Original Papers
found for formulation 1. Finally, the release profile of curcumin
from this formulation showed an interesting prolonged and sus-
tained release profile of curcumin, with the possible application
as a drug delivery system.
Compound 5: Weight: 2.341 g, yield: 99%, yellow oil; 1H‑NMR
(400 MHz, CH3OD) δ 7.25 (m, 5 H), 3.60 (s, 2 H), 3.22 (t, 4 H,
J = 6.4), 2.76 (t, 4 H, J = 6.8), 2.70 (t, 4 H, J = 6.4), 2.40 (t, 4 H, J = 6.4).
Compound 6: Weight: 0.8461 g, yield: 91%, yellow oil; TLC:
AcOEt/MeOH (10/0.8) Rf = 0.4; AcOEt/MeOH (10/1) Rf = 0.2;
1
AcOEt/MeOH (10/1.2) Rf = 0.1; H‑NMR (400 MHz, CDCl3) δ 7.21
Materials and Methods
!
(m, 5 H), 3.58 (s, 2 H), 3.56 (s, 12 H), 3.20 (m, 4 H), 2.74 (t, 4 H,
J = 6.4), 2.68 (t, 8 H, J = 6.8), 2.44 (t, 4 H, J = 6), 2.34 (m, 12 H).
Reagents and standard solutions
Benzylamine and ReagentPlus® 99% were from Sigma-Aldrich;
Buffer saline (PBS) was from Euroclone Company; Cell line MCF-
7 were from American Type Culture Collection. Culture medium
DMEM, Gibco®, was purchased from European Division; Curcuma
e.s 95% curcumin, PM = 368.39, was from Sigma-Aldrich; Dimeth-
yl sulfoxide (DMSO, 99.9%) and bovine serum albumin were from
Sigma-Aldrich. Ethylenediamine ≥ 99.5% by GC was from Sigma-
Aldrich; FBS and glutamine were from the Euroclone Company;
Isoton solution for the electronic cell counter was a Beckman-
Coulter sample. Methyl acrylate 99%, containing ≤ 100 ppm
monomethyl ether hydroquinone as an inhibitor, was from Sig-
ma-Aldrich. Phosphotungistic acid and copper grid of 200 mesh
coated with a carbon film was from Società Italiana Chimici. Po-
tassium carbonate, ≥ 99.0%; Potassium permanganate, ACS re-
agent, ≥ 99.0%; Silica gel, Merk F-254; Single-use material for cell
cultures were from Costar and Sarstedt. SRB and trichloroacetic
acid were from Sigma Chemical Co. TRIS (hidroxymethylamino-
methane) was from Merck.
Cytotoxicity evaluation by the sulforhodamine-B assay
Cytotoxicity of dendrimer G0.5 was evaluated 72 h after exposure
of the cells to various concentrations in a range between 10−3 and
3 × 10−8 M. The MCF-7 cells, in the exponential growth phase,
were diluted in complete DMEM medium to obtain a density of
5000 cells/100 µL and distributed in plates of 96 wells. Subse-
quently, the cells were incubated for 24 h at 37°C in a humidified
atmosphere at 5% CO2 to allow adhesion before exposure. All ex-
periments were performed in triplicate. Inhibition of cell growth
was assayed by sulforhodamine-B (SRB), which is capable of
binding cellular proteins and developing a color directly propor-
tional to cell viability [20].
Briefly, MCF-7cells were fixed with 10% trichloroacetic acid and
incubated for 1 h at + 4°C; then the microplates were washed
with tap water 5–6 times and air-dried. The fixed cells were
stained with 0.4% SRB solution dissolved in 1% acetic acid and
kept at room temperature for 30′. Unbound SRB was removed by
washing with 1% acetic acid solution and the dye bound to the
proteins was extracted with 10 mM unbuffered TRIS base in a
volume of 150 µl/well at room temperature under stirring. Opti-
cal density was read in an automatic 96-well microplate reader
interfaced with the software Microplate Manager/PC version 4.0
(Bio-Rad Laboratories) for automatized analysis of the results at
540 nm to maximize the value of SRB absorption. The results
were processed using the program GraphPad 5 and were com-
pared with those obtained from blank wells that represented
the untreated control.
Synthesis of dendrimer 6
The preparation of dendrimer 6 was carried out using a method
reported in the literature [19] and modified according to our
"
needs. In particular, product 3 (l Fig. 1) was prepared starting
from a solution of benzylamine 1 (0.88 mL; 8 mmol) in methanol
(20 mL) to which methylacrylate 2 (2.6 mL; 28.8 mmol) was
added in a dropwise fashion. The resulting reaction mixture was
stirred under argon for 72 h at 34°C. After this time, the solvent
was removed under reduced pressure and the residue obtained
confirmed to be pure 3 via 1H NMR and was used in the following
step without further purification.
Morphological analysis of G0.5 nanoparticles
by transmission electron microscopy
"
Compound 5 (l Fig. 1) was prepared starting from a solution of 3
NP dispersions were analyzed in terms of morphology and mean
diameter by TEM (Jeol Jem 1010). Ten µL of NP dispersion diluted
10-times was applied to a carbon film-covered copper grid. Most
of the dispersion was blotted from the grid with filter paper to
form a thin film specimen, which was stained with a phospho-
tungstic acid solution 1% w/v in sterile water. The samples were
dried for 3 min and then were examined under a JEOL 1010 elec-
tron microscope and photographed at an accelerating voltage of
64 kV.
Characterization of the G0.5 NPs was done in terms of particle
size, polydispersity index, and ζ-potential. The particle size of
the developed NPs was measured by a DLS, Zetasizer Nano series
ZS90 (Malvern Instruments) equipped with a JDS Uniphase
22 mW He-Ne laser operating at 632.8 nm, an optical fiber-based
detector, a digital LV/LSE-5003 correlator, and a temperature con-
troller (Julabo water-bath) set at 25°C. Time correlation functions
were analyzed to obtain the hydrodynamic diameter of the par-
ticles (Zh) and the particle size distribution (polydispersity index,
PD) using ALV-60X0 software V.3.X provided by Malvern. Auto-
correlation functions were analyzed by distribution, fitting a
multiple exponential to the correlation function to obtain par-
ticle size distributions. In particular, polydispersity values were
calculated for each peak as peak width/mean diameter. Scatter-
(2.235 g; 8 mmol) in methanol (4 mL) to which ethylendiamine 4
(9.95 mL; 148.8 mmol) was added. The reaction mixture was
then stirred for 72 h at 34°C. The solvent was removed under re-
duced pressure and the residue obtained was confirmed to be the
desired 5 by 1H NMR and was used in the following step without
further purification.
"
Compound 6 (l Fig. 1) was prepared following the same proce-
dure reported for the synthesis of product 3. Hence, to a solution
of 5 (459 mg; 1.37 mmol) in methanol (3.4 mL), methylacrylate 2
(1.24 mL; 13.68 mmol) was added dropwise. The reaction mix-
ture was maintained at 34°C for 72 h. After this time, the solvent
was removed under reduced pressure, and the residue was puri-
fied by column chromatography on silica gel 60 (Merck 70–230
mesh) using EtOAc/MeOH at different gradients, starting from
(10/0.8), (10/1), (10/1.2). Pure 6, so obtained, was characterized
by 1H NMR. All TLC (Merck F-254) were visualized via UV fluores-
cence.
Characterization of compounds 3, 5, and 6
Compound 3: Weight: 2.235 g, yield: 99%, yellow oil; 1H‑NMR
(400 MHz, CDCl3) δ 7.26 (m, 5 H), 3.64 (s, 6 H), 3.58 (s, 2 H), 2.79
(t, 4 H, J = 6.8), 2.47 (t, 4 H, J = 7.2).
Falconieri MC et al. New Dendrimer-Based Nanoparticles… Planta Med
Falconieri, Maria Cristina
