Supplementary Materials
Fig S1. 1H nuclear magnetic resonance (NMR) spectroscopic analysis of 2-propylacrylic acid and
tripolymer poly(BMA-co-PAA-co-DMAEMA).
1
Fig S2. 1H nuclear magnetic resonance (NMR) of blend particles made from PLGA and tripolymer with the
indicated weight fractions.
2
Fig. S3. Effect of blend particles on the viability of DC2.4 cells. DC2.4 cells were incubated with blend
particles at different concentration (0, 5, 10, 20, 50 g/ml) for 4 h at 37 oC. DC2.4 were then stained with Fixable
Viability Dye eFluor780 (ebioscience). Cell viability was determined by using flow cytometry and expressed as the
percentage of control group (cells without any particle). Blend particles did not induce cell death below 50 g/ml.
The asterisk indicates p<0.05 with respect to control group without particles and is considered as statistically
significant.
3
Fig. S4. Co-localization of endosomal/lysosomal compartment and particles in DC2.4 cells. Cells were
incubated with HiLyte647-labeled blend particles for 4 h at 37°C. Carboxylate-modified polystyrene beads
(200nm) were used as controls. All types of blend particle stayed inside the endosomal/lysosomal compartments
labeled by LAMP-2, similar to PS control. Blue: nucleus, green: LAMP-2, red: particles. Scale bar = 10 µm. The
images are representative of two independent experiments. Three to five regions for each sample were taken by
confocal microscopy.
4
Fig. S5. Optimization of the time of incubation and concentration of digitonin for extracting cytosolic
contents. 4 x 106 DC2.4 cells were treated with varying concentrations of digitonin (20, 30, 40 μg/ml) on ice for 5,
10 or 20 min. Lysis buffer and extraction buffer without digitonin were used as controls. (A) Fraction of
permeabilized cells. Treated cells were stained with trypan blue and counted under microscopy. Black:
permeabilized cells; white: membrane-intact cells. (B) Activity of N-acetyl-β-D-glucosaminidase (NAG) was
measured with assay kits and expressed as units per 10 5 permeabilized cells. The asterisk indicates p<0.05
versus the cells treated with lysis buffer and is considered as statistically significant.
5
Table S1. Molecular weight of a selection of endosomal/lysosomal enzymes
Enzyme type
Enzyme name
Molecular weight (kDa)
Literature Reference
Cathepsin A
mature form: 32 ; proform:55
(1)
Cathepsin B
mature form: 25; proform:43
(2)
Cathepsin D
mature form: 34 ; proform:52
(3)
mature form: 42;
Cathepsin E
(4)
proform:46,dimeric: 86
Lysosomal
Proteases
Nucleases
Carbohydrate
digesting
enzymes
Lipid digesting
enzymes
Cathepsin G
31
(5)
Cathepsin H
50 (two-chain form)
(6)
Cathepsin L
mature form: 27; proform: 40
(7)
Cathepsin K
mature form: 29; proform:37
(8)
Cathepsin O
mature form: 23 ; proform: 36
(9)
Cathepsin S
mature form: 24 ; proform: 36
(10)
Legumain
mature form: 36 ; proform: 56
(11)
Deoxyribonuclease II
23,30
(12)
N-acetyl-β-D-glucosaminidase
140
(13)
α-1,6-D-mannosidase
120
(14)
α-L-fucosidase
110
(15)
sphingomyelin phosphodiesterase
200
(16)
6
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