Ion transport-promoted unwanted death of normal cells remains a major concern for ion therapy. So, to minimize the apoptosis of the normal cells and selectively target the cancer cells, in this work photo-triggered ion transporters has been used which can give site-selective precise control over the functioning of the ion transporters. Herein, a C2 symmetric guanidinium-based macrocycle was synthesised. 13 The ion binding scaffold and the hydrophobic domain impart lipophilicity to transport the ions. To further explore the concept of a photocleavable proanionophore, the o-nitrobenzyl (ONB) group with the guanidinium-N moiety was attached. 14
Figure 3.1. Schematic representation of the photomediated generation of ionophore from ONB-linked proanionophore.
Initially, thiourea-based macrocyclic intermediate was synthesized, and then desulfurization of thiourea moiety with alkyl amines resulted in the expected guanidinium-based macrocyclic compounds. To investigate the importance of hydrophobicity in ion transportation, alkyl amines of different chain lengths (n = 4, 8, and 10) were used. Compound 3.2 showed better binding with phosphate ion and Cl- rather than NO3-. Eventually the ion transport properties of the anionophores (3.1-3.2) were explored in EYPC/CHOL⊃lucigenin using the fluorescence-based assay. The phosphate transport efficacy of compound 3.2 was higher than the compounds 3.1, 3.3, and 3.4. The higher transport proficiency of compound 3.2
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could be attributed to its subtle balance of hydrophilicity and membrane residence aptitude. The anion selectivity of compound 3.2 was performed by lucigenin assay dispersing the LUVs in an aqueous of 225 mM NaxAy salt (where NaxAy = NaH2PO4, Na2SO4, NaOAc, NaClO4, and NaNO3). The selectivity studies revealed that compound 3.2 is more selective towards phosphate ions than the other tested oxyanions. To comprehend the mechanistic pathway, the cation selectivity of compound 3.2 was checked in the presence of Na+ and K+ ions. The Cl-ISE-based assay showed compound 3.2 mediated transport of phosphate/Cl─ ions across the lipid bilayers.
Additionally, when Na2SO4 was kept outside, no change in fluorescence intensity was observed due to inability of the anionophore to facilitate the SO42─/phosphate antiport process as the selectivity for SO42─ is too low for compound 3.2. This result also supports the independent nature of the transport mechanism with respect to the cations. The non-involvement of H+ in the mechanistic pathway could be assured from the enhancement of the HPTS fluorescence intensity when FCCP was added along with the transporter. The only persisting pathway is the phosphate/Cl− antiport mechanism. Moreover, the U-tube assay also propounds a similar result.
Gradually with progression of time, NaCl and NaH2PO4 steadily increases in the opposite arms of the U-Tube. This result also indicates carrier mediated pathway followed by the transporters. This outcome was moreover concluded with the help of temperature-dependent DPPC assay. The transport activities across the Tb(III)- complex-based fluorescent chemosensor (of phosphate ion) LUVs also demonstrated the phosphate transport activities of compound 3.2. The transmembrane transport of phosphate ions in the presence of compound 3.2 using giant unilamellar vesicles (GUVs) was deployed. Furthermore, 31P NMR experiments were done to provide undeniable proof of the phosphate/Cl− antiport process carried by compound 3.2.
After incubation with compound 3.2, the 31P peak decreased which can only be feasible after the phosphate transport, mediated by the transporter 3.2. The molecular dynamics (MD) simulation studies with compound 3.2 and phosphate ion embedded inside a preequilibrated dipalmitoyl phosphocholine (DPPC)/water lipid bilayer system studies revealed the retention of the compound within the bilayer and expulsion of phosphate ion from the complex suggesting the successful transport of the phosphate ion by the compound. To assess the photo-triggered cleavage of proanionophore 3.4 and in-situ generation of anionophore 3.2 the 1H NMR-based
study was performed. After photo irradiation with an LED lamp (365 nm), a new peak was seen at δ = 10.25 ppm, which implies the formation of 4-formyl-3- nitrosobenzoic acid, a plausible result of the photocleavage of the proanionophore 3.4. The successful generation of the anionophore was also studied in the membrane environment. The ion transport activities of proanionophore 3.4 were recorded before and after photo irradiation. It was perceived that the transport efficacy increased by 60%. Consequently, by both the NMR and fluorescence-based methods, the resurgence of the anionophore 3.2 from proanionophore 3.4 was be affirmed.
Figure 3.2. Photoinduced release of anionophore 3.2 from proanionophore 3.4 was monitored by 1H NMR titration experiment in DMSO-d6 solvent.
The perturbation of chloride-phosphate ion homeostasis in the cancer cells could induce apoptosis or other cell death pathways. The MTT-based cell viability assay revealed that compound 3.2 induced moderate toxicity (IC50 = 20 µM) to the HeLa cells. However, the proanionophore 3.4 showed negligible toxicity even after treatment with 60 µM concentration. Interestingly, photo irradiation (UV 365 nm, 5 min) of proanionophore 3.4 promoted moderate toxicity to the HeLa cells (IC50 = 36
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µM). Whereas only photo irradiation (UV 365 nm, 5 min) without the treatment of proanionophore 3.4 did not significantly alter the viability of the HeLa cells. Hence, the lower viability after the photo irradiation could be due to cleavage of proanionophore 3.4 and in-situ generation of active anionophore 3.2 under the cellular environment. Overall, these results successfully established that photoinduced disruption of chloride-phosphate ions homeostasis induces cancer cell death.
Chapter 4
4. Targeted Delivery of Pro-anionophore: Photo-induced transport of Cl- ion