A novel synthesis of chitosan nanoparticles in reverse emulsion.
ABSTRACT 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.
- SourceAvailable from: umn.edu[show abstract] [hide abstract]
ABSTRACT: Polyelectrolyte protected beta-carotene nanoparticles (nanosuspensions) with average diameter of <100 nm were achieved by turbulent mixing and flash nanoprecipitation (FNP). Three types of multi-amine functional polyelectrolytes, epsilon-polylysine (epsilon-PL), poly(ethylene imine) (PEI), and chitosan, were investigated to electrosterically protect the nanoparticles. Particle size and distribution were measured by dynamic light scattering (DLS); particles were imaged via scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (cryo-TEM). Low pH and high polyelectrolyte molecular weight gave the smallest and most stable particles. High drug loading capacity, >80 wt%, was achieved by using either PEI or chitosan. X-ray diffraction (XRD) patterns showed that beta-carotene nanoparticles were amorphous. These findings open the way for utilization of FNP for preparation of nanoparticles with enhanced bioavailability for highly water insoluble drugs.Journal of Pharmaceutical Sciences 02/2010; 99(10):4295-306. · 3.13 Impact Factor
Subscriber access provided by UNIV CLAUDE BERNARD LYON 1
Langmuir is published by the American Chemical Society. 1155 Sixteenth Street
N.W., Washington, DC 20036
A Novel Synthesis of Chitosan Nanoparticles in Reverse Emulsion
Fabrice Brunel, Laurent Ve#ron, Laurent David, Alain Domard, and Thierry Delair
Langmuir, 2008, 24 (20), 11370-11377 • DOI: 10.1021/la801917a • Publication Date (Web): 06 September 2008
Downloaded from http://pubs.acs.org on December 3, 2008
More About This Article
Additional resources and features associated with this article are available within the HTML version:
Access to high resolution figures
Links to articles and content related to this article
Copyright permission to reproduce figures and/or text from this article
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
Ba ˆt. 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.
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
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
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 biocom-
Chitosan is a ?,(1f4)-linked copolymer of 2-amino-2-deoxy-
?-D-glucan (GlcN) and 2-acetamido-2-deoxy-?-D-glucan (GlcNAc)
obtained by heterogeneous deacetylation of chitin.5-7Chitosan
can be obtained by reacetylation in homogeneous conditions of
a fully deaceylated chitosan.8Chitosan exhibits a high degree
for intravenous and oral administration.9-11Chitin 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,
in humans, yet.16Chitosan 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.17,18In addition, some studies have
polymorphonuclear cells,20suppressing tumor growth,21,22
promoting resistance to infections by microorganisms,23,24and
enhancing both humoral and cell-mediated immune re-
sponses.25-28For 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
* Corresponding author. E-mail: Thierry.Delair@eu.biomerieux.com.
‡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.
(8) Sorlier,P.;Denuzie `re,A.;Viton,C.;Domard,A.Biomacromolecules2001,
(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.
(12) Tomihata, K.; Ikada, Y. Biomaterials 1997, 18(7), 567–575.
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,
(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.
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.
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.
Langmuir 2008, 24, 11370-11377
10.1021/la801917a CCC: $40.75
2008 American Chemical Society
Published on Web 09/06/2008
state, with a simple electrolyte of polyanionic nature such as
polyanion and chitosan. Many anionic polymers were used such
konjac glucomannan.36Besides 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
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,38,39epichlorhydrine,
dodecylsulfate (SDS)42or lecithin,43were also used to form
emulsion of chitosan was mixed with a second reverse emulsion
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
appeared to be an interesting, though challenging, alternative.
of the chitosan solubility in the medium, in a such a way that
solvent-segment interactions are reduced to favor segment-
segment interactions, and (ii) the polymer concentration to be
a network can be formed. Reducing chitosan solubility will be
achieved by decreasing the repulsive electrostatic interactions
the charge density of chitosan or by modifying the dielectric
was obtained by increasing the degree of acetylation (DA) up
to 70%, as studied by Roberts and Moore45and Vachoud et al.46
In this case, the presence of a large number of N-acetyl groups
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.47Later, the same team reported an approach toward
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
aspects of the process is the absence of any cross-linker, the
2. Materials and Methods
capric/caprilic acid (Miglyol 812 N, dynamic viscosity: 24 mPa·s))
purchased from SASOL (Germany) and a surfactant, sorbitan
by Mahtani Chitosan PVT, Ltd., India, batch 124 (DA ∼ 5%, Mw
∼ 400 000 g·mol-1). Its molecular characteristics were determined
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,
3 to 0.22 µm), precipitation with an ammonia/methanol mixture
(3/7, v/v), rinsing with deionized water until neutrality, and freeze-
A purified chitosan of high molar mass was N-acetylated with
acetic anhydride in homogeneous medium to reach different DAs.
to the procedure previously described by Vachoud et al.46After
and then freeze-dried.
The nitrous deamination was carried out to produce low molar
mass polymers.49,50For 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
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 by1H NMR spectroscopy (Varian, 500 MHz),
according to the method developed by Hirai et al.51
The weight-average molecular weight (Mw), the z-average root-
mean-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
(29) Berthold, A.; Cremer, K.; Kreuter, J. J. Controlled Released 1996, 39,
(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),
(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,
(39) Jia, Z.; Wang, Y.; Lu, Y.; Luo, G. React. Funct. Polym. 2006, 66, 1552–
(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:
& Cosmetic Applications” European symposium held in Rennes, France, June
(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.
(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,
(47) Montembault, A.; Viton, C.; Domard, A. Biomaterials 2004, 26, 933–
(48) Montembault, A.; Viton, C.; Domard, A. Biomacromolecules 2005, 6,
(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.
ReVerse Emulsion Synthesis of Chitosan NPs Langmuir, Vol. 24, No. 20, 2008 11371
(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
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
(TGA; DuPont Instrument 2950, Twin Lakes, WI).
2.3. Viscosimetry. Intrinsic viscosity measurements were per-
formed at 25 ( 0.1 °C using an automatic Ubbelhode capillary
viscometer with an inner diameter of 0.53 mm (Viscologic TI.1,
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 approxima-
tion C* × [η] ) 1.
Dynamic viscosities of chitosan solution in pure water were
measured using a stress rheometer: AR2000 (TA Instruments, New
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-1in shear
2.4. Interfacial Tension Measurement. The interfacial tension
by the drop shape analysis (DSA) method with a contact angle
measuring system, KRU¨SS G10 (Hamburg, Germany). A droplet
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.54,55The
volume of the droplet was the highest possible volume before the
fall of the drop. The system was calibrated (camera focus,
in air (71.5 mN.m).
2.5. Nanoparticles Synthesis. Nanoparticles were prepared in
(5 mL, chitosan: 1% w/v) was emulsified in 25 mL (23.75 g) of
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
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
the sample with the highest molar mass was significantly degraded
g·mol-1down to 190 000 g·mol-1(Table 1).
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-1pH ) 4.5) by slow stirring overnight,
and the final pH was measured between 5.5 and 6.5.
the purified particles pellets were dispersed with deionized water
2.6. NanoparticleCharacterization.2.6.1. ColorimetricAssay.
Orange II, an anionic dye interacting with ammonium groups of
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 × 10-4mol·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
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 × 10-4mol·L-1([NH3+]
titrated with an Orange II solution of 2.5 × 10-4mol·L-1). The
absorbance at 485 nm was plotted against [NH3+]. The chitosan
concentration was determined from the absorbance data via the
and DAs as those used for particle synthesis. In this assay, there is
(52) Sorlier, P.; Rochas, C.; Morfin, I.; Viton, C.; Domard, A. Biomacro-
molecules 2003, 4(4), 1034–1040.
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.
2005, 12, 95.
(58) Maghami, G. G.; Roberts, G. A. F. Makromol. Chem. 1988, 189(10),
Table 1. Effect of Ultrasound on the Molar Mass Distributions of Chitosana
molar mass distribution of chitosan before sonication
405400 ( 8800304800 ( 5800
117300 ( 110076200 ( 740
91700 ( 53056260 ( 230
43950 ( 8024970 ( 80
11 ( 305630 ( 40
of the degree of polymerization (DPn) Mn/M0with M0being the average molar mass of chitosan repeat units).
molar mass distribution of chitosan after sonication
197600 ( 1200114200 ( 100
145440 ( 900 90900 ( 200
97440 ( 410 53570 ( 200
41700 ( 7025520 ( 50
12410 ( 406500 ( 30
11372 Langmuir, Vol. 24, No. 20, 2008Brunel et al.
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:
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
where 203 and 161 are the molar masses of N-acetylglucosamine
and glucosamine residues, respectively.
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 self-
correlation function was expanded in a power series (Cumulants
methods).59The polydispersity value provided by the software is
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
126.96.36.199H 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
d4(TMSP-d4) was added.1H NMR spectra were recorded with a
Bruker 200 MHz spectrometer at 50 °C in order to decrease the
according to the peak of the TMSP-d4corresponding to 9 protons
and then, to an intensity of 9 au. The amount of chitosan in the
particles was obtained from integration of the1H NMR spectrum
using the following equation:
where M0is 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
constant of the dispersion medium imposed a steric stabilization
of emulsion droplets by a nonionic surfactant.62According to
the hydrophilic-lipophilic balance (HLB) concept introduced
value ranging between 4 to 6.64For 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
and the surfactant. After the first centrifugation, the reaction
one and the water one, plus the pellet containing the particles.
The mass of the recovered organic phase was around 20 g,
The crude particles were washed and centrifuged twice with
The supernatants of the washings with ethanol were evaporated
under reduced pressure, and1H 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 by1H NMR and a colorimetric
assay using Orange II (see Section 2.6.1. Colorimetric Assay).
(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.
2000, 38, 79.
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.
Scheme 1. Reaction Diagram of Chitosan Complexation by an Anionic Dye (Orange II)
ReVerse Emulsion Synthesis of Chitosan NPsLangmuir, Vol. 24, No. 20, 2008 11373
solvent clearly showed the presence of chitosan and some traces
of Miglyol 812N at 1.29 ppm (methylene protons). To allow the
must be lower than its solubility limit. The solubility limit of
Miglyol was determined by1H NMR as follows. At 5 mg·mL-1
in the solvent phase (D2O/DMSO-d6) was determined using a
The solubility limit was found to be 1.2 mg·mL-1. For our
were clear; hence the residual oil concentration was lower than
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.
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
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
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).
11374 Langmuir, Vol. 24, No. 20, 2008Brunel et al.
first centrifugation (between the organic phase and the particle
pellet). This aqueous phase was lyophilized and analyzed by1H
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
The amount of chitosan in the particles was obtained (i) from
1H NMR via the intensities of the peak of chitosan glucosidic
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
In the case of high molar mass chitosan, discrepancies between
the two methods are more important. The lower value obtained
by1H 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
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.
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
temperature was 20 °C, corresponding to the lowest average
diameter and polydispersity index. According to the Eo ¨tvo ¨s
decreases with increasing temperature, reaching a value of 0 at
a critical temperature. This could explain why the particle size
20 °C, the increase in particle mean size could result from the
of the decrease of the viscosity of the oil phase. Another reason
for the observed destabilization at high temperature could be a
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.68Moreover, 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
fixed at 20 °C.
Size Distribution. On increasing the Span 80 concentration in
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
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.
of the surfactant concentration (see inset of Figure 4). When the
critical micelle concentration (CMC) was reached, i.e., at
(65) Eo ¨tvo ¨s, R. Ann. Phys. Chem. 1886, 263(3), 448.
(66) Villiers, D.; Platten, J. K. J. Phys. Chem. 1988, 92(14), 4023.
New York, 1995.
Table 2. Chitosan Recovery in Particles, Obtained by Colorimetric and NMR Assaysa
chitosan recovered in the
particles (mg) (particles
chitosan recovery (%) ) chitosan
recovered in the particles/chitosan
dry chitosan used
(mg) (% yield)
68 ( 3%
46 ( 5%
Mw) 12 410 ( 30 g·mol-1
Mw) 405 400 ( 8800 g·mol-1
aResults are the average value of three experiments.
DA ) 4%, and wt % Span 80 ) 1%).
ReVerse Emulsion Synthesis of Chitosan NPsLangmuir, Vol. 24, No. 20, 2008 11375
tension. The CMC value was deduced from the intercept of the
straight lines for the linear concentration-dependent section and
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
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
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
to the solvent viscosity.71The presence of surfactant at the gas/
rate, then diminish the efficiency of the ultrasound.72
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,8Schatz,53Lama-
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 con-
counterbalanced); hence its physicochemical properties remain
more or less constant. (iii) For DA > 50%, hydrophobic
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
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
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
after the ultrasound treatment (see Table 1). A critical Mwvalue
around 50 000 g·mol-1could 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
(69) Grant, J.; Cho, J.; Allen, C. Langmuir 2006, 22, 4327.
(70) Dalmazzone, C. Oil Gas Sci. Technol.-ReV. IFP 2000, 55(3), 281.
(71) Dzubiella, J. J. Chem. Phys. 2007, 126, 194504.
(72) Shchukin, D. G.; Mo ¨hwald, H. Phys. Chem. Chem. Phys. 2006, 8, 3496–
(73) Lamarque, G.; Lucas, J.-M.; Viton, C.; Domard, A. Biomacromolecules
2005, 6, 131.
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.
nanoparticles at different surfactant concentrations (Mw ) 400 000
g·mol-1DA ) 4%, and Te) 20 °C).
Figure 4. Interfacial tension between a chitosan aqueous solution and
fraction for different chitosan concentrations.
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%).
11376 Langmuir, Vol. 24, No. 20, 2008Brunel et al.
dispersions were broadly distributed. So, the best-defined
nanogels, in terms of particle size and size distribution, could
be obtained with the lower molar mass samples. The polysac-
charide concentration used during the manufacturing of the
particles was Cexp) 10 mg·mL-1; comparing this value to the
chain entanglement concentration, C* ) 16 mg·mL-1for
be used under C*. This would be a major difference with
macroscopic gels for which working above C* is a prerequisite
for forming physical gels, in order to increase the number of
junctions between macromolecular chains, and then to induce a
percolating process of gelation.48But this result should be
tempered considering the impact of the chemical nature of the
buffer used for C* determination. Indeed, the measurement of
acetate buffer. According to the Manning theory,75a fraction of
the counter-ions condense on the macromolecular chain to
decrease the effective charge density of the polymer, thus the
another. However, the formation of independent droplets of
chitosan solution in the organic phase during the emulsification
step requires that chains disentangle. The critical molar mass of
would correspond to a limit in the molecular dimension of the
enough to allow this separation.
The molar masses of the polysaccharides also impact the
viscosities their solutions.73Many authors studied the effect of
the dispersed phase viscosity on the droplets size. Walstra64
observed that the average diameter of emulsion droplets was
effect of the viscosity of the dispersed phase on the emulsion
size distribution using the Oshnsorge number:77Oh ) ηd/
(γFddmax)1/2. For low Oh (ηd< 10 mPa·s),
For high Oh (ηd. 10 mPa·s),
with ε the energy density (W·m-3), γ the interfacial tension
(mN/m), Fdthe density of the dispersed phase, dmaxthe droplet
size, and ηdthe dispersed phase viscosity. In both cases, the
decrease in the dispersed phase viscosity leads to a decrease in
As seen in Table 3, the viscosity of the chitosan solutions, at
identical polysaccharide mass concentrations (10 mg·mL-1),
decreased when the molar mass of chitosan decreased, and only
solutions with lower viscosity allowed control of the polydis-
in particle size and polydispersity corresponded to a viscosity
ratio ηd/ηc(ηc, viscosity of the continuous phase, ) 24 mPa·s)
molar mass increased), the droplet disruption energy probably
increased with the viscosity of the aqueous dispersed phase,
leading to ill-defined materials.
Finally, the sharp decrease in particle size and polydispersity
under a critical Mwcould also be explained by the reported high
mechanism of low molar mass chitosans. Further investigations
of the particles morphology and nanostructure are needed to
confirm, or disprove, this hypothesis.
In this work, we described a new approach toward polysac-
charide-based physical nanogels. A formulation designed with
ingredients generally regarded as safe led to the first physical
colloidal gels of chitosan, obtained in the absence of any cross-
linker. The optimum temperature of the emulsification process
was 20 °C, corresponding to a balance between various effects
such as the viscosity of the dispersed phase, the surface tension,
and the thermal motion of emulsion droplets. The investigations
formation process showed that the dispersions were better
controlled, in terms of size and size distribution, when the DA
a behavior of a strong polyelectrolyte. This particular behavior
was related to the viscosity of the dispersed phase; the lower the
viscosity, the better the control over particle size distribution
though the crystallinity of chitosan oligomers could favor the
Though further investigations are still needed to fully
understand the formation of the particles, this work afforded
new materials obtained from naturally occurring polymers, thus
gels, with numerous potential applications.
Acknowledgment. We thank J. M. Lucas for his assistance
in gel chromatography and viscosimetry experiments, D. Gillet
(77) Karbstein, H.; Schubert, H. Chem. Eng. Proc. 1995, 34, 205.
(78) Belamie, E.; Domard, A.; Chanzy, H.; Giraud-Guille, M.-M. Langmuir
1999, 15, (1549)
Figure 6. Average diameter (b) and PDI (9) of nanoparticles as a
function of chitosan molar mass distribution (DA ) 4%, Te) 20 °C,
and % Span 80 ) 1%).
Table 3. Intrinsic Viscosities ([η]), the Chain Entanglement
Concentration (C*), and Dynamic Viscosity (η) of Chitosan
Samples of Different Molar Massesa
405400 ( 8800 1430
117300 ( 1100691
91700 ( 530430
43950 ( 80214
11350 ( 3062
ReVerse Emulsion Synthesis of Chitosan NPs Langmuir, Vol. 24, No. 20, 2008 11377