Polymer coated liposomes for dental drug delivery--interactions with parotid saliva and dental enamel.

The interactions between pectin coated liposomes and parotid saliva and dental enamel were studied to investigate their potential to mimic the protective biofilm formed naturally on tooth surfaces. Different pectin coated liposomes with respect to pectin type (LM-, HM- and AM-pectin) and concentration (0.05% and 0.2%) were prepared. Interactions between the pectin coated liposomes and parotid saliva were studied by turbidimetry and imaging by atomic force microscopy. The liposomes were adsorbed to hydroxyapatite (HA) and human dental enamel using phosphate buffer and parotid saliva as adsorption media. A continuous flow was imposed on the enamel surfaces for various time intervals to examine their retention on the dental enamel. The results were compared to uncoated, charged liposomes. No aggregation tendencies for the pectin coated liposomes and parotid saliva were revealed. This makes them promising as drug delivery systems to be used in the oral cavity. In phosphate buffer the adsorption to HA of pectin coated liposomes was significantly lower than the negative liposomes. The difference diminished in parotid saliva. Positive liposomes adsorbed better to the dental enamel than the pectin coated liposomes. However, when subjected to flow for 1h, no significant differences in the retention levels on the enamel were found between the formulations. For all formulations, more than 40% of the liposomes still remained on the enamel surfaces. At time point 20 min the retention of HM-pectin coated and positive liposomes were significantly higher. It was concluded that pectin coated liposomes can adsorb to HA as well as to the dental enamel. Their ability to retain on the enamel surfaces promotes the concept of using them as protective structures for the teeth.

Solubilization of the novel anionic amphiphilic photosensitizer TPCS 2a by nonionic Pluronic block copolymers.

The influence of four nonionic Pluronic block copolymers (L44, F68, P123 and F127) on the solubilization and aggregation of the novel anionic amphiphilic photosensitizer TPCS(2a), intended for use in the technology of photochemical internalization (PCI), was evaluated in aqueous media as part of pharmaceutical preformulation studies. The evaluation was performed by use of UV-Vis spectroscopy for diluted samples of TPCS(2a) (3×10(-6)M), and capillary viscosimetry, freezing point depression measurements and atomic force microscopy (AFM) at pharmaceutical relevant concentrations (2; 10 or 30 mg/ml TPCS(2a)). The critical micelle concentration (CMC) of the Pluronics in presence of TPCS(2a) was determined spectrophotometrically. The Pluronic block copolymers solubilized the photosensitizer above CMC at ambient temperature by formation of vehicle-drug complexes apparently organized in networks of varying viscosity and morphology, which were sensitive towards the addition of neutral and charged excipients.

Air-filled polymeric microcapsules from emulsions containing different organic phases.

Air-filled polymeric microcapsules for use as a contrast agent in ultrasonography have been prepared by the freeze-drying of different oil-in-water emulsions. The water phases consisted of a block copolymer in water. The organic phases consisted of a biodegradable polyester dissolved in (-)-camphene, cyclooctane, cyclohexane or tricyclene, which were relatively poor solvents for the polyester. A polymeric wall was, therefore, precipitated at the droplet surface early in the process, i.e. during freezing. Removing the solvent during freeze-drying, resulted in air-filled microcapsules. The microcapsules were suspended in saline after freeze-drying. All the suspensions contained echogenic microcapsules with a volume mean diameter of approximately 5-7 microm. Microscopic investigations showed that the microcapsules were spherical and hollow. Tricyclene and, to some degree, (-)-camphene were found unsuitable for industrial production due to melting points above 30 degrees C. Cyclooctane and cyclohexane were investigated as replacements for the initially chosen (-)-camphene, since they are liquids over a wider temperature range. These solvents gave improved yields, measured both as particle volume concentration per amount of polymer in suspension and acoustic attenuation at 3.5 MHz per amount of polymer in suspension, although the freeze-drying cycle was not optimized for these systems.

Preparation of polymeric microcapsules: formulation studies.

Air-filled microcapsules were prepared by freeze-drying different oil-in-water emulsions containing biodegradable polyester as the wall-forming material. The aim of this work was to find an acceptable formulation with respect to the microcapsule suspension and the stability of the emulsion during the production process. The influence of various formulation parameters (concentrations of mannitol, polymer, and surfactant; pH; oil-in-water phase ratio) was investigated in a factorial design. The results were treated by ordinary least-square (OLS) regression and partial least-square regression (PLSR). In a previous work, air-filled microcapsules were successfully made using human serum albumin as the surfactant in the emulsion (1). In the present work, a new block copolymer based on poly(ethylene glycol) (PEG) was implemented as the surfactant to replace human serum albumin. It was found that the new block copolymer is a suitable replacement for human serum albumin. The concentration of the polymer in water and the concentration of the surfactant in the oil phase and the interaction between these variables had a significant influence on the stability of the emulsion at 60 degrees C. A surfactant concentration of approximately 2% (w/v) in water was necessary when the concentration of the wall-forming polymer was below 5% (w/v) in (-)-camphene. The concentration of the polymer in the oil phase influenced the yield, measured as the volume concentration of particles in suspension per milligram of polymer added and as acoustic effect per milligram of polymer. Low levels of polymer concentration in (-)-camphene (< 5% w/v) gave the highest yield. Excess polymer in the oil phase did not form microcapsules, but precipitated in the suspension or was included in the wall of the microcapsules. Addition of mannitol protected the microcapsules from being destroyed during freeze-drying and resulted in freeze-dried products with few cracks, little shrinkage, and higher suspension yield.

Formulation and characterisation of primaquine loaded liposomes prepared by a pH gradient using experimental design.

The effect of different formulation factors (lipid type, cholesterol, charge, internal buffer capacity, drug-to-lipid incubation ratio) on the encapsulation efficiency and size of primaquine liposomes (SUV's) in response to a pH gradient was investigated by a fractional factorial screen ing design. Three of the factors (charge, internal buffer capacity, drug -to-lipid incubation ratio) were further studied in a Box--Behnken optimisation design. The lipid type was the most important parameter followed by the drug-to-lipid incubation ratio, buffer capacity, cholesterol and charge. Several of the interactions wer e important. In the optimisation design a robust region with high encapsulation efficiency (>95%) was obtained for DSPC: 33.33 mol% cholesterol-liposomes at high internal citrate concentration (200 mM) by maintaining the drug-to-lipid incubation ratio below 0.15:1 (mol:mol) and varying the charge incorporation between 2 and 10%. In order to achieve long-term stability and sterility, the liposomes were lyophilised followed by gamma irradiation. The pH gradient was maintained during this treatment with little chemical degradation of the substances. The final preparation consisted of three separate vials with lyophilised liposomes, solid state primaquine and hydration medium.

Toxicity of gamma irradiated liposomes. 1. In vitro interactions with blood components.

Gamma irradiation is a potential technique for sterilisation of liposome suspensions. Unfortunately, gamma irradiation may result in chemical degradation of the phospholipids and the toxicological aspects have to be considered. The effects of liposome composition and gamma irradiation on the interactions of the liposomes with the hemostatic mechanisms (hemolysis, aggregation and coagulation) were studied. Non-irradiated liposome suspensions showed no hemolysis of erythrocytes. After irradiation, up to 3.1% hemolysis was measured. Least hemolysis was observed with irradiated liposomes composed of unsaturated or charged phospholipids. The negatively charged DSPG-liposomes (both non-irradiated and irradiated) induced aggregation of platelets as observed by the spectrophotometric method. However, no aggregates were seen in the microscope or measured by the aggregometer. Negatively charged liposomes also affected the coagulation cascade where prolonged coagulation times were measured. Irradiation of the liposome suspensions resulted in even longer coagulation times. The prolonged coagulation times correlated to some extent with the measured binding and depletion of calcium from plasma by the negatively charged liposomes.

On the rôle of human salivary micelle-like globules in bacterial agglutination.

The hypothesis to be tested in this in vitro study was that the salivary micelle-like globules (SMGs) have a rôle in the agglutination of some oral bacteria. An attempt to determine the mechanisms for the interactions involved was also carried out. 4 laboratory and 4 native streptococci strains were tested. Human whole (HWS) and parotid (HPS) saliva was collected from 4 subjects, and SMGs were isolated from both salivas, and agglutination was recorded in the various bacterial suspensions over time. HPS, HWS and SMGs isolated from HPS and HWS caused typical agglutination patterns for the mutans strains. Salivary supernatants (without SMGs) caused a much delayed or no agglutination. Electron microscopy showed SMG-like structures on the surface of the agglutinated bacteria. Addition of pyrophosphate to HPS prevented agglutination, whereas guanidine HCl prevented normal agglutination of a sanguis strain, and urea had no obvious effect. Together, these results indicate that the SMGs are important in the agglutination of streptococci, and that both calcium-dependent, electrostatic and hydrophobic interactions may be involved.

Fractionation of salivary micelle-like structures by gel chromatography.

Globular structures have been demonstrated in human parotid saliva by transmission electron microscopy and photon correlation spectroscopy. The aim of this study was to fractionate these salivary globular structures for analytical and preparative purposes using a gel-filtration material capable of separating spherical particles up to 300-400 nm in diameter. Freshly obtained parotid saliva was applied to a Sephacryl S-1000 column. Peak fractions were collected and prepared for transmission electron microscopy (TEM) or for amino acid analysis. Bovine milk was included as the casein micelles by TEM appear to be similar to the salivary aggregates and their elution profiles are known. The salivary globular structures were eluted in one major peak. TEM of negatively stained samples from the peak fractions demonstrated globular protein aggregates consistent with the salivary structures in parotid saliva. Amino acid analysis showed characteristic amino acid profiles with unusual high levels of proline, 40-45%. The casein micelles were eluted in one major peak and separated from the whey proteins. This study indicates that the salivary globular structures can be isolated by gel chromatography. The amino acid analysis indicates that proline-rich proteins may be an important fraction of the salivary globular structures.

Zeta potentials of human enamel and hydroxyapatite as measured by the Coulter DELSA 440.

The zeta potential of human enamel is of physiological importance for interactions between enamel surfaces and the surrounding aqueous medium of saliva. The zeta potentials of both enamel and hydroxyapatite (HA) have been examined previously by various techniques. In this study, we examined the zeta potential of human enamel and HA using the Coulter DELSA 440, which, by a laser, makes independent Doppler shift measurements of moving particles in an electric field at 4 different angles, providing advantages over previous techniques. The enamel and HA particles were suspended directly in different phosphate buffers, or first incubated for 2 hrs in parotid (PS) or whole saliva (HWS) and then suspended in the same buffers. The enamel and HA particles exhibited an overall net surface potential of -15 to -30 mV, depending on the buffer content. Incubation in PS and HWS gave less negative potentials of -8 to -14 mV. In our previous studies, the salivary micelle-like structures (SMSs), seen in TEM of parotid saliva, were observed to have a zeta potential of -9 mV (Rykke et al., 1996). The zeta potential determinations in this study support the concept of an adsorption of mostly SMSs to the enamel surfaces, with a change of the zeta potential of the enamel and HA toward that of the SMSs.

The mechanism of particulate contamination in soft polyvinyl chloride infusion fluid bags.

In the seventies, it was shown that particles were generated in soft polyvinyl chloride (PVC) infusion fluid bags when they were shaken. In this investigation an exponentially modified log-normal distribution (EMLN) is fitted to the particle data. Using the formula for the volume of a sphere, the number-density distribution is converted into a volume-density distribution. The total particle load in the samples is estimated by integration for total particle volume. The results are expressed as volume concentration in plain SI-units (microliters/l). A four-factor analysis of variance demonstrates that the mechanism of the contamination process is most probably an emulsification of low molecular weight additives in the PVC plastic, i.e. di(2-ethylhexyl) phthalate (DEHP), epoxidized vegetable oils (EVO), and others.

The covariation of chemical contamination, particulate matter and turbidity in soft polyvinyl chloride infusion fluid bags.

About 200 samples of normal saline, isotonic glucose and Ringer acetate infusions in soft polyvinyl chloride (PVC) bags obtained from three manufacturers have been analyzed by conductometric particle counting, turbidimetry and gas liquid chromatography (GLC). The particle counts were fitted to an exponentially modified log-normal model and integrated for total particle-volume. Di(2-ethylhexyl) phthalate (DEHP) and epoxidized vegetable oils (EVO), which are the main water insoluble contaminants in PVC fluid bags, were determined by GLC. There was a strong linear correlation between turbidity and GLC results. The correlation between particle-volume concentration and the DEHP and EVO concentrations was fairly good. The results seem to verify that an emulsion of plastic additives is formed when soft PVC infusion fluid bags are shaken.

Determination by gas-liquid chromatography of trace amounts of soft polyvinyl chloride plastic additives in aqueous solutions. I. Epoxidized vegetable oils.

A gas-liquid chromatographic method for the determination of epoxidized vegetable oils (EVO), such as epoxidized soybean oil and epoxidized linseed oil, in aqueous solutions is described. The EVOs are extracted with n-hexane and transesterified to the methyl esters by sodium methoxide in methanol. 3% OV-210 is used as the stationary phase. The weakest standard solution corresponds to 5 micrograms/l (5 ppb) of EVO in an aqueous sample. The reproducibility of a single analysis is 5%. The method is used for the determination of EVO in intravenous fluids stored in flexible polyvinyl chloride bags.

Determination by gas-liquid chromatography of trace amounts of soft polyvinyl chloride plastic additives in aqueous solutions. II. Di(2-ethylhexyl) phthalate, epoxidized vegetable oils and stearates.

A method for the determination of di(2-ethylhexyl) phthalate (DEHP), epoxidized vegetable oils (EVO) and stearates in aqueous solutions is described. A stepwise extraction procedure is employed to separate DEHP and EVO from the stearates, using n-hexane as extraction solvent. EVO is transesterified to methyl esters by sodium methoxide in methanol. The stearates are derivatized by methanol containing sulfuric acid. The alkyl esters are analyzed by gas-liquid chromatography, using 3% OV-210 as the stationary phase. The concentrations of the weakest standard solutions correspond to 10, 5 and 8 micrograms/l (ppb) of DEHP, EVO and stearates, respectively, in the aqueous samples. The method is used for the determination of DEHP, EVO and stearates in intravenous solutions stored in flexible polyvinyl chloride bags.

The influence of product factors on the migration in soft polyvinyl chloride infusion fluid bags.

The concentration of epoxidized vegetable oils (EVO) and di(2-ethylhexyl) phthalate (DEHP) in infusion fluids in soft polyvinyl chloride (PVC) bags has been determined by gas-liquid chromatography prior to and after agitation. Normal saline, isotonic glucose and Ringer acetate from four different manufacturers were investigated. Agitation resulted in an increased concentration of EVO and DEHP. Significant differences in contamination level were observed between solutions of different compositions and in synonymous preparations from different manufacturers. The results indicated that the total amount of migrating EVO and DEHP was affected by the pH of the solution and by the age of the plastic material. It is suggested that the migration of EVO and DEHP during agitation is influenced by the concentration of plastic degradation products.

The influence of mono(2-ethylhexyl) phthalate and stearates on the migration in soft polyvinyl chloride infusion fluid bags.

The influence of mono(2-ethylhexyl) phthalate (MEHP) and stearates on the migration of di(2-ethylhexyl) phthalate (DEHP) and epoxidized vegetable oils (EVO) in soft polyvinyl chloride infusion fluid bags during agitation has been investigated by gas-liquid chromatography. Normal saline, isotonic glucose and Ringer acetate from 3 different manufacturers were investigated. A covariation was observed between the MEHP concentration in the solution and the pH. The MEHP concentration was not influenced by agitation. There was no correlation between the MEHP concentration in the solution and the migration of EVO and DEHP, or between the stearate concentration and the EVO and DEHP migration.

Migration of plastic additives from soft polyvinyl chloride bags into normal saline and glucose infusions.

Samples of soft polyvinyl chloride (PVC) fluid bags containing normal saline and glucose 50 mg/ml were analyzed for plastic additives. The bags were shaken for 24 hours before analysis. The PVC plastic materials contained di(2-ethylhexyl)phthalate, epoxidized vegetable oils and stearates as the main additives. The same components were found in the solutions. Mono(2-ethylhexyl)phthalate was detected only in the solutions.