Design, Synthesis And In Vitro Biological Activities Of Phthalocyanine-Based Targeting And Near-Infrared Photosensitizers

Photosensitizer (PS) is one of the three elements of photodynamic therapy (PDT). During the photodynamic process, photosensitizer could be excited by light with appropriate wavelength, and then transfers its energy to the oxygen, generating reactive oxygen species (ROS). The ROS plays an important role in the oxidation of nearby biomolecule, causing apoptosis and death of cell. Photosensitizer is the key factor for energy transfer in the process of photodynamic therapy. Therefore, the properties of photosensitizer determine the photodynamic therapy effect directly. In 1955, hematoporphyrin derivatives (HpD) were first synthesized by Schwartz et al. So far, photosensitizers have undergone three generations. HpD is the first generation PS that was used clinically for PDT, but its relatively short-wavelength absorption, complicated composition and slow in vivo metabolism limit its application in PDT; Second generation of photosensitizers overcome the shortages of HpD, with longer wavelength absorption and fast in vivo metabolism; and the third generation of photosensitizers improve the selectivity towards tumor cells based on the second generation of photosensitizers. In general, the ideal photosensitizer should have strong absorption in the red to NIR region along with a high extinction coefficient; be stable and soluble in water to some extent; low dark toxicity and high phototoxicity; efficient selectivity towards cancer cells and fast in vivo metabolism. So far, improving the selectivity and long-wavelength absorption has being attracted more and more attention of researchers. Phthalocyanine(Pc) is the synthetic derivative of porphyrin. Its high extinction coefficient in the red and near-infrared region, high singlet oxygen generation and fast metabolism, make it one of the most promising second-generation PS. Meanwhile, Pcs often exhibit high aggregation tendency and poor solubility in aqueous solution. Therefore, how to modify the structure of phthalocyanine is the focus of researchers.Peptides are biologically occurring short chains of amino acid linked by amide bonds, which biological activity is related to its sequence. It was reported, the peptides with specific sequence could selectively bind to different integrins that are expressed on the surface of cells. For example, peptides with an RGD (arginine-glycine-aspartic) sequence could selectively bind to the αvβ3 integrin that is over-expressed on the surface of a variety of cancer cells and tumor vascular, while low-expression in the normal cells. As a result, modification of Pc with RGD peptide could enhance the tumor selectivity of the photosensitizer.In this thesis, zinc phthalocyanines with RGD motif and different substituents were designed and synthesized. Their photophysical photochemical and in vitro properties are investigated. In addition, we also studied the in vitro biological activity of a NIR photosensitizer based on naphthalocyanine. The detailed information of this thesis are as follows:1. An unsymmetrical phthalocyanine with one carboxyl acid moiety is designed and synthesized, and then conjugated with c(RGDyK) via amide bond. The HPLC and high-resolution MALDI-TOF/MS results identify the high purity of the product. Photophysical, photochemical properties and in vitro photodynamic activities of the RGD conjugate are studied along with the non-conjugated one. The results show that the conjugated RGD motif has little influence on the photophysical and photochemical properties of the zinc phthalocyanine, such as the electronic absorption and fluorescence spectra, and singlet oxygen quantum yield. The conjugate exhibits good solubility in the cell culture medium with the presence of Cremophor EL. In vitro cell studies are carried on DU145 prostate cancer cells that with high expression of αvβ3 integrin. The extraction method demonstrates that the conjugation of RGD enhance the cellular uptake, which is corresponding with the intracellular fluorescence results. The conjugate shows higher cytotoxicity under the illumination, with an IC50 of 0.04 μM, comparing to the non-conjugated one. These results indicate that this phthalocyanine-RGD conjugated is a promising photosensitizer for targeted PDT.2. Three tertbutyl moieties are introduced to the phthalocyanine-RGD ring. The unsymmetrical phthalocyanine is also conjugated to c(RGDyK). The HPLC and high-resolution MALDI-TOF/MS results show the high purity of the product. The conjugate is well soluble in DMF and cell-culture medium formulate with 0.1% Cremophor EL as evidenced by UV-Vis and fluorescence spectra. Low fluorescence quantum yield and high singlet oxygen quantum yield were observed from both the RGD-conjugated and non-conjugated Pcs. The conjugation of c(RGDyK) enhance the cellular uptake of Pc greatly in both DU145 and PC3 prostate cancer cells as comparing to the non-conjugated one. Although bright fluorescence emitted by the Pc is observed in mitochondria, the conjugate shows negligible cytotoxicity either in dark or upon exposure to red-light with dose up to 12 J·cm-2. These results suggest that this conjugate is a promising multifunctional imaging probe for mitochondria and cancer cells.3. With expanded π-conjugation relative to phthalocyanine, the absorption of naphthalocyanine red shifts to the near infrared region. We designed and synthesized a water-soluble zinc naphthalocyanine with four carboxylate acid moieties. Its biological activities were studied along with its Pc counterpart. The naphthalocyanine showed good solubility in DMF, and generated singlet oxygen efficiently upon exposure to both light of λ> 610 nm and λ=750 nm. Aggregation was observed for naphthalocyanine in culture medium. The cellular uptake of naphthalocyanine towards HeLa cells is about a half of that of Pc as evidenced by the extraction method in DMF, which is in accordance with the relatively weaker intracellular fluorescence than Pc. Similar phototoxicity were observed for both naphthalocyanine and phthalocyanine under the same irradiation, while remains non-toxic in the dark. Therefore, this novel water-soluble naphthalocyanine could be potentially a NIR photosensitizer for photodynamic therapy.