Brain targeting of Baicalin and Salvianolic acid B combination by OX26 functionalized nanostructured lipid carriers

Journal Pre-proofs Brain targeting of Baicalin and Salvianolic acid B combination by OX26 functionalized nanostructured lipid carriers Yumei Wu, Xunan Song, Dereje Kebebe, Xinyue Li, Zhifeng Xue, Jiawei Li, Shouying Du, Jiaxin Pi, Zhidong Liu PII: DOI: Reference:

S0378-5173(19)30799-9 https://doi.org/10.1016/j.ijpharm.2019.118754 IJP 118754

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

1 August 2019 17 September 2019 29 September 2019

Please cite this article as: Y. Wu, X. Song, D. Kebebe, X. Li, Z. Xue, J. Li, S. Du, J. Pi, Z. Liu, Brain targeting of Baicalin and Salvianolic acid B combination by OX26 functionalized nanostructured lipid carriers, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.118754

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Brain targeting of Baicalin and Salvianolic acid B combination by OX26 functionalized nanostructured lipid carriers Yumei Wua,b,c, Xunan Songa,b, Dereje Kebebea,b,d, Xinyue Lia,b, Zhifeng Xuea,b, Jiawei Lia,b,e, Shouying Duf, Jiaxin Pia,b,*, Zhidong Liua,b,* a Engineering Research Center of Modern Chinese Medicine Discovery and Preparation

Technique, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China. b

Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of

Traditional Chinese Medicine, Tianjin 301617, China. c School

of Pharmacy, Zunyi Medical University, Zunyi 563000, China.

d

School of Pharmacy, Institute of Health Sciences, Jimma University, Jimma, Ethiopia.

e

Department of Experimental Department, Tianjin University of Traditional Chinese

Medicine, Tianjin 301617, China. f

School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing

100029, China.

Corresponding Authors: *

Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese

Medicine, No. 10 Poyang Lake Road, Jinghai District, Tianjin 301617, China. E-mail addresses: [email protected] (J. Pi) and [email protected] (Z. Liu)

1

ABSTRACT In order to deliver Salvianolic acid B (Sal B) and Baicalin (BA) to the brain tissue to repair neuron damage and improve cerebral ischemia-reperfusion injury (IRI), in our previous study, a nanostructured lipid carrier (NLC) containing BA and Sal B, and modified by the transferrin receptor monoclonal antibody OX26 (OX26-BA/Sal BNLC) was constructed. The present study is to evaluate its in vitro release behavior, in vitro and in vivo targeting ability, in vitro pharmacodynamics and brain pharmacokinetics. The results showed that the release mechanism of the formulation was in line with the Weibull model release equation. The in-vitro and in-vivo targeting ability study exhibited that OX26 modified formulations was obviously higher than that of non-modified and solution groups. The results of in vitro preliminary study to investigate the protective effect of OX26-BA/Sal B-NLC on oxygen-glucose deprivation/ reperfusion injured cells showed that it could decrease the injury. Furthermore, the results of brain microdialysis study showed that the OX26-modified preparation group could significantly increase the content of BA in the brain. In the solution group and the unmodified group, Sal B can only be detected at few time points, while OX26-modified BA/Sal B-NLC could be detected within 4 h. These results indicating that OX26-modified NLC can promote the brain delivery of Sal B and BA combination.

Keywords: Nanostructured lipid carriers; Baicalin; Salvianolic acid B; Monoclonal antibody OX26; Brain microdialysis; Cellular uptake 2

1. Introduction Stroke remains one of the leading causes of global disability and death (Collaborators, 2018). Despite many advances in the acute treatment of ischemic stroke, the proportion of patients eligible for this medical and intravascular intervention is low, and even for many patients receiving acute treatment, the results are not effective. In addition, the global population ageing will lead to an increase in the absolute number of cerebrovascular events. The World Health Organization (WHO) estimates that by 2030, the prevalence of primary strokes is expected to increase to 23 million per year if there were no more population interventions (Béjot and Touzé, 2016). Cerebral ischemia is the main type of stroke, accounting for 60-80% of stroke cases (Zhai et al., 2019). The damage of ischemic stroke is mainly due to the over expression of intermediate factors such as sudden hypoxia and excitatory glutamate release, free radicals and inflammatory products, which synergistically cause brain cell death (Li et al., 2017). Ischemic reperfusion injury is a common feature of ischemic stroke, which occurs when the blood supply is restored after a period of ischemia (Lin et al., 2016). During ischemia-reperfusion, neuronal damage caused by stroke is associated with overproduction of reactive oxygen species and reactive nitrogen (RNS), excitatory amino acidosis and inflammatory response (Zhai et al., 2019). Reperfusion is the only effective treatment for acute ischemic stroke; however, it causes excessive inflammatory reactions and aggravates brain damage. Therefore, additional therapy is needed for the inflammation caused by reperfusion (Li et al., 2017). Traditional Chinese medicine (TCM) has rich theoretical knowledge and clinical 3

experience in the treatment of brain injury and its sequelae. TCM is widely used in China and other Asian countries with a heavy burden of ischemic stroke (Liu et al., 2018). Currently, many drugs isolated from Chinese medicine have been reported to play a neuroprotective role in cerebral ischemia. The water-soluble phenolic acid Salvianolic acid B (Sal B) from Salvia miltiorrhiza Bge. and the flavonoids Baicalin (BA) from Scutellaria baicalensis Georgi have been reported to exert protective effects on cerebral ischemia-reperfusion injury and neuron injury. Studies have shown that BA can activate the bHLH transcription factor and inhibit Notch signaling, thus playing a role in limiting the fate of neural stem cell differentiation (Zhuang, 2011). BA has a protective effect on ischemic brain injury, can inhibit the elevation of glutamate and aspartate in the brain after focal cerebral ischemic injury in rats (Li et al., 2003), and protect rat primary neurons from damage caused by glutamate/n-methyl-d-aspartate (NMDA) or glucose deprivation (Lee et al., 2003). BA also has a protective effect on oxygen-glucose deprivation (OGD)-induced hippocampal ischemic or NMDA-induced excitotoxicity in rats (Ge et al., 2007), increasing the number of blast cells in the hippocampal dentate gyrus after cerebral ischemia/reperfusion (Zhuang et al., 2013). Sal B has been shown to have antiinflammatory and neuroprotective effects in vitro and in vivo, and is associated with inhibition of the TLR4 signaling pathway and up-regulation of mtCx43 via the PI3K/AKT pathway (Hou et al., 2016; Wang et al., 2016). Recently, Fan et al. demonstrated that Sal B has neuroprotective effects on ischemia-reperfusion-induced brain injury in a mouse model of middle cerebral artery occlusion (MCAO) (Fan et al., 4

2018). The compatibility of effective components of the TCM is based on the theory of TCM and activity relationship, guided by modern scientific thought, and modern pharmacological methods are used to study the optimal compatibility of effective component dose (Zhang et al., 2015). Sal B combined with BA (molar ratio 5:3) can synergistically promote nerve regeneration in vitro, improve learning and memory ability of cerebral ischemic rats, increase the number of hippocampal neurons, and have a significant therapeutic effect on the recovery of ischemic stroke (Zhuang, 2011). Although Sal B and BA have broad application prospects in cerebral ischemia, their clinical application is limited due to their poor stability and low bioavailability (Grossi et al., 2017; Luan et al., 2015). In addition, the blood-brain barrier (BBB) is the main barrier for drugs to enter the brain (Upadhyay, 2014). At present, the main clinical treatments of brain diseases are brain implantation, intraventricular and intracerebral injection, which bring great pain to patients. Therefore, the development of brain-targeted drug delivery systems has important clinical implications. In the past three decades, different methods have been developed to overcome BBB. Among them, encapsulation of drugs in nanoparticles is capable of passing through the BBB and is considered to be one of the most promising methods (Dal Magro et al., 2017). Nanostructured lipid carriers (NLC) is a new generation of nanoparticle delivery systems based on solid lipid nanoparticles (SLN). Compared with SLN, NLC consists of liquid lipids instead of some solid lipids, which makes the particles exist in amorphous or crystalline defects, thus solving the problems of low encapsulation efficiency, poor stability and small drug loading (Ghasemiyeh and 5

Mohammadi-Samani, 2018). However, nanoparticles cannot diffuse freely through the BBB, and receptor-mediated translocation of the brain capillary endothelium is required to deliver its contents to the brain parenchyma. Therefore, receptor ligand or antibody can be coupled to the surface of the nanoparticle as a targeting molecule to construct a targeted nano-preparation, which helps the drug enter the brain through the specificity of

receptor

binding. This

type

of

transport

is

called

receptor-mediated

endocytosis/transcytosis (RMT) (Olivier, 2005). A variety of receptors are highly expressed on endothelial cells that form BBB, such as the transferrin receptor (TfR), insulin receptor, insulin-like growth factor and leptin. There have been many reports on the use of transferrin or antibodies against TfR as specific ligands to transport nanocarriers through BBB (Liu et al., 2013). Studies have shown that TfR antibody (OX26)-conjugated nanoparticles (NPs) enhance drug delivery in the cellular uptake pathway. To that end, the aim of this work was to evaluate the in vitro release behavior of OX26modified nanostructured lipid carriers containing BA and Sal B (OX26-BA/Sal BNLC), and to study the uptake of bEnd.3 in brain microvascular endothelial cells using coumarin-6 (Cou-6) as a fluorescent probe. In vitro brain IRI cell model was constructed by using OGD/R treatment with human neuroblastoma cell SH-SY5Y. After administration, cell viability was determined by CCK-8 method, and in vitro targeting research and pharmacodynamics preliminary evaluation were performed. The distribution of DiR preparation in nude mice was investigated by in vivo imaging system to evaluate the in vivo targeting ability of OX26 modified preparations. 6

Meanwhile, the rat model of cerebral ischemia was established by a suture method, and the distribution of OX26-FITC and OX26-FITC modified BA/Sal B-NLC in the brain was investigated. The brain concentrations of BA and Sal B were analyzed using the brain microdialysis sampling technology combined with UPLC-MS/MS analysis technology, so as to explore the possibility of OX26-BA/Sal B-NLC improving BBB permeability and brain targeting, and to explore the feasibility of OX26 to improve the bioavailability of BA and Sal B in the treatment of brain diseases to promote the followup study on brain targeted preparations. 2. Material and methods 2.1 Materials Salvianolic acid B standard and Baicalin extract were purchased from Zhongxin Pharmaceuticals (purity > 98%, Tianjin, PR China). Baicalin standard and dialysis bag (cut off: 7000 Da) were supplied by Shanghai yuanye Bio-Technology Co., Ltd.. Salvianolic acid B extract was obtained from Chengdu Herbpurify Co., Ltd.. Soy lecithin S100 was purchased from Lipoid (German). mPEG-MAL and mPEG-OH were obtained from YareBio. Monoclonal antibody OX26 for the transferrin receptor was obtained from Bio-Rad and Sanata company, and OX26-FITC was purchased from BD Biosciences. 2-iminothiolane and coumarin-6 were supplied by Sigma, and Hitrap Desalting column was purchased from GE Healthcare. 1,10-Dioctadecyl-3,3,3,3tetramethyl indotricarbocyanine iodide (DiR), near-IR lipophilic carbocyanine dye, was obtained from Biotium Inc. (Hayward, CA). Paraformaldehyde (4%) was obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). 4,6-Diamidino-27

phenylindole (DAPI) was purchased from Beyotime (Haimen, China). 0.25% TrypsinEDTA (1×), penicillin (10,000 units/mL) and streptomycin (10,000 μg/mL) were purchased from Life Technologies. Cell Counting Kit 8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). DMEM medium, fetal bovine serum were purchased from Gibco. CMA/12 probe (membrane length 2 mm, interception 20000 Dalton) and probe catheter (CMA, Sweden). Mouse brain microvascular endothelial cell line (bEnd.3) was purchased from ATCC (American) and cultured in DMEM supplemented 10% fetal bovine serum (Gibco), 100 units/mL penicillin and 100 mg/mL streptomycin at 37 ℃ in a humidified atmosphere containing 5% CO2. Human neuroblastoma cell SH-SY5Y cell line was a kind gift from the Institute of Basic Medicine, Peking Union Medical College, and the same culturing condition as bEnd.3 cells was used. BALB/c nude mice (male, 6-8 weeks, 20-22 g) were procured from Beijing HFK Bioscience Co., Ltd. (Beijing, China), and healthy Sprague Dawley rats (male,230-280 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed at 25 ± 1 ℃ with access to food and water ad libitum in a specific pathogen-free (SPF) environment. All animal experiments were carried out in accordance with protocols evaluated and approved by the ethics committee of Tianjin University of Traditional Chinese Medicine (document number: TCM-LAEC2018025). 2.2 Preparation of NLC BA/Sal B-loaded unconjugated NLC (BA/Sal B-NLC) was prepared by emulsification and solvent evaporation method (Liu et al., 2015). Briefly, BA and lecithin were 8

dissolved in ethanol, Compritol® 888 ATO, mPEG-MAL, mPEG-OH and MCT 812 were dissolved in chloroform, the above two systems were mixed to get the oil phase. The aqueous phase was prepared by dissolving the prescribed quantity of Myrj 52 in deionized water, and then the oil phase was quickly added into the aqueous phase under magnetic stirring at 75℃. The mixture was kept under stirring for 2 h to evaporate the organic solvent and solidified in the refrigerator at 4℃ followed by characterization of size, drug concentration and encapsulation efficiency. For preparation of OX26-BA/Sal B-NLC, OX26 was thiolated using 2-iminothiolane (Traut’s reagent) and incubated with BA/Sal B-NLC for 8 h under nitrogen flow at room temperature (Liu et al., 2015). For the preparation of Cou-6-NLC, OX26-Cou-6NLC, DiR-NLC, OX26-DiR-NLC and OX26-FITC-BA/Sal B-NLC, the same methods mentioned above were used. 2.3 Determination of particle size and encapsulation efficiency The particle size of the above formulations was measured by photon correlation spectroscopy using Nano ZS (Malvern Instruments, Malvern, UK). Furthermore, the entrapment efficiency (EE) was determined by measuring the concentration of unloaded drug in dispersion collected after centrifugation of nanoparticle dispersion at 4000 r/min and 4 ℃ for 20 min, and analysis was conducted using the validated highperformance liquid chromatography (HPLC) methods. 2.4 In vitro release study The dialysis method was used to study the in vitro release behavior of BA/Sal B-Sol (solution), BA/Sal B-NLC and OX26-BA/Sal B-NLC. The artificial cerebrospinal fluid 9

(ACSF) and normal saline added with vitamin C (Vc-NS) were selected as the release medium. One mL of the formulation and 4 mL release medium were placed in the dialysis bag. Both ends of the dialysis bag were fastened, and the bag was placed into the vessel containing 250 mL release medium. The rotation speed was set at 100 r/min and the temperature was maintained at 37±1 ℃. 5 mL sample was taken at 0.17, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10, 24 h, and then blank release medium with the same volume and temperature was added. The sample was filtered with 0.22 μm filter membrane, and the filtrate was taken to determine the contents of BA and Sal B according to the validated HPLC methods. BA and Sal B in release samples were analyzed by an HPLC (LC-20AT, Shimadzu) equipped with SPD-20A UV-detector. Chromatographic separations were achieved using a Diamond DIKMA ODS C18 （4.6×200 mm, 5 μm) column. The mobile phase comprised of (A) water (containing 0.2% phosphoric acid) (55%), (B) methanol and acetonitrile (the ratio of methanol to acetonitrile was 1:1) (45%). The column oven temperature was maintained at 30 ℃ and a flow rate of 1.0 mL/min was used to achieve separation from interferences. The injected volume was 20 μL and monitored at a wavelength of 280 nm. Method validation was performed for the specificity, linearity, precision and stability. 2.5 Cellular uptake study 2.5.1 UPLC method validation Cou-6 in NLC was analyzed by a UPLC (Waters, American) equipped with TUVdetector. The chromatographic separation of Cou-6 was performed on an ACQUITY 10

UPLC® BEH C18 column (1.7 µm, 2.1×50 mm) with the column temperature at 40 ℃. Chromatographic separation was achieved using a mobile phase comprised of water (A) and acetonitrile (B) (5:95). Efficient and symmetrical peak was obtained at a flow rate of 1.0 mL/min. The injected volume was 2 μL and monitored at a wavelength of 448 nm. Method validation was performed for the specificity, linearity, precision, stability, repeatability and recovery. 2.5.2 Determination of Cou-6 in NLC 0.2 mL of Cou-6-NLC or OX26-Cou-6-NLC was precisely added into a 10 mL volumetric flask, followed by 10 min of methanol ultrasonic emulsion breaking, which was filtered by 0.22 µm filter membrane, and the contents of Cou-6 in the formulations were determined according to the validated UPLC methods. 2.5.3 Cytotoxicity assay The cytotoxicity of different formulations was evaluated by CCK-8 kit. bEnd.3 cells in the logarithmic growth phase were seeded in 96-wells plate and cultured for 24 h, and DMEM medium without serum was used to synchronize the cells for 12 h. Different Cou-6 formulations were added with a serial concentration (282, 211.5, 141, 70.5, 35.2 and 17.6 ng/mL) which were prepared with DMEM without fetal bovine serum. Twenty-four hours later, the medium containing drugs was removed by pipettor and the cells were washed three times with PBS. A hundred microliters of 10% CCK-8 dilution was then added into each well, after incubation for 1.5 h at 37 ℃, the absorbance at 450 nm wavelength was read by a multifunctional microplate reader (Multiskan MK3; Thermo Scientific, Atlanta, GA) and the cell viability (%) was calculated. 11

2.5.4 Cellular uptake bEnd.3 cells in the logarithmic growth phase were seeded in 96-wells plate and after 24 h, DMEM medium without serum was used to synchronize the cells for 12 h. Cou-6Sol, Cou-6-NLC and OX26-Cou-6-NLC were added into the wells which concentrations were determined according to cytotoxicity assays and incubated with cells at 37 ℃, 5% CO2 condition for 2.0, 4.0, 8.0 and 12 h, respectively. The adsorptive and free particles were removed by washing with ice-cold PBS three times. The cells were fixed with 4% paraformaldehyde for 20 min and the nuclei were stained by 0.4 μg/mL of DAPI solution for 10 min. Subsequently, the cellular internalization of Cou6 was analyzed by High Content Cell Imaging Analysis System (GE InCell Analyzer 2000, Fairfield, CT). 2.6 Preliminary study on the effects of the formulations on SH-SY5Y in OGD/R injury model in vitro 2.6.1 Establishment of OGD/R model Investigation of oxygen-glucose deprivation time The SH-SY5Y cells were planted in 96-well plates and incubated in a 5% CO2, 37 °C incubator. When the cell adherent culture fusion degree was greater than 80%, the original medium was aspirated, washed three times with PBS, and 150 μL of medium without oxygen and glucose was added. And then the plates were placed in an anaerobic incubator, OGD induction lasted for 3 h, 4 h, 5 h, 6 h, 7 h. The hypoxic liquid was aspirated when the hypoxia process completed, and DMEM complete medium was added to carry out the reperfusion process in a normal cell incubator (5% CO2, 37 °C) 12

for 24 h, and finally the cell activity was measured. Anoxic agent concentration investigation When the degree of SH-SY5Y cells growth fusion was more than 80%, the original medium was replaced by molding solution (2 mmol /L, 5 mmol/L, 10 mmol/L and 20 mmol/L Na2S2O4 sugar-free DMEM medium), and the blank control group was given an equal volume serum-free DMEM medium and cultured in a normal cell incubator (5% CO2, 37 °C) for 24 h, and then the cell activity was measured. 2.6.2 Cytotoxicity study The SH-SY5Y cells were cultured as above method, and then were incubated with BA/Sal B-Sol, BA/Sal B-NLC and OX26-BA/Sal B-NLC at different concentrations （0.05，0.1，0.5，1.0 μg/mL）for 24 h. The culture medium was discarded and the diluent of 100 μL 10% CCK-8 was added to each well, and then the cell activity was measured. 2.6.3 Evaluation of in vitro anti-OGD/R injury The SH-SY5Y cells were cultured as above method, and then were incubated with 0.5 μg/mL BA/Sal B-Sol, BA/Sal B-NLC and OX26-BA/Sal B-NLC for 24 h. 2 mmol /L Na2S2O4 sugar-free DMEM medium was replaced as molding solution, and the blank control group was given an equal volume serum-free DMEM medium, the culture was continued for 4 h in an anaerobic incubator at 37 °C, 95% N2, 5% H2. And then the medium was replaced with DMEM complete medium and cultured in a normal cell incubator (5% CO2, 37 °C) for 24 h, and cell activity was measured. 2.7 Study on the in vivo targeting ability of OX26 modified NLC 13

2.7.1 Study on the distribution of OX26-DiR-NLC in nude mice Nine nude mice were randomly divided into 3 groups, three in each group, and DiR-sol (DiR solution), DiR-NLC and OX26-DiR-NLC were respectively administered intravenously with a dose of 2.0 mg /kg. The animals were anesthetized by isoflurane gas at 2, 4 and 24 h after injection, respectively. Fluorescence imaging was obtained by using in vivo imaging system (CRi Maestro™ 2 Maestro™ EX-RRO, Americam) under the same exposure intensity and time. 2.7.2 Study on the distribution of OX26-FITC and OX26-FITC-NLC in the brain Preparation and evaluation of middle cerebral artery ischemia model The middle cerebral artery ischemia (MCAO) model was prepared by the suturing method. The rats were anesthetized with Zoletil 50 (100 mg/kg, i.p.) and placed on a heating pad set at 37 ℃ to maintain the rats’ body temperature throughout the experiment. After a midline neck incision, tissue and muscles were separated from the right common carotid artery, and then the external carotid artery (ECA) and internal carotid artery (ICA) were found. A small incision was made in the common carotid artery 4 mm away from the bifurcation and a silicone wrapped suture (diameter 0.26 mm, head diameter 0.34±0.02 mm, Beijing Cinontech Co. Ltd.) was introduced into the ECA lumen and extended into the ICA (18.5±0.5 mm) to block the origin of the middle cerebral artery (MCA). All the animals were subjected to neurological evaluation according to Longa’s five-point scale (Longa et al., 1989). Administration and observation of fluorescence distribution OX26-FITC (0.25 mg/rat) or OX26-FITC-BA /Sal B-NLC (1.7 mL/rat) were 14

administered intravenously in the normal group and MACO model group, in which the model group was administered immediately after modeling. The rats were anesthetized with Zoletil 50 (50 mg/kg, i.p.) after 2 h, and the brain was taken followed perfusion, and stored in 4 % paraformaldehyde. The brain tissue was dehydrated, OCT embedded, sectioned and observed under a fluorescence microscope. The FITC excitation and the emission wavelength were set as 465-495nm and 515-555 nm, respectively. 2.8 Brain pharmacokinetic study 2.8.1 BA and Sal B analysis in dialysate by UPLC-MS/MS Instrumentation, chromatographic and mass spectrometry conditions of UPLC-MS/MS Dialysate samples were analyzed by a UPLC-MS/MS system consisting of an Agilent series 1290 UPLC system and an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation of analytes was performed on an ACQUITY UPLC® HSS T3 column (2.1×100 mm,1.8μm particle size) with the column temperature at 35 °C. Mobile phase comprised of 0.05% formic acid in water (A) and acetonitrile (B). The UPLC gradient program was set as follows: 15 %→50 % B at 0.0-5.0 min; 50 %→95 % B at 5.0-6.0 min. Efficient and symmetrical peaks were obtained at a flow rate of 0.3 mL/min. Injection volume of dialysate samples was 5 µL. The detection of the analytes was in the multiple reaction monitoring mode (MRM). The ESI configuration was as follows: gas temperature 350 °C; gas flow rate 10 L/min; nebulizer 40 psi; capillary 4000 V (+) and 3500 V (-) (Agilent Technologies). The quantitative parameters are listed in Table 1. Data acquisition and elaboration were performed using the Agilent Mass Hunter Workstation. 15

Table 1. Mass spectrometry parameters of BA and Sal B Fragmentor Compound

Mode

Ionization

Collision Energy MS1→MS2

（V）

（eV）

Sal B

MRM

ESI-

144

717.14→518.7

17

BA

MRM

ESI-

100

445.36→268.8

17

Preparation of calibration standards Stock solutions of BA and Sal B were prepared in methanol. The subsequent standard solutions were prepared from the stock solutions using ACSF and acetonitrile (1:1). Method validation Method validation was performed for the specificity, linearity, precision and stability. The animals were implanted with probes after surgery, and the blank dialysate was collected as a negative control sample after the equilibrium. The specificity of the method was to ascertain whether brain microdialysate samples contain any other analytes that could elute and interfere with the peak pattern of BA and Sal B. The standards BA and Sal B were precisely weighed and dissolved in methanol to prepare the stock solutions. The appropriate volume of the stock solutions was measured and diluted with acetonitrile-ACSF (1:1) for the standard curve. The concentration of BA was 0.6240, 1.248, 2.496, 9.984, 19.97, 79.87, 159.7 and 319.5 ng/mL, and the concentration of Sal B was 0.9180, 1.836, 3.672, 14.69, 29.37, 117.5, 235.0 and 470.0 ng/mL. The standard curve was plotted using concentration as abscissa, and the peak area as vertical axis. The intra-day precision was determined by repeated analysis of each sample with low, 16

medium and high concentrations of mixed standard solutions on the same day (n = 5). Acetonitrile-ACSF (1:1) was used to prepare mixed reference solutions containing BA and Sal B at concentrations of 1.248 and 1.836 ng/mL, 39.94 and 58.75 ng/mL, 255.6 and 376.0 ng/mL, respectively (n=5). The samples were injected at 0, 1, 2, 3, 4, 6 and 8 h, respectively, to investigate the stability of the samples placed in the sample room. After UPLC-MS/MS analysis of the mixed reference substance at the highest concentration of the standard curve, the blank brain microdialysis fluid was immediately injected and the residual effect of the method was repeated for 3 times. 2.8.2 In vivo recovery of the probe Normal feeding was resumed for 3 days after the brain probe was implanted into the rat brain. The pump flow was set at 1.5 µL/min and the probe was flushed 1 h using blank ACSF. A series concentration of BA and Sal B in ACSF (8.85 and 6.89 ng/mL, 16.68 and 13.85 ng/mL, 37.43 and 29.99 ng/mL, 72.07 and 57.46 ng/mL, 151.60 and 122.80 ng/mL, 310.20 and 273.56 ng/mL) were used as the perfusion fluid, and the samples began to be collected for every 30 min after 1 h of balance. 2.8.3 Microdialysis procedure The rats were anesthetized with Zoletil 50 (50 mg/kg, i.p.), and then the microdialysis procedure was carried out as reported previously by our research group with some necessary changes (Liu et al., 2014). The procedure was described as follows: a pretreated probe was inserted into the right hippocampal region with coordinates: AP: 3.84 mm, ML: +3.20 mm and DV: -4.96 mm relative to Bregma and the angle is 29 degrees, which were adopted from the Paxinos and Waston rat atlas (Miao et al., 2015). 17

Perfusion medium was infused through the microdialysis probe and three blank samples were collected every 30 min as a control. After intravenously administered, microdialysis samples were collected for 30 min at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 6.0, 8.0, 10.0, 12.0, 15.0, 21.0, 24.0 h. The concentrations of BA and Sal B in microdialysate samples were determined by UPLC-MS/MS (Supplementary Figure S1). 2.8.4 Verification of probe insertion location Brain slices were made after the sampling to verify the sampling location of the probe, and the insertion location of the probe was observed. 2.10 Statistical analysis The quantitative data were presented as mean ± standard deviation (SD). Statistical significance was analyzed using one-way analysis of variance with the p-value <0.05 indicating significance. 3. Results and discussion 3.1 Determination of particle size and encapsulation efficiency The particle size of Cou-6-NLC, OX26-Cou-6-NLC, DiR-NLC, OX26-DiR-NLC and OX26-FITC-BA/Sal B-NLC were found in the range of 14 nm to 30 nm with EE between 60%-100%. It showed that the prepared formulations had small particle size, uniform distribution and high encapsulation efficiency. 3.2 In vitro release study 3.2.1 HPLC method validation The method validation results showed that the method has good specificity. The regression equations for BA and Sal B prepared with Vc-normal saline were Y=80.89X18

1248.71, Y=21.70X-1305.60, and the correlation coefficients (r) were 0.999 7 and 0.999 9, respectively. The regression equations for BA and Sal B prepared with ACSF were Y=74.82X-343.80, Y=23.67X-1754.93, and the correlation coefficients (r) were 1.000 0 and 0.999 5, respectively. It indicated that BA and Sal B showed a good linear relationship with peak area in the range of 38.27-2449 ng/mL and 70.08-4485 ng/mL, respectively. The intra-day RSD (%) of BA and Sal B solution were 0.28 % and 0.36 %, respectively. The inter-day RSD (%) of BA and Sal B solution were 0.65 % and 2.00 %, respectively, which indicate the method was precise enough for the determination of BA and Sal B. The RSD (%) of the stability of BA and Sal B in Vc-normal saline for 24 h were 1.02 % and 1.85 % respectively, and were 1.87 % and 1.92 % respectively in ACSF, indicating that BA and Sal B had good stability in 24 h. The validation results showed that the established content determination method was specific and simple, and can be used for the analysis of components in the release liquid. 3.2.2 In vitro release Fitting of the release curve The cumulative release (Qn/μg) and cumulative release rate (CRR) (%) at each time point were calculated using the following formula. 𝑛―1

𝑄𝑛 = （𝐶𝑛 × 250 +

∑ 𝐶 × 5） 𝑖

𝑖=1

Cumulative release rate (%)=Qn/total×100% Where, Cn is the drug mass concentration (μg/mL) measured at the n sampling point; Ci is the drug mass concentration (μg/mL) measured at the i (i≤n-1) sampling point. The curve fitting was performed for the release of BA/Sal B-Sol, BA/Sal B-NLC and 19

OX26-BA/Sal B-NLC using OriginPro 8.6 (Figure 1). As can be seen from Figure 2, in BA/Sal B-Sol group, the release of BA and Sal B close to equilibrium at 4 h and 6 h, respectively. However, in BA/Sal B-NLC group, Sal B has exhibited a sustained release behavior in both release media without sudden release. BA in BA/Sal B-NLC and OX26-modified BA/Sal B-NLC released faster in both release media, and the modified formulation group released faster than the unmodified group. Fitting the release equation DDSolver 1.0(Zhang et al., 2010) was used to fit the release data to: zero-order kinetics, first-order kinetics, Higuchi, korsmeyer-peppas, hixson-crowell and Weibull release equations for each group. The goodness of fit was judged by correlation coefficient (r), Akaike information criterion (AIC) and model-selection criteria (MSC). According to the correlation coefficient close to 1, AIC minimum and MSC maximum principles (generally greater than 2 or 3), the release behavior of BA and Sal B in BA/Sal B-Sol, BA/Sal B-NLC and OX26-BA/Sal B-NLC all conformed to the Weibull model release equation, and r all higher than 0.995, and Sal B has a certain hysteresis in the release of Vc-normal saline. 3.3 Cellular uptake study 3.3.1 UPLC method validation The calibration curve showed a good linearity over concentration ranges (0.3950~25.31 µg/mL) with correlation coefficient (r) of 0.999 9 and the standard curve equation was Y=40073X+3857.1. The intra-day RSD of low, medium and high concentrations of 20

Cou-6 solution were 2.14 %, 0.15 % and 0.09 %, respectively. The inter-day RSD (%) was 0.91 %, 1.82 % and 1.23 %, respectively. The stability RSD (%) of the sample was 1.23 % within 24 h, indicating that Cou-6 was stable within 24 h. The RSD (%) value of repeatability was 1.22 %, indicating that the method had good repeatability. The average recoveries of low, medium and high concentrations of Cou-6 were 98.29 %, 99.15% and 99.04 %, respectively, and the RSD (%) values were 1.79 %, 2.18 %, and 2.48%, respectively, which indicated that this sample processing method was appropriate and in line with the methodological requirements of content determination. 3.3.2 Determination of Cou-6 in NLC The Cou-6 content in Cou-6-NLC and OX26-Cou-6-NLC was determined to be 126.11 μg/mL and 104.5 μg/mL, respectively. 3.3.3 Cytotoxicity assay In comparison with the control group, both Cou-6-NLC and OX26-Cou-6-NLC groups showed significant cytotoxicity at concentrations of 211.5 ng/mL, 282 ng/mL and 141 ng/mL (P<0.05). At concentration of 70.5 ng/mL, the cell survival rate was above 80%, and there was no significant difference with the cell activity of the control group (Figure 2). Therefore, 70.5 ng/mL was selected as the administration concentration of the cell uptake experiment. 3.3.4 Cellular uptake The results showed that when bEnd.3 cells were incubated with each group of Cou-6 preparations for 24 h, the fluorescence intensity of OX26-Cou-6-NLC group was significantly higher than that of Cou-6-NLC group and Cou-6-Sol group (P < 0.05). 21

Moreover, the uptake of OX26-modified Cou-6-NLC in all time points was significantly higher than that in the unmodified group and the solution group (P < 0.05) (Figure 3), indicating that OX26-modified NLC can promote the cellular uptake of the drug through receptor-mediated endocytosis. The results also showed that the fluorescence signal of Cou-6-Sol group was very weak during the whole incubation period, and the uptake amount was very small. 3.4 Preliminary study on the effects of the formulations on SH-SY5Y in OGD/R injury model in vitro 3.4.1 Establishment of OGD/R model According to the results of OGD/R model establishing study (Figure 4-A), at 4 h of the oxygen glucose deprivation time, the cell survival rate was about 60%, which was suitable as the oxygen glucose deprivation time. However, at longer time of oxygen glucose deprivation, the cell death rate was high, and the morphological changes were severe and cannot be recovered. The concentration of the anoxic agent (Na2S2O4) was selected based on the result of their cell viability study (Figure 4-B). Accordingly, the concentration of 2 mmol/L Na2S2O4 was used as the anoxic agent because 2 mmol/L of Na2S2O4 could quickly remove oxygen from the medium and had no obvious toxicity to cells. 3.4.2 Cytotoxicity study The toxicity of different groups to SH-SY5Y cells are shown in Figure 5-A. As it can be seen from the figure, at the concentration of 1.0 μg/mL of the three groups, it was found significantly more toxic to SH-SY5Y cells as compared with the control group 22

(**P<0.01). Therefore, the maximum concentration that is not toxic to the cells, i.e., 0.5 μg/mL, was selected as the concentration for subsequent efficacy studies. 3.4.3 Evaluation of in vitro anti-OGD/R injury From the results (Figure 5-B), BA/Sal B-Sol, BA/Sal B-NLC and OX26-BA/Sal BNLC 3 groups could repair the damage of OGD/R on SH-SY5Y cells to varying degrees, but there was no significant difference among the three groups. 3.5 Study on the in vivo targeting ability of OX26 modified NLC 3.5.1 Study on the distribution of OX26-DiR-NLC in nude mice As shown in Figure 6, no obvious fluorescence signals were detected in the brain regions (head and neck) of the DiR-sol group and DiR-NLC group during the whole imaging process. However, in the OX26-DiR-NLC group, the fluorescence signal was clearly seen at 4 h and 24 h, suggesting that OX26-conjugated DiR-NLC is promoted through the BBB by receptor-mediated endocytosis. The figure also shows that DiR solutions injected intravenously into living animals are quickly eliminated in vivo. 3.5.2 Study on the distribution of OX26-FITC and OX26-FITC-NLC in the brain According to the Longa’s rule, the MCAO modeled rat rotated to the opposite side, and tilted to the opposite side when walking, that indicated the ischemia was successfully developed. FITC-labeled OX26 and OX26-FITC-modified BA/Sal B-NLC were intravenously injected into normal rats and cerebral ischemia model rats. The rats were sacrificed after 2 h followed by brain perfusion, and frozen sections were made and the hippocampal areas of the rats were observed under a fluorescence microscope. 23

As shown in Figure 7, FITC fluorescence was detected in rat brain sections of OX26FITC and OX26-FITC-BA/Sal B-NLC, and the fluorescence intensity was comparable, indicating the access of OX26 and NLC into the brain. 3.6 Brain pharmacokinetic study 3.6.1 UPLC-MS/MS method validation for BA and Sal B determination Typical chromatograms of blank dialysate, reference solution and brain microdialysis sample from tested rats are shown in Supplementary Figure S2. No endogenous interference was observed at the retention time of the analytes in blank dialysate, which proved the assay specificity. Regression equations, linear ranges and correlation coefficients are listed in the Supplementary Table S1. All calibration curves showed good linearity with correlation coefficients more than 0.999 8, and the accuracy of each point was within the range of 80 %~120 %. The intra-day precision of BA and Sal B are shown in Supplementary Table S2, the RSD of the three components were less than 10.0 %, suggesting that precision meets requirements. The results showed that BA had good stability within 8 h, and Sal B had good stability within 4 h (Supplementary Table S3). Therefore, in the process of injection, the sample should be placed in the autosampler and analyzed within 4 h to ensure the accuracy of the measurement. The in vivo recovery of the probe was calculated by reverse dialysis method, and Cd-Cp was plotted against Cp according to the definition of relative recovery (R), and the slope 24

of the obtained straight line was the recovery rate (R) of the probe. Where, Cp is the concentration of the drug in the perfusate, and Cd is the concentration of the drug in the dialysate (Shi et al., 2007). The regression equation, correlation coefficient and recovery rate of BA were found to be Y=-0.3968X+3.0856, 0.999 4 and 39.68 %, respectively. The regression equation, correlation coefficient and recovery rate of Sal B were found to be Y=-0.3351X-1.7254, 0.996 3 and 33.51 %, respectively. 3.6.2 Verification of probe insertion location As can be seen from Supplementary Figure S3, the probe insertion position was the rat hippocampal region, and the data can be used for pharmacokinetic analysis. 3.6.3 Brain pharmacokinetic study The peak area values of each group of samples were substituted into the standard curve equations to calculate the drug concentration. OriginPro 8.6 was used to draw the concentration-time curve of the drug, and the concentration-time curve of BA and Sal B are shown in Figure 8. The pharmacokinetic parameters were calculated by Winnolin 7.5 pharmacokinetic software, and the pharmacokinetic parameters of each group are shown in Table 2 and 3. According to the results, the Cmax and AUCall of BA in OX26BA /Sal B-NLC group were 60.62 ± 49.44 ng/mL and 209.25 ± 103.53 h·ng/mL, respectively. The Cmax and AUCall of BA in BA/Sal B-NLC group were 26.98 ± 21.75 ng/mL and 106.21 ± 48.60 h·ng/mL, respectively. The Cmax and AUCall of BA in solution group were 20.26 ± 15.76 ng/mL and 104.32 ± 28.76 h·ng/mL, respectively. The Cmax and AUCall of the OX26-BA /Sal B-NLC group were 2.99 times (P<0.05) and 2.00 times (P<0.05) higher than solution group, respectively, and 2.25 times and 1.97 25

times of the BA/Sal B-NLC group (P<0.05), indicating that the OX26-modified preparation group could significantly increase the content of BA in the brain. In the solution group and the unmodified group, Sal B could only be detected at individual time points, while the OX26-modified BA/Sal B-NLC could be detected within 4 h, but hardly at the later time point. Table 2. The pharmacokinetic parameters of BA in brain dialysate after intravenous injection (n=6)

Group

t1/2/h

Cmax

AUCall/

(ng/mL)

(h·ng/mL)

tmax/h

BA/Sal B-Sol

18.77 ± 5.96

1.92 ± 1.66

20.26 ± 15.76

104.32 ± 28.76

BA/Sal B-NLC

29.44 ± 18.83

1.67 ± 1.03

26.98 ± 21.75

106.21 ± 48.60

OX26-BA/Sal B-NLC

12.42 ± 4.34#

2.00 ± 2.28

60.62 ± 49.44*

209.25 ± 103.53*#

Compared with BA/Sal B-Sol, *P<0.05,

**P<0.01;

Compared with BA/Sal B-NLC, #P<0.05,

##P<0.01.

Table 3. The pharmacokinetic parameters of Sal B in brain dialysate after intravenous injection (n=6)

Group

OX26-BA/Sal B-NLC

t1/2/h

1.24 ± 0.65

Cmax

AUCall/

(ng/mL)

(h·ng/mL)

53.05 ± 44.61

138.63 ± 209.80

tmax/h

1.00 ± 0.55

Generally, BA crosses the gastrointestinal tract and BBB mainly in the form of aglycone BE after desugarization, and its metabolites are mostly derivatives of BE (Tarrago et al., 2008). However, relevant studies have also shown that BA can be directly detected 26

in cerebrospinal fluid and dialysis fluid via BBB after intravenous administration (Huang et al., 2008). In addition, the results of cell transmembrane transport experiment show that BA could cross the nerve cell membrane and enter the intracellular space in vitro, but the transporter needed to be involved, which was an active transport. There was no obvious conversion to BE during the transport process, suggesting that BA can directly act on the relevant receptor targets in the form of sugar binding to produce biological effects (Chai et al., 2013; Zhao et al., 2012). However, studies have shown that after intravenous injection of BA, the conversion rate of metabolic conversion to BE is 26.5%; after intravenous injection of BE, the conversion rate of metabolic conversion to BA is 35% (Yang et al., 2017). In this study, the content of BE in brain microdialysis solution was also detected, but a small quantity of BE could be detected only at certain time points, and therefore it could not be used for data analysis. It has been reported that BA is unstable in plasma due to oxidation and endogenous substances, and the addition of antioxidants and 1 mol/L HCl to plasma can improve the stability of BA (Wang et al., 2008). Sal B is a mixture of three molecules of Danshensu and one molecule of caffeic acid. Its chemical structure is unstable, containing unsaturated bonds and phenolic hydroxyl groups, which are weakly acidic. Sal B is stable within pH 1.0 to 6.0, and is most stable between pH 2.5 to 5.0. It is highly degradable when pH ≥ 6.5. The blood is slightly alkaline under normal conditions, and the pH is 7.45±0.05, while Sal B is easily degraded in an alkaline environment (Zhuang et al., 2010). Other studies have shown that blood cells are one of the main factors 27

leading to the rapid degradation of Sal B (Zhang et al., 2013). In the BA/Sal B-Sol and BA/Sal B-NLC groups, Sal B was almost impossible to detect, and some time points could be detected before 4 h, but statistical analysis could not be performed due to the insufficient data. 4. Conclusions In this study, OX26-Sal B/ BA-NLC with small particle size, uniform dispersion, and good stability was prepared, and the drugs were completely released within 24 h in Vcnormal saline and ACSF, and the release was in line with the Weibull model release equation. The fluorescence intensity in cell uptake study indicates that OX26-modified NLC could promote the uptake of drugs by cells through receptor-mediated endocytosis. The distribution of DiR-Sol, DiR-NLC and OX26-DiR-NLC in BALB/c-nude mice showed that the fluorescence signal was clearly observed in brain region at 4 h and 24 h in the OX26-DiR-NLC group. This suggests that OX26 has the potential for improving drug delivery to the brain. The results also show that the DiR solution was quickly eliminated from the body after intravenous injection. The detection of FITC fluorescence in rat brain sections after i.v administration of OX26-FITC and OX26FITC-BA/Sal B-NLC indicates the access of OX26 and NLC in the brain. Moreover, a significantly higher fluorescence intensity of FITC was observed in the hippocampus of the ischemic model than that in normal rats. The results of brain pharmacokinetics study show that OX26 modified BA/Sal B-NLC could significantly increase the content of BA and Sal B in the brain. The Cmax and AUCall of BA were significantly different from those of the solution group and the unmodified group. In the solution group and 28

the unmodified group, Sal B can only be detected at few time points, while BA/Sal BNLC modified by OX26 can be detected within 3 h, suggesting that OX26 can mediate BA/Sal B-NLC across BBB. The preliminary study of in vitro anti-OGD/R injury effect show that BA/Sal B-Sol, BA/Sal B-NLC and OX26-BA/Sal B-NLC 3 groups could protect the damage of SH-SY5Y due to glucose deprivation and reperfusion to a certain extent. Finally, OX26 modified NLC could be a promising approach for targeting drugs, and treating brain diseases. Acknowledgments This work was supported by National Natural Science Foundation of China (81673605). The authors also thank Dongli Qi, Nan Li, Pan Guo, Yuanlu Cui, Xiang Fan, Hong Guo, Hai Wang and Weijie Xie for their kind help. Declaration of Competing Interest The authors report no conflicts of interest in this work.

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Figure captions Figure 1. The release curves of BA and Sal B in vitro. A. BA in Vc-normal saline; B. Sal B in Vcnormal saline; C. BA in ACSF; D. Sal B in ACSF. Figure 2. Cytotoxicity of each group of Cou-6 formulations on bEnd.3 cells. Compared to the control group, *P<0.05, **P<0.01. Figure 3. (A) Fluorescence intensity of different Cou-6-loaded formulation in bEnd.3 cells. (B) Cellular uptake of Cou-6 formulations at different time intervals. Intra-group comparison, compared with 2h, ★P<0.05; Compared to 4 h,

■P<0.05;

Compared to 8 h,

P<0.05; Comparison between groups, compared with Cou-6-Sol, NLC,

▲P<0.05;

◇P<0.05;

Compared to 12 h,

●

compared with Cou-6-

○P<0.05.

Figure 4. Effects of different oxygen glucose deprivation time (A) and the effect of different anoxic agent concentrations on the survival rate of SH-SY5Y cells (B). Compared with the control group, *P<0.05, **P<0.01.

Figure 5. Toxicity of each group of BA/Sal B preparations to SH-SY5Y cells (A) and effects of BA/Sal B preparations on the cells after SH-SY5Y OGD/R injury (B). Compared with the control group, *P<0.05,

**P<0.01;

BA/Sal B-Sol group,

Compared with the model group, #P<0.05,

◇P<0.05, ◇◇P<0.01;

##P<0.01;

Compared with

Compared with BA/Sal B-NLC group,

△P<0.05, △△

P<0.01. Figure 6. The in vivo imaging of the nude mouse after administration of DiR-loaded formulations.A. 35

DiR-Sol; B. DiR-NLC; C. OX26-DiR-NLC Figure 7. Fluorescence observation of FITC in rat hippocampus (×400). A. OX26-FITC-BA/Sal-BNLC normal group; B. OX26-FITC normal group; C. OX26-FITC-BA/Sal-B-NLC model group Figure 8. Concentration-time curve of BA and Sal B in brain dialysate after intravenous injection (n=6). A. BA; B. Sal B.

Graphical abstract

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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