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Molecular pharmaceutics [journal]
- Stable and Efficient Transfection of siRNA for Mutated KRAS Silencing using Novel Hybrid Nanoparticles. [JOURNAL ARTICLE]
- Mol Pharm 2014 Oct 23.
siRNA is currently the most widely studied form of RNAi, and has a promising therapeutic potential in not just cancer but in other diseases such as autoimmune and infectious diseases. However, efficient delivery of siRNA to target cells is being limited by lack of an effective delivery system that ensures efficient transfection into cells while protecting the encapsulated siRNA from nuclease. We hypothesized that a hybrid nanoparticle system composing human IgG and poloxamer-188, a stealth polymer will efficiently deliver mutated KRAS siRNA to A549 cells leading to an efficient knockdown of mutated siRNA while protecting the siRNA from serum nuclease. We also hypothesized that the nanoparticles will not elicit an immunostimulatory effect in murine macrophages and also avoid clearance by macrophages. These nanoparticles were found to efficiently deliver siRNA to the cytoplasm and nuclease of A549 cells in a controlled and sustained manner while avoiding recycling by endosomes. An effective knockdown of mutated KRAS was achieved which subsequently led to an increased sensitivity to erlotinib. These nanoparticles successfully avoided uptake by murine macrophages and reduced immune responses normally associated with siRNA / nanoparticle therapy. These results demonstrate that the novel hybrid nanoparticles could potentially serve as a platform for efficient delivery of siRNA to cells for stable gene knockdown.
- Peptide-Coated Liposomal Fasudil Enhances Site Specific Vasodilation in Pulmonary Arterial Hypertension. [JOURNAL ARTICLE]
- Mol Pharm 2014 Oct 21.
We have proposed a liposomal delivery system of fasudil - an investigational drug for the treatment of pulmonary arterial hypertension (PAH) - that preferentially accumulates in the pulmonary arterial hypertensive lungs. Liposomal fasudil was prepared by film hydration method and the drug was encapsulated by active loading. Liposome surface was coated with the targeting moiety, CARSKNKDC, a cyclic peptide. The liposomes were characterized for size, polydispersity index, zeta potential, and storage and nebulization stability. The in vitro drug release profiles, and uptake by TGF-β activated proliferative pulmonary arterial smooth muscle cells (PASMC) and alveolar macrophages were evaluated. The pharmacokinetics was monitored in male Sprague Dawley® rats and the pulmonary hemodynamics was studied in acute and chronic PAH rats. The size, polydispersity index (PDI), and zeta potential of the liposomes were 206-216 nm, 0.058-0.084, and -20-42.7 mV, respectively. The formulations showed minimal change in structural integrity when nebulized with a commercial microsprayer. The optimized formulation was stable for >4 weeks when stored at 4C. Fasudil was released in a continuous fashion over 120 hours with a cumulative release of 76%. Peptide-linked liposomes were taken up at a higher degree by TGF-β activated PASMCs; but alveolar macrophages failed to entrap peptide coated liposomes. The formulations did not injure the lungs and the half-life of liposomal fasudil was 34-fold greater than that of plain fasudil after intravenous administration. Peptide-linked liposomal fasudil, as opposed to plain liposomes, reduced the mean pulmonary arterial pressure (mPAP) by 35 to 40%, without influencing the mean systemic arterial pressure (mSAP). This study establishes that CAR-conjugated inhalable liposomal fasudil offer favorable pharmacokinetics and produces pulmonary vasculature specific vasodilatation.
- Vaginal expression of efflux transporters and the potential impact on the disposition of microbicides in vitro and in rabbits. [JOURNAL ARTICLE]
- Mol Pharm 2014 Oct 19.
In order to reach sufficiently high tissue concentrations and thus be effective, vaginally applied anti-HIV microbicides that are active at the level of the immune cells must permeate across the cervicovaginal mucosal layer. Cellular efflux transporters, such as Pgp, BCRP and MRP-2, have been demonstrated to greatly affect drug disposition at different sites in the body including the intestine and the blood-brain barrier; their possible role on drug uptake from the female genital tract, however, has not been elucidated yet. In the present study, the protein expression of Pgp, BCRP and MRP-2 in endocervical and vaginal tissue of premenopausal women was confirmed by western blot analysis. To enable the assessment of transporter effects in vitro, the identification of an appropriate cervicovaginal cell line was pursued. The cervical SiHa cell line was observed to express mRNA of the 3 studied transporters, but only MRP-2 was found to be active. Consequently, the established Caco-2 cell line was utilized as an alternative in which the interaction of 10 microbicide candidates with the efflux transporters was studied. Darunavir, saquinavir and maraviroc were identified as Pgp and MRP-2 substrates. The impact of Pgp on in vivo drug disposition was further examined for the model Pgp substrate talinolol in rabbits. Its vaginal uptake was significantly reduced by Pgp-mediated efflux when formulated in a neutral, but not in an acidic gel. Our findings indicate the expression of a functional Pgp transporter in the vaginal mucosa that may severely reduce the vaginal uptake of Pgp substrates, including certain microbicide candidates, especially in women with an increased vaginal pH.
- The mechanisms of nanoparticle internalization and transport across an intestinal epithelial cell model: effect of size and surface charge. [JOURNAL ARTICLE]
- Mol Pharm 2014 Oct 19.
This study investigated the effect of nanoparticle size and surface charge on their interaction with Caco-2 monolayers as a model of the intestinal epithelium, including cell internalization pathways and the level of transepithelial transport. Initially, toxicity assays showed that cell viability and cell membrane integrity were dependent on the surface charge and applied mass, number and total surface area of nanoparticles, as tested in two epithelial cell lines, colon carcinoma Caco-2 and airway Calu-3. This also identified suitable nanoparticle concentrations for subsequent cell uptake experiments. Nanoparticle application at doses below EC50 revealed that the transport efficiency (ratio of transport to cell uptake) across Caco-2 cell monolayers is significantly higher for negatively charged nanoparticles compared to their positively charged counterparts (of similar size), despite the higher level of internalization of positively charged systems. Cell internalization pathways were hence probed using a panel of pharmacological inhibitors aiming to establish whether the discrepancy in transport efficiency is due to different uptake and transport pathways. Vesicular trans-monolayer transport for both positively and negatively charged nanoparticles was confirmed via inhibition of dynamin (by dynasore) and microtubule network (via nocodazole), which significantly reduced the transport of both nanoparticle systems. For positively charged nanoparticles a significant decrease in internalization and transport (46% and 37%, respectively) occurred in the presence of a clathrin pathway inhibitor (chlorpromazine), macropinocytosis inhibition (42%; achieved by 5-(N-ethyl-N-isopropyi)-amiloride) and under cholesterol depletion (38%; via methyl-β-cyclodextrin), but remained unaffected by the inhibition of lipid raft associated uptake (caveolae) by genistein. On the contrary, the most prominent reduction in internalization and transport of negatively charged nanoparticles (51% and 48%, respectively) followed the inhibition of lipid raft-associated pathway (caveolae inhibition by genistein), but was not significantly affected by the inhibition of clathrin pathway.
- Mechanism of Amorphous Itraconazole Stabilization in Polymer Solid Dispersions: Role of Molecular Mobility. [JOURNAL ARTICLE]
- Mol Pharm 2014 Oct 17.
Physical instability of amorphous solid dispersions can be a major impediment to their widespread use. We characterized the molecular mobility in amorphous solid dispersions of itraconazole (ITZ) with each polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose acetate succinate (HPMCAS) with the goal of investigating the correlation between molecular mobility and physical stability. Dielectric spectra showed two mobility modes: α-relaxation at temperatures above the glass transition temperature (Tg) and β-relaxation in the sub-Tg range. HPMCAS substantially increased the α-relaxation time, with an attendant increase in crystallization onset time and a decrease in crystallization rate constant, demonstrating the correlation between α-relaxation and stability. The inhibitory effect on α-relaxation as well as stability was temperature dependent and diminished as the temperature was increased above Tg. PVP, on the other hand, affected neither the α-relaxation time nor the crystallization onset time, further establishing the link between α-relaxation and crystallization onset in solid dispersions. However, it inhibited the crystallization rate, an effect attributed to factors other than mobility. Interestingly, both of the polymers acted as plasticizers of β-relaxation, ruling out the latter's involvement in physical stability.
- Moxifloxacin Loaded Nanoemulsions having Tocopheryl Succinate as Integral Component Improves Pharmacokinetics and Enhances Survival in E Coli Induced Complicated Intra Abdominal Infection. [JOURNAL ARTICLE]
- Mol Pharm 2014 Oct 15.
In the present work a novel nanoemulsion laden with moxifloxacin has been developed for effective management of complicated Intra-abdominal infections. Moxifloxacin nanoemulsion fabricated using high pressure homogenization was evaluated for various pharmaceutical parameters, pharmacokinetics and pharmacodynamics in rats with E coli induced sepsis. The developed nanoemulsion MONe6 (size 168±28 d.nm and ZP -24.78±0.45mV respectively) was effective for intracellular delivery and sustaining the release of MOX. MONe6 demonstrated improved plasma (AUC MONe6/MOX= 2.38 fold) and tissue pharmacokinetics of MOX (AUC MONe6/MOX= 2.63 and 1.47 times in lung and liver respectively). Calculated PK/PD index correlated well with reduction in bacterial burden in plasma as well as tissues. Enhanced survival on treatment with MONe6 (65.44 %) and as compared to control group (8.22 %) was result of reduction in lipid peroxidation, neutrophil migration and cytokine levels (TNF-α and IL1b) as compared to untreated groups in rat model of E coli induced sepsis. Parenteral nanoemulsions of MOX hold a promising advantage in the therapy of E. coli induced complicated intra-abdominal infections and is helpful in the prevention of further complication like septic shock and death.
- Amorphous formulations of indomethacin and griseofulvin prepared by electrospinning. [JOURNAL ARTICLE]
- Mol Pharm 2014 Oct 15.
Following a series of optimisation experiments, two series of electrospun polyvinylpyrrolidone (PVP) fibres were prepared. One set of fibres contained various loadings of indomethacin, known to form stable glasses, and the other griseofulvin (a poor glass former). Drug loadings of up to 33 % w/w were achieved. Electron microscopy data showed the fibres largely to comprise smooth and uniform cylinders, with evidence for solvent droplets in some samples. In all cases, the drug was found to exist in the amorphous physical state in the fibre on the basis of X-ray diffraction and differential scanning calorimetry (DSC) measurements. Modulated temperature DSC showed that the relationship between a formulation's glass transition temperature (Tg) and the drug loading follows the Gordon-Taylor equation, but not the Fox equation. The results of Gordon-Taylor analysis indicated that the drug/polymer interactions were stronger with indomethacin. The interactions between drug and polymer were explored using molecular modelling simulations, and found to be stronger with indomethacin; the presence of such intermolecular interactions was further confirmed using IR spectroscopy. The amorphous form of both drugs was found to be stable after storage for 8 months in a desiccator (relative humidity < 25 %). Finally, the functional performance of the fibres was studied; in all cases, the drug-loaded fibres released their drug cargo very rapidly, offering accelerated dissolution over the pure drug.
- Stabilization of Ostwald Ripening in Low Molecular Weight Amino Lipid Nanoparticles for Systemic Delivery of siRNA Therapeutics. [JOURNAL ARTICLE]
- Mol Pharm 2014 Oct 15.
Lipid nanoparticles (LNPs) represent the most clinically advanced technology for the systemic delivery of therapeutic siRNA in vivo. Toward this end, a novel class of LNPs comprising low molecular weight (MW) ionizable amino lipids having asymmetric architecture was recently reported.1 LNPs of these amino lipids, termed asymmetric LNPs, were shown to be highly efficacious and well tolerated in vivo; advances were enabled by improved endosomal escape, coupled with enhanced amino lipid metabolism and clearance. In this work, we show that, in contrast to their desirable pharmacological performance, asymmetric LNPs present a significant pharmaceutical developability challenge, namely physical instability limiting extended shelf life. Using orthogonal characterization methods, we identify the mechanism of LNP instability as Ostwald ripening and establish it to be driven predominantly by the asymmetric amino lipid component. Through rational optimization of LNP physical and macromolecular properties, we are able to significantly attenuate or entirely eliminate the Ostwald ripening instability. Modulation of LNP size, for example, effectively halts particle growth. Similarly, optimization of LNP macromolecular packing through deliberate selection of structurally matched colipids significantly diminishes the rate of ripening. This later experimental observation is substantiated by molecular dynamics simulations of LNP self-assembly, which establish a quantitative dependence of LNP macromolecular order on colipid structure. In totality, the experimental and molecular dynamics outcomes of this work support the rational design of LNP physical and chemical properties leading to effective Ostwald ripening stabilization and enable the advance of asymmetric LNPs as a clinic-ready platform for siRNA therapeutics.
- Biodistribution and in vivo activities of new tumor-associated macrophage-targeting nanoparticles incorporated with doxorubicin. [JOURNAL ARTICLE]
- Mol Pharm 2014 Oct 14.
Tumor-associated macrophages (TAMs) are increasingly considered a viable target for tumor imaging and therapy. Previously, we reported that innovative surface-functionalization of nanoparticles may help target them to TAMs. In this report, using poly (lactic-co-glycolic) acid (PLGA) nanoparticles incorporated with doxorubicin (DOX) (DOX-NPs), we studied the effect of surface-modification of the nanoparticles with mannose and/or acid-sensitive sheddable polyethylene glycol (PEG) on the biodistribution of DOX and the uptake of DOX by TAMs in tumor-bearing mice. We demonstrated that surface-modification of the DOX-NPs with both mannose and acid-sensitive sheddable PEG significantly increased the accumulation of DOX in tumors, enhanced the uptake of the DOX by TAMs, but decreased the distribution of DOX in mononuclear phagocyte system (MPS), such as liver. We also confirmed that the acid-sensitive sheddable PEGylated, mannose-modified DOX-nanoparticles (DOX-AS-M-NPs) targeted TAMs, because depletion of TAMs in tumor-bearing mice significantly decreased the accumulation of DOX in tumor tissues. Furthermore, in a B16-F10 tumor-bearing mouse model, we showed that the DOX-AS-M-NPs were significantly more effective than free DOX in controlling tumor growth, but had only minimum effect on the macrophage population in mouse liver and spleen. The AS-M-NPs are promising in targeting cytotoxic or macrophage-modulating agents into tumors to improve tumor therapy.
- NQO2 is a reactive oxygen species generating off-target for acetaminophen. [JOURNAL ARTICLE]
- Mol Pharm 2014 Oct 14.
The analgesic and antipyretic compound acetaminophen (paracetamol) is one of the most used drugs worldwide. Acetaminophen overdose is also the most common cause for acute liver toxicity. Here we show that acetaminophen and many structurally related compounds bind quinone reductase 2 (NQO2) in vitro and in live cells establishing NQO2 as a novel off-target. NQO2 modulates the levels of acetaminophen derived reactive oxygen species, more specifically superoxide anions, in cultured cells. In humans, NQO2 is highly expressed in liver and kidney, the main sites of acetaminophen toxicity. We suggest that NQO2 mediated superoxide production may function as a novel mechanism augmenting acetaminophen toxicity.