3486
Langmuir 2008, 24, 3486-3492
Lysozyme-Dextran Core-Shell Nanogels Prepared via a Green Process Juan Li, Shaoyong Yu, Ping Yao,* and Ming Jiang The Key Laboratory of Molecular Engineering of Polymer and Department of Macromolecular Science, Fudan UniVersity, Shanghai 200433, China ReceiVed September 8, 2007. In Final Form: December 12, 2007 A novel method has been developed for preparing nanogels with a lysozyme core and dextran shell. The method involves the Maillard dry-heat process and heat-gelation process. First, lysozyme-dextran conjugates were produced through the Maillard reaction. Then, the conjugate solution was heated above the denaturation temperature of lysozyme to produce nanogels. The nanogels are of spherical shape having a hydrodynamic diameter of about 200 nm and swelling ratio of about 30. The nanogel solutions are stable against long-term storage as well as changes in pH and ionic strength. Ibuprofen has been used as a drug model to study the electrostatic and hydrophobic interactions with these nanogels at different pH values. The study reveals that the nanogels are more suitable for loading protonated ibuprofen. We have verified that the knowledge of the formation mechanism of lysozyme-dextran nanogels can be applied to prepare other globular protein-dextran nanogels.
Introduction Many studies have focused on the preparation of core-shell nanoparticles and the application of nanoparticles in the encapsulation of pharmaceuticals ranging from small molecules to protein and DNA.1-8 Generally, the core made from hydrophobic segments serves as a cargo space for lipophilic drugs, while the shell composed of hydrophilic segments stabilizes the particles in water. It is known that a “stealth” property is necessary for a nanoparticle shell for the purpose of avoiding reticuloendothelial recognition and subsequent elimination of the nanoparticle so that its circulation time in the bloodstream is prolonged.1,4 Shells are composed of two kinds of polymers. One is a group of synthetic polymers, especially poly(ethylene glycol);1-5,8 the other is polysaccharides, such as dextran,6,7,9 chitosan,10 and hydroxyethyl cellulose.11 It has been reported that polysaccharides can also provide nanoparticles with a stealth property and decrease the adsorption of plasma protein.6 Besides, ligands can be attached to polysaccharides to enable nanoparticles to target specific cells.6 Protein nanoparticles, of albumin,12-17 gelatin,18 and β-lactoglobulin,19 have been studied in pharmaceuticals and in the * To whom correspondence should be addressed. E-mail: yaoping@ fudan.edu.cn. Fax: 86-21-65640293. (1) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263, 1600-1603. (2) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf., B 1999, 16, 3-27. (3) Vinogradov, S. V.; Bronich, T. K.; Kabanov, A. V. AdV. Drug DeliVery ReV. 2002, 54, 135-147. (4) Otsuka, H.; Nagasaki, Y.; Kataoka, K. AdV. Drug DeliVery ReV. 2003, 55, 403-419. (5) Riley, T.; Govender, T.; Stolnik, S.; Xiong, C. D.; Garnett, M. C.; Illum, L.; Davis, S. S. Colloids Surf., B 1999, 16, 147-159. (6) Lemarchand, C.; Gref, R.; Couvreur, P. Eur. J. Pharm. Biopharm. 2004, 58, 327-341. (7) Lemarchand, C.; Couvreur, P.; Besnard, M.; Costantini, D.; Gref, R. Pharm. Res. 2003, 20, 1284-1292. (8) Harada, A.; Kataoka, K. Macromolecules 1998, 31, 288-294. (9) Tang, M. H.; Dou, H. J.; Sun, K. Polymer 2006, 47, 728-734. (10) Hu, Y.; Jiang, X. Q.; Ding, Y.; Ge, H. X.; Yuan, Y. Y.; Yang, C. Z. Biomaterials 2002, 23, 3193-3201. (11) Dou, H. J.; Jiang, M.; Peng, H. S.; Chen, D. Y.; Hong, Y. Angew. Chem., Int. Edit. 2003, 42, 1516-1519. (12) Irache, J. M.; Merodio, M.; Arnedo, A.; Camapanero, M. A.; Mirshahi, M.; Espuelas, S. Mini-ReV. Med. Chem. 2005, 5, 293-305. (13) Langer, K.; Balthasar, S.; Vogel, V.; Dinauer, N.; Von Briesen, H.; Schubert, D. Int. J. Pharm. 2003, 257, 169-180.
field of nutrition. There are many advantages of protein-based nanoparticles; they are biodegradable, nontoxic, and nonantigenic.12,17 Proteins are polyampholytes; their charges and hydrophobic/hydrophilic properties are pH dependent. Proteinbased nanoparticles offer several possibilities for surface modifications and covalent attachments of drugs.17 For example, nanoparticles consisting of human serum albumin coupled with the antibody trastuzumab enable its delivery to tumor cells;15 PEG-modified human serum albumin nanoparticles have a reduced plasma protein adsorption on the particle surface compared to the unmodified particles.14 A common method for the preparation of protein nanoparticles is desolvation followed by cross-linking with glutaraldehyde.12-18 Gelation of proteins has been well utilized in food science and technology and has been extensively studied.20 The gelation process undergoes three steps following the initial heat denaturation of protein.21 The first is the formation of aggregates via hydrophobic interaction, second the stiffening of the aggregates through sulfhydryl-disulfide reaction, and last a large increase in elasticity resulting from the formation of multiple hydrogen bonds upon cooling. Recently, we have developed a novel method to prepare chitosan-ovalbumin, ovalbumin-lysozyme, and ovalbumin-ovotransferrin nanogels through heat-induced gelation of proteins.22-24 The advantages of this method are simple and conformable to green chemistry. However, these nanogels (14) Lin, W.; Garnett, M. C.; Schacht, E.; Davis, S. S.; Illum, L. Int. J. Pharm. 1999, 189, 161-170. (15) Steinhauser, I.; Spankuch, B.; Strebhardt, K.; Langer, K. Biomaterials 2006, 27, 4975-4983. (16) Wartlick, H.; Spankuch-Schmitt, B.; Strebhardt, K.; Kreuter, J.; Langer, K. J. Controlled Release 2004, 96, 483-495. (17) Weber, C.; Coester, C.; Kreuter, J.; Langer, K. Int. J. Pharm. 2000, 194, 91-102. (18) Coester, C.; Kreuter, J.; Von, Briesen, H.; Langer, K. Int. J. Pharm. 2000, 196, 147-149. (19) Chen, L. Y.; Subirade, M. Biomaterials 2005, 26, 6041-6053. (20) Oakenfull, D.; Pearce, J.; Burley, R. W., Protein Gelation. In Food Proteins and Their Applications; Damodaran, S., Paraf, A., Eds.; Marcel Dekker: New York, 1997; Vol. 80, pp 111-142. (21) Mine, Y. Trends Food Sci. Technol. 1995, 6, 225-232. (22) Yu, S. Y.; Yao, P.; Jiang, M.; Zhang, G. Z. Biopolymers 2006, 83, 148158. (23) Yu, S. Y.; Hu, J. H.; Pan, X. Y.; Yao, P.; Jiang, M. Langmuir 2006, 22, 2754-2759. (24) Hu, J. H.; Yu, S. Y.; Yao, P. Langmuir 2007, 23, 6358-6364.
10.1021/la702785b CCC: $40.75 © 2008 American Chemical Society Published on Web 02/27/2008
Lysozyme-Dextran Core-Shell Nanogels
aggregate at a certain pH range and high salt concentrations due to the lack of enough hydrophilic groups on their surface. Therefore, it is interesting to develop a novel green method to prepare protein-polysaccharide nanogels that are dispersible and stable over a broad range of pH, especially at the physiological pH and ionic strength. Lysozyme is one of the most studied proteins.21 It has a molecular weight of 14 300, an isoelectric point (pI) of 10.7, 129 amino acid residues, and 4 disulfide bridges. Water-soluble dextran is a family of 1f6-R-D-glucans which has many applications in the food and pharmaceutical industries.9,25 In this paper, we study lysozyme-dextran nanogels prepared by a twostep heating process. First, we prepared lysozyme-dextran conjugates through the Maillard dry-heat reaction. Then, we employed a heat-gelation process to prepare nanogels with a core-shell structure. The nanogels were characterized by various physicochemical techniques. Ibuprofen was used as a drug model to study the loading behavior of the nanogels through electrostatic and hydrophobic interactions. Materials and Methods Materials. Hen egg white lysozyme was from Sino-American Biotechnology Co. Dextrans with molecular weights of 10 000, 35 000, and 62 000 were from Amresco Inc. and Amersham Pharmacia Biotech. Pyrene from Aldrich (98%) was recrystallized twice from benzene. Ibuprofen (>98%) was supplied by Shanghai Shunqiang Biotechnology Co. Ltd. o-Phthaldialdehyde of chemical grade was from Sino-China Pharm Co. All solutions were prepared using deionized water having a resistance of 18 MΩ. Preparation of Lysozyme-Dextran Conjugates. Lysozyme and dextran with the desired molecular weight and molar ratio were dissolved together in water. The pH of the mixture was adjusted to 7-8 using 0.1 mol/L NaOH, and the mixture was lyophilized. The lyophilized powder was heated at 60 °C under 79% relative humidity in a desiccator containing saturated KBr solution for 18-24 h.26 The resultant Maillard products (lysozyme-dextran conjugates) were kept at -20 °C before use. Preparation of Lysozyme-Dextran Nanogels. Lysozymedextran conjugates were dissolved in water to reach the desired concentration (1-10 mg/mL), the pH was adjusted to the desired values with 0.1 mol/L NaOH, and the solution was heated at 80 °C for 30 min. The produced lysozyme-dextran nanogel solution and its lyophilized powder were kept at 4 °C before use. The concentrations of the conjugate and nanogel solutions were denoted in lysozyme concentrations that were measured by 280 nm absorbance (Lambda 35, Perkin-Elmer).27 The efficiency of the nanogel formation was estimated by measuring the lysozyme concentration in the ultrafiltrate obtained by ultrafiltration with a high-flow membrane (cutoff molecular weight 100 000; MicroconYM-100, Millipore). The individual lysozyme molecules not taking part in the nanogel formation flowed through the membrane and were collected as an ultrafiltrate, while the nanogels remained in the vessel. Ibuprofen Loading. Ibuprofen was added to the nanogel solution in the absence or presence of 0.15 mol/L NaCl. Then, 1.0 mol/L HCl was added to the solution dropwise to reach pH 3.2, 5.2, and 7.4 separately. The nanogel and ibuprofen concentrations were 1 and 0.2 mg/mL, respectively. The resultant mixture was stirred at room temperature for 24 h. To determine the amount and efficiency of the loading, the unloaded ibuprofen was separated from the nanogels by ultrafiltration (cutoff molecular weight 10 000; MicroconYM10, Millipore). The loaded ibuprofen was calculated by subtracting (25) Heinze, T.; Liebert, T.; Heublein, B.; Hornig, S. AdV. Polym. Sci. 2006, 205, 199-291. (26) Nakamura, S.; Kato, A.; Kobayashi, K. J. Agric. Food Chem. 1991, 39, 647-650. (27) Tani, F.; Murata, M.; Higasa, T.; Goto, M.; Kitabatake, N.; Doi, E. J. Agric. Food Chem. 1995, 43, 2325-2331.
Langmuir, Vol. 24, No. 7, 2008 3487 the free ibuprofen in the ultrafiltrate from the initial ibuprofen in the nanogel solution. At pH 3.2, insoluble ibuprofen was separated from the nanogel solution by 450 nm membrane filtration. Thereafter, 1 mL of the filtrate was dialyzed (cutoff molecular weight 3500) against 50 mL of 0.1 mol/L pH 7.4 phosphate buffer at 37 °C for 2 days to release ibuprofen completely. The unloaded ibuprofen in the filtrate was separated from the nanogels by ultrafiltration (cutoff molecular weight 10 000). Loaded ibuprofen was calculated from the difference between the ibuprofen detected in the dialysate and the ultrafiltrate. The ibuprofen concentration was determined by its absorbance at 222 nm (Lambda 35, Perkin-Elmer) according to the working curve measured using standard ibuprofen solutions.28 The working curve shows a good linear relationship (R ) 0.99992) in the ibuprofen concentration range of 0.001-0.025 mg/mL: A (absorbance) ) 0.0115 + 43.314C (ibuprofen concentration, mg/mL). All ibuprofen samples for absorbance analysis were prepared in 0.1 mol/L pH 7.4 phosphate buffer. The loading data were averaged by analyzing at least two batches of loading samples. Gel Electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on a gel electrophoresis apparatus (JM250, JM-X Scientific Co.) to analyze the molecular weights of lysozyme and lysozyme-dextran conjugates.29 The gel was stained with Coomassie Brilliant Blue. o-Phthaldialdehyde (OPA) Assay. The degree of conjugation of lysozyme-dextran conjugates was analyzed through an OPA assay based on the loss of free amino groups of lysozyme after the Maillard reaction as previously reported.30,31 Briefly, lysozymedextran conjugates were dissolved in water, mixed with OPA reagent (freshly prepared), and incubated at room temperature for 3 min, and finally the absorbance of the mixture was measured at 335 nm (Lambda 35, Perkin-Elmer) immediately. The working curve was measured at the same condition using L-leucine as a standard. Dynamic Laser Scattering (DLS) Measurements. DLS measurements were carried out on a Malvern Autosizer 4700 (Malvern Instruments) equipped with a multi-τ digital time correlator (Malvern PCS7132) and a solid-state laser (Compass 315M-100, Coherent Inc.; output power ∼100 mW, λ ) 532 nm). The measurements were performed at 25 °C and a fixed scattering angle of 90°. The measured time correlation functions were analyzed by the automatic program equipped with the correlator. The apparent z-average hydrodynamic diameter (Dh) and polydispersity index (PDI, 〈µ2/Γ2〉) were obtained by CONTIN mode analysis.32,33 The concentration of the samples for DLS measurement was 0.1 mg/mL. ζ-Potential Measurements. ζ-potential measurements were performed at 25 °C on a ZetaSizer Nano ZS90 (Malvern Instruments) equipped with a 4 mW He-Ne laser (λ ) 633 nm) using the technique of laser Doppler electrophoresis. ζ-potentials were calculated by Dispersion Technology software provided by Malvern according to Smoluchowski approximation in an automatic mode.34 Each sample was analyzed three times. Atomic Force Microscopy (AFM) Measurements. The nanogel solution for AFM measurement was produced at pH 10.7 and 1 mg/mL lysozyme concentration and diluted to 0.01 mg/mL. AFM samples were prepared by drying the nanogel solution on a freshly cleaved mica surface in a desiccator containing dried silica gel at room temperature for 3 days. Images were acquired in tapping mode on a Digital Instruments Nanoscope IV (Veeco Instruments). Silicon tips (RTESP7 Veeco nanoprobe) with a curvature radius of 5-10 nm were used. (28) Mohammed, A. R.; Weston, N.; Coombes, A. G. A.; Fitzgerald, M.; Perrie, Y. Int. J. Pharm. 2004, 285, 23-34. (29) Laemmli, U. K. Nature 1970, 227, 680-685. (30) Pan, X. Y.; Mu, M. F.; Hu, B.; Yao, P.; Jiang, M. Biopolymers 2006, 81, 29-38. (31) Dukes, B. C.; Butzke, C. E. Am. J. Enol. Vitic. 1998, 49, 125-134. (32) Zhang, W. A.; Zhou, X. C.; Li, H.; Fang, Y.; Zhang, G. Z. Macromolecules 2005, 38, 909-914. (33) Yuan, X. F.; Harada, A.; Yamasaki, Y.; Kataoka, K. Langmuir 2005, 21, 2668-2674. (34) Sarmiento, F.; Ruso, J. M.; Prieto, G.; Mosquera, V. Langmuir 1998, 14, 5725-5729.
3488 Langmuir, Vol. 24, No. 7, 2008
Li et al.
Fluorescence Measurements. Fluorescence emission spectra were recorded at 25 °C on a fluorescence spectrophotometer (FLS-920, Edinburgh). The nanogel solutions were first adjusted to the desired pH values with HCl or NaOH followed by the addition of pyrene/ acetone stock solution (2 × 10-5 g/mL). The final pyrene and the nanogel concentrations were 2 × 10-7 and 1 mg/mL, respectively. The resultant mixtures were incubated at 4 °C for 72 h before measurement. The emission spectra were recorded with an excitation wavelength of 335 nm, and three scans were accumulated for each measurement.23
Results and Discussion Oakenfull et al. reported that heating a lysozyme solution at pH close to its pI resulted in insoluble aggregates.20 The same phenomenon was observed when we heated 1 mg/mL lysozyme solution at pH 10.7 and 80 °C. For the mixture of lysozyme and dextran (1 mg/mL lysozyme, 1:2 molar ratio of dextran to lysozyme, and MW 62 000 dextran), coagula also appeared after a heat treatment at pH 10.7 and 80 °C (data shown in the Supporting Information), suggesting that free dextran cannot suppress lysozyme coagulation on heating. However, we found that the transparent solution of lysozyme-dextran conjugates prepared from the same mixture changed to a homogeneous particle solution with a Dh of about 200 nm after the same heat treatment, suggesting the formation of nanogels. Therefore, a two-step heating process was used in this study to prepare nanogels. In the first step, lysozyme-dextran conjugates were prepared through the Maillard dry-heat reaction. In the second step, the resultant conjugates were dissolved in water, the pH was adjusted to the desired value, and the solution was heated at 80 °C for 30 min to induce gelation of lysozyme. Factors influencing the nanogel formation were investigated as follows. Preparation of Lysozyme-Dextran Conjugates. Effect of the Maillard Reaction Time. The Maillard reaction has been studied extensively; being a natural and nontoxic process, it conjugates polysaccharide and protein by linking the reducing end carbonyl group in the former to the amino group in the latter.26,30,35-38 Lysozyme-dextran Maillard conjugates were first reported by Nakamura et al. in 1991, and the conjugates showed significant antimicrobial activities and excellent emulsifying properties.26 Afterward, the conjugates were studied by other researchers for improving functional properties such as solubility and heat stability.38 In the present study, the Maillard reaction was carried out at 60 °C, pH 7-8, and 79% relative humidity as reported by others26,38 and ourselves.30 Figure 1 shows SDSPAGE analysis of the mixture of lysozyme and dextran before and after the Maillard reaction. Before the reaction, lysozyme shown in lane 2 is a single band. After the reaction, a new smear band with much higher molecular weight appears, indicating the formation of lysozyme-dextran conjugates. As the reaction time increases, this smear band becomes clearer while the lysozyme band becomes faint, suggesting more lysozyme-dextran conjugates produced. Our control experiment has excluded the possibility that the smear band is aggregates of individual lysozymes (data shown in the Supporting Information). Figure 1 shows a faint band of lysozyme dimer after the Maillard reaction. The degree of conjugation of the conjugates was analyzed using an OPA assay from the loss of free amino groups of lysozyme.30 There are seven amino groups in each lysozyme (35) Maillard, L. C. C. R. Hebd. Seances Acad. Sci. 1912, 154, 66-68. (36) Fayle, S. E; Gerrard, J. A. The Maillard reaction; Royal Society of Chemistry: Cambridge, U.K., 2002; pp 1-3. (37) Campbell, L.; Raikos, V.; Euston, S. R. Nahrung 2003, 47, 369-376. (38) Aminlari, M.; Ramezani, R.; Jadidi, F. J. Sci. Agric. Food 2005, 85, 2617-2624.
Figure 1. SDS-PAGE analysis of native lysozyme (lane 1), a mixture of lysozyme and dextran (lane 2), lysozyme-dextran conjugates (lanes 3-9) prepared with Maillard reaction of 6, 12, 18, 24, 48, 72, and 120 h, respectively, and protein marker (lane 10). The molar ratio of dextran (MW 62 000) to lysozyme was 1:2. In each of lanes 1-9, 20 µL of sample with a lysozyme concentration of 1 mg/mL was loaded. Table 1. OPA Analysis of Lysozyme-Dextran Conjugates Prepared with Different Maillard Reaction Times and DLS Result of the Resultant Nanogelsa av no. of free Maillard reaction amino groups intensity per lysozyme (kcounts/s) time (h) 6 12 18 24 48 72 120 168
7.0 ( 0.2 7.0 ( 0.3 6.7 ( 0.1 6.6 ( 0.1 6.5 ( 0.2 6.3 ( 0.1 6.3 ( 0.0 6.3 ( 0.1
79 ( 4 64 ( 14 55 ( 1 56 ( 3 50 ( 2 43 ( 5 30 ( 8 32 ( 6
Dh (nm)
PDI
217 ( 1 191 ( 16 180 ( 24 182 ( 4 182 ( 2 172 ( 5 164 ( 2 160 ( 1
0.11 ( 0.03 0.10 ( 0.01 0.10 ( 0.04 0.11 ( 0.01 0.14 ( 0.04 0.12 ( 0.04 0.16 ( 0.03 0.18 ( 0.02
a The conjugates were prepared with MW 62 000 dextran, and 1:2 molar ratio of dextran to lysozyme. The nanogels were produced at pH 10.7 and 1 mg/mL lysozyme concentration.
molecule.26 After the Maillard reaction, some of the amino groups of lysozyme conjugate with dextran, resulting in a decrease of free amino groups. Table 1 shows the average decrease of free amino groups as the reaction time increases. The molar ratio of dextran to lysozyme was 1:2 in the feed, but more than 0.5 mol of free amino groups was lost on average after 72 h or longer of Maillard reaction. This may result from the cross-linking and other side reactions taking place in the final stages of the Maillard reaction.36 To control the reaction in its early stagesAmadori rearrangements,36 a suitable Maillard reaction time is 24 h or less; the resultant products are white and dissolve well in water, indicating that the side reactions are insignificant. In the following study, we chose a Maillard reaction of 18 h to prepare the conjugates, in which about 0.3 mol of dextran is attached to 1.0 mol of lysozyme on average (Table 1) and lysozyme keeps its nativelike secondary and tertiary structure (circular dichroism spectra shown in the Supporting Information). Preparation of Lysozyme-Dextran Nanogels. Effect of the Maillard Reaction Time. The conjugates prepared with different Maillard reaction times as studied above were used to prepare nanogels by adjusting the conjugate solutions to pH 10.7 and then heating the solutions at 80 °C for 30 min. The size distributions of the resultant nanogels were characterized using
Lysozyme-Dextran Core-Shell Nanogels
Langmuir, Vol. 24, No. 7, 2008 3489
Table 2. Effects of the Molecular Weight of Dextran and Molar Ratio of Dextran to Lysozyme (MR) on the Size Distributions of Lysozyme-Dextran Nanogelsa Dh (nm)
a
PDI
MR
MW 10 000
MW 35 000
MW 62 000
MW 10 000
MW 35 000
MW 62 000
1:8 1:4 1:2 1:1 2:1 4:1 8:1
1125 ( 115 906 ( 112 415 ( 34 189 ( 25 107 ( 52 101 ( 56 86 ( 44
439 ( 67 195 ( 10 155 ( 9 133 ( 27 120 ( 32 113 ( 19 110 ( 15
207 ( 50 195 ( 19 172 ( 23 146 ( 21 146 ( 17 140 ( 7 151 ( 8
0.67 ( 0.47 0.75 ( 0.19 0.54 ( 0.05 0.24 ( 0.04 0.22 ( 0.10 0.37 ( 0.05 0.24 ( 0.01
0.40 ( 0.07 0.09 ( 0.01 0.11 ( 0.01 0.19 ( 0.05 0.14 ( 0.05 0.20 ( 0.09 0.35 ( 0.01
0.09 ( 0.04 0.06 ( 0.01 0.11 ( 0.07 0.10 ( 0.02 0.11 ( 0.06 0.21 ( 0.15 0.21 ( 0.01
The nanogels were produced at pH 10.7 and 1 mg/mL lysozyme concentration.
Table 3. Effects of the pH and Concentration of Lysozyme-Dextran Conjugate Solution on the Formation of Lysozyme-Dextran Nanogels DLS result intensitya (kcounts/s) Dh (nm) PDI efficiency of nanogel formation (%)
a
pH
1 mg/mL conjugate
2 mg/mL conjugate
5 mg/mL conjugate
10 mg/mL conjugate
9.5 10.0 10.5 9.5 10.0 10.5 9.5 10.0 10.5 9.5 10.0 10.5
39 ( 18 26 ( 15 43 ( 22 81 ( 9 89 ( 1 166 ( 15 0.60 ( 0.12 0.31 ( 0.10 0.16 ( 0.01 55 85 95
51 ( 9 21 ( 9 37 ( 5 77 ( 11 87 ( 4 181 ( 10 0.51 ( 0.05 0.30 ( 0.07 0.13 ( 0.02
48 ( 7 57 ( 28 66 ( 20 63 ( 10 116 ( 20 225 ( 30 0.50 ( 0.02 0.21 ( 0.03 0.11 ( 0.01
82 ( 14 108 ( 19 58 ( 12 70 ( 9 153 ( 9 266 ( 45 0.37 ( 0.13 0.20 ( 0.05 0.12 ( 0.01 100 100 100
The slit for DLS measurement was 300, 100, and 35 µm for the samples of pH 9.5, 10, and 10.5, respectively.
DLS. Table 1 shows that the intensity and Dh decrease whereas the PDI increases with an increase of the Maillard reaction time. As reported previously,39,40 for surfactant-free particles the average surface area stabilized by one hydrophilic moiety should be a constant. A longer Maillard reaction time results in more dextran chains conjugated to lysozyme; more dextran chains can stabilize a larger surface area, resulting in smaller nanogels. Therefore, when the reaction time increases, the decrease of the Dh and intensity can be explained. On the other hand, when the Maillard reaction time is longer than 24 h, cross-linking and other side reactions become substantial and aggregates exist in the conjugate solution (data not shown). This may result in the increase of the PDI. Effects of Dextran Molecular Weight and the Molar Ratio of Dextran to Lysozyme. The influence of different dextrans with molecular weights of 10 000, 35 000, and 62 000 as well as the molar ratio of dextran to lysozyme (1:8, 1:4, 1:2, 1:1, 2:1, 4:1, and 8:1) in the feed in the Maillard reaction on nanogel formation was investigated. The data in Table 2 show that increasing the dextran molar ratio leads generally to a decrease of the nanogel size. This can also be explained by the fact that more hydrophilic dextran conjugated to lysozyme can stabilize a larger surface area, which can lead to smaller nanogels. For the conjugates prepared with MW 10 000 and 35 000 dextran, coagula appear at low molar ratio, implying that the hydrophilicity of the conjugates is too weak to form homogeneously dispersed nanogels. For the conjugates prepared with MW 62 000 dextran and in the molar ratio range of 1:8 to 2:1, the resultant nanogels have a relatively narrow PDI (about 0.1) and a Dh of about 140-210 nm. Increasing the dextran molar ratio to 4:1 and 8:1, the nanogels obtained show lower intensity and larger PDI values, (39) Li, M.; Jiang, M.; Wu, C. J. Polym. Sci., Part B: Polym. Phys. 1997, 35, 1593-1599. (40) Zhang, G. Z.; Li, X. L.; Jiang, M.; Wu, C. Langmuir 2000, 16, 92059207.
indicating that the higher hydrophilicity disturbs the nanogel formation. In the following study we used MW 62 000 dextran with a 1:2 molar ratio of dextran to lysozyme to prepare the nanogels. Effects of Heating Temperature and Time in the Second Heating Process. The denaturation temperatures of lysozyme solution at pH 7.0 and 10.3 are about 75 and 60 °C, respectively.41 The turbidity change of the conjugate solution (1 mg/mL) at pH 10.7 was monitored at 500 nm absorbance as a function of heating time at 80 °C. The results (Supporting Information) show that the change of turbidity levels off after 10 min of heating, suggesting that the nanogels have formed in the first 10 min of the heating process. In this study, we heated the conjugate solutions at 80 °C for 30 min to induce heat denaturation and aggregation of lysozyme to prepare the nanogels. Circular dichroism spectra (Supporting Information) show that the secondary and tertiary structures of the lysozyme in the nanogels are almost destroyed, confirming that the lysozyme molecules in the nanogels are denatured. Effects of pH and Concentration of the Conjugate Solution in the Second Heating Process. Table 3 shows the result of DLS of the nanogel solutions prepared at different pH values and protein concentrations. At pH 9.0 the intensity is too weak to be detected. When pH increases from 9.5 to 10.5, the Dh and intensity increase, while the PDI decreases, indicating that more homogeneous nanogels form when the pH is closer to the pI of lysozyme. In the process of gelation, there is a balance between hydrophobic association and electrostatic repulsion.20 A protein carries zero net charge at its pI and carries more net charges when the pH of the solution is farther from its pI. The pI of lysozyme is 10.7.21 When the pH decreases from 10.5 to 9.5 or less, the increase in the positive charge of lysozyme causes an increase of electrostatic repulsion, which keeps lysozyme (41) Petersen, S. B.; Jonson, V.; Fojan, P.; Wirnmer, R.; Pedersen, S. J. Biotechnol. 2004, 114, 269-278.
3490 Langmuir, Vol. 24, No. 7, 2008
Figure 2. Size distributions of lysozyme-dextran nanogels (a) as prepared, (b) after 7 months of storage, and (c) after lyophilization and then rehydration. The nanogels were produced at pH 10.7 and 1 mg/mL lysozyme concentration. The nanogels were stored at 4 °C.
Li et al.
Figure 3. AFM image of lysozyme-dextran nanogels produced at pH 10.7 and 1 mg/mL lysozyme concentration.
molecules apart from each other and makes nanogel formation unfavorable. A higher lysozyme concentration can partly overcome the electrostatic repulsion and can cause the molecules to come closer to each other, favoring nanogel formation. Table 3 shows that at a protein concentration of 1 mg/mL the efficiency of nanogel formation decreases with the decrease of pH, the efficiency being only 55% at pH 9.5. However, at a protein concentration of 10 mg/mL, the efficiency reaches 100% at the pH values of our studies. Lysozyme-dextran nanogels prepared by us are very stable against long-term storage as the size distribution of the nanogels does not change after 7 months of storage at 4 °C (Figure 2). The nanogels can also be stored as lyophiled powder because the nanogel size is almost unchanged after lyophilization and rehydration. The size distributions of the rehydrated and original nanogel solutions are somewhat different, perhaps a limited dissociation and aggregation occurring during the process of lyophilization and rehydration. Besides, the nanogel size does not change when the solution is diluted to 0.01 mg/mL. This result confirms that the cross-linking structure of the nanogels can suppress dissociation upon dilution. All these valuable characteristics can offer significant benefits for the practical applications of the nanogels. Characterization of Lysozyme-Dextran Nanogels. Morphology and Swelling Ratio. The morphology of the nanogels was observed by AFM. Figure 3 exhibits nanogels that are spherical with a smooth surface. Miksa et al. reported that vertical data measured in tapping mode are close to the real values and the lateral dimensions measured are larger than the real values because of the broadening effect of the tip.42 The average radius (r) of the nanogels in Figure 3 is about 56 ( 23 nm, obtained by subtracting 7 ( 2 nm (tip radius) from 64 ( 21 nm (measured radius). The average height (h) is 14 ( 7 nm. The average size of the nanogels in Figure 3 is much smaller than the hydrodynamic radius, about 80 nm (Dh ) 160 nm), measured by DLS. As we know, DLS provides the data for the particles swollen in solution, whereas AFM shows the images of the particles spread and dried on a mica surface. The h value is much smaller than the r value, suggesting that the nanogels are very soft and collapsed on the mica surface. The swelling ratio of the nanogels is estimated from the ratio of average volumes, VDLS/VAFM. The nanogels measured by DLS are supposed to be a sphere. The nanogels in
the AFM image are supposed to be a part of the sphere, so the average volume is estimated using the equation VAFM ) πh2[(r2 + h2)/2h - h/3]. The estimated swelling ratio of about 30 indicates that the nanogels have a low-density structure and can contain a large amount of water. ζ-Potential. The ζ-potential directly relates to the net charges on the surface of the macromolecules and particles.43 Figure 4 displays the pH dependence of ζ-potentials of lysozyme, the mixture of lysozyme and dextran, and the nanogels. The applicable particles for ζ-potential measurement using Malvern NanoSizer ZS90 are in the size range of 3 nm to ∼10 µm (Zetasizer Nano Series User Manual). A native lysozyme molecule is 3.8 × 2.4 × 2.2 nm (PDB code 2LYZ), which is too small to be detected. An aggregated lysozyme solution produced by heating native lysozyme solution at neutral pH and 80 °C for 30 min was used in ζ-potential measurement,22 as was the mixture of lysozyme and dextran. Therefore, ζ-potential data of lysozyme and the mixture in Figure 4 can only be regarded as a rough approximation. The zero ζ-potential of lysozyme appears at pH 10.0, which is close to the pI of lysozyme. Lysozyme shows positive and negative ζ-potential values when the pH of the solution is lower and higher than 10.0, respectively. The mixture of lysozyme and
(42) Miksa, B.; Slomkowski, S.; Marsault, J. P. Colloid Polym. Sci. 1998, 276, 34-39.
(43) Murray, M. J.; Snowden, M. J. AdV. Colloid Interface Sci. 1995, 54, 73-91.
Figure 4. pH dependence of ζ-potentials of lysozyme, the mixture of lysozyme and dextran, and lysozyme-dextran nanogels produced at pH 10.7 and 1 mg/mL lysozyme concentration. In both the mixture and the nanogels, the molar ratio of dextran (MW 62 000) to lysozyme was 1:2.
Lysozyme-Dextran Core-Shell Nanogels
Figure 5. pH dependence of the Dh (circles) and PDI (squares) of lysozyme-dextran nanogels without (open) and with (filled) the addition of 0.15 mol/L NaCl. The nanogels were produced at pH 10.7 and 1 mg/mL lysozyme concentration.
Langmuir, Vol. 24, No. 7, 2008 3491
Figure 6. I1/I3 ratio of pyrene fluorescence in lysozyme-dextran nanogel solutions at 25 °C as a function of pH. The nanogels were produced at pH 10.7 and 1 mg/mL lysozyme concentration. Scheme 1. Chemical Structure of Ibuprofen
dextran also shows positive ζ-potentials when the pH is lower than 10.0 and negative ζ-potentials when the pH is higher than 10.0, but absolute values are smaller than those of individual lysozyme. However, it is important to note that the ζ-potential curve of the nanogels is different from those of lysozyme and the mixture; i.e., the ζ-potential values of the nanogels are about zero over the whole pH range we studied. It clearly shows that the lysozyme molecules are buried inside the nanogels. Furthermore, it indicates that it is the hydrophilic dextran chains, not the charges, that stabilize the nanogels in aqueous solution. In the paper on polyion complex micelles prepared from lysozyme and poly(ethylene glycol)-poly(aspartic acid) block copolymer (PEG-P(Asp)) through electrostatic interaction in aqueous medium,8 a coreshell structure with a polyion complex core and a PEG corona was suggested from an extremely low absolute value of the ζ-potential. Riley et al. studied ζ-potentials of poly(lactic acid)block-poly(ethylene glycol) (PLA-PEG) micellar-like nanoparticles and PLA homopolymer.5 They found that the ζ-potentials of the nanoparticles were negligible compared with the ζ-potential of PLA because the carboxyl acid end groups of the PLA chains were capped by the PEG segment.5 In our study, lysozyme gelates after the heat treatment, while the dextran which is conjugated to lysozyme can provide a barrier to prevent lysozyme from macroscopically coagulating. Besides, the conjugating reaction is not homogeneous (Figure 1). Therefore, we tend to suggest that the nanogels have lysozyme-core and dextran-shell structure; i.e., the lysozyme molecules conjugating with dextran locate on the surface of the nanogel core, while the lysozyme molecules without conjugation locate inside the core. Stability at Different pH Values. After the nanogels were prepared at pH 10.7, the pH of the nanogel solution was adjusted to different values. Figure 5 shows almost unchanged Dh and PDI values of the nanogels in a broad pH range of 2.0-12.0. Besides, the Dh and PDI values of the nanogels do not show a significant change in the presence of 0.15 mol/L NaCl in this pH range. Oakenfull et al.20 pointed out that globular protein molecules form a random aggregate gel structure after a heat treatment at pH close to its pI. In such a protein gel, every molecule is cross-linked by hydrophobic interaction, disulfide bonds, and hydrogen bonds. In addition, the dextran shell shields the electrostatic interaction between the nanogel cores. Therefore, the changes of solution pH and ionic strength cannot cause the nanogels to shrink/swell and aggregate. Our previous studies found that the nanogels prepared from lysozyme-ovalbumin or
ovalbumin-ovotransferrin form reversible secondary aggregates when the net changes on the surface of the protein nanogels are close to zero.22,24 In the present study, lysozyme-dextran nanogels are stable against a change of the solution pH. This result further verifies that the nanogels have dextran-shell and lysozymecore structure. Hydrophobicity. Pyrene was used as a probe to characterize the hydrophobic environment of the nanogels. Pyrene has a much lower solubility in water (about 10-6 mol/L) than in hydrocarbons (7.5 × 10-2 mol/L). It migrates from the water phase into hydrophobic regions once the latter are formed, causing remarkable changes in the photophysical character.44,45 Such changes include the increase of the quantum yield and the decrease of the intensity ratio of the first to third band (I1/I3) in its emission spectrum (Supporting Information). The I1/I3 ratio of pyrene in water is about 1.9, and in a poly(styrene-ethylene oxide) block copolymer micelle aqueous solution it is about 1.2.45 The I1/I3 ratio in native lysozyme and lysozyme-dextran conjugate solution is about 1.7-1.8, slightly lower than the ratio in water. Figure 6 shows the I1/I3 values in the nanogel solutions are around 1.4 in the pH range of 2.0-12.0. This clearly indicates a relatively hydrophobic environment of pyrene in the nanogels. Ibuprofen Loading of Lysozyme-Dextran Nanogels. Ibuprofen (Scheme 1) is a nonsteroidal drug and is widely used to treat inflammation. It has a low solubility at pH 1.0-3.0 (0.020.05 mg/mL) and higher solubility at pH 12.0 (30 mg/mL) in water.46 The drug loading behavior of the nanogels at different pH values was investigated using ibuprofen as a model drug because it has hydrophobic and electrostatic interactions with the nanogels. The loading amounts (the weight ratio of loaded ibuprofen to lysozyme in the nanogels) at pH 3.2, 5.2, and 7.4 are shown in Figure 7. In the absence of NaCl and at a weight ratio of ibuprofen to lysozyme of 1:5 in the feed, the largest loading amount, 8.8%, appears at pH 3.2; the loading efficiency (the weight ratio of (44) Kalyanasundaram, K. J. Am. Chem. Soc. 1977, 99, 2039. (45) Wilhelm, M.; Zhao, C. L.; Wang, Y. C.; Xu, R. L.; Winnik, M. A.; Mura, J. L.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 1033-1040. (46) Jiang, B. B.; Hu, L.; Gao, C. Y.; Shen, J. C. Int. J. Pharm. 2005, 304, 220-230.
3492 Langmuir, Vol. 24, No. 7, 2008
Figure 7. Loading amount of ibuprofen in lysozyme-dextran nanogels as a function of pH. The nanogels were produced at pH 10.7 and 1 mg/mL lysozyme concentration.
loaded ibuprofen to ibuprofen in the feed) is 43%. The loading amount decreases to 5.5% and 2.0% at pH 5.2 and 7.4, respectively. As studied above, the hydrophobicity of the nanogels does not change in the pH range of 2-12 (Figure 6), but the net positive charges of lysozyme decrease when the pH of the solution is changed from 3.2 to 7.4 (Figure 4). For ibuprofen, as its pKa is 5.2-5.6,46 its ionization degree is about 1%, 50%, and 99% at pH 3.2, 5.2, and 7.4, respectively; its hydrophobicity decreases and the solubility in water increases with the increase of pH. The hydrophobic interaction between ibuprofen and the nanogels is the strongest and the electrostatic interaction between them is the weakest at pH 3.2; i.e., the hydrophobic interaction is a leading interaction in the loading process at pH 3.2. Compared to pH 3.2, the hydrophobic interaction decreases and the electrostatic interaction increases between ibuprofen and the nanogels at pH 5.2; both hydrophobic and electrostatic interactions are involved in the loading process. At pH 7.4, the loading amount is the lowest, about 2%, and the loading efficiency is only 10%. This may be explained by the fact that the positive charge of lysozyme and the hydrophobicity of ibuprofen are smaller at pH 7.4 compared to those at pH 3.2 and 5.2. Both the electrostatic and hydrophobic interactions between ibuprofen and the nanogels are not strong. Figure 7 shows the changes of the loading amount in the presence of 0.15 mol/L NaCl, which can screen the electrostatic interaction between ibuprofen and the nanogels. The loading amount does not change significantly at pH 3.2 in the presence of NaCl, supporting the conclusion made above that the electrostatic interaction is not important while the hydrophobic interaction is the leading interaction between ibuprofen and the nanogels at pH 3.2. At pH 5.2, the loading amount decreases to 2% in the presence of NaCl, verifying that the hydrophobic and electrostatic interactions coexist at this pH. The loading amount is only 0.4% at pH 7.4 in the presence of NaCl, confirming that both hydrophobic and electrostatic interactions are not strong. The difference in the ibuprofen loading ability of the nanogels at different pH values and in the absence/presence of 0.15 mol/L NaCl indicates that the nanogels are more suitable for loading protonated ibuprofen. Both DLS and TEM studies show that the
Li et al.
size distributions of loaded nanogels (loading amount 8.8%) and unloaded nanogels are very similar (Supporting Information). After lyophilization, the loaded nanogels are redispersible in water without any detectable secondary aggregates. Preparation of Other Protein-Dextran Nanogels. The formation mechanism of lysozyme-dextran nanogels is universal. We have verified other globular proteins, such as ovalbumin, transferrin, and bovine serum albumin, can also be used to produce protein-dextran conjugates and nanogels through the Maillard reaction and heat-gelation process (Supporting Information). The detailed conditions for the preparation of other protein-dextran conjugates and nanogels may be somewhat different from those of lysozyme-dextran conjugates and nanogels because the properties of these proteins are different from those of the lysozyme.
Conclusion Lysozyme and dextran, two natural biomacromolecules, were used to prepare nanogels using a green method. The method involves the Maillard dry-heat process and heat-gelation process. An optimum condition was developed for the preparation of the nanogels. With a molar ratio of dextran (MW 62 000) to lysozyme of 1:2 and using an 18 h Maillard reaction to produce lysozymedextran conjugates, lysozyme-dextran nanogels were produced by heating the conjugate solution at pH 10.7 and 80 °C for 30 min. The hydrodynamic diameter of the nanogels is around 200 nm. The nanogels are of spherical shape having a lysozyme core and dextran shell structure and a swelling ratio of about 30. The nanogel solutions are very stable against long-term storage at 4 °C, even with pH and ionic strength changes. Besides, the nanogels can be stored as lyophilized powder. As a drug model, ibuprofen can be loaded into the nanogels by virtue of their electrostatic and hydrophobic interactions. The largest loading amount (8.8%) and the best efficiency (43%) of ibuprofen loading were achieved in its protonated form. The knowledge gained from these studies on the mechanism of formation of lysozymedextran nanogels is significant, and the strategies developed thereby can be effectively applied to prepare other globular protein-dextran nanogels. Acknowledgment. The financial support of the National Natural Science Foundation of China (NSFC Project 50673020 and 50333010) and Science and Technology Committee of Shanghai Municipality (Grant 07DJ14004) is gratefully acknowledged. Supporting Information Available: Turbidity change and DLS result of the individual lysozyme, the mixture of lysozyme and dextran, and the solution of lysozyme-dextran conjugates after a heat treatment at pH 10.7 and 80 °C for 30 min; SDS-PAGE analysis of the individual lysozyme as well as the mixture of lysozyme and dextran after a similar Maillard heating process; circular dichroism and pyrene fluorescence spectra of the individual lysozyme, the mixture, and the conjugate and nanogel solutions; TEM images and DLS result of the nanogels before and after ibuprofen loading; and DLS result of the nanogels prepared from different proteins. This information is available free of charge via the Internet at http://pubs.acs.org. LA702785B
