POLYSACCHARIDE BASED NANOCARRIERS FOR ANTICANCER DRUG AND PHOTOSENSITIZER DELIVERY TO ENHANCE CHEMOTHERAPEUTIC EFFICIENCY AND PHOTODYNAMIC THERAPY OF CANCERS

• Main Authors: Tilahun - Ayane Debele, Tilahun Ayane Debele
• Other Authors: Hsieh-Chih Tsai
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/49081657825127025779

博士 === 國立臺灣科技大學 === 應用科技研究所 === 105 === Cancer is a multi-gene, multi-step devastating disease with an altered DNA sequence (mutation) which grow uncontrollable by disregarding the normal rule of cell division. The current available treatment methods of cancers are primarily centered on Surgery, Irradiation and the Systemic administration of Chemotherapeutics agents. Recently, photodynamic therapy (PDT) is also an alternative, non-invasive combinatorial therapeutic modality using light, photosensitizer (PS), and oxygen for the treatment of cancer and other diseases. However, those conventional chemotherapy and photosensitizers (PSs) used nowadays have certain limitations (a) Low (non) water solubility, (b) Lack of selectivity and (c) Multidrug resistance (MDR). Hence, a technology platform that can effectively increase PSs and chemotherapeutic agent’s solubility and confer targeting potential is highly sought after. Nano-formulating of chemotherapeutic agents and PSs with polymeric nanoparticles poses as potential strategy to satisfy the requirements of an ideal chemotherapy and PDT system. In this dissertation, polysaccharide based nanocarrier were synthesized and characterized to deliver anticancer drugs (Doxorubicin) and PSs (Zinc phthalocyanine) that have been used to enhance the chemotherapeutic efficacy and photodynamic therapy against cancers. Polysaccharide-based nanocarriers have a great interest as a vesicle of several anti-cancer drugs and PSs due to their unique multi-functional groups in addition to their physicochemical properties, including biodegradability, water solubility and biocompatibility. The presence of multi-functional groups on the polysaccharide backbone allows facile biochemical or chemical modification to synthesize polysaccharide based nanocarriers with miscellaneous structures. Hence, in this abstract several heparin derivitized based nanoparticle (i.e. nanogels and micelles) as the carrier of PS and anticancer drug were summarized session by session to clearly investigate its role in enhancing PDT activity and chemotherapeutic efficiency. In the first work, bioreducible heparin polyethyleneimine (HPC) nanogel composed of heparin, branched polyethyleneimine (PEI) and L-cysteine was synthesized and characterized. 1H-NMR and FTIR analysis confirmed the formation of HPC nanogels while TEM and dynamic light scattering revealed uniform spherical nanoparticles with average diameter of <200 nm. Zinc phthalocyanine (ZnPc) was encapsulated via the dialysis method and the drug is released in vitro from disulfide-containing HPC nanogels in a redox-sensitive manner. Additionally, HPC nanogels possess bright blue fluorescence which eliminates the use of additional probing agent in image-guided drug delivery. Moreover, singlet oxygen detection revealed that nanogels prevented ZnPc aggregation thus enhancing 1O2 generation and photodynamic therapy (PDT) efficacy. These results showed that disulfide crosslinked HPC nanogels are promising vehicles for stimulated photosensitizer delivery in advanced PDT. Even though, the cytotoxicity of PEI are highly improved, the synthesized HPC nanogels has minimum amount of drug loading capacity (~ 42%). Therefore, to enhance drug loading capacity and to minimize cytotoxicity we were designed our second works based on biocompatible heparin and phospholipids. In the second work, we describe the synthesis of a stable, pH-sensitive micelle from heparin, 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine, and L-histidine (HDH) through EDC/NHS chemistry. 1H-NMR and FTIR analysis confirmed the formation of HDH copolymers and DLS measurements indicated a particle size of 111.57 ± 12.36 nm and zeta potential of -59.8 ± 5.2 mV for the nanoparticles. The drug-loading and encapsulation efficiency of the micelles were 14.52 ± 1.2% and 65.47 ± 1.87%, respectively. Drug release studies showed approximately 91% zinc phthalocyanine (ZnPc) release from micelles in acidic conditions (pH 5.0) in comparison with 63% release physiological conditions (pH 7.4) after 96 h of incubation. Singlet oxygen detection revealed that the micelles prevented ZnPc aggregation and enhanced 1O2 generation. Cellular uptake of ZnPc-loaded micelles (ZnPc-HDH) was observed using confocal microscopy. Phototoxicity experiments in HeLa cells showed that ZnPc-loaded micelles had higher toxicity than the same concentration of free ZnPc had. Hence, pH-sensitive HDH micelles are a promising carrier for hydrophobic ZnPc and improvement of PDT efficacy. Although there are several clinical attempts to treat tumors (including surgery, radiotherapy, chemotherapy and combined therapy) at the primary site there are still a little available therapy to inhibit the spread of metastatic cancer. In the third work, a redox sensitive Heparin-β-Sitosterol micelle was synthesized as the carrier of pharmaceutical agents (Dox) and also antimetastasis activities of the micelles was investigated in vitro using scratch assay. 1H-NMR and FTIR analysis confirmed the formation of bHSC copolymers while DLS used to measure particle size (145.07 ± 2.97 nm) and zeta potentials (-56.1 ± 2.16 mV) of micelle. Spherical like surface morphology of bHSC micelle also investigated using FE-SEM. Both MTT and Flow cytometry analysis were confirmed less toxicity of synthesized micelle. Dox was encapsulated via the dialysis method and the Dox loading and encapsulation efficiency was 16.49 ± 1.2% and 58.47 ± 1.87%, respectively. In vitro Dox release study was evaluated by mimicking the intracellular levels of GSH (5 mM) and approximately 89% and 52% of the Dox was released in the 48 h of incubation in the presence and absence of GSH respectively, which clearly shows synthesized micelle was a redox sensitive. The cellular internalization of Dox-loaded bHSC nanoparticles was studied using confocal laser scanning microscope and the strong fluorescence intensity signals (red fluorescence) was observed mainly in the cell’s nucleus. This confirms the Dox loaded bHSC was up taken by HeLa cells and the Dox were released from bioreducible bHSC micelle. In addition to this, antimetastasis and hemocompatibility of bHSC was evaluated via scratch and hemolysis assays respectively. F-actin fluorescence microscopy result shows, heparin and bHSC treated HeLa cells had poorly oriented stress fibers. In summary, due to its less toxicity, an excellent hemocompatibility and antimetastasis effects, the synthesized bHSC micelle is the best candidate carriers in the drug delivery system and can be used to inhibit metastatic cancers as well.