Supporting Information. Supramolecular Host-Guest Chemistry-based Folate/Riboflavin Functionalization and. Cancer Cell Labeling of Nanoparticle

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1 Supporting Information Supramolecular Host-Guest Chemistry-based Folate/Riboflavin Functionalization and Cancer Cell Labeling of Nanoparticle Suman Pal, 1 Chumki Dalal 1 and Nikhil R. Jana 1, * 1 Centre for Advanced Materials, Indian Association for the Cultivation of Science, 2A & 2B Raja S. C. Mullick Road, Kolkata , India *Corresponding authors camnrj@iacs.res.in. S-1

2 Table S1. Concentrations of different coexisting chemicals used for studying the stability of host-guest complex. Bio elements Concentration used (mg/l) Potassium (K + ) chloride 60 Sodium (Na + ) chloride 2500 Calcium (Ca 2+ ) chloride 100 Iron (Fe 3+ ) chloride 120 Zinc (Zn 2+ ) chloride 1.5 Glutathione 300 L-cysteine 11.8 Vitamin E 10 Vitamin C 10 S-2

3 Table S2. Different endocytosis inhibitors used in this study along with their final concentration. Inhibitors (abbreviation) Concentration used Endocytosis inhibited Sucrose 0.4 (M) Clathrin Methyl-β-Cyclodextrin 10 (mm) Lipid raft (MBCD) Genistein (GEN) 50 (µm) Caveolae Amiloride 50 (µm) Macropinocytosis S-3

4 Figure S1. Determination of the number of primary amines and CD per QD. (a) Fluorescence spectra obtained after adding excesses fluorescamine to the solutions of QD(NH 2 ) 100 and QD(CD) 70. Both sample solutions have same concentration of QD but different extents of primary amines. Fluorescence spectra were measured after quenching the QDs with HCland then neutralizing with base. Fluorescence intensity is used to estimate the primary amine concentration (see reference 11 for details) (b) Absorption spectra obtained by anthrone test for QD(NH 2 ) 100 and QD(CD) 70. CD per QD was estimated using the calibration graph obtained for varied concentration of CD. The linear equation is as follows: Y = 2.5x10 4 X +0.1 with R 2 =0.99. (X = concentration of β-cd- NH 2 and Y = absorbance at 620 nm). S-4

5 (a) (b) 3.0 Absorbance QD(NH 2 ) folate folate PL Intensity (a.u.) Absorbance QD(NH 2 ) riboflavine riboflavine Wavelength (nm) Wavelength (nm) Figure S2. a) Evidence of insignificant interaction of folate with QD(NH 2 ) 100. Folate is incubated with colloidal solution of QD(NH 2 ) 100 and then absorption/emission spectrum of folate is measured. Insignificant changes in absorption and fluorescence intensity suggest insignificant interaction. Other experimental conditions are similar to Figure 2. b) Evidence of insignificant interaction of riboflavin with QD(NH 2 ) 100. Riboflavin is incubated with colloidal solution of QD(NH 2 ) 100 and then absorption spectra of riboflavin is measured. No significant changes in absorption intensity suggest insignificant interaction. Other experimental conditions are similar to Figure 2. S-5

6 Figure S3. Evidence of insignificant change of fluorescence intensity of QD in QD-folate under varying solution ph (a) and in presence of different biological substituents (b). X- axis represents variables/substituents and Y-axis represents fluorescence intensity of QD in QD-folate at 580 nm (410 nm excitation). Details of the concentrations of various substituents are summarized in Table S1. Figure S4. Fluorescence intensity of folate at 450 nm after forming the host-guest complex, in presence of various biological substituents. X-axis represents substituents and Y-axis represents fluorescence intensity of folate in QD-folate at 450 nm (370 nm excitation). Experimental conditions are similar to Figure 2 and concentrations of various substituents are summarized in Table S1. Insignificant change in the fluorescence intensity suggests that the host-guest complex is stable under complex bioenvironment. S-6

7 Figure S5. Fluorescence image of KB (a) and A431 (b) cells after 3h incubation with QD(NH 2 ) 100 and QD(CD) 70 showing that they do not label cells without folate/riboflavin functionalization. Images are captured in bright field (BF) and fluorescence (F) mode. Scale bar represents 50 µm. S-7

8 Figure S6. Fluorescence image of CHO cells incubated with folate functionalized QD. (a) Colloidal QD(CD) 70 is incubated with cell for 3h and then washed cells are used for bright field (BF) and fluorescence (F) imaging. (b) Colloidal QD(CD) 70 is incubated with folate keeping the molar ratio of QD bound CD to folate is at 20. Next, cells are incubated with QD sample for 3h and then washed cells are used for bright field (BF) and fluorescence (F) imaging. (c) Colloidal QD(CD) 70 is incubated with folate keeping the molar ratio of QD bound CD to folate at 2. Next, cells are incubated with QD sample for 3h and then washed cells are used for bright field (BF) and fluorescence (F) imaging. Results show that none of these conditions can label cells. Scale bar represents 50 µm. S-8

9 Figure S7. Fluorescence image of CHO cells incubated with riboflavin functionalized QD. Colloidal QD(CD) 70 is incubated with riboflavin keeping the molar ratio of QD bound CD to riboflavin at 30. Cells are incubated with QD sample for 3h and then washed cells are used for bright field (BF) and fluorescence (F) imaging. Results show that this condition cannot label cells. Scale bar represents 50 µm. S-9

10 Figure S8. a) Fluorescence image of KB cells treated with mixture of folate and QD(NH 2 ) 100, showing insignificant cell labeling. b-d) Fluorescence image of KB cells treated with a mixture of folate and γ-cd functionalized QD with varied molar ratio of γ-cd to folate of 2 (b), 20 (c) and 40 (d). Insignificant cell labeling indicates that folate functionalization via host-guest chemistry is inefficient. In all cases cells are incubated with samples for 3h and washed cells are used for imaging. Scale bar represents 50 µm. S-10

11 Figure S9. Riboflavin concentration dependent labeling of KB cells by QD(CD) 70. Colloidal QD(CD) 70 is incubated with varied concentration of riboflavin where the molar ratio of QD bound CD to riboflavin is kept at 2 (a), 5 (b), 30 (c) and 50 (d). Next, cells are incubated with QD samples for 3h and washed cells are used for bright field (BF) or fluorescence (F) imaging. Results show that labeling performance is best for CD to riboflavin molar ratio of 30. Scale bar represents 50 µm. S-11

12 Figure S10. Quantitative image analysis of per cell fluorescence intensity using Image J software showing that average fluorescent intensity per cell increases with increasing the incubation time. Images are taken from Figure 5a and marked cells (shown by white arrows) are used for intensity measurements. Scale bar represents 20 µm. S-12

13 Figure S11. Evidence that simultaneous addition of QD(CD) 70 and folic acid in a culture media can lead to labeling of KB cells. (a) KB cells are incubated with QD(CD) 70 for 3 h and no labeling is observed. (b) KB cells are first incubated with QD(CD) 70 and then folic acid is added, maintaining the molar ratio of CD : folate as 20:1. Significant labeling indicates hostguest complexation and labeling occurs in biological environment. Scale bar represents 50 µm. S-13

14 Figure S12. Evidence that host-guest complex formation allows entry of both QD and riboflavin into A431 cells. We have replaced CdSe-ZnS by Mn-ZnS based QD and prepare similar type riboflavin functionalized QD via host-guest chemistry. In this system QD and riboflavin can be tracked simultaneously, as they have different excitation/emission. a) Cells are incubated with sample for 3 h and washed cells are used for imaging. Blue excitation is used for riboflavin imaging (green color), UV excitation is used for Mn-ZnS imaging (red color) and merged yellow image conclude that Mn-ZnS and riboflavin are colocalized. b,c) Fluorescence spectra of A431 cells labeled with Mn-ZnS-riboflavin for 3h (b) and 9h (c) showing the signature of both riboflavin and Mn-ZnS. Excitation of labeled cells at 440 nm gives the emission of riboflavin in the nm range and excitation of labeled cells at 310 nm gives the emission of narrow Mn-ZnS at nm range. S-14

15 Figure S13. High magnification fluorescence image of a) A431 cells labeled with riboflavin functionalized QD along with nuclear probe (hoechst) and b) KB cells labeled with folate functionalized QD along with nuclear probe (hoechst). Cells are imaged at different z planes (top to bottom with consecutive Z-axis slices of 0.75 µm starting from Z-1 to Z-10). Here cells are incubated with sample for 9 h and washed cells are further used for imaging. From the images it is found that the emission from the QD and nuclear probe are coming from the same Z plane, suggesting that QDs and the nucleus are in the same plane. Scale bar represents 20 µm. S-15

16 Figure S14. Colocalization study of folate functionalized QD in KB cells (a) and riboflavin functionalized QD in A431 cells (b), showing that they localize partially with lysozome. Typically cells are incubated with nanoprobe for 9h. Next, cells are incubated with lysotrakerredfor 30 min and washed cells are used for imaging. Green excitation is used for imaging cell lysotracker (red color) and blue excitation is used for imaging of QD (green color). Yellow colour indicates colocalization of lysotracker and QD nanoprobe. Scale bar represents 20 µm. S-16

17 Figure S15. Colocalization study of folate functionalized QD in KB cells (top panel) and riboflavin functionalized QD in A431 cells (bottom panel), showing that they localize partially at Golgi apparatus. In addition it is also shown that Golgi tracker bleaches within 30 sec but QD probes remain stable. Typically cells are incubated with nanoprobe for 9 h. Next, cells are incubated with Golgi tracker for 30 min and washed cells are used for imaging. Blue excitation is used for imaging Golgi tracker (green color) and green excitation is used for imaging of QD (red color). Yellow color indicates colocalization of Golgi tracker and QD. Scale bar represents 50 µm. S-17

18 Cell viability (%) µm 0.25 µm 0.6 µm 0 Figure S16. MTT based cytotoxicity assay of QD(NH 2 ) 100, QD(CD) 70, and folate functionalized QD (with CD : folate ratio of 20:1). KB cells are incubated with 3 difference concentrations of QD samples for 24 h and cell viability was calculated assuming 100 % viability for cell without any nanoparticle. The mean ± SD of three determinations (n = 3) are represented in bars. S-18