Synthesis of PEGMA-SH compound
Concentrations of 1.0 mmol 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and 2.0 mmol dicyclohexylcarbodiimide (DCC) were dissolved in 50 mL of dichlormethane, followed by the addition of 2.2 mmol 4-dimethylaminopyridine (DMAP) and 2.0 mmol PEGMA. The mixture was degassed with nitrogen and then stirred for 48 h at room temperature. After filtration, the filtrate was washed sequentially with water, 5% acetic acid, and water. Then, the organic phase was dried over magnesium sulfate, filtered, and evaporated to dryness. The product was dissolved in 100 mL of water/ethanol (V/V, 4/1) with the addition of 2 mL of 1 M sodium borohydride (NaBH4) and stirred for 2 h, and was used without further purification.
Synthesis of Aurod@pNIPAAm-PEGMA nanogel
AuNRs with a length of 50 nm were synthesized using the seed-mediated growth method as reported previously . Subsequently, 0.1 mmol PEGMA-SH was added to 25 mL of the as-prepared AuNRs suspension (1.6 × 10−6 μmol) and continuously stirred for 5 h at room temperature. Aurod@PEGMA was collected by centrifugation at 9,500 rpm for 12 min and then re-dispersed in 15 mL of the deionized water, followed by the addition of 1.8 mmol NIPAAm, 0.2 mmol PEGMA, 86.69 μmol sodium dodecyl sulfate (SDS), and 12.97 μmol N,N-methylenebisacrylamide (BIS). The mixture was heated to 75°C with stirring and maintained in vacuum. After equilibration for 1 h, the polymerization was initiated by adding 109.6 μmol ammonium persulfate (APS). The reaction was allowed to proceed for 4 h at 75°C and terminated by opening the system to air. The resulting Aurod@pNIPAAm-PEGMA nanogels were purified by repeated centrifugation (9,000 rpm for 12 min) and subsequently lyophilized for further use.
The optical properties of AuNRs and Aurod@pNIPAAm-PEGMA nanogels were characterized by an UV–vis spectrophotometer (DUTM800, Beckman Coulter, Brea, CA, USA) with a scanning speed of 1,200 nm/min from 400 to 1,000 nm. The transmission electron microscopy (TEM) images were obtained from a JEM 2100 microscope (JEOL Ltd., Tokyo, Japan) operating at an acceleration voltage of 200 kV. Raman spectra were performed on an UV-1000x instrument (Renishaw, Wotton-under-Edge, UK) (path length = 200 nm) using a red light-emitting diode laser (λ = 785 nm, 0.5 mW). A Fourier transform interferometer (AVATAR360, Nicolet Instrument Corporation, Madison, WI, USA) was used to record the absorption spectra of AuNRs and Aurod@pNIPAAm-PEGMA nanogels between 400 and 4,000 cm−1 at a spectral resolution of 4 cm−1.
LCST measurement of Aurod@pNIPAAm-PEGMA nanogel
In order to investigate the thermal property of the Aurod@pNIPAAm-PEGMA nanogel, nanogels with different molar ratios of NIPAAm/PEGMA (1:0, 18:1, 12:1, 9:1, 6:1, 4.5:1) were synthesized. LCSTs of nanogels were measured through turbidimetric measurement. The concentration for each Aurod@pNIPAAm-PEGMA nanogel in the deionized water was maintained at 1 mg/mL. The light transmittances at 600 nm were then measured by an UV–vis spectrophotometer (TU-1901, Beijing Purkinje General Instrument Co. Ltd, Beijing, China) equipped with a temperature-controlled sample holder, and the heating rate was set at 0.1°C/min. The LCST was defined as the initial break point in the resulting transmittance versus temperature curves.
ZnPc4 loading and NIR-mediated ZnPc4 release
Two milligrams of Aurod
@pNIPAAm-PEGMA nanogels and 2 mg of ZnPc4
were dispersed in 10 mL of N
-dimethyl formamide (DMF) and stirred for 24 h at room temperature. The ZnPc4
@pNIPAAm-PEGMA nanogels were then collected by centrifugation (9,000 rpm for 12 min). To determine the amount of unloaded ZnPc4
, the supernatant was analyzed by an UV–vis spectrophotometer (DUTM800, Beckman Coulter) at 680 nm where ZnPc4
has a maximum absorption. The loading efficiency was calculated according to the following formula:
where Wt represents the total amount of ZnPc4 and W0 represents the unloaded amount of ZnPc4.
For the NIR-mediated ZnPc4
release, 5 mL of the ZnPc4
@pNIPAAm-PEGMA nanogel suspension (1 mg/mL) was placed into dialysis bags (molecular weight cutoff, 8 to 14 kDa) and irradiated by an 808-nm laser (0 to 400 mW/cm2
) for different times (0 to 60 min). To determine the amount of ZnPc4
released, the dialysate was removed and subsequently analyzed by an UV–vis spectrophotometer (DUTM800, Beckman Coulter). The release efficiency was calculated as follows:
where Wr represents the released amount of ZnPc4 and Wl represents the loaded amount of ZnPc4.
Singlet oxygen detection
The generation of singlet oxygen (1O2) from ZnPc4 loaded in the Aurod@pNIPAAm-PEGMA nanogel was determined by the transformation of 9,10-dimethylanthracene (DMA) which exhibits a maximum absorption at 377 nm . The DMA can react irreversibly with 1O2 to yield an endoperoxide. The reaction could be monitored by recording the decrease in the absorption at 377 nm. In a typical experiment, 0.105 mg of the Aurod@pNIPAAm-PEGMA nanogel loaded with 0.0135 μmol ZnPc4 was dispersed in 3 mL of DMF, and then, 0.45 μmol DMA was added. Pure ZnPc4 (0.0135 μmol) was used as a control. The solutions were then irradiated with a LED lamp (680 nm, 10 mW/cm2) or a NIR laser (808 nm, 400 mW/cm2). The absorption measurements followed by irradiation were carried out every 5 min.
Light-induced in vitro PDT effect
Hela cells were seeded into 24-well cell culture plates (1 × 105 cells/well) and incubated for 24 h. After being treated with ZnPc4-loaded Aurod@pNIPAAm-PEGMA nanogels (300 μg/mL) in serum-free medium at 37°C for 22 h, chloroquine (10 mg/mL) was added into every well for another 2 h to promote endosomal escape . Then, Hela cells were washed with PBS and incubated in a nanogel-free medium and treated with an 808-nm laser at 400 mW/cm2 for 15 min and a 680-nm LED lamp at 10 mW/cm2 for 40 min. For cell survival test, the irradiated plates were returned to the incubator, and cell viability was colorimetrically measured 48 h later with MTT assay .