Lee, Siu Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 89-91). / Abstracts in English and Chinese. / Content --- p.i / Acknowledgments --- p.iv / Abstract --- p.V / Abbreviations and Acronyms --- p.vii / Publications Originated from the Work of this Thesis --- p.ix / Chapter Chapter 1- --- Introduction / Chapter 1.1 --- Nanoparticle-based drug delivery --- p.1 / Chapter 1.2 --- Magnetic nanoparticle --- p.5 / Chapter 1.3 --- Iron oxide nanoparticle --- p.6 / Chapter 1.3.1 --- Coprecipitation --- p.7 / Chapter 1.3.2 --- Hydrothermal reaction --- p.7 / Chapter 1.3.3 --- Sol-gel reaction --- p.8 / Chapter 1.3.4 --- Solvothermal reaction --- p.8 / Chapter 1.3.5 --- Architecture of iron oxide nanoparticles as drug carriers --- p.9 / Chapter 1.4 --- Supramolecular chemistry involved in controlled release drug delivery system --- p.10 / Chapter 1.5 --- Nano valve --- p.15 / Chapter 1.6 --- Aim of project --- p.17 / Chapter Chapter 2- --- Stimuli-Responsive Drug Delivery Nanosystems based on Fe3O4@SiO2@crown ether Nanoparticles / Chapter 2.1 --- Background --- p.19 / Chapter 2.2 --- Synthesis of the dibenzo-crown ethers --- p.21 / Chapter 2.3 --- Synthetic method of functionalized nanoparticles --- p.22 / Chapter 2.4 --- Characterization of dibenzo-crown ethers --- p.25 / Chapter 2.4.1 --- Nuclear magnetic resonance (NMR) spectroscopy --- p.25 / Chapter 2.4.2 --- Mass spectrometry (MS) --- p.27 / Chapter 2.4.3 --- Infrared (IR) spectroscopy --- p.28 / Chapter 2.5 --- Characterization of nanoparticles --- p.29 / Chapter 2.5.1 --- Transmission electron microscopy (TEM) --- p.29 / Chapter 2.5.2 --- Energy-dispersive X-ray (EDX) spectroscopy --- p.34 / Chapter 2.5.3 --- IR spectroscopy --- p.39 / Chapter 2.5.4 --- Thermogravimetric analysis (TGA) --- p.41 / Chapter 2.5.5 --- Nitrogen absorption/desorption isotherms --- p.44 / Chapter 2.6 --- Biological study of functionalized nanoparticles --- p.45 / Chapter 2.6.1 --- Cytotoxicity study --- p.45 / Chapter 2.6.2 --- Cell adhesion study --- p.46 / Chapter 2.6.3 --- Cell proliferation study --- p.47 / Chapter 2.6.4 --- Cellular uptake of nanoparticles --- p.50 / Chapter 2.7 --- Drug loading under different stimuli --- p.54 / Chapter 2.8 --- Drug release profile of nanoparticles --- p.61 / Chapter 2.9 --- MRI study of nanoparticles --- p.67 / Chapter 2.10 --- Conclusion --- p.69 / Chapter Chapter 3- --- Experimental Procedures / Chapter 3.1 --- General Information --- p.72 / Chapter 3.2 --- General procedure of synthesis of polyethers 3a-b --- p.73 / Chapter 3.2.1 --- Synthesis of 3a --- p.74 / Chapter 3.2.2 --- Synthesis of 3b --- p.74 / Chapter 3.3 --- General procedure of synthesis of diesters 4a-b --- p.75 / Chapter 3.3.1 --- Synthesis of 4a --- p.75 / Chapter 3.3.2 --- Synthesis of 4b --- p.76 / Chapter 3.4 --- General procedure of synthesis of dibenzo crown ether esters 5a-c --- p.77 / Chapter 3.4.1 --- Synthesis of 5a --- p.77 / Chapter 3.4.2 --- Synthesis of 5b --- p.78 / Chapter 3.4.3 --- Synthesis of 5c --- p.78 / Chapter 3.5 --- General procedure of synthesis of dibenzo-crown ethers la-c --- p.79 / Chapter 3.5.1 --- Synthesis of la --- p.80 / Chapter 3.5.2 --- Synthesis of lb --- p.80 / Chapter 3.5.3 --- Synthesis of lc --- p.81 / Chapter 3.6 --- Preparation of superparamagnetic Fe3O4 nanoparticle with an average diameter 120 nm --- p.81 / Chapter 3.7 --- Preparation of core/shell Fe3O4@SiO2 nanoparticle --- p.82 / Chapter 3.8 --- Preparation of Fe3O4@SiO2@meso(CTAB)-Si02 nanoparticle --- p.82 / Chapter 3.9 --- Preparation of Fe3O4@SiO2@meso(CTAB)-SiO2-NH2 nanoparticle --- p.83 / Chapter 3.10 --- Preparation of Fe3O4@SiO2@meso-SiO2@crown ether(a-c) nanoparticles --- p.83 / Chapter 3.11 --- Protocol of biological study of functionalized nanoparticles --- p.84 / Chapter 3.11.1 --- MTT protocol --- p.84 / Chapter 3.11.2 --- Cytotoxicity study --- p.84 / Chapter 3.11.3 --- Cell adhesion study --- p.85 / Chapter 3.11.4 --- Cell proliferation study --- p.85 / Chapter 3.11.5 --- Cellular uptake of functionalized nanoparticles --- p.85 / Chapter 3.12 --- Drug loading of functionalized nanoparticles --- p.86 / Chapter 3.13 --- Drug release profile of functionalized nanoparticles --- p.87 / Chapter 3.14 --- MRI study of nanoparticles --- p.87 / References --- p.89 / Appendix / List of Spectra --- p.A-1

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.

The invasive nature and undesirable side-effects related to conventional cancer therapy, such as surgery and chemotherapy, have led to the development of novel drug delivery systems (DDS). A minimally invasive DDS using near-infrared (NIR) light as a trigger for drug release is investigated to reduce the adverse side-effects triggered by systemic delivery of chemotherapeutic drugs. The low tissue absorbance in the NIR region, λ = 650–2500 nm, allows the irradiation to penetrate through tissues to release cisplatin from a NIR-sensitive nanocomposite of Au-Au₂S. Our laboratory has recently shown that cisplatin can be effectively released from Au-Au₂S upon NIR irradiation. Cisplatin was loaded onto Au-Au₂S through its adsorption on COOH-functionalized alkanethiols coated on Au-Au₂S. The current work focuses on the development of methods to control the release of cisplatin. Drug release is controlled by either the irradiation parameters or the type of coatings. The effect of different coatings on NIR sensitivity and drug release is investigated. Molecular layers of HS-(CH₂)n-COOH and HS-CH₂-COO-CH₂(CH₂CH₂O)xCH₂-COOH have been successfully coated onto Au-Au₂S. The effect of different surface layers on drug adsorption is being examined. In addition, a mathematical model has been developed to describe the thermal effects of different irradiation parameters on soft tissues. / Singapore-MIT Alliance (SMA)

Glioblastoma multiforme (GBM) is the most common malignant central nervous system tumor. The ability of glioma cells to rapidly disperse and invade healthy brain tissue, coupled with their high resistance to chemotherapy and radiation have resulted in extremely poor prognoses among patients. In recent years, nitric oxide (NO) has been discovered to play a ubiquitous of role in human physiology and studies have shown that, at sufficient concentrations, NO is able to induce apoptosis as well as chemosensitization in tumor cells. This thesis discusses the synthesis and characterization of targeted NO donors for the treatment of GBM. Two glioma targeting biomolecules, Chlorotoxin (CTX) and VTWTPQAWFQWVGGGSKKKKK (VTW) were reacted with NO gas to synthesize NO donors. These NO donors, CTX-NO and VTW-NO, released NO for over 3 days and were able to induce cytotoxicity in a dose dependent manner in glioma cells. The biggest advantage, a result of the targeted delivery of NO, was that the NO donors did not have toxic effects on astrocytes and endothelial cells. To characterize the chemosensitizing effects of CTX-NO, cells were incubated with CTX-NO prior to exposure to temozolomide (TMZ) or carmustine (BCNU). These drugs are the most popular chemotherapeutics used in the treatment of GBM, but have only shown modest improvements in patient survival. Viability studies showed that CTX-NO selectively elicited chemosensitivity in glioma cells, whereas the chemosensitivty of astrocytes and endothelial cells remained unaffected. Further investigation showed that CTX-NO pretreatment decreased O6-methylguanine DNA methyltransferase (MGMT) and p53 levels, suggesting that a decrease in DNA repair ability may be the mechanism by which chemosensitivity is induced. Lastly, the effects of CTX-NO on glioma cell invasion and migration were studied using Boyden chamber and modified scratch assays. Non-toxic doses of CTX-NO decreased glioma cell invasion in a dose dependent manner. Studies quantifying matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) surface expression demonstrated that while MMP-2 expression was decreased by both CTX and CTX-NO, MMP-9 expression was decreased only by CTX-NO. Furthermore quantifying MMP-2 and MMP-9 activity levels showed that NO and CTX work synergistically to decrease the activity of the enzymes. These studies demonstrate that the decrease in glioma invasion resulting from CTX-NO treatment was partially a consequence of decreased levels of surface and activated MMP-2 and MMP-9. The work presented in this thesis describes a novel approach to treating GBM that can be modified to develop treatments for various other tumors. Furthermore this is the first study to develop glioma-targeting NO donors.

Microemulsions have gained interest from the pharmaceutical industry due to their ability to co-solubilize hydrophilic and lipophilic drugs, and to provide enhanced drug penetration. In this work, thermosensitive gelatin- and poloxamer 407-stabilized microemulsion-based gels (MBGs) were formulated using alcohol-free, low toxicity and low viscosity lecithin-based linker microemulsions. The addition of gelatin to water-rich bicontinuous microemulsions induced the formation of clear viscoelastic gels containing an oil-rich microemulsion as the gelatin seemed to dehydrate the original microemulsion. The addition of poloxamer 407 to water-continuous microemulsions produced MBGs with different gelation temperatures. High concentrations of lipophilic components in the microemulsion, particularly the oil, reduced sol-gel transition temperature, while hydrophilic components increased sol-gel transition temperature. Gelatin and poloxamer MBGs provided desirable viscoelastic properties for ophthalmic and transdermal applications with minimal impact on the transport properties of the original microemulsions.

Microemulsions have gained interest from the pharmaceutical industry due to their ability to co-solubilize hydrophilic and lipophilic drugs, and to provide enhanced drug penetration. In this work, thermosensitive gelatin- and poloxamer 407-stabilized microemulsion-based gels (MBGs) were formulated using alcohol-free, low toxicity and low viscosity lecithin-based linker microemulsions. The addition of gelatin to water-rich bicontinuous microemulsions induced the formation of clear viscoelastic gels containing an oil-rich microemulsion as the gelatin seemed to dehydrate the original microemulsion. The addition of poloxamer 407 to water-continuous microemulsions produced MBGs with different gelation temperatures. High concentrations of lipophilic components in the microemulsion, particularly the oil, reduced sol-gel transition temperature, while hydrophilic components increased sol-gel transition temperature. Gelatin and poloxamer MBGs provided desirable viscoelastic properties for ophthalmic and transdermal applications with minimal impact on the transport properties of the original microemulsions.

No description available.

Targeted intracellular delivery is a goal of many novel drug delivery systems to treat site-specific diseases thereby increasing the effectiveness of drugs and reducing side effects associated with current drug administration. The development of ultrasound-enhanced delivery is aimed at providing a targeted means to deliver drugs and genes intracellularly by utilizing ultrasound s ability to non-invasively focus energy into the body and generate cavitation, which has been found to cause transient poration of cells. To address some of the current issues in this field, the goals of this study were (i) to develop a measurement of cavitation to correlate with cellular bioeffects and (ii) to evaluate the ability of ultrasound to target delivery into cells in viable tissue. In addition, this study sought to exploit the shear-based mechanism of cavitation by (iii) developing a simplified device to expose cells to shear stress and cause intracellular uptake of molecules. This study has shown that broadband noise levels of frequency spectra processed from cavitation sound emissions can be used to quantify the kinetic activity of cavitation and provide a unifying parameter to correlate with the cellular bioeffects. We further demonstrated that ultrasound can target delivery of molecules into endothelial and smooth muscle cells in viable arterial tissue and determined approximate acoustic energies relevant to drug delivery applications. Lastly, we developed a novel device to expose cells to high-magnitude shear stress for short durations by using microfluidics and demonstrated the ability of this method to cause delivery of small and macromolecules into cells. In conclusion, this work has advanced the field of ultrasound-enhanced delivery in two major areas: (i) developing a real-time non-invasive measurement to correlate with intracellular uptake and viability that can be used as means to predict and control bioeffects in the lab and potentially the clinic and (ii) quantitatively evaluating the intracellular uptake into viable cells in tissue due to ultrasound that suggest applications to treat cardiovascular diseases and dysfunctions. Finally, by using shear forces generated in microchannels, we have fabricated a simple and inexpensive device to cause intracellular uptake of small and large molecules, which may have applications in biotechnology.

Traditional methods of drug delivery to the eye include topical application, intraocular injection and systemic administration; however, each method has its limitation to efficiently delivery drugs to the back of the eye. In this study, microneedles were tested to provide targeted drug delivery into the eye in a minimally invasive way. To better interpret subsequent microneedle studies, we first quantified lateral drug diffusion profile within the sclera, by carrying out a diffusion study of a model compound, sulforhodamine, through human cadaver sclera, and developing a theoretical model for prediction of drug delivery kinetics and distribution. The results showed that measurable amounts of sulforhodamine were detected at distances of 5 and 10 mm from the sulforhodamine donor reservoir at 4 h and 3 days, respectively. The effective lateral diffusivity of sulforhodamine was determined to be 3.82 x 10-6 cm2/s, which is similar in magnitude to the transverse diffusivity. We next assessed the capability of using coated solid metal microneedles to deliver drugs into the ocular tissue in both in vitro and in vivo scenarios. The in vitro insertion tests showed that these microneedles were mechanically strong enough to penetrate into human cadaver sclera, and the coating solution rapidly dissolved off the needles after insertion and had been deposited within the tissue. In the in vivo experiments, microneedle delivery exhibited elevated fluorescein levels in the rabbit eye 60 times greater than that delivered by topical application of the equivalent dose. Similarly, microneedle delivery of pilocarpine caused rapid and extensive pupil constriction. Safety exams reported no inflammatory responses in the eye after microneedle administrations. We also used hollow glass microneedles to infuse solutions into the sclera tissue in vitro and examined the physiological barriers for flow. On average, 18 microliters of sulforhodamine solution and a solution containing nanoparticles was delivered into the sclera upon retraction of the microneedle. Successful delivery of micron-sized particles into the sclera could be improved by breaking down tightly packed collagen and GAG fibers using either collagenase or hyaluronidase.

Currently, biopharmaceuticals including vaccines, proteins, and DNA are delivered almost exclusively through the parenteral route using hypodermic needles. However, injection by hypodermic needles generates pain and causes bleeding. Disposal of these needles also produces biohazardous sharp waste. An alternative delivery tool called microneedles may solve these issues. Microneedles are micron-size needles that deliver drugs or biopharmaceuticals into skin by creating tiny channels in the skin. This thesis focuses on dissolving microneedles in which the needle tips dissolve and release the encapsulated drug or vaccine upon insertion. The project aimed to (i) design and optimize dissolving microneedles for efficient drug and vaccine delivery to the skin, (ii) maintain vaccine stability over long-term storage, and (iii) immunize animals using vaccine encapsulated microneedles. The results showed that influenza vaccine encapsulated in microneedles was more thermally stable than unprocessed vaccine solution over prolonged periods of storage time. In addition, mice immunized with microneedles containing influenza vaccine offered full protection against lethal influenza virus infection. As a result, we envision the newly developed dissolving microneedle system can be a safe, patient compliant, easy to-use and self-administered method for rapid drug and vaccine delivery to the skin.