WO2022056880A1

WO2022056880A1 - Ros-responsive nanocarrier, preparation and uses thereof

Info

Publication number: WO2022056880A1
Application number: PCT/CN2020/116318
Authority: WO
Inventors: Guixue Wang; Li Luo; Haijun Zhang; Wei Wu; Kunshan YUAN; Xian QIN
Original assignee: Chongqing University; Branden Medical Device Co., Ltd.
Priority date: 2020-09-18
Filing date: 2020-09-18
Publication date: 2022-03-24

Abstract

Provided is a ROS-responsive NPs coated with CXCR4 over expression primary mouse thoracic aortic endothelial cell membrane which can specifically target to the ischemic site, controllable drug release and long circulation, so that it will bring about considerable breakthroughs in the technology of the field for the benefit of a large group of people in need thereof.

Description

ROS-responsive nanocarrier, preparation and uses thereof

BACKGROUND OF THE INVENTION

Stroke is one of the most important fatal diseases in the world due to its rapid onset and high fatality rate, and there still has been a lack of effective treatment. Due to reperfusion following cerebral ischemia, excessive production of reactive oxygen species (ROS) induced the necrosis and detachment of the cerebrovascular endothelium at the end of the embolized part due to hypoxia, eventually leads to irreversible severe oxidative damage through neuroinflammation and apoptosis. Except the major challenges in the safety profile and pharmacokinetic effects of pre-clinic therapy, the problem of diminish neuroinflammation and attenuate reperfusion injury in stroke have still remained.

Nanoparticles (NPs) can be used clinically to deliver drugs (such as anticancer drugs) to target sites, and thus are considered to have high potential in the field of nanotechnology and medicine. However, the blood-brain barrier (BBB) can also impedes the delivery of therapeutic agents to specific region of the brain. Meanwhile, traditional NPs for the context of neurological disease and targeting brain parenchyma do not well selectively targeting post-ischemic inflammation and injury location. On the other hand, conventional NPs often suffer from poor biocompatibility, poor stability, and degradation before reaching the target site.

SUMMARY OF THE INVENTION

The present disclosure provides a ROS-responsive NPs coated with CXCR4 overexpression (using lentiviral transcription) primary mouse thoracic aortic endothelial cell membrane, aiming to develop a smart bioengineered drug delivery system. A facile and efficient strategy for treatment of reperfusion injury after stroke has been put forward: mTOR inhibitor rapamycin (RAPA) was loading in the micelles via self-assembly of ROS-responsive nanocarrier (HBA-OC-PEG ₂₀₀₀) then encapsulated into the bioengineered membrane. The masterpiece of perfect combination of several advantages including: due to the CD47 from endothelial cell membrane, no acute or long-term immune responses against the NPs, because of active and effective targeting of bioengineered membrane highly expressed CXCR4, and the ability of intravenously administered anti-ischemic agents to cross BBB and reach ischemic lesion, ROS scavenging polymer to integrate antioxidation. When the nanomedicine was actively delivered to the ischemic area, thus achieving effective therapeutic efficacy for blood perfusion after ischemic stroke (Figure. 23) . By utilizing mechanism of ROS activation, the present disclosure achieves a more rapid and wide-spread response to the stimulus. This provides for an increased sensitivity and efficacy in sensing diseased environments, or other targeted site, and then releasing and/or activating bioactive agents at the targeted site.

Accordingly, in one aspect, the present disclosure provides a ROS-responsive nanocarrier, comprising a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups comprises a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity.

In some embodiments, said hydrophilic segment is formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment is formed by copolymerization of antioxidant groups and ROS labile groups.

In some embodiments, said polymer backbone comprises a copolymerized oxalate backbone.

In some embodiments, said antioxidant groups comprise p-Hydroxybenzyl alcohol (HBA) .

In some embodiments, said hydrophilic groups comprise poly- (ethylene glycol) .

In some embodiments, said poly- (ethylene glycol) comprises poly- (ethylene glycol) ₂₀₀₀ (PEG ₂₀₀₀) .

In some embodiments, said ROS-labile groups comprise oxalyl chloride (OC) .

In some embodiments, said hydrophobic segment is a compound of formula (I) , wherein, x is selected from 1 to 100.

In some embodiments, said hydrophilic segment is a compound of formula (II) , wherein, y is selected from 1 to 100.

In some embodiments, said polymer backbone is a compound of formula (III) , wherein, x and y are independently selected from 1 to 100.

In some embodiments, said polymer backbone is a compound of formula (III) , wherein, x and y are independently selected from 1 to 25.

In some embodiments, said polymer backbone is a compound of formula (III) , wherein x=3 and/or y=7.

In some embodiments, said polymer backbone is assembled into stable micelles in an aqueous solution.

In another aspect, the present disclosure provides a ROS-responsive nanoparticle, wherein comprising said ROS-responsive nanocarrier and bioactive agents, and said ROS responsive nanocarrier and bioactive agents are autonomously installed as nanoparticles.

In some embodiments, said bioactive agents comprise nano-drug and/or nanoprobe.

In some embodiments, said nano-drug comprises mTOR inhibitor.

In some embodiments, said mTOR inhibitor comprises Rapamycin (RAPA) .

In some embodiments, said nanoprobe comprises fluorescent nanoprobe.

In some embodiments, said fluorescent nanoprobe comprises DiD nanoprobe.

In some embodiments, said ROS-responsive nanoparticle is the uniform spherical nanoparticle with the dehydrated size of 170～220 nm.

In another aspect, the present disclosure provides a nano-drug delivery system, comprising said ROS-responsive nanocarrier coated with bio-engineered cell membrane.

In some embodiments, said bio-engineered cell membrane specifically targets to the ischemic site.

In some embodiments, said bio-engineered cell membrane can specifically cross the blood-brain barrier in ischemic brain.

In some embodiments, said bio-engineered cell membrane has a high initial brain uptake and/or the ability to expand circulation in the brain.

In some embodiments, said bio-engineered cell membrane expresses a receptor targeting the normal or highly expressed protein of the ischemic region.

In some embodiments, said normal or highly expressed protein of the ischemic region comprises SDF-1.

In some embodiments, said bio-engineered cell membrane expresses CXCR4.

In some embodiments, said bio-engineered cell membrane overexpresses CXCR4.

In some embodiments, said bio-engineered cell membrane expresses “don’t eat me” protein making the bio-engineered cell membrane have immune evasion ability.

In some embodiments, said “don’t eat me” protein comprises CD47.

In some embodiments, said bio-engineered cell membrane comprises vascular endothelial cell membrane.

In some embodiments, said bio-engineered cell membrane comprises the primary vascular endothelial cell membrane.

In some embodiments, said bio-engineered cell membrane comprises arterial endothelial cell membrane.

In some embodiments, said bio-engineered cell membrane comprises endothelial cell membrane of the thoracic aorta.

In another aspect, the present disclosure provides a neuroprotective agent, comprising said nano-drug delivery system, wherein said ROS-responsive nanocarrier loaded with nano-drug.

In some embodiments, said nano-drug comprises mTOR inhibitor.

In some embodiments, said mTOR inhibitor comprises Rapamycin (RAPA) .

In some embodiments, said neuroprotective agent is the uniform spherical nanoparticle with the dehydrated size of 200～230 nm.

In another aspect, the present disclosure provides a ROS-responsive nanoprobe, comprising said nano drug delivery system, wherein the ROS-responsive nanocarrier loaded with nanoprobe.

In some embodiments, said nanoprobe comprises fluorescent nanoprobe.

In some embodiments, said fluorescent dyes comprises DiD nanoprobe.

In another aspect, the present disclosure provides a method for preparing said ROS-responsive nanocarrier, comprising: synthesizing a polymer skeleton containing antioxidant groups and hydrophilic groups through a one-step polycondensation reaction with ROS labile groups.

In some embodiments, said antioxidant groups comprises p-Hydroxybenzyl alcohol (HBA) .

In some embodiments, said hydrophilic groups comprises poly- (ethylene glycol) .

In some embodiments, said ROS-labile groups comprises oxalyl chloride (OC) .

In some embodiments, said method for preparing ROS-responsive nanocarrier comprising: synthesizing HBA-OC-PEG ₂₀₀₀ containing PEG ₂₀₀₀ and HBA through a one-step polycondensation reaction with oxalyl chloride, and obtaining the obtained after repeated precipitation into cold ether, purifying the collected copolyoxalate, drying under high vacuum, and then obtained HBA-OC-PEG ₂₀₀₀.

In another aspect, the present disclosure provides a method for preparing ROS-responsive nanoparticle, wherein said bioactive agents are loaded into micelles through the self-assembly of ROS-responsive nanocarrier to obtain nanoparticles.

In another aspect, the present disclosure provides a method for preparing nano-drug delivery system, comprising the method of physically extruding the bio-engineered cell membrane to coat said ROS-responsive nanocarrier, wherein said bio-engineered cell membrane is obtained by lentiviral transcription.

In some embodiments, said method for preparing the bio-engineered cell membrane specifically comprises: using lentivirus to transcribe vascular endothelial cells to obtain the bio-engineered cell membrane with high expression of CXCR4.

In another aspect, the present disclosure provides a method for preparing neuroprotective agent, comprising the method of physically extruding the bio-engineered cell membrane to coat said ROS-responsive nanoparticle, wherein said bio-engineered cell membrane is obtained by lentiviral transcription.

In another aspect, the present disclosure provides a method for preparing ROS-responsive nanoprobe, comprising the method of physically extruding the bio-engineered cell membrane to coat the ROS-responsive nanoparticle, wherein said bio-engineered cell membrane is obtained by lentiviral transcription.

In another aspect, the present disclosure provides a use of said ROS-responsive nanocarrier in the preparation of a medicine for the treatment and/or diagnosis of ROS-mediated diseases.

In another aspect, the present disclosure provides a use of said ROS-responsive nanoparticle in the preparation of a medicine for the treatment and/or diagnosis of ROS-mediated diseases.

In another aspect, the present disclosure provides use of said nano-drug delivery system in the preparation of a medicine for targeted treatment of ischemic stroke.

In another aspect, the present disclosure provides a use of said nano-drug delivery system in the preparation of a kit for diagnosing ischemic stroke.

In another aspect, the present disclosure provides a use of said neuroprotective agent in the preparation of a medicament for targeted treatment of ischemic stroke.

In another aspect, the present disclosure provides a use of said ROS-responsive nanoprobe in the preparation of a kit for diagnosing ischemic stroke.

In another aspect, the present disclosure provides a pharmaceutical composition, comprising said ROS-responsive nanocarrier, said nanoparticle, said nano-drug delivery system or said neuroprotective agent.

In another aspect, the present disclosure provides a diagnostic kit, comprising said ROS-responsive nanocarrier, said nanoparticle of, said nano-drug delivery system or said ROS-responsive nanoprobe.

In another aspect, the present disclosure provides a method of treating or preventing ischemic diseases comprising the administration of an effective amount of said neuroprotective agent to a patient in need thereof.

In some embodiments, said ischemic diseases comprise reperfusion injury after stroke.

Additional aspects and advantages of the present invention will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present invention are shown and described. As will be realized, the present invention is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENC

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIGURE. ” herein) , of which:

Figure. 1 is the characterizations of bioengineered cell membrane coated NPs.

Figure. 2A-2E illustrate results of HOP、 MHOP and BMHOP on intracellular ROS elimination.

Figure. 3A-3F show the Targeting ability and accumulation into the ischemic brain of NPs.

Figure. 4A-4G show the results of cellular uptake the cell membrane coated NPs

Figure. 5A-5E illustrate the capability of HOP、 MHOP and BMHOP in antioxidative in vivo.

Figure. 6A-6H illustrate the in vivo circulation behavior of different NPs formulations.

Figure. 7 illustrates the NMR results of HBA-OC-PEG ₂₀₀₀.

Figure. 8 illustrates the identification of lentivirus transfection rate in cells.

Figure. 9 illustrates the results of q-RCR and WB of the 8th-generation bioengineered primary cells.

Figure. 10 illustrates the results of cell migration experiment.

Figure. 11 illustrates the DLS results of nanoparticles in PBS and in PBS containing H ₂O ₂.

Figure. 12 shows the CLSM images of cellular uptake of DiD@NPs in HUVEC and SDF-1 overexpression HUVEC.

Figure. 13 illustrates the flow cytometry results and quantitative analysis of DiD@NPs.

Figure. 14 illustrates the CLSM results of fluorescence co-location of DiD@BMHOP.

Figure. 15 illustrates the biocompatibility of HOP, MHOP and BMHOP.

Figure. 16 illustrates the intracellular ROS production after DiD@BMHOP treatment at different time points were measured by flow cytometry and quantitative analysis.

Figure. 17 shows the CLSM images results of 3 different DiD@NPs on intracellular ROS elimination at different time points.

Figure. 18 illustrates the inhibitory effect of BMHOP on cell proliferation of RAW264.7 cells.

Figure. 19 illustrates the radiant efficiency of fluorescence intensity of different DiD@NPs in major organs after 12 h post-injection.

Figure. 20 shows cell transmembrane resistance was measured by R/V Meter of Epithelium.

Figure. 21 shows the properties of Primary mouse thoracic aortic endothelial cells identified by immunofluorescence.

Figure. 22 illustrates the standard curve of EB.

Figure. 23 shows the strategy of the present disclosure for treatment of reperfusion injury after stroke.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

Definitions

The term “ROS-responsive” refers to the sensitivity of a composition or chemical linkage to reactive oxygen species relevant signals. A “ROS-responsive” composition (e.g., polymer backbone, nanocarrier or nanoparticle) can undergo structural and/or morphological changes in response to a one or more reactive oxygen species stimulus. For example, a “ROS-responsive” nanocarrier can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity. A “ROS-responsive” linkage" refers to a bivalent chemical moiety that contains one or more bonds (e.g., one or more covalent bonds) that are cleaved and/or transformed in the presence of reactive oxygen species stimulus. In some embodiments, the linkage is cleaved. In some embodiments, the ROS-responsive composition or linkage is sensitive to reactive oxygen species stimulus present in a tissue.

Reactive oxygen species comprise a variety of chemically reactive molecules and free radicals derived from molecular oxygen, such as H ₂O ₂, superoxide anion (O ^2-) , hydroxyl radical (HO·) , and hypochlorite ion (OCl ^-) . Due to reperfusion following cerebral ischemia, excessive production of reactive oxygen species (ROS) induced the necrosis and detachment of the cerebrovascular endothelium at the end of the embolized part due to hypoxia, eventually leads to irreversible severe oxidative damage through neuroinflammation and apoptosis. ROS is an acronym for reactive oxygen species and for the research purposes herein hydrogen peroxide (H ₂O ₂) is used as a representative ROS chemical reagent.

The term “nanocarrier” or “nanocarriers” refers to an assembly of polymers, having a structure comprising at least one region or characteristic dimension with a dimension of between 1-500 nm and having any suitable shape, e.g., a rectangle, a circle, a sphere, a cube, an ellipse, or other regular or irregular shape. Non-limiting examples of nanocarriers may comprise liposomes, poloxamers, microemulsions, micelles, dendrimers and other phospholipid-containing systems, and perfluorocarbon nanoparticles. The term “nanocarriers” can comprise nanospheres, nanorods, nanoshells, and nanoprisms. Without limitations, the nanocarriers used herein can be any nanocarrier available in the art or available to one of skill in the art. The term “nanocarriers” may also refer to particles that are between 1 nm and 500 nm in diameter. Some of the novel properties associated with nanocarriers, which differentiate them from bulk materials, is generally associated with their size being less than 300 nm. A nanocarrier of the present disclosure may be used to indicate there is no payload associated with the nanocarrier, and the term “nanoparticle” or other forms of the word, such as “nanoparticles” 、 “NP” or “NPs” refers to nanocarrier with a payload.

The term "payload" refers to one or more bioactive agent. In some embodiments, the payload is a therapeutic payload.

The term “bioactive agent” refers to any molecule or molecules that are administered by a practitioner to produce an effect within a patient. A bioactive agent may comprise a pharmaceutical agent, such as a drug, and/or an imaging agent. The term “bioactive agent” is not meant to be restricted to a single type of agent, and a bioactive agent associated with a nanocarrier as used herein may also can comprise populations and/or combinations of bioactive agents with one, two, or a plurality of components, and each of the components may be capable of acting, or functioning as a bioactive agent, by itself.

The term “antioxidant” refers to both compounds that act directly to oxidize or reduce ROS and also to compounds that inhibit generation of ROS.

The term “Hydrophobic groups” means water-hating Chemical groups that tend to make substances hydrophobic comprise -CH ₂-chains and rings (hydrocarbons) . These substances lack the ability to hydrogen bond and their surface free energy is relatively low. Water does not tend to wet hydrophobic surfaces; rather, the droplets stay beaded up with high values of contact angle. The opposite of hydrophobic is hydrophilic, water-loving. “nanocarrier” and “nanoparticle” of present disclosure contain both hydrophobic and hydrophilic groups on the same molecules.

The term “CXCR4” refers to a G-protein-coupled receptor, and its naturally occurring ligand, stromal cell-derived factor-1 (SDF-1; CXCL12) , are a chemokine receptor-ligand pair. CXCR4 is consiutively or over-expressed in a wide variety of human cancers SDF-1, the only known ligand of CXCR4, is highly expressed in tumor microenvironments, as well as in bone marrow, lung, liver, and lymph nodes, i.e., organ sites most commonly involved in tumor metastasis. CXCR4/SDF-1 interaction plays important roles in multiple stages of tumorigenesis, comprising tumor growth, invasion, angiogenesis, and metastasis (Furusato, et al., Pathology International 2010, 60, 497-505) . The CXCR4/SDF1 axis also serves a role in attraction multiple leukocyte subsets and stimulation B cell production and myelopoeisis, all of which are implicated in autoimmune diseases (Chong and Mohan, Expert Opin. Ther. Targets 2009, 13 (10) , 1147-1153) .

The term “degradation” or other forms of the word, such as “degraded” or “degrading” means that all or substantially the degradation of the nanocarrier or nanoparticle, accompanying substantial release antioxidant groups and/or bioactive agent. As used herein in this instance, the “substantial release” may refer to the release of approximately 50%or more; the release of approximately 60%or more; the release of approximately 70%or more; the release of approximately 80%or more; the release of approximately 90%or more; the release of approximately 95%or more; or the release of approximately 99%or more of the antioxidant groups and/or bioactive agent.

The term “Don't-eat-me protein” refers to the proteins on the cell surface can tell macrophages not to destroy them, such as CD47, which when expressed on the surface of a cell, inhibit phagocytosis of that cell, by activating SIRP-alpha receptors on the phagocyte. Endothelial cells widely expressed CD47 can bind to signal regulatory protein (SIRP) on the surface of macrophages, and then recruit SHP-1 protein to produce a series of cascade reactions to inhibit the phagocytosis of macrophages.

The term “treating” or other forms of the word, such as “treated” or “treatment” refers to administering a composition to an organism afflicted with an abnormal condition, such as a cell proliferative disorder, where the administration of the composition has a therapeutic effect and at least partially alleviates or abrogates the abnormal condition. Note that the treatment needs not provide a complete cure and the treatment will be considered effective if at least one symptom is improved or eradicated. The treatment may reduce mortality. Furthermore, the treatment need not provide a permanent improvement of the medical condition or other abnormal condition.

The terms “prevent” or other forms of the word, such as “preventing” or “prevention, ” refers to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

The terms “administration” or “administering” refers to a method of incorporating a compound into the cells or tissues of an animal, preferably a mammal, in order to treat or prevent an abnormal condition. When the composition of the invention is provided in combination with one or active agents, the terms “administration” or “administering” comprise sequential or concurrent introduction of the composition with the other agent (s) . For cells harbored within the organism, many techniques exist in the art to administer compounds, comprising (but not limited to) oral, injection, parenteral, dermal, and aerosol applications.

The term “patient” refers to a human subject who has presented at a clinical setting with a particular symptom or symptoms suggesting the need for treatment. A patient's diagnosis can alter during the course of the disease, condition, or abnormal condition, such as development of further symptoms, or remission of the disease, condition, or abnormal condition, either spontaneously or during the course of a therapeutic regimen or treatment. The term “patient” may also broadly refer to non-human organisms, such as a mouse, rat, rabbit, guinea pig, goat, cow, horse, pig or other domestic animals.

In one aspect, the present disclosure provides a ROS-responsive nanocarrier may comprise a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups may comprise a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be completely degraded in the presence of ROS and release antioxidant groups with pharmacological activity.

In some embodiments, the hydrophilic segment may be formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment may be formed by copolymerization of antioxidant groups and ROS labile groups.

In some embodiments, the polymer backbone may comprise a copolymerized oxalate backbone.

In some embodiments, the antioxidant groups may comprise p-Hydroxybenzyl alcohol (HBA) .

In some embodiments, the hydrophilic groups may comprise poly- (ethylene glycol) .

In some embodiments, the poly- (ethylene glycol) may comprise poly- (ethylene glycol) ₂₀₀₀ (PEG ₂₀₀₀) .

In some embodiments, the ROS-labile groups may comprise oxalyl chloride (OC) .

In some embodiments, the ROS-responsive nanocarrier may comprise a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups may comprise a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said hydrophilic segment may be formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment may be formed by copolymerization of antioxidant groups and ROS labile groups; and said polymer backbone may comprise a copolymerized oxalate backbone; and said antioxidant groups may comprise p-Hydroxybenzyl alcohol; and said hydrophilic groups may comprise poly- (ethylene glycol) ; and said ROS-labile groups may comprise oxalyl chloride.

As an example, said ROS-responsive nanocarrier, comprising a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups comprises a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said polymer backbone comprises a copolymerized oxalate backbone; and said hydrophilic segment is formed by copolymerization of poly- (ethylene glycol) ₂₀₀₀ and oxalyl chloride, and said hydrophobic segment is formed by copolymerization of p-Hydroxybenzyl alcohol and oxalyl chloride.

In some embodiments, said hydrophobic segment may be a compound of formula (I) ,

wherein, x may be selected from 1 to 100.

In some embodiments, said hydrophobic segment may be a compound of formula (I) , wherein, x may be selected from 1 to 25.

In some embodiments, said hydrophobic segment may be a compound of formula (I) , wherein, x=3.

In some embodiments, the ROS-responsive nanocarrier may comprise a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups may comprise a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said hydrophilic segment may be formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment may be formed by copolymerization of antioxidant groups and ROS labile groups; and said polymer backbone may comprise a copolymerized oxalate backbone; and said antioxidant groups may comprise p-Hydroxybenzyl alcohol; and said hydrophilic groups may comprise poly- (ethylene glycol) ; and said ROS-labile groups may comprise oxalyl chloride; and said hydrophobic segment may be a compound of formula (I) ,

wherein, x may be selected from 1 to 100.

As an example, said ROS-responsive nanocarrier, comprising a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups comprises a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said polymer backbone comprises a copolymerized oxalate backbone; and said hydrophilic segment is formed by copolymerization of poly- (ethylene glycol) ₂₀₀₀ and oxalyl chloride, and said hydrophobic segment is formed by copolymerization of p-Hydroxybenzyl alcohol and oxalyl chloride; and said hydrophobic segment is a compound of formula (I) , wherein, x=3.

In some embodiments, wherein said hydrophilic segment may be a compound of formula (II) ,

wherein, y may be selected from 1 to 100.

In some embodiments, wherein said hydrophilic segment may be a compound of formula (II) , wherein, y may be selected from 1 to 25.

In some embodiments, wherein said hydrophilic segment may be a compound of formula (II) , wherein, y =7.

In some embodiments, the ROS-responsive nanocarrier may comprise a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups may comprise a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said hydrophilic segment may be formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment may be formed by copolymerization of antioxidant groups and ROS labile groups; and said polymer backbone may comprise a copolymerized oxalate backbone; and said antioxidant groups may comprise p-Hydroxybenzyl alcohol; and said hydrophilic groups may comprise poly- (ethylene glycol) ; and said ROS-labile groups may comprise oxalyl chloride; and said hydrophilic segment may be a compound of formula (II) ,

wherein, y may be selected from 1 to 100.

As an example, said ROS-responsive nanocarrier, comprising a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups comprises a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said polymer backbone comprises a copolymerized oxalate backbone; and said hydrophilic segment is formed by copolymerization of poly- (ethylene glycol) ₂₀₀₀ and oxalyl chloride, and said hydrophobic segment is formed by copolymerization of p-Hydroxybenzyl alcohol and oxalyl chloride; and said hydrophilic segment is a compound of formula (II) , wherein, y=7.

In some embodiments, wherein said polymer backbone may be a compound of formula (III) ,

wherein, x and y may be independently selected from 1 to 100.

In some embodiments, x or y may be a single integer greater than 1. In some embodiments, x or y may be a single integer between1 and 3, 1 and 4, 1 and 5, 1 and 6, 1 and 7, 1 and 8, 1 and 9, 1 and 10, 1 and 11, 1 and 12, 1 and 13, 1 and 14, 1 and 15, 1 and 16, 1 and 17, 1 and 18, 1 and 19, 1 and 20, 1 and 21, 1 and 22, 1 and 23, 1 and 24, 1 and 25, 1 and 26, 1 and 27, 1 and 28, 1 and 29, 1 and 30, 1 and 35, 1 and 40, 1 and 45, 1 and 50, 1 and 60, 1 and 70, 1 and 80, 1 and 90, or 1 and 100.

In some embodiments, said polymer backbone may be a compound of formula (III) , wherein, x and y may be independently selected from 1 to 25.

In some embodiments, said polymer backbone may be a compound of formula (III) , wherein x=3.

In some embodiments, said polymer backbone may be a compound of formula (III) , wherein y=7.

In some embodiments, said polymer backbone may be a compound of formula (III) , wherein x=3 and y=7.

In some embodiments, the ROS-responsive nanocarrier may comprise a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups may comprise a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said hydrophilic segment may be formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment may be formed by copolymerization of antioxidant groups and ROS labile groups; and said polymer backbone may comprise a copolymerized oxalate backbone; and said antioxidant groups may comprise p-Hydroxybenzyl alcohol; and said hydrophilic groups may comprise poly- (ethylene glycol) ; and said ROS-labile groups may comprise oxalyl chloride; and said polymer backbone may be a compound of formula (III) , wherein x=3 and y=7.

As an example, said ROS-responsive nanocarrier, comprising a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups comprises a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said polymer backbone comprises a copolymerized oxalate backbone; and said hydrophilic segment is formed by copolymerization of poly- (ethylene glycol) ₂₀₀₀ and oxalyl chloride, and said hydrophobic segment is formed by copolymerization of p-Hydroxybenzyl alcohol and oxalyl chloride; and said polymer backbone is a compound of formula (III) , wherein x=3 and y=7.

In some embodiments, said polymer backbone may be assembled into stable micelles in an aqueous solution.

In some embodiments, the ROS-responsive nanocarrier, comprising a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups comprises a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said hydrophilic segment may be formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment may be formed by copolymerization of antioxidant groups and ROS labile groups; and said polymer backbone may comprise a copolymerized oxalate backbone; and said antioxidant groups may comprise p-Hydroxybenzyl alcohol; and said hydrophilic groups may comprise poly- (ethylene glycol) ; and said ROS-labile groups may comprise oxalyl chloride; and said polymer backbone may be a compound of formula (III) , wherein x=3 and y=7; and said polymer backbone may be assembled into stable micelles in an aqueous solution.

As an example, said ROS-responsive nanocarrier, comprising a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups comprises a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said polymer backbone comprises a copolymerized oxalate backbone; and said hydrophilic segment is formed by copolymerization of poly- (ethylene glycol) ₂₀₀₀ and oxalyl chloride, and said hydrophobic segment is formed by copolymerization of p-Hydroxybenzyl alcohol and oxalyl chloride; and said polymer backbone is a compound of formula (III) , wherein x=3 and y=7; and said polymer backbone is assembled into stable micelles in an aqueous solution.

In another aspect, the present disclosure provides a ROS-responsive nanoparticle may comprise said ROS-responsive nanocarrier and bioactive agents, and said ROS responsive nanocarrier and bioactive agents may be autonomously installed as nanoparticles.

In some embodiments, said bioactive agents may refer to any compound of interest that can be incorporated into the ROS responsive nanocarrier of the present invention. Non-limiting examples of bioactive agents comprise chemicals such as drugs, pharmaceutical agents, and/or radioactive elements; a bioactive agent can also comprise proteins such as antibodies, antibody fragments, antigens, cytokines; a bioactive agent can also comprise nucleic acids, comprising DNAs, RNAs, siRNAs, antisense oligonucleotides; a bioactive agent can also comprise detectable labels, such as fluorescent compounds (e.g., rhodamine dyes or fluorescent proteins) ; and/or a bioactive agent can also comprise a cocktail that comprises more than one compound (e.g., a pharmaceutical agent and an antibody) .

In some embodiments, said bioactive agents may comprise nano-drug and/or nanoprobe.

In some embodiments, said nano-drug may comprise mTOR inhibitor (e.g., Rapamycin) .

In some embodiments, ROS-responsive nanoparticle may comprise said ROS-responsive nanocarrier and Rapamycin (named HOP) , and said ROS responsive nanocarrier and Rapamycin may be autonomously installed as nanoparticles; and said ROS-responsive nanocarrier may comprise a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups may comprise a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said hydrophilic segment may be formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment may be formed by copolymerization of antioxidant groups and ROS labile groups; and said polymer backbone may comprise a copolymerized oxalate backbone; and said antioxidant groups may comprise p-Hydroxybenzyl alcohol; and said hydrophilic groups may comprise poly- (ethylene glycol) ; and said ROS-labile groups may comprise oxalyl chloride; and said polymer backbone may be a compound of formula (III) , wherein x=3 and y=7; and said polymer backbone may be assembled into stable micelles in an aqueous solution.

As an example, ROS-responsive nanoparticle comprises said ROS-responsive nanocarrier and Rapamycin (named HOP) , and said ROS responsive nanocarrier and Rapamycin are autonomously installed as nanoparticles; and said ROS-responsive nanocarrier comprises a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups comprises a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said polymer backbone comprises a copolymerized oxalate backbone; and said hydrophilic segment is formed by copolymerization of poly- (ethylene glycol) ₂₀₀₀ and oxalyl chloride, and said hydrophobic segment is formed by copolymerization of p-Hydroxybenzyl alcohol and oxalyl chloride; and said polymer backbone is a compound of formula (III) , wherein x=3 and y=7; and said polymer backbone is assembled into stable micelles in an aqueous solution; and said ROS-responsive nanoparticle is the uniform spherical nanoparticle with the dehydrated size of 170～220 nm.

In some embodiments, said nanoprobe may comprise fluorescent nanoprobe (e.g., DiD nanoprobe) .

In some embodiments, ROS-responsive nanoparticle may comprise said ROS-responsive nanocarrier and DiD nanoprobe (named DiD@HOP ) , and said ROS responsive nanocarrier and DiD nanoprobe may be autonomously installed as nanoparticles; and said ROS-responsive nanocarrier may comprise a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups may comprise a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said hydrophilic segment may be formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment may be formed by copolymerization of antioxidant groups and ROS labile groups; and said polymer backbone may comprise a copolymerized oxalate backbone; and said antioxidant groups may comprise p-Hydroxybenzyl alcohol; and said hydrophilic groups may comprise poly- (ethylene glycol) ; and said ROS-labile groups may comprise oxalyl chloride; and said polymer backbone may be a compound of formula (III) , wherein x=3 and y=7; and said polymer backbone may be assembled into stable micelles in an aqueous solution.

As an example, ROS-responsive nanoparticle comprises said ROS-responsive nanocarrier and DiD nanoprobe (named DiD@HOP ) , and said ROS responsive nanocarrier and DiD nanoprobe are autonomously installed as nanoparticles; and said ROS-responsive nanocarrier comprises a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups comprises a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said hydrophilic segment is formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment is formed by copolymerization of antioxidant groups and ROS labile groups; and said polymer backbone is a copolymerized oxalate backbone; and said antioxidant groups comprise p-Hydroxybenzyl alcohol; and said hydrophilic groups comprise poly- (ethylene glycol) ; and said ROS-labile groups comprise oxalyl chloride; and said polymer backbone is a compound of formula (III) , wherein x=3 and y=7; and said polymer backbone is assembled into stable micelles in an aqueous solution; and said ROS-responsive nanoparticle is the uniform spherical nanoparticle with the dehydrated size of 170～220 nm.

In another aspect, the present disclosure provides a nano-drug delivery system may comprise said ROS-responsive nanocarrier coated with bio-engineered cell membrane.

In some embodiments, said bio-engineered cell membrane may specifically target to the ischemic site.

In some embodiments, said bio-engineered cell membrane may specifically cross the blood-brain barrier in ischemic brain.

In some embodiments, said bio-engineered cell membrane may have a high initial brain uptake and/or the ability to expand circulation in the brain.

In some embodiments, said bio-engineered cell membrane may express a receptor targeting the normal or highly expressed protein of the ischemic region.

In some embodiments, said normal or highly expressed protein of the ischemic region may comprise SDF-1.

In some embodiments, said bio-engineered cell membrane may express or overexpress CXCR4.

In some embodiments, said bio-engineered cell membrane may express “don’t eat me” protein making the bio-engineered cell membrane have immune evasion ability.

In some embodiments, said bio-engineered cell membrane may express or overexpress CD47.

In some embodiments, said bio-engineered cell membrane may comprise vascular endothelial cell membrane.

In some embodiments, said bio-engineered cell membrane may comprise the primary vascular endothelial cell membrane.

In some embodiments, said bio-engineered cell membrane may comprise arterial endothelial cell membrane.

In some embodiments, said bio-engineered cell membrane may comprise endothelial cell membrane of the thoracic aorta.

In some embodiments, said nano-drug delivery system may comprise said ROS-responsive nanocarrier coated with bio-engineered cell membrane; and said ROS-responsive nanocarrier may comprise a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups may comprise a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said hydrophilic segment may be formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment may be formed by copolymerization of antioxidant groups and ROS labile groups; and said polymer backbone may comprise a copolymerized oxalate backbone; and said antioxidant groups may comprise p-Hydroxybenzyl alcohol; and said hydrophilic groups may comprise poly- (ethylene glycol) ; and said ROS-labile groups may comprise oxalyl chloride; and said polymer backbone is a compound of formula (III) , wherein x=3 and y=7; and said polymer backbone is assembled into stable micelles in an aqueous solution; and said bio-engineered cell membrane specifically targets to the ischemic site and has a high initial brain uptake and/or the ability to expand circulation in the brain, said bio-engineered cell membrane can specifically cross the blood-brain barrier in ischemic brain; said bio-engineered cell membrane comprises primary endothelial cell membrane of the thoracic aorta; said bio-engineered cell membrane expresses or overexpresses CXCR4 and CD47.

In another aspect, the present disclosure provides a neuroprotective agent may comprise said nano-drug delivery system, wherein the ROS-responsive nanocarrier loaded with nano-drug.

As an example, said neuroprotective agent may comprise said nano-drug delivery system, wherein the ROS-responsive nanocarrier loaded with Rapamycin (named MHOP) ; said ROS responsive nanocarrier and Rapamycin may be autonomously installed as nanoparticles; and said ROS-responsive nanocarrier may comprise a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups may comprise a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said polymer backbone may be a compound of formula (III) , wherein x=3 and y=7; and said polymer backbone may be assembled into stable micelles in an aqueous solution; and said bio-engineered cell membrane may specifically target to the ischemic site and have a high initial brain uptake and/or the ability to expand circulation in the brain, said bio-engineered cell membrane may specifically cross the blood-brain barrier in ischemic brain; said bio-engineered cell membrane may comprise primary endothelial cell membrane of the thoracic aorta; said bio-engineered cell membrane may express CXCR4 and CD47.

As an example, said neuroprotective agent may comprise said nano-drug delivery system, wherein the ROS-responsive nanocarrier loaded with Rapamycin (named BMHOP) ; said ROS responsive nanocarrier and Rapamycin may be autonomously installed as nanoparticles; and said ROS-responsive nanocarrier may comprise a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups may comprise a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said polymer backbone may be a compound of formula (III) , wherein x=3 and y=7; and said polymer backbone may be assembled into stable micelles in an aqueous solution; and said bio-engineered cell membrane may specifically target to the ischemic site and have a high initial brain uptake and/or the ability to expand circulation in the brain, said bio-engineered cell membrane can specifically cross the blood-brain barrier in ischemic brain; said bio-engineered cell membrane may comprise primary endothelial cell membrane of the thoracic aorta; said bio-engineered cell membrane may overexpress CXCR4 and express CD47.

In another aspect, the present disclosure provides a ROS-responsive nanoprobe may comprise said nano drug delivery system, wherein the ROS-responsive nanocarrier loaded with nanoprobe.

As an example, said ROS-responsive nanoprobe may comprise said nano drug delivery system, wherein the ROS-responsive nanocarrier loaded with DiD nanoprobe (named DiD@MHOP) ; said ROS responsive nanocarrier and DiD nanoprobe may be autonomously installed as nanoparticles; and said ROS-responsive nanocarrier may comprise a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups may comprise a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone may be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said polymer backbone may be a compound of formula (III) , wherein x=3 and y=7; and said polymer backbone may be assembled into stable micelles in an aqueous solution; and said bio-engineered cell membrane may specifically target to the ischemic site and have a high initial brain uptake and/or the ability to expand circulation in the brain, said bio-engineered cell membrane may specifically cross the blood-brain barrier in ischemic brain; said bio-engineered cell membrane may comprise primary endothelial cell membrane of the thoracic aorta; said bio-engineered cell membrane may express CXCR4 and CD47.

As an example, said ROS-responsive nanoprobe may comprise said nano drug delivery system, wherein the ROS-responsive nanocarrier loaded with DiD nanoprobe (named DiD@BMHOP) ; said ROS responsive nanocarrier and DiD nanoprobe are autonomously installed as nanoparticles; and said ROS-responsive nanocarrier comprises a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups comprises a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity; and said polymer backbone is a compound of formula (III) , wherein x=3 and y=7; and said polymer backbone is assembled into stable micelles in an aqueous solution; and said bio-engineered cell membrane specifically targets to the ischemic site and has a high initial brain uptake and/or the ability to expand circulation in the brain, said bio-engineered cell membrane can specifically cross the blood-brain barrier in ischemic brain; said bio-engineered cell membrane comprises primary endothelial cell membrane of the thoracic aorta; said bio-engineered cell membrane overexpresses CXCR4 and expresses CD47.

In another aspect, the present disclosure provides a method for preparing said ROS-responsive nanocarrier may comprise: synthesizing a polymer skeleton containing antioxidant groups and hydrophilic groups through a one-step polycondensation reaction with ROS labile groups.

In some embodiments, said antioxidant groups may comprise p-Hydroxybenzyl alcohol (HBA) .

In some embodiments, said hydrophilic groups may comprise poly- (ethylene glycol) .

In some embodiments, said ROS-labile groups may comprise oxalyl chloride (OC) .

In some embodiments, said method for preparing said ROS-responsive nanocarrier, may comprise: synthesizing HBA-OC-PEG ₂₀₀₀ containing PEG ₂₀₀₀ and HBA through a one-step polycondensation reaction with oxalyl chloride, and obtaining the obtained after repeated precipitation into cold ether, purifying the collected copolyoxalate, drying under high vacuum, and then obtained HBA-OC-PEG ₂₀₀₀.

In some embodiments, the preparation method of HBA-OC-PEG ₂₀₀₀ may comprise the following steps:

a) HBA (p-hydroxybenzyl alcohol) , PEG ₂₀₀₀ (Polyethylene glycol ₂₀₀₀) were dissolved into CH ₂Cl ₂ (methylene chloride) , Mixed the dissolved HBA with PEG ₂₀₀₀, and the whole reaction process is operated in an ice bath;

b) OC (oxaloyl chloride) was rapidly added into the mixture solution of step a) , and reacted at room temperature and stays overnight, then the HBA-OC-PEG ₂₀₀₀ were obtained.

In another aspect, the present disclosure provides a method for preparing said ROS-responsive nanoparticle, wherein said bioactive agents may be loaded into micelles through the self-assembly of ROS-responsive nanocarrier to obtain nanoparticles.

In some embodiments, the preparation method of ROS-responsive nanoparticle may comprise the following steps:

a) HBA-OC-PEG ₂₀₀₀ and RAPA were respectively dissolved in the THF (tetrahydrofuran) , under the action of full agitation, appropriate amount of RAPA THF solution was dropped into the HBA-OC-PEG ₂₀₀₀ THF solution which were mixed with H ₂O;

b) The mixture of step a) was further dialyzed using dialysis bag (molecular weight cut-off, MWCO: 3500 Da) against water to remove the free RAPA and THF, then the HOP were obtained.

In another aspect, the present disclosure provides a method for preparing said neuroprotective agent may comprise the method of physically extruding the bio-engineered cell membrane to coat the ROS-responsive nanoparticle, wherein said bio-engineered cell membrane may be obtained by lentiviral transcription.

In some embodiments, the method for preparing the bio-engineered cell membrane specifically may comprise: using lentivirus to transcribe vascular endothelial cells to obtain the bio-engineered cell membrane with normal expression or overexpression of CXCR4.

In some embodiments, the preparation method of neuroprotective agent may comprise the following steps:

a) Rapamycin-loaded (HBA-OC-PEG ₂₀₀₀/RAPA, named HOP) was assembled using dialysis method;

b) Extrusion method was used to prepare different NPs formulations, comprising mouse aortic endothelial cell membrane coated HBA-OC-PEG ₂₀₀₀/RAPA (named MHOP) and CXCR4 overexpression mouse aortic endothelial cells, namely bioengineered cell membrane coated HBA-OC-PEG ₂₀₀₀/RAPA (named BMHOP) .

In another aspect, the present disclosure provides a method for preparing said ROS-responsive nanoprobe may comprise the method of physically extruding the bio-engineered cell membrane to coat said ROS-responsive nanoparticle, wherein said bio-engineered cell membrane is obtained by lentiviral transcription.

a) DiD-loaded (HBA-OC-PEG ₂₀₀₀/DiD, named DiD @HOP) was assembled using dialysis method;

b) Extrusion method was used to prepare different NPs formulations, comprising mouse aortic endothelial cell membrane coated HBA-OC-PEG ₂₀₀₀/DiD (named DiD@MHOP) and CXCR4 overexpression mouse aortic endothelial cells, namely bioengineered cell membrane coated HBA-OC-PEG ₂₀₀₀/DiD (named DiD@BMHOP) .

In another aspect, the present disclosure provides a use of said ROS-responsive nanocarrier、 said ROS-responsive nanoparticle、 said nano-drug delivery system and/or said neuroprotective agent in the preparation of a medicine for targeted treatment of ischemic stroke.

In another aspect, the present disclosure provides a use of said ROS-responsive nanocarrier、 said ROS-responsive nanoparticle、 said nano-drug delivery system and/or said ROS-responsive nanoprobe in the preparation of a kit for diagnosing ischemic stroke.

In another aspect, the present disclosure provides a method of treating or preventing ischemic diseases may comprise the administration of an effective amount of said neuroprotective agent to a patient in need thereof.

In some embodiments, said ischemic diseases may comprise reperfusion injury after stroke.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete invention and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; and the like.

Example 1: Synthesis of HBA-OC-PEG ₂₀₀₀, HBA-OC-PEG ₂₀₀₀/RAPA (HOP) , mouse aortic endothelial cell membrane coated HBA-OC-PEG ₂₀₀₀/RAPA (MHOP) , bioengineered cell membrane coated HBA-OC-PEG ₂₀₀₀/RAPA (BMHOP)

p-Hydroxybenzyl alcohol (HBA) , Oxalyl chloride (OC) , Rapamycin (RAPA) and poly- (ethylene glycol) ₂₀₀₀ (PEG ₂₀₀₀) were obtained from Shanghai Aladdin Bio-Chem Technology Co, Ltd. (Shanghai, China) . HBA-OC-PEG ₂₀₀₀ containing PEG ₂₀₀₀ and HBA was synthesized through a one-step condensation polymerization with oxalyl chloride.

Primary mice thoracic aorta endothelial cells were isolated and cultured from C57BL/6 mice (Figure. 21) , mouse mononuclear macrophage leukemia cell (RAW264.7) , mouse brain capillary endothelial cells (BECE) and Human umbilical vein endothelial cells were obtained from Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) . MTAEC and BCEC cells were cultured in primary endothelial cell medium, RAW264.7 cells were cultured in DMEM medium and HUVEC cells were cultured in RPMI 1640 medium at 37℃ in a 5%CO2 humidified incubator. All media contained 10%FBS, 100 U mL-1 penicillin and 100 μg mL-1 streptomycin. Eight-week old C57BL/6 mice (male) were purchased from SPF (Beijing) Biotechnology Co, Ltd. (Beijing, China) .

Figure. 21: Primary mouse thoracic aortic endothelial cells were extracted and the cell properties were identified by immunofluorescence: CD31 (the endothelial cell specific marker protein) . (A) The third generation cell original figure; (B) DAPI for nuclear staining; (C) immunofluorescence for CD31; (D) merge for endothelial cell identification (Scale bar = 200 nm) .

MTAEC cells were cultured by primary cell complete culture medium (ScienceCell. Beijing Yuhengfeng Biotech Co, Ltd. ) , comprising 500 mL of basal medium, 25 mL of FBS (fetal bovine serum) , 5 mL of endothelial cell growth supplement and 5 mL of penicillin/streptomycin solution, in the 37 ℃ 5%CO2 saturated humidity incubator. The lentivirus vector (A5585-1) for CXCR4 (mouse) overexpression were designed and chemically synthesized from Shanghai GenePharma Co, Ltd. (Shanghai China) . The sequence of target gene CXCR4 comes from Genebank accession NO: NM_009911.3. The lentiviral vector with green fluorescence protein (GFP) was used as the identification of successful transfection marker (Figure. 8) . Following the experimental instructions, CXCR4 was successfully overexpressed in cells after transfection for 96 h at MOI=100. The transfection effect from 5 different time periods was shown in the above figure: 12 h, 24 h, 48 h, 72 h, 96 h , and GFP was the green fluorescence indicator (Scale bar = 200 nm) .

Ⅰ. Synthesis of HBA-OC-PEG ₂₀₀₀

The polymerization was proceeded in dry tetrahydrofuran (THF) under low temperature environment to generate the corresponding copolymers. And the obtained copolyoxalate was purified through repeated precipitation into cold hexane and obtained as pale yellow transparent colloidal solids after drying under high vacuum oven.

HBA-OC-PEG ₂₀₀₀ was prepared by chemical synthesis, and the preparation method of HBA-OC-PEG ₂₀₀₀ comprises the following steps:

a) HBA (p-hydroxybenzyl alcohol) , PEG ₂₀₀₀ (Polyethylene glycol- ₂₀₀₀) were dissolved into CH ₂Cl ₂ (methylene chloride) , Mixed the dissolved HBA with PEG ₂₀₀₀, and the whole reaction process was operated in an ice bath; and

The chemical structure of HBA-OC-PEG ₂₀₀₀ was confirmed by the 1H NMR (Figure. 7) . The NMR results of HBA-OC-PEG ₂₀₀₀. The chemical structure of polymers was identified with a 400 MHz 1H NMR spectrometer (Bruker ADVANCE500) . 1H NMR in deuterated DMSO-d6 on a 400 MHz spectrometer. The chemical structure of HBA-OC-PEG ₂₀₀₀ was confirmed by the 1H NMR. Comparing it to the resonance signal of the integrated methoxy group of the PEG chain at ～3.5 ppm, the resonance peaks at ～4.3 ppm correspond to the methylene protons adjacent to oxalate ester linkages, two multiplet aromatic proton peaks appear at 6.7 and 7.2 ppm. These results demonstrated successful polymerization from the condensation reaction between oxalyl chloride and six PEG ₂₀₀₀ and one HBA, generating polyoxalate containing peroxalate ester linkages.

Ⅱ. Synthesis of HOP

Rapamycin-loaded HBA-OC-PEG ₂₀₀₀ (HBA-OC-PEG2 ₀₀₀/RAPA, named HOP) were prepared by nanoprecipitation method comprising the following steps:

a) HBA-OC-PEG ₂₀₀₀ and RAPA were respectively dissolved in the THF (tetrahydrofuran) , under the action of full agitation, appropriate amount of RAPA THF solution was dropped into the HBA-OC-PEG ₂₀₀₀ THF solution which were mixed with H ₂O; and

Ⅲ. Synthesis of MHOP

The cell membrane of MTAEC and HBA-OC-PEG ₂₀₀₀/RAPA NPs were fused to prepare MHOP by an extrusion method comprising the following steps:

a) The extraction of MTAEC cell membrane comprised reagents A melt at room temperature and PMSF (Phenylmethylsulfonyl fluoride) was added; A few minutes before use to make the final concentration of PMSF 1mM; Culture about 20-50 million cells, wash them with PBS, and scrape the cells with a cell scraper; Cells were collected by centrifugation, and cell precipitation was left for later use after supernatant was removed; Add 1 mL working solution to 20-50 million cells, gently and fully suspend the cells, and leave in an ice bath for 10-15 min; The samples were successively frozen and thawed three times in liquid nitrogen and room temperature. Remove nuclei and unfragmented cells: centrifuge at 4 ℃, 700g, for 10 min, carefully collect the supernatant into a new centrifuge tube; When the supernatant is absorbed, do not contact with the precipitation to precipitate cell membrane fragments: centrifuge at 4 ℃ for 14000×g for 30 min to precipitate cell membrane fragments.

b) The cell membrane was extracted from the MTAEC cells and HBA-OC-PEG ₂₀₀₀/RAPA were mixed; and the mixture solution was extruded using an Avestin mini-extruder (Avestin, LF-1, Canada) through a 200 nm polycarbonate porous membrane for 10 times to harvest the MHOP.

Ⅳ. Synthesis of BMHOP

The preparation method of BMHOP comprises the following steps:

a) Lentivirus was used to transcribe vascular endothelial cells to obtain the bio-engineered cell membrane with high expression of CXCR4:

(1) . The well-grown MTAEC cells were digested and re-suspended, and an appropriate amount of cells were inoculated into a 24-well plat (Corning) and placed in a 37 ℃ incubator overnight;

(2) . The negative control virus was diluted with the medium at 1: 10, 1: 100, 1: 1000, and the total volume was about 500 μL; Polybrene was added with a final concentration of 5 μg/mL;

(3) . The original culture medium was removed from the 24-well plate and replaced with negative control virus gradient diluting solution;

(4) . After 24 h, the diluent of negative control virus was removed and replaced with 500 μL fresh medium;

(5) . The results were observed and recorded 48 hours later under an inverted fluorescence microscope. According to the instructions and preliminary experiments, the optimal transfection rate was finally determined as the highest multiplicity of infection (MOI=100) . At different time points of 12, 24, 48 and 72 h, the efficiency of transfection was measured by the fluorescence microscope. The expression levels of target gene CXCR4 (mouse) was confirmed by using q-PCR and western blotting after 3 days (Figure. 9) ; As shown in Figure. 9, the DNA of the 8th-generation bioengineered primary cells were extracted and comparing with the unmodified cells for detection by q-PCR, and it was found that CXCR4 had significant high expression at the molecular level. Meanwhile, the expression level of cxcr4 was confirmed by using western blotting. (A) The results of q-PCR (Values represent mean ± SD, n = 3, ***P < 0.01) . (B) GPF of the 8th-generation bioengineered primary cells observed by microscopy. (C) The WB results of cxcr4 expression. (D) The analysis of WB gray value (Values represent mean ± SD, n = 3, ***P < 0.01) (Scale bar = 200 nm) .

b) The extraction of cell membrane comprised reagents A melt at room temperature and PMSF (Phenylmethylsulfonyl fluoride) was added. A few minutes before use to make the final concentration of PMSF 1mM. Culture about 20-50 million cells, wash them with PBS, and scrape the cells with a cell scraper. Cells were collected by centrifugation, and cell precipitation was left for later use after supernatant was removed. Add 1 mL working solution to 20-50 million cells, gently and fully suspend the cells, and leave in an ice bath for 10-15 min. The samples were successively frozen and thawed three times in liquid nitrogen and room temperature. Remove nuclei and unfragmented cells: centrifuge at 4 ℃, 700g, for 10 min. Carefully collect the supernatant into a new centrifuge tube. When the supernatant is absorbed, do not contact with the precipitation to precipitate cell membrane fragments: centrifuge at 4 ℃ for 14000×g for 30 min to precipitate cell membrane fragments.

c) The cell membrane of MTAEC and HBA-OC-PEG ₂₀₀₀/RAPA NPs were fused to prepare MHOP by an extrusion method. Briefly, the cell membrane was extracted from the MTAEC cells and HBA-OC-PEG ₂₀₀₀/RAPA were mixed. Then, the mixture solution was extruded using an Avestin mini-extruder (Avestin, LF-1, Canada) through a 200 nm polycarbonate porous membrane for 10 times to harvest the MHOP.

Transmission electron microscopy (TEM) observation showed that all the nano-assemblies exhibit the uniform spherical nanoparticle with the dehydrated size of ～170 nm (HOP) , ～200 nm (MHOP) and ～200 nm (BMHOP) . An outer layer was clearly observed in MHOP and BMHOP samples indicating the membrane-formed shell structure of the NPs (Figure. 1A) . Average hydrodynamic diameter of HOP was 172.5 nm, MHOP and BMHOP were slightly increased to 219.6 nm and 227.8 nm (Figure. 1B) .

The ζ potentials of MHOP and BMHOP were respectively changed to -18.1 mV and -20.5 mV compared to the HOP with ζ potential of -15.7 mV was observed by dynamic light scattering (DLS) (Figure. 1C) . In respect to the relatively increased particle size and surface ζ potential suggested that the membranes were translocated onto the surface of NPs.

The protein ingredients of cell membrane of CXCR4 high expression mouse thoracic aorta endothelia cells were analyzed using sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) . The cell-membrane (CM) modification strategy rendered BMHOP with the similar exterior structures of CXCR4 overexpression mouse aortic endothelial cells (Figure. 1D) , the extracted CM and CM-coated NPs resulted in highly consistent in protein bands, matching closely with the whole mouse aortic endothelial cells. The SDS-PAGE results suggested that the proteins of CM in MHOP were significantly retained.

Representative adhesion proteins CXCR4 were evaluated by Western blot assay (WB) (Figure. 1E) to further confirm the modification of the specific targeting proteins on BMHOP. Cell migration experiment was carried out with the following steps: (1) . Mouse thoracic aortic endothelial cells and bioengineering cells were respectively removed from the culture flashs and resuspended at 0.5×106 cells/mL in serum free culture medium. (2) . SDF-1 (100 ng/mL) was placed in the lower wells. Test cells were then placed in the upper chamber for 60 min. (3) . After incubation, the upper surface of the transwell membrane was wiped gently with a cotton swab to remove non-migrating cells. Cells which migrated to the lower surface of the membrane were stained using DAPI. (4) . Migrated cells were counted in 3 different fields of a defined size (5×0.25 mm ²) using a phase contrast microscope and the mean cellular migration rate was calculated. As shown in Figure 1E, both the HCM and BMHOP exhibited the obvious expression of CXCR4 compared with lentivirus transfected mouse aortic endothelial cells (Figure 10) , further demonstrating the SDF-1 targeting potential of the modification cell membrane coated NPs. And the presence of CD47 on CM, MHOP, CXCR4 overexpression CM (HCM) and BMHOP exhibited the obvious CD47 band demonstrating that the membrane coated onto the NPs, these proteins, specifically CXCR4 and CD47, were shown to retain their functionality.

Hydrolysis profiles were investigated in 0.01M PBS (pH 7.4) to test the stability of HOP, MHOP, BMHOP for evaluation of their ROS-responsive behaviors. For seven consecutive days, the average size of HOP remained at 181.81 ± 9.16nm, for MHOP was 220.91 ± 11.16nm and BMHOP was 236.4±2.70nm. The HOP, MHOP and BMHOP exhibited an excellent stability without obvious changes in the size over a period of one week at room temperature. Significant particle size changes at 37 ℃ after one week (Figure. 1F) suggested that the nanocarrier formulation kept a better stability under relative low temperature.

Figure 1. Characterizations of bioengineered cell membrane coated NPs. (A) TEM image of HOP, MHOP and BMHOP (scale bar = 200 nm) . (B) Hydrodynamic size distribution and (C) ζ potential of HOP, MHOP and BMHOP (Values represent mean ± SD, n = 3) . (D) SDS-PAGE protein analysis and (E) western blot. (F) The stability of NPs in 0.01 M PBS at room temperature and (G) drug release of different NPs (Values represent mean ± SD, n = 3) .

As shown in Figure. 10, in order to verify the target ability of bioengineered cells, the 6-well cell culture plates transwell chamber (aperture 8 μm) were performed on cell migration experiments to evaluate it. This cell migration experiment mechanism: transwell Chambers located in the culture plate, due to the permeability of polycarbonate membrane between apical side and basolateral side, the ingredients of the lower culture medium can affect the cell movement in the upper chamber. Appropriate SDF-1 (100 ng/mL) was added to the lower culture medium as a chemokine for the cells on apical side, it is observed that some cells migrated to the basolateral side of the membrane after 1h. In contrast, four other groups for experimental control were set: (A) bioengineered cells + SDF-1 (100ng/mL) ; (B) normal cells + culture medium (without SDF-1) ; (C) bioengineered cells + culture medium (without SDF-1) ; (D) bioengineered cells + SDF-1 (100 ng/mL) + AMD 3100 (100 ng/mL) : Plerixafor (AMD 3100) is a selective CXCR4 antagonist; (E) normal cells + SDF-1 (100 ng/mL) however, comparing with treatment group there is no significant cell migration occurred (Scale bar = 200 nm) (Values represent mean ± SD, n = 3, ***P < 0.01) .

Cell migration experiment:

1) . Mouse thoracic aortic endothelial cells and bioengineering cells were respectively removed from the culture flashs and resuspended at 0.5×106 cells/mL in serum free culture medium.

2) . SDF-1 (100 ng/mL) was placed in the lower wells. Test cells were then placed in the upper chamber for 60 min.

3) . After incubation, the upper surface of the transwell membrane was wiped gently with a cotton swab to remove non-migrating cells. Cells which migrated to the lower surface of the membrane were stained using DAPI.

4) . Migrated cells were counted in 3 different fields of a defined size (5×0.25 mm2) using a phase contrast microscope and the mean cellular migration rate was calculated.

Example 2: Evaluation of the stability and ROS responsiveness of HOP, MHOP, BMHOP

DLS was used to monitor the change in the particle size of the HOP, MHOP and BMHOP in PBS with/without 1 mM H ₂O ₂ to test the physical stability of the cell membrane coated NPs. The size of NPs in H ₂O ₂ containing PBS gradually increased in 12 h comparing with the NPs still exhibiting stable particle size in PBS, revealing their feasible ROS responsive degradation for potentially enhancing cargo release locally (Figure. 11) .

ROS are over-produced and accumulated triggering a rapid release of RAPA from the stimulate-NPs in the course of stroke ischemia-reperfusion. To study the drug release profile in vitro, HOP, MHOP and BMHOP nanoparticle solutions (1 mg/mL, 1 mL) were added to disposable dialysis cups (Slide-A-Lyzer MINI Dialysis Units, MWCO: 3500 Da, Thermo Scientifc) in PBS (10 mL) . At different time points, the external drug release buffers were collected and an equivalent amount of PBS was added. Quantitative study (Figure. 1G) showed that approximately 27.3%of Rapamycin were found at the beginning 2 h in 1 mM H ₂O ₂ within PBS. The concentration of RAPA gradually enhanced with time, and reached the drug release limit point when the degradation time is more than 12 h. In the absence of H ₂O ₂, the HOP, MHOP and BMHOP exhibited a stability without obvious size increase in 8 h, and there was slight release (13.2%) of drug in 8 h and no significant change in the accumulative release of RAPA under drug release more than 8 h, indicating their favorable stability for reducing premature cargo leaky during the blood circulation. While HOP, MHOP and BMHOP were hydrolyzed in the medium within 2 h in the presence of H ₂O ₂ (Figure. 1G) , along with it the physical stability of the NPs was broken.

Example 3: Evaluation of the ability of NPs to specifically target SDF-1 overexpression cells

As shown in (Figure. 2A) , the primary mode of action of BMHOP was through specific CXCR4/SDF-1 targeting to the SDF-1 overexpression microenvironment. Meanwhile, NPs accumulated in cells whose high level of ROS efficiently triggered BMHOP for ROS-scavenging and antioxidation. The released RAPA stimulated endothelium survival and functional anti-inflammation, giving rise to a significant therapeutic effect in ischemic stroke.

DiD@HOP、 DiD@MHOP、 DiD@BMHOP were prepared by the same protocol. RAPA-loaded NPs were substituted by using DiD encapsulation for fluorescence analysis.

The HUEVC cells either before or after oxLDL-stimulated (overexpression of SDF-1) were treated with DiD@NPs to evaluate the ability of NPs to specifically target SDF-1 overexpression cells. The results showed that DiD@BMHOP were preferentially accumulated in oxLDL-stimulated HUEVC (Figure. 2B) .

Quantitative flow cytometry analysis showed that oxLDL-induced HUEVC exhibited significantly stronger DiD@BMHOP fluorescence than normal cells compared with DiD@HOP and DiD@MHOP (Figure. 12) . In contrast with normal NPs, a 72.4-fold increase in DiD@BMHOP accumulation was observed when HUEVCs cultured with NPs for 2 h (Figure. 13) indicating that CXCR4/SDF-1 axis is involved in the enhancement of migration with BMHOP in vitro. As shown in Figure. 12, the red fluorescence of DiD loading into the DiD@HOP, DiD@MHOP and DiD@BMHOP. For the targeting of DiD@BMHOP, there was a significant difference between the two different HUVEC (normal cells and SDF-1 overexpression) during the first 1 h. Nuclear exhibited blue (dyeing with DAPI) (Scale bar = 20 μm) .

Figure. 13 A. The flow cytometry results of DiD@HOP, DiD@MHOP and DiD@BMHOP. B. The quantitative analysis of red fluorescence of different DiD@NPs (Values represent mean ± SD, n = 3, **P < 0.05) .

Colocation studies were used to identify the subcellular locations of DiD@BMHOP against SDF-1 overexpression HUVEC cells. The red fluorescence of DiD colocalized well with the green fluorescence of DiO, and it accumulated well with surroundings of nucleus stained with DAPI (Figure. 14) . As shown in Figure. 14, the red fluorescence of DiD loading into the DiD@HOP, the bioengineered cell membrane labeled with the green fluorescence of DiO coated the DiD@HOP, and it accumulated well with surroundings of nuclear dyeing with DAPI (Scale bar = 50 nm) .

Example 4: Assessment of the cytotoxicity of BMHOP

The cytotoxicity of BMHOP was evaluated using HUEVC by MTS assays at the concentration of not more than 10 μg/mL. The results showed no significant reduction in cell viability by different doses (0.01, 0.1 or 1 μg/mL) of BMHOP, neither at a higher concentration tested (5 μg/mL) had a notable effect on cell survival (Figure. 15, Values represent mean ± SD, n = 6, ***P < 0.01) ) . Treatment of HUVEC cells with H ₂O ₂ (300 μM) significantly increased the production of ROS. The BMHOP (5 μg/mL) suppressed the H ₂O ₂-induced cytotoxicity of cells, moreover, H ₂O ₂-induced cytotoxicity was inhibited by co-treatment at 5 μg/mL with HOP or MHOP. Compared with untreated HUVEC, treatment of cells with BMHOP significantly restored cell viability was shown in Figure. 2C suggesting that BMHOP attenuated the intracellular ROS level, suppressed H ₂O ₂-induced oxidative stress, and ultimately reversed H ₂O ₂-induced cytotoxicity in cells.

Example 5: Examination of the scavenging ratio of the generation of ROS by BMHOP

Intracellular ROS overexpression was constructed by following steps. The cells were plated at a density of 5 × 105 cells/well in six-well Biocoat plates and grown for 24 h in complete medium that 1640 culture medium supplemented with 10%fetal bovine serum (FBS) , at 37 ℃ in a normoxia with 5%CO2 atmosphere. Cells were washed twice in 1640 without FBS (Life Technologies, Carlsbad, CA, USA) switched to 1640 without FBS supplemented with 300 μM H2O2 and placed in modular incubator chambers (BillupsRothenberg, Del Mar, CA, USA) for 24 h. Intracellular ROS levels were detected with 10 μM dihydroethidium (DHE) to examine the scavenging ratio of the generation of ROS by BMHOP. The CLSM results illustrated that BMHOP (5 μg/mL) exhibited significant inhibitory effects on ROS generation in a time-dependent manner, 1 h later the intracellular ROS levels were decreased along with the expression of red fluorescence in the cells was reduced (Figure. 16) , indicating that the level of ROS in the cells was decreased in the presence of BMHOP. Reduced red fluorescence intensities tend to disappear and almost cannot be visualized after 6 h (Figure. 2D) . In another result, fluorescence microscopic images of DHE-stained cells indicated consumption of intracellular ROS in vitro is consistent for 3 diverse NPs (Figure. 17) .

Figure. 16: (A) The intracellular ROS production after DiD@BMHOP treatment at different time points were measured by flow cytometry. (B) The quantitative analysis of red fluorescence of DiD@BMHOP at different time points (Values represent mean ± SD, n = 3, ***P < 0.01) .

Figure. 17: CLSM images results of 3 different DiD@NPs on intracellular ROS elimination at different time points (Scale bar = 50 nm) .

Example 6: Evaluation of the anti-inflammatory properties of BMHOP using RAW264.7

The anti-inflammatory properties of BMHOP were evaluated using RAW264.7 based on the ROS-eliminating and cell protective capacity of NPs. The MTS assay (Figure. 18) verified that cell proliferation of RAW264.7 cells were significantly inhibited by BMHOP (5 μg/mL) . At the same concentration, apart from cell apoptosis was initial observed after 0.5 h under the treatment of BMHOP, significant necrosis was even emerged followed by 2 h to 6 h (Figure. 2E) .

Figure 2. (A) Scheme of the mechanisms of ischemic location treatment by BMHOP. (B) CLSM images of cellular uptake of DiD@NPs in SDF-1 overexpression HUVEC (scale bar = 200 μm) . (C) The effects of BMHOP on H2O2-induced HUVEC and untreated HUVEC (Values represent mean ± SD, n = 6, ***P < 0.01) . (D) CLSM images results of BMHOP on intracellular ROS elimination (Scale bar = 50 μm) . (E) The results of flow cytometry analysis images of cell apoptosis gating on Annexin V-FITC/PI staining of BMHOP effects on RAW264.7.

Figure. 18: Inhibitory effect of BMHOP (5 μg/mL) on cell proliferation of RAW264.7 cells (Values represent mean ± SD, n = 6, ***P < 0.01) .

The results showed biocompatibility and the dominating role of the ROS scavenging and the additive effect of RAPA, demonstrating the potent antioxidant and anti-inflammatory activity of BMHOP in stroke therapy.

Example 7: Evaluation of the enhancement of the targeted delivery from NPs to the ischemic brain caused by CXCR4 overexpression cell membrane

NPs accumulation into the ischemic brain was studied after in vivo administration of NPs encapsulation an infrared fluorescent dye of DiD, comprising BMHOP and HOP and MHOP. Fluorescence of DiD in major organs (heart, brain, lung, kidney, spleen) was observed 12 h after MCAO surgery reperfusion along with tail vein injection (Figure. 3A) , NPs appeared in the liver, lung and kidneys may be on account of the damage in these organs caused by ischemia. DiD@BMHOP showed prominent accumulation in the ischemic brain area compared with HOP group which had no cell membrane coating or MHOP NPs which had low CXCR4 targeting affinity (Figure. 3B) .

As shown in Figure. 19, Radiant efficiency of fluorescence intensity of different DiD@NPs in major organs after 12 h post injection (Values represent mean ± SD, n = 3, ***P < 0.01) . The large amount of aggregation of nanoparticles in the liver is consistent with the previous research. As SDF-1 are highly express in the inflammation site caused by stroke of some organs, it could be possible for large numbers of BMHOP to accumulate in the liver and spleen serosa and in the stomach serous membrane.

The targeted accumulation of DiD@BMHOP into the ischemic side in first 2 h after NPs in vivo administration was confirmed by IVIS Lumina II imaging (Figure. 3C) . Quantification of the fluorescence intensity in the brain indicated that the accumulation of DiD@BMHOP significantly increased when injected at the early stage of ischemic stroke, the fluorescence intensity was obviously reduced after injection by time points at 6 h, 12 h, 24 h (Figure. 3D) . The accumulation of fluorescence signal and wider biodistribution of DiD@BMHOP was still present on the brain by 24 h post-injection suggesting that DiD@BMHOP efficiently crossed the BBB in the ischemic brain with precise specificity, demonstrating not only high initial brain uptake but also good accumulation by the extended circulation ability in the brain.

CD31 immunostaining of brain sections of MCAO mice after DiD@BMHOP injection was performed to profile the nanodrug carriers penetrate the blood-brain barrier (BBB) to further investigate DiD@BMHOP distribution within the ischemic area (Figure. 3E) . A large number of red fluorescence signal were found away from CD31 staining area within the ischemic brain indicating that the NPs could further cross the BBB then diffused into and stay in the brain parenchyma. Red fluorescence signal in the ipsilateral brain hemisphere also showed enhanced accumulation of DiD@BMHOP compared with the contralateral side having a low expression level of SDF-1 (Figure. 3F) .

Figure 3. Targeting ability and accumulation into the ischemic brain of NPs. (A) Ex vivo IVIS imaging of major organs from MCAO mice 12 h after i. v. injection of DiD@HOP, DiD@MHOP and DiD@BMHOP. (B) Quantification of the radiant efficiency of NPs in the brain by IVIS Lumina imaging software performed by drawing a region of interest (ROI) (Values represent mean ± SD, n = 3, ***P < 0.01) . (C) Ex vivo IVIS imaging of targeting accumulation of DiD@BMHOP into the brain at different time points (2 h, 6 h, 12 h and 24 h) after i. v. injection, as the control group, naive healthy mice were injected with DiD@BMHOP. (D) Quantification of the radiant efficiency of DiD@BMHOP in the brain by IVIS Lumina imaging software performed by drawing a region of interest (ROI) (Values represent mean ± SD, n = 3, **P < 0.05 and ***P < 0.01) . (E) Distribution of DiD@BMHOP in ischemic penumbra (blue: DAPI for nucleus; green: CD31 for blood vessels; red: DiD@BMHOP) . White arrow indicate DiD@BMHOP associated with blood vessels; greyish-green arrows indicate DiD@BMHOP diffused away from blood vessels (white scale bar = 200 μm) . (F) Scheme of the expression of SDF-1 in the ischemic or normal brain.

Example 8: Study of BBB transport using transwell system seeded with brain capillary endothelial cells (BCECs)

The effectiveness in crossing the BBB is one critical problems of the application of nanobiotechnology. The transwell system seeded with brain capillary endothelial cells (noted as BCECs) is the most commonly used and convenient approach currently available for studying BBB transport (Figure. 4A) .

BCEC cells were maintained at 37 ℃ and with 5%CO2 in primary cell complete culture medium (ScienceCell. Beijing Yuhengfeng Biotech Co, Ltd. ) in a humidified incubator. The BBB (brain blood barrier) model was constructed by a polycarbonate 12-well Transwell membrane of 1.0 mm mean pore size with 0.33 cm ² surface areas (FALCON Cell Culture Insert, Becton Dickinson Labware, USA) , in which BCECs were seeded at a density of 104 cells/well and cultured for 4 days. An epithelial voltohmmeter (Millicell-RES, Millipore, USA) was used to measure the transendothelial electrical resistance (TEER) of cell monolayers, and those above 200 Ω cm ² were selected for experiments to ensure the permeability to inorganic ions (Figure. 20) . Flow cytometry data of DiD@BMHOP and DiD@MHOP showed more than 250-fold difference in cellular uptake the cell membrane coated NPs to DiD@HOP at the first 2 h (Figure. 4B, 4C) . This could be attributed to the excellent monodispersity, furthermore, surface effects and the effect of interface commensurability on the transport behavior of cell membrane coated NPs.

Figure 4. (A) Schematics of the BCEC transwell model in vitro for constructing the BBB permeability. (B) Flow cytometry analysis of BCECs after being co-incubated with DiD@NPs for 2 h in vitro and (C) quantification of BBB crossing of DiD@NPs in BCECs (Values represent mean ± SD, n = 3, ***P < 0.01) . (D) Establishment of MCAO mice model and schematic illustration of tail intravenous injection into brain tissue for stroke treatment. (E) Digital photographs of brains stained by permeating EB and (F) corresponding representative CLSM images of the brain slices showing red fluorescences of EB under the excitation at a wavelength of 405 nm (scale bar = 200 μm) . (G) EB micrograms per gram of brain tissue calculated from the EB exudation (Values represent mean ± SD, n = 3, **P < 0.05 and ***P < 0.01) .

Example 9: Cerebral uptake experiments of NPs carried out on healthy mice and MCAO mice model to demonstrate the BBB crossing and prevention of the BBB damage capability

The permeability of BBB was investigated by the traditional Evans Blue (EB) staining assay. 0.5%EB was injected into mice as a BBB permeability tracer at the dose of 16 mL/kg within 24 h of stroke, after circulation for 2 h, the mice chest walls were opened to perfuse with saline through the left ventricle. The brains were carefully removed and weighed (Table S1) , and then taking photos by a digital camera. Meanwhile, the permeability of EB was observed by CLSM under 405 nm excitation on the frozen section of brain tissue. To further quantify the amount of EB, brain tissue was homogenized in PBS (1 mL) using a homogenizer and centrifuged for 15

Table S1. The effects of different treatments on brain weight in MCAO mice.

min at 1000×g. The precipitate was discarded and the supernatant was collected, added to acetone at a 1: 9 proportion (supernatant: acetone) , and kept for 24 h after mixed well. Then taken out for absorbance measurement at 620 nm, and the content of the extraction of EB was calculated via a standard concentration line (Figure. 22) .

As shown in the digital photographs (Figure. 4E) , the left half of the brain in both MCAO and RAPA groups in which EB cannot penetrate through the complete BBB to stain brain tissue exhibiting a bright blue color compared with healthy mice (sham) . All of NPs groups (HOP, MHOP and BMHOP) showed much less significant EB staining than the MCAO group, and the brain EB staining of BMHOP-treated mice is the slightest, which in accordance with the results of EB fluorescent signal detection in brain slices by confocal microscopy (Figure. 4F) . To measure the amount of extravasated EB from blood into the brain, quantitative analysis of EB exudation by spectrophotometry (Figure. 4G) showed that the amount of EB uptake in the brain of BMHOP treatment was the lowest in all. The results demonstrated that BMHOP have a role in maintaining the integrity of the BBB integrity, revealing that CXCR4 overexpression targeting can effectively accumulate within the cerebral ischemic microenvironment and prevent BBB from breakdown by ROS scavenging and degradation of excessive ROS levels.

In vitro simulation experiment, the activated HUVEC cells that H ₂O ₂-triggered upregulation of intracellular ROS were inoculated on the cell slide in proportion 1×10 ⁵ per cm ² and cultured in 24-well plates, 5 μg/mL BMHOP were added into each well and the 24-well plates cultured under the condition of 37 ℃ and 5%CO ₂. And then the solution of 10 μM DHE as an superoxide anion fluorescence detection probe was added in each well at different time periods of 0.5, 1, 2, 6 h during the treatment. Finally, after co-incubation with DHE solution for 20 min, the cells were immediately resuspended in 1 mL of PBS and were examined using flow cytometry (BD LSRFortessa) . ROS levels were detected by fluorescent probe of DHE in the brain after ischemia induction for to investigate the antioxidant protective effect of BMHOP. As shown in the Figure. 5A, the red fluorescent signal indicated ROS level in the BMHOP-treated brain frozen sections was markedly reduced compared to that in the MCAO and another groups, exhibiting the highly efficient ROS elimination in vivo.

Middle cerebral artery occlusion (MCAO) model was established to mimic I/R injury in ischemic stroke according to a previous protocol with minor modification. Male C57 mice (24～28 g) were used following the previous studies. Mice were anesthetized with 4%pentobarbital sodium in saline via intraperitoneal injection. The external carotid artery (ECA) was exposed and the monofilament was inserted into the internal carotid artery (ICA) through the ECA until it reached the middle cerebral artery (MCA) , causing a blockage of blood flow. The MCAO monofilament was gently withdrawn 2 h later for reperfusion. Mice having the same procedure without monofilament blocking was used as sham group. Mice were housed separately in a temperature-and humidity-controlled room under a 12 h light-dark cycle with free access to food and water. Animal handing procedures were in accordance with Chongqing Medical University animal care guidelines for all in vivo experiments. All animal housing, care, and experiments were performed according to the guidelines and regulations of the Institutional Animal Care and Use Committee.

The therapeutic efficacy of NPs was assessed by the expression level of inflammation factors in MCAO mice model brain using the ELISA assays. Similar results were obtained with the expression level of pro-inflammatory (tumor necrosis factor-α, TNF-α and interleukin 6, IL-6) that BMHOP had a tendency to down-regulate anti-inflammatory factors, on the other hand, BMHOP caused a significantly increasing the anti-inflammatory factors (interleukin 10, IL-10) (Figure. 5B) . The results revealed that the effect of mTOR inhibition incurred by RAPA achieve similar anti-inflammation therapeutic effects, via pro-inflammatory M1 phenotype microglia within the neurovascular unit were directly convert into anti-inflammatory M2 phenotype activation state, attenuate neuroinflammation injury. Meanwhile, in response to inhibition of mTOR pathway by RAPA treatment, autophagy was induced to enable neuron cellular survival.

MCAO mice brain infarct area was stained to substantiate the strategy of NPs therapy. After stroked for 24 h, the cerebral infarct volume was measured by triphenyltetrazolium chloride (TTC) staining, showing the stained normal tissue in red and the unstained infarction in white. After the experiment, the mice were anesthetized with 5%chloral hydrate and then being decapitated. Brain tissue was quickly removed and rinsed with cold saline and then refrigerated at -20℃ for 20 min. Taking the brain tissue from the refrigerator before staining and quickly slice it with a razor blade every 2 mm for TTC staining. The tissue sections were placed in a 12-well plate containing TTC staining solution, and incubated at 37 ℃ in dark for 15～20 min. The color changes of the samples were observed while incubating. After dyeing, the used TTC staining solution were removed, and then take photos after fixed it with 4%paraformaldehyde for 6 h.

The examination of the ischemic brain in Figure. 5C showed that BMHOP exerted an obvious protective effect of the salvageable ischemic penumbra compared to the large ischemic area in the MCAO group. Quantification studies were used to evaluate the protection effects of HOP, MHOP and BMHOP at the same injection dose of 0.05 mg/kg, the MCAO mice treated with BMHOP decreased down to 10.7 ± 1.3%, had fewer infarct area percent than those treated with another NPs, suggesting better recovery of the ischemic injuries of BMHOP (Figure. 5E) . In addition, the neuroscore were evaluated 72 h after the ischemic reperfusion. The BMHOP-treated MCAO mice began to be able to maintain their balance and walk in a straight line as well as Sham group (Figure. 5D) and showed significantly lower scores than another NPs-treated groups (a higher score indicates a more severe injury) . Statistical analysis used one-way student’s t-test or two-way analysis of variance (ANOVA) test using GraphPad Prism 6.0. Values were expressed as means ± SD and p < 0.05 is considered statistically significant. (Figure. 5E) .

Figure 5. (A) CLSM images of red fluorescent signals of DHE for detecting ROS levels in ischemic brain (scale bar = 200 μm) . (B) Relative expression of pro-inflammatory (tumor necrosis factor-α, TNF-α and interleukin 6, IL-6) and anti-inflammatory factors (interleukin 10, IL-10) in brain tissue (Values represent mean ± SD, n = 3, **P < 0.05 and ***P < 0.01) . (C) Representative TTC-stained brain sections of Sham-operated group and MCAO group (administration with 5%sucrose group) , HOP group, MHOP group, BMHOP group. The nonischemic region is observed as red, and the infarct region is shown in white. (D) Representative images of the balance and behavior of different NPs-treated mice after 72 h. (E) Quantification of brain infarct volume at 24 h in different groups mice and (F) Mean neurological scores of MCAO mice during the treatment (Values represent mean ± SD, n = 3, **P < 0.05 and ***P < 0.01) . A higher score indicates a more severe injury. Statistical analysis used one-way ANOVA test.

Example 10: In vivo circulation behavior of different NPs formulations was evaluated using male C57 mice as the animal model

The mice were intravenously administered different formulations at the same dose of 0.05 mg/kg DiD@NPs. Blood samples were taken at different interval time, and the fluorescence intensity in the plasma was assessed for quantitation. The results showed in Figure. 6A that longer circulation time (over 48 h) was observed in both DiD@MHOP and DiD@BMHOP groups compared to the DiD@HOP group, attributing to the immune evasion ability of “don’t eat me” protein CD47 on the bioengineered cell membranes. At 24 and 48 h, there was about 36.5%and 30.6%of the DiD@BMHOP remaining in the blood circulation. All the NPs were found to be rapidly eliminated and could not be detected after 72 h. DiD@HOP showed much lower retention in blood with comparison to the other groups, indicating the stealth capability of the cell membrane, confirming the successful coating of cell membranes onto the NPs.

MCAO mice could not survive more than 5 days without treatment. The treatment of BMHOP increased the survival rate to 85.7 % (Figure. 6B) . The body weights of the MCAO mice treated with all NPs dropped sharply compared with Sham group in the first 4 days before gradually recovering, but the MCAO mice treated with BMHOP recovered and became like normal until the 18th day (Figure. 6C) . The results indicated that reductions in stroke-caused mortality may have occurred by the more effective therapy of BMHOP.

Figure 6. (A) In vivo pharmacokinetics of HOP, MHOP, and BMHOP (n = 3) . Error bars indicated SD. (B) Survival rate and (C) weight of mice receiving different NPs treatments (Values represent mean ± SD, n = 5) . (D-G) The biochemical assays of hepatic and kidney functions. AST, aspartate aminotransferase; ALT, alanine aminotransferase; URN, blood urea nitrogen; and CRE, creatinine. (Values represent mean ± SD, n = 5) . (H) Histochemistry analysis of brain, heart, liver, spleen, lung, and kidney tissue sections stained with hematoxylin-eosin.

Example 11: Establishment of a preliminary in vivo safety profile of different types of NPs

HOP, MHOP, and BMHOP were injected to the healthy mice by tail vein injection. The serum biochemical analyses and histopathology evaluation were performed to monitor the potential toxicity. The results showed that there were no significant abnormalities in the serum chemistry tests (AST (aspartate aminotransferase) , ALT (alanine aminotransferase) , BUN (blood urea nitrogen) , and creatinine levels) for renal and hepatic functionality analysis after daily administration for 1 week (Figure. 6D-G) . H&E staining of the tissue samples comprising liver, heart, spleen, kidney and lung after the treatment with 5%sucrose, HOP, MHOP, and BMHOP was conducted. Results showed that there was no abnormal and inflammatory cell infiltration in tissue sections (Figure. 6H) , demonstrating the good biocompatibility of the nanoparticle in vivo.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A ROS-responsive nanocarrier, comprising a polymer backbone, the polymer backbone combined with antioxidant groups and hydrophilic groups comprises a hydrophilic segment and a hydrophobic segment, wherein the polymer backbone can be degraded in the presence of ROS and release antioxidant groups with pharmacological activity.
The ROS-responsive nanocarrier of claim 1, wherein said hydrophilic segment is formed by copolymerization of hydrophilic groups and ROS labile groups, and said hydrophobic segment is formed by copolymerization of antioxidant groups and ROS labile groups.
The ROS-responsive nanocarrier of any one of claims 1-2, said polymer backbone comprises a copolymerized oxalate backbone.
The ROS-responsive nanocarrier of any one of claims 1-3, wherein said antioxidant groups comprise p-Hydroxybenzyl alcohol.
The ROS-responsive nanocarrier of any one of claims 1-4, wherein said hydrophilic groups comprise poly- (ethylene glycol) .
The ROS-responsive nanocarrier of claim5, wherein said poly- (ethylene glycol) comprises poly- (ethylene glycol) ₂₀₀₀.
The ROS-responsive nanocarrier of any one of claims 2-6, wherein said ROS-labile groups comprise oxalyl chloride.
The ROS-responsive nanocarrier of any one of claims 1-7, wherein said hydrophobic segment is a compound of formula (I) ,

wherein, x is selected from 1 to 100.
The ROS-responsive nanocarrier of any one of claims 1-8, wherein said hydrophilic segment is a compound of formula (II) ,

wherein, y is selected from 1 to 100.
The ROS-responsive nanocarrier of any one of claims 1-9, wherein said polymer backbone is a compound of formula (III) ,

wherein, x and y are independently selected from 1 to 100.
The ROS-responsive nanocarrier of claim 10, wherein, x and y are independently selected from 1 to 25.
The ROS-responsive nanocarrier of any one of claims10-11, wherein x=3 and/or y=7.
The ROS-responsive nanocarrier of any one of claims 1-12, wherein said polymer backbone is assembled into stable micelles in an aqueous solution.
A ROS-responsive nanoparticle, wherein comprising said ROS-responsive nanocarrier of any one of claims 1-13 and bioactive agents, and said ROS responsive nanocarrier and bioactive agents are autonomously installed as nanoparticles.
The ROS-responsive nanoparticle of claim 14, wherein said bioactive agents comprise nano-drug and/or nanoprobe.
The ROS-responsive nanoparticle of claim 15, wherein said nano-drug comprises mTOR inhibitor.
The ROS-responsive nanoparticle of claim 16, wherein said mTOR inhibitor comprises Rapamycin.
The ROS-responsive nanoparticle of claim 15, wherein said nanoprobe comprises fluorescent nanoprobe.
The ROS-responsive nanoparticle of claim 18, wherein said fluorescent nanoprobe comprises DiD nanoprobe.
The ROS-responsive nanoparticle of any one of claims 13-19, said ROS-responsive nanoparticle is the uniform spherical nanoparticle with the dehydrated size of 170～220 nm.
A nano-drug delivery system, comprising said ROS-responsive nanocarrier of any one of claims 1-13 coated with bio-engineered cell membrane.
The nano-drug delivery system of claim 21, wherein said bio-engineered cell membrane specifically targets to the ischemic site.
The nano-drug delivery system of any one of claims 21-22, wherein said bio-engineered cell membrane can specifically cross the blood-brain barrier in ischemic brain.
The nano-drug delivery system of any one of claims 21-23, wherein said bio-engineered cell membrane has a high initial brain uptake and/or the ability to expand circulation in the brain.
The nano-drug delivery system of any one of claims 21-24, wherein said bio-engineered cell membrane expresses a receptor targeting the normal or highly expressed protein of the ischemic region.
The nano-drug delivery system of claim 25, wherein said normal or highly expressed protein of the ischemic region comprises SDF-1.
The nano-drug delivery system of any one of claims 21-26, wherein said bio-engineered cell membrane expresses CXCR4.
The nano-drug delivery system of any one of claims 21-27, wherein said bio-engineered cell membrane overexpresses CXCR4.
The nano-drug delivery system of any one of claims 21-28, wherein said bio-engineered cell membrane expresses “don’t eat me” protein making the bio-engineered cell membrane have immune evasion ability.
The nano-drug delivery system of claim 29, wherein said “don’t eat me” protein comprises CD47.
The nano-drug delivery system of any one of claims 21-30, wherein said bio-engineered cell membrane comprises vascular endothelial cell membrane.
The nano-drug delivery system of any one of claims 21-31, wherein said bio-engineered cell membrane comprises the primary vascular endothelial cell membrane.
The nano-drug delivery system of any one of claims 21-32, wherein said bio-engineered cell membrane comprises arterial endothelial cell membrane.
The nano-drug delivery system of any one of claims 21-33, wherein said bio-engineered cell membrane comprises endothelial cell membrane of the thoracic aorta.
A neuroprotective agent, comprising said nano-drug delivery system of any one of claims 21-34, wherein said ROS-responsive nanocarrier loaded with nano-drug.
The neuroprotective agent of claim 35, wherein said nano-drug comprises mTOR inhibitor.
The neuroprotective agent of claim 36, wherein said mTOR inhibitor comprises Rapamycin.
The neuroprotective agent of any one of claims 35-37, said neuroprotective agent is the uniform spherical nanoparticle with the dehydrated size of 200～230 nm.
A ROS-responsive nanoprobe, comprising said nano drug delivery system of any one of claims 21-34, wherein the ROS-responsive nanocarrier loaded with nanoprobe.
The ROS-responsive nanoprobe of claim 39, wherein said nanoprobe comprises fluorescent nanoprobe.
The ROS-responsive nanoprobe of claim 40, wherein said fluorescent dyes comprises DiD nanoprobe.
A method for preparing said ROS-responsive nanocarrier of any one of claims 1-13, comprising: synthesizing a polymer skeleton containing antioxidant groups and hydrophilic groups through a one-step polycondensation reaction with ROS labile groups.
The method for preparing said ROS-responsive nanocarrier of claim 42, wherein said antioxidant groups comprises p-Hydroxybenzyl alcohol.
The method for preparing said ROS-responsive nanocarrier of any one of claims 42-43, wherein said hydrophilic groups comprises poly- (ethylene glycol) .
The method for preparing said ROS-responsive nanocarrier of claim 44, wherein said poly- (ethylene glycol) comprises poly- (ethylene glycol) ₂₀₀₀.
The method for preparing said ROS-responsive nanocarrier of any one of claims 42-45, wherein said ROS-labile groups comprises oxalyl chloride.
The method for preparing said ROS-responsive nanocarrier of any one of claims 42-46, comprising: synthesizing HBA-OC-PEG ₂₀₀₀ containing PEG ₂₀₀₀ and HBA through a one-step polycondensation reaction with oxalyl chloride, and obtaining the obtained after repeated precipitation into cold ether, purifying the collected copolyoxalate, drying under high vacuum, and then obtained HBA-OC-PEG ₂₀₀₀.
A method for preparing said ROS-responsive nanoparticle of any one of claims 14-20, wherein said bioactive agents are loaded into micelles through the self-assembly of ROS-responsive nanocarrier to obtain nanoparticles.
A method for preparing said nano-drug delivery system of any one of claims 21-34, comprising the method of physically extruding the bio-engineered cell membrane to coat the ROS-responsive nanocarrier of any one of claims 1-13, wherein said bio-engineered cell membrane is obtained by lentiviral transcription.
The method for preparing said nano-drug delivery system of claim 49, wherein the method for preparing the bio-engineered cell membrane specifically comprises: using lentivirus to transcribe vascular endothelial cells to obtain the bio-engineered cell membrane with high expression of CXCR4.
A method for preparing said neuroprotective agent of any one of claims 35-38, comprising the method of physically extruding the bio-engineered cell membrane to coat the ROS-responsive nanoparticle of any one of claims 14-17、20, wherein said bio-engineered cell membrane is obtained by lentiviral transcription.
The method for preparing said neuroprotective agent of claim 51, wherein the method for preparing the bio-engineered cell membrane specifically comprises: using lentivirus to transcribe vascular endothelial cells to obtain the bio-engineered cell membrane with high expression of CXCR4.
A method for preparing said ROS-responsive nanoprobe of any one of claims 39-41, comprising the method of physically extruding the bio-engineered cell membrane to coat the ROS-responsive nanoparticle of any one of claims 14、 15、 18、 19、 20, wherein said bio-engineered cell membrane is obtained by lentiviral transcription.
The method for preparing said ROS-responsive nanoprobe of claim 53, wherein the method for preparing the bio-engineered cell membrane specifically comprises: using lentivirus to transcribe vascular endothelial cells to obtain the bio-engineered cell membrane with high expression of CXCR4.
A use of the ROS-responsive nanocarrier of any one of claims 1-13 in the preparation of a medicine for the treatment and/or diagnosis of ROS-mediated diseases.
A use of the ROS-responsive nanoparticle of any one of claims 14-20 in the preparation of a medicine for the treatment and/or diagnosis of ROS-mediated diseases.
A use of the nano-drug delivery system of any one of claims 21-34 in the preparation of a medicine for targeted treatment of ischemic stroke.
A use of the nano-drug delivery system of any one of claims 21-34 in the preparation of a kit for diagnosing ischemic stroke.
A use of the neuroprotective agent of any one of claims 35-38 in the preparation of a medicament for targeted treatment of ischemic stroke.
A use of the ROS-responsive nanoprobe of any one of claims 39-41 in the preparation of a kit for diagnosing ischemic stroke.
A pharmaceutical composition, comprising said ROS-responsive nanocarrier of any one of claims 1-13, said nanoparticle of any one of claims 14-17、 20, said nano-drug delivery system of any one of claims 21-34 or said neuroprotective agent of any one of claims 35-38.
A diagnostic kit, comprising said ROS-responsive nanocarrier of any one of claims 1-13, said nanoparticle of any one of claims 14、 15、 18、 19、 20, said nano-drug delivery system of any one of claims 21-34 or said ROS-responsive nanoprobe of any one of claims 39-41.
A method of treating or preventing ischemic diseases comprising the administration of an effective amount of said neuroprotective agent of any one of claims 35-38 to a patient in need thereof.
The method of claim 63, said ischemic diseases comprise reperfusion injury after stroke.