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Treatment of traumatic brain injury using dye-loaded polymeric nanoparticles

Periodic Reporting for period 1 - NEUROTARGET (Treatment of traumatic brain injury using dye-loaded polymeric nanoparticles)

Reporting period: 2018-12-01 to 2020-11-30

Traumatic brain injury (TBI) is the number one cause of death and disability in children and young adults. Despite well-known mechanisms of secondary brain damage after TBI effective treatments have not been established yet. Therefore, novel approaches to develop therapeutics which have translational potential are at urgent need. One promising strategy is exploiting nanoparticles (NPs) to deliver the pharmacological substance into the brain in high concentration. However, the blood-brain barrier (BBB) remains very challenging for nanoparticles to cross and therefore their bio-distribution in the brain is a particularly important topic in this field. Obviously, the lack of methods for the direct observation of NPs’ makes it significantly harder to establish brain targeting. Hence, the detection of NPs at the single-particle level with good spatial-temporal resolution is a key approach for the future development of central nervous system (CNS) drug-delivery systems (DDS).
The aim of this project was to establish a nanoscale platform able to deliver pharmacologically active substance into the injured brain for the treatment of TBI that has the potential for clinical translation.
The project addressed the following questions: 1) To create and optimize an NP-based platform for drug delivery to the brain (WP1), including tracking NP trajectories in healthy and injured brain. 2) To analyse the ability of the NP platform to transport cargo across the BBB (WP2), including establishing a Forster-resonance energy transport (FRET) nanoscale system to track cargo release in healthy and injured brain. 3) To evaluate the effectiveness of targeted treatment after TBI (WP3).
During the project the following work was performed:

1) Establishing a super-bright nanoscale platform
2) Establishing a TBI model including behavioural tests
3) Achieving advanced imaging methods through in vivo 2-photon microscopy (2PM), confocal and light-sheet microscopy
4) Establishing a tissue clearing protocol for visualization of NPs in the brain
5) Visiting the Secondment at University of Strasbourg, France
6) Establishing the protocols for visualization of single NPs at real-time in vivo and ex vivo
7) Analyzing the NPs distribution inside healthy and injured brain in vivo and ex vivo
8) Establishing a FRET nanoscale platform for tracking the cargo release
9) Establishing the protocols for visualization of FRET NPs at real-time in vivo
10) Analyzing the cargo release in real-time in vivo in healthy and injured brains

During the project the following results have been achieved so far:

1) Result. We visualize for the first time in real-time NPs trajectories in healthy and injured brain using a combination of super-bright polymeric NPs and intravital 2PM.
Exploitation. The use of the currently developed ultrabright NPs pushes the boundary of the detection of NPs’ in living organisms significantly, thus allowing rapid and precise assessment of the biodistribution and bioavailability of NPs in real-time. We demonstrated how the combination of NPs' design and the latest imaging technologies can help to enhance the precision of nano-formulation and nano-biodistribution thereby providing direct evidence how NPs cross biological barriers and distribute within living tissue. Using our approach researches will be able to track NPs with various targeting systems and will be able to investigate brain targeting of NPs in real time.
Dissemination. Conference: in 11th European and Global Summit for Clinical Nanomedicine, Targeted Delivery and Precision Medicine. Basel, Switzerland, 2018.
Article: Khalin I et al. Ultrabright Fluorescent Polymeric Nanoparticles with Stealth Pluronic Shell for Live Tracking in Mouse Brain. ACS Nano 2020, 14, 8, 9755–9770; doi:10.1021/acsnano.0c01505
2) Result. We demonstrate that Poloxamer-188, a specific coating agent of NPs, stabilizes FDA approved Poly (Lactic-co-Glycolic Acid) (PLGA) NPs in cell culture and in mouse blood, and increases elimination from blood. Using our novel experimental approach, we demonstrate that uncoated PLGA NPs are mainly taken up by liver and spleen macrophages, while Poloxamer-188 coating shifts the uptake towards brain capillary endothelial cells where NPs accumulate in late endosomes.
Exploitation. The combination of novel, highly fluorescent PLGA NP with high resolution in vivo and ex vivo imaging, and strategies to avoid unspecific autofluorescence allowed us to unambiguously detect the biodistribution of NPs down to the subcellular level with so far unprecedented accuracy, specificity, and resolution. This may help to significantly close the existing translational gap between the preclinical and clinical evaluation of NPs.
Dissemination. Conference: 11th European and Global Summit for Clinical Nanomedicine, Targeted Delivery and Precision Medicine, Basel, Switzerland, 2018 as well as at Centre for Stroke and Dementia Research, Noon Seminar, Munich, 2020.
Article: Khalin I et al. Highly fluorescent biodegradable PLGA nano-carriers allow real-time tracking of individual particles in vivo. bioRxiv 2020.11.19.385062; doi: https://doi.org/10.1101/2020.11.19.3850623
3) Result. We established a nanoscale platform able to track the release of cargo in real-time in living animal. We first demonstrate that using a FRET system it is possible to detect NP degradation in real-time in vivo. This allowed us for the first time to understand when and where exactly NPs released their cargo in tissue.
Exploitation. We addressed the major issue in the field how to differentiate between intact and decomposed NPs. We prepared NPs carrying two different fluorescent molecules optimized for 2-photon excitation which undergo FRET when being in close proximity to each other. Upon degradation of the NPs the distance between the dyes increases and their fluorescence properties change creating a shift in fluorescence emission. This system serves as a universal platform for imaging DDS and may be of high interest for scientists pursuing drug discovery.
Dissemination. Conference: Society for Neuroscience Annual Meeting, Chicago, IL, USA, October 19-23, 2019.
Our new technology of real-time single NPs tracking in living mouse brain has great potential for basic medical NP research. Using our approach researches will be able to track NPs with various targeting systems under physiological as well as under pathological conditions and will be able to investigate brain targeting of NPs in real time. Our data referred to NPs distribution in healthy and injured brains will help to establish new approaches to target different brain compartments after the brain injury through nanoscale systems. This will help to significantly reduce the translational gap in the field of nanomedicine. In the future, our approach may help to develop novel nano-medicines for the treatment of TBI. Apart from this, our results may have the potential to support the development of novel therapeutics for neurodegenerative diseases and to improve brain imaging.
Conclusion. The current inter-disciplinary project elegantly combined the latest knowledge from pharmaceutical science, nanotechnology, neuroscience and bioimaging thereby developing a novel, multi-functional nanoscale platform that helps to visualize NPs in vivo at real-time. Thereby we were able to develop a clinically relevant strategy how to precisely target drug carriers to the brain.
Super-bright nanoparticles open new treatments approaches for CNS diseases