Study On The Mechanism Of Oligophenylacetylene Derivatives Across Cell Membranes And Their Application In Radiopharmaceuticals

Photodynamic inactivating bacteria uses light stimulate the light-sensitive molecules to produce singlet oxygen or reactive oxygen species components to cause cytotoxicity.Photosensitizer plays the key role in photodynamic therapy,most of which are porphyrins,cyanines,and recent research in OPEs.To be noted,previous research focused on quaternary ammonium salt,and the other forms of amine are rarely tested such as tertiary amine and primary amine compounds,meanwhile,highly toxic and volatile compound methyl iodide was used to produce quaternary ammonium.Therefore,it is necessary to develop new OPEs,specifically,tertiary amines will be very interesting since these structures are relatively easy to be synthesized and possess high singlet oxygen quantum yields.Through this research,we hope to obtain the structure-activity relationship of OPE and its cytotoxicity.For the surface of cancer cells is also negative charged,the properties of which membranes are similar to those of bacteria in some ways,so the bacteria was used to preliminary screening OPEs for targeting radio-therapy of cancer.Based on the structure of PPEs,we synthesized six symmetrical OPEs by using Sonogashira coupling method,in which,3 are neutral tertiary amines and 3 are corresponding positive charged quaternary amines.And their cytotoxicity was examined and compared both in dark and under visible light irradiation.In the dark condition,no bacteria toxicity was observed.While the bacteria toxicity of neutral OPEs is much better than that of positive charged OPEs under visible-light irradiation(9 mw/cm2),the difference is more than 100 times.It is generally assumed that the light-induced bacteria toxicity is related to their singlet oxygen yield,which is related to the structure of OPEs.However,the alkyl length of OPEs affected the bacteria toxicity and the 1O2 yield more or less,but no significant difference was observed.Due to the limited operation range of 1O2,the relative position between 1O2 generator-OPEs and bacteria is the key point for bacteria toxicity.Only getting close enough to the bacteria,the effective antibacterial properties of OPE could work.So the penetrating-membrane ability of OPEs plays the key role in their bacteria toxicity.So the interaction of cell membrane model and OPEs was studied by establishing phospholipid vesicles and phospholipid monolayer membranes,and proposed the possible penetration membrane mechanism of OPEs on molecular level.DOPG,and E.coli total lipids was chosen as membrane mimics for Gram positive bacteria,and Gram negative bacteria to simulate the interaction of OPEs with cell membrane.The results showed that neutral OPEs can strongly lead to the fluorescence leakage,by contrast,the fluorescence leakage for positive chargedOPEs was much less.We further investigated surface pressure(SP)in the system of the above-mentioned lipids monolayer,and we found that SP became very significant,while positive charged OPEs just induced very little SP enhancement.These results indicated that the membrane-crossing ability of neutral OPEs is much stronger than that of positive charged OPEs.Combined the result of cytotoxicity,it is safely concluded that The tertiary ammonium of neutral OPE is protonated in aqueous solution(pH 7.4),present as a positively charged molecule,which promotes its rapid adsorption onto a bacterial surface,since protonation process is reversible,the hydrophobicity will increase once proton dissociates and cause the increase of entropy by the release of interfacial water through the binding of neutral OPEs to membranes,thus drive neutral OPEs internalization in the outer envelopes of bacteria.Upon irradiation of visible light,the interfacial generated singlet oxygen will produce great antibacterial activity.For the better hydrophily of positive charged OPEs,they could get close to the bacteria by electrostatic interaction,while they could not get into the hydrophobic membrane.In addition,Gram negative bacteria E.coli showed more resistance to OPEs that Gram positive bacteria S.aureus(more than 10 times difference),which is induced by the difference of membrane structure and composition.In conclusion,the irradiation is necessary in the sterilization,and the bacteria toxicity presents positive correlation to the optical density and irradiation time.The structure of OPEs affects the hydrophilic-hydrophobic property of OPEs and interaction with the membrane.Based on the study of symmetric OPEs,we synthesized 4 asymmetric OPEs for connecting tumor targeting peptide.For this 4 asymmetric OPEs,their antibacterial ability and interaction with membrane model were also investigated,which further verified that the antibacterial property of neutral OPE is better than that of positive charged OPE.Regarding to the mammalian cytotoxicity,the normal liver cell L02,normal lung cell MRC-5,normal kidney cell HEK-293 and cancer cell PC-3 were selected for this study.In dark condition,the cytotoxicity could be obtained with increasing the concentration of neutral OPEs.It is worth noting that PC-3 cell showed less tolerance than other 3 normal cells,which is probably induced by the negative charged surface of PC-3 cell.Overall,bacteria showed much more tolerance to OPEs than mammalian cells.Cellular internalization is not only the requirement for light-induced antimicrobial drugs,but for therapeutic radiopharmaceuticals.Agonists were broadly studied in the last decades due to their efficient internalization into cells;however,their drawbacks were also obvious,such as high uptake in pancreas,side effects,and potential risk of cancer.Antagonists,on the other hand,with higher cancer cell affinity,uptake in cancer cells,and fast clearance in normal tissue have attracted much research interest in radiopharmaceutical development.How to take advantage of their virtues and avoid the drawbacks becomes a great challenge for the radiopharmaceutical development.Combining the strong cell-penetrating ability and high affinity of Gastrin-releasing peptide which is overexpressed in the PC-3 cells,the thesis designed and synthesized asymmetric OPEs followed by the coupling reaction with RM26 peptide to obtain the OPE-RM26 and NOTA-OPE-RM26.The two OPE-peptide derivates were radiolabeled with 1-131 and Lu-177,respectively,to give high radiolabeling yields with optimized radio-labeling condition.Considering that the multi-properties being targeting +membrane crossing + crossing fire of radioactive elements of 131I-OPE-RM26 and 177Lu-RM26-OPE.The interaction experiment between the radiolabeling compound and PC-3 cells shows that OPEs do not affect the specific binding of RM26 to cells significantly,but increase the cell internalization ability.It is expected that the molecules may fast travel to the target tissue and bind to GRP receptor and internalized into cancer cells.The cascade process will give rise to its cancer cell toxicity due to longer retention and more toxic to cancer cells.In conclusion,a new class of biocides were developed,namely neutral OPEs,and neutral OPEs were found to be more toxic to cells than positive charged OPEs,which have been explained by cell membrane penetrating ability of neutral OPEs.Positive charges on the biocides have long been recognized as a must.The trend can be understood as the good water solubility,thus a relatively high concentration can be obtained,more importantly,positive charges make these compound more absorptive to cell membrane.However,results in this thesis have indicated that the penetrating ability of positive charged OPEs were much weaker in the system.Thus,it becomes very important to balance its lipophilicity and charge properties of OPE to tune OPEs cytotoxicity.We further explored and extended the application of the OPEs in radiopharmaceuticals by combining the cell penetrating units and cancer targeting peptide while focusing on its cell-penetrating properties to obtain a multi-functional prodrug.Using this strategy is expected to overcome the risk that peptide agonists promote cancer cell mitosis,also overcome the problem of peptide antagonists can't internalize in cancer cells,therefore,a better performance can be expected.