A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science in Medicine 2015 / The dissertation aims to discuss the disulphide and thiol chemistry for use in drug delivery. In particular it focuses on the use of the modified native ovalbumin polymer as a vehicle for the thiol containing captopril. The binding capabilities of thiols expand the area in which peptides and proteins can be used as potential therapeutic drug carriers. It is important that drug delivery systems enhance drug storage stability and in vivo particle stability while delivering the drug efficiently. As part of the developing novel drug delivery systems, thiol-based chemical reactions are distinctive role players in stabilizing disulphide bioconjugated nanostructures for use as efficient drug carrier vehicles in vivo. A review of the current approaches for designing, optimizing and functionalizing nanostructures and conjugates by thiol chemistry modifications was explored. Captopril (Cp) is an Angiotensin-Converting Enzyme (ACE) inhibitor, which acts as an anti-hypertensive, structurally contains a free reactive thiol that binds variably via the thiol/disulphide reaction. A single dose of captopril can regulate hypertension for up to eight hours and the duration of the antihypertensive action of a single dose of 35-75 mg would be taken at 8 hour intervals for 24 hours. Hence the necessities in developing a sustained controlled release ovalbumin carrier system to maintain relatively constant blood pressure levels for 24 hours. The research focused on the construction, characterization and optimization of the thiol conjugated complex for sustained oral drug delivery. The thiol/disulphide-functionalized captopril-ovalbumin conjugate complex was assessed in terms of the structural characteristics and the thiol-disulphide covalent substitution reaction. For analysis of the conjugation complex, the Fourier Transmission IR-spectroscopy (FTIR), H+ NMR and Differential Scanning Calorimetry (DSC) was performed and used to confirm conjugation. Preliminary studies focused on a comparative study of sodium alginate, polyvinyl alcohol and hydroxypropylmethylcellulose hydrogel formulations for the release testing and drug entrapment of the ovalbumin-captopril conjugate complex. Utilizing this data, a series of process variables were used to achieve an optimized formulation through a Box- Behnken statistical design. Furthermore the drug release profiles of the optimised formulation were then analyzed in vitro and in vivo. The captopril released from the formulation was high with a cumulative release of 82%. In vivo analysis was the final testing to verify the validity of the ovalbumin-captopril conjugate complex encapsulated in sodium alginate and utilized a pig model. Ultra Performance Liquid Chromatography (UPLC) blood analysis revealed increased blood levels of captopril (Cmax Cp=33.2ng/mL) in relation to conventional dosage forms validating prolonged (24 hour) site-specific release and increased bioavailability. In conclusion, our validated method was successfully applied to the pharmacokinetic studies of captopril in the blood plasma samples.

The aim of this research was to formulate, characterise and assess the feasibility of a novel drug delivery system known as the in situ gelling matrix (ISGM) where a hydrophilic polymer is suspended in a non-aqueous solvent that converts into a gel when injected subcutaneously or intramuscularly thus giving a controlled release matrix for a drug. Although the concept has been patented with claims that this kind of drug delivery is achievable in theory for a wide variety of candidate substances, actual formulation studies for making a commercially viable product for this technology are completely lacking in practice. The research embodied in this thesis addresses this lack. Initial studies involved conducting a biocompatibility study using the HET-CAM (hens egg test - chorioallantoic membrane) test on a range of possible ingredients for the delivery system. The materials deemed biocompatible were then carried through to a screening process where the physical stability of the hydrophilic polymers in non-aqueous solvents was monitored. It was found that the hydrophilic polymers tested sedimented rapidly in the non-aqueous solvents indicating such a system was not physically stable. Consequently, density-inducing or viscosity-inducing agents were added to the non-aqueous solvents to retard the sedimentation rate. The addition of polycarbophil, a viscosity-inducing agent, clearly increased the viscosity of the system. However, undesirable formation of polycarbophil globules occurred during the manufacturing process, which caused batch-to-batch variations in the viscosity of the continuous phase. Various manufacturing methods were tested before arriving at the optimum procedure to prevent globule formation using a high speed dispersion tool. A final physical sedimentation analysis of candidate continuous phases and hydrophilic polymers was conducted for determining the ideal combination of ingredients to use in the system. These investigations finally led to the adoption of an optimum mix of components consisting of 10% (w/w) hydroxypropyl methylcellulose (HPMC) (the hydrophilic polymer) suspended in a continuous phase of propylene glycol (the non-aqueous solvent) containing 0.67% (w/w) polycarbophil (the viscosity inducing agent). Using this mix of components, the in situ gelling matrix system was then subjected to various characterisation studies including infrared (IR), differential scanning calorimetry (DSC), ultraviolet-visible (UV-Vis) spectrophotometry and redispersion studies. The chemical stability of the hydrophilic polymer and the continuous phase (the non-aqueous solvent and polycarbophil) was monitored and were found to be chemically stable over a 9 month period. The feasibility of the in situ gelling matrix technology as a controlled release device was assessed using the drug propranolol. In vitro drug release studies were conducted using a custom-built dissolution apparatus. The effect of various parameters such as the concentration of the hydrophilic gelling agent on the drug release rate was investigated. Increasing the concentration of the gelling agent in the formulation resulted in a slower rate of release. The drug release data were modelled using the Higuchi relationship and a power law relationship to compare the effects of the various parameters on the release rate Stability studies on the drug in the in situ gelling matrix system were carried out by storing samples in accelerated ageing conditions of 40 C / 75% relative humidity for 4 weeks. During this time, the samples were analysed each week by high performance liquid chromatography (HPLC). These demonstrated that no apparent drug degradation had occurred over the 4-week period. This indicates that the drug propranolol in the in situ gelling matrix system is stable under ambient conditions for at least 4 weeks. The results of this study demonstrated that the in situ gelling matrix technology is potentially viable as a drug delivery system and provide a practical methodology for the commercial development of such systems.

Hydrogels are three dimensional networks of hydrophilic homopolymers or copolymers generally covalently or ionically crosslinked. They interact with aqueous media by swelling to some equilibrium value by retaining the aqueous media in their structures. This study concerns the investigation of the swelling and the controlled drug release behaviour of hydrogels synthesized via the photopolymerisation process. The study of hydrogels in this project was oriented towards their biomedical applications as controlled drug delivery devices. It is a known fact that the complete conversion of monomers to polymers may not be achieved in the polymerisation process thus there is always a certain component of unreacted toxic monomers still remained in the polymer matrix. These monomers have the tendency to leach out of the polymer matrices when the polymers are in contact with an aqueous medium thus rendering the hydrogel to be nonbiocompatable. The polymers synthesized in this work were washed thoroughly in milli-Q-water and then evaluated in vitro for any possible toxic effect on human keratinocyte (HaCaT)v cells using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diaphenyl tetrazolium bromide (MTT) cell proliferation assay. The cytotoxicity results indicated that the hydrogels understudy sustained and allowed a positive growth of the HaCat cells in the duration of the cytotoxicity experiment, thus proving to be satisfactorily compatible. / Doctor of Philosophy (PhD)

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 86-92).

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Titles called 1,2,3, and 4. Includes bibliographical references (leaves 276-286). Also available on the World Wide Web.

Nanomedicine, the medical application of nanotechnology, uses nanoscale objects that exist at the interface between small molecule and the macroscopic world for medical diagnosis and treatment. One of the healthcare applications of nanomedicine is drug delivery: the development of nanoscale objects to improve therapeutics' bioavailability and pharmacokinetics. Shell crosslinked knedel-like nanoparticles (SCKs), that are self assembled from amphiphilic block copolymers into polymeric micelles and then further stabilized with crosslinkers isolated throughout the peripheral shell layer, have been investigated for drug delivery applications that take advantage of their core-shell morphology and tunable surface chemistry. SCKs are attractive nanocarriers because the cores of the SCKs are used for sequestering and protecting guests. The readily adjustable shell crosslinking density allows for gating of the guest transport into and out of the core domain, while retaining the structural integrity of the SCKs. Moreover, the highly functionalizable shell surface provides opportunity for incorporation of targeting ligands for enhanced therapeutic delivery. The optimization of nanoparticle size, surface chemistry, composition, structure, and morphology has been pursued towards maximization of the SCKs' therapeutic efficacy. With distinctively different dimensions, compositions and structures of the core and shell domains of SCKs, and an ability to modify each independently, probing the effects of each is one of the major foci of this dissertation. Utilization of a living radical polymerization technique, reversible addition-fragmentation chain transfer (RAFT) polymerization, has allowed for facile manipulation of the block lengths of the polymer precursors and thus resulted in various dimensions of the nanoparticles. SCKs constructed from poly(acrylic acid)-b-polystyrene (PAA-b-PS) with various chain lengths, have been investigated on the loading and release of doxorubicin (DOX). The effect of PEGylation on paclitaxel (PTX) loaded SCKs on the cell internalization and killing was investigated. Apart from chemotherapies, the SCKs were explored as antimicrobial agents by incorporating silver species. Conjugation of the SCK surface with a protein adhesin through amidation chemistry to promote epithelial cell targeting and internalization was developed. Nanoscale assemblies with complex morphologies constructed from a linear triblock copolymer was investigated. Furthermore, a highly multifunctional nanodevice for imaging and drug delivery functionalized with a chelator for radio-labeling, polyethylene glycol (PEG) for improved biodistribution, targeting ligands, a chromophore and a therapeutic agent was evaluated in vivo as active-targeted delivery of therapeutics.

The interest in microemulsions as transdermal delivery systems have been motivated by their large surface area for mass transfer, their high solubilization capacity of hydrophobic actives, and their ability to improve skin penetration. Lecithins (mixtures of phospholipids similar to those find in the skin) have been proposed as ideal surfactants in microemulsions due to their skin compatibility. Unfortunately, their incorporation into microemulsions used to require toxic medium-chain alcohols or viscous polymeric co-surfactants. Recently, microemulsion-base “green solvents” were formulated with lecithin and linker molecules. The main objective of this dissertation was to test this concept of linker-based lecithin microemulsions in transdermal delivery. In the first part of this study, linker-based lecithin formulations were developed using soybean lecithin as main surfactant, sorbitol monooleate as lipophilic linker, and caprylic acid/sodium caprylate as hydrophilic linkers. These additives, at the suggested concentration, are safe for cosmetic and pharmaceutical applications. The low toxicity of these formulations was confirmed in cultured human skin tissues. The solubilization and permeation of a common anaesthetic, lidocaine, was evaluated. The concept of “skin” permeability was introduced to account for the differences in solvent-skin partition when comparing different delivery systems. The linker-based lecithin microemulsion produced a substantial absorption of lidocaine into the skin, when compared to a conventional pentanol-lecithin microemulsion. The second part of this study takes advantage of the lidocaine adsorbed in the skin with the linker-based lecithin microemulsion as reservoir for in situ skin patches. The in situ patches were able to release 90% of the lidocaine over 24 hours, which is comparable to the release profile obtained from conventional polymer or gel-based patches. In the third part of this work, the role of surfactant droplets on the transport of lidocaine was studied. A mass balance model that accounted for mass transfer and partition coefficients was introduced. The parameters generated from the model confirm that in most cases the transport through the skin limits the overall penetration of lidocaine. Besides the conventional diffusion mechanism, the results suggest that surfactant droplets, carrying lidocaine, also penetrate into the skin and contribute to the accumulation of the lidocaine in the skin.

Polymeric nanoparticles have gained increased popularity for drug delivery as they not only overcome the problem of limited aqueous solubility of many hydrophobic drug molecules, but also have the potential to improve the pharmacologic properties of anticancer drugs by increasing their in vivo half-life. A series of biodegradable poly(D,L-lactide-co-2-methyl-2-carboxytrimethylene carbonate), P(LA-co-TMCC), was first synthesized by Sn(Oct)2 catalyzed bulk polymerization. In order to obtain the polymer product with a better-defined composition, the polymer synthesis was improved by using organo-catalytic ring-opening copolymerization. The copolymer molar mass and composition were controlled by varying the monomer to initiator ratio and the monomer feed ratio. By grafting amine-terminated polyethylene glycol (PEG-NH2) to the carboxylate groups on the copolymer backbone, amphiphilic copolymers were formed and self-assembled to form nanoparticles with narrow size distribution. The nanoparticle size was observed to be influenced by the polymer composition and the self-assembly conditions. To gain greater insight into the stability of these nanoparticles in blood, they were tested in both fetal bovine serum and individual serum protein solutions. By encapsulating Förster resonance energy transfer (FRET) pairs and following their release by fluorescence, these micelles demonstrated strong thermodynamic and kinetic stability in the presence of serum. By incorporating functional groups (azide or furan) on the PEG chains, either cell adhesive peptides (i.e. alkyne-functionalized GRGDS) or targeting antibodies (i.e. maleimide-modified trastuzumab) were coupled to the surface of the nanoparticles through Huisgen 1,3-dipolar cycloaddition reaction or Diels-Alder chemistry, respectively. The GRGDS modified nanoparticles showed specific binding affinity to rabbit corneal epithelial cells that express αvβ1 integrin receptors.

Polymeric nanoparticles have gained increased popularity for drug delivery as they not only overcome the problem of limited aqueous solubility of many hydrophobic drug molecules, but also have the potential to improve the pharmacologic properties of anticancer drugs by increasing their in vivo half-life. A series of biodegradable poly(D,L-lactide-co-2-methyl-2-carboxytrimethylene carbonate), P(LA-co-TMCC), was first synthesized by Sn(Oct)2 catalyzed bulk polymerization. In order to obtain the polymer product with a better-defined composition, the polymer synthesis was improved by using organo-catalytic ring-opening copolymerization. The copolymer molar mass and composition were controlled by varying the monomer to initiator ratio and the monomer feed ratio. By grafting amine-terminated polyethylene glycol (PEG-NH2) to the carboxylate groups on the copolymer backbone, amphiphilic copolymers were formed and self-assembled to form nanoparticles with narrow size distribution. The nanoparticle size was observed to be influenced by the polymer composition and the self-assembly conditions. To gain greater insight into the stability of these nanoparticles in blood, they were tested in both fetal bovine serum and individual serum protein solutions. By encapsulating Förster resonance energy transfer (FRET) pairs and following their release by fluorescence, these micelles demonstrated strong thermodynamic and kinetic stability in the presence of serum. By incorporating functional groups (azide or furan) on the PEG chains, either cell adhesive peptides (i.e. alkyne-functionalized GRGDS) or targeting antibodies (i.e. maleimide-modified trastuzumab) were coupled to the surface of the nanoparticles through Huisgen 1,3-dipolar cycloaddition reaction or Diels-Alder chemistry, respectively. The GRGDS modified nanoparticles showed specific binding affinity to rabbit corneal epithelial cells that express αvβ1 integrin receptors.

The interest in microemulsions as transdermal delivery systems have been motivated by their large surface area for mass transfer, their high solubilization capacity of hydrophobic actives, and their ability to improve skin penetration. Lecithins (mixtures of phospholipids similar to those find in the skin) have been proposed as ideal surfactants in microemulsions due to their skin compatibility. Unfortunately, their incorporation into microemulsions used to require toxic medium-chain alcohols or viscous polymeric co-surfactants. Recently, microemulsion-base “green solvents” were formulated with lecithin and linker molecules. The main objective of this dissertation was to test this concept of linker-based lecithin microemulsions in transdermal delivery. In the first part of this study, linker-based lecithin formulations were developed using soybean lecithin as main surfactant, sorbitol monooleate as lipophilic linker, and caprylic acid/sodium caprylate as hydrophilic linkers. These additives, at the suggested concentration, are safe for cosmetic and pharmaceutical applications. The low toxicity of these formulations was confirmed in cultured human skin tissues. The solubilization and permeation of a common anaesthetic, lidocaine, was evaluated. The concept of “skin” permeability was introduced to account for the differences in solvent-skin partition when comparing different delivery systems. The linker-based lecithin microemulsion produced a substantial absorption of lidocaine into the skin, when compared to a conventional pentanol-lecithin microemulsion. The second part of this study takes advantage of the lidocaine adsorbed in the skin with the linker-based lecithin microemulsion as reservoir for in situ skin patches. The in situ patches were able to release 90% of the lidocaine over 24 hours, which is comparable to the release profile obtained from conventional polymer or gel-based patches. In the third part of this work, the role of surfactant droplets on the transport of lidocaine was studied. A mass balance model that accounted for mass transfer and partition coefficients was introduced. The parameters generated from the model confirm that in most cases the transport through the skin limits the overall penetration of lidocaine. Besides the conventional diffusion mechanism, the results suggest that surfactant droplets, carrying lidocaine, also penetrate into the skin and contribute to the accumulation of the lidocaine in the skin.