Formulation development and toxicity assessment of triacetin mediated nanoemulsions as novel delivery systems for rapamycin

The aim of this investigation was to design and develop nanoemulsions [NEs] as novel delivery systems for rapamycin. Phase behavior of quaternary systems composed of Traicetin [as oil], various surfactants and co-surfactants and water at different surfactant/co-surfactant weight ratios was investigated by the construction of phase diagrams. Formulations were taken from the o/w NE region of the phase diagrams, depending upon the extent of NE domain. The spontaneous emulsification method was used to prepare various formulations containing 1 mg/mL of the drug. The NEs were characterized and subjected to stability tests at various temperatures over 9-12 months. Cumulative drug release from the selected formulations was determined for a period of 48 h using a dialysis sac. The assay of rapamycin was carried out using an HPLC technique. The effect of NEs on the viability of SKBR-3 cells was evaluated by MTT assay. The integrity of Caco-2 cell monolayers was measured by Transepithelial Electrical Resistance [TEER] and the transport of rapamycin-loaded NEs across Caco-2 cell monolayers was then assessed. The uptake of NEs by SKBR-3 cells was also investigated using florescence microscopy. Maximum drug release was observed in case of 4 formulations prepared with Tween 80 and Tween 20. MTT test results revealed different toxicity of NEs for SKBR-3 cell line and TEER demonstrated that formulations containing Tween 20 caused a more considerable decrease in cell integrity in comparison with those prepared with Tween 80. The results obtained from cellular uptake experiments were in consistent with those obtained from TEER and cytotoxicity experiments

Study on the effect of solution conditions on heat induced-aggregation of human alpha interferon

A major problem in the formulation of therapeutic proteins is the irreversible protein aggregation. Recombinant human interferon alpha2b [rhIFN alpha 2b] has poor stability and undergoes physical degradation. The aim of this study was to investigate the effect of solution conditions on the heat-induced aggregation of rhIFN alpha 2b. The protein was incubated for 1 h at 40-70 [degree sign]C and for up to 240 h at 50 [degree sign]C and its aggregation tendency was then studied using optical density [at 350 nm], SE-HPLC, dynamic light scattering and SDS-PAGE methods. The effect of various pH [5, 6 and 7] and buffer concentrations [10, 55 and 100 mM] on the aggregation of protein following incubation at 50 [degree sign]C for 72 h was also evaluated. The results obtained for samples incubated at 50 [degree sign]C for up to 240 h showed that OD[350] and the amount of higher molecular weight aggregates [HMW] increased and the monomer content decreased significantly [p<0.05] as the incubation time increased. Following incubation at various temperatures, a significant increase in OD[350], drop in monomer content and increase in the amount of HMW aggregates were observed [p<0.05]. Data obtained from incubation of samples at 50 [degree sign]C for 72 h confirmed that regardless of the buffer concentration, the percentage of monomer at pH 6 was significantly higher than that at pH 7 and pH 5 [p<0.05]. At constant pH, although not significant, the same trend was observed when the buffer concentration increased to 100 mM. In conclusion, the change in solution conditions can influence the aggregation extent of rhIFN alpha 2b

Challenges to design and develop of DNA aptamers for protein targets. I. optimization of asymmetric PCR for generation of a single stranded DNA library

Aptamers, or single stranded oligonucleotides, are produced by systematic evolution of ligands by exponential enrichment, abbreviated as SELEX. In the amplification and regeneration step of SELEX technique, dsDNA is conversed to ssDNA. Asymmetric PCR is one of the methods used for the generation of ssDNA. The purpose of this study was to design a random DNA library for selection of aptamers with high affinity for protein targets and develop an efficient asymmetric PCR amplification. Thus, the influence of factors including annealing temperature, number of amplification cycles, primer ratio, Mg[2+] concentration and the presence of a PCR enhancer on the amplification of the desired product were evaluated. Results obtained by agarose gel electrophoresis showed that the annealing temperature of 64 [degree sign]C, Mg[2+] concentration of 0.25 mM, reverse to forward primer ratio of 15:1, amplification cycle of 25 and the presence L-ectoin as a PCR enhancer with the concentration of 0.4 M were the optimal conditions. Our results supported that the yield of this type of ssDNA production is sufficient for combinatorial screening of aptamers

Challenges to improve the stability and efficacy of an intravesical BCG product

The aim of this investigation was to improve the storage stability and survival rate of an intravesical BCG product, manufactured with an attenuated strain of Mycobacterium bovis [Pasteur strain 1173P2 of BCG] in the presence of sodium glutamate. Formulations with various concentrations of trehalose [a known protectant] were developed as liquid and lyophilized forms. Formulations were evaluated by different methods, including optical density measurement, safety assessment, skin reaction test, moisture content determination, viability assay, bacterial and fungal contaminations and the results were compared with those obtained for sodium glutamate-containing formulations. The stability tests were also carried out in various storage durations and different temperatures. To develop the lyophilization protocol, glass transition temperatures in the presence of both stabilizers were determined using differential scanning calorimetry. In general, results showed that trehalose could considerably increase the stability of the product against freezing and drying processes, increase the survival rate even in the liquid formulations, as well as the production of an acceptable cake. However, further studies are required to optimize the product characteristics

reversed phase high performance liquid chromatographic method for determination of rapamycin

Easily degradating and various isomeric forms of rapamycin [Sirolimus] face the determination of this compound to many challenges. In this study, we developed and validated the isocratic reversed phase high performance liquid chromatographic [RP-HPLC] method for rapamycin. Separation was performed on a C[8] column [MZ, 15 × 4.6 mm, 5 [micro]m particle size] using methanol:water [80:20 v/v] as the mobile phase with the flow rate of 1 mL/min. The column temperature was set at 57degreeC and the detection was carried out at the wavelength of 277 nm. The method was linear over a concentration range of 0.025-2 [micro]g/mL. The coefficient of variation of intra- and inter-day, assessed at three concentration levels of 0.075, 0.3 and 0.900 [micro]g/mL, was less than 2%. Limit of quantification [LOQ] was found 25 ng/mL. The method with high percent recovery and short retention time of rapamycin, was found to be simple, rapid and reproducible

Evaluation of the effect of pegylated single-walled carbon nanotubes on viability and proliferation of jurkat cells

Among the numerous nanosized drug delivery systems currently under investigation, carbon nanotubes [CNTs], regardless of being single or multiple-walled, offer several advantages and are considered as promising candidates for drug targeting. Despite the valuable potentials of CNTs in drug delivery, their toxicity still remains an important issue. After the PEGylation of single-walled CNTs [SWCNTs] with phospholipid-PEG [Pl-PEG] conjugates to prepare water-dispersible nanostructures, the present study was designed to evaluate whether the functionalization with Pl-PEG derivatives could alter the cytotoxic response of cells in culture, affect their viability and proliferation. In-vitro cytotoxicity screens were performed on cultured Jurkat cells. The SWCNTs samples used in this exposure were pristine SWCNTs, Pl-PEG 2000/5000-SWCNTs at various concentrations. Jurkat cells were first incubated for 3 h at 37°C with test materials and seeded in 6-well culture plates at a given concentration. The plates were then incubated for 24, 48 and 72 h at 37°C in a 5% CO[2] humidified incubator. Cell Viability and proliferation assay were performed using trypan blue exclusion test and the cell cycle kinetic status of Jurkat cells was analyzed by flow cytometry. Cell morphology was finally studied using double staining technique and a fluorescence microscope. We found that, regardless of the duration of exposure, functionalized SWCNTs were substantially less toxic, compared to pure SWCNTs and that the molecular weight of Pl-PEGs played an important role at higher concentrations. In conclusion, our noncovalent protocol seemed to be effective for increasing SWCNTs biocompatibility

Separation of somatropin and its degradation products by high-performance liquid chromatography using a reversed-phase polymeric column

The accurate prediction of protein stability is one of the most challenging goals in protein formulation and delivery. In this study, a gradient RP-HPLC method is described for the separation of human growth hormone [hGH] variants as deamidated and oxidized forms. The methodology employed a polymeric poly [styrene-co-divinylbenzene] column and a 1mL/min flow rate of a linear gradient of 0.1% v/v TFA/acetonitrile and TFA/Water [pH=2.0] mixture as the mobile phase. The overall run time of this method was 12 min and the average retention times were about 8.7 min for the native somatropin, 7.2 min for the deamidated form and 1.6 and 5.3 min for oxidized variants. The method was also validated in terms of selectivity, linearity, intra- and inter-day variations. In conclusion the method was found to have the potential for being applied as an initial and rapid evaluation method for assessing the quality and quantity of hGH during downstream processing, formulation and storage