Preparation and characterization of solid lipid nanoparticles containing zataria multiflora boiss essential oil by high-shear homogenization and ultrasound method

Background: One of the new methods to overcome problems with using essential oils [such as instability, evaporation and decomposition in the environmental conditions[ in pharmaceutical, food and agricultural industries is use of Solid Lipid Nanoparticles [SLNs] carrier systems

Objective: Preparation of SLNs containing the Zataria multifloni Boiss essential oil. Methods: In this regard, an experiment was performed on the preparation of SLNs containing the Zataria multiflora Boiss essential oil at Nanotechnology Research Center of School of Pharmacy, Mashhad University of Medical Sciences. SLNs containing Zataria multiflora Boiss essential oil was prepared using high tensile pressure homogenization and ultrasound

Components of SLNs include lipids, Zataria multiflora Boiss essential in fat phase and poloxamers 188 in the aqueous phase

The mean particle size and zeta potential, essential oil encapsulation percentage and thermal analysis were measured using particle size analysis instruments, gas chromatography and differential scanning calorimetry [DSC], respectively. Moreover, an electron microscope was used for imaging of SLNs

Results: The results showed that particle size, polydispersity index and zeta potential of the above formulations were respectively about 486 nm, 0.296, -27.2 mv. The results obtained from transmission electron microscopy [TEM] showed particle size less than 300 nm and particles were spherical

Thermal analysis by DSC, confirmed the presence of solid particles in the prepared SLN. Also, the essential oil encapsulation percentage was 95.2 percent. Stability studies of particle size and zeta in four months showed SLNs containing essential oils had relatively good stability

Conclusion: In general, the results of the present research showed that SLNs composed of stearic acid, was good carriers for Zataria multifloni Boiss essential oil

Nanolipoparticles-mediated MDR1 siRNA delivery: preparation, characterization and cellular uptake

Lipid-based nanoparticles [NLP] are PEGylated carriers composed of lipids and encapsulated nucleic acids with a diameter less than 100 nm. The presence of PEG in the NLP formulation improves the particle pharmacokinetic behavior. The purpose of this study was to prepare and characterize NLPs containing MDR1 siRNA and evaluate their cytotoxicity and cellular uptake. MDR1 siRNA could be used in multidrug resistance reversal in cancer therapy. siRNAs were encapsulated into NLPs consisted of mPEG-DSPE/DOTAP/DOPE [10:50:40 molar ratio] by the detergent dialysis method. The particle diameters of NLPs and their surface charge were measured using dynamic light scattering. siRNA encapsulation efficiency was determined by an indirect method via filtration and free siRNA concentration determination. NLPs cytotoxicity was investigated by MTT assay. The ability of NLPs for siRNA delivery checked in two human cell lines [MCF-7/ADR and EPP85-181/RDB] by fluorescence microscopy and compared with oligofectamine. NLPs containing MDR1 siRNA were prepared with the stable size of 80-90 nm and the zeta potential near to neutral. The siRNA encapsulation efficacy was more than 80%. These properties are suitable for in vivo siRNA delivery. NLPs cytotoxicity studies demonstrated they were non-toxic at the doses used. NLPs improved siRNA localization in both cell lines. NLPs containing MDR1 siRNA can be a good candidate for in vivo siRNA delivery studies

Seroprevalence of Neospora caninum infection in dairy cattle in Tabriz, northwest Iran

The aim of this study was to determine the seroprevalence of antibody to Neospora caninum in healthy and aborted dairy cattle in Tabriz, capital of East-Azarbaijan in northwest of Iran. In this cross-sectional study serum samples were collected from 266 healthy and aborted Holestein-Feriesisnc cows from September 2008 to August 2009. The sera were analyzed to detect of antibody against N. caninum using the commercially ELISA kit. Seroprevalence of antibody to N. caninum was 10.5% in Tabriz dairy cattle. Also the abortion rate in all cattle sampled was 33.6% but percentage of seropositive aborted cattle was 18.4%. Neosporosis could be one of the possible causes of abortion in dairy cattle in Tabriz and regarding the distribution in dogs as definitive host for the parasite, further studies in dog and cattle are recommended

role of liposomal CpG ODN on the course of L major infection in BALB/C mice

Historically, leishmanization is the most effective protective measure against Cutaneous Leishmaniasis [CL], CL lesion induced by leishmanization sometimes takes a long time to heal. Manipulation of leishmanization inoculums needed to induce a mild and acceptable CL lesion. The aim of this study was to explore if liposomal form of CpG ODN [Cytosin phosphate Guanin Oligodeoxynu-cleotides] mixed with Leishmania major would induce a milder lesion size in Balb/c mice. Methods: This study was performed in Biotechnology Research Center, Mashhad, and Center for Research and Training in Skin Diseases and Leprosy, Tehran, Iran during 2008-2009. Different groups of BALB/c mice were subcutaneously [SC] inoculated with L. major mixed with liposomal form of CpG ODN, or L. major plus free CpG ODN, or L. major mixed with empty liposomes or L. major in PBS. The lesion onset and the size of lesion were recorded; the death rate was also monitored. Footpad thickness was significantly [P<0.01] smaller, death rate was also significantly [P<0.05] lower in the mice received L. major mixed with liposomal CpG ODN or free CpG ODN than control groups received L. major in PBS or L. major plus liposomes, also mice which received L. major mixed with CpG ODN in soluble form showed a significantly [P <0.001] smaller lesion size than control groups. CpG ODN seems to be an appropriate immunopotentiator mixed with Leishmania stabi-late in leishmanization

Selective inhibitory effect of adenosine A1 receptor agonists on the proliferation of human tumor cell lines

In this study, the effects of three structural analogues of adenosine upon proliferation of human tumor cells were investigated. Previous research showed a cytotoxic effect of adenosine via A3 receptor and A[1] receptor and sometimes this effect was receptor independent. The researches showed a differential cytotoxic effect of adenosine and its A[3] agonists on cancerous cells, while other studies demonstrated tumor promoting effect of adenosine and its A[1] agonists. The purpose of the present study was the evaluation of the possible selective anti-tumor effect of A1 receptor agonists on cancerous cells. The substances of N6-cyclohexyl-adenosine [CHA, A[1] agonist], R-isomer of N6-phenylisopropyladenosine [R-PIA, A[1] agonist] and N5-ethylcarboxamido-adenosine [NECA, adenosine A[1]-A[2] non-specific agonist] were tested for their anti-proliferative effect using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT] assay method. Hep G2, Hep2, CACO2, ACHN and L929 cell lines were used in this assay. CHA inhibited cell proliferation in three cell lines [in concentration of 5-50 micro M] and R-isomer of R-PIA in one cell line [in concentration of 10-50 micro M]. These effects were inhibited partially by addition of 1,3-Dipropyl-8-cyclopentylxanthine [A1 antagonist]. The NECA analogue had no inhibitory effect on the cell proliferations. All of the substances had no cytotoxic effect on L929 cells [mouse connective tissue fibroblast cell line]. CHA and R-PIA had inhibitory effect on the proliferation of human tumor cell lines partially via A1 receptor, while they didn't show such effect on fibroblast cells. These results suggest that A[1] adenosine receptor agonists have a good potential of specific anti-tumor activity

Preparation, characterization and mucoadhesive evaluation of chitosan coated liposomes containing cyclosporine A

The aim of this study was to prepare and characterize chitosan coated liposomes containing cyclosporine A [CyA]. For this purpose, negatively charged liposomes containing CyA were prepared by solvent evaporation method. Liposomes were then added dropwise to chitosan solution [0.25% w/w] for coating. Morphology, mean size and encapsulation efficiency of chitosan coated liposomes were evaluated. To assess the mucoadhesive properties of this drug delivery system, percent of mucin adsorption onto the surface of coated liposomes was determined. The in vitro immunosuppressive effects of CyA encapsulated in the formulated liposomes were also determined on human T-cells by MTT [3- [4, 5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide] test. Liposomes were multilamellar vesicles [MLVs]. Mean diameter of chitosan coated liposomes was 2.76 micro m and zeta potential of them was positive [45.3 mV]. Encapsulation efficiency of coated liposomes was 86.11% +/- 2.36 and they were stable during two months. The average of IC50 or the half maximal inhibitory concentration for MLV liposomes was 3.08 2-10 M. According to the mucin adsorption results, this particulate system showed suitable mucoadhesive properties. From these results, it was concluded that the surface modification of liposomes by chitosan coating could increase the prospects of their usefulness as oral drug delivery systems for CyA

[Immune response evaluation against autoclaved leishmania major ncapsulated in liposome with different Tm in murine model]

Leishmaniasis is a parasitic disease which is caused by different species of Leishmania. Protozoa of the Leishmania species cause leishmaniasis. The protective immunity against leishmaniasis is the cell-mediated immunity [CMI]. Autoclaved Leishmania major [ALM] has been used as vaccine in clinical trial; however, its efficacy has been low. Liposomes are microscopic vesicles consisting of phospholipid bilayers which enclose aqueous compartments and are used as an immunoadjuvant. The aim of this study was to evaluate the immune response of ALM entrapped in liposome with different phase transition temperature [Tm] in BALB/c mice. Liposomes containing ALM were prepared as dehydration-rehydration vesicles [DRV] and composed of dipalmitoylphosphatidylcholine [DPPC, Tm 41°C] and cholesterol or phosphatidylcholine [PC, Tm -10°C] and cholesterol in a molar ratio of 7:2 DRV-DPPC-Chol- ALM [180 microg], DRV-PC-Chol-ALM [180 microg], ALM alone [180 microg], PBS, and a control empty liposome were injected separately subcutaneously [SC] in female BALB/c mice [10 per group], 3 times in three weeks interval. The mice were tested for DTH with freeze-thawed L. major promastigotes [1.5 × 10[7]] SC to the left footpad and PBS to the right footpad for control at 3 weeks after the last booster. The footpad swellings were measured after 24, 48 and 72 hours. Blood samples were collected before second and third vaccination and also before DTH test to titrate the anti-Leishmania antibodies [IgG total, IgG1 and IgG2a] by ELISA method. The sizes of liposome were between 0.5-3 microm. Percent encapsulation of ALM in DRV-DPPC- Chol-ALM and DRV-PC-Chol-ALM was 46 and 43 respectively. The results of DTH showed that footpad swelling in DRV-DPPC-Chol-ALM and DRV-PC-Chol-ALM is significantly more than control groups. The results of ELISA showed that the titer of IgG total in PBS, control empty liposomes and ALM alone is low and it is significantly less than DRV-DPPC- Chol-ALM and DRV-PC-Chol-ALM. There were no differences in the titer of IgG1 in the different groups; however, the titer of IgG2a was significantly higher in DRV-DPPC-Chol- ALM compared to DRV-PC-Chol-ALM and control groups. The results indicated that liposome prepared by higher Tm phospholipid seems to be a suitable immunoadjuvant for ALM in to improve the CMI

Preparation of biodegradable microspheres encapsulated with Cyclosporine A and evaluation of some factors affecting microsphene properties

Poly [D, L lactide-co-glycolide][PLGA] is a biocompatible-biodegradable polymer used as a drug delivery carrier. Also, Cyclosporine A [CyA] is the immunosuppressant that its commercial dosage forms have some disadvantages such as low bioavailability, kidney, liver and neural toxicity and variation of blood concentration after adminstration. The aim of this study was to prepare microspheres containing CyA by using different grades of PLGA.Microspheres were prepared by Solvent Evaporation Method using three grades of PLGA including PLGA [50:50], PLGA [65:35] and PLGA [85:15]. Various characteristics of microspheres such as morphology, size and encapsulation efficiency were evaluated. The mean diameter and particle size distribution of microspheres were measured by particle size analyzer. Release profile of CyA from microspheres was also studied. Complementary studies were carried out by IR [Infrared Spectroscopy] and DSC [Differential Scanning Calorimetery] to evaluate the drug and polymer interaction.SEM studies showed that microspheres were spherical in shape and the small CyA was loaded as islands on the surface of microspheres. Microsphere size was varied between 1 to 25 um with microspheres of PLGA [50:50] having the minimum size. Encapsulation efficiency was varied from 75% to 90% and encapsulation efficiency of PLGA [50:50] microspheres was different compared to other grades significantly. Profile of release was biphasic, with an initial rapid phase during first 5 days followed by a continuous and slower rate thereafter. Microspheres made from grades 50:50 and 85:15 showed the highest and the lowest amount of release, respectively. IR spectra for drug, polymer and microsphere did not indicate any chemical interaction between the components of microsphere and DSC thermograms suggested that CyA is present in its amorphous state.In conclusion, in this study suitable microspheres especially with PLGA [50:50] were prepared which allow the controlled release of CyA over a prolonged period of time and could be used as a slow release particulate drug deliverysystem and according to microsphere size intact absorption of microspheres after oral administration is expected

Evaluation of in vitro methods for the determination of sun protection factor of the homosalte standard sunscreen lotion

Exposure to the sunlight can have harmful effects on the human skin; such as, skincancers, hyperpigmentation and photoaging. Therefore, it is essential to use sunscreen productsto prevent these adverse effects. The photoprotection provided by a sunscreen product isassessed in terms of its sun protection factor [SPF]. There are in vivo and in vitro methods forthe SPF determination. In vivo methods have some problems; therefore, in vitro methods areused more often. In this study we compared 3 in vitro methods for the determination ofSPF of homosalte PDA standard sunscreen lotion. The SPF of the homosalte standard lotion by the method of solution in ethanol was8.23 +/- 0.13, by the method of solution in methanol method was 6.17 +/- 1.02 and by transpore tapemethod was 5.88 +/- 0.12. According to these results and statistical tests, transpore tape was the best method

[Formulation of topical liposomes encapsulated with triamcinolone and comparison of their anti-inflammatory effects with available conventional topical ointment in mice]

Triamcinolone is a steroidal anti-inflammatory drug widely used in the treatment of skin diseases. Liposomes are microscopic vesicles that composed of lipid bilayers enclosing aqueous compartments. In this study, different liposomal formulations encapsulated with triamcinolone were prepared and their anti-inflammatory effects were compared with a conventional topical ointment. In this study liposomes containing 0.1% triamcinolone were prepared by the fusion method using lecithin, cholesterol and penetration enhancers. Encapsulation efficiency was determined by UV spectrophotometry. Liposome size was examined by optical microscopy. The anti-inflammatory effect of liposomal formulations was evaluated by "xylene-induced ear edema" method in mice and then results were compared with a conventional topical ointment. Among different formulations only two formulations were stable and suitable regards to encapsulation efficiency, size, and the lack of triamcinolone precipitation. The first formulation did not have penetration enhancer and the second one contained a penetration enhancer. Liposome size was varied from 2 to 5 micron, and encapsulation efficiency in the first and second formulation was 80.33 +/- 3.51% and 90.50 +/- 2.78%, respectively. In vivo study showed that both conventional ointment and liposomal triamcinolone decrease ear edema compared to the control liposome [p<0.01]. The percent of edema inhibition [Mean +/- SEM] in comparison with control was 44% +/- 6.0, 71% +/- 6.4 and 78% +/- 5.4 for conventional ointment, first and second liposomal formulations respectively. The anti-inflammatory of liposomal formulations were significantly more than conventional triamcinolone ointment [p<0.05]. Results show that liposomal triamcinolone have more anti-inflammatory effect than conventional triamcinolone ointment. Thus, to provide the same effect as conventional triamcinolone ointment, the lower concentration of triamcinolone in liposome formulation is needed, this in turn will cause less side effects

Immune response evaluation against leishmaniasis using liposomes containing rLmSTI1 in murine model

Leishmaniasis is a parasitic disease spread by the bite of infected sand flies. Protozoa of the Leishmania species cause leishmaniasis. The protective immunity against leishmaniasis is the cell-mediated immunity [CMI]. LmSTI1 is a candidate for the development of vaccine against cutaneous leishmaniasis [CL]. Liposomes are microscopic vesicles consisting of phospholipid bilayers which enclose aqueous compartments and are used as an immunoadjuvant. The aim of this study was to formulate liposome preparations containing recombinant rLmSTI1 to induce Th1 response in BALB/c mice against infection with L. major. Liposomes containing rLmSTI1 were prepared as dehydration-rehydration vesicles [DRV] and composed of distearoylphosphatidylcholine [DSPC] and cholesterol [CHOL] in a molar ratio of 2:1 [DSPC/CHOL-rLmSTI1]. The average size of liposome formulations was 1.1micro m checked by light microscope and particle size analyzer. DSPC/CHOL-rLmSTI1 [2micro g]; soluble rLmSTI1 [2micro g]; PBS, and a control empty liposome were injected separately subcutaneously [SC] in female BALB/c mice [10 per group], 3 times in three week intervals. The mice were challenged with L. major promastigotes [1.5 X 10[6]] SC to the left footpad and PBS to the right footpad for control at 3 weeks after the last booster. The footpad swellings were measured weekly for 12 weeks. Blood samples were collected on the day before and 12 weeks after challenge to titrate the anti-Leishmania antibodies [IgG total, IgG1 and IgG2a] by ELISA method. The parasite burden in spleen was determined at 15weeks after challenge. The results showed that in the group that received DSPC/CHOL-rLmSTI1, footpad thickness was significantly less; IgG2a titer was higher with very few parasites in the spleen compared to the other groups. The results indicated that encapsulation of rLmSTI1 in liposome seems to be a suitable tool to improve the CMI and rate of protection in murine model of leishmaniasis

Evaluation of Leishmanicidal effect of Euphorbia myrisinites extract by in vitro antileishmanial assay using promastigote of Leishmania.major

In this study, the antileishmanial effect of different extracts of Euphorbia myrsinites aerial part was evaluated on promastigotes of Leishmania major in vitro. Dried and ground aerial parts of the plant were extracted using either maceration in 80% ethanol or soxhlet in methanol. Then 5 different concentrations of each extract, one positive control, one negative control, and one solvent control were prepared and placed in a 24-well plate containing 40, 000 parasites/well. The extract concentrations were 0.06, 0.12, 0.25, 0.5 and 1 mg/ml. Amphotricin B [0.5 mg/ml] was used as positive control while negative control contained only culture medium. The plate was incubated at 25°C for six days, and the amount of parasites in each well was determined on days 2, 4, and 6 of experiment microscopically using Neubar chamber. It was observed that amphotricin B and both macerated and soxhlet extracts at concentration of 1 mg/ml killed all parasites. Lower doses exhibited a dose-dependent antileishmanial activity. The ECso for macerated and soxhlet extracts in DMSO was between 0.5 and 0.25 mg/ml. The control solvents had no significant effect on the L. major promastigotes. These results indicated that both macerated and soxhlet extracts of E. myrsinites have favorable leishmanicidal activity