Biomedical subjects

Martin Brandl

Publications and source records attributed to Martin Brandl.

18 recordsLinked to original sources

PubMed2006

Drug permeability across a phospholipid vesicle based barrier: 3. Characterization of drug-membrane interactions and the effect of agitation on the barrier integrity and on the permeability.

Recently, we reported on the development and structural characterization of a phospholipid vesicle based barrier useful for medium throughput screening of passive drug permeability. Here, we investigate the physical and functional integrity of the phospholipid vesicle based barriers to agitation by stirring or shaking, and whether agitation affects drug permeability of sulpiride, metoprolol and testosterone. In addition, three drugs (caffeine, naproxen and sulphasalazine) which were shown in a previous study to affect the electrical resistance of the barriers, were investigated for their influence on the permeability of a simultaneously applied hydrophilic marker (calcein), and on the thermotropic phase transition of the phospholipid bilayers using differential scanning calorimetry (DSC). Electrical resistance measurements indicated that the barriers should withstand shaking speeds up to 150rpm without losing their integrity, but significant release of phospholipids from the membrane barriers to the donor and acceptor chambers was observed under agitation >or=150rpm. When using agitation up to 100rpm no increase in permeability was observed for sulpiride, metoprolol and testosterone. The phospholipid vesicle-based barrier thus differ from other permeability models in that agitation does not lead to an increase in permeability, not even for highly lipophilic drugs such as testosterone. This is explained by the different morphology of the vesicle-based barrier which is containing a 100microm thick layer of mostly aqueous compartments immobilised within a matrix of phospholipids vesicles. Sulphasalazine and naproxen were shown to decrease the electrical resistance and increase the permeability of the hydrophilic marker calcein. The DSC experiments showed that these two drugs probably interact with the head groups of the phospholipids. In contrast, caffeine gave an increase in electrical resistance and a decrease in permeability of calcein. From the DSC experiments no signs of interaction of caffeine with the phospholipid bilayer could be observed.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2006

Drug permeability across a phospholipid vesicle-based barrier 2. Characterization of barrier structure, storage stability and stability towards pH changes.

Recently we reported on the development of a phospholipid vesicle-based barrier as a medium throughput method for screening of drug permeability. The aim of this present study is to characterize the barrier structure, including an estimation of the amount of phospholipid within it, its storage stability and its stability over various pH ranges found in different parts of the gastrointestinal tract. The amount of lipid in the barrier was quantified using a colorimetric phospholipase D-based assay. The total amount averaged 3.30mg phospholipid per barrier. The preparation process comprises the consecutive deposition of two types of liposomes on a filter support. We estimated that the smallest liposomes, with a mean diameter of 298nm, would fill the pore volume of the filter when tightly packed. The volume of the bigger liposomes, deposited on top of the filter, was calculated to generate a 0.1mm thick layer. Visualisation of fluorescently labelled liposomes by confocal laser-scanning microscopy confirmed that the pores of the filter were completely filled with liposomes and that there was a liposome layer on top. Small angle X-ray scattering (SAXS) analysis was used to study the lamellarity of the liposomes. The liposomes contained oligo- and/or multilamellar structures before and after deposition. The functionality of the barriers during storage at three different temperatures was examined for a period of up to 4 weeks by measuring the permeability of the hydrophilic marker calcein across them. The conclusion was that the phospholipid vesicle-based barriers could be stored at -80 degrees Celsius for up to 2 weeks without significant changes. The stability of the barriers in a pH range from 2.0 to 8.0 was investigated by performing permeation studies with fluorescein at different pH values. It was found that the phospholipid vesicle-based barrier did not lose its integrity within this range. Thus, the barriers appear suitable for further studies to provide insight into segmental absorption in the human gastrointestinal tract. Furthermore, because the phospholipid vesicle-based barrier can be stored, larger batches can be produced. This makes the phospholipid vesicle-based barrier more appropriate for high throughput screening.

Crystallography, X-Ray↗

PubMed2006

Fluidized bed adsorbent systems for extracorporeal liver support.

Acute liver failure based on acute-on-chronic liver failure (AoCLF) or on acute severe damage of the liver caused by different etiologies includes complex mechanisms resulting in severe disturbances of principle liver functions. In order to compensate the liver's function of detoxification as efficiently as possible, fluidized bed absorbent systems have been designed. In these systems, small particles with specific adsorption properties for toxins related to acute liver failure are applied. A special technology based on adsorbents in suspension has been developed under the guidance of our group and is prepared for clinical application during the coming year. This technology is called microspheres-based detoxification system (MDS) and is based on microadsorbents with a diameter of 1-10 microm which are recirculated in suspension. The safety of the MDS is guaranteed by the use of fluorescently labeled magnetic microparticles, which in case of a membrane-leakage are detected in the blood circuit by an optical system equipped with a magnetic trap. In vitro tests with two kinds of microadsorbents (a combination of a hydrophobic neutral resin and an anion exchange resin) showed excellent efficiency of the system with respect to adsorption capacity as well as to the kinetics of elimination of albumin-bound substances (e.g. unconjugated bilirubin or cholic acid) and of non-protein-bound substances (e.g. phenol). Moreover, using a plasma filter or the Albuflow filter as membrane filters in the blood circuit, the MDS technology offers the possibility to remove inflammatory mediators such as tumor necrosis factor-alpha (TNF) by additional use of specific adsorbents.

Adsorption↗

PubMed2005

Drug permeability across a phospholipid vesicle based barrier: a novel approach for studying passive diffusion.

The aim of this study was to develop a novel predictive medium-throughput screening method for drug permeability, with use of a tight barrier of liposomes on a filter support. To our knowledge no one has succeeded in depositing membrane barriers without the use of an inert solvent such as hexadecane. The first part of the study involved development of a protocol for preparation of these barriers, which were made of liposomes from egg phosphatidylcholin in phosphate buffer pH 7.4 with 10 % (v/v) ethanol. The liposomes were deposited into the pores and onto the surface of a filter support (mixed cellulose ester) by use of centrifugation. Solvent evaporation and freeze-thaw cycling were then used to promote fusion of liposomes. A tight barrier could thus be obtained as shown with calcein permeability and electrical resistance. In the second part of the study the model was validated using 21 drug compounds, which cover a wide range of physicochemical properties and absorption (F(a)) in humans (13-100%). The drug permeation studies were carried out at room temperature with phosphate buffer (pH 7.4) in both acceptor and donor chambers. The apparent permeability coefficients obtained from the phospholipid vesicle based model correlated well with literature data on human absorption in vivo, which suggests that its performance is adequate and that the method is suitable for rapid screening of passive transport of new chemical entities. The results obtained from our model were compared with polar surface area (PSA) and experimental logD and with results obtained by established permeability screening methods such as immobilized liposome chromatography (ILC), the PAMPA models and the Caco-2 model. Our approach seems to model the in vivo absorption better than PSA, experimental logD, the ILC and PAMPA models, when similar conditions are used as in our assay, and equally well as the Caco-2 model and the Double Sink PAMPA (DS-PAMPA) model.

Cell Membrane Permeability↗

PubMed2005

Particle leakage in extracorporeal blood purification systems based on microparticle suspensions.

AIM: The newly developed 'Microspheres based Detoxification System' (MDS) designed for any extracorporeal adsorption therapy uses microparticles as adsorbents characterized by a size of 1-20 microm in diameter which are recirculated in the secondary (filtrate) circuit connected to a hollow fiber filter located in the primary (blood) circuit. In the case of a leakage or rupture in the hollow fiber filter, microspheres can enter patients' blood circuits and cause embolic episodes in different organs with varying degrees of clinical relevance. Aim of this study was to determine the amount of particles infused to a patient during a long-term treatment under different failure conditions of the filter. METHODS: The filters were prepared by cutting single hollow fibers. Fresh-frozen plasma (FFP) and a mixture of glycerol and water were used as a medium together with microparticles potentially used in the MDS. The amounts of particles transferred from the filtrate into the primary circuit were measured. RESULTS: The analysis of particle transfer in the case of a single cut hollow fiber inside the membrane results in particle volumes of up to 26 ml calculated for 10 h. CONCLUSION: Particle leakage in microparticle suspension based detoxification systems can lead to considerable particle transfer to the patient. Therefore, a particle detection unit which is able to detect critical amounts of particles (<1 ml particle volume/treatment) in the extracorporeal blood line is necessary for patient safety.

Extracorporeal Circulation↗

PubMed2005

Development and in vitro evaluation of a liposome based implant formulation for the decapeptide cetrorelix.

Semisolid phospholipid dispersions of vesicular morphology, so-called vesicular phospholipid gels (VPGs), were prepared by high-pressure homogenisation and tested in vitro for their suitability as implantable sustained release system for the decapeptide cetrorelix, a potent LH-RH antagonist. The VPGs contained 300-500 mg/g egg phosphatidylcholine (E80) and 0.5-10 mg/g cetrorelix acetate (CXA). The in vitro release experiments showed a wide variability of the system in release, ranging from complete release within less than 24 h (0.5mg/g CXA; 400 mg/g E80) to a predicted 80% sustained release over 3 months (8.6 mg/g CXA; 280 mg/g E80). Erosion of the phospholipid matrix, i.e. release of phospholipid vesicles was found to be the main release mechanism, following zero order or first order kinetics depending on the composition of the VPG. CXA-concentration dependent drug-drug or drug-lipid interactions are assumed to be responsible for the change in release kinetics and the decrease of CXA release at high concentrations of the peptide. Multivariate analysis revealed that both lipid concentration and peptide concentration and also the interactions between the two factors are significant factors for the release rate of the peptide. In summary: based on the presented in vitro release data sustained release of therapeutically relevant CXA doses over up to 6 weeks appears feasible. VPGs are thus considered as a promising new approach for the sustained release of peptide hormones.

Chemistry, Pharmaceutical↗

PubMed2005

Protein O-mannosylation is crucial for cell wall integrity, septation and viability in fission yeast.

Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans, which are of fundamental importance in eukaryotes. The PMT family, which is classified into PMT1, PMT2 and PMT4 subfamilies, is evolutionarily conserved. Despite the fact that PMTs are crucial for viability of baker's yeast as well as of mouse, recent studies suggested that there are significant differences in the organization and properties of the O-mannosylation machinery between yeasts and mammals. In this study we identified and characterized the PMT family of the archaeascomycete Schizosaccharomyces pombe. Unlike Saccharomyces cerevisiae where the PMT family is highly redundant, in S. pombe only one member of each PMT subfamily is present, namely, oma1+ (protein O-mannosyltransferase), oma2+ and oma4+. They all act as protein O-mannosyltransferases in vivo. oma1+ and oma2+ form heteromeric protein complexes and recognize different protein substrates compared to oma4+, suggesting that similar principles underlie mannosyltransfer reaction in S. pombe and budding yeast. Deletion of oma2+, as well as simultaneous deletion of oma1+ and oma4+ is lethal. Characterization of the viable S. pombe oma1Delta and oma4Delta single mutants showed that a lack of O-mannosylation results in abnormal cell wall and septum formation, thereby severely affecting cell morphology and cell-cell separation.

Cell Proliferation↗

PubMed2004

Camptothecin-catalyzed phospholipid hydrolysis in liposomes.

Hydrolysis of phospholipid (PL) within camptothecin (CPT)-containing liposomes was studied systematically, after elevated lyso-phosphatidylcholine (LPC)-concentrations in pH 5, CPT-containing liposomes (22.1+/-0.9 mol%) relative to control-liposomes (7.3+/-0.5 mol%) occasionally had been observed after four months storage in fridge. Liposomes were prepared by dispersing freeze-dried PL/CPT mixtures in 25 mM phosphate buffered saline (PBS) of varying pH (5.0-7.8) and CPT concentrations (0, 3 and 6 mM). PL-hydrolysis was monitored by HPTLC, quantifying LPC. In an accelerated stability study (60 degrees C), a catalytic effect of CPT on PL-hydrolysis was observed after 40 h, but not up to 30 h of incubation. The pH profile of the hydrolysis indicated a stability optimum at pH 6.0 for the liposomes independent of CPT. The equilibrium point between the more active lactone- and the carboxylate-form of CPT was found to be pH 6.8. As a compromise, pH 6.0 was chosen, assuring >85% CPT to be present in the lactone form. At this pH, both control- and CPT-liposomes showed only minor hydrolysis after autoclaving (121 degrees C, 15 min). Storage at room temperature and in fridge (2 months), as well as accelerated ageing (70 degrees C, 25 h), gave a significant elevation of LPC content in CPT-liposomes relative to control-liposomes. This study demonstrates a catalytic effect of CPT on PL-hydrolysis, the onset of which seems to require a certain threshold level of hydrolytic degradation.

Camptothecin↗

PubMed2004

Effect of hydroxypropyl-beta-cyclodextrin-complexation and pH on solubility of camptothecin.

The influence of both pH and complexation by hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on the overall solubility of camptothecin (CPT) was studied, with particular focus on the equilibrium between its lactone- and carboxylate-form. Phase solubility studies at therapeutically relevant pH values (pH 5.5-7.0) and physiologically acceptable HP-beta-CD-concentrations (0-25% (w/v)) were performed, and amounts of solubilized CPT quantified by HPLC. The solubility of CPT increased with both increasing pH and HP-beta-CD-concentration. The apparent complexation constant (KC) decreased with increasing pH (245 M(-1) at pH 5.5; 184 M(-1) at pH 7.0). The lactone-carboxylate equivalence point shifted from a pH value of 6.8-7.0 and 7.1 with 0, 10 and 25% HP-beta-CD, respectively. The lactone-carboxylate-ratios from the equilibrium study were applied to the phase-solubility data, and the lactone- and carboxylate concentrations at 0, 10 and 25% HP-beta-CD calculated. Separate complexation constants (KC) for the carboxylate-CPT and lactone-CPT could thus be derived, and found to be 113 +/- 7 and 260 +/- 18 M(-1), respectively. This allows the prediction of amounts of both lactone- and carboxylate-CPT solubilized at any HP-beta-CD concentration and pH-combination.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2004

A method to determine the incorporation capacity of camptothecin in liposomes.

The purpose of this study was to establish a new experimental approach to determine the maximum amount of camptothecin (CPT) that can be incorporated in liposomes, and to use this method to compare the CPT-incorporation capacity of various liposome formulations. Small, CPT-saturated liposomes were prepared by dispersing freeze-dried blends of lipids and drug in phosphate buffer, and subsequent probe-sonication. Excess precipitated CPT could be separated from the liposomes by ultracentrifugation. The small and homogeneous liposome size obtained gave a good and reproducible recovery of liposomes in the supernatant (>80%), whereas the acidic pH (pH 6.0) kept CPT in its hydrophobic lactone form, which is poorly soluble in the buffer. The maximum CPT-incorporation capacity of 12 different liposome formulations was investigated, using the described method, and was found to vary widely. With liposomes made of neutral and anionic phospholipids, the solubility of CPT in the buffer was improved by approximately a factor of 10 (from 2.7 to 15-50 microg/mL) as compared with buffer. With cationic liposomes containing 1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP), a maximum CPT-solubilization of 100-fold, the buffer solubility was reached, probably owing to an electrostatic interaction between the cationic lipids and the carboxylate-CPT isomer. Increasing DOTAP fractions within egg-phosphatidylcholine (EPC)/DOTAP liposomes reached a CPT-incorporation plateau at 20 mol% DOTAP. The presented approach appears suitable to study the incorporation capacity of any drug component within small vesicles as long as the liposome incorporation is high relative to the intrinsic water solubility of the drug.

Camptothecin↗

PubMed2004

Development and validation of a HPLC method for routine quantification of the decapeptide Cetrorelix in liposome dispersions.

The development and validation of an HPLC method for the quantification of the decapeptide Cetrorelix (acetyl-D-2-naphthylalanyl-D-4-chlorphenylalanyl-D-3-pyridylalanyl-seryl-tyrosyl-D-citrullyl-leucyl-arginyl-prolyl-d-alaninamide), a potent antagonist of the luteinising hormone-releasing hormone in liposome dispersions is described. An isocratic reversed phase method with UV-detection appeared most appropriate. Several detergents were tried to disrupt liposomes. Furthermore, detergents turned out to be useful, because they minimised unwanted loss of Cetrorelix due to adsorption to the vial surfaces. Triton X-100 was found most effective, while sodium cholate led to quantification problems. In the presence of 2.5% Triton X-100 calibration curves with a high degree of linearity were achieved in the desired range of 0.2-10 microg/ml. The limits of detection and quantification of Cetrorelix were calculated from the peak-to-noise ratio to be 11 and 37 ng/ml, respectively. The repeatability of the method in presence of phospholipid and Triton was good with relative standard deviations (R.S.D.) ranging from 0.8% (at 0.05 microg/ml) to 1.5% (at 0.2 microg/ml). The presence of liposomes at phospholipid contents of up to 0.25mg/ml did not significantly affect the slope or linearity of the calibration curve, nor the peak-to-noise ratio.

Chromatography, High Pressure Liquid↗

PubMed2004

Adsorption of the decapeptide Cetrorelix depends both on the composition of dissolution medium and the type of solid surface.

High performance liquid chromatography (HPLC) analysis of increasing amounts of the decapeptide Cetrorelix, a potent antagonist of the luteinising hormone-releasing hormone, in distilled water resulted in a poor and variable response when solutions of low concentration (0.2-4microg/ml) were analysed. Rinsing experiments revealed loss of analyte due to adsorption to the vial surfaces as the main reason for this. The adsorption of Cetrorelix was found to follow a Langmuir isotherm reaching a plateau at 0.4microg/cm(2) and to be influenced by both the dissolution medium and the type of vial used. The adsorption tendency of Cetrorelix was reduced by: (a) a more lipophilic solvent (ethanol), (b) a more acidic pH (acetic acid) inducing repulsive charges (c) a micellar solution of various tensides. With all of these media the HPLC response was higher (up to five times) and less variable. Adsorption of Cetrorelix to solid surfaces decreased in the rank order: glass > polypropylene = polyethylene > poly-(tetrafluoroethylene), with considerable differences between the glass vials of various suppliers.

Acetic Acid↗

PubMed2004

Cytotoxic effect of different camptothecin formulations on human colon carcinoma in vitro.

Two innovative 20-S-camptothecin (CPT) formulations, previously found suitable to achieve therapeutically relevant CPT concentrations, were assessed for their in vitro cytotoxic potential as compared to an aqueous CPT solution, using the MTT assay. The formulations, cationic CPT-containing liposomes (CPT-Lip), hydroxypropyl-beta-cyclodextrin (HP-beta-CD) complexed CPT (CPT-CD) and a saturated aqueous CPT solution (CPT-Sol), were diluted in culture medium to appropriate CPT concentrations (4.7-300 ng/ml), and incubated with HT-29 and SW-480 human colon carcinoma cell lines. IC50 values were calculated after 48 and 72 h incubation for the HT-29 and SW-480 cell lines, respectively, and were found to be of the same magnitude for all formulations, with only a slight difference (CPT-Sol<CPT-CD<CPT-lip). The cells obtained apoptotic morphology after 36 h incubation with CPT-CD and were demonstrated to be active caspase-3 immuno-positive. Both formulations investigated, CPT-CD and CPT-Lip, showed significant cytotoxicity in vitro relative to CPT-Sol and warrant investigation for future therapeutic application.

Camptothecin↗

PubMed2003

Quantification of various phosphatidylcholines in liposomes by enzymatic assay.

The purpose of this research was to adapt a colorimetric, phospholipase D-based serum-phospholipid assay for the quantification of phosphatidylcholine (PC) in liposomes using a microtitre plate reader. PC from natural egg PC liposomes was quantified reliably. In contrast, poor sensitivity was found for liposomes composed of saturated PCs (di-palmitoyl-phosphatidylcholine [DPPC], hydrogenated egg PC). Triton X-100 was then added to the liposomes followed by heating above the phase transition temperature. This modified sample preparation resulted in recoveries of 102.6% +/- 1.0%, 104.4% +/- 7.6%, and 109.4% +/- 3.2% for E80, E80-3/cholesterol, and DPPC liposomes, respectively. Absolute quantification of unknown PCs against a choline chloride standard is feasible, but relative measurements against the very same PC are recommended whenever possible. Validation experiments revealed an absolute quantification limit of 1.25 microg per assay, a good linearity in the range of 25 to 1000 microg/mL PC (r2> or = 0.9990) and a quite high accuracy (99.8%-101.4% of theory) and precision (relative standard deviation < or = 3.2%) for all 3 PCs studied. The method is thus regarded as suitable for sensitive, rapid, and reliable routine quantification of PCs in liposomes.

Clinical Enzyme Tests↗

PubMed2003

Assessing the accuracy of routine photon correlation spectroscopy analysis of heterogeneous size distributions.

The aim of the current study was to investigate the ability of a fixed-angle routine photon correlation spectrometer (PCS) to resolve bimodal size distributions. The focus was on dispersions consisting of a majority of smaller and a minority of bigger particles. Monodisperse latex beads of sizes from 21 to 269 nm were measured first as single-size dispersions and then with various binary blends. For single-size dispersions, the mean diameters obtained were as indicated by the manufacturer, except for 21- and 34-nm particles, which were somewhat smaller. PCS analysis of blends of 21 + 102-nm and 34 + 102-nm particles resulted in bimodal distributions with particle diameters of the 2 peaks in the expected magnitude down to critical blending ratios of 0.002% and 0.08% of bigger particles, respectively. At these ratios, PCS results became inconsistent, and an increased number of monomodal results and/or high residuals were seen. For 21 + 102-nm blends, at even smaller ratios (0.001%), more consistent results were obtained again with predominantly monomodal distributions in the size range of the smaller particles (ie, the bigger particles were neglected). PCS analysis of blends of 21 + 269-nm particles yielded bimodal distributions with diameters within the expected magnitude as long as the content of bigger particles did not exceed 0.005%. Above this ratio, predominantly monomodal results with mean diameters in the magnitude of the bigger particles were obtained (ie, the smaller particles were neglected). In conclusion, a routine PCS instrument can resolve bimodal size distributions of colloidal dispersions only at certain ratios of the 2 subpopulations. Both low and high ratios lead to 1 of the 2 subpopulations being neglected.

Chemistry, Physical↗

PubMed2002

Pharmacokinetics and antitumor activity of vincristine entrapped in vesicular phospholipid gels.

In vivo antitumoral activity, pharmacokinetics (PK) and biodistribution of a new liposomal formulation of vincristine (VCR-Lip) were compared to VCR in aqueous solution (VCR-Conv). VCR was entrapped into a vesicular phospholipid gel (VPG) consisting of densely packed liposomes. Redispersed VCR-containing VPG (VCR-Lip) consisted of 54% liposomally entrapped and 46% free VCR. In vivo efficacy of VCR-Lip versus VCR-Conv was tested using the s.c. growing human small cell lung carcinoma LXFS 650 and the human mammary carcinoma MX1. PK and biodistribution were evaluated using radiolabeled drug and lipid in LXFS 650 tumor-bearing mice. VCR-Lip at a dose of 1.0 mg/kg (dose near the maximum tolerated dose) led to partial remissions in the MX1 tumor xenograft model (T/C=3.9%). VCR-Conv at an equitoxic dose of 0.6 mg/kg produced only a tumor growth inhibition (T/C=7.0%). In LXFS 650 tumor-bearing mice, VCR-Lip was highly active at doses of 0.75 (T/C=0.7%) and 1.0 (T/C=0.0%) mg/kg, and complete tumor regressions were observed. In contrast, equitoxic doses of VCR-Conv (0.6 mg/kg) resulted only in less pronounced tumor remissions (T/C=4.1%). The PK study revealed that VCR-Lip achieved an over 10-fold higher plasma AUC (22.6 microg x h/ml) than VCR-Conv (2.16 microg x h/ml). Moreover, tumor drug levels were 2.3-fold higher when VCR was injected as VCR-Lip in comparison to VCR-Conv. In some cases, however, VCR-Lip as well as blank VPG appeared to be toxic. We conclude that VCR-Lip is an effective VCR delivery system with superior antitumor activity compared to VCR-Conv. The enhanced efficacy can be explained by sustained release and passive tumor targeting.

Animals↗

PubMed2002

Determination of the size distribution of liposomes by SEC fractionation, and PCS analysis and enzymatic assay of lipid content.

In this study, small liposomes obtained by high-pressure homogenization were fractionated according to their particle sizes by size exclusion chromatography (SEC). The subfractions were analyzed by photon correlation spectroscopy (PCS) as well as enzymatic phosphatidylcholine (PC) assay for their particle sizes and lipid contents, respectively. For small egg PC-liposomes, a size range of 15 nm to 60 nm was found, with 80% of the vesicles being smaller than 30 nm in size. This is in contradiction to a mean size of 85 +/- 32 nm as indicated by PCS without fractionation. The PCS technique appears to underestimate very small particles below 30 nm if (few) bigger particles are present. The PCS particle size analysis of unfractionated hydrogenated egg PC/cholesterol-liposomes (2:1, mole/mole) by PCS did not yield any significant results. On fractionation, however, a particle size range of 40 nm to 120 nm was determined in a reproducible manner. Our results indicate that the combination of size exclusion fractionation with subsequent photon correlation spectroscopic particle size analysis and enzymatic PC assay can give both more detailed and more reliable insight into the particle size distribution of small liposomes than PCS alone.

Chemical Fractionation↗

PubMedDate not supplied

Liposome size analysis by dynamic/static light scattering upon size exclusion-/field flow-fractionation.

The aim of the current study was to analyse the particle size distribution of a liposome dispersion, which contained small egg phosphatidylcholine vesicles and had been prepared by high-pressure homogenisation, by various size analysis techniques. Such liposomes were chosen since they can be looked at as a prototype of drug nano-carriers. Three sub-micron particle size analysis techniques were employed: (1) fixed-angle quasi-elastic laser light scattering or photon correlation spectroscopy (PCS), (2) size exclusion chromatographic (SEC) fractionation with subsequent (off-line) PCS size-analysis and quantification of the amount of particles present in the sub-fractions, and (3) field-flow-fractionation coupled on-line with a static light scattering and a refractive index (RI)-detector. When designing liposome-based drug carrier systems, a reliable and reproducible analysis of their size and size distribution is of paramount importance: Not only does liposome size influence the nanocarrier's in-vitro characteristics such as drug loading capacity, aggregation and sedimentation but also it is generally acknowledged that the pharmacokinetic behaviour and biodistribution of the carrier is strongly size-dependent. All three approaches of liposome size analysis used here were found to yield useful results, although they were not fully congruent. PCS indicated either a broad, mono-modal, log-normal size distribution in the range of below 20 to over 200 nm in diameter, or alternatively, a bimodal distribution with two discrete peaks at 30 to 70 nm and 100 to over 200 nm. Which of the two distribution models represented the best fit depended primarily on the data collection times used. In contrast, both fractionating techniques revealed a size distribution with a large, narrow peak well below 50 nm and a minor, broad, overlapping peak or tail extending to over 100 nm in diameter. The observed differences in liposome size distribution may be explained by the inherent limitations of the different size analysis techniques, such as the detection limit and the fact that PCS is overemphasizing bigger particle sizes.

Chemical Fractionation↗