A cell nanoinjector based on carbon nanotubes

Chen et al. 10.1073/pnas.0700567104.

Supporting Information

Files in this Data Supplement:

SI Figure 5
SI Movie 1
SI Figure 6
SI Figure 7
SI Table 1
SI Figure 8
SI Figure 9
SI Figure 10
SI Scheme 1
SI Text

SI Figure 5

Fig. 5. Control experiment. MWNT-AFM tips were incubated directly with QDot streptavidin without prior coating with linker 1. (A) TEM image of a MWNT-AFM tip before incubation with QDot streptavidin conjugates. (B) TEM image of the MWNT-AFM tip after incubation with QDot streptavidin conjugates. In this case, no QDot streptavidin conjugates were observed on the MWNT surface. The dark shape is the AFM tip.

SI Figure 6

Fig. 6. Functionalization of MWNT-AFM tips with QDot streptavidin conjugates via linker 2. (A) TEM image of a MWNT-AFM tip before functionaliztion. (B) TEM image of the MWNT-AFM tip after functionalization with 2 and then conjugated with QDot streptavidin. The resulting MWNT-AFM tip appeared similar to MWNT-AFM tips bearing the disulfide-bound conjugates. The dark shape is the AFM tip.

SI Figure 7

Fig. 7. Control experiments: QDot streptavidin conjugates were loaded onto the MWNTs using linker 2 (SI Fig. 6), which possesses pyrene and biotin moieties but lacks a disulfide bond. Similar nanoinjection experiments were carried out using HeLa cells but in this case no QDot streptavidin conjugates were released (with >5 different MWNT-AFM tips in >10 injection experiments). (A) Fluorescence image of the cells before nanoinjection. (B) Combined bright-field and fluorescence image of the cells before nanoinjection. The inserted arrow indicates the target cell. The dark shape in the upper right corner is the AFM cantilever. (C) Fluorescence image of the cells after the nanoinjection, showing no fluorecent QDot streptavidin conjugates released inside the target cell. (D) Combined bright-field and fluorescence image of the cells after the nanoinjection. No released QDot streptavidin conjugates were observed. The dark shape in the lower right corner is the retracted AFM cantilever. (E) Combined bright-field and fluorescence image of another four examples of HeLa cells after nanoinjection of QDot streptavidin, in none of which fluorecent QDot streptavidin conjugates were released. In all cases, fluorescence images were acquired with l_ex = 415 nm and data collection with a 655-nm filter. Images are 70 ´ 70 mm in A-D and 30 ´ 30 mm in E.

SI Figure 8

Fig. 8. Calcein AM assay. (A) Bright-field image of the cells before nanoinjection. The inserted arrow indicates the target cell. (B) Fluorescence image (FITC filter set) of the cells before nanoinjection. The cells were loaded with Calcein AM and the green fluorescence indicates the viable cells. (C) Fluorescence image (Qdot-655 filter set) of the cells after the nanoinjection, showing fluorecent QDot streptavidin released inside the target cell. (D) Fluorescence image (FITC filter set) of the cells after the nanoinjection, in which no detectable loss in fluorescence was observed up to 10 h after the injection.

SI Figure 9

Fig. 9. Annexin V-FITC/PI assay for apoptosis. (A) Bright-field image of the cells before nanoinjection. The inserted arrow indicates the target cell. (B) Fluorescence image (Qdot-655 filter set) of the cells after the nanoinjection, showing fluorecent QDot streptavidin released inside the target cell. (C) Fluorescence image (FITC filter set) of the cells before nanoinjection. The cell after nanoinjeciton did not stain with Annexin V-FITC, indicating nanoinjeciton did not induce apoptosis. (D) Fluorescence image (Rhodamine filter set) of the cells after the nanoinjection. No PI staining was observed up to 10 h after the injection, indicating nanoinjeciton does not induce apoptosis. (E and F) Positive control: Apoptosis was induced by incubation with 5% ethanol for 60 min (E and F: FITC and Rhodamine filter sets, respectively).

SI Figure 10

Fig. 10. Tracking the movement of a quantum dot cluster in the cytosol. (A) A combined bright-field and fluorescence image of part of a HeLa cell with the trajectory of a quantum dot cluster indicated in blue. (B) The measured mean-square displacement versus time. The line is best fit to <Dr²> = 4DDt, where D is the diffusion coefficient.

SI Scheme 1

Scheme 1. Synthesis of linkers.

SI Movie 1

Movie 1. Movement of the injected quantum dots in the cytosol of HeLa cells. After 20 s, the exogenous background light was dimmed in order to highlight the quantum dots.

Table 1. Trypan blue test of cells after nanoinjecton

	Trypan blue test
Cells after nanoinjection of the QDot streptavidin conjugates	Unstained (alive)
Cells penetrated with nanoneedle without loading of cargo	Unstained (alive)
Cells penetrated with nanoneedle loaded with QDot streptavidin conjugates using linker 2	Unstained (alive)
Positive control: cells penetrated with AFM tips with micron scale tip	Stained (dead)

SI Text

Cell Viability Studies

Trypan Blue Test. Trypan blue stain solution (0.4% in PBS buffer) was purchased from Invitrogen.

Trypan blue is a vital dye. It is a negatively charged chromophore that penetrates only cells with damaged membranes.

Immediately after nanoinjection, or penetration with nanoneedles, the cells were washed with serum-free medium. The dye was then added (0.08% in medium) and the cells were monitored for 10 h thereafter. No trypan blue inclusion or reduction in cell viability was observed during this time period (SI Table 1). Indeed, in some experiments we held the nanoneedle inside the cell for >1 h without any effect on membrane integrity and cell viability. In a positive control experiment, the cells were intentionally damaged with unmodified AFM tips to induce cell death.

Calcein AM Test. Calcien acetoxymethylester (calcein AM) was purchased from Invitrogen.

Calcein AM is the acetoxymethyl ester derivative of the fluorescent indicator calcein. Calcein AM is membrane-permeant and thus can be introduced into cells via coincubation in media. Once inside the cells, calcein AM is hydrolyzed by endogenous esterases into the highly negatively charged green fluorescent dye calcein, which is retained in the cytoplasm. Calcein AM is an excellent tool for the studies of cell membrane integrity and for long-term cell tracing.

HeLa cells were incubated with calcein AM (2 mM, PBS, 1% FBS) at room temperature for 1 h. After washing with buffer, the cells were imaged (Figure S5B) by fluorescence microscopy. Nanoinjection was then performed on the target cell with calcien retained inside and the fluorescence was monitored during the injection and afterward. No detectable loss in fluorescence was observed even several hours after the injection. (SI Fig. 9D).

Apoptosis Assay Using Annexin V-FITC/Propidium Iodide Test. The annexin V-FITC/propidium iodide apoptosis detection kit was obtained from BD Biosciences.

Annexin V-FITC is widely used as a marker for the early stages of apoptosis. Cells undergoing apoptosis stain positively with annexin V-FITC but not with the vital dye propidium iodide (PI), whereas dead or necrotic cells stain with both annexin V-FITC and propidium iodide (PI). Loss of plasma membrane integrity is one of the earliest features of apoptosis. In apoptotic cells, the membrane phopholipid phophatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thus exposing PS to the external cellular environment. Annexin V is a phospholipid-biding protein that has a high affinity for PS. Annexin V-FITC staining can identify apoptosis at an early stage as well as the late stages of cell death resulting from either apoptotic or necrotic processes.

Immediately after nanoinjection or 2 h after nanoinjection, the cells were incubated in the dark in 300 ml of binding buffer containing 60 ml of annexin V-FITC stock solution and 60 ml of PI stock solution. The cells were monitored for 10 h thereafter. In a positive control experiment, apoptosis was intentionally induced by incubation with 5% ethanol for 60 min.

Synthetic Procedure of Compounds Used in This Work (SI Scheme 1)

General. All chemical reagents were of analytical grade, obtained from commercial suppliers and used without further purification. Flash chromatography was performed using Merck 60-Å 230-400 mesh silica gel. Analytical TLC was performed on Analtech Uniplate silica gel plates and visualized by staining with ceric ammonium molybdate or by absorbance of UV light at 254 nm. ¹HNMR, and ¹³CNMR spectra were obtained with Bruker AMX-400 or Bruker DRX-500 MHz spectrometers. ¹H and ¹³C chemical shifts (d) are reported in parts per million (ppm) referenced to TMS (0 ppm) and were measured relative to the residual solvent peak. Coupling constants (J) are reported in hertz (Hz). High-resolution fast atom bombardment (FAB) mass spectra were obtained at the UC Berkeley Mass Spectrometry Laboratory.

N-((pyren-1-yl)butanoyl)cystamine (3). To a solution of cystamine dihydrochloride (1.00 g, 4.40 mmol) and 1-pyrenebutyric acid (1.27 g, 4.40 mmol) in 40 ml of anhydrous MeOH were added TBTU (2.80 g, 8.80 mmol), HOBt (0.890 g, 6.60 mmol), and triethylamine (1.82 ml, 13.2 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was evaporated, and 1 M NaH₂PO₄ was added (10 ml, pH 4.2). The aqueous was washed with ether. The aqueous solution was then basified to pH 9 by 10 M NaOH and extracted with EtOAc (5 ml ´ 6). The combined organic phases were dried over MgSO₄, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography eluted with MeOH:CH₂Cl₂ (1:4) to give the product (950 mg, 51%). R_f = 0.40 (MeOH:CH₂Cl₂ = 1:4). ¹H NMR (CD₃OD, 400 MHz) d 8.13 (d, J = 9.3 Hz, 1 H), 8.02 (d, J = 7.5 Hz, 2H), 7.94 (d, J = 8.4 Hz, 2 H), 7.84-7.76 (m, 3 H), 7.70 (d, J = 7.8 Hz, 1 H), 3.43 (t, J = 6.6 Hz, 2 H), 3.20 (t, J = 7.5 Hz, 2 H), 2.8 (t, J = 6.3 Hz, 2 H), 2.71 (t, J = 6.6 Hz, 2 H), 2.65 (t, J = 6.3 Hz, 2 H), 2.25 (t, J = 7.2 Hz, 2 H), 2.10-1.98 (m, 2 H). ¹³C NMR (CD₃OD, 100 MHz) d 176.0, 137.3, 132.8, 132.3, 131.3, 129.9, 128.6, 128.5, 128.4, 127.7, 127.0, 126.2, 126.1, 126.0, 125.8, 124.4, 41.8, 41.2, 39.6, 38.6, 36.8, 33.8, 29.0. FAB-HRMS calcd for C₂₄H₂₇N₂OS₂ [MH]⁺: m/z 423.1565; found 423.1562.

N-(pyren-1-yl)butanoyl-N'-(biotinyl)cystamine (1). To a solution of biotin N-hydroxysuccinimide (290 mg, 0.850 mmol) in 4 ml of DMF were added N-((pyren-1-yl)butanoyl)cystamine (360 mg, 0.850 mmol), and triethylamine (0.350 ml, 2.55 mmol). The reaction mixture was stirred at room temperature for 7 h. DMF was then removed under high vacuum. The resulting residue was purified by silica gel column chromatography eluting with MeOH:CH₂Cl₂ (1:9) to give the product (520 mg, 94%). R_f = 0.64 (MeOH:CH₂Cl₂ = 1:4). ¹H NMR (CDCl₃ + CD₃OD₃, 500 MHz) d 8.14 (d, J = 9.0 Hz, 1 H), 8.04-7.98 (m, 2 H), 7.96 (d, J = 8.0 Hz, 2 H), 7.89-7.81 (m, 3 H), 7.72 (d, J = 7.5, 1 H), 4.22-4.17 (m, 1 H), 3.99-3.93 (m, 1 H), 3.37 (t, J = 6.5 Hz, 2 H), 3.33 (t, J = 6.6 Hz, 2 H), 3,27-3.23 (m, 2H), 2.82-2.77 (m, 1 H), 2,68-2.63 (m, 4 H), 2.61 (dd, J = 12.5, 4.5 Hz, 1 H), 2.50 (d, J = 12.5 Hz, 1 H), 2.22 (t, J = 7.5 Hz, 2 H), 2.11-1.98 (m, 4 H), 1.49-1.35 (m, 4 H), 1.21-1.09 (m, 2 H). ¹³C NMR (CDCl₃ + CD₃OD, 125 MHz) d 174.5, 174.2, 164.0, 135.9, 131.3, 130.8, 129.9, 128.6, 127.4, 127.3, 126.7, 125.9, 125.0, 124.8(9), 124.8(6), 124.8, 124.7, 123.3, 62.0, 60.3, 55.4, 53.4, 40.1, 38.4, 38.3, 37.7, 37.5, 35.8, 35.5, 32.8, 28.2, 27.9, 27.5, 25.4. FAB-HRMS calcd for C₃₄H₄₁N₄O₃S₃ [MH]⁺: m/z 649.2341; found 649.2323.

N-(pyren-1-yl)butanoyl-N'-biotinyl-3,6-dioxaoctane-1,8-diamine (2). To a solution of 1-pyrenebutyric acid N-hydroxysuccinimide (55.0 mg, 0.140 mmol) in 2 ml of DMF were added EZ-Link Biotin-PEG₂-Amine (Pierce, IL) (50.0 mg, 0.130 mmol), and triethylamine (20.0 ml, 0.140 mmol). The reaction mixture was stirred at room temperature for 5 h. DMF was then removed under high vacuum. The resulting residue was purified by silica gel column chromatography eluting with MeOH:CH₂Cl₂ (15:85) to give the product (75.0 mg, 89%). R_f = 0.62 (MeOH:CH₂Cl₂ = 1:4). ¹H NMR (CDCl₃, 400 MHz) d 8.25 (d, J = 9.2 Hz, 1 H), 8.11 (dd, J = 7.6, 2.4 Hz, 2 H), 8.05 (d, J = 8.8 Hz, 2 H), 7.97-7.92 (m, 3 H), 7.81 (d, J = 7.6, 1 H), 6.67 (br, 2 H, NH), 6.57 (br, 1 H, NH), 5.72 (br, 1 H, NH), 4.21-4.18 (m, 1 H), 3.96-3.93 (m, 1 H), 3.50-3.27 (m, 14 H), 2.84-2.78 (m, 1 H), 2.63 (dd, J = 12.8, 4.4 Hz, 1 H), 2.56 (d, J = 11.6 Hz, 1 H), 2.28 (t, J = 7.2, 2 H), 2.19-2.13 (m, 2 H), 2.08 (t, J = 7.2, 2 H), 1.58-1.43 (m, 4 H), 1.28-1.17 (m, 2 H). ¹³C NMR (CDCl₃, 100 MHz) d 173.6, 173.3, 164.4, 136.2, 131.5, 131.0, 130.0, 128.9, 127.7, 127.6, 127.5, 126.8, 126.1, 125.2, 125.1, 125.0, 125.9, 123.6, 70.2, 70.1, 70.0, 69.9, 61.8, 60.3, 55.7, 50.8, 40.5, 39.4, 39.2, 36.0, 32.9, 28.3, 28.1, 27.6, 25.7. FAB-HRMS calcd for C₃₆H₄₅N₄O₅S [MH]⁺: m/z 645.3111; found 645.3092.