No description available.

No description available.

No description available.

No description available.

Despite the scientific progress in the last decades in terms of therapeutic agents to fight cancer there is still the need of developing safer and more effective drugs. Developing an innovative drug is not only very expensive, but also highly time consuming; furthermore, the number of anticancer agents that fail in clinical trials with high attrition rates mainly caused by unexpected toxicity and lack of efficacy far outweighs those considered effective, which indicates that drug screening processes require further improvements. In this project the application of Fourier transform infrared microspectroscopy is evaluated in order to develop a spectral based model that could be used to describe the drug-cell interaction and also to discriminate between the metabolic modifications due to a particular drug and the inherent cell cycle of a cell. A computational method was built using the FTIR spectra from a highly resistant renal cell carcinoma cell line, Caki-2, in order to discriminate between the phases of the continuous cell cycle that cells undergo while proliferating in vitro. Such model enabled the discrimination of early events of the cell cycle (G0/G1 phase cells) from G2/M phase cells with a prediction accuracy of 90% and 92.9% respectively. On the other hand, when the RMieS-corrected FTIR spectra corresponding to G0/G1, S and G2/M phases were modelled, the algorithm was able to retrieve each stage of proliferation with 82.3%, 71.8% and 84.4% accuracy respectively. Although the average accuracy yielded by the method was relatively low compared with what has previously been reported in the literature, these results emphasize the need to correct the data from physical distortions due to size and prove the principle that it is possible to create a method for identifying different events of the cell cycle based on the data that the FTIR spectroscopy provides, as well as using the scattering profile characteristic of each phase of development. Once the underlying biochemistry of proliferating Caki-2 cells were characterised by FTIR, the cells were treated with 5-Fluorouracil and paclitaxel, two widely used cytotoxic agents known to induce cellular damage at S or G2/M phase of the cell cycle respectively. The FTIR spectra collected were analysed via multivariate and bivariate techniques. Results demonstrated that, after 24 hours of treatment at the IC50 concentration of each drug, Caki-2 cells displayed spectral features consistent with early stages of apoptosis. These spectral characteristics did not appear to be linked either to the drug’s mode of action or the cell’s cycle phase. The cell’s proliferation stage was not the main classification trend among the drug-treated spectra; nevertheless, the cell cycle phase of each drug-treated population was successfully retrieved by an optimized model capable to classify such phases with an average accuracy of 77.98%. Altogether, this study offers a new perspective when analysing FTIR data from single cells as a function of the cell cycle and also when investigating the biochemical response of a cell line to a given anticancer agent.

Doxorubicin (DOX) and Pluronic-PAA interaction was investigated using isothermal titration calorimetry (ITC). DOX/polymer interaction is governed primarily by electrostatic interaction. The uptake of DOX results in the formation of insoluble polymer/DOX complex. Addition of salt weakens the interaction of drug and polymer by charge shielding effect between positive ionized amino group on DOX and oppositely charged polymer chains. However high drug-loading capacity in high salt condition implied that self-association property of DOX also play a role in the drug loading process. / Singapore-MIT Alliance (SMA)

Self-assembling peptides have emerged as new nanobiomaterials in the areas of nanoscience and biomedical engineering. In this category are self-assembling, ionic-complementary peptides, which contain a repeating charge distribution and alternating hydrophobic and hydrophilic residues in the amino acid sequence, leading to a unique combination of amphiphilicity and ionic complementarity. These peptides can self-assemble into stable nanostructures or macroscopic membranes that can withstand conditions of high temperature, extreme pH, many digesting enzymes and denaturation agents. Moreover, they exhibit good biocompatibility with various cultured mammalian cells, and do not have detectable immune responses when introduced into animals. These properties make them ideal materials for tissue scaffolding, regenerative medicine and drug delivery. This thesis focuses on the utilization of self-assembling peptides for hydrophobic anticancer drug delivery. The hydrophobic anticancer agent ellipticine was selected as a model drug. The studies include: (i) characterization of the photophysical properties of ellipticine in different environments; (ii) study of the formation of peptide-ellipticine complexes and the release kinetics; (iii) investigation of the cellular toxicity of the complexes and ellipticine uptake; (iv) study of the peptide sequence effect on the complex formation and in vitro delivery. Prior to applying ellipticine to the peptide-based delivery system, the fundamental studies on the effect of solution conditions, especially solvent polarity and hydrogen bonding, on the fluorescence of ellipticine were carried out. Ultraviolet (UV) absorption and fluorescence emission of ellipticine were found to be solvent/environment dependent. The absorption and emission maxima shifted to higher wavelengths (red shift) with increased solvent polarity. Large Stokes’ shifts were due to intramolecular charge transfer (ICT), which was enabled by large solvent polarity and hydrogen bonding of ellipticine with the solvents. The photophysical response of ellipticine to changes in solvent polarity and hydrogen bond formation could be used to infer the location of ellipticine in a heterogeneous medium, such as liposomes and cultured cells. EAK16-II, a model self-assembling peptide, was found to be able to stabilize ellipticine in aqueous solution. The equilibration time required to form peptide-ellipticine complex suspensions was found to be peptide concentration-dependent and related to the peptide critical aggregation concentration (CAC, ~0.1 mg/mL). With different combinations of EAK16-II and ellipticine concentrations, two molecular states (protonated or crystalline) of ellipticine could be obtained in the complexes. The release kinetics of ellipticine from the complex into egg phosphatidylcholine (EPC) vesicles (cell membrane mimics) was also affected by the peptide concentration used in the drug formulation. A higher peptide concentration resulted in a faster transfer rate, in relation to the size of the resulting complexes. Subsequent cellular studies on two cancer cell lines, A549 and MCF-7, showed that the complexes with protonated ellipticine were more effective against both cell lines, but their dilutions were not very stable. In addition, it was found that ellipticine uptake in both cell lines was very fast and through direct membrane permeation. Three peptides, EAK16-II, EAK16-IV and EFK16-II, either having a different charge distribution (EAK16-II vs. EAK16-IV) or hydrophobicity (EAK16-II vs. EFK16-II), were tested for the complexation and in vitro delivery of ellipticine. It was found that EAK16-II and EAK16-IV were able to stabilize protonated or crystalline ellipticine depending on the peptide concentration; EFK16-II, on the other hand, could stabilize neutral ellipticine molecules and ellipticine (micro)crystals. The viability results showed that the charge distribution of the peptides seemed not to affect the complex formation and its therapeutic efficacy in vitro; however, the increase in hydrophobicity of the peptides significantly altered the states of stabilized ellipticine and increased the stability of the complexes. This work provides essential information for peptide sequence design in the development of self-assembling peptide-based delivery of hydrophobic anticancer drugs.

Cisplatin (CisPt) is an important drug that is used against various cancers, including testicular, ovarian, lung, head, and neck cancer. However, its effects are limited by cellular resistance. The resistance is believed to be multifactorial, and may be mediated to varying degree by multiple systems in cells, one of the proposed systems being the copper (Cu) transporting system. The Cu-importer Ctr1 has proven importance for cellular sensitivity to CisPt by regulating its influx, while the Golgi-localized Cu-ATP:ases ATP7A/B can putatively mediate CisPt efflux and/or drug sequestration. Atox1 is a small Cu-chaperone that normally transfers Cu between Ctr1 and ATP7A/B, prior to delivery of Cu to the proteins in the secretory pathway. Since Ctr1 and ATP7A/B are reportedly involved in CisPt-resistance, CisPt interaction with Atox1 was the focus of the project this thesis is based upon.   Using a variety of techniques, Atox1 was found to bind CisPt, also simultaneously with Cu. The Atox1-CisPt complexes were further probed using selected mutants in studies demonstrating that only the two cysteines (Cys12 and Cys15) in the Cu-binding site of Atox1 are essential for CisPt interactions. A proposed Atox1 di-metal complex containing both Cu and CisPt was found to be monomeric, and no loss of Cu was observed. In vitro experiments demonstrated that CisPt could also bind to metal-binding domain 4 of ATP7B (WD4), and that the drug could be transferred from Atox1 to the domain. These findings indicated that Atox1 may transfer CisPt to ATP7A/B in vivo, utilizing the same transport pathway as Cu. However, the CisPt-bound Atox1 complexes were not stable over time; upon incubation, protein unfolding and aggregation were observed. Thus, in vivo, Atox1 might alternatively be a dead-end sink for CisPt.   The effects of the ligands around the Pt-center of Pt-based anticancer drugs and drug derivatives on Atox1 binding and unfolding were also investigated. The ligands’ chemistry and geometry were shown to dictate the extent and rate of the Pt-based substances interactions with Atox1. Finally, the occurrence of Atox1-CisPt interactions in a biological environment was demonstrated by developing and applying an antibody-based method allowing analysis of metals associated with Atox1 extracted from CisPt-treated cells.   The findings presented in this thesis show that CisPt binds to Atox1 and WD4, also simultaneously with Cu, in vitro. The results support the hypothesis that Cu-transporting proteins can mediate cellular resistance to CisPt in vivo, and provide a deeper chemical understanding of the interactions between the proteins and the drug.

Self-assembling peptides have emerged as new nanobiomaterials in the areas of nanoscience and biomedical engineering. In this category are self-assembling, ionic-complementary peptides, which contain a repeating charge distribution and alternating hydrophobic and hydrophilic residues in the amino acid sequence, leading to a unique combination of amphiphilicity and ionic complementarity. These peptides can self-assemble into stable nanostructures or macroscopic membranes that can withstand conditions of high temperature, extreme pH, many digesting enzymes and denaturation agents. Moreover, they exhibit good biocompatibility with various cultured mammalian cells, and do not have detectable immune responses when introduced into animals. These properties make them ideal materials for tissue scaffolding, regenerative medicine and drug delivery. This thesis focuses on the utilization of self-assembling peptides for hydrophobic anticancer drug delivery. The hydrophobic anticancer agent ellipticine was selected as a model drug. The studies include: (i) characterization of the photophysical properties of ellipticine in different environments; (ii) study of the formation of peptide-ellipticine complexes and the release kinetics; (iii) investigation of the cellular toxicity of the complexes and ellipticine uptake; (iv) study of the peptide sequence effect on the complex formation and in vitro delivery. Prior to applying ellipticine to the peptide-based delivery system, the fundamental studies on the effect of solution conditions, especially solvent polarity and hydrogen bonding, on the fluorescence of ellipticine were carried out. Ultraviolet (UV) absorption and fluorescence emission of ellipticine were found to be solvent/environment dependent. The absorption and emission maxima shifted to higher wavelengths (red shift) with increased solvent polarity. Large Stokes’ shifts were due to intramolecular charge transfer (ICT), which was enabled by large solvent polarity and hydrogen bonding of ellipticine with the solvents. The photophysical response of ellipticine to changes in solvent polarity and hydrogen bond formation could be used to infer the location of ellipticine in a heterogeneous medium, such as liposomes and cultured cells. EAK16-II, a model self-assembling peptide, was found to be able to stabilize ellipticine in aqueous solution. The equilibration time required to form peptide-ellipticine complex suspensions was found to be peptide concentration-dependent and related to the peptide critical aggregation concentration (CAC, ~0.1 mg/mL). With different combinations of EAK16-II and ellipticine concentrations, two molecular states (protonated or crystalline) of ellipticine could be obtained in the complexes. The release kinetics of ellipticine from the complex into egg phosphatidylcholine (EPC) vesicles (cell membrane mimics) was also affected by the peptide concentration used in the drug formulation. A higher peptide concentration resulted in a faster transfer rate, in relation to the size of the resulting complexes. Subsequent cellular studies on two cancer cell lines, A549 and MCF-7, showed that the complexes with protonated ellipticine were more effective against both cell lines, but their dilutions were not very stable. In addition, it was found that ellipticine uptake in both cell lines was very fast and through direct membrane permeation. Three peptides, EAK16-II, EAK16-IV and EFK16-II, either having a different charge distribution (EAK16-II vs. EAK16-IV) or hydrophobicity (EAK16-II vs. EFK16-II), were tested for the complexation and in vitro delivery of ellipticine. It was found that EAK16-II and EAK16-IV were able to stabilize protonated or crystalline ellipticine depending on the peptide concentration; EFK16-II, on the other hand, could stabilize neutral ellipticine molecules and ellipticine (micro)crystals. The viability results showed that the charge distribution of the peptides seemed not to affect the complex formation and its therapeutic efficacy in vitro; however, the increase in hydrophobicity of the peptides significantly altered the states of stabilized ellipticine and increased the stability of the complexes. This work provides essential information for peptide sequence design in the development of self-assembling peptide-based delivery of hydrophobic anticancer drugs.

Ellipticine (EPT), a natural plant polyphenolic compound, has long been known for its significant anticancer and anti-HIV activities. Recent study on its photophysical properties has revealed that ellipticine has three molecular states: protonated, neutral and crystalline. Further in vitro cytotoxicity tests indicated that protonated ellipticine exhibited much higher anticancer activity than the other two states. To maximize drug therapeutic effect, a small library of ariginine-rich ionic complementary peptides derived from EAK, including EAR8-II, EAR8-a, ELR8-a, and EAR16-II, were investigated as a potential carrier to deliver prescribed protonated ellipticine for treatment of cancer. Fluorescence study demonstrated that all four peptides were able to solubilize and stabilize protonated ellipticine in aqueous solution at 5:1 mass ratio of peptide-to-ellipticine (0.5: 0.1 mg/mL) even upon 4000 times dilution. Physicochemical characteristics of peptides self-assemblies and peptide-ellipticine complexes such as particle size, surface charge, secondary structure and morphology were determined by dynamic light scattering (DLS), zeta potential, circular dichroism (CD) , atomic force microscopy (AFM) and transmission electron microscopy (TEM), respectively. Then the ellipticine maximum suspension was determined by ellipticine UV-absorption. With the help of the peptides and mechanical stirring overtime, up to 100% ellipticine could be uptaken and stabilized in the solution as protonated ellipticine. In vitro cytotoxicity tests indicated that the peptides were demonstrating significant biocompatibility without affecting the survival of two cancer cell lines, human lung carcinoma cell line A549 and breast cancer cell line MCF-7, whereas the complexes with protonated ellipticine were found to show great anticancer activity to the two cancer cell lines. The IC50 values were obtained for each of four different peptide-ellipticine complexes ranged from 0.36±0.12 to 18.90±0.46 μM. It is worth noting that the IC50 value of EAR16-ellipticine complex to MCF-7 was over 50 times higher than that one to A549, which presented that EAR16-ellipticine complex has a selective targeting activity to A549, with the lowest IC50 value of 0.36±0.12 μM among all four complexes. Such a result indicated that this library of novel arginine-rich ionic complementary peptides had a great potential to encapsulate prescribed protonated ellipticine and exhibited an excellent anticancer activity upon serial dilution in aqueous solution. Overall, the charge distribution and increased hydrophobicity of the short (8 amino acids length) peptides seemed not to affect the complex formation and its therapeutic efficacy in vitro; however, the increase in length of the peptides significantly altered the nanostructure of peptides and its complexation with ellipticine, increased the therapeutic efficacy of EAR16-EPT to A549. This work provides essential information for peptide sequence design in the development of self-assembling peptide-based delivery of hydrophobic anticancer drugs.