Exploring the potential to enhance drug distribution in the brain subregion via intranasal delivery of nanoemulsion in combination with borneol as a guider

Abstract

The number of people with Alzheimer's disease (AD) is increasing annually, with the nidus mainly concentrated in the cortex and hippocampus. Despite of numerous efforts, effective treatment of AD is still facing great challenges due to the blood brain barrier (BBB) and limited drug distribution in the AD nidus sites. Thus, in this study, using vinpocetine (VIN) as a model drug, the objective is to explore the feasibility of tackling the above bottleneck via intranasal drug delivery in combination with a brain guider, borneol (BOR), using nanoemulsion (NE) as the carrier. First of all, the NE were prepared and characterized. In vivo behavior of the NE after intranasal administration was investigated. Influence of BOR dose, BOR administration route on drug brain targeting behavior was evaluated, and the influence of BOR addition on drug brain subregion distribution was probed. It was demonstrated that all the NE had comparable size and similar retention behavior after intranasal delivery. Compared to intravenous injection, improved brain targeting effect was observed by intranasal route, and drug targeting index (DTI) of the VIN—NE group was 154.1%, with the nose-to-brain direct transport percentage (DTP) 35.1%. Especially, remarkably enhanced brain distribution was achieved after BOR addition in the NE, with the extent depending on BOR dose. VIN brain concentration was the highest in the VIN-1-BOR-NE group at BOR dose of 1 mg/kg, with the DTI reaching 596.1% and the DTP increased to 83.1%. BOR could exert better nose to brain delivery when administrated together with the drug via intranasal route. Notably, BOR can remarkably enhance drug distribution in both hippocampus and cortex, the nidus areas of AD. In conclusion, in combination with intranasal delivery and the intrinsic brain guiding effect of BOR, drug distribution not only in the brain but also in the cortex and hippocampus can be enhanced significantly, providing the perquisite for improved therapeutic efficacy of AD.

Graphical abstract

1. Introduction

According to statistics, about 1.5 billion people in the world are suffering from different degree of central nervous system diseases, and they have become major diseases endangering human being's life and health [1]. Among them, Alzheimer's disease (AD) is a more common neurodegenerative disease characterized by memory and cognitive decline, endangering over 46 million people around the world. The number of people suffering from AD increases every year and is likely to triple in the next 40 years [2]. The pathogenesis of AD is complicated. The nidus of AD is mainly concentrated in the cortex and hippocampus, which are responsible for learning, memory and cognition [3]. Despite of numerous efforts, effective treatment of AD is still facing great challenges. On one hand, the penetration of drugs into the central nervous system is restricted by the blood brain barrier (BBB), on the other hand, even for the drugs entering the brain, a random, non-specific brain distribution may lead to drug concentration in the AD nidus sites, the cortex and hippocampus, under the therapeutic level [4]. Therefore, new strategies should be explored to overcome the above limitations for better AD therapy.

Anatomically, there are unique olfactory and trigeminal neural pathway between the nasal cavity and the cranial cavity, which makes intranasal drug delivery an effective way for brain targeting [5]. Compared with other routes of administration, intranasal drug delivery is a non-invasive way to achieve intracerebral delivery of drugs with high safety [6], a rapid onset of action and good patients convenience [7]. Studies have demonstrated that it can increase drug distribution in the brain for better central nervous diseases therapy. For example, compared with the intravenous injection solution group, after nasal administration of risperidone solid lipid nanoparticles, the drug AUC_brain value increased by nearly 10 times [8].

Moreover, to enhance drug brain targeting after intranasal delivery, the selection of appropriate drug carrier is of special importance, especially for poorly soluble drugs. Contributed to its excellent solubilizing ability, high drug dispersion, easy to be absorbed with simple preparation process, nanoemulsion has been extensively studied to enhance drug delivery from nose to the brain [9]. Additionally, it has been demonstrated that nanoemulsion may inhibit P-gp efflux pump [10], extend drug residence time in the nasal mucosa, facilitate drug penetration through the nasal epithelia, promote drug influx from nose to brain [9], thus reducing the therapeutic dose and avoiding or reducing peripheral toxicity of drugs [11]. Therefore, nanoemulsion is a promising carrier for brain targeting via intranasal route.

In addition, since the niduses of AD are mainly concentrated in cortex and hippocampus [3], therefore for AD therapy, just increasing drug concentration in the brain is not sufficient. After brain targeting being achieved, specific brain subregion targeting to cortex and hippocampus is highly desired for efficient AD therapy. Fortunately, borneol (BOR), a traditional Chinese medicine, might be a good candidate, which can not only increase drug transport efficiency into the brain [12,13], but also enhance drug distribution to brain subregions [14]. Especially, previous studies have demonstrated that by taking BOR orally, doxorubicin distribution in cortex and hippocampus was selectively increased after intravenous injection of doxorubicin liposome [15]. Thus, in combination with the brain targeting characteristics of intranasal drug delivery, the intrinsic brain guiding effect of BOR, and the subregional distribution tendency of BOR, we assume that drug distribution not only in the brain but also in the cortex and hippocampus can be enhanced significantly after intranasal delivery, which provides a perquisite for improved therapeutic efficacy of AD. However, to the best of our knowledge, no such related research is available yet, it is not clear whether and at which dose BOR can improve drug reginal distribution in the brain.

Vinpocetine (VIN), a phosphodiesterase inhibitor (PDEIs), can be used clinically to treat AD, vascular dementia and other dementia, improve and restore memory [16]. However, its metabolism in the liver is up to 75%, with oral bioavailability only 6.7%, with insufficient drug concentration in the brain, limiting its application in clinic [17]. Therefore, using VIN as a model drug, the objective of this study is to inquire into whether VIN combination with BOR can increase VIN delivery to the brain more efficiently after intranasal administration, and the potential influence on VIN distribution in the nidus sites of AD, such as cortex and hippocampus. First of all, VIN and BOR were used in combination to prepare nanoemulsion. The nanoemulsion was characterized for particle size, drug content, and morphology. In vivo behavior of the nanoemulsion after intranasal administration was investigated. Influence of BOR dose, BOR administration route on drug brain targeting behavior was evaluated, and the influence of BOR addition on drug brain subregion distribution was further explored.

2. Materials and methods

2.1. Materials

Vinpocetine (VIN) was purchased from Haide Pharmacy Ltd. (Liaoning, China). Borneol (BOR) was from Aladdin Biochemical Technology Ltd. (Shanghai, China). Diethyleneglycol monoethyl ether (Transcutol P) was provided by Gattefosse (France). Oleic acid (OA) was from Yongda Chemical Reagent Ltd. (Tianjin, China). Polyethylene glycol lithium dodecyl stearate (HS15) was provided by BASF (Ludwigshafen, Germany). Progesterone (PRG) was from Renfu Pharmaceutical Ltd. (Hubei, China). DiR was from Dalian Meilun Biotechnology Ltd. (Liaoning, China). All the other reagents were of analytical or HPLC grade.

Sprague-Dawley (SD) rats (male, 220 ± 20 g) were provided by the Laboratory Animal Center of Shenyang Pharmaceutical University. All the animal experiments were approved by the Ethics Committee of Shenyang Pharmaceutical University and followed the Principle of Laboratory Animal Care.

2.2. Preparation and characterization of the nanoemulsions

The nanoemulsions were prepared by ultrasonic emulsification method. Briefly, specific amount of OA, HS15, and diethylene glycol monoethyl ether (Transcutol P) were weighed in a ratio of 1:3:3, and placed in a vial, stirred at room temperature to mix evenly, thereafter 0.6% VIN and a certain amount of BOR were added to the vial, keep stirring for 30 min to completely dissolve VIN and BOR in the mixture, and specific amount of aqueous solution was added dropwise under magnetic agitation. Then, the mixture was ultrasonized at 300 W, and a series of VIN nanoemulsions containing different doses of BOR were obtained.

DiR labelled nanoemulsion was prepared by dissolving DiR together with VIN and BOR in the oil phase, all the other procedures were exactly the same as described above and the content of DiR in the preparation was 0.05%.

The mean particle size and size distribution of the nanoemulsions were measured by using Malvern Zetasizer Nano ZS90 (Malvern Instruments, UK) at 25 °C and a scattering angle of 90^., based on the principle of photon correlation spectroscopy (PCS). Before measurement, the nanoemulsion samples were diluted 20 times with distilled water. The size distribution was obtained from the correlation function via the CONTIN algorithm.

The content of VIN in the nanoemulsions was measured using HPLC method (Agilent 1100 HPLC, USA) after breaking the nanoemulsions in methanol at a ratio of 1:99 (v/v), under the following conditions: Diamonsil C₁₈ chromatographic column (4.6 mm × 200 mm, 5 μm) was used, the detection wavelength was set to 273 nm, the mobile phase was a mixture of methanol:water (80:20, v/v), and the flow rate was 1.0 ml/min.

Similarly, after the nanoemulsions being broken with ethyl acetate at a ratio of 1:4 (v/v), vortexed for 5 min and centrifuged at 10000 r/min for 10 min, the BOR content in the supernatant was measured by GC method (Agilent 7890B GC, USA), with quartz capillary column using 5% diphenyl-95% dimethyl polysiloxane as stationary phase (0.25 mm × 30 m, 0.25 μm). The carrier gas was N₂, and flow rate was 1 ml/min.

Morphology of the nanoemulsions was observed using transmission electron microscopy (TEM, JEM-1200EX, Japan). Negative staining of the samples was performed as follows: the nanoemulsions were diluted 50 times, 10 μl the diluted preparation was dropped onto a copper grid (300 mesh) coated with carbon, after air-drying, the samples were stained with 10 μl 2% phosphotungstic acid for 1 min and dried at room temperature, then the samples were observed under TEM at an accelerated voltage of 120 kV.

2.3. Intranasal retention of the nanoemulsions

Intranasal retention study of the nanoemulsions was carried out in male SD rats (220 ± 20 g). The SD rats were randomly divided into five groups, the dose of DiR in each group was given at 0.125 mg/kg, correspondingly, the dose of BOR was given at 0, 0.5, 1, 2, 3 mg/kg respectively, for the ease of description, these DiR-labelled nanoemulsions containing different dose of BOR were recorded as DiR-NE, DiR-0.5-BOR-NE, DiR-1-BOR-NE, DiR-2-BOR-NE, DiR-3- BOR-NE, respectively.

Before administration, the SD rats were anesthetized by intraperitoneal injection of 4% chloral hydrate (7.5 ml/kg), then a micro syringe with a hose at the front was slowly inserted into nose of the anesthetized rats with a fixed insertion depth of about 6 mm, the nanoemulsion was delivered to both nostrils as described previously [18]. At specific time points (0, 2, 4, 6, 8 and 10 h) after the nanoemulsions administration, the rats were observed by the In vivo imaging system (Carestream FX PRO, USA), both the background image in bright field and the fluorescence image of the spontaneous specific photon DiR in the dark field (λex = 720 nm, λem = 790 nm) were photographed.

2.4. Pharmacokinetic and brain distribution study

Influence of BOR dose on the pharmacokinetic behavior and brain distribution of different VIN nanoemulsions was studied in rats. The dose of VIN in each group was given at 1.5 mg/kg, correspondingly, the dosage of BOR was given at 0, 0.5, 1, 2, 3 mg/kg respectively, these VIN nanoemulsions containing different dosage of BOR were recorded as VIN—NE, VIN-0.5-BOR-NE, VIN-1-BOR-NE, VIN-2-BOR-NE, VIN-3-BOR-NE, respectively. The rats were fasted for 12 h before experimentation with free access to water, then were randomly divided into six groups (n = 6 for pharmacokinetic study, n = 3 for brain distribution study in each group), VIN—NE intravenous injection group as control, and VIN—NE, VIN-0.5-BOR-NE, VIN-1-BOR-NE, VIN-2-BOR-NE, VIN-3-BOR-NE intranasal administration groups.

In addition, to investigate the influence of BOR administration route on its brain distribution inducing behavior, in another rats group, BOR was dissolved in PEG 400 and administered by intragastric administration at a dose of 300 mg/kg [17], 20 min after BOR oral administration, VIN—NE was administered intranasally at a dose of VIN 1.5 mg/kg, this group was recorded as i.n VIN-NE+i.g 300-BOR.

For the pharmacokinetic study, approximately 0.5 ml blood samples were withdrawn from orbital venous plexus at 3, 5, 10, 20, 30, 45, 60, 120 and 180 min post-dosing and centrifuged at 10,000 r/min for 10 min. The supernatant plasma samples were stored at −20°C until analysis. Pharmacokinetic parameters including the maximum drug concentration (C_max), the time to reach C_max (T_max) and AUC_0-t were calculated from the plasma concentration time profiles using DAS 2.0 software. The absolute bioavailability (F) of the intranasally administrated nanoemulsions was calculated by comparing the (AUC_0-t)_plasma with that of intravenous injection.

In the brain distribution study, the administration group, administration method and dose were exactly the same as described above. The rats were sacrificed at 10, 30, 60 and 180 min after dosing, then the whole brain was collected after cardiac perfusion using 0.9% NaCl solution. VIN concentration in the brain was measured by homogenizing the brain tissue with 2 times ice cold 0.9% NaCl solution before analysis.

For brain targeting evaluation, drug targeting index (DTI) and nose-to-brain direct transport percentage (DTP) were calculated according to the following equations:

$$\begin{array}{ccc}\hfill \text{DTI}\%& =& \left[\frac{{(AU{C}_{brain}/AU{C}_{plasma})}_{i.n.}}{{(AU{C}_{brain}/AU{C}_{plasma})}_{i.v.}}\right]\times 100\%\hfill \\ \hfill \text{DTP}\%& =& \left(\frac{B_{i.n.}-B_x}{B_{i.n.}}\right)\times 100\%\hfill \\ \hfill B_x& =& \left(\frac{B_{i.v.}}{P_{i.v.}}\right)\times P_{i.n.}\hfill \end{array}$$

where B_i.v. and P_i.v. represent the AUC_0-t in brain tissue and plasma after intravenous administration, respectively, B_i.n. and P_i.n. represent the AUC_0-t in brain tissue and plasma after nasal administration, respectively, and B_x indicates the proportion of drug absorbed into the body and then crossing the blood-brain barrier into the brain after intranasal administration. When DTI>100%, brain targeting is considered to be achieved; when DTP>0, it is considered that compared to the drug crossing the blood-brain barrier through the systemic circulation, the direct pathway between nose and brain is a more effective channel for drug brain delivery.

2.5. In vivo analysis of VIN

Based on previously reported method, HPLC method was utilized to analyze VIN concentration in plasma and brain [17,19]. Interference from endogenous substances was not observed. Good linear relationship between drug/internal standard peak area ratio and drug concentration was established (plasma: A_VIN/A_PRG = 0.0026C - 0.013, R² = 0.9993; brain: A_VIN/A_PRG = 0.0068C - 0.0091, R² =0.9991). Mean extraction recoveries were 88.19% and 85.08% for plasma and brain samples, respectively. Mean method recoveries were 91.81% and 94.25% for plasma and brain samples, respectively. The detection limits of plasma and brain samples were both 20 ng/ml. The quantitative limits of plasma and brain samples were both 50 ng/ml. The RSDs of intra-day and inter-day precision were both below 10.0%.

2.6. Ex vivo imaging study of brain tissue and brain subregions distribution

Ex vivo imaging was used to observe brain distribution of different formulations using DiR labelled nanoemulsions. The SD rats were randomly divided into six groups: DiR-NE, DiR-0.5-BOR-NE, DiR-1-BOR-NE, DiR-2-BOR-NE, DiR-3-BOR-NE and i.n DiR-NE+i.g 300-BOR. Among them, the DiR-NE, DiR-0.5-BOR-NE, DiR-1-BOR-NE, DiR-2-BOR-NE, DiR-3-BOR-NE groups were administrated according to the method described in nasal retention part by keeping DiR dose at 0.125 mg/kg. The i.n DiR-NE+i.g 300-BOR group was administrated using the same method as that of i.n VIN-NE+i.g 300-BOR group described in pharmacokinetic and brain distribution study, also keeping DiR dose at 0.125 mg/kg. The rats were sacrificed 30 min after administration, and the brain tissue was completely removed, rinsed with normal saline. After excess water was removed, the brain tissue was put into petri dish, the in vivo imaging system was used to capture the background image and the spontaneous specific photon DiR fluorescence map of brain tissue (λex = 720 nm, λem = 790 nm), and the fluorescence intensity of each brain tissue was detected.

Moreover, brain subregions distribution was further observed after intranasal administration of the following two representative groups: DiR-NE group and DiR-1-BOR-NE group, as described above. The DiR dose was kept at 0.125 mg/kg. 30 min after dosing, the rats were sacrificed and their whole brains were quickly collected after cardiac perfusion using 0.9% NaCl solution. After rinsing, the brain tissue was divided into olfactory bulk, cortex, cerebellum, hippocampus, hypothalamus and the residual. The in vivo imaging system was used to capture the background image and the fluorescence images, and detect the fluorescence intensity of these six brain subregions.

2.7. Drug distribution in brain subregions

To further explore the influence of BOR on brain subregions distribution, the rats were randomly divided into two groups. VIN—NE and VIN-1-BOR-NE were given at a dose of VIN 1.5 mg/kg through intranasal route respectively. The rats were sacrificed at 30 min after dosing and the integral brain was collected after cardiac perfusion using 0.9% NaCl solution. The brain tissue was quickly divided into olfactory bulk, hippocampus, hypothalamus, cortex, cerebellum and the residual. The six brain subregions tissue samples were collected immediately, washed with 0.9% NaCl solution, weighed and homogenized before analysis.

2.8. Statistical analysis

All the experimental results were expressed as mean ± standard deviation (SD) from at least three measurements. Significance of difference was evaluated using one-way ANOVA at the probability level of 0.05.

3. Results and discussion

3.1. Preparation and characterization of the nanoemulsions

It have been demonstrated that nanoscale drug delivery systems can increase drug nose-to-brain delivery compared to equivalent drug solution formulations [20,21]. Nanoemulsion is a promising nanocarrier for intranasal drug delivery while it can not only increase the solubility of poorly soluble drugs to satisfy the delivered dose requirement in limited volume [22], its small size and large surface area may also enhance drug absorption across the nasal mucosa for better systemic action [23]. Here, first of all, the solubility of VIN in different types of oil phase, surfactants and cosurfactants was investigated (Table 1S), and finally OA was selected as the oil phase, HS15 as the surfactant, Transcutol P as the co-surfactant. In the selected concentration range, by keeping the oil phase at 3%, influence of HS15 and Transcutol P concentration on the particle size and size distribution of the nanoemulsion was investigated (Fig. 1SA&1SB). Oil phase 3%, HS15 9% and co-surfactant Transcutol P 9% was selected for the following studies, with particle size 55.35 ± 0.186 nm, PDI 0.246 ± 0.014.

Influence of VIN addition on properties of the nanoemulsion was investigated. As shown in Fig. 1SC, when the concentration of VIN was in the range of 0.1%−0.6%, no influence on particle size and size distribution was observed, and a significant increase in both size and size distribution was noted when VIN concentration increased to 0.7%. Thus VIN 0.6% nanoemulsion was selected in this research. The effect of BOR addition on the properties of the VIN nanoemulsion was also investigated. As shown in Fig. 1SD, when the concentration of BOR was in the range of 0–1.2%, no apparent change on particle size and size distribution was observed.

Table 1 listed the particle size, polydispersity index (PDI) and drug content of the nanoemulsions used in this study with different BOR percentage (0, 0.2%, 0.4%, 0.8%, 1.2%). All the particle size was around 60–70 nm, and no significant difference in particle size among these five nanoemulsions was found, which provided the prerequisite to compare the influence of BOR dose on its brain inducing effect, while it is well known that the particle size of nanocarriers affects their interaction with cells and thus the cellular internalization and intracellular localization [24]. Stability of the nanoemulsions was also investigated, as shown in Fig. 2SA&2SB, no significant difference in particle size and PDI was found after storage at room temperature for 7 d.

Table 1.

Properties of the nanoemulsions used in this study.

Percentage of BOR in the nanoemulsion (%)	Particle size (nm)	PDI	VIN (%)	BOR (%)
0	62.83±2.58	0.19±0.02	0.60±0.01	0
0.2	61.59±1.96	0.21±0.02	0.60±0.01	0.20±0.01
0.4	60.19±3.49	0.20±0.019	0.60±0.01	0.41±0.03
0.8	66.30±0.75	0.21±0.02	0.60±0.01	0.80±0.02
1.2	71.02±5.02	0.23±0.02	0.60±0.01	1.20±0.09

Morphology of the nanoemulsions was observed by transmission electron microscopy (TEM). As shown in Fig. 1A-1E, all the nanoemulsion particles were well dispersed with spherical shape, no influence of BOR dose on the particle size and appearance was observed. In addition, compared with the results of dynamic light scattering (DLS), the particle size of the five nanoemulsions measured by TEM decreased slightly (about 50 nm). While DLS measures hydrodynamic particle size, and the hydrophilic group of surfactant HS15 could form a hydrophilic layer on the particle surface, but the preparation of TEM samples requires negative dying and drying, and the hydrophilic layer of the nanoemulsion particles will shrink during the drying process, leading to a smaller particle size measured by the TEM [25].

Fig. 1.

Morphology of the nanoemulsions containing different percentage of BOR observed by TEM. (A) 0% BOR; (B) 0.2% BOR; (C) 0.4% BOR; (D) 0.8% BOR; (E) 1.2% BOR.

3.2. Nasal mucosa retention of the nanoemulsions

The cilia of the nasal mucosa have a clearing effect, which can influence drug retention and therefore in vivo absorption of intranasally delivered drugs [18,26]. The above studies demonstrated that the nanoemulsion formulations with different BOR percentage had similar particle size and size distribution, the influence of BOR on the nasal mucosa retention was further investigated to better elucidate the influence of BOR dose on the in vivo behavior of VIN. Here, for the ease of observation, DiR labelled nanoemulsions were used in this study. As shown in Table 2S, it has been demonstrated that the addition of DiR had no statistical influence on particle size and size distribution of the nanoemulsions. Therefore, the DiR labeled nanoemulsions can be used as a fluorescent marker to track the in vivo behavior.

Fig. 2A shows evolution of the fluorescence change in the nasal cavity along with time for the five nanoemulsions investigated (DiR-NE, DiR-0.5-BOR-NE, DiR-1-BOR-NE, DiR-2-BOR-NE and DiR-3-BOR-NE), Fig. 2B is the quantitative description of the remaining fluorescence intensity in the nasal cavity at different time points. It was noted that the fluorescence intensity decreased remarkably at 2 h, followed by almost linear decrease and the fluorescence intensity at 10 h was less than 10%. No significant difference in the residual fluorescence intensity at the nasal mucosa was found among the five nanoemulsions at each time point, implying BOR dose had no influence on the nasal retention behavior of the nanoemulsions. Therefore, the potential influence of particle size and nasal retention difference on the in vivo behavior can be omitted, which provided the background to elucidate the potential influence of BOR dose on the in vivo absorption and brain targeting effect.

Fig. 2.

(A) Clearance of five nanoemulsions and (B) change of remaining fluorescence intensity in the nasal cavity after intranasal administration of five nanoemulsions in rats (n = 3).

3.3. Influence of intranasal administration on the in vivo behavior of the nanoemulsion

First of all, taking VIN—NE as an example, the advantages of intranasal route for enhanced drug absorption and brain targeting were explored. After intravenous injection and intranasal administration of VIN—NE at the same dose, blood and brain were collected at predetermined time points. The plasma and brain drug concentration-time profiles of the two administration routes are shown in Fig. 3, and the main pharmacokinetic parameters are presented in Table 2.

Fig. 3.

Concentration-time profiles of vinpocetine in plasma (A) and brain (B) after intravenous injection (i.v) and intranasal (i.n) administration of VIN—NE in rats (n = 6 for plasma, n = 3 for brain).

Table 2.

The pharmacokinetic parameters in plasma and brain of VIN—NE intravenous (i.v) administration group, five nanoemulsions containing different doses of BOR by intranasal (i.n) administration and i.n VIN—NE+i.g 300-BOR administration group in rats, n = 6 for plasma, n = 3 for brain.

Parameters	i.v VIN—NE	VIN—NE	VIN-0.5-BOR-NE	VIN-1-BOR-NE	VIN-2-BOR-NE	VIN-3-BOR-NE	i.n VIN—NE+i.g 300-BOR
(AUC0-t)plasma (ng·min/ml)	58,270.0 ± 2568.5	50,098.6 ± 5127.8	34,424.1 ± 5895.1#,*	23,833.0 ± 5230.8#	29,047.1 ± 2344.3#	29,962.6 ± 2231.2#,*	45,050.8 ± 5368.4*
F(%)	100	86.0 ± 8.8^	59.1 ± 10.1#,*	40.9 ± 9.0#	49.8 ± 4.0#	51.4 ± 3.8#,*	77.3 ± 9.2*
(AUC0-t)brain (ng·min/g)	10,806.8 ± 1939.6	14,317.4 ± 381.5^	18,611.0 ± 1823.8#,*	26,350.3 ± 2747.9#	21,138.3 ± 2405.6#	19,094.8 ± 2160.1#,*	18,754.1 ± 2077.9#,*
(Cmax)plasma (ng/ml)	1825.3 ± 93.9	1026.0 ± 107.2^	788.3 ± 93.5#,*	626.9 ± 65.8#	669.7 ± 119.7#	814.9 ± 101.4#,*	965.5 ± 83.7*
(Cmax)brain (ng/g)	120.6 ± 27.5	168.5 ± 30.4	214.6 ± 17.8*	312.7 ± 10.9#	236.0 ± 4.0#,*	220.1 ± 5.1#,*	214.2 ± 30.6*
(Tmax)plasma (min)	/	10 ± 0	7.5 ± 2.7	6.7 ± 2.6#	5.8 ± 2.0#	6.7 ± 2.6#	9.2 ± 2.0
(Tmax)brain (min)	30 ± 0	30 ± 0	30 ± 0	30 ± 0	30 ± 0	30±0	30±0
DTI (%)	/	154.1 ± 4.1	291.5 ± 28.6#,*	596.1 ± 62.2#	392.4 ± 44.7#,*	343.6 ± 38.9#,*	224.5 ± 24.9#,*
DTP (%)	/	35.1 ± 1.8	65.5 ± 3.2#,*	83.1 ± 1.7#	74.3 ± 2.8#,*	70.6 ± 3.4#,*	55.1 ± 5.2#,*

^{^}P < 0.05 compared to the i.v VIN—NE administration group.

^#P < 0.05 compared to the VIN—NE administration group.

^⁎P < 0.05 compared to the VIN-1-BOR-NE administration group.

As shown in Fig. 3A, after intravenous injection of VIN—NE, significantly high drug concentration was achieved in the plasma and it decreased rapidly with time. In contrast, after intranasal administration of VIN—NE, the maximum drug absorption occurred at about 10 min (T_max) and the C_max was significantly lower than that of intravenous injection. This study demonstrated that the drug can be absorbed into systemic circulation at a fast rate after being administered through the nasal cavity, with the potential to decrease high concentration related side effect such as in the case of intravenous injection. After VIN—NE intranasal administration, the plasma absolute bioavailability reached 86%, while the oral bioavailability of VIN is just 6.7% [17], implying intranasal delivery is a promising non-invasive route for systemic drug absorption with fast onset of action, which can be used as the alternative of intravenous injections for some drugs [27].

Whether intranasal drug delivery can increase drug distribution in the brain was further investigated. As shown in Fig. 3B, indeed, after intranasal administration, VIN concentration in the brain was higher in the initial 60 min compared with that of the intravenous injection group, and the (C_max)_brain of the intranasal administration group was about 1.40 times higher than that of the intravenous injection group. In addition, the (AUC_0-t)_brain increased by about 32.5% after intranasal administration compared with that of intravenous injection group (Table 2). The brain-targeting index (DTI) of the intranasal group was 154.1%, with the nose-to-brain DTP 35.1%. This study demonstrated that better brain targeting effect can be achieved by nasal route, and at least 35% of the drug was transported through the direct pathway between nose and brain, instead of entering the systemic circulation first and then crossing the blood-brain barrier into brain. After intranasal administration, the delivered drug may enter the brain tissue through either the olfactory pathway or the trigeminal nerve pathway, thus bypassing the blood-brain barrier, leading to better brain targeting effect [28].

3.4. Influence of BOR dose on drug brain targeting behavior

The above study demonstrated that intranasal administration is an efficient way to enhance drug brain targeting. Here, the addition of brain message guider, BOR, and its dose on the pharmacokinetics and brain-targeting behavior of VIN was further explored after intranasal administration of different nanoemulsions.

First of all, ex vivo imaging was used to study the effect of BOR dose on brain tissue distribution using DiR labelled nanoemulsions, 30 min after dosing, fluorescence intensity in brain tissues from both ventral and dorsal side was recorded and presented in Fig. 4A&4B is quantitative analysis of the fluorescence intensity in the brain tissue. Compared to the DiR-NE group without BOR, the addition of BOR in the formulation increased drug distribution in the brain remarkably, with the extent in a BOR dose dependent manner, the highest intensity increase was observed for the DiR-1-BOR-NE group with BOR dose of 1 mg/kg, thereafter the fluorescence intensity in the brain tissue decreased with the further increase of BOR dose. No significant difference in fluorescence intensity between DiR-2-BOR-NE and DiR-3-BOR-NE group was found (P>0.05), which was considerably lower than that of DiR-1-BOR-NE group (P<0.05), but higher than the DiR-0.5-BOR-NE group (P<0.05). This study demonstrated that the brain inducing effect of BOR was its dose-dependent and should be optimized, similar results have been reported previously [29]. The above ex vivo imaging study illustrated that when the dosage of BOR was 1 mg/kg, the best brain targeting effect could be achieved.

Fig. 4.

(A) The ex vivo imaging of DiR fluorescence signals and (B) sum fluorescent intensity in the brain of SD rats 30 min after intranasal administration of five nanoemulsions from both dorsal and ventral side (n = 3). ^#P < 0.05 compared to the DiR-NE administration group, *P < 0.05 compared to the DiR-1-BOR-NE administration group.

To confirm the results from the above ex vivo imaging study, and further explore the influence of BOR dose on the drug systemic exposure and brain targeting effect, VIN concentration in both plasmas and brain was measured quantitatively by HPLC analysis. The drug concentration-time profiles, the DTI and DTP values of different administration groups are shown in Fig. 5, and the main pharmacokinetic parameters are summarized in Table 2.

Fig. 5.

Concentration time profiles of VIN in plasma (A), brain (B), and DTI (C), DTP (D) parameters after intranasal administration of the nanoemulsions containing different doses of BOR in rats, n = 6 for plasma, n = 3 for brain. ^#P < 0.05 compared to the VIN—NE group, *P < 0.05 compared to the VIN-1-BOR-NE group.

As shown in Fig. 5A and Table 2, compared with the VIN—NE group, addition of BOR in the nanoemulsion can increase VIN absorption rate but decrease the absorption extent, it was noted that the drug plasma peak time (T_max)_Plasma was shortened, from 10 min in VIN—NE group to 5 min in VIN-2-BOR-NE group, implying BOR might function as an absorption enhancer and increase VIN absorption across the nasal mucosa into systemic circulation more quickly [14]. Contrary to the accelerated absorption rate, after adding BOR, VIN exposure in the body decreased, and the (AUC_0-t)_plasma of VIN-0.5-BOR-NE, VIN-1-BOR-NE, VIN-2-BOR-NE and VIN-3-BOR-NE groups decreased to 68.7%, 47.6%, 58.0% and 59.8% that of the VIN—NE group. Correspondingly, the absolute bioavailability (F) decreased from 86.0% of VIN—NE to 59.1%, 40.9%, 49.8% and 51.4%, respectively.

In agreement with the decreased drug concentration in the plasma, increased drug concentration in the brain was observed in a BOR dose dependent manner. As shown in Fig. 5B, compared to the VIN—NE group, VIN distribution in the brain tissue increased in all the BOR-added nanoemulsion groups, especially at the initial 30 min, statistical enhanced brain concentration in VIN-1-BOR-NE, VIN-2-BOR-NE and VIN-3-BOR-NE groups was observed. In agreement with the ex vivo imaging studies, VIN brain concentration was the highest in the VIN-1-BOR-NE group (p<0.05), while no statistical difference was found among VIN-0.5-BOR-NE, VIN-2-BOR-NE and VIN-3-BOR-NE groups. Compared with the VIN—NE group, the (AUC_0-t) _brain of VIN-0.5-BOR-NE, VIN-1-BOR-NE, VIN-2-BOR-NE and VIN-3-BOR-NE groups increased by 1.30, 1.84, 1.48, 1.33 times, respectively. In combination with the decreased bioavailability of VIN in the plasma, this study implied that more drug might be transported from blood to brain in the presence of BOR, probably BOR as a brain distribution inducer could help drug transport from plasma into brain through enhancing BBB permeability [30], and thus improve drug nose-to-brain direct transport, leading to better brain targeting effect [31]. Moreover, DTI and DTP values (Fig. 5C-5D) further confirmed that BOR can increase drug distribution in the brain, and the VIN-1-BOR-NE group exhibited the highest brain targeting index, with the DTI reaching 596.1%. The DTP was 65.5%, 83.1%, 74.3% and 70.6% for VIN-0.5-BOR-NE, VIN-1-BOR-NE, VIN-2-BOR-NE and VIN-3-BOR-NE groups respectively.

The dose-dependent brain targeting effect of BOR was also reported previously when it was given via oral route [29]. This phenomenon might be explained by bidirectional regulation of BOR on BBB permeability. BOR could increase BBB permeability by inhibiting nuclear factor κB (NF-κB), down-regulating P-gp and then decreasing efflux [32]. It can also improve the permeability of BBB by promoting pinocytosis of BBB [33]. On the contrary, BOR can inhibit the expression of interleukin-1β (IL-1β) and matrix metalloproteinase-9 (MMP-9) [34,35], resist the degradation effect of IL-1β and MMP-9 on BBB vascular outer basal membrane and tight junctions [36], then reduce BBB permeability. The dose of BOR may determine the direction of its effect on BBB permeability. When the dose of BOR is moderate, it can promote BBB opening, and the increase of BBB permeability was dose-dependent with BOR, while when the dose of BOR is too high, the effect of BOR on BBB will be opposite, which shows permeability reduction. In this experiment, VIN-1-BOR-NE group exhibited the highest brain targeting efficiency, therefore, VIN-1-BOR-NE group with BOR 1 mg/kg was selected in the following studies.

3.5. Influence of BOR administration route on its brain distribution inducing behavior

The above studies demonstrated that when delivered intranasally, BOR dose 1 mg/kg was sufficient to achieve the best brain inducing distribution. Our previous studies indicated that the oral dose of BOR to promote the best brain inducing distribution in rats was 300 mg/kg [17]. It is still unclear, when BOR was delivered per oral at the optimized dose and VIN nanoemulsion was delivered intranasally, or BOR was delivered together with VIN via intranasal route, which can achieve the best brain inducing effect.

To answer this question, first of all, ex vivo imaging was used to compare the brain distribution of i.n DiR-NE+i.g 300-BOR and i.n DiR-1-BOR-NE group by comparing their fluorescence intensity difference in brain tissues from both ventral and dorsal side 30 min after administration. As shown in Fig. 6A-6B, the brain tissue fluorescence intensity of the i.n DiR-1-BOR-NE group was significantly higher than that of the i.n DiR-NE+i.g 300-BOR group, either observed from ventral or dorsal side, despite of the fact the dose of BOR in the i.n DiR-NE+i.g 300-BOR group was 300 times higher than that in the i.n DiR-1-BOR-NE group.

Fig. 6.

(A) The ex vivo imaging of DiR fluorescence signals and (B) sum fluorescent intensity in the brain of SD rats at 30 min post administration after treated with i.n DiR-NE+i.g 300-BOR and i.n DiR-1-BOR-NE in both dorsal and ventral side (n = 3). *P < 0.05 compared to the i.n DiR-NE+i.g 300-BOR administration group. The VIN concentration time profiles in plasma (C) and brain (D) after treated with i.n VIN—NE+i.g 300-BOR and i.n VIN-1-BOR-NE administration groups in rats, n = 6 for plasma, n = 3 for brain. ^#P < 0.05 compared to the i.n VIN—NE+i.g 300-BOR administration group.

To confirm the results from ex vivo imaging studies, VIN concentration in both plasma and brain tissue was measured after administration of i.n VIN-NE+i.g 300-BOR and i.n VIN-1-BOR-NE. The drug plasma concentration-time profile is shown in Fig. 6C, and the main pharmacokinetic parameters are listed in Table 2. It was noted that i.n VIN-1-BOR-NE group had significantly lower drug concentration in the plasma compared with the i.n VIN-NE+i.g 300-BOR group, and the (AUC_0-t)_plasma of the i.n VIN-NE+i.g 300-BOR group was 1.89 times that of the i.n VIN-1-BOR-NE group although the same dose of VIN was used in this study, implying system drug exposure can be decreased by using i.n VIN-1-BOR-NE group.

Moreover, drug concentration in the brain was also compared. As shown in Fig. 6D, significantly higher drug concentration in the brain was observed in the i.n VIN-1-BOR-NE group compared with that of the i.n VIN-NE+i.g 300-BOR group after 10 min and 30 min administration (P <0.05). And the (AUC_0-t) _brain of i.n VIN-1-BOR-NE group was 1.41 times higher than that of i.n VIN-NE+i.g 300-BOR group. The brain targeting effect was also evaluated using DTI and DTP index. Especially, the DTI of i.n VIN-1-BOR-NE group was 2.66 times of the i.n VIN-NE+i.g 300-BOR group, implying its better brain inducing effect. Also, the DTP of i.n VIN-1-BOR-NE group was 83.1%, while it was only 55.1% for the i.n VIN-NE+i.g 300-BOR group, indicating BOR could lead to increased nose to brain delivery when administrated via intranasal route together with the drug. This can probably be explained by the fact that, BOR, as a brain "guider", can guide the drug VIN to enter the brain tissue more effectively. When the "guider" BOR and the "tourist" VIN are administered in the same route, the distance between them is short, which is more conducive for the guider to play a leading role for tourists, thus BOR exerts the brain inducing effect to a greater extent at lower dose.

3.6. Influence of BOR incorporation on drug distribution in brain subregion

Some recent studies have shown that BOR can not only increase the efficiency of drug transport into brain [13], but also has regional specificity to enhance drug distribution in some brain subregions [15]. For AD therapy, it is highly desirable that the drug can be concentrated in the hippocampus and cortex nidus areas [3]. Therefore, the potential of BOR added in the nanoemulsions to increase drug subregion distribution, especially in the hippocampus and cortex area, was further explored. For this purpose, drug subregion distribution after intranasal administration of VIN—NE (DiR-NE) and VIN-1-BOR-NE (DiR-1-BOR-NE) was compared.

First of all, ex vivo imaging experiments were carried out, 30 min after intranasal administration of DiR-NE and DiR-1-BOR-NE, fluorescence intensity in olfactory bulk, cortex, cerebellum, hippocampus, hypothalamus and residual, were measured. As shown in Fig. 7A and 7B, compared with the DiR-NE group, significantly higher fluorescence signal was observed in DiR-1-BOR-NE group in olfactory bulk, cortex, cerebellum, hippocampus and hypothalamus (P<0.05), especially, in DiR-1-BOR-NE group, the sum fluorescence intensity in the olfactory bulk, cortex, hippocampus was almost five times higher than the DiR-NE group (Fig. 7C), demonstrating the strong capacity of BOR to increase drug distribution in these brain subregions.

Fig. 7.

(A) The ex vivo imaging of DiR fluorescence signals, (B) sum fluorescent intensity and (C) sum fluorescent intensity ratio of DiR-1-BOR-NE and DiR-NE in six brain sub-areas (olfactory bulk, hippocampus, hypothalamus, cortex, cerebellum and residual) at 30 min after intranasal administration of DiR-NE and DiR-1-BOR-NE (n = 3). *P < 0.05 compared to the DiR-NE administration group. (D) Vinpocetine concentration in the six brain sub-areas at 30 min post administration after intranasal administration of VIN—NE and VIN-1-BOR-NE (n = 6). ^#P < 0.05 compared to the VIN—NE administration group.

To further confirm the above conclusion, VIN concentration in six brain subregions (olfactory bulk, hippocampus, hypothalamus, cortex, cerebellum and residual) 30 min after intranasal administration of VIN-1-BOR-NE and VIN—NE groups was measured. As shown in Fig. 7D, in agreement with the results from ex vivo imaging studies, compared to the VIN—NE group, significantly higher VIN concentration in olfactory bulk, hippocampus and cortex was observed in the VIN-1-BOR-NE group (P<0.05). Therefore, both ex vivo imaging semi-quantitative experiment and quantitative study demonstrated that after intranasal delivery, BOR can remarkably enhance drug distribution in both hippocampus and cortex, the nidus areas of AD, which may provide a new strategy for the effective therapy of AD.

As shown in Fig. 7A and 4A, after nasal administration, the fluorescence distribution in brain tissue showed consistency. As the brain frontend, the olfactory bulk area had the strongest fluorescence distribution, with the transition to the brain backend, in the order of cortex, hippocampus, hypothalamus, the fluorescence intensity decreased gradually. However, it was noted that the fluorescence intensity in the cerebellum, which is in the cerebellar backend but near the brain stem area, obviously increased. The reason for this phenomena may be caused by the special nose to brain direct pathway: olfactory pathway and trigeminal nerve pathway. As shown in Fig. 8A, the olfactory pathway mainly depends on the olfactory nerve, the olfactory nerve is one of the cell types that make up the olfactory region epithelium, the olfactory area is located at the roof of the nasal cavity below the cribriform plate, next to the olfactory bulb, which interlocks the nose with the brain [37]. After nasal administration, the nose-to-brain drug transport via the olfactory pathway mainly through the following three mechanisms: (i) axonal transport through internalization into olfactory neurons, (ii) paracellular transport across the intercellular space, especially through channels next to olfactory neurons, (iii) transcellular transport via trans-basal epithelial cells (Fig. 8B) [38]. As shown in Fig. 8D-8E), the drug or drug delivery system may reach the olfactory bulb through internalization into the olfactory neurons, and then enter other brain regions connected with olfactory bulb (such as the piriform cortex, hypothalamus, hippocampus, etc.) [5]. It is also possible to enter the cerebrospinal fluid near the olfactory bulb through the paracellular route first, and then spread to the entire brain tissue with the cerebrospinal fluid [37]. Therefore, the fluorescence intensity of olfactory bulb was the strongest in the experimental results, it became weaker towards backend of the brain. In addition, it was noted that there was a fluorescence increase at the cerebellum backend, near the brainstem area, which may be caused by the trigeminal nerve pathway. The trigeminal nerve pathway is another nose to brain direct pathway. As shown in Fig. 8C, some trigeminal nerve extends from the brainstem to respiratory mucosa epithelial layer of nasal cavity, communicating the nasal cavity with the brainstem [5]. Therefore, it was speculated that the nanoemulsion was absorbed through the respiratory mucosa and then be transported to brainstem through the trigeminal nerve, afterwards, transmitted to the cerebellum near the brainstem through the cerebrospinal fluid or brain tissue neurons, then increased the fluorescence distribution in cerebellum backend, as schematically described in Fig. 8D-8E.

Fig. 8.

(A) Location of the olfactory and trigeminal nerve in the nasal cavity (B) Schematic illustration of the olfactory pathway and possible uptake mechanisms for direct drug transport from the nose to the brain; (C) Schematic illustration of the trigeminal nerve pathway; (D) Schematic illustration of the different absorption routes from the nose to the brain after intranasal delivery in rat, VIN-1-BOR-NE can reach different parts of the brain through the trigeminal (green) and olfactory (red) pathways; (E) Schematic showing two different routes from the nose to the brain, whereas drugs can reach different parts of the brain through the trigeminal (green) and olfactory (red) pathways. Note: (A), (B) and (C) are reproduced with permission from [37]. Copyrights 2018 Elsevier Ltd.

The significant drug content increase in the olfactory bulk region may be the result of BOR increasing drug transport through the olfactory pathway between nose to brain, while the enhancement in hippocampus and cortex regions may be related to the BOR's pharmacokinetic behavior in vivo. BOR showed a specific distribution in the brain subregions, with the highest concentration in cortex, followed by the hippocampus and hypothalamus, and the lowest in striatum [39]. The role of BOR in promoting drug distribution into the brain is dose-dependent [29]. BOR has higher distribution in some brain subregions after entering the brain, then it could open the intercellular tight connection in this subregion, regulating the efflux of P-gp and increasing the number and volume of cell swallowing vesicles, then leading to the distribution increase in this brain subregion of the guided drugs [14].

4. Conclusion

In this study, to overcome the BBB and limited drug distribution in the niduses for better AD therapy, VIN nanoemulsions with different BOR content were prepared. It was demonstrated that BOR dose had no influence on the particle size, morphology and nasal retention behavior of the nanoemulsions. Better brain targeting effect can be achieved by nasal route, and DTI of the VIN—NE intranasal group was 154.1%, with the DTP 35.1%. Incorporation of BOR in the nanoemulsion led to significantly increased drug distribution in the brain, and the optimal BOR dose for intranasal administration was 1 mg/kg (VIN-1-BOR-NE group), with the DTI reaching 596.1% and the DTP 83.1%. The best BOR administration method was to deliver together with the drug via intranasal route. Both ex vivo imaging and quantitative study demonstrated that after intranasal delivery, BOR can remarkably enhance drug distribution in both hippocampus and cortex, the nidus area of AD. This study may provide a new strategy for the effective therapy of AD and also shed light to the central nerves system related diseases therapy.

Conflicts of interest

The authors have declared no conflict of interest.