Controlled cellular uptake and drug efficacy of nanotherapeutics.

DOX-tethered surface-modified AuNPs The average core size obtained by transmission electron microscopy (TEM) is approximately 20 nm for all the AuNPs. The concentration of standard AuNP stock solution (×1) is controlled at 1 mM of Au ( Supporting Information ). The concentration of the standard AuNP solution is 2.4 × 10 12 AuNPs/mL. Then the number of DOX per unit AuNP is controlled as 1.15 × 10 3 DOX/AuNP. The carboxyl groups of the designed spacers are activated by N -hydroxysuccinimide and N , N ′-dicyclohexylcarbodiimide. The activated acid is then reacted with the amine group of DOX. All the reaction mixtures are dialyzed against Milli-Q water for purification. For respective AuNP, the spacers containing 0.05 mg DOX (M w : 543.52, 9.2 ×10 −8 mole DOX, 5.5×10 16 DOX molecules) are introduced to a 20 mL standard citrate-covered AuNP stock solution (1 mmol·L −1 of Au). Then, the number of DOX per unit AuNP is controlled as 1.15×10 3 DOX/AuNP. The completion of the reaction is confirmed by the absence of the thiol-containing DOX-conjugated spacers in the reaction solvent (confirmed by UV-vis spectrum and fluorescence, data is not shown). To the aforementioned DOX-conjugated AuNPs, negatively and positively charged thiol ligands are further introduced to modify the surface properties of the DOX-conjugated AuNPs: thioglycolic acid (SH-CH 2 COOH, designated as AuNP 1) and 6-thioguanine (SH-C 5 H 4 N 5 , designated as AuNP 2). The obtained AuNP solutions are purified by dialysis for more than 2 days. The detailed procedures are provided in Supporting Information .

Cell preparation

A549 cells are cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. A low-serum growth supplement containing 20% fetal bovine serum and 1% penicillin-streptomycin is also added. The cells are cultured in a humidified atmosphere with 5% CO 2 at 37°C. A trypsin (0.25%)-EDTA solution is used to detach the cells from the culture flask. Detailed procedures are in Supporting Information . SEM−EDXS The cells are loaded onto a cover glass and then cultured for 24 h. Thereafter, the cells are treated with a series of surface-modified AuNPs for the designated time. The samples are fixed with 2.5% glutaraldehyde in Dulbecco's phosphate buffered saline for 20 min at room temperature, washed thrice with deionized water, and then air-dried. The samples are Ni-coated using a coater (Quorum Technology, SC7640 model). SEM images are captured using an XL30SFEG (Philips) SEM connected to an EDXS system at an acceleration voltage of 20 kV. SEM and EDXS measurements are performed using a XL30SFEG electron probe microanalyzer equipped with a Genesis System EDAX detector. The detailed procedures are described in Supporting Information . TPM An upright microscope (BX51; Olympus) is used to perform TPM. The system is equipped with a Ti:sapphire femtosecond laser (Chameleon; Coherent) with a tuning range of 680 nm to 1020 nm. To generate the two-photon effect, the laser source is operated at 80 MHz repetition rate and 140 fs pulse width. The beam first passes through a half-wave plate and then through a polarizer for power control. The beam is expanded twice by passing it through a pair of plano-convex lenses, and then the expanded and excited light are passed through a resonant x - y scanner (counter rotation scanner, GSI Lumonics) operating at 8 KHz scanning speed. Details are provided in Supporting Information .

Zebrafish preparation and cancer cell microinjection

Zebrafish is maintained in E3 embryo media (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO 4 , 1 mM CaCl 2 , 0.15 mM KH 2 PO 4 , 0.05 mM Na 2 HPO 4 , 0.7 mM NaHCO 3 , 10–5% methylene blue; pH 7.4) containing 1-phenyl 2-thiourea (Sigma-Aldrich) to prevent pigmentation at 29°C. Red fluorescence labeled cancer cell pellet and matrigel (BD Biosciences) are mixed with 1/1 (vol/vol) at 4°C. To this call-gel mixture, doxorubicin or doxorubicin-tethered surface-modified AuNP solutions are mixed with 3/1 (vol/vol). This cell suspension mixture containing 1000 cells is loaded into a borosilicate glass needle and implanted into each zebrafish of 4 day-post-fertilization embryo through the yolk in a single injection by using an electronically regulated air-pressure microinjector (Eppendorf, FemtoJet express). After injection, zebrafish is washed once with E3 embryo media and examined the presence of fluorescent cells. Forty zebrafishes are selected and transferred to 24-well plate containing 1 mL of fresh E3 embryo media for photography. E3 embryo media is changed daily and maintained under normal fish husbandry conditions for 3 days. For the work with zebrafish, all the experiments were performed in accordance with relevant guidelines and regulations.