Cellular delivery of PEGylated PLGA nanoparticles.

OBJECTIVES: The objective of this study was to investigate the efficiency of uptake of PEGylated polylactide-co-gycolide (PLGA) nanoparticles by breast cancer cells. METHODS: Nanoparticles of PLGA containing various amounts of polyethylene glycol (PEG, 5%-15%) were prepared using a double emulsion solvent evaporation method. The nanoparticles were loaded with coumarin-6 (C6) as a fluorescence marker. The particles were characterized for surface morphology, particle size, zeta potential, and for cellular uptake by 4T1 murine breast cancer cells. KEY FINDINGS: Irrespective of the amount of PEG, all formulations yielded smooth spherical particles. However, a comparison of the particle size of various formulations showed bimodal distribution of particles. Each formulation was later passed through a 1.2 µm filter to obtain target size particles (114-335 nm) with zeta potentials ranging from -2.8 mV to -26.2 mV. While PLGA-PEG di-block (15% PEG) formulation showed significantly higher 4T1 cellular uptake than all other formulations, there was no statistical difference in cellular uptake among PLGA, PLGA-PEG-PLGA tri-block (10% PEG), PLGA-PEG di-block (5% PEG) and PLGA-PEG di-block (10% PEG) nanoparticles. CONCLUSION: These preliminary findings indicated that the nanoparticle formulation prepared with 15% PEGylated PLGA showed maximum cellular uptake due to it having the smallest particle size and lowest zeta potential.

Interactive roles of Ets-1, Sp1, and acetylated histones in the retinoic acid-dependent activation of guanylyl cyclase/atrial natriuretic peptide receptor-A gene transcription.

Cardiac hormones atrial and brain natriuretic peptides activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which plays a critical role in reduction of blood pressure and blood volume. Currently, the mechanisms responsible for regulating the Npr1 gene (coding for GC-A/NPRA) transcription are not well understood. The present study was conducted to examine the interactive roles of all-trans retinoic acid (ATRA), Ets-1, Sp1, and histone acetylation on the transcriptional regulation and function of the Npr1 gene. Deletion analysis of the Npr1 promoter and luciferase assays showed that ATRA enhanced a 16-fold Npr1 promoter activity and greatly stimulated guanylyl cyclase (GC) activity of the receptor protein in both atrial natriuretic peptide (ANP)-dependent and -independent manner. As confirmed by gel shift and chromatin immunoprecipitation assays, ATRA enhanced the binding of both Ets-1 and Sp1 to the Npr1 promoter. The retinoic acid receptor α (RARα) was recruited by Ets-1 and Sp1 to form a transcriptional activator complex with their binding sites in the Npr1 promoter. Interestingly, ATRA also increased the acetylation of histones H3 and H4 and enhanced their recruitment to Ets-1 and Sp1 binding sites within the Npr1 promoter. Collectively, the present results demonstrate that ATRA regulates Npr1 gene transcription and GC activity of the receptor by involving the interactive actions of Ets-1, Sp1, and histone acetylation.

Preparation and in-vitro/in-vivo evaluation of surface-modified poly (lactide-co-glycolide) fluorescent nanoparticles.

OBJECTIVE: The aim was to develop biodegradable nanoparticles suitable for cellular delivery of chemotherapeutic drugs. METHODS: Poly (lactide-co-glycolide) (PLGA) nanoparticles were prepared using a modified solvent evaporation method. Chitosan and calcium chloride were tested as surface modifiers. Coumarin-6 was incorporated into some formulations as a fluorescent marker. KEY FINDINGS: The median size of the particles was between 400 nm and 7 microm, and scanning electron microscope pictures showed that the particles were smooth and spherical. The zeta potentials of the particles with and without surface modifier ranged between -25.7 mV and -7.0 mV, respectively. Fluorescence microscopy and flow cytometry (FACS) analysis showed that smaller surface-modified particles were efficiently internalised by neoplastic 4T1 cells. Image analysis of frozen tissue sections from Balb/c mice given nanoparticles via the tail vein showed that the particles were distributed preferentially into the lungs, followed by the liver, spleen, kidney and heart. CONCLUSIONS: Chitosan-modified PLGA nanoparticles showed significant uptake by neoplastic 4T1 cells, and were distributed to several major organs frequently seen as sites of cancer metastasis in mice.

Regulation of natriuretic peptide receptor-A gene expression and stimulation of its guanylate cyclase activity by transcription factor Ets-1.

ANP (atrial natriuretic peptide) exerts its biological effects by binding to GC (guanylate cyclase)-A/NPR (natriuretic peptide receptor)-A, which generates the second messenger cGMP. The molecular mechanism mediating Npr1 (coding for GC-A/NPRA) gene regulation and expression is not well understood. The objective of the present study was to elucidate the mechanism by which Ets-1 [Ets (E twenty-six) transformation-specific sequence] contributes to the regulation of Npr1 gene transcription and expression. Chromatin immunoprecipitation and gel-shift assays confirmed the in vivo and in vitro binding of Ets-1 to the Npr1 promoter. Overexpression of Ets-1 enhanced significantly Npr1 mRNA levels, protein expression, GC activity and ANP-stimulated intracellular accumulation of cGMP in transfected cells. Depletion of endogenous Ets-1 by siRNA (small interfering RNA) dramatically decreased promoter activity by 80%. Moreover, methylation of the Npr1 promoter region (-356 to +55) reduced significantly the promoter activity and hypermethylation around the Ets-1 binding sites directly reduced Ets-1 binding to the Npr1 promoter. Collectively, the present study demonstrates that Npr1 gene transcription and GC activity of the receptor are critically controlled by Ets-1 in target cells.