International Journal of Pharmaceutics 455 (2013) 219–228

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Chitosan nanoparticles: Preparation, size evolution and stability

Antonio Rampino a , Massimiliano Borgogna a , Paolo Blasi b,∗∗ ,
Barbara Bellich a , Attilio Cesàro a,∗
a
Laboratory of Physical and Macromolecular Chemistry, Dept. Life Sciences, University of Trieste, Via Giorgieri 1, 34127 Trieste, Italy
b
Dept. of Chemistry and Technology of Drugs, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Purpose: Characterisation of chitosan-tripolyphosphate nanoparticles is presented with the aim of corre-
Received 2 May 2013 lating particle shape and morphology, size distribution, surface chemistry, and production automatisation
Received in revised form 9 July 2013 with preparation procedure, chitosan molecular weight and loaded protein.
Accepted 10 July 2013
Methods: Nanoparticles were prepared by adding drop wise a tripolyphosphate-pentasodium solution to
Available online 22 July 2013
chitosan solutions under stirring. Trehalose, mannitol and polyethylene-glycol as bioprotectants were
used to prevent particle aggregation and to reduce mechanical stress during freezing and drying pro-
Keywords:
cesses.
Chitosan nanoparticles
Ionotropic gelation
Results: As a novel result, time evolution of the particle size distribution curve showed the presence of a
Nanoparticle ageing bimodal population composed of a fraction of small particles and of a second fraction of larger particles
Freeze-drying attributed to the rearrangement of particles after the addition of tripolyphosphate. Storage for 4 weeks
Spray drying resulted in a slight increase in average size, due to the continuous rearrangement of small particles.
Protein carriers Improvement of nanoparticle stability after lyophilisation and spray-drying was observed in the presence
of all bioprotectants. Trehalose was the best protectant for both methods. Finally, in vivo tests using chick
embryos assessed the biocompatibility of chitosan, tripolyphosphate and the nanoparticles.
Conclusion: The simple ionotropic gelation method with low-MW chitosan was effective in achieving
reproducible nanoparticles with the desired physico-chemical and safety characteristics.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction 2001; Prego et al., 2005) and to modulate drug pharmacokine-

The study of novel pharmaceutical formulations is gradually
abandoning the parenteral route and is developing medicines
administered by alternative routes in order to avoid the typical
disadvantages of the former. In particular, among the non-invasive
administration routes, the mucosal route, such as the nasal, pul-
monary, oral and vaginal routes, is attracting a great deal of interest,
thanks to its enabling local delivery to target tissues or its deliv-
ering the active pharmaceutical ingredient (API) to the systemic
circulation (Andrews et al., 2009).
Micro and nanoparticles are the most promising of the deliv-
ery systems showing potential for the mucosal delivery of drugs
and antigens. First described for pharmaceutical applications by
Birrenbach and Speiser (1976), nanostructured carriers of appro-
priate size and surface charge should be able to protect drugs
from enzymatic degradation (Des Rieux et al., 2006), to improve
their penetration across the mucosal epithelium (Lamprecht et al.,
∗ Corresponding author. Tel.: +39 0405583684; fax: +39 0405583691.
∗∗ Corresponding author. Tel.: +39 0755852057; fax: +39 0755855123.
E-mail addresses: paolo.blasi@unipg.it (P. Blasi), cesaro@units.it (A. Cesàro).
tics, thus improving efficacy and reducing drug toxicity (Kammona
and Kiparissides, 2012; Ricci et al., 2004; Rosen and Abribat, 2005;
Shmulewitz et al., 2006; Zhang et al., 2008).
A wide range of materials, such as natural and synthetic
polymers, lipids, and surfactants, have been employed to prepare
drug-containing nanocarriers (Blasi et al., 2007; Duncan, 2003;
Kumari et al., 2010). Among the materials proposed for mucosal
delivery, polysaccharides have received increasing attention
because of their outstanding physical and biological properties (Liu
et al., 2008). In particular, cationic polymers such as chitosan and
its derivatives generate particular interest. It is a natural biopoly-
mer consisting of ␤-1 → 4 linked 2-amino-2-deoxy-glucopyranose
(GlcN) and 2-acetamido-2-deoxy-␤-d-glucopyranose (GlcNAc)
residues, manufactured commercially on a large scale by alka-
line N-deacetylation of chitin, an abundant biopolymer isolated
from the exoskeleton of crustaceans, such as crabs and shrimps
(Tømmeraas et al., 2002). The preparation of chitosan with
controlled properties by means of micro-organism (i.e. fungi) fer-
mentation is of increasing interest (Niederhofer and Müller, 2004;
Wang et al., 2008). Chitosan offers several advantages for mucosal
delivery, such as low toxicity and good biodegradability (Lee et al.,
1995), as well as immunostimulating (Nishimura et al., 1986) and

0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2013.07.034
220 A. Rampino et al. / International Journal of Pharmaceutics 455 (2013) 219–228
high mucoadhesive properties (Lehr et al., 1992). Chitosan has
been used as an excipient in different formulations, such as tablets,
matrix, micro- and nanoparticles (Dash et al., 2011).
The cationic amino groups of chitosan complex with metal
anions or small multiple-charged anionic molecules, such as
sulphates, citrates, and phosphates (Dambies et al., 2001). The for-
mation of particles using ionic gelation is advantageous because
the process is relatively simple and mild and avoids the use of
organic solvents and high temperatures, thus allowing the success-
ful encapsulation of fragile molecules, such as proteins (Al-Qadi
et al., 2012; Berger et al., 2004; Nasti et al., 2009; Xu and Du,
2003). In particular, many researchers have explored the potential
pharmaceutical usage of tripolyphosphate (TPP)-chitosan com-
plexes after Bodmeier and Pramar reported the preparation of
beads by dropping chitosan into a TPP solution (Bodmeier et al.,
1989). The prepared hydrogels provide the opportunity to for-
mulate carriers with tunable geometries, positive surface charge,
and, generally, with a good capacity of API encapsulation. How-
ever, nanoparticles (NPs) prepared by ionotropic gelation methods
are usually characterised by aggregation/particle fusion immedi-
ately after preparation or by limited physical/chemical stability
when nanoparticle suspensions are stored for an extended period
of time. In addition, the technological development of nanocarri-
ers is paralleled by the increasing concern about their safety and
possible toxic effect: both the carrier and the materials used must
be biocompatible and biodegradable and particular attention is
placed on the side effects of nanoentities (Aggarwal et al., 2009;
Bystrzejewska-Piotrowska et al., 2009). To accomplish this aim, it
is worth exploring the possibility of using biopolymeric nanopar-
ticles for oral drug delivery applications (Food Safety Authority of
Ireland, 2008).
The aim of this work was the preparation and characterisa-
tion of chitosan-TPP nanoparticles as mucosal drug carriers using
the ionotropic gelation method, as well as their optimisation by
monitoring particle shape and morphology, size distribution, sur-
face charge, and drug loading capacity. Different proteins have
been encapsulated as model compounds and attempts to scale
up the production methodology and to produce re-dispersible dry
powders by freeze-drying and spray-drying have been performed
as well. Finally, chitosan and chitosan NPs biocompatibility was
investigated by chorioallantoic membrane (CAM) assay to fulfil the
current request for nanoparticle safety assessment.
2. Materials and methods
2.1. Materials
Low molecular weight (LMW) chitosan, tripolyphosphate pen-
tasodium (TPP), albumin from bovine serum (BSA), albumin from
chicken egg white (OVA), trehalose from corn starch, mannitol
and polyethylene glycol 10000 (PEG 10000) were purchased from
Sigma–Aldrich Co. (St. Louis, Missouri). Very low molecular weight
(VLMW) chitosan was prepared by controlled sodium nitrite degra-
dation, starting from medium molecular weight chitosan (MMW)
(Sigma–Aldrich Co. St. Louis, Mo), as elsewhere reported (Janes and
Alonso, 2003) with slight modifications. Briefly, a 1% (w/v) NaNO2
solution was added to a 1% (w/v) chitosan acid solution (ratio 1:15)
at room temperature under magnetic stirring. The reaction was
maintained for 1 h. Zinc-free human insulin (HI) was donated by
Novo Nordisk, (Copenhagen, Denmark). All other chemicals and
reagents were of the highest purity grade commercially available.
2.2. Polymers characterisation
The two polymer samples, LMW-chitosan and VLMW-
chitosan have been fully characterised for their chemical and
macromolecular properties as reported elsewhere (Rampino,
2011). The experiments and the analysis of the polymer conforma-
tional features (molecular weight, hydrodynamic radius, intrinsic
viscosity and viscosity exponent parameter) were carried out with
a size exclusion chromatography (SEC) system (SEC3, Viscotek,
USA) in the laboratory of UFT – Centre for Environmental Research
and Sustainable Technology, Bremen, Germany. The description of
the SEC3 system experiments and of the analysis of raw data has
already been described elsewhere (Weinhold et al., 2009). A full
report of the methods and analysis of the results of the solution
conformational properties of the two chitosan samples and some
chitosan derivatives will be reported shortly (Rampino et al., 2013,
in preparation).
2.3. Preparation of the nanoparticles
The NPs were prepared following the procedure described by
Calvo et al. (1997). A 2.5 mg/mL chitosan solution was prepared by
dissolving LMW or VLMW chitosan in a 0.05% (v/v) acetic acid solu-
tion and leaving it under stirring for 24 h. The pH was adjusted to 5.5
with a 0.5 M sodium hydroxide solution and diluted in deionised
water to the final desired concentrations. TPP was dissolved in
deionised water to a final concentration of 0.25 mg/mL. TPP and
chitosan solutions were filtered through a 0.45 ␮m membrane (Mil-
lipore). Then, the TPP solution was added to the chitosan solution
drop wise (0.3 mL/min) at different TPP:chitosan ratios under vig-
orous magnetic stirring at room temperature (Calvo et al., 1997).
The resulting suspension was then left to gelify for 30 min.
The dynamic evolution of particle formation was investigated by
monitoring particle size (as described in Section 2.6.1) over time
on the formulation with the best characteristics. After the com-
plete addition of TPP, one set of samples was left under stirring and
another was removed from the stirrer, and both were left at room
temperature. Particle size was measured on freshly prepared parti-
cles, after 2 and 24 h from preparation. Moreover, the possibility of
a scale-up has been explored increasing both the final volume (10
times) and the component concentration (3 times).
Protein-loaded nanoparticles were prepared as follows: differ-
ent volumes of the protein stock solution (4 mg/mL) in deionised
water were added to the chitosan solution, obtaining a final protein
concentration of 0.2, 0.4, and 0.6 mg/mL. Only LMW chitosan was
employed to prepare nanoparticles loaded with the three differ-
ent proteins: bovine serum albumin (BSA), ovalbumin (OVA) and
human insulin (HI). BSA and OVA were added directly to the chi-
tosan solution, while HI was added to the TPP solution and then
dripped. The dispersion was left under constant stirring for 30 min
at room temperature, in order to allow a homogeneous gelation.
2.4. Nanoparticle yield
To remove the excess unreacted chitosan, the suspension was
centrifuged and the supernatant was recovered for further anal-
ysis. The best separation condition, giving the highest amount of
particle recovery, was 3270 RCF (Relative Centrifugal Force) for 2 h.
By increasing the speed or time of centrifuging, higher amounts of
precipitate could be obtained, but it was not possible to recover
nanoparticles even using an ultrasonic bath or an ultrasonic probe.
Moreover, the addition of a glycerol bed, generally used to prevent
particle aggregation due to centrifuge packing, was not suitable for
the complete recovery of nanoparticles.
Chitosan quantitative determination in the supernatant was
performed by employing the colorimetric protocol set-up by
Muzzarelli (1998) with very slight modifications. The buffer solu-
tion was prepared with a final pH of 3 and the standard solutions
of chitosan were prepared starting from a 0.5 mg/mL stock solu-
tion containing 0.05% acetic acid. Absorbance was measured at
 A. Rampino et al. / International Journal of Pharmaceutics 455 (2013) 219–228
575 nm with a UV–visible spectrophotometer (Cary 4E) in triplicate
and results were expressed as mean value and standard deviation
(±SD).
2.5. Nanoparticle recovery and drying
NP suspensions were centrifuged for 2 h at 3270 RCF and the
obtained sediment was resuspended in deionised water, analysed
and lyophilised or alternatively spray dried, using trehalose, man-
nitol and PEG 10000 as cryoprotectants. Dried particles were
redispersed and characterised in terms of size, surface charge and
morphology.
To prepare spray-dried nanoparticles a new kind of spray-
dryer was used, the Nano Spray Dryer B-90 (Buchi, Switzerland).
The spray cap with a 4.0 ␮m mesh hole size was used in order
to obtain the smallest droplet size. The operating conditions
were set up as follows: inlet temperature at 120 ◦ C, drying air
flow rate = 100 L/min, pressure, 50 mbar; pump, 1; spray, 100%
(Schoubben et al., 2013).
2.6. Nanoparticle characterisation
2.6.1. Particle size and morphology
NP size and ␨ potential were determined using a Malvern
Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK).
Diluted samples were placed in disposable polystyrene cuvettes
and the scatter intensity was measured at 25 ◦ C. Size values were
reported as mean hydrodynamic diameter (MHD), standard devi-
ation (SD), and polydispersity index (PDI). Chitosan nanoparticles
were observed by transmission electron microscope (TEM) (Philips
EM 208 microscope, Eindhoven, Netherlands). All samples for TEM
analysis were prepared by allowing a single drop of nanoparticle
suspension to dry overnight at room temperature on a Formvar®
coated 200-mesh copper grid (TAAB Laboratories Equipment Ltd.,
Aldermaston, England). Spray-dried chitosan nanoparticles were
observed by scanning electron microscopy (SEM) (Philips XL30
SEM, Eindhoven, Netherlands). The dry powder was placed onto
an aluminium specimen stub covered with a double-sided carbon
adhesive disc (Taab, Berkshire, UK) and sputter coated with gold
(20 kV for 3 min).
2.6.2. Fourier transform-infrared (FT-IR) spectroscopy
IR spectra (range 5000–600 cm−1 ) were recorded on lyophilised
samples using a Vertex 70 (Bruker Optics GmbH) spectrophotome-
ter (spectral resolution of 4 cm−1 ) equipped with a MIRacleTM
ATR device (Pike Optics) with a single reflection diamond crys-
tal (1.8 mm spot size) and using a MCT detector (HgCdTe,
mercury-cadmium-tellurium) cooled with liquid nitrogen. Both
raw materials and formulations were analysed by attenuated total
reflectance (ATR). The samples were deposited on the top of a dia-
mond crystal and secured with a high-pressure clamp.
2.7. Determination of protein loading efficiency
The loading efficiency was obtained by determining the concen-
tration of the protein in the supernatant using the bicinchoninic
acid (BCA) colorimetric assay (Sigma–Aldrich Co., St. Louis, Mis-
souri). The amount of protein loaded in the NPs was calculated as
the difference between the target loading and the protein recovered
in the supernatant. The supernatant of unloaded nanoparticles was
used as a blank, in order to subtract the interference of the compo-
nents of loaded samples with the BCA. Loading efficiency (LE) was
calculated using the following equation:
target loading − unloaded protein
LE(%) = × 100
target loading
221
2.8. Chorioallantoic membrane (CAM) assay
The in vivo biocompatibility of the raw materials and NPs was
evaluated using chick embryo chorioallantoic membrane assay, a
test that has been used in the past and is receiving renewed atten-
tion (Saw et al., 2008; Vargas et al., 2007). Fertilised eggs were first
disinfected with alcohol 70◦ before being placed in the incubator
at 38 ◦ C with 60% humidity. At incubation day 3, a window opening
was punctured at the blunt end of the egg and living embryos were
selected for the experiment. The opening was then covered with
a polyethylene film glued with albumen to avoid water loss and
contamination. At incubation day 6, solid samples (6 mm tablets of
raw materials and blank nanoparticles) were applied directly on the
CAM (Ribatti et al., 1996; Schoubben et al., 2013). A Leica WILD M32
stereomicroscope (equipped with a WILD PLAN 1X lens), connected
to a Leica DFC 320 camera system, was used to follow the evolution
of any effects on the materials on the CAM. After 24 h, all the eggs
were examined again and all the acquired images were compared
with the previous ones for qualitative acute toxicity evolution.
3. Results and discussion
3.1. Nanoparticles preparation and characterisation
Chitosan nanoparticles were prepared by ionotropic gelation
with the dropwise addition of tripolyphosphate (TPP) to a chitosan
solution (concentration 2.5 mg/mL). Preliminary experiments were
addressed to the selection of the optimal ratios for the formation
of nanoparticles of TPP and of two chitosan samples with different
molecular weights. For this purpose, the appearance of opalescence
was used as an indicator of nanoparticle formation, which was also
confirmed by means of dynamic light scattering. Low molecular
weight (LMW) chitosan had a weight-average molecular weight
(Mw) of 150 kDa, with an intrinsic viscosity [] of 2.37 dL/g and
a hydrodynamic radius Rh of 15.8 nm in a 0.3 M aqueous acetate
buffer solution at pH 4.5. Very low molecular weight (VLMW) chi-
tosan showed a Mw of 32 kDa, with an intrinsic viscosity of 0.6 dL/g
and a hydrodynamic radius of 6.4 nm, in the same solvent.
The effect of chitosan Mw and ratio with respect to TPP on par-
ticle characteristics is reported in Table 1. Nanoparticle formation
began at chitosan:TPP weight ratio of 6:1. The ability of chitosan
to gel rapidly upon contact with TPP relied on the formation of
inter- and intramolecular cross-linkages mediated by the anionic
molecule (De Campos et al., 2001; Xu and Du, 2003). By increas-
ing the amount of TPP, the nanoparticle suspension became more
and more turbid (chitosan NP formation) but particle aggregation
occurred rapidly and drastically. When the available quantity of
TPP was high, the dominantly inter- and intramolecular cross-links
were associated with TPP which enabled NPs to form larger par-
ticles and large flocculating aggregates (chitosan/TPP mass ratio
lower than 4:1). The suspensions formed in the range between
the appearance of the opalescence and flocculation were deeply
analysed in terms of particle size and reproducibility. Some of
them gave unpredictable results in terms of particle formation or
flocculation: this is a common behaviour, as with polysaccharide
preparations, batch-to-batch variation appears to be an unavoid-
able consequence of the polydispersity of the macromolecules, and
has a determinant impact on the physicochemical properties of the
final product (Morris et al., 2011).
The interaction at molecular lever between chitosan chains and
TPP was evidenced by ATR FT-IR spectra of chitosan NPs compared
with those of native chitosans (data not shown). The results were
consistent with previous published data (Dudhani and Kosaraju,
2010). A broad peak between 3350 and 3270 cm−1 was attributed
(1)
to a combination of stretching modes of O H and N H bonds in
222 A. Rampino et al. / International Journal of Pharmaceutics 455 (2013) 219–228

Table 1
Mean hydrodynamic diameter (MHD) and surface charge of VLMW and LMW chitosan-TPP NP prepared at different chitosan:TPP mass ratios.

Chitosan:TPP ratio (w/w) VLMW chitosan NPs LMW chitosan NPs

MHD ± S.D. (nm) PDI ␨-pot ± S.D. (mV) MHD ± S.D. (nm) PDI ␨-pot ± S.D. (mV)

3:1 –* –*
4:1 –* –*
5:1 151 ± 10 0.185 24 ± 4 200 ± 24 0.227 25 ± 3
6:1 164 ± 12 0.198 28 ± 3 193 ± 28 Not reproducible
*
Aggregation took place and no particles were recovered.

chitosan matrix. In the sample of chitosan nanoparticles this band
becomes wider and shifts to lower wavenumbers, indicating an
enhancement of the hydrogen bond interactions (Yu et al., 1999). In
addition, the 1523 cm−1 peak of the NH2 bending vibration of chi-
tosan samples shifted to 1533 cm−1 in the NPs. A similar result has
been observed in literature on chitosan-TPP NPs (Xu and Du, 2003)
and on chitosan films treated with phosphate (NaH2 PO4 ); the shift
was attributed to the interaction between the amino group and the
phosphate anion (Knaul et al., 1999). The other peaks observed in
the sample of nanoparticles were those of the P O stretching at
1201 cm−1 and P O bending at 885 cm−1 of the TPP.
The results of these preparations and characterisations indicate
that NPs prepared with a chitosan/TPP ratio of 5:1 turned out to
be the best formulations for both chitosan Mws, with an average
size of 151 nm in the case of VLMW chitosan. The increase of chi-
tosan Mw to 150 kDa provoked an increase in the average particle
size from 151 to 200 nm. These results were consistent with find-
ings that higher Mw chitosan produced larger nanoparticles (Csaba
et al., 2009; Luangtana-anan et al., 2005; Tang et al., 2003). The poly-
dispersity index (PDI) was low for all the preparations evaluated,
indicating that a homogeneous dispersion was obtained. The PDI
changed from 0.185 to 0.227 in correspondence to the increase in
chitosan Mw (Table 1); a higher variety of particle size was obtained
using a solution of larger polymer chains. The distribution curve
indicates the presence of two different particle size populations:
indeed, the bimodal curve consists of a fraction of small particles
(around 40 nm) and a second population of around 250 nm. The
presence of two populations was also confirmed by TEM. Size anal-
ysis was performed also on NPs deriving from a scale-up procedure.
When changing the component concentration, the measurements
indicated that tripling the total concentration gave larger particles,
increasing the size from 200 to 273 nm (SD 18), in agreement with
previous results (Gan et al., 2005; Hu et al., 2008). When increasing
of 10 times the preparation volume, a smaller variation in dimen-
sion was measured (average size of 237 ± 27 nm).
Regarding the surface charge, both chitosan samples gave pos-
itively charged nanoparticles. When chitosan and TPP were mixed
with each other, they spontaneously formed compact complexes
with an overall positive surface charge, as confirmed by measures
of zeta potential values. In accordance with literature (Fan et al.,
2012; Janes and Alonso, 2003), by increasing TPP concentration,
the zeta potential decreased (Table 1). Analysing the differences
between the two chitosan samples, the same trend as noticed for
particle size, could also be observed as a function of the molecular
mass: compared to VLMW chitosan samples, LMW chitosan sam-
ples with the same chitosan:TPP mass ratio had a slightly higher
zeta potential. Chitosan samples with different Mws should have
different accessibility to cationic groups, even if they have the same
degree of deacetilation: 87% in the studied chitosans. The result
is a higher surface charge for particles formed by polymers with
a longer chain, in accordance with results obtained by Gan et al.
(2005).
Although there is a large number of studies on chitosan nanopar-
ticle formation in relation to the different polymer amounts used,
very few of these studies incorporated results on the production
method yield. Here, the amount of chitosan in the supernatant
after centrifugation was assayed using a colorimetric determina-
tion (Fig. 1). By adding TPP to the chitosan solution, a slight change
in the amount of precipitate could be noticed: this could be due to
the presence of small nanoparticles that cannot be easily separated
from the suspension by centrifuging because of their light weight.
By increasing the addition of TPP, a sudden increment of the precip-
itate can be observed in correspondence to 3.5 mL of TPP solution
(0.875 mg, chitosan to TPP ratio 7.14:1). This volume corresponds to
the start of the opalescence in the chitosan solution, signalling the
formation of aggregates of a dimension comparable to the incident
light. The amount of precipitate increases with the addition of over
1.25 mg of TPP (5 mL of anionic solution, chitosan:TPP ratio 5:1),
where larger aggregates are formed: at this point, the amount of
precipitated chitosan was 0.7 mg out of 6.25 mg in the original solu-
tion. This result indicates that the yield of nanoparticles at this stage
is low (11.2%). It is reasonable to assume that some nanoparticles
remain in the suspension and are not separated under the mild cen-
trifuge conditions (3270 RFC for 2 h). However, while increasing TPP
amounts and/or centrifugation conditions (speed or time) generate
flocculation or particle packing, recovering unreacted chitosan and
adding further TPP are also worth exploring.
3.2. Nanoparticles’ evolution over time
As previously mentioned, the size distribution curve of freshly
prepared samples is characterised by two populations of parti-
cles, one around 40 nm and the other around 250 nm. This typical
distribution is mainly observed in freshly prepared samples (Ing
et al., 2012); if samples were kept under stirring and analysed
after 6 h, the smallest particle population disappeared. Indeed the
bimodal curve became a single Gaussian curve, spread up to a few
hundreds of nanometers. This phenomenon could be explained by
Fig. 1. Quantity of recovered nanoparticles (in mg) as a function of the amount of
TPP added (in mg).
 A. Rampino et al. / International Journal of Pharmaceutics 455 (2013) 219–228
Fig. 2. (a–b) TEM image of chitosan nanoparticles in two enlargements. (c) The
image b taken after few minutes: the fusion process is clearly visible.
the fact that the small particles aggregate during stirring to form
a more homogeneous population of particles. This phenomenon
is clearly visible in TEM images, where particles range from few
tens to few hundreds of nanometers (Fig. 2). TEM provides the
size of almost-dehydrated particles, while dynamic light scatter-
ing measurements yield an ensemble average of the particle size in
suspension.
Taking into account dynamic light scattering and TEM results,
a mechanism of various steps could be presented. Chitosan
223
in solution has a random coil conformation (Weinhold et al.,
2009). TEM images provide a chitosan chain dimension of around
20–40 nm, consistent with a hydrodynamic radius of 15.8 nm,
as reported in section 3.1. Moreover, the chitosan concentration
value of 2.5 mg/mL is close to the limit of the critical concentration
of coil overlap (C*). This means that chitosan chains are quite close
to one another, some of them statistically in contact with others.
By adding a dilute solution of TPP (0.25 mg/mL) a double effect
is obtained. On the one hand, a dilution of the solution generates
more randomly distributed chitosan coils; on the other, anionic
TPP starts interacting with few cationic groups on chitosan chains
folding over themselves (mainly through intramolecular links).
The dimensions of the new entities are comparable to the random
coil chain ones because they are the result of few bent chains
grafted together or even a single gelified random coil which has
increased in size due to the presence of TPP (osmotic pressure).
Adding further TPP onto chitosan leads to both the formation
of new particles, supported by the constant size value, and also
their rearrangement towards a more compact particle structure. At
the end of the addition of polyanions, the negatively charged TPP
interacts with the chitosan surface groups leading to the primary
aggregates (charged chitosan monomoles exceed TPP by 10 to 1).
The partially neutralised positive charges of chitosan still present in
the primary aggregates, favour a rearrangement of chains and the
formations of more compact particles with a smaller size. A further
rearrangement of nanoparticles is obtained with time by an effi-
cient sharing of neutralising anionic charges, producing particles
described by the presence of a Gaussian distribution curve instead
of a bimodal one. Although such a phenomenon is often described
in literature to be a function of the effective charge density and
of the flexibility of the polymer, a quantitative elucidation is still
lacking.
TEM images confirm this hypothesis: particles appear as small
and individual, with a diameter of around 30–40 nm; larger parti-
cles are due to the aggregation of single small particles that tend
to fuse together generating a larger entity. This aspect is clearly
shown by subsequent TEM images (Fig. 2). Air-dried samples are
not completely desiccated, especially not nanogels as chitosan-TPP
particles. During TEM analysis, when magnifying a NP aggregate as
much as possible, a very fast fusion (seconds/minutes) of single par-
ticles into one entity was observed: the heat of the electron beam
promoted intermolecular links thanks to the still present aqueous
environment inside the gel-network. In order to confirm that this
behaviour was due to a linking agent, TEM images of chitosan solu-
tion were collected showing small particles, of around 40 nm in
diameter, but with a completely different behaviour: magnifying on
several close entities, their structure did not change and no fusion
occurred even if the ray was kept on them for long time (data not
shown).
The dimensions of freshly formed single particles were clearly
visible in TEM images of samples with paraformaldehyde. This
agent was added immediately after the TPP dribble, there-
fore before the complete rearrangement of chains and particles.
Paraformaldehyde blocks the particles’ interactions by limiting
their mobility. TEM images showed that most particles were single
isolated ones and only few of them were grouped together: this
is because there was not enough time for a complete TPP redis-
tribution and particle rearrangement (data not shown). This was
also confirmed by particle size analysis showing the inhibition of
particle growing during ageing.
Size stability as a function of time has been studied for both
LMW and VLMW chitosan nanoparticles. All samples presented an
increase in size without leading to the formation of large aggregates
(micrometric aggregates) (Fig. 3). As regards the particles prepared
with LMW chitosan, after 1 month a slight increase in size was mea-
sured, showing a 2-step behaviour: a growth in size was observable
224 A. Rampino et al. / International Journal of Pharmaceutics 455 (2013) 219–228

Table 3
Mean hydrodynamic diameter (MHD), surface charge and loading efficiency of
protein-loaded chitosan nanoparticles.

Protein amount Loading MHD ± SD ␨-pot ± SD

(␮g/mL) efficiency ± SD (nm) (mV)
(%)

BSA
200 39 ± 9 200 ± 11 27 ± 3
400 40 ± 9 225 ± 21 23 ± 8
600 60 ± 6 224 ± 25 21 ± 6
OVA
200 48 ± 4 195 ± 13 17 ± 1
400 57 ± 5 202 ± 17 17 ± 1
600 76 ± 4 215 ± 13 16 ± 2
HI
200 55 ± 8 330 ± 36 30 ± 4

(Gan et al., 2005). Samples left at room temperature showed a ten-

dency to aggregate after two weeks. The total amount of chitosan
was in excess compared to TPP (charged monomole: TPP ratio 10
Fig. 3. Particle size as a function of storage time: different Mws of chitosan were
used. Square points refer to LMW chitosan NPs and triangle points to VLMW chitosan
to 1), and waiting for a more efficient rearrangement of particles
NPs. Size increase is expressed as 100 × (d − d0 )/d0 (d refers to diameter at time t and led to the production of large aggregates: in fact, the formation of
d0 to the initial diameter). larger nanoparticles is favoured at a higher chitosan concentration
(Kaloti and Bohidar, 2010).
after the first hour, completing the particle formation over the next
3.3. Effect of protein loading on size and surface charge
24 h: size was increased by 5%. Then, after 1 month, the size slightly
increased by 8% in total. On the other hand, particles prepared using
Three different proteins were loaded onto chitosan nanopar-
VLMW chitosan showed a linear increase in diameter size, with a
ticles: bovine serum albumin (BSA), ovalbumin (OVA) and human
growth of 7 and 24% after 7 and 28 days, respectively.
insulin (HI). BSA and OVA, with a pI of 4.8 and 4.7 respectively, were
The size evolution during storage is ascribed to many fac-
added directly to the cationic solution and then TPP was dripped
tors, such as particle aggregation, which provides a more efficient
on. HI (pI = 5.6), on the other hand, was diluted with TPP (pH 7.5)
rearrangement, the interaction of free polymer chains with the
and then added to the cationic solution, because it has a neutral
particle network, that leads to a reorganisation of intermolecu-
net charge in the chitosan solution (pH 5.5) leading to precipita-
lar entanglements, syneresis, and swelling, due to the presence
tion. As shown in Table 3, the presence of the model protein has a
of TPP that generates an inflow of water by osmosis (López-León
small influence on particle size, increasing it by few nanometers.
et al., 2005; Tang et al., 2003). The size change is usually due to
This can be ascribed to the ionic interaction and ionic cross-linking
the balance between the above-mentioned forces. The time span
between negatively charged BSA (or OVA) and positively charged
in which the growing stage finished depended on the size of the
chitosan under the preparation conditions (∼pH 6). This generates
initial nanoparticle, which in turn depended on the molecular
a reduction in the electrical repulsion among biopolymers, where
weight of the chitosan. Larger NPs, prepared with a chitosan of
modifications in the electrical state cause the particle to shrink.
higher molecular weight, have more intermolecular entanglements
Moreover, TPP links intra- and intermolecular chitosan chains giv-
and inter/intra-molecular hydrogen bondings, producing a stable
ing a more compact structure.
nanogel which therefore needs more time to change the size of the
The decrease in the zeta potential after the addition of pro-
nanoparticle.
tein is reasonably due to the partial deposition of the negatively
The aggregation level increased rapidly within the first 2 h and
charged protein on the particle surface, reducing the total net
continued after 24 h. The polydispersity index, on the contrary,
charge. Similarly, in the case of HI, an ionic interaction of chi-
increased after 2 h (Table 2) but decreased after 24 h. Particles left
tosan occurs with TPP and HI. The incorporation of HI increases
without stirring took longer to reach the equilibrium. Without stir-
particle size, probably due to concurrent competition between TPP
ring, the particle size did not increase significantly, growing by 4%
and HI in their interaction with chitosan, thus causing the particle
after 2 h and 5% after 24 h (Table 2). A small particle population
to swell: when HI is added, it is negatively charged and interacts
was still present after 24 h and the polydispersity index was 0.186,
with -NH3 + groups of chitosan, competing with TPP. After parti-
lower than that of the samples kept under stirring. The particle size
cle formation, the HI net charge is next to neutrality as its pI of
population was less variable because the system was slowly looking
5.6 is very close to the pH of the particle suspension (≈6): this
for the best equilibrium and few large nanoparticles were present
generates, on the one hand, a weaker interaction with chitosan (a
swollen particle) and on the other, less influence on the total surface
Table 2 charge (higher zeta potential compared to chitosan/TPP particles)
Effect of time and stirring on particle size. Size growth is expressed as percentage (Table 3).
of size increase.

Hours after Stirring No stirring 3.4. Lyophilisation

preparation

Size PDI Size PDI Lyophilisation is one of the most commonly used technologies
increase ± SD increase ± SD that ensures a long-term conservation of polymeric nanoparticles.
(%) (%) Indeed, nanoparticles stored as an aqueous suspension undergo
0 0 0.282 0 0.198 solubilisation and/or degradation of the polymers, drug leakage,
2 31 ± 1 0.456 4±1 0.195 desorption or degradation. Dried nanoparticles are usually easily
24 77 ± 1 0.341 5±1 0.186
redispersible, although in some cases a complete redispersion after
 A. Rampino et al. / International Journal of Pharmaceutics 455 (2013) 219–228
Fig. 4. Effect of cryoprotectants on particle size. Each bar represents the average
size and error bars refer to SD. In the absence of cryoprotectants, the freeze-drying
of untreated samples produces collapsed NPs (see text).
lyophilisation may be difficult to achieve, due to the aggregation
or irreversible fusion of nanoparticles. Furthermore, the crystalli-
sation of ice can induce a mechanical stress on nanoparticles,
leading to their destabilisation. Thus, the addition of a suitable
lyo/cryo protective agent before freezing, prevents nanoparticles
from aggregating. It is well established that sugars are employed
for this purpose. The mechanism by which sugars protect particles
resides in the interaction with the solute via hydrogen bonding,
which maintains the solute in a “pseudo hydrated” state during
the dehydrating step, thus providing protection from damage
during dehydration and subsequent rehydration. Such a protective
interaction is made possible by the ability of the sugars to remain
amorphous during freeze-drying.
All the chitosan nanoparticles prepared were characterised by
a difficult redispersion after lyophilisation (Dyer et al., 2002; Lee
et al., 2004), therefore the effect of adding trehalose, mannitol and
PEG 10000 was investigated (Blasi et al., 2013; Luangtana-anan
et al., 2010). Following the addition of cryoprotectants, all samples
could be re-dispersed properly after lyophilisation. NPs contain-
ing different concentrations of trehalose, mannitol and PEG 10000
were evaluated in terms of size, after lyophilisation (Fig. 4). A com-
mon behaviour was found, indeed lyophilised nanoparticles were
more similar to freshly prepared nanoparticles with increasing con-
centrations of cryoprotectant. From these results, it emerged that a
complete re-dispersion, that is a balance between the average size
and polydispersion index, was obtained when sugars or PEG were
added to the nanoparticle suspension at a concentration of at least
5% (w/v).
Comparing the different cryoprotectants, suspensions contain-
ing mannitol or PEG were characterised by a greater increase in size,
especially at low concentrations. Therefore the best results were
obtained with trehalose. This could be related to its peculiar char-
acteristics, which are low hygroscopicity, the absence of internal
hydrogen bonds thus allowing a more flexible formation of hydro-
gen bonds with nanoparticles, a very low chemical reactivity and,
finally, a higher glass transition temperature Tg (Crowe et al., 1996).
It must be taken into account that the crystallisation of cryopro-
tectants and the formation of a eutectic with ice can cause phase
separation in the cryo-concentrated portion of the frozen nanopar-
ticle suspension with no opportunity for a stabilising interaction
with nanoparticles. Individual nanoparticles in the nanoparticle-
rich phase can interact and form aggregates. Moreover, the growing
225
Fig. 5. SEM images of microparticles derived from the sintering of unprotected
chitosan nanoparticles after spray-drying in two magnifications.
crystals of water and sugar may exert mechanical forces on the
nanoparticles leading to their fusion (Cesàro et al., 2008). There-
fore, in order to avoid this situation, it is mandatory that at least
some sugar molecules remain molecularly dispersed throughout
the amorphous nanoparticle phase (Abdelwahed et al., 2006). It is
suggested that sugars isolate individual particles in the unfrozen
fraction, thereby preventing aggregation during freezing above Tg .
In this case, vetrification is not required for this effect and the spa-
tial separation of particles within the unfrozen fraction is sufficient
to prevent aggregation (Allison et al., 2000).
3.5. Spray drying
The effect of spray-drying on the size of nanoparticles was eval-
uated; the three selected cryoprotectants were added to the NP
preparation at the concentration of 0.5% (w/v). This concentration,
which gave an important increase in size after lyophilisation, was
chosen in order to compare the effect of the two processes. The
effect of the addition of the three selected cryoprotectants was
investigated before and after spray-drying. The sole addition of cry-
oprotectants in NP suspension slightly increased the average size
with respect to blank nanoparticles.
The drying process causes particle aggregation, an aspect that
was clearly visible in the SEM picture of unprotected nanoparticles
where they can be seen to be fused together, generating micro-
particles (Fig. 5). After spray-drying, an increase in size was found
for all cryoprotectants. Trehalose and PEG gave nanoparticles of
a larger size but they were still nano-sized and re-dispersible;
however particles containing PEG were characterised by a high
226 A. Rampino et al. / International Journal of Pharmaceutics 455 (2013) 219–228
Fig. 6. CAM image taken from the top of the egg after deposition of prepared tablet. (a–b) Effect of chitosan and NPs on chick embryo after 24 h from deposition, respectively.
(c) Effect of TPP on chick embryo: after 2 h from deposition, a haemorrhagic event occurs with the death of the embryo.
polydispersity index after spray-drying. On the contrary, nanopar-
ticles with mannitol remained in suspension as macroaggregates
and it was not possible to properly redisperse the sample after
spray-drying. A comparison of the spray-drying results with the dif-
ferent excipients showed that trehalose is preferable with respect
to PEG and mannitol, as it was also for lyophilisation. Thus,
trehalose is believed to be a preferable protectant against NP aggre-
gation, as it is for other biomolecules in cell bioprotection (Crowe
et al., 1996).
The comparison between lyophilised and spray dried particles
treated with the same amount of protector (0.5%, w/v) shows a
similar increase of the average size. Since spray drying is faster,
and cost-effective, than lyophilisation, this technique is recom-
mended for the dehydration of chitosan NP (Schoubben et al.,
2010).
3.6. Chorioallantoic membrane assay
Renewed interest has been shown in the chorioallantoic mem-
brane (CAM) assay, as this can be carried out at a much lower
cost with greater efficiency and faster measurements than other
in vivo assays (Saw et al., 2008; Schoubben et al., 2013; Vargas
et al., 2007). Thus, the CAM assay was used for studying the bio-
compatibility of materials and NPs, analysing the inflammatory
and neo-angiogenic effect (Fig. 6) after 24 h from the treatment.
In the case of the raw material (chitosan), no significant changes
occurred after 24 h of incubation. In fact, all the embryos were
alive and there was no sign of any vascular change in the CAM,
e.g. haemorrhaging, neoangiogenesis or the occurrence of vessels
devoid of blood flow (ghost vessels). This is in good agreement with
similar works reported in literature, confirming that our materials
are non-toxic, as expected and described: chitosan is a polymer
approved for dietary applications in Japan, Italy and Finland and
it is approved by the FDA for use in wound dressings (Kean and
Thanou, 2010).
Moreover, the prepared nanoparticles did not cause any death or
inflammatory response. This is an important point because, unlike
raw materials, they are not always predictable: a great deal of atten-
tion is paid to nanosized carriers, concerning the effect (toxicity) of
the shape/surface structure of the new nanoentities on cells and
tissues. These results were in accordance with the in vitro toxicity
test (data not shown).
The only sample that gave an adverse effect was the TPP tablet
(Fig. 6c). This was an expected result, because the tripolyphosphate
salt increases the pH of the chorioallantoic membrane drastically,
generating a haemorrhagic event and the death of the embryo. On
the other hand, the amount of TPP present in nanoparticles was sig-
nificantly lower, its availability limited by chitosan ionic interaction
and no sign of any damage was noticed. Furthermore, low amounts
of TPP are commonly used as a food additive to preserve foods such
as red meats, poultry, and seafood, helping them to retain their
tenderness and moisture.
4. Conclusions
All the results on the preparation, characterisation and size sta-
bility of chitosan-TPP nanoparticles as drug carriers rely on the
attempt to modulate the experimental conditions and to identify
the appropriate procedures. Three main follow-out are worth to be
mentioned:
(1) The simple ionotropic gelation method was shown to be effec-
tive although involved a complicated form of dynamic intra-
and inter-molecular complexes. The concentration and time
dependence of the TPP addition suggest that average particle
size depends not only on the TPP concentration, but also on
a rearrangement of the small compact particles towards larger
particles. It is also worth noting the beneficial effect of quiescent
conditions, as opposed to the stirring conditions, in providing
smaller and homogeneous particles and in delaying the aggre-
gation of the final suspension.
(2) The exploration of appropriate techniques of particle drying
(i.e., lyophilisation and spray-drying) was carried out with the
aim of achieving both the stability of the nanoparticles’ char-
acteristics and also the best protection of labile drugs, such as
therapeutic peptides and proteins. The addition of bioprotec-
tants in the suspension of nanoparticles proved very effective
in reducing surface attraction and maintaining the nanopow-
der in a dispersed form. The best results were obtained by using
trehalose, which has been advocated in many cases, despite
the still controversial interpretation of the underlying mech-
anism of its action (Cesàro, 2006; Golovina et al., 2010; Sakurai
et al., 2008). An in-depth physicochemical study of the pref-
erential distribution (i.e. solvation) of trehalose surrounding
the chitosan nanoparticles may add some useful results to the
“trehalose story”.
(3) Finally, out of all the main positive results, emphasis has been
given to the assessment of the safety of nanoparticles for
biological membranes with in vivo tests. This point is often
overlooked in the experiments producing nanoparticles for
human or animal use, despite the great attention nowadays
assigned in Europe through the Nanosafety Cluster activities
(http://www.nanosafetycluster.eu/). As an alternative to mam-
malian cells for in vivo tests, the use of chick embryos confirmed
the biocompatibility of both chitosan and nanostructures, and
encouraged future studies using this simple and direct tech-
nique.
Acknowledgment
This work has been partially carried out within the Euro-
pean Project FP6 NanoBioPharmaceutics (NMP 026723-2). Antonio
Rampino was the recipient of a grant from MIUR (Rome) during
his Ph.D. studies on “Polysaccharide-based nanoparticles for drug
delivery”.
 A. Rampino et al. / International Journal of Pharmaceutics 455 (2013) 219–228
The authors wish to thank Dr. G. Birarda (Advanced Technology
and Nanosciences – TASC INFM, Area Science Park, Trieste) and Miss
F. Virgilio (University of Trieste) for providing help with the ATR-
FTIR and Prof. Carlo Cirotto and Lanfranco Barberini for support in
the CAM tests.
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