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Quantification of in-vivo confocal neuroimaging of fluorescent Polyvinylpyrrolidone nanoparticles’ kinetics at the neurovascular interface



Abstract

Nanoparticulate systems can serve as vehicles for drug delivery across the blood-brain barrier (BBB), or, when loaded with biomarkers, for diagnostics of neuropathological changes. However, the kinetics of such nanoparticles (NPs) are complex, because it is necessary to differentiate between the loaded carrier and the load released from it. Therefore, we developed a quantitative image analysis workflow for in-vivo and ex-vivo confocal neuroimaging data, which provides a better understanding of the nanoparticles’ kinetics. Here we used nanoparticles that were double-labeled with fluorescent dyes and followed their distribution with in-vivo imaging of the retina. Using the blood-retina barrier (BRB) as a surrogate to the BBB, we utilized live in-vivo confocal neuroimaging (ICON) for the retina and compared our results with ex-vivo wholemount retina preparation.

We fabricated polymeric NPs from Polyvinylpyrrolidone (PVP-NPs) as a new nano-carrier-system and investigated their ability to cross the BRB. PVP-NPs were loaded with fluorescent markers as active compounds and were injected intravenously into the tail vein of rats. Our analysis indicates that linking a hydrophobic compound to the shell of the PVP-NPs can induce their passage into brain tissue and we demonstrated that the distribution of the nano-carriers, with or without hydrophobic surface ligand, have significantly a different distribution in the blood compartment.  To In sum up, we revealed that quantitative analyses of in-vivo confocal imaging data can provide new information about the kinetics of nanoparticles at the neuro-vascular barrier. 

Keywords: blood−retina barrier, in-vivo imaging, ex-vivo imaging, polyvinylpyrrolidone nanoparticles, blood−brain barrier, Arivis Vision4D.

Central nervous system (CNS) diseases’ such as stroke, Morbus Parkinson, or Morbus Alzheimer are on the rise. and t They represent the second largest category of life-threatening diseases (1, 2). Pharmacological treatment of such diseases is, however, hampered by a major obstacle which is the blood-brain-barrier (BBB) (3). The latter BBB is a limiting barrier with complex mechanisms to separate the CNS parenchyma from the blood vessel compartment. It strictly controls the transport of cells and compounds to maintain the delicate brain homeostasis and to protect the brain from harmful substances. However, the existence of this tight barrier is also the reason why approximately about 98% of all therapeutic or diagnostic compounds cannot be delivered into brain tissue. (4-7). Because of these protection mechanisms, only hydrophilic substances lower than 150 Da and lipophilic compounds under a threshold of 400-500 Da can cross the BBB (8). One way to overcome this limitation is to use nanoparticles as a vehicle to cross the BBB, a potential solution for drug delivery to the brain that has long been missing (9, 10).

Polymeric NPs have emerged as a safe nano-carrier system to target the brain because of their biocompatibility and biodegradability. Especially polymeric NPs such as polybutylcyanoacrylate (PBCA), polylactic acid (PLA) and poly-lactic-co-glycolic acid (PLGA) were extensively studied in this context (11-13).

A new polymeric material to synthesize NPs is polyvinylpyrrolidone (PVP).  PVP is a non-toxic, non-ionic and bulky polymer with C=O, C–N and CH2 functional groups that is widely used as a stabilizer in metallic NPs synthesis (14).  

Kuskov et al. (DATE) produced nanoparticles synthesized from PVP using an emulsion method. These NPs have a hydrophobic core with a hydrophilic surface which renders it easy for surface modification and which may be ideal for delivering hydrophobic drugs. They have delivered indomethacin, an anti-inflammatory, hydrophobic drug with a loading efficiency of up to 95% and with high indomethacin content (35%). Hemolysis and cytotoxicity tests showed no significant acute toxicity for 14 days. Furthermore, PVP-NPs showed an exceptionally good stability for at least up to 3 months in saline or when freeze-dried (15, 16). 

Based on these considerations, we have now investigated the PVP-NPs as carriers of fluorescent markers and followed their distribution in different compartments at the blood-retina barrier (BRB), i.e., blood cells, vessel lining and retina tissue. Because the retina is ontogenetically derived from the brain, the BRB is very similar to the BBB and can therefore serve as a BBB model (17, 18). With the ICON technique combined with our new image analysis workflow, we were able to study the NPs’ fate and passage from the vessel lumen into brain tissue to identify and characterize the multifactorial influences, which determine the distribution of NPs at biological barriers (19-21). We hypothesized that the interaction of NPs with peripheral compartments and molecules significantly influences their distribution (22). Therefore, we performed in-vivo confocal neuroimaging (ICON) to monitor the retina in living rats (23, 24), developed and implemented image analysis workflows based on the Arivis Vision 4D software. This allows us to quantitatively analyze the co-localization of the double-labelled particles and to follow the spatial separation of the markers. There are many tools for image analysis available in open sources (e.g. https://imagej.net/Introduction) and commercial software packages. However, putting together a sequence of specific image analysis steps to form a complete workflow is a tricky task. The Arivis Vision4D visualization and analysis software package helps to select and assemble numerous steps of the whole analysis workflow in one flexible pipeline. The user can choose from many functional components ranging from pre-processing, filtering, background correction, several segmentation tools, and further processing of the detected objects as well as export of the data into Excel or CSV files. No programming skills are required for setting up such a pipeline according to the required workflow and the user can easily focus on developing a creative analysis strategy for the specific task. This pipeline is applicable to many data sets reducing bias and ensuring comparability. In addition, analysis pipelines can be shared among different users. The present study is the first to evaluate the PVP as a nano-carrier to target the CNS.