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Langmuir 2007, 23, 6358-6364
Stable Amphoteric Nanogels Made of Ovalbumin and Ovotransferrin via Self-Assembly Jinhua Hu, Shaoyong Yu, and Ping Yao* The Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan UniVersity, Shanghai 200433, China ReceiVed NoVember 24, 2006. In Final Form: February 8, 2007 Ovalbumin and ovotransferrin are two proteins in hen egg white with isoelectric points of 4.8 and 6.8, respectively. A convenient and green method was developed in this study to prepare ovalbumin-ovotransferrin nanogels: a mixture of the two proteins was adjusted to a certain pH and then heated. Heat induced denaturation and gelation of the proteins, but the negative charges of ovalbumin prevented the proteins from coagulating. Dynamic light scattering, transmission electron microscopy, and atomic force microscopy studies reveal the nanogels have a spherical shape in both the swell and dry forms. Their apparent hydrodynamic diameters are in the range of 100-220 nm depending on the protein concentration in the nanogel preparation process. The nanogels display an amphoteric property: they carry net positive charges at pH lower than 5.5 and net negative charges at pH higher than 5.5. They form redispersible secondary aggregates at pH 5.0-6.0. The nanogels are stable in the pH ranges of 2.0-4.0 and 7.0-11.0, and they exhibit pH unchangeable but thermoreversible hydrophobicity. Benzoic acid was used as a model drug to study the loading ability. The native ovalbumin and ovotransferrin cannot bind with benzoic acid, whereas the nanogels with the network structure and hydrophobic binding sites can load benzoic acid through hydrophobic and electrostatic interactions.
Introduction Molecular self-assembly is a process in which molecules spontaneously form ordered aggregates through noncovalent binding.1,2 The self-assembly of biomacromolecules has attracted considerable attention recently becasue it leads to new materials and scaffolds3,4 which can find many applications in nanotechnology and medical technology.5,6 Food proteins have many functions, such as gelation, emulsification, and lipid and flavor binding and retention.7 Therefore, food proteins are ideal building blocks to fabricate functional biomaterials. Gelation as an important property of proteins has been well studied.8 The gel properties of proteins are sensitively affected by various factors, including pH, ionic strength, temperature, and solvent.9,10 During the heating process, heat-induced protein denaturation causes proteins to lose their compact structure, to expose their hydrophobic residues to the surface, and to exchange their disulfide bonds, resulting in intermolecular hydrophobic interactions and disulfide bonds.11 The gelation process follows three steps after the initial heat denaturation of food proteins: * To whom correspondence should be
[email protected]. Fax: 86-21-65640293.
addressed.
E-mail:
(1) Whitesides, G. M.; Boncheva, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4769-4774. (2) Olenyuk, B.; Whiteford, J. A.; Fechtenkotter, A.; Stang, P. J. Nature 1999, 398, 796-799. (3) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (4) Alivisatos, A. P.; Johnsson, K. P.; Peng, X. G.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P.; Schultz, P. G. Nature 1996, 382, 609-611. (5) Langer, R.; Vacanti, J. P. Science 1993, 260, 920-926. (6) Hubbell, J. A. Curr. Opin. Biotechnol. 1999, 10, 123-129. (7) Damodaran, S. In Food Proteins and Their Applications; Damodaran, S., Paraf, A., Eds.; Marcel Dekker, Inc.: New York, 1997; pp 1-24. (8) Oakenfull, D.; Pearce, J.; Burley, R. W. In Food Proteins and Their Applications; Damodaran, S., Paraf, A., Eds.; Marcel Dekker, Inc.: New York, 1997; pp 111-142. (9) Handa, A.; Takahashi, K.; Kuroda, N.; Froning, G. W. J. Food Sci. 1998, 63, 403-407. (10) Reddy, T. T.; Lavenant, L.; Lefebvre, J.; Renard, D. Biomacromolecules 2006, 7, 323-330. (11) Doi, E.; Kitabatake, N. In Food Proteins and Their Application; Damodaran, S., Paraf, A., Eds.; Marcel Dekker Inc.: New York, 1997; pp 325340.
the formation of aggregates via hydrophobic interaction, the stiffening of the aggregates through sulfhydryl-disulfide reaction, and a large increase in elasticity resulting from the formation of multiple hydrogen bonds upon cooling.12 It is reasonable to think that food protein hydrogels with a nano- or microsize are ideal candidates for loading and releasing drugs because the low density and network structure and rapid pH response property offer space and reversible binding sites to load and release drugs and the cross-linking property can suppress dissociation upon dilution. Ovalbumin (OVA) and ovotransferrin (OT) account for 54% and 13% of the egg white proteins.13 In detail, OVA is a monomeric phosphoglycoprotein, it consists of 385 amino acid residues, the molecular weight is 47 000, and the isoelectric point (pI) is 4.8.11 The OVA molecule has one internal disulfide bond and four free sulfydryl groups.12 OT is a single polypeptide protein, it consists of 686 residues and 15 disulfide bonds, the molecular weight is 78 000,11 and the pI is 6.8.14 When heating a transparent OVA solution, one can get a turbid suspension, a transparent gel, a translucent gel, or an opaque gel depending on the temperature, concentration of OVA, and pH and ionic strength of the solution.11,15 OT is the most heat labile protein in egg white; heating egg white at a temperature near 60 °C causes OT coagulation.16 Such a heat sensitivity of OT may be the reason that egg white forms a soft opaque gel at a lower temperature of around 65 °C. The interactions between OVA and OT on heating were investigated, and it was found that OVA had an inhibiting capacity against OT coagulation.17,18 It was presumed that OVA interacted with OT by an electrostatic attractive force (12) Mine, Y. Trends Food Sci. Technol. 1995, 6, 225-232. (13) Stadelman, W. J.; Cotterill, O. J. Egg Science and Technology, 2nd ed.; Avi Publishing: Westport, CT, 1977. (14) Desert, C.; Guerin-Dubiard, C.; Nau, F.; Jan, G.; Val, F.; Mallard, J. J. Agric. Food Chem. 2001, 49, 4553-4561. (15) Tani, F.; Murata, M.; Higasa, T.; Goto, M.; Kitabatake, N.; Doi, E. J. Agric. Food Chem. 1995, 43, 2325-2331. (16) Yamashita, H.; Ishibashi, J.; Hong, Y. H.; Hirose, M. Biosci. Biotechnol. Biochem. 1998, 62, 593-595. (17) Watanabe, K.; Nakamura, Y.; Xu, J. Q.; Shimoyamada, M. J. Agric. Food Chem. 2000, 48, 3965-3972. (18) Matsudomi, N.; Oka, H.; Sonoda, M. Food Res. Int. 2002, 35, 821-827.
10.1021/la063419x CCC: $37.00 © 2007 American Chemical Society Published on Web 04/18/2007
Stable Amphoteric Nanogels Made of OVA and OT
and then the two proteins unfolded together by heating and then formed soluble aggregates through intermolecular forces such as hydrophobic and disulfide bonds.18 In this study, we used a convenient method to assemble OVA and OT to amphoteric stable nanogels. The nanogels were characterized using a combination of techniques. The loading property of the nanogels for a model compound, benzoic acid, with changeable charges and hydrophobicity was studied. Materials and Methods Materials. Egg white OVA (grade V) and OT (substantially ironfree) from Sigma were used without further purification. All other reagents were analytical grade, were purchased commercially, and were used as received. All samples were prepared using water which was deionized and heated at 85-90 °C for at least 120 min and then filtered with 0.45 µm filters to obtain dust-free and aseptic solutions. Nanogel Preparation. OVA and OT aqueous solutions were prepared separately, and their concentrations were in the range of 0.1-5 mg/mL. Freshly prepared solutions were used. The following experiments were performed in an ice bath. The same volume of OVA solution with the desired concentration was added dropwise to the same concentration of OT solution to reach a weight ratio (WR) of OVA to OT of 1:1. After gentle shaking for 15 min, the pH of the mixture was adjusted to 7.0 or 7.5 with 0.1 M NaOH, and then the mixture was heated immediately at 80 °C for 30 min in a water bath to obtain a homogeneously dispersed nanogel solution. The nanogel samples were kept at 4 °C before characterization. The procedure shown here is a standard one; the other conditions used in this study will be specially indicated. ζ Potential Measurement. The ζ potential measurement was carried out at 25 °C on a ZetaSizer Nano ZS90 (Malvern Instruments) equipped with an MPT-2 autotitrator and a 4 mW He-Ne Laser (λ0 ) 633 nm) on the basis of the technique of laser doppler electrophoresis. The electrophoresis mobility UE was measured, and the ζ potential was calculated by the Henry equation,19 UE ) (2ζ/ 3η)[f(ka)], where , η, f(ka) are the dielectric permittivity of the solvent, the viscosity of the solution, and Henry’s function. The value of f(ka) here was determined to be 1.5 according to the Smoluchowski approximation.20,21 Dynamic Light Scattering (DLS) Measurement. A commercial laser light scattering instrument (Malvern Autosizer 4700, Malvern Instruments) equipped with a multi-τ digital time correlator (Malvern PCS7132) and a diode-pumped, solid-state laser (Compass 315M100, Coherent Inc.; output power ∼100 mW, λ0 ) 532 nm) as the light source was used to measure the nanogel size. The sample concentration was 0.1 mg/mL. The line width Γ distribution function G(Γ) was calculated from the Laplace inversion of the measured intensity-intensity time correlation function by a CONTIN program. The polydispersity index (PDI; 〈µ2/Γ2〉, where µ2 is the second cumulant of the correlation functions)22 was also obtained by the CONTIN program. G(Γ) can be converted into the translational diffusion coefficient distribution G(D) via Γ/q2 ≈ D, where q is the scattering vector. The hydrodynamic radius distribution f(Rh) can be obtained via the Stokes-Einstein equation, Rh ) kBT/6πηD, where kB, T, and η are the Boltzmann constant, the absolute temperature, and the solvent viscosity, respectively. DLS measurements were performed at a fixed scattering angle of 90° and a temperature of 25 °C; thus, the hydrodynamic diameter obtained was an apparent z-averaged hydrodynamic diameter, 〈Dh〉, simply written as hydrodynamic diameter or Dh. The angular dependence of the scattering in the angle range of 15-90° was investigated.23 Other measured temperatures in this paper will be specially indicated. The Dh and (19) Deshiikan, S. R.; Papadopoulos, K. D. Colloid Polym. Sci. 1998, 276, 117-124. (20) Sarmiento, F.; Ruso, J. M.; Prieto, G.; Mosquera, V. Langmuir 1998, 14, 5725-5729. (21) Gonzalez-Perez, A.; Ruso, J. M.; Prieto, G.; Sarmiento, F. Colloid Polym. Sci. 2004, 282, 351-356. (22) Yuan, X. F.; Harada, A.; Yamasaki, Y.; Kataoka, K. Langmuir 2005, 21, 2668-2674.
Langmuir, Vol. 23, No. 11, 2007 6359 PDI values were obtained by averaging at least two batches of the samples and three measurements of each sample. The standard deviations of Dh are less than 5%; the error bars of PDI are less than (0.03. Fluorescence Measurement. Fluorescence emission spectra were recorded on a fluorescence spectrophotometer, FLS-920 (Edinburg Instruments), at 25 and 50 °C. Recrystallized pyrene was dissolved in acetone to prepare a concentration of 2 × 10-5 g/mL stock solution. Nanogel solutions were first adjusted to different pH values with HCl or NaOH, then a pyrene/acetone solution was added, and the resultant solutions were equilibrated at 4 °C for 4 days. The final concentrations of pyrene and nanogels were 2 × 10-7 and 0.1 mg/ mL, respectively. The emission spectra with an excitation wavelength of 335 nm were recorded. The spectral resolution was 1 nm for both excitation and emission; three scans were accumulated for each measurement. The intensity ratio of the first to third band (I1/I3) was averaged with two batches of the samples; the largest error bar is (0.02. Transmission Electron Microscopy (TEM) Measurement. TEM observations were conducted on a Philips CM 120 electron microscope at an accelerating voltage of 80 kV. The samples were prepared by dropping nanogel solutions onto copper grids coated with thin films of Formvar and carbon. The samples were dried naturally. Atomic Force Microscopy (AFM) Measurement. AFM samples were prepared by drying the solution naturally on a freshly cleaved mica surface at room temperature. Images were acquired in tapping mode on a Digital Instruments Nanoscope IV equipped with a silicon cantilever of 125 µm and an E-type vertical engage piezoelectric scanner. Quantitative Analysis of Benzoic Acid. The maximum absorbance of benzoic acid in the range of 224-230 nm at different pH values and ionic strengths was recorded on a UV-vis spectrometer (Perkin-Elmer). The benzoic acid concentration was determined according to the working curves obtained using standard benzoic acid solutions with the desired pH and ionic strength. Encapsulation of Benzoic Acid. Benzoic acid was mixed with the nanogel solution with the desired pH and ionic strength; the final benzoic acid concentration was 3 mg/mL and that of the nanogels 5 mg/mL in the feed. After 24 h of stirring performed in the dark, free benzoic acid was separated from the nanogels by a high-flow ultrafiltration membrane (molecular weight cutoff 30 000, Millipore). Then the benzoic acid concentration was determined as described above. Control experiments were performed with the same condition except the addition of the nanogels. Data were obtained by analyzing and averaging at least three batches of samples.
Results and Discussion OVA-OT Nanogel Preparation. Our study found that heating OVA or OT solutions individually at different concentrations and pH values cannot fabricate nanogels. We produced a nanogel dispersion by warming an egg white powder aqueous dispersion by chance. Considering that OVA and OT are the main proteins in egg white, we speculated that OVA and OT might form nanogels on heating their mixture solution. Therefore, we investigated the influence of the pH, WR of OVA to OT, heating temperature and time, protein concentration, and stirring time on nanogel formation as follows. Heating the Mixture at pH 5.0-6.5. The pI is 4.8 for OVA and 6.8 for OT as mentioned above. The electrostatic attraction between these two proteins exists in the pH range of 4.8-6.8 because they carry opposite charges. Coagulation appeared after the OVA and OT mixture was heated at pH 5.0, 5.5, 6.0, and 6.5 separately. This can be explained by the fact that the charges of the two proteins are neutralized and the net charges are not enough to prevent the mixture from coagulating on heating. (23) Zhang, W. A.; Zhou, X. C.; Li, H.; Fang, Y. E.; Zhang, G. Z. Macromolecules 2005, 38, 909-914.
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Table 1. Influence of the WR of OVA to OT on the Scattering Light Intensity, Dh, and PDI of the Nanogelsa WR
Intensity (kcounts)
Dh (nm)
PDI
5:1 3:1 1:1 1:2 1:3 1:4 1:5 1:6 1:7
68 86 237 280 286 367 368 425 118
166 143 206 216 250 214 264 275 155
0.79 0.40 0.15 0.12 0.12 0.12 0.15 0.15 0.40
a The samples were prepared at pH 7.0 with a protein concentration of 0.1 mg/mL.
Heating the Mixture at pH 7.0. At this pH, OVA carries negative charges and OT carries about zero net charge. OVA may interact with OT through electrostatic attraction because of the heterogeneous charge distribution of the protein.24,25 Our DLS study showed that homogeneous nanogels formed at this pH on heating. The influence of WR on nanogel formation was investigated. Table 1 shows the relationship of WR and scattering light intensity/ Dh/PDI. The protein concentration was 0.1 mg/mL in all the samples. When WR decreases from 5:1 to 3:1, the nanogel size and intensity do not change very much, but PDI decreases significantly. In the WR range of 1:1 and 1:6, the intensity increases with the decrease of WR, the size is in the range of 205-275 nm, and PDI is in the range of 0.12-0.15; the stability study showed that the nanogels prepared with WR ) 1:1 are most stable and the stability decreases with a decrease of WR. When WR is further decreased to 1:8 or less, coagula appear. These results suggest that the negative charges of the OVA molecules prevent the mixture from coagulating. Increasing the protein concentration to 5 mg/mL with WR ) 1:1 can produce nanogels with a size of 118 nm and a PDI of 0.18. Further increasing the protein concentration to 10 mg/mL cannot give a homogeneous nanogel dispersion; possibly, the protein molecules are too crowded to form homogeneous nanogels at this concentration. Heating at 80 °C for 30 min is enough to make OVA and OT gelate as prolonging heating or increasing the temperature does not affect the nanogel size and scattering light intensity significantly. Heating the Mixture at pH 7.5-8.4. In this pH range, both OVA and OT carry negative charges, so electrostatic repulsion exists between them, and the repulsion is larger at pH 8.4 than that at pH 7.5. Coagula appeared when the mixture was heated at a protein concentration of 1 mg/mL or less, suggesting that the electrostatic repulsion keeps the protein molecules away from each other, and OVA cannot prevent OT from coagulating as OT is the most heat labile protein in egg white. However, when the protein concentration is increased to 5 mg/mL, homogeneous nanogels can be obtained. At pH 7.5 and 8.4, the nanogel sizes are 137 and 126 nm and the PDI values are 0.17 and 0.14, respectively. This indicates that the high protein concentration partly overcomes the electrostatic repulsion and causes the molecules to become closer, favoring nanogel formation. The scattering light intensity of the nanogels produced at pH 8.4 is weaker than that of the nanogels produced at pH 7.5; possibly, the stronger electrostatic repulsion at pH 8.4 reduces the nanogel formation. The stirring time prior to heat treatment of the protein mixture at pH 7.0 was found to have effects on the nanogel formation. (24) Cooper, C. L.; Dubin, P. L.; Kayitmazer, A. B.; Turksen, S. Curr. Opin. Colloid Interface Sci. 2005, 10, 52-78. (25) Yu, S. Y.; Hu, J. H.; Pan, X. Y.; Yao, P.; Jiang, M. Langmuir 2006, 22, 2754-2759.
Figure 1. Time course of Dh and PDI of the nanogels. The sample was produced at pH 7.0 with a protein concentration of 0.1 mg/mL.
Figure 2. Size distributions of nanogels before and after lyophilization. The nanogels were produced at pH 7.0 with a 5 mg/mL protein concentration.
The DLS result displayed that the Dh and scattering light intensity did not change significantly but PDI increased when the stirring time was changed from 0 to 30 min; when the stirring time was further increased to 60 min, coagula appeared. We noticed that during the stirring process turbidity and then coagula appeared for the individual OT solution at pH 7.0, while the individual OVA solution was kept transparent. It is obvious that stirring causes OT denaturation and aggregation. To avoid OT denaturation before the heat treatment, the nanogel preparation was performed in an ice bath without stirring. The observations above reveal a suitable condition for OVAOT nanogel preparation. In the following study, the nanogels used were prepared with this standard method: the same volume and same concentration of OVA and OT solutions (OVA and OT concentrations in the range of 0.1-5 mg/mL) were mixed to reach WR ) 1:1, and the mixture was adjusted to pH 7.0 or 7.5 and then heated at 80 °C for 30 min. It is interesting to find that a reproducible f(Dh) of the nanogels was obtained when it was monitored over a period as long as 90 days (Figure 1). Another advantage is that the nanogels can be stored as a lyophiled powder because the sample does not change its size distribution significantly after lyophilization and rehydration (Figure 2). These characters of the nanogels provide a possibility for practical applications. Nanogel Properties. To characterize the structure of the nanogels in aqueous solution, the angular dependence of the diffusion coefficient D of the nanogels was measured using DLS.23 Figure 3 shows a linear relation between Γ and q2 in the angle range of 15-90°, indicating that the nanogels have an isotropic
Stable Amphoteric Nanogels Made of OVA and OT
Figure 3. Angular dependence of the diffusion coefficient D of the nanogels in aqueous solution measured by DLS. The nanogels were produced at pH 7.0 with a 2 mg/mL protein concentration.
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Figure 5. AFM image of the nanogels prepared at pH 7.5 with a protein concentration of 5 mg/mL.
Figure 6. pH dependence of the ζ potentials of the nanogels and individual OT and OVA. The concentration for ζ potential measurement is 0.1 mg/mL in each sample. The nanogels were prepared at pH 7.0 with a protein concentration of 0.1 mg/mL.
Figure 4. TEM image of the nanogels prepared at pH 7.5 with a protein concentration of 5 mg/mL.
diffusive behavior; i.e., the nanogels have a spherical structure.26 The TEM image (Figure 4) verifies that the nanogels are spherical. The statistics on all of the nanogels in Figure 4 gives an average diameter of about 40 nm, which is much smaller than 137 nm measured by DLS. This is attributed to the shrinkage of the nanogels after water evaporation because DLS provides the data for the nanogels swollen in solution, while TEM shows the image of dried nanogels. The swelling ratio of the nanogels is about 40 calculated by the ratio of the wet volume to the dry volume (137 nm)3/(40 nm)3. The high shrinkage indicates that the nanogels have a low-density structure and contain a large amount of water. The nanogel morphology can also be verified by AFM imaging. Figure 5 exhibits that the nanogels are spherical with a smooth surface. The average diameter of the nanogels in Figure 5 is about 195 nm, the height about 38 nm. This can be explained by the fact that the nanogels were not dry enough and the soft nanogels collapsed under the force of the tip. On the other hand, our TEM experiment was carried out at an 80 kV accelerating voltage and in a high vacuum. In such conditions, only the dry sample was usable. (26) Hui, T. C.; Chen, D. Y.; Jiang, M. Macromolecules 2005, 38, 58345837.
The ζ potential relates to the net charges on the surface of the macromolecules and particles.27 Figure 6 displays the pH dependence of the ζ potentials of OVA, OT, and nanogels. The zero ζ potential values of OVA and OT appear at pH 4.9 and 6.4, respectively, which are close to their pI values. The ζ potentials of the nanogels are between the ζ potentials of OVA and OT. This result implies that the nanogel surface is occupied by both OVA and OT molecules. This is different from the nanogels made of OVA and lysozyme at pH 10.3 whose surface is mainly occupied by OVA molecules.28 The difference in the surface charge properties between the OVA-OT nanogel and the OVA-lysozyme nanogel may result from the difference in the pI values of OT and lysozyme and the difference in the OVA-OT interaction and OVA-lysozyme interaction. The zero ζ potential value of OVA-OT nanogels appears at pH 5.5; i.e., the nanogels carry net positive charges and net negative charges when the solution pH is lower and higher than 5.5, respectively. This indicates that we obtained amphoteric nanogels. The hydrophobicity/hydrophilicity of the nanogels was investigated using pyrene fluorescence. Pyrene has a much lower solubility in water (about 10-7 M) than in hydrocarbons (0.075 M). It migrates from the water phase into hydrophobic regions once the latter form in aqueous solution, causing remarkable photophysical changes.29-32 The hydrophobicity/hydrophilicity of the nanogels prepared at pH 7.0 was investigated by examining (27) Murray, M. J.; Snowden, M. J. AdV. Colloid Interface Sci. 1995, 54, 73-91. (28) Yu, S. Y.; Yao, P.; Jiang, M.; Zhang, G. Z. Biopolymers 2006, 83, 148158. (29) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 20392044.
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Figure 7. I1/I3 ratio of pyrene fluorescence in a nanogel solution as functions of pH and temperature. The nanogels were prepared at pH 7.0 with a protein concentration of 0.1 mg/mL.
Figure 8. Temperature and pH dependence of the nanogel hydrodynamic diameter. The nanogels were prepared at pH 7.0 with a 0.1 mg/mL protein concentration. T25-1, T37, and T50 denote the nanogels at 25, 37, and 50 °C, respectively. T25-2 denotes the nanogel at 25 °C again after a heat treatment at 50 °C.
the intensity ratio of the first to third band (I1/I3) in the pyrene fluorescence emission spectrum. Figure 7 shows that the I1/I3 values do not change significantly when the pH of the nanogel solution is changed from 7.0, but the I1/I3 values decrease when the solution temperature increases. The I1/I3 values are about 1.5 and 1.2 at 25 and 50 °C, respectively, indicating that the nanogels are relatively hydrophobic at 25 °C and more hydrophobic at 50 °C. It is interesting to find that when the temperature of the nanogel solution returns to 25 °C, the I1/I3 values return to about 1.5; i.e., the nanogels exhibit a thermoreversible change of the hydrophobicity. Figure 8 shows the nanogel size changes with pH and temperature. The nanogel Dh values decrease about 30% when the temperature increases from 25 to 50 °C, and they almost turn back when the temperature returns to 25 °C. Figure 7 has proven that the nanogels have a thermoreversible hydrophobicity change. Perhaps the increase of hydrophobicity at higher temperature results in an enhancement of the intermolecular interaction and the shrinkage of the nanogels; therefore, the nanogels exhibit a thermoreversible size change. Figure 8 shows the nanogels form secondary aggregates when the solution pH is in the range of 5.0-6.0, but these aggregates (30) Turro, N. J.; Lei, X. G.; Ananthapadmanabhan, K. P.; Aronson, M. Langmuir 1995, 11, 2525-2533. (31) Li, M.; Jiang, M.; Wu, C. J. Polym. Sci., Part B: Polym. Phys. 1997, 35, 1593-1599. (32) Kogej, K.; Skerjanc, J. Langmuir 1999, 15, 4251-4258.
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are redispersible when the solution pH is out of this range. Figure 6 exhibits that the zero ζ potential of the nanogels appears at pH 5.5, where the nanogels do not carry net charges on their surface, resulting in secondary aggregation. When the solution pH is out of the range of 5.0-6.0, the nanogels carry more net charges so that the nanogels become redispersible. The DLS measurement shows that the nanogel size increases slightly when the solution pH decreases from 4.0 to 2.0 or increases from 7.0 to 11.0 (Figure 8), but PDI does not increase, excluding the possibility of aggregation of the nanogels. OVA and OT carry the same positive and negative charges when the solution pH is lower than 4.8 and higher than 6.8, respectively, and they carry more charges when the solution pH is away from their pI values. In the pH ranges of 2.0-4.0 and 7.0-11.0 electrostatic repulsion between the protein molecules and the nanogels exists. The electrostatic repulsion between the protein molecules causes the nanogels to swell, while the electrostatic repulsion between the nanogels influences the mutual diffusion coefficients, resulting in a measurement deviation of Dh. In the presence of 10 mM NaCl, the effect of the electrostatic repulsion on Dh can be eliminated; the Dh values measured at pH 11.0 are very close to the values measured at pH 7.0. However, from pH 11.0 to pH 12.0, the nanogel size increases more than 2 times in the absence of NaCl, but the scattering light intensity decreases to a very small value, suggesting that most of the nanogels have dissociated due to the strong electrostatic repulsion between the molecules at pH 12.0. To suppress the secondary aggregation of the nanogels at pH 5.0-6.0, dextran (Mw ) 10 000) was conjugated to OVA using the Maillard reaction, which links the reducing end of the polysaccharide to the amines in the protein (terminus and amino group of lysine) as reported previously.33 The molar ratio of dextran to OVA in the feed was 2:1 in the conjugation reaction. Conjugate-OT nanogels were prepared using the same method as OVA-OT nanogel preparation. DLS measurement showed that conjugate-OT nanogels with a Dh of 200 nm do not aggregate at pH 5.0-6.0, suggesting that the hydrophilic dextran prevents the secondary aggregation of the nanogels. Loading Ability of Nanogels. The efficiency of the nanogel formation for the sample prepared at pH 7.0 with 5 mg/mL protein was estimated by measuring the protein concentration of the filtrate that was obtained by ultrafiltration with a high-flow membrane (cutoff molecular weight 100 000; MicroconYM-100, Millipore). The individual protein molecules, which did not take part in the nanogel assembly, flowed through the membrane and were collected as a filtrate, while the nanogels remained in the vessel. The 280 nm absorption measurement showed that almost no protein exists in the filtrate. Therefore, we presume all of the protein molecules have assembled to nanogels. The fluorescence probe study shows that the nanogels are somewhat hydrophobic and their hydrophobicity does not change at the pH range we studied (Figure 7). The ζ potential study shows the nanogels have an amphoteric property (Figure 6). The software Antheprot 4.3 was used to estimate the charges of OVA and OT carried at different pH values, and then the charges at different pH values were estimated for 1 mol of proteins with WR ) 1:1, i.e., 1 mol of proteins composed of 0.624 mol of OVA and 0.376 mol of OT (Table 2). To investigate the drug loading ability of the nanogels at different pH values, benzoic acid (Scheme 1) was studied as a model drug. The pKa of benzoic acid is 4.204,34 and (33) Mu, M. F.; Pan, X. Y.; Yao, P.; Jiang, M. J. Colloid Interface Sci. 2006, 301, 98-106. (34) Dean, J. A. Lange’s Handbook of Chemistry, 15th ed.; Beijing World Publishing Corp./McGraw-Hill: Beijing, China, 1999; p 8.31.
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Table 2. Benzoic Acid Amounts Loaded in the Nanogels at Different pH Values and without/with 0.15 M NaCla
pH
ionization deg of benzoic acid (%)
amt of net charges of 1 mol of proteinsb (mol)
2.0 4.0 7.4 10.0
0.6 39 100 100
+65 +35 -9 -38
molar ratio of loaded benzoic acid to proteins in the nanogels
weight ratio of loaded benzoic acid to nanogels in the feed (%)
0 M NaCl
0.15 M NaCl
0 M NaCl
0.15 M NaCl
27.6 49.2 17.4 15.2
39.1 18.8 11.6 15.5
5.75 ( 1.18 10.24 ( 1.72 3.62 ( 0.79 3.17 ( 0.46
8.14 ( 0.79 3.91 ( 1.31 2.41 ( 0.38 3.22 ( 0.38
a The nanogels were prepared at pH 7.0 with a 5 mg/mL protein concentration. b One mole of proteins composed of 0.624 mol of OVA and 0.376 mol of OT.
Scheme 1. Structure of Benzoic Acid
the solubility in water is 3.6 mg/mL at room temperature.35 The ionization degree of benzoic acid, molar ratio of loaded benzoic acid to proteins in the nanogels, and loading amounts (weight ratio of loaded benzoic acid to nanogels in the feed) at pH 2.0, 4.0, 7.4, and 10.0 without and with the addition of 0.15 M NaCl are shown in Table 2. In our loading study, the loading efficiency (weight ratio of loaded benzoic acid to benzoic acid in the feed) is in the range of 4.7-19%. At pH 2.0, the ionization degree of benzoic acid is smallest; therefore, the hydrophobic interaction between the nanogel and benzoic acid is strongest and the electrostatic interaction is weakest compared to those at other pH values we studied. The loading amount is about 5.7%; i.e., the molar ratio of loaded benzoic acid to proteins in the nanogels is 27.6. When 0.15 M NaCl was added, the loading amount increased to 8.1%. It is obvious that the interaction between the nanogel and benzoic acid increases in the presence of 0.15 M NaCl at pH 2.0. NaCl may influence the interaction between benzoic acid and protein by two mechanisms: (1) NaCl may screen the electrostatic interaction, and (2) a high concentration of NaCl may alter the structural organization of water molecules, which alters the strength of the hydrophobic interaction between nonpolar groups.36 The study of surface aromatic hydrophobicity of whey and isolate soybean proteins using the hydrophobic fluorescence probe ANS (1anilino-8-naphthalenesulfonate) showed that in the presence of 0.15 M NaCl the surface aromatic hydrophobicity of the proteins increases significantly.37 Cooper and Dubin et al. reported that the hydrophobicity interaction between the polyelectrolyte and protein becomes stronger at a high salt concentration.24 It is possible that 0.15 M NaCl increases the hydrophobic interaction between benzoic acid and the nanogel, resulting in an increase of the loading amount at pH 2.0. The electrostatic attraction between the nanogels and benzoic acid is strongest at pH 4.0, where the ionization degree of benzoic acid is 39% and 1 mol of proteins in the nanogels carries about 35 mol of positive charges. The largest loading amount, 10.2%, was obtained at pH 4.0 in the absence of NaCl, which means 1 mol of protein molecules in the nanogels loads 49.2 mol of benzoic acid molecules. In the presence of 0.15 M NaCl, the loading amount decreases to 3.9%; i.e., 1 mol of proteins in the nanogels can load only 18.8 mol of benzoic acid. In the process of loading, the nanogel concentration is 5 mg/mL; i.e., the total net charges (35) Stephen, H.; Stehphen, T. Solubilities of Inorganic and Organic Compounds, 2nd ed.; Pergamon Press: Oxford, 1963. (36) McClements, D. Food emulsions: principles, practice and techniques; CRC Press: New York, 1999. (37) Mitidieri., F. E.; Wagner., J. R. Food Res. Int. 2002, 35, 547-557.
of the nanogels are about 3.0 mM at pH 4.0. Therefore, 0.15 M NaCl can largely screen the electrostatic attraction between the nanogel and benzoic acid, causing a sharp decrease of the loading amount. At pH 7.4 and 10.0, the loading amounts are 3.6% and 3.3%, respectively, which are much lower than that at pH 4.0. The reasons are that at pH 7.4 and 10.0 the nanogels and benzoic acid carry the same negative charges and benzoic acid is more hydrophilic as it is about 100% charged. The loading amount at pH 7.4 decreases to 2.4% in the presence of 0.15 M NaCl. This may be explained by the heterogeneous distribution of charges on the protein surface,24,25 which leads to the electrostatic attraction between the nanogel and benzoic acid. At pH 10.0, NaCl does not influence the loading amount significantly, suggesting that neither electrostatic interaction nor hydrophobic interaction plays an important role at this pH. We also investigated the benzoic acid binding ability of native OVA and OT at pH 2.0 and 4.0 with and without 0.15 M NaCl. The binding condition is the same as the benzoic acid loading of the nanogels except that the nanogels were replaced by native OVA and OT. After ultrafiltration of the mixtures of benzoic acid and OVA and OT using a high-flow membrane (cutoff molecular weight 30 000), all of the benzoic acid can be found in the filtrate, implying that there is no significant interaction between benzoic acid and the native proteins. This indicates that the network structure and hydrophobic binding sites formed after protein denaturation enhance the electrostatic and hydrophobic interactions between benzoic acid and the nanogel.
Conclusions Natural biomacromolecules, OVA and OT, were used to fabricate nanogels using a heating process without organic solvent and chemicals except an alkali. The optimum conditions of the nanogel preparation were developed. At pH 7.0, heat treatment induces protein denaturation and gelation, but the negative charges of the OVA molecules prevent the OVA and OT mixture from coagulating when the protein concentrations are in the range of 0.1-5 mg/mL and the weight ratio of OVA to OT is 1:1. At pH 7.5-8.4, higher protein concentration can partly overcome the electrostatic repulsion between the protein molecules and facilitates the formation of nanogels. DLS, TEM, and AFM measurements reveal that the nanogels have a spherical shape in both the swell and dry forms; the swelling ratio is about 40. The ζ potential result indicates the nanogels carry net positive charges at pH lower than 5.5 and net negative charges at pH higher than 5.5. The nanogels are stable in the pH ranges of 2.0-4.0 and 7.0-11.0, and they display pH unchangeable but thermoreversible hydrophobicity. The nanogels form redispersible secondary aggregates at pH 5.0-6.0 due to lack of net charges on their surface. The nanogels dissociate at pH 12.0 as a result of strong electrostatic repulsion within the nanogels.
6364 Langmuir, Vol. 23, No. 11, 2007
Benzoic acid was used to study the loading ability. The nanogels can load benzoic acid at pH 2.0 through hydrophobic interaction. At pH 4.0, 39% of the benzoic acid molecules ionize, and the hydrophobic interaction decreases but the electrostatic attraction increases between the nanogel and benzoic acid, so the largest loading amount appears at this pH. At pH 7.4 and 10.0, neither strong electrostatic attraction nor strong hydrophobic interaction exists between the nanogel and benzoic acid, and the loading amounts decrease sharply. A control experiment verified that the
Hu et al.
native OVA and OT cannot bind with benzoic acid at pH 2.0 and 4.0. It is the network structure and hydrophobic binding sites of the nanogels that play important roles in benzoic acid loading. Acknowledgment. The financial support of the National Natural Science Foundation of China (NSFC Projects 50673020 and 50333010) is gratefully acknowledged. We thank Professor Ming Jiang for the comments on this paper. LA063419X
