Dissertations / Theses on the topic 'Integrin-targeting'

Abstract Nowadays, systemic administration of cytotoxic agents is one of the main keystones of modern anticancer chemotherapy. However, many drugs do not selectively localize into solid tumors, as they normally accumulate also in healthy organs (e.g. liver and kidney). As a result, severe side effects are caused by the suboptimal biodistribution profile, in combination with the non-specific mode of action of cytotoxic agents, preventing the drug administration at therapeutic doses. The targeted delivery of anticancer agents into solid tumors is emerging as encouraging approach to overcome the intrinsic drawbacks of cytotoxic drugs. For example, some antibodies with high affinity for accessible tumor markers have been used as vehicles for the targeted drug delivery (Antibody-Drug Conjugates, ADCs). Since they may have limitations due to their high molecular weight, to their possible immunogenicity and to their high production costs, the development of smaller ligands (e.g. vitamins, peptides and peptidomimetics) capable of binding efficiently to tumor-overexpressed receptors, may show some significant therapeutic advantages. Peptides and peptidomimetics with the Arg-Gly-Asp tripeptide (RGD) are known to bind integrin αvβ3, a heterodimeric transmembrane receptor which shows low expression on healthy tissues, while being upregulated in a variety of cancer cells. For these reasons, integrin ligands have been widely explored as homing devices for the selective delivery of anticancer agents. In this PhD thesis, the synthesis and the biological evaluation of new small molecule-drug conjugates (SMDCs) targeting αvβ3 integrin are described. This work started with the preparation of a SMDC for the integrin-targeted delivery of Camptothecin, containing a disulfide linker and a naphtalimide moiety for the real-time monitoring of the cellular uptake (Chapter II). While the design of this complex molecular structure was carried out to reproduce literature data, the biological evaluation of this compound revealed a complex scenario. For this reason, the biological properties of all the three moieties (ligand, drug and linker) have been deeply investigated individually. Firstly, in Chapter III we established the role and the behavior of the ligand, trying to understand its action mechanism. Moreover, modifications of the ligand unit have been carried out to deface the affinity for the receptor and to unambiguously evaluate the capability of the original ligand to selectively target cancer cells that (over)express αvβ3. Later on, linker-related structural features (e.g. stability, linker cleavage experiments, kinetics of drug release, etc.) have been studied and improved (Chapter IV). Finally, the development of other integrin-targeted SMDC products featuring peptide linkers (with their biological evaluation) is reported in Chapter V. In Chapter VII, all the experimental details of synthetic and biological procedures are included, together with spectroscopic data and HPLC profiles of the newly synthesized compounds.

Peptides and peptidomimetics bearing the Arg-Gly-Asp peptide sequence have been demonstrated to bind with high affinity to αvβ3 Integrin, a heterodimeric transmembrane receptor overexpressed in several tumor cells. For these reasons, integrin ligands have been coupled to a variety of anticancer drugs, aiming at the selective delivery of the payload at the tumor site. This PhD work describes the conjugation of the peptidomimetic αvβ3 Integrin ligand cyclo[DKP-RGD] to different anticancer drugs (i.e. paclitaxel, daunorubicin and camptothecin) through peptide and disulfide linkers. The resulting small molecule-drug conjugates (SMDCs) are selective αvβ3 binders and are able to release the drug upon exposure to lysosomal enzymes (e.g. cysteine proteases) or intracellular reducing agents (e.g. glutathione). Cell proliferation assays against isogenic human cancer cells expressing αvβ3 at different levels (αvβ3 +/αvβ3 −) have been performed to evaluate the selective cytotoxic activity of RGD-based SMDCs against integrin-positive cancer cells. Fairly effective integrin targeting was displayed by the cyclo[DKP-RGD]-Val-Ala-PTX conjugate (compound 80), which was found to differentially inhibit proliferation in antigen-positive CCRF CEM αvβ3 versus antigen-negative isogenic CCRF-CEM cells. Next-generation cyclo[DKP-RGD]-Drug conjugates were prepared, aiming at improving the targeting effect shown by the cyclo[DKP-RGD]-Val-Ala-PTX conjugate as well as to deeply analyze the SMDC’s interactions with cancer cells.

In this PhD work, a variety of new SMDCs were designed and synthesized featuring different types of linkers and cytotoxic payloads. All of them were characterized and conjugated to peptidomimetic ligands bearing the RGD sequence (namely, the cyclo[DKP-RGD] and cyclo[RDGfK] compounds) aimed at targeting αVβ3 integrin receptor, which is overexpressed in many human cancers. Firstly, six new conjugates containing peptide linkers prone to cleavage in intracellular vesicles (such as the lysosomes) were synthesized and evaluated in vitro. The loss of potency generally displayed by these SMDCs in antiproliferative assays prompted us to the design of new conjugates bearing peptide linkers susceptible to proteolysis in the extracellular environment. This strategy has got credits from literature data and it has been proposed as a promising alternative to internalizing conjugates. Based on this, two new conjugates containing aminoacid sequences recognized and cleaved by MMP-2 were synthesized. The conjugates’ binding ability was studied by competitive binding assays on the integrin αVβ3 receptor was studied, and the effective cleavage of the peptide linker in the presence of recombinant human MMP-2 was observed. Finally, two new conjugates that may be activated both in intracellular compartments and in extracellular milieu, by means of the ubiquitous tumor-associated enzyme β-glucuronidase, were synthesized and evaluated by their ability of inhibit biotinylated vitronectin (binding assays). Further evaluation of the efficient drug release from these non-internalizing prodrugs, brought at the diseased site by the RGD affinity for tumor-expressed integrins, will potentially support the clinical investigation of this anticancer devices.

Current chemotherapeutics pose many di sadvantages due to their lack of specificity and low therapeutic index. To overcome these challenges, research has focused its attention on the development of nano-based delivery systems that can penetrate the leaky vasculature of tumor endothelium, use site-directed ligands that can bind with high affinity and specific ity to tumor cells, physically entrap poorly soluble drugs, and deliver these cytotoxic agents directly to the tumor site. One approach to nanosystem drug delivery is with the use of peptide amphiphiles (PAs) that are conjugated with the Arginine-Glycine-Aspartic Acid (RGD) motif to actively target a αVβ3 integrin receptors on cancer cells or tumor endothelium. The current work is focused on mechanistic studies to evaluate the uptake of novel RGD amphiphi les with varying alkyl chain lengths (palmitic acid : Cl 6 and stearic acid: C 18) and hydrophilic linkers, 8-amino- 3,6-dioxaoctonoic acid (ADA) or glucose, as micellar delivery systems of hydrophobic anticancer agents. PAs were confirmed for their self-assembling properties and further evaluated for their RGD-mediated binding specificity to purified αVβ3 integrin through a competitive binding fluorescence polarization assay (with novel RGD micelles displacing an integrin-bound fluorescent RGD probe by as much as 63.03%). Ultimately, these nanocarriers were assessed for their ability to deliver phys ically entrapped fluorescein isoth iocyanate (FITC) to A2058 cells overexpressing αVβ3 integrin receptors. Results from confocal microscopy indicate that uptake of RGD micelles was driven by an energy-dependent mechanism, as statistically significant levels of FITC internalization was seen at 37°C versus 4°C (p-value<0.05 for all treatment groups); moreover, intracellular fluorescence was notably higher (as much as 4-fold) when delivered through novel RGD conjugates as opposed to its free form. Regardless of chain length and the number of hydrophilic linkers, all RGD PAs showed promising results as micellar carriers that can effectively deliver their payload to the target tumor site via receptor mediated endocytosis.

Stroke is the 5th leading cause of death and the leading cause of disability in the United States, but there are only two available therapies, tissue plasminogen activator and endovascular thrombectomy. As both therapies focus on removal of the clot, the subsequent pathologic processes, i.e. inflammation, cerebrovascular breakdown, ATP depletion, etc. are left untreated, contributing to worsened patient outcome. Many clinical trials have unsuccessfully attempted to address these mechanisms. The blood-brain barrier (BBB), a system of non-fenestrated endothelial cells, extracellular matrix, and astrocytic endfeet, is significantly impacted after ischemic stroke in its role of preventing the free movement of proteins from the blood into the brain. In fact, BBB dysfunction is viewed as one of the major facilitators of damage following ischemic stroke, leading to increased infarct volumes and worsened patient outcomes. Interestingly, a family of endothelial integrins, the b1 integrins, have been shown to regulate tight junction proteins preventing the free movement of molecules. When expression of the tight junctions are decreased, this results in increased BBB permeability. To test this concept, our laboratory has previously shown the knockout of the particular β1 integrin, α5β1, is neuroprotective following ischemic stroke through BBB stabilization. To determine if therapeutically targeting integrin a5b1 was feasible, we first determined if brain integrin a5b1 expression increases after experimental mouse ischemic stroke model, specifically tandem/transient common carotid artery/middle cerebral artery occlusion. We found that integrin a5b1 does increase acutely, by post-stroke day (PSD)2, and continued in an exponential fashion through PSD4. Next, we determined if integrin a5b1 was therapeutically accessible by systemic treatment (i.e. intraperitoneal or intravenous) by being located on the inside (luminal surface) of vasculature. We found that location of integrin a5b1 was dependent on the area relative to the stroke injury. The core, or area of direct impact, demonstrated expression of integrin a5b1 on the outside vasculature (abluminal surface), while per-infarct expression was localized to the lumen. Lastly, to determine the activity of integrin a5b1 following ischemic stroke, we showed that the potential ligands (binding partners), plasma fibronectin, fibrinogen, and amyloid-b, do not bind integrin a5b1 after ischemic stroke. Next, we determined the therapeutic potential of targeting integrin a5b1 with the small peptide, ATN-161. ATN-161 has undergone clinical trials in solid tumors, with limited side effects reported. First, we determined that intraperitoneal (IP) injection of ATN-161 was safe after ischemic stroke, showing no changes in heart rate, pulse distention (blood pressure), or body temperature. Next, we found that IP administration of ATN-161 after experimental ischemic stroke reduced infarct volumes, edema, and functional deficit. Furthermore, these results were due to reduction of BBB permeability and anti-inflammatory effects. Interestingly, ATN-161 reduced cytokine production, prevented leukocyte infiltration, and leukocyte recruitment. Collectively, these results suggest that targeting integrin a5b1 with ATN-161 is 1) feasible, 2) safe and 3) effective, suggesting that ATN-161 may be a novel therapeutic treatment for ischemic stroke.

Integrins are a large family of heterodimeric transmembrane glycoprotein receptors, composed by two non-covalently associated subunits (α and β). Integrins αVβ3 and αVβ5 are overexpressed on blood vessels in human tumors but not on vessels in normal tissues and, for this reason, they have become attractive targets for pharmacological studies in oncology. Thus, in this PhD thesis, the synthesis of new Small Molecule-Drug Conjugates (SMDCs) targeting αvβ3 integrin is described. The structure of such SMDCs consists in the connection of three core components: i) LIGAND - the cyclo[DKP-RGD] peptidomimetic, developed by the Gennari and Piarulli group, has been used as integrin-targeting moiety in all the new constructs; ii) CYTOTOXIC PAYLOAD - three different cytotoxic agents (i.e. paclitaxel, monomethyl auristatin E and monomethyl auristatin F) have been included as anticancer drugs; iii) LINKER - specific functional groups (i.e. peptides) have been used to connect the drug and ligand, aiming at the selective drug release in the intra or extracellular tumor environment. These new SMDCs have been subjected to a panel of biochemical and biological assays, for assessing both their structural features (e.g. stability, kinetics of drug release, etc.) and biological activity (e.g. affinity for the purified integrin αVβ3 receptor, selective cytotoxicity against αvβ3-expressing or non-expressing cells, etc.). As a first project, Chapter II describes the development of a new library of multimeric cyclo[DKP-RGD]-PTX conjugates (monomeric, dimeric, trimeric and tetrameric conjugates) bearing lysosomally-cleavable linkers and their full in vitro biological evaluation. The results achieved with these first compounds prompted the design of next-generation cyclo[DKP-RGD]-PTX conjugates, reported in the following chapters. Chapter III describes the synthesis and in vitro evaluation of cyclo[DKP-RGD]-PTX conjugates bearing extracellularly-cleavable peptide linkers, capable of releasing the payload in the tumor microenvironment, rather than inside cancer cells: this mechanism can be promoted by tumor-associated enzymes present in the tumor stroma (e.g. elastase), which can efficiently cleave the linker and set the drug free to diffuse within the tumor mass. Finally, Chapter IV consists in the development of a small library of conjugates containing the cyclo[DKP-RGD] ligand, a lysosomally-cleavable peptide linker and the highly potent toxins monomethyl auristatin E or F (MMAE and MMAF) as the cytotoxic payloads, which are state-of-the-art tools for targeted anticancer therapy.

In the first part of the Thesis, the synthesis of different classes of peptidomimetics targeting integrin receptors is described. Both simple ligands and drug conjugates were synthesized and tested to assess their biological activity. In cancer therapy, peptides and peptidomimetics targeting integrins are employed as carriers to deliver cytotoxic drugs at the tumor site, taking advantage of integrin over-expression on the surface of tumor cells. Within this frame, synthetic efforts have been focused on the synthesis and on the conjugation to the cytotoxic agent paclitaxel of isoDGR-based integrin ligands to effect drug-targeting to integrin over-expressing tumor cells. The second part of the Thesis deals with the vascular endothelial growth factor receptors and their ligands. In particular, the interaction of VEGF-C with VEGFRs has been considered, focusing the attention on the reason why this growth factor was considered a privileged candidate for the preparation of a small library of VEGFR-selective peptides. These peptides have been synthesized by introducing systematic modifications in the natural portion of the growth factor and evaluated as VEGFR binders and anti-angiogenic agents in vitro.

Specific interactions of ligands with receptors is one of the approaches for active targeting of anticancer drugs to cancer cells. Over expression of integrin receptors is a physiological manifestation in several cancers and is associated with cancer progression and metastasis, which makes it an attractive target for cancer chemotherapy. The peptide sequence for this integrin recognition is the Arg-Gly-Asp (RGD). Self-assembly offers a unique way of presenting ligands to target receptors for recognition and binding. This study focuses on development of integrin specific peptide amphiphile self-assemblies as carriers for targeted delivery of paclitaxel to α v β 3 integrin overexpressing cancers. Amphiphiles composed of conjugates of different analogs of RGD (linear, cyclic or glycosylated) and aliphatic fatty acid with or without 8-amino-3,6-dioxaoctanoic acid (ADA) as linker were synthesized and characterized. The amphiphiles exhibited Critical Micellar Concentration in the range of 7-30 μM. Transmission electron microscopy images revealed the formation of spherical micelles in the size range of 10-40 nm. Forster Resonance Energy Transfer studies revealed entrapment of hydrophobic dyes within a tight micellar core and provided information regarding the cargo exchange within micelles. The RGD micelles exhibited competitive binding with 55% displacement of a bound fluorescent probe by the cyclic RGD micelles. The internalization of fluorescein isothiocynate (FITC) loaded RGD micelles was significantly higher in A2058 melanoma cells compared to free FITC within 20 minutes of incubation at 37°C. The same micelles showed significantly lower internalization at 4°C and on pretreatment with 0.45M sucrose confirming endocytotic uptake of the RGD micellar carriers. The IC50 of paclitaxel in A2058 melanoma cells was lower when treated within RGD micelles as compared to treatment of free drug. On the other hand, IC50 values increased by 2 to 9 fold for micellar treatment in comparison to free drug in Detroit 551 cells. In A2058 melanoma xenograft mice model, the Paclitaxel-RGD micelles exhibited a significant inhibition of tumor growth in comparison to control treatment for both alternate day and twice weekly treatments. The studies showed the feasibility of using the non covalent peptide based self-assemblies as vehicles for targeted delivery in cancer.

Fibronectin (Fn) is an adhesive extracellular matrix protein assembled by fibroblasts into fibrils within ECMs of developing and remodeling tissues. Fn is sensitive to mechanical forces exerted by contractile cells, and can alter its structural conformations in response to mechanical strain within Fn fibrils. We developed probes (both peptide and antibody) to Fn to detect mechano-sensitive perturbations of Fn conformation. Probes were characterized for their binding characteristics (affinity, epitope, mechano-sensitivity) and validated on multiple in vitro and in vivo ECM models. Furthermore, we showed that the mechano-sensitive H5 antibody that we have developed have utility in detection of early molecular signatures of fibrosis in vivo in a mouse model of pulmonary fibrosis. Using the H5 antibody, we also report detection of a conformational switch within the integrin binding FnIII9-10 region. Modulation of Fn’s integrin switching behavior may help in the development of controllable “smart” biomaterials, as well as to the development of conformation-specific imaging probes to detect early molecular signatures of tumor and fibrotic ECMs.

Ce travail est axé sur l’amélioration des propriétés de biospécificité de sondes polymères fluorescentes, d’architectures contrôlées synthétisées par polymérisation RAFT, pour deux applications principales : le ciblage de tumeurs cancéreuses in vivo et le marquage de protéines pour des études in cellulo. Pour une imagerie ciblée de l’angiogénèse tumorale in vivo, des systèmes de ciblage multivalents à deux niveaux ont été élaborés en combinant à la fois i) des polymères bien contrôlés synthétisés par polymérisation RAFT et par le procédé PISA, ii) des clusters peptidiques tétravalents présentant une forte affinité pour les intégrines αvβ3 et iii) des fluorophores émettant dans le rouge lointain/proche-infrarouge pour un suivi in vitro et in vivo par microscopie optique. Deux types de sondes ont été synthétisés, des conjugués linéaires et des nanoparticules chevelues. La présentation multivalente du cluster peptidique permet d’augmenter considérablement l’affinité pour les intégrines αvβ3. Les premières évaluations biologiques indiquent une internalisation cellulaire des sondes polymères médiée par les clusters peptidiques ainsi qu’un marquage sélectif des cellules sur-exprimant les intégrines αVβ3. Pour le marquage de protéines, deux stratégies ont été explorées : le marquage de protéines natives par couplage covalent de sondes ω-fonctionnelles et le marquage de protéines recombinantes par des sondes porteuses d’un ligand spécifique. Pour la première stratégie, une fonction ester activé a été introduite en extrémité ω de sondes polymères par chimie thiol-ène pour marquer les résidus lysines des protéines natives. Cette approche a abouti à un poly-marquage difficile à contrôler mais offrant une brillance élevée. Pour la seconde stratégie, un groupement acide nitrilotriacétique (NTA) a été introduit en extrémité α des sondes polymères afin de marquer spécifiquement les protéines taguées Histidines. Cette approche a permis un marquage efficace de différentes protéines et permet de contrôler précisément le nombre de sondes par protéine ainsi que leur site de fixation sur la protéine. Finalement, suite à ces travaux, une nouvelle stratégie de synthèse de polymères séquencés par addition successive de monomères hétéro-bifonctionnels en utilisant des réactions chimiques très efficaces, sélectives et orthogonales a été proposée et validée<br>This work is focused on improving the biospecificity properties of fluorescent polymer probes, with controlled architectures, for two main applications: the in vivo targeting of cancer tumors and the labeling of proteins for in cellulo studies. For a targeted imaging of tumor angiogenesis in vivo, targeting systems presenting two levels of multivalency were developed by combining both i) well-controlled polymers synthesized by RAFT polymerization and the PISA process, ii) peptide tetravalent clusters exhibiting a high affinity for the αvβ3 integrins and iii) fluorophores emitting in the far red / near-infrared for a monitoring in vitro and in vivo by optical microscopy. Two types of probes were synthesized, linear conjugates and hairy nanoparticles. Multivalent presentation of the peptide cluster induced a significant increase of the affinity for αvβ3 integrins. The first biological evaluations also indicated an efficient cellular internalization of polymer probes mediated by the peptide clusters and a selective labeling of cells over-expressing αvβ3 integrins. For protein labeling, two strategies were explored: the labeling of native proteins by covalent coupling of ω-functional polymer probes and the labeling of recombinant proteins by probes bearing a specific ligand at one chain-end. For the first strategy, an activated ester function was introduced at the ω-end of polymer probes by thiol-ene chemistry to label the lysine residues of native proteins. This approach resulted in a poly-labeling, difficult to control but providing highly bright bioconjugates. For the second strategy, a nitrilotriacetic acid group (NTA) was introduced at the α-end of polymers probes to specifically label Histidine tagged proteins. This approach enabled an efficient labeling of different proteins with a more precise control of the number of probes per protein and of the binding site. Finally, following this work, a new synthetic strategy of sequenced polymers by successive addition of hetero-bifunctional monomers using highly efficient, selective and orthogonal chemical reactions was proposed and validated

碩士<br>國立臺灣大學<br>生化科技學系<br>105<br>In tradition, drugs for cancer treatment always belong to a dump release system. Furthermore, people are devoted to developing the new drug delivery for cancer therapy. Recently, liposomes, primarily composed of phospholipids, are the major drug delivery system. However, liposomes still have some disadvantages as follows. In recent years, there are many research for find new drug delivery, and the ferritin is the one of them. Ferritin is composed of 24 subunits and able to self-assemble to form a nanocage structure with external and internal diameters of 12 and 8 nm, respectively. Ferritin can be easily modified and conjugated with various molecules onto the surface, including dye, peptides, siRNA or antibodies etc. the cavity of ferritin can be loaded drugs. Recent studies demonstrated RGD-modified ferritin had potential in cancer diagnosis and therapy. The RGD peptide has high affinity to integrin αVβ3, a tumor angiogenesis biomarker, and is currently applied to clinical cancer imaging. However, the RGD peptide has disadvantages that the peptide also targets to endothelial cells, leading to stimulation of tumor growth and angiogenesis. Hence, in this study, we wanted to construct a new drug delivery and applied on molecular imaging and cancer therapy. We intended to set up the integrin-targeted ferritin with the DGEA (Asp-Gly-Glu-Ala) peptide which has high affinity to integrin α2β1. In the thesis, we not only made the construct of DGEA-ferritin but also tried the 2xDGEA-ferritin to assess the effect of increasing the copy number of DGEA on the surface of ferritins. Next, we estimated the tumor targeting ability of DGEA-ferritin and 2xDGEA-ferritin on U-87 MG, glioblastoma cell line, or PC-3, human prostate cancer cell line, by incubating cells with FITC-labeled protein. After confirming that proteins could recognize cancer cells through the DGEA peptide, we loaded the doxorubicin into 2xDGEA-ferritin. Through the MTT assay, we demonstrated that the 2xDGEA-ferritin could kill cancer cells and improved drug efficiency, which could use less amount of doxorubicin than free doxorubicin to achieve the same cellular toxicity effect. In the future, we will do in vivo experiments to confirm the potential of developing this drug delivery. If the system succeeds, it will be a new, safe and efficient technology and has great potential in clinical application. Furthermore, multiple peptides could be constructed on the ferritin or other drugs be loaded into the ferritin, and apply these modified-ferritins on molecular imaging and cancer therapy.

This thesis reports on the synthesis, characterisation and biological evaluation of a number of metal complexes designed to interact with the α vβ3 integrin receptor, an important biological target that is heavily involved in angiogenesis, and thus cancer related processes. Two approaches were used to synthesise the integrin-avid targets. The first was to attach a variety of bifunctional chelators (BFC's) for the incorporation of different metal centres to a known integrin antagonist, L-748,415, developed by Merck. The BFC's used were the hydrazinonicotinamide (HYNIC) and monoamine monoamide dithiol (MAMA) systems for coordination to Tc-99m and rhenium of which was used as a characterization surrogate for the unstable Tc core. The 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA) BFC was attached for the inclusion of copper and lutetium. This 'conjugate' approach was designed to yield information on how the BFC and the linker length would affect the affinity for the integrin receptor. The second approach was an 'integrated' method where the chelation moiety was integral to the biologically relevant part of the molecule, which in the case of the α vβ3 integrin receptor, is the arginine-glycine-aspartic acid (RGD) mimicking sequence. Two complexes were created with a modified MAMA derivative placed between a benzimidazole moiety (arginine mimick) and the aspartic acid mimicking terminal carboxylic acid to see how it would affect binding while keeping the molecular weight relatively low. The molecules were tested in vitro against purified human α vβ3 integrin receptor protein in a solid phase receptor binding assay to evaluate their inhibition constants against a molecule of known high affinity and selectivity in [I125]L-775,219, the I125 labelled α vβ3 integrin antagonist. The radiolabelled analogues were also tested in vivo against the A375 human melanoma cell line transplanted into balb/c nude mice as well as Fischer rats implanted with MAT BIII rat mammary adenocarcinoma cells. Animals were imaged on a SPECT camera at various time points and compared to normal tissue to yield tumour/non-tumour uptake ratios.

A novel, lipid-based platform nanotechnology has been designed to overcome limitations of in vivo fluorescent imaging, multidrug resistance (MDR) phenotypes hindering breast cancer chemotherapy, and shortcomings of magnetic resonance imaging (MRI) thermometry. Using this platform, three nanoparticle systems have been developed: QD-SLN (quantum dot-loaded solid lipid nanoparticles), DMsPLN (doxorubicin and mitomycin C co-loaded polymer-lipid hybrid nanoparticles), and HLN (hydrogel-lipid hybrid nanoparticles). Stealth, near-infrared emitting QD-SLN were developed for deep tissue fluorescence imaging, which were capable of extending the depth of penetration beyond 2 cm, with near complete probe clearance and good tolerability in vivo. The QD-SLN was used to evaluate the biodistribution of non-targeted SLN and actively targeted RGD-conjugated SLN. Non-targeted SLN accumulated in breast tumors and evaded liver uptake. The RGD-SLN showed prolonged retention in breast tumor neovasculature at the cost of lesser tumor accumulation due to enhanced liver uptake. With this information, a long circulating, non-targeted DMsPLN with a synergistic cancer chemotherapeutic combination of doxorubicin and mitomycin C was formulated to overcome MDR, enhancing breast cancer chemotherapy. Extensive tumor cell uptake and perinuclear trafficking of DMsPLN overcame the MDR phenotype of breast tumor cells in vitro. The DMsPLN provided the most efficacious chemotherapy reported in literature against aggressive mouse mammary tumors in vivo with significant reduction in whole animal and cardiotoxicity as compared to clinically applied liposomal doxorubicin. In establishing our tumor models, the impact of Matrigel™ on the tumor microenvironment was investigated, demonstrating altered tumor vascular and lymphatic anatomy and physiology, and significantly impacting nanomedicines assessment in mouse models of cancer. In all in vivo studies, tumors were established without use of Matrigel™. To guide thermotherapy of solid tumors, a novel HLN was formulated for use in MRI thermometry, presenting the first contrast agent capable of indicating a tunable, absolute two-point temperature window. In using specific limitations of therapeutic and imaging modalities to inform rational nanoparticle design, this lipid-based platform nanotechnology has extended the application of fluorescence imaging in vivo, enhanced the utility of nanoparticulate chemotherapeutics against breast cancer independent of MDR status, and provided novel functionality for MRI thermometry.