Acid Degradable Biocompatible Polymeric Nanoparticles for the ...
Supporting Information
Acid Degradable Biocompatible Polymeric Nanoparticles for the Potential co-Delivery of Therapeutic Agents Hien T.T. Duong,a Christopher P. Marquis,b Michael Whittaker, a Thomas P. Davis, a* Cyrille Boyer a* Australian Centre for NanoMedicine (ACN) b
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, 2052 NSW, Sydney, Australia
School of Chemical Engineering, The University of New South Wales, 2052 NSW, Sydney, Australia; Emails: [email protected]; [email protected].
Content: 1- Abbreviations 2- Characterization methods 3- Figures S1-S18
S1
1. Abbreviations: AIBN: 2,2’-Azobisisobutyronitrile BSPA: 3-(benzylsulfanylthiocarbonylsulfanyl)-propionic acid (BSPA Da: Dalton DLS: Dynamic light scattering DMF: N,N’-Dimethylformamide DTT: DL-Dithiothreitol FITC: fluorescein isothiocyanate GPC: Gel permeation chromatography MTS: Methanethiosulfonate OEG-A: Oligo(ethylene glycol) methyl ether acrylate PDI: Polydispersity P(OEG-A): Poly(oligo(ethylene glycol) methyl ether acrylate) P(OEG-A)-b-P(VBC-co-PFP-A): poly(oligo(ethylene glycol) methyl ether acrylate)-blockpoly(vinyl benzyl chloride-co-pentafluorophenyl acrylate) RAFT: Reversible addition fragmentation transfer VBC: Vinyl benzyl chloride VBS: Vinyl benzyl chloride methane thiosulfonate TFA: Trifluoric acid
S2
2. Characterization methods Gel permeation chromatography (GPC) measurements. DMAc GPC analyses of the polymers were performed in N,N-dimethylacetamide [DMAc; 0.03% w/v LiBr, 0.05% 2, 6– di-butyl-4-methylphenol (BHT)] at 50 °C (flow rate = 1 mL.min-1) using a Shimadzu modular system comprised of an SIL-10AD auto-injector, a PL 5.0-mm bead-size guard column (50 × 7.8 mm) followed by four linear PL (Styragel) columns (105, 104, 103, and 500 Å) and an RID-10A differential refractive-index detector. The SEC calibration was performed with narrow-polydispersity polystyrene standards ranging from 168 to 106 g.mol-1. A total of 50 µL of polymer solution (2 mg.mL-1 in DMAc) was injected for every analysis. Nuclear Magnetic Resonance (NMR). Structures of the synthesized compounds were analyzed by 1H NMR, 13C NMR and 19F NMR spectroscopy using a Bruker DRX 300 MHz spectrometer at 300 MHz for hydrogen nuclei and at 75 MHz for carbon nuclei. OEG-A conversion was calculated using the following equation: αOEG-A = (∫ICH=CH2
5.5-6.5 ppm
/ ∫IOCH3
3.6ppm
) × 100, where ∫ICH=CH2
5.5-6.5 ppm
and ∫IOCH3
3.3 ppm
correspond to intensities from acrylate bond of OEG-A and methyl ether. VBC conversion was calculated using the following equation: αVBC = [2 × ∫ICH=CH2 5.5-6.5 ppm/ 3 × ∫ICH2Cl 4.5ppm] × 100, where ∫ICH=CH2 5.5-6.5 ppm and ∫ICH2Cl 4.5 ppm
correspond to intensities from OEG-A (methyl ester) and BSPA RAFT agent (benzyl
group), molar mass of monomer and RAFT agent, respectively. PFP-A conversion was calculated using 19F NMR analysis and the following equation: αPFPA-A = [∫-158.0
ppm
/ (∫I-158.0 ppm + ∫-152.5
ppm
)] × 100
UV-visible spectroscopy. UV-visible spectra were recorded using a CARY 300 spectrophotometer (Bruker). Dynamic light scattering (DLS). DLS measurements were performed using a Malvern Zetasizer Nano Series running DTS software and using a 4 mW He-Ne laser operating at a
S3
wavelength of 633 nm and an avalanche photodiode (APD) detector. The scattered light was detected at an angle of 173o. The temperature was stabilized to +/- 0.1oC of the set temperature. To reduce the influence of larger aggregates the number-average hydrodynamic particle size is reported. The polydispersity index (PDI) is used to describe the width of the particle size distribution, as calculated from the DTS software using a cumulate analysis of the measured intensity autocorrelation function; it is related to the standard deviation of the hypothetical Gaussian distribution (i.e. PDI = s2/ZD2, where s is the standard deviation and ZD is the Z average mean size). Transmission Electron Microscopy (TEM). The sizes and morphologies of the un-crossliked and cross-linked polymers were observed using a transmission electron microscopy JEOL1400 TEM at an accelerating voltage of 100 kV. The particles were dispersed in water (1 mg.mL-1) and deposited onto 200 mesh, holey film, copper grid (ProSciTech). Osmium vapor (OsO4) staining was applied.
S4
3. Figures S1-S19
A a c
a
b
c b
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm B
a
a
b c
-155.0
c
b
-160.0
ppm Figure S1. A- 1H NMR and B- 19F NMR spectra of pentafluorophenyl acrylate recorded in CDCl3 at 20 oC.
S5
b c d
a
d
b c
a
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm Figure S2. 1H NMR spectrum of compound 1 (scheme 1 in the main text) recorded in CDCl3 at 20 oC.
S6
d
b a c d c
7.0
6.0
5.0
4.0
b
3.0
a
2.0
1.0
ppm Figure S3. 1H NMR spectrum of compound 2 (scheme 1 in the main text) recorded in CDCl3 at 20 oC.
S7
d a e b f c d f
e
a
b
c
-150.0
-155.0
-160.0
ppm Figure S4. 19F NMR spectra of crude polymerization reaction of P(OEG-A)-b-P(VBC-coPFP-A) copolymer obtained via RAFT polymerization technique, recorded in CDCl3 at 20 o C.
Note: After polymerization, the signals associated to pentafluorophenyl acrylate shift from 152.5, -156.8 and -162.5 ppm to -153.0, -158.0 and -162.5 ppm. Pentafluorophenyl acrylate monomer conversion was calculated using the following equation: αPFPA-A = [∫-158.0
ppm
/ (∫I-158.0 ppm + ∫-152.5
ppm
)] × 100
S8
d
d
a e e b f c b a
-140
f
c
-150
-160
-170
-180
ppm Figure S5. 19F NMR spectra of crude reaction of P(OEG-A)-b-P(VBC-co-PFP-A) in the presence of compound (2) recorded in CDCl3 at 20 oC.
Note: The reaction of poly(pentafluorophenyl acrylate) in the presence of amino-compound results by the formation of pentafluorophenol as indicated by the signals at -167, -168 and 169 ppm. The yield was calculated using 19F NMR spectroscopy and the following equation: Yield (%) = ∫-167.0
ppm
/ (∫I-162.0ppm + ∫-167.0
ppm
) × 100
S9
a g
b c
d
e
e f
f
× HDO
d c a
b
g
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm Figure S6. 1H NMR spectrum of P(OEG-A) homopolymer obtained via RAFT polymerization recorded in CDCl3 at 20 oC, Mn (NMR) = 11 000 g.mol-1. Note: Mn (NMR) = (5 × ∫ICH2O 4.0 ppm / 2 × ∫ICH 7.2 ppm) × MWOEG-A + MWBSPA, where ∫ICH3O 3.6 ppm
, ∫ICH2O 4.0 ppm, MWMA and MWBSPA correspond to intensities from OEG-A (methyl ester)
and BSPA RAFT agent (benzyl group), molar mass of monomer and RAFT agent, respectively.
S10
A
d d
a
b
a b c
0
-50
-100
-150
c
-200
PPM B e
e f
f
10.0
5.0
PPM
Figure S7. A- 19F NMR and B- 1H NMR spectra of P(OEG-A)-b-(VBC-co-PFP-A) copolymer recorded in CDCl3 with trifluoric acid (TFA, 5v-% in CDCl3) at 20 oC. PFTP-A composition was calculated using TFA as an internal reference in 1H NMR and 19F NMR, and by the following equation. PFTP-A composition = [B/(A+B)] × 100, A = I4.5ppm / (2 × I11.0ppm), with I11.0ppm and I4.5ppm corresponding to signal intensities at 11.0ppm and 4.5ppm attributed to TFA and VBC, respectively. B = (3 × I-153ppm)/ (2 ×I-75ppm), with I-153ppm and I-75ppm corresponding to signal intensities at 153ppm and -75ppm attributed to PFP-A and TFA, respectively.
S11
Refractive Index (RI)
20
30
40
20
30 Retention Time (min)
40
Figure S8. GPC traces of (-) P(OEG-A) homopolymer, Mn (GPC) = 10 200 g.mol-1; (-) purified P(OEG-A)-b-P(VBC-co-PFP-A) block copolymer, Mn (GPC) = 15 500 g.mol-1.
S12
-150.0
-155.0
-160.0
-165.0
Figure S9. Overlay of 19F NMR spectra of P(OEG-A)-b-(VBC-co-PFP-A) copolymer before and after modification with sodium methanethiosulfonate.
S13
Intensity distribution Volume distribution (%) (%)
25 20 15 10 5 0 1
10
100
1000
10
100
1000
20 15 10 5 0 1
Particle size (nm)
Figure S10. DLS data of uncross-linked nanoparticles obtained by self-assembly of block copolymer in water, concentration 1 mg.mL-1: volume and intensity distribution.
S14
Figure S11. DLS data of cross-linked nanoparticles obtained by self-assembly of block copolymer in water and compound (2), concentration 1 mg.mL-1: volume and intensity distribution.
S15
Number distribution (%)
30 25 20 15 10 5 0 1
10
100
1000
Particle size (nm) Figure S12. DLS data of (-) uncross-linked nanoparticles obtained by self-assembly of block copolymer in water and (-) cross-linked nanoparticles in pH = 5.0 (acidic water), concentration 1 mg.mL-1.
S16
1.0
Absorbance
0.8 0.6 0.4 0.2 0.0 300
400
500
600
Wavelength (nm) Figure S13. UV-visible spectra of cross-linked nanoparticles modified with thiol-fluorescein (-) before purification and (-) after purification by dialysis against water, and (-) UV-visible spectrum of purified of cross-linked nanoparticles modified with no functional-fluorescein. Comment: The reaction yield between thiol-modified FITC and methane thiosulfonate was calculated using the following equation: FITC yield (%) = (λFITC/ εFITC)/ [VBC], with λFITC, εFITC and [VBC] corresponding to absorbance at 484 nm and extension coefficient of FITC, and concentration of VBC, respectively. εFITC
=
78
000
L/mol/cm,
value
obtained
from
the
following
website:
http://www.nlv.ch/Molbiology/sites/Fluorescence1.htm.
S17
Figure S14. A- DLS data of cross-linked nanoparticles obtained by self-assembly of fluorescein modified block copolymer in water after purification by dialysis against water using membrane cut off 3 500 Dalton (inset: picture of the solution); B- TEM micrographs of cross-linked cross-linked nanoparticles prepared by self-assembly of fluorescein modified block copolymer (scale bar = 100 nm).
S18
Absorbance
0.4
0.2
0.0 200
300
400
500
600
Wavelength (nm) Figure S15. (-) UV-visible spectrum of purified cross-linked nanoparticles modified with thiol-modified FITC and (-) UV-visible spectrum of purified cross-linked nanoparticles modified with thiol-modified FITC after treatment with dithiothreitol (DTT) (20 mM) at 37 o
C for 24 h.
Comment: UV-visible spectrum of cross-linked nanoparticles modified with thiol-modified FITC treated with dithiothreitol (DTT) was obtained after dialysis against water. UV-visible confirms the release of FITC from the nanoparticles.
S19
Number distribution (%)
25 20 15 10 5 0 1
10
100
1000
Particle size (nm) Figure S16. DLS data of purified cross-linked nanoparticles obtained by self-assembly of block copolymer in the presence of Nile red, inset picture of the solution.
S20
0.6
Absorbance
0.4
0.2
0.0
300
400
500
600
700
800
Wavelength (nm) Figure S17. (-) UV-visible spectrum of purified cross-linked nanoparticles containing Nile Red and (-) UV-visible spectrum of cross-linked nanoparticles treated with acid (pH =5.0) at 37 oC for 24 h (0.1 mg.mL-1). Comment: The decrease of the Nile red absorbance is attributed to the precipitation of the dye in water. According UV-visible, 10% of dye is still encapsulated in the block copolymer due to the presence of hydrophobic interaction.
S21
Figure S18. Cellular uptake of nanoparticles by fluorescence microscopy using NIH-3t3 cells. (A) cells treated with uncross-linked FITC nanoparticles (B) cells treated with crosslinked FITC nanoparticles. Note: As a control experiment, untreated NIH-3t3 cell lines were analyzed by fluorescence microscopy, no signal was detected.
S22
Figure S19. Cellular internalization of uncross-linked and cross-linked nanoparticles of NIH3T3 cells after 48h studied by flow cytometry (λemission = 521 nm).
S23
Acid Degradable Biocompatible Polymeric Nanoparticles for the Potential co-Delivery of Therapeutic Agents Hien T.T. Duong,a Christopher P. Marquis,b Michael Whittaker, a Thomas P. Davis, a* Cyrille Boyer a* Australian Centre for NanoMedicine (ACN) b
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, 2052 NSW, Sydney, Australia
School of Chemical Engineering, The University of New South Wales, 2052 NSW, Sydney, Australia; Emails: [email protected]; [email protected].
Content: 1- Abbreviations 2- Characterization methods 3- Figures S1-S18
S1
1. Abbreviations: AIBN: 2,2’-Azobisisobutyronitrile BSPA: 3-(benzylsulfanylthiocarbonylsulfanyl)-propionic acid (BSPA Da: Dalton DLS: Dynamic light scattering DMF: N,N’-Dimethylformamide DTT: DL-Dithiothreitol FITC: fluorescein isothiocyanate GPC: Gel permeation chromatography MTS: Methanethiosulfonate OEG-A: Oligo(ethylene glycol) methyl ether acrylate PDI: Polydispersity P(OEG-A): Poly(oligo(ethylene glycol) methyl ether acrylate) P(OEG-A)-b-P(VBC-co-PFP-A): poly(oligo(ethylene glycol) methyl ether acrylate)-blockpoly(vinyl benzyl chloride-co-pentafluorophenyl acrylate) RAFT: Reversible addition fragmentation transfer VBC: Vinyl benzyl chloride VBS: Vinyl benzyl chloride methane thiosulfonate TFA: Trifluoric acid
S2
2. Characterization methods Gel permeation chromatography (GPC) measurements. DMAc GPC analyses of the polymers were performed in N,N-dimethylacetamide [DMAc; 0.03% w/v LiBr, 0.05% 2, 6– di-butyl-4-methylphenol (BHT)] at 50 °C (flow rate = 1 mL.min-1) using a Shimadzu modular system comprised of an SIL-10AD auto-injector, a PL 5.0-mm bead-size guard column (50 × 7.8 mm) followed by four linear PL (Styragel) columns (105, 104, 103, and 500 Å) and an RID-10A differential refractive-index detector. The SEC calibration was performed with narrow-polydispersity polystyrene standards ranging from 168 to 106 g.mol-1. A total of 50 µL of polymer solution (2 mg.mL-1 in DMAc) was injected for every analysis. Nuclear Magnetic Resonance (NMR). Structures of the synthesized compounds were analyzed by 1H NMR, 13C NMR and 19F NMR spectroscopy using a Bruker DRX 300 MHz spectrometer at 300 MHz for hydrogen nuclei and at 75 MHz for carbon nuclei. OEG-A conversion was calculated using the following equation: αOEG-A = (∫ICH=CH2
5.5-6.5 ppm
/ ∫IOCH3
3.6ppm
) × 100, where ∫ICH=CH2
5.5-6.5 ppm
and ∫IOCH3
3.3 ppm
correspond to intensities from acrylate bond of OEG-A and methyl ether. VBC conversion was calculated using the following equation: αVBC = [2 × ∫ICH=CH2 5.5-6.5 ppm/ 3 × ∫ICH2Cl 4.5ppm] × 100, where ∫ICH=CH2 5.5-6.5 ppm and ∫ICH2Cl 4.5 ppm
correspond to intensities from OEG-A (methyl ester) and BSPA RAFT agent (benzyl
group), molar mass of monomer and RAFT agent, respectively. PFP-A conversion was calculated using 19F NMR analysis and the following equation: αPFPA-A = [∫-158.0
ppm
/ (∫I-158.0 ppm + ∫-152.5
ppm
)] × 100
UV-visible spectroscopy. UV-visible spectra were recorded using a CARY 300 spectrophotometer (Bruker). Dynamic light scattering (DLS). DLS measurements were performed using a Malvern Zetasizer Nano Series running DTS software and using a 4 mW He-Ne laser operating at a
S3
wavelength of 633 nm and an avalanche photodiode (APD) detector. The scattered light was detected at an angle of 173o. The temperature was stabilized to +/- 0.1oC of the set temperature. To reduce the influence of larger aggregates the number-average hydrodynamic particle size is reported. The polydispersity index (PDI) is used to describe the width of the particle size distribution, as calculated from the DTS software using a cumulate analysis of the measured intensity autocorrelation function; it is related to the standard deviation of the hypothetical Gaussian distribution (i.e. PDI = s2/ZD2, where s is the standard deviation and ZD is the Z average mean size). Transmission Electron Microscopy (TEM). The sizes and morphologies of the un-crossliked and cross-linked polymers were observed using a transmission electron microscopy JEOL1400 TEM at an accelerating voltage of 100 kV. The particles were dispersed in water (1 mg.mL-1) and deposited onto 200 mesh, holey film, copper grid (ProSciTech). Osmium vapor (OsO4) staining was applied.
S4
3. Figures S1-S19
A a c
a
b
c b
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm B
a
a
b c
-155.0
c
b
-160.0
ppm Figure S1. A- 1H NMR and B- 19F NMR spectra of pentafluorophenyl acrylate recorded in CDCl3 at 20 oC.
S5
b c d
a
d
b c
a
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm Figure S2. 1H NMR spectrum of compound 1 (scheme 1 in the main text) recorded in CDCl3 at 20 oC.
S6
d
b a c d c
7.0
6.0
5.0
4.0
b
3.0
a
2.0
1.0
ppm Figure S3. 1H NMR spectrum of compound 2 (scheme 1 in the main text) recorded in CDCl3 at 20 oC.
S7
d a e b f c d f
e
a
b
c
-150.0
-155.0
-160.0
ppm Figure S4. 19F NMR spectra of crude polymerization reaction of P(OEG-A)-b-P(VBC-coPFP-A) copolymer obtained via RAFT polymerization technique, recorded in CDCl3 at 20 o C.
Note: After polymerization, the signals associated to pentafluorophenyl acrylate shift from 152.5, -156.8 and -162.5 ppm to -153.0, -158.0 and -162.5 ppm. Pentafluorophenyl acrylate monomer conversion was calculated using the following equation: αPFPA-A = [∫-158.0
ppm
/ (∫I-158.0 ppm + ∫-152.5
ppm
)] × 100
S8
d
d
a e e b f c b a
-140
f
c
-150
-160
-170
-180
ppm Figure S5. 19F NMR spectra of crude reaction of P(OEG-A)-b-P(VBC-co-PFP-A) in the presence of compound (2) recorded in CDCl3 at 20 oC.
Note: The reaction of poly(pentafluorophenyl acrylate) in the presence of amino-compound results by the formation of pentafluorophenol as indicated by the signals at -167, -168 and 169 ppm. The yield was calculated using 19F NMR spectroscopy and the following equation: Yield (%) = ∫-167.0
ppm
/ (∫I-162.0ppm + ∫-167.0
ppm
) × 100
S9
a g
b c
d
e
e f
f
× HDO
d c a
b
g
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm Figure S6. 1H NMR spectrum of P(OEG-A) homopolymer obtained via RAFT polymerization recorded in CDCl3 at 20 oC, Mn (NMR) = 11 000 g.mol-1. Note: Mn (NMR) = (5 × ∫ICH2O 4.0 ppm / 2 × ∫ICH 7.2 ppm) × MWOEG-A + MWBSPA, where ∫ICH3O 3.6 ppm
, ∫ICH2O 4.0 ppm, MWMA and MWBSPA correspond to intensities from OEG-A (methyl ester)
and BSPA RAFT agent (benzyl group), molar mass of monomer and RAFT agent, respectively.
S10
A
d d
a
b
a b c
0
-50
-100
-150
c
-200
PPM B e
e f
f
10.0
5.0
PPM
Figure S7. A- 19F NMR and B- 1H NMR spectra of P(OEG-A)-b-(VBC-co-PFP-A) copolymer recorded in CDCl3 with trifluoric acid (TFA, 5v-% in CDCl3) at 20 oC. PFTP-A composition was calculated using TFA as an internal reference in 1H NMR and 19F NMR, and by the following equation. PFTP-A composition = [B/(A+B)] × 100, A = I4.5ppm / (2 × I11.0ppm), with I11.0ppm and I4.5ppm corresponding to signal intensities at 11.0ppm and 4.5ppm attributed to TFA and VBC, respectively. B = (3 × I-153ppm)/ (2 ×I-75ppm), with I-153ppm and I-75ppm corresponding to signal intensities at 153ppm and -75ppm attributed to PFP-A and TFA, respectively.
S11
Refractive Index (RI)
20
30
40
20
30 Retention Time (min)
40
Figure S8. GPC traces of (-) P(OEG-A) homopolymer, Mn (GPC) = 10 200 g.mol-1; (-) purified P(OEG-A)-b-P(VBC-co-PFP-A) block copolymer, Mn (GPC) = 15 500 g.mol-1.
S12
-150.0
-155.0
-160.0
-165.0
Figure S9. Overlay of 19F NMR spectra of P(OEG-A)-b-(VBC-co-PFP-A) copolymer before and after modification with sodium methanethiosulfonate.
S13
Intensity distribution Volume distribution (%) (%)
25 20 15 10 5 0 1
10
100
1000
10
100
1000
20 15 10 5 0 1
Particle size (nm)
Figure S10. DLS data of uncross-linked nanoparticles obtained by self-assembly of block copolymer in water, concentration 1 mg.mL-1: volume and intensity distribution.
S14
Figure S11. DLS data of cross-linked nanoparticles obtained by self-assembly of block copolymer in water and compound (2), concentration 1 mg.mL-1: volume and intensity distribution.
S15
Number distribution (%)
30 25 20 15 10 5 0 1
10
100
1000
Particle size (nm) Figure S12. DLS data of (-) uncross-linked nanoparticles obtained by self-assembly of block copolymer in water and (-) cross-linked nanoparticles in pH = 5.0 (acidic water), concentration 1 mg.mL-1.
S16
1.0
Absorbance
0.8 0.6 0.4 0.2 0.0 300
400
500
600
Wavelength (nm) Figure S13. UV-visible spectra of cross-linked nanoparticles modified with thiol-fluorescein (-) before purification and (-) after purification by dialysis against water, and (-) UV-visible spectrum of purified of cross-linked nanoparticles modified with no functional-fluorescein. Comment: The reaction yield between thiol-modified FITC and methane thiosulfonate was calculated using the following equation: FITC yield (%) = (λFITC/ εFITC)/ [VBC], with λFITC, εFITC and [VBC] corresponding to absorbance at 484 nm and extension coefficient of FITC, and concentration of VBC, respectively. εFITC
=
78
000
L/mol/cm,
value
obtained
from
the
following
website:
http://www.nlv.ch/Molbiology/sites/Fluorescence1.htm.
S17
Figure S14. A- DLS data of cross-linked nanoparticles obtained by self-assembly of fluorescein modified block copolymer in water after purification by dialysis against water using membrane cut off 3 500 Dalton (inset: picture of the solution); B- TEM micrographs of cross-linked cross-linked nanoparticles prepared by self-assembly of fluorescein modified block copolymer (scale bar = 100 nm).
S18
Absorbance
0.4
0.2
0.0 200
300
400
500
600
Wavelength (nm) Figure S15. (-) UV-visible spectrum of purified cross-linked nanoparticles modified with thiol-modified FITC and (-) UV-visible spectrum of purified cross-linked nanoparticles modified with thiol-modified FITC after treatment with dithiothreitol (DTT) (20 mM) at 37 o
C for 24 h.
Comment: UV-visible spectrum of cross-linked nanoparticles modified with thiol-modified FITC treated with dithiothreitol (DTT) was obtained after dialysis against water. UV-visible confirms the release of FITC from the nanoparticles.
S19
Number distribution (%)
25 20 15 10 5 0 1
10
100
1000
Particle size (nm) Figure S16. DLS data of purified cross-linked nanoparticles obtained by self-assembly of block copolymer in the presence of Nile red, inset picture of the solution.
S20
0.6
Absorbance
0.4
0.2
0.0
300
400
500
600
700
800
Wavelength (nm) Figure S17. (-) UV-visible spectrum of purified cross-linked nanoparticles containing Nile Red and (-) UV-visible spectrum of cross-linked nanoparticles treated with acid (pH =5.0) at 37 oC for 24 h (0.1 mg.mL-1). Comment: The decrease of the Nile red absorbance is attributed to the precipitation of the dye in water. According UV-visible, 10% of dye is still encapsulated in the block copolymer due to the presence of hydrophobic interaction.
S21
Figure S18. Cellular uptake of nanoparticles by fluorescence microscopy using NIH-3t3 cells. (A) cells treated with uncross-linked FITC nanoparticles (B) cells treated with crosslinked FITC nanoparticles. Note: As a control experiment, untreated NIH-3t3 cell lines were analyzed by fluorescence microscopy, no signal was detected.
S22
Figure S19. Cellular internalization of uncross-linked and cross-linked nanoparticles of NIH3T3 cells after 48h studied by flow cytometry (λemission = 521 nm).
S23
