Formulation, Characterization, and Optimization of a Topical Gel Containing Tranexamic Acid to Prevent Superficial Bleeding: In Vivo and In Vitro Evaluations.

Objectives: Tranexamic acid (TXA) is used systemically to stop bleeding, but it can lead to thromboembolism. Trials have revealed the efficacy of topical TXA on local hemorrhages. However, there is a need for an efficient delivery system that can keep the drug at the site of action. Materials and Methods: To develop a gel containing TXA (3%) optimized in terms of viscosity and dispersibility, the central composite design based on two factors-three levels [carbopol 940 and hydroxypropyl methylcellulose (HPMC), 1-1.5% and 1-2%, respectively] was applied. The spreadability and viscosity were assessed using glass slide and rheometer, respectively. To confirm the compatibility of TXA with the gel, fourier transform-infrared (FTIR) spectroscopy was performed. Drug content uniformity was analyzed by a spectroscopy method. An ex vivo mice model using Franz cells was applied to evaluate the permeation of TXA through the skin. To investigate the effect of topical TXA gel on bleeding time, IVY human method was performed. Results: HPMC/carbopol 940 (1:1, w/w) gel showed the highest quality in terms of viscosity and dispersibility (3.982 ± 17.6 and 6.052 ± 3.562, respectively). FTIR absorption spectrum showed that all the TXA index peaks appeared without displacement. The complete-encapsulated TXA content was uniformly dispersed throughout the gel. In vitro TXA cumulative release reached 90% in 4 h. The bleeding time determined in vivo for TXA gel was significantly lower than that for TXA solution and control. Conclusion: The results confirm the importance of further studies on this formulation as a potential medication to stop acute superficial bleeding.

Sphingomyelin liposome bearing whole Leishmania lysate antigens induce strong Th2 immune response in BALB/c mice.

OBJECTIVES: Whole Leishmania lysate antigens (WLL) has been shown to be effective to tackle leishmaniasis in murine models. Although liposomes can be considered as promising vaccines, the activity of phospholipase-A (PLA) in WLL, breeds difficulties to preparing stable liposomal WLL. One strategy to overcome this shortcoming is to use lipids such as sphingomyelin (SM) which is resistant against PLA. This study aim is formulating stable SM liposomes containing WLL and comparing their adjuvant effects with another first generation vaccine , i.e. solube Leishmania Antigen (SLA) liposomes in BALB/c mice. MATERIALS AND METHODS: BALB/c mice were immunized subcutaneously, three times with 2-week intervals, with Empty-liposome (E-lipo), Particulate WLL, Liposome-WLL, Liposome-SLA and control Buffer, three times every 2-week. Protection was assessed through measuring the swollen footpads and the load of parasites in the spleen. Other factors were used to assess the response of immune system by means of IgG subclasses, IL-4 and IFN-γ levels and intracellular cytokine assay in cultured splenocytes. RESULTS: Although liposomal WLL were stable in terms of physicochemical properties, mice received Liposome-WLL did not reduce footpad swelling. The load of parasites in spleen and levels of IL-4- were also higher compared to other immunized groups. In terms of IgG isotypes, no considerable difference observed in mice received Liposome-WLL or other formulations. CONCLUSION: Liposome-WLL could be a suitable vaccine delivery system when a Th2 response is desired. Also, further studies are warranted to fully understand the role of sphingomyelin in inducing an immune response.

A Phospholipase-A Activity in Soluble Leishmania Antigens Causes Instability of Liposomes.

AIM: This study aimed to investigate the existence of phospholipase-A (PLA) activity in Soluble L. major Antigens (SLA) because of no reports for it so far. Liposomes were used as sensors to evaluate PLA activity. OBJECTIVES: Liposomal SLA consisting of Egg Phosphatidylcholine (EPC) or Sphingomyelin (SM) were prepared by two different methods in different pH or temperatures and characterized by Dynamic Light Scattering (DLS) and Thin Layer Chromatography (TLC). METHODS: Lipid hydrolysis led to the disruption of EPC liposomal SLA in both methods but the Film Method (FM) produced more stable liposomes than the Detergent Removal Method (DRM). RESULT: The preparation of EPC liposomal SLA at pH 6 via FM protected liposomes from hydrolysis to some extent for a short time. EPC liposomes but not SM liposomes were disrupted in the presence of SLA. CONCLUSION: Therefore, a phospholipid without ester bond such as SM should be utilized in liposome formulations containing PLA as an encapsulating protein.

Chitosan Nanoparticles Loaded with Whole and Soluble Leishmania Antigens, and Evaluation of Their Immunogenecity in a Mouse Model of Leishmaniasis.

BACKGROUND: Although there have been numerous attempts to develop vaccines for Leishmaniasis, no vaccine can be found against Leishmania in routine use for an effective global vaccination. It seems that one of the reasons for the low efficacy of such vaccines is the lack of a suitable adjuvant. OBJECTIVE: To evaluate the effects of chitosan nanoparticles containing whole Leishmania lysate antigen (WLL) and soluble leishmania antigens (SLA), a first generation Leishmania vaccine, on the type of immune response generated in BALB/c in a murine model of leishmaniasis. METHODS: The optimum coating ratio between the polymer and antigens was determined according to their physico-chemical properties such as particle size and zeta potential. Chitosan nanoparticles were loaded with antigens via ionic gelation method. BALB/c mice were immunized subcutaneously three times with various nanoparticulate and free antigens with 2-week intervals. RESULTS: There was no significant (P > 0.05) difference concerning the footpad thickness of mice immunized with nanoparticulate formulations containing either SLA or WLL during the experiment period; these formulations induced a strong mixed Th1/Th2 type immune response characterized by the production of IFN-γ and IL-4, and high levels of IgG2a IgG1 anti-Leishmania antibody. CONCLUSION: Nanoparticulate formulations (CHT: SLA and CHT: WLL) are not suitable candidates for preferential induction of a pure Th1-type immune response and immunization against Leishmania infection. However, it might be a good strategy in other infectious diseases where a mixed Th1/Th2 immune response is required.

Coadminstration of L. major amastigote class I nuclease (rLmaCIN) with LPD nanoparticles delays the progression of skin lesion and the L. major dissemination to the spleen in BALB/c mice-based experimental setting.

Human cutaneous leishmaniasis is a disease caused by eukaryotic single-celled Leishmania species, the developmental program of which relies upon blood-feeding adult female sand flies and their dominant mammal blood sources, namely wild rodents in area where human beings exert more or less transient activities. The recourse to model rodents - namely laboratory mice such as C57BL/6 mice - has allowed extracted the immune signatures that account for the healing of the transient cutaneous lesion that develops at the site where Leishmania major promastigotes were delivered. Indeed, if the latter mice are exposed to a second inoculum of L. major promastigotes, no lesion will develop in the secondary skin site remodeled as a niche for a low size intracellular L. major amastigote population. Moreover, IFN-γ dominates over IL-10 in the supernatant of cultures of PBMCs -prepared from blood sampled from human beings who healed from a cutaneous lesion- and incubated with L. major class I Nuclease LmaCIN, a protein highly expressed in the cell-cycling amastigote population which is dominant by macrophages. Altogether, these datasets were strong incentive to promote research aimed to design and monitor efficacy of L. major amastigote protein-based vaccines in pre-clinical settings. Using L. major enzyme class I nuclease (LmaCIN) expressed in the L. major cell-cycling amastigote population hosted by macrophages, BALB/c mice were immunized three times with either rLmaCIN plus LPD nanoparticles (LPD-rLmaCIN), or rLmaCIN-CpG DNA or free rLmaCIN and dextrose. The following parameters: footpad swelling, splenic L. major load, L. major binding IgGs and cytokine profiles of rLmaCIN- reactive T lymphocytes were then compared. Once coadminstered with LPD, rLmaCIN allow BALB/c mice to display delayed onset of skin lesion at the challenge inoculation site and delayed L. major dissemination from the challenged site to the spleen. Thus, the LPD-rLmaCIN is shown to display some promising features out of three formulations inoculated to the BALB/c mouse immunization.

Sphingomyelin Liposomes Containing Soluble Leishmania major antigens Induced Strong Th2 Immune Response in BALB/c Mice.

OBJECTIVE(S): Soluble Leishmania antigens (SLA) provide suitable protection against leishmaniasis in murine model when delivered by an appropriate delivery system. Liposomes have been shown to be suitable vaccine delivery systems against leishmaniasis, however, the phospholipase-A (PLA) activity of SLA is a drawback to prepare a stable liposomal SLA. One strategy to overcome this problem might be using a lipid which is resistant to PLA activity of SLA such as sphingomyelin (SM). The aim of this study was to evaluate the effect of stable SM liposomes containing SLA on the immune response induced against leishmaniasis in BALB/c mice . MATERIALS AND METHODS: BALB/c mice were immunized subcutaneously, three times with 2-week intervals, with SLA, SM-liposome-SLA, empty liposome or buffer. As criteria for protection, footpads swelling at the site of challenge and foot parasite loads were assessed. The immune responses were also evaluated by determination of IgG subtypes and the level of IFN-γ and IL-4 in cultured splenocytes. RESULTS: The group of mice receiving SM-liposome-SLA, showed a significant large footpad swelling, higher parasite burden in foot and higher IL-4 level compared to the group immunized with buffer. In terms of IgG and IgG isotypes, there was no significant difference between the mice receiving SM-liposome-SLA and the mice that received buffer. Moreover, the immune response induced by SM-liposome-SLA showed no significant difference compared with the one caused by SLA alone. CONCLUSION: It is concluded that SM-liposome-SLA is not an appropriate strategy to induce Th1 immune response and protect the mice against Leishmaniasis; however, SM-liposomes could be suitable vaccine delivery systems when a Th2 response is needed.