Journal of Polymer Science, Part A: Polymer Chemistry
Supporting Information
Biocompatible Stimuli-Responsive Nanogels for Controlled Antitumor Drug Delivery
Garbiñe Aguirrea,#, Eva Villar-Alvarezb,#, Adrián Gonzálezb, Jose Ramosa, Pablo Taboadab,*, Jacqueline
Forcadaa,*
1
POLYMAT, Bionanoparticles Group, Department of Applied Chemistry, UFI 11/56, Faculty of Chemistry,
University of the Basque Country UPV/EHU, Apdo. 1072, Donostia-San Sebastián, 20080, Spain.
2
Condensed Matter Physics Department, Faculty of Physics, 15782 Campus Sur, Universidad de Santiago
de Compostela, Santiago de Compostela, Spain.
#
These authors contributed equally to this work.
Correspondence to: Pablo Taboada (E-mail: pablo.taboada@usc.es) and Jacqueline Forcada (E-mail:
jacqueline.forcada@ehu.eus)
Experimental section
Materials
N-vinylcaprolactam (VCL, Sigma-Aldrich), 2-(diethylamino) ethyl methacrylate (DEAEMA, SigmaAldrich), poly(ethylene glycol) methacrylate (PEGMA, Sigma-Aldrich), dextran T06 (M r ~ 6,000,
Sigma-Aldrich), glycidyl methacrylate (GMA, Sigma-Aldrich), 4-(N,N´-dimethylamino) pyridine
(DMAP, Sigma-Aldrich), 2,2´-azobis(N,N´-dimethyleneisobutyramidine) dihydrochloride (ADIBA,
Wako Chemical Gmbh), hexadecyltrimethylammonium bromide (HDTAB, Sigma-Aldrich),
hydrochloric acid (HCl, Fluka), dimethyl sulfoxide (DMSO, Scharlab), doxorubicin hydrochloride
(DOXO, Sigma-Aldrich), breast cancer cell line (MDA-MB-321, Cell Biolabs), cervical cancer cell
line (HeLa, Cell Biolabs), Oregon green (Molecular Probes), Rhodamine 6G (R6G, Sigma-Aldrich),
4´-6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen), bodipy phalloidin (Invitrogen),
were used as supplied. Different cationic buffers were used to control the pH (all of them
obtained from Sigma-Aldrich): glycine (pH 3), γ -aminobutyric acid (pH 4), pyridine (pH 5–5.9),
bis-tris base (pH 6–7), trizma-HCl (pH 7.1–9), and triethylenamine (pH 10). “Milli-Q” grade water
was used throughout work.
Synthesis and characterization of dextran-methacrylates (Dex-MA)
Different dextran-methacrylates were synthesized and characterized as described in our previous work.1
In this work two dextran-methacrylates were prepared with an average molecular weight of 40,000 and
86 methacrylate groups per chain and with 6,000 g/mol and 1 methacrylate group per chain,
respectively (i.e.; Dex40MA86 and Dex06MA1).
Synthesis of PVCL-based nanogels
PVCL-based nanogels were synthesized by emulsion polymerization in a batch reactor by following the
procedure and recipe described elsewhere.1 Two amounts (6 and 50 wt% with respect to VCL) of
Dex40MA86 macro-cross-linker were used to produce two different nanogels NG1 and NG2,
respectively.
1
Synthesis of PDEAEMA-based nanogel
The synthesis of PDEAEMA-based nanogel (NG3) was carried out following the procedure described
previously,2 using in this case a certain amount of PEGMA as stabilizer. Briefly, 21 g of DEAEMA as main
monomer, 0.37 g of Dex40MA86, 2.1 g of PEGMA and 170 g of DDI water were placed into a 250 mL
jacketed glass reactor. The reactor content was heated at 70 °C, stirred at 300 rpm and purged with
nitrogen for 40 min before starting the polymerization reaction. After adding the initiator (2 wt% of
ADIBA with respect to the main monomer) in 10 g of DDI water, the polymerization reaction was
allowed to continue under nitrogen atmosphere while stirring for 2 h. The reaction mixture was
subsequently cooled to 25 °C maintaining the stirring, and the final dispersion was filtered through glass
wool.
Synthesis of PVCL/PDEAEMA-based core-shell nanogel
PVCL/PDEAEMA-based core-shell nanogel (NG4) was prepared by means of a batch seeded emulsion
polymerization. Firstly, PDEAEMA-based nanogel core was synthesized by batch emulsion
polymerization using dextran-methacrylate (Dex06MA1) and poly(ethylene glycol) methacrylate
(PEGMA) as stabilizers. Briefly, 21 g of DEAEMA as main monomer, 0.37 g of Dex40MA86, 2.1 g of
Dex06MA1, 2.1 g of PEGMA and 170 g of DDI water were placed into a 250 mL jacketed glass reactor.
The reactor content was heated at 70 °C, stirred at 300 rpm and purged with nitrogen for 40 min before
starting the polymerization reaction. After adding the initiator (2 wt% of ADIBA with respect to the main
monomer) in 10 g of DDI water, the polymerization reaction was allowed to continue under nitrogen
atmosphere while stirring for 2 h. The reaction mixture was subsequently cooled to 25 °C maintaining
the stirring. The final nanogels particles obtained were dialyzed against acidic distilled water and
lyophilized using a Telstar Cryodos-80 lyophilizer in order to obtain dry nanogels.
Secondly, taking the advantage of the existence of hydroxyl groups in Dex-MA chains, polymerizable
methacrylate groups were incorporated onto the surface of the previously synthesized PDEAEMA-based
nanogel core following the procedure described in our previous work.1 In brief, 6 g of lyophilized
PDEAEMA-based nanogels were dissolved in 60 mL of DMSO under nitrogen atmosphere. After
dissolution of 0.12 g of DMAP, 0.12 g of GMA was added. The solution was stirred at 30 oC for 48h, after
which adding an equimolar amount of concentrated HCl to neutralize DMAP stopped the reaction. The
final nanogels particles obtained were dialyzed against distilled water and lyophilized.
After modification of the surface of PDEAEMA-based nanogels, a PVCL-based shell was added by means
of a batch seeded emulsion polymerization. 1 g of modified PDEAEMA-based nanogel, 0.5 g of VCL and
90 g of DDI water were placed into a 100 mL jacketed glass reactor, fitter with a reflux condenser,
stainless steel stirrer, sample device, and nitrogen inlet tube reactor. The pH value of the nanogels
dispersion was adjusted to be ~6. The reactor mixture was heated at 40 oC, stirred at 300 rpm and
purged with nitrogen for 1 h before starting the polymerization reaction. After adding the initiator (1 wt
% M) in 10 g of DDI, the polymerization reaction was allowed to continue with stirring for 5 h under
nitrogen atmosphere at 40 oC. The reaction mixture was subsequently cooled to 25 oC and the final
dispersion was filtered through glass wool. Prior to colloidal characterization nanogels was dialyzed
against distilled water.
Colloidal characterization of nanogels
Colloidal characteristics of the nanogels particles synthesized, such as the average hydrodynamic
particle diameters at different temperatures and pHs were measured by photon correlation
spectroscopy (PCS, Zetasizer Nano ZS instrument, Malvern Instruments). In all the measurements, the
2
pH was controlled using different buffered media at an ionic strength of 10 mM. In order to study the
pH-sensitivity, measurements were carried out at 25 oC from pH 3 to pH 10 taking three measurements
every pH unit at a 0.05 wt% particle concentration. The optimized stabilizing time of measurements was
10 min. The volume phase transition pH (VPTpH) was determined as the pH corresponding to the
inflection point in the average hydrodynamic radius versus pH curve.
To study the thermal behavior, measurements were carried out from 10 to 55 oC, taking measurements
every 2 oC, except from 30 oC to 40 oC that they were carried out per grade.
ζ -potential values were calculated from electrophoretic mobility measurements conducted by
electrophoretic light scattering (ELS, Zetasizer Nano ZS instrument, Malvern Instruments) using
Smoluchwski´s equation.3 Previously dialyzed and lyophilized nanogel particles were suspended in
buffered water solutions at a 0.05 wt% particle concentration. Each sample was subjected to three
measurements at 25 oC.
3
Table S1 ζ -potential values for the different nanogels.
ζ-potential (mV)
Reaction
pH 5.2
pH 7.4
NG1
4.12 ± 0.13
2.89 ± 0.05
NG2
3.27 ± 0.08
2.23 ± 0.09
NG3
14.46 ± 0.57
8.36 ± 0.32
NG4
12.20 ± 0.56
6.01 ± 0.30
Table S2 Fitting parameters for Peppas and Peppas model for the different nanogels.
Parameters
Buffers
Nanogel
k
n
NG1
0.22 ± 0.03
0.31 ± 0.04
NG2
0.13 ± 0.02
0.38 ± 0.04
NG3
0.32 ± 0.02
0.22 ± 0.02
NG4
0.28 ± 0.03
0.27 ± 0.02
NG1
0.13 ± 0.01
0.40 ± 0.02
NG2
0.16 ± 0.01
0.35 ± 0.02
NG3
0.18 ± 0.02
0.33 ± 0.02
NG4
0.15 ± 0.02
0.38 ± 0.03
PBS
SA
4
Table S3 Fitting parameters for Peppas-Sahlin model for the different nanogels.
Parameters
Buffers
Nanogel
k1
k2
m
NG1
0.16 ± 0.03
-0.0066 ± 0.0026
0.63 ± 0.06
NG2
0.09 ± 0.02
-0.0022 ± 0.0008
0.63 ± 0.06
NG3
0.29 ± 0.02
-0.0231 ± 0.0033
0.35 ± 0.03
NG4
0.21 ± 0.02
-0.0121 ± 0.0023
0.47 ± 0.02
NG1
0.10 ± 0.01
-0.0027 ± 0.0007
0.56 ± 0.04
NG2
0.14 ± 0.01
-0.0048 ± 0.0008
0.48 ± 0.04
NG3
0.14 ± 0.02
-0.0050 ± 0.0013
0.50 ± 0.04
NG4
0.09 ± 0.01
-0.0025 ± 0.0005
0.06 ± 0.01
PBS
SA
Hydrodynamic radius (nm)
200
150
100
50
0
10
20
30
40
50
60
Temperature (ºC)
Figure S1 Average hydrodynamic radii as a function of temperature. ■NG1; ●NG2.
5
300
Hydrodynamic radius (nm)
Hydrodynamic radius (nm)
1500
(a)
1250
1000
750
500
100
50
0
2
4
6
8
(b)
)
250
200
150
100
0
10
10
20
30
40
50
60
Temperature (ºC)
pH
Figure S2 Average hydrodynamic radii as a function of pH at 25 oC (a) and as a function of
temperature at pH 9 (b). ■NG3; ●NG4.
100
(a)
80
Cell viability (%)
Cell viability (%)
100
60
40
20
0
(b)
80
60
40
20
0
1 mg/mL
0.1 mg/mL
0.01 mg/mL
1 mg/mL
0.1 mg/mL
0.01 mg/mL
Figure S3 HeLa (a) and MDA-MB-231 (b) cell viability in the presence of nanogels at different
concentrations and at an incubation time of 24 h. ■NG1; ■NG2; ■NG3.
Figure S4 Reconstructed fluorescence image of DOXO-loaded NG2 in HeLa cells stained with DAPI after 6
h of incubation.
6
a1
a2
a3
a4
b1
b2
b3
b4
c2
c3
c4
(a)
c1
Figure S5 Fluorescence microscopy images of DOXO-loaded NG2 in MDA-MB-321 cells stained with DAPI
after 2 (a), 6 (b) and 12 (c) h of incubation. For each panel, the images from left to right show differential
interference contrast (DIC), DOXO fluorescence in cells (red), cell nuclei stained by DAPI (blue), and the
combination of the previous three images.
120
120
(a)
100
Cell viability (%)
(c)
Cell viability (%)
(b)
80
60
40
20
0
(b)
100
80
60
40
20
0
0
20
40
60
80
DOXO concentration (M)
100
0
20
40
60
80
100
DOXO concentration (M)
Figure S6 Cell viability of HeLa (a) and MDA-MA-231 (b) cells as a function of DOXO concentration at
different incubation periods. ▼6 h; ♦ 12 h; ◄ 24 h.
References
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2 A. Pikabea, J. Ramos, J. Forcada, Part. Part. Syst. Charact., 2014, 31, 101-109.
3 F. Brunel, N. E. E. Gueddari, B. M. Moerschbacher, Carbohydr. Polym., 2013, 92, 1348-1356.
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