Development of early diagnosis of Parkinson's disease on animal models based on the intranasal administration of α-methyl-p-tyrosine methyl ester in a gel system.

The fight against neurodegenerative diseases, including Parkinson's disease (PD), is a global challenge of this century. The effectiveness of current PD therapy is limited, since it is diagnosed many years after the onset, following the death of most nigrostriatal dopaminergic neurons regulating motor function. PD treatment could be greatly improved if it was started at an early (preclinical) stage. For this purpose, it is necessary to develop an early diagnosis of PD, which is the goal of our study. We have developed an early diagnosis of PD on animal models using a provocative test by intranasal administration of α-methyl-p-tyrosine methyl ester (αMPTME), a reversible inhibitor of dopamine synthesis. First, we produced the provocative agent, αMPTME in gel, and showed its safety and penetration into the brain bypassing the blood-brain barrier. Then, the optimal dose of αMPTME and time after administration were selected, at which the level of dopamine in the striatum of intact animals decreases, but does not reach the 30% threshold for the appearance of motor disorders in PD patients. Finally, we proved on animal models that intranasal administration of αMPTME can serve as a diagnostic test for preclinical PD. Indeed, intranasal administration of αMPTME to mice in a model of PD at the preclinical stage reversibly reduced the dopamine level in the striatum to the 30% threshold causing short-term motor disorders. Thus, using animal models of PD, we have developed a provocative test for the preclinical diagnosis of PD, a fundamentally new technology in neurology.

How subtle differences in polymer molecular weight affect doxorubicin-loaded PLGA nanoparticles degradation and drug release.

Aims: To evaluate the influence of minor differences in molecular weights of commercially available low molecular weight PLGA grades on the kinetics of doxorubicin release from the nanoparticles.Methods: Three low-molecular weight 50/50 PLGA polymers were thoroughly characterised concerning intrinsic viscosity, molecular weight (Mw), acid value, and residual monomer content. The doxorubicin-loaded nanoparticles prepared using these polymers were evaluated concerning the kinetics of drug release and hydrolytic degradation.Results: The Mw of the polymers were slightly different: 10.2, 10.3, and 4.7 kDa. The nanoparticles obtained from the polymer with Mw of 4.7 kDa exhibited considerably higher rates of drug release and polymer degradation.Conclusion: In the case of low molecular weight PLGA grades even a few kilodaltons could be important for the batch-to-batch reproducibility of the nanoformulation parameters. These results bring forward the importance of in-house characterisation of the polymers to be used for the nanoparticle preparation.

Doxorubicin-loaded PLGA nanoparticles for the chemotherapy of glioblastoma: Towards the pharmaceutical development.

Brain delivery of drugs by nanoparticles is a promising strategy that could open up new possibilities for the chemotherapy of brain tumors. As demonstrated in previous studies, the loading of doxorubicin in poly(lactide-co-glycolide) nanoparticles coated with poloxamer 188 (Dox-PLGA) enabled the brain delivery of this cytostatic that normally cannot penetrate across the blood-brain barrier in free form. The Dox-PLGA nanoparticles produced a very considerable anti-tumor effect against the intracranial 101.8 glioblastoma in rats, thus representing a promising candidate for the chemotherapy of brain tumors that warrants clinical evaluation. The objective of the present study, therefore, was the optimization of the Dox-PLGA formulation and the development of a pilot scale manufacturing process. Optimization of the preparation procedure involved the alteration of the technological parameters such as replacement of the particle stabilizer PVA 30-70 kDa with a presumably safer low molecular mass PVA 9-10 kDa as well as the modification of the external emulsion medium and the homogenization conditions. The optimized procedure enabled an increase of the encapsulation efficiency from 66% to >90% and reduction of the nanoparticle size from 250 nm to 110 nm thus enabling the sterilization by membrane filtration. The pilot scale process was characterized by an excellent reproducibility with very low inter-batch variations. The in vitro hematotoxicity of the nanoparticles was negligible at therapeutically relevant concentrations. The anti-tumor efficacy of the optimized formulation and the ability of the nanoparticles to penetrate into the intracranial tumor and normal brain tissue were confirmed by in vivo experiments.

Delivery of doxorubicin-loaded PLGA nanoparticles into U87 human glioblastoma cells.

The paramount problem in the therapy of brain tumors is the inability of most drugs to cross the blood-brain barrier. PLGA nanoparticles overcoated with poloxamer 188 could overcome this problem and enabled a high anti-tumoral effect against the very aggressive intracranial 101.8 glioblastoma in rats that closely resembles human grade IV glioblastomas. The basis for the transport of these particles across the blood-brain barrier appears to be adsorption of blood apolipoproteins (ApoE or ApoA-I) on the nanoparticle surface caused by the poloxamer 188-coating, followed by receptor-mediated transcytosis of the nanoparticles. The objective of the present study is the elucidation of the mechanism by which the poloxamer 188-coated nanoparticles then enter the brain tumor cells. Their intracellular fate, therefore, was investigated using the U87 human glioma cell line. The main mechanism of the PLGA nanoparticle internalization by U87 cells was clathrin-mediated endocytosis. Within 1h free doxorubicin was released from late endosomes and could reach its target site, i.e. the DNA in the nuclei without degradation, whereas the PLGA nanoparticles, which were labeled with Cy5.5, still were observed in the endo-lysosomal compartment. These results demonstrate that the underlying mechanism of action in the brain cells is by diffusive doxorubicin release from the nanoparticles rather than by their intracellular degradation.

Water-soluble N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride as a nucleic acids vector for cell transfection.

To endow the cationic polysaccharides with solubility in the whole pH-range without loss of functionality of the amino groups, different chitosan samples were treated with glycidyltrimethylammonium chloride. Each modified unit of the exhaustively alkylated quaternized chitosan (QCht) contained both quaternary and secondary amino groups. The intercalated dye displacement assay and ζ-potential measurements implied stability of QCht polyplexes at physiological conditions and protonation of the secondary amino groups in slightly acidic media which is favorable for transfection according to proton sponge mechanism. The cytotoxicity and transfection efficacy increased with the chain lengthening. Nevertheless, the longest chains of QCht, 250 kDa were less toxic than PEI for COS-1 cells and revealed comparable and even significantly higher transfection activity of siRNA and plasmid DNA, respectively. Thus, highly polymerized QCht (250 kDa) provided the highest level of the plasmid DNA transfection being 5 and 80 times more active than QCht (100 kDa) and QCht (50 kDa), respectively, and 4-fold more effective than PEI, 25 kDa. The established influence of QCht molecular weight on toxicity and transfection efficacy allows elaborating polysaccharide vectors that possess rational balance of these characteristics.