Multidrug resistance (MDR) is a major obstacle limiting chemotherapeutic efficacy. The purpose of these studies was to investigate the potential application of injectable paclitaxel (PX) and doxorubicin (Dox)-loaded nanoparticles (NPs) engineered from oil-in-water microemulsion precursors for overcoming P-glycoprotein (P-gp)- mediated drug resistance in solid tumors. An in-vitro study was performed to test whether the oil (stearyl alcohol and cetyl alcohol) used to make lipid nanoparticles could be metabolized. The results showed that the concentrations of the fatty alcohols within nanoparticles, which were quantitatively determined over time by gas chromatography, decreased to only 10-20% of the initial concentration after 15-24 h of incubation with horse liver dehydrogenase (HLADH) and NAD+ at 37ºC. Moreover, the surfactant Brij 78 (polyoxyethylene 20-sterayl ether) in the nanoparticles influenced the activity of the enzyme. Novel Cremophor EL-free paclitaxel-loaded nanoparticles were developed using experimental design combining Taguchi array and sequential simplex optimization. The resulting PX G78 and PX BTM NPs were stable at 4ºC over five months and in PBS at 37ºC over 102 h. Release of PX from PX NPs was slow and sustained without initial burst release. Interestingly, PX BTM NPs could be lyophilized without cryoprotectants and without changing any physiochemical properties and bioactivities. Cytotoxicity studies in breast cancer MDA-MB-231 cells showed that PX NPs have similar anti-cancer activities compared to Taxol. Optimized Dox-loaded NPs were prepared using an ion-pair agent, sodium tetradecyl sulfate (STS), to mask Dox charge and to enhance its entrapment in NPs. In-vitro cytotoxicity studies were carried out in both sensitive and resistant human cancer cells treated with PX and Dox-loaded NPs. All of drug-loaded NPs decreased IC50 values by 6-13-fold in resistant cells compared to free drugs. A series of in-vitro assays were used to understand the underlying mechanisms. The results, in part, showed that the NPs inhibited P-gp and transiently depleted ATP, leading to enhanced uptake and prolonged retention of the drugs in P-gp-overexpressing cancer cells. Finally, in-vivo anti-cancer efficacy studies were performed using pegylated PX BTM NPs after intravenous (i.v.) injection and showed marked anti-cancer efficacy in nude mice bearing resistant NCI/ADR-RES tumors versus all control groups. These results suggest that NPs may be used to both target drug and biological mechanisms to overcome MDR.

<p>The synthesis of new macromolecular diagnostic imaging agents has been a growing field in polymeric chemistry research. Dendrimers provide a viable scaffold for such applications due to their unique, defined macromolecular architecture. The precise structural control afforded via the step-wise synthesis of dendrimers yields exceptional and precise macromolecules that can be functionalized to include necessary imaging moieties with the same degree of precision.</p> <p>We have herein contributed to this growing field by attempting the synthesis of a series of PEGylated poly(2,2-bis[hydroxymethyl]propanoic acid) PMPA dendrons using thiol-ene "click" chemistry. The series consisted of three dendritic architectures peripherally functionalized with poly(ethylene glycol) (PEG) chains of varying length (n= 3, 8, 16), with the goal of determining the effect of PEG chain length on blood circulation times. Synthesis of these conjugates began first with functionalization of the dendron periphery to incorporate alkene functionalities using anhydride-mediated esterification chemistry.</p> <p>The core of the alkene PMPA dendrons was then modified to introduce a metal chelating bis-pyridyl functionality, which has been observed to chelate the radionuclide technetium-99m (^99mTc) with high binding affinity. ^99mTc is the most widely used diagnostic radioisotope in diagnostic medicine due to its ideal isotopic properties, widespread availability, low cost, and its ability to be traced, in real time, <em>in vivo</em> using Single Photon Emission Computed Tomography (SPECT).</p> <p>PEGylation at the periphery was performed by thiol-ene “click” chemistry using thiol-terminated PEG chains. Metallation of the core of each PEGylated dendron was then attempted according to literature procedures for ^99mTc radiolabeling with the bis-pyridyl chelate.</p> / Master of Science (MSc)

This diploma thesis brings new data about design, synthesis, physico-chemical characterisation and biological efficacy of the novel star-like HPMA-based conjugates intended for treatment of solid tumors. Recently, many different water-soluble drug delivery systems based on N-(2- hydroxypropyl)methacrylamide (HPMA) copolymers have been described. Here, we report synthesis and physico-chemical characterisation of high molecular weight star-like HPMA- based polymer carriers with low polydispersity prepared by controlled grafting of HPMA copolymers onto PAMAM dendrimer core. With the aim to keep the polydispersity of drug delivery system as low as possible, reversible Addition-Fragmentation Chain Transfer (RAFT) polymerisation was used for HPMA-based polymer precursor preparation. The end groups of the polymer presursors was afterwards used for grafting using carbodidimide condensation reaction or copper free click chemistry on polyamidoamine (PAMAM) dendrimers resulting in a formation of star-like high-molecular-weight (HMW) drug carriers. Described synthetic procedure provided preparation of star-like HMW drug carriers with Mw between 1.105 - 3.105 g/mol and narrow distribution of Mw. The model drug, doxorubicin (Dox), was attached to the hydrazide group containing polymer cariers by pH- sensitive...

Contrast agents for magnetic resonance imaging (MRI) are but one of a variety of nanosystems that have incredible potential for the detection and diagnosis of cancer. Nanosystems share a common disadvantage: they are quickly sequestered by biological processes that clear foreign material from the body, requiring ever larger doses to accumulate in targets, and reducing their overall effectiveness and viability. This thesis explores a pair of strategies for nanomaterials to boost their evasiveness from these defensive systems in the context of lanthanide MRI contrast agents, in an attempt to increase their probability to collect in cancerous tissue. Chapter 1 provides precedent and rationale for the modification of two parameters regarding novel nanosystem design: aspect ratio and zeta potential. Chapter 2 details the controlled syntheses and analysis of sodium dysprosium fluoride nanomaterials at a range of aspect ratios. Chapter 3 concerns the construction of tunable zwitterionic polymer coatings for synthesized nanomaterials to demonstrate control over the zeta potential in aqueous dispersion. Chapter 4 tests polymer-coated spherical nanoparticles and nanorods for internalization into or adsorbance onto a cancerous cell line. Chapter 5 summarizes the work of the previous chapters and suggests future research approaches. Though internalization or adsorbance onto HeLa cells was not observed for prepared nanomaterials, control over their aspect ratio at the synthetic level and zeta potential via constructed zwitterionic polymers was demonstrated, with implications for application to a plethora of nanosystems. / Graduate

Cette étude porte sur le développement de nanoparticules pour différentes applicationsbiologiques. Trois systèmes de nanoparticules ont été mis au point : des clusters de nanoparticulesmagnétiques pour l’extraction par magnétophorèse d’objets biologiques, des agents de contrastemultimodaux (IRM, fluorescence dans le proche infrarouge) pour le diagnostic de l’athérosclérose etdes nanoparticules de silice fluorescentes doublement marquées pour la détection de tumeurs in vivo.Au cours de cette étude, une stratégie de greffage de surface de silice par des macromolécules depoly(oxyde d’éthylène) (PEG) permettant d’atteindre de hautes densités de greffage. Cette PEGylationpermet, d’annuler les interactions non spécifiques dans le cadre de l’extraction magnétique rendantainsi ce système plus efficace, et de conférer aux nanoparticules des propriétés de furtivité vis-à-vis dusystème immunitaire dans le cadre du marquage de tumeurs. Le contrôle du nombre de biomoléculesgreffées régiosélectivement sur les nanoparticules (Annexine A5, ou fragments d’anticorps) ainsi quel’étude des interactions biomoléculaires par des techniques de biophysique (SPR, QCM-D) ont permisd’optimiser la propriété de reconnaissance des nano-objets pour leurs cible respective. Enfin, les nanoobjetsont été évalués dans le cadre de leur application. / The aim of this study was the design of nanoparticles for three different biologicalapplications: magnetic nanoparticles cluster for magnetic extraction of biological materials,multimodal contrast agents (MRI and near infrared fluorescence imaging) for atherosclerosisdiagnosis, and fluorescent silica nanoparticles with two different dyes for in vitro and in vivo tumorlabeling. One part of the project dealt with the developement of a new grafting method ofpoly(ethylene oxide) macromolecules onto nanoparticle’s silica surfaces (PEGylation) in order toobtain a high grafting densities. The obtained results have shown that this PEGylation reduces the nonspecificprotein adsorption allowing a better extraction and sorting efficiency, and also permitsnanoparticles to escape the surveillance of the immune system for in vivo tumor labeling. Therefore,the biomolecular recognition of the nanoparticles has been optimized by controlling the number ofconjugated biomolecules and by studying this biomolecular recognition using biophysical methods(SPR, QCM-D). Finally, the different nano-objects were evaluated in the context of their respectiveapplication.

Utveckling av polymer baserade läkemedelsbärare i nanostorlek har blivit allt viktigare för att effektivisera behandling och diagnosering av olika sjukdomar, speciellt cancer. Flera läkemedel som används i kemoterapi har bristfälliga egenskaper som låg löslighet i vatten, oönskad nedbrytbara till dess inaktiva form, och distribution i stora volymer till oönskade organ p.g.a. dess icke-selektiva förmåga. Nanopartiklar är små partiklar med diameter 1-500 nm som genom passiv/aktiv transport kan passera olika biologiska barriärer och transportera läkemedel i optimala mängder till specifika celler. Denna selektiva transport bidrar till ökad terapeutiskt index och minskning av toxiska effekter i övriga delar av kroppen. Hyperförgrenade linjär-dendritiska hybrider är en subgrupp av dendritiska polymer som har stor potential att användas som byggstenar i utvecklingen av läkemedelsbärare. I detta projekt producerades ett bibliotek av hyperförgrenade linjär-dendritiska material via Fischer esterifikation reaktionen som är en snabb, billig och uppskalningsbar produktionsmetod. Vidare post funktionaliserades materialen med allyl grupper för produktion av nano geler genom UV-inducerad korslänkning och vidare funktionalisering. Samtliga producerade hyperförgrenade linjär-dendritiska material hade förmågan att bilda miceller i vatten. Materialen med bäst micelle bildningsförmåga användes för att kemiskt korslänka dem och producera nano geler. Nano gelernas inre del funktionaliserades framgångsrikt med tre olika funktionella grupper; katjoniska, anjoniska och hydrofoba via resterande fria allyler. Detta påvisar att dessa dendritiska nano geler har potential att bära olika material som hydrofobiska läkemedel eller genetiskt material. Dom producerade nano gelerna hade en hydrodynamisk volym inom intervallet 124-200 nm. Detta är fördelaktigt då dem kan transporteras till tumörområdet via ökad permeabilitet och retention, också kallad EPR effekten, utan att initiera ett immunologiskt svar eller filtreras från blodomloppet via njuren. / The development of nano- based drug carriers is of high importance in anti-cancer treatment as anticancer drugs suffers from limitations as low aqueous solubility, non-selective targeting, off-target degradation and low therapeutic concentrations at target site. Hyperbranched polymers are potential candidates as drug carrier due to its unique properties as globular shape, high number of functional groups and high degree of branching. In addition, hyperbranched polymers are synthesized via one-step polymerization reaction with high yields, low costs and good scale-up possibilities. In this project a library of hyperbranched linear-dendritic hybrid materials based of 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) and monofunctional poly (ethylene glycol) (mPEG) was synthesized via the Fischer esterification reaction. The materials were then post functionalised with hydrophobic allyl groups. The materials self-assembled into micelles in water and candidates with best self-assembly ability were used to fabricate dendritic nanogels by UV-induced cross-linking. The formed dendritic nanogels obtained a hydrodynamic volume between 124-200 nm, which indicates that these dendritic nanogels can be used as drug carrier and accumulate at target-site via the enhanced permeability and retention (EPR) effect. The dendritic nanogels inner core was also successfully attached with cationic, hydrophobic and anionic groups respectively. This confirmed that the dendritic nanogels have the potential to encapsulate different types of cargo such as DNA or hydrophobic drugs in the inner core.

Polymer-based drug delivery systems represent one of the promising strategies for successful tumor treatment. Conjugation of a low-molecular-weight drug to a syn- thetic polymer carrier enables targeted drug delivery to tumor tissue/cells and limited systemic toxicity of the drug. The conjugates show extended circulation time, and preferentially accumulate in tumor tissue due to the Enhanced Permeability and Re- tention (EPR) effect. The EPR effect depends on a structural anomaly in tumor neovasculature, and vasodilators were shown to enhance the EPR effect via an in- crease of blood supply in the tumor. Polymer drug carriers based on water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) benefit from variable architecture, drug loading and controlled release. HPMA-based conjugates with cancerostatics have al- ready proved high anti-tumor activity, inducing complete tumor regression followed by resistance to a second tumor challenge in experimental murine models. Three HPMA-based conjugates with organic nitrates (labeled 1, 2, and 3) were pre- pared as polymer donors of nitric oxide (NO) with the aim to intensify the EPR effect, thereby enhancing accumulation of co-administered macromolecular cancerostatics in the tumor. In this study, the conjugates were non-toxic to cancer cells and did not potentiate...