Hybrid nanoreservoirs based on dextran-capped dendritic mesoporous silica nanoparticles for CD133-targeted drug delivery

In this study, the chemical features of dendritic mesoporous silica nanoparticles (DMSNs) provided the opportunity to design a nanostructure with the capability to intelligently transport the payload to the tumor cells. In this regard, doxorubicin (DOX)-encapsulated DMSNs was electrostatically surface-coated with polycarboxylic acid dextran (PCAD) to provide biocompatible dextran-capped DMSNs (PCAD-DMSN@DOX) with controlled pH-dependent drug release. Moreover, a RNA aptamer against a cancer stem cell (CSC) marker, CD133 was covalently attached to the carboxyl groups of DEX to produce a CD133-PCAD-DMSN@DOX. Then, the fabricated nanosystem was utilized to efficiently deliver DOX to CD133+ colorectal cancer cells (HT29). The in vitro evaluation in terms of cellular uptake and cytotoxicity demonstrated that the CD133-PCAD-DMSN@DOX specifically targets HT29 as a CD133 overexpressed cancer cells confirmed by flow cytometry and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. The potentially promising intelligent-targeted platform suggests that targeted dextran-capped DMSNs may find impressive application in cancer therapy.     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60-65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S(0.5) values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S(0.5) ATP increased by 330%. Assay at 10 degrees C also elevated S(0.5) ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

An optimized protocol for the in vitro generation and functional analysis of human PD1/PD-L1 signal

Programmed cell death-1 (PD1) is an inhibitory receptor expressed on the activated T and B cells. Binding of PD1 to its ligands, PD-L1 and PD-L2 has led to deliver an inhibitory signal into the activated T cells. Recently, blocking PD1/PD-L1 pathway has emerged as a new treatment paradigm across a broad spectrum of malignancies. Remarkable clinical responses of monoclonal antibodies specific for PD-1 or its ligands in patients with many different types of cancer, attracted several pharmaceutical companies and researchers to investigate the agents that block PD1/PD-L1 signal. The safety and efficacy of the agents are needed to examine in the preclinical studies. In this study, we optimized a facile and cost-effective protocol for in vitro generation and functional analysis of human PD1/PD-L1 pathway. Activation of CD8?+?CD279?+?T cell was performed by anti-CD3 and D28 antibodies and the recombinant PD-L1 was used for inactivation of T cells through PD1/PD-L1 pathway. In this protocol, T-cell cytokine production (IL-2 and IFN-γ) and proliferation assay confirmed that a measurable PD1/PD-L1 signal was generated. We expected that in vitro PD1/PD-L1 signal that has been optimized in this study will serve as a valuable protocol for preclinical studies involving PD1/PD-L1 pathway.     

PEG-PLA nanoparticles decorated with small-molecule PSMA ligand for targeted delivery of galbanic acid and docetaxel to prostate cancer cells

Prostate cancer (PCa) is one of the most prevalent non-drug delivery system cutaneous malignancies. Undoubtedly, introducing novel treatment options to achieve higher therapeutic index will be worthwhile. In this study, we report for the first time, a novel targeted self-assembled based on PEG-PLA nanoparticles (PEG-PLA NPs) containing galbanic acid (GBA) and docetaxel, which was targeted using ((S)-2-(3-((S)-5-amino-1-carboxypentyl) ureido) pentanedioic acid (ACUPA), a small molecule inhibitor targeting prostate-specific membrane antigen (PSMA), in prostate cancer cell line. The prepared NPs were characterized by different analytical methods. The MTT assay was used to compare the anti-proliferation of drugs-loaded PEG-PLA NPs and ACUPA-PEG-PLA against LNCaP (PSMA⁺) and PC3 (PSMA⁻) cells. PEG-PLA NPs with an average size of 130–140 nm had an enhanced release of GBA and docetaxel at pH 5.5 compared with pH 7.5. Spectrofluorometric analysis suggested that ACUPA-modified PEG-PLA could effectively enhance the drug uptake in PSMA⁺ prostate cancer cells. Cytotoxicity studies showed that the targeted NPs loaded with different concentrations of GBA and fixed concentration of docetaxel (4 nM) have shown higher toxicity (IC₅₀ 30 ± 3 µM) than both free GBA (80 ± 4.5 µM) and nontargeted NPs (IC₅₀ 40 ± 4.6 µM) in LNCaP cells. Collectively, these findings suggest that ACUPA-conjugated PEG-PLA nanosystem containing GBA and docetaxel is a viable delivery carrier for various cancer-targeting PSMA that suffer from short circulation half-life and limited therapeutic efficacy.     

Formulation and evaluation of anticancer and antiangiogenesis efficiency of PLA–PEG nanoparticles loaded with galbanic acid in C26 colon carcinoma,in vitro and in vivo

Galbanic acid (GBA) is an active sesquiterpene coumarin derivative, with various medicinal benefits, including anticancer properties. However, the low solubility of GBA is the main limitation of its clinical applications. In this study, we used a nanosystem based on poly (D, l -lactide)–polyethylene glycol (PLA–PEG), for the delivery of GBA to C26 colon carcinoma cells. The physicochemical characteristics of nanoparticles (NPs) prepared by the emulsification–evaporation method were evaluated. MTT assay was used to compare the anticell proliferation of GBA and PLA–PEG–GBA against C26 cell lines. PLA–PEG-NPs with an average size of about 140 nm had an enhanced release of GBA at a pH of 5.5 compared with a pH of 7.4. Cytotoxicity studies showed that the IC ₅₀ of the PLA–PEG–GBA NPs (8 µM) was significantly lower than free GBA (15 µM). In the in vivo study, PLA–PEG–GBA NPs exhibited remarkable efficacy and reduced in vivo toxicity in C26 colon carcinoma tumor-bearing female BALB/c mice. To study the antiangiogenesis effect of the NPs, tumor sections were stained with an anti CD34 antibody. The results show the CD34 (+) vessels were decreased in the GBA and PLA–PEG–GBA treated mice by more than 75% and 90%, respectively. These results suggest that the encapsulation of GBA into the PLA–PEG could potentially be used for the treatment of colorectal cancer.     

Proteomics screening of molecular targets of curcumin in mouse brain

Aims

Curcumin is one of the most important constituent of Curcuma longa L. with antioxidant, anti-inflammatory and anticancer effects. In this study, we investigated potential intracellular targets of curcumin by affinity chromatography based on target deconvolution. Identification of curcumin interacting proteins may help in evaluating biological and side effects of this natural compound.

Main methods

Curcumin was immobilized through a linker to sepharose beads as solid matrix. Pull down assay was performed by passing tissue lysate of mouse brain through the column to enrich and purify curcumin interacting proteins. Then proteins were separated using two-dimensional gel electrophoresis and identified using MALDI/TOF/TOF mass spectrometry.

Key findings

Our results show that curcumin physically binds to a wide range of cellular proteins including structural proteins, metabolic enzymes and proteins involved in apoptosis pathway.

Significance

Finding curcumin interacting proteins may help in understanding a part of curcumin pharmacological effects.     

Skeletal muscle hexokinase: regulation in mammalian hibernation

The perfused rat liver responds in several ways to NAD⁺ infusion (20–100 μM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption and gluconeogenesis are some of the effects that were observed. Extracellular NAD⁺ is also extensively transformed in the liver. The purpose of the present work was to determine the main products of extracellular NAD⁺ transformation under various conditions and to investigate the possible contribution of these products for the metabolic effects of the parent compound. The experiments were done with the isolated perfused rat liver. The NAD⁺ transformation was monitored by HPLC. Confirming previous findings, the single-pass transformation of 100 μM NAD⁺ ranged between 75% at 1.5 min after starting infusion to 95% at 8 min. The most important products of single-pass NAD⁺ transformation appearing in the outflowing perfusate were nicotinamide, ADP-ribose, uric acid, and inosine. The relative proportions of these products presented some variations with the time after initiation of NAD⁺ infusion and the perfusion conditions, but ADP-ribose was always more abundant than uric acid and inosine. Cyclic ADP-ribose (cADP-ribose) as well as adenosine were not detected in the outflowing perfusate. The metabolic effects of ADP-ribose were essentially those already described for NAD⁺. These effects were sensitive to suramin (P2_XY purinergic receptor antagonist) and insensitive to 3,7-dimethyl-1-(2-propargyl)-xanthine (A2 purinergic receptor antagonist). Inosine, a known purinergic A3 agonist, was also active on metabolism, but uric acid and nicotinamide were inactive. It was concluded that the metabolic and hemodynamic effects of extracellular NAD⁺ are caused mainly by interactions with purinergic receptors with a highly significant participation of its main transformation product ADP-ribose.     

A fluorescent aptasensor for potassium ion detection-based triple-helix molecular switch

Here, a biosensor based on a quadruplex-forming aptamer for the determination of potassium ion (K⁺) is presented. The aptamer was used as a molecular recognition element; it was adjacent to two arm fragments and a dual-labeled oligonucleotide serving as a signal transduction probe (STP) that is complementary of the arm fragment sequence. In the presence of K⁺, the aptamer was displaced from the STP, which was accompanied by decreased signal. The quenching percentage of fluorescence intensity was proportional to the concentration of K⁺ in the range of 0.05 to 1.4 mM. A detection limit of 0.014 mM was achieved. Furthermore, other metal ions, such as Na⁺, Li⁺, NH₄⁺, Mg²⁺, and Ca²⁺, caused no notable interference on the detection of K⁺.