marine drugs

Article
Controlling Properties and Cytotoxicity of Chitosan
Nanocapsules by Chemical Grafting
Laura De Matteis 1, *, Maria Alleva 1 , Inés Serrano-Sevilla 1,2 , Sonia García-Embid 1 ,
Grazyna Stepien 1 , María Moros 3 and Jesús M. de la Fuente 2, *
1 Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, Edificio I+D, calle Mariano
Esquillor s/n, 50018 Zaragoza, Spain; mariaalleva1234@gmail.com (M.A.); inessersev@gmail.com (I.S.-S.);
sonia.garcia.embid@gmail.com (S.G.-E.); gstepien@unizar.es (G.S.)
2 Instituto de Ciencia de Materiales de Aragón (ICMA), CSIC-Universidad de Zaragoza, Edificio I+D,
calle Mariano Esquillor s/n, 50018 Zaragoza, Spain
3 Istituto di Scienze Applicate e Sistemi Intelligenti “E. Caianiello”, Consiglio Nazionale delle Ricerche,
Pozzuoli 80078, Italy; m.moros@isasi.cnr.it
* Correspondence: lauradem@unizar.es (L.D.M.); jmfuente@unizar.es (J.M.d.l.F.);
Tel.: +34-876-555-433 (L.D.M.); +34-606-949-073 (J.M.d.l.F.)

Academic Editors: David Harding and Hitoshi Sashiwa

Received: 28 July 2016; Accepted: 20 September 2016; Published: 30 September 2016

Abstract: The tunability of the properties of chitosan-based carriers opens new ways for the
application of drugs with low water-stability or high adverse effects. In this work, the combination
of a nanoemulsion with a chitosan hydrogel coating and the following poly (ethylene glycol) (PEG)
grafting is proven to be a promising strategy to obtain a flexible and versatile nanocarrier with an
improved stability. Thanks to chitosan amino groups, a new easy and reproducible method to obtain
nanocapsule grafting with PEG has been developed in this work, allowing a very good control and
tunability of the properties of nanocapsule surface. Two different PEG densities of coverage are
studied and the nanocapsule systems obtained are characterized at all steps of the optimization
in terms of diameter, Z potential and surface charge (amino group analysis). Results obtained are
compatible with a conformation of PEG molecules laying adsorbed on nanoparticle surface after
covalent linking through their amino terminal moiety. An improvement in nanocapsule stability in
physiological medium is observed with the highest PEG coverage density obtained. Cytotoxicity
tests also demonstrate that grafting with PEG is an effective strategy to modulate the cytotoxicity of
developed nanocapsules. Such results indicate the suitability of chitosan as protective coating for
future studies oriented toward drug delivery.

Keywords: chitosan; hydrogel; surface grafting; nanocapsules; stability

1. Introduction
In the last few decades, many kinds of nanocarriers have been developed for delivery and
targeting of therapeutic or diagnostic agents, thanks to some important advantages that they offer
depending on their physico-chemical properties [1,2].
Depending on the specific needs, the nanocarrier type and formulation process must be chosen
on the basis of therapy goals and administration route [3]. The most common nanocarriers can
be classified as follows: solid (inorganic or organic) nanoparticles [4,5], nanospheres (polymeric
matrices or hydrogels) [6–8], or nanocapsules (usually liposomes, emulsion-based, or protein-based
nanocapsules) [9–11].
According to Vrignaud and co-workers, nanocapsules are vesicular systems, composed of an oily
or an aqueous core that can be considered as a reservoir in which the drug is confined to a cavity,

Mar. Drugs 2016, 14, 175; doi:10.3390/md14100175 www.mdpi.com/journal/marinedrugs

Mar. Drugs 2016, 14, 175 2 of 15

surrounded by a polymeric shell [12]. Nanocapsules can be obtained combining nanoemulsion and a
polymeric coating. Nanoemulsion particles are stable colloidal suspensions obtained by mixing an
organic phase containing oil and a lipophilic surfactant with an aqueous one containing a hydrophilic
surfactant, resulting in a particle size ranging from 20 to 600 nm. The characteristics of the obtained
particles depend on the spontaneity of the emulsification process that is affected by the nature of the
single components of the reaction mixture and also by the rate of the mixing process [13,14]. Depending
on the desired application, a coating is necessary to further stabilize the nanoscaled particles resulting
from this spontaneous process and improve surface properties. The most commonly used coatings are
natural polymers, which are deposited on the nanoemulsion template surface to produce a rigid and
dense shell [15]. The shell can be easily tailor-made to achieve desired characteristics and its surface
chemistry can be tuned to obtain a proper functionalization for biological targeting [16–18].
Natural polymers are among the most used for these kind of coatings since they usually provide a
high colloidal stability in water suspensions. Active research is now focused on the use of hydrophilic
biopolymers as carrier coatings because of their biocompatibility and biodegradability [19,20]. Chitosan
(CS) has been used for the development of sustained release carriers, mucoadhesive formulations,
and peptide drug absorption systems [21–24]. It is currently employed to prepare nanomaterials
with mucoadhesive properties since its positive charges allow the interaction of particles with the
negative charge of mucin, resulting in a better interaction with mucosal tissues and with epithelial cells.
Moreover, it is known that the positive charge of the polymer can promote the paracellular transport
by tight-junction regulation [25,26].
In this work, core-shell nanocapsules made of a nanoemulsion core and a chitosan shell were
synthesized and characterized with the aim of obtaining a multipocket nano-reservoir carrier to be
used in future applications for sustained release of different drugs. The secondary effects—toxicity,
poor solubility, and bioavailability—of new drugs lead to the need of their encapsulation to protect
them from degradation and to enhance their stability and solubility [27,28].
Thanks to the presence of chitosan amino groups, the surface of the obtained nanocarrier can also
be grafted with specific moieties in order to tune the net charge to introduce specific functional groups
and/or improve the carrier stability in biological media and physiologic solutions for intravenous
administration [29–33]. The development of a smart nanocarrier is strictly related with controlling
its surface properties since they are responsible for specific recognition of targeted sites but also for
non-specific adsorption of serum proteins. A decrease in protein adsorption leads to a reduced uptake
by the mononuclear phagocytic system, leading to a prolonged circulation time in the blood stream and
to a higher residence time of the encapsulated drug. Poly (ethylene glycol) (PEG) coatings are known to
prevent aggregation and serum protein adsorption by steric and hydration repulsions leading to more
stable colloidal suspensions of nanocapsules in physiological media [33–35]. In this work, the surface
of the developed chitosan-coated nanocapsules was grafted covalently with PEG molecules through
a novel, simple, and reproducible strategy based on the use of a homobifunctional crosslinker, bis
(sulfosuccinimidyl) suberate (BS3 ), that links aminated PEG molecules to amino groups on nanocapsule
surface. Nanocapsule behavior in different media was evaluated in terms of aggregation degree before
and after grafting and the effect of PEG on their cytotoxicity was also assessed.

2. Results

2.1. Chitosan-Coated Nanocapsules

A nanoemulsion method was developed to obtain small nanoparticles (smaller than 200 nm) to
be used as a template for the following polymer coating and reinforcement. The aim was to obtain
capsules with a lipophilic core and a hydrophilic cationic shell of chitosan hydrogel.
The synthesis of the nanocapsules was carried out in two steps. The formation of the nanoemulsion
template particles was carried out simply by adding a water-miscible organic solution of Span®
85/oleic acid (Croda International PLC, Cowick Hall Snaith, Goole, East Yorkshire, UK) to a Tween® 20
Mar. Drugs 2016, 14, 175 3 of 15

Mar. Drugs 2016, 14, 175  3 of 14 
(Croda International PLC, Cowick Hall Snaith, Goole, East Yorkshire, UK) aqueous solution under
stirring. under 
solution  Optimal ratiosOptimal 
stirring.  betweenratios 
components
between have been found
components  have adapting a method
been  found  adapting reported by
a  method 
Bouchemal et al. [14]. The particles are immediately and spontaneously formed. Chitosan has been
reported by Bouchemal et al. [14]. The particles are immediately and spontaneously formed. Chitosan 
chosen for the coating of the nanoemulsion template since it is one of the richest in amino groups’
has been chosen for the coating of the nanoemulsion template since it is one of the richest in amino 
natural polymers.
groups’ natural polymers. 
The presence of such functional groups is responsible for the ability of the polymer to gelify in
The presence of such functional groups is responsible for the ability of the polymer to gelify in 
presence of multi-anions leading to the formation of hydrogel. Moreover, the amino groups exposed
presence of multi‐anions leading to the formation of hydrogel. Moreover, the amino groups exposed 
on hydrogel shell surface allow the easy functionalization of the nanocapsule with the desired moiety.
on hydrogel shell surface allow the easy functionalization of the nanocapsule with the desired moiety. 
Chitosan is directly added to the nanoemulsion that is subsequently mixed with a sodium sulfate
Chitosan is directly added to the nanoemulsion that is subsequently mixed with a sodium sulfate 
solution to obtain the coating with a chitosan shell. This treatment has been used in several works to
solution to obtain the coating with a chitosan shell. This treatment has been used in several works to 
obtain chitosan particles [36–38]. In our case, the method allowed to obtain a hydrogel polymer shell
obtain chitosan particles [36–38]. In our case, the method allowed to obtain a hydrogel polymer shell 
as a result of the interaction of chitosan polyelectrolyte structure with sodium sulfate. Sodium sulfate
as a result of the interaction of chitosan polyelectrolyte structure with sodium sulfate. Sodium sulfate 
acts as a bridge promoting interactions between polymeric chains.
acts as a bridge promoting interactions between polymeric chains. 
The result of the synthesis is a water-stable suspension of chitosan-coated nanocapsules (CS-NCs).
The result of the synthesis is a water‐stable suspension of chitosan‐coated nanocapsules (CS‐NCs). 
A schematic representation of the hypothesized nanocapsule structure is reported in Figure 1A.
A schematic representation of the hypothesized nanocapsule structure is reported in Figure 1A. 
Moreover, to 
Moreover,  to investigate 
investigate the 
the morphology 
morphology of  of the 
the obtained 
obtained material, 
material, an 
an electron 
electron microscopy 
microscopy
characterization was
characterization  was carried out on
carried  out chitosan-coated nanocapsules
on  chitosan‐coated  using bothusing 
nanocapsules  Brightboth 
Field Bright 
Transmission
Field 
Electron Microscopy (BF-TEM) and Environmental Scanning Electron Microscopy (ESEM) (Figure
Transmission  Electron  Microscopy  (BF‐TEM)  and  Environmental  Scanning  Electron  Microscopy  1B,C
respectively). Due to the sensitive nature of the sample common in soft materials, previous fixation,
(ESEM) (Figure 1B,C respectively). Due to the sensitive nature of the sample common in soft materials, 
dehydration, dyeing and resin embedding were necessary.
previous fixation, dehydration, dyeing and resin embedding were necessary. 

 
Figure 1. Morphological characterization of chitosan‐coated nanocapsules (CS‐NCs). (A) Schematic 
Figure 1. Morphological characterization of chitosan-coated nanocapsules (CS-NCs). (A) Schematic
representation 
representation of 
of a 
a section 
section of 
of aa  nanoemulsion-based
nanoemulsion‐based  and  chitosan‐coated  nanocapsule; 
and chitosan-coated nanocapsule; (B) 
(B) Bright 
Bright
Field Transmission Electron Microscopy (BF‐TEM) image and (C) Environmental Scanning Electron 
Field Transmission Electron Microscopy (BF-TEM) image and (C) Environmental Scanning Electron
Microscopy (ESEM) image of nanocapsules from a section of the epoxy resin block; (D) representation 
Microscopy (ESEM) image of nanocapsules from a section of the epoxy resin block; (D) representation
of frequency count analysis of size distribution. 
of frequency count analysis of size distribution.

The sample turned out to be composed of spherical capsules of a quite homogeneous size. The 
The sample turned out to be composed of spherical capsules of a quite homogeneous size.
polymeric shell can be appreciated in BF‐TEM and ESEM images (Figure 1B,C). ESEM estimation of 
The polymeric shell can be appreciated in BF-TEM and ESEM images (Figure 1B,C). ESEM estimation
the  diameter  distribution  is  reported  in  Figure  1D.  The  number  of  capsules  of  different  sizes,  as 
of the diameter distribution is reported in Figure 1D. The number of capsules of different sizes,
percentages over the total number of measured capsules, is reported in the graph as a function of the 
as percentages over the total number of measured capsules, is reported in the graph as a function of
diameter. The calculated mean diameter of NCs is 104 nm. 
the diameter. The calculated mean diameter of NCs is 104 nm.
As optimization of the process, the importance of the sonication treatment during hydrogel shell 
formation  was  evaluated  by  substituting  it  with  a  gentle  stirring  while  adding  nanocapsules  to 
Na2SO4 solution. The elimination of the sonication step in the synthesis process could represent an 
Mar. Drugs 2016, 14, 175 4 of 15

As optimization of the process, the importance of the sonication treatment during hydrogel
Mar. Drugs 2016, 14, 175  4 of 14 
shell formation was evaluated by substituting it with a gentle stirring while adding nanocapsules to
Na2 SO4 solution. The elimination of the sonication step in the synthesis process could represent an
advantage  in  terms  of  applicability  of  the  process  for  future  applications  to  industrial  production 
advantage in terms of applicability of the process for future applications to industrial production with
with  high  levels  of  scale‐up.  Nanocapsules  obtained  with  the  modified  process  have  been 
high levels of scale-up. Nanocapsules obtained with the modified process have been characterized
characterized in terms of hydrodynamic diameter and surface charge and they have been compared 
in terms of hydrodynamic diameter and surface charge and they have been compared with the
with the sonicated ones. In Figure 2, Dynamic Light Scattering (DLS) measures of the hydrodynamic 
sonicated ones. In Figure 2, Dynamic Light Scattering (DLS) measures of the hydrodynamic diameter
diameter of the nanoemulsion template just before chitosan coating (A), nanocapsules obtained by 
of the nanoemulsion template just before chitosan coating (A), nanocapsules obtained by sonication
sonication  (sCS‐NCs)  (B), and  non‐sonicated  nanocapsules  obtained  by  stirring (nsCS‐NC) (C)  are 
(sCS-NCs) (B), and non-sonicated nanocapsules obtained by stirring (nsCS-NC) (C) are reported
reported and compared. 
and compared.

 
Figure 2. Dynamic Light Scattering (DLS) measures reported as percentages of frequency counts at
Figure 2. Dynamic Light Scattering (DLS) measures reported as percentages of frequency counts at 
each hydrodynamic diameter: nanoemulsion before chitosan coating (A); nanocapsules obtained by
each hydrodynamic diameter: nanoemulsion before chitosan coating (A); nanocapsules obtained by 
sonication (sCS-NCs) (B); and non-sonicated nanocapsules obtained by stirring (nsCS-NC) (C).
sonication (sCS‐NCs) (B); and non‐sonicated nanocapsules obtained by stirring (nsCS‐NC) (C). 

Hydrodynamic diameter of chitosan–coated nanocapsules always increases with respect to

Hydrodynamic diameter of chitosan–coated nanocapsules always increases with respect to the 
the nanoemulsion template (Figure 2A). Nevertheless, in the case of sonicated capsules the final
nanoemulsion  template  (Figure  2A).  Nevertheless,  in  the  case  of  sonicated  capsules  the  final 
hydrodynamic diameter is much higher than the non-sonicated sample (Figure 2B). The value is also
hydrodynamic diameter is much higher than the non‐sonicated sample (Figure 2B). The value is also 
higher than the diameter distribution obtained from SEM images referring to sonicated nanocapsules.
higher than the diameter distribution obtained from SEM images referring to sonicated nanocapsules. 
Moreover, the presence of a significant percentage of big aggregates is observed in the sCS-NC.
Moreover, the presence of a significant percentage of big aggregates is observed in the sCS‐NC. The 
The increase in the diameter observed can be attributed to aggregation phenomena, indicating a lower
increase in the diameter observed can be attributed to aggregation phenomena, indicating a lower 
stability of this sample in water suspension. In the case of non-sonicated samples, a certain amount
stability of this sample in water suspension. In the case of non‐sonicated samples, a certain amount 
of aggregates are detected too, even though such aggregates have a smaller diameter than the ones
of aggregates are detected too, even though such aggregates have a smaller diameter than the ones 
obtained by sonication. In any case, the presence of a low percentage (i.e., 5%–10%) of aggregates
obtained by sonication. In any case, the presence of a low percentage (i.e., 5%–10%) of aggregates 
could be considered acceptable for future purposes. The hydrodynamic diameter of the nanocapsules
could be considered acceptable for future purposes. The hydrodynamic diameter of the nanocapsules 
stored in water suspension has been found to be reproducible over at least two months, indicating the
stored in water suspension has been found to be reproducible over at least two months, indicating 
suitability of chitosan to successfully stabilize the nanoemulsion.
the suitability of chitosan to successfully stabilize the nanoemulsion. 
It is supposed that the hydrogel formation treatment affects the properties of the polymer-coated
It is supposed that the hydrogel formation treatment affects the properties of the polymer‐coated 
surface of the nanocapsules depending on the degree of the interaction that could be established
surface  of  the  nanocapsules  depending  on  the  degree  of  the  interaction  that  could  be  established 
between –NH and –OH on polymer chains and Na2 SO4 molecules. Moreover, it should be taken
between –NH22 and –OH on polymer chains and Na2SO 4 molecules. Moreover, it should be taken into 
into account that the presence of salts can also promote hydrophobic interactions between polymer
account that the presence of salts can also promote hydrophobic interactions between polymer chains 
chains themselves. As a consequence of the establishment of such hydrophobic interactions, the
themselves. As a consequence of the establishment of such hydrophobic interactions, the exposure 
exposure amino groups on the surface of the nanocapsule would be favored. To evaluate how the
amino groups on the surface of the nanocapsule would be favored. To evaluate how the sonication 
sonication during hydrogel formation can improve the interactions and so affect the nanocapsule
during  hydrogel  formation  can  improve  the  interactions  and  so  affect  the  nanocapsule  surface 
surface properties, sonicated and non-sonicated nanocapsules have been compared in terms of surface
properties,  sonicated  and  non‐sonicated  nanocapsules  have  been  compared  in  terms  of  surface 
potential. In particular, analysis of Z-potential and amino group spectrophotometric determination
potential. In particular, analysis of Z‐potential and amino group spectrophotometric determination 
analysis are reported. Both capsules showed a positive potential when measured in a 10 mM KCl
analysis are reported. Both capsules showed a positive potential when measured in a 10 mM KCl 
solution, being slightly more positive the surface of sCS-NC (+21.1 mV) than the surface of nsCS-NC
solution, being slightly more positive the surface of sCS‐NC (+21.1 mV) than the surface of nsCS‐NC 
(+13.8 mV). The observed variation in the surface potential depending on the sonication treatment
(+13.8 mV). The observed variation in the surface potential depending on the sonication treatment 
could be explained in this case by the presence of a higher number of amino groups exposed on
could be explained in this case by the presence of a higher number of amino groups exposed on the 
the outer surface of chitosan shell in the case of sCS-NC. This hypothesis was confirmed by the
outer  surface  of  chitosan  shell  in  the  case  of  sCS‐NC.  This  hypothesis  was  confirmed  by  the 
quantification of amino groups of the chitosan shell of nanocapsules by the spectrophotometric method
quantification  of  amino  groups  of  the  chitosan  shell  of  nanocapsules  by  the  spectrophotometric 
method of Orange II dye, previously reported and already optimized for inorganic nanoparticles [39]. 
Briefly,  the  method  is  based  on  a  pH‐dependent  interaction  between  positively  charged  amino 
groups and –SO3− group of Orange II dye. This spectrophotometric method is simple, inexpensive, 
and easy, and its most important advantage over other methods for amino quantification consists in 
Mar. Drugs 2016, 14, 175 5 of 15

of Orange II dye, previously reported and already optimized for inorganic nanoparticles [39]. Briefly,
the method is based on a pH-dependent interaction between positively charged amino groups and
–SO3− group of Orange II dye. This spectrophotometric method is simple, inexpensive, and easy, and
its most important advantage over other methods for amino quantification consists in the use of a
molecule with low steric hindrance—an especially important aspect for porous materials materials.
Moreover, the relationship between amino groups and reactant is in this case of 1:1, allowing a direct
and reliable quantification [40]. In the present work, the method has been slightly modified to use
syringe filters as support for the separation of nanocapsules from the solution during all the washing
steps. Results from the spectrophotometric assay for the measurement of amino groups through the
interaction with Orange II dye also proved the presence of a high number of positively charged amino
groups in the case of sCS-NC (0.35 µmol·mg−1 ) while a value of amino groups of only 0.2 µmol·mg−1
was obtained in the case of nsCS-NC.
It should be taken into account that results from Orange II interaction only represented the amount
of amino groups available for the interaction with dye molecules and that the amount of these groups
could be lower than the total amount of –NH2 of the nanocapsules. Data from the Orange II assay
were in good agreement with Z potential analysis. sCS-NC presented a number of moles mg−1 of
–NH2 groups almost double with respect to the moles mg−1 of –NH2 of nsCS-NC.
Thermo Gravimetric Analysis (TGA) of chitosan-based nanocapsules sonicated or not during
the synthesis process is reported in Figure S3 of the Supplementary Materials. The weight loss
corresponding to chitosan shell can be appreciated at ~200 ◦ C and it corresponded to 50% weight of the
sCS-NC. Surprisingly in the case of nsCS-NC this percentage is even higher, where chitosan represents
65% of the total weight of the sample. This evidence demonstrated that the observed decrease of
amino groups on the surface of non-sonicated nanocapsules was not due to a decrease in the chitosan
total amount. Consequently, it is reasonable to suppose that the organization of chitosan layer and
interactions between polymer chains themselves and with the surface of the nanoemulsion template
are slightly different depending on the condition applied during the synthesis process.

2.2. Grafting of the Surface of Chitosan-Coated Nanocapsule

PEG coating has been chosen in this work for the further optimization of the carrier. As previously
stated in the introduction and well documented in the literature, the prevention of unspecific
adsorption of serum proteins on carrier surface represents a very important goal to allow a prolonged
circulation time in the blood stream. From this point of view, pegylation is reported to be one of the
most successful strategies [33–35].
Nanocapsules obtained without sonication during the synthesis have been selected for the process
of grafting of the surface due to their better stability in water suspension. The surface was grafted with
α-methoxy-ω-amino poly (ethylene glycol) (5000 Da) (aminated PEG) through a previous reaction
with the homobifunctional linker bis (sulfosuccinimidyl) suberate (BS3 ) (Scheme 1). As the colloidal
stability of nanocapsules strongly depends on their surface properties, which in turn greatly affect
nanocapsule cytotoxicity in animal cells, the possibility of obtaining nanocapsule surface with a
low-density coverage of PEG molecules (5 nmol/mg initially added) (ldCS-NC) and a high-density
coverage (100 nmol/mg initially added) (hdCS-NC) was explored. Consecutively, the final properties
of the obtained materials were studied. A schematical representation of the strategy used for grafting
is reported in Scheme 1.
The determination of the accessible amino groups was fundamental for the optimization of the
grafting protocol. In fact, the method utilized here implied the use of a homobifunctional linker
in which the main drawback lays in the possibility of crosslinking of amino groups from different
nanocapsules and production of aggregates if the amount of reactants is not strictly controlled. For this
reason, the value of accessible amino groups has been used as the starting point to adjust the amount
of reactants in the following steps of the process. In particular, the amount of BS3 has been always
maintained below the total moles of functional groups of nanocapsules to reduce the possibility of
 process of grafting of the surface due to their better stability in water suspension. The surface was 
grafted  with  α‐methoxy‐ω‐amino  poly  (ethylene  glycol)  (5000  Da)  (aminated  PEG)  through  a 
previous reaction with the homobifunctional linker bis (sulfosuccinimidyl) suberate (BS3) (Scheme 1). 
As the colloidal stability of nanocapsules strongly depends on their surface properties, which in turn 
Mar. Drugsaffect 
greatly  2016, 14,nanocapsule 
175 6 of 15
cytotoxicity  in  animal  cells,  the  possibility  of  obtaining  nanocapsule 
surface with a low‐density coverage of PEG molecules (5 nmol/mg initially added) (ldCS‐NC) and a 
high‐density coverage (100 nmol/mg initially added) (hdCS‐NC) was explored. Consecutively, the 
crosslinking between capsules. To complete the process, the amount of aminated PEG exceeded twice
final properties of the obtained materials were studied. A schematical representation of the strategy 
the used amount of BS3 in all the experiments to assure the reaction with all the reactive groups of BS3
used for grafting is reported in Scheme 1. 
on the surface.

 
Scheme 1. Chitosan‐coated nanocapsule grafted with aminated polyethylene glycol (PEG) through 
Scheme 1. Chitosan-coated nanocapsule grafted with aminated polyethylene glycol (PEG) through
BS33 linking. 
BS linking.

The presence of PEG on nanocapsule surface has been evaluated by Fourier Transform Infrared
Spectroscopy (FTIR) analysis (data reported in Supplementary Materials). The properties of
chitosan-coated nanocapsules before and after the grafting have been compared and the FTIR
spectra of all the single components of nanoemulsion-based nanocapsules are reported respectively in
Figures S4 and S5 of the Supplementary Materials.
In the FTIR spectrum of nsCS-NC (Figure S4A), it is possible to recognize the presence of the
four starting components, meaning that the final composition is compatible with the hypothesized
structure of the nanocapsule (Figure 1A). Moreover, reported in Figure S6 of the Supplementary
Materials a comparison of sCS-NCs and nsCS-NC demonstrates that the sonication process is not
affecting the chemical interaction between nanoemulsion template and chitosan shell.
The grafting process consists of three steps. The first one is the incubation with the linker to
provide the surface with the sulfosuccinimidyl ester group sensitive to the linking of PEG. During the
second step the ligand is added and finally the grafted nanocapsules are incubated with Tris-HCl buffer
to quench the free sulfosuccinimidyl ester groups eventually not linked to any PEG molecule. This last
step is fundamental to avoid the crosslinking between nanocapsules due to the reaction between free
sulfosuccinimidyl ester groups and amino group on a different capsule.
To determine if the grafting with PEG successfully occurred, the FTIR spectrum of the intermediate
step of nanocapsules after the incubation with BS3 is also reported (Figure S4B). It should be noted
that at this intermediate step the sample is incubated with Tris-HCl buffer after the linking of BS3 to
avoid the crosslinking with amino groups on other nanocapsule surface through the reactive groups
still available for ligand reaction. In the case of samples incubated with ligand in the second step,
Tris-HCl was added at the end of the process (after incubation with the ligand) but in this case it can be
supposed that BS3 , which should have reacted previously with PEG, is not available to react with Tris.
This hypothesis was confirmed by comparing FTIR spectra of nanocapsules after the incubation with
BS3 (Figure S4B) and spectra in Figure S4C,D referring to nanocapsules grafted with a low density and
high density of PEG, respectively. Peaks at 3180 and 3100 cm−1 , as well as peaks at 1630 and 1550 cm−1
referring to Tris are present in the intermediate step and (in ldCS-NC with lower intensities) but they
completely disappeared in hdCS-NC spectrum.
Differences in the spectra could also be appreciated, comparing peaks and intensities ratios
between 1150 and 1030 cm−1 . In this region the C-O-C peak (1105 cm−1 ) presents an increased
intensity, compared to other peaks in the same region, especially in the hdCS-NC spectrum. Moreover,
in this sample peak at 1055 cm−1 is lost. This peak is supposed to refer to R-SO3− group that is released
from BS3 in the linking reaction. In the BS3 intermediate step the peak is still appreciable and it can
be observed as a low intensity peak in ldCS-NCs too. It is possible that in these samples not all BS3
reactive groups are quenched after Tris-HCl incubation and that some of them are still present on
Mar. Drugs 2016, 14, 175 7 of 15

the surface. In any case this state did not represent a problem for the quality of the sample since any
appreciable crosslinking and consequent aggregation are observed.
The successful grafting on nanocapsule surface was proved also by measuring the Z potential
of the material surface before and after the modification with PEG. Using both a low amount and
Mar. Drugs 2016, 14, 175  7 of 14 
high amount of PEG, a decrease in the potential was observed, indicating a decrease in the free amino
groups A on the surface and
confirmation  an increase
of  this  evidence  in the totalsmall 
is  the  electronegativity
difference  in of the 
the surface.
decrease  of  amino  groups 
A confirmation of this evidence is the small difference in the decrease of amino groups (measured
(measured by Orange II spectrophotometric assay) registered between low‐density and high‐density 
by Orange II spectrophotometric
PEG‐covered  nanocapsules.  The  assay) registered
measured  between
amounts  of low-density
amino  groups  andare 
high-density PEG-covered
0.1  and  0.08  μmol/mg 
nanocapsules. The measured amounts of amino groups are 0.1 and
respectively,  corresponding  to  50%  and  40%  of  the  amounts  of  amino  groups  on  0.08 µmol/mg respectively,
ungrafted 
corresponding to 50% and 40% of the amounts of amino groups on ungrafted nanocapsules.
nanocapsules. These values are considered as the apparent disappearing of amino groups probably 
These values
due  to  are considered
a  screening  effect as thethe 
for  apparent
presence disappearing of amino groups
of  PEG  molecules  on  the probably
surface. due to a screening
Orange  molecule 
effect for the presence of PEG molecules on the surface. Orange molecule
interaction with –NH3+ groups would be hampered by the presence of the polymer chains since, in  interaction with –NH3 +
groups would
the  case  be hampered
of  low‐density  by the
PEG  presence
coverage,  it  of the
can  be polymer
supposed  chains
that since, in the
polymer  case of low-density
molecules  stand  in  a 
PEG coverage, it can be supposed that polymer molecules stand in a conformation that lays around
conformation that lays around nanocapsule surface, probably thanks to a possible hydrogen bond 
nanocapsule surface, probably thanks to a possible hydrogen bond interaction of the PEG chain with
interaction of the PEG chain with positively charged amino groups on the nanocapsule surface (see 
positively charged amino groups on the nanocapsule surface (see Scheme 1). The tendency to form
Scheme 1). The tendency to form such interactions could also be responsible for a lower than expected 
such interactions could also be responsible for a lower than expected efficiency of linking in the case of
efficiency of linking in the case of high‐density coverage sample. 
high-density coverage sample.
The effect of the grafting on the behavior of nanocapsules in physiological medium was further 
The effect of the grafting on the behavior of nanocapsules in physiological medium was further
evaluated by measuring the degree of aggregation of grafted and not‐grafted nanocapsules in water 
evaluated by measuring
and  phosphate  the degree
saline  buffer  (PBS) of aggregation
by  of grafted and diameter 
means  of  hydrodynamic  not-grafted nanocapsulesUngrafted 
measurement.  in water
and phosphate saline buffer (PBS) by means of hydrodynamic diameter measurement. Ungrafted
nanocapsules are sensitive to the presence of salts in the medium and they tend to aggregate during 
nanocapsules are sensitive to the presence of salts in the medium and they tend to aggregate during
incubation in physiological media like PBS. The grafting with PEG chains is a common strategy to 
incubation in physiological media like PBS. The grafting with PEG chains is a common strategy to
improve the stability of nanoparticles in physiological and in vitro culture media [33]. Both phosphate 
improve theproteins 
ions  and  stabilitycan 
of nanoparticles
adsorb  onto innanocapsule 
physiologicalsurface 
and in vitro culture
due  to  media [33].
the  presence  of Both phosphate
amino  groups, 
ions and proteins can adsorb onto nanocapsule surface due to the presence of
producing the crosslinking between different capsules. Moreover, the presence of salts can produce amino groups, producing
the crosslinking
aggregation  between
due  different capsules.
to  a  salting‐out‐like  Moreover,
effect.  the presence
The  presence  of  PEG ofon 
salts can produce
nanocapsule  aggregation
surface  would 
due to a salting-out-like effect. The presence of PEG on nanocapsule surface would screen the amino
screen the amino groups on the surface from interaction with salts in the medium. All samples were 
groups on the
measured  in surface fromPBS 
water  and  interaction with
and  their  salts in the
diameters  medium.
were  All samples
compared  were
to  assess  measured
the  effect  of inPEG 
wateron 
and PBS and their diameters were
prevention of aggregation (Figure 3).  compared to assess the effect of PEG on prevention of aggregation
(Figure 3).

 
Figure 3. Hydrodynamic diameters of chitosan-coated nanocapsules before (A) and after grafting with
Figure  3.  Hydrodynamic  diameters  of  chitosan‐coated  nanocapsules  before  (A)  and  after  grafting 
different amounts of PEG ((B) and (C), respectively referring to low-density coverage (ldCS-NC) and
with different amounts of PEG ((B) and (C), respectively referring to low‐density coverage (ldCS‐NC) 
high-density coverage (hdCS-NC)).
and high‐density coverage (hdCS‐NC)). 

In
In Figure
Figure 3A,
3A, aa strong
strong aggregation
aggregation effect
effect inin PBS
PBS isis reported
reported for
for ungrafted
ungrafted chitosan-based
chitosan‐based 
nanocapsules. The mean hydrodynamic diameter changed from 103 nm in water to 471 nm in
nanocapsules. The mean hydrodynamic diameter changed from 103 nm in water to 471 nm in PBS. 
PBS. On the contrary, the diameter and the PDI of grafted nanocapsules are maintained in PBS, even in
On the contrary, the diameter and the PDI of grafted nanocapsules are maintained in PBS, even in 
the case of ldCS-NCs, confirming that the grafting successfully occurred in both cases and that it was
the case of ldCS‐NCs, confirming that the grafting successfully occurred in both cases and that it was 
effective for nanocapsule stabilization.
effective for nanocapsule stabilization. 
To  further  demonstrate  the  stabilizing  effect  of  PEG  grafting  on  nanocapsule  surface,  an 
aggregation  test  has  been  carried  out  by  measuring  the  hydrodynamic  diameter  of  grafted  and 
ungrafted nanocapsules in the presence of increasing concentrations of Bovine Serum Albumin (BSA). 
As in the case of the above reported stability assay in presence of PBS, the high density of amino 
groups  on  nanocapsule  surface  can  be  considered  responsible  for  the  adsorption  of  proteins  and 
aggregation observed.   
The comparison of the behaviors of ungrafted nanocapsules, ldPEG‐CS NC and hdPEG‐CS NC, 
 Mar. Drugs 2016, 14, 175 8 of 15

To further demonstrate the stabilizing effect of PEG grafting on nanocapsule surface,

an aggregation test has been carried out by measuring the hydrodynamic diameter of grafted and
ungrafted nanocapsules in the presence of increasing concentrations of Bovine Serum Albumin (BSA).
As in the case of the above reported stability assay in presence of PBS, the high density of amino
groups on nanocapsule surface can be considered responsible for the adsorption of proteins and
aggregation observed.
The comparison of the behaviors of ungrafted nanocapsules, ldPEG-CS NC and hdPEG-CS NC,
Mar. Drugs 2016, 14, 175  8 of 14 
has been reported in Figure 4.

 
Figure 4. Hydrodynamic diameters of chitosan-coated nanocapsules before and after grafting
Figure 4. Hydrodynamic diameters of chitosan‐coated nanocapsules before and after grafting with 
with different amounts of PEG measured in presence of different concentrations of Bovine Serum
different amounts of PEG measured in presence of different concentrations of Bovine Serum Albumin 
Albumin (BSA).
(BSA). 

Data reported in the graph demonstrated without any doubt the strong stabilizing effect of PEG 
Data reported in the graph demonstrated without any doubt the strong stabilizing effect of PEG
on nanocapsules in the presence of proteins. The grafting with PEG, and so the strong decrease of the 
on nanocapsules in the presence of proteins. The grafting with PEG, and so the strong decrease of the
free amino groups on the surface lead to a very good stability of the nanocapsules in a protein-rich
free amino groups on the surface lead to a very good stability of the nanocapsules in a protein‐rich 
medium. Both ldPEG-CS NC and hdPEG-CS NC showed a significant increase in the hydrodynamic
medium. Both ldPEG‐CS NC and hdPEG‐CS NC showed a significant increase in the hydrodynamic 
diameter only at very high concentrations of proteins (higher than 0.1 mg/mL). Comparatively, the
diameter only at very high concentrations of proteins (higher than 0.1 mg/mL). Comparatively, the 
concentration of BSA one order of magnitude lower is enough to produce the same increase in the
concentration of BSA one order of magnitude lower is enough to produce the same increase in the 
diameter in the case of ungrafted NCs.
diameter in the case of ungrafted NCs. 
2.3. Cell Viabily and Internalization Assays
2.3. Cell Viabily and Internalization Assays 
The cytotoxicity of chitosan-based nanocapsules before and after the grafting was tested on
The cytotoxicity of chitosan‐based nanocapsules before and after the grafting was tested on Vero 
Vero cells through MTT spectrophotometric assay. Cells were incubated for 24 h with different
cells  through  MTT 
concentrations spectrophotometric 
of nsCS-NC, assay.  nanocapsules
selected as ungrafted Cells  were  incubated  for  24 ones
over the sonicated h  with 
due todifferent 
their
concentrations of nsCS‐NC, selected as ungrafted nanocapsules over the sonicated ones due to their 
better stability in water, and with the same concentrations of ldCS-NC and hdCS-NC. In Figure 5,
better stability in water, and with the same concentrations of ldCS‐NC and hdCS‐NC. In Figure 5, the 
the comparison between the grafted nanocapsules and the ungrafted ones is reported in terms of
comparison 
percentage between  the 
of viability grafted 
of cell nanocapsules 
culture and  the  ungrafted  ones  is  reported  in  terms  of 
after 24 h incubation.
percentage of viability of cell culture after 24 h incubation. 
It can be observed from Figure 5 that the grafting significantly improved the safety of the
nanocapsules, especially when using high concentrations of nanocapsules (greater than 0.15 mg/mL).
Both types of grafted nanocapsules were less toxic to the cells, especially the high density ones
(at high concentrations).
Using an inverted microscope, it can be observed that non-sonicated nanocapsules lead to bigger
vesicles inside cells than both types of grafted nanocapsules (Figure 6), which could be related with
the higher toxicity).
 The cytotoxicity of chitosan‐based nanocapsules before and after the grafting was tested on Vero 
cells  through  MTT  spectrophotometric  assay.  Cells  were  incubated  for  24  h  with  different 
concentrations of nsCS‐NC, selected as ungrafted nanocapsules over the sonicated ones due to their 
better stability in water, and with the same concentrations of ldCS‐NC and hdCS‐NC. In Figure 5, the 
comparison  between  the  grafted  nanocapsules  and  the  ungrafted  ones  is  reported  in  terms 
Mar. Drugs 2016, 14, 175
of 
9 of 15
percentage of viability of cell culture after 24 h incubation. 

Mar. Drugs 2016, 14, 175  9 of 14 

 
Using an inverted microscope, it can be observed that non‐sonicated nanocapsules lead to bigger 
Figure 
Figure 5. 
5. Viability 
Viability test 
test with 
with chitosan‐coated 
chitosan-coated nanocapsules 
nanocapsules before 
before and 
and after 
after grafting 
grafting with 
with low 
low and 
and
vesicles inside cells than both types of grafted nanocapsules (Figure 6), which could be related with 
high‐density coverage of PEG. 
high-density coverage of PEG.
the higher toxicity). 
It  can  be  observed  from  Figure  5  that  the  grafting  significantly  improved  the  safety  of  the 
nanocapsules, especially when using high concentrations of nanocapsules (greater than 0.15 mg/mL). 
Both types of grafted nanocapsules were less toxic to the cells, especially the high density ones (at 
high concentrations). 

 
Figure 6. Inverted microscope images from (A) control cells; and cells incubated for 24 h with (B) non‐
Figure 6. Inverted microscope images from (A) control cells; and cells incubated for 24 h with
sonicated nanocapsules; (C) ldCS‐NC; and (D) hdCS‐NC. Scale bars correspond to 20 μm. 
(B) non-sonicated nanocapsules; (C) ldCS-NC; and (D) hdCS-NC. Scale bars correspond to 20 µm.

The test proved not only that aminated PEG was definitively linked to nanocapsule surface, but 
The test proved not only that aminated PEG was definitively linked to nanocapsule surface,
also that the conformation of polymer chains obtained using the developed method of grafting for 
but also that the conformation of polymer chains obtained using the developed method of grafting
ldCS‐NC  hdCS‐NC  is  effective  for  significantly  improving  the  cytotoxicity  of  chitosan‐based 
for ldCS-NC hdCS-NC is effective for significantly improving the cytotoxicity of chitosan-based
nanocapsules. Moreover, it should be noted that in the case of PEG‐grafted nanocapsules the decrease 
nanocapsules. Moreover, it should be noted that in the case of PEG-grafted nanocapsules the decrease
in the cytotoxicity is still associated with a very high degree of internalization, indicating that the 
in the cytotoxicity is still associated with a very high degree of internalization, indicating that the
developed material can be considered a very effective carrier for drug delivery applications. 
developed material can be considered a very effective carrier for drug delivery applications.
3. Discussion 
3. Discussion
Among all
Among all  kinds 
kinds of  nanocarriers, 
of nanocarriers, nanocapsules 
nanocapsules are  frequently 
are frequently the  of
the material material  of biomedical
choice for choice  for 
biomedical applications since they offer the advantage of providing a good stability of encapsulated 
applications since they offer the advantage of providing a good stability of encapsulated drugs and
adrugs and a favorable pharmacokinetic. The aim of the work was to develop nanocapsules to be used 
favorable pharmacokinetic. The aim of the work was to develop nanocapsules to be used in
in the future as potential drug reservoir and whose composition could allow maximum flexibility of 
the future as potential drug reservoir and whose composition could allow maximum flexibility of
application  together  with  great  feasibility  for  the  administration  by  different  routes  (intravenous 
injection, oral administration, and inhalation). 
In this work, chitosan—a biocompatible polymer—has been used to obtain a potentially smart 
nanocarrier whose surface properties could be properly tuned depending on the desired application. 
A nanoemulsion‐based nanocapsule has been used as template to be coated with a chitosan shell. 
Mar. Drugs 2016, 14, 175 10 of 15

application together with great feasibility for the administration by different routes (intravenous
injection, oral administration, and inhalation).
In this work, chitosan—a biocompatible polymer—has been used to obtain a potentially smart
nanocarrier whose surface properties could be properly tuned depending on the desired application.
A nanoemulsion-based nanocapsule has been used as template to be coated with a chitosan shell.
The process of chitosan hydrogel coating of a nanoemulsion template was studied in terms of
exposure of amino groups on the surface depending on the interaction produced during the sonication
process. It could be hypothesized that the sonication promoted interactions and produced stronger
hydrophobic interaction between polymer chains, leading to a condensation of the structure that would
lead to a higher exposure of amino groups and so to a higher availability for the interaction with the
dye. On the other hand, it was also supposed that a different amount of polymer could be incorporated
on the surface of the nanoemulsion template when sonication is used during the synthesis, leading to
a different total amount of glucosamine units (and so amines) in the shell. The characterization of the
material and the comparison between the sonicated and not sonicated one alloowed finally to confirm
that the sonication process lead to a higher amount of amino groups exposed on nanocapsule surface.
Chitosan shell offers the possibility of an easy functionalization through amino groups on its
surface so chitosan-based nanocapsules have been further coated with PEG since this is known to be
a very effective strategy to stabilize surfaces in biologically relevant media. The possibility to tune
the surface properties by varying the PEG density coverage has been explored and results obtained
are compatible with a conformation of PEG molecules laying adsorbed on nanoparticle surface after
covalent linking through their aminated terminal.
It is reported in literature that the attempt to produce high-density coverage of surfaces with
PEG can result in a loss of efficiency of grafting. In fact, similarly to what reported for the grafting
of other surfaces, it is reasonable to suppose that not all PEG molecules react immediately with BS3
linker on nanocapsule surface. Once they are linked, their tendency is to assume a mushroom-like
conformation until the density of PEG molecules on the surface does not reach a value high enough
to produce a change into a brush-like conformation [41]. In the case of high-density PEG-covered
nanocapsules, the decrease in amino groups on the surface was found to be not proportional to the
reactants, differently to what happened in the case of low-density ones, indicating that eventually a
conformation of PEG molecules laying adsorbed on nanoparticle surface was hampering the grafting
of masked amino groups.
Despite the fact that the obtained grafting degree was slightly lower than the one expected,
a highly improved stability in physiological medium (resistance to salting out effect) was observed
with both low and high PEG-covered nanocapsules (although at different degrees) indicating that the
presence of PEG on the surface will allow the use of the grafted capsules in physiological media for
biomedical applications.
We also demonstrated that the presence of PEG on chitosan surface of nanocapsules was effective
in avoiding the unspecific adsorption of proteins. This issue is of the utmost importance as, following
intravenous injection, non-passivated nanoparticles tend to adsorb plasma proteins (protein corona),
changing the charge and size of them. This binding of plasma components will be responsible of the
final fate of the nanocapsules, and can greatly influence the biodistribution and therapeutic efficacy [42].
To date, PEG is the most widely used polymer to prevent this protein corona formation, although the
final effect depends on different parameters such as the molecular weight or grafting density [43].
Following the encouraging results obtained in physiological media and protein-rich media,
preliminary tests with cell cultures have been carried out showing very interesting results.
The obtained coating demonstrated effectiveness in tuning the cytotoxicity of chitosan-coated
nanocapsules. Moreover, the nanocapsules showed a high degree of internalization in Vero cells,
a useful property for the potential application as drug reservoirs directed to cytoplasm release. Finally,
the tunability of all the properties of the synthesized chitosan-based nanocapsules was obtained by
Mar. Drugs 2016, 14, 175 11 of 15

a very easy and reproducible chemical grafting, indicating that the developed nanocarrier was very
promising for further studies oriented toward drug encapsulation for biomedical application.

4. Materials and Methods

Tween® 20 (Croda International PLC, Cowick Hall Snaith, Goole, East Yorkshire, UK), absolute
ethanol, sodium sulfate anhydrous (99%–100.5%), and sodium chloride (99%), were purchased
from Panreac. Span® 85 (sorbitanetrioleate) (Croda International PLC, Cowick Hall Snaith, Goole,
East Yorkshire, UK), oleic acid (90%), chitosan (medium molecular weight), Orange II sodium salt,
and Bovine Serum Albumine (BSA) were obtained from Sigma-Aldrich. Bis(sulfosuccinimidyl)
suberate (BS3) was purchased from Pierce Biotechnology and α-methoxy-ω-amino poly(ethylene
glycol) (PEG-MW 5000 Dalton) from IRIS Biotech GmbH. Water (double processed tissue culture) used
in all nanocapsule synthesis was from Sigma. Millipore Biomax 300 kDa Ultrafiltration Discs were
purchased from Merck Millipore.
Vero cells (monkey kidney epithelial cells) were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA, number CCL-81). Dulbecco’s modified Eagle’s medium
(DMEM), Phosphate-Buffered Saline (PBS), Dulbecco’s Phosphate-Buffered Saline (DPBS) were
purchased from Lonza. MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide), penicillin,
streptomycin, glutamine solutions, and 40 ,6-diamidino-2-fenilindolphenylindol (DAPI) were purchased
from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA).
For the preparation of chitosan-based nanocapsules an organic solution containing 400 mg oleic
acid and 86 mg Span® 85 in 40 mL of absolute ethanol was added to the aqueous one, containing
136 mg Tween® 20 solved in 80 mL water, under magnetic stirring during 15 min for the formation of
the nanoemulsion. Then 25 mg from a 5 mg/mL chitosan solution in acetic acid 1% (v/v) were added
and again the mixture was left under stirring 15 min. Finally, the chitosan-coated nanoemulsion
was added to 200 mL of 50 mM Na2 SO4 under sonication (or stirring in the case of optimized
nanocapsules). Capsules were separated from Na2 SO4 through ultracentrifugation (30 min, 69673 G,
10 ◦ C), washed with 100 mL of water, centrifuged again, and resuspended in water. The concentration
of the nanocapsules in water suspension was obtained by measuring the weight of 1 mL of sample
after freeze-drying.
For the grafting of nanocapsule surface suspensions, 20 mg of nanocapsules at a concentration
of 2 mg/mL in borate buffer 10 mM pH 8.3 were added with different amounts of the linker
bis(sulfosuccinimidyl) suberate (BS3) (20–100 nmol/mgNC) and they were kept under stirring for
30 min. Then a double amount of α-methoxy-ω-amino poly (ethylene glycol) (MeO-PEG-NH2 ) was
added and the mixture was kept under stirring for 2 h at 37 ◦ C. Finally, 20 mL of Tris-HCl buffer 10 mM
pH 8.0 was added to quench the linker that eventually did not react with PEG. Grafted nanocapsules
were filtered using an Amicon Ultrafiltration unit using Millipore Biomax 300 kDa Ultrafiltration Discs
to separate them from unreacted PEG. After a washing with fresh water nanocapsules, they were
concentrated to a final volume of 2 mL.
Several techniques have been used for the characterization of chitosan nanocapsules.
DLS analysis has been carried out using a Brookhaven 90Plus DLS instrument, by means of
the Photo-Correlation Spectroscopy (PCS) technique. Nanoparticle hydrodynamic diameter and
polydispersity index (PDI) have been measured in water at the concentration of 0.05 mg/mL.
Electrophoretic mobility (Z Potential) of nanoparticles at different pH values has been determined
by measuring the potential of a 0.05 mg/mL nanoparticle suspension in 10 mM KCl with a Plus Particle
Size Analyzer (Brookhaven Instruments Corporation).
Nanocapsule composition was analyzed by Fourier Transform Infrared Spectroscopy analysis in
a JASCO FT/IR—4100 Fourier transform infrared spectrometer in a frequency range of 600–4000 cm−1
with a resolution of 2 cm−1 and a scanning number of 32.
Thermogravimetric analysis was performed in a TASTD 2960 thermogravimetric analyzer,
by heating the sample at 10 ◦ C/min under air atmosphere.
Mar. Drugs 2016, 14, 175 12 of 15

Environmental Scanning Electron Microscopy (ESEM) images were obtained using a

QUANTA-FEG 250 microscope in Scanning Transmission Electron Microscopy (STEM) mode.
Bright Field Transmission Electron Microscopy (BF-TEM) analysis was carried out in a FEI Tecnai
T20 microscope operating at 200 kV. Due to the sensitive nature of the sample, previous fixation,
dehydration, and epoxy resin embedding were necessary for both techniques. This process can be
briefly achieved as follows. A fresh sample was synthesized and it was fixed with glutaraldehyde
0.25% in phosphate buffer 10 mM at pH 7.4 for 2 h. It was washed three times with buffer and
incubated with 1% osmium tetroxide in PBS for further fixation and staining. Finally, the sample was
accurately washed with water and resuspended in 5% gelatin. The sample was centrifuged to obtain a
pellet and was incubated overnight at 4 ◦ C. The obtained solid sample was cut in very small pieces
before undergoing the subsequent steps. The dehydration of the samples was carried out using the
following steps: incubation in ethanol 30%, ethanol 50%, and incubation overnight in ethanol 70%;
incubation in ethanol 90% for 1 h; and finally incubation three times in absolute ethanol. After that,
samples were incubated overnight in a 1:1 mixture absolute ethanol/epoxy resin (r.t.). The mixture
was then removed, changed with absolute epoxy resin and samples were left for impregnation for 8 h
at room temperature. After another change of the medium, the final incubation in epoxy resin was
carried out overnight at 60 ◦ C to obtain the polymerization.
From different ESEM images (Figures S1 and S2 of Supplementary Materials), an estimation of
the diameter distribution has been obtained using Digital Micrograph® (Gatan Inc., Pleasanton, TX,
USA) and OriginLab® (OriginLab, Northampton, MA, USA) softwares to measure the diameters of
more than 100 nanocapsules and for the frequency count statistical analysis respectively.
Nanocapsule amino content was measured by the Orange II spectrophotometric assay [34,35].
0.2 mg of nanocapsules were put in contact with 1 mL of 2 mM Orange II sodium salt acidic solution
(pH 3) and kept under stirring for 30 min at 37 ◦ C. Capsule suspension was passed through a syringe
membrane filter (Millex syringe-driven filter unit, PVDF filter with 0.22 µm pores, purchased from
Merck Millipore) to adsorb nanocapsules in the membrane and keep them retained in order to separate
them from the Orange II solution. After that, an acidic solution (pH 3) was passed several times
through the same filter until all the unbound dye was removed from the nanocapsules (verified by
measuring spectrophotometrically the supernatant content). Then they were washed with an alkaline
solution (pH 12) to desorb the bound dye from the amino groups on nanocapsules. The washing
fractions were collected, the pH was adjusted at 3 and the amount of unadsorbed and desorbed dye
was measured at a wave length of 480 nm with a Varian Cary 50 UV/V is spectrophotometer after
carrying out a calibration curve.
Resistance of nanocapsules to aggregation has been determined by incubating nanocapsules
(3 mL of a 0.15 mg/mL suspension) at different concentrations of albumin from bovine serum (BSA).
The hydrodynamic diameter of the capsules under the incubation conditions was measured after
10 min using a Brookhaven 90Plus DLS instrument.
In vitro cell viability test was carried out to determine the cytotoxicity of nanocapsules
using 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Vero cells
were grown at 37 ◦ C in a 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 µg/mL),
and glutamine (2 mM). 7500 cells were seeded using a standard 96-well plate (five replicates per
sample). After 24 h of incubation in a humidified atmosphere containing 5% CO2 , the medium was
replaced with new medium containing five different concentrations of nanocapsules and a negative
control containing no capsules (non-treated cells). After 24 h of incubation, the medium was replaced
with fresh medium containing MTT dye solution (0.5 mg/mL in DMEM). After 2 h of incubation at
37 ◦ C and 5% CO2 , the medium was removed and the formed crystals were dissolved in 200 µL of
DMSO. The absorbance was read on a ThermoScientificMultiskan GO TM microplate reader at 570 nm.
The relative cell viability (%) related to control cells without nanocapsules was calculated using the
Mar. Drugs 2016, 14, 175 13 of 15

percentage ratio between absorbance of the sample and the absorbance of the control. Experiments
were carried out in triplicate.
To perform optical microscopy analysis, 3 × 104 cells were seeded on glass coverslips in a 24-well
plate at 37 ◦ C. 24 h later, nanocapsules were added at 50 µg/mL in DMEM and incubated for 24 h at
37 ◦ C. Non-internalized nanocapsules were removed, washing with DPBS twice. Cells were fixed with
4% paraformaldehyde for 20 min at 4 ◦ C, washed twice with DPBS, and incubated for 10 min at room
temperature with 40 ,6-diamidino-2-fenilindolphenylindole (DAPI) for nucleus labeling. The coverslips
were mounted on glass microscope slides using ProLong® (Thermo Fisher Scientific Inc., Waltham, MA,
USA) Gold Antifade. Optical microscopy was performed using an inverted microscope (Nikon Eclipse
Ti-E), and images were analyzed using NIS-Elements Advanced Research software.

Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/14/10/175/

s1, Figure S1: ESEM image of nanocapsules, Figure S2: ESEM image of nanocapsules, Figure S3: TGA analysis of
nanocapsules, Figure S4: FTIR analysis of grafted nanocapsules, Figure S5: FTIR analysis of starting compounds,
Figure S6: FTIR analysis of nanocapsules.
Acknowledgments: Authors would like to acknowledge the public funding from Fondo Social de la DGA
(grupos DGA), Ministerio de la Economía y Competitividad del Gobierno de España for the public founding
of ProyectosI+D+i—ProgramaEstatal de Investigación, Desarrollo e InnovaciónOrientada a los Retos de la
Sociedad (project n. SAF2014-54763-C2-2-R), the European Seventh Framework Program (NAREB Project 604237),
LLP/Erasmus fellowship 2013/2014, INA fellowship “Iniciación a la Investigación” 2014 and 2015, and the
European Union’s Horizon 2020 research and innovation program for MCSA Fellowship (Grant Agreement
No. 660228). The authors also acknowledge José Antonio Ainsa and Ainhoa Lucía for the fruitful discussions
as well as Rodrigo Fernandez-Pacheco and Alfonso Ibarra from Advanced Microscopy Laboratories of the
Universidad de Zaragoza and Iñigo Echaniz for their technical support. The costs to publish in open access have
been covered by funds from NAREB Project (see before).
Author Contributions: L.D.M. and J.M.d.l.F. conceived and designed the experiments; L.D.M., M.A., I.S.-S. and
S.G.-E. performed the experiments; L.D.M., M.M., G.S. and J.M.d.l.F. analyzed the data; L.D.M. wrote the paper;
all the authors revised the paper besides contributing to the work performance.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.

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