In vitro characterisation of Span 65 niosomal formulations containing proteins.

Proteins can be encapsulated in niosomes as they are known to protect proteins against the surrounding environment. Niosomes of Span 65/cholesterol/pluronic F -127 were prepared by thin film hydration method. Insulin and lysozyme were chosen as model proteins. Niosomes were characterised for morphology by Transmission Electron Microscopy (TEM) and vesicles size using Dynamic Light Scattering. The entrapment efficiency of protein in niosomes was determined by complete vesicle disruption using 50:50% isopropanol:buf fer, followed by analysis of the resulting solutions by HPLC method. Thermal behaviour of the niosomes was investigated using Differential Scanning Calorimetry (DSC). Protection of proteins against simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were also assessed. The results showed that niosomes prepared with different molar ratios % of Span 65, cholesterol and pluronic F-127 successfully produced with insulin and lysozyme. For insulin containing niosomes, the ratio % of 64.7 (Span 65): 32.3 (cholesterol): 3.0 (pluronic F - 127) produced the highest protein encapsulation efficiency and the smallest vesicle size of 653.6 nm. For lysozyme containing niosomes, the maximum protein encapsulation was found in 72.75/24.25/3.00% molar ratio of Span 65/cholesterol/pluronic F -127 niosomes with vesicle size of 627.3 nm. The release study of proteins from the niosomal preparations in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) revealed that insulin and lysozyme efflux from the niosomes is a biphasic process. The results indicate that Span 65 niosomes could be developed as controlled release dosage forms for delivery of peptides and proteins such as insulin and lysozyme.

Compare and contrast the effects of surfactants (PluronicF-127 and CremophorEL) and sugars (ß-cyclodextrin and inulin) on properties of spray dried and crystallised lysozyme.

The stabilisation of proteins using different excipients in dried forms for possible therapeutic use is extensively studied. However, the effects of excipients on proteins in crystallised forms are sparsely documented. Therefore, the influences of PluronicF-127 and CremophorEL (as surfactants) and ß-cyclodextrin and inulin (as sugars) on stability and biological activity of lysozyme, a model protein, in spray dried and crystallised forms were investigated. Spray dried and crystallised lysozyme were prepared in absence and presence of the mentioned excipients in a concentration of 0.05% w/v. The protein formulations were characterised in both solution state (using biological assay, particle size analysis and protein concentration determination) and solid state (employing yield determination, scanning electron microscopic (SEM) examination, Fourier transform infrared (FT-IR) spectroscopy for secondary structure analysis and Differential Scanning Calorimetry (DSC) for thermal study). Also, protein samples were assayed for their biological activities after exposing to storage stability study for 20 weeks in solid states at 24 °C/76% relative humidity (RH) and in aqueous states at 24 °C. The results showed that lysozyme crystals with CremophorEL, PluronicF-127, ß-cyclodextrin and inulin maintained protein thermal stability (as indicated by DSC) to greater extent compared with spray dried protein formulations. Also, PluronicF-127 was competent to recover 100% lysozyme from crystallisation protein solutions (as confirmed by yield determination); this surfactant was able to prevent aggregate formation within spray dried lysozyme (as demonstrated by particle size analysis). The presence of PluronicF-127, ß-cyclodextrin and inulin preserved the protein biological activity in freshly prepared spray dried and crystallised samples. PluronicF-127 was competent to protect lysozyme in both spray dried and crystallised forms after storage. PluronicF-127 has proved to be a promising protectant of proteins. The improved stability of the spray dried and crystallised protein containing PluronicF-127 shows promise for delivery of proteins via inhalation (in a spray dried form which has particle size range suitable for inhalation as revealed by particle size analysis and SEM) and injectable routes (in spray dried and crystallised forms). The way excipients react with proteins is different in the case of spray drying and crystallisation techniques, hence the choice of the additives and the processing techniques play a great role in controlling protein properties, activity and stability as shown in this study.