Project: Nanoparticle Transfer Through Endothelial Barrier
Call for proposal: M-ERA.NET Call 2018
Contract number: No. 184.108.40.206/ERANET/19/07
Implementation period: 1 October 2019 – 31 August 2023
Project coordinator: University of Lodz
University of Alcala, Faculty of Sciences, Department of Organic and Inorganic Chemistry
Riga Stradiņš University
Latvian Biomedical Research and Study Centre
Leader of Latvian team: Dr. biol., Juris Jansons
Total Costs: 170 000.00 EUR
Many brain-associated diseases (multiple sclerosis, Alzheimer’s disease, stroke, etc.) remain undertreated by effective therapies because therapeutic molecules cannot cross the endothelial barrier, which is an important part of the blood-brain barrier. Some nanoparticles (NP) ‒ gold NP, dendrimers, dendrons ‒ represent a promising new tool for crossing the endothelial and blood-brain barriers and delivering into the brain the biologically active compounds, as drugs and immunotherapeuticals.
As we do not know what properties they have to possess to be able to cross the barriers, we cannot meaningfully design them. All the NPs described in literature were chosen empirically after numerous candidates had been screened. Therefore, the main aim of present project is to elucidate the mechanisms by which the most promising NPs: gold NPs, dendrimers, dendrons and their complexes with drugs are transferred through the endothelial barrier. The in vitro experiments will use an endothelial barrier model: primary human aortic endothelial cells (HAEC). Isolation of primary cells encounters many obstacles, among them the variability of the reagents and procedures and access to appropriate ethically sourced donor tissue type, therefore, we decided to use the commercially available sources.
The mechanisms of NP transfer (uptake, cytotoxicity and efflux) and the functional state of the cells (active/passive transport, proliferation, morphology) will be examined on the basis of the size, charge, hydrophobicity, and flexibility of the NP alone and when loaded with drugs or immunotherapeuticals. The same mechanisms will be analyzed under cell stress conditions using oxidative stress and an acidic medium (related to stroke), and in the presence of amyloid-beta peptides (related to AD). The data obtained will: (i) elucidate the mechanisms of NP transfer through the endothelial barrier depending on their nature, key functional groups, and attached drug(s) and/or immunotherapeutical(s), and (ii) identify the functioning of the endothelial barrier during transfer of NPs and their complexes with drugs/immunotherapeuticals(s) under physiological and pathological conditions. The results will provide a basis for systematic screening of NPs for the delivery of active compounds/substances into the brain.
The in vivo experiments will use the murine models of AD and brain ischemia to study the therapeutic potential of NP complexes with drugs, nucleic-acids based immunotherapeuticals.
The objects for study will be gold NPs of 2-4 different types, the newly synthesized dendrimers of 1st-4th generations, and their corresponding dendrons; and complexes of NPs with drugs (laquinimod), endothelial cell targeting proteins (transferrin, wheat germ agglutinin (WGA)) or short-chain nucleic acids (including siRNA, and immunomodulating short double stranded DNAs and single stranded RNAs). The complexes will be labelled with fluorescent probes to follow up the transfer.
The subject for investigation will be the endothelial barrier model (commercially available primary HAEC) and murine models of Alzheimer’s disease and brain ischemia in experiments in vivo. Primary cells are isolated directly from the donor tissue and therefore maintain tissue characteristics that are closer to in vivo conditions.
The tasks are to:
(1) Study the uptake and efflux of NPs through the endothelial barrier and their cytotoxicity using molecular and cell biology techniques; (2) Estimate the functional state of the endothelial barrier cells in the presence of NPs and their complexes; (3) Repeat (1) and (2) under conditions of endothelial barrier stress induced by oxidative stress and acidification (related to brain ischemia) and the presence of amyloid-beta peptides (related to Alzheimer’s disease); (4) Establish murine models of brain ischemia and AD; and (5) Approve the therapeutic potential of NP complexes of drugs/nucleic acids tested above in in vivo system.
Progress of the project
October 1, 2019 – December 31, 2019
During the reporting period, the Project Consortium Agreement has been agreed and signed. Work is underway on the development of the website, the necessary information is being collected from the consortium partners.
Information published on 31.12.2019.
1 January 2020 – 31 March 2020
During the reporting period, an application is being prepared for the Ethics Commission of the Food and Veterinary Service for a pilot project permit for the use of animals in experiments planned in the project. Work on the website is continuing.
Information published on 31.03.2020.
April 1, 2020 – June 30, 2020
During the reporting period, the development of the project website http://nanotendo.pl/, was completed and it is supplemented with information from the consortium partners. An experimental design was developed in consultation with the consortium partners, taking into consideration the situation with the Covid19 pandemic and the available infrastructure. The ethical aspects of the research were analyzed.
Information published on 30.06.2020.
1 July 2020 – 30 September 2020
During the reporting period, the theoretical basis of the project was supplemented with information from current scientific publications on ischemic stroke and Alzheimer’s disease treatment methods, the possibilities for testing in mouse models of selected therapeutic agents in a complex with nanoparticles (dendrimers) have been evaluated.
Information published on 30.09.2020.
1 October 2020 – 31 December 2020
During the reporting period, experiments on the formation of complexes of dendrimer nanoparticles and therapeutic agents and testing of the stability and other properties of the obtained complexes in in vitro experiments were performed. Based on the results, the siRNA that specifically induces silencing of MAPT genes involved in the development of the disease was selected as a treatment agent for Alzheimer’s disease.
Information published on 31.12.2020.
January 1, 2021 – March 31, 2021
During the reporting period, a strategy has been developed for the labeling of dendrimers and transportable substances with fluorescent markers, which allows to study the distribution of injected drugs in the body and the ability to cross the blood-brain barrier in animal experiments. Due to the situation in Covi19 in Spain and Poland, the schedule of experiments was significantly delayed, thus the project partners agree to extend the project implementation deadline.
Information published on 31.03.2021.
April 1, 2021 – June 30, 2021
During the reporting period, experiments were performed to optimize the stability of complexes of dendrimers and labeled siRNA during storage, transportations and at physiological conditions. The extension of the project duration has been approved, the consortium agreement and the work plan have been amended in accordance with the new project implementation schedule.
Information published on 30.06.2021.
July 1, 2021 – September 30, 2021
Studying the stability of dendrimers and labeled siRNA complexes during transportation under different conditions, it was concluded that the obtained complexes are sensitive to temperature fluctuations. Therefore, the decision was made to transport dendrimers in a dry form and to prepare complexes with siRNA in Latvia just before performing in vivo experiments. Work is underway to transfer the method of preparation of dendrimers and labeled siRNA complexes from the Polish team to the Latvian team.
Information published on 30.09.2021.
1 October 2021 – 31 December 2021
During the reporting period, we worked on mastering the method of preparation of dendrimers and labeled siRNA complexes. The protocol for the preparation of the complexes as well as the necessary raw materials were transferred from the Polish team to Latvia. The properties and stability of the obtained complexes were tested by physical chemistry methods. In parallel, the work was continued preparing of an application to the Ethics Commission of the Food and Veterinary Service for a pilot project permit for the use of animals in experiments planned in the project.
Information published 30.12.2021.
1 January 2022 – 31 March 2022
During the reporting period, work was continued on mastering the method of preparation of dendrimers and labeled siRNA complexes. The biological properties of the obtained complexes (ability to effectively cross the cell membrane) were tested in cell culture experiments. In parallel, the work was continued preparing of an application to the Ethics Commission of the Food and Veterinary Service for a pilot project permit for the use of animals in experiments planned in the project.
Information published 31.03.2022.
1 April 2022 – 30 June 2022
During the reporting period, work was done on the development of mouse models to test the efficacy of the dendrimers and therapeutic agents. Transgenic mice with the introduced human APOE4 gene were selected as the model of the Alzheimer’s disease. The surgical occlusion of the middle cerebral artery in mice was selected as an ischemic stroke model. A protocol was developed to determine the absorption, distribution, and localization of dendrimers and labeled siRNA complexes in mice. The work was continued preparing of an application to the Ethics Commission of the Food and Veterinary Service for a pilot project permit for the use of animals in experiments planned in the project.
Information published 30.06.2022.
July 1, 2022 – September 30, 2022
During the reporting period, work was done on the development of mouse models to test the efficacy of the complexes of the dendrimers and therapeutic agents. The brain, liver, kidney, lungs, and heart murine tissues containing the labeled dendrimers were obtained and sent to the RSU pathologists for biodistribution studies.
For Alzheimer disease model from different kinds of genetically modified mice expressing APOE4 gene the APOE4 KI strain was selected. The genotype and phenotype of the animals was studied for the experiment planning, to choose the optimal age of the animals to show effectiveness of treatment. For the ischemic stroke model the phenotypic changes discriminable analyzing tissues by immunohistology were studied. The work was continued preparing of an application to the Ethics Commission of the Food and Veterinary Service for a pilot project permit for the use of animals in experiments planned in the project.
Information published on 30.09.2022.
Progress of the project
1 October 2022 – 31 December 2022
In consultation with the project coordinator, it was decided to use the drug Laquinimod in complex with dendrimers for the treatment of the ischemic stroke consequences in a mouse model. Carbosilane and polymer-coated gold dendrimer nanoparticles were selected as drug carriers. The characteristics of the required mouse model were specified. The theoretical basis of the project was supplemented with information from current scientific publications on the use of nanoparticles to overcome the endothelium blood-brain barrier, considering the specifics of laboratory animal models. The work was continued preparing of an application to the Ethics Commission of the Food and Veterinary Service for a pilot project permit for the use of animals in experiments planned in the project.
Information published 30.12.2022.
Progress of the project
1 January 2023 – 31 March 2023
After additional consultation with the project coordinator, as a therapeutic agent for the treatment of brain ischemic stroke was chosen siRNA, which blocks the synthesis of metalloproteinase MMP12 in damaged tissues and thus reduces the negative effect of inflammation. An experimental scheme was developed, which includes histopathological and biochemical tests of tissues and behavioral tests of experimental animals to determine the effect of therapy. It is planned to inject the labeled MMP12 siRNA in a complex with two types of nanoparticles – carbosilane dendrimers and dendrimers with a gold core in mice with artificially induced cerebral ischemia and healthy animals.
A model of ischemic cerebral stroke was created – surgical occlusion of the middle cerebral artery in mice. A protocol for collection and processing of brain tissue for subsequent histological analyzes has been developed. The application of the pilot project for permit to use animals in experiments planned in the project has been prepared and submitted to the Ethics Commission of the Food and Veterinary Service.
Information published 31.03.2023.
1 April 2023 – 30 June 2023
Experiments were performed with a mouse model of ischemic stroke. Mice with surgically induced cerebral ischemia were intravenously injected with labeled MMP12 siRNA complexed with two types of nanoparticles – carbosilane dendrimers and dendrimers with a gold core. Behavior tests characterizing the functions of the locomotor system were performed on the animals to evaluate the therapeutic effect of the administered substances. Animal tissue samples have been collected and given to RSU Department of Pathology researchers for histological analysis to determine the effect of the administered substances and their biodistribution among the animal’s organs. The obtained experimental results are analyzed.
Permission has been obtained from the Food and Veterinary Service Ethics Commission for the use of animals in experiments with the Alzheimer’s disease mouse model planned in the project.
Information published 30.06.2023.
Progress of the project
1 July 2023 – 31 August 2023
Experiments were performed using a mouse model of Alzheimer’s disease. The genetically modified mice with introduced human APOE4 gene, which increases the risk of developing Alzheimer’s disease, were used for the experiments. Labeled APOE4 siRNA complexed with two types of nanoparticles – carbosilane dendrimers and dendrimers with a gold core – were injected intravenously into mice. Using the IVIS imaging technique, the distribution of the administered substance in the animal organs was determined. To evaluate the therapeutic effect of the administered substances, the animals were subjected to behavioral tests that characterize cognitive functions. Animal tissue samples have been collected for histological analysis and determination of APOE4 gene expression levels.
Information published 31.08.2023.