Particle size analysis of concentrated phospholipid microemulsions: II. Photon correlation spectroscopy

The solvated droplet size of concentrated water-in-oil (w/o) microemulsions prepared frome egg and soy lecithin/water/isopropyl myristate and containing short-chain alcohol cosurfactants has been determined using photon correlation spectroscopy (PCS). The effect of increasing the water volume fraction (from 0.04 to 0.26) on the solvated size of the w/o droplets at 298 K has been investigated at 4 different surfactant/cosurfactant weight ratios (K _m of 1∶1, 1.5∶1, 1.77∶1, and 1.94∶1); in all cases the total surfactant/cosurfactant concentration was kept constant at 25% w/w. In the case of the microemulsions prepared from egg lecthin, the diffusion coefficients obtained from PCS measurements were corrected for interparticulate interactions using a hard-sphere model that necessitated estimation of the droplet volume fractions, which in the present study were obtained from earlier total intensity light-scattering (TILS) studies performed on the same systems. Once corrected for hard-sphere interactions, the diffusion coefficients were converted to solvated radii using the Stokes-Einstein equation assuming spherical microemulsion droplets. For both egg and soy lecithin systems, no microemulsion droplets were detected at water concentrations less than 9 wt% regardless of the alcohol and K _m used, suggesting that at low concentrations of added water, cosolvent systems were formed. At higher water concentrations, however, microemulsion droplets were observed. The changes in droplet size followed the expected trend in that for a fixed K _m the size of the microemulsion droplets increased with increasing volume fraction of water. At constant water concentration, droplet size decreased slightly upon increasing K _m. Interestingly, only small differences in size were seen upon changing the type of alcohol used. The application of the hard-sphere model to account for interparticulate interactions for the egg lecithin systems indicated that the uncorrected diffusion coefficients underestimated particle size by a factor of slightly less than 2. Reassuringly, the corrected droplet sizes agreed very well with those obtained from our earlier TILS study.     

Lipid nanocapsule size analysis by hydrodynamic chromatography and photon correlation spectroscopy

Particle size and the size distribution are known to be important factors in the overall behavior of nanoparticulate drug delivery systems and an exact determination of these parameters is highly important. Several techniques are applied in the size determination of nanoparticulates, particularly dynamic light scattering. Also other methods have been proposed, among them hydrodynamic chromatography (HDC). In order to characterize a HDC method for nanosized carriers, differently sized lipid nanocapsules having a diameter of 25–100 nm were analyzed and results were compared with measurements from photon correlation spectroscopy (PCS) and atomic force microscopy. Results from atomic force microscopy studies were generally not in line with determinations from the other techniques due to a particle shape loss by the drying step. PCS and HDC led to comparable results in simple size determination of lipid nanocapsules. However, slight differences were found during the characterization of more complex samples. HDC was able to detect micelles as a byproduct of nanocapsule preparation while in PCS the sample dilution turned micelles undetectable. HDC analysis was able to characterize mixed samples of particle batches differing in their average size similar to PCS. HDC was found to be an excellent tool for the determination of size and average size distribution of lipid nanocapsules.     

Particle size analysis of concentrated phospholipid microemulsions: I. Total intensity light scattering

Water-in-oil phospholipid microemulsions prepared from a constant total surfactant/cosurfactant concentration of 25 wt% at four different lecithin/alcohol weight ratios (K _m of 1∶1, 1.5∶1, 1.77∶1, and 1.94∶1) and containing water concentrations (or volume fractions) ranging from 2.0 to 26 wt% (or 0.04 to 0.26) have been examined at 298 K using total intensity light scattering. The data obtained were analysed using the hard-sphere model of Percus-Yevick, modified to account for the partitioning of the alcohol between the various phases. The light-scattering results showed that, regardless of the K _m or the alcohol used, a minimum water concentration of at least 9 wt% was required for the formation of a microemulsion; although this value was reasonably constant for each of the alcohols investigated, there was a tendency for a slightly higher concentration of water to be required for microemulsion formation at higher K _m values. Simple calculations suggested that a microemulsion was formed only when sufficient water was present to satisfy the hydration of both the phospholipid head groups and the hydroxyl groups of the cosurfactant associated with the droplet. At water concentrations lower than this minimum value, a cosolvent system was observed. In all systems above this minimum concentration, as the concentration of water increased, the size of the microemulsion droplets also increased. Surprisingly, however, there was little difference in the size of the microemulsion droplets obtained with the different alcohols, regardless of the K _m, although for a particular alcohol there was some indication that the higher K _m systems produced the slightly smaller droplets for an equivalent water concentration. There was also a suggestion that the more hydrophobic alcohols produced slightly smaller droplets than the more polar alcohols at the same K _m.     

Particle size determination of phase-inverted water-in-oil microemulsions under different dilution and storage conditions

Laser light scattering was used to monitor particle size and polydispersity of several water-in-oil (w/o) microemulsion formulations upon dilution with excess of the dispersed (aqueous) phase to induce phase inversion and generate oil-in-water (o/w) and/or water-in-oil-in-water (w/o/w) emulsions. Factors affecting particle size, such as, the extent and temperature of dilution, as well as, sample storage conditions were investigated. In addition, the particle size of diluted formulations incorporating a peptide (SK& F 110679) was determined and compared to that of the peptide-free microemulsions. The extent of dilution had a pronounced effect on particle size. Dilution at ambient temperature or 37°C produced particles with similar size unless the microemulsion was solid at ambient temperature where significant effect on particle size upon dilution was observed. As expected from the non-ionic nature of the investigated microemulsions, using different physiologically relevant diluent solutions the particle size of the diluted microemulsion was found to be unaffected by pH and/or ionic strength. Dilution with a micellar sodium deoxycholate doubled the particle size and polydispersity of the diluted microemulsion, presumably as a result of physical interactions. The presence of a small peptide SK& F 110679 (Mol. Wt = 850) in different microemulsions prior to dilution, at levels varied from 0.8 to 3.0 mg/ml of formulation, had no major effect on the size of the inverted particle. Microemulsions which have been stored at various temperatures for up to 70 days and then diluted, showed no significant changes in particle size whereas the polydispersity was increased upon storage.     

Two-time window and multiangle photon correlation spectroscopy size and zeta potential analysis--highly sensitive rapid assay for dispersion stability

Polysaccharide-stabilized iron oxide particles are highly potent contrast agents in magnetic resonance imaging. After intravenous injection, the size of these small or ultrasmall particles strongly influences their distribution in the body. Knowledge about the uniformity of particle size distribution within this particle size range is not accessible by laser diffraction (lower edge of detection range), and photon correlation spectroscopy (PCS) data only yield insufficient information, the so-called polydispersity index. A combination of two-time window and multiangle analysis makes detailed characterization of particle size distribution and particle aggregation feasible, which was shown using five different iron oxide dispersions. Additional particle charge characterization yielded conclusions about the type of stabilization present in the dispersion - electrostatic or steric stabilization. Thus, this thorough particle size and charge analysis is a tool for quick detection of broad particle distributions or aggregates.     

Enhancing the sensitivity of fluorescence correlation spectroscopy by using time-correlated single photon counting

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.     

Particle size analysis of microcapsules

To standardize the preparation of three-walled w/o/w microcapsules it was necessary to assess their particle size distribution, since they occur as both unicored and multicored types. The log-normal law, reported to be obeyed by many particulate systems, including microcapsules, was not applicable to the size data. A curve-fitting computer program using least squares minimization was used to assess the size distribution data. The models tested were the unimodal and bimodal log-normal distributions, the bimodal form being the most appropriate. Using the equations to the curves of best fit, the modal size and standard deviation of each population were estimated, and the relative percentages of unicored and multicored microcapsules could be deduced from an analysis of the bimodal curves in which they were represented as the sum of two constituent unimodal distributions.     

Release profiles and morphological characterization by atomic force microscopy and photon correlation spectroscopy of 99mTechnetium-fluconazole nanocapsules

Several classes of antifungal have been employed in candidiasis treatment, but patients with advanced immunodeficiency can present unsatisfactory results after therapy. In these cases, high doses of drugs or the use of multiple agents are sometimes used, and hence increasing the risk of serious side effects. Considering theses difficulties, the encapsulation of antifungal agents in nanoparticulate carriers has been used with the objective of modifying the pharmacokinetic of drugs resulting in more efficient treatments with less side effects. The purpose of this work was the preparation, characterization and the investigation of the release profiles of radiolabeled fluconazole nanocapsules. The size, homogeneity and zeta potential of NC preparations were determined with a Zetasizer 3000HS. The morphology and the structural organization were evaluated by atomic force microscopy (AFM). The release study in vitro of NC was evaluated in physiologic solution with or without 70% mouse plasma. The labeling yield of fluconazole with 99mTc was 94% and the radiolabeled drug was stable within 24h period. The encapsulation percentage of 99mTc-fluconazole in PLA-POLOX NC and PLA-PEG NC was approximately of 30%. The average diameter calculated by photon correlation spectroscopy (PCS) varied from 236 to 356 nm, while the average diameter determined by AFM varied from 238 to 411 nm. The diameter/height relation decreased significantly when 25% glutaraldehyde was used for NC fixation on mica. The zeta potential varied from -55 to -69 nm and surface-modified NC showed lower absolute values than conventional NC. The in vitro release of 99mTc-fluconazole in plasma medium of the conventional and surface-modified NC was greater than in saline. The drug release in plasma medium from conventional NC was faster than for surface-modified NC. The results obtained in this work suggest that the nanocapsules containing fluconazole could be used to identify infectious foci, due to the properties, such as size, zeta potential and controlled release of (99m)Tc-fluconazole. The surface-modified nanocapsules could constitute a long-circulating intravenous formulation of fluconazole for treating sepsis caused by disseminated form of candidiasis. However, in vivo studies should be considered and are under investigation.     

Particle size and charge distribution analysis of pharmaceutical aerosols generated by inhalers

Aerosol particles generated by inhalers for respiratory drug delivery acquire electrostatic charge during the dispersion process. The electrostatic charge distribution of the particles can affect the efficiency of drug delivery by influencing both the transport and deposition of inhaled particles in the human lung. To analyze the electrostatic charge acquired by the aerosol particles, two sets of metered-dose inhaler (MDI) and dry powder inhaler (DPI) devices were investigated. Both the particle size and charge distributions were measured simultaneously by using an electrical single-particle aerodynamic relaxation time (E-SPART) analyzer. The analyzer was calibrated with particles of known size, which were generated by a vibrating orifice aerosol generator (TSI Inc.) and charge using the Faraday cup method. The charge distributions of the pharmaceutical aerosols from both the DPI and MDI devices were bipolar in nature. Although the net charge-to-mass ratio was less than 0.2 microC/g, the individual particles were charged with a relatively high charge: -2 to + 2 microC/g. The count mean aerodynamic diameter of the aerosols generated from these devices was 3-5 microm.     

Particle size analysis of nanocrystals: Improved analysis method

The influence of optical parameters, additional techniques (e.g. PIDS technology) and the importance of light microscopy were investigated by comparing laser diffraction data obtained via the conventional method and an optimized analysis method. Also the influence of a possible dissolution of nanocrystals during a measurement on the size result obtained was assessed in this study. The results reveal that dissolution occurs if unsaturated medium or microparticle saturated medium is used for the measurements. The dissolution is erratic and the results are not reproducible. Dissolution can be overcome by saturating the measuring medium prior to the measurement. If nanocrystals are analysed the dispersion medium should be saturated with the nanocrystals, because the solubility is higher than for coarse micro-sized drug material. The importance of using the optimized analysis method was proven by analysing 40 different nanosuspensions via the conventional versus the optimized sizing method. There was no large difference in the results obtained for the 40 nanosuspensions using the conventional method. This would have led to the conclusion, that all the 40 formulations investigated are physically stable. However, the analysis via the optimized method revealed that from 40 formulations investigated only four were physically stable. In conclusion an optimized analysis saves time and money and avoids misleading developments, because discrimination between “stable” and “unstable” can be done reliably at a very early stage of the development.     

Particle size distributions of inert spheres and pelletized pharmaceutical products by image analysis

Image analysis was used to measure particle size distributions (PSDs) of ensembles of 425 to 1400 microm-size materials. Repeatability of a measurement, suitable sample sizes, and methods of sampling were assessed. Two lots of inert spheres were compared prior to drug layering in a Glatt GPCG-5 rotor. The differences in PSD in the starting materials were reflected in the rotor-granulated products. Such detailed information was not available from sieving with U.S. standard wire mesh sieves. The products from the rotor process were polymer-coated in a Wurster process in a Glatt GPCG-3, 4-in. Wurster. The resolution of the technique was sufficient to measure differences in diameter equating to 4-microm coat thickness, which resulted from applying 2% polymer coat weight. The utility of the technique for monitoring commercial scale processes was demonstrated by measuring diameter after layering drug onto nonpareils in a Glatt RG-150 rotor, and by measuring the diameter after application of a polymer solution in a Glatt 46-in. Wurster coating process. The similarity of samples removed from the sample port in situ and samples from the batch suggested that processes in the fluid bed are intensively mixed and inherently random.     

Coagulation and flocculation in suspensions of griseofulvin and polystyrene latex

Coagulation and flocculation mechanisms have been examined in suspensions of the drug griseofulvin containing anionic surfactants. It has been shown that both mechanisms can give suspensions which are readily redispersible but that flocculation may produce pharmaceutically undesirable effects. The differences between the two phenomena are discussed.     

DNA measurements by using fluorescence correlation spectroscopy and two-color fluorescence cross correlation spectroscopy

FCS and FCCS measurements provide two important analytical parameters, the average number of molecules in the detection area and the translational diffusion constant of the molecules at the single molecule level. Considering these properties, FCS and FCCS have been applied to analysis of the cellular environment and dynamic processes of molecules in the living cell. More recently, a systematic approach for the analysis of macromolecule complex formation has focused on the new field of single molecule detection in the post-genome era. In this work, we tested the sensitivities of FCS and FCCS based on the distance between fluorophores in DNA as a model macromolecule complex. The results show that FCCS is not limited by the size of the macromolecular complex even in a very small detection area.     

Atomic force microscopy and photon correlation spectroscopy: two techniques for rapid characterization of liposomes.

The direct evaluation of the heterogeneity of the particle population of nanometric drug delivery systems as liposomes is difficult to achieve owing to the dimension and the carrier characteristics. The influence of the lipidic ratio and composition on the physical stability of liposomes during their storage was investigated using atomic force microscopy (AFM) and photon correlation spectroscopy (PCS). Liposomes were made by a mixture of different lipids and obtained using distinct methods of preparation. AFM images, acquired immediately after the deposition of the sample on mica surface, clearly showed the spherical shape of the lipidic vesicles. In all the 7 months of the experiment, the average sizes of the different liposomes evaluated using the two techniques were comparable. According to PCS analysis, AFM images confirmed that almost all the diversified vesicular systems tended to form aggregates during their storage; this loss of stability was strengthened by the increase of polydispersity index value. The different behaviours observed were to ascribe to the lipidic composition more than the methods of liposome preparation. In conclusion, AFM technique owing to the relative simplicity cold be useful for the technological control of size distribution profile according to the preparative factors and moreover to the batch-to-batch reproducibility.     

Particle size analysis of gelatin-acacia coacervate and ethylcellulose walled microcapsules

The Coulter counter has become one of the methods of choice for the measurement of small particle sizes. However, many colloidal walled microcapsules are prone to hydration when dispersed in the electrolyte solution used in Coulter measurements. This hydration causes the walls to swell, producing different size analyses dependent upon the time of measurement. In the present work the change in size with time was studied for microcapsules with gelatin-acacia coacervate, or ethylcellulose, walls. The former, although rendered insoluble with formalin, still hydrated; the latter were almost unaffected by water. A sample size of 30 mg was required to prevent blocking of the orifice with larger samples, or too few particles to permit measurement in smaller samples. The coacervate coated microcapsules showed a bimodal distribution, in part due to aggregate of smaller microcapsules, and this distribution changed significantly over the time of measurement. Although the peak due to larger microcapsules did not disappear, the number of large microcapsules fell significantly, most probably because of the disintegration of aggregated microcapsules. Microcapsules with ethyl cellulose walls did not show a bimodal distribution and the particle size analysis did not alter significantly with time.     

PLA-PEG nanocapsules radiolabeled with 99mTechnetium-HMPAO: release properties and physicochemical characterization by atomic force microscopy and photon correlation spectroscopy.

The present work describes the preparation, characterization and labelling of conventional and surface-modified nanocapsules (NC) with 99m Tc-HMPAO. The size, size distribution and homogeneity were determined by photon correlation spectroscopy (PCS) and zeta potential by laser doppler anemometry. The morphology and the structural organization were evaluated by atomic force microscopy (AFM). The stability and release profile of the NC were determined in vitro in plasma. The results showed that the use of methylene blue induces significant increase in the encapsulation efficiency of 99m Tc-HMPAO, from 24.4 to 49.8% in PLA NC and 22.37 to 52.93% in the case of PLA-PEG NC (P<0.05) by improving the complex stabilization. The average diameter of NC calculated by PCS varied from 216 to 323 nm, while the average diameter determined by AFM varied from 238 to 426 nm. The AFM analysis of diameter/height ratios suggested a greater homogeneity of the surface-modified PLA-PEG nanocapsules compared to PLA NC concerning their flattening properties. The in vitro release of the 99m Tc-HMPAO in plasma medium was faster for the conventional PLA NC than for the surface-modified NC. For the latter, 60% of the radioactivity remained associated with NC, even after 12h of incubation. The results suggest that the surface-modified 99m Tc-HMPAO-PLA-PEG NC was more stable against label leakage in the presence of proteins and could present better performance as radiotracer in vivo.     

流动注射微乳液化学发光法测定片剂和注射剂中磷酸可待因的含量

目的:建立测定磷酸可待因的化学发光快速分析方法。方法:在酸性条件下,磷酸可待因被质子化后与阴离子AuCl4-形成离子缔合物,该缔合物被二氯甲烷带入鲁米诺的十六烷基三甲基氯化铵(CTAC)微乳液中,离解出来的AuCl4-立即与鲁米诺产生化学发光。在一定浓度范围内,发光强度与磷酸可待因的含量成线性关系,从而间接测定磷酸可待因的含量。结果:在优化的试验条件下,磷酸可待因的检测线性范围为0.001～15μg·mL-1,检出限(3σ)为0.03 ng·mL-1,对浓度为1.0μg·mL-1的磷酸可待因进行11次平行测定,其RSD为1.2%。结论:该法快速、简便,已成功用于片剂、针剂和尿样中磷酸可待因的测定。     

Electrophoretic properties of acrylic latex suspensions (Kollicoat MAE 30 D) and ibuprofen

We have compared the electrophoretic properties (measured on the electrical surface) of the commercial latex Kollicoat MAE 30 D and the non-steroidal anti-inflammatory drug (NSAID) ibuprofen in preparation for attempts to develop a suitable vehicle for the NSAID to obtain a modified release formulation. Electrophoretic mobility of the latex and the active principle was measured in solutions containing different concentrations of inorganic electrolytes (NaCl, CaCl2 and AlCl3) at different pH values. This was considered an indispensable first step for further characterization of the substance's electrical properties. Suspensions of both the latex and the drug had negative mobility values throughout the range of pH values studied here. Of the electrolytes, neither NaCl nor CaCl2 led to positive mobility, and no isoelectric point could be determined. However, AlCl3 at a concentration of 10(-3) M led to the greatest reduction in mobility. We therefore found that trivalent cations were more effective than divalent cations, which in turn were more effective than monovalent cations, in reducing mobility.