NL2033447B1

NL2033447B1 - Brain-targeting erythrocyte membrane-enveloped salvianolic acid b nanoparticles as well as preparation method and application thereof

Info

Publication number: NL2033447B1
Application number: NL2033447A
Authority: NL
Inventors: Zhou Yuan; Zhang Shanshan; Fan Xiang; Li Ruoqi
Original assignee: Univ Zhejiang Chinese Medical
Priority date: 2022-04-21
Filing date: 2022-11-02
Publication date: 2023-11-07
Also published as: CN114569576A; CN114569576B

Abstract

Disclosed are a preparation method and application of brain— targeting RBCM wrapped salvianolic acid B (SAB) nanoparticles. The preparation. method, includes the following steps: (1) dissolving bovine serum. albumin (BSA) in water and mixing BSA. with SAB, 5 adding ethanol and glutaraldehyde, and performing stirring, rotary evaporation and filtration on the mixture to obtain SABNPS; and (2) mixing the SABNPS with RGD—PEG—DSPE modified RBCMS, and performing coextrusion through a 800 nm polycarbonate membrane to prepare the brain—targeting erythrocyte membrane—enveloped SAB 10 nanoparticles. The brain—targeting erythrocyte membrane—enveloped SAB nanoparticles can prolong the circulation time in vivo of the SAB and increase the ability of SAB across the blood brain barrier on an ischemic side. Moreover, the nanoparticles improve the concentration of the SAB in an ischemic brain, and improve the 15 effect of the SAB in treating acute ischemic cerebral stroke.

Description

BRAIN-TARGETING ERYTHROCYTE MEMBRANE-ENVELOPED SALVIANOLIC ACID B

NANOPARTICLES AS WELL AS PREPARATION METHOD AND APPLICATION

THEREOF

TECHNICAL FIELD

The application relates to the technical field of traditicnal

Chinese medicine and pharmaceutic preparations, and in particular to a preparation method and application of brain-targeting eryth- rocyte membrane-enveloped salvianolic acid B nanoparticles.

BACKGROUND ART

Stroke is the second leading cause of death and the third leading cause of disability in the world, and ischemic stroke ac- counts for over 87% of stroke. Deficiency of oxygen and glucose resulting from the occlusion of the cerebral artery will lead to occurrence of ischemic stroke. At present, recombinant tissue plasminogen activator is the only drug approved by the US Food and

Drug Administration (FDA) for treating ischemic stroke, which may dissolve thrombosis quickly and restore the cerebral blood flow.

Reperfusion injury induced by recovery of the cerebral blood flow is the main obstacle to the recovery of neurological function af- ter thrombolytic therapy. It is no doubt that neuroprotection is very significant for the treatment of ischemic stroke. The FDA has not approved any neuroprotective agents for the treatment of is- chemic stroke due to the complexity of the pathological mechanisms of stroke, the limitations of the blood-brain barrier (BBB), and the lack of specific targeting.

Salvianolic acid B (SAB) is a water soluble phenolic acid ex- tracted from a traditional Chinese medicine Salviae Miltiorrhizae.

SAB plays a role of resisting cerebral ischemia/reperfusion injury by scavenging free radicals, improving energy metabolism and re- ducing cell apoptosis and inflammations by the activation of SIRT1 signaling pathway. Meanwhile, SAB can promote proliferation of neural stem cells/ progenitor cells via the PI3K/Akt signal path- way, so as to improve cognition impairment in a rat transient forebrain ischemia model. However, the clinical application of SAB is hindered due to the poor stability of SAB in plasma and the ex- istence of BBB. Therefore, how to improve the bioavailability of

SAB in vivo and the targeting ability to the ischemic brain is crucial to clinical application of SAB in the treatment of ischem- ic stroke.

It has been reported that the constructed transferrin recep- tor monoclonal antibody 0X26 modified lipid carrier (OX26-BA/Sal

B-NLC) could promote the delivery of SAB to the brain. However, the circulation time of the nanoparticles in vivo is restricted by a reticuloendothelial system (RES), resulting in the distribution of most nanoparticles in liver and spleen rather than disease sites. The short circulation time in vivo is also not conducive to the aggregation of nanoparticles in the target site of disease.

Pegylation (PEG modified) is a common strategy to prolong the cir- culation time of the nanoparticles in vivo, while the encapsula- tion of nanoparticles with hydrophilic materials also reduces the interaction with target cells. Therefore, a strategy of “do not eat us” is established to intercept RES so as to prolong the cir- culation time of the nanoparticles in vivo. Red blood cell mem- brane (RBCM) has been widely applied to a nano delivery system due to its immune escape capability. CD47, an integral membrane pro- tein embedded in RBCM, could interact with a phagocytic cell and release a signal of “do not eat me”. Meanwhile, other membrane proteins also contribute to representing a drug nanocarrier in form of “itself” to the immune system, thus preventing the drug nanocarrier from being scavenged by RES. Therefore, RBCM coating can prolong the circulation time of the nanoparticles and reduce scavenging of the nanoparticles by the RES, thereby promoting the delivery of the nanoparticles to the brain.

The present invention designs a biomimetic drug delivery sys- tem (RRE@SABNPs), including two parts: 1) SAB-loaded BSA nanoparti- cles (SABNP) as the inner core. BSA is one of the most potential materials in a nano drug delivery system due to its biocompatibil- ity, biodegradability, nontoxicity and non-immunogenicity. 2) The

Arg-Gly-Asp (RGD) functioned RBCM as the outer shell, RGD-RBCM acts as a bionic camouflage with a targeted ligand to prolong na-

noparticle circulation time and target ischemic BBR actively, and thus improve the bicavailability of SAB and the concentration of

SAB in the ischemic brain.

SUMMARY

In order to overcome the deficiencies in the prior art, the present invention is intended to provide a preparation method and application of brain-targeting erythrocyte membrane-enveloped SAB nanoparticles. The brain-targeting erythrocyte membrane-enveloped

SAB nanoparticles prepared in the present invention can prolong the in vivo circulation time of SAB and increase the ability of

SAB across ischemic BBB. Moreover, the nanoparticles improve the concentration of the SAB in the ischemic brain, and thus improve the effect of the SAB in treating acute ischemic stroke. Compared with conventional SAB for treating acute ischemic stroke, the pre- sent invention improves the bioavailability of SAB and the effect of SAB for treating cerebral stroke.

The objective of the present invention is realized by the following technical solution:

The first aspect provides a preparation method of brain- targeting erythrocyte membrane-enveloped SAB nanoparticles, spe- cifically including the following steps: (1) dissolving BSA and SAB at a weight ratio of (5-20):1 in water 2-10 times in volume, adjusting the pH value to 8.0, adding ethanol 6 times of water volume at 1.0 mL/min, stirring the mix- ture for 30 min, adding 2% glutaraldehyde 0.021 time of water vol- ume, continuously stirring the mixture for 12 h, and filtering the mixture to obtain SAB nanoparticles (SABNPs); and {2} putting 1-5 parts by volume of 10 mg/mL SABNPs PBS solu- tion and 1 part by volume of RBCM in 1 part by volume of PBS, and performing full mixing and extrusion to prepare erythrocyte mem- brane-enveloped SAB nanoparticles (R@GSABNPs).

Further, in the step (2), the RBCM is prepared by the follow- ing steps: taking mouse whole blood, centrifugalizing the whole blood (2000 rpm, 10 min) at 4°C, and discarding serum and white matters at a boundary; adding a pre-cooled 1xPBS buffer solution, perform-

ing centrifugal washing many times, and finally discarding the su- pernatant to obtain free red blood cells; adding a pre-cooled 0.25xPBS buffer solution at a volume ratio of 1:10, and allowing the red blood cells to swell and break in a 4°C refrigerator for 30 min; and performing centrifugation (12,000 rpm, 30 min) at 4°C, discarding the supernatant, and repeating the above steps till the supernatant is colorless to obtain the RBCM (pink gellike).

Further, the method further includes following steps: per- forming ultrasonic treatment on the collected RBCM for 3 min, and enabling the colorless supernatant to respectively pass through a 800 nm polycarbonate membrane and a 400 nm polycarbonate membrane many times by using a mini extruder.

Further, the RBCM is an RGD modified RBCM.

Further, the RGD modified RBCM is prepared by the following steps: mixing the RGD-polyethylene glycol-phospholipid RGD-PEG-DSPE with the RBCM at a ratio of 1:1-10 (parts by weight ug to parts by volume ul), incubating the mixture at 37°C for 30 min, centrifu- galizing the mixture, discarding the supernatant, and re- dissolving precipitates to obtain the brain-targeting RBCM.

Further, the brain-targeting erythrocyte membrane-enveloped

SAR nanoparticles are RGD modified, and have brain-targetability.

The second aspect provides brain-targeting erythrocyte mem- brane-enveloped SAB nanoparticles prepared by the method according to the first aspect.

The third aspect provides an application of the brain- targeting erythrocyte membrane-enveloped SAB nanoparticles accord- ing to the second aspect in preparation of a drug for treating stroke.

In some embodiments, the stroke includes ischemic stroke and hemorrhagic stroke.

The fourth aspect provides a stroke therapeutic drug, includ- ing an effective dose of the brain-targeting erythrocyte membrane- enveloped SAB nanoparticles according to the first aspect.

The present invention has the following beneficial effects: the brain-targeting erythrocyte membrane-enveloped SAB nanoparti-

cles of the present invention can prolong the circulation time in vivo of SAB and increase the ability of the SAB across ischemic

BBB. Moreover, the nanoparticles improve the concentration of the

SAB in an ischemic brain, and thus improve the effect of the SAB 5 in treating acute ischemic stroke. Compared with conventional SAB for treating acute ischemic stroke, the present invention improves the bioavailability of SAB and the effect of SAB for treating stroke. Furthermore, the present invention features simple prepa- ration method, mild process conditions and low manufacturing cost, has the pharmacoeconomics advantage for treating stroke, and is suitable for clinical application and market promotion.

BRIEF DESCRIPTION OF DRAWINGS

The present invention will be further described below in com- bination with the accompanying drawings and embodiments.

FIG. 1: characterization of RRESABNPs. (A) SDS-PAGE electro- phoretic protein analysis of red blood cells and RBCM; (B) size distribution and zeta potential of SABNPs, RE@SABNPs and RRESABNPs; (C) laser scanning confocal image of RRESABNPs; (D) TEM image of

SABNPs, (E) TEM image of RE@SABNPs; and (F) TEM image of RRESABNPs.

FIG. 2: stability and biocompatibility of RRESABNPs. (A)

Placing stability result of RRESABNPs; (B) plasma stability result of SAB solution and RRE@SABNPs at 37°C; (C) release curves of SAB solution and RRE@SABNPs in a PBS solution at 37°C; and (D) hemolytic assay of RRESABNPs.

FIG. 3: The cytotoxicity of SABNPs, RESABNPs, and RRE@SABNPs to (A) bend.3 cells, (B) RAW 264.7 cells, and (C) PCl2 cell.

FIG. 4: long circulation of RRESABNPs. (A) Fluorescence imag- es of uptake of RAW 264.7 cells to FITC-loading SABNPs and

RRE@SABNPs, scale: 50 um; (B) flow cytometry histogram of uptake of

RAW 264.7 cells; {C) guantitative analysis (n=3) result of mean fluorescence intensity of flow cytometer; and (D) pharmacokinetic results (n=6) of SAB in SAB solution, SABNPs and RR@SABNPs intra- venously administrated to rats; *** P<0.001.

FIG. 5: uptake of bEnd.3 cells to RRESABNPs. (A) Representa- tive fluorescence image of uptake of bEnd.3 cells to different preparations, scale: 50 um; (B) flow cytometry histogram of uptake of bEnd.3 cells (Control, SABNPs, R@SABNPs and RRESABNPs from left to right); (C) quantitative analysis result (n=3) of mean fluores- cence intensity of flow cytometer; ***P < 0.001.

FIG. 6: ability of RRESABNPs targeting an ischemic brain tis- sue. (A) Living real-time image at different time points after in- travenous injection of PBS, Dir-loaded SABNPs or RRESABNPs to

MCAC/R mice; (B) representative fluorescence image of anatomical organs of the mice; (C) quantitative analysis results of fluores- cence intensities of different organs; (D) representative TTC stained brain slice of MCAO/R mice and fluorescence image of the representative stained brain slice; (E) quantitative result of fluorescence intensity in the brain; (F) comparison result (n=3) of fluorescence intensities on an ischemic side/a normal side of the brain slice; ***P < 0.001.

FIG. 7: influence of RRE@SABNPs on brain infarct volume and neurological function of MCAO/R model mice. (A) Flowchart of an experimental scheme; (B) TTC stained brain tissue slice; (C) quantative analysis result (n=10) of the brain infarct volume; (D) mNSS test result; (E) foot fault test (n=12) result; (F) brain slice image of the MCAO/R model mice; (G) H&E staining analysis result; scale, 50 um; (H) Nissl's staining analysis result; scale, 250 um. * P<0.05, **P<0.01, or ***P<0.001.

FIG. 8: neuroprotective effect of RRESABNPs on OGD/R impaired

PC12 cells. (A) LDH release result of the OGD/R impaired PC12 cells; (B) representative fluorescence image of ROS in the OGD/R impaired PC12 cells; scale, 250 um; (C) representative fluores- cence image of MMP in the OGD/R impaired PC12 cells; scale, 250 pm. ** P<0.01, ***P<0.001.

FIG. 9: neuroprotective effect of RRESABNPs on H:0; impaired

PC12 cells. (A) LDH release result of the H;0, impaired PC12 cells; (B) representative fluorescence image of morphology of the H:0; im- paired PC12 cells; (C) representative fluorescence image of ROS in the H;0, impaired PC12 cells; scale, 250 pm; (D) representative fluorescence image of MMP in the H:0; impaired PC12 cells; scale, 250 um. ** P<0.01, ***P<0.001.

FIG. 10: in vivo safety of RRESABNPs; H&E staining analysis result; scale, 50 um.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention provides brain-targeting erythrocyte membrane-enveloped SAB nanoparticles, including a main ingredient

SAB. A nano system of the brain-targeting RBCM includes BSA, a

RBCM and RGD-PEG-DSPE.

The preparation method of the present invention includes the following steps: (1) Preparation of SABNPs

BSA and SAB at a weight ratio of 5-20:1 are dissolved in wa- ter 2-10 times in volume, the pH value was adjusted to 8.0, etha- nol 6 times of water volume was added at 1.0 mL/min, the mixture was stirred for 30 min, 2% glutaraldehyde 0.021 time of water vol- ume was added, the mixture was continuously stirred for 12 h, ro- tary evaporation was performed to remove ethanol, and the mixture was passed through a 0.8 um filter membrane. (2) Preparation of R@SABNPs

Mouse whole blood was taken and centrifuged (2,000 rpm, 10 min) at 4°C, and serum and white matters at a boundary were dis- carded. A pre-cooled 1xPBS buffer solution was added, and the mix- ture was washed and centrifuged 3 times, and finally the superna- tant was discarded to obtain free red blood cells. A pre-cooled 0.25xPBS buffer solution was added at a volume ratio of 1:10, and the red blood cells were allowed to swell and break in a 4°C re- frigerator for 30 min. Centrifugation (12,000 rpm, 30 min) was performed at 4°C, the supernatant wad discarded, and the above steps were repeated till the supernatant was nearly colorless to obtain the RBCM (pink jellylike). Ultrasonic treatment was per- formed on the collected RBCM for 3 min. The obtained congenital vesicle of the RBCM was extruded through a 800 nm polycarbonate membrane and a 400 nm polycarbonate membrane (respectively 20 times) by using a mini extruder to obtain the RBCM. 1-5 parts by volume of 10 mg/mL SABNPs and 1 part by volume of extracted RBCM were put in PBS and mixed, and the mixture was coextruded through the 800 nm polycarbonate membrane 10 times by using a mini extruder, and passed through a 0.8 um filter membrane to prepare RE@SABNPs.

(3) Preparation of RRESABNPs

The RGD-PEG-DSPE was mixed with the RBCM at a weight-to- volume ratio of 1:1-10, incubated at 37°C for 30 min and centri- fuged, the supernatant wad discarded, precipitates were re- dissolved, the RBCM was treated according to the mode in (2) to obtain the brain-targeting RBCM, and the RBCM and the SABNPs were coextruded by the similar method to prepare RRESABNPs.

Example 1:

Preparation and representation of brain-targeting erythrocyte membrane-enveloped SAB nanoparticles 1. Preparation of SAENPs 20 mg of BSA was precisely weighed and dissolved in 1 mL of water, 2.0 mg of SAR was added, the pH value was adjusted to 8.0, 6 mL of ethanol was added at 1.0 mL/min, the mixture was stirred for 30 min, 21 pl of 2% glutaraldehyde was added, the mixture was continuously stirred for 12 h, rotary evaporation was performed to remove ethanol, and the mixture was passed through a 0.8 um filter membrane to obtain SABNPs. 2. Preparation of RESABNPs

Extraction of RBCM: mouse whole blood was taken and centri- fuged (2,000 rpm, 10 min) at 4°C, and serum and white matters at a boundary were discarded. A pre-cooled 1xPBS buffer solution was added, and the mixture was washed and centrifuged 3 times, and fi- nally the supernatant was discarded to obtain free red blood cells. A pre-cooled 0.25xPBS buffer solution was added at a volume ratio of 1:10, and the red blood cells were allowed to swell and break in a 4°Crefrigerator for 30 min. Centrifugation (12,000 rpm, min) was performed at 4°C, the supernatant was discarded, and the above steps were repeated till the supernatant was nearly col- 30 orless to obtain the RBCM (pink jellylike). Ultrasonic treatment was performed on the collected RBCM for 3 min. The obtained con- genital vesicle of the RBCM was extruded through a 800 nm polycar- bonate membrane and a 400 nm polycarbonate membrane {respectively 20 times) by using a mini extruder to obtain the RBCM.

Identification: the free red blood cells and the RBCM were added into a centrifuge tube, a proper amount of protein lysate

(RIPA:PMSF=100:1) was added therein to allow pyrolysis on ice for 30 min, then the mixture was subjected to 100W ultrasonic treat- ment to be fully pyrolyzed, centrifugation at 4°C was performed (10,000 rpm, 10 min), and the supernatant was taken for later use.

A total protein concentration of sample was measured according to description of a BCA protein quantitative kit. An SDS loading buffer solution was added into the sample, and the mixture was boiled for 10 min to obtain a loaded sample. After spacer gel and 10% separation gel were prepared, the sample was loaded according to 30 pg protein/well, a 80V was used first for 30 min, and then a 120V was used for 90 min for protein separation. After electropho- resis was finished, the gel was taken out, stained with Coomassie brilliant blue for 30 min and then washed with an eluent till a protein stripe was clear, and finally the gel was imaged in a gel imaging system. The results are as shown in FIG. 1A. The protein stripes of the red blood cell and the RBCM are similar, indicating that extraction of the RBCM does not damage the integrity of the

RBCM proteins, which is beneficial to obtaining intrinsic func- tions of the red blood cells by the membrane coated nanoparticles.

Preparation of RESABNPs: 0.5 mL of 10 mg/mL SABNPs, 0.25 mL of RBCM extracted from the red blood cells, and 0.25 mL of PBS was mixed, the mixture was coextruded through the 800 nm polycarbonate membrane 10 times by using a mini extruder, and the mixture was passed through to a 0.8 pm filter membrane for later use. 3. Preparation and identification of RRESABNPs

Preparation: 50 ug of RGD-PEG-DSPE was mixed with 100 pl of

RBCM, incubated at 37°C for 30 min and centrifuged, the supernatant was discarded, and precipitates were re-dissolved to obtain the brain-targeting RBCM, and the RBCM and SABNPs were coextruded to obtain the RRESABNPs.

In order to represent an integral coating of the RBCM and successful modification of the RGD, RGD-PEG-DSPE was replaced by

FITC-PEG2000-DSPE, FITC-RESABNPs were prepared according to the above method, centrifugation was performed to remove free FITC, the mixture was washed two times, and the precipitates were re- dissolved and imaged with laser scanning confocal microscope. The results are as shown in 1C. Green fluorescence appears around SAB-

NPs, indicating that DSPE can be inserted into the cell membrane and SABNPs are fully covered by RBCM. FITC-PEG2000-DSPE is suc- cessfully inserted, similarly indicating that RGD can be modified in this way. 4: Representation of physical and chemical properties of

RR@SABNPs

Particle size, polydispersity index and zeta potentials: par- ticle sizes, polydispersity indexs (PDI) and zeta potentials of different nanoparticles were measured by using a dynamic light scattering method.

As shown in FIG. 1B, compared with R@SABNPs, the particle size and zeta potential of RRESABNPs are slightly increased re- spectively. Specific parameters include particle size, polydisper- sity index, zeta potential, EE and DL, as shown in Table 1.

Table 1. Characteristics of different nanoparticles

Nanoparticles Particlesize PDL Potential (mV) EE(%) DL(%) (nm)

SABNPs 1285%42 0.050#0.004 -26.0:0.5 OO 69.6644,3 7414041

R@SABNPs 168.4+7.5 0.119+0.039 -33.4+1.2 65.4735 6.7310.38

RR@SABNPs 174.8+5.9 0.13710.010 -29.011.5 63.4715.1 6.68+0.47 © Encapsulation efficiency (EE) and drug loading capacity (DL): 100 pL of SABNPs, R@CSABNPs and RRESABNPs were diluted by acetoni- trile, subjected to ultrasonic treatment for 10 min and then cen- trifuged (12,000 rpm, 10 min). The content of SAB in the superna- tant was measured by using high performance liquid chromatography (HPLC). A moving phase is methanol-acetonitrile (v/v, 1: 1): 0.2% phosphoric acid solution, the proportion being 60: 40 (v/v) and the flow rate being 1 ml/min. A stationary phase is a C18 chroma- tographic column (250x4.6 mm, 5 pm). A sample size is 30 pL, and a detection wavelength is 280 nm. Calculating formulae of DL and EE of the nanoparticles are as follows:

EE (%)=weight of SAB in SABNPs/weight of the initial SAB x100

DL {%)=weight of SAB in SABNPs/weight of nanoparticlesx100

The results are as shown in Table 1. Compared with RE@SABNPs,

EE and DL of RRE@SABNPs have no significant changes.

Transmission electron microscopy (TEM) analysis: the nanopar-

ticles were dripped onto a copper grid with a carbon membrane coating, and then several drops of phosphotungstic acid solution were added. The morphology of the nanoparticles was observed with

TEM. The results are as shown in FIG. 1D, 1E and 1F. SABNPs in the

TEM image are uniformly spherical, and obvious core-shell struc- tures of RESABNPs and RRESABNPs further indicate that there is a

RBCM coating on the surface of SABNPs.

In vitro drug release: the in vitro release quantity of SAB in an RRESABNPs or SAB solution was measured by using a dynamic dialysis technique. Normal saline containing 5% (5 g of vitamin

C/100 mL of normal saline) vitamin C was used as a dialyzate to simulate a plasma environment. 1 ml of solution containing

RRE@SABNPs or SAB (the content of SAB in a preparation was 1 mg/mL) was placed in a dialysis bag (3.5 KDa MWCO), the dialysis bag was placed in 50 mL of release medium, and the mixture was continuous- ly stirred at 37°C (400 rpm). 0.5 ml of sample was taken at a fixed interval, and the sample was replaced by equivalent dialyza- te preheated to 37°C. After centrifugation (12,000 rpm, 5 min), the supernatant of the sample was collected and extracted, and an- alyzed by HPLC. The SAB release curve indicates that RR@SABNPs has a better releasing characteristic than the SAB solution (FIG. 2C), which can guarantee minimum leakage of SAB in circulation and pre- vent SAB from being degraded.

Stability research: ® Stability of SAB in plasma: 0.2 ml of mouse plasma was mixed with 1.8 ml of RRE@SABNPs or SAB solution {the content of SAB in the preparation is 1 mg/mL), and incubated at 37°C. 0.1 ml of sample was extracted at a predetermined time interval and mixed with acetonitrile to precipitate proteins. Cen- trifugation (12,000 rpm, 10 min) was performed, and the concentra- tion of SAB in the supernatant was measured by HPLC. @ Placing stability: SABNPs and RRESABNPs were placed at 4°C, and the parti- cle sizes and PDI were measured at a predetermined time interval.

As shown in FIG. 2A, for 72 h at 4°C, the particle size change of the nanoparticles is very small, indicating that the time-varying stability is quite good. Compared with the SAB solution, degrada- tion of RRESABNPs to SAB has an obvious protective effect (FIG.

2B).

In vitro hemolysis test: the red blood cells after centrifu- gation (3,000 rpm, 5 min) were collected and suspended in normal saline to obtain a 2 vols red blood cell suspension. 10 mg/mL SAB-

NPs, RESABNPs or RR@CSABNPs were mixed with 2 vol % red blood cell suspension at a volume ratio of 1: 1, and incubated at 37°C for 4 h. Negative control is normal saline and positive control is Tri- zol. The mixture was centrifuged (3,000 rpm, 5 min), and the ab- sorbance of the supernatant was measured at a wavelength of 570 nm with microplate reader. As shown in FIG. 2D, the nanoparticles were free of obvious hemolysis.

In vitro cytotoxicity test: the bEnd.3, PC12 or RAW 264.7 cells (purchased from cell bank, CAS) were seeded into 96-well plates at a density of 1x10° cells/well and cultured for 24 h. The culture medium was replaced by 100 pL of DMEM culture medium con- taining SABNPs, RE@SABNPs and RRESABNPs nanoparticles (concentra- tions were 0, 5, 10 and 20 mg/mL). After 24 h, the culture medium was washed with PBS two times, and the cell viability was detected by a CCK8 assay kit. The absorbance was measured at the wavelength of 570 nm with microplate reader. The results indicated that no obvious cytotoxicity has been observed in SABNPs, RE@SABNPs and

RRE@SABNPs groups (FIG. 3), indicating that the SABNPs, RESABNPS and RRESABNPs groups have good biocompatibility.

Example 2

Researches on pharmacokinetics of brain-targeting erythrocyte membrane-enveloped SAB nanoparticles and phagocytic uptake in vitro 1. Research on pharmacokinetics

Healthy SD rats were randomly divided into three groups (6 rats in each group). SABNPs, RRESABNPs (the content of SAB in the preparation was 40 mg/mL) and the SAB solution (40 mg/kg SAB) were administrated through caudal veins. At an appointed time after ad- ministration, blood sample was collected in a 0.5 ml hepariniza- tion tube, and centrifuged (4,000 rpm, 10 min) to obtain plasma.

The collected plasma was mixed with isometric acetonitrile (HCL lvols), and centrifuged (12,000 rpm, 10 min). The concentration of

SAB in the supernatant was measured by using HPLC. Pharmacokinetic parameters were analyzed by using PK solver 2.0. Concentration- time distribution of SAB in plasma is as shown in FIG. 4D, and pa- rameters are as shown in Table 2. After intravenous injection of 40 mg/kg SAB, the concentration of SAB in the plasma was dramati- cally decreased from 33.51 pg/ml to 3.8 pg/ml within 10 min, and gradually decreased to 0.25 pg/ml within 6 h. Compared with the

SAB solution, the plasma concentration of SAB in the nanoparticles was significantly increased, which mainly contributed to protec- tive effect of the nanoparticles to protease degradation of SAB. ti/20 of SABNPs and ti; of RRE@SABRNPs were respectively 5.71 and 7.00 times of that of the SAB solution. Compared with the SAB so- lution and the SABNPs group, the AUCO-inf value of RRESABNPs was respectively increased by 132.09 and 37.04 times. These results indicate that SAB in RRESABNPs has more chances to accumulate in the target site rather than being degraded or scavenged directly by RES.

Table 2. Pharmacokinetic parameters of solution, SABNPs and

RRE@SABNPs (n=6) “pharmacokinetic parameters ~~ SABsolution ~~ SABNPs RR@SABNPs

Cmalmg/mi) 3351#10.72 59.16+4.61* 80.27+1808 tipa(h) 0.014+0.007 0.080+0.052* 0.098+0.171" t2 (bh) 0.55810.236 1.828+1.178* 30.53+15.64"

AUCq.nt (ug/ ml*h) 10.9245.77 38.94+15.48* 1442.46+629.76"

MRT (mg/kg/ (ug/ml) 0.47+0.32 2.46+1.94% 43.63+22.45" ~~ Notes: *P<0.05 compared with SAB solution; #P<0.05 compared with SABNPs. Data presented as mean SD. 2. Research on anti-phagocytic experiment in vitro

The RAW 264.3 cells were seeded into 24-well plates at a den- sity of 5x10°% cells/well and cultured for 24 h. Then the culture medium was replaced by 500 uL of DMEM culture medium containing

FITC-loading SABNPs or RRESABNPs (FITC 20 pg/mL). After incubation for 1 h, the culture medium was removed, and the RAW 264.3 cells were washed with PBS two times. A phagocytic condition of the na- noparticles in the RAW 264.3 cells was measured with a reversed fluorescence microscope and a flow cytometer. As shown in FIG. 4A, the fluorescence intensity of the RRESABNPs group is obviously lower than that of the SABNPs group, indicating that RBCM modifi- cation can reduce elimination of the nancparticles by the RES sys- tem. The guantitive results of the flow cytometry histogram show that compared with the SABNPs group, the uptake of macrophages to

RRESABNPs was reduced by about 50% (FIG. 4B, FIG. 4C). Reduction in the uptake of macrophages means prolonging of the in vivo cir- culation time, which is favorable for SAB to arrive at a target region. 3. Research on cellular uptake in vitro

The bEnd.3 cells were seeded into 24-well plates at a density of 5x10% cells/well and cultured for 24 h. Then the culture medium was replaced by 500 pL of DMEM culture medium containing FITC- loading SABNPs or RR@SABNPs (FITC 5 pg/mL). After incubation for 4 h, the culture medium was removed, and the bEnd.3 cells were washed with PBS two times. The cellular uptake of the nanoparti- cles in the bEnd.3 cells was measured with a reversed fluorescence microscope and a flow cytometer.

As shown in FIG. 5A, fluorescence of the RRESABNPs group is higher than that of SABNPs and RESABNPs groups, indicating that

RRESABNPs can enter the bEnd.3 cells more effectively. Flow cytom- etry analysis further shows a similar uptake condition. The mean fluorescence intensity of the bEnd.3 in the RRESABNPs group is nearly 1.4 times that of the SABNPs group (FIG. 5B and FIG. 5C).

These results indicate that RR@CSABNPs haves high affinity to in- tegrin avp3 expressed cells, which is favorable for RRESABNPs to specifically deliver SAB to the ischemic brain region where the integrin ovp3 is highly expressed.

Example 3

Pharmacodynamic evaluation experiment of the brain-targeting erythrocyte membrane-enveloped SAB nanoparticles of the present invention on cerebral ischemia-reperfusion injury 1. Establishment of mouse MCAO/R model

Adult male C57BL/6J mice (body weight: 23-25 g) were induced anesthesia with 3% isoflurane, and then maintained with 1.0-1.5% isoflurane. In the operation, a heating pad was used to maintain the body temperature at 36.5-37°C. Skin and subcutaneous tissues were dissected in the middle of the neck, blunt dissection of mus-

cles was performed under a microscope, and the left common carotid artery (CCA), the left external carotid artery (ECA) and the in- ternal carotid (ICA) were exposed. CCA and ECA were ligated, and

ICA was clamped with a microvacular aneurysm clip. A V-shaped small incision was cut at the distal end of ECA, and 6-0 filament coated with silica gel were mildly inserted into ICA to block mid- dle cerebral artery. Cerebral blood flow changes were monitored by using a laser Doppler blood flow monitoring system (MoorVMS-LDF2).

Over 70% of blood flow drop was regarded as successful establish- ment of the MCAO model. After 1.5 h of ischemia, the filament were pulled out to achieve reperfusion. An arterial exposure operation was performed on sham-operated mice without artery ligation and filament embolization. All mice surviving to the endpoint were in- cluded in the experiment. 2. Living imaging

PBS, DiR-loaded SABNPs and RR@SABNPs (Dir 10 pg/mL) were in- travenously administrated to the MCAO/R model mice after 16 h of reperfusion, and imaging was performed at different time points (2, 4 and 6 h) by using an IVIS spectral in vivo imaging system (PerkinElmer, US). After 6 h, the mice were perfused with normal saline and sacrificed. Major organs (heart, liver, spleen, lung, kidney and brain) were collected and imaged. In addition, the brain was cut into five slices which were detected by the IVIS spectral in vivo imaging system. As shown in FIG. 6A, compared with the control group, the RRESABNPs and SABNPs groups have obvi- ous fluorescence, indicating that the nanoparticles were accumu- lated in the brain. Distribution of the nanoparticles in the brain was markedly increased along with time, and fluorescence was the strongest at 6 h after administration (22 h after reperfusion).

Meanwhile, compared with the SABNPs group, fluorescence in the brain region of the mice in the RRESABNPs group was enhanced, in- dicating that the coating of RGD modified RBCM is beneficial to the accumulation of RR@SABNPs in the brain. After 6 h, the mice were dissected, and major organs were collected and imaged through the imaging system (FIG. GB). FIG. 6C is a fluorescent quantita- tive graph of the major organs, and the results indicate that flu- orescence of liver in the SABNPs group is the strongest. Although high level integrin ovp3 is expressed in liver, RGD modification in RR@SABNPs does not increase distribution thereof in liver but decreases the same, which contributes to the inherent immune es- cape capability of the RBCM.

In order to evaluate whether RR@GSABNPs were targeted to is- chemic brain tissues more intuitively, the collected brain tissues were dissected and analyzed. As shown in FIG. 6D, both the ischem- ic region and the non-ischemic region have fluorescence distribu- tion. The fluorescence intensity of the RRESABNPs group is much higher than that of the SABNPs group (nearly four times) (FIG. 5D and FIG. 5E). In addition, the fluorescence intensity of the is- chemic region of the RRE@SABNPs group is obviously higher than that of the SABNPs group, indicating that SAB was accumulated more in the ischemic region (FIG. 6D). Specifically, after injection of

RRESABNPs, the fluorescence ratio of the ischemic side to the nor- mal side is increased up to 1.5 (FIG. 6F), indicating that the cerebral ischemic target region after injection of the RGD modi- fied RRESABNPs is obviously enhanced, which may be related to the integrin ovp3. 3. Pharmacodynamic evaluation 3.1 Experimental grouping and administration

The adult male C57BL/6J mice (body weight: 23-25 g) were ran- domly divided into a sham-operated group, a model group, an SAB solution group and an RRESABNPs group. Except the sham-operated group, all groups were subjected to MCAO/R operation, and the nor- mal saline, the SAB solution (10 mg/kg) and the RRE@SABNPs (10.6 mg/kg) were continuously administrated through caudal vein for three days immediately after reperfusion. 3.2 Behavioral evaluation

Neurological function and sensorimotor function of the MCAO/R model mice before and after operation were respectively evaluated with a modified neurological severity score (mNSS) and a foot fault test. Specifically, mNSS score integrally evaluates the in-

Jury degree of the neurological function of the mice from four as- pects: motion, feeling, balance and reflection. The foot fault test was used for evaluating the placing accuracy of forepaws on a grid, that is, a percentage of falling step number of damaged forepaws in stroke in total step number. The paw of the mice was placed on the surface of the grid (30x35x31lcm), with an opening of 2.5 cm’. In a case that one paw moving on the surface of the grid slid, one fall was recorded. Compared with the MCAO/R model group, the neurological score of the RRESABNPs is obviously reduced (FIG. 7D) and the foot fault step number on the affected side is marked- ly reduced (FIG. 7E), indicating that the neurological function and the sensorimotor function are markedly improved. 3.3 TTC staining and brain infarct volume quantitive analysis

As shown in FIG. 7A, heart perfusion was performed on the mice at 24 h after last administration, the brain was taken on ice, and the brain was cut into seven 1 mm thick coronary slices.

The brain slices were then stained with 1.5% TTC. After TTC stain- ing, the brain slices were photographed, and the infarct volume was analyzed with an ImageJ software (NIH). The infarct volume was calculated by using the following formula: infarct volume ($)=[ (hemispherical volume of opposite side)- (hemispherical volume of non-ischemic same side)] x100/ hemispherical volume of opposite side. Compared with the sham-operated group, the brain infarct volume after MCAO/R is markedly increased, indicating successful establishment of the MCAO/R model (FIG. 7B). Compared with the

MCAO/R model group, the infarct volume of the RRESABNPs is obvi- ously reduced, and is markedly decreased from 48% to 38% (FIG. 7B,

FIG. 7C). 3.4 HE staining and Nissl staining

Heart perfusion was performed on the mice at 24 h after last administration , 4% paraformaldehyde was perfused for fixation, and the brain of the mice was collected, fixed in 4% paraformalde- hyde at room temperature for 24 h and dehydrated in a saccharose solution. Then frozen slices were cut into 5 pm thick continuous coronal sections using a microtome. The brain sections were stained with hematoxylin and eosin (H&E) at room temperature for 5 min. Histological observation was performed under an optical mis- croscope. In addition, the brain sections were stained with a

Nissl staining solution for 20 min. The sections were dehydrated in 70%, 95% and 100% ethanol, and ethanol was scavenged in xylene and was then covered with a neutral resin. Then the section were scanned, and histological observation was performed through a physical and chemical scanner.

FIG. 7F is a representative image of the brain sections of the MCAO/R model mice, pathological characteristics after treat- ment being evaluated by selecting regions around infarct (Cortex) and dentate gyrus (DG). As shown by H&E staining in FIG. 7G, the quantity of cells in ischemic DG and Cortex regions of the mice in the MCAO/R group is severely decreased, and the cell density in the ischemic DG and Cortex regions of the mice in the RRESABNPs group is markedly increased. Compared with the sham-operated group, an obvious cavity can be seen in the ischemic region of the

MCAC/R group, and Nissl bodies are obvious in deficiency (FIG.7H).

After treatment of RRE@SABNPs, the cavity shrinks, and the quantity of the Nissl bodies in an ischemic penumbra is increased (FIG. 7H). The results show that RRESABNPs can effectively inhibit loss of brain neurons of the MCAO/R mice. 4. In vitro neuroprotective effect of RRESABNPs 4.1 Neuroprotective effect of RRESABNPs on an OGD/R impaired

PC12 cell

LDH content detection: the PCl2 cells were seeded into 96- well plates at a density of 1x10° cells/well and incubated till 80% of the cells were fused. The cells were randomly divided into three groups: the control group, the model group and the RRESABNPs group. The PC12 cells were incubated at 37°C for 4 h in a dedicated anaerobic incubator which contained an anaerobic gas mixture (95% of N; and 5% of CO,), then the RRESABNFs were administrated (the content of SAB in the preparation was 10 ug/ml), and the cells were then oxygenated for 20 h. The culture solution was collected and centrifuged at 2,000 rpm for 5 min, and the LDH content in a supernatant was measured with an LDH detection kit. The absorbance was measured at 560 nm with microplate zeader. As shown in FIG.

SA, compared with the control group, OGD/R markedly increases re- lease of LDH from the PCl2 cells. The PCl2 cells treated by

RRESABNPs may reverse increase of LDH release.

Measurement of intracellular ROS level and mitochondrial mem- brane potential (MMP): the above OGD/R impaired PC12 cell model was established. After the OGD/R experiment was finished, ROS and

MMP of the PC12 cells were respectively detected by DHE and JC-1 staining. A reversed fluorescence microscope detected cell fluo- rescence of DHE and JC-1, and MMP was represented by a red and green fluorescence intensity ratio. As shown in FIG. 8B, compared with the control group, OGD/R markedly increases fluorescence in- tensity of the PC12 cells. The increase of fluorescence intensity is reversed by RR@SABNPs, indicating that RRE@SABNPs can reduce rise of intracellular ROS level induced by OGD/R. A JC-1 fluores- cence probe was used to evaluate whether RRESABNPs could inhibit mitochondrial dysfunction by increasing the mitochondrial membrane potential after OGD/R impairment. As shown in FIG. 8C, JC-1 in the control group emits stronger red fluorescence, indicating that JC- 1 exists in normal mitochondria in an aggregated state. After

OGD/R impairment, red fluorescence is decreased and green fluores- cence is increased, indicating that MMP of the PC12 cells is re- duced. Compared with the OGD/R group, MMP of the RR@SABNPs group is obviously raised. 4.2 Neuroprotective effect of RRESABNPs on H;0, impaired PC12 cells

Generation of excessive ROS is related to mitochondrial dys- function and cellular damage. In order to further study the abil- ity of RRESABNPs to reduce the intracellular ROS level and main- tain the MMP, an H;0, induced oxidative damage model was estab- lished. The PC12 cells were seeded into 96-well plates at a densi- ty of 1x10% cells/well and incubated till 80% of the cells were fused. The cells were randomly divided into three groups: the con- trol group, the model group and the RRESABNPs group. After the

PC1l2 cells in the RRESABNPs group were pre-treated by RRESABNPs (SAB 10 pg/ml) for 20 h, the model group and the RRESABNPs group were treated by 600 uM H;0: for 4 h. The LDH release amount, the intracellular ROS level and the MMP level in the culture solution were detected by using the above-mentioned method.

The results are as shown in FIG. 9. The protective effect of

RRE@SABNFs on H.0, induced oxidative damage of the PCl2 cells is similar to that of the OGD/R model. The results are verified again in the H:0; induced PC12 cell oxidative damage model. 5. In vivo safety evaluation of RRESABNPs

In order to illustrate the biological safety of the nanopar- ticles, after continuous administration to the male C57BL/6J mice through intravencus injection for three days (the content of SAB in the preparation was 10 mg/kg), heart perfusion was performed on the mice, 4% paraformaldehyde was perfused for fixation after the liquid was clear, the mice were dissected, and major organs (heart, liver, spleen, lung, kidney and brain) of the mice were collected, fixed in 4% paraformaldehyde at room temperature for 24 h and dehydrated in a saccharose solution to produce frozen slic- es. Then the frozen slices were cut into 5 pm thick continuous coronal slices by using a slicer. The organ slices of heart, liv- er, spleen, lung, kidney and brain were stained with hematoxylin and eosin (H&E) at room temperature for 5 min. Histological obser- vation and H&E staining analysis were performed under an optical miscroscope. As shown in FIG. 10, compared with the control group, there are no obvious histological changes in the RRE@SABNPs. These results indicate that RRESABNPs have good biocompatibility and safety in vivo.

The above embodiments are used for explaining the present in- vention, rather than limiting the present invention. Any modifica- tion and variation made to the present invention under the spirit of the present invention and within the protection scope of the claims shall fall into the protection scope of the present inven- tion.

Claims

CONCLUSIONS

1. Preparation method for brain targeting of erythrocyte membrane-coated salvianolic acid B (SAB) nanoparticles, which specifically comprises the following steps: (1) dissolving bovine serum albumin (BSA) and SAB in a weight ratio of (5 -20): 1 in water, 2-10 times the volume, adjust the pH value to 8.0, add ethanol 6 times in the water volume at 1.0 ml/min, stir the mixture for 30 minutes, 2% add glutaraldehyde at 0.021 times the volume of water, stir the mixture continuously for 12 hours, and filter the mixture to obtain SABNPs; and (2) Place 1-5 parts by volume of 10 mg/ml SABNPs PBS solution and 1 part by volume of RBCM in 1 part by volume of PBS, and perform complete mixing and extrusion to prepare RE@SABNPs.

The preparation method according to claim 1, wherein in step (2) the RBCM is prepared by the following steps: taking whole blood from mice, centrifuging the whole blood at 4°C and discarding serum and white matter at a border; adding a pre-chilled 1x PBS buffer solution, performing centrifugal washing many times, and finally discarding the supernatant to obtain free red blood cells; adding a pre-chilled 0.25 x PBS buffer solution with a volume ratio of 1:10, and allowing the red blood cells to swell and break for 30 minutes in a 4°C refrigerator; and performing centrifugation at 4°C, discarding the supernatant, and repeating the above steps until the supernatant is colorless to obtain the RBCM.

The preparation method according to claim 2, further comprising the step of: allowing the RBCM to pass through an 800 nm polycarbonate membrane and a 400 nm polycarbonate membrane many times respectively by using a mini-extruder.

The preparation method according to claim 1, wherein the RBCM is an RGD-modified RBCM.

Preparation method according to claim 4, wherein the RGD-modified RBCM is prepared by the following steps: mixing the RGD-PEG-DSPE with the RBCM in a weight-to-volume ratio of 1:1-10, the mixture for 30 incubate at 37°C for minutes, centrifuge the mixture, discard the supernatant, redissolve the precipitates, and perform extrusion and filtration to obtain the RGD-modified RBCM.

The preparation method according to claim 1, wherein in step (2) the extrusion specifically comprises the following step: performing co-extrusion through the 800 nm polycarbonate membrane many times using a mini-extruder.

7. Brain-targeted erythrocyte membrane-coated SAB nanoparticles prepared by the method of any one of claims 1 to

6.

Use of the brain-targeted erythrocyte membrane-coated SAB nanoparticles according to claim 7 in the preparation of a medicament for the treatment of a cerebral infarction.