A FRET assay for the quantitation of inhibitors of exonuclease EcoRV by using parchment paper inkjet-printed with graphene oxide and FAM-labelled DNA.

A graphene oxide (GO)-based cost-effective, automatted strip test has developed for screening of inhibitors of endonuclease EcoRV. The method involves the use of GO and a DNA substrate for EcoRV that contains both an ssDNA region for binding of GO and a fluorescein amidite (FAM)-labelled dsDNA. All the components were inkjet printed on a piece of parchment paper. The ssDNA region binds to the surface of GO and anchors so that the fluorescence of FAM is quenched. The parchment paper strip is then incubated with a sample containing EcoRV which causes enzymatic hydrolysis, and dsDNA was separated from the GO. As a result, green fluorescence is generated at the reaction spot. Enzyme activity can be measured in the presence and absence of aurintricarboxy acid acting as an EcoRV inhibitor. This method excels by its need for 2-3 orders less reagents compared to the standard well plate assay. Thus, it is an efficient platform for GO-based screening of EcoRV enzyme inhibitors. Graphical abstract A graphene oxide (GO)-based endonuclease EcoRV inhibition FRET assay using inkjet printing was developed. Printing of GO along with assay reagents has a beneficial effect on the enzymatic reaction on paper. This method was successfully applied to evaluate EcoRV inhibitor activity.

Liposomal co-delivery-based quantitative evaluation of chemosensitivity enhancement in breast cancer stem cells by knockdown of GRP78/CLU.

Resistance to chemotherapy is a key factor in the inefficacy of various forms of treatments for cancer. In the present study, chemo-resistant proteins, including glucose-regulated protein 78 (GRP78)/clusterin (CLU) targeted 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) liposomes, were developed as a delivery system for co-delivery of camptothecin (CPT) and GRP78 siRNA/CLU siRNA. Their drug/gene co-deliveries were quantitatively assessed in cancer stem cells (CSC) and MCF-7 cells. DOTAP-CPT/siRNA were prepared via electrostatic interaction on GRP78 siRNA or CLU siRNA. The size and ζ-potential of liposomes and lipoplexes were measured by dynamic light scattering techniques and electrophoretic light scattering spectrophotometry. The lipoplexes formation was tested by using gel electrophoresis. Immunofluorescence analysis showed that the expression level of CLU and GRP78 were significantly elevated in CSC compared to MCF-7 cells. Transfection and drug-delivery efficiency of DOTAP-CPT/siRNA were quantitatively compared with Lipofectamine 2000. Compared to free CPT, DOTAP-CPT-siCLU delivery in CSC and MCF-7 cells increased transfection efficiency and chemo-sensitivity by 4.1- and 5.9-fold, respectively. On the other hand, DOTAP-CPT-siGRP78 delivery increased transfection efficiency and chemo sensitivity by 4.4- and 6.2-fold in CSC and MCF-7 cells, respectively, compared to free CPT. It is significant that 3 ± 1.2-fold increase in transfection efficiency was achieved by lipofectamine. Consequently, an increase in anti-cancer/gene silencing efficacy was quantitatively observed as an effect of DOTAP-CPT/siRNA treatment, which was relatively higher than lipofectamine treatment. Conclusively, our experimental data quantitatively demonstrate that using DOTAP-CPT-siRNA specifically targeting (CSCs) chemo-resistant protein in vitro offers substantial potential for synergistic anti-cancer therapy.

Biotin-conjugated PEGylated porphyrin self-assembled nanoparticles co-targeting mitochondria and lysosomes for advanced chemo-photodynamic combination therapy.

The combination of chemotherapy and photodynamic therapy (chemo-PDT) has been suggested as an alternative therapy for drug-resistant cancers. In this study, biotin-conjugated PEGylated photosensitizer (PS) self-assembled nanoparticles (meso-tetraphenylporphyrin (TPP)-PEG-biotin SANs) were prepared via a self-assembly process to serve as nanocarriers for chemo-drugs as well as PSs. Electron microscopy results reveal the spherical shape of the nanoparticles (NPs). In the NPs, conjugated biotin plays a key role in selective tumor targeting. In vitro cellular experiments revealed the rapid cellular uptake of the TPP-PEG-biotin conjugates by MCF-7 cells that overexpress the biotin receptor, and verified that the conjugates were much more effective PSs than TPPS used as control in the cytotoxicity test. Interestingly, subcellular localization studies showed that the conjugates and their self-assembled NPs were localized mainly in mitochondria and partially in lysosomes, whereas TPPS was localized only in lysosomes. With the exclusive localization in mitochondria, high-content cell based assay showed that the TPP-PEG-biotin SANs induced rapid mitochondrial membrane potential transition (MPT), leading to cellular apoptosis. The chemo-drug doxorubicin (DOX) was successfully encapsulated in the TPP-PEG-biotin SANs (DOX@TPP-PEG-biotin) and had synergistic effects with enhanced cytotoxicity after PDT action. Collectively, the DOX@TPP-PEG-biotin SANs have promising potential as an effective anticancer agent in targeted combination therapy.

Novel ruthenium(II) triazine complex [Ru(bdpta)(tpy)]2+ co-targeting drug resistant GRP78 and subcellular organelles in cancer stem cells.

Ruthenium(II/III) metal complexes have been widely recognized as the alternative chemotherapeutic agents to overcome the drug resistance and tumor recurrence associated with platinum derivatives. In this work, a novel ruthenium(II) triazine complex namely, 1 ([Ru(bdpta)(tpy)]2+) was synthesized and spectroscopically characterized. Drug resistant cancer stem cells (CSCs) were used to evaluate the cytotoxicity of Ru(II) complex 1. The complex 1 showed a greater cytotoxic potential with IC50 values lower than that of cisplatin. The intracellular localization assay confirmed that the complex 1 was effectively distributed into mitochondria as well as endoplasmic reticulum (ER), and executed a ROS-mediated calcium and Bax/Bak dependent intrinsic apoptosis. Interestingly, direct interaction between complex 1 and glucose regulated protein 78 (GRP78), a protein associated with drug resistance caused the ROS-mediated ubiquitination of GRP78. Notably, western blot and confocal microscopy analysis confirmed that complex 1 significantly reduced the protein levels of GRP78. Dose-dependent in vivo antitumor efficacy against CD133+HCT-116 CSCs derived tumor xenograft further validated that complex 1 could be an effective chemotherapeutic agent.

Inkjet printing-based ß-secretase fluorescence resonance energy transfer (FRET) assay for screening of potential ß-secretase inhibitors of Alzheimer's disease.

Amyloid-ß (Aß) is generated by proteolytic processing of amyloid precursor protein (APP) by beta-secretase (BACE-1) and gamma-secretase. Amyloid-ß is responsible for the formation of senile plaques in Alzheimer's disease (AD). Consequently, inhibition of ß-secretase (BACE-1), a rate-limiting enzyme in the production of Aß, constitutes an attractive therapeutic approach to the treatment of AD. This paper reports an inkjet printing-based fluorescence assay for high throughput screening of ß-secretase inhibitors achieved by employing a BACE-1 FRET substrate (Rh-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-Quencher). This peptide substrate is known to be a readily available and suitable substrate for proteolytic activity, and it has high affinity to BACE-1. The BACE-1 peptide substrate printed on parchment paper was effectively cleaved by BACE-1, which was printed on the same spot. The amount of enzyme and substrate required for this inkjet printing-based BACE-1 assay can be less than 1.4ⅹ103, permitting the evaluation of inhibitor activity with femtomolar potency. The inkjet-printing-based BACE-1 inhibitory assay revealed inhibitory effects of inhibitor IV and STA on BACE-1 with an IM50 of 1.00 × 10-15 mol and 1.01 × 10-14 mol, respectively. These data confirm that both BACE-1 inhibitors (inhibitor IV and STA) actively inhibited the BACE-1 proteolysis of BACE-1 substrate on parchment paper. It important to note that the number of mole of BACE-1-substrate and enzyme utilized in the printing-based enzymatic assay are 1.4ⅹ103 smaller than the amount used in the conventional well-plate assay. The inkjet printing-based inhibitory assay constitutes a versatile high throughput technique and the IM50 values of the inhibitors were obtained with satisfactory reproducibility, suggesting that this inkjet-printing BACE-1 inhibitory assay could be quite suitable for the screening of new potential BACE-1 inhibitors for AD.

Inkjet printing-based photo-induced electron transfer reaction on parchment paper using riboflavin as a photosensitizer.

In this study, we report the photo-induced electron transfer (PET) on parchment paper using riboflavin as a photo inducer and ultraviolet lamp (362 nm) as the light source. To this end, a conventional inkjet printer equipped with 4 cartridges was used. Parchment paper was found to be a favorable substrate due to its insignificant self-absorption while assisting efficient sample interaction. Upon UV-irradiation, riboflavin generated superoxide anion radical (O2-·) and it was available to interact with superoxide dismutase present on the same spot. A decrease in NBT formazan in the reaction spot indicates increased O2-· scavenging activity of molecule. It was estimated that the well-plate based-colorimetric method used 12.5 µM (1.25 × 10-9 mole) of riboflavin and 0.25 mM (2.50 × 10-8 mole) of NBT to react with different superoxide dismutase or drug concentrations, while the printing technique consumed 3.19 × 10-13 mole of NBT and 2.98 × 10-13 mole of riboflavin to react with gradient superoxide dismutase or drug concentration. In contrast to the conventional well plate method, inkjet printing-based molecular assay provides automatic delivery in the nanoliter range with precise time, which ensures four-to five-order lower reagent consumption. The inkjet printing-based quantitative measurement specifies the amount of a particular drug/enzyme printed on a surface. Therefore, applicability of inkjet printing technique conjoined radical scavenging assay will be more competent to determine the radical scavenging potential of natural plant products. In addition, this inkjet printing approach offers easy, fast, cost-effective, and less time-consuming method to determine PET reaction on paper.

Paper-based inkjet bioprinting to detect fluorescence resonance energy transfer for the assessment of anti-inflammatory activity.

For the first time, a paper-based fluorescence resonance energy transfer (FRET) determination with cyclic AMP (cAMP)-specific phosphodiesterase 4B (PDE4B) inhibitory assay using an inkjet-printing technique is proposed. Non-fabricated parchment paper is found to constitute a unique substrate to measure fluorescent energy transfer, due to its insignificant self-absorption, and enables efficient sample interaction. Here, we report the responsive FRET signals generated on paper, upon sequentially printing reaction components on parchment paper using a conventional inkjet printer equipped with four cartridges. After printing, the energy emitted by Eu chelate was transferred by FRET to ULight molecule on paper, detected at 665 nm. In the absence of free cAMP, a maximum FRET signal was achieved on paper, while a decrease in FRET signals was recorded when free cAMP produced by PDE4B inhibitors compete with Eu-cAMP, binding with ULight-mAb. The IM50 value was determined as 2.46 × 10-13 mole for roliparm and 1.86 × 10-13 mole for roflumilast, to effectively inhibit PDE4B activity. Inkjet printing-based FRET signal determination utilizes components that are less than the femtomole range, which was four-orders less than the standard assay method. The methodology reported here constitutes an innovative approach towards the determination of FRET signals generated on paper.

Hypermulticolor Detector for Quantum-Antibody Based Concurrent Detection of Intracellular Markers for HIV Diagnosis.

Antiretroviral treatment can reduce the death rate of human immunodeficiency virus (HIV) infection, and its effectiveness is maximized at the early stage of HIV infection. The present protocol demonstrates an early stage high-content HIV diagnosis based on multicolor concurrent monitoring of CD4, CD8, and CD3 coreceptors and F-actin cytoskeleton using quantum dot (Qdot)-antibody conjugates at the single cell level. Artificial HIV infection of peripheral blood mononuclear cells (PBMCs) can be achieved by treating PBMCs with gp120. Using the present methodology, we can determine the CD4-CD8 ratios of normal PBMCs and artificial HIV-infected PBMCs. In addition, this protocol enables monitoring of structural changes of actin filament alignments in PBMCs bound to gp120 proteins using the multicolor single cell imaging system. Overall, this approach presents a new model for accurate early stage HIV diagnosis. Simultaneously the approach provides information on actin cytoskeleton and subtypes of PBMCs as well as their CD4-CD8 ratios.

Inkjet-Printing Enzyme Inhibitory Assay Based on Determination of Ejection Volume.

An accurate, rapid, and cost-effective methodology for enzyme inhibitor assays is highly needed for large-scale screening to evaluate the efficacy of drugs at the molecular level. For the first time, we have developed an inkjet printing-based enzyme inhibition assay for the assessment of drug activity using a conventional inkjet printer composed of four cartridges. The methodology is based on the determination of the number of moles of the drug on the printed surface. The number of moles was quantified through the volume of substance ejected onto the printed surface. The volume ejected on the reaction spot was determined from the density of reagent ink solution and its weight loss after printing. A xanthine oxidase (XOD) inhibition assay was executed to quantitatively evaluate antioxidant activities of the drug based on the determination of the number of moles of the drug ejected by inkjet printing. The assay components of xanthine, nitro blue tetrazolium (NBT), superoxide dismutase (SOD)/drug, and XOD were printed systematically on A4 paper. A gradient range of the number of moles of SOD/drug printed on A4 paper could be successfully obtained. Because of the effect of enzyme activity inhibition, incrementally reduced NBT formazan colors appeared on the paper in a number-of-moles-dependent manner. The observed inhibitory mole (IM50) values of tested compounds exhibited a similar tendency in their activity order, compared to the IC50 values observed through absorption assay in well plates. Inkjet printing-based IM50 assessment consumed a significantly smaller reaction volume (by 2-3 orders of magnitude) and more rapid reaction time, compared to the well-plate-based absorption assay.

Cell death mechanistic study of photodynamic therapy against breast cancer cells utilizing liposomal delivery of 5,10,15,20-tetrakis(benzo[b]thiophene) porphyrin.

5,10,15,20-Tetrakis(benzo[b]thiophene) porphyrin (BTP) is a newly synthesized hydrophobic photosensitizer with fluorescence quantum yield in toluene: ΦF=0.062. Previously, its limitations in solubility had hindered scientific experimentation regarding its photodynamic effects on cancer cells. By utilizing various compositions of liposomes in order to alter the solubility of BTP, the photocytotoxicity, reactive oxygen species generation, and subcellular localization of the liposomal BTP were identified in this work. DNA fragmentation and high content screening assays were performed in order to shed light on the tumoricidal mechanism of the liposomal photosensitizer. The MTT assay results showed promising results in the irradiation specific PDT activity against MCF-7 cells in all liposomal compositions. Production of ROS was confirmed in the liposomal BTP treated MCF-7 cells after irradiation in a concentration dependent manner. The subcellular localization assays revealed that the localization of BTP was dependent on both the photosensitizer's chemical properties and the properties of the delivery agent encapsulating aforesaid substance. Significant DNA fragmentation was observed in both nucleus localizing liposomal BTP, BTP encapsulated DOPC and DOPE (DOPC-BTP and DOPE-BTP), treated MCF-7 cells. All liposomal-BTPs were successful in inducing mitochondrial permeability transition, an increase in the permeability of the mitochondrial membrane, and activating caspase-3/7. ER localizing BTP were able to significantly increase the cytosolic calcium levels by photodynamic therapy, confirming the photodynamic ability of ER localized BTP to damage the ER membrane. The application of liposomes in delivering a novel hydrophobic photosensitizer, BTP, and photodynamic therapy treatment against MCF-7 cells were successful. It was confirmed that the MCF-7 cell death pathway via photodynamic therapy was altered in a controlled manner by controlling the intracellular localization of the photosensitizer through lipid composition adjustment.