Biopharmaceutic aspects of buccal absorption of insulin

Absorption of insulin through buccal mucosa was investigated using rabbits as animal model. The effects of various dose sizes, pH of the insulin solutions and a series of sorption enhancers or absorption promoters on the hypoglycemic response were studied using a buccal cell. In the absence of sorption promoters, the various dose sizes and pH of the administered insulin solution did not show any significant hypoglycemic response, indicating that substantial amounts of insulin were not absorbed through the buccal mucosa. In the presence of sorption enhancers, the extent of hypoglycemia was significantly enhanced in terms of AUC and Cmax. Among the sorption enhancers investigated, Brij 35 was the most satisfactory one, followed by sodium taurocholate, sodium laurylsulfate, sodium deoxycholate, sodium methoxysalicylate, sodium dextransulfate and EDTA. An inverse correlation was found between extent of hypoglycemia and light scattering intensity of insulin solution. The extent of hypoglycemia in presence of Brij 35 was concentration-dependent although a plateau was observed at high concentration range. The maximum pharmacological availability obtained from buccal administration of insulin solution was about 12%, while that obtained with the tablet formulation was about 4.3%.     

Production of solid lipid submicron particles for protein delivery using a novel supercritical gas-assisted melting atomization process

A supercritical carbon dioxide micronization technique based on gas-assisted melting atomization has been designed to prepare protein-loaded solid lipid submicron particles. The supercritical process was applied to homogeneous dispersions of insulin in lipid mixtures: (1) tristearin, Tween-80, phosphatidylcholine and 5 kDa PEG (1:0.1:0.9:1 and 1:0.1:0.9:2 weight ratio); and (2) tristearin, dioctyl sulfosuccinate and phosphatidylcholine (1:1:0.5 weight ratio). Optimized process conditions yielded dry nonagglomerated powders with high product recovery (70%, w/w). Dynamic light scattering and transmission electron microscopy showed that two size fractions of particles, with 80-120 and 200-400 nm diameters, were produced. In all final products, dimethylsulfoxide used to prepare the insulin/lipid mixture was below 20 ppm. Protein encapsulation efficiency increased up to 80% as the DMSO content in the insulin/lipid mixture increased. Compared to the particles without PEG, the polymer-containing particles dispersed rapidly in water, and the dispersions were more stable under centrifugation as less than 20% of suspended particles precipitated after extensive centrifugation. In vitro, the protein was slowly released from the formulation without PEG, while a burst and faster release were obtained from the formulations containing PEG. Subcutaneous injection to diabetic mice of insulin extracted from the particles showed that the supercritical process did not impair the protein hypoglycemic activity.     

Effects of rhIGF-I and insulin-induced hypoglycaemia on cardiovascular parameters recorded with telemetry in the conscious dog

Using telemetry, the effects on cardiovascular parameters after single intravenous administration (0.5 mg/kg) of recombinant human insulin-like growth factor-I (rhIGF-I) were studied in conscious and unrestrained dogs. Insulin (1.1 IU/kg) was used as a reference compound. Telemetry transmitters were implanted subcutaneously with a pressure catheter in the femoral artery and electrodes for ECG subcutaneously. Cardiovascular effects and changes in blood glucose levels induced with rhlGF-I were similar to those induced with insulin. Heart rate increased continuously for about 90 min. after treatment, regardless of compound. Thereafter, heart rate slowly decreased but did not fully reach predose values 4 hr after treatment. Both systolic and diastolic blood pressure decreased continuously for about 90 min. and remained low for up to 4 hr after treatment. Treatment with rhIGF-I or insulin did not influence dp/dt values. Treatment with glucose intravenously to abolish the rhIGF-I-induced hypoglycaemia reduced the heart rate, but caused a substantial increase in dp/dt and a slight increase in blood pressure. RhIGF-I and insulin induced an almost identical onset and degree of hypoglycaemia. Blood glucose reached a minimum level 1 hr after treatment and was almost returned to normal 4 hr after treatment. There was an increase in the amplitude of the T-waves, though this effect occurred earlier and was longer lasting with insulin than with rhIGF-I. After 4 hr, the T-wave amplitude was normal with rhIGF-I but remained high with insulin. These T-wave effects were probably due to an increase in the transport of potassium or calcium since they decreased in plasma. This suggests that the effects of rhIGF-I and insulin are highly comparable and that the cardiovascular changes induced by rhIGF-I are likely caused by its insulin-like activity. As shown by the recorded cardiovascular responses induced by rhIGF-1 and insulin, the telemetric recording system makes it possible to evaluate the effects of different drugs in a continuous way that is not possible with conventional techniques. This new telemetric technique can be of significant importance in the process of future drug development.     

Effects of rhIGF‐I and Insulin‐Induced Hypoglycaemia on Cardiovascular Parameters Recorded with Telemetry in the Conscious Dog

Abstract: Using telemetry, the effects on cardiovascular parameters after single intravenous administration (0.5 mg/kg) of recombinant human insulin‐like growth factor‐I (rhIGF‐I) were studied in conscious and unrestrained dogs. Insulin (1.1 IU/kg) was used as a reference compound. Telemetry transmitters were implanted subcutaneously with a pressure catheter in the femoral artery and electrodes for ECG subcutaneously. Cardiovascular effects and changes in blood glucose levels induced with rhIGF‐I were similar to those induced with insulin. Heart rate increased continuously for about 90 min. after treatment, regardless of compound. Thereafter, heart rate slowly decreased but did not fully reach predose values 4 hr after treatment. Both systolic and diastolic blood pressure decreased continuously for about 90 min. and remained low for up to 4 hr after treatment. Treatment with rhIGF‐I or insulin did not influence dp/dt values. Treatment with glucose intravenously to abolish the rhIGF‐I‐induced hypoglycaemia reduced the heart rate, but caused a substantial increase in dp/dt and a slight increase in blood pressure. RhIGF‐I and insulin induced an almost identical onset and degree of hypoglycaemia. Blood glucose reached a minimum level 1 hr after treatment and was almost returned to normal 4 hr after treatment. There was an increase in the amplitude of the T‐waves, though this effect occurred earlier and was longer lasting with insulin than with rhIGF‐I. After 4 hr, the T‐wave amplitude was normal with rhIGF‐I but remained high with insulin. These T‐wave effects were probably due to an increase in the transport of potassium or calcium since they decreased in plasma. This suggests that the effects of rhIGF‐I and insulin are highly comparable and that the cardiovascular changes induced by rhIGF‐I are likely caused by its insulin‐like activity. As shown by the recorded cardiovascular responses induced by rhIGF‐1 and insulin, the telemetric recording system makes it possible to evaluate the effects of different drugs in a continuous way that is not possible with conventional techniques. This new telemetric technique can be of significant importance in the process of future drug development.     

Preparation and release characteristics of insulin and insulin-like growth factor-one from polymer nanoparticles

Two methods of preparing polymer nanoparticles containing (a) insulin and (b) insulin-like growth factor-1 were compared and the influence of process parameters on size and release characteristics was determined. Poly(lactide-glycolide)co-polymer (50:50) was used in both methods. Method one used a salting-out process; while method two used a solvent evaporation/double emulsion procedure forming a w/o/w secondary emulsion. Particles were separated by centrifugation and dried under vacuum. Particle size was analysed by scanning electron microscopy and protein release by dissolution and high pressure liquid chromatography. Method one produced particles of diameter 0.3–0.8?µm, whereas method two gave larger particles of 0.76–1.05?µm and in both procedures reducing pH also decreased particle size. Optimal emulsifying speed was below 4?000?rpm and scanning electron micrographs showed smooth spherical particles. Release characteristics of insulin and IGF-1 in method one and two were similar releasing 60% in 10 days but in method one release was diminished to 8% over a similar time period. Method one proved successful in producing spheres of the required size range but hampered protein loading by denaturation resulting in a low release rate. Method two provided an acceptable release rate but produced particles with diameters of about one micron.     

Preparation and release characteristics of insulin and insulin-like growth factor-one from polymer nanoparticles

Two methods of preparing polymer nanoparticles containing (a) insulin and (b) insulin-like growth factor-1 were compared and the influence of process parameters on size and release characteristics was determined. Poly(lactide-glycolide)co-polymer (50:50) was used in both methods. Method one used a salting-out process; while method two used a solvent evaporation/double emulsion procedure forming a w/o/w secondary emulsion. Particles were separated by centrifugation and dried under vacuum. Particle size was analysed by scanning electron microscopy and protein release by dissolution and high pressure liquid chromatography. Method one produced particles of diameter 0.3-0.8 microm, whereas method two gave larger particles of 0.76-1.05 microm and in both procedures reducing pH also decreased particle size. Optimal emulsifying speed was below 4 000 rpm and scanning electron micrographs showed smooth spherical particles. Release characteristics of insulin and IGF-1 in method one and two were similar releasing 60% in 10 days but in method one release was diminished to 8% over a similar time period. Method one proved successful in producing spheres of the required size range but hampered protein loading by denaturation resulting in a low release rate. Method two provided an acceptable release rate but produced particles with diameters of about one micron.     

Insulin Nanoparticles: A Novel Formulation Approach for Poorly Water Soluble Zn-Insulin

PURPOSE: To determine the feasibility of using wet milling technology to formulate poorly water soluble zinc-insulin as a stable, biologically active, nanoparticulate dispersion. METHODS: The feasibility of formulating zinc-insulin as a nanoparticulate dispersion using wet milling technology was studied. An insulin nanoparticulate formulation was reproducibly obtained after milling zinc-insulin in the presence of F68, sodium deoxycholate and water at neutral pH. The physical and chemical properties of these peptide particles were studied using electron microscopy, laser light scattering, HPLC and SDS-PAGE. To verify efficacy, hyperglycemic rats were dosed subcutaneously and intraduodenally with nanoparticles or solubilized insulin. Glucose and insulin levels were monitored on blood samples collected throughout the study. RESULTS: Zn-insulin (mean size = 16.162 microm) was processed using milling technology to form an aqueous-based nanoparticle dispersion with a mean particle size of less than 0.150 microm. The formulation was homogeneous and exhibited a unimodal particle size distribution profile using laser light diffraction techniques. Insulin, processed as a peptide-particle dispersion, was shown to be comparable to unprocessed powder using HPLC and SDS-PAGE. In addition, HPLC analyses performed on samples, heat-treated at 70 degrees C for 100 minutes, demonstrate that under conditions which effect the solubilized peptide, formulated as a peptide-particle dispersion, insulin was chemically stable. Also, when stored refrigerated, the insulin dispersion was chemically and physically stable. Finally, peptide particles of insulin, dosed subcutaneously and intraduodenally, were effective at lowering blood glucose levels of hyperglycemic rats. CONCLUSION: Water insoluble Zn-insulin can be formulated as a stable, biologically active nanometer-sized peptide particle dispersion using wet media milling technology.     

Physico-chemical characterization of insulin-loaded poly(isobutylcyanoacrylate) nanocapsules obtained by interfacial polymerization.

Insulin could be encapsulated very efficiently in oily containing poly(isobutylcyanoacrylate) nanocapsules obtained by interfacial polymerization. In addition, these nanocapsules showed unexpected biological activity after intragastric administration. The hypoglycemic effect was characterized by a lag time period of 2 days and a prolonged effect over a period of 20 days. To explain, the high encapsulation rate of insulin achieved in these nanocapsules and the biological effect, this work was focused on the characterization of the nanocapsules and on the study of the mechanism of nanocapsule formation. Results showed that insulin was found unmodified during the nanoencapsulation process. This was due to the large amount of ethanol used in the preparation of the nanocapsules that initiated the polymerization of isobutylcyanoacrylate preserving the peptide from a reaction with the monomer. Results also showed that insulin was located inside the core of the nanocapsules and not simply adsorbed onto their surface.     

A time-course study of submandibular kallikrein, blood glucose and insulin of alloxan-diabetic and streptozotocin-diabetic rats

A time-course study was carried out of the levels of submandibular kallikrein, serum insulin and blood glucose of rats rendered diabetic by alloxan or streptozotocin. The permanent hyperglycemia seen one day after treatment and the subsequent relative changes in the levels of blood glucose recorded over the following nine days were in agreement with previous observations. A significant reduction in the concentration of submandibular kallikrein did not become apparent until ten days following the injection of alloxan or streptozotocin. Thus the enzyme would not appear to have played either a causal or a preventative role in the production of the hyperglycemic state. Furthermore, in disagreement with previous speculation, the submandibular gland had not compensated for the deficiency of insulin with an increase in production of kallikrein. The fall in the level of serum insulin had preceded the decrease in submandibular kallikrein. However, administration of exogenous insulin over a period of three days did not bring about an increase in the concentration of submandibular kallikrein of the diabetic rats. Thus insulin would not seem to be involved in controlling the level of kallikrein in the submandibular gland.     

Preparation of Polymeric Submicron Particle-Containing Microparticles Using a 4-Fluid Nozzle Spray Drier

Purpose We studied a novel method for preparing polymeric submicron particle-containing microparticles using a 4-fluid nozzle spray drier. Method Ethylcellulose (EC) and poly(lactic-co-glycolic acid) (PLGA), either alone or in combination with polyethylenimine (PEI), were used as polymers to produce submicron particles, and mannitol (MAN) was used as a water-soluble carrier for the microparticles. The polymer and MAN solutions were supplied through different liquid passages of a 4-fluid nozzle and then dried to obtain MAN microparticles containing EC or PLGA submicron particles. The polymer/MAN ratio was controlled by changing the concentration of the polymer and MAN solutions. EC or PLGA microparticles were observed via scanning electron microscopy, and the size of microparticles was determined by image analysis. The particle size distribution of EC or PLGA submicron particles was measured with a super dynamic light scattering spectrophotometer. Results The method generated submicron-sized (<1 μm) particles of EC and PLGA. The mean diameters of EC and PLGA particles at a polymer/MAN ratio of 1:10 were 631 and 490 nm, respectively. The mean diameter of PLGA particles decreased as the PLGA/MAN ratio was reduced, reaching ∼200 nm at a PLGA/MAN ratio of 1:100. The mean diameter of PLGA/PEI particles at PLGA/PEI/MAN ratios of 1:0.5:10 and 1:0.5:100 were 525 and 223 nm, respectively, and their zeta potentials were +50.8 and +58.2 mV, respectively. The size of EC submicron particles could be controlled by varying the spray conditions. Conclusions This study demonstrated that it is possible to prepare polymeric submicron particles dispersed in MAN microparticles in a single process using the 4-fluid nozzle spray drying method. Cationic PLGA particles with a diameter of ∼200 nm could be prepared by adding PEI, suggesting the possibility of its use as a carrier for delivering DNA into cells. The precipitation of EC may occur by the mutual dispersion and mixing of solvents after collision of EC and MAN mists by antisolvent effect, thereby producing MAN microparticles containing EC submicron particles.     

Development of a spray congealing process for the preparation of insulin-loaded lipid microparticles and characterization thereof.

A spray congealing process for the preparation of protein-loaded microparticles was developed. The influence of the process parameters atomization pressure and spraying temperature on particle size and process yield was investigated by experimental design. The employed spray congealing technique enabled the production of microparticles with yields ranging from 79% to 95% and median particle sizes (d(0.5)) from 182.2 to 315 microm. Insulin lipid microparticles could be prepared without any loss of insulin during the preparation process and the protein stability was not affected by the spray congealing process as investigated by HPLC-MS analysis. The stability of insulin encapsulated in lipid microparticles under release conditions over 28 days was assessed by investigating the residual insulin content. Starting after 3 days of release, a continuous increase of desamidoinsulin in the remaining particles of up to 7.5% after 28 days was observed. An additional degradation product was detected by HPLC and HPLC-MS analysis and identified as a covalent insulin dimer by MALDI-ToF. The microparticles did not show a burst release and testing the insulin lipid microparticles in a fibrin gel chondrocyte culture revealed that the released insulin was bioactive and had a significant effect on chondrocyte extracellular matrix production.     

Effect of pretreatment with 0.9% sodium chloride or insulin solutions on the delivery of insulin from an infusion system

The effect of pretreating a polyvinyl chloride i.v. administration system with sodium chloride or insulin solution on the delivery of insulin was studied. Insulin labeled with iodine 125 was added to human insulin, which was added to 0.9% sodium chloride injection packaged in flexible polyvinyl chloride containers and to 0.9% sodium chloride injection placed in empty ethylene vinyl acetate containers. Samples were tested for insulin content by gamma spectrometry after storage in the bags and after infusion through four different polyvinyl chloride administration sets at different flow rates. Effluent samples were collected at 10 times (6-50 minutes) after the start of the infusion. The 0.9% sodium chloride injection had a conditioning effect on the polyvinyl chloride administration sets, indicating an electrostatic sorption mechanism for insulin. Sorption to the untreated polyvinyl chloride sets and the ethylene vinyl acetate bags was substantial and followed a Langmuir adsorption isotherm. Insulin sorption to the untreated administration sets was greatest from the first 100 mL of effluent and did not differ by flow rate or type of set investigated. Storing the sodium chloride injection in the tubing for one hour or flushing the tubing with 100 mL of sodium chloride injection or 100 mL of the insulin admixture decreased sorption by half. Storing the insulin admixture in the tubing for 30 minutes caused sorption to be reduced by a factor of three. When either of the solutions was stored in the set and then the set was flushed with the solution, sorption was even further suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Galactosamine hepatotoxicity: effect of galactosamine on glutathione resynthesis in rat primary hepatocyte cultures.

The effect of galactosamine on the resynthesis of glutathione in rat primary hepatocyte cultures was investigated. Cultured rat hepatocytes were treated with galactosamine (4 mM) 1.5 hr prior to concurrent with, or 1.5 hr after cell attachment; total cellular glutathione was then measured over time. Addition of galactosamine at any of these times suppressed methionine-enhanced glutathione resynthesis in the cultures after a lag period of about 120 min. The lag period was not due to slow uptake of galactosamine by the cultured cells, since cellular UTP levels fell to less than 10% of controls within 60 min, a time frame comparable to that observed in vivo. Neither was the lag period a result of interference with cellular uptake of methionine or with conversion of methionine to cysteine, since the phenomenon was observed regardless of whether methionine or cysteine was used to promote glutathione resynthesis. Addition of uridine, which protects against galactosamine hepatotoxicity in vivo by replenishing hepatic UTP levels, did not prevent the suppression of glutathione resynthesis. The data indicate that (a) galactosamine inhibits the time-dependent resynthesis of glutathione in primary hepatocyte cultures, (b) a lag period exists for this response, and (c) this effect is not directly related to depletion of cellular UTP stores.     

Sustained release of insulin from insoluble inhaled particles

Conventional slow‐acting insulin preparations for subcutaneous injection, e.g., suspensions of the complex with protamine and/or zinc, were reformulated as dry powders for inhalation and the insoluble aerosol tested for providing sustained insulin plasma levels. Large porous particles made of lactose, albumin, and dipalmitoylphosphatidylcholine, and incorporating insulin, protamine, and/or zinc chloride were prepared using spray‐drying. Integrity of insulin after spray‐drying and insulin insolubilization in spray‐dried particles was verified in vitro. The pharmacokinetic profile of the formulation delivered by inhalation and subcutaneous injection was assessed in vivo in the rat. The formulation process of insulin as dry powders did not alter insulin integrity and did not impede, in most cases, insulin insolubilization by protamine and/or zinc. Large porous insulin particles presented 7 μm mass mean geometric particle diameters, 0.1 g/cm³ bulk powder tap densities and theoretical aerodynamic diameters suitable for deep lung deposition (in the range of 2.2–2.5 μm). The dry powders exhibited 40% respirable fractions in the Andersen cascade impactor and 58–75% in the Aero‐Breather™. Insoluble inhaled insulin provided sustained insulin plasma levels for half a day, similar to injected insulin, and exhibited a bioavailability of 80.5% relative to subcutaneous injection of the same formulation. Drug Dev. Res. 48:178–185, 1999. ©1999 Wiley‐Liss, Inc.     

Determination of the particle size of realgar nano-particles by scanning 

目的：建立纳米雄黄的粒度分析研究方法。方法：利用扫描电镜对纳米雄黄表观形态进行直接观察测定,用激光光散射法对纳米雄黄的粒度分布范围进行分析测定。结果：经扫描电镜和激光光散射颗粒度测定仪的测量,粒径在100 nm以下的纳米雄黄约达90%,其中较大颗粒又是由粒径在5~30 nm左右的细小晶粒和其周围的非晶体聚集而成。结论：扫描电镜法和激光光散射法快速、简便、准确,可用于纳米雄黄的粒度检测。     

The fate of diesel hydrocarbons in soils and their effect on the germination of perennial ryegrass

Hydrocarbon contamination in soils may be toxic to plants and soil microorganisms and act as a source of groundwater contamination. The objective of this study was to evaluate the fate of diesel in soils with or without added nutrients. The soils examined either had or had not a previous history of hydrocarbon contamination. Particular aspects examined were soil respiration, changes in microbial population, breakdown of diesel hydrocarbons, and phytotoxicity to the germination of perennial ryegrass. Soil respiration was measured as evolved CO2. Bacterial population was determined as colony forming units in dilution plates and fungal activity was measured as hyphal length. The fate of individual hydrocarbons was determined by gas chromatography-mass spectrometry after extraction with dichloromethane. When diesel was added to soil with no previous history of hydrocarbon contamination at rates up to 50 mg/g, the respiration response showed a lag phase of 6 days and maximum respiration occurred at day 11. The lag phase was 2 days and maximum respiration occurred at day 3 in soil with a previous history of hydrocarbon contamination. After the peak, respiration decreased up to about 20 days in both soils. Thereafter, respiration become more or less constant but substantially greater than the control. N and P addition along with diesel did not reduce the lag phase but increased the respiration over the first 20 days of incubation. Diesel addition with or without N and P increased the bacterial population 10- to 100-fold but fungal hyphal length did not increase. Diesel addition at a rate of 136 mg/g did not increase the microbial population. Removal of inhibition to germination of perennial ryegrass was linked to the decomposition of nC10 and nC11 hydrocarbons and took from 11 to 30 days at diesel additions up to 50 mg/g depending on the soil. Inhibition to germination of perennial ryegrass persisted to more than 24 weeks at the 136 mg/g of diesel addition.     

Using Image Attributes to Assure Accurate Particle Size and Count Using Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) obtains particle size by analysis of particle diffusion through a time series of micrographs and particle count by a count of imaged particles. The number of observed particles imaged is controlled by the scattering cross-section of the particles and by camera settings such as sensitivity and shutter speed. Appropriate camera settings are defined as those that image, track, and analyze a sufficient number of particles for statistical repeatability. Here, we test if image attributes, features captured within the image itself, can provide measurable guidelines to assess the accuracy for particle size and count measurements using NTA. The results show that particle sizing is a robust process independent of image attributes for model systems. However, particle count is sensitive to camera settings. Using open-source software analysis, it was found that a median pixel area, 4 pixels², results in a particle concentration within 20% of the expected value. The distribution of these illuminated pixel areas can also provide clues about the polydispersity of particle solutions prior to using a particle tracking analysis. Using the median pixel area serves as an operator-independent means to assess the quality of the NTA measurement for count.     

Solid lipid micro-particles carrying insulin formed by solvent-in-water emulsion-diffusion technique

The study aimed to produce solid lipid insulin-loaded micro-particles by the solvent-in-water emulsion-diffusion technique, using isobutyric acid as solvent phase, glyceryl monostearate or cetyl palmitate as lipid, soya lecithin and taurodeoxycholate as emulsifiers. Isobutyric acid, a partially water-miscible solvent with low toxicity, was used due to its high insulin-solubilization capacity. Solid lipid micro-particles of spherical shape were prepared by simple dilution of the emulsion with water. To increase the lipid load the process was conducted at 50 degrees C, and in order to reach sub-micron size, a high-shear homogeniser was used. Insulin encapsulation efficiency was about 80%. Analysis of microsphere content after processing showed that insulin did not undergo any chemical modification within the micro-particles. The in vitro release of insulin from the micro-particles was very low, and an initial burst effect of 20% of the dose was observed. After treatment of the solid lipid micro-particles with pepsin solution, an insulin loss of about 24% of the total englobed insulin was observed. The solid lipid micro-particles appear to have interesting possibilities as delivery systems for oral administration of insulin.     

Structures of Nanoparticles Prepared from Oil-in-Water Emulsions

Hydrophobic substances were dissolved in an organic solvent and emulsified with an aqueous solution at very high shear. Droplets of very small sizes (50–100 nm) were obtained by using surfactants which were combinations of lecithins and bile salts. After emulsification, the organic solvent was removed by evaporation, yielding stable dispersions of solid particles. The sizes, shapes, and structures of the particles were examined through quasi-elastic light scattering, small-angle neutron scattering and cryotransmission electron microscopy. Cholesteryl acetate particles stabilized by lecithin and bile salts were found to be platelets of 10–20 nm thickness and 80 nm diameter. Cholesteryl acetate particles stabilized with POE-(20)-sorbitan monolaurate were dense spherical globules of diameter 100 nm. Particles with a composition similar to the endogenously occurring lipoprotein, LDL, were large spherical globules studded with small vesicles. The subsequent evolution of the Cholesteryl acetate dispersion upon aging was examined. There was no transfer of cholesteryl acetate between particles nor to large crystals. However, some aggregation of the particles was observed when the volume fraction of the particles in the aqueous dispersion exceeded 0.05. Thus, the structure of the nanoparticles obtained through deswelling of emulsion droplets changes according to the nature of the emulsifiers and to the composition of the hydrophobic substances which they contain.     

Generation of Micro-Particles of Proteins for Aerosol Delivery Using High Pressure Modified Carbon Dioxide

Purpose. To investigate the feasibility of using the Aerosol Solvent Extraction System (ASES) to generate microparticles of proteins suitable for aerosol delivery from aqueous-based solutions. Methods. The ASES technique using high- pressure carbon dioxide modified with ethanol was utilised for the generation of microparticles of proteins (lysozyme, albumin, insulin and recombinant human deoxyribonuclease (rhDNase)) from aqueous solutions. Particle size, morphology, size distributions and powder aerosol performance were examined. The biochemical integrity of the processed proteins was assessed by testing the level of molecular aggregation using size exclusion chromatography and by bioassay technique for lysozyme. Results. Proteins were precipitated as spherical particles ranging in size from 100 to 500 nm. The primary nano-sized particles agglomerated to form micron-sized particles during the precipitation process. The median size of the particles was a function of the operating conditions. In-vitro aerosol performance tests showed that the percent fine particle mass (< 5m) was approximately 65%, 40% and 20% for lysozyme, albumin and insulin, respectively. Negligible loss in the monomer content or biological activity was observed for lysozyme. Insulin exhibited slight aggregation and 93% of the monomer was retained after processing. Albumin was affected by processing and only 50-75% of the monomer was retained compared with 86% in the original material. However, rhDNase was substantially denatured during processing as shown by the significantly reduced monomer content. Conclusions. Micron-sized particles of lysozyme, albumin and insulin with satisfactory inhalation performance were successfully generated from aqueous solutions using the modified ASES technique. The biochemical integrity of the processed proteins was a function of the operating conditions and the nature of the individual protein.