Targeted therapy is a significant area of research in pharmaceutical and biomedical science. Its overall aim is to achieve maximum impact on malignant cells with minimum side effects to healthy tissue. In this thesis the capabilities of magnetic microbubbles as targeted therapeutic delivery vehicles are explored. New characterisation techniques were developed in order to understand and improve the current magnetic microbubble formulation. Electron microscopy was used to analyse the nanoscale structure of microbubble shells and observe nanoparticles attached to the shell surface. A new flow phantom was developed and the targeting of magnetic microbubbles against flow conditions corresponding to those in the human body was found to be feasible in numerous vessel sizes and flow conditions. Magnetic targeting of microbubbles was also observed in a perfused porcine liver model. Magnetic targeting was then attempted against flowing blood and a decrease in targeting efficiency observed. This was also seen for biochemical targeting and collisions with red blood cells identified as the most likely cause. Importantly, the number of magnetically targeted microbubbles significantly exceeded those targeted via biochemical interactions in both blood and water. In the second part of the thesis new types of magnetic microbubble were developed. The first exploits the fusion of nano-scale magnetic droplets with phospholipid microbubbles. In the second magnetic nanoparticles were incorporated directly into the lipid shell. The new magnetic microbubble formulation could be magnetically targeted, observed via contrast ultrasound and was successfully used to deliver siRNA to neuroblastoma cells.

Master of Science / Department of Chemistry / Santosh Aryal / Nanomedicine-based therapeutics have exhibited clear benefits when compared to unmodified drugs, which include improved pharmacokinetics, drug retention, targeting efficiency, and minimizes toxicity. Every year thousands of bone cancer cases are diagnosed in the United States. Moreover, development of bone metastasis occurs in over 80% to 90% of various cancers that metastasize and signals the entry of the disease into an incurable phase. Cancer in bones can cause pain, fractures, hypercalcemia, and compression of the spinal cord, due to deposits that can erode into the bone using bone-absorbing cells. Bisphosphonates are drugs that reduce the activity of bone-absorbing cells and targets overexpressed calcium. They are characterized pharmacologically to inhibit bone resorption, skeletal distribution, and renal elimination. In addition, they can target bone microenvironment and bind strongly with calcium. The goal of this thesis is to engineer targeted nanomedicine drug with the ability to spatiotemporally control therapeutics delivery to the bone. Herein we synthesized biopolymers with functional end group moieties as alendronate (a molecular member of bisphosphate), which can target overexpressed calcium ions at the vicinity of the bone lesion where bone resorption takes place. In order to achieve our goal, a ring opening polymerization of cyclic L-lactide initiated by ALE in the presence of catalytic amount of stannous octoate was conducted in an inert environment. Thus, formed polymers are characterized for their chemistry and physicochemical properties using various analytical tools. These polymers were characterized by nuclear magnetic resonance (¹H-NMR) and Fourier Transfer Infrared Spectrometer (FT-IR), which shows monomer conversion and the presence of amide and phosphate moiety. Thereafter we engineered bone-homing polymeric nanoparticles of 80nm diameter by nanoprecipitation for controlled delivery of Dox, a first line anticancer drug used in clinics. The in-vitro results show that the nanoparticles have the ability to accumulate and internalized into the bone cancer cells, deliver drugs efficiently, and are least toxic. Therefore, innovative and efficient bisphosphonate functionalized Poly-l-lactide polymers were synthesized to target bone microenvironment.

Sonoporation, enhanced by ultrasound contrast agents has been explored as a promising non-viral technique to achieve gene transfection and targeting drug delivery in recent years. However, the short lifespan of traditional ultrasound contrast agents like Optison® microbubbles under moderate intensity ultrasound exposure limits their application. Liposomes, as drug carriers consisting of curved spherical closed phospholipid bilayer shells, have the following characteristics: 1) The ability to encapsulate and carry hydrophilic or hydrophobic molecules. 2) The biocompatibility with cell membranes. 3) The nanometer size and the relative ease of adding special ligands to their surface to target a specific disease site. 4) The stability in the blood stream. 5) Targeted ultrasound irradiation can induce rupture of liposomes letting the drug encapsulated in them leak out to achieve controlled release of the therapeutic agents at a certain concentration and a delivery rate. In this thesis, several liposome synthesis methods are presented. Liposomes synthesized in our laboratory were characterized acoustically and optically. Anti rabbit IgG conjugated with Alexafluor 647 was delivered into Jurkat cells in a suspension containing liposomes by 10 % duty cycle ultrasound tonebursts of 2.2 MHz (the in situ spatially averged and temporally averaged intensity, ISATA = 80W/cm2) with an efficiency of 13 %. It has been experimentally shown that liposomes may be an alternative stable agent to Optison® to cause sonoporation. Furthermore, a type of nanometer-sized liposome (<300nm) was synthesized to explore the feasibility of ultrasound-triggered release from drug encapsulated lipsomes. It has been demonstrated encapsulated fluorescence materials (FITC) can be released from liposomes with an average diameter of 210 nm when exposed to high intensity focused ultrasound (HIFU) at 1.142MHz (ISPTA= 900 W/cm2). Rupture of relatively large liposomes (>100nm) and porelike defects in the membrane of small liposomes due to the excitation of HIFU were the main causes of the content release. The great enhancement of HIFU-mediated release in the nanometer-sized liposomes may prove useful for clinical applications. The presence of fine particles in Martian and lunar soil poses a significant threat to NASA’s viable long-term exploration and habitation of either the moon or Mars. It has been experimentally shown that the acoustic levitating radiation force produced by a 13.8 kHz 128 dB sound-level standing wave between a 3 cm-aperture acoustic tweeter and a reflector separated by 9 cm is strong enough to overcome the van der Waals adhesive force between the dust-particles and the reflector-surface. The majority of fine particles (> 2μm diameter) on a reflector surface can be dislodged and removed by a technique combining acoustic levitation and airflow methods. This dust removal technique may be used in space-stations or other enclosures for habitation.

In the field of biomaterials, naturally-derived and synthetic polymers are utilized individually or in combination with each other, to create bio-inspired or biomimetic materials for various bioengineering applications, including drug delivery and tissue engineering. Natural polymers, such as proteins and polysaccharides, are advantageous due to low or non-toxicity, sustainable resources, innocuous byproducts, and cell-instructive properties. Synthetic polymers offer a variety of controlled chemical and physical characteristics, with enhanced mechanical properties. Together, natural and synthetic polymers provide an almost endless supply of possibilities for the development of novel, smart materials to resolve limitations of current materials, such as limited resources, toxic components and/or harsh chemical reactions. Herein is discussed the synthetic-biological material formation for cell-instructive tissue engineering and controlled drug delivery. We hypothesized that the combination of hydrogel-based scaffold and engineered nanomaterials would assist in the development or regeneration of tissue and disease treatment. Chemically-modified alginate was formed into alginate-based nanoparticles (ABNs) to direct the intracellular delivery of proteins (e.g., growth factors) and small molecular drugs (e.g., chemotherapeutics). The ABN surface was modified with cell-targeting ligands to control drug delivery to specific cells. The ABN approach to controlled drug delivery provides a platform for studying and implementing non-traditional biological pathways for disease (e.g., osteoporosis, multiple sclerosis) and cancer treatment. Through traditional organic and polymer chemistry techniques, and materials engineering approaches, a stimuli-responsive alginate-based smart hydrogel (ASH) was developed. Physical crosslinks formed based on supramolecular networks consisting of β-cyclodextrin-alginate and a tri-block amphiphilic polymer, which also provided a reversible thermo-responsiveness to the hydrogel. The hydrogel was shear-thinning, and recovered physical crosslinks, i.e., self-healed, after un-loading. The ASH biomaterials provide a platform for injectable, therapeutics for tissue regeneration and disease treatment. Indeed, various hydrogel constituents and tunable mechanical properties created cell-instructive hydrogels which promoted tissue formation.

Pneumothorax, or a collapsed lung, is a serious medical condition resulting when air or fluid escapes the lung into the chest cavity and prevents the lung from inflating. Few viable means of sealing the damaged and leaking tissues are currently available, leading to longer hospital stays, multiple interventions, and increasing costs of care. The motivation of this dissertation is to engineer a novel polysaccharide-based therapeutic surgical sealant, which can be utilized to seal trauma-induced damage to the outer lining of the lung, i.e. pleura, preventing or reversing lung collapse to restore normal breathing function. The use of polysaccharides, such as alginate and hyaluronan, has become increasingly prevalent in biomedical and tissue engineering applications due to the ability to add functionality through chemical modification, allowing for tunable mechanical and physical properties. These hydrophilic polymer chains can be crosslinked to form hydrogels, which can retain large volumes of water and can mimic the properties of tissues found within the body. In this work, polysaccharide hydrogel sealants were engineered with well-regulated gelation and mechanical properties, and further modified to achieve adhesion to biological tissues. This was accomplished by mimicking the mechanical and physical properties of the complex tissues, and crosslinking the hydrogels in situ using a visible light-initiated system. Methacrylated alginate and oxidized alginate were successfully synthesized and utilized to fabricate adhesive sealant patches, which can adhere and seal damaged tissues in vivo. Methacrylation was implemented to allow covalent photo-crosslinking between adjacent polymer chains in solution. Here, a novel anhydrous chemistry was developed to allow for precise control over the degree of methacrylation and thus tune the mechanical properties of the resulting hydrogels by modulating the number of crosslinkable side-groups attached to the polysaccharide chain. To increase the adhesive properties of the resulting hydrogels, oxidation of the polysaccharide chain was subsequently implemented to form functional aldehyde groups capable of protein interactions through the formation of imine bonds on biological tissue surfaces. To test the performance of this multifunctional material, burst pressure testing was executed, revealing the relationship between the two distinct chemical modifications performed and the mechanical and adhesive properties of the resulting sealant. In addition, methacrylated alginate was utilized to synthesize therapeutic, drug-encapsulating hydrogel nanoparticles, which when incorporated within the polysaccharide-based surgical sealant allow for local drug release. The ability to control drug release at the site of application further broadens the potential uses of this surgical sealant patch and will be discussed further within this dissertation.

The main challenge in disease treatment is no more the discovery of new therapeutic drugs, but to provide targeted delivery of therapeutic drugs to specific sites without incurring systemic toxicity effects. An efficient way of reducing the toxicity is by encapsulating the drug with a biodegradable matrix that can provide controlled release of the drug along with local heating of the drug. Local heating can be obtained by incorporating magnetic iron oxide particles that heat upon exposure to AC electromagnetic fields. The magnetic iron oxide nanoparticles are also gaining much attention as MRI contrast agents. Thus it would be of potential benefit if a drug delivery system is designed to encapsulate the drug as well as the magnetic iron oxide nanoparticles within a biodegradable matrix, thereby providing a dual modal imaging and therapeutic delivery system. The key step in the design of a dual modal drug delivery system is the encapsulation of the magnetic iron oxide nanoparticles with polymer of choice. The magnetic iron oxide nanoparticles were encapsulated into a robust poly (styrene-co-vinylbenzylchloride-co-divinylbenzene) (PSVBDVB) to study these synthetic variations upon encapsulation with a polymer. The next step to the design of drug delivery system was to replace the PSVBDVB polymer by a biocompatible and biodegradable polymer- Poly (lactide-co-glycolide) (PLGA). The PLGA composites containing the Fe@FeOx core shell nanoparticles and the drug analog [Ru(bpy) dye] was prepared by oil-in water emulsion solvent evaporation technique. The local heating of the PLGA composites was also achieved by irradiating the Fe@FeOx nanoparticles with 2.45 GHz microwave radiations. Higher Ru(bpy) dye release from the composites by locally heating the sample with 2.45 GHz microwave pulse compared to externally heating the composite sample was achieved. The final step was the design of controlled release drug delivery system with dual modal imaging and therapeutic capabilities. To obtain narrow sized PLGA composites the Fe@FeOx nanoparticles were replaced by chloroform based ferrofluid. The ferrofluid was synthesized by novel thermolysis technique. The release of the dye from the PLGA composites when placed in the Rf induction coil was determined by fluorescence spectroscopy and a linear increase in the fluorescent intensity was observed with time. Also, the controlled release of the dye from the composites was achieved by a pulsed Rf treatment. Magnetic resonance imaging was also performed using the PLGA composites which showed enhancement in the T2-weighted image contrast and thus negligible reduction in the contrast capabilities of the iron oxide particles (R2 = 58.7 s-1mM-1). The PLGA composites containing the drug analog and the iron oxide nanoparticles thus constitute a controlled release drug delivery system with dual modal imaging and therapeutic capabilities.

Ocular diseases such as glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa require drug management in order to prevent blindness and affecting millions of adults in US and worldwide. There is an increasing need to develop devices for drug delivery to address ocular diseases. This research focused on an implantable ocular drug delivery device design, simulation and experiments with design requirements including constant diffusion rate, extended period of time operation, the smallest possible volume of device and reservoir. The drug delivery device concept uses micro-/nano-channels module embedded between top and bottom covers with a drug reservoir. Several microchannel design configurations were developed and simulated using commercial finite element software (ANSYS and COMSOL), with a goal to investigate how the microchannel dimensions affect the diffusion characteristics. In addition to design simulations, various microchannel configurations were fabricated on silicon wafer using photolithography techniques as well as 3D printing. Also, the top and bottom covers of the device were fabricated from PDMS through replica molding techniques. These fabricated microchannel design configurations along with top and bottom covers were all integrated into the device. Both single straight microchannels (nine different sizes of width and depth) as well as four micro-channel configurations were tested using citric acid (pH changes) and Brimonidine drug (concentration changes using the Ultra-Violet Visible Spectrophotometer) for their diffusion characteristics. Experiments were conducted to obtain the diffusion rates through various single micro-channels as well as micro-channel configurations using the change in pH neutral solution to verify the functionality and normalized diffusion rate of microchannels and configurations. The results of experimental data of diffusion rate were compared with those obtained from simulations, and a good agreement was found. The results showed the diffusion rate and the optimum size of microchannel in conjunction with the required drug release time. The results obtained also indicate that even though specific diffusion rates can be obtained but delivering the drug with constant amount needs a mechanism at the device outlet with some control mechanism. For future studies, this result may be used as a baseline for developing a microfabricated device that allows for accurate drug diffusion in many drug delivery applications.

Common therapy for diabetes mellitus is subcutaneous administration of insulin that is subject to serious disadvantages, such as patient noncompliance and occasional hypoglycemia. Hence, oral administration of insulin could be more convenient and serve as a desired route. However, oral administration of insulin is severely limited by the low bioavailability of insulin through the gastrointestinal tract. In this study, a semi-interpenetrating network gelatin fiber scaffold (sIPN GF) was fabricated for oral mucosal delivery of insulin as an alternative route. This sIPN GF was engineered from an electrospun gelatin fiber scaffold (GF), which was further crosslinked with polyethylene glycol diacrylate (PEG-DA) to enhance its stability. Within the crosslinking process, eosin Y served as a photoinitiator, and the ratio of PEG-DA to eosin Y was optimized with respect to cytocompatibility and degradation rate. The results showed that the fabricated scaffold morphology, mechanical properties, and degradation rate were significantly enhanced after the crosslinking process. This optimized formulation was used to fabricate sIPN gelatin-co-insulin fiber scaffold (sIPN GIF). Enzyme-linked immunosorbent assay (ELISA) was used to monitor the insulin releasing kinetics of sIPN GIF. Western blot analysis showed that sIPN GIF activated intracellular AKT phosphorylation in a releasing time-dependant manner. Oil red O staining confirmed the released insulin was able to induce 3T3-L1 preadipocyte differentiation. The permeability of insulin from sIPN GIF was determined on the order of 10^-7 cm/s using a vertical Franz diffusion cell system mounted with porcine buccal mucosa. These findings suggest that sIPN GIF holds a great potential for oral mucosal delivery of insulin.

Topical medication is a standard treatment for glaucoma. However, frequent dosing makes the therapy inconvenient and patient unfriendly. There is a great need to develop new topical formulations that provide long lasting noninvasive drug release. In this thesis, novel clickable dendrimer hydrogels for anti-glaucoma drug delivery were synthesized and characterized. Polyamidoamine (PAMAM) dendrimers have been widely applied for drug delivery. The physical characteristics they possess include monodispersity, water solubility, encapsulation ability, and a large number of surface groups. Polycationic PAMAM dendrimer G3 was surface modified with alkyne-PEG5-acid and then reacted with polyethylene glycol bisazide (PEG-BA, 1100 gmol-1) through click chemistry to form a cross-linked hydrogel. The resulting hydrogels were characterized in terms of mechanical properties, swelling, structural morphology, pH-dependent degradation, anti-glaucoma drugs (brimonidine tartrate and timolol maleate) release and cytotoxicity. To fully explore PAMAM dendrimers to make clickable hydrogels, polyanionic PAMAM dendrimer G4.5 was also surface modified with propargylamine to possess alkyne groups and successfully formed a hydrogel with PEG-BA. The work conducted in the thesis shows that clickable dendrimer hydrogels were successfully developed and shown to possess desired properties for delivery of anti-glaucoma drugs.

Dendrimers are a class of polymers with a highly branched, three-dimensional architecture comprised of an initiator core, several interior layers of repeating units, and multiple active surface terminal groups. Dendrimers have been recognized as the most versatile compositionally and structurally controlled nanoscale building blocks for drug and gene delivery. Polyamidoamine (PAMAM) dendrimers have been most investigated because of their unique structures and properties. Polycationic PAMAM dendrimers form compacted polyplexes with nucleic acids at physiological pH, holding great potential for gene delivery. Folate receptor (FRα) is expressed at very low levels in normal tissues but expressed at high levels in cancers in order to meet the folate demand of rapidly dividing cells under low folate conditions. Our primary aim was to investigate folic acid (FA)-conjugated PAMAM dendrimer generation 4 (G4) conjugates (G4-FA) for targeted gene delivery. The in vitro cellular uptake and transfection efficiency of G4-FA conjugates and G4-FA/DNA polyplexes were investigated in Chapter 4. It was found the cellular uptake of G4-FA conjugates and G4-FA/DNA polyplexes was in a FR-dependent manner. Free FA competitively inhibited the cellular uptake of G4-FA conjugates and G4-FA/DNA polyplexes. G4-FA/DNA polyplexes were preferentially taken up by FR-positive HN12 cells but not FR-negative U87 cells. In contrast, the cellular uptake of G4 dendrimers and G4/DNA polyplexes was non-selective via absorptive endocytosis. G4-FA conjugates significantly enhanced cytocompatibility and transfection efficiency compared to G4 dendrimers. This work demonstrates that G4-FA conjugates allow FR-targeted gene delivery, reduce cytotoxicity, and enhance gene transfection efficiency. The in vivo biodistribution of G4-FA conjugates and anticancer efficacy of G4-FA/siRNA polyplexes were investigated in Chapter 5. Vascular endothelial growth factor A (VEGFA) is one of the major regulators of angiogenesis, essential for the tumor development. It was found G4-FA/siVEGFA polyplexes significantly knocked down VEGFA mRNA expression and protein release in HN12 cells. In the HN12 tumor-bearing nude mice, G4-FA conjugates were preferentially taken up by the tumor and retained in the tumor for at least 21 days following intratumoral (i.t.) administration. Two-dose i.t. administration of G4-FA/siVEGFA polyplexes significantly inhibited tumor growth by lowering tumor angiogenesis. In contrast, two-dose i.t. administration of G4/siVEGFA polyplexes caused severe skin lesion, presumably as a result of local toxicity. Taken together, this work shows great potential for the use of G4-FA conjugates in targeted gene delivery and cancer gene therapy. We also explored polyanionic PAMAM dendrimer G4.5 as the underlying carrier to carry camptothecin (CPT) for glioblastoma multiforme therapyin Chapter 6. "Click" chemistry was applied to improve polymer-drug coupling reaction efficiency. The CPT-conjugate displayed a dose-dependent toxicity with an IC50 of 5 μM, a 185-fold increase relative to free CPT, presumably as a result of slow release. The conjugated CPT resulted in G2/M arrest and cell death while the dendrimer itself had little to no toxicity. This work indicates highly efficient "click" chemistry allows for the synthesis of multifunctional dendrimers for sustained drug delivery. Immobilizing PAMAM dendrimers to the cell surface may represent an innovative method of enhancing cell surface loading capacity to deliver therapeutic and imaging agents. In Chapter 7, macrophage RAW264.7 (RAW) was hybridized with PAMAM dendrimer G4.0 (DEN) on the basis of bioorthogonal chemistry. Efficient and selective cell surface immobilization of dendrimers was confirmed by confocal microscopy. It was found the viability and motility of RAW-DEN hybrids remained the same as untreated RAW cells. Furthermore, azido sugar and dendrimer treatment showed no effect on intracellular AKT, p38, and NFκB (p65) signaling, indicating that the hybridization process neither induced cell stress response nor altered normal signaling. This work shows the feasibility of applying bioorthogonal chemistry to create cell-nanoparticle hybrids and demonstrates the noninvasiveness of this cell surface engineering approach. In summary, these studies indicate surface-modification of PAMAM dendrimer G4 with FA can effectively target at FR-positive cells and subsequently enhance in vitro transfection efficiency and in vivo gene delivery. G4-FA conjugates may serve as a versatile targeted gene delivery carrier potentially for cancer gene therapy. PAMAM dendrimers G4.5 may serve as a drug delivery carrier for the controlled release of chemotherapeutics. The immune cell-dendrimer hybrids via bioorthogonal chemistry may serve as an innovative drug and gene delivery carrier potentially for cancer chemotherapy. Taken together, engineering of PAMAM dendrimers may advance anticancer drug and gene delivery.