Inulin-based polymer coated SPIONs as potential drug delivery systems for targeted cancer therapy

Article abstract of DOI:10.1016/j.ejpb.2014.09.008

This paper deal with the synthesis and characterization of PEGylated squalene-grafted-inulin amphiphile capable of self-assembling and self-organizing into nanocarriers once placed in aqueous media. It was exploited as coating agent for obtaining doxorubicin loaded superparamagnetic iron oxide nanoparticles (SPIONs) endowed with stealth like behavior and excellent physicochemical stability. Inulin was firstly modified in the side chain with primary amine groups, followed in turn by conjugation with squalenoyl derivatives through common amidic coupling agents and PEGylation by imine linkage. Polymer coated SPIONs were so obtained by spontaneous self-assembling of inulin copolymer onto magnetite surface involving hydrophobic-hydrophobic interactions between the metallic core and the squalene moieties. The system was characterized in terms of hydrodynamic radius, zeta potential, shape and drug loading capacity. On the whole, the stealth-like shell stabilized the suspension in aqueous media, though allowing the release of the doxorubicin loaded in therapeutic range. The cytotoxicity profile on cancer (HCT116) cell line and in vitro drug uptake were evaluated both with and without an external magnetic field used as targeting agent and uptake promoter, displaying that magnetic targeting implies advantageous therapeutic effects, that is amplified drug uptake and increased anticancer activity throughout the tumor mass.

Full text of DOI:10.1016/j.ejpb.2014.09.008

European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 695–705
Contents lists available at ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics
journal homepage: www.elsevier.com/locate/ejpb
Research paper
Inulin-based polymer coated SPIONs as potential drug delivery systems
for targeted cancer therapy
C. Scialabba a, M. Licciardi a, , N. Mauro a, F. Rocco b, M. Ceruti b, G. Giammona a
⇑
a Laboratory of Biocompatible Polymers, Department of ‘‘Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche’’ (STEBICEF), University of Palermo, Palermo, Italy
b Department of Scienza e Tecnologia del Farmaco, University of Torino, Torino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 June 2014
Accepted in revised form 22 September 2014
Available online 2 October 2014
This paper deal with the synthesis and characterization of PEGylated squalene-grafted-inulin amphiphile
capable of self-assembling and self-organizing into nanocarriers once placed in aqueous media. It was
exploited as coating agent for obtaining doxorubicin loaded superparamagnetic iron oxide nanoparticles
(SPIONs) endowed with stealth like behavior and excellent physicochemical stability. Inulin was firstly
modified in the side chain with primary amine groups, followed in turn by conjugation with squalenoyl
derivatives through common amidic coupling agents and PEGylation by imine linkage. Polymer coated
SPIONs were so obtained by spontaneous self-assembling of inulin copolymer onto magnetite surface
involving hydrophobic–hydrophobic interactions between the metallic core and the squalene moieties.
The system was characterized in terms of hydrodynamic radius, zeta potential, shape and drug loading
capacity. On the whole, the stealth-like shell stabilized the suspension in aqueous media, though allow-
ing the release of the doxorubicin loaded in therapeutic range. The cytotoxicity profile on cancer
(HCT116) cell line and in vitro drug uptake were evaluated both with and without an external magnetic
field used as targeting agent and uptake promoter, displaying that magnetic targeting implies advanta-
geous therapeutic effects, that is amplified drug uptake and increased anticancer activity throughout
the tumor mass.
Keywords:
SPIONs
Inulin copolymer
Doxorubicin
Magnetic targeting
Squalene
Pegylated
Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction
effects. These therapeutic limitations are mainly due to the lack
of selective drug accumulation into the tumor site, thus affecting
Therapeutic approaches used to eradicate tumors involve inva-
sive processes, long term largely ineffective, such as surgical
removal of neoplastic mass [1]. The surgical therapy often needs
further treatments during the follow-up, encompassing chemo-
therapy or radiotherapy [2]. The latter, even though it is the most
localized technique, entails several drawbacks, among which,
repeated treatments and secondary effects (e.g., sterility and side
tumor genesis) [3]. In addition, current chemotherapy fails in many
cases because of the recurrence of metastases or heavy toxic
healthy cells as well [4,5]. The use of nanocarriers, designed to
enhance drugs efficiency, permits to overcome some of these mat-
ters being drugs released into a specific body district. Several sys-
tems have been developed so as to act as drug delivery systems
useful for targeted therapy, such as polymeric micelles [6–8] lipo-
somes [9], polymersomes [10], polymeric nanoparticles [11,12],
and magnetic nanoparticles [1].
Magnetic nanoparticles represent an innovative class of nano-
carrier which has proved capable of targeting anticancer drugs,
avoiding the most part of limitations attributable to conventional
therapeutic tools. Such systems imply improved bioavailability in
the tumor site by means of a double targeting effect, namely
enhanced permeability retention effect (EPR) [13,14] (passive)
and the magnetic targeting owing to the attraction to an external
magnetic field. The advantages above reported lead to other bene-
fits, including reduced toxicity toward healthy cells and the ability
to locally deliver a high drug payload into tumor mass. Superpara-
magnetic iron oxide nanoparticles (SPIONs) are a unique family of
magnetic nanoparticles endowed with additional advantageous
Abbreviations: SPIONs, superparamagnetic iron oxide nanoparticles; EDA,
ethylenediamine; BNPC, bis-nitrophenyl carbonate; SqCOOH-C27, 1,10,2-tris-nor-
squalene acid; INU-Sq, inulin-1,10,2-tris-nor-squalene; INU-Sq-PEG2000, inulin-
1,10,2-tris-nor-squalene-PEG2000; IC-SPIONs, INU-Sq-PEG2000 coated SPIONs;
Doxo-IC-SPIONs, doxorubicin loaded IC-SPIONs; DD, derivatization degree.
⇑
Corresponding author. Laboratory of Biocompatible Polymers, Department of
Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), University
of Palermo, Via Archirafi, 32 90123 Palermo, Italy. Tel.: +39 09123891927; fax: +39
091 6100627.
E-mail address: mariano.licciardi@unipa.it (M. Licciardi).
http://dx.doi.org/10.1016/j.ejpb.2014.09.008
0939-6411/Ó 2014 Elsevier B.V. All rights reserved.

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C. Scialabba et al. / European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 695–705
physicochemical properties. Specifically, they show magnetic
interactions that do not persist once the external magnetic field
is removed (superparamagnetism) [15], and hence site specific
magnetic targeting can be efficiently obtained applying onto the
tumor mass removable and nontoxic magnetic fields as external
drawing force.
The 1H NMR spectra were recorded using a Bruker Avance II 300
spectrometer operating at 300 MHz. Fluorescence spectroscopy
was performed by using a Shimadzu RF-5301 PC spectrofluorime-
ter. Centrifugations were performed using a Centra MP4R IEC
centrifuge. Size exclusion chromatography (SEC) was carried out
using
a
Phenomenex PolySep-GFC-P3000 column (California,
Water 2410 refractive index detector.
SPIONs consist of
a
solid core made up of iron oxides
USA) connected to a
(magnetite, Fe3O4 and/or maghemite, Fe2O3) mostly coated with
biocompatible polymers. Typically, the coating agent plays a dou-
ble role, which is to protect nanoparticles from oxidation and
increase their stability when dispersed in aqueous media. Indeed,
the presence of a hydrophilic shell avoids hydrophobic–hydropho-
bic interactions that provoke SPIONs aggregation, so nullifying all
advantages related to the nanomeric dimensions of the starting
carrier [16]. Many natural and synthetic polymers such as
poly(hydroxyethyl) aspartamide derivative [17,18], chitosan, ethyl
cellulose, poly(lactide) acid, poly(lactide-co-glycolide) acid and
poly(ethylene)glycol have been employed as coating agents for
enhancing SPIONs stability and conferring surface functional
groups available for further reactions [19,20]. However, some of
these either are not sufficiently bioeliminable or degrade to acidic
products often yielding to local inflammation. Therefore, new cost-
effective polymers provided with excellent biocompatibility and
versatility should be synthesized and studied in view of clinical
applications [20–22].
In this study we propose inulin-based copolymer as coating
agent for SPIONs. Inulin is a natural, biocompatible, bioeliminable
and biodegradable [23] polysaccharide consisting of linear chains
of b-(2-1) fructose units and typically has a glucose unit attached
at the reducing end. It is water soluble and exhibits high hydroxyl
functional groups available for common coupling reactions [24].
Various studies have dealt with its chemical modification in order
to obtain biocompatible drug delivery systems (DDS), including
hydrogels [25], nanoparticles [26], micelles [6,27], and macromo-
lecular bioconjugates [28]. Being inulin versatile and highly func-
tionalizable [29,30], here we focused our attention on the
synthesis of an amphiphilic inulin derivative capable of coating
SPIONs once placed in aqueous media. We demonstrated that the
polymer-coated SPIONs loaded significant amounts of doxorubicin
during the self-assembling process, which was released at physio-
logical conditions in a controlled fashion so acting as an excellent
anticancer system. Under the effect of external magnetic stimuli,
it was also assessed the ability of the system to be accumulated
into a specific area mimicking its potential targeting effect in the
human body.
Phosphate buffer pH 6.5/methanol 9:1 (v/v) solution was used as
eluent at 35 °C with a flux of 0.6 mL/min, and pullulan standards
(112.0–0.18 kDa, Polymer Laboratories Inc., USA) were used to
set up calibration curve.
2.2. Synthesis of 1,10,2-tris-nor-squalene acid (SqCOOH-C27): (4E,8E,
12E,16E)-4,8,13,17,21-pentamethyl-4,8,12,16,20-docosapentaenoic
acid
1,10,2-Tris-nor-squalene aldehyde [31–33] (1.58 g, 4.12 mmol)
was dissolved in diethyl ether (20 mL) at 0 °C. Separately, sulfuric
acid (2.3 mL) was added at 0 °C to distilled water (20 mL) with stir-
ring, followed by potassium dichromate (1.21 g, 4.12 mmol) to
obtain chromic acid. It was then added at 0 °C within 20 min to
the solution of 1,10,2-tris-nor-squalene aldehyde, previously pre-
pared, and left to react for 2 h at 0 °C with stirring. The reaction
mixture was extracted with diethyl ether (50 mL ꢀ 3), washed
with saturated brine, dried over anhydrous sodium sulfate and
evaporated in vacuum. The completion of the reaction was
revealed by silica gel TLC with light petroleum/diethyl ether/meth-
anol, 70:23:7. The crude product was purified by flash chromatog-
raphy with light petroleum, then light petroleum/diethyl ether,
95:5 as eluent, to give 578 mg of 1,10,2-tris-nor-squalene acid
(35% yield), as a colorless oil.
1H NMR spectra were recorded on a Bruker 300 ultrashield
instrument (Karlsruhe, Germany) for samples in CDCl3 solution at
room temperature, with Me4Si (TMS) as internal standard. Cou-
pling constants (J) are given in Hz. Mass spectra were recorded
on a Finnigan MAT TSQ 700 spectrometer (San Jose, CA). The reac-
tions were monitored by TLC on F254 silica gel precoated sheets
(Merck, Damstadt, Germany); after development, the sheets were
exposed to iodine vapor. Flash-column chromatography was per-
formed on 230–400 mesh silica gel.
1H NMR (CDCl3): d, 1.55–1.63 (m, 18 H, allylic CH3), 1.90–2.05
(m, 16 H, allylic CH2), 2.26 (t, 2 H, CH2CH2COOH), 2.38 (t, 2 H,
CH2CH2COOH), 5.00–5.19 (m, 5 H, vinylic CH), 12.20 (broad, 1 H,
COOH). MS (EI): m/z 400 (M+, 5), 357 (3), 331 (5), 289 (3), 208
(6), 136 (3), 81 (100).
2.3. Synthesis of Inulin-1,10,2-tris-nor-squalene (INU-Sq)
2. Experimental part
SqCOOH-C27 (12 mg, 1.5 ꢀ 10ꢁ2 mmol) was solubilized in DMF
(1.2 mL), and then EDC-HCl (3.6 mg, 1.9 ꢀ 10ꢁ2 mmol) was added.
2.1. Materials and methods
After that, NHS (2.2 mg, 1.9 ꢀ 10ꢁ2 mmol) and TEA (3
lL,
Squalene (purity > 99%) was purchased from VWR (Italy). Inulin-
(2-aminoethyl)-carbamate (INU-EDA), used as starting copolymer,
was synthesized as previously reported [6]. Inulin, triethylamine
(TEA), ethylenediamine (EDA), Bis(4-nitrophenyl)carbonate (BNPC),
doxorubicin hydrochloride (DOXO-HCl), iron oxide (Fe3O4) mag-
netic nanoparticles (10 1 nm) in water, iron(III)chloride, ferrozine
(3-(2-pyridyl)-5,6-bis(phenyl sulfonic acid)-1,2,4-triazine), and
neocuproine (2,9-dimethyl(1,10-phenanthroline)), were purchased
from Aldrich (Milan, Italy). N-hydroxysuccinimide (NHS), 1-ethyl-3-
(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCl),
2.1 ꢀ 10ꢁ2 mmol) were added to the reactive mixture at once
together and the reaction was kept 4 h at 40 °C. The resulting solu-
tion was added to 4 mL of a INU-EDA solution in DMF (25 mg/mL)
drop-wise and left to react at 25 °C for 18 h. The INU-Sq conjugate
was then precipitated in diethyl ether/dichloromethane mixture
(2:1 vol/vol) and collected by centrifuging at 5 °C for 5 min, at
9000 rpm. The solid residue was then washed four times with
the same mixture. The obtained solid product was dried under vac-
uum and weighted. INU-Sq was obtained with a yield of 95% (w/w)
based on the starting inulin. The obtained copolymer was charac-
terized by 1H NMR (300 MHz, D2O/DMF-d7 (5:1): d 1.12–1.52
(18H, squalenoyl allylic CH3), 1.91–2.21 (16H, squalenoyl
O-[2-(6-oxocaproylamino)-ethyl]-O0-methylpolyethylene
glycol
2000 (PEG-CH@O), Sephadex G-15, anhydrous dimethylformamide
(DMF), were purchased from Fluka (Switzerland). All reagents were
of analytic grade, unless otherwise stated. SpectraPor dialysis tubing
was purchased from Spectrum Laboratories, Inc. (Italy).
allylic CH2), 2.9–3.1 (4HEDA
(5HINU ACH2AOH; ACHACH2AOH; ACACH2AOA), 4.02–4.19
(2HINU, ACACHAOH; ACHAOH).
, ANHACH2ACH2ANHA), 3.55–4.0
,

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697
2.4. Synthesis of = inulin-1,10,2-tris-nor-squalene-PEG2000
2.7.3. FT-IR analysis
(INU-Sq-PEG2000) graft copolymer
Investigation of the general and quantitative composition of
polymer coated iron oxide nanoparticles were carried out via infra-
red spectroscopy (FT-IR). The spectra of INU-Sq-PEG2000 copoly-
mer, IC-SPIONs and solid SPIONs were recorded in KBr pellets in
the frequency range of 4000–400 cmꢁ1 by using a Perkin-Elmer
Spectrum RX I FT-IR system spectrophotometer. Spectra were
recorded in transmittance scale (%T) with a resolution of 1 cmꢁ1
and a number of scans = 100.
PEG-CH@O (21 mg, 1.05 ꢀ 10ꢁ2 mmol) was added to 4 mL of
INU-Sq aqueous solution (22.5 mg/mL) at pH 6.8. The pH of the reac-
tion mixture increased slowly, and consequently the pH of the reac-
tion was kept at 6.8 using 0.1 N hydrochloric acid. The mixture was
left with stirring at room temperature overnight. Then it was dried
by vacuum and the crude reaction was washed up with diethyl
ether/dichloromethane mixture 2:1 vol/vol (4 ꢀ 40 mL). The solid
product was dried under vacuum yielding a white solid. Yield 85%
with respect to the starting inulin. The obtained graft copolymer
was characterized by 1H NMR (300 MHz, D2O/DMF-d7 (2:1): d
1.12–1.53 (18H, squalenoyl allylic CH3), 1.96–2.22 (16H, squalenoyl
2.7.4. Total iron determination
The iron content in IC-SPIONs and Doxo-IC-SPIONs was mea-
sured using the Ferrozine-based spectrophotometric iron estima-
tion method [17]. For this method freeze-dried IC-SPIONs (5 mg)
or Doxo-IC-SPIONs (5 mg) were dispersed in 1.4 N hydrochloric
acid (1 mg/mL) and kept for 2 h at 60 °C in a water bath to allow
the disaggregation of nanoparticles and complete dissolution of
the Fe3O4 nanoparticles. After that, 1 mL of iron-detection reagent
(6.5 mM ferrozine, 6.5 mM neocuproine and 1 M acid ascorbic dis-
solved in acetate buffer at pH 4.5) was added to the iron solution
and incubated for 30 min at room temperature. Then, the optical
density of the sample was measured at 560 nm using a Shimadzu
UV-2401PC spectrophotometer, and the concentration of Fe3O4
was calculated by comparing this value with a calibration curve
obtained by recording the absorbance of FeCl3 standard solutions
allylic CH2), 2.93–3.07 (4HEDA
, ANHACH2ACH2ANHA), 3.65
(176HPEG2000, AOACH2ACH2AOA), 3.67–3.88 (5HINU, ACH2AOH;
ACHACH2AOH; ACACH2AOA), 3.90–4.20 (2HINU, ACACHAOH;
ACHAOH).
2.5. Preparation of INU-Sq-PEG2000 coated SPIONs (IC-SPIONs)
To an aqueous suspension of Fe3O4 (5 mg/mL) INU-Sq-PEG2000
copolymer (50 mg) was added with gentile stirring. The obtained
mixture was sonicated in an ultrasonic bath for 10 min, thus
diluted to 5 mL with double distilled water and, finally, placed into
dialysis tube (Spectra Por, Float-A-Lyzers) with a nominal molecu-
lar weight cut-off (NMWCO) 8–10 kDa. After 48 h IC-SPIONs were
freeze-dried and obtained as a brownish powder. Yield 100%.
(ranging from 0.5 to 5
was used as blank.
lg/mL). Water with iron-detection reagent
2.7.5. Determination of drug payload into Doxo-IC-SPIONs and drug
release studies
2.6. Preparation of doxorubicin loaded IC-SPIONs (Doxo-IC-SPIONs)
The amount of doxorubicin loaded into Doxo-IC-SPIONs was
determined by UV spectroscopy measuring the absorbance at
480 nm of the sample, prepared by dispersing known amounts of
these in double distilled water at pH ꢄ 6. A calibration curve was
obtained for serially diluted concentrations of doxorubicin hydro-
chloride in bidistilled water at pH ꢄ 6. The content of drug loaded
into the system was expressed as the amount of loaded doxorubi-
cin per unit mass of dry nanoparticles, and resulted to be
11.6 0.5% (w/w), while the encapsulation efficiency was calcu-
lated to be 58.5%.
For drug release studies, an appropriate amount of freeze dried
Doxo-IC-SPIONs (5 mg) was suspended in PBS at pH 7.4 (5 mL) and
placed into floating dialysis tubing with a MWCO 1 kDa. This dial-
ysis membrane was immersed into PBS at pH 7.4 (15 mL) and incu-
bated at 37 °C under continuous stirring (100 rpm) in a Benchtop
808C Incubator Orbital Shaker model 420, for 48 h. At scheduled
time intervals, aliquots of the external medium (1 mL) were with-
drawn from the outside of the dialysis membrane and replaced
with equal amount of fresh medium. The withdrawn samples were
analyzed by HPLC in order to determine the released drug. Profile
releases were determined by comparing the amount of released
drug as a function of incubation time with the total amount of drug
loaded into the Doxo-IC-SPIONs. The same analysis was carried out
using PBS at pH 5.5. All release data were compared with the dif-
fusion profile of doxorubicin hydrochloride alone (0.62 mg)
obtained by using the same procedure.
Analogous procedure was adopted for the preparation of doxo-
rubicin loaded magnetic nanoparticles (Doxo-IC-SPIONs). For the
drug loading, 1 mL of an aqueous doxorubicin hydrochloride solu-
tion (10 mg/mL, 1.7 ꢀ 10ꢁ2 mmol), previously treated with TEA
(10
l
L, 7 ꢀ 10ꢁ2 mmol), was added to the polymer/Fe3O4 mixture
before starting the sonication. The obtained mixture was sonicated
in an ultrasonic bath for 10 min and then placed into dialysis tube
(Spectra Por, Float-A-Lyzers) with a nominal molecular weight cut-
off (NMWCO) 8–10 kDa. After 48 h Doxo-IC-SPIONs were freeze-
dried and obtained as a brownish powder. Yield 70%.
2.7. Characterization of the nanosystems
2.7.1. Scanning electron microscopy (SEM)
For the morphology studies, freeze-dried IC-SPIONs were visu-
alized using a scanning electron microscope, ESEM Philips XL30.
Samples were dusted on a double sided adhesive tape previously
applied on a stainless steel stub. The IC-SPIONs were then sput-
ter-coated with gold prior to microscopy examination.
2.7.2. Dynamic Light Scattering (DLS) measurement and Z-potential
analysis
DLS studies and Z-potential measurements (mV) were per-
formed at 25 °C using a Malvern Zetasizer NanoZS instrument, fit-
ted with a 532 nm laser at a fixed scattering angle of 173°. Aqueous
solutions of IC-SPIONs and Doxo-IC-SPIONs (0.2 mg/mL), were ana-
Data were corrected taking in account the dilution procedure.
Each experiment was carried out in triplicate and the results were
in agreement within 5% standard error.
lyzed after filtration through a 5 lm cellulose membrane filter. The
intensity-average hydrodynamic diameter and polydispersity
index (PDI) were obtained by cumulants analysis of the correlation
function. The zeta potential (mV) was calculated from the electro-
phoretic mobility using the Smoluchowski relationship and assum-
ing that K ꢂ a ꢃ 1 (where K and a are the Debye–Hückel parameter
and particle radius, respectively).
2.8. Cytotoxicity assay on human colon cancer (HCT116) cells
The cytotoxicity was assessed by the MTS assay on human colon
cancer (HCT116) cell lines (purchased from Istituto Zooprofilattico
Sperimentale della Lombardia e dell’ Emilia Romagna, Italy), using a
commercially available kit (Cell Titer 96 Aqueous One Solution Cell

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C. Scialabba et al. / European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 695–705
Proliferation assay, Promega). Cells were seeded in 96 well plate at
a density of 2.5 ꢀ 104 cells/well and grown in Dulbecco’s Minimum
Essential Medium (DMEM) with 10% FBS (fetal bovine serum)
and 1% of penicillin/streptomycin (10,000 U/mL penicillin and
10 mg/mL streptomycin) at 37 °C in 5% CO2 humidified atmo-
micelles. Herein, being hydrophobic interactions useful only to sta-
bilize the polymeric coating by the London dispersion forces, the
synthesis of this copolymer was basically adapted in order to
obtain a copolymer bearing squalenoyl branches as biocompatible
hydrophobic moieties, conferring appropriate hydrophobicity still
maintaining a good biocompatibility. [34]
sphere. After 24 h the medium was replaced with 200 lL of fresh
culture medium containing empty IC-SPIONs and Doxo-IC-SPIONs
Inulin (a-D-glucopyranosyl-[b-D-fructofuranosyl](n-1)-D-fructo-
at a concentration per well equal to 0.13, 0.27, 0.54, 0.81 and
1.08 mg/mL, corresponding to 25, 50, 100, 150 and 200 lM of
doxorubicin. Moreover, the same experiment was performed
applying a permanent magnets (attraction force of 250 g) at the
bottom of the wells, and using a concentration of empty IC-SPIONs
furanoside), a linear polysaccharide consisting of glucopyranose
end-capped fructose units (b-1,2), was used as main biocompatible
polymeric backbone. First of all, it was partially functionalized with
EDA so as to introduce primary amine pendants that can be utilized
as versatile functional groups allowing further functionalization [6].
and Doxo-IC-SPIONs of 0.27 mg/mL (equivalent to 50
l
M of doxo-
A derivatization degree in EDA of 28% was obtained (DDEDA
,
rubicin). Additionally, cells treated with the same magnet were
used as negative control for exposition to external magnetic field.
28 mol%, related to inulin monomer units).
A squalene derivative (SqCOOH-C27) carrying a carboxylic func-
tional group available for additional coupling reactions was syn-
thesized starting from 1,10,2-tris-nor-squalene aldehyde. After the
usual work-up, SqCOOH-C27 was obtained in 35% yield. From the
1H NMR analysis, the presence of a characteristic signal of the car-
boxylic acid at 12.20 ppm, along with the observation of a shift of
the vicinal hydrogens (CH2ACH2ACOOH) observed at 2.26 ppm
and 2.38 ppm respectively, confirmed the feasibility of the reac-
tion. Furthermore, the molecular ion of the product (M+) observed
at 400m/z by means of EI-MS analysis confirmed the hypothesis of
the structure expected for the product.
After 24 and 48 h, DMEM was replaced with 100
lL of fresh med-
ium, and 20 L of a MTS solution was added to each well. Plates
l
were incubated for additional 2 h at 37 °C. Then, the absorbance
at 490 nm was measured using a microplate reader (Multiskan,
Thermo, UK). Doxorubicin hydrochloride solutions at the same
concentrations were used as a positive control, as pure cell med-
ium was used as a negative control.
Results were expressed as percentage reduction of the control
cells. All culture experiments were performed in triplicates.
In a second step, INU-EDA was functionalized with the squale-
noyl derivative (SqCOOH-C27) via amidic bond. In particular,
SqCOOH-C27 was primarily activated using a mixture of EDC and
NHS as coupling agents (Scheme 1). The stoichiometric conditions
employed (moles of SqCOOH-C27/moles of amino pendant groups
in INU-EDA = 0.1; moles of EDC-HCl/moles of SqCOOH-C27 = 1.2
and moles of NHS/moles of SqCOOH-C27 = 1.2) were fixed to obtain
a quantitative conversion of the SqCOOH-C27 carboxylic functions
to amide.
2.9. Cell drug uptake studies
The cellular uptake of the Doxo-IC-SPIONs was evaluated by
fluorescence microscopy analysis (Zeiss ‘‘AXIO Vert. A1’’ Micro-
scope Inverted). HCT116 cell line, 2.5 ꢀ 104 cells/well, maintained
in normal medium were cultured in a 46 well plate at 37 °C in
an atmosphere of 5% CO2 for 24 h. After 24 h the medium was
replaced with 200
IC-SPIONs or free doxorubicin hydrochloride at final drug concen-
tration of 50 M, and thus cells were incubated for 4, 24 and 48 h.
lL of fresh culture medium containing Doxo-
The 1H NMR allowed an easy quantification of the SqCOOH-C27
linked to inulin, by comparing the integral of the peaks at d
1.12–1.52 and at 1.91–2.21, ascribable to squalenoyl allylic CH3
and allylic CH2 respectively, to that belonging to the protons of
INU backbone at d comprised between 3.55 and 4.19. The molar
percent of SqCOOH-C27 covalently linked to inulin (DDSq%) was
equal to 1.7 0.5 mol% referred to the inulin repeating units. The
INU-Sq conjugates still maintain a large number of primary amine
groups available for some potential subsequent side chain func-
tionalization (about 90 mol% with respect to the total amount of
amines), such as PEGylation. Hence, the last synthetic step was
the synthesis of PEGylated INU derivative (INU-Sq-PEG2000). In that
reaction a semitelechelic aldehyde functionalized mono-methoxy
PEG, having an weigh average molecular weight of 2000 Da, was
selected to easily react in a slightly acidic aqueous medium with
the primary amine groups available in the polymer side chain,
yielding to the immine bounds (see Scheme 2).
l
This experiment was performed with or without an external mag-
netic field. For the experiments in the presence of an external mag-
netic field, permanent magnets with an attraction force of 250 g,
were fixed to the bottom of the wells containing the cells. After
each incubation period, the medium was removed and the cell
monolayer was washed twice with DPBS and fixed with 4% formal-
dehyde for 30 min. Subsequently, the formaldehyde solution was
removed, and the cells washed with DPBS and the nuclei were
stained with 40,6-diamidino-2-phenylindole (DAPI). After incuba-
tion, DAPI solution was removed, and the cells were washed with
DPBS and observed by fluorescence microscope. The images were
recorded using an Axio Cam MRm (Zeiss).
2.10. Statistical analysis
A one way analysis of variance (ANOVA) was applied to com-
pare different groups. Data were considered statistically significant
with a value of P < 0.05 and differences between the groups were
compared using a posteriori Bonferroni t-test. All values are the
average of three experiments standard deviation.
INU-Sq-PEG2000 copolymer was characterized by means of 1H
NMR spectroscopy, which confirmed the introduction of PEG2000
chains on the INU-Sq backbone and allowed the calculation of
the molar derivatization degree (DDPEG%). The DDPEG%, indicated
as percentage of linked polyethylene chains in comparison with
the repeating units of inulin, was calculated by comparing the inte-
gral of the peak related to protons at d 3.65 assigned to ethylene
protons of PEG, with the integral of the peaks related to protons
of INU backbone at d comprised between 3.67 and 4.20. The
DDPEG% was equal to 2.7 0.5 mol% referred to the inulin repeating
units.
3. Results and discussion
3.1. Synthesis of inulin-graft co-polymers
INU-Sq-PEG2000 is an amphiphilic polysaccharide based graft-
copolymer useful for the preparation of self-assembled nanocarri-
ers or as coating material of colloidal nanoparticles. In a previously
published article [6], a similar copolymer carrying ceramide tails
have been already studied for the preparation of polymeric
Weight average molecular weights (Mw) and polydispersity of
the synthesized copolymers were determined by SEC analyses
and data are reported in Table 1. The relative Mw of the polymers
was obtained using a calibration curve of monodisperse pullulan

C. Scialabba et al. / European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 695–705
699
Scheme 1. Synthesis of INU-Sq.
standards with Mw ranging from 200 to 10,000. Definitively, the
molecular weight increment of INU-Sq and INU-Sq-PEG2000 with
respect to the starting INU-EDA copolymer confirm the derivatiza-
+16 5 mV) involves the adsorption of the latter on the surface
of the iron oxide nanoparticle, thus acting as hydrocolloidal coating
yielding to the stabilization of SPIONs in aqueous media. The draw-
ing force that allows the IC-SPIONs formation is the ionic interac-
tion established between SPIONs and INU-Sq-PEG2000 copolymer,
as the Van der Waals interactions, with particular emphasis to the
London dispersion forces, imply the assembling of the hydrophobic
moieties of the amphiphiles adsorbed onto the nanoparticles,
which stabilize and consolidate the nano-aggregates.
Fig. 1 shows the morphology of IC-SPIONs obtained by high res-
olution SEM analysis. SEM micrographs showed a homogeneous
spherical nanoparticles population and with a diameter of about
50 nm.
These data were toughen up by DLS measurements which
proved that the particle size observed by SEM have an average
hydrodynamic diameter value of 54.79 nm and a PDI of 0.12 in
aqueous dispersion as well. Zeta potential measures were also
performed to establish the potential stability and polarity of the
nanoparticles. It was found that the IC-SPIONs possessed positively
charged surfaces with values of zeta potential of 21 8 mV,
probably due to the presence of protonated amines onto the nano-
particle surface. This value guarantees a good shelf stability of the
tion of INU-EDA with Sq firstly and, subsequently, with PEG2000
.
After the derivatization of inulin with EDA, a drastic molecular
weight abatement was observed, passing from 4.9 kDa to 2.5 kDa.
This might be ascribed to several factors. In first instance, confor-
mational changes of the polymer chain, caused by the introduction
of ionisable amine groups into the polymer backbone, imply vari-
ations in the hydrodynamic radii of the copolymers. On the other
hand, microwave assisted reactions can entail the degradation of
the main polymer chain, thus leading to a polymer with lower
molecular weight [6]. Similar behavior was observed after the
introduction of Sq moieties in the main backbone.
3.2. Preparation and characterization of IC-SPIONs
Polymeric coated iron oxide nanoparticles were obtained adding
INU-Sq-PEG2000 copolymer to iron oxide nanoparticles water dis-
persion dropwise. The simultaneous presence in the aqueous phase
of both SPIONs, which are negatively charged (Z-potential = ꢁ29.2
4 mV), and the cationic amphiphilic copolymers (Z-potential =

700
C. Scialabba et al. / European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 695–705
Scheme 2. Synthesis of INU-Sq-PEG2000 (n = 45).
The drug loading procedure was carried out adding the aqueous
doxorubicin hydrochloride solution, previously treated with TEA,
to the polymer/Fe3O4 mixture. A molar excess of TEA was added
to drug solution in order to obtained doxorubicin free base, with
a less water solubility, so becoming more affine to the hydrophobic
portion of the coating consisting of the squalenoyl tails. The
amount of drug loaded evaluated by UV analysis was found to be
11.6 0.5% (w/w), while the encapsulation efficiency was calcu-
lated to be 58.5%. DLS measurements carried out on Doxo-IC-SPI-
ONs showed a reduction in size upon doxorubicin loading into
nanoparticles, perhaps due to the formation of hydrophobic
domains consisting of Sq moieties and aromatic portions of doxo-
rubicin. Such interactions also explain well the higher polydisper-
sity observed for the Doxo-IC-SPIONs (see Table 2). Being
hydrophobic interactions concentration dependent phenomena,
during the first step nanoparticles had available a large number
of doxorubicin molecules which gave rise to compacted nanoparti-
cles with high drug payload. After drug dilution, lesser drug–Sq
interactions brought about bigger nanoparticles with a lower
payload thus yielding to a high polydisperse system. As the lack
Table 1
Molar derivatization degree values of EDA, Sq and PEG linked to inulin and molecular
weight (Mw) and dispersity of the correspondent copolymers.
Copolymers
Composition
aDDEDA%
–
bMw
(kDa)
bDispersity
Mw/Mn
aDDSq%
aDDPEG%
Inulin
–
–
–
–
–
4.9
2.5
3.1
4.3
1.3
1.8
1.3
1.2
INU-EDA
INU-Sq
INU-Sq-
PEG2000
28
28
28
1
1
1
1.7 0.5
1.7 0.5
2.7 0.5
a
Calculated by means of 1H NMR spectroscopy.
Calculated by SEC analysis.
b
system, along with the proper superficial charge helpful for the cel-
lular up-take, which usually requires positively charged systems.
Doxorubicin loaded IC-SPIONs were then prepared to establish
the ability of the system to act as a drug-delivery system. Doxoru-
bicin was used as anticancer drug model because it is also
endowed with fluorescence properties, which can be exploited to
evaluate its uptake into cells by means of fluorescence microscopy.

C. Scialabba et al. / European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 695–705
701
Fig. 1. SEM images of IC-SPIONs.
of significant Z potential variations, which affects nanoparticles
surface, endorsed the hypothesis that all changes provoked by
doxorubicin involved the Doxo-IC-SPIONs core.
The chemical composition of the hybrid nanoparticles, that is
Doxo-IC-SPIONs and IC-SPIONs, was assessed by ferrozine colori-
metric iron determination assay and for the latter also by FTIR
spectroscopy. The total iron oxide amount in IC-SPIONs and
Doxo-IC-SPIONs is revealed by the ferrozine assay, and was found
to be equal to 6.5 1% and 7.7 1% on a weight basis respectively.
The existence of the polymer coating on the IC-SPIONs surface was
further investigated by FTIR analysis. In Fig. 2 the spectra are
reported in comparison with Fe3O4 and INU-Sq-PEG2000 copolymer
alone. The IR spectrum of Fe3O4 showed only the characteristic
broad band of FeAO stretching at 579 cmꢁ1
, whereas the
INU-Sq-PEG2000 copolymer displays characteristic stretching bands
of carbonyl functions at about 1650 and 1740 cmꢁ1, coupled with
some broad bands at 1100 cmꢁ1, together with typical bands in
the region of 1500–1200 cmꢁ1. The spectrum of IC-SPIONs clearly
showed the vibrating bands of the polymer consistent with the
existence of a polymer coating on magnetite core.
Fig. 2. FTIR spectra of magnetite (Fe3O4), INU-Sq-PEG2000 copolymer and IC-SPIONs.
(For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
The superparamagnetic behavior of prepared IC-SPIONs was
already evidenced macroscopically by the effect of an external
magnetic field on their water dispersion. In Fig. 3, photograph of
IC-SPIONs dispersion is shown (a), clearly displaying that nanopar-
ticles are well dispersed throughout the sample. It can be noticed
that, after the application of an external magnetic field for some
minutes, nanoparticles moved toward the magnet (b), whereas a
homogeneous dispersion was obtained after the removal of the
magnetic stimulus (c).
3.3. Drug release studies
To evaluate the capability of Doxo-IC-SPIONs to release the
loaded model drug, release experiments were carried out in two
media simulating different human body compartments. In
particular, PBS solution at pH 7.4, which mimics physiological
medium (Fig. 4a) and pH 5.5, mimicking endosomal and lysosomal
Fig. 3. Photographs of IC-SPIONs water dispersion before (a) during (b) and after (c)
the application of an external magnetic field overnight. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of
this article.)
Table 2
DLS data and zeta potential values of IC-SPIONs and Doxo-IC-SPIONs samples in
aqueous medium at a final copolymer concentration of 0.2 mg/mL.
Sample
Hydrodynamic diameter
(nm)
PDI
Zeta potential (mV)
environments were used (Fig. 4b). The release experiments at the
two pH conditions consisted into quantify the amount of released
doxorubicin in the external medium, at pre-determined time
intervals and until 48 h. In Fig. 4a and b the amount of released
IC-SPIONs
Doxo-IC-
SPIONs
54.19
44.15
0.16
0.46 19.8
21
8
2

702
C. Scialabba et al. / European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 695–705
For IC-SPIONs, negligible effect and no significant differences
120
90
60
30
0
(a)
both after 24 and 48 h in cell viability were detected, at the
whole concentration range tested, with respect the untreated
control, indicating a good cytocompatibility of the empty system
(Fig. 5b).
120
(a)
24 h
48 h
100
0
10
20
30
40
50
60
80
60
40
20
0
Time (hours)
Doxo
Doxo-IC-SPIONs
120
90
60
30
0
(b)
control
25 μM
50 μM
100 μM
150 μM
200 μM
120
100
80
60
40
20
0
(b)
24 h
48 h
0
10
20
30
40
50
60
Time (hours)
Doxo
Doxo-IC-SPIONs
Fig. 4. Drug release of Doxo-IC-SPIONs and doxorubicin hydrochloride in PBS
solution at pH 7.4 (a) and at pH 5.5 (b).
doxorubicin, referred to the total amount of doxorubicin loaded
into the micelles, is shown as function of the incubation time.
The nanoparticles proved capable of releasing doxorubicin in the
intact form for a prolonged period and without a first ‘‘burst
effect’’. For instance, after 48 h about 12% of doxorubicin payload
was slowly released at the two pH conditions, providing good find-
ings so as to guarantee the slow drug release once the site of action
will be reached. Additionally, the release profile has not been
affected by the pH of the medium.
120
100
80
60
40
20
0
(c)
24 h
48 h
3.4. In vitro biological evaluations
The effectiveness of the nanocarrier here prepared was estab-
lished indirectly by evaluating the cytotoxicity of IC-SPIONs,
Doxo-IC-SPIONs and doxorubicin hydrochloride on human colon
cancer cells (HCT116), a cancer cell line used to investigate the
anti-cancer activity of drugs and the associated mechanism of
action [35], by MTS assay. MTS assay is based on the ability of
mitochondrial dehydrogenases in viable cells to convert the MTS
salt into a colored formazan product, which can be quantitatively
detected by spectrophotometry at a kmax of 490 nm. The obtained
absorbance is directly proportional to the number of living cells
in culture. In Fig. 5 are reported the cytotoxicity profiles at 24 h
and 48 h of incubation time of the plain drug at concentration in
(d)
120
100
80
60
40
20
0
24 h
48 h
*
*
**
**
the range of 25–200 lM (5a), IC-SPIONs at concentration within
the range 0.13–1.08 mg/mL (5b), and Doxo-IC-SPIONs (5c) at the
same concentration above reported for IC-SPIONs, corresponding
to the amount the doxorubicin tested in the positive control (5a).
Fig. 5a shows that doxorubicin alone displayed a dose-dependent
cytotoxicity profile passing from 32% of cell viability at the concen-
Fig. 5. Cell viability % (MTS assay) on colon cancer cells (HCT116) of: doxorubicin
hydrochloride (a), IC-SPIONs (b), Doxo-IC-SPIONs (c) and all the samples previously
mentioned in the presence and in the absence of the external magnet (d).
Incubation times was 24 and 48 h. The results are reported as the mean SD (n = 6).
⁄p = 0.0016, ⁄⁄p = 0.00018.
tration of 25
incubation.
lM to 0% at the concentration of 200 lM and 48 h of

C. Scialabba et al. / European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 695–705
703
Fig. 6. Fluorescence microscopy images of HCT116 cells incubated with free doxorubicin hydrochloride (line A), Doxo-IC-SPIONs (line B) and Doxo-IC-SPIONs with the
application of the external magnet (line C). incubation times were 4 (panel a), 24 (panel b) and 48 h (panel c). Doxorubicin (DOXO) is visualized in red; cell nuclei were stained
with DAPI (blue). Magnification is 40ꢀ for all images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
While, the cytotoxicity profile obtained for increasing dose of
Doxo-IC-SPIONs makes viewable a quasi-dose-independent trend
of effectiveness (Fig. 5c), in particular after 48 h of incubation.
The variation in cytotoxicity observed at different concentration
was indeed within range of standard errors. Worthy of note is that
the cytotoxicity observed after 48 h is higher than that revealed
after 24 h of incubation, thus suggesting that nanoparticles provide
an intracellular reservoir of the drug, which is gradually released
and internalized within nuclei. Moreover, even at the lower
dosage, namely at 25 and 50 lM, the activity observed for Doxo-
IC-SPIONs was remarkable higher, after 48 h of incubation, if
compared with the same dose of plain drug, varying from 9% and
8% (Fig. 5c) to 20% and 16% (Fig. 5a) of viability respectively. This
behavior clearly confirms that the loaded drug was locally and

704
C. Scialabba et al. / European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 695–705
Fig. 6 (continued)
efficiently internalized into the cytosol compartment and there
slowly released. Differently, free doxorubicin is rapidly internal-
ized but also promptly expelled from the above compartment by
the activation of the cell drug resistance mechanism, such as, for
instance, those due to the efflux pump activity [36].
The same experiment was performed applying a permanent
magnetic field at the bottom of the wells as shown in Fig. 5, in
order to evaluate the effect of the magnetic field on cells viability.
In particular, IC-SPIONs at concentration of 0.27 mg/mL and
Doxo-IC-SPIONs at the same concentration, which corresponds to
concentration of 50 lM, both with and without the application of
the external magnetic field. Fig. 6 shows the image of cells after
4 (a), 24 (b) and 48 h (c) of incubation. These studies showed that
free doxorubicin was rapidly accumulated in HCT116 nuclei just
after 4 h of incubation (see line A of Fig. 6a), and no substantial dif-
ferences in intracellular trafficking were observed after 24 and
48 h, apart from the reduction of live cells (line A of Fig. 6b and
c). Differently, in the case of Doxo-IC-SPIONs incubated without
the external magnet, the higher fluorescence intensity is visualized
in the cell cytoplasm after 4 h of incubation (see line B of Fig. 6a).
The same result was obtained with Doxo-IC-SPIONs incubated
with the external magnet (see line C of Fig. 6a). After 24 h of
incubation, a diffuse red fluorescence appeared inside cell nuclei
also in cells treated with Doxo-IC-SPIONs (lines B and C of
Fig. 6b), because doxorubicin was slowly released from nanoparti-
cles. It is interesting to note that, in agreement with the cytotoxic-
ity results above reported, the number of cells incubated with the
external magnet appeared drastically reduced after 48 h of incuba-
tion (see line C of Fig. 6c), endorsing the hypothesis that the
presence of the external magnetic force improves nanoparticles
uptake. Moreover, it is well visible a red circle around the cell
nuclei (see line C of Fig. 6c) presumably due to accumulation of
Doxo-IC-SPIONs nanoparticle. This finding may confirmed the
hypothesis that the magnetic force enhanced the Doxo-IC-SPIONs
uptake and created a reservoir of the drug available inside cells
that is slowly released into the nucleus, sustaining doxorubicin
activity and eluding all possible drug resistance mechanisms.
50 lM in doxorubicin hydrochloride, were incubated for 24 h and
48 h either in the presence of a magnetic field or not. Results are
reported in Fig. 5d with respect to cells treated with the magnetic
field alone and cells treated with doxorubicin hydrochloride at
concentration of 50 lM. All results are reported as function of
the control, which is the cell viability of untreated cells. As can
be seen from the Fig. 5d, the magnetic field applied both to the
untreated cell culture and the cells treated with the IC-SPIONs
did not affect cell viability, since the cell viability was definitely
comparable to that of the control. As, the cytotoxic effect on
HCT116 cells increases by about 50% when cells were incubated
with Doxo-IC-SPIONs for 24 h in the presence of the external mag-
net, while the effectiveness was lower if compared with the same
dose of free doxorubicin hydrochloride. On the other hand, the per-
sistence of the external field for 48 h enhanced the effectiveness of
the Doxo-IC-SPIONs (3.4 0.1% of viability observed), being exten-
sively higher than that expressed by the same dose of free doxoru-
bicin hydrochloride (16.6 1.7% in Fig. 5a). These results prove that
the attractive force of the magnetic field on the magnetic particles
that increase the particles penetration into cells [17,37], in combi-
nation with the slow drug release into cells above discussed, aids
to an important improvement of the anticancer activity of
doxorubicin.
4. Conclusions
In this work, the synthesis of squalene-grafted-inulin copoly-
mer bearing PEG2000 moieties to give an amphiphilic copolymer
with stealth like behavior is reported. This copolymer was easily
obtained by in turn conjugating squalenoyl derivative and semite-
lechelic aldehyde terminated PEG chains of average weight molec-
ular weight 2000, without employing drastic synthetic conditions.
Finally, the ability of IC-SPIONs to release doxorubicin payload
into HCT116 cells was confirmed by fluorescence microscopy
analysis, incubating HCT116 cells with Doxo-IC-SPIONs, at drug

C. Scialabba et al. / European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 695–705
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allowed obtaining a copolymer able to self-assemble into orga-
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with average diameter of about 50 nm. We demonstrated that, in
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Conflict of interest
Authors declare that there are no conflict of interest involving
this study.
Acknowledgements
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We thank the MIUR, the University of Turin (Ricerca locale
2013-14) and University of Palermo (FFR 2012) for the financial
support to this research.
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