Cationic immune stimulating complexes containing soluble leishmania antigens: preparation, characterization and in vivo immune response evaluation

Background: Cationic immune stimulating complexes [PLUSCOMs] are particulate antigen delivery systems. PLUSCOMs consist of cationic immunostimulatory complexes [ISCOMs] derivatives and are able to elicit in vivo T cell responses against an antigen

Objective: To evaluate the effects of PLUSCOMs containing Leishmania major antigens [SLA] on the type of immune response generated in the murine model of leishmaniasis

Methods: PLUSCOMs consisting of 1, 2-dioleoyl-3-trimethylammonium-propane [DOTAP] were used as antigen delivery system/immunoadjuvants for soluble SLA. BALB/c mice were immunized subcutaneously, three times in 2-week intervals. Footpads swellings at the site of challenge and parasite loads were assessed as a measure of protection. The immune responses were also evaluated by determination of IgG subclasses and the level of IFN- gamma and IL-4 in cultured splenocytes

Results: There was no significant difference [p<0.05] between the sizes of lesions in mice immunized with different formulations. Also, there was no significant difference in the number of parasites in the footpad or spleen of all groups compared with the control group. The highest level of IFN- gamma secretion was observed in the splenocytes of mice immunized with PLUSCOM/SLA [p<0.001] and lower amounts of IL-4 was observed in PLUSCOM group [p<0.001] as compared to negative control

Conclusion: Our results indicated that SLA in different formulations generated an immune response with mixed Th1/Th2 response that was not protective enough despite the activation of CD4+ T cells with secreting IFN- gamma in groups which received PLUSCOM with antigen

Optimization of a method to prepare liposomes containing HER2/Neu-derived peptide as a vaccine delivery system for breast cancer

The purpose of this study was to optimize a method for the encapsulation of P5 peptide, a new designed peptide containing MHC class I epitopes from rat HER2/neu protein, into liposomes as an approach for breast cancer vaccine formulation. The efficiency of liposomal encapsulation of peptides is generally low and development of an optimized method to increase encapsulation efficiency is a big challenge. In this study, P5 peptide was encapsulated into liposomes using the following three different methods based on film-hydration procedure. In method A, the lipid film containing P5 was hydrated using buffer and then extruded to 100 nm using polycarbonate filter. In method B all the steps were the same as method A, except that the lipid film was hydrated in buffer containing 10% [v/v] of DMSO and P5 peptide. In method C, P5 peptide was added to preformed liposomes [40 mM] in the presence of ethanol [30% v/v] and incubated at 25 [degree sign]C for 1h. The highest peptide encapsulation efficiency was achieved using method C [44%]. The presence of P5 peptide in purified liposomes was also confirmed using SDS- PAGE analysis. Investigation on the effects of procedure parameters of method C on encapsulation efficiency demonstrated that method is an optimized procedure for encapsulating P5 peptide. Maximal recovery from liposomes for the accurate quantification of peptide was discovered using acidified isopropanol at 1:2 of sample to solvent ratio [v/v]. In conclusion, the optimal methods of encapsulation and peptide content determination in liposomes can accelerate the development of liposomal vaccine formulations

Evaluation of infection course in mice induced by L. major in presence of positively charged liposomes containing CpG ODN

An inoculation of virulent Leishmania major is known as leishmanization [LZ] which is proven to be the most effective control measure against Cutaneous Leishmaniasis [CL]. However, using LZ is restricted due to various side effects such as uncontrolled lesion development. In the present research, the efficacy of cationic nanoliposomes containing CpG oligodeoxynucleotides [CpG ODN] as an improved adjuvant delivery system was studied to diminish the lesion development and infection course of L. major after inoculation into the mice. BALB/c mice were inoculated subcutaneously [SC] with L. major plus empty DSPC, DSPC [CpG ODN], DSPC [Non CpG ODN], empty DMPC, DMPC [CpG ODN], DMPC [Non CpG ODN] or HEPES buffer. The results showed that group of mice received DMPC [CpG ODN] nanoliposomes developed a significantly smaller lesion and showed minimum number of L. major in the spleen and draining lymph nodes. In addition, using DMPC [CpG ODN] liposomes resulted in a Th1 type of immune response with a preponderance of IgG2a isotype which is concurrent with the production of DMPC [CpG] induced IFN-gamma in the spleen of the mice. Taken together, the results suggested that immune modulation using DMPC [CpG ODN] nanoliposomes might be a practical approach to improve the safety of LZ

efficacy of isotretinoin-loaded solid lipid nanoparticles in comparison to Isotrex on acne treatment

Topical retinoids are considered as the first line therapy in the treatment of acne vulgaris, but they are associated with cutaneous irritation. In this study, isotretinoin-loaded solid lipid nanoparticles [IT-SLN] were prepared to treat the mild to moderate acne. Also using IT-SLN would minimize IT adverse effects in comparison to commercial product, Isotrex. This study was conducted to prepare and characterize IT-SLN and assessing the efficiency of IT-SLN comparing to Isotrex acne. IT-SLN was prepared using hot high pressure homogenization method. IT-SLN contained 0.05% IT in 5% of lipid phase [Glyceryl monostearate- GMS] and tween 80 [2.5% w/v] was used as surfactant in the aqueous phase. IT-SLN was characterized by particle size analyzing, differential scanning calorimetry and transmission electron microscopy. Encapsulation efficacy was also obtained using spectrophotometry. The efficacy of IT-SLN was evaluated in a randomized, single-blind, parallel-group study and compared with Isotrex. Forty patients encountered in the study and divided in two groups. Treatment regimen was once-nightly topical administration accompanied with topical administration of clindamycin 2% solution twice a day for 8 weeks. The particle size of IT-SLN was around 60 nm with PDI of 0.4 and zeta potential was about -40 mV. Encapsulation efficacy of IT in SLN in crystalline form was 84 +/- 0.21%. IT-SLN produced significantly better treatment than Isotrex in both non-inflammatory and inflammatory lesions according to its recovery percent after 8 weeks. Also IT-SLN gained better global assessment scores. Our results showed that IT-SLN had higher efficacy than Isotrex to clear non-inflammatory and inflammatory lesions

effect of cationic liposomes encapsulating pcDNA3.1+PA plasmids on humoral immune response in mice

DNA vaccines are third generation vaccines which have made promises to combat infectious diseases. Cationic liposomes are used as effective delivery systems for DNA vaccines to generate stronger immunity. Encapsulation of pcDNA3.1+PA plasmid, encoding protective antigen [PA] of Bacillus anthracis [B. anthracis] into cationic liposomes, and evaluation of its effect on specific humoral specific immunity against PA were aimed. The liposomes containing pcDNA3.1+PA plasmids were prepared with phosphatidylcholine [PC], dioleoyl phosphatidylethanolamine [DOPE] and 1,2-dioleoyl-3-trimethylammonium-propane [DOTAP] using dehydration-rehydration method. BALB/c mice were immunized by intramuscular [IM] injection to investigate the immunogenicity of the formulations. The resulting specific antibodies against PA, total IgG, IgG1, IgG2a and IgG2b isotypes, were evaluated by enzyme linked immunosorbent assay [ELISA] method. A higher concentration of specific IgG against PA was found in sera of a group immunized with the encapsulated plasmid compared with the naked plasmid alone. This difference was significant for IgG1 isotype

Optimization of Anti-Rh D immunoglobulin stability in the lyophilization processes

Anti-Rh D IgG is used for the prevention of anti-D antibody production in Rh- individuals who have been exposed to Rh+ red blood cells. The stability of IgG preparations as a solution is low, with a shelf life of a year or more. Formulation of anti-Rh D IgG as a lyophilized preparation would decrease its degradation rate and increases its shelf life. The objective of this study was to formulate the anti-Rh D as a lyophilized preparation using different formulations and optimize the lyophilization processes. The effect of various formulations on the stability of anti-Rh D was evaluated using accelerated stability test. In this method the amount of transmittance [T%] at 585 nm for the lyophilized preparations had inverse relationship with aggregation of anti-Rh D. To improve stability, the most stable formulation was selected and different concentrations of sucrose in the presence of sodium-potassium phosphate buffer 25 mM pH 7.5. Then, the bioactivity was determined, using the ELAT test and also, the amount of moisture measured in this formulation. Among different formulations, the one with anti-Rh D 5 mg/ml, tween 80 0.1%, glycine 0.15 M, manitol 7% and sucrose 60 mM in sodium-potassium phosphate buffer 25 mM pH 7.5 was the most stable formulation [P < 0.05]. The result of biological test of ELAT showed that bioactivity of more than 93% meets the requirement set by British Pharmacopoeia. The amount of moisture measured in this formulation was less than 3%. It was concluded that this formulation could be introduced as a candidate for the formulation of anti-Rh D in a lyophilized dosage form

Isolation and in vitro cultivation of human skin keratinocytes and preparation of epidermal sheet

Human keratinocytes have been cultivated in vitro using different methods. Using small size skin, large epidermal sheets have been produced. These epidermal sheets have been used for the treatment of injured epidermis, especially burn and other skin clinical disorders, and also for research purposes. The objective of this study was isolation and in vitro cultivation of human skin keratinocytes and preparation of epidermal sheet without using mouse 3T3 fibroblast feeder layer. Small size human skin with a relative thickness was divided to small pieces and the epidermis was separated from dermis using warm trypsin. Then keratinocytes were isolated from epidermis and cultured with high density in DMEM containing FBS and other supplements like cholera toxin and epidermal growth factor. The resulting epidermal sheet was separated using dispase grade II and transferred to vaseline gauze. Histological studies were also carried out on the epidermal sheet. Keratinocytes grew as multiple colonies and produced a thin confluent epidermal sheet consisted of a few layers of cells. With time, number of cell layers was increased and a thicker epidermal sheet was produced. Fibroblasts, which were present in the original cell suspension, grew in the culture when the growth of keratinocyte was slow or when a low density of keratinocytes was used. The histological examination of epidermal sheet showed the presence of germinative basal cells, suprabasal cells with relative differentiation and also melanocytes. The dimension of epidermal sheet was decreased when it was separated from tissue culture flask using dispase. Cultured epidermal autografts have several clinical and investigative indications. Even though the cultivation of keratinocytes with high density is easy, however, the surface area of resulting epidermal sheet is limited and its subculture is difficult. In this method there is the risk of growth of dermal fibroblasts. To produce thicker epidermal sheet with more cellular layers, it is better to separate it within two to three weeks after cultivation