Preparation and Characterization of Multimodal Hybrid Organic and Inorganic Nanocrystals of Camptothecin and Gold

We demonstrate a novel inorganic-organic crystalline nanoconstruct, where gold atoms were imbedded in the crystal lattices as defects of camptothecin nanocrystals, suggesting its potential use as simultaneous agents for cancer therapy and bioimaging. The incorporation of gold, a potential computed tomography (CT) contrast agent, in the nanocrystals of camptothecin was detected by transmission electron microscope (TEM) and further quantified by energy dispersive X-ray spectrometry (EDS) and inductively coupled plasma-optical emission spectrometers (ICP-OES). Due to gold\u27s high attenuation coefficient, only a relatively small amount needs to be present in order to create a good noise-to-contrast ratio in CT imaging. The imbedded gold atoms and clusters are expected to share the same biological fate as the camptothecin nanocrystals, reaching and accumulating in tumor site due to the enhanced permeation and retention (EPR) effect

NANOCRYSTALS OF CHEMOTHERAPEUTIC AGENTS FOR CANCER THERANOSTICS: DEVELOPMENT AND IN VITRO AND IN VIVO EVALUATION

The majority of pharmacologically active chemotherapeutics are poorly water soluble. Solubilization enhancement by the utilization of organic solvents often leads to adverse side effects. Nanoparticle-based cancer therapy, which is passively targeted to the tumor tissue via the enhanced permeation and retention effect, has been vastly developed in recent years. Nanocrystals, which exist as crystalline and carry nearly 100% drug loading, has been explored for delivering antineoplastic agents. Additionally, the hybrid nanocrystal concept offers a novel and simple way to integrate imaging agents into the drug crystals, enabling the achievement of theranostics. The overall objective of this dissertation is to formulate both pure and hybrid nanocrystals, evaluate their performance in vitro and in vivo, and investigate the extent of tissue distribution and tumor accumulation in a murine model. Pure and hybrid nanocrystals of several model drugs, including paclitaxel (PTX), camptothecin, and ZSTK474, were precipitated by the antisolvent method in the absence of stabilizer, and their size was further minimized by homogenization. The nanocrystals of PTX, which is the focus of the study, had particle size of approximately 200 nm and close-to-neutral surface charge. Depending on the cell type, PTX nanocrystals exerted different level of cytotoxicity. In human colon and breast cancer xenograft models, nanocrystals yielded similar efficacy as the conventional formulation, Taxol, at a dose of 20 mg/kg, yet induced a reduced toxicity. Biodistribution study revealed that 3H-PTX nanocrystals were sequestered rapidly by the macrophages upon intravenous injection. Yet, apparent toxicity was not observed even after four weekly injections. The sequestered nanocrystals were postulated to be released slowly into the blood circulation and reached the tumor. Tritium-labeled-taxol, in contrast, was distributed extensively to all the major organs, inducing systemic toxicity as observed in significant body weight loss. The biodistribution results obtained from radioactive analysis and whole-body optical imaging was compared. To some degree, the correlation was present, but divergence in the quantitative result, due to nanocrystal integrity and limitations associated with the optical modality, existed. Despite their promising properties, nanocrystal suspensions must be securely stabilized by stealth polymers in order to minimize opsonization, extend blood-circulation time, and efficiently target the tumor

TiO2@BSA nano-composites investigated through orthogonal multi-techniques characterization platform.

Abstract Biocompatible coating based on bovine serum albumin (BSA) was applied on two different TiO2 nanoparticles (aeroxide P25 and food grade E171) to investigate properties and stability of resulting TiO2@BSA composites, under the final perspective to create a "Safe-by-Design" coating, able to uniform, level off and mitigate surface chemistry related phenomena, as naturally occurring when nano-phases come in touch with proteins enriched biological fluids. The first step towards validating the proposed approach is a detailed characterization of surface chemistry with the quantification of amount and stability of BSA coating deposited on nanoparticles' surfaces. At this purpose, we implemented an orthogonal multi-techniques characterization platform, providing important information on colloidal behavior, particle size distribution and BSA-coating structure of investigated TiO2 systems. Specifically, the proposed orthogonal approach enabled the quantitative determination of bound and free (not adsorbed) BSA, a key aspect for the design of intentionally BSA coated nano-structures, in nanomedicine and, overall, for the control of nano-surface reactivity. In fact, the BSA-coating strategy developed and the orthogonal characterisation performed can be extended to different designed nanomaterials in order to further investigate the protein-corona formation and promote the implementation of BSA engineered coating as a strategy to harmonize the surface reactivity and minimize the biological impact

Development and Characterization of Multifunctional Nanoparticles for Drug Delivery to Cancer Cells

Lipid and polymeric nanoparticles, although proven to be effective drug delivery systems compared to free drugs, have shown considerable limitations pertaining to their uptake and release at tumor sites. Spatial and temporal control over the delivery of anticancer drugs has always been challenge to drug delivery scientists. Here, we have developed and characterized multifunctional nanoparticles (liposomes and polymersomes) which are targeted specifically to cancer cells, and release their contents with tumor specific internal triggers. To enable these nanoparticles to be tracked in blood circulation, we have imparted them with echogenic characteristic. Echogenicity of nanoparticles is evaluated using ultrasound scattering and imaging experiments. Nanoparticles demonstrated effective release with internal triggers such as elevated levels of MMP-9 enzyme found in the extracellular matrix of tumor cells, decreased pH of lysosome, and differential concentration of reducing agents in cytosol of cancer cells. We have also successfully demonstrated the sensitivity of these particles towards ultrasound to further enhance the release with internal triggers. To ensure the selective uptake by folate receptor- overexpressing cancer cells, we decorated these nanoparticles with folic acid on their surface. Fluorescence microscopic images showed significantly higher uptake of folate-targeted nanoparticles by MCF-7 (breast cancer) and PANC-1 (pancreatic cancer) cells compared to particles without any targeting ligand on their surface. To demonstrate the effectiveness of these nanoparticles to carry the drugs inside and kill cancer cells, we encapsulated doxorubicin and/or gemcitabine employing the pH gradient method. Drug loaded nanoparticles showed significantly higher killing of the cancer cells compared to their non-targeted counterparts and free drugs. With further development, these nanoparticles certainly have potential to be used as a multifunctional nanocarriers for image guided, targeted delivery of anticancer drugs

Multiscale Biodistribution Analysis of Lipophilic, Poorly Soluble Drugs.

Clofazimine is a poorly soluble drug that accumulates as solid deposits in the body during prolonged oral administration. At the outset, we hypothesized that clofazimine accumulated intracellularly by a passive and spontaneous crystallization, and in various levels of experimental set-ups, from a tissue culture to mouse models. We found that clofazimine readily formed amorphous inclusions in complexes with intracellular membranes in MDCK cells, while different types of inclusions were found in the tissue macrophages of clofazimine-diet fed mice. Most of the inclusions in vivo appeared as vibrant red, birefringent, 10 – 20 µm length crystal-like structures; however, their physicochemical and morphological characteristics were inconsistent with those of pure clofazimine crystals. Most remarkably, among the inclusions from macrophages, we discovered a new cytoplasmic structure delimited by double membranes with internal supramolecular organizations resembling stacks of lipidic lamellae. Upon prolonged dosing, the intact clofazimine was redistributed from adipose tissues to the lymphatic organs paralleled by anti-inflammatory responses such as splenomegaly, liver microgranulomas, and an expansion of macrophage populations. In conclusion, instead of passive intracellular crystallization hypothesis, I propose that clofazimine accumulates in vivo by active sequestration in the immune system. By constructing intracellular crystal- and organelle-like “polyhedrosomes”, the macrophages can impact the clofazimine’s systemic pharmacokinetics and biodistribution, from micro to macro scale.Ph.D.Pharmaceutical SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91404/1/jsbaik_1.pd

Assessment of Cellular Alignment and Proliferation on Cellulose Nanofibril(CNF) Films

Research on novel biomaterials to address current limitations associated with bone grafting is currently of high demand. Current treatment options for bone regeneration are facing challenges such as donor site morbidity and limited availability, immune rejection, and disease transmission. Research conducted on natural polymers to mimic Extracellular matrix (ECM)structure of bone tissue demonstrated their strong potential to spur bone regeneration. Engineered scaffolds that are inspired by oriented collagen/Hydroxyapatite (HA)structure in natural bone are promising biomaterials that can provide an ECM like structure for primitive mesenchymal cells to grow and differentiate to the bone cells. Cellulose nanofibril (CNF) is a natural and biocompatible polymer that is widely used in tissue engineering and wound healing. CNF has the same size features of collagen and due to its biocompatibility and anisotropy is a potential candidate to recapitulate bone microstructure. Hydroxyapatite (HA) is the major mineral compound of the bone which improves bioactivity and tissue integration attributes of implanted biomaterials. Current study suggests a methodology to induce fiber alignment in CNF/HA nanocomposites and evaluates the potential of oriented CNF/HA nanocomposite material to support cell growth and redirect pre osteoblasts as precursors to osteocytes. This study lays the foundation for the development of synthetic bone mimic scaffolds with affordable cost and relatively simple method

Mechanics of Biomaterials

The mechanical behavior of biomedical materials and biological tissues are important for their proper function. This holds true, not only for biomaterials and tissues whose main function is structural such as skeletal tissues and their synthetic substitutes, but also for other tissues and biomaterials. Moreover, there is an intimate relationship between mechanics and biology at different spatial and temporal scales. It is therefore important to study the mechanical behavior of both synthetic and livingbiomaterials. This Special Issue aims to serve as a forum for communicating the latest findings and trends in the study of the mechanical behavior of biomedical materials

Fabrication, Characterization and Properties of Hiercchically Ordered Nanocomposites

The objectives of this research are to study the structure, morphology and properties of poly(ethylene-oxide) (PEO)-clay nanocomposites using various imaging techniques. Optical Microscopy, Scanning Electron Microscopy, Environmental Scanning Electron Microscopy and Atomic Force Microscopy are used to image structures on all length scales. Complementary scattering experiments are discussed along with preliminary differential scanning calorimetry and Fourier Transform Infrared spectroscopy results. In particular we investigated the multilayered structures of PEO-Laponite and PEO-Montmorillonite multilayered films made from solution. The shear orientation of a polymer-clay network in solution combined with simultaneous solvent evaporation leads to supramolecular multilayer formation in the film. The resulting films have highly ordered structures with sheet-like multilayers on the micrometer length scale. The polymer covered clay platelets were found to orient in interconnected blob-like chains and layers on the nanometer length scale. Inside the blobs, scattering experiments indicate the polymer covered and stacked clay platelets orient in the plane of the film. The effect of clay on polymer crystallinity in multilayered films containing different concentrations of clay is inferred from preliminary DSC studies. Overall our results suggest the re-intercalation of clay platelets in films made from exfoliated polymer-clay solutions as well as the supramolecular order and hierarchical structuring on the nanometer, via micrometer to the centimeter length scale

A Regenerable Biosensing Platform for Bacterial Toxins

Waterborne diarrheal diseases such as travelers’ diarrhea and cholera remain a threat to public health in many countries. Rapid diagnosis of an infectious disease is critical in preventing the escalation of a disease outbreak into an epidemic. Many of the diagnostic tools for infectious diseases employed today are time-consuming and require specialized laboratory settings and trained personnel. There is hence a pressing need for fit-for-purpose point-of-care diagnostic tools with emphasis in sensitivity, specificity, portability, and low cost. We report work toward thermally reversible biosensors for detection of the carbohydrate-binding domain of the Escherichia coli heat-labile enterotoxin (LTB), a toxin produced by enterotoxigenic E. coli strains, which causes travelers’ diarrhea. The biosensing platform is a hybrid of two materials, combining the optical properties of porous silicon (pSi) interferometric transducers and a thermoresponsive multivalent glycopolymer, to enable recognition of LTB. Analytical performance of our biosensors allows us to detect, using a label-free format, sub-micromolar concentrations of LTB in solution as low as 0.135 μM. Furthermore, our platform shows a temperature-mediated “catch-and-release” behavior, an exciting feature with potential for selective protein capture, multiple readouts, and regeneration of the sensor over consecutive cycles of use

Development of 2-Dimensional Photonic Crystal Sensors and Pure Protein Organogel Sensing and Biocatalytic Materials

We developed responsive hydrogels, organogels, and ionogels for chemical sensing and catalysis applications. Gels have two components, polymer networks and solvent mobile phases. Hydrogels contain an aqueous mobile phase; organogels an organic solvent; and ionogels an ionic liquid. Different solvent types were required to target different applications, e.g. gas sensing requires solvents that resist evaporation. Colorimetric chemical sensors utilize our 2-Dimensional Photonic Crystals (2DPC) technology. 2DPC are arrays of self-assembled polystyrene nanoparticles that have close-packed, hexagonal crystal structures. 2DPC diffract wavelengths of light into discrete angles according to the 2D Bragg equation. Diffraction depends on 2DPC particle spacing and ordering. 2DPC—embedded into gels that were designed such that analytes actuate polymer volume phase transitions (VPT)—change particle spacing with the VPT, shifting diffraction angles. VPT occur when analytes cause Gibbs free energy changes, ∆G. 2DPC surfactant sensors utilized poly(N-isopropylacrylamide) (PNIPAAm) hydrogels. PNIPAAm hydrogels swell when the hydrophobic tail of ionic surfactants bind to the PNIPAAm isopropyl group. A Donnan potential created by bound charges induces ∆GIonic, causing swelling that red shifts the diffraction. 2DPC gas sensors for humidity and ammonia utilized poly(hydroxyethylmethacrylate)-based polymers in the ionic liquid ethylguanidinium perchlorate (EGP). Ionogels are suitable gas sensors—ionic liquids have negligible vapor pressures, delivering mobile phases that don’t evaporate. ∆GMixing occurs when EGP absorbs water vapor, causing ionogel shrinking that blue shifts the diffraction. Ammonia sensors incorporated acrylic acid into the polymer. Ammonia absorbed by EGP deprotonated the carboxyl groups, causing swelling that red shifts the diffraction. Responsive pure protein organogels were fabricated from protein hydrogels by exchanging water with ethylene glycol. 2DPC albumin organogels swell when the proteins bind ligands, enabling water insoluble analyte detection that utilizes protein selectivity. Organophosphorus Hydrolase organogels catalyze hydrolysis of organophosphate nerve agents ~160 times faster than their monomers in organic solvent. Organic solvents typically denature proteins. Crosslinked organogel proteins mostly retain their native protein reactivity because the proteins are immobilized—i.e. stabilized—during hydrogel polymerization. Protein polymer phase separation that accompanies the solvent exchange irreversibly changes the polymer morphology, however the proteins retain their secondary structure and solvation shell waters in pure ethylene glycol