Design and characterization of protein-quercetin bioactive nanoparticles
© Fang et al; licensee BioMed Central Ltd. 2011
Received: 27 January 2011
Accepted: 17 May 2011
Published: 17 May 2011
The synthesis of bioactive nanoparticles with precise molecular level control is a major challenge in bionanotechnology. Understanding the nature of the interactions between the active components and transport biomaterials is thus essential for the rational formulation of bio-nanocarriers. The current study presents a single molecule of bovine serum albumin (BSA), lysozyme (Lys), or myoglobin (Mb) used to load hydrophobic drugs such as quercetin (Q) and other flavonoids.
Induced by dimethyl sulfoxide (DMSO), BSA, Lys, and Mb formed spherical nanocarriers with sizes less than 70 nm. After loading Q, the size was further reduced by 30%. The adsorption of Q on protein is mainly hydrophobic, and is related to the synergy of Trp residues with the molecular environment of the proteins. Seven Q molecules could be entrapped by one Lys molecule, 9 by one Mb, and 11 by one BSA. The controlled releasing measurements indicate that these bioactive nanoparticles have long-term antioxidant protection effects on the activity of Q in both acidic and neutral conditions. The antioxidant activity evaluation indicates that the activity of Q is not hindered by the formation of protein nanoparticles. Other flavonoids, such as kaempferol and rutin, were also investigated.
BSA exhibits the most remarkable abilities of loading, controlled release, and antioxidant protection of active drugs, indicating that such type of bionanoparticles is very promising in the field of bionanotechnology.
Over the last several decades, the development of nanoparticles as drug delivery systems has gained considerable interest. Nanotoxicology research has indicated that  not only pharmacological properties but also the biodegradability, biocompatibility, and nontoxicity should be considered in such new systems. Therefore, synthetic macromolecules, such as the amphiphilic hyperbranched multiarm copolymers (HPHEEP-star-PPEPs) , poly(2-ethyl-2-oxazoline)-b-poly(D,L-lactide) , and polyethylene glycol , are often investigated; replacing these synthetic materials with natural proteins, which are more likely to be accepted by people, has become the focus of many research studies [5–9]. However, the microstructure of natural substances is generally complex and difficult to control; progress largely depends on knowledge of the physiochemical properties of the materials.
The potential therapeutic usefulness of albumin, such as bovine serum albumin (BSA), is high; it possesses the ability to transport fatty acids and many other endogenous or exogenous compounds throughout the body [10, 11]. Using a coacervation process, i.e., desolvation with ethanol and then solidification with glutaraldehyde, BSA can form nanoparticles . Hydrophilic drugs, such as phosphodiester oligonucleotide, 5-fluorouracil, and sodium ferulate, among others, can be incorporated into the matrix or adsorbed on the surface of nanoparticles [7–9]. However, the molecular sizes obtained from such a process are often larger than 70 nm; such particles cannot be used to entrap hydrophobic drugs, thereby restricting the development of bio-nanocarriers.
In the present study, the Q binding and releasing capacity of Lys and Mb are compared with those of BSA. The salting out method was combined with UV-Vis spectrometry to determine the binding capacity of the proteins. The release of Q from nanocarriers was detected in acidic and neutral conditions. The antioxidant properties of the bound Q in proteins were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals. Raman, fluorescence, and UV-Vis spectroscopy were combined to study the secondary and tertiary structures of the protein aggregates.
Results and Discussion
Size and Zeta Potential Measurements
Laser Raman spectroscopy
Intensitiesa of Raman Band of BSA system
BSA + DMSO (10%)
BSA + DMSO (30%)
BSA + DMSO (50%)
BSA + DMSO (70%)
Intensitiesa of Raman Band of Lys system
Lys + DMSO (10%)
Lys + DMSO (30%)
Lys + DMSO (50%)
Lys + DMSO (70%)
Intensitiesa of Raman Band of Mb system
Mb + DMSO (10%)
Mb + DMSO (30%)
Mb + DMSO (50%)
Mb + DMSO (70%)
Intensitiesa of Raman Band in BSA
D-BSA + Q2
D-BSA + Q6
D-BSA + Q10
Intensitiesa of Raman Band in Lys
With the addition of Q, fluorescence quenching was observed in D-BSA, D-Lys, and D-Mb; simultaneous slight blue shifts also occurred (Figures 13B, 13B', and 13B''). Quenching processes usually involve two modes, dynamic and static. Dynamic quenching occurs when the excited fluorophore experiences contact with an atom or molecule that can facilitate non-radiative transitions to the ground state, while static quenching implies either the existence of a spherical region of effective quenching, or the formation of a ground-state non-fluorescent complex. In many cases, the fluorophore can be quenched both by collision and by complex formation with the same quencher [42, 43]. The binding of Q with BSA, Lys, or Mb was static, as Q was less than 1.5 × 10-5 mol/L. The mode was determined by comparing the fitting results of the dynamic, static, and the combination modes to the D-BSA-Q, D-Lys-Q, and D-Mb-Q systems (See Additional File 1: Fitting results of the different modes on the experimental data). In this case, the binding constant (K a ) is equivalent to the quenching constant, which was determined by fitting Eq. 1 to the experimental data.
Binding parameters between Q and the three proteins
K a (L/mol)
7.34 × 104
7.92 × 104
2.93 × 104
3.44 × 104
3.72 × 104
4.13 × 104
Binding and Release Capacity of Proteins
Antioxidant Activity of Quercetin
Comparing the results acquired from the BSA, Lys, and Mb systems, BSA exhibited the best functional features, such as loading, controlled release, and particularly antioxidant protection of active drugs. Other commercially available flavonoids, such as kaempferol and rutin, were also investigated in order to produce a more general statement and conclusive study of such bionanoparticles. Similar to Q, the thermodynamic, i.e., ΔG, values of kaempferol and rutin were negative (both about -30 kJ/mol), and their ΔH and ΔS were positive (about 6 kJ/mol and 113 J/mol·K for kaempferol, 13 kJ/mol and 130 J/mol·K for rutin, respectively), indicating that these substances could be hydrophobically loaded by BSA since the size of the bionanosystem is less than 30 nm. One BSA could bind 12 kaempferl molecules and 5 rutin molecules. The main features of the oxidation kinetics of kaempferol and rutin in the BSA system were very similar to those of Q under the same conditions.
In this work, we demonstrated that proteins, such as BSA, Lys, and Mb be used to fabricate bioactive nanoparticles resulting from the secondary and tertiary structure transformations promoted by DMSO to deliver hydrophobic drugs such as Q. The adsorption of Q on proteins was mainly hydrophobic, particularly occurring in the region of Trp residues. BSA exhibited the highest binding capacity of Q, indicating that H-bonding and MWs also contribute to enhancing binding capacity. The formation of a hydrophobic core surrounded by a hydrophilic outer layer was therefore promoted. Protein nanocarriers can not only transport Q molecules, they also provide a protective effect on the activity of Q in both acidic and neutral conditions. The antioxidant activity of Q was also preserved by entrapment by the nanocarrier. Through the formation of complex aggregates composed of proteins, especially the BSA system, DMSO, and Q, such bio-nanoparticles with improved properties could be potentially efficient drug-carriers. Confirmed by further studies on kaempferol and rutin, this approach of protein nanoparticle preparation may provide a general and conclusive way to deliver hydrophobic drugs.
BSA (Fraction V) (A-0332) was purchased from AMRESCO (Amresco Inc., OH, USA); its MW was 67, 200 Da, and its purity was 98%. Myoglobin (Mb, M0630) was purchased from Sigma Aldrich, Inc. (St. Louis, MO, USA); its MW was 17, 800, and its purity was > 95%. Lysozyme (Lys) was purchased from Sanland Chemical Co. (LTD, LA, USA); its MW was 14, 400 Da. The isoelectric point (pI) of Lys in this work was about 7.0 as determined by zeta potential measurements. The stock solutions of BSA, Lys, and Mb (1.5 × 10-3 mol/L) were prepared with Milli-Q water and stored in the refrigerator at 4°C prior to use. 1-Diphenyl-2-picrylhydrazyl (DPPH, D9132-1G), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, A-1888), and dimethyl sulfoxide (DMSO) were all purchased from Sigma Aldrich, Inc. (St. Louis, MO, USA). The purity of DMSO was 99.5%. Quercetin (3,3',4',5,7-pentahydroxyflavone hydrate, Q-100081) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); its purity was 97.3%, as detected by high performance liquid chromatography. The stock solution of Q (1.5 × 10-3 mol/L) was prepared with DMSO, and stored in the refrigerator at 4°C prior to use. All other reagents used were of analytical grade or purer.
Preparation of DMSO-inducing protein nanoparticle (D-BSA, D-Lys, and D-Mb)
BSA, Lys, and Mb stock solutions (1.5 × 10-3 mol/L) were diluted to 1.5 × 10-5 mol/L; various volumes of DMSO were added. The total volume of the solution was kept at 10 mL, and the concentrations of DMSO were 1%, 10%, 20%, 30%, 40%, 50%, 60%, and 70%. The solution was mixed thoroughly for 5 min. Freeze-drying was used to remove DMSO  and obtain the nanoparticles.
Preparation of Quercetin-loaded protein nanoparticle (D-BSA-Q, D-Lys-Q, and D-Mb-Q)
BSA, Lys, and Mb stock solutions (1.5 × 10-3 mol/L) were diluted to 1.5 × 10-5 mol/L, and various volumes of Q were added. The total volume of the solution was kept at 10 mL, and the concentration of DMSO was kept at 10%; the concentration of Q was adjusted from 1.5 × 10-5 to 1.5 × 10-4 mol/L. The solution was mixed thoroughly for 5 min. Freeze-drying was used to remove DMSO  and obtain the nanoparticles.
Scanning Transmission Electron Microscopy (STEM)
Ten microliter samples were deposited onto a copper TEM grid for 5 s, after which the excess solutions were absorbed. Phosphotungstic acid was used to stain the sample. The observations were performed with a HITACHIS-5500 STEM (Hitachi High-Technologies America, Inc. IL, USA) at 30 KV. Images (1280 × 960 pixels) were acquired using a Gatan high-angle annular bright field (HAABF) scintillating detector.
Dynamic Light Scattering (DLS) Measurements
Hydrodynamic sizes and zeta potentials were determined by means of photon correlation spectroscopy using a Delsa Nano Particle Analyzer (A53878, Beckman Coulter, Inc., CA, USA). The size measurements were performed at 25°C and at a 15° scattering angle. Size was recorded for 400 μs for each measurement, and the accumulation time was 3 times. In dynamic light scattering, when the hydrodynamic size was measured, the fluctuations in the time of scattered light from particles in Brownian motion were measured. The zeta potential measurements were performed at 25°C. The accumulation time was 70 times, and equilibration time was 60 sec.
Raman Spectroscopy Measurements
The solution samples were prepared as in the section on sample preparation. Raman spectral data were collected with a HORIBA Jobin Yvon HR800 spectrometer (HORIBA Jobin Yvon S.A.S., Villeneuve Dáscq, France), with 785 nm excitation. Spectral differences were recorded in the 400-2000 cm-1 wave-number range. To increase the signal-to-noise ratio, at least 10 scans of each sample were collected to obtain averaged spectral data. The averaged spectral were baseline-corrected, and smoothed using ORIGIN software (version 8.0). The relative intensities were normalized to the phenylalanine band at 1002 or 1008 cm-1.
Fluorescence Spectrometry Measurements
The fluorescence intensities were recorded with a Cary Eclipse fluorophotometer (Varian, Inc., CA, USA). The widths of the excitation and emission slits of BSA, Lys, and Mb were set to 2.5/5.0, 5.0/5.0, and 10.0/20.0 nm, respectively. All the operations were carried out at 27 and 37°C. Fluorescence spectra were then measured in the range of 200-500 nm at an excitation wavelength of 280 nm. Each spectrum was background-corrected by subtracting the spectrum of the Milli-Q water and DMSO blank.
UV-Vis Spectrometry Measurements
All the samples were scanned on a Varian Cary 50 UV-visible spectrophotometer (Varian Medical Systems, Inc., CA, USA) at wavelength range of 300-500 nm. The operations were carried out at room temperature, 25°C. The scan rate was 600.00 nm/min. The data interval was 1.00 nm, and the average time was 0.10 sec. All the absorptions of the protein (BSA, Lys, and Mb) were near 280 nm. In the case of Mb, another weak absorption appeared at 420 nm.
Determination of Quercetin Loading Capacity (Salting Out Analysis)
The Q entrapped by nanocarriers was separated from the free Q through the salting out method as described below. A 5 mL sample was placed in a beaker. Excess ammonium sulphate was added to the beaker, and the mixture was stirred for 10 min and then left to stand for 20 min. A 2 mL solution was transferred to a centrifuge tube, and then centrifuged for 30 min at 15,000 rpm, at 4°C. The absorbance (Abs) of free Q in supernatant was detected at 367 nm by a Varian Cary 50 UV-Vis spectrophotometer (Varian Medical Systems, Inc., CA, USA), and the concentration of free Q was calculated by the standard curve method. The entrapped Q was calculated by determining all the Q in a sample and then subtracting the free Q. All measurements were performed in triplicate.
Quercetin Stability and Release Study In Vitro (UV-Vis Spectrometry Analysis)
The pH conditions of the release buffer were controlled using phosphate buffer (pH 7.4) or HCl (pH 1.2). The experiment was carried out using an improved method of Arnedo  as described below. A 90 mL sample was separated into 30 tubes, placed in an incubator at 37°C, and then wagged at 100 rpm. The tubes were successively detected at predetermined intervals by means of UV-Vis spectrometry. All measurements were performed in triplicate.
Antioxidant Activity Evaluation
The ABTS radical cation decolorization test is a spectrophotometric method widely used for the assessment of antioxidant activity of various substances. The experiment was carried out by the method of Re . In brief, 140 mmol/L ABTS stock solution was diluted in water to a concentration of 14 mM. A mixture of 500 μL 14 mM ABTS diluent and 500 μL 4.9 mM potassium persulfate (KPS) stock solution was placed in a 1.5 mL tube, and then left to stand in the dark at room temperature for at least 12 h before use. To study the samples, the ABTS· solution was diluted with the sample buffer to an absorbance of 0.70 ± 0.02 at 734 nm. After the addition of 900 μL of diluted ABTS· solution to 100 μL of sample, the absorbance (A Sample ) reading was taken after exactly 4 min. A sample buffer blank (A Control ) was run in each assay. All determinations were carried out in triplicate. The percent radical scavenging activity (% RSC) was calculated using Eq. 5.
This research was supported by the National Scienceand Technology Support Program (No. 2011BAD23B04). Prof. Yunjie Yan (Beijing National Center for Electron Microscopy, Department of Materials Science and Engineering, Tsinghua University), Prof. Wei Qi (Chemical Engineering Research Center, School of Chemical Engingeering and Technology, Tianjin University, Tianjin, China), Engr. Ke Zhu (Institute of Physics, Chinese Academy of Sciences, Beijing, China), and Dr. Yanhong Liu (Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, China) are acknowledged for their technical advice.
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