In the past decades, a series of small peptides, Protein Transduction Domain (PTD), were discovered to be able to facilitate the delivery of small proteins into living cells. With the specific feature, researchers have successfully delivered some functional proteins into living cells. To fully explore and understand the functions and structures of intracellular proteins, more powerful tools are under demand. Recently, an increasing number of rabbit monoclonal antibodies (RabMAbs) have been approved to able to recognize subtle distinctions between the changes of intracellular proteins status. They could be good tools for researchers with the ability to traverse through cell membrane into living cells. In this dissertation, a novel delivery technology for RabMAbs was established. Transcriptional activator of transcription (TAT) peptide was utilized as a delivery carrier for RabMAbs. It was demonstrated that RabMAbs could be delivered into living cells by conjugating with TAT peptide. Different cell lines, including adherent and suspension cells, were tested for the delivery of RabMAbs. The delivery process was studied in terms of incubation concentration and time, and an optimal delivery condition was established. To investigate the biological function of delivered RabMAbs inside cytoplasm, three RabMAbs against actin, procaspase-3 and NF-κB respectively were studied. Their binding activities after delivery were verified via sandwich-ELISA data. The immunofluorescent staining of the delivered RabMAb against actin showed it specifically bound to the actin filament in its native morphology. The quantitative analysis of the delivered RabMAb against procaspase-3 showed that approximately 60% of delivered antibody bound to the antigen proteins. The delivered RabMAb against NF-KB apparently blocked the nuclear translocation of NF-KB introduced by TNF-a. The success of delivering the three rabbit monoclonal antibodies with binding or inhibiting functions demonstrated the feasibility of delivering various RabMAbs into living cells by TAT peptide for studying the biological functions of intracellular proteins. Furthermore, to overcome the efficiency and cost issues of the RabMAb delivery system, a universal delivery platform for RabMAbs was developed. This platform uses goat-anti-rabbit polyclonal antibody conjugated with TAT peptide as delivery vehicle. It was confirmed that the goat-anti-rabbit polyclonal antibody modified with TAT peptide was able to capture RabMAbs and deliver RabMAbs into living cells by the conjugated TAT peptide. The results provide a promising delivery platform for all RabMAbs.

Microencapsulation of 5-fluorouracil was successfully accomplished with poly(ortho esters) polymer by emulsification-solvent evaporation method. While actual drug loading increased with increasing drug load (5 to 15% w/w), the entrapment efficiency remained essentially unaffected under a given set of experimental conditions. Incorporation of sorbitan sesquioleate enhanced entrapment efficiency, decreased the volume-surface mean diameter of the poly(ortho esters) microspheres and provided controlled release of 5-fluorouracil. The volume of aqueous phase was more important than the concentration of polyvinyl alcohol in it. The entrapment efficiency improved from 13 to 33% (w/w) when the volume of the aqueous phase was increased from 20 to 80 ml. The volume of organic phase (methylene chloride) and the concentration of polymer in it played an important role. The use of smaller volumes of more concentrated polymer solution enhanced actual drug loading and entrapment efficiency, and produced larger microspheres. The release of 5-FU from the microspheres prepared with sorbitan sequioleate was found to be nearly independent of the initial drug load with a mean zero-order rate constant of 0.0063 % per hr. The data suggested that drug release was largely a diffusional process with contributions from dissolution and polymer degradation.

Underexplored environments such as soil samples continue to be an untapped source of bacterial strains with great potential to produce secondary metabolites for medicinal applications. As a result, these microorganisms represent a broad and yet unknown reservoir of new strains capable of producing these novel compounds. The current research primarily seeks to perform the isolation, purification, and characterization of secondary metabolites from a soil bacterium (Janthinobacterium lividum TAJX1901). The isolated soil bacterium was successfully cultured on rich media agar plates, followed by extraction using methanol and chloroform. The purification methods utilized include flash column chromatography, preparative thin-layer chromatography, and high-performance liquid chromatography. For structural elucidation, UV-Vis analysis, infrared spectroscopy, and nuclear magnetic resonance spectroscopy were employed. The extraction resulted in a dominant violet pigment soluble in methanol. Results revealed the presence of highly conjugated, polar, and aromatic compounds (violacein or relatives of violacein) and dioctyl phthalate (a contaminant).

Photodynamic Therapy (PDT) of cancer involves radiating photosensitizing drugs with light in tumors, which results in generating active singlet oxygen that kills cancer cells. Photosensitizers currently used in PDT are of low quantum yield and require high energy radiation, normally laser. Therefore there is always need for more effective PDT drugs. In this project we synthesized new derivatives of phenothiazinium for potential applications in PDT. Phenothiazinium was synthesized and derivatized by linking it to side groups containing imidazole rings. These derivatives are also expected to catalyze certain hydrolytic reactions. Such ôhydrolase modelsö use molecular recognition based on ??? stacking between the phenothiazinium ring and aromatic rings of specific substrates, such as anthracene monophosphate, while imidazole groups catalyze the hydrolysis of the phosphate ester by general acid-base mechanism.

Microencapsulated forms of procainamide hydrochloride (PA) were formulated and evaluated for in vitro release against Procan SR('R) tablet. Both ethylcellulose as well as Eudragit-S microcapsules were prepared by a phase separation coacervation technique. The ethylcellulose or Eudragit-S coat-to-core (PA) ratios studied were 1:1, 1:1.33, 1:2 and 1:4, while the coat-to-polyethylene ratios investigated were 3:1, 5:1, 10:1 and 30:1. Treatment of microcapsules with four different concentrations (5, 10, 15 and 20% w/v) of paraffin wax in cyclohexane was also evaluated for effects on drug release. Ethylcellulose microcapsules and Eudragit-S microcapsules with coat to core ratio of 1:4 and 1:2 respectively and a coat-to-polyethylene ratio of 30:1, after treatment with 5% paraffin wax solution, were selected for a biphasic in vitro release study in hydrochloric acid buffer, pH 1.2 for first hour, and phosphate buffer, pH 7.8 for the remaining period. The mixed blends of ethylcellulose and Eudragit-S microcapsules in the proportions of their PA content were also considered for biphasic in vitro release study. While ethylcellulose microcapsules were insensitive to pH changes, Eudragit-S microcapsules gave faster drug release in phosphate buffer pH 7.8 as expected. The time for release of 50% PA was 2.05, 5.85, 2.30, 2.70, 3.00 and 3.90 hours for Procan SR tablets, ethylcellulose microcapsules, Eudragit-S microcapsules, mixed blends MX-1, MX-2 and MX-3 respectively. The ethylcellulose microcapsules (XXI) and mixed blend (MX-3) provided more effective sustained release of PA over several hours than commercially available product Procan SR tablets. Three promising formulations (XXI, XXII and MX-3) were further evaluated for in vivo bioavailability in dogs. On the basis of area under the plasma concentration-time curve (0-10 hrs.) and total cumulative amount of drug excreted (0-30 hrs.), three formulations were found to be bioequivalent to Procan SR('R) tablets. The plasma concentration-time curve data for each formulation was analyzed pharmacokinetically by an open, one-compartment model. Mechanism of drug release from these microcapsules was also studied. The analysis of in vitro release data strongly suggests a diffusion-controlled release of PA from ethylcellulose microcapsules and possibly Procan SR tablets. The release of PA from Eudragit-S microcapsules was shown to be dissolution-controlled.

In view of their native medicinal uses, a systematic investigation of the phytochemistry and Hippocratic screening of three members of the Saururaceac namely, Anemopsis californica, Saururus cernuus and Houttuynia cordata was undertaken.

The antioxidant activity of novel drugs has been of increasing interest in recent years. Free radicals are linked as a cause to many diseases such as atherosclerosis and cancer,1 so development of drugs that can scavenge and break down free radicals is needed. One such potential solution is using dopamine, which is water-soluble and an antioxidant. However, the tendency of antioxidant drugs reacting undesirably with proteins and other biochemical compounds is a big issue for the drugs' antioxidant potential. One solution is by encapsulating the antioxidant compound in biocompatible polymer nanoparticles. In this project, dopamine is bound to the polymer polyacrylic acid (PAA) and spherical PAA-dopamine nanoparticles were synthesized. Following their synthesis, the nanoparticles were characterized by Dynamic Light Scattering (DLS), Transmission Electron Microscope (TEM), and Fourier-Transform Infrared (FT-IR) spectroscopy and were shown to have an average size of 90 nm after dialysis cleaning. Finally, their hydroxyl radical (OH·) scavenging ability was tested through pH changes and fluorescence, and the data acquired suggests possible radical scavenging potential.

An inclusion complex between β-cyclodextrin and insoluble guest compounds has been reported by several researchers. The main purpose of forming an inclusion complex between β-CD and sparingly soluble guests is to enhance the guest’s solubility and mask its undesirable properties. Preliminary studies have shown that when conjugated with target-specific moieties, these inclusion complexes can be used in pharmaceutical applications for drug delivery. β-Sitosterol, a plant sterol, has been well documented to reduce tumor cell growth and migration as well as exhibit apoptotic characteristics. An issue with this plant sterol and most pharmaceutical compounds is their lack of solubility. In this study, we propose that an inclusion complex will enhance the solubility of this sterol and change the physicochemical properties of the sparingly soluble guest. We first prepared the β-CD:β-Sitosterol inclusion complex and characterized the samples in both solid and solution state. The complex was characterized using FT-IR, DSC, SEM and NMR. IR studies of the inclusion complex and physical mixture revealed changes in the characteristic peaks of the inclusion complex suggestive of the formation of a new compound. 1HNMR studies revealed an upfield resonance shift of β-CD internal protons (H3 and H5) as an equal molar ratio of β-Sitosterol is introduced into the β-CD mixture. 2D NOESY NMR studies suggest that the initial sites of interaction of β-CD and β-Sitosterol occur between the aliphatic tail of β-Sitosterol and H3 of β-CD. 2D ROESY NMR reveals that the cyclic head of β-Sitosterol also interacts with the cavity of β-CD suggesting that β-Sitosterol may be completely encapsulated inside β-CD’s cavity. From these initial studies, we hypothesize that the β-CD-PEG-FA will facilitate absorption of β-Sitosterol and increase the drug delivery vehicle’s solubility as a whole. Since most tumor cells over-express folic acid, inclusion of folic acid in the construct of the vehicle will direct these sterols to tumor sites. β-cyclodextrin-PEG, a precursor to the bio-conjugate for antitumor delivery of sterols, was synthesized and characterized.

The proteasome, a multiprotease complex, is clinically validated as an anticancer target by the FDA approval of bortezomib and carfilzomib for the treatment of multiple myeloma. The emergence of resistance to proteasome inhibitors however remains a major clinical challenge. Recently, distinct types of proteasomes termed ‘intermediate proteasomes’, which contain unconventional mixtures of catalytic subunits, have been implicated with drug resistance of tumor cells. In elucidating the role of intermediate proteasomes in drug resistance, a crucial step is to unequivocally determine the subunit composition of intermediate proteasomes in cells. With this in mind, the goal of the studies reported in this dissertation is to develop novel chemical tools which can facilitate the investigation of intermediate proteasomes via two complementary approaches: a FRET-based approach and a bifunctional cross-linking approach. Chapter 2 describes the structure-based design, synthesis, and characterization of a peptide epoxyketone-based fluorescent probe, named as LKS01-B650, which selectively targets the immunoproteasome subunit β5i/LMP7. In addition to its utility in determining the identity of intermediate proteasomes as FRET-based probe, this imaging agent may also serve as a valuable tool in visualizing the immunoproteasome in living cells. Chapter 3 describes the design and synthesis of various epoxyketone-based bifunctional agents. The ability of these bifunctional agents to cross-link different catalytic subunits within a proteasome complex is shown by mobility shift assays.These bifunctional agents may provide important information in determining the subunit composition of proteasomes. Chapter 4 describes a systematic study of the relationship between the proteasome inhibitor structure and the inhibitory activity against critical subunits of the proteasome. Given the reported role of β5i/LMP7 in autoimmune diseases, this study may provide useful insights in developing therapeutic agents for autoimmune diseases as well as other diseases. Chapter 5 describes a separate study which is not related to proteasome biology. A concise approach to synthesize 1-azaspirocyclic ring systems is developed by utilizing a novel semi-pinacol/Beckmann rearrangement. Additionally, an environmentally benign, microwave-assisted, and solvent-free self-condensation of carbonyl compounds is reported.

Fibroblast growth factors (FGFs) are a family of growth factors which includes twenty three proteins. FGFs work as modulators for various cellular activities like mitosis, differentiation and survival. Among the FGF family, human fibroblast growth factor-1 (hFGF-1), which is also known as acidic fibroblast growth factor, is a potent angiogenic agent, involved in the formation of new blood vessels in various tissues. hFGF-1 is regarded as a prototype of the FGF family. It serves as one of the potential targets in tumor inhibition and obesity due to its involvement in new blood vessel formation in cancerous regions and adipose tissues. In general, FGFs exert their action by binding to heparin, forming FGF-heparin complex, which can then bind to fibroblast growth factor receptors (FGFRs). Inhibition of FGF dependent signal transduction by heparin mimicking compounds has shown promising results in control and treatment of tumor growth. Naturally occurring glycoside called phloridzin found to have anticancer property. Phloridzin (2-glucoside of phloretin) has structural resemblance to heparin; it is a natural antioxidant, widely known for its antidiabetic activity, besides controlling tumor growth. Phloridzin can mimic heparin and compete with it for FGF binding. This binding can be agonistic or antagonistic in nature on FGF signal transduction. In the present study, we investigated the molecular level interaction between phloridzin and hFGF-1 using various biophysical techniques like steady state fluorescence, limited trypsin digestion and protein-NMR spectroscopy. hFGF-1 needed for the study was expressed in recombinant Escherichia coli cells. The expressed protein was then purified using heparin sepharose affinity chromatography. Both expression and purification were monitored using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Conformational stability of purified hFGF-1 was assessed through steady state fluorescence. Purified hFGF-1 is in, its native, properly folded conformation. Interaction studies, such as thermal unfolding and limited trypsin digestion were performed to assess the thermal stability and solvent accessibility of hFGF-1 in the presence of phloridzin respectively. It was found from interaction studies that hFGF-1 in the presence of phloridzin shown increased thermal stability and increased resistance against trypsin digestion. In order to locate the sites of interaction on hFGF-1 surface, a protein-NMR study was performed. Exact sites of interaction of phloridzin on hFGF-1 surface were found. In future, isothermal titration calorimetry will be performed to determine kinetics of the enthalpy change and dissociation constant of phloridzin-hFGF-1 interaction. In vivo studies will also be performed after completion of in vitro studies, which will give an insight about possibility of phloridzin and hFGF-1 interaction under physiological condition