Alf Lamprecht

Size-Dependent Bioadhesion of Micro- and Nanoparticulate Carriers to the Inflamed Colonic Mucosa

AbstractPurpose. The size-dependent deposition of microparticles and nanoparticles after oral administration to rats using an experimental model colitis was examined. Local delivery of an entrapped drug could reduce side effects and would be a distinct improvement compared with existing colon delivery devices. Methods. Ulcerative colitis was induced in Lewis rats with trinitrobenzenesulfonic acid. Fluorescent polystyrene particles with a size of 0.1, 1, or 10 μm were administered for 3 days. The animals then were sacrificed and their guts resected. Particle distribution in the colon was imaged by confocal laser scanning microscopy and quantified by fluorescence spectrophotometry. Results. In the inflamed tissue, an increased adherence of particles was observed at the thicker mucus layer and in the ulcerated regions. A size dependency of the deposition was found, and an increased number of attached particles to the colon was determined compared with the control group. For 10-μm particles, only fair deposition was observed (control group: 1.4 ± 0.6%; colitis: 5.2 ± 3.8% of administered particle mass). One-micrometer particles showed higher binding (control group: 2.0 ± 0.8%; colitis: 9.1 ± 4.2%). Highest binding was found for 0.1-μm particles (control group: 2.2 ± 1.6%; colitis: 14.5 ± 6.3%). The ratio of colitis/control deposition increased with smaller particle sizes. Conclusions. The use of submicron-sized carriers holds promise for the targeted delivery of drugs to the inflamed colonic mucosal areas in inflammatory bowel disease.

A new generation of anticancer, drug-loaded, colloidal vectors reverses multidrug resistance in glioma and reduces tumor progression in rats

By focusing on rat glioma, we elucidated whether new lipid nanocapsules (LNC) were able to improve anticancer hydrophobic drug bioavailability while also overcoming multidrug resistance. Blank LNCs and LNCs loaded with the antineoplastic agent paclitaxel were formulated by an emulsion inversion phase process. Expression of efflux pumps by rat glioma cells was assessed by reverse transcription-PCR, Western blot, and immunohistochemistry, and their activity was followed using the tracer 99Tcm-methoxyisobutylisonitrile. Modalities of LNC action were addressed by using confocal microscopy detection of fluorescently labeled LNCs, fluorescence-activated cell sorting, high-performance liquid chromatography measurement of paclitaxel release, and analysis of tumor cell growth. This revealed an interaction between LNCs and efflux pumps that resulted in an inhibition of multidrug resistance in glioma cells, both in culture and in cell implants in animals. LNCs were able to target the intracellular compartment of glioma cells, a mechanism that was abrogated by using intracellular cholesterol inhibitors but not by clathrin-coated pit or caveolae uptake inhibitors. This result can be correlated to the LNC inhibitory effects on efflux pump activity that is itself known to be stimulated by intracellular cholesterol. In parallel, we showed that paclitaxel-loaded LNCs were active reservoirs from which paclitaxel could be released. Finally, we established that paclitaxel-loaded LNCs were more efficient than the commercially available paclitaxel formulation (Taxol) for clinical use, thus reducing tumor expansion in vitro and in vivo. Considering the physiologically compatible nature of LNC excipients, these data may represent an important step towards the development of new clinical therapeutic strategies against cancers. [Mol Cancer Ther 2006;5(7):1710–22]

The preparation and evaluation of poly(ϵ-caprolactone) microparticles containing both a lipophilic and a hydrophilic drug

An original dosage form for oral delivery based on the encapsulation of both, lipophilic and hydrophilic drugs, in poly(epsilon-caprolactone) (PCL) microparticles prepared either by the oil-in-water (o/w) or the water-in-oil-in-water (w/o/w) solvent evaporation method was developed. Microparticles were characterized in terms of morphology, size, encapsulation efficiency and drug release. The physical state of the drugs and the polymer was determined by scanning electron microscopy (SEM), X-ray powder diffractometry, and differential scanning calorimetry (DSC). Nifedipine (calcium antagonist) and propranolol HCl (beta-blocker), used for the treatment of hypertension, were chosen as lipophilic and hydrophilic drugs. The microparticles were spherical with diameters in the range of 191-351 microm by the o/w-method, and in the range of 302-477 microm by the w/o/w-method. The encapsulation efficiency (EE) was 91% for nifedipine and 37% for propranolol HCl with the o/w-method, and 83% for nifedipine and 57% for propranolol HCl with the w/o/w-method. DSC and X-ray diffraction studies showed that PCL maintained its semi-crystalline structure, while the drugs were either dispersed or dissolved in the polymer. In vitro release studies revealed a controlled release of nifedipine and propranolol HCl from microparticles prepared by the o/w-method; a burst release of propranolol HCl was observed from microparticles prepared by the w/o/w-method. In conclusion, microparticles containing both a hydrophilic and a lipophilic drug were successfully prepared.

Biodegradable monodispersed nanoparticles prepared by pressure homogenization-emulsification

The aim of the present work was to investigate the preparation of nanoparticles (NP) as potential drug carriers for proteins. The hydrophilic protein bovine serum albumin (BSA) was chosen as the model drug to be incorporated within NP. Owing to the high solubility of the protein in water, the double emulsion technique has been chosen as one of the most appropriate method. In order to reach submicron size we used a microfluidizer as a homogenization device with a view to obtaining NP with a very high grade of monodispersity. Two different biodegradable polymers, poly[D, L-lactic-co-glycolic acid] 50/50 (PLGA) and poly[epsilon-caprolactone] (PCL) has been used for the preparation of the NP. The drug loading has been optimized by varying the concentration of the protein in the inner aqueous phase, the polymer in the organic phase, the surfactant in the external aqueous phase, as well as the volume of the external aqueous phase. The BSA encapsulation efficiency was high (>80%) and release profiles were characterized by a substantial initial burst release for both PLGA and PCL NP. A higher release was obtained at the end of the dissolution study for PLGA NP (92%) compared with PCL NP (72%).

Influences of process parameters on nanoparticle preparation performed by a double emulsion pressure homogenization technique

The preparation of nanoparticles (NP) as an improved colloidal carrier system for proteins was investigated. Bovine serum albumin (BSA) was used as model drug. Owing to the high solubility of the protein in water, the double emulsion technique has been chosen as one of the most appropriate method. In order to both reaching submicron size as well as increasing the grade of monodispersity compared to previous preparation techniques, a microfluidizer as homogenization device was used. All experiments were performed using two biodegradable polymers, poly[D,L-lactic-co-glycolic acid] 50/50 (PLGA) and poly[epsilon-caprolactone] (PCL). The homogenization procedure has been optimized with regard to particle size and monodispersity by studying the influence of the homogenization time as well as the amount of polymer and surfactant in the external aqueous phase. The drug loading has been improved by varying the concentration of the protein in the inner aqueous phase. By increasing the protein concentration in the inner aqueous phase the polydispersity was slightly higher, while the particle size was not influenced significantly. The BSA encapsulation efficiency decreased with higher protein concentration in the inner aqueous phase. All release profiles were characterized by a initial burst effect, a higher release rate was obtained after 4 weeks for PLGA NP (60%) compared with PCL NP (47%).

Characterization of microcapsules by confocal laser scanning microscopy : structure, capsule wall composition and encapsulation rate

The potential of confocal laser scanning microscope (CLSM) has been evaluated for characterizing microcapsules. The aim was to visualize the polymer distribution within the particle wall, and to localize and to quantify the encapsulated oil phase. Microcapsules were prepared by complex coacervation: the oil phase, gelatine, and arabic gum were labelled with fluorescent markers. For all compounds it was proved that fluorescence labelling did not alter physico-chemical properties critical to the encapsulation process. Labelling of the inner oil phase allowed us to identify and to localize, three-dimensionally, the encapsulated compound. A homogeneous distribution for both gelatine and arabic gum throughout the capsule wall was observed. The addition of fluorescently labelled casein as a macromolecular model compound to the coacervate resulted in an inhomogeneous distribution of casein within the wall material, the highest concentration of casein was found at the oil-wall interface. To determine the encapsulation rate, CLSM pictures of the microcapsule samples were acquired using different fluorescence labels for the microcapsule wall polymers and the incorporated oil phase, respectively. By applying computational image analysis, the volumes of the different phases were calculated. Comparing the results of non-destructive image analysis with those obtained by degradation, extraction and chemical analysis, a linear relation was found with correlation coefficients better than 0.980.

Targeted drug-delivery approaches by nanoparticulate carriers in the therapy of inflammatory diseases

Limitations in therapy induced by adverse effects due to unselective drug availability and therefore the use of potentially too high doses are a common problem. One prominent example for this dilemma are inflammatory diseases. Colloidal carriers allow one to improve delivery of drugs to the site of action and appear promising to overcome this general therapeutic drawback. Specific uptake of nanoparticles by immune-related cells in inflamed barriers offers selective drug targeting to the inflamed tissue. Here we focus on nanocarrier-based drug delivery strategies for the treatment of common inflammatory disorders like rheumatoid arthritis, multiple sclerosis, uveitis or inflammatory bowel disease.

Microsphere design for the colonic delivery of 5-fluorouracil

The treatment of colon cancer has been aimed by approaches of oral drug administration. 5-Fluorouracil is the standard treatment still nowadays and would be a candidate to be delivered orally to the colon. A pH-sensitive polymer Eudragit P-4135F was used to prepare microspheres by a simple oil/water emulsification process. Process parameters were analyzed in order to optimize the drug loading and release profiles. In further attempts mixtures with Eudragit RS100 were prepared to prolong drug release. Scanning electron microscopy and confocal laser scanning microscopy permitted a structural analysis. The solvent extraction was preferable over solvent evaporation with a view to the encapsulation rate (extraction: 37%; evaporation: 19%) due to the hydrophilic character of the drug while release pattern were nearly unchanged. Eudragit P-4135F, pure or in mixture, was found to retain drug release at pH 6.8 lower than 35% within 6 h. At pH 7.4, nearly immediate release (within 30 min) was observed for pure P-4135F, while mixtures enabled to prolong the release slightly. Analysis of the morphology led to an inhomogeneous polymer distribution of P-4135F and RS100 throughout the particle core. A capsule-like structure was concluded which allowed only slight changes of the release kinetics by the addition of RS100. However, the formulation proved its applicability in-vitro as a promising device for pH-dependent colon delivery of 5-fluorouracil.

The targeting of surface modified silica nanoparticles to inflamed tissue in experimental colitis.

One aspect in the emerging field of nanomedicine is site specific drug delivery via nanoparticles. The use of nanoparticles allows for increased therapeutic efficiency with a lowered risk for and extent of adverse reactions resulting from systemic drug absorption. 5-Amino salicylic acid (5ASA) loaded silica nanoparticles (SiNP) are proposed here as drug delivery system for specific accumulation in inflamed colonic tissues allowing for selective medication delivery to such inflammation sites. The drug was covalently bound to SiNP by a four-step reaction process. In-vitro toxicity of modified SiNP was tested in appropriate cell culture systems, while targeting index and therapeutic efficiency were evaluated in a pre-existing colitis in mice. Particle diameter was around 140 nm after final surface modification. In-vitro drug release demonstrated significant drug retention inside the NP formulation. Toxicity of the different formulations was evaluated in-vitro cell culture exhibiting a lowered toxicity for 5ASA when bound to SiNP. In-vivo, oral SiNP were found to accumulate selectively in the inflamed tissues allowing for significant amounts of drug load. SiNP demonstrated their therapeutic potential by significantly lowering the therapeutically necessary drug dose when evaluating clinical activity score and myeloperoxidase activity (untreated control: 28.0+/-5.0 U/mg; 5ASA-solution (100mg/kg): 8.2+/-3.4 U/mg 5ASA-SiNP (25mg/kg): 5.2+/-2.4 U/mg). SiNP allow to combine advantages from selective drug targeting and prodrugs appearing to be a promising therapeutic approach for clinical testing in the therapy of inflammatory bowel disease.

Influences of process parameters on preparation of microparticle used as a carrier system for O - 3 unsaturated fatty acid ethyl esters used in supplementary nutrition

Microparticles were prepared by complex coacervation to encapsulate eicosapentaenoic acid ethyl ester (EPA-EE) for incorporation into foods as a nutrition supplement. Gelatin and acacia were used in the coacervation process. With an increasing oil/polymer ratio, both yield and encapsulation rate decreased; with an increasing homogenization time, the yield remained constant while the encapsulation rate slightly increased. Several particle hardening techniques were examined and their influence on particle structure, yield and encapsulation rate were examined. Ethanol hardening was compared to cross-linking with dehydroascrobic acid with respect to both yield and encapsulation rate. The particle diameters for both formulations were similar (ethanol: 38.4 +/- 4.1 microm; cross-linking: 41.8 +/- 3.0 microm). Spray-drying of the coacervates led to the smallest particles (5.2 +/- 1.1 microm), lowest yield and encapsulation rate. All microencapsulation products were assayed for their storage stability over 4 weeks with respect to the oxidation of the encapsulated omega - 3 unsaturated fatty acid ester inside the particles. Hardening with ethanol showed the lowest amount of peroxides: particle wall cross-linking by dehydroascorbic acid and spray-drying were observed to be less protective. All microparticles were characterized for their internal structure with confocal laser scanning microscopy (CLSM) after fluorescence labelling of the polymers, in order to localize the oil phase and visualize the distribution of the polymers in the coacervates. With increasing homogenization time, the internal structure changed stepwise from a capsule structure (core/wall) towards a matrix structure. For all experiments, a homogeneous distribution for both polymers, gelatin and acacia was observed inside the particle wall. No influence of the different particle hardening procedures on the polymer distribution was found.