Supporting information for In Situ Gelation-Induced Death of Cancer Cells Based on Proteinosomes Yuting Zhou, Jianmin Song, Lei Wang*, Xuting Xue, Xiaoman Liu, Hui Xie*, and Xin Huang* MIIT Key Laboratory of Critical Materials Technology for New Energy Conversion and Storage, State Key Laboratory of Robotics and Systems, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China. E-mails: leiwang_chem@hit.edu.cn, xiehui@hit.edu.cn, xinhuang@hit.edu.cn. These authors contributed equally to this work. Characterization of PNIPAAm, BSA-NH 2, BSA-P Determination of primary amine group on the surface of BSA-NH 2 by TNBSA Measurement. 2,4,6-trinitrobenzene sulfonic acid (TNBSA) is a rapid and sensitive assay reagent for the determination of free primary amino groups. Primary amines, upon reaction with TNBSA, form a highly chromogenic derivative, which can be measured at 345 nm by UV-vis spectroscopy. Briefly, sample solutions of BSA-NH 2 were prepared in 0.1 M sodium bicarbonate buffer (ph 8.5). 0.4 ml of sample solution was prepared, followed by the addition of 250-time diluted 5% TNBSA solution (in 0.1 M bicarbonate buffer, ph 8.5) and incubation for 2 h at 37 o C. Then, 0.2 M HCl (0.65 ml) was added to each sample to stop the reaction, followed by the UV-vis detection of each sample solution (Fig. S2). The determination of amine concentration was realized based on the same procedure using glycine as a standard compound (Fig. S2a-b). As a control, the original primary amine groups on the surface of BSA were also tested accordingly (Fig. S2c-d). Compared with BSA, the number of linked primary amine groups per BSA-NH 2 was determined to be 35 in average (Table S1-2). Determination of the BSA-NH 2 /PNIPAAm conjugate composition. The composition of the BSA-NH 2 /PNIPAAm conjugate was determined by UV spectroscopy. Aqueous solutions of protein-polymer conjugate were prepared at a range of known concentrations and in each case the BSA concentration was determined using calibration curves obtained by measuring the concentration-dependent characteristic UV absorbance of the native protein at 276 nm (Fig. S3). The PNIPAAm content in each case was then determined from the difference between the known conjugate concentration and determined BSA concentration (Table S3). The average number of PNIPAAm chains per BSA was 2.0. Evaluation of esterase-like activity of BSA The esterase-like activity of BSA towards p-nitrophenyl acetate was performed according to previous procedure. Briefly, 0.1 ml of BSA or BSA-NH 2 /PNIPAAm 1
conjugate solution ([BSA] = 0.3 mm) in phosphate buffer (ph 8.0), and 10 μl of p-nitrophenyl acetate were dissolved in acetonitrile (10 mm) and rapidly mixed with 0.9 ml of phosphate buffer solution (ph 8.0). After incubating at room temperature for 20 min, the absorbance at 405 nm was measured for each sample to evaluate the activity, and normalized compared with native BSA. The activity measurements were conducted with two different samples in triplicates. The results represent the average of 6 measurements ± standard deviation. Fig. S1 1 H NMR spectrum of mercaptothiazoline-activated PNIPAAm (Mn 15000 g/mol) in CDCl 3. 2
Fig. S2 (a) UV-Vis spectra obtained for TNBS/glycine control assay at different concentrations; (b) corresponding calibration curve based on plotting the absorbance at 345 nm against the concentration of primary amine; UV-Vis spectra obtained for TNBS/BSA (c) and TNBS/BSA-NH 2 (d) assays at different protein concentrations. Supplementary Table S1. Number of primary amine groups in BSA. Concentration of BSA A (345 nm) The number of primary amine per BSA Test 1 0.2 mg/ml 0.296098 30.5 Test 2 0.8 mg/ml 0.564731 32.3 Test 3 1.0 mg/ml 0.652013 32.4 Test 4 1.5 mg/ml 0.81416 29.4 Test 5 2.0 mg/ml 0.892466 25 Supplementary Table S2. Number of linked primary amine groups in BSA-NH 2. Concentration BSA-NH 2 of 3 A (345 nm) The number of primary amine per BSA Test 1 0.05 mg/ml 0.254982 61.9 Test 2 0.1 mg/ml 0.30266 66 Test 3 0.15 mg/ml 0.357144 70.6 Test 4 0.5 mg/ml 0.681021 68.8 Test 5 0.75 mg/ml 0.809941 58.5
Fig. S3 (a) UV-Vis spectra obtained for BSA control assay at different concentrations; (b) corresponding calibration curve based on plotting the absorbance at 276 nm; (c) UV-Vis spectra obtained for BSA-P at different concentrations. Supplementary Table S3. Number of linked polymers in BSA-NH 2 /PNIPAAm Concentration of A (276 nm) PNIPAAm Content (chains per BSA-NH 2 /PNIPAAm BSA-NH 2 /PNIPAAm) Test 1 0.1 mg/ml 0.029198 2.96 Test 2 0.25 mg/ml 0.065746 1.7 Test 3 0.6 mg/ml 0.215787 2.3 Test 4 0.8 mg/ml 0.295458 2 Demonstration of the water-in-oil Proteinosomes. 5 μl of the hydrophilic fluorescence dye (FITC-Dextran) (5 mg/ml, dissolved in water) was added to 0.06 ml of aqueous BSA-NH 2 /PNIPAAm (20.0 mg/ml, ph 8.5, sodium carbonate buffer), and then mixed with 1.0 ml of 2-ethyl-1-hexanol containing 0.5% of hydrophobic fluorescence dye (Nile Red). Followed by a certain time of sonication, the product proteinosomes were observed using optical and fluorescence microscopy. Fig. S4 Optical (a) and fluorescence (b, c) microscopy images of proteinosomes in oil; the fluorescence originates from encapsulating fluorescein isothiocyanate labeled dextran (FITC-Dextran, Mw 150 kda) in water and Nile Red in oil. (d) The merged image of (b) and (c). 4
Fig. S5 AFM image (a) of a dried proteinosome, and the corresponding height plot (b) to the white line in (a). Scale bar is 200 nm. Optimization of the concentration of BSA-NH 2 /PNIPAAm, sonication time, and sonication power for the fabrication of proteinosomes i). The concentration of BSA-NH 2 /PNIPAAm conjugates 0.06 ml of aqueous BSA-NH 2 /PNIPAAm (ph 8.5, sodium carbonate buffer) were mixed with 1.0 ml of the 2-ethyl-1-hexanol. Proteinosomes were prepared by sonication. The concentration of the protein-polymer building blocks increased from 5, 10, 15, 20 to 25 mg/ ml, respectively. The time and power of sonication remained same. 5
Fig. S6 (a-e) Size distribution of proteinosomes prepared at different concentrations of BSA-NH 2 /PNIPAAm (5, 10, 15, 20, and 25 mg/ml, respectively). Median values and standard deviations (s.d.) were calculated by fitting Gaussians to the histograms in a-e. (f) Plot showing mean size (diameter, bars) and s.d. (lines on bars) of proteinosomes dispersed in oil phase, and prepared at different concentrations of BSA-NH 2 /PNIPAAm from 5 to 25 mg/ml. ii). Sonication time 0.06 ml of aqueous BSA-NH 2 /PNIPAAm (ph 8.5, sodium carbonate buffer) were mixed with 1.0 ml of the 2-ethyl-1-hexanol. Proteinosomes were prepared by sonication. The time of sonication was increased from 1, 2, 3, 4 to 5 min in turn. The concentration of the BSA-NH 2 /PNIPAAm conjugate and the power of sonication remained same. 6
Fig. S7 (a-e) Size distribution of proteinosomes prepared at different time of sonication (5, 4, 3, 2, and 1 minutes, respectively). Median values and standard deviations (s.d.) were calculated by fitting Gaussians to the histograms in a-e. (f) Plot showing mean size (bars) and s.d. (lines on bars) of proteinosomes dispersed in oil and prepared at different time of sonication from 1 to 5 minutes. iii). Sonication power 0.06 ml of aqueous BSA-NH 2 /PNIPAAm (ph 8.5, sodium carbonate buffer) were mixed with 1.0 ml of the 2-ethyl-1-hexanol. Proteinosomes were prepared by sonication. The power of sonication was set as 100, 150, 200, 250 and 300 W, respectively. The concentration of the BSA-NH 2 /PNIPAAm conjugate and the time sonication remained same. 7
Fig. S8 (a-e) Size distribution of proteinosomes prepared at different power of sonication (100, 150, 200, 250, and 300 W, respectively). Median values and standard deviations (s.d.) were calculated by fitting Gaussians to the histograms in a-e. (f) Plot showing mean size (bars) and s.d. (lines on bars) of proteinosomes dispersed in oil and prepared at different sonication power from 100 to 300 W. 8
Fig. S9 Esterase activity of BSA after treatments under various conditions (normalized using native BSA). Fig. S10 (a-e) Optical microscopy images of BSA-NH 2 /PNIPAAm proteinosomes cross-linked by different final concentrations of NHS-PEG16-DS disulfide ester (2.5, 7.5, 12.5, 20 and 25 mg/ml, respectively). (f) The production of proteinosomes prepared at different concentrations of cross-linker from 2.5 to 25 mg/ml. The insets are photos of proteinosomes dispersed in water, where the red circles showing the location of proteinosomes. For details, the crosslinker of NHS-PEG16-DS disulfide ester, with the final concentration of 2.5, 7.5, 12.5, 20 and 25 mg/ml, was utilized in the crosslinking process, respectively. From the results, we can see that when the concentration was lower than 12.5 mg/ml the yield of the produced proteinosomes decreased obviously. Furthermore, when the concentration was over 12.5 mg/ml, some aggregations of proteinosomes appeared. Therefore, 12.5 mg/ml was selected as the optimum concentration in our system. 9
Fig. S11 The effect of preparation temperature on the formation of the proteinosomes. The temperature from (a) to (e) are 25, 30, 32, 37 and 40 respectively. (f) The yield of proteinosomes prepared at different temperature after 10 minutes (purple bar) and one week (orange bar). Fig. S12 (a) Optical microscopy images of BSA-NH 2 /PNIPAAm proteinosomes containing sodium alginate from 25 to 40 showing the effect of temperature on the proteinosomes. The formation process of hydrogel. Proteinosomes with the size around 20 µm were prepared by shaking the mixture of an aqueous BSA-NH 2 /PNIPAAm solution and 2-ethyl-1-hexanol. 0.06 ml of aqueous BSA-NH 2 /PNIPAAm (10.0 mg/ml, ph 8.5, sodium carbonate buffer) were mixed with 1.0 ml of oil. The components encapsulated in proteinosomes, including NaAlg, were added into the water phase before mixing with the oil phase. The proteinosomes were then crosslinked in the continuous oil phase by the addition of NHS-PEG16-DS disulfide ester. The crosslinked proteinosomes were transferred into a continuous water phase. Then Ca 2+ ions were added to proteinosomes solution. The concentration was optimized according to its effect on the live cell percentage, shown in Figure S13. After 10 min, GSH was added to the above solution. 10
Fig. S13 The cell viability of cancer cells under different concentrations of Ca ions. Fig. S14 Effect of different concentrations of calcium ions on formation of the hydrogel. (a) Images of five vials containing sodium alginate, with the addition of calcium ions of different concentrations (from left to right: 2.5, 2.0, 1.5, 1.0 and 0.5 mg/ml, respectively); (b) Images of five inversed vials after the addition of different concentrations of calcium ions for 30 s. The red color comes from red ink, for better observation. Considering the time scale and the gel quantity, 1.5 mg/ml was selected as the optimized concentration for calcium ions. 11
Fig. S15 Optical (a) and fluorescence (b) microscopy images of proteinosomes with loaded CaAlg hydrogel after drying in air; SEM (c) and TEM (d) images of proteinosomes with loaded CaAlg. Scale bars are 2 μm in the insets of a-b, and 500 nm in the insets of c and d. Fig. S16 (a) FL spectra of FITC-Dextran at different concentration and FITC-Dextran released from proteinosomes with GSH (2 mm) after 240 min (the red line). (b) Determination of the FITC-Dextran content of FITC-Dextran-loaded proteinosomes ( ) by using a dilution of FITC-Dextran ( ). 12
Fig. S17 (a) FL spectra of FITC-Dextran released from proteinosomes at different time with GSH (2 mm). (b) Plots showing GSH-mediated release of FITC-Dextran from BSA-NH 2 /PNIPAAm proteinosomes cross-linked by NHS-PEG16-DS disulfide ester by using different concentration of GSH from 0, 0.5, 2.0 and 5.0 mm, respectively. 13
Fig. S18 Concentration-dependent cellular uptake behaviors of HepG2 cells. (a-e) The HepG2 cells were incubated in culture medium with the presence of FITC-Dextran-loaded proteinosomes at the concentration from 0.3125, 0.625, 1.25, 2.5 to 5.0 µm, respectively. 14
Fig. S19 Concentration-dependent cellular uptake behaviors of NIH 3T3 cells. (a-e) The NIH 3T3 cells were incubated in culture medium with the presence of FITC-Dextran-loaded proteinosomes at the concentration from 0.3125, 0.625, 1.25, 2.5 to 5.0 µm, respectively. Experimental Operation The cell sample was first placed and the probe was found under an optical microscope. The distance of the probe from the bottom of the sample is about 30 μm. The laser sensitivity of the probe cantilever was calibrated on the surface of the dish. Determine the sensitivity of the probe Q. The location of the samples was determined using optical microscopy. Then, the pull-off experiment was conducted, with more than 10 times random experiment for each point obtained in the plot, to ensure the accuracy of 15
the experimental data. The optical image of experimental setup was shown in Fig. S20, which showed the relative position between the tip and sample cells. Fig. S20 The optical image of experimental setup and sample cells under the tip. The scale bar is 50 µm. Typical force-distance curves Young s modulus was calculated mainly from the data of force-distance curve. Figure S21a was the force-distance curve of normal cancer cells, with the calculated Young s modulus of 457.40 Pa. Figure S21b showed the force-distance curve of cancer cells engulfed capsules containing calcium alginate, with the calculated Young s modulus of 5862.04 Pa. Similarly, all the data in this paper was calculated using the above method. Figure S22 was the obtained profile of pure proteinosomes containing calcium alginate. Fig. S21 Typical force-distance curves of cancer cells (a), and cancer cells with proteinosomes containing calcium alginate (b). 16
Fig. S22 Young s modulus statistical histogram of proteinosomes containing calcium alginate. Fig. S23 MTT toxicity assay with NIH 3T3 cells. Inhibitory effect of proteinosomes in PBS solution, proteinosomes-naalg, and proteinosomes-caalg within NIH 3T3 cells, after 24 h (a), 36 h (b) and 48 h (c). (n = 3, error bar = SD). 17