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Molecular pharmaceutics [journal]
- Design and characterization of novel recombinant listeriolysin O-protamine fusion proteins for enhanced gene delivery. [JOURNAL ARTICLE]
- Mol Pharm 2014 Dec 18.
To improve the efficiency of gene delivery for effective gene therapy, it is essential that the vector carries functional components that can promote overcoming barriers in various steps leading to the transport of DNA from extracellular to ultimately nuclear compartment. In this study, we designed genetically engineered fusion proteins as a platform to incorporate multiple functionalities in one chimeric protein. Prototypes of such a chimera tested here contain two domains: one that binds to DNA; the other that can facilitate endosomal escape of DNA. The fusion proteins are composed of listeriolysin O (LLO), the endosomolytic pore-forming protein from Listeria monocytogenes, and a 22 amino acid sequence of the DNA-condensing polypeptide protamine (PN), singly or as a pair: LLO-PN and LLO-PNPN. We demonstrate dramatic enhancement of the gene delivery efficiency of protamine-condensed DNA upon incorporation of a small amount of LLO-PN fusion protein and further improvement with LLO-PNPN in vitro using cultured cells. Additionally, the association of anionic liposomes with cationic LLO-PNPN/protamine/DNA complexes, yielding a net negative surface charge, resulted in better in vitro transfection efficiency in the presence of serum. An initial, small set of data in mice indicated that the observed enhancement in gene expression could also be applicable to in vivo gene delivery. This study suggests that incorporation of a recombinant fusion protein with multiple functional components, such as LLO-protamine fusion protein, in a non-viral vector is a promising strategy for various non-viral gene delivery systems.
- One Platform Comparison of Estrone and Folic acid Anchored Surface Engineered MWCNTs for Doxorubicin Delivery. [JOURNAL ARTICLE]
- Mol Pharm 2014 Dec 17.
Our main aim in the present investigation was to assess and compare the in vitro and in vivo cancer targeting propensity of doxorubicin (DOX) loaded folic acid (FA)-, and estrone (ES)-anchored PEGylated multi-walled carbon nanotubes (MWCNTs) employing tumor bearing Balb/c mice. The DOX was loaded into the developed functionalized MWCNTs after proper characterization using dialysis diffusion methods. The in vitro, ex vivo and in-vivo studies were performed on MCF-7 cell line for assessment of the cancer targeting propensity. Both qualitative and quantitative cell uptake studies indicated the preferential higher uptake of estrone anchored nanotubes formulation compared to other formulations and free DOX owing to the over-expression of estrogen receptors (ERs) on human breast MCF-7 cells. Similarly, the pharmacokinetic and increased anti-tumor activities also confirmed the elevated cancer targeting propensity of the estrone and folic acid anchored MWCNTs formulations. The DOX/ES-PEG-MWCNTs has also shown significantly longer survival span (43 days), than free DOX (18 days) and control group (12 days). Present outcomes from the ex vivo and in vivo studies is deemed to be of great scientific value and shall assist targeted drug delivery formulations scientists for selection of the targeting moieties in the treatment of human breast cancer.
- Next Generation Carbon Nanoparticles for Efficient Gene Therapy. [JOURNAL ARTICLE]
- Mol Pharm 2014 Dec 16.
In a pursuit to develop a commercially exploitable and traceable gene delivery vehicle, here, we develop next generation carbon nanoparticle-DNA complex (CNPLex). CNPLexes were used to transfect green fluorescent protein (GFP) reporter gene containing plasmid DNA (pDNA) pEGFP-N1 targeting breast cancer cells MCF-7 and MDA-MB231. Prepared CNPs were optimized for particle size, surface potential, polymer surface decoration, absorbance efficiency, fluorescence efficiency, IR spectroscopic signatures and DNA loading and release efficiencies. Rigorous biophysical methods were employed to determine the variations in physico-chemical properties of CNPs after surface decoration with polymers followed by complexation with pDNA. Optimized CNPLexes were used to deliver pEGFP-N1 plasmid and efficiency of GFP was followed by fluorescence microscopy and quantified by flow assisted cell sorting. Lipofectamine2000® was used as positive control according to manufacturer's protocol and found to be comparative in transfection efficiency with one of our novel formulations. Further evaluation of cell toxicity and cell viability was performed by LDH activity and MTT assay, respectively. It was found that cell toxicity furnished by polymer decorated carbon nanoparticles was significantly low compared to the parent polymer (polyethylenimine, PEI). Similarly cell viability was found to be much higher with CNPLexes compared to PEI alone. This established the developed particles as better transfecting agents for reporter gene plasmid pEGFP-N1 compared to PEI and showed similar efficacy to one of the best known commercial transfection agents Liofectamine2000® in breast cancer cells MCF-7 and MDA-MB231.
- Divalent Folate Modification on PEG: An Effective Strategy for Improving the Cellular Uptake and Targetability of PEGylated Polyamidoamine-Polyethylenimine Copolymer. [JOURNAL ARTICLE]
- Mol Pharm 2014 Dec 16.
The stability and targeting ability of nanocarrier gene delivery systems are necessary conditions to ensure the good therapeutic effect and low nonspecific toxicity of cancer treatment. Poly(ethylene glycol) (PEG) has been widely applied for improving stability and as a spacer for linking ligands and nanocarriers to improve targetability. However, the cellular uptake and endosomal escape capacity of nanocarriers has been seriously harmed due to the introduction of PEG. In the present study, we synthesized a new gene delivery vector by coupling divalent folate-PEG (PEG3.4k-FA2) onto polyamidoamine-polyethylenimine (PME) copolymer (PME-(PEG3.4k-FA2)1.72). Both PEG and monovalent folate-PEG (PEG3.4k-FA1) modified PME were prepared as control polymers, which were named as PME-(PEG3.5k)1.69 and PME-(PEG3.4k-FA1)1.66, respectively. PME-(PEG3.4k-FA2)1.72 exhibited strong DNA condensation capacity like parent polymer PME which was not significantly influenced by PEG. PME-(PEG3.4k-FA2)1.72/DNA complexes at N/P = 10 had a diameter ∼143 nm and zeta potential ∼13 mV and showed the lowest cytotoxicity and hemolysis and the highest transfection efficiency among all tested polymers. In folate receptor positive (FR-positive) cells, the cellular uptake and transfection efficiency were increased with the increase in the number of folates coupled on PEG; the order was PME-(PEG3.4k-FA2)1.72 > PME-(PEG3.4k-FA1)1.66 > PME-(PEG3.5k)1.69. Folate competition assays showed that PME-(PEG3.4k-FA2)1.72 complexes had stronger targeting ability than PME-(PEG3.5k)1.69 and PME-(PEG3.4k-FA1)1.66 complexes due to their higher folate density per PEG molecule. Cellular uptake mechanism study showed that the folate density on PEG could change the endocytosis pathway of PME-(PEG3.5k)1.69 from clathrin-mediated endocytosis to caveolae-mediated endocytosis, leading to less lysosomal degradation. Distribution and uptake in 3D multicellular spheroid assays showed that divalent folate could offer PME-(PEG3.4k-FA2)1.72 complexes stronger penetrating ability and higher cellular uptake. With these advantages, PME-(PEG3.4k-FA2)1.72 may be a promising nonviral vector candidate for efficient gene delivery. This study also indicates that divalent folate modification on PEG can serve as an efficient strategy to improve the cellular uptake and targeting ability of PEGylated cationic polymers for gene delivery.
- Molecular properties determining unbound intracellular and extracellular brain exposure of CNS drug candidates. [JOURNAL ARTICLE]
- Mol Pharm 2014 Dec 15.
In the present work we sought to gain a mechanistic understanding of the physicochemical properties that influence the transport of unbound drug across the blood-brain barrier (BBB) as well as the intra- and extracellular drug exposure in the brain. Interpretable molecular descriptors that significantly contribute to the three key neuropharmacokinetic properties related to BBB drug transport (Kp,uu,brain), intracellular accumulation (Kp,uu,cell) and binding and distribution in the brain (Vu,brain) for a set of 40 compounds were identified using partial least squares (PLS) analysis. The tailoring of drug properties for improved brain exposure includes decreasing the polarity and/or hydrogen bonding capacity. The design of CNS drug candidates with intracellular targets may benefit from an increase in basicity and/or the number of hydrogen bond donors. Applying this knowledge in drug discovery chemistry programs will allow designing compounds with more desirable CNS pharmacokinetic properties.
- Ligand-Directed Stearic acid Grafted Chitosan Micelles to Increase Therapeutic Efficacy in Hepatic Cancer. [JOURNAL ARTICLE]
- Mol Pharm 2014 Dec 11.
Targeted delivery system would be an interesting platform to enhance the therapeutic effect and to reduce the side effects of anticancer drugs. In this study, we have developed lactobionic acid (LA)-modified chitosan-stearic acid (CS-SA) to deliver doxorubicin (DOX) to hepatic cancer cells. The average particle size of CSS-LA/DOX was ~100 nm with a high entrapment efficiency of >95%. Drug release studies showed that DOX release from pH-sensitive micelles is significantly faster at pH 5.0 than at pH 7.4. The LA conjugated micelles showed enhanced cellular uptake in HepG2 and BEL-7402 liver cancer cells than free drug and unconjugated micelles. Consistently, CSS-LA/DOX showed enhanced cell cytotoxicity in these two cell lines. Annexin-V/FITC and PI based apoptosis assay showed that the number of living cells greatly reduced in this group with marked presence of necrotic and apoptotic cells. LA-conjugated carrier induced typical chromatic condensation of cells; membrane blebbing and apoptotic bodies were begin to appear. In vivo, CSS-LA/DOX showed an excellent tumor regression profile with no toxic side effects. The active targeting moiety, long circulation profile, and EPR effect contributed to its superior anticancer effect in HepG2 based tumor. Our results showed that polymeric micelles conjugated with lactobionic acid (LA) increased the therapeutic availability of DOX in the liver cancer cell based solid tumor without any toxic side effects. The active targeting ligand conjugated nanoparticulate system could be a promising therapeutic strategy in the treatment of hepatic cancers.
- Modeling the drug release from hydrogel-based matrices. [JOURNAL ARTICLE]
- Mol Pharm 2014 Dec 11.
In this work the behavior of hydrogel-based matrices, the most widespread systems for oral controlled release of pharmaceuticals, has been mathematically described. In addition, the calculations of the model have been validated against a rich set of experimental data obtained working with tablets made of HydroxyPropyl-MethylCellulose (an hydrogel) and Theophylline (a model drug). The model takes into account water up-take, hydrogel swelling, drug release, and polymer erosion. The model was obtained as an improvement of a previous code, describing the diffusion in concentrated systems, and obtaining the erosion front (which is a moving boundary) from the polymer mass balance (in this way, the number of fitting parameters was also reduced by one). The proposed model was found able to describe all the observed phenomena, and then it can be considered a tool with predictive capabilities, useful in design and testing of new dosage systems based on hydrogels.
- Drug permeation through skin is inversely correlated with carrier gel rigidity. [JOURNAL ARTICLE]
- Mol Pharm 2014 Dec 11.
Controlled release plays an essential role in formulating topical and transdermal drug delivery systems. In this study, we correlated the skin permeation of Sesamin, a lipophilic drug, with the rheological properties of two different organogel carriers, i.e., low molecular weight gelling agent N-lauroyl-L-glutamic acid di-n-butylamide (GP-1) and Carbopol polymeric gels. Although these two gels have distinct network structures, they share the same trend: the more rigid the gel network and the higher the gelator concentration, the lower the steady flux of Sesamin through skin. This negative correlation lies in the fact that organogel network hinders the diffusion of drug to the gel-skin interface, as a result the depletion zone near the interface is non-negligible and contributes to the resistance of the whole diffusion system, thus the permeation flux is reduced. More interestingly, the dependence of the steady flux against gel complex modulus at the linear viscoelastic region followed a 'universal' power law regardless of the gel types, i.e., 1/J = 1/J0 + a.(G*)ε/C0 with a = 11.25, ε = 0.21 ± 0.03 for GP-1 gels, and a = 0.16, ε = 1.05 ± 0.06 for Carbopol gels, J0 is the steady flux without gel (G* = 0), C0 is the initial concentration of drug in gels. The empirical formulae are crucial in developing transdermal organogel systems with controlled release of drug content through readily obtainable data of their rheological properties. The explanation for the power law dependence of the steady flux on gel complex modulus is discussed.
- Assessment of the Amorphous "Solubility" of a Group of Diverse Drugs Using New Experimental and Theoretical Approaches. [JOURNAL ARTICLE]
- Mol Pharm 2014 Dec 11.
The supersaturation potential of poorly water soluble compounds is of interest in the context of solubility enhancing formulations for enhanced bioavailability. In this regard, the amorphous "solubility", i.e. the maximum increase in solution concentration that can be obtained relative to the crystalline form, is an important parameter, albeit a very difficult one to evaluate experimentally. The goal of the current study was to develop new approaches to determine the amorphous "solubility" and to compare the experimental values to theoretical predictions. A group of six diverse model compounds was evaluated using the solvent exchange method to generate an amorphous phase in situ, determining the concentration at which the amorphous material was formed. The theoretical estimation of the amorphous "solubility" was based on the thermal properties of the crystalline and amorphous phases, the crystalline solubility, and the estimated concentration of water in the water-saturated amorphous phase. The formation of an amorphous precipitate could be captured transiently for all six compounds and hence the amorphous "solubility" determined experimentally. A comparison of the experimental amorphous "solubility" values to those calculated theoretically showed excellent agreement, in particular when the theoretical estimate method treated the precipitated phase as a supercooled liquid, and took into account heat capacity differences between the two forms. The maximum supersaturation ratio in water was found to be highly compound dependent, varying between 4 for ibuprofen and 54 for sorafenib. This information may be useful to predict improvements in biological exposure for poorly water soluble compounds formulated as amorphous solid dispersions or other formulations that rely on supersaturation.
- Effect of Anionic PEGylated Polypeptide on Gene Transfection Mediated by Glycolipid Conjugate Micelles. [JOURNAL ARTICLE]
- Mol Pharm 2014 Dec 9.
To improve the gene transfection efficiency mediated by chitosan-g-stearic acid (CS) micelles, poly(ethylene glycol)-b-poly(γ-glutamic acid) (PG) was incorporated into a CS-based gene delivery system. CS/PG/pDNA complexes were prepared by ionic interaction. CS and PEGylated CS (PCS) micelles were introduced to prepare binary complexes for use as controls. CS/PG/pDNA complexes possessed similar sizes and presented as irregular spheroids in shape. The incorporation of PG into CS/pDNA complexes did not affect the ability of CS to compact pDNA and also showed a protective effect against DNase I-based degradation of pDNA. Importantly, PG could increase gene transfection efficiency, which was also affected by the mixing methods used for the preparation of CS/PG/pDNA ternary complexes. The transfection efficiencies mediated by CS/PG/pDNA complexes against HEK293 and EC-1 cells reached up to 40.8% and 11.6%, respectively, which were much higher than those of CS/pDNA complexes (1.3% and 4.0%) and PCS/pDNA complexes (0.8% and 2.4%). In addition, the incorporation of PG into CS/pDNA complexes significantly enhanced cellular uptake in HEK293 and EC-1 cells and, additionally, improved endosomal escape and intracellular vector unpacking. However, the incorporation of PG reduced the cellular uptake of CS/PG/pDNA complexes in macrophages (RAW264.7 cells). It was further demonstrated that, in addition to a non-specific charge-mediated binding to cell membranes, a γ-PGA-specific receptor-mediated pathway was involved in the internalisation of CS/PG/pDNA complexes. These results indicated that PG played multiple important roles in enhancing the transfection efficiency of CS/PG/pDNA complexes.