacy. The new systems are based on proteins, natural existing polymers, that have the intrinsic property of binding small molecules with high affinity and specificity. The project is structured in two phases. In the first phase I applied FuncLib, an automated method that uses phylogenetic analysis and Rosetta design calculations, to design mutants with higher affinity toward two selected chemotherapeutic agents. In the second phase, fifty-five designs were selected according to G Rosetta energy score and structural diversity, synthesised and assembled using a repertoire of molecular biology techniques.

Cancer is a vast group of diseases caused by mutations in the human DNA that result in an uncontrollable growth of cells which can spread around the body. Cancer is one of the leading causes of mortality worldwide. Advancements in CTXs allowed the development of effective treatments. However, most CTX agents present poor pharmacokinetic and safety profiles. Development of appropriate drug carriers is one of the potential solutions for counteracting these drawbacks. The aim of this thesis is to make advancements in the engineering of a protein based carrier, with higher binding affinity to the CTX agents. Furthermore, explore the expression of fusion proteins with the aim of improving the characteristics of the delivery system. In the first phase of the project, 55 genes, corresponding to human serum albumin (HSA) mutants previously engineered to have higher binding affinity to doxorubicin or 9-aminocamptothecin, were cloned by Gibson assembly (GA). 52 of these mutants were then successfully expressed in a yeast display system, which will be used for future binding affinity experiments. For the second part of this thesis, a cloning system using restriction enzymes (RE) was designed in order to produce a series of HSA oligomers. The HSA dimer was successfully cloned, expressed, and purified. However it showed a complete degradation over time. Therefore, alternative cloning strategies or changes in the design of the linker between the monomers should be employed.

Niosomes are synthetic microscopic vesicles consisting of an aqueous core enclosed in a bilayer consisting of cholesterol and one or more nonionic surfactants. They are made of biocompatible, biodegradable, non-toxic, non-immunogenic and non-carcinogenic agents which form closed spherical structures (self assembly vesicles) upon hydration. With high resistance to hydrolytic degradation, niosomes are capable of entrapping many kinds of soluble drugs while exhibiting greater vesicle stability and longer shelf life. In this work, a potential drug delivery system has been designed, synthesized and characterized. For the synthesis of niosomes, a hydration process was developed with varying design parameters such as mass per batch, angle of evaporation, rotation speed of vacuum rotary evaporator and nitrogen flowrate to produce uniform thin film in 50 ml round bottom flask. The rehydration process was developed by varying the choice of solvents (H2O, phosphate buffer solution (PBS) and PBS/5(6)-carboxyfluorescein (CF) as a drug model) and hydrating temperature of below and above gel transition temperature. Lastly, a sonication process to produce unilamellar vesicles was partially optimized based on the particle distribution and the number of vesicles formed with sonication time. As a result of this process, unilamellar and multilamellar vesicles were formed with the combination of different nonionic surfactants (sorbitan monostearate-Span 60, sorbitan monopalmitate-Span40 and sorbitan monolaurate-Span20), cholesterol and an electrostatic stabilizer (dicetyl phosphate). The vesicles were examined using light scattering optical microscopy and UV microscopy. Optical sensing technology (Particle Sizing System) is used to determine the vesicles' size distribution. Gel exclusion chromatography (GEC) is discussed as a method to separate unencapsulated CF while retaining vesicle integrity. Particle Sizing System and luminescence spectrophotometer were used to determine CF encapsulation percentage and leakage. Result: Span 20, Span 40 and Span 60/Niosomes were made with mean particle size of 0.95-0.99 micro (mu)m. Typical concentrations of vesicle per ml/per mass of surfactant used were in the range of 1.46-1.79x108 . Typical encapsulation efficiencies were in the range of 48.8-62.9% for all three Span/Niosome systems. Niosomes were found to be stable for 9 days. The largest vesicles were observed with Span 60 with highest entrapment efficiency as compared to Span 20 and Span 40.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado, 2006.
Typescript. Includes bibliographical references (leaves 144-161). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Daily injections for basal insulin therapy are far from ideal resulting in hypo/hyperglycemic episodes associated with fatal complications in type-1 diabetes patients. The purpose of this study was to develop a thermosensitive copolymer-based in situ depot forming delivery system to provide controlled release of insulin for extended duration following a single subcutaneous injection, closely mimicking physiological basal insulin requirement. Size and nature of the incorporated therapeutic were observed to affect the release profile of insulin. Modification with zinc and chitosan preserved thermal, conformational, and chemical stability of insulin during the entire duration of storage (up to 9 months at 4 °C) and release (up to 3 months at 37 °C). In vivo, daily administration of long-acting insulin, glargine, resulted in fluctuating blood glucose levels between 91 – 443 mg/dL in type 1 diabetic rats. However, single administration of oleic acid-grafted-chitosan-zinc-insulin complexes incorporated in copolymer formulation demonstrated slow diffusion of insulin complexes maintaining peak-free basal insulin level of 21 mU/L for 91 days. Sustained release of basal insulin also correlated with efficient glycemic control (blood glucose <120 mg/dL), prevention of diabetic ketoacidosis and absence of cataract development, unlike other treatment groups. The suggested controlled basal insulin delivery system has the potential to significantly improve patient compliance by improving glycemic control and eliminating life-threatening diabetes complications. Furthermore, oleic acid-grafted-chitosan (CO) nanomicelles were investigated as a non-viral vector to deliver plasmid DNA encoding short hairpin RNA (shRNA) against pro-inflammatory cytokines to adipose tissue macrophages and adipocytes for the treatment of insulin resistance. Nanomicelles modified using mannose (COM) and adipose homing peptide (AHP) (COA) showed significantly higher uptake and transfection efficiency in inflamed macrophages- adipocytes co culture owing to glucose transporter-1 and prohibitin receptor mediated internalization, respectively. Ligand modified nanomicelles loaded with shRNA against tumor necrosis factor alpha (COM-TNFα) and monocyte chemoattractant protein-1 (COA-MCP1) demonstrated significant attenuation of pro-inflammatory cytokines and improved insulin sensitivity and glucose tolerance in obese-diabetic mice for six weeks post treatment with single dose of optimized formulation. Overall, chitosan nanomicelles mediated targeted gene therapy can help attenuate inflammation, the chief underlying cause of insulin resistance, thereby helping reverse the progression of diabetes.
National Institutes of Health (NIH) grant R15GM114701
ND EPSCoR seed award FAR0030636

Thesis (Ph.D.)--Biomedical Engineering, Georgia Institute of Technology, 2008.
Committee Chair: Dr. Niren Murthy; Committee Member: Dr. Carson Meredith; Committee Member: Dr. Julia Babensee; Committee Member: Dr. Mark Prausnitz; Committee Member: Dr. Ravi Bellamkonda.

Purpose: This thesis aimed to gain a better understanding of the effects of salts in modifying in vitro phase behaviour of lecithin and cholesterol solid implants and to obtain further information on in vitro protein release and stability. Methods: Raman spectroscopy and partial least squares regression (PLSR) were used to investigate lecithin-cholesterol molecular interactions as a function of method of preparation. Lipid-salt interactions were studied by attenuated total reflectance Fourier transform infrared (ATR-FTIR) and Raman spectroscopy using principal component analysis (PCA). In vitro release of bovine serum albumin (BSA), a model protein, from lecithin and lecithin:cholesterol implants comprising 10 and 30% NaCl and CaCl₂ were performed. Size exclusion (SE) HPLC was used for quantitative and qualitative analysis of the released BSA. On hydration, changes in phase behaviour and implant morphology were studied by ATR spectroscopy and light microscopy. SE-HPLC, ATR and fluorescence spectroscopy were used to evaluate the structure of unreleased BSA. Protein adsorption on lipid films was studied by flow through ATR spectroscopy. Increased amide II peak area upon recirculation of BSA in salt solutions over hydrated lecithin and lecithin:cholesterol films cast on ZnSe prisms was used to quantify the deposition of BSA onto the lipid surfaces. Results: Shifts in the Raman spectra suggested the lecithin headgroup may be involved in lecithin-cholesterol interactions. Greater R� and root mean square error of cross validation in the calibration curves of physical mixing and heating (120�C) methods reflected poor mixing in these preparations. The mean absolute residue and mean Mahalanobis distance values from the physical mixing and granulation methods indicated their spectral similarity and comparable level of lecithin-cholesterol interactions. Calcium exhibited stronger affinity for phospholipids than sodium and it induced headgroup hydration and reorganisation upon binding. PCA of ATR spectra was sensitive to cholesterol addition, calcium binding and method of preparation whilst PCA of Raman spectra only differentiated the presence of cholesterol. In vitro release of BSA from implants produced from wet granulation mixtures of lecithin and lecithin:cholesterol in the absence of salt showed retention of a high monomer content and the release profiles were similar to the literature. Cholesterol increased the swelling, induced phase transformation of lecithin and, subsequently, reduced the BSA release. Salts only slightly modified the BSA release from the lecithin implants. In contrast, for lecithin:cholesterol matrices salts greatly enhanced implant swelling, induced the formation of hydrated lecithin of heterogeneous size and inhibited the in vitro BSA release. Analyses of the protein showed increased aggregation of BSA with a high retention of native structure while retained within the swollen matrices. ATR spectra suggested that salts promoted protein adsorption onto hydrated lecithin surfaces and the effects depend on salt types (NaCl > CaCl₂) and concentration (0.1 M > 1.0 M) but not on lecithin:cholesterol surfaces. Conclusion: PLSR and PCA can be used to investigate molecular interactions in the solid lipid matrices. In lecithin:cholesterol implants, salts modified the phase behaviour of lecithin which resulted in enhanced swelling, formation of hydrated lecithin of altered morphology and inhibition of in vitro BSA release.

Since the early 80’s the forward steps in genetics and proteomics, have led a particular interest to biotech products, such as DNA and proteins. Although difficult, their large-scale production enabled the therapeutic use of this compounds. Proteins and DNA sequences can be very interesting therapeutic molecules owing to their high selectivity/affinity for the receptor or the specific site of action. Unfortunately, some issues still limit their pharmaceutical use, such as the susceptibility to enzymatic degradation, rapid renal clearance and immunogenicity. To overcome these limitations, many researchers are seeking solutions in the field of drug delivery systems (DDSs). In this respect, many systems have been developed and conjugation with PEG (polyethylene glycol) can be considered one of the leading approaches. PEGylation brings to the conjugated molecule great solubility and stability to proteolytic digestion, furthermore it reduces the tendency to aggregate and reduces the immunogenicity. Thanks to these advantages and the particular characteristics of PEG, to date, there are on the market 12 pegylated compounds: 9 are proteins, one peptide, one aptamer and a liposomal formulation, containing doxorubicin. The improvements in the pharmacokinetic profile of these drugs, thanks to the use of drug delivery systems, can be also applied in the field of tissue engineering, where the same issues are of fundamental importance for the development of scaffolds for cells capable of releasing growth factors. In the last years various polymers have been studied by many research groups to find an alternative to PEG, but its excellent biocompatibility and the know-how in its use has not brought any polymer to be truly competitive against PEG. Nevertheless, PEG presents some limits such as its non-biodegradability and in some case there are reports of antibodies against PEG. Therefore, there is an increased need for a PEG substitute. In the first section of this work hyaluronic acid (HA) has been studied as a candidate polymer for bioconjugation of proteins (HAylation). HA, being biodegradable can compensate this limit of PEG. HA, is also present in humans and is metabolized by hyaluronidase. Moreover, HA has the advantage of a high loading compared to PEG, thanks to the presence of repetitive functional groups in each monomer. This part of the work was focused on the study of HA conjugation (HAylation) to two model enzymes, trypsin and Ribonuclease A, and then to an interesting protein in pharmaceutical field, insulin. In order to avoid cross-linking phenomena, only a fraction of all carboxyl groups of the polymer has been modified to aldehyde allowing the conjugation with the amino groups of the protein models. Furthermore, by modulating the pH of reaction two protein-HA conjugates were obtained, selective N-terminal (pH 6) or random (pH 8), this taking advantage of the different pKa values of the amino groups in the proteins. The first products obtained with the enzymes Ribonuclease A and trypsin were tested verifying the residual activity compared to the native proteins. All conjugates, in particular those obtained by N-terminal selective conjugation, maintain a good activity on small substrates (30% decrease); only the HA-derived trypsin retains about 60% of residual activity against the substrate with a high weight molecular. Furthermore, enhanced stability over time was found for HA-trypsin respect to the free enzyme (45% on average) and also susceptibility to hyaluronidase was confirmed for both conjugates. Polymer validation as potential protein carrier was then evaluated by preparing conjugates with bovine insulin, as an example of pharmacologically active protein. Two conjugates were synthesized by N-terminal selective conjugation starting from polymers with different degree of aldehyde derivatization, 4% and 21%, yielding products with a protein loading of 17% and 32% (w/w), respectively. The therapeutic efficacy of the conjugates in comparison with insulin was tested in Sprague Dawley rats with induced diabetes. The conjugate with a lower protein loading was more effective and with a longer pharmacodynamic effect on the reduction on blood glucose level. The second section of the work was focused on an innovative strategy of enzymatic PEGylation of oligonucleotides. Briefly, the method investigated on model oligonucleotides is composed of two steps: the first consists in the chemical conjugation of a short oligonucleotide to a PEG chain, the second step is the enzymatic-mediated conjuagation of the PEGylated oligonucleotide with a DNA sequence by the DNA T4 ligase. To study the enzymatic PEGylation, 4 oligo sequences have been prepared as ligation model: two complementary pairs ending with sticky-ends in turn complementary (18-mer + 21-mer and 16-mer + 19-mer). The 18-mer has a thiol group in 5’-ending, in order to perform the coupling with PEG. Applying some modifications to ligation classical protocols, excellent results were obtained: PEGylated portion completely ligate the other ds-DNA and no undesired products were found. To further confirm the effective ligation, the ligated and PEGylated sequence was restricted with EcoRI. Indeed, the EcoRI recognized a sequence that is present only the ligated DNA. Complete restriction was found in absence and even in the presence of the polymer, further confirming the successes of ligation. Furthermore it was investigated if a reduced number of bases coupled to PEG can still preserve the requirements for the ligase enzyme activity. Thus, the pair of the complementary sequences then coupled to PEG has been reduced to half (9-mer + 12-mer). Even with a shorter PEGylated sequence a complete ligation was obtained. In conclusion in this thesis it has been demonstrated that HA can be a valid alternative to PEG for protein conjugation. In the field of oligonucleotide delivery an enzymatic approach of oligonucleotide conjugation can open new horizons that so far have not been completely explored.
Dai primi anni 80 i passi avanti fatti nel campo della genetica e della proteomica, hanno portato ad un particolare interesse nei confronti dei prodotti biotecnologici, quali DNA e proteine. L’utilizzo terapeutico di queste entità, seppur non privo di difficoltà, è stato facilitato dalla loro produzione su larga scala. Proteine e sequenze oligonucleotidiche si sono rivelate interessanti come agenti terapeutici essendo molecole dotate d’elevatissima selettività/affinità per il recettore o il sito d’azione specifico. L’impiego farmaceutico può evidenziare alcuni svantaggi che ne possono limitare l’utilizzo, come ad esempio la suscettibilità alla degradazione da parte di proteasi e DNasi, la rapida clearance renale e l’immunogenicità. Per affrontare tali limiti, molti ricercatori hanno cercano soluzioni nel campo dei drug delivery sistems (DDSs). A tal proposito, sono stati sviluppati molti sistemi e la coniugazione al PEG (polietilen glicole) è risultata essere una delle più promettenti. La PEGhilazione, infatti, conferisce alle molecole coniugate maggiore solubilità e stabilità nei confronti della digestione proteolitica, una ridotta tendenza all’aggregazione ed una ridotta immunogenicità. Grazie a questi vantaggi ed alle particolari caratteristiche del PEG, ad oggi sono presenti nel mercato 12 composti PEGhilati: 9 sono proteine, un peptide, un aptamero ed una formulazione liposomiale (contenente doxorubicina). Le migliorie apportate ai profili farmacocinetici di questi farmaci biotech grazie all’uso di DDSs possono essere anche impiegate nel campo dell’ingegneria tessutale, dove le medesime problematiche sono di basilare importanza per lo sviluppo di scaffold per cellule, in grado di rilasciare fattori di crescita. Il polietilen glicole (PEG) è il polimero leader per la coniugazione di proteine. Negli ultimi anni diversi polimeri sono stati studiati per trovare una valida alternativa a questo polimero, ma la sua eccellente biocompatibilità e la conoscenza nel suo utilizzo non ha ancora portato nessun polimero ad essere realmente competitivo nei suoi confronti. Nonostante tutto, anche l’utilizzo del PEG presenta alcuni limiti, quali la non-biodegradabilità e la documentata presenza di anticorpi anti-PEG sviluppati in alcuni casi specifici. Per questo motivo si è alla ricerca di un polimero che possa validamente sostituire il PEG. Nella prima parte di questo lavoro di tesi è stato studiato l’acido ialuronico (HA) per la bioconiugazione di proteine (HAylation). Essendo biodegradabile, l’HA può essere vantaggioso rispetto al PEG. L’HA è un polimero endogeno ed è metabolizzato dalle ialuronidasi, inoltre ha il vantaggio di poter raggiungere una capacità di loading elevate rispetto al PEG, grazie alla presenza di gruppi funzionali ripetitivi in ciascun monomero. In questa parte del lavoro di tesi, la ricerca si è concentrata sullo studio della coniugazione dell’HA a due enzimi modello, Ribonuclease A e tripsina, e poi ad un interessante proteina per uso farmaceutico, l’insulina. Per evitare fenomeni di cross-linking, solo una parte dei gruppi carbossilici del polimero è stata coniugata ad uno spacer aldeidico, consentendo la coniugazione con i gruppi amminici delle proteine. Inoltre, modulando il pH di reazione si sono potuti ottenere coniugati con legame selettivo all’N-terminale (pH 6) oppure random (pH 8), sfruttando la differente pKa degli ammino gruppi nelle proteine. I primi coniugati ottenuti con gli enzimi Ribonuclease A e tripsina sono stati studiati verificandone l’attività residua rispetto alle proteine native. Tutti i coniugati, in particolare quelli ottenuti per legame selettivo all’N-terminale, mantengono una buona attività su piccoli substrati (diminuzione del 30%); solo il derivato HA-tripsina mantiene circa il 60% di attività residua nei confronti del substrato ad alto peso molecolare. Inoltre, sempre per HA-tripsina, si è trovata una maggiore stabilità nel tempo rispetto l’enzima nativo (mediamente 45%) e si è confermata la suscettibilità di entrambe i coniugati nei confronti della ialuronidasi. La valutazione del polimero come potenziale carrier per proteine è proseguita preparando dei coniugati con l’insulina bovina, come esempio di proteina farmacologicamente attiva. Sono stati sintetizzati due coniugati con modalità selettiva all’N-terminale a partire da polimeri con diverso grado di modifica con gruppi aldeidici, pari a 4 e 21% e si sono ottenuti prodotti con il 17 e 32% (p/p), rispettivamente, di loading proteico. L’efficacia terapeutica dei coniugati in comparazione con l’insulina è stata testata su ratti Sprague Dawley con diabete indotto. Il coniugato con un minore loading proteico si è rivelato essere più efficace e con una riduzione dei livelli di glucosio nel sangue più prolungata. Nella seconda parte di questo lavoro di tesi si è studiata un’innovativa strategia di PEGhilazione enzimatica di sequenze oligonucleotidiche al fine di sviluppare questo approccio per il delivery di oligonucleotidi. Il metodo è stato messo a punto con sequenze nucleotidiche modello e l’approccio è stato il seguente: una breve sequenza oligonucleotica viene legata chimicamente ad una catena di PEG. Poi, mediante l’azione catalitica della T4 DNA ligase la porzione di PEG-DNA viene coniugata ad un’altra sequenza oligonucleotidica. Per lo studio di PEGhilazione enzimatica si è ideato un modello costituito da 4 sequenze oligonucleotidiche di riferimento: due coppie complementari terminanti con sticky-ends complementari a loro volta (18-mer + 21-mer e 16-mer + 19-mer). L’oligo di 18 nucleotidi portava in posizione 5’ una funzione tiolica, che è stata impiegata per la coniugazione col polimero. Dopo aver apportato alcune variazioni ai protocolli classici di ligazione si sono ottenuti ottimi risultati: completa ligazione del modello PEGhilato ed assenza di prodotti indesiderati. Un’ulteriore conferma di ligazione del modello PEGhilato si è ottenuta tramite digestione con EcoRI. Infatti, solamente dopo la ligazione è possibile trovare nella sequenza oligonucleotidica il sito di restrizione dell’enzima. In presenza o in assenza di polimero la restrizione è avvenuta completamente. Si è poi voluto indagare se una sequenza PEGhilata con un numero di basi ridotto potesse comunque mantenere i requisiti per essere substrato della T4 DNA ligase. Così, la coppia di sequenze complementari designata alla PEGhilazione è stata ridotta alla meta della sua lunghezza (9-mer + 12-mer). Anche con la sequenza PEGhilata così accorciata la ligazione è avvenuta completamente. In conclusione questo lavoro di tesi ha dimostrato che l’HA può essere una promettente alternativa al più noto PEG per la modifica di proteine. Nell’ambito del delivery di oligonucleotidi lo sviluppo di un approccio enzimatico di coniugazione può aprire nuovi orizzonti in questo settore il cui potenziale non è stato ancora esplorato.

The biopharmaceutical market has been on the rise for the past two decades and is expected to continue to excel, currently presenting a growing rate of more than double than conventional pharma. Traditionally this growth has been hindered by multiple formulation issues such as poor bioavailability and poor stability. Consequently, the drive to optimise the stability of protein drug candidates via formulation impels the need for development of novel excipients. Novel glycopolymer excipients were reported to confer improved protein stability in selected cases. Nonetheless,their structure-function relationship and wider applicability remain largely unknown. Here we report the synthesis of glycopolymers with different molecular architectures based on mannose, galactose, arabinose, N-acetyl glucosamine, lactose and trehalose, and nvestigate their utility as excipients for the solution formulation of a monoclonal antibody (mAb). In this thesis work the physical stability of selected antibodies was measured as the unfolding transition temperature (Tm) and aggregation onset temperature (Tagg), as a function of glycopolymer properties, such as the nature of sugar repeating unit, macromolecular architecture and concentration. Results show that, in contrast to the stabilising effect of the corresponding mono- and di-saccharide constituents, both linear and 4-arm star glycopolymers generally destabilised the antibody, decreasing both Tm and Tagg. Accelerated stability studies of a concentrated mAb solution followed the same trend, where an increasing glycopolymer:mAb molar ratio generally decreased the percentage monomer(i.e. increased soluble aggregates). Importantly, trehalose-based glycopolymers further generated visible aggregates that could not be predicted from Tm or Tagg data. The data demonstrate a complex interplay of sugar chemistry and solution concentration of synthetic glycopolymers on their modulation of protein conformational stability and aggregation propensity. The mechanisms involved in protein:glycopolymer interaction, both in solution and dry state were further investigated, thus unravelling the behaviour reported in terms of protein stabilisation. Finally, the glycopolymers were studied as drug delivery systems, acting as solubility enhancers for hydrophobic species in aqueous solutions, through the use of extrinsic fluorescent dyes.

Protein therapeutics have great potential in treating human disease, especially for those caused by alternations in the functions of intracellular proteins. However, clinical use of protein by intracellular delivery has been hampered by the instability due to proteins' physicochemical properties, and some barriers in the delivery pathway. This study was to prepare and test a novel intracellular protein delivery system - magnesium phosphate nanoparticles with cationic lipid coating for catalase intracellular delivery (LP MgP NP-CAT), and investigate whether it can release the encapsulated catalase to cytosol. LP MgP NP-CAT was designed, prepared and characterized, showing that it had an average diameter around 300 nm and zeta potential around +40mV. The pH - triggered catalase release from LP MgP NP-CAT was determined by a hydrogen peroxide degradation assay, where the concentration of remaining hydrogen peroxide was measured by UV-Vis spectroscopy, indicating catalase was released in response to the drop of pH, which was confirmed by the morphology change of LP MgP NP-CAT observed by transmission electron microscopy. The in vitro catalase release behavior was conducted on MCF-7 cells and EA.hy926 cells. LP MgP NP-CAT was delivered into MCF-7 cells and the release behavior was determined by the resultant resistance of the cells against hydrogen peroxide using MTS cell viability assay. The delivery of LP MgP NP-CAT into EA.hy926 cells was determined by the decrease of the reactive oxygen species level. Both of the studies showed that catalase was successfully delivered and released which is supported by the reduction of hydrogen peroxide.

Tuberculosis (TB) is an infectious, deadly disease, caused by Mycobacterium tuberculosis (M.tb). In 2010, there were 8,8 million incident cases of TB globally. South Africa currently has the third highest TB incident cases worldwide. In an attempt to address the challenges facing TB chemotherapy, among which frequent dosing and long duration of therapy resulting in poor patient compliance, a novel poly(DL-lactic-co-glycolic) acid (PLGA) nanoparticulate drug delivery system (DDS) encapsulating anti-TB drugs was developed. It is hypothesised that this nanoparticulate DDS will address the challenges mentioned by enabling decreased dosing frequency, shortening duration of therapy and minimising adverse side effects. Therefore, favourable modification of pharmacodynamic (PD) and pharmacokinetic (PK) properties of the conventional anti-TB drugs was demonstrated. Furthermore, the nanoparticles will provide a platform for drug delivery to macrophages that serve as hosts for M.tb. The study design was based on determining specific physicochemical properties of the nanoparticulate DDS to elucidate the hypothesis. Spray-dried PLGA nanoparticles were prepared using the double emulsion solvent evaporation technique. In vivo analysis of macrophage uptake and possible immunological response in mice were evaluated. In vitro protein-binding assays of PLGA nanoparticles encapsulating anti-TB drugs isoniazid (INH) and rifampicin (RIF) were performed with subsequent in vivo tissue distribution assays to support protein-binding data generated. Finally, PK/PD analyses were conducted to evaluate the effect of nanoencapsulation on the anti-TB drugs. These involved in vitro assays to determine if sufficient drug was released from the nanoparticles to exhibit minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC). Furthermore, in vivo drug distribution and drug release kinetics assays of encapsulated RIF, INH, pyrazinamide (PZA) and ethambutol (ETB) in a mouse model were performed. The results confirmed that the PLGA nanoparticles (<250 nm, low positive zeta potential) were taken up by macrophages in vivo with no significant immunological effect. Furthermore the nanoparticles were present in the brain, heart, kidneys, lungs, liver and spleen for up to 7 days following once-off oral dosing at 13.23± 0.11%, 16.81± 0.11%, 54.89± 0.95%, 15.61± 1.15%, 48.48± 2.28% and 5.73± 0.21%, respectively. This was further confirmed by drug analysis demonstrating the presence of INH, RIF and ETB at different time points up to 7 days in the lungs, kidneys, liver and spleen. However, PZA was not detected. Nanoencapsulated RIF and INH exhibited MICs and MBCs in vitro over 14 days and these drugs were also observed in plasma for up to 7 days post once-off oral dosing. ETB and PZA were observed up to 3 days. From the results generated, it can be concluded that the nanoparticles were taken up by macrophages without eliciting an immune response. This provides a platform for drug delivery to specific sites. Furthermore, the nanoparticulate DDS exhibited sustained drug release in vitro and in vivo over a number of days above the MIC for the drugs analysed. Sustained drug distribution was also observed. It can therefore be concluded that the hypothesised reduction in dose frequency and duration of therapy for this DDS is a possibility
Thesis (PhD (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013

Tuberculosis (TB), caused by Mycobacterium tuberculosis is the leading infectious disease epidemic that claims over 1.6 million lives, while 10 million fell ill in 2017. South Africa is burdened with the third highest global incidences following India and China with high rates of co-infections with HIV and highest numbers of multi-drug resistant (MDR) and extremely resistant (XDR) TB per capita. The current treatment regimen is decades old and requires a prolonged period of 6 months. The lack of efficient TB therapy and the emergence of MDR and XDR TB, there is an urgent need to find new drug targets for TB therapy through understanding the complex host-pathogen interactions. This may then lead to pathogen, host-directed therapies (HDT) or adjunct therapies as well as the development of effective drugs and drug formulations for the treatment of TB. Here we aimed to investigate potential targets for pathogen-and host-directed therapies for TB. We screened the anti-mycobacterial activity of 172 minor groove binder (MGB) compounds that selectively bind to AT-rich regions of the minor groove of bacterial DNA with the helical structure matching that of DNA in Mtb culture. Of the 172 total compounds screened 17 hits were identified, of which 2, MGB 362 and MGB 364 displayed intracellular mycobactericidal activity against Mtb HN878 at an MIC50 of 4.09 and 4.19 μM, respectively, whilst being non-toxic. Encapsulation of MGBs into non- ionic surfactant vesicles (NIVs) demonstrated a 1.6- and 2.1-fold increased intracellular mycobacterial activity, similar to that of rifampicin when compared with MGB alone. Treatment with MGB 364 or MGB 364 formulation did not cause DNA damage in murine infected macrophages as displayed by low expression of γ-H2Ax compared to H2O2 and DMSO. Intranasal administration of MGB 364 and MGB-NIV 364 formulation showed one log reduction in bacterial burden with improved pathology and immune cytokine production when in formulation. However, intranasal administration of 10 mg/kg MGB 362 together with rifampicin had no effect on bacterial loads. In summary, the data demonstrate the potential of MGB as a novel class of drug/chemical entity in anti-TB therapy and NIVs as an effective delivery system in a novel anti-TB formulation. Using deep CAGE and small RNA (CHIP-seq) technologies, International Center for Genetic Engineering and Biotechnology’s Cytokines and Diseases lab in collaboration with the RIKEN Center for Integrative Medical Sciences (Yokohama, Japan) performed a novel transcriptomics study approach by conducting a genome-wide transcriptional analyses of RNA transcripts from classically activated macrophages (caMph) and alternatively activated macrophages (aaMph) during Mtb infection. We identified host target genes that may play a role in host immune subverting mechanism by Mtb to hide away from host effector functions providing a possible target for host-directed therapy for tuberculosis. It is postulated that Mtb modulates the transcriptional landscape of IL-4/IL13 alternatively activated macrophages (aaMph) to escape killing by reactive nitrogen intermediates (NO) and reactive oxygen species (ROS) functions by IFN-γ stimulated classically activated macrophages (caMph). Here we report on the immunoregulatory role of IL-4i1, a candidate gene that was upregulated in aaMph during Mtb infection. IL-4i1 is a secreted L-amino oxidase with antibacterial properties. The enzyme converts Phenylalanine (Phe) into phenylpyruvate releasing toxic products ammonia and hydrogen peroxide (H2O2) which in-turn cause immunosuppression of effector T-cells by directly inhibiting polarization, proliferation and function or by promoting the generation of Foxp3 T-regulatory cells. Thus suggesting that IL-4i1 is involved in immune-regulatory mechanisms and may be implicated in immune evasion mechanisms by the pathogen. Here we report on the role of IL-4i1 on tissue localized T-cell activation and proliferative status thus maintaining immune local immune homeostasis. Thus showing that the absence of IL-4i1 could cause autoimmunity. To determine the functional role of IL-4i1 during Mtb infection, IL-4i1 deficient mice and wild-type littermate controls were infected with H37Rv and hypervirulent HN878 Mtb strain. IL-4i1 deficient mice were highly resistant to both strains of Mtb at 12- and 21-days post-infection as denoted by significant reduction in bacterial loads, reduced inflammation, reduced tissue iNOS expression reduced recruitment of interstitial macrophages, pro-inflammatory cytokines showed a trend for reduction. Interestingly there was a significant increase in NO production in infected tissues. There was an increase in M1-like macrophages that correlated with increased pro-inflammatory cytokines and chemokines. These data suggested that IL-4i1 regulates macrophage-mediated inflammatory responses during acute Mtb infection thus showing potential as an immunomodulatory target for TB HDT therapy. The study thus provides a framework for new drug targets for the development of new effective drugs and vaccines for TB therapy.

ABSTRACT

This project aimed at developing a drug delivery system (DDS) able to enhance the stability and

residence time in vivo of antibodies (Abs). The system will deliver drug by the subcutaneous

route (SC), while ensuring accurate control of the drug release and the resulting plasmatic level. This technology platform will allow to reduce frequency of injection, potentially decrease side effects and maintain high concentration of Abs which will improve life of patient having chronic disease such as autoimmune and inflammatory disease. Biodegradable synthetic polymer-based formulations (polylactide-co-glycolide (PLGA)) were selected as carriers for encapsulated Abs. This was because they offer good protection for the Abs and allow sustained release of the Abs for a controlled period of time. After the evaluation of different encapsulation methods such as the water-oil-in-water (w/o/w) and the solid-in-oil-inwater

(s/o/w) processes, the encapsulation of the Ab in solid state (s/o/w) appeared to be more appropriate for producing Ab-loaded PLGA microspheres (MS). It allowed us to maintain the

Ab in a monomeric conformation and to avoid the formation of unsoluble aggregates mainly present at the water/oil interface. The first part of the project was the optimization of both the method for producing the Ab solid particles (spray-drying process) and the encapsulation of these Ab solid particles into the polymeric MS (s/o/w process) by design of experiment (DoE). These optimizations were carried out using a bovine polyclonal immunoglobulin G (IgG) as model molecule. In further optimization of the spray-drying process by (DoE), aqueous Ab solutions were spray-dried using a mini Spray-Dryer assembly with a 0.7 mm spray nozzle. In accordance with the particle size (d(0.5) ~5 μm), the stability (no loss of monomer measured by

size exclusion chromatography (SEC) and the yield of the spray-drying process (> 60 % w/w), the process parameters were set of follow: 3 mL/min as liquid feed flow rate, 130°C /75°C as inlet temperature (inlet T°) / outlet temperature (outlet T°), 800 L/h as atomization flow rate and

30 m3/h as drying air flow rate. For the s/o/w, the methylene chloride (MC) commonly used for

an encapsulation process was replaced by ethyl acetate (EtAc), which was considered as a more

suitable organic solvent in terms of both environmental and human safety. The effects of several processes and formulation factors were evaluated on IgG:PLGA MS properties such as: particle size distribution, drug loading, IgG stability, and encapsulation efficiency (EE%). Several formulations and processing parameters were also statistically identified as critical to get reproducible process (e.g. the PLGA concentration, the volume of the external phase, the emulsification rate, and the quantity of IgG microparticles). The optimized encapsulation

method of the IgG has shown a drug loading of up to 6 % (w/w) and an encapsulation efficiency

of up to 60 % (w/w) while preserving the integrity of the encapsulated antibody. The produced MS were characterized by a d(0.9) lower than 110 μm and showed burst effect lower than 50 %(w/w). In the second part of the project, the optimized spray-drying and s/o/w processes

developed with the IgG were applied to a humanized anti-tumor necrosis factor (TNF) alpha

MAb to confirm the preservation of the MAb activity during these processes. The selected s/o/w method allowed us to produce MAb-loaded PLGA MS with an appropriate release profile up to 6 weeks and MAb stability. In order to maintain the Abs’ activity, both during encapsulation and

dissolution, the addition of a stabilizer such as trehalose appeared to be crucial, as did the

selection of the PLGA. It was demonstrated that the use of a PLGA characterized by a 75:25

lactide:glycolide (e.g. Resomer ® RG755S) ratio decreased the formation of low molecular weight species during dissolution, which led to preserve Abs activity through its release from the

delivery system. Furthermore, the release profile was adjusted according to the type of polymer

and its concentration. E.g. 10 % w/v RG755S allowed Ab MS with a release time of 6 weeks to

be obtained. The optimization of both the formulation and the encapsulation process allowed

maximum 13 % w/w Ab-loaded MS to be produced. It was demonstrated that the Ab-loaded PLGA MS were stable when stored at 5°C for up to 12 weeks and that the selection of the appropriate type of PLGA was critical to assuring the stability of the system. The better stability observed when using a PLGA characterized by a 75:25 lactide:glycolide ratio was attributed to

its slower degradation rate. Finally, the sustained release of Ab from the developed MS and the preservation of its activity was confirmed in vivo in a pharmacokinetic (pK) study realized in

rats. In conclusion, the application of the concept of entrapment into a polymer matrix for

stabilization and sustained release of biological compounds was demonstrated through this work.

RÉSUMÉ

Ce projet a pour but de développer un système de délivrance de médicament capable d’augmenter la stabilité et le temps de résidence in vivo des anticorps. Ce système sera administré par voie sous-cutanée et permettra un control précis de la libération du produit et de son niveau plasmatique. Cette plateforme technologique nous permettra de réduire la fréquence d’injection, de réduire potentiellement les effets secondaires et de maintenir des concentrations élevées en anticorps tout en améliorant la vie des patients atteints de maladies chroniques autoimmunes ou inflammatoires. Les formulations à base de polymères synthétiques, biodégradables (PLGA) ont été sélectionnés comme véhicules pour encapsuler les anticorps. Ils offrent en effet une bonne protection pour les anticorps and permettent une libération contrôlée de ceux-ci pendant une période définie. Après l’évaluation de différents méthodes d’encapsulation tels que les procédés d’eau-dans-huile-dans-eau (w/o/w) et solide-dans-huile-dans-eau (s/o/w), l’encapsulation des anticorps sous forme solide apparaissait plus apporpriée pour produire des microsphères de polymère chargées en anticorps. Cette technique nous permettait de maintenir l’anticorps sous sa forme monomérique et d’éviter la formation d’agrégats insolubles qui apparaissaient principalement à l’interface eau/huile. La première partie du projet a été d’optimiser à la fois la méthode nous permettant d’obtenir les anticorps sous forme de particules solides (spray-drying) et la méthode d’encapsulation de ces particules d’anticorps dans les microsphères de polymères. Cela a été réalisé par des plans d’expérience en utilisant une IgG bovine polyclonale comme molécule modèle. Durant l’optimisation du procédé de spray-drying,

les solutions aqueuses d’anticorps ont été atomisées en utilisant le mini Spray-Dryer assemblé avec une buse de pulvérisation d’un diamètre de 0.7 mm. En accord avec la taille particulaire (d(0.5) ~5 μm), la stabilité (absence de perte en monomère mesurée par chromatographie d’exclusion de taille et le rendement d’atomisation (> 60 % w/w), les paramètres d’atomisation ont été fixés: 3 mL/min pour le débit de liquide, 130°C /75°C pour la température d’entrée / température de sortie, 800 L/h pour le débit d’air d’atomisation et 30 m3/h pour le débit d’air de séchage. Pour le s/o/w, le dichlorométhane communément utilisé dans les procédés d’encapsulation a été remplacé par l’acétate d’éthyle qui est considéré comme un meilleure solvant organique en terme d’environnement et de sécurité. Les effets de plusieurs paramètres de fabrication ou de formulation ont été évalués sur les propriétés des microsphères polymériques d’anticorps (distribution de taille particulaire, taux de charge en anticorps, stabilité de l’anticorps et efficacité d’encapsulation). Plusieurs paramètres de fabrication et de formulation ont été statistiquement identifiés comme critiques pour obtenir un procédé reproductible (par exemple. La concentration en PLGA, le volume de phase externe, la vitesse d’émulsification et la quantité d’anticorps). La méthode d’encapsulation ainsi optimisée permettait d’obtenir un taux

de charge jusqu’à 6% (w/w) avec une efficacité d’encapsulation jusqu’à 60 % (w/w) tout en

préservant l’intégrité de l’anticorps encapsulé. Les microsphères produites étaient caractérisées

par un d(0.9) inférieur à 110 μm et montraient une libération après 24 h inférieure à 50 % (w/w).

Dans le seconde partie du projet, les procédés d’atomisation et d’encapsulation développés avec

l’IgG ont été appliqués à un anticorps monoclonal anti-TNF alpha humanisé pour confirmer la

conservation de l’activité de l’anticorps pendant ces procédés. La méthode s/o/w sélectionnée

permettait de produire des microsphères de PLGA chargées en anticorps avec un profil de libération jusqu’à 6 semaines et un maintien de la stabilité de l’actif. Afin de maintenir l’activité de l’anticorps, à la fois pendant le procédé d’encapsulation et pendant la libération, l’ajout d’un stabilisant tel que le tréhalose est apparu crucial ainsi que le choix du type de PLGA. Il a été démontré que l’utilisation du PLGA caractérisé par un ratio lactide :glycolide de 75 :25 (par exemple, Resomer ® RG755S) diminuait la formation d’espèces de faible poids moléculaire

pendant la dissolution. Cela contribuait à préserver l’activité de l’anticorps durant la libération à partir des microsphères. De plus, le profil de libération était modulé en fonction du type de polymère et de sa concentration. Par exemple, l’utilisation d’une solution à 10 % w/v RG755S conduisait à la production de microsphères d’anticorps avec un temps de libération sur 6

semaines. L’optimisation de la formulation et du procédé d’encapsulation a permis de produire

des microsphères avec des taux de charge en anticorps de maximum 13 % w/w. Il a été démontré

que ces microsphères, stockées à 5°C, étaient stables jusqu’à 12 semaines et que la sélection du

type de PLGA était critique pour assurer la stabilité du système. La meilleure stabilité a été

obtenue en utilisant le PLGA caractérisé par un ratio lactide :glycolide de 75 :25. Cela a été

attribué à sa plus faible vitesse de dégradation. Enfin, la libération contrôlée de l’anticorps à

partir de ces microsphères et la conservation de son activité ont été confirmées in vivo lors d’une

étude pharmacocinétique réalisée chez le rat. En conclusion, ce travail a permis de démontrer

l’application du concept d’ « emprisonnement » des composés biologiques dans des matrices

polymériques afin de les stabiliser et contrôler leur libération.
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Background: Spinal cord injury (SCI) is a devastating condition for which current treatment strategies provide no cure. Delivery of growth factors at the injury site may stimulate endogenous stem cells for nerve regeneration. Biocellulose (BC) was reported to be biocompatible, abundant and have adjustable mechanical properties. However, BC has not been tested for the treatment of SCI. Hypothesis: Composite microsphere loaded BC tubes can have a sustained protein release profile with high encapsulation efficiency and low initial burst rendering it suitable for spinal cord regeneration. Methods: Bovine serum albumin loaded poly (lactic-co-glycolic acid) microspheres were fabricated and characterized while studying the effect of different process parameters on encapsulation efficiency, release profile and morphology. Microspheres were loaded to BC tubes and were characterized morphologically and mechanically. Results: Inner phase volume and the drug:polymer ratio are the main factors impacting microsphere protein encapsulation. Furthermore, presence of different osmotic agent concentrations in the aqueous phase produced a smooth morphology while eliminating the initial burst. Finally, the composite BC tubes were fabricated, and mechanical properties were suitable for SCI applications. Contexte : Les lésions de la moelle épinière sont une maladie dévastatrice que les stratégies de traitement actuelles ne permettent pas de guérir. L'administration de facteurs de croissance sur le site de la lésion peut stimuler les cellules souches endogènes pour la régénération des nerfs. La biocellulose est biocompatible, abondante et possède des propriétés mécaniques ajustables. Cependant, la biocellulose n'a pas été testée pour le traitement des lésions de la moelle épinière. Hypothèse : Les microsphères en composite situées dans les tubes de biocellulose peuvent avoir un profil de libération soutenue de protéines avec une grande efficacité d'encapsulation ainsi qu’un faible taux de libération initial, ce qui les rend appropriés pour la régénération de la moelle épinière. Méthodes : Des microsphères de poly (acide lactique-co-glycolique) chargées d’albumine de sérum bovin ont été fabriquées et caractérisées tout en étudiant l'effet de différents paramètres du processus sur l'efficacité de l'encapsulation, le profil de libération et la morphologie. Les microsphères ont été mises dans des tubes de biocellulose et ont été entièrement caractérisées. Résultats : Le volume de la phase interne et le ratio médicament : polymère sont les principaux facteurs qui influent sur l'encapsulation des protéines en microsphères. De plus, la présence de différentes concentrations de sel dans la phase aqueuse a produit une morphologie lisse tout en éliminant la libération initiale. Enfin, les tubes de biocellulose en composite ont été fabriqués et les propriétés mécaniques étaient adaptées pour l’application sur des lésions de la moelle épinière.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirement for the degree Master of Science (MSc) in Chemistry. Johannesburg, March 2017.
A series of [Pt(diimine)(Ln-O,S)]Cl complexes, where Ln-O,S represents a series of N,N dialkyl-N’-acylthiourea ligands and diimine represents (1,10-Phenanthroline; 5,6-dimethyl 1,10-phenanthroline or [3,2-d:2’,3’-f]-quinoxaline were successfully synthesised and characterised. A new crystal structure was obtained for N,N-di(2-hydroxy)-N’ benzoylthiourea which revealed an interesting herringbone crystal packing arrangement. The cytotoxicity of the series of complexes was evaluated on H1975 lung cancer cell lines at 50 µM and 5 µM. All the complexes were highly cytotoxic with cell death of 90-98% at 50 µM. However, at 5 µM there were much more variations on cell viability percentages. Although the structure–activity relationship can only be established when the IC50 (the concentration of an inhibitor where the response is reduced by half) values are determined, it is clear that the complexes containing the methyl substituents on the 5 and 6 positions of the phenanthroline moiety were the most cytotoxic with almost 98% cell death at 5 µM. The solubility of the complexes did improve by using N,N-dialkyl-N’-acylthiourea as ancillary ligands, however aqueous solubility remains a major problem. Sulphobutyl-ether-β-cyclodextrin (captisol) and low-molecular-weight surfactant micelles as drug delivery systems were considered in attempt to improve the solubility. DOSY NMR Spectroscopy revealed that there was no inclusion complex formation between the complex and capstiol, although the chemical shift trend suggested that there is at least some interaction. The low-molecular-weight surfactant micelles were considered as an alternative, which showed some promise as a drug delivery system, since the aqueous solubility improved and a colloidal suspension was obtained.
LG2018

The objective of this thesis is to design nanoparticle (NP)-based drug delivery systems suitable for treatment of bone diseases. Two types of nanocarriers, (1) polymer coated bovine serum albumin (BSA) NPs and (2) lipid based NPs (micelles and liposomes) were investigated. The BSA NPs were prepared by a coacervation method and stabilized with a polymer coating approach. For bone-specific delivery of bone morphogenetic protein-2 (BMP-2), a copolymer polyethyleneimine-graft-poly(ethylene glycol) conjugated with 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (PEI-PEG-thiolBP) was synthesized and used for coating the BSA NPs. The particle size and ζ-potential of the NPs could be effectively modulated by the processing parameters. All the NPs showed no or low cytotoxicity (except for a high concentration of PEI), and the NP encapsulated BMP-2 displayed full retention of its bioactivity. By encapsulating 125I-labeled BMP-2, the polymer-coated NPs were assessed for hydroxyapatite (HA) affinity; all NP-encapsulated BMP-2 showed significant affinity to HA as compared with free BMP-2 in vitro, and the PEI-PEG-thiolBP coated NPs improved the in vivo retention of BMP-2 compared with uncoated NPs. However, the biodistribution of NPs after intravenous injection in a rat model indicated no beneficial effects of thiolBP-coated NPs for bone targeting. Alternatively, micelles and liposomes were prepared with a conjugate of distearoylphosphoethanolamine-polyethyleneglycol with thiolBP (DSPE-PEG-thiolBP) to create mineral-binding nanocarriers. The thiolBP-decorated liposomes also displayed a stronger binding affinity to HA and a collagen/HA (Col/HA) scaffold and gave increased retention in the scaffold in a subcutaneous implant model in rats. Taking advantage of the high HA affinity of the BP-liposomes, a sustainable release system was developed by sequestering the liposomal drugs in the Col/HA scaffolds. Three different model drugs, carboxyfluorescein, doxorubicin and lysozyme, were used to evaluate the drug release profiles from the liposome-loaded scaffolds, and all showed a slowing effect of the BP on the release of the liposome-encapsulated drugs from the Col/HA scaffolds. This liposome-scaffold combination will provide a platform for the application of various therapeutic agents for bone regeneration. In conclusion, the BP-modified NPs showed strong mineral-binding affinity. Although the systemic bone targeting was limited by physiological barriers, these NPs are promising in local delivery and controlled release of bioactive molecules for treatment of bone diseases.
Chemical Engineering

Protein therapeutics offer numerous advantages over small molecule drugs and are rapidly becoming one of the most prominent classes of therapeutics. Unfortunately, they are delivered almost exclusively by injection due to biological obstacles preventing high bioavailability via the oral route. In this work, numerous approaches to overcoming these barriers are explored. PH-Responsive poly(itaconic acid-co-N-vinylpyrrolidone) (P(IA-co-NVP)) hydrogels were synthesized, and the effects of monomer ratios, crosslinking density, microparticle size, protein size, and loading conditions were systematically evaluated using in vitro tests. P(IA-co-NVP) hydrogels demonstrated up to 69% greater equilibrium swelling at neutral conditions than previously-studied poly(methacrylic acid-co-N-vinylpyrrolidone) hydrogels and a 10-fold improvement in time-sensitive swelling experiments. Furthermore, P(IA-co-NVP) hydrogel microparticles demonstrated up to a 2.7-fold improvement in delivery of salmon calcitonin (sCT) compared to methacrylic acid-based systems, with a formulation comprised of a 1:2 ratio of itaconic acid to N-vinylpyrrolidone demonstrating the greatest delivery capability. Vast improvement in delivery capability was achieved using reduced ionic strength conditions during drug loading. Use of a 1.50 mM PBS buffer during loading yielded an 83-fold improvement in delivery of sCT compared to a standard 150 mM buffer. With this improvement, a daily dose of sCT could be provided using P(IA-co-NVP) microparticles in one standard-sized gel capsule. P(IA-co-NVP) was also tested with larger proteins urokinase and Rituxan. Crosslinking density provided a facile method for tuning hydrogels to accommodate a wide range of protein sizes. The effects of protein PEGylation were also explored. PEGylated sCT displayed lower release from P(IA-co-NVP) microparticles, but displayed increased apparent permeability across a Caco-2 monolayer by two orders of magnitude. Therefore, PEG-containing systems could yield high bioavailability of orally delivered proteins. Finally, a modified SELEX protocol for cellular selection of transcellular transport-initiating aptamers was developed and used to identify aptamer sequences showing enhanced intestinal perfusion. Over three selection cycles, the selected aptamer library showed significant increases in absorption, and from an initial library of 1.1 trillion sequences, 5-10 sequences were selected that demonstrated up to 10-fold amplification compared to the naïve library. These sequences could provide a means of overcoming the significant final barrier of intestinal absorption.
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Biophysics has seen unprecedented progress, applying concepts from physics to study intriguing biological phenomena. Further advances in this field require fundamental understanding of various processes at the nanoscale and development of appropriate methods and models for different applications. Molecular simulation is playing an ever-increasing role for these purposes. In this thesis, I have examined the structure, dynamics and thermodynamics of various biomolecules of interest using molecular simulation and theoretical modeling. The thesis is organized as follows. In the 1st chapter, I briefly introduce various bioactive molecules and relevant biological phenomena. The 2nd chapter consists of detailed descriptions of simulation methodologies and theoretical frameworks. These include classical molecular dynamics (MD) simulations, advanced sampling techniques for free energy calculations, and various entropy calculation methods. In chapter 3, we propose a carbon nanotube (CNT)-based drug-delivery method. One of the major challenges of nanomedicine and gene therapy is the effective delivery of drugs and genes across cell membranes. Generally, bioactive molecules used as drugs or drug-delivery vehicles cannot passively pass through the cell membrane due to the high penalty associated with membrane rupture. We show via MD simulations that molecules of various shapes, sizes and chemistries can spontaneously enter a membrane-spanning CNT nanopore. We study the thermodynamics of entry of several molecules of interest, such as dendrimers, asiRNA, ssDNA and ubiquitin protein. We show that another free CNT can spontaneously enter the CNT nanopore and eject the encapsulated molecule out of the nanopore. In this way, a macromolecule can be translocated across the cell membrane. We also verify the thermodynamic feasibility of the proposed method. This method should work for other molecules as well, and hence could be potentially useful for drug-delivery applications. The fourth chapter deals with the understanding of complex force-dependent protein unfolding kinetics. For some proteins, e.g., ubiquitin, the unfolding rate at very low forces doesn't vary much up to a critical force, after which the rate increases exponentially by increasing the force further. This crossover in the unfolding rate can be due to one of the following two scenarios. First, there are two unfolding pathways for the protein and pathway-switch occurs after the critical force. Second, the unfolding pathway can change continuously due to force-dependent modifications in the free-energy landscape. By performing nonequilibrium MD simulations of ubiquitin at forces ranging 20–800 pN, we find a crossover in the unfolding rate and show that the crossover is due to the second scenario. We rationalize the results by using multidimensional transition state theory. The findings from this chapter will have implications in understanding the folding/unfolding kinetics of protein which is one of the outstanding problems of the current century. In the 5th chapter, we decipher the molecular mechanism and thermodynamic driving force for lower critical solution temperature (LCST) phase behavior of the aqueous solution of proteins induced by multivalent ions, observed recently in experiments. LCST phase behavior manifests itself as phase separation of the protein–salt solution upon heating. This has been attributed to entropy effects. We use MD simulation along with the two-phase thermodynamic method for entropy calculation. Our simulations reveal two key steps that help in explaining the LCST phase behavior. First, the cations binding to the protein: this requires the release of tightly bound water molecules from the solvation shells of cations and partial desolvation of the protein surface residues, which are indeed entropy driven. Second, the protein-bound cations attract other proteins present in the solution, whose binding is again entropy driven, resulting in LCST behavior. By performing series of simulations of protein in chloride solutions of various cations (Na+, Ca2+, Mg2+ and Y3+), at temperatures ranging 283–323 K, we suggest that multivalent cation binding to any negatively charged surface can be entropy driven. These findings have direct implications for tuning the phase behavior of soft matter systems, such as reentrant condensation and protein crystallization. In a broader context, molecular-level understanding of interactions of heavy metals—usually not found in healthy cells—with different biomolecules can provide insights for carcinogenicity and neurotoxicity induced by exposure to such environmental contaminants. In chapter 6, we provide a molecular-level understanding of how intercalation of a drug affects DNA mechanics. Most of the anticancer drugs are known to intercalate in-between two consecutive base-pairs of a double-stranded DNA (dsDNA). These DNA-intercalators are believed to hinder DNA replication and transcription, eventually leading to cell death—thus acting as anticancer drugs. We probe, using MD simulations, change in the mechanical properties of the intercalated drug–DNA complexes for two intercalators, daunomycin and ethidium. We find that, upon drug intercalation, the persistence length and bending modulus of dsDNA don’t change significantly, whereas its stretch modulus increases by as much as 65%. Steered MD simulations also reveal that it requires higher forces to stretch the drug-intercalated dsDNA complexes than the bare dsDNA. Adopting various pulling protocols to study force-induced DNA melting, we find that dissociation of the dsDNA complex becomes difficult in the presence of intercalators. The results obtained here provide a plausible mechanism of action of the anticancer drugs—i.e., via modifying the mechanical properties of DNA. Finally, in chapter 7, I summarize all the results with concluding remarks and future outlooks

Recent advances in molecular biology have led to an explosive growth in the number of peptide and protein drugs derived from both recombinant technology and conventional peptide drug design. However, development of peptide and protein therapeutics has proven to be very challenging because of inadequate physical and chemical stability. In recent years, particle engineering processes have become promising approaches for enhancement of protein stability as well as provide options for more delivery routes. In this research program, spray freezing into liquid (SFL) process was developed and optimized in order to achieve broad platform and application in protein and peptide drug delivery systems. The overall goal of this research was to produce stabilized protein and peptide microparticles for various drug delivery systems by using SFL particle engineering technology. Firstly, the use of the SFL process to produce peptide microparticles was investigated. Insulin microparticles produced by the SFL process were highly porous, low tap density and narrow particle size distribution. The influence of the SFL process parameters and excipients on the physicochemical properties of peptide microparticles was determined and compared to the widely used particle formation technique--freeze-drying. The SFL process was further used to produce protein microparticles. In the study, bovine serum albumin (BSA), a medium sized protein, was used as a model drug. The influence of SFL process parameters and excipients on the stability of BSA was studied. Very low monomer loss of BSA was found in this study even though the specific surface area of the powder was very high. Results also demonstrated that the SFL process had minimal influence on protein structure. The SFL process was further investigated by comparing the SFL process to spray freeze drying process (SFD), which is a relatively new process to produce protein and peptide microparticles. The influence of atomization, freezing and drying on the stability of lysozyme was investigated for both the SFL and SFD process. This study tested the hypothesis that the SFL process is a better process than SFD process because of avoiding air-liquid interface and minimum interfacial surface absorption of protein in SFL process. The particle size of protein and peptide microparticles produced by SFL process was further reduced to nanoparticles by sonication or homogenization processes in organic solvent. In this study, the influence of process parameters on the particle size and enzyme activity of lysozyme was investigated. The results showed that sonication or homogenization did not influence the enzyme activity of lysozyme. Lastly, insulin and insulin/dextran microparticles produced by SFL the process was encapsulated into polymer microspheres for oral delivery. Complexation and polymer composition was studied in order to optimize release and stability of insulin. Insulin nanoparticles in microspheres minimized the release of insulin in acid with high drug loading compared to other studies. The stability of insulin was decreased by complexation to dextran sulfate. The results of this research demonstrated that the SFL process offers a highly effective approach to produce protein and peptide powders suitable for different drug delivery systems. The microparticles produced by the SFL process had desirable characteristics such as narrow particle size distribution and high porosity. The stability of protein and peptide was well maintained through the SFL process. Therefore, SFL process is an effective particle engineering process for protein and peptide pharmaceuticals.
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博士
國防醫學院
生命科學研究所
91
ABSTRACT Microparticles containing ovalbumin as a model protein and using poly(L-lactide; PLA) or poly(DL- lactic-co-glycolic acid; PLGA) as polymer material were prepared by a water-in oil-in water emulsion solvent evaporation technique. The particle solidifying time, protein leakage pattern, protein distribution in the microparticle, and protein loading efficiency were measured with time functions of microparticle formation to clarify the particle-formatting pathway. Compared adding sodium chloride or not in the external phase of w/o/w multiple emulsion evaluated the affected particle solidification process, the pathway of protein loss during drug encapsulation, and developed a novel surface-indented microparticle with higher protein loading and larger surface adsorption capacity were obtained. A gas chromatography analytical method was established to determine the evaporative behavior of dichloromethane (DCM) during the solidifying process. The analytical method had a minimum quantitative concentration of DCM 7.3 mM and good linearity from 7.5 to 75 mM with coefficient of variations 1.2%~3.9%. The OVA-loaded PLA microparticle from semi-solid dispersion changing to solid dispersion formation characteristics happened within a 10-minute period. The disappearances of DCM levels in total mixtures during the solidifying process were described as a function of time by zero-order (0~15 min) and first-order (after 15 min) kinetics. In this period, the remaining water and particle size of microparticle significantly decreased. Total protein leakage amount was reduced after adding NaCl in the continuous aqueous phase, which resulted from reducing the particle solidifying time and protein leakage rate during particle formation periods. The microparticle of loading efficiency, yield, total content and deep layer protein content all obviously increased. OVA-loaded PLGA microparticles prepared by with and without sodium chloride in the continuous phase were evaluated the particle formatting process. When adding NaCl in the external phase resulted in a faster solidifying crust-like structure formed as a barrier to remarkably reduce OVA loss and improve OVA content from 40.1 to 72.8 μg/mg. The total entrapment efficiency also highly increased from 43% to 72%. However, the microparticle formation of the crust-like surface structure was affected by entrapped drugs, salt content in the external phase and aqueous volume in the inner phase.

碩士
國立宜蘭大學
生物技術研究所碩士班
97
Since the injection administration as the peptides and proteins delivery system, it imposes discomfort and inconvenience on patients, particularly for a long-term treatment. Therefore, the goal of this thesis is to find out an oral delivery system for improving the absorption of therapeutic protein drugs. Emulsion is one of strategies known delivery vehicles for those oral medicines. Water-in-oil -in-water (W/O/W) multiple emulsion has been known to be possible to protect protein drugs from enzyme degradation and enhance the absorption of hydrophilic drugs to systemic circulation. Therefore, we tried to use granulocyte colony-stimulating factor (G-CSF) as the indicator to test the emulsion formulation for the oral delivery system in the study. In this two-step emulsification procedure, the data of the droplet size (2 μm of diameter), encapsulation efficiency (40±5%), stability (at pH 1.2 for 2 hrs and 60°C for 5 days), permeability (into Caco-2 cell monolayer with 30 min) and in vitro cytotoxicity studies all showed that the emulsion formulation was indicated to promote the absorption of proteins drugs by oral administration. Thus, we used the E. coli expression system to express and purify the recombinant human G-CSF (rhG-CSF). And then, rhG-CSF was incorporated into the inner aqueous phase of the emulsion for the test of oral bioavailability. After 6 hrs of oral administration of various dosages of rhG-CSF in solution or in emulsion for 7 days, the proliferations of leukocytes in the two treatments with the highest dose of rhG-CSF (2,500 μg/kg/day) were significantly higher than the blank emulsion (p < 0.01). However, two of them, rhG-CSF in emulsion and not in emulsion, were not different. After 12 hrs of oral administration, mice, administrated by 50, 500, and 2500 μg/kg/day rhG-CSF emulsion, all presented a significant elevation in leukocyte numbers when compared to the blank emulsion (p < 0.01). Finally, it was indicated about the advantages of the W/O/W multiple emulsions with encapsulation efficiency, penetrating ability, and stability to resist acid pH (1.2) and high temperature (60°C). In the future, it could be potential for protein drugs delivery by oral administration.

Targeted delivery aids in minimizing most of the drug-originated systemic toxic effects as well as improving the pharmacokinetic properties of anticancer therapeutics. Tumor targeting using hyaluronic acid (HA) as the targeting ligand has attracted a great deal of interest among a host of strategies developed to target the overexpressed tumor specific receptors. HA is an endogenous molecule that possesses a lot of biological functions in the human body. The role of HA synthases, HA degrading enzymes and the interaction of HA with its primary receptor CD44 in tumor metastasis and angiogenesis is really complex and controversial to date. However, overexpression of CD44receptors on tumor surface has been well studied, which have been utilized to direct tumor targeted drugs. Most of the HA based targeting systems were HA drug conjugates and surface modified colloidal carriers which required covalent modification. The lack of accurate structural characterization of these systems resulted in modification of HA binding sites that could affect the efficient cellular uptake. LbL technique is a simple and facile method to incorporate several materials into polyelectrolyte assemblies for drug delivery applications. HA being a negatively charged polysaccharide can be easily incorporated into such systems without any covalent modification. Although HA based polyelectrolyte multilayer films and microcapsules have been reported in combination with polycations like PAH, PLL and chitosan, their application as targeted drug delivery systems have not yet been explored. Herein, two LbL architectures with HA as the terminal layer have been investigated as targeted drug carriers, which can recognize overexpressed CD44 receptors in metastatic breast cancer cells. In the first part of the thesis, a novel polyelectrolyte nanocapsule system composed of biopolymers HA and protamine sulphate (PR) as the wall components was prepared and characterized. These pH and enzyme responsive nanocapsules were then utilized for efficient loading and release of anticancer drug doxorubicin (dox). Higher drug release was observed in simulated intracellular conditions like acidic pH and presence of hyaluronidase enzyme as compared to physiological pH. In the second part of the thesis, dox incorporated bovine serum albumin (BSA) nanoparticles modified with HA-Poly(l-Lysine) multilayers were developed and characterized. The drug release pattern of the dox loaded BSA nanoparticles was found to depend on the presence of a protease enzyme trypsin than pH variations. Both of these drug delivery systems were then evaluated for their cell targeting efficiency and cytotoxicity in CD44+ positive metastatic breast cancer cell line MDA MB 231. The final layer HA facilitated targeted delivery of these drug carriers via CD44 receptor mediated endocytosis. The enhanced cellular uptake followed by sustained delivery of dox by virtue of slow intracellular enzymatic degradation of the drug carriers resulted in their improved cytotoxicity as compared to free dox. Further in vitro biodistribution and tumor suppression efficiency of both the systems were studied in breast cancer xenograft models using BALB/c nude mice. Enhance accumulation of dox in the tumor tissue and significant tumor reduction were observed when treated with encapsulated dox using the HA based nanocarriers as opposed to free dox.