Polymeric nanoparticles improved Curcumin brain delivery and its therapeutic ecacy against intracerebral hemorrhage

Conclusion: Cur-NPs represent a potentially effective strategy to enhance Cur brain delivery and therapeutic efficacy in the treatment of ICH.


Background
Intracerebral hemorrhage (ICH) is a severe type of stroke, which leads to 15-25% of strokes [1]. The survival rate of 1-year in ICH patients is less than 40% [2].
The specific mechanism of severe neurological injury occurring following ICH is not clear. During the acute phase of ICH, hemoglobin is quickly degraded into iron, carbon monoxide and biliverdin. Large amounts of iron are thus released into the extracellular space [3]. Accumulating evidence suggested that iron overload could be a potent contributor to perihematomal edema, peroxide accumulation and cell death [4].
Ferroptosis is a new form of regulated cell death triggered by lipid peroxidation in an iron-dependent manner [5]. Resent studies demonstrated that ferroptosis plays an indispensable role in the pathological process of ICH [6].
Targeting ferroptosis may represent a novel therapeutic strategy for the treatment of ICH [7].
Although a degree of efficacy can be achieved through the surgical evacuation of hematoma in ICH patients, the clinical applications of surgery is limited by its high costs and significant risks [8][9][10]. Unfortunately, effective pharmaceutical approaches for ICH are lacking at present [11]. One of the challenges in drug development is the poor oral bioavailability and brain accumulation [12,13].To resolve these puzzles, extensive research efforts have been pursued in the quest for novel compounds from herbal medicines [14].
Curcumin (Cur), a widespread phenolic compound, is derived from the rhizome of Curcuma longa. Recent studies have reported that Cur has widely pharmacological functions including anti-oxidation, anti-inflammation and neuroprotective effects [15][16][17]. Crucially, Cur effectively attenuated the hematoma volume and neurological injury in ICH model mice [18]. Nevertheless, its poor solubility in water, low oral bioavailability and difficulty in transporting across physiological barriers reduce the efficacy [19]. Multiple efforts have been devoted to improve Cur performance via encapsulating Cur in liposomes, polymeric micelles, microspheres and solid lipid nanoparticles [20][21][22]. These strategies increased Cur bioavailability to some degrees, but no studies showed the pharmacokinetics of drugs and whether they can cross biological barriers remains not elucidated. So, it is urgent to develop a new strategy which can increase the oral bioavailability and brain accumulation of Cur in ICH treatment .
Polymer Nanoparticles (NPs) are extensively used drug delivery matrix material with good biocompatibility, which have been approved by the US Food and Drug Administration [23,24]. Previous work reported that NPs can improve the efficacy, solubility, bioavailability and pharmacokinetic profiles of drugs [25,26]. Importantly, NPs have many advantages over other delivery platforms with multifunctionality, less toxicity and lower immune response [27]. Furthermore, NPs are prepared by an anti-solvent precipitation method, which is cheap and easy to perform [28,29].
In this work, we utilized an anti-solvent precipitation to make Cur polymer nanoparticles (Cur-NPs). Madin Darby canine kidney (MDCK) cells have tight junctions and polarized mucus layersare, which are similar to intestinal epithelial cells [30]. So we employed them as an in vitro drug absorption model. The endothelial tight junction-based blood-brain barrier (BBB) in zebrafish is similar to that of higher vertebrates [31]. Therefore, we used zebrafish to investigate the in vivo distribution and elimination process of Cur-NPs. Furthermore, we analyzed the plasma and brain pharmacokinetics of Cur-NPs in mice and assessed their neuroprotective effects on ICH model mice. Finally, a vitro study was conducted to evaluate the anti-ferroptosis effect of Cur-NPs on erastin-induced HT22 mouse hippocampal cells.

Cur-NPs characterization
In this study, Cur-NPs were made by an anti-solvent precipitation method (Fig.1A).
Cur-NPs were predominantly spherical and ranged from 70 nm to 100 nm in diameter under transmission electron microscope (TEM) (Fig. 1B). The dynamic light scattering (DLS) detection showed a particle size of 127.31 ± 2.73nm, a PDI of 0.21±0.01 (Fig. 1C) and a zeta potential of-0.25±0.02 mV (Fig. 1D). The results of powder X-ray diffraction (XRD) showed that there were no distinctive Cur peaks at 17°in Cur-NPs, potentially due to the larger Cur-NPs percentage resulting in masking of Cur peaks (Fig. 1E).

Cur-NPs cellular uptake and distribution assessment
The cellular uptake progress of Cur-NPs into the MDCK cells was in a time-dependent and concentration-dependent manner ( Fig. 2A). To determine the cellular uptake mechanism of Cur-NPs in vitro, MDCK cells were incubated with different kinds of endocytosis inhibitors. Among these inhibitors, Chlorpromazine

Analysis of Cur-NPs biodistribution in vivo
To determine the in vivo biodistribution of Cur-NPs after oral administration, we incubated zebrafish with free C6 or C6/Cur-NPs. In the C6/Cur-NPs group, from 15 min to 60 min, the fluorescence in the intestine and brain was significantly enhanced in a time-dependent manner. In contrast, the free C6 group showed minimal fluorescence. These results clearly suggested the higher absorption of the fluorescence delivered as the NPs, which strongly indicated that the NPs can cross BBB and lead to superior brain accumulation. Interestingly, C6/Cur-NPs were distributed to the eyes, which indicated that NPs can also cross blood retinal barrier. On the basis of these results, NPs were able to improve the absorption and brain accumulation of Cur (Fig.   3A). The concentration of Cur-NPs in the plasma of C57BL/6 mice was significantly higher than that received Cur alone (Fig. 3B). These findings provided strong evidence that NPs loading of Cur could significantly increase the systemic circulation because of the advantageous particle size and surface properties of NPs. The concentration of Cur-NPs in the brains was remarkably higher than that of the control group, reaching a peak at 8 h, which indicated that the prolonged sustained release of Cur-NPs in the brain lead to better brain accumulation (Fig. 3C). Moreover, the concentration of Cur-NPs in heart, liver, spleen, lung and kidney were also significantly higher than those in the control, which may be related to the prolonged sustained release of Cur-NPs and its high plasma exposure rate ( Fig. 3D-H) [33].

Cur-NPs attenuated ICH induced behavioral deficits
In the rotarod test, ICH lead to an obvious drop in the latency to fall, while Cur-NPs caused an obvious increase in latency to fall. Besides, Cur-NPs effectively reversed the ICH-induced increase in the number of drops (Fig. 4A). In the pole climbing test, ICH lead to a marked increase in turn downward (T-turn) and the total time (T-total).
While Cur-NPs treatment reversed these effects (Fig. 4B). Cut-NPs were significantly more effective than free Cur as a means of mitigating behavioral deficits. Overall, these findings suggested that Cur-NPs can significantly attenuated behavioral deficits in mice.

Cur-NPs decreased the hematoma volumes in the brain of mice
Cur-NPs treatment obviously decreased the hematoma volumes in the brain of mice compared with the ICH and Cur groups (Fig. 5A, 5B). Moreover, the results of Hematoxylin eosin (HE) staining showed that ICH lead to significant neural loss in the perihematoma brain tissues, while Cur-NPs treatment effectively mitigated the neural damage (Fig. 5C).

Fig.5
Cur-NPs decreased the hematoma volumes in the brain of mice. A Images of hematoma of ICH mice brain sections. B The hematoma volumes of ICH mice in each group (means ± SD, n = 6). C HE staining of the brain tissues in each group.

Cur-NPs attenuated ICH induced neurological injury
Nissl staining showed that ICH resulted in obvious neuronal degeneration in the perihematoma brain tissues of ICH mice. In contrast, Cur-NPs significantly attenuated the neuronal degeneration in the perihematoma brain tissues of ICH mice. Cur-NPs exhibited a better curative effect than Cur. Prussian blue staining was conducted to assess the intracellular iron accumulation and distribution in perihematoma brain tissues. The results of Prussian blue staining showed that Cur-NPs markedly decreased the number of Prussian blue positive cells compared with the ICH and Cur groups, suggesting that Cur-NPs was effective to reduce iron deposition in the perihematoma brain tissues. Glutathione peroxidase 4 (GPX4) is a subtype of glutathione peroxidase that can detoxify lipid peroxidase [34]. It is a molecular marker of ferroptosis which can inhibit ferroptosis through decreasing the lipid peroxidization in cells [35,36]. In this study, it was observed that Cur-NPs administration reduced the compensatory overexpression of GPX4 in the perihematoma region after ICH (Fig. 6). These results thus suggested that Cur-NPs treatment significantly attenuated ferroptosis in ICH.

Cur-NPs inhibited ferroptosis induced by erastin in HT22 cells
MTT assay showed that conventional-dose Cur-NPs (not higher than 20 μM) existed no obvious toxic effect on the cell viability (Fig.7A). We utilized erastin, a ferroptosis inducer to investigate the anti-ferroptosis effects of Cur-NPs [37]. MTT test showed that Cur-NPs treatment effectively enhanced the cell viability relative to the erastin group and Cur-PM group (Fig.7B). Likewise, the live/dead cell staining assay revealed the consistent results that Cur-NPs dramatically increased the survival rate of HT22 cells (Fig.7C).

Conclusions
In this study, Cur-NPs were made by an anti-solvent precipitation method. Cur-NPs can draw into cells through a number of nonspecific endocytosis mechanisms, mainly mediated via clathrin and plasma membrane microcapsules. Cur-NPs tended to accumulate in the endoplasmic reticulum and lysosome. Moreover, Cur-NPs could transport across physiological barriers and improve Cur accumulation in the plasma and brain. Importantly, Cur-NPs were an effective treatment for ICH through inhibiting ferroptosis. Taken together, Cur-NPs represent a potentially effective strategy to enhance Cur brain delivery and therapeutic efficacy in the treatment of ICH.

Materials
Cur and PVP k90 was purchased from Nantong Feiyu Biological Technology Co., Ltd.

Cur-NPs preparation and characterization
In our research, Cur-NPs were made by an anti-solvent precipitation method. An organic phase: Cur (20 mg/ml) and mPEG(2K)-PTMC(16K) 20 mg/ml were dissolved into acetone. An aqueous phase was prepared through dissolving PVP k90 (2 mg/ml) into water. Next, 0.2 mL organic phase was poured into 10 mL aqueous phase, and the mixture was subjected to 1000 rpm/min stirring to make the Cur-NPs.
Additionally, C6 and mPEG-PTMC were mixed in the organic phase at a ratio of 1:30 to make C6-NPs. To remove the residual solvent, the obtained C6-NPs were stirred in the dark at room temperature for 2 h. The physicochemical properties of Cur-NPs were analyzed, including morphology by a TEM, the particle size, zeta potential and crystalline patterns by XRD.

Endocytosis mechanism detection of Cur-NPs
To analyze the endocytic mechanisms of Cur-NPs, MDCK cells were incubated with five different endocytosisinhibitors including EIPA, LY, CPZ, HS and MβCD for 30 min [38]. Next, the C6-NPs were incubated with MDCK cells for 1 h. Then the liquid was removed and the cells were washed three times in PBS. After that, the samples were fixed with 4% paraformaldehyde (PFA) for 5 min. At last, the images of the samples were obtained by using a confocal laser scanning microscopy (CLSM; TCS SPEⅡ, Leica, Germany).

In vitro cellular uptake of Cur-NPs
MDCK cells were seeded on coverslips at a cell density of 5×10 4 in 24-well plates.
Next, we cultured C6-NPs with complete medium to 0.5 μg/mL, 1.0 μg/mL and 2.0 μg /mL respectively. The incubation of medium was removed at different time points (10, 30, 60 min). Subsequently, we fixed the samples with 4% PFA. At last, the images were acquired using a fluorescence microscope (model DMi8, Leica, Germany) at 488 nm. In the present work, we selected Lyso, ER and Mito as detection organelles. The cells were mixed with Lyso tracker, ER tracker and Mito tracker for 120 min [39]. Sequentially, the samples were rinsed with serum-free medium for 5 min three times and fixed with C6-NPs. The colocalization of C6-NPs with the organelles were imaged using a confocal laser scanning microscopy (CLSM; TCS SPEⅡ, Leica, Germany).

In vivo biodistribution of Cur-NPs in zebrafish
In this study, we obtained zebrafish (Danio rerio) from the China Zebrafish Resource Center (Wuhan, China). The zebrafish were housed on a 14 h/10 h light/dark cycle.
Male and female zebrafish were separated at a 1:2 ratio in a 1 L tank overnight when they were mature. The screen was separated and embryos that had been fertilized were collected after the light cycle. Pigment formation was blocked by 1-phenyl-2thiourea. To determine the biodistribution of Cur-NPs after oral administration, we incubated zebrafish embryos at 7dpf in C6/Cur-NPs solutions with a C6 concentration of 400 ng/ml for 15 and 60 min. After the treatment, the biodistribution of C6/Cur-NPs were observed and imaged using a fluorescence microscope (DMi8, Leica, Germany).

Pharmacokinetic analysis of Cur-NPs in mice
Male C57BL/6 mice (8 weeks old) were utilized to carry out the Cur-NPs pharmacokinetics analysis. The animals were housed under a 12 h day/night cycle at 25°C with 55% relative humidity. They had free access to water and standard diet.

Establishment of ICH mouse model
Male C57BL/6 mice (8-10 weeks old) were provided by the Experimental Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China). We established an ICH animal model according to previously discribed as following [41]: The mice were placed in a prone position and the head was stabilized using a stereotaxic frame after anesthetization. We made a 1 mm burr hole by a dental

Drug treatment
The mice were randomly divided into four groups: Control group, ICH group, Cur group and Cur-NPs group (n=6/group). The Cur solution or Cur-NPs solution was administered to the mice in the Cur group and Cur-NPs group respectively by gastric gavage 2 hours after ICH injury onset. To achieve 20mg/kg/day dosage, the drug administration was conducted twice daily successively for the 3 days after operation. The remaining two groups received an equal volumes of saline through oral gavage.

Behavioral test
We assessed the impact of Cur-NPs on motor ability of ICH mice by a rotarod test and a pole climbing test. In the rotarod test, the mice were positioned on a rotarod at a speed of 20 rounds per minute for 120 s. The latency to fall and the number of drops within 120 s were recorded. In the pole climbing test, the mice were placed on the top of a pole with a diameter of 0.9 cm and a height of 50 cm, and allowed to climb down at 5-min intervals without interference. The time a mouse required to turn downward (T-turn) and the total time (T-total) a mouse required to reach the bottom were recorded.

Hematoma assessment
After the behavioral tests, the animals were sacrificed and the whole brains were carefully removed and cut into 1 mm thick brain sections after perfusion with PBS.
Sequentially, the images were acquired by an Epson Perfection V370 photo scanner (Epson China, Beijing, China). The hematoma volume of each section measured by using ImageJ software. Hematoma volume in cubic millimeters was calculated as the mean of the summation of the hematoma areas multiplied by the interslice distance (1 mm).

Brain paraffin slice preparation
The mice were perfused transcardially with PBS followed by 4 %PFA. Next, the animals were decapitated and the whole brains were removed and stored in 4% PFA.
The brains were then paraffin-embedded and cut into 5 μm thick sections for the following analysis.

HE staining
HE staining was conducted to investigate the effect of Cur-NPs on neuronal loss in the perihematoma brain tissues of mice. The paraffin sections were deparaffinized in xylene and rehydrated with different concentrations of alcohol and distilled water. The sections were stained with hematoxylin solution for 5 min and eosin solution for 1 min. Next, the sections were dehydrated with alcohol, cleared with xylene and mounted with neutral gum. The images were acquired using an optical microscope (DMi8, Leica, Germany).

Nissl staining
Nissl staining was performed to evaluate the impact of Cur-NPs on neuronal degeneration in the perihematoma brain tissues of mice. The paraffin sections were dewaxed in xylene and rehydrated using different concentrations of ethanol. Next, the sections were stained with 1% toluidine blue for 10 min. After that, the sections were dehydrated with alcohol gradient and xylene and blocked by neutral gum. The neuronal Nissl bodies in the samples were imaged using an optical microscope (DMi8, Leica, Germany).

Prussian blue staining
Prussian blue staining was conducted to detect iron accumulation in brain tissues around hematoma. Paraffin-embedded sections were dewaxed with xylene and rehydrated with gradient ethanol. Next, the samples were stained with Perls Prussian blue stain for 15 min and hematoxylin for 30 s. The samples were then subjected to gradient ethanol dehydration, dimethyl benzene transparency and mounting on neutral resin cover slides. Finally, the sections were observed and imaged with an optical microscope (DMi8, Leica, Germany).

Immunofluorescence staining
The brain 30 μm thick frozen tissue sections were saturated and permeabilized in 0.1% Triton X-100 and blocked with the goat serum at room temperature. Then the samples were incubated with anti-GPX4 at 4 ℃ overnight. The sections were then incubated with goat anti-rabbit IgG (Alexa Fluor 594) in the dark for 1 hour. After DAPI nuclear staining, images were captured using a fluorescent microscope (DMI8, Leica, Germany) and the positive cells enumerated from 3-4 different views in each sample.

Cell viability assay
MTT assay was conducted to determine the effect of Cur-NPs on cell viability. In our present work, different concentrations of drugs were added to HT22 cells treated with erastin for 24 h. Then the samples were inculated with 90 μL DMEM and 10 μL MTT solution for 4 h. Next, the supernatant was discarded and each well was added with 150 μL DMSO. Sequentially, the plate was shaken on an oscillator for 10 min. The cell viability was determined by calculating the absorbance at 490 nm using a microplate reader (Multiskan FC, Thermo Scientific, United States).

Live / dead cell staining assay
Live and dead cell staining assay test was performed to analyze the impact of