Self-calibrated fluorescent probe resembled as an indicator of the lysosomal phosphatase pertaining to the cancer cells.

A self-calibrated fluorescent probe Lyso-Phos has synthesized followed by a straightforward synthetic pathway. Lyso-Phos acts as an indicator for lysosomal phosphatase. Its photophysical property including cellular imaging was described. Lyso-Phos showed ratiometric UV-Vis- absorption changes from λabs 370nm to λabs 450nm in the presence of alkaline phosphatase (ALP). On the other hand, fluorescence intensity λem 560nm of Lyso-Phos has increased around 45-fold in the presence of ALP. The probe Lyso-Phos was found to be highly chemoselective toward the phosphatase compared with other ubiquitous entities in cellular milieu. The non-toxic nature of the Lyso-phos has accounted by observing higher cell viability in prostate cancer- LnCap, fibrosarcoma HT1080 and normal mouse embryo fibroblast NIH3T3 cells. Further, the probe Lyso-Phos was utilized for tracking of cellular phosphatase in live-cells. Lyso-Phos enabled to track cellular phosphatase by the extent of fluorescence labeling of LnCap cells which showed reasonable uptake efficiency in the presence of Lyso-Phos as indicated by the intracellular fluorescence. The phosphoester bond in the probe was cleaved by intracellular alkaline phosphatase leading to turn on fluorescence of the fluorescent probe Lyso-Phos. Finally, cellular colocalization with Lyso-Tracker empowered our speculation that Lyso-Phos can track endogenous phosphatase in the lysosomes. Altogether these findings suggest that Lyso-Phos would be powerful probe to detect phosphates in cancer cells.

New Insight into a Cancer Theranostic Probe: Efficient Cell-Specific Delivery of SN-38 Guided by Biotinylated Poly(vinyl alcohol).

An optically modulated "turn-on" theranostic prodrug TP1 has been explored and formulated with biotinylated poly(vinyl alcohol) (biotinPVA) to obtain desired pharmacokinetics. TP1, consisting of the antineoplastic camptothecin analogue SN-38, and the fluorescent dye rhodol green have been covalently conjugated through a disulfide bond. Glutathione triggering the release of drug and fluorophore has been well established by UV-vis measurements through mass spectral analysis in physiological conditions. The biocompatible biotinPVA formulated prodrug (PTP1) showed remarkably higher stability against blood serum and cell-specific activation in contrast to that of TP1. Significantly, PTP1 permits monitoring of the delivery and release of well-known topoisomerase I inhibitor SN-38 by modulating fluorescence signal at λem 550 nm within intracellular milieus. Moreover, theranostic probe PTP1 exhibited dose-dependent antiproliferative activity against receptor-positive HeLa cells, whereas it did not show such an effect against receptor-negative NIH3T3 cells. Finally, the cell-specific antiproliferative activity of PTP1 via the apoptotic pathway is an efficient approach in cancer theranostics. Thus, futuristic PTP1 could be a promising agent in which diagnostic and prognostic data will be monitored synergistically.

Elastin-like recombinamers with acquired functionalities for gene-delivery applications.

In this work, well-defined elastin-like recombinamers (ELRs) were studied as a choice to the existing nonviral vectors due to their biocompatibility and ease of scale-up. Functional motifs, namely penetratin and LAEL fusogenic peptides were incorporated into a basic ELR sequence, and imidazole groups were subsequently covalently bound obtaining ELRs with new functionalities. Stable polyplexes composed of plasmid DNA and ELRs were formed. A particle size around 200 nm and a zeta potential up to nearly +24 mV made them suitable for gene delivery purposes. Additionally, viability and transfection assays with C6 rat glioma cell line showed an increase in the cellular uptake and transfection levels for the construction containing the LAEL motif. This study highlights the importance of controlling the polymer functionality using recombinant techniques and establishes the utility of ELRs as biocompatible nonviral systems for gene-therapy applications.

Enhanced intracellular uptake and endocytic pathway selection mediated by hemocompatible ornithine grafted chitosan polycation for gene delivery.

Nanotechnology is adopted in gene therapy research to create gene vectors that will facilitate gene transfer to cells with utmost efficacy and safety. For vector design, polymers are the preferred nonviral colloidal systems as they are feasible for any chemical modifications. In this study, chitosan, a versatile biopolymer has been subjected to chemical conjugation with the amino acid ornithine to generate chitosan-ornithine conjugate (CON) for gene delivery. With the help of FTIR and (1)H NMR spectra the chemical composition of the chitosan derivative was confirmed. Buffering capacity was found enhanced with the synthesised chitosan derivative when compared to the parent unmodified chitosan. The cationic derivative formed nanoparticles when mixed with negatively charged DNA. The nanoparticles showed good DNA retardation ability in agarose gel electrophoresis and sizes were ascertained by DLS and TEM observations. The derivative on interaction with blood plasma showed negligible protein adsorption and did not cause either hemolysis or RBC aggregation in blood. In vitro cell culture also revealed the CON derivative to be nontoxic to cells and capable of transfection with an explicit increase in cellular uptake of nanoparticles. An uptake study in the presence of endocytosis inhibitors indicated the specific pathway used for cell entry. The results revealed that the clathrin mediated pathway and dynamin played a role in the internalisation of these specific nanoparticles.

Spermine grafted galactosylated chitosan for improved nanoparticle mediated gene delivery.

Despite multitude of beneficial features, chitosan has poor water solubility and transfection ability which affect its gene delivery efficacy. The two features are improved when certain chemical modifications are incorporated into the chitosan parent backbone. This strategy is adopted here, by coupling galactose and spermine into the chitosan backbone. The conjugation was determined with FTIR and (1)H NMR and nanoparticle morphology was assessed by TEM and AFM techniques. Particle size, zeta potential, buffering capacity and DNA binding ability gave encouraging result of enhanced solubility and stability. In vitro studies of GCSM in HepG2 cell lines displayed low cytotoxicity and improved transfection. We also identified the preference of receptor mediated internalization for nanoparticles cellular uptake by treating with cellular uptake inhibitors. The results evidently led us to comprehend that galactosylated chitosan-g-spermine could be considered as a promising chitosan derivative for conducting nanoparticle mediated gene delivery.

Differential expression of metallothionein type-2 homologues in leaves and roots of Black pepper (Piper nigrum L)

Black pepper (Piper nigrum L.), member of the family Piperaceae is indigenous to India and is one of the most widely used spices in the world. In this paper we report the results of our attempts to identify a set of genes differentially expressed in the leaves of Piper nigrum, which could facilitate targeted engineering of this valuable crop. A PCR-based Suppression Subtractive Hybridization (SSH) technique was used to generate a leaf-specific subtracted cDNA library of Piper nigrum. A tester population of leaf cDNA was subtracted with a root derived driver cDNA. The efficiency of subtraction was confirmed by PCR analysis using the housekeeping gene actin. On sequence analysis, almost 30 percent of the clones showed homology to metallothionein type-2 gene. The predominance of metallothionein transcripts in the leaf was further confirmed using Real-Time PCR analyses and Northern blot. The possible role of metallothionein type-2 homologues in the leaf is discussed along with the feasibility of using SSH technique for identification of more number of tissue-specific genes from Piper nigrum.