SUPPORTING INFORMATION
pH-Responsive Near-Infrared Emitting Conjugated Polymer Nanoparticles for Cellular
Imaging and Controlled-Drug Delivery
Jousheed Pennakalathil,a,b Alp Özgün,b İrem Durmaz,c Rengül Çetin-Atalay,c Dönüs Tuncel*a,b
a
Department of Chemistry, Bilkent University, 06800 Ankara, Turkey.
E-mail: dtuncel@fen.bilkent.edu.tr
b
Institute of Material Science and Nanotechnology, Bilkent University, 06800 Ankara,
Turkey.
c
Department of Molecular Biology and Genetics, Bilkent University, 06800 Ankara, Turkey.
* Correspondence to: dtuncel@fen.bilkent.edu.tr
Scheme S1. Reaction scheme for P1. (a) 2-(thiophen-3-yl)ethanol, NBS, EtOAc, 25 C, 12 h,
60% (b) acetic anhydride, pyridine, 25 C, 12 h, 90% (c) THF/toluene/H2O (1:1:1, v/v),
K2CO3 (aq.), tetrabutylammonium bromide (TBAB), Pd(Ph3)4, 80 C, 72 h, 46%.
Figure S1. 1H-NMR (400 MHz, CDCl3, 25 C) spectra of of 2-(2,5-dibromothiophen-3yl)ethanol (M1) and 2-(2,5-dibromothiophen-3-yl)ethyl acetate (M2).
Figure S2a. 13C-NMR (100 MHz, CDCl3, 25 C) spectrum of of 2-(2,5-dibromothiophen-3yl)ethyl acetate (M2).
Figure S2b. Mass spectrum of 2-(2,5-dibromothiophen-3-yl)ethyl acetate (M2).
Figure S3. 13C-NMR (100 MHz, CDCl3, 25 C) spectrum of of P1.
Figure S4. FT-IR (KBr pellet) spectra of of 2-(2,5-dibromothiophen-3-yl)ethanol (M1) and 2(2,5-dibromothiophen-3-yl)ethyl acetate (M2).
Figure S5. FT-IR spectrum of P1 (KBr pellet).
r
Figure S6. GPC data for polymer P1.
Preparation of Nanoparticles of P1:
In a typical procedure, P1 (1.0 mg, 3.3 × 10 -3 mmol, based on per repeating unit) was dissolved
in dry THF (1 mL). The solution was sonicated for ~15 min and then injected rapidly to 20 mL
of DD water. The sonication was continued for a further 30 min. THF was removed from the
solution under reduced pressure.
Details for the preparation of nanoparticles of Polymer P1.

A stock solution of polymer P1 (1 mg/mL in THF) was prepared and used for the
preparation of Nanoparticles in different concentrations. To optimize the size of the
nanoparticles with their concentration, 8 samples of nanoparticles with different
concentrations of polymer solutions were prepared as described in table S1. Size of the
nanoparticles with varying concentrations were tabulated in table S2.

THF was dried by passing over silica and distilled by rotary evaporator, prior to the
preparation of polymer solution.

Polymer was well dissolved in THF by ~15 minutes sonication and filtered through syringe
filter (0.40 µm) prior to nanoparticles preparation.

Double distilled water was used for NP preparaion.
Table S1: Nanoparticle preparation with varying concentration of polymer solution.
Sample
S1
S2
S3
Nanoparticles preparation method
1 mL of polymer stock solution was injected to 20 mL of DD water
1 mL of polymer stock solution was made upto 2 mL by THF and injected to
20 mL of DD water
1 mL of polymer stock solution was made upto 4 mL by THF and injected to
20 mL of DD water
S4
1 mL of polymer stock solution was injected to 15 mL of DD water
S5
1 mL of polymer stock solution was injected to 10 mL of DD water
S6
1 mL of polymer stock solution was injected to 5 mL of DD water
S7
S8
0.5 mL of polymer solution was diluted to 2 mL with THF and 1 mL of this
solution was injected to 20 mL of DD water.
0.5 mL of polymer solution was diluted to 2 mL with THF and 0.5 mL of this
solution was injected to 20 mL of DD water.
Table S2: NP preparation from P1 and DLS data of P1NPs for different samples
Sample
Average size in nm
PdI
Zeta Potetntial, ζ, in mV
S1
59.18 ± 2.97
0.11 ± 0.04
-20 .3 ± 0.3
S2
56.28 ± 3.21
0.07 ± 0.02
-27.3 ± 3.6
S3
57.58 ± 2.14
0.08 ± 0.01
-23. 4 ± 2.0
S4
65.98 ± 1.94
0.22 ± 0.08
-29.3 ± 5.1
S5
76.87 ± 2.73
0.15 ± 0.05
-19.6 ± 3.8
S6
85.40 ± 3.54
0.22 ± 0.08
-27.8 ± 2.6
S7
44.45 ± 2.01
0.10 ± 0.07
-23.5 ± 3.8
S8
21.06 ± 1.13
0.31 ± 0.05
-25.8 ± 2.2
Figure S7. Z-average size of the P1 nanoparticles by varying the concentration of polymer
solution.
zeta potential
z- average size
SET 1
SET 2
SET 3
SET 4
SET 5
SET 6
SET 7
SET 8
Figure S8. DLS data and zeta potential of P1 nanoparticles of different samples with different
concentrations of polymer solutions.
70
z-average size, d (nm)
z-average size, d (nm)
70
60
50
40
30
20
60
50
40
30
20
0
5
10
15
20
25
Days
30
35
0
5
10
15
20
25
30
35
Days
Figure S9. Stability tests for the nanoparticles of P1 in water (left panel) and PBS buffer (right
panel) (at pH 7.4) over time.
Experimental details of exposing nanoparticles to aqueous buffer solutions of pH 5 and 8
•
Acetate buffer (for pH 5) and Sorenson's Buffer (for pH 8) are used.
•
Equal amount of NP dispersion and buffer solution were mixed and collected the
samples in each intervals of time.
•
Just after the addition of buffer solution to the NP dispersion, the solution was
sonicated for less than 2 min (1-2 minutes).
•
After well mixing the solution, the solution was kept in RT.
•
2 mL of each of the solution was taken by particular intervals of time and measured
size and zeta potential (and made the sample for SEM)
Figure S10. Z-average diameter of NP-P1 measured by DLS after incubating in the buffer of
pH 5.
Figure S11. DLS and Zeta potential histograms of NP-P1 after 1 h and 2 exposure of pH 5.
Figure S12. DLS and Zeta potential histograms of NP-P1 after 1 h exposure to pH 8.
Figure S13. IR spectra of (a) P1, (b) P1 NP after treating at pH 8.0 and (c) P1 NP after
treating at pH 5.0.
Figure S14. Absorption and emission spectra of polymer in different solvents, dispersion of
polymer nanoparticles in water.
0.5
180
Absorbance
Excitation at 324 nm
Excitation at 544 nm
731 nm
140
120
544 nm
0.3
160
100
80
0.2
60
PL Intensity
Absorbance (a.u)
0.4
40
0.1
20
0.0
300
400
500
600
700
800
900
0
1000
Wavelength (nm)
Figure S15. Absorption and emission spectra of polymer in film drop-casted on a fused slica
substrate.
Preparation of drug loaded nanoparticles: determination of drug loading capacity and
entrapment efficiency
Seven sets of drug-loaded nanoparticles were prepared with the drug to polymer ratios of 1:0.5
(w/w), 1:1 (w/w), 1:2 (w/w), 1:3.125 (w/w), 1:6.25 (w/w), 1:12.5 (w/w), and 1:25 (w/w).
The following is a typical procedure for the preparation of the drug loaded nanoparticles (the
drug to polymer ratio: 1:25 (w/w)): P1 (1.0 mg, 3.3 × 10-3 mmol, based on per repeating unit)
and CPT (0.04 mg, 0.115 × 10-3 mmol) was dissolved in THF (1 mL). The resulting solution
was sonicated for 30 min and then injected rapidly into autoclaved double distilled water (20
mL). It was stirred with sonication for further 30 min. THF was removed under reduced
pressure and the CPN dispersion was concentrated to 10 ml and dialyzed against water using a
14 kDa MWCO regenerated cellulose membrane for 24 h to remove any remaining free CPT.
Tween 20 (0.2 %, v/v) was added to dialysate to solubilize free CPT. UV-Vis absorbance
spectra (λmax: 366 nm) of dialysates were recorded to determine the concentration of CPT.
Samples at known concentrations of CPT dissolved in water containing Tween 20 (0.2 %, v/v)
were used to obtain calibration curves of CPT.
Preparation of Drug Loaded NPs with different ratios of drug: Polymer: - Description of
samples
Table S3: Description of CPT loaded P1 Nanoparticle samples: Ratios of CPT:Polymer.
Sample
Ratio of CPT:Polymer
Amount of drug:polymer
Set 1
1:25
40 µg:1 mg
Set 2
1:12.5
80 µg:1 mg
Set 3
1:6.25
160 µg:1 mg
Set 4
1:3.125
320 µg:1 mg
Set 5
1:2
500 µg:1 mg
Set 6
1:1
1mg:1 mg
Set 7
1:0.5
2mg:1 mg
The amount of loaded drug was determined by UV-VIS absorption spectrophotometry by
measuring the absorbance at λ=366 nm (absorbance of CPT) and calculated from the calibration
curve of CPT (Figure S16). Samples at known concentrations of CPT dissolved in water
containing Tween 20 (0.2 %, v/v) were used to obtain calibration curves of CPT.
Figure S16. Calibration curve of known concentrations of CPT for the calculation of amount
of drug loaded in P1NPs.
Table S4: Amount of CPT loaded to P1 nanoparticles with different ratios of drug:polymer.
Sample
SET 1
Amount of
CPT used
Polymer
for loading
1 mg
40 µg
CPT
CPT loaded in µg
unloaded in
For each
µg
expt.
a. 23.51
a. 16.49
b. 24.69
b. 15.31
c. 23.73
c. 16.27
Average
16.02 ± 0.63
SET 2
SET 3
SET 4
SET 5
SET 6
SET 7
1 mg
1 mg
1 mg
1 mg
1 mg
1 mg
80 µg
160 µg
320 µg
500 µg
1000 µg
2000 µg
a. 35.27
a. 44.73
b. 38.75
b. 41.25
c. 32.43
c. 46.57
a. 58.71
a. 101.29
b. 63.54
b. 96.46
c. 57.43
c. 102.57
a. 208.35
a. 111.65
b. 216.38
b. 103.62
c. 209.58
c. 110.42
a. 401.75
a. 98.25
b. 398.45
b. 101.55
c. 391.14
c. 108.86
a. 898.75
a. 101.25
b. 899.65
b. 100.35
c. 882.68
c. 117.32
a. 1893.75
a. 106.25
b. 1900.75
b. 99.25
c. 1885.14
c. 114.86
Figure S17. Amount of CPT loaded with different ratios of drug:polymer
44.18 ± 2.70
100.11 ±
3.22
108.56 ±
4.33
102.89 ±
5.43
106.31 ±
9.55
106.78 ±
7.82
Table S5: Entrapment and Loading Efficiencies of P1NPs with diff. concentrations of CPT
Average Size, nm
Poly:Drug
ratio
Amt. of CPT
Unloaded
Loaded
NPs
NPs
loaded, µg
EEa (%)
LEb (%)
25 : 1
57.41 ± 2.4
16.02 ± 0.63
40.06 ± 1.57
1.58 ± 0.06
12.5 : 1
56.47± 1.5
44.18 ± 2.70
55.23 ± 3.38
4.23 ± 0.25
6.25 : 1
55.94 ± 2.4
100.11 ± 3.22
62.57 ± 2.01
9.10 ± 0.27
57.12 ± 4.1
108.56 ± 4.33
33.93 ± 1.35
9.79 ± 0.35
2:1
64.51 ± 3.2
102.89 ± 5.43
20.58 ± 1.09
9.33 ± 0.45
1:1
69.74 ± 3.5
106.31 ± 9.55
10.63 ± 0.95
9.60 ± 0.78
0.5 : 1
70.49 ± 2.9
106.78 ± 7.82
5.34 ± 0.39
9.65 ± 0.64
3.125 : 1
56.28 ±
3.21
a: Entrapment Efficiency =
wt.of loaded drug
wt.of total drug
× 100
wt.of loaded drug
b: Drug Loading Efficiency = total wt.of system × 100
In vitro drug-release studies: 10 mM phosphate buffer saline (PBS) containing 0.2 % Tween
20 was used as a release medium to improve the drug solubility. After the dialysis (to remove
the unloaded CPT) the dispersion of drug loaded P1NPs was concentrated to 5 mL. Then it
was mixed with 5 mL of phosphate buffer and transferred in to a cellulose tubular membrane
with molecular weight cut-off 14 kDa. The dialysis tube was placed into a beaker containing
100 ml of PBS and 0.2 wt% Tween 20 and shaken at 60 RPM, 37 °C in an incubator. 2 ml of
aliquot was withdrawn from the dialysate at different time intervals during the 300 hours and
at each time equal amount of fresh PBS was added to the dialysate beaker to maintain the sink
condition. The concentration of CPT in the release medium was measured by using absorption
spectrophotometer (λmax: 366 nm).
The in vitro drug release studies of the nanoparticles with two different CPT contents as
the CPT to P1NP ratios of 1:23 and 1:10 (determined after dialysis) were carried out at
37 C at pH 5.0 (acetate buffer) and pH 7.4 (PBS). The time-dependent release profiles
of CPT from the nanoparticles were measured by the absorption at 366 nm with UV-vis
spectrophotometry. Fig. S11 shows the release profile of CPT loaded P1NPs (CPT to
P1NP ratio of 1:23).
Figure S18. Percentage of cumulative release of CPT from NP1 at different pH. The release of
drug molecules was monitored using a UV-vis spectrophotometer (1:23 Loading Ratio of
CPT/P1NP).
FIGURE S19. SEM images of blank nanoparticles (left), loaded nanoparticles (right)
Preparation of cells and culture: Human hepatocellular carcinoma cell line Huh7 was
maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen GIBCO), with
10% fetal bovine serum (FBS) (Invitrogen GIBCO), nonessential amino acids, and 1%
penicillin (Biochrome). Huh7 cells were incubated at 37 °C with 5% CO2. DMSO (Sigma)
was used as a solvent for camptothecin (Calbiochem) at a concentration less than 1% in the
cell culture medium.
Cytotoxicity Assay: In order to assess the real-time cell analysis (RT-CES, ACEA
Biosciences), first a steady impedance value was obtained by adding 50 μL of cell culture
media to each well of the 96X e-plate (Roche Applied Sciences). Then, human liver cancer
cells (Huh7) were inoculated (2000cell/well). The attachment, spreading, and proliferation of
the cells were monitored every 30 minutes using the RT-CA in a cell culture incubator. 24
hours later, as the cell reach their log growth phase, they were treated with the following
samples with varying concentrations:
Table S6: Samples tested for their cytotoxicity and cell growth inhibition.
1
2
CPT
P1NP-A blank (NP
concentrations)
3
P1NP-B blank
P1NP-A-CPT (drug
loaded)
(1:62, CPT/NP)
P1NP-B-CPT (drug
loaded)
(1:10.4, CPT/NP)
4
5
0.1 µM
6.2 µM
0.2 µM
12.4 µM
0.4 µM
24.8 µM
CPT
1.04 µM
0.1 µM
2.08 µM
0.2 µM
4.16 µM
0.4 µM
NP
6.2 µM
12.4 µM
24.8 µM
CPT
NP
0.1 µM
1.04 µM
0.2 µM
2.08 µM
0.4 µM
4.16 µM
For the control, only DMSO or only ddH2O was added to the wells. The experiment was
conducted in triplicate. The electronic readout (cell-sensor impedance) was displayed as an
arbitrary unit called the cell index (CI). The CI value was noted every 10 minutes for the first
24 hours and then every 30 minutes. The cell inhibition rate (%) = (1 − CItreated cells/CIDMSO) ×
100.
Cell staining and imaging: Huh7 cells plated on coverslips and treated with CPT, CPTloaded CPNs, DMSO-only and CPNs-only for 24 h and 72h were MeOH (100%, ice-cold)
fixed prior to staining. Following 30min dark incubation, cells were washed with PBS and
then counter-stained with 1 μg/mL Hoechst 33258. Coverslips de-stained with ddH2O were
mounted on glass slides and examined under a fluorescence microscope (Nikon ECLIPSE
50i).
Figure S20. Real-time growth inhibitory effect of loaded and blank P1NPs with camptothecin
on the human liver (Huh7) cancer cell line for 24, 48 h, 72 h, 96 and 120, 144 h incubation
periods as well as the full profiles of the CPT, P1NP (high and low concentrations), P1NP-ACPT and P1NP-B-CPT exposed cells over 144 h duration were determined by RT-CES. The
concentrations are based on CPT concentrations but in brackets the concentrations of
nanoparticles were also given. (0.1, 02, 04 µM CPT concentrations for 1:62 CPT/NP ratios
correspond to 6.2, 12.4, 24.8 µM NP and and for 1:10.4 CPT/NP ratios, 1.04, 2.08, 4.16 µM
NP concentrations, respectively). The experiments were conducted in triplicate.
(A)
(B)
Figure S21. Fluorescence images of Huh7 cells plated on coverslips and treated with red
emitting P1NP-A-CPT with the concentration of 0.1 µM after (A) 24 h and (B) 72 h
incubations. The blue fluorescence arises from the nuclear stain Hoechst. Images were
acquired with a 20-objective.