European Journal of Pharmaceutical Sciences 60 (2014) 1–9

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Novel hydrophobin-coated docetaxel nanoparticles for intravenous delivery: In vitro characteristics and in vivo performance Guihua Fang, Bo Tang, Zitong Liu, Jingxin Gou, Yu Zhang, Hui Xu, Xing Tang ⇑ School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China

a r t i c l e

i n f o

Article history: Received 7 March 2014 Received in revised form 14 April 2014 Accepted 25 April 2014 Available online 9 May 2014 Keywords: Hydrophobin Docetaxel Nanoparticles Drug delivery Chemical compounds studied in this article: Docetaxel (PubChem CID: 148124) Paclitaxel (PubChem CID: 36314) Methanol (PubChem CID: 887) Tween 80 (PubChem CID: 6364656) Ter-Butyl methyl ether (PubChem CID: 15413) Formic acid (PubChem CID: 284) Acetonitrile (PubChem CID: 6342)

a b s t r a c t Novel hydrophobin (H star ProteinÒ B, HPB)-coated docetaxel (DTX) nanoparticles were designed for intravenous delivery. DTX-HPB nanoparticles (DTX-HPB-NPs) were prepared using a nanoprecipitation–ultrasonication technique. The physicochemical properties in terms of particle size, size distribution, zeta potential, morphology, crystalline state of the drug, in vitro release and plasma stability were evaluated. To investigate the drug–hydrophobin interaction, FTIR analysis was carried out. The pharmacokinetics of DTX-HPB-NPs and Taxotere were compared after i.v. administration to rats. The optimized formulations have a high drug loading (>25%) and nanoparticle yield (>93%), small particle size with a narrow distribution, and exhibit delayed release. X-ray diffraction (XRD) demonstrated that the drug is present in a crystalline state. FTIR analysis suggested that the interaction of DTX and HPB involved hydrogen bonding. In vitro hemolysis study confirmed the safety of these nanoparticles. In plasma, DTX-HPB nanoparticles exhibited a significantly enhanced Cmax (1300.618 ± 405.045 ng/mL vs 453.174 ± 164.437 ng/mL, p < 0.05), and AUC0t (409.602 ± 70.267 vs 314.924 ± 57.426 lg/L h, p < 0.05), and a significantly reduced volume of distribution (36.635 ± 15.189 vs 95.199 ± 40.972 L/kg, p < 0.05) compared with the Taxotere. These results demonstrated that hydrophobin has the potential to be used as a novel biocompatible biomaterial for drug delivery. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Over the last few decades, an increasing number of newly developed drugs have been found to be poorly water-soluble and, as a result, these so called ‘brickdust drugs’ often exhibit poor bioavailability (Porter et al., 2007; Rabinow, 2004). Different methods and formulations have been developed to overcome this problem and there has been considerable interest in the development of novel drug delivery systems using nanotechnology. For example, polymeric nanocarriers (Ernsting et al., 2012; Jones and Leroux, 1999) and lipid nanocarriers (Liu et al., 2011; Lowery et al., 2011) are now widely used. Numerous synthetic and natural polymers have been extensively investigated as polymeric materials for drug delivery applications. In any case, to be considered as a suitable material to deliver drugs in vivo, a polymer needs to fulfill several basic requirements (Nair and Laurencin, 2007). Firstly, it should be biocompatible and any potential degradation products should not have ⇑ Corresponding author. Tel.: +86 24 23986343; fax: +86 24 23911736. E-mail address: [email protected] (X. Tang). http://dx.doi.org/10.1016/j.ejps.2014.04.016 0928-0987/Ó 2014 Elsevier B.V. All rights reserved.

toxic or immunogenic effects. Secondly, the material should still have an acceptable long-term stability and it should possible to carry out some processing procedures during the preparation. Natural polymers, such as natural polysaccharides and their derivatives (Ernsting et al., 2012; Liu et al., 2008), gelatin (Lu et al., 2004), albumin (Han et al., 2010; Sebak et al., 2010) are described in detail in the literature for the preparation of drug delivery systems. Biosurfactants are an alternative to common natural polymers, are produced by microorganisms, and have pronounced surface and emulsifying activities due to their amphiphilicity (Mulligan, 2005). Hydrophobins are a family of low molecular weight proteins with a characteristic pattern of cysteine residues that form four disulfide bonds, and are exclusively found in fungi. They selfassemble to form robust polymeric monolayer films that are highly amphipathic and play an important role in fungal growth and development, forming protective films and their action is mediated by the attachment of fungi to solid surfaces (Hektor and Scholtmeijer, 2005; Sunde et al., 2008). Secreted hydrophobins have the ability to convert hydrophobic surfaces to hydrophilic ones and hydrophilic surfaces to hydrophobic ones by self-assembling into

2

G. Fang et al. / European Journal of Pharmaceutical Sciences 60 (2014) 1–9

an amphipathic protein membrane. On the basis of differences in hydropathy patterns and aqueous solubility of their assembled films, hydrophobins are classified into Class I and Class II hydrophobins (Linder et al., 2005). Hydrophobins, as fungal protein have attracted more attention in a number of pharmaceutical fields. Class I hydrophobin SC3 has been used to increase oral drug bioavailability (Haas Jimoh Akanbi et al., 2010), and Class II hydrophobin HFB II-coated porous silicon nanoparticles have been used to improve the biocompatibility and change the biodistribution after intravenous injection (Sarparanta et al., 2012). In addition, HFB II can be genetically modified to bioengineer nanoparticles, offering special bindings domain for target adhesion (Valo et al., 2011). Hydrophobins generally are considered to be safe because they are present in button mushrooms and other edible fungi (Paslay et al., 2013). As far as we know, to date, no reports have described whether in vivo pharmacokinetics can be modified by hydrophobin-coated drug nanoparticles after intravenous administration. Docetaxel (DTX), an analog of paclitaxel, is an effective and widely used anticancer drug in clinical practice (Rowinsky, 1997). However due to its high lipophilicity and low solubility, the main commercial formulation (TaxotereÒ) that is used clinically is formulated in Tween 80 and ethanol, which requires dilution in normal saline or 5% dextrose solution prior to intravenous administration. Tween 80 carries a high risk of producing serious side effects in patients, such as allergic reactions, neurotoxicity, and nephrotoxicity (Clarke and Rivory, 1999). Therefore, the development of an alternative drug delivery system for DTX is urgently required. In this work we describe the design and developed hydrophobin-coated drug nanoparticle to investigate the in vivo pharmacokinetic characteristics after intravenous administration, using DTX as a model hydrophobic drug. DTX-HPB nanoparticles were prepared by a nanoprecipitation–ultrasonication technique, and the physicochemical properties were characterized. The particle size distribution was determined by dynamic light scattering (DLS) and the morphology of the nanoparticles was characterized by transmission electron microscopy (TEM). The drug state in the nanoparticles was characterized by X-ray diffraction (XRD) and the drug–hydrophobin interaction was investigated by Fourier transform infrared spectroscopy (FTIR). An in vitro drug release study was conducted using an equilibrium dialysis method to evaluate the influence of hydrophobin on the release behavior of DTX. Finally, hemolysis test was carried out and the in vivo pharmacokinetics of DTX-HPB nanoparticles was evaluated to demonstrate that hydrophobin-coated drug nanoparticles can be used as an intravenous drug delivery system for hydrophobic drug.

2. Materials and methods 2.1. Materials and animals Docetaxel and paclitaxel were purchased from (Shanghai sanwei Pharma Ltd. Co., Shanghai, China), H Star ProteinÒ B, from now on abbreviated as HPB (18.8 kDa; IEP: 5.9; purity: 96%), is recombinant hydrophobins and is a gift from BASF, Ludwigshafen (Germany), and it belongs to Class I hydrophobins. While tert-butyl methyl ether (TBME, Sinopharm Chemical Reagent Ltd. Co., Shenyang, China), formic acid (Dima Technology Inc., Richmond Hill, USA), methanol, acetonitrile and dehydrated alcohol (Tianjin Concord Technology Ltd., Co., Tianjin, China) were obtained from the sources indicated, all other chemicals and reagents were of analytical or chromatographic grade. Male Sprague–Dawley rats (200 ± 20 g) were obtained from the Experimental Animal Center of Shenyang Pharmaceutical University and housed at 22 ± 2 °C with access to food and water ad libitum. The protocol for the animal experiments was approved

by the Shenyang Pharmaceutical University Institutional Animal Care and Use Committee. 2.2. Preparation of hydrophobin solution Prior to use, to ensure complete dissolution, a weighed amount of hydrophobin was dissolved in pure deionized water with magnetic stirring for about 2 days at room temperature. Then, the protein solution was passed through a 0.8 lm filter membrane to remove impurities in the protein solution. Finally, a clear protein solution was obtained which was used in all subsequent studies. 2.3. Preparation of hydrophobin-coated drug nanoparticles and bare drug nanoparticles A nanoprecipitation–ultrasonication technique (He et al., 2013) was used to prepare the nanoparticles. Methanol was used as a solvent for DTX and HPB aqueous solution was obtained as described above. Then, 0.5 mL of DTX solution was added dropwise to 10 mL of HPB aqueous solution. The resulting mixed solution was ultrasonicated using probe sonication (Sonics & Material Vibra Cell, 750 W, 20 kHz) at 53% amplification (400 W) for 10 min with a 3 s pulse-on period and a 3 s pulse-off period in an ice bath to keep the temperature low. The methanol was removed by rotary vacuum evaporation and then the nanoparticle suspension was centrifuged (8000 rpm, 10 min) and passed through a 0.8 lm cellulose ester filter membrane to remove the uncoated drug and any impurities. Bare drug nanoparticles were prepared as for the hydrophobin-coated nanoparticles except that no hydrophobin was present. 2.4. Particle size and zeta potential measurements The average particle size, size distribution, and zeta potential of the nanoparticles were determined by dynamic light scattering (PSS NICOMP 380, USA) at room temperature. He–Ne was used as light source, a laser beam at a wavelength of 632.8 nm, and the scattering angle was set at 90° when particle size measurements were conducted. Zeta potential measurements were performed at a laser beam wavelength 633 nm and a scattering angle 18.9°. Prior to making these measurements, all samples were diluted with deionized water to a suitable concentration, avoiding any multiscattering. Each parameter was measured three times, and the average values and standard deviations were calculated. 2.5. Determination of hydrophobin nanoparticle yield In order to determine the amount of HPB transformed into nanoparticles, the hydrophobin-coated DTX nanoparticles were separated from the supernatant by centrifugation at 50,000 rpm for 2 h at 4 °C using a high speed refrigerated centrifuge (HITACH, Japan). An aliquot of the supernatant was diluted with deionized water and the amount of the HPB in the supernatant was determined using a standard Coomassie Brilliant Blue protein assay (Sedmak and Grossberg, 1977). The calculations were made on the base of standard curve which was r > 0.996 in the concentration range 20–80 lg/mL. All sample analyses were carried out in triplicate. The yield of nanoparticles (Ynp) could be calculated as the follow equation:

Y np ð%Þ ¼

W totalHPB  W freeHPB  100 W totalHPB

2.6. Particle morphology The morphology of the hydrophobin-coated docetaxel nanoparticles was visualized by transmission electron microscopy (TEM)

G. Fang et al. / European Journal of Pharmaceutical Sciences 60 (2014) 1–9

(Hitachi H-600, Tokyo, Japan). A drop of 100 lg/mL nanoparticle suspension was deposited on a 200 mesh carbon-coated copper grid, then excess solution was wiped away with the aid of filter paper, and all samples were further dried for 30 min at room temperature. Then, the samples were examined under TEM with an accelerating voltage of 100 kV. 2.7. X-ray diffraction (XRD) To evaluate the physical state of DTX in the nanoparticles, fresh sample prepared as described above were lyophilized before the measurements were carried out. XRD data were recorded on a D/ Max-2400 diffractometer (Rigaku Instrument, Japan) using Cu Ka radiation (40 kV and 30 mA) at a scanning rate of 4° per minute over the range of 5–40°. 2.8. Fourier transform infrared spectroscopy (FTIR) To characterize the protein secondary structure and the intermolecular interactions, Fourier-transform infrared (FTIR) spectra were obtained on a BRUKER IFS 55 FTIR system using the KBr disk method. The transmittance spectra were recorded at a resolution of 1 cm1 between 4000 and 400 cm1. 2.9. HPLC analysis of DTX DTX concentrations were determined by high-performance liquid chromatography (HPLC) (Hitachi L-2130 pump, Hitachi L2400 UV–Vis detector). Briefly, a solution of hydrophobin-coated nanoparticles was diluted 100-fold with acetonitrile and sonicated in a water bath to precipitate the protein and dissolve the drug. The resulting solution was centrifuged for 10 min at 12,000 rpm at 4 °C, then the supernatant was transferred to an HPLC vial and analyzed. Chromatographic separation was performed using a reverse-phase ODS Diamonsil C18 column (5 lm, 250  4.6 mm) at room temperature, and acetonitrile and water (60:40, v/v) was selected as mobile phase. The flow rate was set at 1 mL/min and the wavelength of UV detector was 230 nm. Drug loading ratio (DL) was was defined as the percentage of the drug encapsulated in nanoparticle to the total weight of DTX loaded nanoparticles.

3

dialysis bag (MWCO: 3.5 kDa), and the experimental and detection conditions were the same as in the in vitro release study described above, and the in vitro release of nanoparticles and the mixture of Taxotere and plasma were used as a control. 2.12. In vitro hemolysis In order to determine whether the hydrophobin-coated drug nanoparticles are safe for intravenous administration, healthy rabbit blood was used to perform a hemolysis test. Briefly, rabbit blood was obtained from the marginal ear vein, and the fibrinogen was removed by stirring gently with a glass rod. Then, after fibrinogen removal, the blood sample was diluted 10-fold with normal saline, and the supernatant was removed by centrifugation at 1500 rpm for 10 min, and the above process (from dilution to centrifugation) was repeated at least three times until the supernatant was clear. Finally, an appropriate amount of normal saline was added to the erythrocyte pellets to obtain a standard 2% erythrocyte dispersion. Different amounts of nanoparticle dispersion and Taxotere solution with volumes of 0.1, 0.2, 0.3, 0.4 and 0.5 mL were added to five tubes each containing 2.5 mL 2% erythrocyte dispersion. Then, appropriate amounts of normal saline were added to every tube to obtain a final volume of 5 mL. A positive control was prepared by the addition of 2.5 mL water to 2.5 mL of the 2% erythrocyte dispersion instead of the normal saline and nanoparticle dispersion. A negative control was prepared by addition of 2.5 mL normal saline to 2.5 mL the 2% erythrocyte dispersion. After adding all samples, the tubes were incubated at 37 °C for 4 h. Samples were centrifuged at 3000 rpm for 10 min, then the supernatant was analyzed for released hemoglobin by spectrophotometry at 540 nm. The hemolysis ratio (HR%) was calculated according to the following equation and all samples were analysed in triplicate.

HR ð%Þ ¼

Asample  Anegative  100 Apositive  Anegative

where Asample, Anegative and Apositive are the absorbance of the sample, negative control and positive control, respectively.

2.10. In vitro release

2.13. In vivo pharmacokinetics

Drug release studies were performed using a dialysis technique (Gu et al., 2012). The experiments were carried out under sink conditions using phosphate buffer solution (PBS, pH 7.4) containing 0.1% Tween-80 (w/v) as the release medium. In brief, 1 mL of DTX-HPB nanoparticle dispersion, Taxotere solution, DTX ethanol solution and the mixture of Taxotere and HPB solution were sealed in a preswelled cellulose acetate dialysis bag (MWCO: 3.5 kDa) and immersed in 10 mL release medium, then placed in a horizontal water bath shaker maintained at 37 ± 0.5 °C with a shaking speed of 100 rpm. At designated intervals, 10 mL samples were removed for analysis and replaced with the same volume of fresh release medium. After passage through a 0.22 lm membrane filter, the amount of DTX released was evaluated by HPLC under the same HPLC conditions as described above. All sample release studies were carried out in triplicate.

In order to evaluate the potential application of hydrophobin as a biocompatible biomaterial for coating hydrophobic drugs, a study of the in vivo pharmacokinetics of hydrophobin-coated DTX nanoparticles and Taxotere was conducted in Sprague–Dawley rats after tail vein injection. The rats were fasted 12 h before the experiments with free access to water and randomly divided into two groups, then injected with Taxotere and DTX-HPB-NPs at 4 mg/ kg, respectively. At 0.0833, 0.25, 0.5, 1, 2, 4, 8 and 12 h after administration, 0.5 mL blood samples were collected into heparinized tubes. Then, plasma was separated immediately by centrifugation at 4000 rpm for 10 min and stored at 20 °C for analysis. DTX was extracted from the plasma by liquid–liquid extraction method for Ultra-Performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) (Waters Corp., Milford, MA, USA) analysis. Briefly, 20 lL of the internal standard solution (1000 ng/mL PTX methanol solution) was added to 200 lL rat plasma and vortexed for 1 min. Then, 3 mL tert butyl methyl ether was added to the mixture, vortexed another 10 min, and centrifuged at 4000 rpm for 10 min. Then, 2 mL supernatant was collected and evaporated to dryness under nitrogen at 35 °C. The residue was reconstituted with 100 lL of methanol and centrifuged at 12,000 rpm for 10 min, then an aliquot of 5 lL supernatant was injected into the UPLC–MS/MS system for

2.11. In vitro plasma stability To investigate whether plasma enzymes cause degradation of the outer hydrophobin shell of the nanoparticles, an in vitro plasma stability was conducted. Briefly, a mixture of 100 lL hydrophobin-coated nanoparticles dispersion and 900 lL freshly prepared rat plasma was sealed in a preswelled cellulose acetate

4

G. Fang et al. / European Journal of Pharmaceutical Sciences 60 (2014) 1–9

measurement. Analytical data were acquired using MassLynx software. The chromatographic separation was performed using a C18 column (ACQUITY UPLC™ BEH C18, 50 mm  2.1 mm, 1.7 lm, Waters Corp, Milford, MA, USA). The flow rate was set at 0.2 mL/ min, and the column was maintained at 40 °C. The mobile phase consisted of A (acetonitrile) and B (deionized water containing 0.1% formic acid). The elution started with 50%A then the composition was linearly changed to 20%A over 0.9 min and maintained at that level for 0.7 min. Finally, the composition was returned to the initial composition over 0.1 min and maintained there for 0.8 min. Mass spectrometric detection was performed in positive ESI mode with the chromatographic system. The capillary voltage was 2.5 kV, the ion source temperature was 100 °C and the collision energy was 3 V. Quantification was monitored using the MRM of the transitions of m/z 808.25 ? 527.17 for DTX and m/z 854.54 ? 285.96 for PTX with a scan time of 0.2 s. Samples were quantitatively tested against a calibration curve over the range 5–5000 ng/mL. The correlation coefficients were 0.994. All the concentration data were dose-normalized and plotted as plasma drug concentration–time curves. Pharmacokinetic parameters analysis was performed using Drug and Statistics for Windows (DAS ver 2.0) software. Appropriate models fitting the plasma concentrations data were evaluated according to the goodness of fit for each model. In particular, Akaike’s Information Criterion (AIC) Rule was used to determine the appropriate compartment model, and the minimum AIC value confirmed the optimum compartment model. The single compartment model was selected as appropriate, when optimum compartment model and suboptimum compartment model exhibited no significant difference. 2.14. Statistical analysis All the data were expressed as mean ± standard deviation (SD). Comparison between two groups was determined by Student’s ttest. A statistically significant difference was defined as P < 0.05. 3. Results 3.1. Preparation and characterization of hydrophobin-coated drug nanoparticles Because hydrophobin is an amphipathic protein and can adsorb between the interface of hydrophilic and hydrophobic area, hydrophobin can coat drug particles by physical interaction and form stable nanoparticles. Fig. 1B illustrates the establishment of hydrophobin-coated nanoparticles. In order to obtain pure protein solution, pretreatment was carried out. Hydrophobin solubility in water is up to 3%, and at the HPB solubility limit, a clear hydrophobin solution was obtained. Nanoparticles were prepared by combining nanoprecipitation and ultrasonication techniques. The added HPB had been formed into nanoparticles considerably, and a particle yield of above 93% could be obtained. Dynamic light scattering (DLS) and HPLC experiments were carried out to examine the effect of different DTX concentrations on the size, surface charge and drug-loading content of the nanoparticles. As summarized in Table 1, different amounts of DTX can form nanoparticles in the size range of 186–300 nm and with different drug-loading contents. It is clear that each formulation exhibited a narrow particle size distribution (PI < 0.2). The formulation dispersion is light blue in color, caused by light scattering of the nanoprticles in the solution. However, when DTX predissolved in methanol was added into deionized water in the absence of hydrophobin, and the other

Fig. 1. (A) The chemical structure of docetaxel and the potential hydrogen bonds. Red circle: hydrogen acceptor, Blue circle: hydrogen donor; (B) schematic illustration of the nanoprecipitation–ultrasonic preparation of a protein nanoparticle with an inner drug core and an outer protein shell. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

preparation conditions were the same as for hydrophobin-coated nanoparticles, an unstable dispersion was formed followed by immediate precipitation. The photographs of the hydrophobincoated nanoparticles and the bare drug particles were shown in Fig. 2. TEM was used to monitor the nanoparticle morphology at different magnifications, and the TEM images are presented in Fig. 3. Different hydrophobin concentrations produced different particle morphologies. The DTX concentration was maintained at 15 mg/mL, when protein concentration was 0.05%, as shown in Fig. 3A, only a few DTX nanocrystals clustered together forming large aggregates. This image revealed that the protein concentration (0.05%) was not sufficient for full coverage on the drug particle surface to provide enough steric repulsion between the drug particles. Compared with Fig. 3B, when the protein concentration was up to 0.1%, the morphology and contours of nanoparticle were regular and uniform, and the enlarged image in Fig. 3C shows that the nanoparticle surface is smooth. The particle size is about 200 nm, in agreement with our previous dynamic light scattering results. The above TEM images prove that, at a higher protein concentration (0.1%), drug DTX was coated with hydrophobin molecules that are absorbed to its surface, and small particle size was achieved. Of course, too high protein concentration could result in a highly viscous disperse medium solution which would hinder the transmission of the ultrasonic vibration and the diffusion between the solvent and anti-solvent, and, generally, the particle size is greater than 300 nm (data not shown). Finally, taking all factors into consideration, the optimal formulation consisted of 0.1% hydrophobin and 15 mg/mL DTX for further studies.

5

G. Fang et al. / European Journal of Pharmaceutical Sciences 60 (2014) 1–9 Table 1 Physicochemical characterization of hydrophobin coated docetaxel nanoparticles with different concentrations of DTX at an HPB concentration of 0.1% (n = 3). DTX(mg/mL)

Size (nm)

PI

ZP (mv)

DL (%)

Yield (%)

5 10 15 20

244.9 ± 3.8 202.0 ± 6.5 185.6 ± 5.5 303.7 ± 6.7

0.182 ± 0.001 0.149 ± 0.001 0.121 ± 0.020 0.193 ± 0.080

17.9 ± 0.3 16.6 ± 0.2 19.7 ± 0.6 19.1 ± 0.5

9.00 ± 1.88 29.54 ± 5.92 28.50 ± 2.51 17.13 ± 2.21

94.18 ± 0.28 94.40 ± 0.53 93.87 ± 0.11 94.61 ± 0.18

PI, polydispersity index. ZP, zeta potential. DL, drug loading ratio = [(amount of drug loaded in nanoparticles)/(the total amount of drug in nanoparticles and the hydrophobin in the fabrication process)].

Fig. 4. X-ray diffraction (XRD) patterns of DTX (A), HPB (B) and DTX-HPB-NPs (C). Fig. 2. Photographs of DTX-HPB-NPs (A) and bare drug particulates without hydrophobins (B). Red circle represents precipitated drug. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. Drug state in nanoparticles XRD was used to characterize the physical status of DTX present in the nanoparticles. Fig. 4 shows the XRD patterns of pure DTX powder, HPB aqueous solution lyophilized powder, and DTX nanoparticles. DTX showed the typical peaks at 2h values of 8.6°, 10.3°, 11.7°, 12.3°, 13.7°, 14.4°, 15.3°, 18.5°. In the meantime, some diffraction peaks were detected in the diffraction pattern of nanoparticles. The XRD data indicated that DTX was in the crystalline state in nanoparticles. Thus, it could be concluded that DTX in the nanoparticles was in a crystalline state. 3.3. Drug–hydrophobin interaction and hydrophobin secondary conformation The DTX, HPB, and DTX-HPB NPs were characterized by FTIR spectroscopy with two objectives. Firstly, it has been reported that

Class I hydrophobins can self-assemble at the hydrophilic/hydrophobic interface accompanied a change in secondary structure, and FTIR was used to identify the hydrophobin secondary conformation. Important IR bands of DTX and hydrophobin are summarized from Fig. 5A and B as shown in Table 2. It can be seen that the major characteristic peaks of HPB shown in Fig. 5B are strong amide I (1650 cm1) and amide II (1537 cm1) bands corresponding to the m(C@O) and d(NAH) vibrations of the amide bonds between the constituent amino acids of HPB. Generally, the amide I band is closely associated with protein secondary unfold conformation. Analysis was performed using the method of described in the literature (Kendrick et al., 1996). Bands at 1660– 1650 cm1 were assigned to a-helical structures, and those at 1640–1625 cm1 were assigned to b-sheet structures. Fig. 5B shows that solid-state HPB was mainly present in the a-helical structures. In contrast, it was previously reported (Wang et al., 2002) that Class I hydrophobins in aqueous solution mainly present in b-sheet structures, and this conformation difference is probably due to hydrophobin being present different physical forms (solid-state or liquid-state). However, in the interface of water/

Fig. 3. The TEM images of nanoparticles 0.05% HPB (A) and 0.1% HPB (B and C). Magnification is 25,000 (A), 10,000 (B) and 200,000 (C), respectively.

6

G. Fang et al. / European Journal of Pharmaceutical Sciences 60 (2014) 1–9

Fig. 6. In vitro release profile of DTX from Taxotere, DTX-HPB-NPs co-incubated with or without plasma in phosphate buffered saline (0.1% of Tween 80 in PBS, pH 7.4) at 37 ± 0.5 °C (n = 3).

36 h. It is clear that hydrophobin-coated drug nanoparticles exhibit delayed release and only a minor burst release. Fig. 5. FTIR spectra of (A) DTX, (B) HPB and (C) DTX-HPB-NPs.

Table 2 Summary of important bands in the IR spectrum of DTX and hydrophobin (HPB). Sample name

Frequencies (cm1) Functional group present

DTX

3433 1494 1713 2979 707 HPB 3403 1650 1537 DTX-HPB-NPs 3430

OAH and NAH stretching vibration C@C stretching vibration (benzene ring) C@O stretching vibration CAH stretching vibration CAH out-of-plane bend (benzene ring) OAH and NAH stretching vibration C@O stretching vibration NAH in-plane bending vibration OAH and NAH stretching vibration

hydrophobic solid, the protein has an a-helical structure, which is consistent with many reports (Sunde et al., 2008; Wösten, 2001). Secondly, FTIR was used to study the possibility of an interaction between DTX and HPB. Comparing the spectra of Fig. 5C with those of Fig. 5B, the amide I and II bands in the hydrophobin-coated docetaxel nanoparticles were slightly broadened. On the other hand, as shown in Table 2, the position of the peak corresponding to OAH and NAH stretching vibration in the spectra of nanoparticles shifted, compared to that for DTX and HPB. These differences indicated that the interaction between the DTX and hydrophobin involved hydrogen bonding. Indeed, DTX and hydrophobin contain sufficient H-bond donors (e.g. hydroxyl group) and H-bond acceptors (e.g. carbonyl group). 3.4. In vitro release The in vitro release experiments of DTX from nanoparticles were performed in pH 7.4 PBS, and the release results are presented in Fig. 6. It can be seen that DTX ethanol solution released 100% drug within 8 h, and the release behavior of Taxotere coincubation with HPB was the similar with Taxotere (p > 0.05). Hence, the HPB would be considered to negligibly interfere with the drug release. In contrast to the Taxotere solution, burst release was not obvious and there was a delayed release of DTX from the nanoparticles. The Taxotere solution released almost 95% DTX within 24 h, while the nanoparticles released 88% DTX up to

3.5. In vitro plasma stability Plasma contains many enzymes and, potentially, these could induce protein hydrolysis. Nanoparticle co-incubation with plasma was carried out to investigate the in vitro plasma stability of the nanoparticle outer hydrophobin shell. If enzymes in plasma cause any hydrophobin hydrolysis, the outer protein shell would be damaged, then, drug would be rapidly released from the hydrophobincoated nanoparticles. In fact, as shown in Fig. 6, nanoparticle coincubation with plasma did not produce any fast release compared with the control, instead, the release of DTX from the nanoparticle was a little slower than in the control without plasma co-incubation. Morover, the in vitro release behavior of Taxotere co-incubation with plasma was similar with that of Taxotere (p > 0.05). Hence, the plasma almost did not delay the drug release. This shows that plasma protein is adsorbed onto the hydrophobin-coated nanoparticles following co-incubation with plasma and this prevents DTX release from the plasma protein-nanoparticle complex. 3.6. In vitro hemolysis It is known that surfactants have a particular ability to interact with the lipid bilayer of red blood cell membrane (Shalel et al., 2002). Since hydrophobin is a protein surfactant, and regarding its safety following intravenous injection, it is necessary to examine the possibility that hydrophobin may produce hemolysis. As showed by the hemolysis level in Fig. 7, it is clear that the hemolytic activity of hydrophobin coated DTX nanoparticle is almost negligible (