Colorimetric MTT Metabolic Assay
Colorimetric MTT metabolic (Prestoblue) assay was performed to analyze in vitro cytotoxicity to cell lines A549 and MRC5. After confirming complete solubilization of the purple formazan crystals, the absorbance of the samples using a microplate reader was measured under 570 nm. Samples included no treatment (negative control), 10% sodium dodecyl sulfate (SDS, positive control), buforin IIb (5mM~100mM), MV1 (5mM~100mM), and MV2 (5mM~100mM).
(Figure 1.1 - PI in vitro cytotoxicity evaluation via MTT metabolic assay, A549.)
(Figure 1.2 - PI in vitro cytotoxicity evaluation via MTT metabolic assay, MRC5.)
Cell line A549 with no treatment indicated 0.16 procedure-defined unit (pdu), a baseline value indicating no cell deaths herein; 10% SDS treatment indicated 0.4 pdu. Buforin IIb from 5~40 mM indicated approximately 0.16 pdu, excluding the margin of error. However, the cytotoxic activity of buforin IIb was observed at high concentrations (80-100mM) with approximately 0.11 pdu.
Dose-dependent manner of cytotoxicity was also observed under MV1 and MV2 treatments with a comparable trend. No significant cell deaths were observed until 40mM – A549 cell viability was higher under MV1 and MV2 treatments (0.17 pdu). Significant cytotoxicity (<0.15 pdu) was observed at 80-100 mM. A comparable trend was reported under MRC5 treatments.
FITC Specificity Assay (Confocal Microscopy)
PI displaying the highest cell viability – MV1 – was covalently conjugated with FITC for the assessment of MV1 cell specificity and post-internalization intracellular distribution. Fluorophores were excited with an argon laser at 488nm and merged with the DAPI staining image via confocal microscopy.
(Figure 2.1 - PI cancer specificity evaluation via FITC confocal microscopy, A549.)
(Figure 2.2 - PI cancer specificity evaluation of PI via FITC confocal microscopy, MRC5.)
DAPI staining of A549 presented more frequent, smaller nuclei from its counterpart. FITC fluorescence of MV1-FITC synthetic construct was significantly observable under 40mM MV1 (than 20mM MV1) for A549 and MRC5. At MV1 40 mM, MV1-FITC fluorescence accumulated and was detected in A549 cytoplasm and nuclei, presenting high intracellular distribution and transfection efficacy.
At MV1 40mM, MV1-FITC was poorly internalized to the nucleus and cytoplasm of MRC5, comparable to internalization and distribution presented at MV1 20 mM. It was statistically deduced further that internalized MV1-FITC did not display transfection efficacy to yield observable effects.
RT-PCR (Gene Silencing/Transfection Efficacy Analysis)
Following cDNA synthesis via Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV-RT) of CYP1A1 mRNA, polymerase chain reaction (PCR) assay was performed for the analysis of transfection efficacy and target gene silencing of CPP/siRNA complexes. Subsequently, band intensity (agarose gel electrophoresis) quantification was performed via the Image J program.
(Figure 3.1 - Gene silencing/transfection efficacy analysis via RT-PCR, A549.)
(Figure 3.2 - Gene silencing/transfection efficacy analysis via RT-PCR, MRC5.)
(Figure 3.3 - Normalized CYP1A1 expression via RT-PCR, A549 and MRC5.)
Under A549 and MRC5, expressions of gene CYP1A1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, control) were investigated. Samples included a random arrangement of PI (RNAimax Lipofectamine) and siRNA negative control (scrambled siRNA) - control siRNA (20µM) + RNAimax Lipofectamine, CYP1A1 siRNA (20µM) + RNAimax Lipofectamine, CYP1A1 siRNA (20µM) + 40 mM MV1, CYP1A1 siRNA (40µM) + 40 mM MV1, and CYP1A1 siRNA (80µM) + 40 mM MV1.
Under A549, band intensity quantification presented the highest silencing efficacy under CYP1A1 siRNA (20µM) + 40 mM MV1 (0.2 pdu, normalized). Silencing efficacy of CYP1A1 siRNA (40µM) + 40 mM MV1 and CYP1A1 siRNA (80µM) + 40 mM MV1 and control siRNA (20µM) + RNAimax Lipofectamine (0.3 pdu, normalized) and CYP1A1 siRNA (20µM) + RNAimax Lipofectamine (0.4 pdu, normalized) remained comparable. GAPDH expressions were comparable, validating MV1-CYP1A1 siRNA complex viability to other intracellular mechanisms.
Confirmed through literature, band intensity and normalized expression level of CYP1A1 under MRC5 were inherently lower (range: 0.09 pdu - 0.14 pdu, normalized) and CYP1A1 siRNA (20µM) + 40 mM MV1 presented no effect on decreasing the normalized CYP1A1 expression level, further confirming neoplasia specific cytolytic activity of the synthetic construct herein developed.
Colorimetric MTT Metabolic Assay (Cell Death Analysis)
Colorimetric MTT metabolic (Prestoblue) assay was performed to analyze cell death to cell lines A549 and MRC5. After confirming complete solubilization of the purple formazan crystals, the absorbance of the samples using an ELISA reader was measured under 570 nm. Samples included positive and negative controls of siRNA (MV1-DOX and MV1-DOX + siRNA Control) and final synthetic construct (FSC, MV1-DOX + siRNA-CYP1A1).
(Figure 4 - Cell death analysis of CPP-DOX/siRNA complex via MTT metabolic assay, A549 and MRC5.)
Under A549, MV1-DOX and MV1-DOX + siRNA Control displayed higher cell viability (0.125 pdu), and FSC showed the highest cell death efficacy (0.06 pdu). Under MRC5, all samples presented higher viability than their MRC5 counterpart with statistical assimilation at 0.14 pdu, further confirming FSC cytolytic activity specific to the adenocarcinoma cell line.