Novel in vitro and in vivo anti-Helicobacter pylori effects of pomegranate peel ethanol extract.

BACKGROUND AND AIM: Interest in plants with antimicrobial properties has been revived due to emerging problems associated with using antibiotics to eradicate Helicobacter pylori. Accordingly, this study aims to assess the antibacterial effects of Punica granatum and the possible synergistic effect of its extract along with metronidazole against H. pylori. MATERIALS AND METHODS: Pomegranate peel ethanol extracts (PPEE) was tested against a control strain of H. pylori (NCTC 11916) in vitro and in vivo in female Wistar rats. Moreover, the synergistic effect of PPEE in combination with metronidazole was tested in vitro. RESULTS: The PPEE exhibited a remarkable activity against H. pylori with a minimum inhibitory concentration (MIC) of 0.156 mg/mL. Furthermore, the extract exhibited a pronounced urease inhibitory activity (IC50 ~6 mg/mL) against the tested strain. A synergistic effect between PPEE and metronidazole was also observed (fractional inhibitory concentrations <0.5). Oral treatment of rats with PPEE for 8 days produced a significant reduction in H. pylori gastritis and a significant decrease in both lymphocytic and positive chronicity. CONCLUSION: Pomegranate extract is probably safe and represents a potential alternative and complementary therapy for reducing H. pylori associated with gastric ulcers.

Modified-chitosan nanoparticles: Novel drug delivery systems improve oral bioavailability of doxorubicin.

The efficacy of most anticancer drugs is highly limited in vivo due mainly to poor pharmacokinetics behavior including poor bioavailability after extravascular administration. We have developed novel chitosan-modified polymeric nanoparticles for oral as well as i.v. administration. Nanoparticles were developed utilizing the double emulsion solvent evaporation technique for sustained delivery of various anticancer drugs. Chitosan diacetate (CDA) and chitosan triacetate (CTA) polymers were previously modified in our laboratory and used as novel matrix. Nanoparticles, loaded with various anticancer drugs, were characterized for particle size using dynamic light scattering as well as transmission electron microscopy and net surface charge using dynamic light scattering. Particles size was below 100nm in diameter and zeta potential ranged - (25-30). Encapsulation efficiency of anticancer drugs varied considerably and was dependent on the physicochemical characteristics of the encapsulated drug. However, chitosan triacetate nanoparticles showed relatively higher encapsulation efficiency than chitosan diacetate nanoparticles. In vitro release of encapsulated drugs was sustained over a period of 14days. Nanoparticles enhanced cellular accumulation of encapsulated drugs, compared to the free drugs, in vitro in MCF-7 and Caco-II tumor cell lines. In conclusion, diacetate and triacetate chitosan are novel polymers that can be used to formulate nanoparticles which efficiently encapsulated anticancer drugs, and sustained the release and enhanced tumor cellular uptake of these drugs. Further, chitosan triacetate nanoparticles enhanced oral bioavailability of doxorubicin. CDA and CTA nanoparticles can be used to efficiently deliver anticancer drugs and improve their in vivo profile.

A Validated RP HPLC-PAD Method for the Determination of Hederacoside C in Ivy-Thyme Cough Syrup.

A simple reversed phase high-performance liquid chromatographic (RP-HPLC) method coupled with a photodiode array detector (PAD) has been developed and validated for the analysis of hederacoside C, the marker of ivy plant, in Ivy-Thyme cough syrup. Separation of hederacoside C was achieved using a Phenomenex-Gemini C18 column isothermally at 40°C. A mobile phase system constituted of solvent A (water: acetonitrile: orthophosphoric acid (85%), 860 : 140 : 2 v/v) and solvent B (acetonitrile: orthophosphoric acid (85%), 998 : 2 v/v) was used, at gradient conditions, at a flow rate of 1.5 mL/min. Analysis was performed using UV-detection (205 nm). The method was linear over the range (0.03-0.15) mg/mL of hederacoside C (r = 0.9992). Repeatability and intermediate precision were acceptable (RSD <2%). Limits of detection (LOD) and quantitation (LOQ) were 0.011 and 0.032 mg/mL, respectively. Percentage recovery was found to lie between 99.69% and 100.90% (RSD <2%). The method was also proved to be specific (peak-purity coefficient = 0.996).

Nanoparticle-mediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance.

Tumor drug resistance significantly limits the success of chemotherapy in the clinic. Tumor cells utilize multiple mechanisms to prevent the accumulation of anticancer drugs at their intracellular site of action. In this study, we investigated the anticancer efficacy of doxorubicin in combination with photodynamic therapy using methylene blue in a drug-resistant mouse tumor model. Surfactant-polymer hybrid nanoparticles formulated using an anionic surfactant, Aerosol-OT (AOT), and a naturally occurring polysaccharide polymer, sodium alginate, were used for synchronized delivery of the two drugs. Balb/c mice bearing syngeneic JC tumors (mammary adenocarcinoma) were used as a drug-resistant tumor model. Nanoparticle-mediated combination therapy significantly inhibited tumor growth and improved animal survival. Nanoparticle-mediated combination treatment resulted in enhanced tumor accumulation of both doxorubicin and methylene blue, significant inhibition of tumor cell proliferation, and increased induction of apoptosis. These data suggest that nanoparticle-mediated combination chemotherapy and photodynamic therapy using doxorubicin and methylene blue has significant therapeutic potential against drug-resistant tumors.

Single-step surface functionalization of polymeric nanoparticles for targeted drug delivery.

Targeted drug delivery using nanocarriers is achieved by functionalizing the carrier surface with a tissue-recognition ligand. Current surface modification methods require tedious and inefficient synthesis and purification steps, and are not easily amenable to incorporating multiple functionalities on a single surface. In this report, we describe a versatile, single-step surface functionalizing technique for polymeric nanoparticles. The technique utilizes the fact that when a diblock copolymer like polylactide-polyethylene glycol (PLA-PEG) is introduced in the oil/water emulsion used in polymeric nanoparticle formulation, the PLA block partitions into the polymer containing organic phase and PEG block partitions into the aqueous phase. Removal of the organic solvent results in the formation of nanoparticles with PEG on the surface. When a PLA-PEG-ligand conjugate is used instead of PLA-PEG copolymer, this technique permits a 'one-pot' fabrication of ligand-functionalized nanoparticles. In the current study, the IAASF approach facilitated the simultaneous incorporation of biotin and folic acid, known tumor-targeting ligands, on drug-loaded nanoparticles in a single step. Incorporation of the ligands on nanoparticles was confirmed by using NMR, surface plasmon resonance, transmission electron microscopy and tumor cell uptake studies. Simultaneous functionalization with both ligands significantly enhanced nanoparticle accumulation in tumors in vivo, and resulted in greatly improved efficacy of paclitaxel-loaded nanoparticles in a mouse xenograft tumor model. This new surface functionalization approach will enable the development of targeting strategies based on the use of multiple ligands on a single surface to target a tissue of interest.

Nanoparticle-mediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance in vitro.

Drug resistance limits the success of many anticancer drugs. Reduced accumulation of the drug at its intracellular site of action because of overexpression of efflux transporters such as P-glycoprotein (P-gp) is a major mechanism of drug resistance. In this study, we investigated whether photodynamic therapy (PDT) using methylene blue, also a P-gp inhibitor, can be used to enhance doxorubicin-induced cytotoxicity in drug-resistant tumor cells. Aerosol OT (AOT)-alginate nanoparticles were used as a carrier for the simultaneous cellular delivery of doxorubicin and methylene blue. Methylene blue was photoactivated using light of 665 nm wavelength. Induction of apoptosis and necrosis following treatment with combination chemotherapy and PDT was investigated in drug-resistant NCI/ADR-RES cells using flow cytometry and fluorescence microscopy. Effect of encapsulation in nanoparticles on the intracellular accumulation of doxorubicin and methylene blue was investigated qualitatively using fluorescence microscopy and was quantitated using HPLC. Encapsulation in AOT-alginate nanoparticles significantly enhanced the cytotoxicity of combination therapy in resistant tumor cells. Nanoparticle-mediated combination therapy resulted in a significant induction of both apoptosis and necrosis. Improvement in cytotoxicity could be correlated with enhanced intracellular and nuclear delivery of the two drugs. Further, nanoparticle-mediated combination therapy resulted in significantly elevated reactive oxygen species (ROS) production compared to single drug treatment. In conclusion, nanoparticle-mediated combination chemotherapy and PDT using doxorubicin and methylene blue was able to overcome resistance mechanisms and resulted in improved cytotoxicity in drug-resistant tumor cells.

Surfactant-polymer nanoparticles enhance the effectiveness of anticancer photodynamic therapy.

Photodynamic therapy (PDT) is a promising treatment modality for cancer. PDT is based on the concept that photosensitizers, when exposed to light of specific wavelength, generate cytotoxic reactive oxygen species (ROS) capable of killing tumor cells. The effectiveness of PDT has been limited in part by the lack of photosensitizers that accumulate sufficiently in tumor cells and poor yield of ROS from existing photosensitizers. In this report, we investigated whether aerosol OT-alginate nanoparticles can be used as a carrier to enhance the therapeutic efficacy of a model photosensitizer, methylene blue. Methylene blue loaded nanoparticles were evaluated for PDT effectiveness in two cancer cell lines, MCF-7 and 4T1. Encapsulation of methylene blue in nanoparticles significantly enhanced intracellular ROS production, and the overall cytotoxicity following PDT. It also resulted in higher incidence of necrosis. Greater effectiveness of nanoparticles could be correlated with higher yield of ROS with nanoparticle-encapsulated methylene blue. Further, treatment of tumor cells with nanoparticle-encapsulated methylene blue resulted in significant nuclear localization of methylene blue while free drug treatment resulted in its accumulation mainly in the endolysosomal vesicles. In conclusion, encapsulation of methylene blue in aerosol OT-alginate nanoparticles enhanced its anticancer photodynamic efficacy in vitro. Increased ROS production and favorable alteration in the subcellular distribution contribute to the enhanced PDT efficacy of nanoparticle-encapsulated photosensitizer.

Polymer-surfactant nanoparticles for sustained release of water-soluble drugs.

Poor drug encapsulation efficiency and rapid release of the encapsulated drug limit the use of nanoparticles in biomedical applications involving water-soluble drugs. We have developed a novel polymer-surfactant nanoparticle formulation, using the anionic surfactant Aerosol OT (AOT) and polysaccharide polymer alginate, for sustained release of water-soluble drugs. Particle size of nanoparticles, as determined by atomic force microscopy and transmission electron microscopy, was in the range of 40-70 nm. Weakly basic molecules like methylene blue, doxorubicin, rhodamine, verapamil, and clonidine could be encapsulated efficiently in AOT-alginate nanoparticles. In vitro release studies with basic drug molecules indicate that nanoparticles released 60-70% of the encapsulated drug over 4 weeks, with near zero-order release during the first 15 days. Studies with anionic drug molecules demonstrate poorer drug encapsulation efficiency and more rapid drug release than those observed with basic drugs. Further studies investigating the effect of sodium concentration in the release medium and the charge of the drug suggest that calcium-sodium exchange between nanoparticle matrix and release medium and electrostatic interaction between drug and nanoparticle matrix are important determinants of drug release. In conclusion, we have formulated a novel surfactant-polymer drug delivery carrier demonstrating sustained release of water-soluble drugs.

Surfactant-polymer nanoparticles overcome P-glycoprotein-mediated drug efflux.

Nanoparticles enhance the therapeutic efficacy of an encapsulated drug by increasing and sustaining the delivery of the drug inside the cell. We have previously demonstrated that Aerosol OT (AOT)-alginate nanoparticles, a novel formulation developed recently in our laboratory, significantly enhance the therapeutic efficacy of encapsulated drugs like doxorubicin in drug-sensitive tumor cells. The purpose of this study is to evaluate the drug delivery potential of AOT-alginate nanoparticles in drug-resistant cells overexpressing the drug efflux transporter, P-glycoprotein (P-gp). AOT-alginate nanoparticles were formulated using an emulsion-cross-linking process. Rhodamine 123 and doxorubicin were used as model P-gp substrates. Cytotoxicity of nanoparticle-encapsulated doxorubicin and kinetics of nanoparticle-mediated cellular drug delivery were evaluated in both drug-sensitive and -resistant cell lines. AOT-alginate nanoparticles enhanced the cytotoxicity of doxorubicin significantly in drug-resistant cells. The enhancement in cytotoxicity with nanoparticles was sustained over a period of 10 days. Uptake studies with rhodamine-loaded nanoparticles indicated that nanoparticles significantly increased the level of drug accumulation in resistant cells at nanoparticle doses higher than 200 microg/mL. Blank nanoparticles also improved rhodamine accumulation in drug-resistant cells in a dose-dependent manner. Nanoparticle-mediated enhancement in rhodamine accumulation was not because of membrane permeabilization. Fluorescence microscopy studies demonstrated that nanoparticle-encapsulated doxorubicin was predominantly localized in the perinuclear vesicles and to a lesser extent in the nucleus, whereas free doxorubicin accumulated mainly in peripheral endocytic vesicles. Inhibition of P-gp-mediated rhodamine efflux with AOT-alginate nanoparticles was confirmed in primary brain microvessel endothelial cells. In conclusion, an AOT-alginate nanoparticle system enhanced the cellular delivery and therapeutic efficacy of P-gp substrates in P-gp-overexpressing cells.

Surfactant-polymer nanoparticles: a novel platform for sustained and enhanced cellular delivery of water-soluble molecules.

PURPOSE: Nanoparticles, drug carriers in the sub-micron size range, can enhance the therapeutic efficacy of encapsulated drug by increasing and sustaining the delivery of the drug inside the cell. However, the use of nanoparticles for small molecular weight, water-soluble drugs has been limited by poor drug encapsulation efficiency and rapid release of the encapsulated drug. Here we report enhanced cellular delivery of water-soluble molecules using novel AerosolOT (AOT)-alginate nanoparticles recently developed in our laboratory. MATERIALS AND METHODS: AOT-alginate nanoparticles were formulated using emulsion-crosslinking technology. Rhodamine and doxorubicin were used as model water-soluble molecules. Kinetics and mechanism of nanoparticle-mediated cellular drug delivery and therapeutic efficacy of nanoparticle-encapsulated doxorubicin were evaluated in two model breast cancer cell lines. RESULTS: AOT-alginate nanoparticles demonstrated sustained release of doxorubicin over a 15-day period in vitro. Cell culture studies indicated that nanoparticles enhanced the cellular delivery of rhodamine by about two-tenfold compared to drug in solution. Nanoparticle uptake into cells was dose-, time- and energy-dependent. Treatment with nanoparticles resulted in significantly higher cellular retention of drug than treatment with drug in solution. Cytotoxicity studies demonstrated that doxorubicin in nanoparticles resulted in significantly higher and more sustained cytotoxicity than drug in solution. CONCLUSIONS: AOT-alginate nanoparticles significantly enhance the cellular delivery of basic, water-soluble drugs. This translates into enhanced therapeutic efficacy for drugs like doxorubicin that have intracellular site of action. Based on these results, AOT-alginate nanoparticles appear to be suitable carriers for enhanced and sustained cellular delivery of basic, water-soluble drugs.

Nanoparticles for cellular drug delivery: mechanisms and factors influencing delivery.

Polymeric nanoparticles have demonstrated enormous potential as cellular drug delivery vehicles. Nanoparticles improve drug's stability as well as its availability and retention at the target intracellular site of action. Therapeutic efficacy of nanoparticles can be further enhanced by conjugating specific ligands to nanoparticle surface. Ligand conjugation can also be used to favorably modify the intracellular disposition of nanoparticles. A number of ligands are available for this purpose; use of a specific ligand depends on the target cell, the material used for nanoparticle formulation, and the chemistry available for ligand-nanoparticle conjugation. Cellular drug delivery using nanoparticles is also affected by clearance through the reticuloendothelial system. In this paper, we review the recent progress on our understanding of physicochemical factors that affect the cellular uptake of nanoparticles and the different cellular processes that could be exploited to enhance nanoparticle uptake into cells.