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Molecular pharmaceutics [journal]
- Oxidative stress induced by copper and iron complexes with 8-hydroxyquinoline derivatives causes paraptotic death of HeLa cancer cells. [JOURNAL ARTICLE]
- Mol Pharm 2014 Mar 5.
Here, we report the antiproliferative/cytotoxic properties of 8-hydroxyquinoline (8-HQ) derivatives on HeLa cells in the presence of transition metal ions (Cu2+, Fe3+, Co2+, Ni2+). Two series of ligands were tested, the arylvinylquinolinic L1-L8 and the arylethylenquinolinic L9-L16, which can all interact with metal ions by virtue of the N,O donor set of 8-HQ; however, only L9-L16 are flexible enough to bind the metal in a multidentate fashion, thus exploiting the additional donor functions. L1-L16 were tested for their cytotoxicity on HeLa cancer cells, both in the absence and in the presence of copper. Among them, the symmetric L14 exhibits the highest differential activity between the ligand alone (IC50 = 23.7 μM) and its copper complex (IC50 =1.8 μM). This latter, besides causing a significant reduction of cell viability, associates to a considerable accumulation of the metal inside the cells. Metal accumulation is also observed when the cells are incubated with L14 complexed with other late transition metal ions (Fe3+, Co2+, Ni2+), although the biological response of HeLa cells is different. In fact, while Ni/L14 and Co/L14 exert a cytostatic effect, both Cu/L14 and Fe/L14 trigger a caspase-independent paraptotic process, that results from the induction of a severe oxidative stress and the unfolded protein response.
- Amphiphilic biodegradable PEG-PCL-PEI triblock-copolymers for FRET capable in vitro and in vivo delivery of siRNA and quantum dots. [JOURNAL ARTICLE]
- Mol Pharm 2014 Mar 4.
Amphiphilic triblock-copolymers represent a versatile delivery platform, capable of co-delivery of nucleic acids, drugs and/or dyes. Multifunctional cationic triblock-copolymers based on polyethylene glycol, poly-ε-caprolactone and polyethylene imine, designed for the delivery of siRNA, were evaluated in vitro and in vivo. Moreover, a nucleic-acid-unpacking sensitive imaging technique based on quantum dot mediated fluorescence resonance energy transfer (QD-FRET), was established. Cell uptake in vitro was measured by flow cytometry while transfection efficiencies of nano-carriers with different hydrophilic block length were determined in vitro and in vivo by quantitative real time PCR. Furthermore, a prototype FRET pair was established by co-loading with QDs and fluorescently labeled siRNA after the proof of concept was demonstrated by fluorescence spectroscopy/microscopy. The hydrophobic copolymer mediated 5-fold higher cellular uptake and good knockdown efficiency (61±5% in vitro, 55±18% in vivo) as compared to the hydrophilic counterpart (13±6% in vitro, 30±17% in vivo) which exhibited poor performance. FRET was demonstrated by UV-induced emission of the acceptor dye. Upon complex dissociation, which was simulated by the addition of heparin, a dose-dependent decrease in FRET-efficiency was observed. We believe that in vitro/in vivo correlation of structure and function of polymeric nano-carriers as well as sensitive imaging functionality for mechanistic investigations are prerequisites for a more rational design of amphiphilic gene carriers.
- Preparation of Chondroitin Sulfate-g-Poly(ε-Caprolactone) Copolymers as a CD44-Targeted Vehicle for Enhanced Intracellular Uptake. [JOURNAL ARTICLE]
- Mol Pharm 2014 Mar 4.
Chondroitin sulfate-g-poly(ε-caprolactone) (CP) copolymers were synthesized via atom transfer radical addition (ATRA). The CP copolymers self-assembled into micelles in water and the micelles could be used to encapsulate a hydrophobic anticancer drug, camptothecin (CPT), in the core for tumor targeting delivery. The physicochemical properties of the micelles and CPT-loaded micelles were thoroughly characterized. For the in vitro test, the CPT release, the protection of the lactone ring of CPT from hydrolysis, and the cellular uptake of CPT were studied. The cell-killing and apoptosis-inducing effects using the CPT-loaded micelles were significantly better than using free CPT against CRL-5802 cells. The micellar internalization into CRL-5802 cells was primarily via CD44 and clathrin dual-mediated endocytosis. For the in vivo test, the therapeutic efficacy of the CPT-loaded micelles was studied in a non-small cell lung cancer xenograft animal model. The CPT-loaded micelles showed good inhibition in tumor growth as compared with a commercial product, CPT-11, in CRL-5802 tumor-bearing mice. The in vitro and in vivo data suggested the CP-based micelles are promising anticancer drug vehicles for lung cancer targeting.
- Screening methods to identify indole derivatives that protect against reactive oxygen species induced tryptophan oxidation in proteins. [JOURNAL ARTICLE]
- Mol Pharm 2014 Mar 3.
Oxidative damage to proteins is one of the most prominent chemical degradation pathways that are of concern for drug product development in the biotechnology industry. Especially susceptible to oxidation are the Met and Trp residues in proteins. While L-Met and L-Trp have been shown to act as antioxidants typically protecting proteins against Met and Trp oxidation respectively, L-Trp has been shown to be particularly sensitive to light and thereby produces various reactive oxygen species (ROS), including H2O2. There is hence a need to identify non-photosensitive molecules that can protect Trp oxidation in proteins so that they can be easily handled under drug product manufacturing conditions. A combination of screening methods, namely cyclic voltammetry (CV) and hydrogen peroxide generation upon photo irradiation, was used to screen several molecules to identify compounds that can act as antioxidants. Specifically, indole and tryptophan with hydroxy groups on the 6-membered aromatic ring were found to have lower oxidation potentials than the parent compounds and produced the least amount of H2O2 upon light exposure. These derivatives were also found to sufficiently protect tryptophan oxidation in mAb1 against a variety of reactive oxygen species such as alkyl peroxides, hydroxyl radicals and singlet oxygen, and may be useful as part of the formulation toolkit to protect against protein degradation via oxidation.
- Quantitation of Physiological & Biochemical Barriers to siRNA Liver Delivery via Lipid NanoParticle (LNP) Platform. [JOURNAL ARTICLE]
- Mol Pharm 2014 Mar 3.
Effective delivery of small inference RNA (siRNA) requires efficient cellular uptake and release into cytosol where it forms an active complex with RNAi induced silencing complex (RISC). Despite rapid developments in RNAi therapeutics, improvements in delivery efficiency of siRNA are needed to realize the full potential of this modality in broad therapeutic applications. We evaluated potential physiological and biochemical barrier(s) to the effective liver delivery of siRNA formulated in Lipid NanoParticle (LNP) delivery vehicles. The comparative siRNA delivery performance of three LNPs was investigated in rats. They were assembled with either C14 or C18-anchored PEG-lipid(s), cationic lipid(s) and various helper lipid(s) and contained the same siRNA duplex. These LNPs demonstrated differentiated potency with ED50's ranging from 0.02 to 0.25 mg/kg. The two C14-PEG-LNPs had comparable siRNA exposure in plasma and liver, while the C18-PEG-LNP demonstrated a higher plasma siRNA exposure and a slower but sustained liver uptake. RISC bound siRNA within the liver, a more proximal measure of the pharmacologically active siRNA species, displayed loading kinetics that paralleled the target mRNA knockdown profile, with greater RISC loading associated with more potent LNPs. Liver perfusion and hepatocyte isolation experiments were performed following treatment of rats with LNPs containing VivoTag®-fluorescently labeled siRNA. One hour after dosing majority of the siRNA within the liver was associated with hepatocytes and was internalized (within small sub-cellular vesicles) with no significant cell surface association, indicating good liver tissue penetration, hepatocellular distribution and internalization. Comparison of siRNA amounts in hepatocytes and subcellular fractions of the three LNPs suggests that endosomal escape is a significant barrier to siRNA delivery where cationic lipid seems to have a great impact. Quantitation of Ago-2 associated siRNA revealed that after endosomal escape further loss of siRNA occurs prior to RISC loading. This quantitative assessment of LNP-mediated siRNA delivery has highlighted potential barriers with respect to endosomal escape and incomplete RISC loading for delivery optimization efforts.
- Crystallisation and Phase Changes in Paracetamol from the Amorphous Solid to the Liquid Phase. [JOURNAL ARTICLE]
- Mol Pharm 2014 Feb 28.
For the case of paracetamol we show how terahertz time-domain spectroscopy can be used to characterise the solid and liquid phase dynamics. Heating of supercooled amorphous sorbitol from 295 K in a covered sample under vacuum leads to its crystallisation at 330 K. First, form III is formed followed by the transformation of form III to form II at 375 K, to form I 405 K and finally melting is observed around 455 K. We discuss the difference between the featureless spectra of the supercooled liquid and its liquid melt. Lastly, we ustudied the onset of crystallisation from the supercooled liquid in detail and quantified its kinetics based on the Avrami-Erofeev model. We determined an effective rate constant of k=0.056 min(-1) with a corresponding onset of crystallisation at T=329.5 K for a heating rate of 0.4 K min(-1).
- A three-dimensionally engineered biomimetic cartilaginous tissue model for osteoarthritic drug evaluation. [JOURNAL ARTICLE]
- Mol Pharm 2014 Feb 28.
Osteoarthritis (OA) is primarily characterized by focal cartilage destruction and synovitis. Presently, the pathogenesis of OA remains unclear and an effective treatment methodology is an unmet need. To this end, a plethora of animal models and monolayer models have been developed but they are faced with the limitation of high cost and inability to recapitulate a pure hyaline cartilaginous phenotype, which is important in studying the efficacy of therapeutic agents. We have previously developed a living hyaline cartilage graft (LhCG) that accurately presented a pure hyaline cartilage phenotype. Here, through the coculture of lipopolysaccharide (LPS)-activated macrophages with LhCG, we hypothesized that an accurate OA disease model may be developed. Subsequently, this model was evaluated for its accuracy for in vitro drug-testing. Results indicated that chondrocyte proliferation and apoptosis was increased in the disease model. Additionally, ECM synthesis increased as indicated by the increased anabolic gene expression levels, such as collagen type II and aggrecan. Upregulation of matrix metalloproteinase-1 (MMP-1) and MMP-3 genes suggested increased degradative activity, while chondrocytic hypertrophic differentiation was observed. Furthermore, extensive degradation of collagen type II and glycosaminoglycans (GAGs) were also observed. The results of celecoxib treatment on our model showed inhibition of nitric oxide (NO) and prostaglandin E2 (PGE2) production, as well as downregulation of MMP-1 and MMP-3 expression. Taken together, the results suggested that this coculture model was able to sufficiently mimic the native, diseased OA cartilage, while drug-testing results confirmed its suitability as an in vitro model for predicting native cartilage response to drug treatment.
- Enhanced Tumor Delivery of Gemcitabine via PEG-DSPE/TPGS Mixed Micelles. [JOURNAL ARTICLE]
- Mol Pharm 2014 Feb 28.
Gemcitabine is a potent anticancer drug approved for the treatment of pancreatic, non-small-cell lung, breast and ovarian cancers. The major deficiencies of current gemcitabine therapy, however, are its rapid metabolic inactivation and narrow therapeutic window. Herein, we employed polyethylene glycol-b-distearoylphosphatidylethanolamine (PEG-DSPE)/tocopheryl polyethylene glycol 1000 succinate (TPGS) mixed micelles as a delivery system, to improve the pharmacokinetic characteristics of gemcitabine and enhance its antitumor efficacy. By conjugating stearic acid to gemcitabine and subsequently encapsulating stearoyl gemcitabine (GemC18) within PEG-DSPE/TPGS mixed micelles, the deamination of gemcitabine was delayed in vitro and in vivo. Importantly, compared to free gemcitabine, GemC18-loaded micelles pronouncedly prolonged the circulation time of gemcitabine and elevated its concentration in the tumor by 3-fold, resulting in superior antitumor efficacy in mice bearing human pancreatic cancer BxPC-3 xenografts. Our findings demonstrate the promise of PEG-DSPE/TPGS mixed micelles as a nanocarrier system for the delivery of gemcitabine to achieve safer and more efficacious therapeutic outcomes.
- Albumin fusion prolongs the antioxidant and anti-inflammatory activities of thioredoxin in mice with acetaminophen-induced hepatitis. [JOURNAL ARTICLE]
- Mol Pharm 2014 Feb 27.
Overdoses of acetaminophen (APAP) are a major cause of acute liver failure. N-acetylcysteine (NAC) is the standard therapy for the patients with such an overdose because oxidative stress plays an important role in the pathogenesis of APAP-induced hepatitis. However, NAC is not sufficiently efficacious. We previously developed a recombinant human serum albumin (HSA)-thioredoxin 1 (Trx) fusion protein (HSA-Trx), designed to overcome the unfavorable pharmacokinetic and short pharmacological properties of Trx, an endogenous protein with anti-oxidative and anti-inflammatory properties. In this study, we investigated the therapeutic impact of HSA-Trx in mice with APAP-induced hepatitis. The systemic administration of HSA-Trx significantly improved the survival rate of mice treated with a lethal dose of APAP compared with saline. HSA-Trx strongly attenuated plasma transaminases in APAP-induced hepatitis mice compared with HSA or Trx, components of the fusion protein. HSA-Trx also markedly caused a diminution in the histopathological features of hepatic injuries and the number of apoptosis-positive hepatic cells. In addition, an evaluation of oxidative stress markers and plasma cytokine and chemokine levels clearly showed that HSA-Trx significantly improved the breakdown of hepatic redox conditions and inflammation caused by the APAP treatment. HSA-Trx also significantly decreased oxidative and nitrosative/nitrative stress induced by SIN-1 in vitro. Finally, HSA-Trx, but not the NAC treatment at 4 hours after APAP injection, significantly inhibited the elevation in plasma transaminases levels. In conclusion, the findings suggest that HSA-Trx has considerable potential for use as a novel therapeutic agent for APAP-induced hepatitis, due to its long-lasting anti-oxidative and anti-inflammatory effects.
- Enhancing Targeted Tumor Treatment by Near IR Light-Activatable Photodynamic-Photothermal Synergistic Therapy. [JOURNAL ARTICLE]
- Mol Pharm 2014 Mar 6.
For several decades, cancer has been one of the most life-threatening diseases. For enhancing anticancer efficiency with minimum side effects, combination therapy is envisioned. The current manuscript reports for the first time the development of a methylene blue (MB) bound nanoplatform, which is capable of delivering targeted diagnostic and combined synergistic photothermal and photodynamic treatment of cancer. Experimental data found that, once the nanoparticle binds with the target cell surface, it can detect LNCaP human prostate cancer cell selectively using fluorescence imaging. Our result shows that the therapeutic actions can be controlled with external NIR light. No cytotoxicity was observed in the absence of NIR light. Targeted photodynamic and photothermal treatment using 785 nm NIR light indicates that the multimodal treatment enhances the possibility of destroying LNCaP prostate cancer cells in vitro dramatically. We discuss the operating principle for the targeted imaging and possible mechanisms for combined therapeutic actions. Our experimental data show that NIR light activated combined therapy for cancer may become a highly effective treatment procedure in clinical settings.