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Figure 3.1. The Cl─ ion transport activities of the compounds, 3.1a–d and 3.2a-d (1.56 µM = 0.5 mol % with respect to lipid) across the EYPC/CHOL-LUVs was measured by Ion Selective Electrode-based assay at pH 7.2 (A). Anion transport selectivity of compounds 3.1b and 3.2b was measured by a fluorescence-based assay using EYPC/CHOL-LUVs⊃HPTS in the presence of the base pulse in the extravesicular region (B) Chloride ion efflux capability of compound 3.1b (0.56 µM = 0.18 mol % with respect to the lipid) at pH 5.5 and 7.2 (C). The Cl─ ion efflux aptitudes of compound 3.2b (2.29 µM = 0.733 mol % with respect to lipid) across EYPC/CHOL-LUVs were measured by ISE-based assay at pH 5.5 and 7.2 (D). Compound concentrations were 0.56 µM = 0.13 mol %, and 2.29 µM = 0.55 mol % with respect to lipid for compound 3.1b and 3.2b, respectively. DMSO was used as blank. The efflux efficiency was measured after 450 sec of compound addition. All experiments were performed in triplicate.
numerous well known biological experiments such as MTT in the presence of HBSS buffer, clonogenic, and comet assay. The immunoblot analysis of the caspases and PUMA in the presence of the potent compound 3.2b confirmed the mitochondria-mediated
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these ion transporters in therapeutics, in vivo studies were also executed in an established murine Ehrlich ascites carcinoma (EAC) solid tumor model (Figure 3.3D-F). The growth of the tumors was remarkably regressed in the presence of the potent compound (3.1b and 3.2b). The treatment of potent compound 3.2b also showed trivial toxicity to the kidney and spleen (Figure 3.3F). So overall, the in vivo studies also confirmed its potential role in the treatment of cancer.
In brief, at the end of this chapter, the selectivity between the cancer cells and the normal cells was successfully increased with the promising result both from in vitro and in vivo studies.22
Figure 3.2. Cell viability of the compounds was measured (10 μM) in HCT-116 (a colon cancer cell line), OVCAR8 (an ovarian cancer cell line), MDA-MB-231 (a triple-negative breast cancer cell line), MCF-7 (a luminal A breast cancer cell line), and MCF-10A (a nontumorigenic breast epithelial cell line) (A). The MTT assay was performed after 48 h of compound treatment. Viability of MCF-7 cells in HBSS buffer with or without Cl− ions at different concentrations of compound 3.2b (0−20 μM). The MTT assay was performed after 24 h of incubation (B). All measurements were performed in triplicate.
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Figure 3.3. Fluorescent activated cell sorting (FACS) analysis of MCF-7 cells grown in the absence and presence of compound 3.2b. The cell cycle distribution of the MCF-7 cell line was examined by staining the cells with propidium iodide (PI) in the absence and presence of the compounds. Cells were treated with the test compounds for 24 hours.
Histograms were analyzed using Cell Quest Pro software (A). The compound induces DNA damage in MCF-7 cells. Comet assays for the determination of DNA damage in single cells following the treatment with compound 3.2b (B). Whole-cell protein extracts of MCF-7 cells grown in the absence and presence of compounds (10 μM) for 24 h and immunoblotted for the indicated proteins (C). -Tubulin was used as a loading control.
The progression of a solid tumor in the female Swiss albino mice in the absence and presence of compounds 3.1b and 3.2b (D). The tumor was induced in Swiss albino mice by injecting EAC cells into the thigh muscle. The extent of tumor growth inhibition (E).
Immunotoxicity assessment of spleen by hematoxylin and eosin staining after compound 3.2b treatment (F) showed negligible toxicity.
Chapter 4
Development of Sulphonium Based Water Soluble Proanionophores with Potential Prospects in Therapeutic Applications
At the end of chapter 3, another inadequacy of anionophore was observed which is related to its uptake and cell deliverability. As the anionophore are generally lipophilic molecules, their cell deliverability is quite challenging where excess doses or additional delivery vehicles is required to deliver it to the targeted site. Hence, regulating the
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values. So the stimuli-responsive water-soluble proanionophore concept of hydrophobic anionophores was considered to augment the deliverability and generate the desired lipophilic transporter at specific tissues after the in situ cleavage of the hydrophilic appendages in the presence of certain stimuli, which is present at a higher amount in cancer cell than normal cells. In this regard, the glutathione (GSH)-responsive anion transport strategy was introduced to promote the controlled transport of chloride ions across lipid bilayers. As the concentration of the GSH in cancer cells is higher than the normal cells, it has immense efficiency towards the cancer cell line over the normal one.23 An inspiration came from the information that the presence of the sulfonium moiety could enhance the cellular uptake of the target molecules.
Figure 4.1. GSH responsive sulphonium-based anionophores and proanionophores (A).
Schematic representation of Chloride ion encapsulation by capsule kind of arrangement (B). X-ray crystal structure of compound 4.3a complexed with Cl─ ion and H2O ((4.3a·Cl─·H2O)2). The TBA+ cations present outside the cavity were omitted for clarity.
The probable mode of transportation in the presence of GSH as stimuli (C). Probable mode of transportation across the membrane in the presence of GSH as stimuli (D).
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Hence, the water-soluble sulfonium derivatives of hydrophobic anionophores could solve the conventional hurdles of solubility and deliverability.24 So, in this regard, a tripodal- based water-soluble GSH responsive proanionophore was synthesized having the sulphonium moiety (Figure 4.1).
Figure 4.2. Comparisons of ion transport activities of the compounds (0.56 mol %) across the EYPC/CHOL-LUVs⊃HPTS (A). Anion selectivity of compound 3 at EC50 = 0.14 mol
% (B). Transport of Cl─ ion by the regenerated compound 4.3a in the presence of GSH at different time intervals (C). Schematic representation of the alkylation/dealkylation strategy (D). Fluorescence microscopic images of the HeLa cells treated with NBD- labelled compounds 4.3b and 4.3g. Green channel illustrating compound uptake (E).
To monitor its uptake efficiency through fluorescence spectroscopy, we have modulated one hand of tripodal molecules with a fluorescence tagged NBD compound 4.3b. Initial anion recognition by 1H NMR and the crystal structure confirmed a capsule kind of arranging (Figure 4.1). Numerous bio-physical and in vitro assays suggested its potential chloride ion transport ability. The uptake of the NBD tagged proanionophore 4.3g, and its corresponding active anionophore 4.3b were performed in HeLa cells and
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NBD tagged anionophore 4.3b, which proved its potential applicability for numerous therapeutic applications (Figure 4.2E).
So at the end of this chapter, it has been concluded that this derivative could be used as a therapeutic agent for the treatment of cystic fibrosis due to its non-toxic nature, which is another highly demanded application of an anionophore. For cystic fibrosis, anion transporter molecules should have non-toxic behavior towards the normal cells, which is very rare to found. Fortunately, this sulphonium-based GSH responsive proanionophore has the criteria to be used as a therapeutic agent for the treatment of cystic fibrosis.
Chapter 5
Development of Nitroreductase Responsive Proanionophores with Potential Prospects in Therapy and Diagnosis
To find out the much more precise strategy for anti-cancer activity of the anionophore, another novel concept was looking to improve the efficacy selectively on cancer cells. The enzyme, light, and glutathione (GSH)-responsive proanionophores were successfully developed to improve the target-specific deliverability, uptake, and selectivity towards cancerous cells over healthy cells. However, the presence of significant esterase and glutathione levels in the healthy cell complicates their further biological applications.
Hence, there is a need for precise stimuli-responsive proanionophores, which should have higher activity under the cancer environment. Besides solving these conventional obstacles associated with the anionophore related research, it is the right time to bolster the importance of the diagnostic tool for early detection of cancerous cells in the tumor microenvironment. For this diagnostic purpose, nowadays, scientists are developing numerous stimuli-responsive fluorescence molecules where the fluorescence property of the molecules will be enhanced in the presence of a certain biomarker present in the cancer environment. In this regard, nitroreductase responsive anionophores would potentially eliminate cancer cells under hypoxic conditions selectively. Nitroreductase (NTR) is a well-known flavin-containing oxidoreductases enzyme, the potential biomarker that has been upregulated in cells under hypoxic stress.25 It is also well acknowledged that cells under the hypoxia environment finally transform into tumor cells, resistant to numerous therapy.
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