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Langmuir 2008, 24, 11370-11377
A Novel Synthesis of Chitosan Nanoparticles in Reverse Emulsion Fabrice Brunel,†,‡ Laurent Ve´ron,† Laurent David,‡ Alain Domard,‡ and Thierry Delair*,† BioMe´rieux, Chemin de l’orme, Marcy l’Etoile 69280, France, and Laboratoire des Mate´riaux Polyme`res et Biomate´riaux, UMR CNRS 5223 ‘IMP’, UniVersite´ Lyon 1, UniVersite´ de Lyon, 15 bd. Andre´ Latarjet Baˆt. ISTIL, F-69622, Villeurbanne Cedex, France ReceiVed June 18, 2008. ReVised Manuscript ReceiVed July 30, 2008 Physical hydrogels of chitosan in the colloidal domain were obtained in the absence of both cross-linker and toxic organic solvent. The approach was based on a reverse emulsion of a chitosan solution in a Miglyol/Span 80 mixture, generally regarded as safe. Temperature and surfactant concentration were optimized, and the impact of the degree of acetylation (DA) and the molar mass of chitosan was investigated. When chitosan had a DA above 30%, only macroscopic gels were obtained, because of the predominance of attractive Van der Waals forces. The lower the molar mass of chitosan, the better the control over particle size and size distribution, probably as a result of either a reduction in the viscosity of the internal aqueous phase or an increase in the disentanglement of the polymer chain during the process. After extraction and redispersion of the colloid in an ammonium acetate buffer, the composition of the particles was around 80% of pure chitosan corresponding to a recovery of 60% of the original input. These new and safe colloids offer wide perspectives of development in further applications.
1. Introduction Drug delivery has become a research topic of major interest in the field of nanomedicine. Indeed, the use of carriers has been considered to overcome many problems encountered with conventional drug therapy such as poor bioavailability, difficulties in specific targeting, and so forth. The role of the carrier is to transport, deliver, and protect molecules of interest (drugs, proteins, DNA) at the targeted site. Drug-carrier systems can either be soluble (water-soluble polymer-drug conjugates)1,2 or colloidal (liposomes, polyplexes, nanoparticles, nanoemulsions).3 In the case of colloids, the bioactive molecule to deliver can be encapsulated into the particles or adsorbed at their surface,4 according to the therapeutic strategy and/or the nature of the bioactive compound. The absence of toxicity, good biocompatibility, and even bioactivity are criteria that the carrier should fulfill, hence, the interest in glycosaminoglycans such as chitosan. Chitosan is a β,(1f4)-linked copolymer of 2-amino-2-deoxyβ-D-glucan (GlcN) and 2-acetamido-2-deoxy-β-D-glucan (GlcNAc) obtained by heterogeneous deacetylation of chitin.5-7 Chitosan can be obtained by reacetylation in homogeneous conditions of a fully deaceylated chitosan.8 Chitosan exhibits a high degree * Corresponding author. E-mail:
[email protected]. † BioMérieux. ‡ Universite´ de Lyon. (1) Duncan, R.; Gac-Breton, S.; Keane, R.; Musila, R.; Sat, Y. N.; Satchi, R.; Searle, F. J. Controlled Release 2001, 74(1-3), 135–146. (2) Duncan, R.; Spreafico, F. Clin Pharmacokinet. 1994, 27(4), 290–306. (3) Kumar, M. N. V.; Hellermann, G.; Lockey, R. F.; Mohapatra, S. S. Expert Opin. Biol. Ther. 2004, 4(8), 1213–1224. (4) Kreuter, J.; Speiser, P. P. Infect. Immun. 1976, 13(1), 204–210. (5) Lamarque, G.; Chaussard, G.; Domard, A. Biomacromolecules 2007, 8(6), 1942–1950. (6) Lamarque, G.; Viton, C.; Domard, A. Biomacromolecules 2004, 5(5), 1899– 1907. (7) Lamarque, G.; Viton, C.; Domard, A. Biomacromolecules 2004, 5(3), 992– 1001. (8) Sorlier, P.; Denuzie`re, A.; Viton, C.; Domard, A. Biomacromolecules 2001, 2(3), 765. (9) VandeVord, P. J.; Matthew, H. W. T.; DeSilva, S. P.; Mayton, L.; Wu, B.; Wooley, P. H. J. Biomed. Mater. Res. 2001, 59(3), 585–590. (10) Domard, A.; Rinaudo, M. Chitosane: structure-properties relationship and biomedical applications. In Polymeric Biomaterials; Dumitriu, S., Ed.; CRC Press: Boca Raton, FL, 2002; Chapter 9. (11) Hirano, S.; Seino, H.; Akiyama, Y.; Nonaka, I. Polym. Mater. Sci. Eng. 1988, 59, 897.
of biocompatibility and a low toxicity in mouse and rabbit models for intravenous and oral administration.9-11 Chitin is degraded in Vitro and in ViVo by lysosyme into N-acetyl glucosamine.12,13 Chitin and chitosan can also be depolymerized with chitinase/ chitosanase or nonspecific enzymes such as lipases, proteases, or carbohydrolases.14,15 Recently a human-specific chitinase was discovered (a chitotriosidase), but no chitosanase has been found in humans, yet.16 Chitosan is known to be mucoadhesive and, although it is no longer a polycation at physiological pH, it is considered to interact as a polycation with negatively charged sialic acid residues in mucin.17,18 In addition, some studies have shown that chitosan exhibits immune stimulating properties such as increasing accumulation and activation of macrophages19 and polymorphonuclear cells,20 suppressing tumor growth,21,22 promoting resistance to infections by microorganisms,23,24 and enhancing both humoral and cell-mediated immune responses.25-28 For all the reasons mentioned above, chitosan has been used extensively as a carrier in delivery systems. In the literature, various strategies were reported to produce chitosan nanoparticles. First, chitosan can be complexed, in the protonated (12) Tomihata, K.; Ikada, Y. Biomaterials 1997, 18(7), 567–575. (13) Varum, K. M.; Myhr, M. M.; Hjerde, R. J.; Smidsrod, O. Carbohydr. Res. 1997, 299, 99. (14) Muzzarelli, R. A. A. Cell. Mol. Life Sci. 1997, 53, 131–140. (15) Prashanth, K. V. H.; Taranathan, R. N. Trends Food Sci. Technol. 2007, 18, 117–131. (16) Renkema, G. H.; Boot, R. G.; Muijsers, A. O.; Donker-Koopman, W. E.; Aerts, J. M. F. G. J. Biol. Chem. 1995, 270(5), 2198–2202. (17) Lehr, C.-M.; Bouwstra, J. A.; Schacht, E. H.; Junginger, H. E. Int. J. Pharm. 1992, 78(1), 43. (18) Chopra, S.; Mahdi, S.; Kaur, J.; Iqbal, Z.; Talegaonkar, S.; Ahmad, F. J. J. Pharm. Pharmacol. 2006, 58(8), 1021–1032. (19) Peluso, G.; Petillo, O.; Ranieri, M. Biomaterials 1994, 15, 1215–1220. (20) Usami, Y.; Okamoto, Y.; Minami, S. J. Vet. Med. Sci. 1994, 56, 761–2. (21) Lifeng, Q.; Zirong, X.; Minli, C. Eur. J. Cancer 2007, 43(1), 184–193. (22) Kim, T. H.; Jin, H.; Kim, H. W.; Cho, M. H.; Cho, C. S. Mol. Cancer Ther. 2006, 5(7), 1723–1732. (23) Hirano, S.; Sagao, N. Agric. Biol. Chem. 1989, 53, 3065–3066. (24) Moon, J.-S.; Kim, H.-K.; Koo, H. C.; Joo, Y.-S.; Nam, H.-m.; Park, Y. H.; Kang, M.-I. Appl. Microbiol. Biotechnol. 2007, 75, 989–998. (25) Marcinkiewicz, J.; Polewka, A.; Knapczyk, J. Arch. Immunol. Ther. Exp. 1991, 39, 127. (26) Seferian, P. G.; Martinez, M. L. Vaccine 2000, 19, 661. (27) Muzzarelli, R. A. A.; Baldassare, V.; Conti, F.; Ferrera, P.; Biagini, G.; Gazzanelli, G.; Vasi, V. Biomaterials 1988, 9, 247. (28) Zaharoff, D. A.; Rogers, C. J.; Hance, K. W.; Schlom, J.; Greiner, J. W. Vaccine 2007, 25, 2085–2094.
10.1021/la801917a CCC: $40.75 2008 American Chemical Society Published on Web 09/06/2008
ReVerse Emulsion Synthesis of Chitosan NPs
state, with a simple electrolyte of polyanionic nature such as sulfates29 or phosphate.30 Polyelectrolyte complexes (PECs) can also be formed by ionic condensation between a macromolecular polyanion and chitosan. Many anionic polymers were used such as dextran sulfate,31 DNA,32,33 alginate,34,35 and carboxymethyl konjac glucomannan.36 Besides electrostatic interactions, the stability of PECs can be enhanced by other types of interactions such as hydrogen bonding or hydrophobic forces.37 A second route toward chitosan nanoparticles was based on water-in-oil (w/o) emulsions, i.e., by dispersion of an aqueous solution of chitosan in a continuous oil phase. Chitosan chemical gels can be formed inside aqueous droplets by a cross-linker diluted in the continuous phase. Many chemical cross-linkers were considered, such as glutaraldehyde,38,39 epichlorhydrine, or ethylene diglycidyl ether.40,41 Ionic surfactants, such as sodium dodecylsulfate (SDS)42 or lecithin,43 were also used to form surfactant-PECs yielding physical hydrogels. Finally, a reverse emulsion of chitosan was mixed with a second reverse emulsion containing a hydrophilic gelation agent (tripolyphosphate (TPP), sulfate ion, sodium hydroxide) dissolved in an aqueous phase. The coalescence of both emulsions led to a localized gelation and the formation of chitosan nanoparticles.44 Most of these processes yield chitosan hydrogels formed by the addition of a covalent or electrostatic cross-linker. As these cross-linkers could potentially present toxicological risks for parenteral administration, the synthesis of pure chitosan nanogels appeared to be an interesting, though challenging, alternative. The formation of physical hydrogels requires (i) the alteration of the chitosan solubility in the medium, in a such a way that solvent-segment interactions are reduced to favor segmentsegment interactions, and (ii) the polymer concentration to be initially above the chain entanglement concentration, C*, so that a network can be formed. Reducing chitosan solubility will be achieved by decreasing the repulsive electrostatic interactions between the macromolecules, and this can be obtained by lowering the charge density of chitosan or by modifying the dielectric constant of the medium. The first physical hydrogel from chitosan was obtained by increasing the degree of acetylation (DA) up (29) Berthold, A.; Cremer, K.; Kreuter, J. J. Controlled Released 1996, 39, 17. (30) Calvo, P.; Remunan-Lopez, C.; Vila-Jato, J. L.; Alonso, M. J. J. Appl. Polym. Sci. 1997, 63, 125. (31) Schatz, C. Chitosane: Comportement en Solution et Formation de Particules; Universite´ Claude Bernard - Lyon 1: Vileurbanne, France, 2003. (32) Mumper, R. J.; Wang, J.; Claspell, J. M.; Rolland, A. P. Proc. Int. Symp. Controlled Relat. Bioact. Mater. 1995, 22, 179. (33) Mumper, R. J.; MacLaughlin, F. C.; Wanf, J.; Tagliaferri, J. M.; Gill, I.; Hinchcliffe, M.; Rolland, A. P. J. Controlled Released 1998, 56, 259. (34) Douglas, K. L.; Tarizian, M. J. Biomater. Sci., Polym. Ed. 2005, 16(1), 43. (35) De, S.; Robinson, D. J. Controlled Released 2003, 89, 101. (36) Du, J.; Sun, R.; Zhang, S.; Zhang, L.-F.; Xiong, C.-D.; Peng, Y.-X. Biopolymers 2005, 78, 1. (37) Schatz, C.; Lucas, J.-M.; Viton, C.; Domard, A.; Pichot, C.; Delair, T. Langmuir 2004, 20(18), 7766–7778. (38) Yang, B.; Chang, S.-q.; Dai, Y.-d.; Chen, D. Radiat. Phys. Chem. 2007, 76, 968–973. (39) Jia, Z.; Wang, Y.; Lu, Y.; Luo, G. React. Funct. Polym. 2006, 66, 1552– 1558. (40) Ohya, Y.; Shiratani, M.; Kobayashi, H.; Ouchi, T. J. Macromol. Sci., Pure Appl. Chem. 1994, A31, 629. (41) Banerjee, T.; Mitra, S.; Kumar, A. S.; Sharma, R. K.; Maitra, A. Int. J. Pharm. 2002, 243, 93. (42) Merkovich, E. ; Mironov, A.; Kildeeva, N.; Babak, V.; Rinaudo, M. Colloid properties of complexes between chitin derivatives and surfactants: Fundamentals and applications. Presented at Polymerix 2000 “Biopolymers: Foods & Cosmetic Applications” European symposium held in Rennes, France, June 7-8, 2000. (43) Sonvico, F.; Cagnani, A.; Rossi, A.; Motta, S.; Di.Bari, M. T.; Cavatorta, F.; Alonso, M. J.; Deriu, A.; Colombo, P. Int. J. Pharm. 2006, 324(1), 67–73. (44) Tokumitsu, H.; Ichikawa, H.; Fukumori, Y.; Block, L. H. Chem. Pharm. Bull. 1999, 47(6), 838.
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to 70%, as studied by Roberts and Moore45 and Vachoud et al.46 In this case, the presence of a large number of N-acetyl groups suggests that hydrophobic interactions are highly involved in the formation of the gel. Montembault et al. were the first to report that gels could be obtained independently of the DA, by substituting water by 1,2-propanediol during a gelation process that consisted of evaporation of the initial hydro-alcoholic solution.47 Later, the same team reported an approach toward macroscopic physical gels of chitosans without using potentially toxic chemicals. The charge density of the chitosan chains was decreased by deprotonation with gaseous ammonia. In this process, hydrogen bonding of macromolecules was responsible for the gel formation, but the authors showed the involvement of hydrophobic interactions as the time to reach the gel point decreased when DA increased.48 In the present work, we report on the elaboration of new chitosan nanogels dispersed in an aqueous phase. One of the key aspects of the process is the absence of any cross-linker, the nanogels being obtained by a purely physical process, in reverse emulsion.
2. Materials and Methods The oil phase was composed of medium-chain triglycerides from capric/caprilic acid (Miglyol 812 N, dynamic viscosity: 24 mPa · s)) purchased from SASOL (Germany) and a surfactant, sorbitan monooleate (Span 80) from Fluka (Germany). Chitosan was provided by Mahtani Chitosan PVT, Ltd., India, batch 124 (DA ∼ 5%, Mw ∼ 400 000 g · mol-1). Its molecular characteristics were determined in our laboratory. We found a DA ) 4 ( 1%, and a molecular weight Mw ) 405 400 ( 8800 g · mol-1. 2.1. Chitosan Preparation. Prior to use, the polymers were purified as follows: dissolution in a 0.1 M acetic acid solution, filtration through Millipore membranes of decreasing porosity (from 3 to 0.22 µm), precipitation with an ammonia/methanol mixture (3/7, v/v), rinsing with deionized water until neutrality, and freezedrying. A purified chitosan of high molar mass was N-acetylated with acetic anhydride in homogeneous medium to reach different DAs. The reaction was performed in a hydro-alcoholic mixture according to the procedure previously described by Vachoud et al.46 After reacetylation, chitosans were neutralized, rinsed with deionized water, and then freeze-dried. The nitrous deamination was carried out to produce low molar mass polymers.49,50 For this purpose, chitosan was dissolved at 0.5% (w/v) in a 0.2 M acetic acid/0.1 M sodium acetate buffer. A 0.15 M sodium nitrite solution was added to the chitosan solution to obtain a nitrite/glucosamine unit molar ratio of 0.5. The reaction was performed under moderate magnetic stirring for various durations (1 to 24 h). Low molar mass chitosans were recovered by precipitation with an ammonia/methanol (3/7, v/v) mixture, purified by several washings with deionized water until neutrality, and lyophilized. 2.2. Chitosan Characterization. The DA was determined on purified chitosans by 1H NMR spectroscopy (Varian, 500 MHz), according to the method developed by Hirai et al.51 The weight-average molecular weight (Mw), the z-average rootmean-square of the gyration radius (RG,z) and the polydispersity index (Ip) were measured by gel permeation chromatography (3000 and 6000 PW TSK gel columns, inner diameter ) 7.8 mm and length ) 300 mm) coupled online with a differential refractometer (45) Moore, G. K.; Roberts, G. A. F. Int. J. Biol. Macromol. 1980, 2, 78. (46) Vachoud, L.; Zydowicz, N.; Domard, A. Carbohydr. Res. 1997, 302, 169–177. (47) Montembault, A.; Viton, C.; Domard, A. Biomaterials 2004, 26, 933– 943. (48) Montembault, A.; Viton, C.; Domard, A. Biomacromolecules 2005, 6, 653–662. (49) Allan, G. G.; Peyron, M. Carbohydr. Res. 1995, 277, 273–282. (50) Allan, G. G.; Peyron, M. Carbohydr. Res. 1995, 277, 257–272. (51) Hirai, A.; Odani, H.; Nakajima, A. Polym. Bull. 1991, 26, 87–94.
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Table 1. Effect of Ultrasound on the Molar Mass Distributions of Chitosana molar mass distribution of chitosan before sonication
molar mass distribution of chitosan after sonication
Mw (g · mol )
Mn (g · mol )
Ip
DPn
Mw (g · mol-1)
Mn (g · mol-1)
Ip
DPn
405400 ( 8800 117300 ( 1100 91700 ( 530 43950 ( 80 11 ( 30
304800 ( 5800 76200 ( 740 56260 ( 230 24970 ( 80 5630 ( 40
1.33 1.54 1.63 1.76 2
1850 460 350 150 35
197600 ( 1200 145440 ( 900 97440 ( 410 41700 ( 70 12410 ( 40
114200 ( 100 90900 ( 200 53570 ( 200 25520 ( 50 6500 ( 30
1.73 1.6 1.82 1.7 1.9
670 540 340 150 39
-1
-1
a Mw is the weight-average molecular mass, Mn is the number-average molecular mass, Ip ) Mw/Mn is the polydispersity index, and DPn is the number-average of the degree of polymerization (DPn ) Mn/M0 with M0 being the average molar mass of chitosan repeat units).
(Waters 410) and a multiangle laser-light-scattering spectrometer (MALLS, Wyatt, Dawn DSP, Santa Barbara, CA) equipped with a 5 mW He/Ne laser operating at λ ) 632.8 nm. Analyses were performed in microbatch mode using the K5 flow cell. Light intensity measurements were derived following the classical Rayleigh-Debye equation allowing us to deduce Mw, and RG,z. A degassed 0.2 M acetic acid/0.15 M ammonium acetate buffer (pH ) 4.5) was used as eluent. The flow rate was maintained at 0.5 mL/mn. Refractive index increments (dn/dc) were determined independently for each sample, in the same solvent, with an interferometer (NFT Scan Ref) operating at λ ) 632.8 nm.8,52,53 The water content was determined by thermogravimetric analysis (TGA; DuPont Instrument 2950, Twin Lakes, WI). 2.3. Viscosimetry. Intrinsic viscosity measurements were performed at 25 ( 0.1 °C using an automatic Ubbelhode capillary viscometer with an inner diameter of 0.53 mm (Viscologic TI.1, SEMATech, Nice, France). The intrinsic viscosity [η] was calculated by extrapolation to zero concentration by the Huggins or Kraemer equation, then considering the average of two results. Chitosans were dissolved (0.1-0.3% (w/w)) in a degassed 0.2 M acetic acid/ 0.15 M ammonium buffer, pH ) 4.5. The critical concentration of chain entanglement C* was determined considering the approximation C* × [η] ) 1. Dynamic viscosities of chitosan solution in pure water were measured using a stress rheometer: AR2000 (TA Instruments, New Castle, DE) with an acrylic parallel plate. The diameter of the acrylic plate is 60 mm, and the lower plate is the constant temperature peltier with a sufficiently larger diameter compared with the acrylic plate. All the measurements were performed at 20 °C, for 1% w:w solutions in water, corresponding to the polymer concentration in the dispersed phase during particle synthesis. To measure the zero shear viscosity, a creep test was used with the AR2000. Steady rate-sweep tests were performed between 0.01 and 100 s-1 in shear rate. 2.4. Interfacial Tension Measurement. The interfacial tension between the chitosan aqueous solution and the oil phase was measured by the drop shape analysis (DSA) method with a contact angle ¨ SS G10 (Hamburg, Germany). A droplet measuring system, KRU of aqueous phase was suspended at the end of a needle (diameter 0.5 mm), which was immersed in the oil phase. The shape of the drop was monitored by a CCD camera, and the surface tension was deduced from the mathematical analysis of this shape.54,55 The volume of the droplet was the highest possible volume before the fall of the drop. The system was calibrated (camera focus, horizontality) by measuring the interfacial tension of deionized water in air (71.5 mN.m). 2.5. Nanoparticles Synthesis. Nanoparticles were prepared in a reverse emulsion process with a chitosan aqueous phase emulsified in oil (Miglyol 812N) containing the surfactant. For a typical process with 1% of surfactant, chitosan (0.05 g) was dissolved under moderate stirring by adding a stoichiometric amount of acetic acid with respect to the free amine moieties for each DA. This aqueous chitosan solution (5 mL, chitosan: 1% w/v) was emulsified in 25 mL (23.75 g) of Miglyol 812N containing Span 80 (0.25 g, 1% w/v) with a sonotrode (Bandelin KE76, Berlin, Germany) operating at 20 kHz with a peakto-peak magnitude of 260 µm under magnetic stirring. The ultrasonic probe (Ø ) 6 × L ) 118 mm) was immersed into the liquid up to 2 cm. The reactor was thermostatted to prevent flocculation and destabilization of the emulsion due to the heating induced by
sonication. After 5 min of ultrasound treatment, a stream of ammonia increased the pH of the medium up to 9, which caused the gelation of chitosan droplets. The ultrasound treatment was maintained for 10 min to prevent droplet coalescence and/or particle aggregation. Chitosan is known to be degraded by means of sonication,56,57 thus we checked whether such degradation occurred during the gelation process. For this purpose, the molar mass distributions, before and after ultrasound treatment, were compared by MALLS coupled with size-exclusion chromatography (SEC-MALLS). Only the sample with the highest molar mass was significantly degraded during sonication, as shown by the drastic Mw reduction from 400 000 g · mol-1 down to 190 000 g · mol-1 (Table 1). Finally, the dispersion was centrifuged at 1000g for 15 min. After elimination of the supernatant, the particles were dispersed in 40 mL of ethanol. This washing cycle was repeated twice with ethanol and twice with water, then particles were dispersed in an ammonium acetate buffer (50 mmol · L-1 pH ) 4.5) by slow stirring overnight, and the final pH was measured between 5.5 and 6.5. The efficiency of the purification procedure was controlled by 1H NMR and a colorimetric assay (for chitosan). For these experiments, the purified particles pellets were dispersed with deionized water and lyophilized. 2.6. Nanoparticle Characterization. 2.6.1. Colorimetric Assay. The concentration of chitosan in nanoparticles was determined with Orange II, an anionic dye interacting with ammonium groups of chitosan (Scheme 1).58 Ten milligrams of freeze-dried particles was dissolved in 10 mL of an acidic buffer (ammonium acetate: 50 mmol · L-1, pH ) 4.5). The obtained solution was diluted 10-fold in the same buffer solution. 100 µL of the latter solution (Vchitosan), 50 µL of Orange II ([OII] ) 2.5 × 10-4 mol · L-1) and 50 µL of the ammonium acetate buffer solution were added and mixed for 5 min (Vtotal ) 200 µL). The dilution factor (f) of the chitosan solution was chosen in order to have excess of Orange II sulfate groups with regard to ammonium moieties from chitosan. After centrifugation (13 200 rpm, 5 min) to remove the insoluble chitosan-Orange II complex, the absorbance was measured at 485 nm (corresponding to the unbound dye) with a UV/vis spectrometer µQuant (Bio-TECK instrument). Prior to the determination of the chitosan content of the particle suspensions, a calibration curve was established with chitosan solutions of concentrations from 0 to 2 × 10-4 mol · L-1 ([NH3+] titrated with an Orange II solution of 2.5 × 10-4 mol · L-1). The absorbance at 485 nm was plotted against [NH3+]. The chitosan concentration was determined from the absorbance data via the calibration curves obtained with chitosans of the same molar masses and DAs as those used for particle synthesis. In this assay, there is (52) Sorlier, P.; Rochas, C.; Morfin, I.; Viton, C.; Domard, A. Biomacromolecules 2003, 4(4), 1034–1040. (53) Schatz, C.; Viton, C.; Delair, T.; Pichot, C.; Domard, A. Biomacromolecules 2003, 4(3), 641. (54) Anastasiadis, S. H.; Chen, J.-K.; Koberstein, J. T.; Siegel, A. F.; Sohn, J. E.; Emerson, J. A. J. Colloid Interface Sci. 1987, 119(1), 55. (55) Girault, H. H. J.; Schriffrin, D. J.; Smith, B. D. V. J. Colloid Interface Sci. 1984, 101(1), 1984. (56) Czechowska-Biskupa, R.; Rokitaa, B.; Lotfyb, S.; Ulanskia, P.; Rosiak, J. M. Carbohydr. Polym. 2005, 60, 175. (57) Cravotto, G.; Tagliapietra, S.; Robaldo, B.; Trotta, M. Ultrason. Sonochem. 2005, 12, 95. (58) Maghami, G. G.; Roberts, G. A. F. Makromol. Chem. 1988, 189(10), 2239.
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Scheme 1. Reaction Diagram of Chitosan Complexation by an Anionic Dye (Orange II)
a linear relationship between the Orange II absorbance and the chitosan concentration. The measurement of the absorbance was performed with a UV/vis spectrometer µQuant from Bio-TECK instrument. The mass of chitosan (mchitosan) was deduced from absorbance measurement according to the following equation:
mchitosan )
[(((
) )⁄ ]
)
Vtotal Aλ)485nm - b × × f 100 × a Vchitosan
M0 DA 1100
where Aλ)485nm is related to [NH3+] with the equation of the calibration plot:
NH+ 3
Aλ)485nm ) a[
]+b
a and b are coefficients determined from the calibration curve, Vtotal ) 200 µL, Vchitosan ) 100 µL, f is the dilution factor of the chitosan solution, and M0, the average molar mass of chitosan repeat units, is defined by
DA DA M0(g · mol-1) ) 203 × + 161 × 1 100 100 DA ) 42 × + 161 100
(
)
( )
where 203 and 161 are the molar masses of N-acetylglucosamine and glucosamine residues, respectively. 2.6.2. Photon Correlation Spectroscopy. The size distribution of the nanogels was determined at 90° with a Malvern Zetasizer HS3000 equipped with a 10 mW He/Ne laser beam operating at λ ) 632.8 nm. All measurements were performed at 25.0 ( 0.2 °C. The selfcorrelation function was expanded in a power series (Cumulants methods).59 The polydispersity value provided by the software is a dimensionless parameter defined by µ2/(Γ)2, where µ2 is the second cumulant of the correlation function, and (Γ) is the average decay rate. Each value is the average of three series of 10 measurements. For a monodisperse colloidal suspension, the polydispersity index should be below 0.05, but values up to 0.5 can be considered for comparison purposes.60 2.6.3. 1H NMR. Ten milligrams of freeze-dried particles was dissolved in 1 mL of D2O with 5 µL of DCl (35 wt % in D2O) by slow stirring overnight at room temperature. Then a mixture of 1 mL of DMSO-d6 with 10 mg of sodium 3-(trimethylsilyl)propionated4 (TMSP-d4) was added. 1H NMR spectra were recorded with a Bruker 200 MHz spectrometer at 50 °C in order to decrease the viscosity of the solution (Figure 1). The intensities were normalized according to the peak of the TMSP-d4 corresponding to 9 protons and then, to an intensity of 9 au. The amount of chitosan in the (59) Koppel, D. E. J. Chem. Phys. 1972, 57, 4814. (60) Coombes, A. G. A.; Scholes, P. D.; Davies, M. C.; Illum, L.; Davis, S. S. Biomaterials 1995, 15(9), 673–680.
particles was obtained from integration of the 1H NMR spectrum using the following equation:
mchitosan )
IH3-H6′ mTMSP × M0 × 5 MTMSP
where M0 is the average molar mass of chitosan repeat units, IH3-H6′ is the normalized integral intensity of the glucosidic protons (∼4 ppm) corresponding to five protons, mTMSP ) 3.54 mg, and MTMSP ) 168 g · mol-1.
3. Results and Discussion 3.1. Nanoparticles Synthesis and Characterization. Miglyol 812N was chosen as the continuous oil phase, since it is generally regarded as a pharmaceutically safe product.61 The low dielectric constant of the dispersion medium imposed a steric stabilization of emulsion droplets by a nonionic surfactant.62 According to the hydrophilic-lipophilic balance (HLB) concept introduced by Griffin,63 the stabilization of w/o emulsions required an HLB value ranging between 4 to 6.64 For this reason, sorbitan monooleate (HLB ) 4.3) was selected. In a typical formulation, the reverse emulsion was composed of a continuous phase (Miglyol (23.75 g), a surfactant, sorbitan monooleate, i.e., Span 80 (0.25 g), and a dispersed phase of water (5 g) containing 0.05 g of chitosan. After gelation of the nanodroplets, the particles were washed by centrifugation, as described in the experimental section, in order to remove Miglyol and the surfactant. After the first centrifugation, the reaction mixture consisted of two phases, from top to bottom, the organic one and the water one, plus the pellet containing the particles. The mass of the recovered organic phase was around 20 g, meaning that about 85% of Miglyol was eliminated at this stage. The crude particles were washed and centrifuged twice with ethanol followed by a new washing with water and centrifugation. The supernatants of the washings with ethanol were evaporated under reduced pressure, and 1H NMR analysis showed that the residue (1 or 2 g) was Miglyol. Thus, globally, the eliminated Miglyol was close to 95%; the missing 5% could be lost during the experiment or could still be in the particles. The particle composition was in turn studied by 1H NMR and a colorimetric assay using Orange II (see Section 2.6.1. Colorimetric Assay). The 1H NMR spectra of chitosan and Miglyol 812N are presented (61) Traul, K. E.; Driedger, A.; Ingle, D. L.; Nakhasi, D. Food Chem. Toxicol. 2000, 38, 79. (62) Graillat, C.; Lepais-Masmejean, M.; Pichot, C. J. Dispersion Sci. Technol. 1990, 11(5), 455. (63) Griffin, W. C. J. Soc. Cosmet. Chem. 1954, 1, 311. (64) Walstra, P. Formation of Emulsions; Marcel Dekker: New York, 1983.
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Figure 1. 1H NMR spectra in D2O + DCl + DMSO-d6 (calibration TMSP-d4) of chitosan (a), Miglyol 812 N (b), and freeze-dried particles (c).
in Figure 1a,b. In Figure 1c, the 1H NMR spectrum of the particles after washing cycles, freeze-drying, and redissolution in the NMR solvent clearly showed the presence of chitosan and some traces of Miglyol 812N at 1.29 ppm (methylene protons). To allow the 1H NMR determination of Miglyol, the residual oil concentration must be lower than its solubility limit. The solubility limit of Miglyol was determined by 1H NMR as follows. At 5 mg · mL-1 of Miglyol in the D2O/DMSO-d6 (50/50, v/v) mixture, two phases separated, as a proof of saturation. The fraction of soluble Miglyol in the solvent phase (D2O/DMSO-d6) was determined using a known amount of TMSP-d4 as an internal reference for integration. The solubility limit was found to be 1.2 mg · mL-1. For our chitosan nanoparticle samples, the solutions in the 1H NMR tubes were clear; hence the residual oil concentration was lower than 1.2 mg · mL-1. In Figure 1c, the intensity of the peak corresponding to Miglyol is too low for an accurate determination, but the
amount of residual Miglyol in the particles could be estimated to 3% (w/w) by integration of the peak at 1.29 ppm. On the same spectrum (Figure 1c), we looked for the presence of surfactant traces. The peaks at 1.29 ppm, which we interpreted as the resonance of protons 13-17 of Miglyol, could also partly result from the aliphatic protons of the saturated hydrocarbon chain of Span 80. But no peak at 4.81 ppm corresponding to the two unsaturated protons could be detected. Hence, to the detection limit of the 1H NMR technique, it can be concluded that no Span 80 remained in the purified particles. The results presented in Table 2 show that the yield of crude freeze-dried particle pellet was around 85% for the low molar mass chitosan and only 70% for the high molar mass chitosan. This means that some chitosan was lost in the process, probably during the washing cycles. Furthermore, a small amount of chitosan was still dissolved in the water phase obtained after the
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Langmuir, Vol. 24, No. 20, 2008 11375
Table 2. Chitosan Recovery in Particles, Obtained by Colorimetric and NMR Assaysa chitosan recovered in the particles (mg) (particles purity %)
Mw ) 12 410 ( 30 g · mol Mw ) 405 400 ( 8800 g · mol-1 -1
a
dry chitosan used for particle synthesis (mg)
crude freeze-dried particle pellet (mg) (% yield)
colorimetric assay
50 50
42 (85%) 35 (70%)
37 (88%) 32 (87%)
chitosan recovery (%) ) chitosan recovered in the particles/chitosan input
H NMR
colorimetric assay
34 (81%) 25 (66%)
74% 61%
1
1
H NMR
68 ( 3% 46 ( 5%
Results are the average value of three experiments.
first centrifugation (between the organic phase and the particle pellet). This aqueous phase was lyophilized and analyzed by 1H NMR in order to deduce the chitosan content: about 3% of the chitosan did not form nanogels and thus was not collected with the particle pellet. Finally, the lower amount of raw freeze-dried particle pellet obtained with the high molar mass chitosan could be due to the high viscosity of this chitosan solution. Indeed, high molar mass chitosan solutions are quite viscous and sticky; thus, it is impossible to transfer the entire solution in the reactor without losses on the walls of the tube used for chitosan solubilization. Two independent techniques were used to determine the purity of the prepared particles, in terms of weight fraction of chitosan. The amount of chitosan in the particles was obtained (i) from 1H NMR via the intensities of the peak of chitosan glucosidic protons (from 3.5 to 4 ppm) in reference to the peak of TMSP-d4, and (ii) from the colorimetric assay using an anionic dye that specifically complexes the amino groups of chitosan, after dissolving lyophilized particles in an ammonium acetate buffer (see experimental section). In every case, particles consisted of 80-90% chitosan. For the low molar mass chitosan, 1H NMR results are in good agreement with the colorimetric assay results. In the case of high molar mass chitosan, discrepancies between the two methods are more important. The lower value obtained by 1H NMR is inherent to the technique itself. The high molar mass chitosan is very viscous, and even at 50 °C the molecular mobility of the chains is impeded as compared to TMSP-d4. Hence, 1H NMR signals are weak, and thus the values are underestimated. The 10-20% of residual matter that constitutes the freeze-dried particle is composed of 3% Miglyol (as explained above) and 5 to 10% water (as determined from the loss of mass at 120 °C measured by TGA). The yield in chitosan recovery is defined as the ratio of the mass of chitosan in the particles, determined by titration (1H NMR or colorimetric assay), to the mass of chitosan initially involved in the synthesis. It varied from 50 to 70%, depending on the molar mass of the polysaccharide. 3.2. Effect of Temperature on the Particle Size Distribution. The temperature during emulsification (Te) was varied between 10 and 50 °C (Figure 2). The average particle size evolved in a complex way: between 500 and 1500 nm, and the size distribution was broad (high polydispersity index). The optimum temperature was 20 °C, corresponding to the lowest average diameter and polydispersity index. According to the Eo¨tvo¨s empirical equation,65 the general trend is that the surface tension decreases with increasing temperature, reaching a value of 0 at a critical temperature. This could explain why the particle size decreased when temperature increased from 10 to 20 °C. Beyond 20 °C, the increase in particle mean size could result from the droplet coalescence induced by thermal motions as a consequence of the decrease of the viscosity of the oil phase. Another reason for the observed destabilization at high temperature could be a (65) Eo¨tvo¨s, R. Ann. Phys. Chem. 1886, 263(3), 448.
Figure 2. Average diameter (b) and polydispersity index (9) of chitosan nanoparticles at different synthesis temperatures (Mw ) 400 000 g · mol-1 DA ) 4%, and wt % Span 80 ) 1%).
reduction of the interactions between the hydrophilic head of the surfactant and the dispersed aqueous phase.66 Indeed, L. Peltonen et al. demonstrated that sorbitan surfactant monolayer films were more expanded as the temperature increased, resulting in a lower monolayer film stability.67 Another factor that is impacted by temperature is the acoustic cavitation intensity, which depends on the vapor pressure of the medium.68 Moreover, the mechanism of emulsification with ultrasound cavitation is not clearly understood yet. Hence, the role of temperature on the emulsification is complex, as many parameters are influenced in an antagonistic way. Therefore, in all the following experiments, the experimental temperature was fixed at 20 °C. 3.3. Effect of the Surfactant Concentration on the Particle Size Distribution. On increasing the Span 80 concentration in the formulation medium, up to 1% (w/w in the continuous phase), the particle size strongly decreased, as seen in Figure 3. The adsorption and organization of Span 80 at the surface of water droplets decreases the interfacial tension, and hence the droplet disruption energy. This phenomenon was confirmed by interfacial tension measurements done by the DSA technique (Figure 4). The interfacial tension of the Miglyol/water interface strongly decreased with the addition of Span 80 in the continuous phase. Initially, the surface tension decreased linearly with the logarithm of the surfactant concentration (see inset of Figure 4). When the critical micelle concentration (CMC) was reached, i.e., at saturation of the available surface, a further increase in surfactant concentration no longer had appreciable influence on the surface (66) Villiers, D.; Platten, J. K. J. Phys. Chem. 1988, 92(14), 4023. (67) Peltonen, L.; Hirvonen, J.; Yliruusi, J. J. Colloid Interface Sci. 2001, 239, 134–138. (68) Brennen, C. E. CaVitation and Bubble Dynamics; Oxford Univeristy Press: New York, 1995.
11376 Langmuir, Vol. 24, No. 20, 2008
Figure 3. Average diameter (b) and polydispersity index (9) of chitosan nanoparticles at different surfactant concentrations (Mw ) 400 000 g · mol-1 DA ) 4%, and Te ) 20 °C).
Figure 4. Interfacial tension between a chitosan aqueous solution and an oil phase (Miglyol 812 N + Span 80) as a function of Span 80 weight fraction for different chitosan concentrations.
tension. The CMC value was deduced from the intercept of the straight lines for the linear concentration-dependent section and the concentration-independent section. Hence, the CMC of Span 80 in Miglyol at 20 °C was measured at about 5% (w/w). After the CMC, the value of the interfacial tension was about 2.3 mN/m and decreased to almost zero in the presence of chitosan in the aqueous phase. This result suggests the existence of an interaction between chitosan and Span 80 at the interface, in accordance with the work by Grant and al.69 A minimum particle size was obtained when the Span 80 concentration was five times lower than the CMC, with 1% chitosan in the aqueous phase. This can be explained by the Gibbs-Marangoni effect, which is a mass transfer due to the interfacial tension difference. This movement of surfactant works as a self-stabilizing mechanism for the emulsion and is responsible for preventing coalescence.70 Another phenomenon could explain the increase in particle size for the highest surfactant concentration. Indeed, the typical magnitude of the growth and collapse velocity of the cavitation bubbles appeared to be given by the ratio of the interfacial tension to the solvent viscosity.71 The presence of surfactant at the gas/ liquid interface of the cavitation bubble could enhance their growth rate, then diminish the efficiency of the ultrasound.72 (69) Grant, J.; Cho, J.; Allen, C. Langmuir 2006, 22, 4327. (70) Dalmazzone, C. Oil Gas Sci. Technol.-ReV. IFP 2000, 55(3), 281.
Brunel et al.
Figure 5. Average diameter (b) and polydispersity index (PDI) (9) of nanoparticles from chitosan of different DAs (Mw ) 400 000 g · mol-1, Te ) 20 °C, and % Span 80 ) 1%).
3.4. Impact of the Physicochemical Properties of Chitosan on the Particle Size Distribution. The influence of the DA of chitosan on the particles size is shown in Figure 5. Nanogels were formed for DA e 30% only. This could be explained by the variation of the conformation and solubility parameters of chitin/chitosan as a function of DA. Sorlier,8 Schatz,53 Lamarque,73 Montembault,47 and Boucard74 demonstrated the existence of a general law of behavior of chitosan in aqueous solutions that exhibits three domains: (i) For DA < 25%, chitosan has a high charge density and therefore displays a strong polyelectrolyte behavior, illustrated by the Manning and Oosawa ionic condensation.75,76 (ii) For 25 < DA < 50%, chitosan is in a transition range (hydrophobic and hydrophilic interactions are progressively counterbalanced); hence its physicochemical properties remain more or less constant. (iii) For DA > 50%, hydrophobic interactions become predominant, which induces polymer chain association and a random coil conformation.73 Particle sizes increased with DA, up to DA ) 30%. Beyond this value, only macroscopic gelation was obtained (Figure 5). This could be due to the increase in the hydrophobicity of the macromolecular chains with DA. The first consequence is the adsorption of the hydrocarbon chains of the surfactant onto the droplet surface, rather than being expanded in the continuous phase. This would lead to a reduction of the steric stabilization of the emulsion droplets, resulting in an aggregation process. A second explanation could be a lower segregation of the hydrophobic chitosan in the droplets. The macromolecular chain could be present, or at least partially solubilized in the organic phase, leading to a macroscopic gelation or an entanglement of polymer chains from different droplets. Figure 6 reports on the impact on particle size of the molar mass of chitosan, taking into account the corrected value measured after the ultrasound treatment (see Table 1). A critical Mw value around 50 000 g · mol-1 could be evidenced, below which the particle size distributions were narrower (PDI around 0.4) and particle size under 500 nm. Above this critical Mw, the polydispersity sharply increased up to 0.8, meaning that the (71) Dzubiella, J. J. Chem. Phys. 2007, 126, 194504. (72) Shchukin, D. G.; Mo¨hwald, H. Phys. Chem. Chem. Phys. 2006, 8, 3496– 3506. (73) Lamarque, G.; Lucas, J.-M.; Viton, C.; Domard, A. Biomacromolecules 2005, 6, 131. (74) Boucard, N.; David, L.; Rochas, C.; Montembault, A.; Viton, C.; Domard, A. Biomacromolecules 2007, 8(4), 1209–1217. (75) Manning, G. S. J. Chem. Phys. 1969, 51(3), 924. (76) Oosawa, F. Biopolymers 1968, 6(11), 1633–1647.
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Langmuir, Vol. 24, No. 20, 2008 11377 Table 3. Intrinsic Viscosities ([η]), the Chain Entanglement Concentration (C*), and Dynamic Viscosity (η) of Chitosan Samples of Different Molar Massesa Mw (g · mol-1)
[η] (mL · mg-1)
C* (mg · mL-1)
η (mPa · s)
405400 ( 8800 117300 ( 1100 91700 ( 530 43950 ( 80 11350 ( 30
1430 691 430 214 62
0.7 1.45 2.3 4 16
N.A. N.A. 32 12
