Hyaluronate Nanoparticles as a Delivery System to Carry Neuroglobin to the Brain after Stroke

Abstract

Therapies against stroke can restore the blood supply but cannot prevent the ischemic damage nor stimulate the recovery of the infarcted zone. The neuroglobin protein plays an important role in the neuro-regeneration process after stroke; however, the method for its effective systemic application has not been identified yet, as neuroglobin is unable to pass through the blood-brain barrier. Previously, we developed different types of sodium hyaluronate nanoparticles, which successfully cross the blood-brain barrier after stroke. In this work, these nanoparticles have been used to carry neuroglobin through the bloodstream to the nerve cells in rats submitted to stroke. We have biosynthesized rat-recombinant neuroglobin and determined the formulation of sodium hyaluronate nanoparticles loaded with neuroglobin, as well as its size and ζ-potential, encapsulation efficiently, in vitro release, and its kinetic of liberation. The results show that the formulation achieved is highly compatible with pharmaceutical use and may act as a delivery system to transport neuroglobin within the blood. We have found that this formulation injected intravenously immediately after stroke reached the damaged cerebral parenchyma at early stages (2 h). Neuroglobin colocalizes with its nanocarriers inside the nerve cells and remains after 24 h of reperfusion. In conclusion, the systemic administration of neuroglobin linked to nanoparticles is a potential neuroprotective drug-delivery strategy after stroke episodes.

Keywords: nanoparticles; neuroglobin; sodium hyaluronate; stroke.

Figure 1

Stroke model. (A): Schematic representation of the stroke model of transient middle cerebral artery occlusion (tMCAO). CCA (Common Cerebral Artery); ECA (External Cerebral Artery); ICA (Internal Cerebral Artery); MCA (Middle Cerebral Artery). (B): Representative image of the brain of a rat summited to tMCAO stained with TTC, showing the infarcted zone (white) within the right hemisphere.

Figure 2

The methodology used to quantify the fluorescence emitted by the different fluorophores used to label NGB, NPs, and cell nuclei in histological sections of the infarcted cerebral parenchyma. A and B: Graphic representation (A) and semiquantitative data (B) of the mean grey values obtained by the confocal software AF (Leica) from fluorophores Cy2 (channel 1), rhodamine (channel 2), and DAPI (channel 3). (C): Merged image from a histological section of the infarcted parietal cortex, as the result of the overly of images (C1–C3), which, respectively, show Cy2, rhodamine, and DAPI fluorescence separately: Cell nuclei appear in blue (DAPI); neuroglobin (NGB) in green (Cy2) and nanoparticles (NPs) in red (rhodamine) are colocalized (light orange/yellow) in the cytoplasm of the nervous cells.

Figure 3

Size distribution of NGB–NPs.

Figure 4

Microscopy characterization of NGB–NPs. (A) Field with different NGB–NPs at a scale of 1 µm (TEM). (B) Amplified field with a unique NP–NGB (500 nm) (TEM). (C) Microphotograph showing a unique NGB–NP with a 500 nm scale (SEM).

Figure 5

In vitro release curve (%) of the encapsulated NGB transferred by the NGB–NPs in the function of time (over 72 h) in the study.

Figure 6

TEM microphotography of a representative lyophilized NP.

Figure 7

Confocal microscopy images of the parietal cortex of animals submitted to tMCAO after 2 h of reperfusion. (A): Cy2 green fluorescence represents NGB. (B): Rhodamine red fluorescence detects NPs. (C): DAPI blue fluorescence marks cell nuclei. Images are representative microphotographs of samples from 8 experimental animals. (D): Merge of images of (A–C). Scale bar: 10 μm.

Figure 8

Confocal microscopy images of the parietal cortex of animals submitted to tMCAO after 24 h of reperfusion. (A): Cy2 green fluorescence represents NGB. (B): Rhodamine red fluorescence detects NPs. (C): DAPI blue fluorescence marks cell nuclei. Images are representative microphotographs of samples from 8 experimental animals. (D): Merge of images of (A–C). Scale bar: 10 μm.

Figure 9

Merged confocal images of the parietal cortex from animals submitted to tMCAO after 2 h of reperfusion. Each image is the result of the overlay of three images taken to visualize NGB (Cy2), NPS (rhodamine), and cell nuclei (DAPI). Images are representative microphotographs of samples from 5 experimental animals. Insert is a zoom taken at higher magnification from the main image. Scale Bar: 30 μm.

Figure 10

Merged confocal images of the parietal cortex from animals submitted to tMCAO after 24 h of reperfusion. Each image is the result of the overlay of three images taken to visualize NGB (Cy2), NPS (rhodamine) and cell nuclei (DAPI). Images are representative microphotographs of samples from 8 experimental animals. Insert is a zoom taken at higher magnification from the main image. Scale Bar: 30 μm.

Figure 11

Estimation of the mean grey values of the fluorescent emission from rhodamine (NPs), Cy2 (NGB), and DAPI (nuclei) 2 h and 24 h after MCAO, respectively. Results are the average values of 10 measurements from 8 experimental animals in each group. * p < 0.05.