Polybenzofulvene derivatives bearing dynamic binding sites as potential anticancer drug delivery systems

Article abstract of DOI:10.1039/c4tb01268b

In order to obtain new advanced functional materials capable of recognizing drug molecules, the polybenzofulvene backbone of molecular brush poly-6-MOEG-9-TM-BF3k has been functionalized with a "synthetic dynamic receptor" composed of two 1-adamantylurea moieties linked together by means of a dipropyleneamino bridge as in Meijer's bis(adamantylurea) pincer (BAUP). This functional material, bearing synthetic receptors potentially capable of recognizing/loading and then delivering drug molecules, was used to prepare colloidal drug delivery systems (by means of soft interaction with BAUP) for delivering the model anti-cancer drug doxorubicin (DOXO). The resulting nanostructured drug delivery systems containing the physically loaded drug were characterized in terms of drug loading and release, dimensions and zeta potential, and in vitro cell activity and uptake on two different cell lines (i.e. the human bronchial epithelial 16HBE and the human colon cancer HCT116). On normal cells, free DOXO was found to be more cytotoxic than DOXO-loaded nanogels at the higher tested concentration and, only on cancer cells, DOXO-loaded nanogels show similar or slightly higher cytotoxicity values than free DOXO, suggesting potential advantages in the treatment of cancer. These results were supported by fluorescence microscopy studies, which suggested that DOXO-loaded nanogels provide an extracellular reservoir of the drug, which is gradually released and internalized within the cells.

Full text of DOI:10.1039/c4tb01268b

Journal of
Materials Chemistry B
View Article Online
View Journal
PAPER
Polybenzofulvene derivatives bearing dynamic
binding sites as potential anticancer drug delivery
Cite this: DOI: 10.1039/c4tb01268b
systems†
a
Andrea Cappelli, Giorgio Grisci,a Marco Paolino,a Vincenzo Razzano,a
*
Germano Giuliani,a Alessandro Donati,a Claudia Bonechi,a Raniero Mendichi,b
Antonella Caterina Boccia,b Mariano Licciardi,c Cinzia Scialabba,c
Gaetano Giammonac and Salvatore Vomeroa
In order to obtain new advanced functional materials capable of recognizing drug molecules, the
polybenzofulvene backbone of molecular brush poly-6-MOEG-9-TM-BF3k has been functionalized with
a “synthetic dynamic receptor” composed of two 1-adamantylurea moieties linked together by means of
a dipropyleneamino bridge as in Meijer's bis(adamantylurea) pincer (BAUP). This functional material,
bearing synthetic receptors potentially capable of recognizing/loading and then delivering drug
molecules, was used to prepare colloidal drug delivery systems (by means of soft interaction with BAUP)
for delivering the model anti-cancer drug doxorubicin (DOXO). The resulting nanostructured drug
delivery systems containing the physically loaded drug were characterized in terms of drug loading and
release, dimensions and zeta potential, and in vitro cell activity and uptake on two different cell lines (i.e.
the human bronchial epithelial 16HBE and the human colon cancer HCT116). On normal cells, free
DOXO was found to be more cytotoxic than DOXO-loaded nanogels at the higher tested concentration
and, only on cancer cells, DOXO-loaded nanogels show similar or slightly higher cytotoxicity values than
free DOXO, suggesting potential advantages in the treatment of cancer. These results were supported by
fluorescence microscopy studies, which suggested that DOXO-loaded nanogels provide an extracellular
reservoir of the drug, which is gradually released and internalized within the cells.
Received 31st July 2014
Accepted 15th October 2014
DOI: 10.1039/c4tb01268b
www.rsc.org/MaterialsB
and physical characteristics as it occurs in conventional phar-
maceutical forms.1,2
Introduction
The most common modied release systems are based on
polymeric supports where the drug can be embedded into the
polymeric matrix (drug–polymer complexes)3–5 or covalently
bonded to the polymeric backbone (drug–polymer conjugates).6
Dendrimers are uniform, nanometer-sized, three-dimen-
sional synthetic polymers, characterized by a branched archi-
tecture and spherical shape.7,8 Owing to their peculiar features
(high level of branching, multivalency, clearly dened molec-
ular weight, globular structure), they are widely used as drug
carriers of both pharmaceuticals and nucleic acids for gene
therapy.9 Polypropylene imine (PPI) dendrimers are polyalkyl
amines which present a core of tertiary amines and primary
amines as surface groups. These terminal amino residues can
be conveniently functionalized with various chemical groups.10
Meijer's research group at Eindhoven Technical University
synthesized adamantylurea-functionalized PPI dendrimers by
making PPI terminal amino groups react with 1-adamantyl
isocyanate. The adamantylurea portion proved to be able to
bind ureidoacetic acids through multiple non-covalent inter-
actions.11 In order to evaluate the possibility of host–guest
interactions between these surface-modied PPI dendrimers
During the past 40 years, research in the pharmaceutical eld
has focused on the development of formulations able to release
drugs into an organism at a controlled quantity and speed,
since there is a direct relationship between a drug's concen-
tration in the blood and its therapeutic action. For this purpose,
non-conventional dosage forms have been developed and
implemented with a modied release (Drug Delivery Systems,
DDS). In such systems the release of the active ingredient and
the relative blood level trends depend on the technological
features of the formulations, and not on the drug's chemical
aDipartimento di Biotecnologie, Chimica e Farmacia and European Research Centre for
`
Drug Discovery and Development, Universita degli Studi di Siena, Via A. Moro, 53100
Siena, Italy. E-mail: andrea.cappelli@unisi.it; Fax: +39-0577-234333; Tel: +39-0577-
234320
bIstituto per lo Studio delle Macromolecole (CNR), Via E. Bassini 15, 20133 Milano,
Italy
cDipartimento di Scienze
e Tecnologie Biologiche, Chimiche e Farmaceutiche
`
(STEBICEF), Universita degli Studi di Palermo, Via Archira 32, 90123 Palermo, Italy
† Electronic supplementary information (ESI) available: 1H NMR spectrum of
poly-6-BAUP-TM-BF3k compared with those of DOXO and of their mixtures. See
DOI: 10.1039/c4tb01268b
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B

View Article Online
Journal of Materials Chemistry B
Paper
Scheme 1 Structures of some key benzofulvene derivatives.
(host) with small molecules (guest), Meijer and coworkers loaded by means of interactions with the bis(adamantylurea)
designed specic ureidoacetic acids capable of establishing pincer (BAUP, synthetic dynamic receptor) anchored to the
simultaneous interactions with the tertiary amine (charge- polybenzofulvene backbone.
assisted H-bond) and ureido groups (H-bonds) of these
macromolecules. In this way they developed an interesting
supramolecular system based on a pincer-shaped “dynamic
synthetic receptor” able to incorporate ureidoacetic acids
Experimental section
Synthesis
Melting points were determined in open capillaries in a Gal-
lenkamp apparatus and are uncorrected. Merck silica gel 60
(230–400 mesh) was used for column chromatography. Merck
TLC plates, silica gel 60 F254, were used for TLC. NMR spectra
were recorded with a Varian Mercury-300, a Bruker DRX-400
AVANCE, or a Bruker DRX-600 AVANCE spectrometer in the
indicated solvents (TMS as internal standard): the values of the
chemical shis are expressed in ppm and the coupling
constants (J) in Hz. An Agilent 1100 LC/MSD operating with an
electrospray source was used in mass spectrometry experi-
ments. The starting polybenzofulvene derivative poly 6-PO-BF3k
(ref. 25) and the azide MOEG-9-N3 (ref. 23) were synthesized as
previously reported.
through the establishment of specic and reversible interac-
tions (ionic interactions, hydrogen bonds and van der Waals
interactions).11–14 Subsequently, such a supramolecular system
was implemented with bioactive arylpiperazine ureidoacetic
acid guests to obtain a prototype of a multivalent supramolec-
ular drug.15
The ten-year work performed in our laboratories demon-
strates that a wide range of benzofulvene derivatives (Scheme 1)
polymerize in the apparent absence of catalyst or initiators by
simple removal of solvents to give polymers showing vinyl
structures stabilized by aromatic stacking interactions.16–26
The corresponding polybenzofulvene derivatives show
intriguing features such as rapid and almost quantitative
formation without side reactions and byproducts, high molec-
ular weight, thermoreversible polymerization/depolymerization
behavior, high solubility in the most common organic solvents,
tunable solubility and aggregation behavior in water, liability to
generate nanostructured aggregates, and susceptibility to
molecular manipulation.16–26
Furthermore, we have recently reported that click chemistry
reactions such as the copper(I)-catalyzed alkyne–azide 1,3-
dipolar cycloaddition (CuAAC) can be used to introduce suitably
designed side chains into the polybenzofulvene macromolec-
ular system to obtain polybenzofulvene derivatives tailored for
specic applications.25
The work described in the present paper was aimed at
developing new advanced functional materials by functionali-
zation of preformed polybenzofulvene derivatives (“graing
onto” approach) with synthetic receptors capable of recognizing
and then delivering drug molecules. For this aim, doxorubicin
(DOXO) was used as a model anticancer drug to obtain nano-
structured drug delivery systems containing the drug physically
N0-Trityl-N000-[3-(tritylamino)propyl]propane-1,3-diamine (3)
A mixture of bis(3-aminopropyl)amine (2, Sigma Aldrich, 1.1
mL, 7.9 mmol) in CH2Cl2 (20 mL) containing TEA (3.2 mL, 23
mmol) and trityl chloride (2.2 g, 7.9 mmol) was stirred at room
temperature for 1.5 h and then washed with water. The organic
layer was dried over sodium sulfate and evaporated under
reduced pressure. Purication of the residue by ash chroma-
tography with ethyl acetate as the eluent gave compound 3 (2.3
g, yield 94%, mp 80–82 ꢀC) as a white crystalline solid. 1H NMR
(400 MHz, CDCl3): 1.64 (m, 4H), 2.18 (t, J ¼ 6.7, 4H), 2.65 (t, J ¼
7.0, 4H), 7.16 (m, 6H), 7.25 (m, 12H), 7.46 (m, 12H). MS (ESI):
m/z 616 (M + H+).
3-[Bis[3-(tritylamino)propyl]amino]propan-1-ol (4)
A mixture of compound 3 (2.3 g, 3.7 mmol) in CH3CN (30 mL)
was heated under reux until complete dissolution and then
Na2CO3 (0.80 g, 7.55 mmol) and 3-bromopropan-1-ol (0.83 mL,
9.2 mmol) were added. The reaction mixture was heated under
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
reux for 4 h and then concentrated under reduced pressure. 27.2, 29.6, 36.5, 38.5, 42.6, 49.6, 50.7, 50.8, 51.7, 157.9. MS (ESI):
The residue was partitioned between CH2Cl2 and H2O and the m/z 569 (M + H+).
organic layer was dried over sodium sulfate and evaporated
under reduced pressure. Purication of the residue by ash
Poly-6-BAUP-TM-BF3k
chromatography with ethyl acetate as the eluent gave
compound 4 (1.5 g, yield 60%) as a waxy solid. H NMR (400
In a microwave tube, a mixture of poly-6-PO-BF3k25 (73 mg, 0.22
mmol in monomer units) in THF (5.0 mL) containing synthon 1
(250 mg, 0.44 mmol), CuBr (6.0 mg, 0.042 mmol), and DIPEA
(0.011 mL, 0.063 mmol) was exposed to microwave irradiation
in a CEM Discover instrument for 40 min (4 ꢁ 10 min, T ¼
1
MHz, CDCl3): 1.63 (m, 6H), 2.12 (t, J ¼ 6.9, 4H), 2.44 (t, J ¼ 7.7,
4H), 2.60 (t, J ¼ 5.5, 2H), 3.72 (t, J ¼ 5.2, 2H), 7.17 (m, 6H), 7.26
(m, 12H), 7.46 (m, 12H). 13C NMR (150 MHz, CDCl3): 28.5, 28.7,
42.4, 52.8, 55.2, 64.8, 71.4, 126.7, 128.4, 129.2, 146.6. MS (ESI):
m/z 674 (M + H+).
ꢀ
60 C, W ¼ 50) and nally evaporated under reduced pressure.
The residue was dissolved with CHCl3 (20 mL) and washed with
water (15 mL) and 33% NH4OH (5.0 mL). The organic layer was
dried over sodium sulfate and concentrated under reduced
pressure. The polymer was puried by precipitation with diethyl
ether from a solution of the nal residue in chloroform and
dried under reduced pressure to obtain poly-6-BAUP-TM-BF3k
as a white solid (0.17 g, yield 86%).
N0-(3-Azidopropyl)-N000-trityl-N0-[3-(tritylamino)propyl]
propane-1,3-diamine (5)
To a solution of compound 4 (0.58 g, 0.86 mmol) in CH2Cl2 (20
mL), TEA (0.40 mL, 2.9 mmol) and CH3SO2Cl (0.20 mL, 2.6
mmol) were added in sequence at room temperature. The
resulting mixture was stirred at room temperature for 30 min
and then washed with water. The organic layer was dried over
Poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1)
sodium sulfate and evaporated under reduced pressure. The In a microwave tube, a mixture of poly-6-PO-BF3k25 (0.13 g, 0.39
residue was dissolved in DMF (15 mL) and NaI (0.50 g, 3.3 mmol in monomer units) in THF (5.0 mL) containing synthon 1
mmol) and NaN3 (0.40 g, 6.15 mmol) were added to the solution. (0.022 g, 0.039 mmol), CuBr (0.011 g, 0.077 mmol), and DIPEA
The reaction mixture was stirred at room temperature for 48 h (0.014 mL, 0.080 mmol) was stirred at room temperature for 24
and then concentrated under reduced pressure. The residue h. Then, 28-azido-2,5,8,11,14,17,20,23,26-nonaoxaoctacosane
was partitioned between CH2Cl2 and a saturated NH4Cl solu- (MOEG-9-N3, see ref. 23, 0.35 g, 0.77 mmol) was added and the
tion. The organic layer was dried over sodium sulfate and resulting mixture was exposed to microwave irradiation in a
evaporated under reduced pressure. Purication of the residue CEM Discover instrument for 40 min (4 ꢁ 10 min, T ¼ 60 ꢀC, W
by ash chromatography with ethyl acetate–petroleum ether ¼ 50). Finally, the reaction mixture was concentrated under
(3 : 7) as the eluent gave compound 5 (0.39 g, yield 65%) as a reduced pressure to give a residue, which was dissolved in
pale yellow oil. 1H NMR (400 MHz, CDCl3): 1.56 (m, 6H), 2.11 (t, CHCl3 and washed with water and 33% NH4OH. The organic
J ¼ 6.8, 4H), 2.38 (m, 6H), 3.17 (t, J ¼ 6.5, 2H), 7.15 (t, J ¼ 7.3, layer was dried over sodium sulfate and concentrated under
6H), 7.24 (t, J ¼ 7.6, 12H), 7.46 (d, J ¼ 7.8, 12H). 13C NMR (150 reduced pressure. The polymer was puried by precipitation
MHz, CDCl3): 27.2, 28.7, 42.7, 50.1, 51.4, 52.9, 71.4, 126.6, 128.6, with n-hexane from a solution of the nal residue in chloroform
129.4, 146.8. MS (ESI): m/z 699 (M + H+).
and dried under reduced pressure to obtain poly-6-MOEG-9-TM-
BF3k-co-6-BAUP-TM-BF3k as
a dark yellow rubbery solid
(0.17 g).
1,10-[3,30-(3-Azidopropylazanediyl)bis(propane-3,1-diyl)]bis(3-
adamantylurea) (1)
SEC-MALS
A mixture of compound 5 (0.30 g, 0.43 mmol) in TFA (4.0 mL)
was stirred at room temperature for 2 h. Then, TFA was removed
under reduced pressure and the residue was diluted with CHCl3
and extracted with water. The aqueous phase was concentrated
under reduced pressure and the residue was dissolved in THF (8
mL). To this solution, 1-adamantyl isocyanate (Sigma Aldrich,
0.245 g, 1.38 mmol) and K2CO3 (0.195 g, 1.41 mmol) were added
and the resulting mixture was stirred at room temperature for
24 h. Finally, the reaction mixture was concentrated under
reduced pressure, diluted with water and extracted with CHCl3.
The MWD characterization of the new polybenzofulvene deriv-
atives was performed by a MALS light scattering photometer
connected to a SEC chromatographic system. The SEC-MALS
system and the corresponding experimental conditions were
identical to those used in our previous studies16,17 and are not
reported in detail here.
Preparation of doxorubicin loaded poly-6-MOEG-9-TM-BF3k-
co-6-BAUP-TM-BF3k (9 : 1) nanogels
The organic layer was dried over sodium sulfate and evaporated DOXO–HCl (5.0 mg, 0.0086 mmol) was dissolved in 1.0 mL of
under reduced pressure. Purication of the residue by ash DMSO and neutralized with 4 equivalents of TEA. The resulting
chromatography with ethyl acetate–methanol (8 : 2) as the DOXO solution was mixed with a polymer dispersion (25 mg in
eluent gave the expected bis(adamantylurea) synthon 1 (0.19 g, 1.5 mL of DMSO) obtained by using an ultrasonic bath, and the
yield 78%, mp 174–176 ꢀC) as a white crystalline solid. 1H NMR mixture was added drop-wise to 10 mL of double distilled water
(600 MHz, CDCl3): 1.65 (s, 16H), 1.73 (m, 2H), 1.96 (s, 12H), 2.04 and sonicated for 10 min. The DOXO-poly-6-MOEG-9-TM-BF3k-
(s, 6H), 2.48 (m, 6H), 3.17 (t, J ¼ 6.7, 4H), 3.33 (t, J ¼ 6.7, 2H), co-6-BAUP-TM-BF3k (9 : 1) dispersion was dialyzed for 24 h
4.67 (br s, 2H), 5.44 (br s, 2H). 13C NMR (150 MHz, CDCl3): 25.8, against 1.0 L of double distilled water using Spectra Por Dialysis
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B

View Article Online
Journal of Materials Chemistry B
Paper
oating tubes with molecular weight cut-off (MWCO) of 500 Da. withdrawn from the outside of the dialysis membrane and
Aer 24 h, the nanogel dispersion was collected, frozen by replaced with an equal amount of fresh medium. The with-
immersion in liquid nitrogen and freeze-dried from water in the drawn samples were analyzed by HPLC in order to determine
presence of threalose as a cryoprotector. DOXO-loaded poly-6- the released intact drug amount, using a Waters Breexe System
MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels were Liquid Chromatograph equipped with a Waters 717 Plus Auto-
obtained with a yield of 90 weight%. Empty nanogels were sampler (50 mL injection volume), and using a Shimadzu UV-vis
prepared by using the above reported procedure in the absence HPLC detector connected to a computerized workstation. As
of DOXO.
column a reversed-phase Gemini C18 Phenomenex (5 mm, 4.6 ꢁ
250 mm column with a pre-column H5ODS-10CS) was used. The
mobile phase consisted of 10 mM of (NH4)HPO4 and 5 mL of
TEA (pH 4.0 adjusted with orthophosphoric acid) and acetoni-
trile (68/32 v/v). The eluate was monitored at 485 nm with a ow
rate of 1.0 mL minꢂ1. A calibration curve of DOXO-HCl was used
for drug quantization. Data were corrected taking into account
the dilution procedure. Each experiment was carried out in
triplicate and the results were in agreement within ꢄ5% stan-
dard error.
Dynamic light scattering (DLS) analysis and z potential
measurements
The mean diameter, width of distribution (polydispersity index,
PDI) and z potential of the nanogels were measured at 25 ꢀC
using a Zetasizer NanoZS instrument tted with a 532 nm laser
at a xed scattering angle of 173ꢀ. The intensity-average
hydrodynamic diameter (size in nm) and polydispersity index
(PDI) of DOXO-loaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-
BF3k (9 : 1) and unloaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-
TM-BF3k (9 : 1) nanogels (0.2 mg mLꢂ1) were measured in
double distilled water. The z potential (mV) was calculated from
the electrophoretic mobility using the Smoluchowsky relation-
ship and assuming that Ka [ 1 (where K and a are the Debye–
Cytotoxicity evaluation
The cytotoxicity was assessed by the tetrazolium salt (MTS)
assay on both human colon cancer (HCT116) and human
bronchial epithelial (16HBE) cell lines (purchased from Istituto
Zooprolattico Sperimentale della Lombardia e dell'Emilia
¨
Huckel parameter and particle radius, respectively). Each
experiment was performed in triplicate.
Scanning electron microscopy (SEM)
Freeze-dried DOXO-loaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-
TM-BF3k (9 : 1) nanogels were visualized using a scanning
electron microscope ESEM Philips XL30. Samples were dusted
on a double sided adhesive tape previously applied on a stain-
less steel stub. The MNPs-FA were then sputter-coated with gold
prior to microscopy examination.
Determination of loaded DOXO amount into nanogels and
drug release studies
The amount of DOXO loaded into poly-6-MOEG-9-TM-BF3k-co-
6-BAUP-TM-BF3k (9 : 1) nanogels was determined by UV spec-
troscopy. Samples were prepared by dispersing known amounts
of DOXO-loaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k
(9 : 1) nanogels in double distilled water acidied at pH ꢃ 4 and
measuring the absorbance of DOXO at 480 nm. A calibration
curve was obtained for serially diluted concentrations of
DOXO–HCl in bidistilled water at pH ꢃ 4. The content of drug
loaded into the nanogels was expressed as the amount of loaded
DOXO per unit mass of dry polymer, and was foundto be 3.5%,
while the encapsulation efficiency was calculated to be 70%.
For drug release studies, an appropriate amount (5 mg) of
freeze dried DOXO-loaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-
TM-BF3k (9 : 1) nanogels and DOXO–HCl (0.2 mg) alone (as
positive control) were suspended in phosphate buffer PBS at pH
7.4 or 5.5 (5 mL) and transferred inside a oating Spectra/Por
dialysis membrane (MWCO 1000 Da). This dialysis membrane
was immersed into PBS at pH 7.4 or 5.5 (10 mL) and incubated
at 37 ꢀC under continuous stirring (100 rpm) in a Benchtop
808C Incubator Orbital Shaker model 420, for 48 h. At sched-
Scheme 2 Synthesis of bis(adamantylurea) synthon 1. Reagents: (i)
trityl chloride, TEA, CH2Cl2; (ii) 3-bromopropan-1-ol, Na2CO3, CH3CN;
(iii) methanesulfonyl chloride, TEA, CH2Cl2; (iv) NaN3, NaI, DMF; (v)
uled time intervals, aliquots of the external medium (1 mL) were TFA; (vi) adamantyl isocyanate, K2CO3, THF.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
Scheme 3 Click chemistry functionalization of poly-6-PO-BF3k with synthon 1. Reagents: (i) synthon 1, CuBr, DIPEA, THF; (ii) CH3(OCH2-
CH2)9N3, CuBr, DIPEA, THF.
Romagna, Italy) using a commercially available kit (Cell Titer 96 in normal medium were cultured in a 96 well plate at 37 ꢀC in an
Aqueous One Solution Cell Proliferation assay, Promega). Cells atmosphere of 5% CO2 for 24 h. Aer 24 h the medium was
were seeded in 96 well plates at a density of 25 ꢁ 104 cells per replaced with 200 mL of fresh culture medium containing
well and grown in Dulbecco's Minimum Essential Medium DOXO-loaded
poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k
(DMEM) with 10% FBS (fetal bovine serum) and 1% of peni- (9 : 1) nanogels and free DOXO at a nal drug concentration of
cillin–streptomycin (10 000 U mLꢂ1 penicillin and 10 mg mLꢂ1 30 mM, and cells were incubated for 4, 24 and 48 h. Aer each
streptomycin) at 37 ꢀC in 5% CO2 humidied atmosphere. Aer incubation period, the medium was removed and the cell
24 h of cell growth the medium was replaced with 200 mL of monolayer was washed twice with DPBS (Dulbecco's phosphate-
fresh culture medium containing free DOXO or DOXO-loaded buffered saline) and xed with 4% formaldehyde for 30 min.
poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels Subsequently, the formaldehyde solution was removed and the
at a drug concentration per well equal to 1, 3, 6, 12 and 30 mM. cells were observed by uorescence microscopy. The images
Empty nanogels were incubated at concentrations correspond- were recorded using an Axio Cam MRm (Zeiss).
ing to 10, 50, 100, 200 and 500 mg mLꢂ1. Aer 4, 24 and 48 h
incubation time, DMEM was replaced with 100 mL of fresh
medium and 20 mL of a MTS solution was added to each well.
Plates were incubated for an additional 2 h at 37 ꢀC. Then, the
absorbance at 490 nm was measured using a microplate reader
(Multiskan, Thermo, UK). Pure cell medium was used as a
negative control. Results were expressed as percentage reduc-
tion of the control cells. All culture experiments were performed
in triplicate.
Statistical analysis
A one-way analysis of variance (ANOVA) was applied to compare
different groups. Data were considered statistically signicant
with a value of P < 0.05 and differences between different groups
were compared using a posteriori Bonferroni t-tests. All values
are expressed as the average of three experiments ꢄ standard
deviation.
Results and discussion
Cell drug uptake studies
Synthesis
The cellular uptake of DOXO and distribution of the polymer
were evaluated by uorescence microscopy (Zeiss “AXIO Vert. The synthetic work began with the preparation of azide syn-
A1” Microscope Inverted) analysis. In particular, for the uptake thon 1 (Scheme 2) designed starting from the supramolecular
assay on HCT116 cell lines, 25 ꢁ 104 cells per well maintained system introduced by Meijer and coworkers as a model.11–14
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B

View Article Online
Journal of Materials Chemistry B
Paper
Table 1 Macromolecular features of the new polybenzofulvene derivatives compared with those shown by poly-6-PO-BF3k (used in the
present work) and by previously reported poly-6-MOEG-9-TM-BF3k-GO (obtained by the “grafting onto” approach)
Mp (kg molꢂ1
)
Mwa (kg molꢂ1
)
Mw/Mn
Rg (nm)
Kd (nm)
ad
b
c
Polymer
Poly-6-BAUP-TM-BF3k
Poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-
BF3k (9 : 1)
716
283
1061
436
3.0
3.6
25.4
20.7
0.0226
0.0596
0.48
0.42
Poly-6-MOEG-9-TM-BF3k-GOe
Poly-6-PO-BF3k
484
1045
821
874
8.9
2.0
33.6
33.5
0.0249
0.0099
0.50
0.58
a
b
c
Mw: weight-average molecular weight. Dispersity where Mn denotes the numeric-average molecular weight. Rg: radius of gyration, i.e. the
d
e
dimension of the macromolecules. K and a: intercept and slope of conformation plot. See ref. 25.
Our design was aimed at introducing (by the click chemistry chloride and reacted with sodium azide in the presence of
reaction CuAAC) multiple dynamic binding sites (pincer-sha- sodium iodide to give azide 5. Aer deprotection with tri-
ped receptors) on a polybenzofulvene backbone with the goal uoroacetic acid (TFA), the terminal amino groups of 5
of creating a polymer host system able to interact with small were made to react with adamantyl isocyanate to obtain
molecules (guests) through specic and reversible bis(adamantylurea) derivative 1.
interactions.
The bis(adamantylurea) pincer was initially developed and
Bis(adamantylurea) synthon 1 was prepared from commer- demonstrated to be intriguingly functional on PPI dendrimer
cially available amine 2, which was selectively protected with surface,11–14 which is generally recognized to be a particular
trityl chloride to obtain 3. Protected amine 3 was alkylated with environment. Moreover, the work performed by Meijer and
3-bromopropan-1-ol in the presence of sodium carbonate as the coworkers suggests a dynamic nature of the dendrimer–guest
base to afford alcohol 4, which was activated with mesyl interaction,
in
which
the
proximity
of
several
Fig. 1 1H NMR spectrum (CDCl3) of newly synthesized poly-6-BAUP-TM-BF3k compared with those of synthon 1 and of previously reported
poly-6-PO-BF3k.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
bis(adamantylurea) pincers and the so-called “de Gennes dense
packing” may play a signicant role.27 Thus, the evaluation of
the effects of the introduction of the bis(adamantylurea) pincer
into a different polymer environment required the development
of a systematic approach based on the preparation of homo-
polymers and copolymers, both equipped with synthetic
receptors but with different densities.
The functionalization of the polybenzofulvene backbone
with BAUP was performed by reaction of clickable macromo-
lecular precursor poly-6-PO-BF3k with synthon 1 (Scheme 3) in
THF in the presence of CuBr and DIPEA to afford the homo-
polymer poly-6-BAUP-TM-BF3k. As previously reported,25 the
optimization of our CuAAC conditions started from the
consideration that poly-6-PO-BF3k is liable to depolymerize
when heated in the presence of solvents. However, in the
above-mentioned conditions, the CuAAC reaction proceeded
even at room temperature and became virtually complete by
microwave exposure without signicant depolymerization.
The preparation of the designed copolymer poly-6-MOEG-9-
TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) was carried out by per-
forming a partial functionalization with synthon 1 at room
temperature followed by a nal and virtually complete
Properties of the new polybenzofulvene derivatives
Homopolymer poly-6-BAUP-TM-BF3k was obtained as a white
solid by precipitation with diethyl ether from a chloroform
solution and found to retain the good solubility features
generally shown by the polybenzofulvene derivatives in the
most common organic solvents, but it was virtually
insoluble in water. This expected behavior agrees perfectly
with the lipophilic character of the graed side chain and
restricted the study of this homopolymer to the organic
environment.
On the other hand, the copolymer poly-6-MOEG-9-TM-BF3k-
co-6-BAUP-TM-BF3k (9 : 1) was obtained as a light brown sticky
solid by precipitation with n-hexane from a solution of the
copolymer in chloroform and, similarly to the homopolymer,
showed good solubility in the most common organic solvents.
In dichloromethane or chloroform the copolymer dissolved very
rapidly, while the interaction with water was found to be the
main distinctive feature between the homopolymer and the
copolymer. Very interestingly, the latter interacts with water
generating a transparent hydrogel, which was believed to
constitute an intriguing aggregation state to evaluate the
potential of BAUP as a synthetic dynamic receptor in the bio-
logical environment.
microwave-assisted graing with
a monomethyl oligo-
(ethylene glycol) (MOEG-9) chain bearing an azide group
(MOEG-9-N3).25
Fig. 2 13C NMR spectrum (CDCl3) of newly synthesized poly-6-BAUP-TM-BF3k compared with those of synthon 1 and of macromolecular
precursor poly-6-PO-BF3k.
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B

View Article Online
Journal of Materials Chemistry B
Paper
Fig. 3 1H NMR spectrum (DMSO-d6) of newly-synthesized poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) compared with those of
synthon 1 (CDCl3) and of previously reported poly-6-MOEG-9-TM-BF3k (CDCl3). In the spectrum of poly-6-MOEG-9-TM-BF3k-co-6-BAUP-
TM-BF3k (9 : 1), m characters mark the signals attributed to low amounts of monomer 6-MOEG-9-TM-BF3k produced by depolymerization and
the round-cornered box encloses the ones attributed to adamantane moieties.
of hydrogen bond donors and acceptors in the bis(adamanty-
lurea) pincer of the homopolymer and the copolymer required
the use of a solvent possessing strong H-bond acceptor features.
In particular, DMF was used for the couple of the newly
synthesized polybenzofulvene derivatives, while an (8 : 2)
mixture of THF–(DMF + 0.01 M LiBr) was employed in the case
of poly-6-MOEG-9-TM-BF3k and THF was sufficient for macro-
molecular precursor poly-6-PO-BF3k.25
The results summarized in Table 1 suggest that the CuAAC
reaction of poly-6-PO-BF3k with synthon 1 produces small
changes in the molecular weight distribution (MWD) of the
homopolymer poly-6-BAUP-TM-BF3k. On the other hand, a
signicant decrease in the molecular weight and an increase
in dispersity Mw/Mn were observed when poly-6-PO-BF3k was
reacted in sequence with synthon 1 (at room temperature)
and then with MOEG-9-N3 with the aid of microwaves without
the production of signicant monomer concentrations.
These observations conrm that the polybenzofulvene
backbone is susceptible to breaking under the conditions
used in the CuAAC reaction,25 but the physicochemical
features of the graed side chains appear to play a role. We
assume that the stability of the polybenzofulvene backbone is
Fig. 4 Structures of doxorubicin (DOXO) and bis(adamantylurea)
pincer (BAUP).
Molecular weight distribution of the new polybenzofulvene
derivatives
The molecular weight distribution (MWD) of both newly
synthesized poly-6-BAUP-TM-BF3k and poly-6-MOEG-9-TM-
BF3k-co-6-BAUP-TM-BF3k (9 : 1) was determined by means of a
multi-angle laser light scattering (MALS) detector connected to
a size exclusion chromatography (SEC) apparatus. The presence
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
Fig. 5 NOESY spectrum (600 MHz, CDCl3–DMSO-d6 1 : 1) of DOXO and poly-6-BAUP-TM-BF3k mixture; m characters mark the signals
attributed to low amounts of monomer 6-BAUP-TM-BF3k produced by depolymerization and the round-cornered box encloses the relevant
cross peaks.
Table 2 Mean diameter (Z-average), polydispersity index (PDI) and zeta potential values of DOXO-loaded poly-6-MOEG-9-TM-BF3k-co-6-
BAUP-TM-BF3k (9 : 1) and unloaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels as measured in ultrapure watera
Sample
Z-av. (nm)
PDI
Z-Pot. (mV)
DOXO-loaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1)
Unloaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1)
213
266
0.52
0.48
3.2
3.3
a
Measurements were performed immediately aer the preparation and also aer 72 h storage at 25 ꢀC in the same aqueous media to verify the
physical stability of the micelles. It is interesting to note that in both cases (loaded and unloaded nanogels) the hydrodynamic diameter of the
samples does not change aer 72 h of incubation.
affected by the intrachain interactions established by the side obtained when the appropriate conditions were used in the
chains that are attractive in the case of homopolymer poly-6- CuAAC graing reaction.25 Thus, the structures of both homo-
BAUP-TM-BF3k and mainly repulsive in poly-6-MOEG-9-TM- polymer poly-6-BAUP-TM-BF3k and copolymer poly-6-MOEG-9-
BF3k-co-6-BAUP-TM-BF3k (9 : 1).
TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) were studied by NMR
spectroscopy in order to assess the correct functionalization of
the macromolecular precursor poly-6-PO-BF3k.
Structures of the new polybenzofulvene derivatives
The 1H NMR spectrum of the homopolymer poly-6-BAUP-
TM-BF3k (Fig. 1) is dominated by the very broad and intense
adamantane signals, testifying that our click chemistry CuAAC
conditions were successful in functionalizing the poly-
benzofulvene backbone.
The structure of the macromolecular precursor poly-6-PO-BF3k
was previously characterized by NMR spectroscopy.25 The NMR
studies conrmed both the retention of the spontaneous vinyl
(1,2) polymerization mechanism and the presence of intact
clickable propargyl groups, which were not involved in the
thermoreversible polymerization/depolymerization mechanism
as conrmed by the depolymerization studies. Moreover, these
studies provided evidence about the exhaustive graing
This result was conrmed by comparing the 13C NMR spec-
trum of the homopolymer with those of parent macromolecule
poly-6-MO-BF3k and synthon 1 (Fig. 2). In this comparison,
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B

View Article Online
Journal of Materials Chemistry B
Paper
Fig. 7 SEM images of DOXO-loaded poly-6-MOEG-9-TM-BF3k-co-
6-BAUP-TM-BF3k (9 : 1) nanogels at magnification of 50 000ꢁ (left)
and 25 000ꢁ (right).
Fig. 6 DLS size distribution histograms of DOXO-loaded poly-6-
MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) (a) and unloaded
poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) (b) nanogels.
possess the structural prerequisites (i.e. H-bond acceptors and
donors and an aromatic moiety, Fig. 4) to interact with the
BAUP dynamic synthetic receptors linked to the aromatic poly-
benzofulvene backbone of poly-6-BAUP-TM-BF3k and poly-6-
MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k.
In order to evaluate a possible interaction between the
therapeutically relevant DOXO and BAUP in the newly
synthesized polybenzofulvene derivatives, 1H NOESY experiments
were performed with homopolymer poly-6-BAUP-TM-BF3k as a
macromolecular model of BAUP in CDCl3–DMSO-d6 (1 : 1) as
the solvent. Preliminary results appeared to support a poten-
tially weak interaction of DOXO alcoholic groups with the ada-
mantylurea moieties. In fact, dipolar cross peaks were observed
between the signals attributed to BAUP adamantane protons
and those attributed to the alcoholic hydroxyl protons of
poly-6-PO-BF3k was used as the model of the polymeric back-
bone and synthon 1 as the model of the side chains.
The 13C NMR spectrum of homopolymer poly-6-BAUP-TM-BF3k
shows indeed dominating signals in the up-eld region and the
comparison with the spectrum of synthon 1 allowed the assign-
ment of these signals to BAUP. On the other hand, the signals
attributable to the carbon atoms belonging to the polymeric
backbone are very broad and appear as baseline modulations,
suggesting that homopolymer poly-6-BAUP-TM-BF3k undergoes a
pronounced self-aggregation that drastically decreased the reso-
lution of carbon atoms belonging to the polymeric backbone. This
result can be explained on the basis of the presence of H-bond
donors and acceptors in the side chains that may establish both
interchain and intrachain interactions.
Conversely, the 1H NMR spectrum of the copolymer poly-6-
MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) is dominated
by the signals attributed to the MOEG-9 side chains (Fig. 3),
as expected on the basis of the higher proportion with respect
to BAUP. However, the comparison shown in Fig. 3 demon-
strates that the signals attributable to the adamantane
moieties are clearly visible in the up-eld region of the
spectrum, conrming the co-monomeric structure of the
copolymer.
Selection of the small drug molecule to be loaded in the newly
synthesized polybenzofulvene derivatives
The ability of polybenzofulvene derivatives to complex high
molecular weight bioactive molecules, such as immunoglobulin
G (IgG), has been already investigated in a physical strong
hydrogel obtained with a polybenzofulvene molecular brush of
rst generation bearing only MOEG-9 side chains.28 In this
system, the protein–polymer interaction was assumed to occur
by aspecic adsorption onto the surface of the hydrogel parti-
cles. Differently, the copolymer poly-6-MOEG-9-TM-BF3k-co-6-
BAUP-TM-BF3k (9 : 1) used in the present study bears BAUP
moieties anchored to the polybenzofulvene backbone that may
potentially act as synthetic dynamic receptors for low molecular
weight drug molecules.
Although BAUP was demonstrated to interact with ureido-
acetic acid derivatives when it was expressed on the PPI den-
Fig. 8 Drug release profiles of DOXO-loaded poly-6-MOEG-9-TM-
BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels (-) and free DOXO (A) in
drimer surface,11–14 doxorubicin (DOXO) was envisioned to PBS solution at pH 7.4 (a) and pH 5.5 (b).
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
Fig. 9 Cell viability % of unloaded (grey), DOXO-loaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels (red) and free DOXO
(blue) on human bronchial epithelial (16HBE) cells (a–c) and human colon cancer (HCT116) cells (d–f), at DOXO concentrations of 1, 3, 6, 12 and 30 mM.
For unloaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels the polymer concentrations correspond to 20, 50, 100, 200 and 500
mg mLꢂ1 respectively. Samples were incubated for 4 h (a and d), 24 h (b and e) and 48 h (c and f). The results are reported as the mean ꢄ SD (n ¼ 6).
doxorubicin (Fig. 5). It should be emphasized that the system interaction occurring between the drug molecules and the
used in these preliminary interaction studies represented a polymeric matrix or, as we presume, with the bis(adamanty-
special case and the results could not be generalized, but they lurea) pincer system. Actually, the unloaded drug molecules are
were considered however promising enough to proceed with the eliminated by diffusion outside the dialysis membrane. With
subsequent steps of the work.
this method, a loaded drug amount equal to 3.5 wt% was
obtained with respect to drug-loaded nanogels. The obtained
nanocarriers were characterized by DLS analysis and showed
colloidal size (DLS data are reported in Table 2) but, as expected,
a large size distribution value (Fig. 6) due to self-aggregation
phenomena. In fact, SEM images (Fig. 7) showed micronized
aggregates of colloidal particles.
Drug release studies were performed by using the same
dialysis method in order to provide experimental proof of the
ability of the poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k
(9 : 1) nanogels to release the encapsulated model drug, i.e.
doxorubicin. Release studies were carried out by simulating pH
conditions upon parenteral administration. Thus, nanogels
were dispersed in bidistilled water, placed into the dialysis bag,
which was immersed in external phosphate buffer PBS solution
both at pH 7.4 (mimicking interstitial uids and plasma pH
value) and at pH 5.5 (mimicking the intracellular environment
pH value). Then, the amount of released DOXO in the external
medium, at pre-determined time intervals, was quantied by
Preparation and characterization of doxorubicin loaded poly-
6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels
In this study, the previously discussed ability of poly-6-MOEG-9-
TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) to interact with low
molecular weight drug molecules was applied to prepare
colloidal drug delivery systems delivering the model anti-cancer
drug doxorubicin. The drug loading procedure was carried out
by using an already described so-interaction method (known
as dialysis method) consisting of mixing the polymer dispersion
with the drug solution in an organic solvent and then placing
this mixture into a dialysis membrane against water.29–31 The
slow diffusion of the organic solvent from the internal
compartment causes the polymer aggregation into nanogels
entrapping the drug molecules. This is related to the lower
solubility of the polymer in water than in the organic solvent.
On the other hand the drug loading is generated by physical
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B

View Article Online
Journal of Materials Chemistry B
Paper
Fig. 10 Fluorescence microscopy images of HCT116 cells incubated with free DOXO (A, C and E), and DOXO-loaded poly-6-MOEG-9-TM-
BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels (B, D and F) for 4 (A and B panel), 24 (C and D panel) and 48 h (E and F panel). DOXO is visualized with
Texas Red filter (red); the polymer is visualized with Fura filter (blue). Magnification is 40ꢁ for all images and the scale bar is 20 mm.
HPLC analysis. The drug release prole was detected for 48 h, as (HCT116). The former is a non-tumoral cell line extensively used
shown in Fig. 8, and the amount of released DOXO was as model normal cells to screen cytotoxicity of novel compounds
expressed as the percentage of the total amount of drug loaded or drug carriers,32,33 the latter is a cancer cell line used to
into the nanogels. These experiments demonstrated that loaded investigate the anti-cancer activity of anti-cancer drugs.34 These
poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels cells were incubated with different amounts of DOXO-loaded
were able to release DOXO in the intact form for a prolonged poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels
period and without a rst burst release, as observed for free corresponding to DOXO concentrations of 1, 3, 6, 12 and 30 mM,
DOXO.
for 4, 24 and 48 h. The results are shown in Fig. 9a–f, in terms of
cell viability (%) as a function of sample concentration.
The obtained results demonstrated that poly-6-MOEG-9-TM-
BF3k-co-6-BAUP-TM-BF3k (9 : 1), as a free polymer, is not cyto-
toxic towards normal and cancer cells at all tested concentra-
tions. Differently, DOXO-loaded poly-6-MOEG-9-TM-BF3k-co-6-
BAUP-TM-BF3k (9 : 1) nanogels show a cytotoxicity depending
on incubation time and concentration of DOXO (corresponding
In vitro biological evaluation of anticancer activity
Cytotoxicity of unloaded and DOXO-loaded poly-6-MOEG-9-TM-
BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels and of free DOXO was
evaluated by the MTS assay on two different cell lines, i.e.
human bronchial epithelial (16HBE) and human colon cancer
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
to the concentration of drug loaded into the nanogels). In systems in the form of nanogel particles showing dimensions
particular, on normal cells (16HBE) free DOXO (used as positive around 200 nm for delivering the model anti-cancer drug
control) was found to be more cytotoxic than DOXO-loaded doxorubicin. The resulting nanostructured drug delivery
poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels systems containing physically loaded DOXO (around 3.5% w/w)
at the higher tested concentration. It is interesting to note that were demonstrated to be able to release the drug in the intact
only on cancer cells (HCT116), DOXO-loaded poly-6-MOEG-9- form for a prolonged period and without a rst burst release.
TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels show similar or Moreover, cytotoxicity studies performed with human bronchial
slightly higher cytotoxicity values than free DOXO (incubation epithelial (16HBE) and human colon cancer (HCT116) cell lines
time ¼ 48 h and DOXO concentration ¼ 30 mM, Fig. 9 panel f). revealed that the copolymer showed a low toxic potential,
These data suggest a potential advantage in the treatment of whereas the DOXO-loaded nanostructured delivery systems
cancer, with the aim to achieve the optimal pharmacological show cytotoxicity depending on the incubation time. Interest-
effect and the lowest secondary effects. Then, in order to ingly, on normal cells, free DOXO was found to be more cyto-
investigate a possible correlation between the cytotoxic activity toxic than DOXO-loaded nanogels at the higher tested
of DOXO-loaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k concentration and, only on cancer cells, DOXO-loaded nanogels
(9 : 1) nanogels and the potential ability of this polymer to show similar or slightly higher cytotoxicity values than free
interact with cell membranes, uorescence microscopy studies DOXO suggesting a potential advantage in the treatment of
were performed. HCT116 cells were incubated with either cancer, with aim of achieving the optimal pharmacological
DOXO-loaded
poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k effect and the lowest secondary effects. These results were
(9 : 1) nanogels or free DOXO, at a drug concentration of 30 mM. substantiated by uorescence microscopy studies, which sug-
Aer pre-determined time intervals, the cells were observed gested that DOXO-loaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-
with a uorescence microscope using different lters (Fura to TM-BF3k (9 : 1) nanogels provide an extracellular reservoir of
visualize the polymer and Texas Red to visualize DOXO). The the drug, which is gradually released and internalized within
images of cells aer 4 h (A and B), 24 h (C and D) and 48 h (E and the cells.
F) of incubation are reported in Fig. 10. They showed that DOXO
is visible in the cell nuclei of HCT116 cells just aer 4 h of
incubation when loaded on poly-6-MOEG-9-TM-BF3k-co-6-
Acknowledgements
BAUP-TM-BF3k (9 : 1) nanogels or as the free drug, and it
Thanks are due to Italian MIUR (Ministero dell'Istruzione,
accumulates progressively aer 24 h and 48 h of incubation.
`
dell'Universita e della Ricerca) for nancial support.
Moreover, at the same time we observed the presence of poly-6-
MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1) nanogels adhered
to the cell membrane (visible as blue spots in the brighteld +
Fura + T-Red images). Probably, the positive zeta potential
values shown by the nanogel particles favoured the interaction
with the negatively charged cellular surface. Actually, DOXO-
loaded poly-6-MOEG-9-TM-BF3k-co-6-BAUP-TM-BF3k (9 : 1)
nanogels provide an extracellular reservoir of the drug, which is
gradually released and internalized within the cells.
References
1 R. Langer, Science, 1990, 249, 1527.
2 R. Langer, Nature, 1998, 392, 5.
3 M. Licciardi, G. Pasut, G. Amato, C. Scialabba, A. Mero,
M. Montopoli, G. Cavallaro, O. Schiavon and
G. Giammona, Eur. J. Pharm. Biopharm., 2013, 84, 21.
4 M. Licciardi, G. Pitarresi, G. Cavallaro and G. Giammona,
Mol. Pharmaceutics, 2013, 10, 1644.
Conclusions
`
5 M. Licciardi, G. Montana, M. L. Bondı, A. Bonura,
The polybenzofulvene backbone of the previously published
amphiphilic polymer brush poly-6-MOEG-9-TM-BF3k has been
functionalized with a “synthetic dynamic receptor” composed
of two 1-adamantylurea moieties linked together by means of a
dipropyleneamino bridge as in Meijer's bis(adamantylurea)
pincer. To evaluate the effects of the introduction of BAUP into
the polybenzofulvene environment, homopolymer poly-6-BAUP-
TM-BF3k and copolymer poly-6-MOEG-9-TM-BF3k-co-6-BAUP-
TM-BF3k (9 : 1) were prepared, both equipped with synthetic
C. Scialabba, M. Melis, C. Fiorica, G. Giammona and
P. Colombo, Int. J. Pharm., 2014, 465, 275–283.
6 G. Cavallaro, M. Licciardi, S. Salmaso, P. Caliceti and
G. Giammona, Int. J. Pharm., 2006, 307, 258–269.
7 A. W. Bosman, H. M. Janssen and E. W. Meijer, Chem. Rev.,
1999, 99, 1665–1688.
8 D. A. Tomalia, Prog. Polym. Sci., 2005, 30, 294–324.
9 S. Svenson and D. A. Tomalia, Adv. Drug Delivery Rev., 2005,
57, 2106–2129.
receptors but with different densities. Homopolymer poly-6- 10 A. D'Emanuele and D. Attwood, Adv. Drug Delivery Rev., 2005,
BAUP-TM-BF3k showed good solubility features in the most 57, 2147–2162.
common organic solvents, but it was virtually insoluble in 11 M. W. P. L. Baars, A. J. Karlsson, A. Karlsson, V. Sorokin,
water, while the copolymer interacts with water generating a
transparent hydrogel, which was considered to be an intriguing
B. F. W. de Waal and E. W. Meijer, Angew. Chem., Int. Ed.,
2000, 39, 4262–4265.
aggregation state to evaluate the potential of BAUP as a 12 M. A. C. Broeren, B. F. M. de Waal, M. H. P. van Genderen,
synthetic dynamic receptor in the biological environment. This
functional material was used to prepare colloidal drug delivery
H. M. H. F. Sanders, G. Fytas and E. W. Meijer, J. Am.
Chem. Soc., 2005, 127, 10334–10343.
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B

View Article Online
Journal of Materials Chemistry B
Paper
13 T. M. Hermans, M. A. C. Broeren, N. Gomopoulos, 23 A. Cappelli, M. Paolino, G. Grisci, G. Giuliani, A. Donati,
A. F. Smeijers, B. Mezari, E. N. M. Van Leeuwen,
M. R. J. Vos, P. C. M. M. Magusin, P. A. J. Hilbers,
M. H. P. Van Genderen, N. A. J. M. Sommerdijk, G. Fytas
and E. W. Meijer, J. Am. Chem. Soc., 2007, 129, 15631–15638.
R. Mendichi, A. C. Boccia, F. Samperi, S. Battiato,
E. Paccagnini, E. Giacomello, V. Sorrentino, M. Licciardi,
G. Giammona and S. Vomero, Polym. Chem., 2011, 2, 2518–
2527.
14 T. M. Hermans, M. A. C. Broeren, N. Gomopoulos, 24 A. Cappelli, M. Paolino, G. Grisci, G. Giuliani, A. Donati,
´
P. P. A. M. Van der Schoot, M. H. P. Van Genderen,
N. A. J. M. Sommerdijk, G. Fytas and E. W. Meijer, Nat.
Nanotechnol., 2009, 4, 721–726.
R. Mendichi, A. C. Boccia, C. Botta, W. Mroz, F. Samperi,
A. Scamporrino, G. Giorgi and S. Vomero, J. Mater. Chem.,
2012, 22, 9611–9623.
15 S. Galeazzi, T. M. Hermans, M. Paolino, M. Anzini, 25 A. Cappelli, G. Grisci, M. Paolino, F. Castriconi, G. Giuliani,
L. Mennuni, A. Giordani, G. Caselli, F. Makovec,
E. W. Meijer, S. Vomero and A. Cappelli,
Biomacromolecules, 2010, 11, 182–186.
16 A. Cappelli, G. Pericot Mohr, M. Anzini, S. Vomero,
A. Donati, S. Lamponi, R. Mendichi, A. C. Boccia, F. Samperi,
S. Battiato, E. Paccagnini, M. Gentile, M. Licciardi,
G. Giammona and S. Vomero, Chem. - Eur. J., 2013, 19,
9710–9721.
A. Donati, M. Casolaro, R. Mendichi, G. Giorgi and 26 A. Cappelli, M. Paolino, G. Grisci, G. Giuliani, A. Donati,
F. Makovec, J. Org. Chem., 2003, 68, 9473–9476.
17 A. Cappelli, M. Anzini, S. Vomero, A. Donati, L. Zetta,
R. Mendichi, M. Casolaro, P. Lupetti, P. Salvatici and
G. Giorgi, J. Polym. Sci., Part A: Polym. Chem., 2005, 43,
3289–3304.
A. C. Boccia, F. Samperi, R. Mendichi and S. Vomero,
Reversible polymerization techniques leading to p-stacked
polymers, in p-Stacked Polymers and Molecules, ed. T.
Nakano, Springer, Osaka, Japan, 2014, pp. 51–149.
27 T. Chang, K. Pieterse, M. A. C. Broeren, H. Kooijman,
A. L. Spek, P. A. J. Hilbers and E. W. Meijer, Chem. - Eur. J.,
2007, 13, 7883–7889.
18 A. Cappelli, G. Pericot Mohr, G. Giuliani, S. Galeazzi,
M. Anzini, L. Mennuni, F. Ferrari, F. Makovec,
E. M. Kleinrath, T. Langer, M. Valoti, G. Giorgi and 28 M. Licciardi, M. Grassi, M. Di Stefano, L. Feruglio,
S. Vomero, J. Med. Chem., 2006, 49, 6451–6464.
19 A. Cappelli, S. Galeazzi, G. Giuliani, M. Anzini, A. Donati,
G. Giuliani, S. Valenti, A. Cappelli and G. Giammona, Int.
J. Pharm., 2010, 390, 183–190.
L. Zetta, R. Mendichi, M. Aggravi, G. Giorgi, E. Paccagnini 29 A. Palumbo Piccionello, G. Pitarresi, A. Pace, D. Triolo,
and S. Vomero, Macromolecules, 2007, 40, 3005–3014.
20 A. Cappelli, S. Galeazzi, G. Giuliani, M. Anzini, M. Aggravi,
P. Picone, S. Buscemi and G. Giammona, J. Drug Targeting,
2012, 20, 433–444.
A. Donati, L. Zetta, A. C. Boccia, R. Mendichi, G. Giorgi, 30 M. Licciardi, G. Cavallaro, M. Di Stefano, C. Fiorica and
E. Paccagnini and S. Vomero, Macromolecules, 2008, 41,
2324–2334.
G. Giammona, Macromol. Biosci., 2011, 11, 445–454.
`
31 E. F. Craparo, G. Teresi, M. L. Bondı, M. Licciardi and
21 A. Cappelli, S. Galeazzi, G. Giuliani, M. Anzini, M. Grassi,
G. Cavallaro, Int. J. Pharm., 2011, 406, 135–144.
R. Lapasin, G. Grassi, R. Farra, B. Dapas, M. Aggravi, 32 M. Licciardi, C. Scialabba, G. Cavallaro, C. Sangregorio,
A. Donati, L. Zetta, A. C. Boccia, F. Bertini, F. Samperi and
S. Vomero, Macromolecules, 2009, 42, 2368–2378.
E. Fantechi and G. Giammona, J. Biomed. Nanotechnol.,
2013, 9, 949–964.
22 A. Cappelli, M. Paolino, P. Anzini, G. Giuliani, S. Valenti, 33 M. Licciardi, M. Di Stefano, E. F. Craparo, G. Amato,
M. Aggravi, A. Donati, R. Mendichi, L. Zetta, A. C. Boccia,
F. Bertini, F. Samperi, S. Battiato, E. Paccagnini and
G. Fontana, G. Cavallaro and G. Giammona, Int. J. Pharm.,
2012, 433, 16–24.
S. Vomero, J. Polym. Sci., Part A: Polym. Chem., 2010, 48, 34 G. Pitarresi, F. S. Palumbo, A. Albanese, C. Fiorica, P. Picone
2446–2461.
and G. Giammona, J. Drug Targeting, 2010, 18, 264–276.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2014