Nanoparticle-based drug delivery systems (NDDS) have emerged as a promising strategy for formulation of anticancer drugs due to their ability to passively target solid tumors via exploitation of the enhanced permeation and retention effect. In particular, nano-sized block copolymer micelles (BCMs) have proven to be a viable delivery vehicle for hydrophobic anticancer drugs. To further enhance the specificity of BCMs towards cancer cells, extensive research has been focused on the formulation of actively targeted BCMs with tumor cell binding antigens conjugated to their surface. However, the in vivo transport of passively and actively targeted BCMs has only been studied to a limited extent. This thesis explores the potential and limitations of passively and actively targeted BCMs, as NDDS for delivery to solid tumors. The in vivo transport of BCMs at the whole body, tumor, and cellular levels is investigated in human breast cancer xenografts. Overall, active targeting of BCMs with epidermal growth factor (EGF) as the tumor cell binding antigen was not found to alter the whole body clearance of the vehicles; however, particle size had a profound effect on their pharmacokinetics and biodistribution profiles. Both passively and actively targeted BCMs exhibited heterogeneous distribution throughout solid tumors, with preferential localization in the tumor periphery and/or highly vascularized regions. In addition, the BCMs were found to exhibit impaired tumor penetration due to limited mobility and/or the binding site barrier. Although active targeting increases the in vivo BCM cellular uptake, the BCMs largely remained in the extracellular compartment, indicating that incomplete BCM delivery to all tumor cells remains as a major biological barrier. Interestingly, EGF-conjugated BCMs induced a potent bystander effect in vitro as a result of the paradoxical apoptotic effect of EGF, which has the potential to treat nearby tumor cells that do not respond directly to BCM treatment in vivo. In this way, EGF-BCMs may be beneficial for rendering the aforementioned in vivo barriers such as limited tumor penetration, as well as heterogeneity in tumor vascularization and receptor expression.

Hydrogels have successfully proved themselves useful for drug delivery applications and several delivery routes have been developed over the years. The particular interest in this work was to design, synthesise and evaluate in situ forming drug eluting hydrogels, which have the potential to ameliorate the healing of cardiovascular diseases. With this aim the anti-inflammatory and immunosuppressant drugs rapamycin (Ra) and dexamethasone (Dex) were made water soluble by conjugation with polyethylene glycol (PEG). Ra was attached pendant from the terminal of PEGs while Dex was incorporated into dendritic structures grown from PEGs. These conjugates were further crosslinked into hydrogels by either conjugate or thiol-ene addition. The gel degradation was tuned to take between 5 and 27 days by using gel building block combinations that induced either 2 or 4 hydrolytically labile bonds per crosslink or by varying the number of crosslinking sites of the building blocks. The use of thiol-ene addition prolonged the degradation time nearly seven folded compared to conjugate addition as a more stable crosslink was formed. Two different formulations for gelling via conjugate addition were used (acrylate-thiol or vinyl sulphone-thiol) to deliver Ra, which was carried by either a 4- or 2-armed PEG. The elution kinetic for the respective gel formulation was of zero order during 15 and 19 days of gel degradation. In addition, Ra was PEGylated via esters, with a distance of either one or two carbons to a nearby thio-ether functionality. The difference in ester conjugation resulted in a slight but significant change in drug-PEG conjugate stability, which was mirrored by the increased time to reach the half amount of total drug elution; from 9.3 to 10.2 days and from 5.1 to 9.7 days for the two gel formulations, respectively. Dexamethasone was incorporated via an ester into dendrons of first and second generation pending from 2- and 4-armed PEGs at loadings of 2, 4 or 6 Dex molecules per carrier molecule. The resulting elution kinetic was of zero order during degradation periods of 5-27 days. Released Dex still possessed biological activity as determined by an in vitro cell assay. The novelties in this thesis are: (A) slow release of rapamycin obtained by covalent incorporation into hydrogels, (B) the use of unique PEG-based dendrimers to incorporate dexamethasone into a hydrogel and (C) zero order sustained release of dexamethasone at physiological pH. / Hydrogeler har framgångsrikt visat sig användbara för att leverera läkemedel och ett flertal metoder har utvecklats de senaste 20 åren. Fokuset i den här avhandlingen ligger på att designa, framställa och utvärdera läkemedelsutsöndrande hydrogeler som spontanhärdar in situ, vilka har potential att förbättra läkningen efter kardiovaskulär sjukdom. Med det syftet gjordes de anti-inflammatoriska och immunsänkande läkemedlen rapamycin (Ra) och dexametason (Dex) vattenlösliga genom att konjugeras med polyetylenglygol (PEG). Ra fästes kovalent längst ut på PEGar medans Dex inkluderades i dendritiska strukturer vilka byggdes från ändpunkten av PEGar. De här konjugaten tvärbands till hydrogeler via antingen konjugerad addition eller radikal polymerisation. Nedbrytningen av gelerna trimmades till att ta mellan 5 och 27 dagar genom att använda kombinationer av gelbyggstenar som bildar antingen 2 eller 4 hydrolyserbara estrar per tvärbindning eller genom att variera antalet tvärbindningspunkter hos byggstenarna. Användandet av radikal polymerisation i sig ledde till att nedbrytningen av geler tog nära sju gånger längre tid jämfört med geler gjorda via konjugerad addition eftersom stabilare tvärbindningar då formas. Två olika kombinationer för härdning via konjugerad addition (akryl-tiol eller vinylsulfon-tiol) användes för att leverera Ra som bars av antingen en 4- eller 2-armad PEG. Utsöndringskinetiken av Ra för de två kombinationerna var av nollte ordningen under de 15 och 19 dagar som gelerna degraderade. Dessutom, Ra PEGylerades via estrar med ett avstånd på antingen ett eller två kol till en närliggande tioeter. Skillnaden i avstånd ledde till en liten men signifikant skillnad i stabiliteten hos Ra-PEG konjugaten, vilket speglades i den förlängda tiden att nå halva mängden av den totala läkemedelsutsöndringen; från 9.3 till 10.2 dagar och från 5.1 till 9.7 dagar för de två respektive gelkombinationerna. Dex kopplades in via en esterbindning till dendroner av första och andra generationen byggda från PEGar med 2 eller 4 armar, vilket resulterade i att 2, 4 eller 6 Dex levererades per bärarmolekyl. Dex eluerade med nollte ordningens kinetik under degraderingsperioder på mellan 5 och 27 dagar. Vidbehålllen biologisk aktivitet av eluerad Dex bekräftades genom cellexperiment in vitro. Nyheterna i den här avhandlingen består av: (A) kontrollerad utsöndring av rapamycin uppnådd genom kovalent inbindning till hydrogeler, (B) användandet av unika PEGbaserade dendrimerer för kovalent inbindning av dexametason till hydrogeler och (C) nollte ordningens utsöndring av dexametason vid fysiologiskt pH. / <p>QC 20130204</p>

A class of mitochondria-penetrating peptides (MPPs) was studied in an effort to optimize their applications in the delivery of bioactive cargo to this therapeutically important organelle. The sequence requirements for mitochondrial entry were monitored, and it was discovered that while an alternating cationic/hydrophobic residue motif is not required, the inclusion of a stretch of adjacent cationic amino acids can impede access to the organelle. In addition, a variety of C-terminal cargos were tested to determine if there are limitations to the lipophilicity, charge, or polarity of compounds that can be transported to mitochondria by MPPs. Furthermore, these systematic studies aided the design and synthesis of a copper-binding MPP for the delivery of copper ions to mitochondria for the potential rescue of disorders associated with copper-deficiency. The results reported demonstrate that MPPs are versatile transporters that may have a wide range of biological applications.

Spinal cord injury and stroke are two devastating neurological events that lack effective clinical treatments. Recent neuroregenerative approaches involving the delivery of cells or drugs to the injured tissue have shown promise, but face critical challenges to clinical translation. Herein, hyaluronan-methylcellulose (HAMC) hydrogels were investigated as a versatile means of overcoming the challenges facing central nervous system cell and drug delivery. HAMC was shown to support the viability of encapsulated human umbilical tissue-derived cells, demonstrating utility as a scaffold for therapeutic cell delivery to the injured spinal cord. In a drug delivery context, release of the neuroregenerative drug cyclosporin A from the hydrogel was tunable over 2-28 days and the drug diffused to the stem cell niche in the brain and persisted for up to 24 days at a stable concentration when the HAMC-based system was implanted epi-cortically. HAMC is thus a promising tool for emerging neuroregenerative therapies.

This research work involves multifunctional magnetically targeted drug delivery microspheres for treatment against breast cancer. A combination therapy approach was followed by encapsulating two chemotherapeutics, 5-Fluorouracil (5-Fu) and cyclophosphamide in poly(D, L-lactide-co-glycolide) (PLGA) microspheres. Magnetite nanoparticles and albumin were also incorporated in the microspheres to achieve targeted treatment. The microspheres were fabricated using oil-in-oil emulsion/solvent evaporation technique. Albumin is attracted to cancer cells and thus it is likely to draw the microspheres towards tumor cells. On application of magnetic field near tumor site, magnetites in the microspheres are likely to guide them to the region of magnetic field. This will allow release of drugs from microspheres in the cancer cells. Also the burst release of drugs and then slow release due to diffusion in the cancer cells lead to effective treatment and also limit excessive spreading of drugs in other regions of the body. Release rate study was carried out using high performance liquid chromatography (HPLC). Invitro and in-vivo study was carried out to check the efficacy of treatment. / Thesis (M.S.)--Wichita State University, College of Engineering, Dept. of Mechanical Engineering.

A class of mitochondria-penetrating peptides (MPPs) was studied in an effort to optimize their applications in the delivery of bioactive cargo to this therapeutically important organelle. The sequence requirements for mitochondrial entry were monitored, and it was discovered that while an alternating cationic/hydrophobic residue motif is not required, the inclusion of a stretch of adjacent cationic amino acids can impede access to the organelle. In addition, a variety of C-terminal cargos were tested to determine if there are limitations to the lipophilicity, charge, or polarity of compounds that can be transported to mitochondria by MPPs. Furthermore, these systematic studies aided the design and synthesis of a copper-binding MPP for the delivery of copper ions to mitochondria for the potential rescue of disorders associated with copper-deficiency. The results reported demonstrate that MPPs are versatile transporters that may have a wide range of biological applications.

Spinal cord injury and stroke are two devastating neurological events that lack effective clinical treatments. Recent neuroregenerative approaches involving the delivery of cells or drugs to the injured tissue have shown promise, but face critical challenges to clinical translation. Herein, hyaluronan-methylcellulose (HAMC) hydrogels were investigated as a versatile means of overcoming the challenges facing central nervous system cell and drug delivery. HAMC was shown to support the viability of encapsulated human umbilical tissue-derived cells, demonstrating utility as a scaffold for therapeutic cell delivery to the injured spinal cord. In a drug delivery context, release of the neuroregenerative drug cyclosporin A from the hydrogel was tunable over 2-28 days and the drug diffused to the stem cell niche in the brain and persisted for up to 24 days at a stable concentration when the HAMC-based system was implanted epi-cortically. HAMC is thus a promising tool for emerging neuroregenerative therapies.

Water-soluble fullerene containing polymers comprising of poly(2-(dimethylamino) ethyl methacrylate)-fullerene (PDMAEMA-C60) with targeting moieties, poly(oligo(ethylene glycol) methyl ether methacrylate)-C60 (POEGMA-C60), nanocrystalline cellulose-fullerene (NCC-C60) and NCC-C60-POEGMA were synthesized and their solution properties were investigated. PDMAEMA-C60 with galactose targeting moiety was prepared by atom transfer radical polymerization (ATRP) and atom transfer radical addition (ATRA) processes. The self-assembly of galactose functionalized PDMAEMA-C60 structure in aqueous solutions was investigated using dynamic light scattering (DLS) at different pHs. A smaller hydrodynamic radius (Rh) was observed at pH 10 than at pH 3 due to electrostatic repulsion at low pH values. In addition, free PDMAEMA chains induced the demicellization of self-assembled nanostructures caused by the formation of charge transfer complex between PDMAEMA and C60. A well-defined poly(di(ethylene glycol) methyl ether methacrylate–stat-oligo(ethylene glycol) methyl ether methacrylate)-block-poly(di(ethylene glycol) methyl ether methacrylate ((PMEO2MA-stat-POEGMA300)-b-PMEO2) was successfully synthesized at room temperature via a two-step ATRP process. The block copolymer exhibited two thermal transitions at ~ 30 and 45 oC, which was believed to be associated with the formation of micelles and larger aggregates. The Rh of the aggregates increased from 47 to 90 nm, the aggregation number increased from 76 to ~9800 and Rg/Rh increased from 0.75 to 1.2 within the temperature range of 34 to 45oC. Well-defined statistical (PMEO2MA-stat-POEGMA300)-C60 was synthesized via ATRP and ATRA. The lower critical solution temperature (LCST) of (PMEO2MA-stat-POEGMA300)-C60 increased with methanol content in water, exhibiting lower LCSTs than PMEO2MA-stat-POEGMA300 for all methanol/water compositions. Higher critical micelle concentration (CMC) and larger spherical micelles were observed for (PMEO2MA-stat-POEGMA300)-C60 with increasing methanol content. The Rh of the micelles remained constant at temperature below the LCST and increased dramatically at temperature greater than the LCST, and (Rg/Rh) increased from ~ 0.75 to ~ 1.0. Nanocrystalline cellulose (NCC) was modified with water-soluble C60-(β-cyclodextrin) and (PMEO2MA-stat-POEGMA300)-C60) through a radical coupling reaction. NCC-C60-(PMEO2MA-stat-POEGMA300) possessed thermal responsive behavior in water and ~3.5 oC hysteresis associated with the heating/cooling cycles. No observable damage to NCC occurred during the radical coupling reaction as determined by TEM. NCC-C60-(β-cyclodextrin) possessed a similar thermal degradation behavior as NCC except it possessed a broader temperature range. Both NCC-fullerene systems demonstrated a radical scavenging activity when screened with the 2,2-diphenyl-1-picrylhydrazyl (DPPH). In addition, the drug loading and delivery using PDMAEMA-C60 with targeting moieties was explored. Two model drugs, namely fluorescein and pyrene were employed to evaluate the location of drug in the self-assembled structure of PDMAEMA-C60. It was found that the hydrophobic drugs were partitioned between the PDMAEMA shells and the hydrophobic fullerene cores. The drug delivery profiles indicated that PDMAEMA-C60 is an efficient drug carrier, however, it was cytotoxic to cells. The gene transfection efficacy of PDMAEMA-C60 to different cell lines was investigated and the results demonstrated that PDMAEMA-C60 exhibited good gene transfection performance. However, the targeting selectivity to liver cells cannot be determined in both cases. This study demonstrates that nanostructures of stimuli-responsive fullerene polymers can be controlled and manipulated by changing the external environments. Several potential applications, such as in drug and gene delivery, and free radical scavenging can be further explored.

Nanoparticle-based drug delivery systems (NDDS) have emerged as a promising strategy for formulation of anticancer drugs due to their ability to passively target solid tumors via exploitation of the enhanced permeation and retention effect. In particular, nano-sized block copolymer micelles (BCMs) have proven to be a viable delivery vehicle for hydrophobic anticancer drugs. To further enhance the specificity of BCMs towards cancer cells, extensive research has been focused on the formulation of actively targeted BCMs with tumor cell binding antigens conjugated to their surface. However, the in vivo transport of passively and actively targeted BCMs has only been studied to a limited extent. This thesis explores the potential and limitations of passively and actively targeted BCMs, as NDDS for delivery to solid tumors. The in vivo transport of BCMs at the whole body, tumor, and cellular levels is investigated in human breast cancer xenografts. Overall, active targeting of BCMs with epidermal growth factor (EGF) as the tumor cell binding antigen was not found to alter the whole body clearance of the vehicles; however, particle size had a profound effect on their pharmacokinetics and biodistribution profiles. Both passively and actively targeted BCMs exhibited heterogeneous distribution throughout solid tumors, with preferential localization in the tumor periphery and/or highly vascularized regions. In addition, the BCMs were found to exhibit impaired tumor penetration due to limited mobility and/or the binding site barrier. Although active targeting increases the in vivo BCM cellular uptake, the BCMs largely remained in the extracellular compartment, indicating that incomplete BCM delivery to all tumor cells remains as a major biological barrier. Interestingly, EGF-conjugated BCMs induced a potent bystander effect in vitro as a result of the paradoxical apoptotic effect of EGF, which has the potential to treat nearby tumor cells that do not respond directly to BCM treatment in vivo. In this way, EGF-BCMs may be beneficial for rendering the aforementioned in vivo barriers such as limited tumor penetration, as well as heterogeneity in tumor vascularization and receptor expression.

Many diseases have been associated with oxidative stress (OS) which is caused when the production of reactive oxygen species (ROS), such as superoxide (O2•-) and hydroxyl radical (•OH), overcome the scavenging efficiency of living organisms. It is known that ROS production is worsened during traumas related to ischemic events and subsequent reperfusion in which the treatment with fast and effective antioxidants is critical to prevent cell and tissue damage. PEG-HCCs are carbon nanoparticles that showed O2•- and •OH scavenging properties according to electron paramagnetic resonance (EPR) experiments and peroxyl scavenging properties based on oxygen radical absorbance capacity (ORAC) assays. The O2•- quenching capability was also examined in vivo using a mild traumatic brain injury (mTBI) model complicated with hypotension. As result of the PEG-HCCs treatment, the cerebral blood flow (CBF) was restored while normalizing O2•- and nitric oxide (NO•) levels, primarily in the cerebral vasculature