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Brain-targeted heptapeptide-loaded exosomes attenuated ischemia–reperfusion injury by promoting the transfer of healthy mitochondria from astrocytes to neurons | Journal of Nanobiotechnology

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Ischemic stroke (IS), characterized by a common acute cerebrovascular disorder, is one of the primary fatal diseases in middle-aged and elderly people [1]. However, recent available effective treatment options are limited and only suitable for a small number of stroke patients. Urokinase and streptokinase are neuroprotective drug candidates that have been clinically applied for the treatment of IS. Although these drugs can activate plasminogen to form plasmin, they can also degrade coagulation factors and fibrinogen. Therefore, they potentiate a high risk of bleeding when used in thrombolytic therapy [2]. In addition, United States Food and Drug Administration (FDA) has proved that recombinant tissue plasminogen activator can be used to recanalize blocked vessels in the treatment of IS, however, its therapeutic efficacy is very limited because of a narrow therapeutic window (< 4.5 h) and an increased risk of intracranial hemorrhage [3]. Therefore, it is urgent to develop effective treatments to reduce IS-induced brain injury.

Astrocytes (AS), the supporting matrix cells in the central nervous system (CNS), have multiple regulatory functions, including buffering extracellular ions, clearing amino acid neurotransmitters, limiting excitatory toxicity and promoting synaptic development [4, 5]. Recently, the crosstalk between astrocytes and neurons has been more fully investigated and clarified [6]. On the one hand, IS induces the transition of astrocytes from the resting state to the reactive state and releases cytokines, interleukins and other potentially cytotoxic molecules at higher levels than that from resting astrocytes, thus destroying synaptic homeostasis and initiating neuronal injury [7]. On the other hand, mitochondria can be secreted from astrocytes and delivered into neurons, modulating the function of damaged neuronal mitochondria to affect neuronal damage [8, 9]. Briefly, when IS occurs, astrocytes are rapidly induced to become activated A1-type astrocytes (A1-AS). As a result, under pathological stress, mitochondrial dynamin-related protein-1 (Drp1) as a key mitochondrial regulatory protein predominantly interacts with fission 1 (Fis1), causing an exaggerated fission process, as evidenced by excessive mitochondrial fragmentation, production of reactive oxygen species (ROS) and loss of mitochondrial membrane potential [10]. Therefore, marked pathological fragmentation of mitochondria is produced, and mitochondrial function is significantly disrupted by decreasing adenosine triphosphate (ATP) and mitochondria membrane potential [11]. Finally, damaged astrocytic mitochondria are further released, enter adjacent neurons and induce fusion with neuronal mitochondria, inducing neuronal mitochondrial dysfunction, amplifying neuronal damage and worsening neurological outcomes [12, 13]. In contrast, after incubation of normal astrocytic mitochondria with oxygen–glucose deprivation (OGD)-injured neurons, astrocytic mitochondria can be detected in neurons, restore ATP levels in injured neurons and enhance cell survival and plasticity [14]. These findings indicate a new mitochondrial crosstalk between astrocytes and neurons that may contribute to neurological regulation after IS. Improving the function of astrocytic mitochondria and promoting the delivery of healthy astrocytic mitochondria into neurons are potential therapeutic targets for reducing IS-induced neuronal injury and neurodegenerative disorders.

To treat IS by modulating mitochondrial crosstalk between astrocytes and neurons, we focused on inhibiting Drp1/Fis1-mediated mitochondrial fission in type A1 astrocytes to reduce the transfer of damaged astrocytic mitochondria into neurons. Heptapeptide (Hep), a Drp1–Fis1 peptide inhibitor P110, alleviates mitochondrial dysfunction and is a key contributor in IS [15]. It selectively inhibits the binding of activated Drp1 to Fis1 and reduces the pathological fission of astrocytic mitochondria without affecting physiological fission [16]. Therefore, treatment with Hep has been shown to be beneficial for reducing IS mediated damage in vitro and in vivo [17]. However, Hep as a protein drug suffers the susceptibility to enzymatic degradation, short circulation half-lives and poor membrane permeability, thus posing significant barriers for effective delivery [18]. To overcome these shortcomings to convey a better therapeutic effect, various carriers, such as liposomes, polymer vesicles, exosomes, dendrimers, and inorganic nanoparticles, have been applied to encapsulate drugs to establish a protein drug-targeted delivery system [19]. Among these carriers, exosomes characterized by low immunogenicity, high biological permeability, and high delivery efficiency have promising potential innate properties for loading protein agents and targeting ischemic regions [20]. As shown in Fig. 1, we prepared Hep-loaded macrophage-derived exosomes (EXO-Hep) to attenuate mitochondrial disorder in astrocytes by inhibiting the Drp1/Fis1 mediated mitochondrial fission in astrocytes. Subsequently, more healthy mitochondria of astrocytes were released and transferred into neurons, resulting in reduced neuronal damage by improving neuronal mitochondrial function. Finally, EXO-Hep alleviated cerebral ischemia–reperfusion injury in a model of transient middle cerebral artery occlusion (tMCAO).

Fig. 1
figure 1

The primary hypothesis of this study. We fabricated brain-targeted multifunctional biomimetic heptapeptide loaded exosomes for the treatment of IS. Heptapeptide loaded macrophage derived exosomes (EXO-Hep) targeted brain and inhibited Drp1/Fis1 interaction to improve the mitochondrial function of astrocyts. As a result, more healthy astrocytic mitochondria were secreted from astrocytes and transferred into neurons for reducing mitochondria-mediated damage of neurons. Finally, EXO-Hep ameliorated IS injury by reducing infarct area and improving neurological performance in tMCAO rats

Materials and animals

Hep, as the Drp1 inhibitor P110 was synthesized from Nanjing Peptide Industry Biotechnology Co., Ltd, PKH26 was obtained from Sigma-Aldrich Co. Antibodies such as Drp1, TOM20, BAX, BCL-2, Fis1 (1:500), were purchased from WanLeiBio (Shenyang, China), and rabbit anti-β-actin was obtained (1:1000, Biogot Technology, Co, Ltd). This study acquired goat anti-rabbit IgG/HRP secondary antibody (1:10,000) from EarthOx Life Sciences (Millbrae, CA, USA). All reagents and chemicals were provided by Sigma (St. Louis, MO, USA).

The murine macrophage RAW264.7 cell line was purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, People’s Republic of China) and cultured in RPMI 1640 medium (HyClone, UT, USA) containing 10% foetal bovine serum (FBS; Gibco, CA, USA) and 1% penicillin–streptomycin (PS). SH-SY5Y cells were purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, People’s Republic of China) and cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% foetal bovine serum (FBS) and 1% PS. The astrocyte cell line HA-1800 was purchased from Guang Zhou Jennio Biotech Co., Ltd. and cultured in Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% of fetal bovine serum (FBS) and 1% PS. To simulate the astrocytic A1 state that occurs in stroke, astrocytes were treated with lipopolysaccharide (LPS) to obtain type A1 astrocytes (A1-AS). Male Sprague–Dawley (SD) rats (250–280 g) were provided by Jinzhou Medical University. The experimental protocol was performed with the approval of the Institutional Animal Care and Use Committee of Jinzhou Medical University and followed the National Guidelines for Animal Protection.

Preparation and identification of EXO-Hep

According to the previous report [21], RAW 264.7 cells cultured in full growth medium were seeded into culture plates (1 × 106 cells). Twenty-four hours later, the culture medium was collected and filtered. After centrifugation at 20,000×g for 30 min, the supernatant was discarded, and exosome pellets were resuspended in phosphate-buffered saline (PBS) and ultracentrifuged again at 100,000×g for 150 min to obtain EXO. To load Hep into EXO, 1 mg/mL Hep was added to 8 mg/mL EXO, and the mixture was sonicated using a sonic dismembrator for 4 cycles of 45 s on/2 min off with a 3 min cooling period between each cycle. After sonication, the mixture was incubated in an ice water bath at 4 °C for 24 h. After centrifugation, EXO-Hep was collected and the remaining Hep in the supernatant was measured by checking its UV absorbance. Encapsulation efficacy (EE) of Hep in EXO was calculated as follow:

$$EE\% = \frac{{W_{total} – W_{free} }}{{W_{total} }} \times 100\%$$

\(W_{total}\) indicated the amount of initial added Hep, while \(W_{free}\) was the amount of Hep remaining in the supernatant.

Identification of EXO was performed by western blotting using anti-Alix (1:2000, Abcam, Cambridge, UK) and anti-CD63 (1:1000, Bioworld, Bloomington, USA) antibodies. The morphology and shape of EXO and EXO-Hep were determined using transmission electron microscopy (TEM, HITACHI HT7800; Hitachi, Ltd). Particle size was measured using a Zetasizer Nano ZS instrument.

Mitochondria membrane potential measurement

To monitor mitochondrial health, JC-1 dye (Solarbio, Cat no #M8650) was used to assess mitochondrial membrane potential. Mitochondria from astrocytes media and SH-SY5Y cells were incubated with JC1 (5 μM or 1 μM) for 30 min at 37 °C. JC1 dye exhibited potential-dependent accumulation in mitochondria, indicated by fluorescence emission shift from green (Ex 485 nm/Em 516 nm) to red (Ex 579 nm/Em 599 nm). Mitochondria membrane potential was determined by calculating the fluorescent ratio with a fluorescent microplate reader.

ATP measurement

Relative intracellular or extracellular ATP levels were determined using ATP Luminescent Cell Viability Assay Kit (US EVERBRIGHT, Cat no #A6103S), which can perform cell lysis and generate a luminescent signal proportional to the amount of ATP present [16]. In brief, for intracellular ATP levels, 96-well plates with cell lysate (50 μl) were prepared. An equal volume of the single-one-step reagent provided by the kit was added to each well and incubated for 30 min at room temperature. For measuring ATP content in extracellular mitochondria, cell supernatant was cleared of cellular debris by centrifugation at 1000×g for 10 min and then centrifuged 13,000×g for 25 min followed by a wash with 1 mL of PBS. The pellet was then resuspended in the 50 μl of serum-free phenol-free DMEM or PBS before an equal volume of the single-one-step reagent provided by the kit with incubation for 30 min at room temperature. ATP content was measured using microplate Reader.

Determination of ROS production

To determine mitochondrial ROS production [17], extracellular mitochondria were treated with 5 μM MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen, Cat no #40778ES50) at the end of the experiment for 20 min at 37 °C according to the manufacturers’protocols. Fluorescence was analyzed at excitation/emission maxima of 510/588 nm. To determine cellular ROS production, cells were incubated with 5 μM CellROX™ oxidative stress reagent (Invitrogen, Cat no #BB-47053) for 30 min along. The fluorescence was analyzed using microplate reader, at excitation/emission maxima of 485/520 nm for CellROX™.

MTT and TUNEL assays

Hep, EXO and EXO-Hep were added to the plates and incubated with A1-AS for 24 h to obtain Hep-treated A1-AS (A1-AS (Hep)), EXO-treated A1-AS (A1-AS (EXO)) and EXO-Hep-treated A1-AS (A1-AS (EXO-Hep)). According to a previous report [16], the culture medium (CM) from A1-AS treated with Hep, EXO and EXO-Hep was removed, and the released mitochondria were collected from the culture medium of A1-AS treated with Hep (Mito/A1-AS (Hep)), EXO (Mito/A1-AS (EXO)) and EXO-Hep (Mito/A1-AS (EXO-Hep)). After the astrocytic mitochondria and culture medium were co-incubated with OGD-stimulated SH-SY5Y cells for 24 h, serum-free medium containing MTT was added and incubated for 4 h. Finally, dimethyl sulfoxide was added to OGD-stimulated SH-SY5Y cells, and absorbance was measured at 490 nm. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay was also used to detect apoptotic cell level.

Distribution of EXO-Hep in ischemic brain tissue

To evaluate the location of EXO and EXO-Hep in astrocytes of the ischemic brain, astrocytes were stimulated with LPS followed by the addition of PKH26-labelled EXO and PKH26-labelled EXO-Hep. The distribution of PKH26-labelled EXO and PKH26-labelled EXO-Hep in activated astrocytes was observed by confocal laser scanning microscopy. To evaluate the ability of EXO-Hep to target ischemic brains in vivo, tMCAO rats were intravenously injected with PKH26-labelled EXO-Hep through the tail vein. One hour later, tMCAO rats were first perfused with PBS for removing the blood and the brains of rats were dissected. An IVIS Spectrum imaging system (PerkinElmer, Waltham, MA, USA) was employed to capture PKH26-labelled EXO-Hep-emitted fluorescence images in the brain. Two hours after administration, tMCAO rats were sacrificed and the brains were cryosectioned. After staining with a C3 antibody to label type A1 astrocytes, the colocalization of PKH26-labelled EXO-Hep in A1 astrocytes was investigated using confocal laser scanning microscopy.

Treatment administration following tMCAO

To establish tMCAO, a 6–0 nylon monofilament suture was inserted into the right internal carotid arteries of SD rats for 2 h and subsequently removed to allow blood reperfusion. After reperfusion, 1 mL PBS, Hep, EXO and EXO-Hep containing Hep at a concentration of 0.75 mg/mL were administered via a single intravenous injection via the tail vein.

Neurological evaluation of EXO-Hep in the tMCAO model

Twenty-four hours after administration of 1 mL PBS, Hep, EXO and EXO-Hep containing Hep at a concentration of 0.75 mg/mL to SD rats subjected to tMCAO, neuroprotective effects were evaluated by performing TTC (2,3,5-triphenyltetrazolium chloride, Sigma–Aldrich) staining and determining neurological scores and immunofluorescence according to previous reports [22].

Immunofluorescence staining

Frozen sections of rat brains were kept at room temperature for 30 min. Then, sections were pre-incubated with goat serum followed by primary anti-C3 (1:100, ZenBio Antibody) and anti-NeuN (1:100, Biolegend Antibody) antibodies overnight at 4 °C. After several rinses with PBS, the sections were incubated with secondary antibody (Texas red-conjugated donkey anti-rabbit IgG or fluorescein isothiocyanate [FITC]-conjugated donkey anti-rat IgG) for 1 h. Then, the sections were incubated with DAPI for 5 min. Images were obtained under a fluorescence microscope.

Western blot assay

Based on a previously reported protocol [23], proteins were extracted from mitochondria, cells and culture medium, and transferred to polyvinylidene fluoride (PVDF) membranes (BioTrace; Pall Corporation, New York, USA). After incubation with primary antibodies such as TOM20, Drp1, Fis1, BAX, BCL-2 (1:500, Wanleibio, Shenyang, China), VDAC (1:1000, Wanleibio, Shenyang, China), β-actin (1:1000, Biogot Technology, Co, Ltd), C3 and cytochrome c (1:1000, Biolegend Antibody), membranes were incubated with a 1:10,000 dilution of the secondary antibody for 1 h at room temperature. The levels of the target proteins were imaged and analysed using a UVP gel analysis system (iBox Scientia 600; UVP, LLC, CA, USA).

Statistical analysis

The results are presented as the means ± standard deviations (SD). Statistical analyses were performed using one-way analysis of variance (ANOVA). P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) were considered statistically significant differences.

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