Supplementary Information
“Side-by-Side Comparison of Dendritic-linear Hybrids and their
Hyperbranched Analogues as Micelle Carriers of Chemotherapeutics”
Yvonne Hed,1 Yuning Zhang,2 Oliver Andrén,1 Xianghui Zeng,2 Andreas M. Nyström2,*, and Michael
Malkoch1,*
Experimental part
Instrumentation
Plate reader: A BioTek SynergyTM MX monochromator-based multi-mode micro plate
reader was applied for UV-Visible absorbance and fluorescence plate reading. The software
Gen5TM was used to operate the equipment and the 490 nm wavelength was used in the UV-
Visible absorbance model and 480/600 (excitation/emission) nm was applied for DOX fluorescence
determination.
Dynamic light scattering (DLS): A Malvern Zetasizer NanoZS instrument was operated with a He-Ne
laser (Max, 5 mW) at 633 nm and 37 °C after 5 minutes equilibration. Size was determined by triple
measurements with 10 scans in each measurement.
MALDI-TOF MS: A Bruker UltraFlex MALDI-TOF MS with a SCOUT-MTP Ion Source (Bruker Daltonics,
Bremen), N2-laser (337nm), and reflector design was used. The intensity of the laser was set to the
lowest possible level to acquire high resolution spectra of the product and all spectra were acquired
using reflector-positive mode. The instrument was calibrated using SpheriCalTM calibrants purchased
from Polymer Factory Sweden AB. The received spectra were analyzed using the software
FlexAnalysis from Bruker Daltonics, Bremen, version 2.2.
1
H NMR and 13C NMR: A Bruker Avance 400 MHz NMR instrument was used. Proton NMR spectra
were acquired with a spectral window of 20 ppm, an acquisition time of 4 seconds and a relaxation
delay of 1 second. 13C NMR spectra were acquired with a spectral window of 240 ppm, an acquisition
time of 0.7 seconds and a relaxation delay of 2 seconds.
Size Exclusion Chromatography (SEC) DMF: A TOSOH EcoSECHLC-8320GPC system equipped with
an EcoSES RI detector and three columns from PSS GmbH was used (PSS PFG 5 μm; Microguard, 100
Å and 300 Å). The mobile phase was DMF with 0.01M LiBr (0.2mL min-1) at 50 °C using a conventional
calibration method with narrow linear poly(ethylene glycol) PEG standards.
Size Exclusion Chromatography (SEC) CHCl3: A Verotech PL-GPC 50 Plus system equipped with a
PL-RI detector and two PLgel 10 µL mixed (300 * 7.5 mm) columns from Varian was used. The mobile
phase was chloroform (1mL min-1) and the calibration was produced using polystyrene standards.
Chemicals and Cells
All chemicals were purchased from Sigma Aldrich and used as received unless otherwise noted.
Trimethylolpropane (TMP) and 2,2 bis(hydroxymethyl)proponic acid (bis-MPA) were kindly donated
by Perstorp. The 32-64 D 60 Å silica gel was purchased from ICN. Ethylacetate, diethylether dimethyl
sulphoxide (DMSO) and heptane were purchased from Merck. Methanol and acetone were
purchased from Fisher.
For cellular based experiments, breast cancer cell line MDA-MB-231 was purchased from the
American Type Culture Collection (ATCC). Dulbecco’s modified Eagle’s medium (DMEM), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetra sodium bromide (MTT) and doxorubicin (DOX) were also
purchased from Sigma Aldrich. Fetal bovine serum (FBS) and penicillin solution were provided by
Hyclone Laboratories.
Calculation of the Mw from 1H-NMR
The Mw of the DL hybrids were calculated by normalizing the spectra according to the theoretical
number of protons represented in a 5k PEG. Thereafter, the peak at 4.55 ppm, corresponding to the CH2OCHPh in the dendron, was integrated giving (Iint). This value was further divided with the
theoretical integral number (Itheo) and both incorporated in the equation below:
𝑀𝑤(𝑁𝑀𝑅) = 𝑀𝑤(𝑃𝐸𝐺 − 𝑎𝑐𝑒𝑡𝑦𝑙𝑒𝑛𝑒) + 𝑀𝑤(𝑑𝑒𝑛𝑑𝑟𝑜𝑛) ×
𝐼𝑖𝑛𝑡
𝐼𝑡ℎ𝑒𝑜
Calculation of degree of branching (DB)
DB is calculated from the integrals of the different quaternary carbons RC(CH2)2OR’ of the bis-MPA
units, that are located at different ppm according if linear(L), terminal (T), or dendritic (D). The
following equation was used::
𝐷+𝑇
𝐷𝐵(%) = (
) × 100
𝐷+𝑇+𝐿
Nomenclature
Ac
Acetonide
Acet
Acetylene/alkyne
bis-MPA
2,2 bis(hydroxymethyl)propionic acid
Bz
Benzylidene
CHCl3
Chloroform
CuAAC
Copper catalyzed Azide Alkyne Cyclo addition
DCC
Dicyclohexylcarbodiimide
DCM
Dichloromethane
DMAP
Dimethylaminopyridine
DMEM
Dulbecco’s Modified Eagle Medium
DMF
Dimethylformamide
DMPA
2,2-Dimethoxypropane
DMSO
Dimethyl sulphoxide
DOX
Doxorubicin
EtOAc
Ethylacetate
FBS
Fetal bovine serum
Hep
Heptane
MeOH
Methanol
MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Na2CO3
Sodium carbonate
NaHSO4
Sodium bisulphate
PBS
Phosphate buffered saline
PMDETA
N,N,N',N',N-Pentamethyldiethylenetriamine
p-TSA
para-toluenesulfonic acid
TEA
Triethylamine
TMP
Trimethylolpropane
THF
Tetrahydrofuran
Synthesis
(1)
DL PEG-[HBG2]-(OH)4 (1) PEG5K mono methyl ether (1 eq, 35.0 g, 7.00 mmol) was added to a twonecked round-bottom flask and heated to 130 °C. The necks were equipped with argon inlet and
distillation apparatus to remove the water and drive the equilibrium towards the product side. BisMPA (3 eq, 2.82 g, 21.0 mmol) was added in two steps, one per generation, along with pTSA (5 wt%,
141 mg). Between each addition argon was flushed through the system for one hour, after the final
addition the argon inlet was closed, the distillation apparatus removed and the reaction vessel was
placed under vacuum overnight. Complete consumption of carboxylic acid was confirmed with IR
(R’COOH carboxylic shift 1625 cm -1), and 13C NMR with the disappearance of the R’COOH at 178
ppm. The crude reaction mixture was collected as a white solid in a yield of 95 % (34.1 g). 1H-NMR
(CDCl3, 400 MHz) δ 1.18-1.08 (m, 9H, -CCH3), 3.34 (m, 3H, CH3OCH2CH2-), 3.61 (m, backbone OCH2CH2O- and 10H -CCH2OCO- and CH2OH), 4.31 (m, 2H, -CH2OCO-) Mw (theoretical) = 5349 g mol-1,
Mw (1H-NMR) = 4900 g mol-1 SEC (DMF) Mw = 5000 g/mol, Ð=1.02)
(2)
DL PEG-[HBG3]-(OH)8 (2) PEG5k mono methyl ether (1 eq, 50.0 g, 10.0 mmol) was added to a twonecked round-bottom flask and heated to 130 °C. The necks were equipped with argon inlet and
distillation apparatus to remove the water and drive the equilibrium towards the product side. . BisMPA (7 eq, 9.39 g, 70.0 mmol) was added in three steps, one per generation, along with pTSA (5 wt%,
470 mg). Between each addition argon was flushed through the system for one hour, after the final
addition the argon inlet was closed, the distillation apparatus removed and the reaction vessel was
placed under vacuum overnight. Complete consumption of carboxylic acid was confirmed with IR
(R’COOH carboxylic shift 1625 cm -1), and the crude reaction mixture was collected as a white solid in
a yield of 96 % (56.2 g). 1H-NMR (CDCl3, 400 MHz) δ 1.20-1.03 (m, 21H, -CCH3), 3.33 (s, 3H,
CH3OCH2CH2-), 3.60 (m, backbone -OCH2CH2O- and 22H -CCH2OCO- and CH2OH), 4.30 (m, 6H, CH2OCO-) 13C-NMR (CDCl3, 100 MHz) δ 17.25, 17.59, 48.01, 48.78, 50.71, 58.60, 60.71, 64.17, 64.32,
70.31, 71.80, 72.85, 173.62, 174.76, 175.75, 177.08. Mw (theoretical) = 5814 g mol-1, Mw (1H-NMR) =
5300 g mol-1 SEC (DMF) Mw = 5000 g/mol, Ð=1.01)
(3)
Bezylidiene protected bis-MPA anhydride (3) was synthesized as previously reported.1
(4)
DL PEG-[HBG3]-(Bz)4 (4) DL PEG-[HBG2]-(OH)4 (1) (5.00g, 0.93 mmol) and DMAP (91 mg, 0.75 mmol)
were dissolved in pyridine (0.90 ml, 11.2 mmol) and DCM (40 mL) in a round-bottom flask. The flask
was cooled to 0 °C, after which benzylidene protected bis-MPA anhydride (3) (1.91 g, 4.48 mmol)
dissolved in a minimal amount of DCM was added slowly. The reaction was allowed to proceed
overnight, and checked by 13C-NMR followed by quenching of the residual anhydride over night by
addition of water (15 mL). The reaction mixture was diluted with DCM (150 mL) and the organic
phase washed five times with aqueous solution NaHSO4 (10 wt %), two times with aqueous solution
Na2CO3 (10 wt %), dried with MgSO4 and evaporated until dry. The product was subsequently
solubilized in minimal amount of DCM and precipitated in diethyl ether. The product was collected as
a white powder in a yield of 74 % (4.30 g). 1H-NMR (CDCl3, 400 MHz) δ 1.18-1.15 (m, 21H, -CCH3),
3.34 (s, 3H, CH3O-) 3.60 (m, backbone -OCH2CH2O- and 8H, -CH2OCPh), 4.64-4.20 (m, 20H, -CH2OCOand -CH2OCPh), 5.48-5.38 (m, 4H, -CHPh-), 7.72-7.28(m, 20H, -Ph), Mw (theoretical) = 6167 g mol-1,
Mw (1H-NMR) = 5500 g mol-1 SEC (DMF) Mw = 5000 g/mol, Ð=1.05)
(5)
DL PEG-[HBG4]-(Bz)8 (5) DL PEG-[HBG3]-(OH)8 (2) (5.00g, 0.86 mmol) and DMAP (168 mg, 1.37 mmol)
were dissolved in pyridine (1.66 ml, 20.6 mmol) and DCM (50 mL) in a round-bottom flask. The flask
was cooled to 0 °C, after which benzylidene protected bis-MPA anhydride (3) (3.52 g, 8.23 mmol)
dissolved in a minimal amount of DCM was added slowly. The reaction was allowed to proceed
overnight, and checked by
13
C-NMR followed by quenching of residual anhyrdide over night by
addition of water (15 mL). The reaction mixture was diluted with DCM (150 mL) and the organic
phase washed five times with aqueous NaHSO4 solution (10 wt %), two times with aqueous Na2CO3
solution (10 wt %), dried with MgSO4 and evaporated until dry. The product was subsequently
solubilized in minimal amount of DCM and precipitated in diethyl ether. The product was collected as
a white powder in a yield of 64 % (4.09 g).1H-NMR (CDCl3, 400 MHz) δ 0.91-1.22 (m, 45H, -CCH3), 3.38
(s, 3H, CH3O-) 3.64 (m, backbone -OCH2CH2O- and 16H, -CH2OCPh), 4.56-4.21 (m, 44H, -CH2OCO- and
-CH2OCPh), 5.47-5.39 (m, 8H, -CHPh-), 7.32-7.40 (m, 40H, -Ph), Mw (theoretical) = 7448 g mol-1, Mw
(1H-NMR) = 6700 g mol-1 SEC (DMF) Mw=5300 g/mol, Ð=1.02.
(6)
Acetylene protected bis-MPA anhydride (6). was synthesized as previously reported.2
(7) and (8)
Azide-[G2]-(OH)4 (7) and Azide-[G3]-(OH)8 (8) dendrons. were synthesized as previously reported.3
(9)
Azide-[G3]-(Bz)4 (9) Azide-[G2]-(OH)4 (7) (500 mg, 1.02 mmol), DMAP (99 mg, 0.81 mmol) and
pyridine (1mL) were dissolved in DCM (4mL) and placed in an ice bath. The benzylidene protected
bis-MPA anhydride (3) (2.08 g, 4.88 mmol) was added and the reaction was left to react over night.
The reaction was followed by MALDI-TOF until complete substitution, quenched with water (5 mL),
washed with NaHSO4, Na2CO3, and brine, dried over MgSO4, filtered and evaporated until dry. The
product was purified with chromatography with a gradually increased concentration of ethyl acetate
in heptane. The concentrated product was obtained as a white powder in a yield of 74 % (1.14 g). 1HNMR (CDCl3, 400 MHz) δ 0.93 (s, 12H, -CH3), 1.04 (s, 3H, -CH3), 1.22 (s, 6H, -CH3), 1.26-1.36 (m, 4H, CH2CH2CH2OCO-), 1.51-1.60 (m, 4H, N3CH2CH2CH2-), 3.21 (t, J = 6.9 Hz, 2H, N3CH2-), 3.59 (d, J = 11.6 Hz,
8H, -CH2OCHPh), 4.01-4.12 (m, 6H, -CH2CH2OCOCCH2-), 4.33-4.40 (m, 8H, -COCH2-), 4.57 (d, J = 11.6
Hz, -CH2OCHPh), 5.40 (s, 4H, -OCH-), 7.29-7.41(m, 20H, -Ph) 13C-NMR (CDCl3, 100 MHz) δ 17.33; 17.72;
25.38; 26.29; 28.32; 28.68; 42.60; 46.47; 46.89; 46.90; 51.28; 65.16; 65.27; 65.97; 73.44; 73.52;
101.69; 126.20; 128.12; 128.14; 128.87; 137.86; 171.91; 172.20; 173.22. MALDI calc [M+Na+] =
1330.55 Da, Obtained [M+Na+] = 1330.84 Da.
(10)
Azide-[G4]-(Bz)8 (10). Azide-[G3]-(OH)8 (8) (0.500 g, 0.52 mmol), DMAP (0.100 g, 0.84 mmol) and
pyridine (2mL) were dissolved in DCM (3mL) and placed in an ice bath. The benzylidene protected
bis-MPA anhydride (3) (2.14 g, 5.02 mmol) was added and the reaction was left to react over night.
The reaction was followed by MALDI-TOF until complete substitution, quenched with deionized
water (5mL), washed with NaHSO4, Na2CO3, and brine, dried in MgSO4, filtered and evaporated until
dry. The product was purified with chromatography with a gradually increased concentration of ethyl
acetate in heptane. The concentrated product was obtained as a white powder in a yield of 52 %
(700 mg). 1H-NMR (CDCl3, 400 MHz) δ 0.91 (s, 24H, -CH3), 1.02 (s, 6H,-CH3), 1.14 (s, 3H, CH3), 1.20 (s,
12H, -CH3),1.26-1.36 (m, 4H, -CH2CH2CH2OCO-), 1.51-1.60 (m, 4H, N3CH2CH2CH2-), 3.21 (t, J = 6.9 Hz,
2H, N3CH2-), 3.56 (d, J = 11.6 Hz, 16H, -CH2OCHPh), 4.05 (m, 6H, -CH2CH2OCOCCH2-), 4.30-4.38 (m,
16H, -COCH2-), 4.57 (d, J = 12 Hz, -CH2OCHPh), 5.40 (s, 4H, -OCH-), 7.28-7.40(m, 40H, -Ph) 13C-NMR
(CDCl3, 100 MHz) 17.34, 17.51, 17.18, 25.08, 26.42, 28.45, 28.80, 42.69, 46.63, 46.97, 51.38, 65.17,
65.49, 65.57, 66.38, 73.55, 73.62, 101.79, 126.30, 128.25, 128.98, 137.97, 171.53, 171.98, 173.30
MALDI calc [M+Na+] = 2612.06 Da, Obtained [M+Na+] = 2612.52 Da.
(11)
Acetylene anhydride (11). was synthesized as previously reported.4
(12)
PEG5k-acetylene (12). mPEG5K-OH (5.00 g, 1.00 mmol), DMAP (24 mg, 0.20 mmol) and pyridine
(1mL) were dissolved in DCM (20 mL) and placed in an ice bath. The acetylene anhydride (11) (0.736
g, 2.50 mmol) was added and the reaction was checked for completion using MALDI-TOF. The
product was purified by precipitation in diethyl ether (500 mL). The product was obtained as a white
powder in a yield of 94 % (4.67 g). 1H-NMR (CDCl3, 400 MHz) δ 2.47 (s, 1H, -CH), 2.61 (m, 4H, OOCCH2CH2COO-), 3.33 (s, 3H, CH3-), 3.42 (t, J = 4.8 Hz, 2H, CH3OCH2-), 3.60 (m, 113H, -OCH2CH2O-),
4.21 (t, J = 4.8 Hz, 2H, CH3OCH2CH2-), 4.65 (d, J = 2.4 Hz, -CH2CCH). 13C-NMR 28.77, 28.81, 51.14,
58.97, 63.85, 70.49, 75.12, 77.36, 171.37, 171.95. Mw (theoretical) = 5139 g mol-1, Mw (1H-NMR) =
5500 g mol-1, Mw (MALDI) = 5302 g mol-1, Ð = 1.005, SEC (DMF) Mw = 5100 g mol-1, Ð=1.02.
(13)
DL PEG-[G3]-(Bz)4 (13) Azide-[G3]-(Bz)4 (9) (0.554 g, 0.42 mmol) and PEG-acetylene (12) (1.737 g, 0.35
mmol) were dissolved in dry DMF and the flask was sealed with a rubber septum and degassed by
three cycles with vacuum and argon. CuBr (15 mg, 0.10 mmol) was added and the flask was degassed
again. PMDETA (18 mg, 0.10 mmol) was added and the reaction was allowed to proceed at 35 °C. The
reaction was monitored by MALDI-TOF until the PEG-acetylene was consumed. The crude mixture
was diluted with THF (200 mL) and passed through a neutral activated aluminium oxide column to
remove the copper. The solution was concentrated and precipitated from DMF into cold diethyl
ether (200 mL), re-dissolved in DCM and precipitated a second time in diethyl ether (200 mL), filtered
and dried under vacuum. The collected solid was slightly coloured from traces of copper and was
therefore dissolved in DCM (150 mL) and extracted three times with 0.5 wt% EDTA solution (30 mL),
dried from DCM under vacuum and finally freeze dried. The product was obtained as a white solid 72 %
(1.60 g) 1H-NMR (CDCl3, 400 MHz) δ 0.93 (s, 12H, -CH3), 1.03 (s, 3H,-CH3), 1.20 (s, 6H, -CH3), 1.27 (m,
4H, -CH2CH2CH2OCO-), 1.53 (m, 4H, triazol-CH2CH2CH2-), 2.64(s, 4H, -OOCCH2CH2COO-), 3.37 (s, 3H, OCH2CH2OCH3), 3.55-3.70 (m, backbone -OCH2CH2O- and -CH2OCHPh), 3.98-4.14 (m, 6H, CH2CH2OCOCCH2-), 4.23 (m, 2H, triazol-CH2CH2-), 4.31-4.38 (m, 8H, -COCH2-), 4.56 (d, J = 12 Hz, 8H, CH2OCHPh), 5.20 (s, 2H, -COOCH2-triazol), 5.40 (s, 4H, -OCH-), 7.29-7.41(m, 20H, -Ph), 7.54 (s, 1H,
triazol-H) 13C-NMR (CDCl3, 100 MHz) δ 17.31, 17.17, 25.22, 26.06, 28.22, 28.93, 29.98, 42.58, 46.45,
46.87, 59.05, 63.89, 65.12, 65.95, 70.56, 73.43, 73.50, 101.66, 126.15, 128.10, 128.85, 137.81, 171.86,
172.16, 172.19, 173.20. Mw (theoretical) = 6448 g mol-1, Mw (1H-NMR) = 5800 g mol-1, Mw (MALDI) =
6510 g mol-1, Ð = 1.003, SEC (DMF) Mw 6000 g mol-1, Ð 1.03
(14)
DL PEG-[G4]-(Bz)8 (14). Azide-[G4]-(Bz)8 (10) (0.450 g, 0.17 mmol) and mPEG5k-acetylene (12) (0.702
g, 0.14 mmol) were dissolved in dry DMF and the flask was sealed with a rubber septum and
degassed by three cycles with vacuum and argon. CuBr (4 mg, 0.028 mmol) was added and the flask
was degassed again. PMDETA (5 mg, 0.028 mmol) was added and the reaction was allowed to
proceed at 35 °C. The reaction was monitored by MALDI-TOF. The crude mixture was diluted in THF
(200 mL) and passed through an aluminium oxide column to remove the copper. The solution was
concentrated and precipitated from DMF into cold diethyl ether (200mL), redissolved in DCM and
precipitated in diethyl ether, filtered and dried under vacuum. The product was obtained as a white
solid 30 % (320 mg). 1H-NMR (CDCl3, 400 MHz) δ 0.91 (s, 24H, -CH3), 1.01 (s, 6H,-CH3), 1.14 (s, 3H, CH3), 1.19 (s, 6H, -CH3) 1.29 (m, 4H, -CH2CH2CH2OCO-), 1.81 (m, 4H, -NCH2CH2CH2-), 2.64 (s, 4H, OOCCH2CH2COO-), 3.38 (s, 3H, -OCH2CH2OCH3), 3.54-3.69 (m, backbone –OCH2CH2O- and -CH2OCHPh),
4.00-4.24 (m, 32H, -COCH2-, -CH2CH2OCOCCH2- and -NCH2-), 4.55 (d, J = 12 Hz, 16H, -CH2OCHPh), 5.19
(s, -COOCH2CHN-), 5.38 (s, 8H, , -OCH-), 7.29-7.40 (m, 40H, -Ph), 7.52 (s, 1H, COOCH2CHN-). 13C-NMR
(CDCl3, 100 MHz) δ 17.22, 17.40, 17.70, 25.22, 26.08, 28.24, 28.90, 28.93, 30.09, 42.57, 46.48, 46.51,
46.85, 59.07, 63.89, 65.04, 65.28, 65.47, 66.24, 70.58, 73.42, 73.48, 101.65, 126.18, 128.12, 128.86,
137.86, 171.41, 171.86, 172.10, 172.20, 173.19. Mw (theoretical) = 7729 g mol-1, Mw (1H-NMR) =
7800 g mol-1, Mw (MALDI) = 7736 g mol-1, Ð = 1.001 m, SEC (DMF) Mw 6300 g mol-1, Ð = 1.02.
Micelle formation
Micelles were formed via a two-phase emulsion method. The polymer materials were dissolved in
DCM (1 mg mL-1), and then the equivalent amount of PBS was added with the aid of a syringe pump
at the rate of 1 mL h-1 under magnetic stirring. The mixed solution was stirred at 100 rpm overnight
to evaporate the organic solvent. The size of the micelles was determined using a Malvern Zetasizer
NanoZS at 37 °C at the polymer concentration of 1 mg mL-1.
Doxorubicin loading
DOX working solution was produced by dissolving 5 mg mL-1 in 1 mL DCM containing TEA (1:3 mol eq
to DOX) combined with sonication. 0.15 mL of the DOX solution were added into 5 mL DI water
containing 2.5 mg materials drop by drop with magnetic stirring.5 The mixed solution was shielded
from the light and stirred (100 rpm) at room temperature for 24 h to remove the organic solvent. The
consequence solution was washed with DI water and free DOX was removed via spin filtration
(MWCO, 3 kDa; 2000 rpm). The concentration of DOX in the micelles was determined by UV-vis
spectroscopy, reading the absorbance at 490 nm after diluting the sample with DMF:H2O (4:1) (five
replicates), and calculated by comparing a standard curve.6 Size of the DOX-NPs were measured by
DLS experiment without sample dilution at 37°C.
In vitro drug release
3 mL DOX or DOX-NPs solutions in PBS were transferred into dialysis cassettes (MWCO 3500, Slide‐A‐
Lyzer, Thermo). Cassettes were suspended in 4 L of preheated 37°C PBS. Sample solution inside the
cassettes was collected in 0.5 eppendorf tubes (triplicates of 10 µL each) at the following time
intervals: 0, 2, 4, 8, 12, 24, 48, and 72 hours. Thereafter, samples were diluted with 100 µL DMF:H 2O
(4:1) and transferred into fluorescence plates. The fluorescence intensity of samples was determined
using a plate reader (BioTek SynergyTM MX) at wavelength 480/600 (excitation/emission) nm.7
Cellular cultivation and Viability assays
Human breast cancer cells MDA-MB-231, were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) (pH 7.4) supplemented with 10 % (v/v) fetal bovine serum (FBS), penicillin/streptomycin
(100 U mL-1 / 100µg/mL), and 2 mM glutamine solution. The cells were incubated at 37 °C with 5 %
CO2 and harvested by trypsin. For the MTT viability tests MDA-MB-231 cells were seeded into 96-well
plates at the concentration of 5×104 cells per well (in 100 µL DMEM). The medium was removed after
24 hours and refilled with 100 µL of fresh medium containing various concentrations of the micelle,
DOX-micelle, and DOX (4 parallel wells for each concentration). The cells were cultured for another
48 hours with samples, and then 10 µL MTT solutions (5 mg mL-1) were introduced into each well.
100 µl SDS solutions (10 %) were added to each well after additional incubation for 4 hours. The
absorbance of the MTT reagent was determined 18 h later using a plate reader at 570 nm.8
Statistical Analysis
Dynamic light scattering, cellular viability tests and drug release studies are presented as mean
values with standard deviation (SD). MTT tests were further analyzed with one-way analysis of
variance (ANOVA) with the threshold value for statistical significant p<0.001 using KaleidaGraph v4.1
(Reading, PA).9
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