Supplementary Material for Revised Manuscript:

Multi-Parametric Surface Plasmon Resonance Platform for Studying Liposome-Serum Interactions and Protein Corona Formation

Otto K. Kari(equal contribution), Tatu Rojalin(equal contribution), Stefano Salmaso, Michela Barattin, Hanna Jarva, Seppo Meri, Marjo Yliperttula, Tapani Viitala(corresponding author), Arto Urtti

Table S1. Physicochemical characterization and endotoxin determination results for DOX, DOX+PEG, OGD and OGD+PEG liposomes.Results are presented asaverage ( standard deviation) for hydrodynamic diameter and ζ-potential. Results for endotoxins are reported in EU/ml and described as positive (+) and negative (-) based on two complementary assays.

Hydrodynamic diameter / Polydispersity index / ζ-potential / Endotoxin
DOX / 98.12  14.80 nm / 0.324 / -1.42  0.16 mV / <0.1 EU/ml (-)
DOX
+PEG / 105.07  2.64 nm / 0.017 / -1.90  0.17 mV / <0.1 EU/ml (-)
OGD / 227.03  19.28 nm / 0.254 / +20.10  0.40 mV / >1.0 EU/ml (+)
OGD
+PEG / 248.43  30.40 nm / 0.328 / -1.84  0.41 mV / >1.0 EU/ml (+)a
a) OGD+PEG liposome tested after the charge-to-charge association process tested positive for endotoxin, but the polyanionic-PEG block-copolymer solution alone contained <0.1 EU/ml of endotoxin.

Table S2. The compiled modeling results of separate experiments for OGD and OGD+PEG liposomes and serum interactions. The calculated values are low and high limits for layer thickness (d) with corresponding refractive index (n) values from all three independent measurements and their average ( standard deviation).Refractive indices extracted during fitting procedure: serum 1.34960, buffer 1.33535.

(I) Liposome / (II) Soft corona / (III) Hard corona
OGD / Low / High / Low / High / Low / High
1 / 39.97 / 51.08 / 32.7 / 94.28 / 9.73 / 16.0
2 / 31.31 / 42.15 / 33.37 / 97.72 / 10.5 / 17.45
3 / 49.88 / 73.27 / 36.61 / 126.05 / 8.59 / 15.79
d / 40.3867 ± 9.29201 / 55.5 ± 16.0239 / 34.2267 ± 2.09104 / 106.017 ± 17.4344 / 9.60667 ± 0.96095 / 16.4133 ± 0.9039
1 / 39.97 / 1.35166 / 1.40034 / 1.37681 / 1.44632 / 1.41075
2 / 31.31 / 1.34876 / 1.40279 / 1.37927 / 1.45346 / 1.41491
3 / 49.88 / 1.34947 / 1.38789 / 1.37619 / 1.43904 / 1.40797
n / 1.35302
± 0.0021 / 1.34996
± 0.00151 / 1.39701
± 0.00799 / 1.37742
± 0.00163 / 1.44627
± 0.0072 / 1.41121
± 0.0035
OGD+PEG / Low / High / Low / High / Low / High
1 / 48.0 / 61.94 / 22.63 / 54.75 / 3.2 / 4.99
2 / 45.44 / 68.51 / 36.53 / 124.19 / 7.73 / 15.99
3 / 39.72 / 52.35 / 7.5 / 10.64 / 2.99 / 4.8
d / 44.3867
± 4.23931 / 60.9333
±8.1269 / 22.22 ± 14.5193 / 63.1933 ± 57.2439 / 4.64 ± 2.67808 / 8.6 ± 6.4
1 / 1.35643 / 1.35295 / 1.38403 / 1.37284 / 1.3802 / 1.36849
2 / 1.35987 / 1.35461 / 1.39426 / 1.38047 / 1.43979 / 1.40062
3 / 1.35406 / 1.35034 / 1.35963 / 1.38709 / 1.36573 / 1.35681
n / 1.35679
± 0.00292 / 1.35263
± 0.00215 / 1.37931
± 0.01779 / 1.38013
± 0.00713 / 1.39524
± 0.03925 / 1.37531
± 0.02269

Table S3. The compiled modeling results of separate experiments for DOX and DOX+PEG liposomes and serum interactions. The calculated values are low and high limits for layer thickness (d) with corresponding refractive index (n) values from all three independent measurements and their average ( standard deviation).Refractive indices extracted during fitting procedure: serum 1.34960, buffer 1.33535.

(I) Liposome / (II) Soft corona / (III) Hard corona
DOX / Low / High / Low / High / Low / High
1 / 104.01 / 161.5 / 39.48 / 88.5 / 3.85 / 7.45
2 / 93.98 / 145.02 / 36.32 / 105.39 / 1.98 / 4.39
3 / 99.16 / 153.05 / 36.63 / 66.41 / 4.26 / 7.81
d / 99.05 ± 5.015905 / 153.19 ± 8.240892 / 37.47667 ± 1.741848 / 86.76667 ± 19.54772 / 3.363333 ± 1.215415 / 6.55 ± 1.879255
1 / 1.36309 / 1.35754 / 1.42127 / 1.4351 / 1.39668 / 1.39222
2 / 1.36128 / 1.35579 / 1.42137 / 1.40966 / 1.39603 / 1.37973
3 / 1.36275 / 1.35706 / 1.41862 / 1.44315 / 1.39048 / 1.38648
n / 1.362373
± 0.000962 / 1.356797
± 0.0009 / 1.41742
± 0.00457 / 1.429303
± 0.017481 / 1.39440
± 0.00341 / 1.38614
± 0.00625
DOX+PEG / Low / High / Low / High / Low / High
1 / 40.8 / 50.64 / 20.27 / 51.26 / 6.65 / 12.17
2 / 31.98 / 45.78 / 27.62 / 48.07 / 8.25 / 13.57
3 / 39.2 / 50.87 / 29.36 / 80.68 / 7.96 / 13.36
d / 37.3267 ± 4.69895 / 49.0967 ± 2.87462 / 25.75 ± 4.8249 / 60.0033 ± 17.9774 / 7.62 ± 0.85247 / 13.0333 ± 0.75501
1 / 1.34788 / 1.34742 / 1.40478 / 1.38021 / 1.41521 / 1.38219
2 / 1.35118 / 1.34706 / 1.39126 / 1.37628 / 1.42977 / 1.39814
3 / 1.35008 / 1.34719 / 1.39528 / 1.3754 / 1.42562 / 1.39527
n / 1.34971
± 0.00168 / 1.34722
± 0.00018 / 1.39711
± 0.00694 / 1.37730
± 0.00256 / 1.42353
± 0.0075 / 1.39187
± 0.0085