Honokiol nanosuspensions: Preparation, increased oral bioavailability and dramatically enhanced biodistribution in the cardio-cerebro-vascular system

Colloids and Surfaces B: Biointerfaces 116 (2014) 114–120

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Honokiol nanosuspensions: Preparation, increased oral bioavailability and dramatically enhanced biodistribution in the cardio-cerebro-vascular system Meihua Han a,1 , Xin Yu a,b,1 , Yifei Guo a,1 , Yanhong Wang b,1 , Haixue Kuang b,∗ , Xiangtao Wang a,∗∗ a

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing 100193, PR China b School of Pharmacy, Heilongjiang University of Chinese Medicine, No. 24, Heping Road, Xiangfang District, Harbin 150040, PR China

a r t i c l e

i n f o

Article history: Received 7 August 2013 Received in revised form 21 December 2013 Accepted 24 December 2013 Available online 3 January 2014 Keywords: Honokiol Nanosuspensions Preparation Bioavailability Biodistribution

a b s t r a c t Honokiol is a phytochemical component with multiple pharmacological activities, but Honokiol’s wider use has been restricted by its poor solubility. Using bovine serum albumin and polyvinylpyrrolidone as stabilisers in a solvent precipitation–ultrasonication method, Honokiol nanosuspensions were prepared with a mean particle size of 116.2 nm (±2 nm), a zeta potential of −44.7 mV (±1.7 mV) and a high drug payload of 50.4 ± 0.6% (w/w). X-ray powder diffraction and differential scanning calorimetry indicated that Honokiol was in an amorphous state in the nanosuspensions, in contrast with bulk Honokiol powder. Honokiol was released faster in vitro from nanosuspensions with no burst release, and the highest 98% cumulative release was after 60 h. Honokiol nanosuspensions improved the oral bioavailability of Honokiol in in vivo studies in rats with a 3.94-fold Cmax and a 2.2-fold AUC(0–t) . Remarkably, in contrast to oral administration, intraperitoneal administration of Honokiol nanosuspensions could dramatically alter the biodistribution of Honokiol, resulting in a much higher drug level and tissue bioavailability in the blood, heart and brain, benefitting the treatment of cardio-cerebro-vascular diseases. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Honokiol (HK) is a biologically active small molecule that was isolated and identified from the stem bark of Magnolia officinalis Rehd. Et Wils. HK exhibits antioxidant [1], antimicrobial [2], antithrombotic [3], anti-inflammatory [4], antianxiety [5] and antitumour [6] activities. However, this molecule’s poor solubility limits its dispersion in aqueous solution, greatly restricting its applications. In recent decades, reducing drug particle size has been found to be able to increase drug dissolution rates. The dissolution rates of drugs can be expressed using the Noyes–Whitney equation. It is well known that reducing particle size increases total surface area, which subsequently increases the dissolution rate [7].

∗ Corresponding author. Tel.: +86 451 82110803; fax: +86 451 82110803. ∗∗ Corresponding author. Tel.: +86 10 57833266; fax: +86 10 57833266. E-mail addresses: [email protected] (M. Han), [email protected] (X. Yu), [email protected] (Y. Guo), [email protected] (Y. Wang), [email protected] (H. Kuang), [email protected], [email protected] (X. Wang). 1 Tel.: +86 10 57833266; fax: +86 10 57833266. 0927-7765/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.056

Nanosuspensions (NSps) are carrier-free colloidal drug delivery systems that contain drug particles and minimal stabilisers. The mean sizes of these drug particles are in the nanometer range, typically between 10 and 1000 nm [8,9]. Nanometer-sized particles can increase drug solubility, the rate of dissolution and mucosal adhesion. These factors are critical in improving the bioavailability of poorly soluble drugs and in determining their effectiveness and stability. Because of their nanometer scale particle size and safe composition, NSps can be delivered through various routes of administration, such as the oral [10], ocular [11] and pulmonary pathways [12]. There are various ways to produce drug NSps, including anti-solvent precipitation. In this method, briefly, a poorly water-soluble drug is dissolved in an organic solvent and the drug solution is then poured into an anti-solvent containing stabiliser. Precipitation occurs immediately due to the rapid desolvation of the drug [13]. To date, there have been no reports of Honokiol nanosuspensions (HK-NSps). The only related preparations are HK liposomes and micelles [14–19], which have drug pay-loads of 25% (w/w) or lower. The objective of the present study was to prepare HK-NSps and to investigate the nanosuspensions’ in vitro properties, as well as in vivo bioavailability and biodistribution. The obtained HK-NSps seem to be a promising new dosage form for clinical application.

M. Han et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 114–120

115

2. Materials and methods

2.6. The stability of lyophilised HK-NSps

2.1. Materials

2.6.1. Stability of HK-NSps in rat plasma The stability of HK-NSps (reconstituted with 0.9% NaCl solution) in rat plasma was evaluated as follows. Reconstituted HK-NSps (0.5, 1 and 2 mg/mL) were mixed with rat plasma (1:4, v/v) and incubated at 37 ◦ C. A 1 mL sample was removed at predetermined time intervals (0, 1, 3 and 5 h) and analysed for changes in the particle size. Each experiment was performed in triplicate.

Honokiol (98.0% purity) was provided by Shaanxi YongYuan Bio-Tech Co., Ltd. (Shaanxi, China). Bovine serum albumin (BSA), polyvinylpyrrolidone (PVP), and mannitol were purchased from Sigma Chemical (St. Louis, MO). Sodium carboxymethyl cellulose (CMC-Na) was obtained from Xilong Chemical Co., Ltd. (Guangzhou, Guangdong, China). Acetonitrile (HPLC grade) was purchased from Fisher Chemical (Loughborough, UK). All other chemicals and solvents were of the highest grade commercially available, and deionised-distilled water was used throughout the study.

2.2. Chromatographic conditions HK concentrations were determined using HPLC (DGP3600RS pump, fluorescence detector, excitation/emission wavelengths 294/380 nm) with a Symmetry® C-18 column (5 ␮m, 4.6 mm × 250 mm). The mobile phase was a mixture of acetonitrile and 0.2% formic acid solution (75/25, v/v). The flow rate was 1 mL/min. The column temperature was maintained at 25 ◦ C. The lower limits of detection and quantification were 1.4 ng/mL and 4.7 ng/mL, respectively.

2.6.2. Stability of HK-NSps in simulated gastrointestinal fluids over a range of pH Reconstituted HK-NSps (containing 2 mg/mL HK in 0.9% NaCl solution) were mixed with 4-fold volume of simulated gastric fluid (containing 1.0% pepsinum (w/v) in pH 1.5 diluted hydrochloric acid) or 4-fold volume of simulated intestinal fluid (containing 1.0% pancreatin (w/v) in pH 6.8 PBS (0.01 M)) and then incubated at 37 ◦ C for a stability test. A sample (1 mL) of the mixture was removed at intervals of 0, 1, 3 and 5 h for particle size analysis. The change in particle size in reconstituted HK-NSps at different pH values (1, 2, 3, 4, 4.5, 5, 5.5, 6, 7 and 8) was also studied in the same way. To improve the stability of HK-NSps in gastric fluid, lyophilised HK-NSps were reconstituted in 0.5 M PBS (pH 8.0, composed of dipotassium phosphate and monopotassium phosphate) to offset the impact of acidic gastric fluid before oral administration or before mixture with the simulated gastric fluid for the stability test. Each experiment was performed in triplicate.

2.3. Preparation of HK-NSps 2.7. Transmission electron microscopy (TEM) HK-NSps were prepared using the precipitation–ultrasonication method. Briefly, HK powder was dissolved in acetone (25 ◦ C, 25 mg/mL). This solution (2 mL) was introduced rapidly into 10 mL of aqueous solution containing 0.25% BSA (w/v) and 0.25% PVP (w/v) under continuous ultrasonication (250 W) and 300 rpm stirring (EURO-ST D S25, IKA, Germany). The resulting HK-NSps were evaporated under vacuum at 40 ◦ C until no residual acetone remained. Both the organic and aqueous phases were filtered before use (0.45 ␮m filter, Xinya Purification Device Factory, Shanghai, China).

2.4. Particle size, polydispersity index and zeta potential determination

The morphology of the drug particles was observed using a JEM1400 electron microscope (JEOL Ltd., Tokyo, Japan). The samples were negatively stained with 2% (w/v) phosphotungstic acid for 30 s and placed on copper grids with image recording. 2.8. X-ray powder diffraction The freeze-dried HK-NSps and bulk powder were analysed by an X-ray diffractometer (DX-2700, China) with Cu K␣ radiation ˚ The scanning speed was 10 ◦ C/min from a 2 of 3–40 ◦ C (1.542 A). at a rate of 2◦ /min, a 0.02◦ step size and 2 s/step at 40 mA and 40 kV. 2.9. Differential scanning calorimetry (DSC)

The particle size, polydispersity index (PDI) and zeta potential of the NSps were determined using dynamic light scattering (DLS) (Zetasizer Nano ZS 90, Malvern Instruments, UK). The instrument was calibrated using a NanosphereTM size standard (500 nm, Duke Scientific Corporation, USA). Samples were diluted with water, and a suitable scattering intensity for the final experimental values was identified. All measurements were repeated in triplicate, and the mean values are reported.

2.5. Lyophilisation of HK-NSps For further physicochemical characterisation, freshly prepared HK-NSps (approximately 5 mg/mL) were lyophilised with 0, 0.2, 0.4, 0.8 and 1.0% (w/v) mannitol as a cryoprotectant. Briefly, HK-NSps were rapidly cooled to −80 ◦ C, maintained at that temperature for 12 h, and transferred to a freeze-dryer (Beijing Sihuan Scientific Instruments Co., Ltd.). Drying was performed at 0.120 mbar and −50 ◦ C for 24 h [20]. The freeze-dried HK-NSps were reconstituted with 0.9% NaCl for intraperitoneal administration or 0.5 M PBS (pH 8.0) for intragastric administration.

The thermal properties of the powder samples were investigated using a differential scanning calorimeter (DSC Q 200, TA Co., USA). Accurately weighed samples (5–8 mg) were placed in perforated aluminium-sealed pans. Each sample was tested from 30 ◦ C to 200 ◦ C at 20 ◦ C/min under a dry nitrogen atmosphere. 2.10. In vitro release behaviour The in vitro release of HK-NSps was analysed using dialysis bag diffusion. HK-NSps (1 mL, 5 mg/mL HK) were placed into a preswelled dialysis bag (5.0 kDa molecular mass cutoff, Sigma, USA). The dialysis bag was then immersed in 50 mL of phosphate buffer (pH 7.4) containing 0.5% (v/v) SDS as the release medium at 37 ◦ C with stirring (100 rpm). Thus, the concentration of HK was kept below its solubility in the release medium throughout the experiment. Periodically, 1 mL of the release medium was withdrawn. The amount of released drug was quantified using HPLC. Coarse suspensions of HK (HK-CS, prepared by suspending HK powder in pure water) were dialysed under the same conditions as a control. The release medium was replaced every 12 h. The results are expressed as the mean ± SD of three test runs.

116

M. Han et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 114–120

Table 1 HK-NSps all physical characteristics (n = 3). Particle size (nm)

PDI

Zeta potential (−mV)

Drug loading (%)

Highest drug concentration (mg/mL)

116.2 ± 1.77

0.12 ± 0.02

44.7 ± 1.7

50.4 ± 0.6

10.18

2.11. Pharmacokinetics and biodistribution 2.11.1. Formulation Bulk HK powder was dispersed in 0.8% (w/v) CMC-Na solutions to make coarse suspensions (HK-CMCS); the resulting suspensions were subjected to continuous ultrasound (Kunshan Ultrasonic Instruments Co., Ltd., Kunshan, China) for 10 min before intragastric (i.g.) administration. The freeze-dried HK-NSps were reconstituted in 0.5 M PBS (pH 8.0) before i.g. administration and in 0.9% NaCl before intraperitoneal (i.p.) administration. 2.11.2. Animals and cell lines Male Sprague-Dawley (SD) rats (220 ± 20 g) and male ICR mice (25 ± 2 g) were obtained from the Experimental Animal Centre of Peking University (Beijing, China). All of the animal experiments complied with the principles of care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Centre. The animals were acclimatised at 25 ± 2 ◦ C, a relative humidity of 70 ± 5% and under natural light/dark conditions for 1 week with food and water ad libitum. The H22 liver tumour cell lines used in the animal experiments were gifts from the Chinese Academy of Medical Science (Beijing, China). 2.11.3. Pharmacokinetics and bioavailability in rats Male Sprague-Dawley rats were randomly divided into two groups of 5 and fasted for 24 h (unrestricted water) before use. HK-NSps (reconstituted in 0.5 M PBS (pH 8.0)) and HK-CMCS were administered i.g. at a dose of 20 mg/kg. Water was freely available, but no food was allowed until 6 h had passed since drug administration. Blood samples were collected via the orbital sinus at 0, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h after administration. After centrifugation at 5000 rpm for 5 min, plasma was separated and stored at −80 ◦ C until analysis. 2.11.4. Biodistribution in tumour-bearing mice ICR mice were inoculated subcutaneously with H22 liver tumour cells (5 × 105 cells per mouse) in the axillary region. H22 liver tumour-bearing mice were randomly sorted into groups of 5 at 8 days after the inoculation (tumours reached a volume of ca. 100 mm3 ). Then, freeze-dried HK-NSps reconstituted in 0.9% NaCl were administered i.p. at a single dose of 20 mg/kg. The same dose of HK-NSps and HK-CMCS was administered i.g. for a comparative biodistribution study. This time, the HK-NSps were reconstituted in 0.5 M PBS (pH 8.0) to offset the impact of the acidic gastric environment on the HK-NSps particle size. Blood samples were obtained from the fossa orbitalis vein at 0.5, 1, 3, 6, 12 and 24 h, placed into heparinised test tubes and centrifuged (5000 rpm for 5 min) to obtain plasma samples. Afterwards, the heart, liver, spleen, lung, kidney, brain and tumour were collected and accurately weighed. After collection, both plasma and tissues were stored at −80 ◦ C until analysis. 2.11.5. Plasma and tissue sample preparation An aliquot of 200 ␮L of plasma was transferred into a clean 2.0 mL test tube, 1 mL of acetonitrile was added, and the mixture was vigorously mixed by vortexing for 30 s to precipitate the protein. After incubation at 25 ◦ C for 10 min and centrifugation at 10,000 rpm for 5 min, the supernatant was transferred to another

tube that contained 50 mg of sodium chloride. After vortexing for 30 s, suspensions were kept for 10 min at 25 ◦ C and centrifuged at 10,000 rpm for 5 min. A sample of 20 ␮L of supernatant was directly injected into the HPLC system for analysis. All tissues were prepared as 25% aqueous homogenates: 1 g of tissue was homogenised with 3 mL of 0.9% NaCl (glass tissue homogeniser, 5 min). Tissue homogenates were processed in the same way as plasma samples and analysed using HPLC. 2.12. Statistical analysis Pharmacokinetic parameters were calculated using noncompartmental methods with WinNonlin (V 4.0, Pharsight Corporation, USA). A statistical analysis of bioavailability and biodistribution in H22 liver tumour-bearing mice was performed using one-way analysis of variance. Data are presented as the mean ± standard deviation for all treatments. 3. Results and discussion 3.1. Preparation of HK-NSps To obtain a smaller particle size and narrower size distribution, PVP and BSA were used as stabilisers to prepare HK-NSps. The mean particle size of the resulting HK-NSps was 116.2 ± 1.77 nm with a PDI of 0.12 ± 0.02; the zeta potential was −44.7 ± 1.7 mV. The drug loading capacity was determined to be 50.4 ± 0.6% (w/w), and the drug concentration reached 10.18 mg/mL (Table 1). With decreasing concentration, HK-NSps became clearer and tended to exhibit a pale blue opalescence, but the particle size and PDI did not significantly change. TEM photomicrographs are presented in Fig. 1. The sizes of the HK-NSps determined using TEM were in good agreement with those determined by DLS, confirming that precipitation–ultrasonication produced submicron particles with a narrow size distribution [21]. 3.2. Lyophilisation of HK-NSps Lyophilisation of NSps can be performed to overcome either physical or chemical instability, permitting the retention of the original particle size after reconstitution. Hydrophobic interactions may cause HK-NSps particles to aggregate during the freeze-drying process. To prevent this, the addition of a cryoprotective agent (mannitol) was tested at various concentrations. The mean particle size and PDI values of HK-NSps reconstituted in 0.9% NaCl were determined. We found that 0.4% mannitol led to the best cryoprotective effect, resulting in reconstituted HK-NSps with nearly the same sizes and PDI values as before lyophilisation. 3.3. Stability of reconstituted HK-NSps The stability of reconstituted HK-NSps (in 0.9% NaCl solution) during storage was studied. After 7 days, the particle size of HKNSps remained nearly unchanged from the size observed before lyophilisation (124 ± 2.4 nm), whether it was stored at 25 ◦ C or 4 ◦ C. The particle size increased significantly under both conditions after 15 days of storage (175.3 ± 1.3 and 147.5 ± 0.5 nm at 25 ◦ C and 4 ◦ C, respectively). These results indicate that reconstituted HK-NSps should be used within 7 days to ensure particle size consistency.

M. Han et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 114–120

117

Fig. 2. (A) Particle size of HK-NSps after incubation in simulated gastrointestinal fluids at 37 ◦ C for 1, 3 and 5 h (n = 3), (B) particle size of HK-NSps before and after incubation at different pHs at 37 ◦ C for 3 h (n = 3) and (C) particle size of HK-NSps (reconstituted with 0.5 M PBS, pH 8.0) incubated in simulated gastric fluid at 37 ◦ C for 1, 3 and 5 h (n = 3).

Fig. 1. TEM of HK-NSps: (A) HK-NSps (20,000×) and (B) HK-NSps (50,000×).

To explore the possibility of intravenous administration, the stability of reconstituted HK-NSps was studied in plasma. The particle size of HK-NSps at all concentrations (0.5, 1 and 2 mg/mL HK) increased with incubation time. The concentration of HK-NSps had a significant effect on the stability in plasma. At 0.5 or 1 mg/mL, the HK-NSps particle size increased by less than 25 nm after 5 h of incubation. At 2 mg/mL, however, the particle size dramatically increased from 129.1 nm to 2709 nm after only 1 h of incubation in rat plasma. This result indicated that reconstituted HK-NSps were not sufficiently stable in plasma and have to be injected slowly at a low dose for intravenous administration. Stability in gastrointestinal fluids determines whether HK-NSps are suitable for oral administration to improve HK dissolution and bioavailability. For this reason, HK-NSps (containing 2 mg/mL HK, reconstituted with 0.9% NaCl) were incubated in simulated gastrointestinal fluids and the particle size was analysed. As shown in Fig. 2A, the particle size did not change, even after the 5 h incubation of HK-NSps in simulated intestinal fluid. However, the HK-NSps particle size increased immediately from 147.6 nm to 230 nm, and then exceeded 500 nm after 1 h of incubation in

artificial gastric fluid. This suggested that HK-NSps were quite stable in intestinal fluid but unstable in gastric fluid. The pH value of HK-NSps, artificial gastric fluid and artificial intestinal fluid is 7.03, 1.90 and 6.80 respectively. When HK-NSps were mixed with artificial gastric fluid, the pH of the micro-environment where HKNSp stay will experience a change from pH 7 to nearly pH 2. It has been demonstrated that during this period of pH change, HK-NSps will aggregate immediately. The reason may be the zeta potential change of the HK-NSps during this process. The zeta potential values of HK-NSps were 5.6, 10.9, 11.9, 7.8, −1.0, −5.6, −11.9, −15.0, −20.3, −24.5 in pH 1, 2, 3, 4, 4.5, 5, 5.5, 6, 7, 8 respectively, and it was found that during the pH change from 4 to 4.5, HK-NSps experience a zero zeta potential phase. It was presumed that it was this zero zeta potential phase that induced HK-NSps aggregation. When incubated of HK-NSps in intestinal fluid (pH 6.80), the pH of the mixture system will maintain nearly 7, so HK-NSps can still have relatively high zeta potential, this is why the obtained HK-NSps are stable in intestinal fluid. Further examination found that HK-NSps were unstable and prone to aggregation within the pH range 3.5–5.5 (Fig. 2B); outside of this range, for example at pH 1–3.5 and pH 5.5–8, HK-NSps were quite stable. Thus, HK-NSps would be able to pass through the stomach and reach the intestinal tract at their original particle size if the pH could be maintained over 5.5 throughout. For this purpose, a series of concentrations of PBS (pH 8.0, ranging from 0.05 M to 2 M) was mixed with 4 times the volume of simulated gastric fluid to determine whether the final pH of the resulting mixture would be maintained above 5.5. Finally, 0.5 M PBS (pH 8.0) was found to be sufficient to buffer the simulated gastric fluid and maintain the particle size of HK-NSps (Fig. 2C). Therefore, for i.g. administration, lyophilised HK-NSps should be reconstituted with 0.5 M PBS (pH 8.0) to maintain the particle size to enable enhanced absorption in the gastrointestinal tract. 3.4. Crystal properties of freeze-dried HK-NSps To study the state of HK in NSps, DSC thermal analysis was made for freeze-dried HK-NSps, HK bulk powder alone and physical mixture of HK bulk powder with excipients (Fig. 3A). HK bulk powder

118

M. Han et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 114–120

Fig. 3. (A) Differential scanning calorimetry thermograms for (top to bottom): freeze-dried HK-NSps, mixed HK bulk powder and excipients, HK bulk powder and (B) X-ray diffraction spectra (top to bottom): HK bulk powder, mixed HK bulk powder and excipients, freeze-dried HK-NSps.

exhibited a sharp melting peak at approximately 100 ◦ C, indicating that the drug was in crystalline form, although the endothermic melting peak was not present for the freeze-dried HK-NSps samples, indicating the absence of crystalline HK in freeze-dried NSps [22,23]. The XRPD spectra of HK bulk powder with and without excipients and freeze-dried HK-NSps are shown in Fig. 3B. The XRPD spectrum of bulk powder exhibited intense peaks indicative of a crystalline compound. The XRPD spectrum of the HK powder–excipient mixture was a composite of individual components, indicating that no new physical state formed. The characteristic diffraction peak of HK bulk powder disappeared in the freeze-dried HK-NSps samples. The profile for freeze-dried HKNSps was very flat with no intense peaks, indicating the loss of the crystalline structure, suggesting that HK may be present in an essentially amorphous state in the freeze-dried NSps [21,23]. 3.5. In vitro release from nanosuspensions This study investigated the rate of HK release from NSps and from CS. Typical cumulative release curves are presented in Fig. 4A. Compared to CS, the release from NSps was markedly enhanced: nearly 94% of the HK was released within 48 h, as opposed to only 27% for CS. Meanwhile, no burst release was observed in either case, with less than 5% cumulative release within 1 h. The enhanced release of HK from NSps can be attributed to the increased surface area and enhanced solubility of HK in NSps. Formulating poorly soluble HK as NSps thus had a dramatic effect on the drug’s solubility and release rate.

Fig. 4. (A) The in vitro release profiles of HK from CS and NSps. HK-NSps solutions (1 mL, 5 mg/mL HK) were placed in a dialysis tube (molecular mass cutoff 5.0 kDa). HK-CS (5 mg/mL HK) was used as a control. The dialysis tubes were incubated in 50 mL of phosphate buffer (pH 7.4) containing 0.5% SDS. The temperature was held at 37 ± 0.5 ◦ C with shaking (100 rpm) and (B) mean plasma concentration–time curves of HK in rats after i.g. administration of a single dose of HK-CMCS and HK-NSps (20 mg/kg). Points and bars represent the mean ± SD (n = 5).

3.6. Pharmacokinetics and oral bioavailability The plasma concentration–time curves of HK-NSps exhibited an obvious improvement in drug absorption over HK-CMCS in SD rats after i.g. administration at a single dose of 20 mg/kg body weight. After the i.g. administration, the concentration–time curves of HKNSps and HK-CMCS mainly differed between 0 and 2 h (Fig. 4B). The bioavailability of the formulations will be more feasible if the HK coarse suspensions and HK nanosuspensions could be intravenously injected or there was HK solution intravenously injectable. However, the HK coarse suspensions are NOT suitable for i.v. administration because of their large particle size and instability in plasma, the HK nanosuspensions are NOT suitable for i.v. administration because of their hemolytic effect. And we have tried many

M. Han et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 114–120

119

Fig. 5. Mean concentration of HK (ng/g of tissue) in mouse tissues following i.g. administration of HK-NSps and HK-CMCS and i.p. administration of HK-NSps (20 mg/kg) (n = 5).

methods and failed to find a formulation to make HK solution that is suitable for i.v. administration. Based on the above consideration, the RELATIVE bio-availability calculated by AUC(oral)/AUC(IP) was used. A pharmacokinetic analysis was performed using Phoenix WinNonlin (version 6.1). The mean pharmacokinetic parameters of HK-NSps were significantly different from those of HK-CMCS (Table 2). The Cmax and AUC(0–t) values of HK-NSps were approximately 3.94-fold and 2.2-fold greater than those of HK-CMCS. The improved oral bioavailability of HK-NSps can be explained as follows. First, the smaller particle size increased the surface area and reduced the thickness of the diffusion layer of HK-NSps against HK bulk powder, leading to enhanced HK dissolution and then enhanced absorption [24,25]. Second, the adhesion between nanoparticles and intestinal villi prolongs the contact of HK-NSps with the absorbing membranes of the gut [26,27]. Third, a small portion of HK-NSps may pass through the Peyer’s patch of the small intestine and then be directly absorbed in nanoparticle form, as previously reported [28]. Adhesion of HK-NSps to intestinal villi could prolong the effective drug absorption time. This may be why HK-NSps showed an extended plasma MRT in comparison with HKCMCS (6.86 h vs. 3.77 h) and a longer t1/2 (23.7 h vs. 6.9 h) after oral administration. 3.7. Biodistribution of HK-NSps The biodistribution of HK-CMCS was investigated following i.g. administration of a single dose to tumour-bearing mice at 20 mg/kg. The biodistribution of HK-NSps was investigated following both i.g. and i.p. administration of a single dose to tumour-bearing mice at 20 mg/kg. HK concentrations in various tissues including the heart,

liver, spleen, lung, kidney, brain, tumour and plasma were determined by HPLC. Fig. 5 shows the mean HK concentration–time profiles in all the tissues and plasma for the above three experiments. The corresponding statistical analysis is reported in Table 3. The Cmax in all tissues was observed at 0.5 h after administration and declined thereafter until 12 h. However, the concentrations in various tissues were markedly different. The highest Cmax and AUC values among all the test tissues were found in the liver, followed by (in descending order) the kidney and lung. The much higher drug level in the kidney than in other organs (Table 3) indicated that the kidney may be the primary route of excretion of HK. Fig. 5 and Table 3 clearly show that i.g. administration of HKNSps resulted in higher AUC values than HK-CMCS in all tissues tested in mice, with a 2.89-fold increase in plasma AUC, a 4.04fold increase in kidney AUC and a less than 2-fold increase in AUC in other tissues. As previously observed in the results of a pharmacokinetic study in rats (Table 2), oral administration of NSps effectively improved not only the blood bioavailability but also the bioavailability in important organs. After i.g. administration, HK was mainly absorbed into blood in the form of free HK. The much higher Cmax and AUC(0–t) values in the liver and kidney than in other organs (Table 3) indicated that free HK may naturally tend to be biodistributed to the liver and kidney. To gain deeper insight into the in vivo characteristics of HKNSps, the biodistribution data in the tissues tested after both i.p. and i.g. administration were compared in Table 3. Compared with i.g. administration, i.p. administration led to a 9.08-fold greater plasma AUC, 3.63-fold greater heart AUC and 14.67-fold greater brain AUC, with a 45.28-fold greater plasma Cmax , 6.19-fold greater heart Cmax and 10.22-fold greater brain Cmax . This dramatically

Table 2 Pharmacokinetic parameters of HK in rats following i.g. administration of single doses of HK-CMCS and HK-NSps (20 mg/kg, n = 5). Parameter

Unit

NSps

CMCS

NSps/CMCS

Cmax Tmax AUC(0–t) t1/2 MRT(0–t)

ng/mL h h ng/mL h h

1456.59 ± 168.59 0.25 2623.40 ± 289.46 23.74 ± 6.23 6.86 ± 0.41

369.27 ± 84.70 0.25 1187.55 ± 152.96 6.87 ± 6.49 3.77 ± 0.63

3.94 2.2

120

M. Han et al. / Colloids and Surfaces B: Biointerfaces 116 (2014) 114–120

Table 3 Mean Cmax and AUC(0−t) (n = 5) of HK in H22 liver tumour-bearing mice following the i.g. and i.p. administration of HK-NSps and the i.g. administration of HK-CMCS (20 mg/kg). Tissue

Plasma Heart Liver Spleen Lungs Kidney Brain Tumour

Mean Cmax (ng/g) i.g. (CMCS)

i.g. (NS)

37.97 177.87 11,860.24 374.33 591.27 601.90 149.37 158.01

66.60 256.68 7518.83 208.07 851.97 3106.08 186.27 162.37

Cmax (NS) (i.p./i.g.) i.p. (NS) 3015.76 1589.93 20,794.62 1510.98 2392.69 4354.84 1903.69 1093.28

Mean AUC(0–t) (h ng/g) i.g. (CMCS)

45.28 6.19 2.77 7.26 2.81 1.40 10.22 6.73

enhanced biodistribution to the blood, heart and brain illustrated that i.p. administered HK-NSps have an excellent potential to benefit the therapy of cardio-cerebro-vascular diseases. At the same dose of 20 mg/kg of HK-NSps, i.p. administration achieved a 9.08-fold greater plasma AUC and a 45.28-fold greater plasma Cmax than i.g. administration; however, the liver AUC (0–t) was almost unchanged (Table 3). The greater HK delivery to the blood, the lack of enhancement in liver biodistribution and the dramatic enhancement in delivery to the cardio-cerebro-vascular system suggested that i.p. administration of HK-NSps may enter the blood in some other form in addition to free HK. Nanoparticles have been reported to pass through endothelia [28], and nanoparticles help to increase drug absorption in the brain via passive diffusion and carrier-mediated transport [29–32]. However, further investigation is required to determine whether and how HK-NSps could be directly absorbed into blood after i.p. administration. Unfortunately, although HK-NSps demonstrated enhanced biodistribution in tumour to a certain extent (1.92-fold greater tumour AUC by i.g. administration, NSps vs. CMCS; 1.85-fold greater tumour AUC, HK-NSps, i.p. vs. i.g.), this enhancement seemed to be insufficient to enable tumour-targeted drug delivery. 4. Conclusions To the best of our knowledge, this is the first report of the preparation of HK-NSps and their testing in in vitro and in vivo studies. HK-NSps formed smooth spheres with a hydrodynamic diameter of approximately 120 nm. Nanosuspensions released HK faster than bulk powder, mainly due to their significantly reduced particle size and the amorphous state of HK in nanosuspensions. In vivo experiments confirmed that nanosuspensions significantly increased the oral bioavailability of HK. In contrast to i.g. administration, i.p. administration of HK-NSps dramatically altered the in vivo HK biodistribution, resulting in a much higher drug level and tissue bioavailability in the blood, heart and brain, making HK-NSps a promising drug delivery system for the treatment of cardio-cerebro-vascular diseases. Acknowledgements This work was supported by the China International Science and Technology Cooperation Program for Key Projects (no. 2008DFA31070), the National Natural Science Foundation of China (no. 81102813) and the National Mega-project for Innovative Drugs (2012ZX09301002-001).

145.64 458.58 37,954.04 1000.89 1899.95 2151.23 95.02 786.37

i.g. (NS) 420.65 765.58 43,068.93 1694.95 3543.95 8684.13 122.61 1512.40

AUC (i.g.) i.p. (NS)

(NS/CMCS)

3820.06 2781.21 43,212.89 3385.73 4503.66 1,1072.22 1798.22 2793.34

2.89 1.67 1.13 1.69 1.87 4.04 1.29 1.92

AUC (NS) (i.p./i.g.)

9.08 3.63 1.00 2.00 1.27 1.27 14.67 1.85

References [1] H. Haraguchi, H. Ishikawa, N. Shirataki, A. Fukuda, J. Pharm. Pharmacol. 49 (1997) 209. [2] W.J. Syu, C.C. Shen, J.J. Lu, G.H. Lee, C.M. Sun, Chem. Biodivers. 1 (2004) 530. [3] M.K. Pyo, Y. Lee, H.S. Yun-Choi, Arch. Pharm. Res. 25 (2002) 325. [4] K.T. Liou, Y.C. Shen, C.F. Chen, C.M. Tsao, S.K. Tsai, Eur. J. Pharmacol. 475 (2003) 19. [5] H. Kuribara, E. Kishi, M. Kimura, S.T. Weintraub, Y. Maruyama, Pharmacol. Biochem. Behav. 67 (2000) 597. [6] X. Bai, F. Cerimele, M. Ushio-Fukai, M. Waqas, P.M. Campbell, B. Govindarajan, C.J. Der, T. Battle, D.A. Frank, K. Ye, E. Murad, W. Dubiel, G. Soff, J.L. Arbiser, J. Biol. Chem. 278 (2003) 35501. [7] A.A. Noyes, W.R. Whitney, J. Am. Chem. Soc. 19 (1897) 930. [8] C.M. Keck, R.H. Müller, Eur. Pharm. Biopharm. 62 (2006) 3. [9] L. Gao, D. Zhang, M.H. Chen, C.X. Duan, W.T. Dai, L.J. Jia, W.F. Zhao, J. Nanopart. Res. 10 (2008) 845. [10] G.G. Liversidge, K.C. Cundy, Int. J. Pharm. 125 (1995) 91. [11] R. Pignatello, C. Bucolo, P. Ferrara, A. Maltese, A. Puleo, G. Puglisi, Eur. J. Pharm. Sci. 16 (2002) 53. [12] C. Jacobs, R.H. Müller, Pharm. Res. 19 (2002) 189. [13] X.S. Li, J.X. Wang, Z.G. Shen, P.Y. Zhang, J.F. Chen, J. Yun, Int. J. Pharm. 342 (2007) 26. [14] X.H. Wang, L.L. Cai, X.Y. Zhang, L.Y. Deng, H. Zheng, J.L. Deng, X. Zhao, Y.Q. Wei, L.J. Chen, Int. J. Pharm. 410 (2011) 169. [15] H. Luo, Q. Zhong, L.J. Chen, X.R. Qi, A.F. Fu, H.S. Yang, F. Yang, H.G. Lin, Y.Q. Wei, X. Zhao, J. Cancer Res. Clin. Oncol. 134 (2008) 937. [16] J. Hu, L.J. Chen, L. Liu, X. Chen, P. Chen, G.L. Yang, W.L. Hou, M.H. Tang, F. Zhang, X.H. Wang, Z. Xia, Y.Q. Wei, Exp. Mol. Med. 40 (2008) 617. [17] Q. Neng, L.L. Cai, D.C. Xie, G.C. Wang, W.S. Wu, Y.K. Zhang, H. Song, H.B. Yin, L.J. Chen, J. Biomed. Mater. 065006 (2010) 1748. [18] C.Y. Gong, X.W. Wei, X.H. Wang, Y.J. Wang, G. Guo, Y.Q. Mao, F. Luo, Z.Y. Qian, Nanotechnology 21 (2010) 215103. [19] C.Y. Gong, Y.J. Wang, X.H. Wang, X.W. Wei, Q.J. Wu, B.L. Wang, P.W. Dong, L.J. Chen, F. Luo, Z.Y. Qian, J. Nanopart. Res. 13 (2011) 721. [20] Y.X. Zhao, H.Y. Hua, M. Chang, W.J. Liu, Y. Zhao, H.M. Liu, Int. J. Pharm. 392 (2010) 64. [21] X. Pu, J. Sun, Y. Wang, X. Liu, P. Zhang, X. Tang, W. Pan, J. Han, Z. He, Int. J. Pharm. 379 (2009) 167. [22] W. Sun, S.R. Mao, Y. Shi, L.C. Li, L. Fang, J. Pharm. Sci. 100 (2011) 3365. [23] L. Gao, G.Y. Liu, X.Q. Wang, F. Liu, Y.F. Xu, J. Ma, Int. J. Pharm. 404 (2011) 231. [24] R.J. Hintz, K.C. Johnson, Int. J. Pharm. 51 (1989) 9. [25] L. Jia, H. Wong, C. Cerna, S.D. Weitman, Pharm. Res. 19 (2002) 1091. [26] X.M. Li, L. Gu, Y.L. Xu, Y.L. Wang, Drug Dev. Ind. Pharm. 35 (2009) 827. [27] A. Lamprecht, P. Koening, N. Ubrich, P. Maincent, D. Neumann, Nanotechnology 17 (2006) 3673. [28] C.N. Grama, D.D. Ankola, M.N.V. Ravi Kumar, Curr. Opin. Colloid Interface Sci. 16 (2011) 238. [29] T.M. Goppert, R.H. Müller, J. Drug Target. 13 (2005) 179. [30] A. Brioschi, F. Zhang, G.P. Zara, Neurol. Res. 29 (2007) 324. [31] J. Huwyler, J. Wang, W.M. Pardridge, J. Pharmacol. Exp. Ther. 282 (1997) 1541. [32] R.H. Müller, C. Jacobs, O. Kayser, Adv. Drug Deliv. Rev. 47 (2001) 3.