Cationic nanoemulsions as potential carriers for intracellular delivery.

Successful cytosolic delivery enables opportunities for improved treatment of various genetic disorders, infectious diseases and cancer. Cationic nanoemulsions were designed using alternative excipients and evaluated for particle size, charge, effect of sterilization on its stability, DNA condensation potential and cellular uptake efficiency. Various concentrations of non-ionic and ionic stabilizers were evaluated to design formula for colloidally stable cationic nanoemulsion. The nanoemulsion comprised of 5% Capmul MCM, 0.5% didodecyldimethylammonium bromide (DDAB), 1% phospholipid, 1% Poloxamer 188 and 2.25% glycerol and possessed particle size of 81.6 ± 3.56 nm and 137.1 ± 1.57 nm before and after steam sterilization, respectively. DNA condensation studies were carried out at various nanoemulsion: DNA ratios ranging from 1:1 to 10:1. Cell uptake studies were conducted on human embryonic kidney (HEK) cell lines which are widely reported for transfection studies. The nanoemulsions showed excellent cellular uptake as evaluated by fluorescence microscopy and flow cytometry. Overall, a colloidally stable cationic nanoemulsion with good DNA condensation ability was successfully fabricated for efficient cytosolic delivery and potential for in vivo effectiveness.

Design and evaluation of self-nanoemulsifying pellets of repaglinide.

The aim of study was to develop self-nanoemulsifying pellets (SNEP) for oral delivery of poorly water soluble drug, repaglinide (RPG). Solubility of RPG in oily phases and surfactants was determined to identify components of self-nanoemulsifying drug delivery system (SNEDDS). The surfactants and cosurfactants were screened for their ability to emulsify oily phase. Ternary phase diagrams were constructed to identify nanoemulsification area for the selected systems. SNEDDS formulations with globule size less than 100 nm were evaluated for in vivo anti-hyperglycemic activity in neonatal streptozotocin rat model. A significant reduction in glucose levels was produced by optimized SNEDDS formulation in comparison to the control group. The optimized SNEDDS formulations were pelletized via extrusion/spheronization technique using microcrystalline cellulose and lactose. SNEP were characterized by X-ray powder diffraction and scanning electron microscopy. X-ray diffraction study indicated loss of crystallinity of RPG in SNEP. The SNEP exhibited good flow properties, mechanical strength and formed nanoemulsion with globule size less than 200 nm. SNEP showed in vitro release of more than 80% RPG in 10 min which was significantly higher than RPG containing reference pellets. In conclusion, our studies illustrated that RPG, a poorly water soluble drug can be successfully formulated into SNEP which can serve as a promising system for the delivery of poorly water soluble drugs.

Characterization of soy polysaccharide and its in vitro and in vivo evaluation for application in colon drug delivery.

The objective of the present investigation was to establish potential of commercially available soy polysaccharide (Emcosoy®) for colon drug delivery. The soy polysaccharide-ethyl cellulose films were fabricated and characterized. The effect of the pectinase enzyme on the tensile strength and surface morphology of the film was evaluated. The permeation of chlorpheniramine maleate (CPM), a model hydrophilic drug from pectinase enzyme treated and untreated films was measured in pH 7.4 buffer. The soy polysaccharide-ethyl cellulose films were also incubated with Lactobacillus sp. culture for a specific duration, and effect on the CPM permeation was evaluated. The CPM capsules were coated with the soy polysaccharide-ethyl cellulose mixture, and Eudragit S100 was applied as a secondary coat. The coated CPM capsules were radiolabelled, and their in vivo transit was evaluated in human volunteers on oral administration. The pectinase enzyme had a significant influence on the tensile strength and surface morphology of the soy polysaccharide-ethyl cellulose films. The permeability of pectinase enzyme-treated and Lactobacillus sp.-treated films was significantly higher than that of untreated films. The CPM capsules were coated with the soy polysaccharide-ethyl cellulose mixture and Eudragit S100 and were successfully radiolabelled by a simple method. Gamma scintigraphic studies in human volunteers showed that the radiolabelled capsules maintained integrity for at least 9 h after oral administration. Thus, the soy polysaccharide has a potential in colon drug delivery.

Polysaccharide and monosaccharide guided liver delivery of Sorafenib Tosylate - A nano-strategic approach and comparative assessment of hepatospecificity.

Hepatospecific delivery by ligand based receptor targeting is an established strategy to augment therapy associated with liver diseases and disorders. Previously, we have investigated the effect of ligand headgroup on cellular uptake mediated by the asialoglycoprotein receptor by in silico and in vitro approach. In this paper, we report the design of agarose based liposomes for delivery to liver cancer cells and provide a proof of concept of the targeting efficiency against galactose liposomes using an in vivo approach. Sorafenib Tosylate loaded targeting liposomes were developed and optimized using factorial design. Comparative evaluation including cell cytotoxicity, pharmacokinetics and biodistribution and hepatospecific uptake was performed for both the liposomal systems. The formulations possessed a particle size of 150 - 180 nm and a zeta potential of 30 - 60 mV depending on the amount of ligand and drug loading, with more than 90% entrapment efficiency. A two-fold increase in cytotoxicity was observed with agarose-based liposomes as compared to galactose based liposomes. In vivo PK evaluation indicated a reduction in half life of drug when loaded in agarose ligand loaded system, probably due to greater uptake in the liver as evidenced in biodistribution study. Intrahepatic disposition revealed a higher PC/NPC uptake ratio with the targeted systems as compared to conventional liposomes, although the agarose-based system resulted in highest uptake ratio. A biocompatible platform for specific delivery of drugs to hepatocytes was established validating a rational approach to design liver targeting systems.

Positively charged polymeric nanoparticles: application in improving therapeutic efficacy of meloxicam after oral administration.

The potential of positively charged polymeric nanoparticles in improving therapeutic efficacy of meloxicam (MLX), a poorly water-soluble anti-inflammatory agent was evaluated. MLX loaded positively charged nanoparticles were prepared by using poly-epsilon-caprolactone (PCL) as a biodegradable polymer and didodecyldimethylammonium bromide (DDAB) as a cationic surfactant. The MLX nanoparticles were characterized for particle size and encapsulation efficiency. MLX loaded PCL nanoparticles and MLX suspension were evaluated for their in vivo anti-inflammatory activity and ulcerogenic potential. MLX loaded PCL nanoparticles had particle sizes of approximately 300 nm and the encapsulation efficiency of MLX was approximately 90%. The polymeric nanoparticles significantly improved the anti-inflammatory activity of MLX (P< 0.01) as compared to that of MLX suspension. The higher anti-inflammatory effect was maintained for a longer duration (6 h). The polymeric nanoparticles also resulted in less ulcerogenicity as compared to that of MLX suspension.

Polysaccharide conjugates surpass monosaccharide ligands in hepatospecific targeting - Synthesis and comparative in silico and in vitro assessment.

Ligands with the polysaccharide headgroups have been recently reported by our group to possess enhanced interaction with asialoglycoprotein receptor (ASGPR) in silico as compared to ligands having galactose moieties. This enhanced interaction is a result of the polymer's backbone support in anchoring the ligand in a specific orientation within the bilayer. In this paper, we have attempted to provide an in vitro proof of concept by performing a comparative evaluation of polysaccharide and monosaccharide-based ligands. Docking was performed to understand interaction with ASGPR in silico. Agarose and galactose conjugates with behenic acid were synthesized, purified, and characterized to yield biocompatible hepatospecific ligands which were incorporated into nanoliposomes. Cellular internalization of these targeted liposomes was studied using confocal microscopy and flow cytometry. The toxicity potential was assessed in vivo. Results indicated that the polysaccharide-based ligand increased cellular uptake due to better interaction with the receptor as compared to ligand bearing a single galactose group. In addition to developing novel liver targeting ligands, the study also established proof of concept that has been suggested by earlier in silico investigations. The approach can be used to design targeting ligands and develop formulations with improved targeting efficacy.

Organic solvent-free approach to single step fabrication of Eudragit nanoparticles using Labrasol.

The present investigation describes organic volatile solvent-free approach for the single step fabrication of Eudragit nanoparticles. The solubility of various grades of Eudragit viz. Eudragit L100-55, Eudragit L100, Eudragit S100, Eudragit EPO, Eudragit RSPO and Eudragit RLPO in Labrasol (Caprylocaproyl macrogol-8 glycerides) was determined. We observed that Labrasol has the ability to solubilize various cationic Eudragits such as Eudragit RSPO, Eudragit RLPO and Eudragit EPO at a concentration as high as 200 mg/ml. We also evaluated the ability of Labrasol to act as a nanoparticle stabilizer due to its amphiphilic nature and high HLB of 14. It was observed that Eudragit EPO, Eudragit RSPO and Eudragit RLPO nanoparticles of size 110-150 nm could be easily fabricated using Labrasol as a solubilizer and nanoparticle stabilizer. Transmission electron microscopy was carried out to confirm the size and morphology of the nanoparticles. We could encapsulate hydrophobic drugs such as repaglinide and triclosan in the Eudragit RLPO nanoparticles as Labrasol also had the ability to solubilize these hydrophobic drugs. The ability of Eudragit RSPO and RLPO nanoparticles to condense plasmid DNA was also established. This is the first report that demonstrates the polymer solubilizing potential of Labrasol.

Lipid nanoconstructs for superior hepatoprotection: In vitro assessments as predictive tool for in vivo translation.

AIM: To investigate comparative in vitro and in vivo performance of lipid vesicular and particulate systems in escalating oral bioavailability for superior hepatoprotection. MATERIALS AND METHODS: Systems were fabricated using easy to scale up process and novel excipients to deliver Silibinin. In vitro characterization followed by pharmacokinetic and pharmacodynamic evaluation in rats was conducted to establish a correlation. RESULTS: Nanoformulations resulted in 20 fold increase in solubilisation and significant increase in permeation. 2.5 fold increase in bioavailability was evident in vivo. Vesicles demonstrated greatest hepatoprotective potential in efficacy study. CONCLUSION: The findings establish a link between in vitro and in vivo performance to rank order lipid nanoartchitects. Concurrently, a significant potential in therapeutic intervention of hepatotoxicity is envisaged as elucidated.

Self-nanoemulsifying granules of ezetimibe: design, optimization and evaluation.

Self-nanoemulsifying granules (SNGs) were formulated with the objective of enhancing the bioavailability of the ezetimibe. Various modified oils, surfactant and cosurfactant mixtures were used and composition of self-nanoemulsifying system (SNS) was optimized. SNS diluted and resultant emulsion was characterized for mean globule size and stability. The self-nanoemulsifying systems were formulated into free flowing self-nanoemulsifying granules using varying proportions of hydrophilic colloidal silicon dioxide as an adsorbing agent. Self-nanoemulsifying granules were characterized by X-ray diffraction pattern, differential scanning calorimetry, dissolution profile and for in vivo performance in rats. X-ray diffraction studies indicated loss of crystallinity and/or solubilisation of ezetimibe in the self-nanoemulsifying granules. It was supported by SEM studies, which did not show evidence of precipitation of the drug on the surface of the carrier. Dissolution studies revealed remarkable increase in dissolution of the drug as compared to plain drug. In vivo evaluation in rats showed significant decrease in the total cholesterol levels as compared to positive control. The SNGs filled into hard gelatin capsules showed two to threefold increase in the dissolution rate as compared to plain drug filled capsules signifying its potential in improved delivery of lipophilic drugs.

Parenteral microemulsions: an overview.

Parenteral delivery of the hydrophobic drugs is a very challenging task. The conventional approaches such as use of co-solvents, oily vehicles and modern approaches such as mixed micelles, liposomes, complexation with cyclodextrins and emulsions have several limitations. Microemulsions have evolved as a novel vehicle for parenteral delivery of the hydrophobic drugs. Their interesting features such as spontaneity of formation, ease of manufacture, high solubilization capacity and self-preserving property make them the vehicle of choice. The review focuses on the excipients available for formulation of the parenteral microemulsions and describes the investigations reported for the various classes of therapeutic agents.

Formulation and in vivo evaluation of self-nanoemulsifying granules for oral delivery of a combination of ezetimibe and simvastatin.

Self-nanoemulsifying granules were formulated with the objective of improving the bioavailability of the ezetimibe and simvastatin when administered together. Composition of self-nanoemulsifying system (SNS) was optimized using various modified oils, surfactant, and cosurfactant mixtures. SNSs were mixed with water and resultant emulsions were characterized for mean globule size and stability. SNSs were adsorbed on hydrophilic colloidal silicon dioxide to give free-flowing self-nanoemulsifying granules. Self-nanoemulsifying granules were characterized by X-ray diffraction pattern, scanning electron microscopy, dissolution profile, and for in vivo performance in hypercholesterolemic rats. X-ray diffraction studies and scanning electron microscopy indicated loss of crystallinity and/or solubilization of both drugs in the self-nanoemulsifying granules. Self-nanoemulsifying granules effected substantial increase in dissolution of the drugs as compared with pure powder of drugs. In vivo evaluation in rats showed significant decrease in the total cholesterol levels and triglyceride levels in rats as compared with positive control confirming potential of self-nanoemulsifying granules as a drug delivery system for the poorly water-soluble drugs.

Potential of cyclodextrin complexation and liposomes in topical delivery of ketorolac: in vitro and in vivo evaluation.

The objective of this investigation was to evaluate the effect of delivery strategies such as cyclodextrin complexation and liposomes on the topical delivery of ketorolac acid (KTRA) and ketorolac tromethamine. Ketorolac acid-hydroxypropyl-beta-cyclodextrin solid dispersions (KTRA-CD) were prepared by kneading method. The liposomes containing ketorolac tromethamine (KTRM) and KTRA-CD were prepared. The in vitro permeation of KTRM solution, KTRA solution, KTRA-CD, and liposomes containing KTRM or KTRA-CD through guinea pig skin was evaluated. The anti-inflammatory activity of the topically applied KTRA-CD gel (containing 1% w/w KTRA) was compared to that of orally delivered KTRM solution. The KTRA-CD demonstrated significantly higher transdermal transport of ketorolac as compared to all other systems whereas liposomes significantly reduced the transport of ketorolac. The anti-inflammatory activity of the topically applied KTRA-CD gel was similar to that of the orally administered KTRM. Thus, cyclodextrin complexation enabled effective transdermal delivery of the ketorolac.

Design and evaluation of self-nanoemulsifying drug delivery systems (SNEDDS) for cefpodoxime proxetil.

Self-nanoemulsifying drug delivery systems (SNEDDS) were developed with the objective to overcome problems associated with the delivery of cefpodoxime proxetil (CFP), a poorly bioavailable high dose antibiotic having pH dependant solubility. Solubility of CFP in oily phases and surfactants was determined to identify components of SNEDDS. Various surfactants and co-surfactants were screened for their ability to emulsify selected oily phases. Ternary phase diagrams were constructed to identify area of nanoemulsification for the selected systems. The influence of CFP and the pH of dilution medium on the phase behavior of selected system were assessed. The globule size of optimized CFP SNEDDS in various dissolution media was determined to check the effect of pH on its behavior. The optimized CFP SNEDDS needed surfactant content less than 40% and yielded nanoemulsion of mean globule size 170 nm, which was not affected by the pH of dilution medium. The optimized SNEDDS released CFP completely within 20 min irrespective of the pH of dissolution medium.

Design, optimization and evaluation of domperidone coevaporates.

Coevaporates of domperidone were prepared using different polymers by solvent evaporation technique. Ethyl cellulose and hydroxypropyl methylcellulose phthalate were used in preparation of coevaporates. The coevaporates were characterized by X-ray diffraction studies, IR spectrophotometry and differential scanning calorimetry. The dissolution behavior of coevaporates was studied using buffer solution of pH 1.2 for the first 2 h and that of pH 6.8 thereafter up to 12 h. A two-factor, three-level design was used to quantitate the effect of polymers on dissolution profile of domperidone. The drug release rate was found to be dependent on the concentration of polymers in the coevaporates. Dissolution of drug in a pH 6.8 buffer improved with increasing concentration of hydroxypropyl methylcellulose phthalate in coevaporates. Bioavailability studies in human volunteers confirmed that domperidone coevaporates sustained drug release.

Sterically stabilized etoposide liposomes: evaluation of antimetastatic activity and its potentiation by combination with sterically stabilized pentoxifylline liposomes in mice.

Cytotoxic activity of chemotherapeutic agents can be enhanced by site-specific delivery or by combination with other less toxic agents. In the present study, enhancement in the antimetastatic activity of etoposide (ETP) by encapsulation in sterically stabilized liposomes was evaluated in the murine experimental B16F10 melanoma model. Further, potentiation of its antimetastatic activity by combination with pentoxifylline (PTX) solution or sterically stabilized PTX liposomes was evaluated in the same animal model. Upon intravenous administration, ETP solution and ETP liposomes inhibited pulmonary tumor nodule formation in a dose-dependent manner. Encapsulation of ETP in liposomes resulted in significant enhancement in its antimetastatic activity at doses of 0.5 mg/kg and 0.75 mg/kg as compared to ETP solution at similar doses. In combination therapy, the effect of sequence of administration of the drugs, ETP and PTX, was evaluated. Enhancement of antimetastatic activity of ETP solution when used in combination with PTX solution was effected by the sequence in which the solutions were administered. However, a combination of ETP liposomes and PTX liposomes led to potentiation of antimetastatic activity in a sequence-independent manner. The results indicate that antimetastatic activity of ETP is significantly enhanced by encapsulation in liposomes. Administration of ETP liposomes with PTX liposomes further potentiated the activity, suggesting the usefulness of this combination in clinical practice for reducing the dose-limiting toxic effects of ETP.

Preparation and evaluation of a liposomal formulation of sodium cromoglicate.

Sodium cromoglicate (SCG) is given by inhalation in prophylactic control of asthma. It was encapsulated in liposomes with a view to improve utilization of the drug when given via pulmonary route. The liposomes were characterized for encapsulation efficiency, shape, size and release rate. Liposomal dispersions were freeze-dried using a cryoprotectant. Freeze-dried liposomal dispersion retained 60% of drug upon reconstitution but increase in size of liposomes was noted. Liposomes exhibited good keeping properties when stored at 4 degrees C. In vivo performance of liposomal SCG was evaluated in sensitized guinea pigs. In one of the studies, differential leukocyte count and total leukocyte count in bronchoalveolar lavage fluid was measured. Liposomal dispersion showed significant inhibition of influx of neutrophils as compared with drug solution at 24 h. However, in the second study, when recovery period required by animal to revert back to normal respiratory pattern from the onset or preconvulsion time was measured, no significant difference was found between drug solution and liposomal dispersion when administered 2 h before allergen challenge.

Preparation and evaluation of liposomal formulations of tropicamide for ocular delivery.

Tropicamide, a mydriatic, cycloplegic drug was entrapped in liposomes. Liposomes were investigated by laser counting studies, transmission electron microscopy and differential scanning calorimetry for characterization. The precorneal clearance of liposomes was compared with solution by gamma-scintigraphy in the rabbit. The neutral liposomes failed to demonstrate significant enhancement in precorneal retention in comparison with aqueous solution. The potential of liposomes as an ophthalmic drug delivery system was investigated by comparing pupil dilatory effect of tropicamide by topical instillation, in the rabbit eye, of the solution and various drug-loaded liposomal forms, i.e. neutral liposomes, positively charged liposomes and neutral liposomes dispersed in 0.25% (w/v) polycarbophil gel. The positively charged liposomal formulation and liposomes dispersed in polycarbophil gel were found to be more effective than neutral liposomal dispersion when data were statistically treated at the 5% level of significance.

Solid dispersion of hydroxypropyl beta-cyclodextrin and ketorolac: enhancement of in-vitro dissolution rates, improvement in anti-inflammatory activity and reduction in ulcerogenicity in rats.

Ketorolac, is a non-steroidal anti-inflammatory drug, with strong analgesic activity. It is practically insoluble in water and has been implicated in causing gastrointestinal ulceration. This study describes the formulation of solid dispersions of ketorolac using hydroxypropyl beta-cyclodextrin (HPbeta-CyD) and beta-cyclodextin (beta-CyD) as carriers, to improve the aqueous solubility of the drug, thus enhancing its bioavailability. Also, reduction in ulcerogenicity was anticipated. Differential scanning calorimetry and X-ray diffraction studies indicated loss of crystalline nature of the drug, in the dispersions prepared with HPbeta-CyD. NMR studies revealed a strong interaction between drug and HPbeta-CyD. Solid dispersions of drug with beta-CyD retained the crystalline nature of the drug. All the solid dispersions showed a remarkable improvement in the rate and extent of dissolution of ketorolac. The kneaded dispersion with HPbeta-CyD prepared using a 1:1 alcohol-water mixture showed promise in reducing the ulcer-inducing effect of ketorolac in rats. Oral administration of this dispersion was found to inhibit carrageenan-induced paw oedema in rats to a significantly greater extent compared with ketorolac or its trometamol salt. Though beta-CyD as a carrier for ketorolac gave faster release of the poorly soluble drug, HPbeta-CyD proved to be superior to beta-CyD, as a carrier in the kneaded dispersion prepared using 1:1 alcohol-water mixture. These results suggest that solid dispersions of ketorolac with HPbeta-CyD aid in faster dissolution and better bioavailability of the drug. The higher solubility of the drug in the presence of HPbeta-CyD also reduces local gastrointestinal side-effects of the drug.

Azo-containing urethane analogues for colonic drug delivery: synthesis, characterization and in-vitro evaluation.

A urethane-based analogue containing an azo aromatic linkage in the backbone was synthesized for use in colon-specific delivery systems by reacting toluene-2,6-diisocyanate with a mixture of an aromatic azo diol, (bis-4-hydroxyphenyl)-4,4'-diazobiphenyl, poly(ethylene glycol) (Mn = 4000; number-average molecular weight) and 1,2-propanediol (propylene glycol). The resultant compounds (UR-1 and UR-2) were characterized by IR spectroscopy, 1H NMR spectroscopy, DSC studies, X-ray diffraction studies and molecular weight determination by gel permeation chromatography. The compounds exhibited low molecular weight, lacked film-forming properties and crystallinity in the structure. An in-vitro bacterial degradation test to demonstrate the susceptibility of azo bond to bacterial enzymes was performed using media inoculated with lactobacillus culture. The results indicated degradation of films by azoreductase. In-vitro permeation of 5-aminosalicylic acid was studied in control and lactobacilli-treated films. The permeability of the lactobacilli treated films was significantly increased suggesting the potential of these compounds for application in colonic targeting.

Enhancement of anti-metastatic activity of pentoxifylline by encapsulation in conventional liposomes and sterically stabilized liposomes in murine experimental B16F10 melanoma model.

Pentoxifylline has been shown to exhibit anti-metastatic activity by inhibiting homing of B16F10 melanoma cells in the murine experimental metastasis model. In this study, the effect of encapsulation of pentoxifylline in conventional and sterically stabilized liposomes on its anti-metastatic activity in the murine experimental metastasis model was investigated. After a single intravenous dose (10, 20 or 40 mg kg(-1)), pentoxifylline solution, as well as conventional pentoxifylline liposomes, significantly reduced the number of pulmonary nodules compared with the untreated control group. Conventional pentoxifylline liposomes showed significantly higher inhibition (69%) of pulmonary tumour nodule formation at a dose of 20mg kg(-1) as compared with pentoxifylline solution (49%) at the same dose. Encapsulation of pentoxifylline in sterically stabilized liposomes prepared by incorporation of monomethoxypolyethyleneglycol (5000)-cholesteryl ester further enhanced the inhibition of pulmonary nodule formation (77%) at a dose of 20 mg kg(-1) as compared with conventional pentoxifylline liposomes. Overall, the results suggest that encapsulation of pentoxifylline in conventional liposomes enhanced its anti-metastatic activity. Steric stabilization of pentoxifylline liposomes also resulted in a two-fold increase in anti-metastatic activity (at dose of 10 mg kg(-1)) as compared with conventional liposomes.