Reverse-phase Evaporation Method Research Articles

Development of a liposome-based immunochromatographic strip assay for the detection of Salmonella

Salmonella species are ubiquitous human pathogens which pose a dangerous threat to the elderly and children worldwide. In this study, to develop a more efficient assay procedure for the rapid detection of Salmonella Typhimurium, an immunochromatographic strip assay was developed using immunoliposome (anti-Salmonella IgG-tagged) encapsulated with sulforhodamine B (SRB). The detection sensitivity of the developed immunochromatographic assay was compared with a commercial immunochromatographic test strip using colloidal gold nanoparticles. The liposomes were prepared through a reverse-phase evaporation method by using a lipid and phospholipid mixture and SRB, a fluorescence dye, which was encapsulated in the phospholipid bilayer. Furthermore, the outer surface of the SRB-encapsulated liposome was conjugated with antibody (affinity-purified polyclonal goat anti-Salmonella IgG) to form an immunoliposome (size, 223 nm), used as the analytical reagent in the developed immunoassay. For this strip assay, a plastic-backed nitrocellulose strip was immobilized with two antibody zones. The lower zone of the strip referred to Salmonella antigen capture zone (test line), while the other zone served as a positive control (control line). The lower zone was coated with affinity-purified polyclonal goat anti-Salmonella IgG, while the upper zone was coated with rabbit anti-goat IgG. During capillary migration of the wicking solution (diluted liposome and Salmonella culture, each 50 μl), Salmonella was captured with surface-bound immunoliposomes at the antigen capture zone, while the unbound liposomes migrated upward and bound to another zone. The color density of the antigen capture zone was directly proportional to the amount of S. Typhimurium in the test sample. As a result, the detection limit of the immunochromatographic strip assay developed in this study against S. Typhimurium was found to be 10(2) CFU/ml, which was significantly higher than the detection limit (10(7) CFU/ml) of the commercial immunochromatographic test strip assay.

Application of liposome encapsulation technique to improve anti-carcinoma effect of resveratrol

Aim: The promising anti-tumor effect of resveratrol (RES) has aroused much interest in recent years, but its clinical application was seriously hindered due to its poor solubility in water. The aim of this study was to improve the water solubility of RES by liposome encapsulation technique for effective tumor treatment.Methods: This study develops two liposomal formulations to solubilize RES by reverse-phase evaporation method with or without poly(ethylene glycol-2000)-grafted distearolyl phosphatidylethanolamine (DSPE-PEG2000). The effect of different formulation factors on the encapsulation efficiency (EE) and the particle sizes were investigated. These factors included the mass ratio of drug to soybean phosphatidylcholine (drug/SPC), the mass ratio of cholesterol to soybean phosphatidylcholine (chol/SPC), the volume ratio of water phase/organic phase and the microfluidization process. The drug release studies were performed in various media, simulating the desired application conditions. The cytotoxicity study was carried out by MTT assay on HeLa and Hep G2 cell lines.Results: The RES EE of 95% was obtained when using drug/SPC (1:40 mass ratio), Chol/SPC (1:10 mass ratio), water phase/oil phase (1:2 volume ratio), microfluidization process (entrance pressure 6 kpa, two times of cycle time). The addition of DSPE-PEG2000 into the formulation showed little effect on the formation and properties of RES liposome. The release of RES was pH-independent. RES liposomes and PEG-modified liposomes performed significant inhibition effects on both cells growth due to the solubilized RES.Conclusion: RES can be effectively loaded into liposomes and its anti-cancer effect was evidently improved by the application of liposome encapsulation technique.

Influence of some physical properties of 5-fluorouracil on encapsulation efficiency in liposomes

The aim of this study is to encapsulate two drugs: 5-fluorouracil (5-FU) with the hydrophobic properties and 1-β-D-arabinofuranosylcytosine (Ara-C) with the amphiphilic properties into liposomes prepared by the modified reverse-phase evaporation method (mREV) from L-α-phosphatidylcholine dipalmitoyl (DPPC). We studied the thermotropic phase behavior of liposome entrapped 5-FU and Ara-C. It is known that the stability of liposomes depends not only on the method of chemical gradient loading, the use of membrane stabilizer such as sterols, but also on the phase transition temperature (Tc) of phospholipids, which undergoes an alteration after encapsulation of drugs to liposomes. The competition of these two drugs entrapped in liposomes was analyzed by the use of two spectroscopies: 1H NMR and UV on the basis of the analysis of the signals of each drug in the liposome—drug system. The percent of encapsulation in DPPC/Ara-C/5-FU liposome obtained by the use of UV spectroscopy amounted 93.84 and 96.05% for 5-FU and Ara-C, respectively. Phase transition temperature Tc of liposomes containing Ara-C did not significantly change while for the liposomes containing 5-FU it increased in comparison with Tc of the reference liposomes formed from DPPC.

Niosomal Delivery of Isoniazid - Development and Characterization

Purpose: To develop a niosomal formulation for the delivery of isoniazid to achieve effective treatment of tuberculosis.Methods: Niosomes were prepared by reverse phase evaporation method and given a charge with a charge-inducing agent, dicetyl phosphate. Drug entrapment efficiency in the niosomes was determined spectrophotometrically. The niosomes were further characterized for their particle size, polydispersity index (PI) and zeta potential as well as by scanning electron microscopy and stability studies. Furthermore, in vitro drug release and cellular uptake studies on the niosomes by macrophage J744 A were undertaken.Results: Suitable isoniazid niosomes were obtained. The niosomes demonstrated a potential to remain in the treated site for prolonged periods and were also capable of maintaining steady drug concentrations for up to 30 h. Cellular uptake of the drug-loaded niosomes by macrophage cells was as high as 61.8 %, a level that is capable of achieving effective treatment of tuberculosis.Conclusion: The isoniazid niosomes developed are capable of reducing drug dose and toxicity as well as dosing frequency which should bring about improved patient compliance. More importantly, macrophage targeting should be feasible at sites where tuberculosis bacteria are harbored.

Integrin receptor‐selective peptide based targeting reduces immunogenicity and improves vascular delivery of streptokinase

Streptokinase (STK) is used in treating thrombosis; however, immunogenicity and uniform distribution in the body with resulting bleeding complications are major limiting factors. We hypothesized that an integrin-receptor-selective peptide based delivery system would reduce immunogenicity and improve vascular delivery of STK. Plain and surface modified ligand anchored liposomes were prepared using reverse phase evaporation method. These were characterized for shape, size, polydispersity index, entrapment efficiency, number of vesicles/mm3 and in vitro drug release. Liposomal surface was modified by attaching RGD peptide as ligand. Modified liposomes exhibited lower entrapment efficiency (17.7 ± 0.3% vs 20.1 ± 0.2% for conventional liposomes) and released more drug in 48 hours (79.8 ± 2.1%) as compared to conventional liposomes (67.8 ± 3.7%). In ex-vivo clot penetration studies using fluorescence imaging, ligand conjugated liposomes showed greater penetration in the clot than control. In in vivo studies (BALB/c mice; 28 days), the antibody titer was reduced (p<0.05) in animals treated with ligand anchored liposomes (0.69 ± 0.14 AU) than those treated with free STK solution (0.81±0.12 AU). In comparison to free STK, the surface modified liposomes resulted in a significant reduction in immunogenicity. In addition the developed delivery system showed higher affinity and better clot penetration than control.

&lt;I&gt;In Vitro&lt;/I&gt; and &lt;I&gt;In Vivo&lt;/I&gt; Anti-Tumor Effects of Gemcitabine Loaded with a New Drug Delivery System

In recent years, much attention has been given to liposomal formulation as an efficient drug loading system (DDS) in chemotherapy of cancer. In this study, the advantages of magnetic nanoparticles and Polyethylene Glyco (PEG) materials were considered to synthesize magnetic gemcitabine long-circulating liposomes (MGLL) and the potential of MGLL as a brand new delivery system was evaluated. MGLL was prepared using the reverse-phase evaporation method. In the optimized preparation, MGLL had an average diameter of 201 nm with a narrow size distribution measured by dynamic light scattering (DLS), which could be easily dispersed in ultrapure water under a stable state for 90 days. The encapsulation efficiency of gemcitabine in MGLL reached 87.2% as determined by HPLC. In vitro MTT assay showed that MGLL had significant cytotoxicity to MCF-7 cells compared with the conventional modalities. In vivo, the inhibition of tumor growth in MGLL group was more remarkable than that of other groups (P < 0.05). In conclusion, MGLL under optimized condition could be used as an effective carrier for tumor-targeted therapy.

Study of TAT-PEG loaded magnetic liposomes crossing blood spinal cord barrier after spinal cord injury in rats

To evaluate the ability of a kind of novel magnetic liposomes modified with polyethylene glycol (PEG) and transactivating-transduction protein (TAT) to cross the blood spinal cord barrier (BSCB) so as to demonstrate whether or not they can accumulate at the lesions of injured spinal cord. The novel liposomes were made through reverse-phase evaporation method modified with polyethylene glycol (PEG) and transactivating-transduction protein (TAT) with an iron core. Thirty-six Wistar rats subject to spinal cord injury (SCI) at T10 were randomly divided into three groups (Groups I, II and III). The rats of Group III were injected with TAT-PEG loaded magnetic liposomes (4.55 mg/kg). The rats of GroupII received an injection of the equivalent PEG loaded magnetic liposomes while those of control group (GroupI) the equivalent normal saline. The accumulation of liposomes was observed by MRI (magnetic resonance imaging), Prussian blue staining, electron microscope and flame atomic absorption spectrophotometer. This kind of TAT-PEG loaded magnetic liposomes could cross the BSCB and enter into the cells around the injured tissue. A low signal of T2WI on MRI could also be found in Group III. The results of flame atomic absorption spectrophotometer showed that the iron content accumulated around the lesion site in Group III was obviously higher than the other two groups (P < 0.05). The TAT-PEG loaded magnetic liposomes may be employed as one kind of novel drug carrier to cross the BSCB and accumulate at tissue cells of spinal cord. It is likely to become a new therapy for SCI.

Effective transcutaneous immunization by antigen-loaded flexible liposome in vivo

BackgroundTranscutaneous vaccines have received wide attention due to their easy-to-use, needle-free, noninvasive delivery. However, the novel barrier function of stratum corneum hinders the transport of antigen and adjuvant in transcutaneous immunization. Novel nanoscale delivery systems employing, for example, liposomes and nanoparticles, have been widely investigated to overcome the penetration barrier of stratum corneum for effective transcutaneous immunization.ObjectiveThe objective of this study was to prepare two types of flexible liposomes and determine their efficacies for the transcutaneous delivery of antigen and the subsequent immune response induced in vivo.MethodsOvalbumin (OVA) liposome-based transcutaneous vaccines were prepared using reverse-phase evaporation and film-dispersion methods. Particle sizes and antigen encapsulating efficiency were then evaluated. After application to bare mouse skin, topical sites were examined for the presence of fluorescence-labeled liposome. The efficacy of the transcutaneously delivered OVA-loaded flexible liposome in activating the immune responses was investigated by detecting serum immunoglobulin G levels. The influence of an adjuvant, imiquimod, in the transcutaneous immunization was also tested.ResultsTwo flexible liposomes with well-encapsulated OVA were successfully prepared by film-dispersion or reverse-phase evaporation methods. The sizes of the prepared flexible liposomes ranged from 200 to 400 nm. In vivo, the fluorescence-labeled liposome was detected in hair-follicle ducts, indicating that the flexible liposome can penetrate the skin barrier through the hair follicles. Upon transcutaneous administration, the OVA-encapsulated flexible liposome elicited a strong immune response similar to that of positive control (ie, OVA solution administrated by subcutaneous injection with Al(OH)3 as an adjuvant). Co-administration of imiquimod with the OVA-loaded liposome expressed a significant enhancement on the transcutaneous immune responses.ConclusionResults of this study highlight the nanoscale formulation, flexible liposome, as a promising carrier for the transcutaneous delivery of antigen proteins. Imiquimod was shown to be an effective adjuvant as a transcutaneous immunization enhancer with the potential for transcutaneous vaccine development.

Liposomes containing glycocholate as potential oral insulin delivery systems: preparation, in vitro characterization, and improved protection against enzymatic degradation

Background:Oral delivery of insulin is challenging and must overcome the barriers of gastric and enzymatic degradation as well as low permeation across the intestinal epithelium. The present study aimed to develop a liposomal delivery system containing glycocholate as an enzyme inhibitor and permeation enhancer for oral insulin delivery.Methods:Liposomes containing sodium glycocholate were prepared by a reversed-phase evaporation method followed by homogenization. The particle size and entrapment efficiency of recombinant human insulin (rhINS)-loaded sodium glycocholate liposomes can be easily adjusted by tuning the homogenization parameters, phospholipid:sodium glycocholate ratio, insulin:phospholipid ratio, water:ether volume ratio, interior water phase pH, and the hydration buffer pH.Results:The optimal formulation showed an insulin entrapment efficiency of 30% ± 2% and a particle size of 154 ± 18 nm. A conformational study by circular dichroism spectroscopy and a bioactivity study confirmed the preserved integrity of rhINS against preparative stress. Transmission electron micrographs revealed a nearly spherical and deformed structure with discernable lamella for sodium glycocholate liposomes. Sodium glycocholate liposomes showed better protection of insulin against enzymatic degradation by pepsin, trypsin, and α-chymotrypsin than liposomes containing the bile salt counterparts of sodium taurocholate and sodium deoxycholate.Conclusion:Sodium glycocholate liposomes showed promising in vitro characteristics and have the potential to be able to deliver insulin orally.

Study of ocular drug delivery system using drug-loaded liposomes

Background:This project aims to formulate and characterize a drug-eluting contact lens to provide controlled release of drug for a longer period of time, using liposomes as drug delivery system.Materials and Methods:Drug delivering contact lenses were created by coating disposable soft contact lenses with ciprofloxacin entrapped in the liposomes. Reverse phase evaporation and lipid film hydration methods were used for the preparation of ciprofloxacin trapping reverse phase evaporation vesicles, that is, unilamellar vesicles (REVs) and multilamellar vesicles (MLVs), respectively. Soya lecithin and cholesterol (CH) were used in the molar ratios of 7:4 and 7:2. The spherical structure of the liposomes, the mean diameter, and their purity were determined by photomicroscopic, transmission electron microscope, and chromatographic analysis, respectively. The prepared liposomes were evaluated for their entrapment efficiency, in vitro drug release, stability, and toxicity.Results:MLVs were larger than REVs with their mean diameter 338.32 nm and also entrapped greater amount of ciprofloxacin. Drug loading and its release from the liposomal vesicles was dependent on CH content. Ciprofloxacin released from the liposomes coated on the contact lenses not only inhibited both Staphylococcus aureus and Pseudomonas aeruginosa on an agar plate but also showed an enhanced antibacterial effect as determined by minimal inhibitory concentrations. Approximately 40% of ciprofloxacin was retained up to a period of 3 months at 4°C. Furthermore, the formulation was found to be nontoxic and also a reduction in toxicity of ciprofloxacin was observed after entrapment when assessed by lymphocyte toxicity assay and chick embryo inoculation.Conclusions:An innovative drug delivery system consisting of drug-loaded liposomes coated onto the surface of contact lenses has been developed. This system is highly specific in terms of localized and sustained application of the drug.

Development and Evaluation of Chitosan-Coated Liposomes for Oral DNA Vaccine: The Improvement of Peyer’s Patch Targeting Using a Polyplex-Loaded Liposomes

The aim of this study was to develop chitosan-coated and polyplex-loaded liposomes (PLLs) containing DNA vaccine for Peyer's patch targeting. Plain liposomes carrying plasmid pRc/CMV-HBs were prepared by the reverse-phase evaporation method. Chitosan coating was carried out by incubation of the liposomal suspensions with chitosan solution. Main lipid components of liposomes were phosphatidylcholine/cholesterol. Sodium deoxycholate and dicetyl phosphate were used as negative charge inducers. The zeta potentials of plain liposomes were strongly affected by the pH of the medium. Coating with chitosan variably increased the surface charges of the liposomes. To increase the zeta potential and stability of the liposome, chitosan was also used as a DNA condensing agent to form a polyplex. The PLLs were coated with chitosan solution. In vivo study of PLLs was carried out in comparison with chitosan-coated liposomes using plasmid encoding green fluorescence protein as a reporter. A single dose of plasmid equal to 100 μg was intragastrically inoculated into BALB/c mice. The expression of green fluorescence protein (GFP) was detected after 24 h using a confocal laser scanning microscope. The signal of GFP was obtained from positively charged chitosan-coated liposomes but found only at the upper part of duodenum. With chitosan-coated PLL540, the signal of GFP was found throughout the intestine. Chitosan-coated PLL demonstrated a higher potential to deliver the DNA to the distal intestine than the chitosan-coated liposomes due to the increase in permanent positive surface charges and the decreased enzymatic degradation.

Preparation, characterization and the stability of ferrous glycinate nanoliposomes

Ferrous glycinate nanoliposomes prepared by the reverse-phase evaporation method (REV) from egg phosphatidylcholine (EPC) were investigated, based on the encapsulation efficiency, transmission electron microscopy (TEM), size distribution, and zeta potential. The nanoliposomes had high encapsulation efficiency, and TEM photomicrographs of nanoliposomes clearly showed their spherical shape. The size distribution and zeta potential indicated the stability of the nanoliposome suspensions. Retention ratio and size distribution of ferrous glycinate nanoliposomes were used to determine the influence of storage period, sonication and boiling water on the stability of ferrous glycinate nanoliposomes. Furthermore, in vitro stability of ferrous glycinate nanoliposomes in simulated gastrointestinal juice was evaluated. The nanoliposomes showed an acceptable stability in simulated gastrointestinal juice at 37 °C for 5 h. According to the results, ferrous glycinate nanoliposomes may be fit for the oral administration of ferrous glycinate and be used for the fortification of foodstuffs.

Thermotropic Phase Behavior of Liposome Entrapped 5-FU and LCV

The aim of this work was to encapsulate two drugs: 5-Fluorouracil (5-FU) with the hydrophobic properties and 5-Formyl-5,6,7,8-tetrahydropteroyl-L-glutamic acid calcium salt (leucovorin) with the hydrophilic properties into liposomes prepared by the modified reverse-phase evaporation method (mREV) from L-α-phosphatidylcholine dipalmitoyl (DPPC). The drugs are used in the anticancer multidrug therapy FLv (5-fluorouracil, leucovorin) and ELF (etoposide, leucovorin, 5-fluorouracil). We studied the competition for their encapsulation in liposomes by the use of two spectroscopies: 1H NMR and UV on the basis of the analysis of the signals of each drug in the liposome – drug system. Liposomes are highly versatile structures for research, therapeutic and analytical applications. We concluded that the liposomes obtained by the mREV method may transport more than one drug simultaneously.

Serine‐ and mannose‐modified liposomal contrast agent for computed tomography: evaluation of the enhancement in rabbit liver VX‐2 tumor model

Kupffer cell imaging is a powerful tool for the detection of liver cancer. This diagnostic procedure depends on the faculty of the reticuloendothelial system (RES) which takes up foreign bodies, including small particles. The current study aimed to develop a novel RES targeting liposomal contrast agent that functionalized with serine or mannose, the moiety specifically binding to a corresponding receptor on phagocytic cells. Liposomes loaded with non-ionic X-ray contrast media, Iohexol, were prepared by supercritical carbon dioxide reverse-phase evaporation method and were intravenously injected to healthy rabbits in order to evaluate the liver parenchymal enhancement in X-ray computed tomography (CT). From 10 to 40 min after injection, the mean enhancement value of the liver parenchyma approached 45 and 34 Hounsfield units (HU) when serine-modified iodinated liposomal contrast agent (ILCA) and mannose-modified ILCA were applied, respectively. The tumor-to-liver contrast values were also evaluated after the administration of the prepared ILCA to rabbits with VX-2 carcinoma. For serine-modified ILCA, tumor-to-liver contrast was 82 HU at 1 min and >24 HU at 10-40 min; for mannose-modified ILCA, the values were 58 HU at 0.5 min and >21 HU at 10-40 min. These vales estimated from the region of intrest and the imaging figures of liver indicate the potential of ILCA for clinical use.

Liposomes Coloaded with Iopamidol/Lipiodol as a RES-Targeted Contrast Agent for Computed Tomography Imaging

The need for computed tomography (CT) of reticuloendothelial system (RES)-rich organs such as the liver is increasing, particularly in patients with suspected hepatic metastasis. CT images of the liver have been improved by encapsulating currently used, water-soluble iodine contrast agent in liposomes. The present study was performed to investigate a possibility to overcome the limitations of entrapped iodine in liposomes by preparing liposomes co-loaded with iopamidol, a water-soluble iodinated compound, and lipiodol, an iodized oil. Iopamidol and lipiodol were simultaneously loaded in liposomes by modified reverse-phase evaporation method. The entrapped iodine concentration, mean particle size and polydispersity index of resulting liposomes were evaluated. Following intravenous injection of these liposomes into rats, CT scanning was performed. Simultaneous loading of iopamidol and lipiodol into liposomes resulted in entrapped iodine concentrations as high as 49.2 iodine mg/ml. The mean particle size was 280 nm, and the mean polydispersity index was 0.230. CT scanning with these iopamidol/lipiodol (I/L) liposomes into rats resulted in more pronounced and more persistent increases in RES-rich organs, liver and spleen, compared with free liposomes or liposomes loaded with iopamidol alone. These findings indicate that I/L liposomes have the potential to allow thorough CT examination of RES-rich organs.

Tissue distribution and pulmonary targeting studies of cefpiramide sodium-loaded liposomes

The aim of this study was to evaluate the pharmacokinetics (PK), tissue distribution, and the specific drug targeting of cefpiramide sodium-loaded liposomes (CPMS-Lips) compared with cefpiramide sodium solution (CPMS-Sol) in mice. CPMS-Lips were prepared by reverse phase evaporation method. In the PK and biodistribution study, mice received a single intravenous (i.v.) injection of 152 mg/kg of either CPMS-Lips or CPMS-Sol. Plasma and tissues were treated using liquid–liquid extraction and determined using reversed-phase high-performance liquid chromatography (RP-HPLC). The results showed that the CPMS-Lips prepared in this study had an average diameter of 7.146 ± 0.29 μm. In the plasma, the bioavailability (F) and the mean residence times (MRT) of the CPMS-Lips were 2.8- and 4.5-fold larger, respectively, than those of CPMS-Sol. CPMS-Lips also showed a significant difference in the tissue distribution profile in mice when compared with the conventional. The value of the intake rate (re) for the lung was 2.97, which was the highest among the tested tissues. Meanwhile, the ratio of targeting efficiency (Teliposome/Teinjection) of lung to that of other tissues for CPMS-Lips elevated significantly. These showed that CPMS-Lips can improve the bioavailability and biodistribution of CPMS in the lung. In conclusion, the liposome was a promising sustained-release and drug-targeting system for antibiotic drugs.

Mannosylated niosomes as carrier adjuvant system for topical immunization

The aim of this study was to develop mannosylated niosomes as a topical vaccine delivery carrier and adjuvant for the induction of both humoral and cellular immunity. Bovine serum albumin (BSA)-loaded niosomes composed of sorbitan monostearate/sorbitan trioleate (Span 60/Span 85), cholesterol and stearylamine as constitutive lipids were prepared by the reverse-phase evaporation method. The niosomes were coated with a modified polysaccharide O-palmitoyl mannan (OPM) to target them to Langerhan's cells, the major antigen presenting cells found in abundance beneath the stratum corneum. Prepared niosomes were characterized in-vitro for their size, shape, entrapment efficiency and ligand binding specificity. The immune stimulating activity was studied by measuring serum IgG titre and its subclasses (IgG2a/IgG1 ratio) following topical application of various niosomal formulations in albino rats. The results were compared with alum-adsorbed BSA following topical application and intramuscular injection. It was observed that niosomal formulations elicited a significantly higher serum IgG titre upon topical application as compared with topically applied alum adsorbed BSA (P<0.05). The serum IgG levels were significantly higher for the mannosylated niosomes as compared with plain uncoated niosomes (P<0.05). All formulations displayed a combined serum IgG2a/IgG1 response, which suggested that the formulations were capable of eliciting both humoral and cellular responses. The study signified the potential of OPM-coated niosomes as a topical vaccine delivery carrier and adjuvant. The proposed system would be simple, stable, and cost effective and might be clinically acceptable.

Development of a topical niosomal preparation of acetazolamide: preparation and evaluation

Orally administered acetazolamide has a limited use in glaucoma due to the systemic side effects associated with its use. No topical formulation of acetazolamide is available, mainly because of it having a limited aqueous solubility and poor corneal permeation. To enhance the bioavailability of acetazolamide by the topical route and to improve the corneal permeability of the drug, niosomes of acetazolamide were prepared (employing span 60 and cholesterol) by different methods. Transmission electron microscopy (TEM) of the selected formulation was carried out to study the morphology. Niosomes were also prepared in the presence of dicetyl phosphate and stearylamine to obtain negatively and positively charged vesicles, respectively. It was found that the reverse-phase evaporation method (REV) gave the maximum drug entrapment efficiency (43.75%) as compared with ether injection (39.62%) and film hydration (31.43%) techniques. Drug entrapment efficiency varied with the charge and the percent entrapment efficiency for the REV method was 43.75, 51.23 and 36.26% for neutral, positively charged and negatively charged niosomes, respectively. Corneal permeability studies, however, showed that the percent permeation and the apparent permeability coefficient for the charged niosomes were less than for the neutral ones. A bioadhesive niosomal formulation of acetazolamide was also prepared and compared with the positively charged formulation, considering that both of them would have a prolonged stay in the cul-de-sac because of their expected interactions with mucin. The formulations were also compared based on their intraocular pressure (IOP)-lowering capacity. The positively charged niosomes (REV2), although showing good corneal permeability and pharmacodynamics, were however found to be inappropriate in terms of the corneal cell toxicity. The bioadhesive coated formulation (REV1bio) compared well with REV2 and also showed a much lesser toxicity. Further, the IOP-lowering effect of the developed formulations was compared with that of a marketed formulation of dorzolamide 2%, a topical carbonic anhydrase inhibitor. The developed niosomal formulations of acetazolamide showed a comparable physiological effect (33% reduction of IOP in REV1bio and 37% reduction in dorzolamide) with a duration of up to 6 h (the duration being 3 h for dorzolamide). Results of the study indicate that it is possible to develop a safe (as indicated by corneal toxicity studies) and physiologically active topical niosomal formulation of acetazolamide relative in efficiency to the newer local carbonic anhydrase inhibitor, dorzolamide. The developed formulations can form a cost effective treatment plan, which is especially important in the treatment of glaucoma, a chronic ailment affecting middle-aged to old patients.

Effect of charge and lipid concentration on in-vivo percutaneous absorption of methyl nicotinate from liposomal vesicles

We have investigated the influence of charge and lipid concentration on the in-vivo percutaneous absorption of a model compound, methyl nicotinate (MN), from liposomal vesicles. MN-loaded liposomes were produced by the reverse-phase evaporation method (REV) using different concentrations of phosphatidyl choline (PC), in association with surfactants such as dioctadecyl dimethyl ammonium bromide (DDAB18) and dicetyl phosphate (DCP), which impart a positive or negative charge to the systems, respectively. The liposomal suspensions were then processed to hydrogels and used to study in-vivo the MN permeation profile. MN was chosen as the model compound since it was capable of causing cutaneous erythema, the intensity and duration of which was proportional to the amount entering the living epidermis over time. The extent of the erythema was monitored by reflectance spectrophotometry, a non-invasive technique. In-vivo findings showed an interesting MN delayed release, which was proportional to the amount of phospholipids in each liposomal formulation. Furthermore, it could be noted that the erythematous effect was more prolonged when MN was delivered from neutral or negatively-charged liposomal forms.

Abstract A130: Effects of a novel liposomal formulation of fenretinide on human neuroblastoma cell growth, apoptosis and angiogenesis

Abstract Neuroblastoma (NB) is an embryonal tumor originating from the simpatico-adrenal lineage of the neural crest and developing virtually at any site of the sympathetic nervous system. It approximately accounts for about 15% of all paediatric oncology deaths. NB is characterized by different clinical presentations and courses. Metastatic tumors at diagnosis remains a clinical challenge, despite advances in multimodal therapy. Retinoids are a class of compounds known to induce both terminal differentiation and apoptosis/necrosis of NB cells. Among them, fenretinide (HPR), thanks to its good safety profile in humans, has been considered one of the most promising synthetic retinoids tested as chemopreventive agent but it is only partially efficacious due to both a scanty oral delivery and a rapid methabolism. Here, we have developed a novel liposomal HPR by which the drug was encapsulated into sterically stabilized liposomes [SL-(HPR)] according to the reverse-phase evaporation method. This procedure led to a higher drug encapsulation and to better size and stability in organic fluids when compared to our previously HPR-entrapped liposome preparation (Pagnan et al., Int.J.Cancer 1999;81:268). Moreover, liposomes were coupled with NGR peptides that target the tumor endothelial cell marker, the aminopeptidase N [NGR-SL-(HPR)] (Pastorino et al, Cancer Res 2006;66:10073). The in vivo sensitivity of NB to the HPR liposomal preparations was tested against an established orthotopic NB xenograft animal model. Tumor-bearing mice treated with NGR-SL-(HPR) lived statistically longer than mice un-treated or treated with free HPR [NGR-SL-(HPR) vs both control and HPR: P&amp;lt;0.0001]. SL-(HPR) resulted in a statistically improved survival [SL-(HPR) vs both control and HPR: P&amp;lt;0.0001] but with a less extent if compared with that obtained with NGR-SL-(HPR) [SL-(HPR) vs NGR-SL-(HPR): P=0.01]. Double staining of primary tumor sections with TUNEL and with antibodies against the NB specific antigen, NB84a, and against either factor VIII or smooth muscle antigens evidenced that HPR reduced NB growth through both antitumor and angiostatic effects, mainly when delivered by NGR-SL-(HPR). These results were confirmed by a marked reduction of intratumoral vessel counts together with a down-modulation of both matrix metalloproteinases MMP2 and MMP9. The use of this novel targeted delivery system for the apoptotic and anti-angiogenic drug, fenretinide, could be considered as an adjuvant tool in the future treatment of NB patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A130.