Detection of Hg²⁺ ion using highly selective fluorescent chemosensor in real water sample and <i>in-vitro</i> cell study upon breast adenocarcinoma (MCF-7)

A novel rhodamine-based chemosensor (R) was designed and synthesised for selective recognition of Hg2+ ion in real water samples collected from different places. The chemosensor was prepared in green condition with high yield. The selectivity of R was examined with various metal ions, among which only Hg2+ was identified selectively with off–on mechanism along with enhancement of fluorescence. Metal ions recognition has been carried out using UV–vis and fluorescence studies taking µM concentration of chemosensor R in HEPES buffer. The detection limit of R was calculated and found to be 4.4 × 10–9 M. Quantum chemical (DFT) calculation was carried out in order to acquire knowledge about the stability of R in presence of Hg2+ ions. Cell viability and fluorescence microscopic experiments showed R as cytocompatible and can be used as a fluorescent probe for detecting Hg2+ in living cells. Detection of Hg2+ ion using highly selective fluorescent chemosensor in real water sample and in-vitro cell study upon breast adenocarcinoma (MCF-7) Ashish Kumara, Sumit Kumar Hirab, Partha Pratim Mannac and Swapan Deya*</p

Histo-pathological analysis of tumor in vascularized organs.

<p>Images of haematoxylin and eosin (H&E) staining of Liver, Lung & Spleen tissues from normal (Control), tumor bearing (DL) or treated groups (Free DOX or DOX+PCL₆₃-<i>b</i>-PNVP₉₀) are shown. In liver infiltrated metastatic lymphoid cell (Red circle) are shown. Loss of normal architecture & recruitment of neutrophils (Red Arrow) & Evidence of haemorrhage and necrosis (yellow arrow) in lungs of treated group compared to normal (blue arrows). Normal spleen is with intact capsule (green arrow), no invasion by malignant cell, with hematopoietic cell (red arrow). In DL, sub-capsular sinus is infiltrated with malignant cell (black arrow) and in treated group, capsule invasion by malignant cells is observed. (A).Tumor metastatic foci/field (arrows) were counted (B & C), mean± SD, n = 4 are shown.</p

Targeted Delivery of Doxorubicin-Loaded Poly (ε-caprolactone)-b-Poly (N-vinylpyrrolidone) Micelles Enhances Antitumor Effect in Lymphoma

<div><p>Background</p><p>The present study was motivated by the need to design a safe nano-carrier for the delivery of doxorubicin which could be tolerant to normal cells. PCL₆₃-b-PNVP₉₀ was loaded with doxorubicin (6 mg/ml), and with 49.8% drug loading efficiency; it offers a unique platform providing selective immune responses against lymphoma.</p><p>Methods</p><p>In this study, we have used micelles of amphiphilic PCL₆₃-b-PNVP₉₀ block copolymer as nano-carrier for controlled release of doxorubicin (DOX). DOX is physically entrapped and stabilized in the hydrophobic cores of the micelles and biological roles of these micelles were evaluated in lymphoma.</p><p>Results</p><p>DOX loaded PCL₆₃-b-PNVP₉₀ block copolymer micelles (DOX-PCL₆₃-b-PNVP₉₀) shows enhanced growth inhibition and cytotoxicity against human (K-562, JE6.1 and Raji) and mice lymphoma cells (Dalton's lymphoma, DL). DOX-PCL₆₃-b-PNVP₉₀ demonstrates higher levels of tumoricidal effect against DOX-resistant tumor cells compared to free DOX. DOX-PCL₆₃-b-PNVP₉₀ demonstrated effective drug loading and a pH-responsive drug release character besides exhibiting sustained drug release performance in in-vitro and intracellular drug release experiments.</p><p>Conclusion</p><p>Unlike free DOX, DOX-PCL₆₃-b-PNVP₉₀ does not show cytotoxicity against normal cells. DOX-PCL₆₃-b-PNVP₉₀ prolonged the survival of tumor (DL) bearing mice by enhancing the apoptosis of the tumor cells in targeted organs like liver and spleen.</p></div

DOX-PCL₆₃-b-PNVP₉₀ hinders colony formation in DL.

<p>Clonogenic assay was performed in six-well plates following seeding of 100 cells with clones produced by DL tumor cells. Untreated controls and PCL₆₃-b-PNVP₉₀ treated cells formed colonies (A and C). Smaller size colonies with less number of cells were observed following doxorubicin (0.5 μM) treatment (B). Cells treated with DOX-PCL₆₃-b-PNVP₉₀ treatment (0.5 μM) unable to form colony (D). Survival analysis of DL cells treated with free DOX (Red Line) or, DOX-PCL₆₃-b-PNVP₉₀ (Blue line), (p = 0.01) (E). DOX-PCL₆₃-b-PNVP₉₀ induced growth inhibition in DL cells is caspase dependent. Growth inhibition study in presence and absence of pan caspase inhibitor ZVAD-FMK for 48 h (n = 3, mean ±SD). MTT assay was performed as described above (G).Data presented as mean ± SD, n = 3.</p

In vivo anti-tumor role of DOX-PCL₆₃-b-PNVP₉₀ micelles.

<p>Therapy started at 96×10⁴ tumor cells (DL) were injected i.p. in to AKR mice (n = 12/group) and referred as day 0. Lymphoma bearing animals were injected with nine doses of free DOX or, DOX-PCL₆₃-b-PNVP₉₀ micelles at a dose of 3 mg kg^-1 of body weight doxorubicin as indicated by the arrows for 3 weeks (A). All together 9 doses were given in PBS; out of 9 doses, 4 doses were given every day (from day 0 to Day 4) and remaining 5 dose were given from day 10 at an interval of 48 h and continued up to day 18. The animals in study responded to the therapy with DOX-PCL₆₃-b-PNVP₉₀ (B). Kaplan-Mayer survival analysis of tumor bearing mice was performed following therapy and was analyzed for the percent survival up to day 50 post tumor transplant by log-rank test using Graph Pad PRISM software (C). Abdominal circumference (D) and body weight (E) are depicted demonstrating the effects of the treatment on the tumor growth of the animals. Representative images of spleen, liver, heart, lung, and kidney from each treatment group (F) and the corresponding weight of excised organ following drug treatment compared to untreated control are shown (G) n = 3.</p

Doxorubicin distribution in organs following therapy with DOX-PCL₆₃-b-PNVP₉₀.

<p>Uptake of free DOX and DOX-PCL₆₃-b-PNVP₉₀ micelles by tumor cells and organs were studied using fluorescence plate reader (A). Data presented as mean ± SD of triplicate determination, n = 3. Distribution profile of DOX in Tumor cell (B), Spleen (C), Liver (D), Lung (E), Kidney (F), and Heart (G) by FACS analysis, n = 3.</p

Cytotoxicity of DOX-PCL₆₃-b-PNVP₉₀.

<p>Cytotoxic effect of DOX-PCL₆₃-b-PNVP₉₀ against parental and DOX-resistant lymphoma cells was determined by 18 h LDH release assay. Cells were incubated with free polymer (carrier), DOX-PCL₆₃-b-PNVP₉₀ micelles or free DOX solution at different concentrations (0.0001, 0.0002, 0.0005, 0.001, 0.01, 0.05, 5 μM) for 18 h followed by measurement of the released LDH according to the manufacture's protocol. (A and B) K-562 and K-562/DOX-R, (C and D) JE6.1 and JE6.1/DOX-R (E and F) Raji and Raji/DOX-R (G and H) DL and DL/DOX-R. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in control versus experimental group) data represents mean ± SD, n = 3.</p

Effect of free DOX and DOX- PCL63-b-PNVP90 on normal Cell viability.

<p>Normal human lymphocytes (A), DC (B), and monocytes (C) were treated with serial concentrations of doxorubicin (0.0001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 μM). Plates were incubated at 37°C, 5% CO₂, for 48 h. The cell viability was measured by XTT assay kit (Cell Signaling, USA). Data shown as mean ± SD, n = 3. Effects on leukocytes number following in vivo administration of free DOX and DOX-PCL₆₃-b-PNVP₉₀ was studied in normal mice (D) tumor bearing mice (E) mice treated with free DOX (F), or DOX- PCL₆₃-b-PNVP₉₀ (G). RBC (H) and differential leukocyte count (I) of individual treatment are shown, mean ± SD, n = 3. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in control versus experimental group).</p

Induction of apoptosis by DOX-PCL₆₃-b-PNVP₉₀ micelles.

<p>The effect of free DOX or, DOX-PCL₆₃-b-PNVP₉₀ on the induction of apoptosis in parental (A) and doxorubicin resistant (DOX-R) (B) tumor cells was quantified by flow cytometric analysis of Annexin V FITC staining in tumor cells following 18 h treatment with indicated drugs. The lower left (LL) quadrant represents live/healthy cells, the lower right (LR) quadrant represents early apoptosis, and the upper right (UR) represents cells in late apoptosis, while the upper left (UL) represents the percent DOX uptake. The percentage of cells undergoing early or, late apoptosis is indicated in the respective quadrate. Data shown are mean ± SD, n = 3.</p

Time dependent uptake of free doxorubicin and DOX-PCL₆₃-b-PNVP₉₀ micelles.

<p>Uptake of free DOX or DOX-PCL₆₃-b-PNVP₉₀ micelles by parental and doxorubicin-resistant K-562 (A). JE6.1 (B) Raji (C) or, DL (D) cells were studied using fluorescence plate reader. Intracellular DOX concentration (μM) was measured in triplicate at different time points up to 30 h. Data presented as Mean ± SD, n = 4. Flow cytometric measurements of cellular DOX levels in parental K-562 (E) JE6.1 (F), Raji (G) and DL (H) (n = 3). Temporal uptake of free DOX or DOX-PCL₆₃-b-PNVP₉₀ micelles into K-562 (I) and DL (J) cells were studied by incubating in 24-well plates at a concentration of 50,000 cells per well. Representative images shown were obtained using a fluorescence microscope Eclipse 80i (Nikon, Japan) (Plan Fluor, 40X, NA 0.75 objective) equipped with blue and red filters for, Hoechst and Doxorubicin respectively (n = 3).</p