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A biomimetic and neuroprotective delivery nanocapsule for the targeted treatment of post-ischemic stroke effects

Periodic Reporting for period 1 - BIONICS (A biomimetic and neuroprotective delivery nanocapsule for the targeted treatment of post-ischemic stroke effects)

Período documentado: 2018-05-01 hasta 2020-04-30

Ischemic stroke occurs when an artery of the brain blocks due to a clot, resulting in reduced blood supply and subsequent depletion of oxygen and nutrients that lead to neuronal cell death. The damaged brain tissue is further affected during the restoration of the blood flow due to the increased production of small molecules called reactive oxygen and nitrogen species (ROS and RNS, respectively) that lead to the so-called oxidative stress. This subsequently leads to the overproduction of the enzyme, matrix metalloproteinase-9 (MMP-9), that further damages the brain due to the loosening of the physical barrier between the brain and the blood, called the blood-brain barrier (BBB). To date, the current treatments for ischemic stroke are limited to substances that break the clot (e.g. thrombolytics like the tissue plasminogen activator, tPA) or methods for its removal like mechanical thrombectomy. Although these treatments can save the life of the patients, they fail further to protect the damaged brain from the produced oxidative stress.

The development of a therapeutic that will ameliorate the post-ischemic stroke effects would improve the quality of life for stroke patients by ameliorating speech, motor, and cognitive problems. Additionally, it would reduce the economic burden attributed to the overall healthcare strategies for stroke survivors. Finally, it has been shown that antioxidants can be used as therapeutics also after a recurrent stroke rendering the proposed system a good alternative for recurrent stroke patients as well.

The main goal of this project was the fabrication of a biocompatible and antioxidant nanosystem that would have the ability to encapsulate specific therapeutics and deliver them into the damaged brain. This antioxidant system would reduce oxidative stress and the overproduced enzyme MMP-9, aiming at improving the post-ischemia symptoms.
In this project, we fabricated lipid-based nanocapsules made of synthetic (e.g. cetyl palmitate) and natural lipids (e.g. sphingomyelin, total brain lipid extract) able to encapsulate a combination of antioxidants, including nanoceria particles (ROS scavengers) and LNIL (a specific inhibitor of the nitric oxide synthesis). To facilitate the delivery of the nanocapsules into the brain, we attached to their surface specific targeting groups (e.g. T7 and angiopep-2 peptides) that could connect to the endothelial cells of the BBB and improve their crossing to the damaged tissue. The first developed system was comprised of cetyl palmitate nanocapsules encapsulating nanoceria (NC-NLCs). Our published data showed that the antioxidant properties of nanoceria were not affected by their encapsulation, suggesting that the system as a whole could act as an antioxidant therapeutic. Further studies showed that these nanocapsules were able to cross an in vitro model of BBB that we fabricated in the lab. In this study, it was also demonstrated that the nanocapsules could protect neuron-like cells undergoing oxidative stress (treatment with an excess of H2O2, which is the most abundant ROS) from dying. Additionally, it was shown that the treatment of these cells with the antioxidant nanocapsules led to an increased number and length of neurites suggesting neurogenic properties.
Based on these results and aiming at making our system more biomimetic, I proceeded in the fabrication of nanocapsules made from commercially available lipids extracted from a porcine brain. The brain lipids were used as a lipid-matrix able to encapsulate nanoceria and the aforementioned specific inhibitor LNIL (LNIL-Ce-BL). This time we attached on the surface of the fabricated LNIL-Ce-BL nanocapsules two different peptides, and we studied if they facilitate their uptake by the cells of the BBB and the neuronal cells. From the tested peptides, only the so-called T7 and angiopep-2 peptides showed to improve the internalization of the nanocapsules. The antioxidant ability was also tested, and it was found that BIONICS (LNIL-Ce-BL-T7) (Figure 1) act better than free nanoceria or LNIL acting by themselves. The in vitro preliminary data (experiments with cells) presented an enhanced therapeutic effect of BIONICS, compared to the control untreated cells or cells treated only with nanoceria or LNIL. It has to be noted that the therapeutic effect of BIONICS against oxidative stress, as well as the level of neuroprotection that BIONICS can offer, was assessed under a modified ischemia/reperfusion model reported in another study. The data showed that BIONICS could reduce oxidative stress derived from overproduced reactive oxygen and nitrogen species, and can protect both the endothelial cells of the BBB as well as the neuron-like cells from dying.
Although additional and more extensive studies are needed, the results derived from this project suggest the potential use of a lipid-based antioxidant treatment as adjuvant therapy for the amelioration of post-ischemia effects.

During this project, a novel idea, on the development of a biomimetic delivery system acting as a versatile theranostic, was generated and is under review for its patentability.

The results of the project were disseminated through a series of conferences (ESB 2019, Nanotoday 2019, EMRS 2018, ESB 2018) and publications (ACS Biom. Sci. & Eng., DOI: 10.1021/acsbiomaterials.8b01033 Adv. Funct. Mater., DOI: 10.1002/adfm.201906283 J. Mater. Chem. B., DOI: 10.1039/D0TB00260G.
Furthermore, a short video-animation in which the causes of ischemic stroke and how BIONICs can help, was created for the non-scientific audience. The video was uploaded in the BIONICs' webpage, as well as in Youtube ( under the name 'Cell Wars-Ischemic Stroke: A New Hope (With Subtitles)'
In this project, we were able to fabricate brain lipid hybrid nanocapsules loaded with inorganic (nanoceria particles, a reactive oxygen species scavenger) and organic (LNIL, an inhibitor of nitric oxide production) components. We were also able to attach on their surface a variety of targeting groups and improve their uptake, under static and dynamic conditions, by specific cells. BIONICS demonstrated that it can reduce the oxidative stress in cells that underwent ischemia/reperfusion using a modified model already described in the literature. Additionally, it demonstrated its ability to protect cells (endothelial and neuronal) from the programmed cell death (apoptosis), as well as specific neurogenic properties since it helped in the faster development of neurites. Two of the main aspects that the project aimed at achieving were a socio-economic impact and prospective societal implications. To date and with the current generated data, the points mentioned above cannot be adequately evaluated. Even though the produced data are encouraging and can support further studies, they are not enough to assess any future impact, unless preclinical studies are carried out. However, the preparation of an invention disclosure form data generated from this project suggests potential commercialization of the system, which may result in future socio-economic changes.