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Apolipoproteins adsorption and brain-targeting evaluation of baicalin nanocrystals modified by combination of Tween80 and TPGS
Accepted Manuscript Title: Apolipoproteins adsorption and brain-targeting evaluation of baicalin nanocrystals modified by combination of Tween80 and TPGS Authors: Yang Liu, Yueqin Ma, Junnan Xu, Yingchong Chen, Jin Xie, Pengfei Yue, Qin Zheng, Ming Yang PII: DOI: Reference:
S0927-7765(17)30649-5 https://doi.org/10.1016/j.colsurfb.2017.10.009 COLSUB 8890
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
7-6-2017 23-8-2017 3-10-2017
Please cite this article as: Yang Liu, Yueqin Ma, Junnan Xu, Yingchong Chen, Jin Xie, Pengfei Yue, Qin Zheng, Ming Yang, Apolipoproteins adsorption and brain-targeting evaluation of baicalin nanocrystals modified by combination of Tween80 and TPGS, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2017.10.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Apolipoproteins adsorption and brain-targeting evaluation of baicalin nanocrystals modified by combination of Tween80 and TPGS Yang Liu 1, #, Yueqin Ma 2,#, Junnan Xu1, Yingchong Chen1, Jin Xie1, Pengfei Yue1,*, Qin Zheng1, Ming Yang1,* 1
Key Lab of Modern Preparation of TCM, Ministry of Education, Jiangxi University
of Traditional Chinese Medicine, Nanchang, China 2 Departments of Pharmacy, 94th Hospital of People’s Liberation Army, Nanchang, China
#
Yang Liu and YueQin Ma contributed to the work equally as joint first authors.
*Corresponding authors at: Key Lab of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, 1688 Meilingdadao Road, Nanchang 330004, China E-mail: [email protected] (P.F. Yue); [email protected](M. Yang) Graphical Abstract
Highlights
A novel formulation strategy of BCL-NS for brain-targeting was designed.
The BCL-NS/TW80+TPGS could adsorb the highest amount of ApoE and least amount of IgG γ and fibrinogen.
The AUC (0-∞) in brain of the BCL-NS/TW80+TPGS was significantly increased.
Combination of TW80 and TPGS was rational choice of nanocrystals for efficient braintargeting.
Abstract To help baicalin pass across BBB and improve its targeting in brain, we designed a novel formulation strategy of baicalin nanocrystals that preferentially adsorbing apolipoprotein E (ApoE) and repelling protein adsorption of opsonins. Intravenous baicalin nanocrystals suspensions (BCL-NS) modified by different surfactant were prepared by high-pressure homogenization. The targeting potential of surfacemodified BCL-NS with mean particles size of about 250nm was assessed by in vitro protein adsorption studies using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), and further evaluated in vivo pharmacokinetics. The protein adsorption results showed that BCL-NS/TPGS, BCL-NS/TW80 and BCL-NS/TPGS+TW80
adsorbed very high amounts of apolipoproteins (ApoA-I, ApoA-Ⅱ, ApoA-IV, ApoCIII, ApoE, ApoJ) and relative low amounts of opsonins (fibrinogen, immunoglobulin heavy chain gamma, immunoglobulin light chain). The pharmacokinetics results demonstrated the AUC (0-∞) in brain of the BCL-NS/TW80+TPGS was 6.67 times as high as that of the BCL solution, and 2.59 times as high as that of the BCL-NS/TW80. It could be attributed to the most ApoE and Apo J adsorption indicative of strong BBB penetration, and least IgG γ and fibrinogen loading minimizing the risk of hepatic uptake. Combination of TW80 and TPGS can be rational choice of surfactants of baicalin nanocrystals for brain-targeting mediated by ApoE adsorption. Keywords: baicalin; nanocrystals; apolipoproteins adsorption; TPGS; brain-targeting 1. Introduction Baicalin (BCL, 7-D-glucuronic acid, 5, 6-dihydroxy flavone, Fig. 1A), a kind of flavonoids, is the main effective component isolated from the root of the traditional Chinese medicinal herb Scutellaria baicalensis Georgi [1, 2], It can protect and improve cerebral ischemia reperfusion and protect nerve and endothelial cells[3,4] , as a potential therapeutic agent for stroke and Alzheimer treatment. BCL can significantly decrease the enzymatic activity of MPO and the expression of iNOS mRNA and COX-2 mRNA in brain, it also significantly inhibited neuronal apoptosis and the expression of cleaved caspase-3 protein after pMCAO[5,6]. And BCL can be distributed in the brain, especially in hippocampus, striatum, cortex and thalamus [7] and has the correlation with dopamine alteration in cerebral nuclei [8]. It is unfortunately that BCL with very poor lipophilicity (logP=-1.42) [9, 10], cannot effectively cross blood-brain-barrier(BBB) and reach an efficacious brain concentration [11-13]. As so far, no formulation has been reported for ehancing the adsorption of BCN in brain. Therefore, there is an urgent need for implementing a new delivery strategy to increase uptake of BCN leading to therapeutic concentrations in the brain.
The BBB is the most important barrier in brain-targeted delivery. Researchers have developed various kinds of strategies to overcome or bypass the BBB, including penetrating through BBB by cellular internalization, opening BBB and intranasal delivery[14]. It is well known that, there are various kinds of transporters or carriers that can mediate the uptake to brain or extrusion from brain of various substances[15, 16]. On the BBB many receptors are overexpressed, including the transferrin (Tf) receptor, insulin receptor, low-density lipoprotein receptor–related protein, ect.[17, 18]. These receptors can specifically bind with corresponding ligands and trigger internalization into cells. Therefore, receptor-mediated transportation is a popular strategy for specifically deliver drug nanocarrier to brain [19]. Nanocrystals suspensions (nanosuspensions, NS) are a liquid sub-micron colloidal dispersion system in the nanoscale (10-1000 nm) with surfactants and/ or polymers as its stabilizers. NS could improve the in vivo fate of poorly soluble drugs [20-23]. Defined coating surfactant of nanocrystals can further be useful for specific blood protein absorbance and improve drug access to the brain. Plasma protein adsorption on drug particles carriers after intravenous administration is generally regarded as the determining factor for the in vivo fate of nanoparticles and microparticles [24]. Opsonins (e.g., immunoglobulin G, complement factors and fibrinogen) can promote phagocytosis by forming a “bridge” between the particles and the phagocyte. Surfactants coatings of nanoparticles that repel protein adsorption have been developed to circumvent the uptake by the mononuclear phagocytic system (MPS) [25, 26]. It is worth mentioning that, polysorbate 80-coated polybutylcyanoacrylate nanoparticles, preferentially adsorbing apolipoprotein E (ApoE) on their surface, showed enhanced delivery of various drugs to the brain[27, 28]. Apolipoprotein E (apoE) is a 34-kDa, 299-amino-acid residue, two-domain protein that mediates the endocytotic uptake and metabolism of triglyceriderich lipoproteins through its highaffinity binding to the low-density lipoprotein receptor (LDLr) and members rich in BBB [29,30]. The protein adsorption might be influenced by the physicochemical properties of nanocrystals, so by suitable surfactant modification the nanocrystals can reach specifically to the brain target.
In this paper, we designed a novel formulation strategy of baicain nanocrystals that preferentially adsorbing apolipoprotein E (ApoE) as liagands of LDLr, and repelling protein adsorption of opsonins from blood. As showed in Fig.1B, the objectives of this study were as follows: (1) Baicalin nanocrystals suspensions (BCL-NS) were prepared by high pressure homogenization technique. The surface of baicalin nanocrystals was modified by using TW80, P188, P407, RH40 and TPGS. The surface-modified BCL-NS were evaluated their physicochemical characterizations. (2) All BCL-NS formulations were compared and evaluated regarding their protein adsorption characterization. To determine the interactions of baicalin nanocrystals with proteins, in vitro protein adsorption studies in plasma were investigated by using 2-D PAGE. (3) After intravenous administration of the surface-modified BCL-NS, the pharmacokinetics evaluation of baicalin in brain and blood tissue was performed to evaluate their brain-targeting potential in vivo. 2. Materials and methods 2.1. Materials Baicalin were purchased from Zelang Co. (Nanjing, China). Polysorbate 80 (Tween80, SHANHE, China) and polyoxyethylene castor oil (Cremophor RH40, YUNHONG, China) were commercially obtained. Poloxamer 188 (P188) and Poloxamer407 (P407) were kindly donated by BASF (Ludwigshafen, Germany). Dtocopherol acid polyethylene glycol 1000 succinic acid (TPGS) was purchased from Xi’an Healthful Biotechnology (Xi’an, People’s Republic of China). Dithioerythritol (DTE), iodoacetamide and Two-dimensional gel electrophoresis standard protein was commercially obtained. IPG buffer, IPG BlueStrips, CHAPS, urea, acrylamide, BisAcrylamide, glycine and Tris were purchased by GE healthcare (Beijing, China). Human plasma was purchased by Beijing Jorferin Bio-Technology Co., Ltd (Beijing, China). Male wistar rats (200±20 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd (Hunan, China). All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the
National Research Council. The protocol was approved by the Institutional Animal Care and Use Committee of Jiangxi University of Traditional Chinese Medicine. 2.2. Production of BCL-NS BCL-NS was prepared by high pressure homogenization. BCL coarse powder (1%, w/v) was firstly dispersed in different surfactant solution (TW80, P188, P407, and TPGS), respectively. The obtained mixture was disintegrated into coarse suspension by a high shear homogenizer (FLUKO® FA25, Essen, Germany) at 19,000 rpm for 2 min. And the coarse suspension was further passed through a piston-gap high pressure homogenizer (AH-1000D, ATS Engineering Inc., Seeker, Canada) at high pressure of 1200 bar for 30 cycles. Then different surface-modified BCL-NS was obtained and further characterized. 2.3. Particle size measurements of BCL-NS The particles size assay was performed on a Mastersizer Micro Plus (Malvern Instruments Limited, Worcestershire, UK). Analysis of the diffraction patterns was done using the Mie model (dispersant refractive index = 1.33, real particle refractive index = 1.670, imaginary particle refractive index = 0.1). From the resulting volume distributions, the median D50 (50% volume percentile), and the average 10% and 90% volume percentile D10 and D90 were determined, as well as the average span of the distribution was calculated: Span=
( D 9 0 -D 1 0 ) D 50
2.4. Zeta potential assay of BCL-NS Zeta potential (ZP) of BCL-NS was determined by using the Zetasizer nano-ZS (Malvern Instruments, UK). The ZP was determined 3 times for each drug and averages and standard deviations were calculated. 2.5. Transmission electron microscope (TEM) of BCL-NS
The morphology of BCL-NSs was observed using TEM (JEM-1200EX, Japan). One drop of BCL suspension was placed on a copper grid. The grid was allowed to dry at room temperature and was examined with the electron microscope. 2.6. In vitro protein adsorption study of BCN-NS The sample was prepared according to the previous report [31, 32]. Briefly, the BCL-NS was incubated in citrate-stabilized human plasma with a volume ration of 1:5 at 37 ± 2°C for 5 minutes. The BCL-NS from excess plasma was separated by centrifugation at 18,000g for 1 h at room temperature (20 ± 2°C). The separated BCLNS was washed three times with 0.05 M (pH 7.4) phosphate buffer, and subsequent centrifuged. Then the separated NS was added into 10 μl of solution containing 10% w/v sodium dodecyl sulfate (SDS) and 2.32% w/v dithioerythritol (DTE) and incubated for 10min in order to desorb the adsorbed proteins from the BCL-NS surface, and the protein lysate containing 4 % w/v CHAPS, urea (8 M), DTE (65 mM) and tris (40 mM) was added. Then the adsorbed protein solutions were obtained and further subjected to the first dimension of the gel electrophoresis. The 2D-PAGE analysis was performed in the first dimension using Multiphor II (GE Healthcare, Germany) on 18 cm IPG BlueStrips with a nonlinear immobilized pH gradient from 3 to10 (J&K Scientific Ltd., China). For the second dimension step, the crosslinked acrylamide gels (linear gradient of 8–16%) were performed using bio-rad PROTEAN II xi Cell (Bio-Rad laboratories, Germany). And the gels were silver stained and scanned using a laser densitometer. The adsorbed proteins were indentified using the SWISS-2DPAGE (ExPASy, Proteomics Server). The spots on gels were recognized by matching the master map of human plasma. The amount of adsorbed protein was analyzed using a semi-quantitative manner based on the spot size and intensity on the gel [33]. The quantitative analysis for the adsorbed protein was described as a percentage (%) of the overall detected amount of protein on the gel.
2.7. Pharmacokinetics study of baicalin in brain and blood after intravenous administration of BCL-NS The pharmacokinetics of baicalin in brain, blood and liver tissue studies were performed to evaluate their brain-targeting potential in rats, after intravenous administration of the surface-modified BCL-NS. The rats were fasted for 12 h prior to the study. One hundred fifty wistar rats with an average weight of 200 ± 20 g were divided randomly into three groups: BCL (BCL saline solution), BCL-NS/TW80 and BCL-NS/TW80+TGPS. All the groups were intravenously administered via tail vein at a single oral dose of 20mg·kg-1. Rats were anesthetized by 1% pentobarbital sodium at 5, 10, 15, 30, 60, 120, 240, 360, 480 and 720min after administration (n=5). Blood samples were collected into heparinised tubes by retro-orbital puncture at predetermined time intervals and centrifuged at 4000 r·min−1 for 15 min at 4℃. The separated plasma was then collected and stored at -80℃ until analysis. After that, the rat hearts were perused with normal saline to exclude any influence of the blood on the results form brain samples, and rat brain and liver samples were subsequently obtained and frozen at -80℃ until analysis. The samples on brain, blood and liver tissue were treated according to the previous method[34, 35], and a validated HPLC/MS/MS system was used to determine the BCL plasma concentrations in brain, blood and liver tissue. Carbamazepine was used as internal standard (IS). An Agilent HPLC system (Agilent 1200, Wilmington, DE) was used. Chromatographic separations were achieved using an Agilent Zorbax SB-C18 column (200 mm × 4.6 mm, 5 mm, Agilent Co. Ltd.) maintained at 35℃. Mobile phase consisted of acetonitrile and 0.1% formic acid (45:55). The constant flow rate was 1mL·min-1. A Quattro Premier XE triple quadrupole mass spectrometer (API3200, AB, Framingham, MA) was interfaced via an electrospray ionization (ESI) source, operating in the negative ion mode with multiple reaction monitoring (MRM). The mass spectrometer was operated in the negative mode of [M−1]− ion at m/z 445/269 for BCL and positive mode of [M+1]+ ion at m/z 237/194 for carbamazepine.
2.8. Statistical analysis The main pharmacokinetics parameters were processed and obtained by DAS 2.0 software (Mathematical Pharmacology Professional Committee of China). ANOVA analysis was carried out using IBM SPSS Statistics 21 software. Differences were considered statistically significant if the p-value was less than 0.05. 2.9. Brain-targeting evaluation Re
( AUC
B
) NS ( AUC
Where Re represents the relative tissue exposure, BCL-NS in tissue,
( AUC
B
)S
B
)S
( AUC
B
) NS
represents the AUC of
represents the AUC of BCL solution in tissue. Re > 1
indicates the brain-targeting ability is good. Te
Where
Te
AUC
T
AUC
NT
represents the targeting efficiency, AUCT represents the AUC of BCL-
NS in target tissue (brain), AUCNT represents the AUC of BCL-NS in non-target tissue (blood). A high Te value suggests efficient (brain) targeting. 3. Results and Discussion 3.1. The particle size and Zeta potential of the BCL-NS According to our previously reported work [36], the intravenous baicalin nanocrystals suspension modified by TW80, RH40, TPGS, P188 and P407 was prepared, respectively. Average 50% volume percentiles (D50), the average span values and Zeta potential (mV) of BCL-NS modified by different surfactants (%, relative weight to drug) were listed in Table 1. The mean particle size of the freshly prepared BCL-NS/TPGS, BCL-NS/TW80, BCL-NS/RH40, BCL-NS/P188, BCLNS/P407 and BCL-NS/TPGS+TW80 was on the range 200-500 nm. And the span for BCL-NS was also 1.314, 1.217, 1.121, 1.243, 1.127 and 1.152, respectively. The morphology images of BCL coarse suspension and surface modified BCL-NSs were
showed in Fig.2. The coarse BCL in suspensions was large needle-shaped crystals (Fig2A), but the BCL nanocrystals in BCL-NSs were near sphere-shaped(Fig2B~G). These results showed that the coarse BCL had been completely disintegrated into nano-sized particles by means of high pressure homogenization technology. The Zeta potential of the BCL-NS was -31.2 mV for BCL-NS/TPGS, -30.2 mV for BCLNS/RH40, -29.5 mV for BCL-NS/TW80, -26.7 mV for BCL-NS/ P188, -27.4 mV for BCL-NS/P407 and -31.5 mV for BCL-NS/TW80+TPGS, respectively. These results indicated that the BCL-NS modified by different surfactants were relatively stable [21, 23]. 3.2. In vitro protein adsorption of BCL-NS The surfactant modification played an important role on the proteins adsorption behavior of different BCL-NS surfaces. The relative amounts of identified proteins on six surface-modified BCL-NSs (BCL-NS/TPGS, BCL-NS/TW80, BCL-NS/RH40, BCL-NS/P188, BCL-NS/P407 and BCL-NS/TW80+TPGS) were showed in Fig.3. The protein adsorption data showed that BCL-NS/TPGS, BCL-NS/TW80 and BCLNS/TPGS+TW80 adsorbed very high amounts of apolipoproteins (ApoA-I, ApoA-Ⅱ, ApoA-IV, ApoC-III, ApoE, ApoJ) and relative low amounts of opsonins (fibrinogen, immunoglobulin heavy chain gamma, immunoglobulin light chain). BCL-NS/TW80 adsorbed very high amount of ApoE(5.4%) and ApoJ(6.4%). It was the reason that the steric effects and hydrophobic effects from surfactants could drastically influence the adsorption behaviors of apolipoproteins and fibrinogens [26, 27]. BCL-NS/TPGS adsorbed the little amount of fibrinogen (2.6%) and IgG γ(9.2%). It was attributed that TPGS had the PEG1000 chains (Fig.1C). TPGS as a PEGylated stabilizer could provide hydrophilic protective layer which could repel the absorption of opsonin proteins (fibrinogen and IgG γ) via steric repulsion forces [37]. Fig.3 also illustrated that BCL-NS/RH40, BCL-NS/P188 and BCL-NS/P407 adsorbed high amount of fibrinogen, immunoglobulin heavy chain gamma and immunoglobulin light chain, but low amount of ApoA-I, ApoA-Ⅱ, ApoA-IV, ApoCIII and ApoJ, and the amounts of protein adsorbed in BCL-NS/P188 were
significantly higher than those in BCL-NS/P407. As illustrated in Fig.1C, it was the reason that the difference of protein amount of nanocrystals was related with hydrophilic polyethylene oxide (PEO) chains or hydrophobic polypropylene (PPO) chains of polymer [38, 39]. It was concluded that the behaviors of adsorbed proteins on different BCL-NS were physical adsorption and influenced by the surface properties of BCL nanocrystals, such as size of nanocrystals (the curvature effects), the chain length and density of surfactant (the steric effects), hydrophobicity/hydrophilicity of surface (the hydrophobic interactions)[25,26]. Among six different BCL-NS, the BCL-NS/TW80+TPGS adsorbed the highest amount of ApoE(6.7%) and ApoJ(7.9%), but the least amount of fibrinogen(0.1%) and Ig γ(6.4%), which was indicative of the strong BBB penetration and minimizing the risk of hepatic uptake. Therefore, BCL-NS/TW80+TPGS was selected for further in vivo testing. 3.3. Pharmacokinetics studies of BCL-NS/TW80+TPGS Pharmacokinetic studies of BCL-NS/TW80+TPGS were performed in order to assess its potential for brain targeting, compared with BCL solution and BCLNS/TW80. The brain concentration versus time curves and pharmacokinetic parameters in brain of the BCL solution, BCL-NS/TW80 and BCL-NS/TPGS was given in Fig. 4A and Table 2, respectively. It can be seen that BCL-NS/TW80+TPGS possessed a Tmax 0.17h and MRT of 4.36 h, and the BCL-solution had a Tmax of 0.083 h and MRT 3.24 h. This indicated that the MRT and Tmax of BCL-NS/TW80+TPGS were not significantly different with those of BCL-NS/TPGS and BCL-TW80 (p>0.05). Table 2 also showed that the Tmax and MRT of BCL-NS/TW80 were 0.17h and 3.22 h, respectively. The MRT and Tmax of BCL-NS/TW80 were not significantly different with those of BCL solution (p>0.05). The mean peak concentration of the BCL-NS/TW80+TPGS (479.74±65.72µg·g-1) was significantly increased (p<0.05), compared with those of BCL-NS/TW80 (241.4±33.67µg·g-1), BCL-NS/TPGS (154.56±34.59µg·g-1) and BCL solution (116.93±44.34µg·g-1). The AUC(0-∞) in brain of BCL solution was 190.47±37.54µg·g-1·h, which indicated that the bioavailability of
BCL in brain is very poor due to the presence of BBB[34, 35]. However, the AUC(0-∞) in brain of BCL-NS/TW80 (300.91±58.16µg·g-1·h) was significantly larger than that of BCL solution(p<0.05). But the AUC(0-∞) in brain of BCL-NS/TPGS (224.52±36.74µg·g-1·h) was not significant different with that of BCL solution(p>0.05). And the AUC (0-∞) in brain of BCL-NS/TW80+TPGS (779.92±102.24µg·g-1·h) was significantly improved, compared with those of BCLNS/TW80 (300.91±58.16µg·g-1·h), BCL-NS/TPGS and BCL solution (p<0.05). The AUC (0-∞) in brain of the BCL-NS/TPGS+TW80 was 6.67 times as high as that of the BCL solution, and 2.59 times as high as that of the BCL-NS/TW80. These results demonstrated that the BCL-NS modified by TW80 could significantly improve the bioavailability of BCL in brain. Fig. 4B and Table 2 showed that the plasma concentration versus time curves and pharmacokinetic parameters in blood of BCL four formulations. It could be seen that the Cmax (1033.45±23.48µg·L-1) and MRT (1.77±0.72 h) of BCL-NS/TW80+TPGS was not significantly different with those (1116.94±52.12 µg·L-1 and MRT 1.22±0.57h) of BCL-solution (p>0.05). But the MRT (2.34±0.27h) of BCL-NS/TPGS was significantly prolonged, compared with those of other formulations (p<0.05). The Cmax of BCL-NS/TW80 were significantly different with those of BCL solution, BCLNS/TPGS and BCL-NS/TW80+TPGS (p<0.05). The Cmax of the BCL-NS/TW80 (803.2±27.52µg·L-1) was significantly decreased (p <0.05), compared with those of BCL-NS/TW80+TPGS, BCL-NS/TPGS and BCL solution. The AUC(0-∞) in blood of BCL solution (707.48±34.61µg·L-1·h) was much larger than that of BCL-NS/TW80 (p<0.05). The AUC (0-∞) in blood of BCL-NS/TW80 (435.26 ± 52.37µg·L-1·h) was significantly decreased, compared with those of BCL-NS/TPGS and BCLNS/TW80+TPGS (p<0.05). It was concluded that in vivo distribution of BCL-NS/TW80 and BCLNS/TW80+TPGS was determined by the protein adsorption. In general, binding of opsonins (e.g., fibrinogen, immunoglobulin heavy chain gamma、immunoglobulin light chain) can promote phagocytosis and removal of the drug carriers from the
systemic circulation by cells of MPS [40, 41]. On the contrary, binding of dysopsonins (e.g., apolipoproteins family ApoA-Ⅱ, ApoA-IV, ApoC-III, ApoE, ApoJ) prolongs circulation time of the drug carriers in the blood [41, 42] and mediated uptake of the drug carriers by the LDLr in BBB [43]. The in vitro protein adsorption study also provided strong evidence that the BCL-NS/TW80+TPGS adsorbed the higher amount of ApoE and ApoJ than those of BCL-NS/TW80 and BCL-NS/TPGS. BCL-NS/TW80 and BCL-NS/TW80+TPGS adsorbing ApoE could mimic the ApoE coated LDLr-particles leading to their brain-uptake by endocytic processes. Therefore, the Cmax(479.74±65.72µg·g-1) and AUC (779.92±102.24µg·g1
·h) in brain of BCL-NS/TW80+TPGS were significantly larger than those of BCL-
NS/TW80 and BCL-NS/TPGS (Table 2). Meanwhile, the BCL-NS/TW80+TPGS and BCL-NS/TPGS adsorbed less amount of fibrinogen and Ig γ, compared with those of BCL-NS/TW80. Therefore, the MRT and Cmax in blood of BCL-NS modified by TPGS were significantly higher than those of BCL-NS/TW80 (p<0.05). But the AUC(0-∞) in blood of BCL-NS/TW80 (435.26 ± 52.37µg·L-1·h) was significantly lower than those of BCL-NS/TPGS and BCLNS/TW80+TPGS (p<0.05), which could be attributed to phagocytosis and removal of some BCL-NS/TW80 from circulation by MPS. This was also evidenced by the distribution results in liver of BCL solution, BCL-NS/TW80, BCL-NS/TPGS and BCL-NS/TW80+TPGS after intravenous administration (Fig.5A). The concentrations in liver of BCL-NS/TW80 were significantly higher at predetermined times than those of other formulations, which indicated that some BCL-NS/TW80 might be transformed into liver from circulation by MPS and resulted in plasma concentration decrease in blood of BCL-NS/TW80. 3.4. Brain-targeting evaluation The brain-targeting potential of BCL-NS/TW80+TPGS was evaluated by the relative tissue exposure (Re) and the targeting efficiency (Te). The Re and Te of BCLNS/TW80+TPGS was showed in Table 3. The results showed that the Re (4.09) of BCL-NS/TW80+TPGS in brain was much higher compared with those of BCL-
NS/TW80 (1.58) and BCL-NS/TPGS (1.18), and was not easily distributed into liver (Re=0.63). Te (1.28) of BCL-NS/TW80+TPGS was more than that (0.69) of BCLNS/TW80 and BCL-NS/TPGS (0.25). Re(1.58, 1.44) for BCL-NS/TW80 in brain and liver was higher than those (1.18, 0.59) for BCL-NS/TPGS. This related with that BCL-NS/TW80 adsorbed higher amounts of apolipoproteins (ApoA-I, ApoA-Ⅱ, ApoA-IV, ApoC-III, ApoE, ApoJ) and lower amounts opsonins (fibrinogen； immunoglobulin heavy chain gamma, immunoglobulin light chain), compared with BCL-NS/TPGS. It was concluded that the BCL-NS/TW80+TPGS possessed most excellent brain-targeting among of all the BCL-NSs, owing to the most ApoE and Apo J adsorption indicative of strong BBB penetration, and the least IgG γ and fibrinogen loading minimizing the risk of hepatic uptake (as illustrated in Fig.5B). Combination of TW80 and TPGS can be rational choice of surfactants of baicalin nanocrystals. 4. Conclusions Baicalin nanocrystals suspensions modified by different surfactants were successfully prepared by using high pressure homogenization technique. BCLNS/TPGS, BCL-NS/TW80 and BCL-NS/TPGS+TW80 adsorbed very high amounts of apolipoproteins (ApoA-I, ApoA-Ⅱ, ApoA-IV, ApoC-III, ApoE, ApoJ) and relative low amounts opsonins (fibrinogen, immunoglobulin heavy chain gamma, immunoglobulin light chain). The differences of protein adsorption might be attributed to the chain length and density of surfactant (the steric effects) and hydrophobicity/hydrophilicity of surface. BCL-NS/TPGS+TW80 possessed most excellent brain-targeting owing to high amount of ApoE and Apo J adsorption indicative of strong BBB penetration, and low amount of IgG γ and fibrinogen loading minimizing the risk of hepatic uptake. The AUC (0-∞) in brain of the BCLNS/TW80+TPGS was significantly increased compared with those of BCL solution, BCL-NS/TW80 and BCL-NS/TPGS. Therefore, combination of TW80 and TPGS can be rational choice of surfactants of nanocrystals for efficient brain-targeting. However, many challenges still stay in this study. To understand how plasma proteins
adsorb on surface of nanocrystals in vivo, better way to control nanocrystals to the target tissue is the next goal in the future. Acknowledgements The authors would like to acknowledge the financial support from the Scientific Research Foundation for the National Natural Science Foundation of China (No. 81560656, 81760715), Fund of distinguished young scientists of Jiangxi Province (No. 20162BCB23033) and the Natural Science Fund of Jiangxi Province (No. 20161BAB205221). Disclosure The authors declare that there are no conflicts of interest. References: [1] Y.C. Shen, W.F. Chiou, Y.C. Chou, C.F. Chen, Mechanisms in mediating the antiinflammatory effects of baicalin and baicalein in human leukocytes, Eur. J. Pharmacol. 465(2003)171. [2] H. Shi, B. Zhao, W. Xin, Scavenging effects of baicalin on free radicals and its protection on erythrocyte membrane from free radical injury, Biochem. Mol. Biol. Int. 35(1995) 981. [3] Z. Zhang, R. Wu, P. Li, Baicalin administration is effective in positive regulation of twenty-four ischemia/reperfusion- related proteins identified by a proteomic study, Neurochem Int. 54 (2009)488. [4] X.K. Tu, W.Z. Yang, S.H. Shi, C.H. Wang, C.M. Chen, Neuroprotective effect of baicalin in a rat model of permanent focal cerebral ischemia, Neurochem Res. 34 (2009) 1626. [5] D.H. Kim, K.H. Cho, S.K. Moon, Y.S. Kim, D.H. Kim, J.S. Choi, H.Y. Chung, Cytoprotective mechanism of baicalin against endothelial cell damage by peroxynitrite [J], J Pharm Pharmacol. 57(2005)1581.
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[43] B. Dehouck, L. Fenart, M.P. Dehouck, A. Pierce, G. Torpier, R. Cecchelli, A new function for the LDL receptor: Transcytosis of LDL across the blood-brain barrier, The Journal of Cell Biology 138(1997) 877. Figure Caption
Figr-1
Figr-2
Figr-3
Figr-4
Figr-5
Figr-7Figure captions:
Fig.1 The chemical structure of baicalin(A), the schematic image on this study(B) and the chemical structure of five surfactants(C) Fig.2 TEM images of BCL(A), BCL-NS/TPGS(B), BCL-NS/RH40(C), BCLNS/TW80(D), BCL-NS/P188(E), BCL-NS/P407(F) and BCL-NS/ TW80+TPGS(G) Fig.3 Amounts of adsorbed proteins on surface modified BCL-NS ( x
s
, n=3)
Fig.4 The mean brain concentration–time curves (A) and plasma concentrationtime curves (B) of BCL solution, BCL-NS/TW80 and BCL-NS/TW80+TPGS (20mg·kg-1) after intravenous administration ( x
s
, n=5)
Fig.5 (A)The concentration in liver of BCL solution, BCL-NS/TW80 and BCLNS/TPGS (20mg·kg-1) after intravenous administration ( x
s
, n=5),
(B) the schematic image on brain-targeting mechanism of BCL-NS/TW80+TPGS
Table 1 The particles size, span and Zeta potential values (ZP) of six BCL-NS formulations ( x TPGS A
RH40
s
, n=3)
TW80
P407
50%
B
50%
C
50%
D
50%
E F
P188
50% 25%
25%
D50(nm)
Span
ZP(mV)
241.6±2.3
1.341±0.023
-20.2±1.6
232.4±1.2
1.217±0.034
-30.2±1.7
247.5±1.1
1.121±0.022
-29.5±2.4
234.6±0.8
1.243±0.017
-26.7±1.6
231.7±0.7
1.127±0.024
-27.4±2.5
247.2±0.6
1.152±0.034
-23.5±2.2
Table 2 Pharmacokinetic parameters in brain and blood of BCL solution, BCL-NS/TW80 and BCL-NS/TPGS (20mg·kg-1) after intravenous administration ( x Tissu
Paramet
es
ers
Units
s
, n=5)
BCL
BCL-
BCL-
BCL-
solution
NS/TW80
NS/TPGS
NS/TW80+TPGS
MRT
h
3.24±0.57
3.22±0.84
3.72±0.75
4.36±0.72
Tmax
h
0.083±0.012
0.17±0.019
0.17±0.023
0.17±0.012
Cmax
µg·g-1
116.93±44.3
241.4±33.6
154.56±34.5
4
7
9
0.033±0.03
0.049±0.037
479.74±65.72 a,b,c
Brain CL
AUC (0-t)
MRT
L·h -1·kg -1
µg·g-1·h
h
0.052±0.011
2
0.013±0.021
190.47±37.5
300.91±58.
224.52±36.7
779.92±102.24
4
16 a
4
a,b,c
1.22±0.57
1.28±0.84
2.34±0.27a,b, c
1.77±0.72
Blood Tmax
h
0.083±0.021
0.083±0.01 7
0.083±0.018
0.083±0.015
Cmax
CL
µg·L-1
L·h -1·kg 1
AUC（0t）
µg·L-1·h
1116.94±52.
803.2±27.5
1171.25±82.
12
2a,c
13b
0.046±0.01
0.019±0.013
2a
b
0.032±0.009
871.56±166.
608.53±101.12a,b
0.028±0.011
707.48±34.6
435.26±52.
1
37a
a
Statistically significant differences from BCL solution (p<0.05).
b
Statistically significant differences from BCL-NS/TW80 (p<0.05).
c
Statistically significant differences from BCL-NS/TPGS (p<0.05)
41b
1033.45±23.48b
Table 3 Targeting parameters of BLC solution, BCL-NS/TW80, BCL-TPGS and BCL-NS/TW80+TPGS after intravenous administration Re Formulation
Te brain
liver
BCL solution
1
1
0.26
BCL-NS/TW80
1.58
1.14
0.69
BCL-NS/TPGS
1.18
0.59
0.25
BCL-NS/TW80+TPGS
4.09
0.63
1.28
30
S0927-7765(17)30649-5 https://doi.org/10.1016/j.colsurfb.2017.10.009 COLSUB 8890
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
7-6-2017 23-8-2017 3-10-2017
Please cite this article as: Yang Liu, Yueqin Ma, Junnan Xu, Yingchong Chen, Jin Xie, Pengfei Yue, Qin Zheng, Ming Yang, Apolipoproteins adsorption and brain-targeting evaluation of baicalin nanocrystals modified by combination of Tween80 and TPGS, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2017.10.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Apolipoproteins adsorption and brain-targeting evaluation of baicalin nanocrystals modified by combination of Tween80 and TPGS Yang Liu 1, #, Yueqin Ma 2,#, Junnan Xu1, Yingchong Chen1, Jin Xie1, Pengfei Yue1,*, Qin Zheng1, Ming Yang1,* 1
Key Lab of Modern Preparation of TCM, Ministry of Education, Jiangxi University
of Traditional Chinese Medicine, Nanchang, China 2 Departments of Pharmacy, 94th Hospital of People’s Liberation Army, Nanchang, China
#
Yang Liu and YueQin Ma contributed to the work equally as joint first authors.
*Corresponding authors at: Key Lab of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, 1688 Meilingdadao Road, Nanchang 330004, China E-mail: [email protected] (P.F. Yue); [email protected](M. Yang) Graphical Abstract
Highlights
A novel formulation strategy of BCL-NS for brain-targeting was designed.
The BCL-NS/TW80+TPGS could adsorb the highest amount of ApoE and least amount of IgG γ and fibrinogen.
The AUC (0-∞) in brain of the BCL-NS/TW80+TPGS was significantly increased.
Combination of TW80 and TPGS was rational choice of nanocrystals for efficient braintargeting.
Abstract To help baicalin pass across BBB and improve its targeting in brain, we designed a novel formulation strategy of baicalin nanocrystals that preferentially adsorbing apolipoprotein E (ApoE) and repelling protein adsorption of opsonins. Intravenous baicalin nanocrystals suspensions (BCL-NS) modified by different surfactant were prepared by high-pressure homogenization. The targeting potential of surfacemodified BCL-NS with mean particles size of about 250nm was assessed by in vitro protein adsorption studies using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), and further evaluated in vivo pharmacokinetics. The protein adsorption results showed that BCL-NS/TPGS, BCL-NS/TW80 and BCL-NS/TPGS+TW80
adsorbed very high amounts of apolipoproteins (ApoA-I, ApoA-Ⅱ, ApoA-IV, ApoCIII, ApoE, ApoJ) and relative low amounts of opsonins (fibrinogen, immunoglobulin heavy chain gamma, immunoglobulin light chain). The pharmacokinetics results demonstrated the AUC (0-∞) in brain of the BCL-NS/TW80+TPGS was 6.67 times as high as that of the BCL solution, and 2.59 times as high as that of the BCL-NS/TW80. It could be attributed to the most ApoE and Apo J adsorption indicative of strong BBB penetration, and least IgG γ and fibrinogen loading minimizing the risk of hepatic uptake. Combination of TW80 and TPGS can be rational choice of surfactants of baicalin nanocrystals for brain-targeting mediated by ApoE adsorption. Keywords: baicalin; nanocrystals; apolipoproteins adsorption; TPGS; brain-targeting 1. Introduction Baicalin (BCL, 7-D-glucuronic acid, 5, 6-dihydroxy flavone, Fig. 1A), a kind of flavonoids, is the main effective component isolated from the root of the traditional Chinese medicinal herb Scutellaria baicalensis Georgi [1, 2], It can protect and improve cerebral ischemia reperfusion and protect nerve and endothelial cells[3,4] , as a potential therapeutic agent for stroke and Alzheimer treatment. BCL can significantly decrease the enzymatic activity of MPO and the expression of iNOS mRNA and COX-2 mRNA in brain, it also significantly inhibited neuronal apoptosis and the expression of cleaved caspase-3 protein after pMCAO[5,6]. And BCL can be distributed in the brain, especially in hippocampus, striatum, cortex and thalamus [7] and has the correlation with dopamine alteration in cerebral nuclei [8]. It is unfortunately that BCL with very poor lipophilicity (logP=-1.42) [9, 10], cannot effectively cross blood-brain-barrier(BBB) and reach an efficacious brain concentration [11-13]. As so far, no formulation has been reported for ehancing the adsorption of BCN in brain. Therefore, there is an urgent need for implementing a new delivery strategy to increase uptake of BCN leading to therapeutic concentrations in the brain.
The BBB is the most important barrier in brain-targeted delivery. Researchers have developed various kinds of strategies to overcome or bypass the BBB, including penetrating through BBB by cellular internalization, opening BBB and intranasal delivery[14]. It is well known that, there are various kinds of transporters or carriers that can mediate the uptake to brain or extrusion from brain of various substances[15, 16]. On the BBB many receptors are overexpressed, including the transferrin (Tf) receptor, insulin receptor, low-density lipoprotein receptor–related protein, ect.[17, 18]. These receptors can specifically bind with corresponding ligands and trigger internalization into cells. Therefore, receptor-mediated transportation is a popular strategy for specifically deliver drug nanocarrier to brain [19]. Nanocrystals suspensions (nanosuspensions, NS) are a liquid sub-micron colloidal dispersion system in the nanoscale (10-1000 nm) with surfactants and/ or polymers as its stabilizers. NS could improve the in vivo fate of poorly soluble drugs [20-23]. Defined coating surfactant of nanocrystals can further be useful for specific blood protein absorbance and improve drug access to the brain. Plasma protein adsorption on drug particles carriers after intravenous administration is generally regarded as the determining factor for the in vivo fate of nanoparticles and microparticles [24]. Opsonins (e.g., immunoglobulin G, complement factors and fibrinogen) can promote phagocytosis by forming a “bridge” between the particles and the phagocyte. Surfactants coatings of nanoparticles that repel protein adsorption have been developed to circumvent the uptake by the mononuclear phagocytic system (MPS) [25, 26]. It is worth mentioning that, polysorbate 80-coated polybutylcyanoacrylate nanoparticles, preferentially adsorbing apolipoprotein E (ApoE) on their surface, showed enhanced delivery of various drugs to the brain[27, 28]. Apolipoprotein E (apoE) is a 34-kDa, 299-amino-acid residue, two-domain protein that mediates the endocytotic uptake and metabolism of triglyceriderich lipoproteins through its highaffinity binding to the low-density lipoprotein receptor (LDLr) and members rich in BBB [29,30]. The protein adsorption might be influenced by the physicochemical properties of nanocrystals, so by suitable surfactant modification the nanocrystals can reach specifically to the brain target.
In this paper, we designed a novel formulation strategy of baicain nanocrystals that preferentially adsorbing apolipoprotein E (ApoE) as liagands of LDLr, and repelling protein adsorption of opsonins from blood. As showed in Fig.1B, the objectives of this study were as follows: (1) Baicalin nanocrystals suspensions (BCL-NS) were prepared by high pressure homogenization technique. The surface of baicalin nanocrystals was modified by using TW80, P188, P407, RH40 and TPGS. The surface-modified BCL-NS were evaluated their physicochemical characterizations. (2) All BCL-NS formulations were compared and evaluated regarding their protein adsorption characterization. To determine the interactions of baicalin nanocrystals with proteins, in vitro protein adsorption studies in plasma were investigated by using 2-D PAGE. (3) After intravenous administration of the surface-modified BCL-NS, the pharmacokinetics evaluation of baicalin in brain and blood tissue was performed to evaluate their brain-targeting potential in vivo. 2. Materials and methods 2.1. Materials Baicalin were purchased from Zelang Co. (Nanjing, China). Polysorbate 80 (Tween80, SHANHE, China) and polyoxyethylene castor oil (Cremophor RH40, YUNHONG, China) were commercially obtained. Poloxamer 188 (P188) and Poloxamer407 (P407) were kindly donated by BASF (Ludwigshafen, Germany). Dtocopherol acid polyethylene glycol 1000 succinic acid (TPGS) was purchased from Xi’an Healthful Biotechnology (Xi’an, People’s Republic of China). Dithioerythritol (DTE), iodoacetamide and Two-dimensional gel electrophoresis standard protein was commercially obtained. IPG buffer, IPG BlueStrips, CHAPS, urea, acrylamide, BisAcrylamide, glycine and Tris were purchased by GE healthcare (Beijing, China). Human plasma was purchased by Beijing Jorferin Bio-Technology Co., Ltd (Beijing, China). Male wistar rats (200±20 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd (Hunan, China). All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the
National Research Council. The protocol was approved by the Institutional Animal Care and Use Committee of Jiangxi University of Traditional Chinese Medicine. 2.2. Production of BCL-NS BCL-NS was prepared by high pressure homogenization. BCL coarse powder (1%, w/v) was firstly dispersed in different surfactant solution (TW80, P188, P407, and TPGS), respectively. The obtained mixture was disintegrated into coarse suspension by a high shear homogenizer (FLUKO® FA25, Essen, Germany) at 19,000 rpm for 2 min. And the coarse suspension was further passed through a piston-gap high pressure homogenizer (AH-1000D, ATS Engineering Inc., Seeker, Canada) at high pressure of 1200 bar for 30 cycles. Then different surface-modified BCL-NS was obtained and further characterized. 2.3. Particle size measurements of BCL-NS The particles size assay was performed on a Mastersizer Micro Plus (Malvern Instruments Limited, Worcestershire, UK). Analysis of the diffraction patterns was done using the Mie model (dispersant refractive index = 1.33, real particle refractive index = 1.670, imaginary particle refractive index = 0.1). From the resulting volume distributions, the median D50 (50% volume percentile), and the average 10% and 90% volume percentile D10 and D90 were determined, as well as the average span of the distribution was calculated: Span=
( D 9 0 -D 1 0 ) D 50
2.4. Zeta potential assay of BCL-NS Zeta potential (ZP) of BCL-NS was determined by using the Zetasizer nano-ZS (Malvern Instruments, UK). The ZP was determined 3 times for each drug and averages and standard deviations were calculated. 2.5. Transmission electron microscope (TEM) of BCL-NS
The morphology of BCL-NSs was observed using TEM (JEM-1200EX, Japan). One drop of BCL suspension was placed on a copper grid. The grid was allowed to dry at room temperature and was examined with the electron microscope. 2.6. In vitro protein adsorption study of BCN-NS The sample was prepared according to the previous report [31, 32]. Briefly, the BCL-NS was incubated in citrate-stabilized human plasma with a volume ration of 1:5 at 37 ± 2°C for 5 minutes. The BCL-NS from excess plasma was separated by centrifugation at 18,000g for 1 h at room temperature (20 ± 2°C). The separated BCLNS was washed three times with 0.05 M (pH 7.4) phosphate buffer, and subsequent centrifuged. Then the separated NS was added into 10 μl of solution containing 10% w/v sodium dodecyl sulfate (SDS) and 2.32% w/v dithioerythritol (DTE) and incubated for 10min in order to desorb the adsorbed proteins from the BCL-NS surface, and the protein lysate containing 4 % w/v CHAPS, urea (8 M), DTE (65 mM) and tris (40 mM) was added. Then the adsorbed protein solutions were obtained and further subjected to the first dimension of the gel electrophoresis. The 2D-PAGE analysis was performed in the first dimension using Multiphor II (GE Healthcare, Germany) on 18 cm IPG BlueStrips with a nonlinear immobilized pH gradient from 3 to10 (J&K Scientific Ltd., China). For the second dimension step, the crosslinked acrylamide gels (linear gradient of 8–16%) were performed using bio-rad PROTEAN II xi Cell (Bio-Rad laboratories, Germany). And the gels were silver stained and scanned using a laser densitometer. The adsorbed proteins were indentified using the SWISS-2DPAGE (ExPASy, Proteomics Server). The spots on gels were recognized by matching the master map of human plasma. The amount of adsorbed protein was analyzed using a semi-quantitative manner based on the spot size and intensity on the gel [33]. The quantitative analysis for the adsorbed protein was described as a percentage (%) of the overall detected amount of protein on the gel.
2.7. Pharmacokinetics study of baicalin in brain and blood after intravenous administration of BCL-NS The pharmacokinetics of baicalin in brain, blood and liver tissue studies were performed to evaluate their brain-targeting potential in rats, after intravenous administration of the surface-modified BCL-NS. The rats were fasted for 12 h prior to the study. One hundred fifty wistar rats with an average weight of 200 ± 20 g were divided randomly into three groups: BCL (BCL saline solution), BCL-NS/TW80 and BCL-NS/TW80+TGPS. All the groups were intravenously administered via tail vein at a single oral dose of 20mg·kg-1. Rats were anesthetized by 1% pentobarbital sodium at 5, 10, 15, 30, 60, 120, 240, 360, 480 and 720min after administration (n=5). Blood samples were collected into heparinised tubes by retro-orbital puncture at predetermined time intervals and centrifuged at 4000 r·min−1 for 15 min at 4℃. The separated plasma was then collected and stored at -80℃ until analysis. After that, the rat hearts were perused with normal saline to exclude any influence of the blood on the results form brain samples, and rat brain and liver samples were subsequently obtained and frozen at -80℃ until analysis. The samples on brain, blood and liver tissue were treated according to the previous method[34, 35], and a validated HPLC/MS/MS system was used to determine the BCL plasma concentrations in brain, blood and liver tissue. Carbamazepine was used as internal standard (IS). An Agilent HPLC system (Agilent 1200, Wilmington, DE) was used. Chromatographic separations were achieved using an Agilent Zorbax SB-C18 column (200 mm × 4.6 mm, 5 mm, Agilent Co. Ltd.) maintained at 35℃. Mobile phase consisted of acetonitrile and 0.1% formic acid (45:55). The constant flow rate was 1mL·min-1. A Quattro Premier XE triple quadrupole mass spectrometer (API3200, AB, Framingham, MA) was interfaced via an electrospray ionization (ESI) source, operating in the negative ion mode with multiple reaction monitoring (MRM). The mass spectrometer was operated in the negative mode of [M−1]− ion at m/z 445/269 for BCL and positive mode of [M+1]+ ion at m/z 237/194 for carbamazepine.
2.8. Statistical analysis The main pharmacokinetics parameters were processed and obtained by DAS 2.0 software (Mathematical Pharmacology Professional Committee of China). ANOVA analysis was carried out using IBM SPSS Statistics 21 software. Differences were considered statistically significant if the p-value was less than 0.05. 2.9. Brain-targeting evaluation Re
( AUC
B
) NS ( AUC
Where Re represents the relative tissue exposure, BCL-NS in tissue,
( AUC
B
)S
B
)S
( AUC
B
) NS
represents the AUC of
represents the AUC of BCL solution in tissue. Re > 1
indicates the brain-targeting ability is good. Te
Where
Te
AUC
T
AUC
NT
represents the targeting efficiency, AUCT represents the AUC of BCL-
NS in target tissue (brain), AUCNT represents the AUC of BCL-NS in non-target tissue (blood). A high Te value suggests efficient (brain) targeting. 3. Results and Discussion 3.1. The particle size and Zeta potential of the BCL-NS According to our previously reported work [36], the intravenous baicalin nanocrystals suspension modified by TW80, RH40, TPGS, P188 and P407 was prepared, respectively. Average 50% volume percentiles (D50), the average span values and Zeta potential (mV) of BCL-NS modified by different surfactants (%, relative weight to drug) were listed in Table 1. The mean particle size of the freshly prepared BCL-NS/TPGS, BCL-NS/TW80, BCL-NS/RH40, BCL-NS/P188, BCLNS/P407 and BCL-NS/TPGS+TW80 was on the range 200-500 nm. And the span for BCL-NS was also 1.314, 1.217, 1.121, 1.243, 1.127 and 1.152, respectively. The morphology images of BCL coarse suspension and surface modified BCL-NSs were
showed in Fig.2. The coarse BCL in suspensions was large needle-shaped crystals (Fig2A), but the BCL nanocrystals in BCL-NSs were near sphere-shaped(Fig2B~G). These results showed that the coarse BCL had been completely disintegrated into nano-sized particles by means of high pressure homogenization technology. The Zeta potential of the BCL-NS was -31.2 mV for BCL-NS/TPGS, -30.2 mV for BCLNS/RH40, -29.5 mV for BCL-NS/TW80, -26.7 mV for BCL-NS/ P188, -27.4 mV for BCL-NS/P407 and -31.5 mV for BCL-NS/TW80+TPGS, respectively. These results indicated that the BCL-NS modified by different surfactants were relatively stable [21, 23]. 3.2. In vitro protein adsorption of BCL-NS The surfactant modification played an important role on the proteins adsorption behavior of different BCL-NS surfaces. The relative amounts of identified proteins on six surface-modified BCL-NSs (BCL-NS/TPGS, BCL-NS/TW80, BCL-NS/RH40, BCL-NS/P188, BCL-NS/P407 and BCL-NS/TW80+TPGS) were showed in Fig.3. The protein adsorption data showed that BCL-NS/TPGS, BCL-NS/TW80 and BCLNS/TPGS+TW80 adsorbed very high amounts of apolipoproteins (ApoA-I, ApoA-Ⅱ, ApoA-IV, ApoC-III, ApoE, ApoJ) and relative low amounts of opsonins (fibrinogen, immunoglobulin heavy chain gamma, immunoglobulin light chain). BCL-NS/TW80 adsorbed very high amount of ApoE(5.4%) and ApoJ(6.4%). It was the reason that the steric effects and hydrophobic effects from surfactants could drastically influence the adsorption behaviors of apolipoproteins and fibrinogens [26, 27]. BCL-NS/TPGS adsorbed the little amount of fibrinogen (2.6%) and IgG γ(9.2%). It was attributed that TPGS had the PEG1000 chains (Fig.1C). TPGS as a PEGylated stabilizer could provide hydrophilic protective layer which could repel the absorption of opsonin proteins (fibrinogen and IgG γ) via steric repulsion forces [37]. Fig.3 also illustrated that BCL-NS/RH40, BCL-NS/P188 and BCL-NS/P407 adsorbed high amount of fibrinogen, immunoglobulin heavy chain gamma and immunoglobulin light chain, but low amount of ApoA-I, ApoA-Ⅱ, ApoA-IV, ApoCIII and ApoJ, and the amounts of protein adsorbed in BCL-NS/P188 were
significantly higher than those in BCL-NS/P407. As illustrated in Fig.1C, it was the reason that the difference of protein amount of nanocrystals was related with hydrophilic polyethylene oxide (PEO) chains or hydrophobic polypropylene (PPO) chains of polymer [38, 39]. It was concluded that the behaviors of adsorbed proteins on different BCL-NS were physical adsorption and influenced by the surface properties of BCL nanocrystals, such as size of nanocrystals (the curvature effects), the chain length and density of surfactant (the steric effects), hydrophobicity/hydrophilicity of surface (the hydrophobic interactions)[25,26]. Among six different BCL-NS, the BCL-NS/TW80+TPGS adsorbed the highest amount of ApoE(6.7%) and ApoJ(7.9%), but the least amount of fibrinogen(0.1%) and Ig γ(6.4%), which was indicative of the strong BBB penetration and minimizing the risk of hepatic uptake. Therefore, BCL-NS/TW80+TPGS was selected for further in vivo testing. 3.3. Pharmacokinetics studies of BCL-NS/TW80+TPGS Pharmacokinetic studies of BCL-NS/TW80+TPGS were performed in order to assess its potential for brain targeting, compared with BCL solution and BCLNS/TW80. The brain concentration versus time curves and pharmacokinetic parameters in brain of the BCL solution, BCL-NS/TW80 and BCL-NS/TPGS was given in Fig. 4A and Table 2, respectively. It can be seen that BCL-NS/TW80+TPGS possessed a Tmax 0.17h and MRT of 4.36 h, and the BCL-solution had a Tmax of 0.083 h and MRT 3.24 h. This indicated that the MRT and Tmax of BCL-NS/TW80+TPGS were not significantly different with those of BCL-NS/TPGS and BCL-TW80 (p>0.05). Table 2 also showed that the Tmax and MRT of BCL-NS/TW80 were 0.17h and 3.22 h, respectively. The MRT and Tmax of BCL-NS/TW80 were not significantly different with those of BCL solution (p>0.05). The mean peak concentration of the BCL-NS/TW80+TPGS (479.74±65.72µg·g-1) was significantly increased (p<0.05), compared with those of BCL-NS/TW80 (241.4±33.67µg·g-1), BCL-NS/TPGS (154.56±34.59µg·g-1) and BCL solution (116.93±44.34µg·g-1). The AUC(0-∞) in brain of BCL solution was 190.47±37.54µg·g-1·h, which indicated that the bioavailability of
BCL in brain is very poor due to the presence of BBB[34, 35]. However, the AUC(0-∞) in brain of BCL-NS/TW80 (300.91±58.16µg·g-1·h) was significantly larger than that of BCL solution(p<0.05). But the AUC(0-∞) in brain of BCL-NS/TPGS (224.52±36.74µg·g-1·h) was not significant different with that of BCL solution(p>0.05). And the AUC (0-∞) in brain of BCL-NS/TW80+TPGS (779.92±102.24µg·g-1·h) was significantly improved, compared with those of BCLNS/TW80 (300.91±58.16µg·g-1·h), BCL-NS/TPGS and BCL solution (p<0.05). The AUC (0-∞) in brain of the BCL-NS/TPGS+TW80 was 6.67 times as high as that of the BCL solution, and 2.59 times as high as that of the BCL-NS/TW80. These results demonstrated that the BCL-NS modified by TW80 could significantly improve the bioavailability of BCL in brain. Fig. 4B and Table 2 showed that the plasma concentration versus time curves and pharmacokinetic parameters in blood of BCL four formulations. It could be seen that the Cmax (1033.45±23.48µg·L-1) and MRT (1.77±0.72 h) of BCL-NS/TW80+TPGS was not significantly different with those (1116.94±52.12 µg·L-1 and MRT 1.22±0.57h) of BCL-solution (p>0.05). But the MRT (2.34±0.27h) of BCL-NS/TPGS was significantly prolonged, compared with those of other formulations (p<0.05). The Cmax of BCL-NS/TW80 were significantly different with those of BCL solution, BCLNS/TPGS and BCL-NS/TW80+TPGS (p<0.05). The Cmax of the BCL-NS/TW80 (803.2±27.52µg·L-1) was significantly decreased (p <0.05), compared with those of BCL-NS/TW80+TPGS, BCL-NS/TPGS and BCL solution. The AUC(0-∞) in blood of BCL solution (707.48±34.61µg·L-1·h) was much larger than that of BCL-NS/TW80 (p<0.05). The AUC (0-∞) in blood of BCL-NS/TW80 (435.26 ± 52.37µg·L-1·h) was significantly decreased, compared with those of BCL-NS/TPGS and BCLNS/TW80+TPGS (p<0.05). It was concluded that in vivo distribution of BCL-NS/TW80 and BCLNS/TW80+TPGS was determined by the protein adsorption. In general, binding of opsonins (e.g., fibrinogen, immunoglobulin heavy chain gamma、immunoglobulin light chain) can promote phagocytosis and removal of the drug carriers from the
systemic circulation by cells of MPS [40, 41]. On the contrary, binding of dysopsonins (e.g., apolipoproteins family ApoA-Ⅱ, ApoA-IV, ApoC-III, ApoE, ApoJ) prolongs circulation time of the drug carriers in the blood [41, 42] and mediated uptake of the drug carriers by the LDLr in BBB [43]. The in vitro protein adsorption study also provided strong evidence that the BCL-NS/TW80+TPGS adsorbed the higher amount of ApoE and ApoJ than those of BCL-NS/TW80 and BCL-NS/TPGS. BCL-NS/TW80 and BCL-NS/TW80+TPGS adsorbing ApoE could mimic the ApoE coated LDLr-particles leading to their brain-uptake by endocytic processes. Therefore, the Cmax(479.74±65.72µg·g-1) and AUC (779.92±102.24µg·g1
·h) in brain of BCL-NS/TW80+TPGS were significantly larger than those of BCL-
NS/TW80 and BCL-NS/TPGS (Table 2). Meanwhile, the BCL-NS/TW80+TPGS and BCL-NS/TPGS adsorbed less amount of fibrinogen and Ig γ, compared with those of BCL-NS/TW80. Therefore, the MRT and Cmax in blood of BCL-NS modified by TPGS were significantly higher than those of BCL-NS/TW80 (p<0.05). But the AUC(0-∞) in blood of BCL-NS/TW80 (435.26 ± 52.37µg·L-1·h) was significantly lower than those of BCL-NS/TPGS and BCLNS/TW80+TPGS (p<0.05), which could be attributed to phagocytosis and removal of some BCL-NS/TW80 from circulation by MPS. This was also evidenced by the distribution results in liver of BCL solution, BCL-NS/TW80, BCL-NS/TPGS and BCL-NS/TW80+TPGS after intravenous administration (Fig.5A). The concentrations in liver of BCL-NS/TW80 were significantly higher at predetermined times than those of other formulations, which indicated that some BCL-NS/TW80 might be transformed into liver from circulation by MPS and resulted in plasma concentration decrease in blood of BCL-NS/TW80. 3.4. Brain-targeting evaluation The brain-targeting potential of BCL-NS/TW80+TPGS was evaluated by the relative tissue exposure (Re) and the targeting efficiency (Te). The Re and Te of BCLNS/TW80+TPGS was showed in Table 3. The results showed that the Re (4.09) of BCL-NS/TW80+TPGS in brain was much higher compared with those of BCL-
NS/TW80 (1.58) and BCL-NS/TPGS (1.18), and was not easily distributed into liver (Re=0.63). Te (1.28) of BCL-NS/TW80+TPGS was more than that (0.69) of BCLNS/TW80 and BCL-NS/TPGS (0.25). Re(1.58, 1.44) for BCL-NS/TW80 in brain and liver was higher than those (1.18, 0.59) for BCL-NS/TPGS. This related with that BCL-NS/TW80 adsorbed higher amounts of apolipoproteins (ApoA-I, ApoA-Ⅱ, ApoA-IV, ApoC-III, ApoE, ApoJ) and lower amounts opsonins (fibrinogen； immunoglobulin heavy chain gamma, immunoglobulin light chain), compared with BCL-NS/TPGS. It was concluded that the BCL-NS/TW80+TPGS possessed most excellent brain-targeting among of all the BCL-NSs, owing to the most ApoE and Apo J adsorption indicative of strong BBB penetration, and the least IgG γ and fibrinogen loading minimizing the risk of hepatic uptake (as illustrated in Fig.5B). Combination of TW80 and TPGS can be rational choice of surfactants of baicalin nanocrystals. 4. Conclusions Baicalin nanocrystals suspensions modified by different surfactants were successfully prepared by using high pressure homogenization technique. BCLNS/TPGS, BCL-NS/TW80 and BCL-NS/TPGS+TW80 adsorbed very high amounts of apolipoproteins (ApoA-I, ApoA-Ⅱ, ApoA-IV, ApoC-III, ApoE, ApoJ) and relative low amounts opsonins (fibrinogen, immunoglobulin heavy chain gamma, immunoglobulin light chain). The differences of protein adsorption might be attributed to the chain length and density of surfactant (the steric effects) and hydrophobicity/hydrophilicity of surface. BCL-NS/TPGS+TW80 possessed most excellent brain-targeting owing to high amount of ApoE and Apo J adsorption indicative of strong BBB penetration, and low amount of IgG γ and fibrinogen loading minimizing the risk of hepatic uptake. The AUC (0-∞) in brain of the BCLNS/TW80+TPGS was significantly increased compared with those of BCL solution, BCL-NS/TW80 and BCL-NS/TPGS. Therefore, combination of TW80 and TPGS can be rational choice of surfactants of nanocrystals for efficient brain-targeting. However, many challenges still stay in this study. To understand how plasma proteins
adsorb on surface of nanocrystals in vivo, better way to control nanocrystals to the target tissue is the next goal in the future. Acknowledgements The authors would like to acknowledge the financial support from the Scientific Research Foundation for the National Natural Science Foundation of China (No. 81560656, 81760715), Fund of distinguished young scientists of Jiangxi Province (No. 20162BCB23033) and the Natural Science Fund of Jiangxi Province (No. 20161BAB205221). Disclosure The authors declare that there are no conflicts of interest. References: [1] Y.C. Shen, W.F. Chiou, Y.C. Chou, C.F. Chen, Mechanisms in mediating the antiinflammatory effects of baicalin and baicalein in human leukocytes, Eur. J. Pharmacol. 465(2003)171. [2] H. Shi, B. Zhao, W. Xin, Scavenging effects of baicalin on free radicals and its protection on erythrocyte membrane from free radical injury, Biochem. Mol. Biol. Int. 35(1995) 981. [3] Z. Zhang, R. Wu, P. Li, Baicalin administration is effective in positive regulation of twenty-four ischemia/reperfusion- related proteins identified by a proteomic study, Neurochem Int. 54 (2009)488. [4] X.K. Tu, W.Z. Yang, S.H. Shi, C.H. Wang, C.M. Chen, Neuroprotective effect of baicalin in a rat model of permanent focal cerebral ischemia, Neurochem Res. 34 (2009) 1626. [5] D.H. Kim, K.H. Cho, S.K. Moon, Y.S. Kim, D.H. Kim, J.S. Choi, H.Y. Chung, Cytoprotective mechanism of baicalin against endothelial cell damage by peroxynitrite [J], J Pharm Pharmacol. 57(2005)1581.
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[43] B. Dehouck, L. Fenart, M.P. Dehouck, A. Pierce, G. Torpier, R. Cecchelli, A new function for the LDL receptor: Transcytosis of LDL across the blood-brain barrier, The Journal of Cell Biology 138(1997) 877. Figure Caption
Figr-1
Figr-2
Figr-3
Figr-4
Figr-5
Figr-7Figure captions:
Fig.1 The chemical structure of baicalin(A), the schematic image on this study(B) and the chemical structure of five surfactants(C) Fig.2 TEM images of BCL(A), BCL-NS/TPGS(B), BCL-NS/RH40(C), BCLNS/TW80(D), BCL-NS/P188(E), BCL-NS/P407(F) and BCL-NS/ TW80+TPGS(G) Fig.3 Amounts of adsorbed proteins on surface modified BCL-NS ( x
s
, n=3)
Fig.4 The mean brain concentration–time curves (A) and plasma concentrationtime curves (B) of BCL solution, BCL-NS/TW80 and BCL-NS/TW80+TPGS (20mg·kg-1) after intravenous administration ( x
s
, n=5)
Fig.5 (A)The concentration in liver of BCL solution, BCL-NS/TW80 and BCLNS/TPGS (20mg·kg-1) after intravenous administration ( x
s
, n=5),
(B) the schematic image on brain-targeting mechanism of BCL-NS/TW80+TPGS
Table 1 The particles size, span and Zeta potential values (ZP) of six BCL-NS formulations ( x TPGS A
RH40
s
, n=3)
TW80
P407
50%
B
50%
C
50%
D
50%
E F
P188
50% 25%
25%
D50(nm)
Span
ZP(mV)
241.6±2.3
1.341±0.023
-20.2±1.6
232.4±1.2
1.217±0.034
-30.2±1.7
247.5±1.1
1.121±0.022
-29.5±2.4
234.6±0.8
1.243±0.017
-26.7±1.6
231.7±0.7
1.127±0.024
-27.4±2.5
247.2±0.6
1.152±0.034
-23.5±2.2
Table 2 Pharmacokinetic parameters in brain and blood of BCL solution, BCL-NS/TW80 and BCL-NS/TPGS (20mg·kg-1) after intravenous administration ( x Tissu
Paramet
es
ers
Units
s
, n=5)
BCL
BCL-
BCL-
BCL-
solution
NS/TW80
NS/TPGS
NS/TW80+TPGS
MRT
h
3.24±0.57
3.22±0.84
3.72±0.75
4.36±0.72
Tmax
h
0.083±0.012
0.17±0.019
0.17±0.023
0.17±0.012
Cmax
µg·g-1
116.93±44.3
241.4±33.6
154.56±34.5
4
7
9
0.033±0.03
0.049±0.037
479.74±65.72 a,b,c
Brain CL
AUC (0-t)
MRT
L·h -1·kg -1
µg·g-1·h
h
0.052±0.011
2
0.013±0.021
190.47±37.5
300.91±58.
224.52±36.7
779.92±102.24
4
16 a
4
a,b,c
1.22±0.57
1.28±0.84
2.34±0.27a,b, c
1.77±0.72
Blood Tmax
h
0.083±0.021
0.083±0.01 7
0.083±0.018
0.083±0.015
Cmax
CL
µg·L-1
L·h -1·kg 1
AUC（0t）
µg·L-1·h
1116.94±52.
803.2±27.5
1171.25±82.
12
2a,c
13b
0.046±0.01
0.019±0.013
2a
b
0.032±0.009
871.56±166.
608.53±101.12a,b
0.028±0.011
707.48±34.6
435.26±52.
1
37a
a
Statistically significant differences from BCL solution (p<0.05).
b
Statistically significant differences from BCL-NS/TW80 (p<0.05).
c
Statistically significant differences from BCL-NS/TPGS (p<0.05)
41b
1033.45±23.48b
Table 3 Targeting parameters of BLC solution, BCL-NS/TW80, BCL-TPGS and BCL-NS/TW80+TPGS after intravenous administration Re Formulation
Te brain
liver
BCL solution
1
1
0.26
BCL-NS/TW80
1.58
1.14
0.69
BCL-NS/TPGS
1.18
0.59
0.25
BCL-NS/TW80+TPGS
4.09
0.63
1.28
30