RESUMO
To unveil the effect of electrolyte concentration, pH and polymer addition on Tween 80 stabilized nanostructured lipid carriers (NLCs, based on dialkyldimethylammonium bromides DxDAB and Na oleate), an in-depth scattering analysis was performed. Dynamic and static light scattering (DLS/SLS) and small-angle neutron scattering (SANS) techniques along with zeta potential studies were exploited to understand the structural evolution and physical stability of NLCs. In these experiments, we varied the salt concentration, pH, and the admixture of Pluronic F127 in order to elucidate their effect on NLC morphologies. In most cases, two populations of different sizes are present which differ by one order of magnitude. The antileprosy drugs (ALD) Rifampicin and Dapsone were encapsulated in NLCs and the vector properties were assessed for a series of DxDAB (where x = 12, 14, 16 and 18) NLCs. The influence of composition on the entrapment and release behavior of NLCs was investigated: The size of NLCs correlates with the release rate of the incorporated drug. The interaction of drug-loaded NLCs with bovine serum albumin was studied to understand the release of ALD in the plasma.
Assuntos
Dapsona/farmacologia , Portadores de Fármacos/química , Hansenostáticos/farmacologia , Nanopartículas/química , Compostos de Amônio Quaternário/química , Rifampina/farmacologia , Animais , Bovinos , Portadores de Fármacos/metabolismo , Liberação Controlada de Fármacos , Cinética , Nanopartículas/metabolismo , Poloxâmero/química , Poloxâmero/metabolismo , Ligação Proteica , Compostos de Amônio Quaternário/metabolismo , Soroalbumina Bovina/metabolismoRESUMO
The presence of anti-BSA antibodies may interfere in serological tests, as ELISA or immunochromatographic assays. BSA is frequently used as a blocking agent or as "inert" carrier of antigens, such as the NT-P-BSA, the semi-synthetic trisaccharide analogue of the PGL-I (phenolic glycolipid-I) antigen from the cell wall of the Mycobacterium leprae. PGL-I was prepared and linked to human serum albumin based in the hypothesis that replacing BSA by a human protein carrier would enhance the performance of leprosy serological tests. A total of 1162 serum samples were tested by ELISA and by the ML Flow rapid test using NT-P-BSA or NT-P-HSA antigens. When grouping leprosy patients as paucibacillary (PB) or multibacillary (MB) according to the Ridley & Jopling classification, ML Flow BSA and ML Flow HSA tests correctly allocated 70.9% and 68.6% of patients in the PB group, and 87% and 81% of patients in the MB group, respectively. Concordant results were found in 82.0% (953/1162) (kappa value=0.637; sd=0.023) of samples between ML Flow tests and 85.7% (996/1162) (kappa value=0.703; sd=0.021) between ELISA tests. ML Flow results were statistically similar and the same was true for ELISA tests using HSA or BSA. However, we noticed a tendency to decreased capacity to detect MB patients and an increased positivity among PB patients, HHC, TB patients and healthy controls by the HSA carrier in both ML Flow and ELISA. The PGL-I serology performed by the ML Flow test with BSA or HSA as antigen carriers can be a useful, friendly auxiliary tool to identify patients with higher bacterial load.
Assuntos
Antígenos de Bactérias/metabolismo , Glicolipídeos/metabolismo , Hanseníase/classificação , Hanseníase/diagnóstico , Mycobacterium leprae/metabolismo , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Antibacterianos/sangue , Carga Bacteriana , Bovinos , Criança , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Reações Falso-Positivas , Feminino , Glicolipídeos/síntese química , Humanos , Imunoglobulina M/metabolismo , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/imunologia , Albumina Sérica/síntese química , Albumina Sérica/metabolismo , Soroalbumina Bovina/síntese química , Soroalbumina Bovina/metabolismo , Adulto JovemRESUMO
BACKGROUND: Silica particles cationized by dioctadecyldimethylammonium bromide (DODAB) bilayer were previously described. This work shows the efficiency of these particulates for antigen adsorption and presentation to the immune system and proves the concept that silica-based cationic bilayers exhibit better performance than alum regarding colloid stability and cellular immune responses for vaccine design. RESULTS: Firstly, the silica/DODAB assembly was characterized at 1 mM NaCl, pH 6.3 or 5 mM Tris.HCl, pH 7.4 and 0.1 mg/ml silica over a range of DODAB concentrations (0.001-1 mM) by means of dynamic light scattering for particle sizing and zeta-potential analysis. 0.05 mM DODAB is enough to produce cationic bilayer-covered particles with good colloid stability. Secondly, conditions for maximal adsorption of bovine serum albumin (BSA) or a recombinant, heat-shock protein from Mycobacterium leprae (18 kDa-hsp) onto DODAB-covered or onto bare silica were determined. At maximal antigen adsorption, cellular immune responses in vivo from delayed-type hypersensitivity reactions determined by foot-pad swelling tests (DTH) and cytokines analysis evidenced the superior performance of the silica/DODAB adjuvant as compared to alum or antigens alone whereas humoral response from IgG in serum was equal to the one elicited by alum as adjuvant. CONCLUSION: Cationized silica is a biocompatible, inexpensive, easily prepared and possibly general immunoadjuvant for antigen presentation which displays higher colloid stability than alum, better performance regarding cellular immune responses and employs very low, micromolar doses of cationic and toxic synthetic lipid.
Assuntos
Adjuvantes Imunológicos/farmacologia , Apresentação de Antígeno , Compostos de Amônio Quaternário/imunologia , Dióxido de Silício/imunologia , Adjuvantes Imunológicos/química , Adsorção , Animais , Formação de Anticorpos , Cátions , Células Cultivadas , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Hipersensibilidade Tardia/imunologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Amônio Quaternário/química , Soroalbumina Bovina/metabolismo , Dióxido de Silício/químicaRESUMO
In this paper, the interaction between florasulam (FU, 2',6',8-trifluoro-5-methoxy [Kragh-Hansen U, Molecular aspects of ligand binding to serum albumin. Pharmacol Rev 33(1):17-53 1981; Carter DC and Ho JX, Structure of serum albumin. Adv Protein Chem 45:153-203 1994; He XM, and Carter DC, Atomic structure and chemistry of human serum albumin. Nature 358(6383):209-215 1992] triazolo [1,5-c]pyrimidine-2-sulfonanilide) and bovine serum albumin (BSA) was investigated by fluorescence, ultraviolet absorption (UV) and Far-UV circular dichroism (CD) spectrometries. A strong fluorescence quenching was observed and the quenching mechanism was considered as static quenching. The binding constant of FU with BSA at 299 and 309 K were obtained as 1.5 x 10(4) and 7.1 x 10(3) l mol(-1), respectively. There was one binding site between FU and BSA. The thermodynamic parameters enthalpy change (DeltaH) and entropy change (DeltaS) were calculated as -57.89 kJ mol(-1) and -113.6 J mol(-1) K(-1), respectively, which indicated that the acting force between FU and BSA was mainly hydrogen bond and Van der Waals force. According to the Förster non-radiation energy transfer theory, the average binding distance between donor (BSA) and acceptor (FU) was obtained (r = 1.59 nm). The investigations of the UV/Vis and CD spectra of the system showed that the conformation of BSA was changed in presence of FU.