Strategic approaches for overcoming peptide and protein instability within biodegradable nano- and microparticles.

This paper reviews the major factors that are closely involved in peptide and protein degradation during the preparation of biodegradable nano- and microparticles. The various means usually employed for overcoming these obstacles are described, in order to bring to the fore the strategies for protein stabilization. Both processing and formulation parameters can be modified and are distinctly considered from a strategic point of view. We describe how partial or full protein stability retention within the carriers and during drug release might be achieved by individual or combined optimized strategies. Additionally, problems commonly encountered during protein quantification, stability determination and release are briefly reviewed. Artefacts that might occur during sampling and analytical procedures and which might hinder critical interpretation of results are discussed.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for quantitation and molecular stability assessment of insulin entrapped within PLGA nanoparticles.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was evaluated for both qualitative and quantitative analysis of insulin entrapped within poly(D,L-lactic-co-glycolic acid) nanoparticles. Quantitation was performed by adding an internal standard (arg-insulin) to defined and unknown sample solutions, in order to reduce point-to-point and sample-to-sample variability. The ratio of the peak height of insulin to the peak height of arg-insulin was plotted against the insulin concentration. In this way, an excellent linear relationship was found (R2 > 0.99). This method of quantitation was compared with classical UV spectroscopy and reverse-phase high-performance liquid chromatography measurements. All methods provided close final drug loading values for the insulin-loaded nanoparticle batches tested. Additionally, with respect to molecular stability, covalent insulin dimers were found only at trace levels in those nanoparticles. Compared with other methods, MALDI-TOF MS is a valuable tool for the characterization of proteins from nanoparticles, because no extensive extraction and complex sampling procedures are required.

Development of a nanoprecipitation method intended for the entrapment of hydrophilic drugs into nanoparticles.

This study investigates formulation and process modifications to improve the versatility of the nanoprecipitation technique, particularly with respect to the encapsulation of hydrophilic drugs (e.g. proteins). More specifically, the principal objective was to explore the influence of such modifications on nanoparticle size. Selected parameters of the nanoprecipitation method, such as the solvent and the non-solvent nature, the solvent/non-solvent volume ratio and the polymer concentration, were varied so as to obtain polymeric nano-carriers. The feasibility of such a modified method was assessed and resulting unloaded nanoparticles were characterized with respect to their size and shape. It was shown that the mean particle size was closely dependent on the type of non-solvent selected. When alcohols were used, the final mean size increased in the sequence: methanol

Nanoprecipitation versus emulsion-based techniques for the encapsulation of proteins into biodegradable nanoparticles and process-related stability issues.

The goal of this study was to investigate the entrapment of 3 different model proteins (tetanus toxoid, lysozyme, and insulin) into poly(D,L-lactic acid) and poly(D,L-lactic-co-glycolic acid) nanoparticles and to address process-related stability issues. For that purpose, a modified nanoprecipitation method as well as 2 emulsion-based encapsulation techniques (ie, a solid-in oil-in water (s/o/w) and a double emulsion (w(1)/o/w(2)) method) were used. The main modification of nanoprecipitation involved the use of a wide range of miscible organic solvents such as dimethylsulfoxide and ethanol instead of the common acetone and water. The results obtained showed that tetanus toxoid and lysozyme were efficiently incorporated by the double emulsion procedure when ethyl acetate was used as solvent (>80% entrapment efficiency), whereas it was necessary to use methylene chloride to achieve high insulin entrapment efficiencies. The use of the s/o/w method or the formation of a more hydrophobic protein-surfactant ion pair did not improve protein loading. The nanoprecipitation method led to a homogenous population of small nanoparticles (with size ranging from approximately 130 to 560 nm) and in some cases also improved experimental drug loadings, especially for lysozyme (entrapment efficiency > 90%). With respect to drug content determination, a simple and quick matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method provided results very close to those obtained by reverse phase-high-performance liquid chromatography. With respect to protein stability, the duration and intensity of sonication were not a concern for tetanus toxoid, which retained more than 95% of its antigenicity after treatment for 1 minute. Only a high methylene chloride:water ratio was shown to slightly decrease toxoid antigenicity. Finally, no more than 3.3% of A21 desamido insulin and only traces of covalent insulin dimer were detected in nanoparticles. In conclusion, both the double emulsion and nanoprecipitation methods allowed efficient protein encapsulation. MALDI-TOF MS allowed accurate drug content determination. The manufacturing processes evaluated did not damage the primary structure of insulin.

Sonication parameters for the preparation of biodegradable nanocapsules of controlled size by the double emulsion method.

The main goal of the present work was to study the influence of the sonication process on the characteristics of poly(lactide-co-glycolide) nanocapsules prepared by the water-in-oil-in-water solvent evaporation method. The duration and intensity of sonication were investigated with respect to their ability to modify the size and distribution of the nanocapsule population. It has been demonstrated that the duration of the second mixing step (leading to the w/o/w emulsion) is of greater influence than that of the first step (water-in-oil emulsion) on the final mean particle size. A three-dimensional response surface was drawn up to show that when the second emulsification time increased, the mean size decreased until reaching a plateau. A threshold in sonication intensity also exists, allowing optimization of both parameters and the formation of nanocapsules of controlled size with a rather narrow distribution. The use of a vortex mixer instead of a sonicator during the first mixing step led to nanocapsules with a similar response surface, supporting the idea that the second step is the decisive one. Finally, nanocapsules loaded with methylene blue, a hydrophilic model compound with a positive charge, were characterized and the encapsulation efficiency calculated. Their size and distribution were similar to that of the blank nanocapsules. Entrapment efficiency was independent of duration, intensity, and type of mixing. From these results, it was concluded that nanocapsules of controlled size could be obtained upon optimizing certain process parameters.