Joerg Kreuter

Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for anti-inflammatory drugs

Chitosan microspheres were prepared by a novel precipitation process using sodium sulfate as precipitant. Low, medium, and high molecular weight chitosan was chosen for the formulation of microspheres. The extent of precipitation was controlled by the concentration of sodium sulfate and monitored by turbidity measurement. The amount of sodium sulfate required for the preparation of the microspheres depended on the molecular weight of chitosan. The particle size was determined by photon correlation spectroscopy (PCS) and centrifugal sedimentation. The morphological characteristics were examined using a scanning electron microscope (SEM). The surface charge was measured by microelectrophoresis. After preparation the loading property with various anti-inflammatory drugs was investigated using spectrophotometry. The influence of surface adsorption on the drug modification was controlled by differential scanning calorimetry (DSC). Drug liberation was tested in vitro using side-by-side diffusion cells with a dialysis membrane made of cellulose acetate. The highest loading (up to 30.5% relative to the polymer mass) was achieved with prednisolone sodium phosphate (PSP). The adsorbed drug was present in an amorphous form. The drug release from the microspheres was dependent on the drug-polymer ratio.

Covalent Linkage of Apolipoprotein E to Albumin Nanoparticles Strongly Enhances Drug Transport into the Brain

Drug delivery to the brain is becoming more and more important but is severely restricted by the blood-brain barrier. Nanoparticles coated with polysorbates have previously been shown to enable the transport of several drugs across the blood-brain barrier, which under normal circumstances is impermeable to these compounds. Apolipoprotein E was suggested to mediate this drug transport across the blood-brain barrier. In the present study, apolipoprotein E was coupled by chemical methods to nanoparticles made of human serum albumin (HSA-NP). Loperamide, which does not cross the blood-brain barrier but exerts antinociceptive effects after direct injection into the brain, was used as model drug. Apolipoprotein E was chemically bound via linkers to loperamide-loaded HSA-NP. This preparation induced antinociceptive effects in the tail-flick test in ICR mice after i.v. injection. In contrast, nanoparticles linked to apolipoprotein E variants that do not recognize lipoprotein receptors failed to induce these effects. These results indicate that apolipoprotein E attached to the surface of nanoparticles facilitates transport of drugs across the blood-brain barrier, probably after interaction with lipoprotein receptors on the brain capillary endothelial cell membranes.

Influence of the type of surfactant on the analgesic effects induced by the peptide dalargin after its delivery across the blood-brain barrier using surfactant-coated nanoparticles

The ability of 12 different surfactants, coated onto the surface of nanoparticles, to facilitate the delivery of a nanoparticle-bound model drug, dalargin, was investigated. The leu-enkephalin analogue hexapeptide dalargin was bound to polybutylcyanoacrylate nanoparticles by sorption for 3 h. Different surfactants were then coated over these nanoparticles and were injected intravenously into mice. Nociceptive analgesia was then measured by the tail-flick text 15, 30, 45 and 90 min after injection. Only nanoparticles that had been coated with polysorbate 20, 40, 60 and 80 yielded a significant effect. The highest effect was observed with polysorbate 80. Maximum effects were found after 15 min, at a dalargin dosage of 10 mg/kg, and after 45 min, with 7.5 mg/kg.

Brain targeting of nerve growth factor using poly(butyl cyanoacrylate) nanoparticles

The nerve growth factor (NGF) is essential for the survival of both peripheral ganglion cells and central cholinergic neurons in the basal forebrain. The accelerated loss of central cholinergic neurons during Alzheimer’s disease may be a determinant cause of dementia, and this observation may suggest a possible therapeutic benefit from treatment with NGF. In recent years, convincing data have been published involving neurotrophic factors for the modulation of dopaminergic transmission within the brain and concerning the ability of NGF to prevent the degeneration of dopaminergic neurons. In this connection, the administration of NGF may slow down the progression of Parkinson’s disease. However, NGF, as well as other peptidic neurotrophic factors, does not significantly penetrate the blood–brain barrier (BBB) from the circulation. Therefore, any clinical usefulness of NGF as a potential CNS therapy will depend on the use of a suitable carrier system that enhances its transport through the BBB. The present study investigates brain delivery of NGF adsorbed on poly(butyl cyanoacrylate) (PBCA) nanoparticles coated with polysorbate 80 and the pharmacological efficacy of this delivery system in the model of acute scopolamine-induced amnesia in rats as well as in the model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonian syndrome. As shown by the passive avoidance reflex (PAR) test, the intravenous administration of the nanoparticle-bound NGF successfully reversed scopolamine-induced amnesia and improved recognition and memory. This formulation also demonstrated a significant reduction of the basic symptoms of Parkinsonism (oligokinesia, rigidity, tremor). In addition, the efficient transport of NGF across the BBB was confirmed by direct measurement of NGF concentrations in the murine brain. These results demonstrate that the PBCA nanoparticles coated with polysorbate 80 are an effective carrier system for the transport of NGF to the central nervous system across the BBB following intravenous injection. This approach may improve the NGF-based therapy of age-related neurodegenerative diseases.

Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model.

Background The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. Methodology/Principal Findings In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. Conclusions/Significance This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.

Influence of the surfactant concentration on the body distribution of nanoparticles.

The rapid reticuloendothelial system (RES) uptake of nanoparticles after i.v. injection, especially by the liver, can be reduced and the body distribution can be altered by coating them with non-ionic surfactants. In the present work 2-14C-poly(methyl methacrylate) nanoparticles were coated with poloxamine 908 and polysorbate 80, and the influence of different surfactant concentrations on the body distribution was investigated. These surfactants were chosen because earlier studies showed that poloxamine 908 was very effective in decreasing the liver uptake and keeping the nanoparticles in circulation, whereas polysorbate 80 was the most effective surfactant to direct the particles to organs that do not belong to the RES. Above nanoparticles were injected i.v. to rats and the animals were sacrificed after 30 min. Below a surfactant concentration of 0.1% the nanoparticle preparations behaved like uncoated particles. At a 0.1% concentration a very sudden and significant change in the body distribution occurred with poloxamine 908. The liver concentration decreased from about 75% of the dose to 13% and stayed at this level at higher surfactant concentrations. This decrease was combined with a similar sudden complementary increase in blood and other organ and tissue concentrations. With polysorbate 80 the decrease in liver concentration and increase in the blood and the other organ levels was gradual and became important only above 0.5% surfactant concentration. The results indicate that the type of interaction and the strength of the adsorptive binding to the nanoparticles are different with different surfactants. This in turn leads to different body distribution patterns after i.v. injection of surfactant coated nanoparticles.

Kinetics of transport of doxorubicin bound to nanoparticles across the blood-brain barrier.

Drug delivery to the brain is restricted due to the blood-brain barrier (BBB). Previously, it has been shown that surfactant-coated doxorubicin-loaded nanoparticles were successful in overcoming the BBB and were effective in the treatment of rat brain tumours. However, drug distribution in brain tissue after crossing the BBB was never determined. To distinguish between the amounts of drug in the whole brain and the fraction of drug in the brain parenchyma after crossing the BBB a capillary depletion technique was employed. For this purpose rats were intravenously treated with a doxorubicin solution in 1% polysorbate 80, or doxorubicin-loaded poly-(n-butyl cyanoacrylate) (PBCA) nanoparticles without and with 1% polysorbate 80 coating, respectively. The dosage of doxorubicin was 5 mg per kg of rat body weight. At 30 min, 2 h, and 4 h following intravenous injection into the tail vein, the rats were sacrificed and their brains removed. Homogenates of the brains were prepared. In addition, one part of the homogenate was separated by centrifugation into a pellet (vascular elements) and supernatant (parenchyma) using a well established capillary depletion technique. The time-dependent distribution of doxorubicin in these brain fractions was studied. Clinically effective concentrations in all investigated brain fractions could only be detected in rats treated with surfactant-coated nanoparticles, indicating a significant transcytosis across the BBB. Only low concentrations were observed after 0.5 and 2 h with the uncoated nanoparticles. No uptake of doxorubicin into the brain was observable after administration of drug solution alone. These observations demonstrate the great potential of surface-coated PBCA nanoparticles for the delivery of drugs to the central nervous system.

In vitro characterization of poly(methyl-methaerylate) nanoparticles and correlation to their in vivo fate

Abstract The organ distribution of poly (methyl methacrylate) nanoparticles surface-modified by the adsorption of surfactants was related to the physicochemical propertics. The nanoparticles were modified by coating with poloxamer, poloxamine, Brij and polysorbate surfactants. They were characterized in terms of coating layer thickness, surface hydrophobicity (hydrophobic interaction chromatography) and interaction with serum components (zeta potential). The liver/spleen uptake of partictes coated with low molecular weight surfactants has been related to their hydrophobic coating layer interacting strongly with serum components. The protective effect of poloxamer 338 and poloxamine 908 could be explained by their ability to render the surfaces of the particles hydrophilic, which made a reduction of serum opsonization possible. The in vitro characterization methods proved to be suitable to select surfactants with high potential to reduce the uptake of nanoparticles by liver and spleen macrophages. The affinity of particles to other sites in the body (bone marrow, lungs) could, however, not be explained using the above-mentioned in vitro characterization methods.

Influence of polybutylcyanoacrylate nanoparticles and liposomes on the efficacy and toxicity of the anticancer drug mitoxantrone in murine tumour models.

Polybutylcyanoacrylate (PBCA) nanoparticles were prepared and loaded with mitoxantrone, a highly effective anticancer drug. The proportion of mitoxantrone bound to the particles was analysed to be about 15 per cent of the initial drug concentration with the incorporation method and about 8 per cent with the adsorption method. Selected nanoparticle formulations were tested in leukaemia- or melanoma-bearing mice after intravenous injection. Efficacy and toxicity of mitoxantrone nanoparticles were compared with a drug solution and with a mitoxantrone-liposome formulation (small unilamellar vesicles with a negative surface charge). Furthermore, influence of an additional coating surfactant, poloxamine 1508, which has been shown to change body distribution of other polymeric nanoparticles, was investigated. It was shown that PBCA nanoparticles and liposomes influenced the efficacy of mitoxantrone in cancer therapy differently: liposomes prolonged survival time in P388 leukaemia, whereas nanoparticles led to a significant tumour volume reduction at the B16 melanoma. Neither nanoparticles nor liposomes were able to reduce the toxic side-effects caused by mitoxantrone, namely leucocytopenia. A slight additional influence of the coating surfactant was observed with only one preparation.

Influence of surface-modifying surfactants on the pharmacokinetic behavior of 14C-poly (methylmethacrylate) nanoparticles in experimental tumor models.

AbstractPurpose. The aim of this study was to investigate the different pharmacokinetic behavior of surface-modified poly(methylmethacrylate) (PMMA) nanoparticles. Methods. The particles were 14C-labeled and coated with polysorbate 80, poloxamer 407, and poloxamine 908. Plain particles served as control particles. In vivo studies were performed in three tumor models differing in growth, localization, and origin. Particle suspensions were administered via the tail vein, and at given time animals were killed and organs were dissected for determination of PMMA concentration. Results. For the PMMA nanoparticles coated with poloxamer 407 or poloxamine 908, high and long-lasting concentrations were observed in the melanoma and at a lower level in the breast cancer model. In an intracerebrally growing glioma xenograft, the lowest concentrations that did not differ between the tumor-loaded and tumor-free hemispheres were measured. Organ distribution of the four investigated batches differed significantly. For instance, poloxamer 407- and poloxamine 908-coated particles circulated over a longer period of time in the blood, leading additionally to a higher tumor accumulation. In contrast, plain and polysorbate 80-coated particles accumulated mainly in the liver. The strong expression of vascular endothelial growth factor and Flk-1 in the melanoma correlated with high concentrations of PMMA in this tumor. Conclusion. The degree of accumulation of PMMA nanoparticles in tumors depended on the particle surface properties and the specific growth differences of tumors.