Structure of Self-Organized Multilayer Nanoparticles for Drug
Delivery
Y. Gerelli,† S. Barbieri,‡ M. T. Di Bari,† A. Deriu,†,* L. Cantu`,§ P. Brocca,§ F. Sonvico,‡
P. Colombo,‡ R. May,| and S. Motta‡
Dipartimento di Fisica, UniVersita` degli Studi di Parma and CNISM, CRS SOFT, INFM-CNR, Parma,
Italy, Dipartimento di Chimica, Biochimica e Biotecnologie per la MedicinasLITA, UniVersita` di Milano
and CNISM, Milano, Italy, Dipartimento Farmaceutico, UniVersita` degli Studi di Parma, Parma, Italy, and
Institut Laue-LangeVin, Grenoble, France
ReceiVed June 25, 2008. ReVised Manuscript ReceiVed July 31, 2008
The combined use of cryo-TEM, dynamic light scattering, and small-angle X-ray and neutron scattering techniques
allows a detailed structural model of complex pharmaceutical preparations of soybean lecithin/chitosan nanoparticles
used as drug vectors to be worked out. Charge-driven self-organization of the lipid(-)/polysaccharide(+) vesicles
occurs during rapid injection, under mechanical stirring, of an ethanol solution of soybean lecithin into a chitosan
aqueous solution. We conclude that beyond the charge inversion region of the phase diagram, i.e., entering the
redissolution region, the initial stages of particle formation are likely to be affected by a re-entrant condensation effect
at the nanoscale. This behavior resembles that at the mesoscale which is well-known for polyion/amphiphile systems.
Close to the boundary of the charge inversion region, nanoparticle formation occurs under a maximum condensation
condition at the nanoscale and the complexation-aggregation process is driven toward a maximum multilamellarity.
Interestingly, the formulation that maximizes vesicle multilamellarity corresponds to that displaying the highest drug
loading efficiency.
Introduction
In health care and pharmaceutical sciences, nanotechnologies
have been indicated as the most promising field for breakthrough
technological innovation in early disease diagnosis and in
improved therapies. In recent years, nanotechnologies have been
proposed to improve drug delivery: specific nanovectors capable
of carrying the biologically active principle have been developed
to protect the encapsulated drug and to modify its distribution,
altering the pharmacokinetics.1,2 In pharmaceutical nanotech-
nologies, colloidal carriers are considered particularly suited for
the administration of drugs with biopharmaceutical problems
such as low bioavailability and poor water solubility. In this
respect different nanosized systems have been proposed to
improve drug bioavailability such as liposomes,3 polymeric
micelles,4 and nanoemulsions and nanoparticles.5,6 Some lipo-
somal and nanoparticle vectors have already been approved for
clinical application, in particular for cancer treatment, while
several others are under evaluation.7 Recently, great attention
has been devoted to colloidal preparations employing lipid
systems: as compared to synthetic polymers, these materials are
considered more biocompatible, biodegradable, and safe.8 In
particular, films and gels based on negatively charged phos-
pholipids and chitosan have been proposed for the delivery of
poorly soluble anticancer drugs.9,10
In this work we address lipid/polysaccharide systems, involving
lecithin and chitosan. Lecithin is a lipid mixture of phospholipids
that has been frequently used for liposome and micelle forma-
tion11,12 and is largely employed in pharmaceutical or nutra-
ceutical formulations. The basic structure of lecithin-based
nanovesicles is common to all membrane-like amphiphile
aggregates;13 however, their stability, physicochemical properties,
and surface characteristics need to be tailored to control the
release of the trapped drug, to adapt the nanocarrier to different
chemical environments, and to direct the nanocarrier toward
specific biological targets. In particular, the surface properties
of nanocarriers are important for their interaction with living
systems; they can be modified using both synthetic6,14 and
natural15,16 polymers. Several polysaccharides have been in-
vestigated for the stabilization of lipid membranes. Among them,
chitosan, a cationic polysaccharide derived from chitin by
deacetylation, has emerged because of its favorable biochemical
characteristics, including low toxicity, high biocompatibility,
biodegradability, bioadhesion, and absorption enhancer proper-
ties.17,18 Chitosan has been used for the production of nano-
particles by ionotropic gelation with tripolyphosphate19,20 as well
* To whom correspondence should be addressed. E-mail: Antonio.Deriu@
fis.unipr.it. Phone: +39 0521 905267. Fax: +39 0521 905223.
† Dipartimento di Fisica, Universita` degli Studi di Parma and CNISM,
CRS SOFT, INFM-CNR.
‡ Universita` di Milano and CNISM.
§ Dipartimento Farmaceutico, Universita` degli Studi di Parma.
| Institut Laue-Langevin.
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ALangmuir XXXX, xx, 000-000
10.1021/la801992t CCC: $40.75  XXXX American Chemical Society
Published on Web 09/24/2008

as for improving the stability of lecithin-based microemul-
sions.21,22 Interestingly, the “coating” of these lipid-based
nanostructures with chitosan, besides increasing their stability,23,24
provides them with mucoadhesion properties25 and enhances
permeation because of its effect of tight junctions between cells.
Thus, lecithin/chitosan nanoparticles are promising carriers for
the delivery of drugs via tranmucosal routes, such as oral, nasal,
or pulmonary administration.
The physical context in which the complexation mechanism
between lecithin and chitosan can be placed is that of
macroion-polyion interactions. Particular attention has been
devoted to complexes formed by lipid vesicles incubated with
polyions, adhering to their surfaces and giving rise to ordered
patterns or coordinating different vesicles. A rich literature exists
on complex structures consisting of amphiphile aggregates
interacting via electrostatic forces with linear polyions, polypep-
tides, or DNA, including lamellar phases or dispersed par-
ticles.26,27 A characterizing feature of the phase behavior of such
systems is charge inversion, corresponding to oppositely charge
polyelectrolytes complexing beyond charge neutralization. Am-
phiphile/polyion coordination reaches its maximum in the region
of charge inversion where large clusters are formed. This behavior
can be described in terms of a re-entrant condensation effect:
extra polyions can be attracted to the surface of the macroion
despite the fact that this surface is already neutralized by the
previously adsorbed polyions. This entropically driven process
is due to an induced attraction between polyions.28 In the present
work we address the complexation mechanism between the
macroion and polyions at the nanoscale and their role in
determining the ultimate structure of the nanoparticles. To this
purpose the lecithin/chitosan nanoparticles described in this paper
were obtained by direct injection of a soybean lecithin alcoholic
solution into a chitosan/water solution. The nanoparticle su-
pramolecular self-organization is contemporarily driven by the
aggregative behavior of the lipid component and by the
electrostatic interaction between the negatively charged fraction
of the lipid material and the positively charged polysaccharide.
A detailed morphological and structural characterization of the
nanoparticle, as a function of the chitosan content in the initial
chitosan/water preparation solution, is essential to optimize the
loading efficiency and to tune the drug release kinetics. Selected
formulations spanning the phase diagram, before and after the
demixing region5 and deep in the re-entrant condensation range,
were chosen for this study. The nanovectors were initially
characterized by cryo-TEM (cTEM), dynamic light scattering
(DLS), and surface charge determination (
potential). Small-
angle X-ray scattering (SAXS) and small-angle neutron scattering
(SANS) experiments were then performed.
Experimental Section
Sample Preparation. The investigated nanoparticles are made
up of two main components. One is Lipoid S45 (a lecithin
manufactured by Lipoid AG, Switzerland), a commercial mixture
of lipids, phospholipids, and fatty acids with an overall negative
charge and with the following composition: (lipid components)
phosphatidylcoline, 47.6%; phosphatidylethanolamine, 16.4%; lyso-
phosphatidylcoline, 2.5%; (acid components) palmitic acid, 15%;
stearic acid, 3%; oleic acid and isomers, 12%; linoleic acid, 5%;
(other minor components) tryriglycerides, 0.1%; DL-R-tocopherol,
0.10%; water (Karl Fisher), 0.6%; ethanol, 0.5%. The other is highly
purified chitosan (provided by Primex, Norway), a polysaccharide,
positively charged at acidic pH, obtained by deacetylation of chitin.
Its average molecular weight, as determined by laser light scattering,
is ∼140 000. The samples were prepared from a lecithin solution
(lecithin in ethanol at 25 mg/mL concentration) and from three
different chitosan/water solutions (chitosan/lecithin ratios 1/80, 1/20,
and 1/5 (w/w), acidic pH). When necessary, as for laser light scattering
measurements, solvents were filtered on 0.2 µm polycarbonate
membranes before nanoparticle preparation; filtration of the final
solutions was avoided. The samples obtained from these starting
formulations are indicated in the following as NCL80, NCL20, and
NCL5. Self-assembled nanoparticles were obtained by rapid injection
(nozzle diameter 0.75 mm, injection rate 40 mL/min), under
mechanical stirring, of the ethanol solution into the chitosan aqueous
solution. The self-assembly process gave rise to a suspension with
a pH of 2.7 and a nanoparticle concentration c ) 0.2% (w/w).5
Lipoid vesicles, used as a reference for the SAXS measurements,
were prepared with a similar procedure, replacing the chitosan/
water solution with water, acidic pH.
The residual chitosan content in the buffer, after nanoparticle
formation, was determined by a colorimetric method, on the basis
of the λ ) 581 nm absorbance of the sample solution, upon addition
of Cibacron Brillant Red 3B-A (Sigma, St.Louis, MO)29 as a specific
dye. SAXS measurements of chitosan in H2O/HCl solution (pH 2.7)
were performed at three different concentrations (0.02%, 0.1%, and
1%).
Cryo-Tem. The nanoparticle morphology was investigated by
electron transmission microscopy on thin (∼5000 Å) aqueous films
vitrified by cooling to liquid nitrogen temperature (cTEM).30 The
limiting factor, when using electrons as probes, is the small difference
between the electron density of the lipids and that of the aqueous
solvent. As a consequence the spatial resolution cannot be higher
than ∼40-50 Å. This corresponds to a typical bilayer thickness;
therefore, the internal structure of the bilayers and other smaller
scale details cannot be resolved by this technique.
Dynamic Laser Light Scattering. The particle size distribution
was determined by DLS using a 200SM apparatus (Brookhaven
Instruments Corp.), equipped with an argon ion laser operating on
the 5145 Å green line; the accessible Q range is 4 × 10-4-3 × 10-3
Å-1. As compared to the other adopted techniques, DLS is the only
one looking at the dynamics of the nanoparticles in Brownian motion
in the solution, rather than at their “static” average scattered intensity.
The average charge of the nanoparticles is expressed in terms of
the
potential31 as obtained from a measure of their mobility under
an electric field with a ZetaPals phase analysis light scattering
apparatus (Brookhaven Instruments Corp.).
Small-Angle X-Ray Scattering. SAXS data were collected at
the ID02 beamline at the European Synchrotron Radiation Facility,
ESRF (Grenoble). Using a wavelength λ)1 Å (instrument resolution
∆E/E ) 2 × 10-4) and a sample-detector distance D ) 1.2 m, a
Q range from 8 × 10-3 to 4 × 10-1 Å-1 was explored corresponding
to a range of length scales from 15 to 800 Å. The particle structure
down to the bilayer length scale is accessible by this technique.
Calibration was performed using a silver behenate standard. The
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