Evaluation Of The Influence Of Substitutions On Photodymic Antitumor Efficacy Of Bacteriochlorin Photosensitizer

The goal of this study is to investigate antitumor efficacy of a series of new stable synthetic bacteriochlorin photosensitizers mediated photodynamic therapy. This study is divided into two parts: the first part mainly using quantitative structure-activity relationship study on 12 new stable synthetic bacteriochlorins compounds for PDT activity. Quantitative structure-activity relationships were derived, and subcellular localization was determined. This combination together with near-infrared absorption gives these bacteriochlorins great potential as photosensitizers for treatment of cancer. Part two investigate a series of four stable synthetic bacteriochlorins was tested for photodynamic therapy(PDT) on He La cells in vitro and in vivo. Cremophor EL(Cr EL) micelles was employed as drug delivery system to reduce aggregation and increased activity. The incorporation of cyano substituents provides an excellent motif for the enhancement of the photoactivity and photostability of bacteriochlorins as PDT photosensitizers.Part one: In vitro photodynamic therapy and quantitative structure-activity relationship(QSAR) studies with stable synthetic near-infrared-absorbing bacteriochlorin photosensitizers.This is the first time study of application of QSAR method on PDT efficacy of synthetic bacteriochlorin photosensitizers.1) UV-visible spectrophotometry was used to study absorption spectrum of bacteriochlorin photosensitizers(BC1-12). The results showed all 12 BC compounds have absorption peaks between 730 nm to 780 nm in nearinfrared range.2) The measured partition coefficient(m Log P) values for relative solubility in octanol versus water were obtained experimentally by the stir-flask method, and the values were also calculated(c Log P) on the basis of the bacteriochlorin structure. The measured partition coefficient(m Log P) values are between-0.5 to 2.5. The results showed that the twelve bacteriochlorins encompassed a range of polarity that extended from rather lipophilic(3 compounds) to more amphipathic(4 compounds) to polar(5 compounds). 3) The cellular uptake of BC1-12 was conducted on Hela cells using fluorescence spectrophotometry. The cellular uptake of 12 compounds measured at the highest concentration(20 μM) was between 0.1-0.4 μM/l04 cells.4) MTT viability assay in Hela cells was employed to determine the PDT efficacy of BC1-12. The results allowed a LD50 concentration(in n M or μM) to be calculated after 10 J/cm2 of light was delivered. The most active compounds have LD50 values of 100 n M or less(BC3, 12, 4, 6, 2). The compounds with intermediate activity have LD50 values between 100 n M and 1 μM(BC11, 1, 10, 9). The least active compounds have LD50 values greater than 1 μM(BC5, 7, 8).5) Three of the bacteriochlorins with different structural features were examined using confocal microscopy. The high subcellular localization of lipophilic BC4 in endoplasmic reticulum and mitochondria, of moderately lipophilic BC6 in the endoplasmic reticulum(and lysosomes), and of low lipophilic BC8 predominantly in lysosomes correlates with the finding that both 4 and 6 are much more active than BC8. 6) To investigate the key feature of structure-function relationship, a new parameter “survival fraction per unit uptake” was obtained to remove the cellular-uptake contribution. A quantitative structure activity relationship(QSAR) was constructed for each of the experiment-derived parameters that reflected PDT effectiveness(LD50, cellular uptake, cell killing per unit uptake) against both c Log P and m Log P. The correlation of PDT toxicity per unit uptake with m Log P is markedly better(R2=0.94) than with c Log P(R2 = 0.61).Conclusion:1) The variations in the effectiveness of the photosensitizers can be due to the differences in subcellular localization(mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, etc.) More hydrophobic photosensitizers may localize in mitochondria and endoplasmic reticulum due to the high lipid bilayer content regions. PDT-induced damage in these critical organelles is most likely to trigger cell death.Part two: Role of Dicyano Peripheral Groups, Central Metal Substitution(2H, Zn, Pd), and Cremophor EL Delivery in stable synthetic bacteriochlorins mediated PDT.The goal was to determine if such substituents and/or the presence or the nature of the central metal ion would enhance photostability and/or alter other physicochemical properties to give even greater efficacy for killing He La human cervical cancer cells in vitro.The study investigated the effect of central metal iron and dicyano peripheral groups on photobleaching and PDT efficacy. We studied whether Cremophor? EL,(CREL) micelle encapsulation improves the water solubility and PDT activity of new synthetic bacteriochlorins in vitro and in vivo. This study will provide experimental basis for clinical application of synthetic bacteriochlorins.1. UV-visible spectrophotometry was used to study absorption spectrum and aggregation of bacteriochlorin photosensitizers with and without CREL encapsulation. The results showed all 4 new BC compounds have absorption peaks between 740 nm to 765 nm in near-infrared range.2. The photostability of the four bacteriochlorins was investigated using UVVis spectrummeter. The results show that(NC)2BC and(NC)2BC-Pd are substantially more photostable than(NC)2BC-Zn and BC and have ￡15% loss of absorbance at 100 J/cm2 illumination in both media.3. Fluorescent probes(SOSG and HPF) were used for evaluaatiom of the generation and diffident types of reactive oxygen species of synthetic bacteriochlorins.The order of activity was(NC)2BC-Pd >(NC)2BC-Zn >(NC)2BC> BC. However, only(NC)2BC-Pd and(NC)2BCproduced significant activation of HPF.4. The thin-film hydration method was applied to prepare Cremophor micellar drug delivery system. Stock concentrations of micellar PS were approximately 500 μM.5. The cell uptake of PS is measured by extraction of cells after incubation with PS and quantification of the dissolved PS in the cell extract by fluorescence spectrophotometry. Cr EL substantially decreased the uptake of(NC)2BC-Pd, and BC, but had mixed effects on the uptake of(NC)2BC and(NC)2BC-Zn.6. MTT viability assay was employed to the phototoxicity of PS. The most active PS delivered by direct dilution,(NC)2BC-Pd, had an LD50 value of 25 n M.(NC)2BCwas intermediate in activity with a LD50 of 60 n M, while the least active PS, BC and(NC)2BC-Zn, had LD50 values equal to or greater than 1 mM. The PDT effectiveness of all four PS was improved by Cr EL encapsulation.(NC)2BC-Zn showed the most substantial improvement in that Cr EL gave a 17-fold decrease in the LD50 concentration from 1 μM to 60 n M, while BC gave a 5-fold decrease from 1.8 μM to 350 n M. Only small(<3-fold) improvements were displayed by the more active PS,(NC)2BCand(NC)2BC-Pd. 7. UV-Visible spectrometer was employed to study the potential effect of Cr EL encapsulation on the rate at which the cells took up the PS and therefore became photosensitive. In all cases, Cr EL encapsulation significantly reduced the incubation time required to produce light-mediated cell killing. 8. Confocal microscopy was used to determine their intracellular localization with the use of fluorescent intracellular tracers for lysosomes, ER and mitochondria for BC,(NC)2BC and(NC)2BC-Zn, which had sufficient detectable fluorescence. AO(labels lysosomes) and Rho 123(labels mitochondria), were employed to determine the location of intracellular damage caused by PDT with(NC)2BC-Pd delivered using direct dilution or Cr EL. Micellar delivery appeared to affect the clustering or organelle distribution/localization of any of the compounds.9. A xenograft model for human cervical cancer in severe combined immunodeficiency(SCID) BALB/c mice was used to evaluate the therapeutic efficacy of(NC)2BC-Pd Cr EL mediated PDT. Mice received tail vein injection of(NC)2BC-Pd Cr EL at dosages of 1 mg/kg body weight. 10.The drug pharmacokinetics in plasma and tumor at different time point was measured by HPLC method. The results showed at 15 min post injection,(NC)2BC-Pd Cr EL reached the highest concentration in plasma and tumor tissue. 11.Changes of tumor vascular perfusion 1 h after PDT treatment were studied using Hoechst 33342 diffusion staining. Compared to control, micellarvascular treatment led to a significant decrease of tumor vascular perfusion. 12.Short drug-light-interval PDT was carried out after 15 mins of the drug administration. The results showed PDT group resulted in a total regression of the principal tumor and stayed in remission for the whole course of observation.Conclusion:The work described herein provides important information to-wards understanding and optimizing structure–function relationships in bacteriochlorin photosensitzers. We showed that dicyano substituents on bacteriochlorins enhance photostability while maintaining or enhancing photoactivity. Inserting palladium as a central metal also enhances photoactivity by increasing the generation of hydroxyl radicals via a type I photochemical pathway. Delivering the compounds in Cr EL micelles further enhances the activity. PDT with(NC)2BC-Pd has significant therapeutic effects in a xenograft tumor model for a vascular-targeted treatment with its Cremophor EL formulation.