Konstantina Karidi

Effective incorporation of insulin in mucus permeating self-nanoemulsifying drug delivery systems

The development of a novel, mucus permeating SNEDDS formulation for oral insulin delivery containing a hydrophobic ion pair of insulin/dimyristoyl phosphatidylglycerol (INS/DMPG) is presented. Three oil/surfactant/cosurfactant combinations and 27 weight ratios of oil, surfactant and cosurfactant for each combination were evaluated with the aid of ternary phase diagrams, for the incorporation of the protein/phospholipid complex. The developed formulation was characterized by an average droplet diameter of 30-45 nm. Depending on the initial protein concentration, the loading of insulin in SNEDDS varied between 0.27 and 1.13 wt%. The therapeutic protein was found to be efficiently protected from enzymatic degradation by intestinal enzymes (i.e., trypsin, α-chymotrypsin). The SNEDDS formulation exhibited increased mucus permeability and did not appear to be affected by ionic strength. The incorporation of INS/DMPG in SNEDDS prevented an initial burst release of insulin. INS/DMPG loaded SNEDDS were found to be non-cytotoxic up to a concentration of 2mg/ml. According to the reported results, the incorporation of the hydrophobic ion pair of INS/DMPG in SNEDDS could be regarded as a promising strategy for the oral delivery of insulin.

PLGA nanoparticles modified with a TNFα mimicking peptide, soluble Leishmania antigens and MPLA induce T cell priming in vitro via dendritic cell functional differentiation

Poly(lactide-co-glycolide) nanoparticles (PLGA NPs) represent a new approach for vaccine delivery due to their ability to be taken up by phagocytes and to activate immune responses. In the present study PLGA NPs were surface-modified with a TNFα mimicking peptide, and encapsulated soluble Leishmania antigens (sLiAg) and MPLA adjuvant. The synthesized PLGA NPs exhibited low cytotoxicity levels, while surface-modified NPs were more efficiently taken up by dendritic cells (DCs). The prepared nanoformulations induced maturation and functional differentiation of DCs by elevating co-stimulatory molecule levels and stimulating IL-12 and IL-10 production. Sensitized DCs promoted T cell priming, characterized by the development of mixed T cell subsets differentiation expressing Th lineage-specific transcriptional factors and cytokine genes. Moreover, PLGA NPs were biocompatible, while they were located in lymphoid organs and taken up by phagocytic cells. Our results suggest that surface-modified PLGA NPs encapsulating sLiAg and MPLA could be considered as an effective vaccine candidate against leishmaniasis.

Identification of BALB/c Immune Markers Correlated with a Partial Protection to Leishmania infantum after Vaccination with a Rationally Designed Multi-epitope Cysteine Protease A Peptide-Based Nanovaccine

Background Through their increased potential to be engaged and processed by dendritic cells (DCs), nanovaccines consisting of Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with both antigenic moieties and adjuvants are attractive candidates for triggering specific defense mechanisms against intracellular pathogens. The aim of the present study was to evaluate the immunogenicity and prophylactic potential of a rationally designed multi-epitope peptide of Leishmania Cysteine Protease A (CPA160-189) co-encapsulated with Monophosphoryl lipid A (MPLA) in PLGA NPs against L. infantum in BALB/c mice and identify immune markers correlated with protective responses. Methodology/Principal Findings The DCs phenotypic and functional features exposed to soluble (CPA160-189, CPA160-189+MPLA) or encapsulated in PLGA NPs forms of peptide and adjuvant (PLGA-MPLA, PLGA-CPA160-189, PLGA-CPA160-189+MPLA) was firstly determined using BALB/c bone marrow-derived DCs. The most potent signatures of DCs maturation were obtained with the PLGA-CPA160-189+MPLA NPs. Subcutaneous administration of PLGA-CPA160-189+MPLA NPs in BALB/c mice induced specific anti-CPA160-189 cellular and humoral immune responses characterized by T cells producing high amounts of IL-2, IFN-γ and TNFα and IgG1/IgG2a antibodies. When these mice were challenged with 2x107 stationary phase L. infantum promastigotes, they displayed significant reduced hepatic (48%) and splenic (90%) parasite load at 1 month post-challenge. This protective phenotype was accompanied by a strong spleen lymphoproliferative response and high levels of IL-2, IFN-γ and TNFα versus low IL-4 and IL-10 secretion. Although, at 4 months post-challenge, the reduced parasite load was preserved in the liver (61%), an increase was detected in the spleen (30%), indicating a partial vaccine-induced protection. Conclusions/Significance This study provide a basis for the development of peptide-based nanovaccines against leishmaniasis, since it reveals that vaccination with well-defined Leishmania MHC-restricted epitopes extracted from various immunogenic proteins co-encapsulated with the proper adjuvant or/and phlebotomine fly saliva multi-epitope peptides into clinically compatible PLGA NPs could be a promising approach for the induction of a strong and sustainable protective immunity.

Lipase activity in Thermus thermophilus HB8: Purification and characterization of the extracellular enzyme

In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity, while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca2+-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract were electrophoresed under denatured and renatured conditions, using α-naphthyl acetate and Fast Blue RR, major bands at 56 kDa or 62 and 32 kDa were observed, respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80°C and pH 7.0, while the T1/2 was 1 h at 100°C. The Km of the partial purified enzyme was 1 mM and the Vmax was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca2+ and Hg2+ stimulated lipase activity, whereas Zn2+, Co2+, or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications.

A Comprehensive Kinetic Investigation of the Ring Opening of L,L-Lactide in the Presence of Multifunctional Polyalcohols

Abstract In the present study, a comprehensive theoretical kinetic investigation of the ring opening polymerization of L,L-lactide in the presence of stannous octoate Sn(Oct)2 as initiator for the synthesis of high molecular weight polylactide is carried out using both the method of moments and the stochastic Monte Carlo (MC) numerical methods. Via the implementation of the MC method, the dynamic evolution of a series of molecular properties (i.e., average molecular weights and molecular weight distributions) of the produced PLLA is accurately predicted. In addition, the effect of different polymerization conditions, such as the polymerization temperature and the monomer to initiator molar ratio on the final polymer properties is investigated. The developed stochastic model is also implemented to assess the effect of the presence of different multifunctional co-initiators (i.e., polyols with a different number of hydroxyl groups: 1,4-butanediol, glycerol, di(trimethylolpropane) (DTMP) and polyglycidol (PG) as co-initiators) in the reacting mixture, on the molecular weight distribution of the produced branched polylactides. The validity of the comprehensive kinetic model and the accuracy of the produced simulation results are verified via a direct comparison with available experimental data.