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.

AuNRs with a length of 50 nm were synthesized using the seed-mediated growth method as reported previously 20 . 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. Au_rod@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 Au_rod@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 Au_rod@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 Au_rod@pNIPAAm-PEGMA nanogels between 400 and 4,000 cm⁻¹ at a spectral resolution of 4 cm⁻¹.

In order to investigate the thermal property of the Au_rod@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 Au_rod@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.

Two milligrams of Au_rod@pNIPAAm-PEGMA nanogels and 2 mg of ZnPc₄ were dispersed in 10 mL of N,N-dimethyl formamide (DMF) and stirred for 24 h at room temperature. The ZnPc₄-loaded Au_rod@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:

Loading efficiency = W t - W 0 W t × 100 % ,

where W _t represents the total amount of ZnPc₄ and W ₀ represents the unloaded amount of ZnPc₄.

For the NIR-mediated ZnPc₄ release, 5 mL of the ZnPc₄-loaded Au_rod@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:

Release efficiency = W r W l × 100 % ,

where W _r represents the released amount of ZnPc₄ and W _l represents the loaded amount of ZnPc₄.

The generation of singlet oxygen (¹O₂) from ZnPc₄ loaded in the Au_rod@pNIPAAm-PEGMA nanogel was determined by the transformation of 9,10-dimethylanthracene (DMA) which exhibits a maximum absorption at 377 nm 21 . 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 Au_rod@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.

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 Au_rod@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 22 . 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 23 .

The synthesis of PEGMA-SH was shown in Figure 1. PEGMA-DTNB compound was firstly gained by the esterification reaction between the terminal hydroxyl group on the PEGMA and the carboxyl group on the DTNB with the DCC as medium and DMAP as catalyst 24 25 . Subsequently, the disulfide bond of PEGMA-DTNB was reduced by NaBH₄ to yield the desired PEGMA-SH compound.

The strategy to prepare the Au_rod@pNIPAAm-PEGMA nanogel involves two steps, growing a PEGMA monolayer on the surface of a AuNR, followed by in situ polymerization and cross-linking of NIPAAm and PEGMA, as depicted in Figure 2. In the first step, the AuNR surface was modified with a PEGMA self-assembled monolayer through a sulfhydryl-gold interaction. In the second step, PEGMA-modified AuNRs could be used as a template for in situ formation of hydrogel by polymerization and cross-linking of NIPAM and PEGMA with BIS as crosslinker, APS as initiator, and SDS as emulsifier. The coating of pNIPAAm-PEGMA on AuNRs can be reflected in the corresponding UV–vis spectra (Figure 3). AuNRs used in this work had a length of about 50 nm with an aspect ratio of approximately 3.2 (Figure 4A) which exhibited the maximum of the plasmon peak of 794 nm (Figure 3a). After the AuNRs were modified with pNIPAAm-PEGMA, a red shift from 794 to 801 nm occurred (Figure 3b). This red shift of SPR and the peak shape widening might be due to a change for AuNRs in the local refractive index produced by the pNIPAAm-PEGMA shell (Figure 4B) 26 .

Raman spectra were also used to identify the synthesis of the Au_rod@pNIPAAm-PEGMA nanogel. The Raman spectrum of the as-prepared AuNRs (Figure 5a) exhibited a band at 190 nm which was ascribable to the Au-Br bond on the surface of AuNRs 27 . This is because the as-prepared AuNRs were stabilized by the cationic detergent cetyltrimethylammonium bromide (CTAB) in the aqueous solution. After being modified with pNIPAAm-PEGMA (Figure 5b), the Au-Br band disappeared, and a band at 320 nm was observed, which was assigned to the Au-S bond 28 . It is thus suggested that PEGMA-SH might replace CTAB to form PEGMA-modified AuNRs through the Au-S bond, and then, PEGMA-SH on the surface of AuNRs might serve as the template for the following polymerization and cross-linking of NIPAAm and PEGMA.

FTIR spectra (Figure 6) were recorded to confirm the structure of the polymer shell. In the FTIR spectrum of PEGMA-modified AuNRs (Figure 6a), the absorption peaks of PEGMA, including ν(C=O) (1,721 cm⁻¹) and ν(C-O-C) (1,105 cm⁻¹), were observed. The spectrum of Au_rod@pNIPAAm-PEGMA nanogels (Figure 6b) exhibited the characteristic peaks of polymerized NIPAAm at 1,650 cm⁻¹ (ν(C=O), amide I) and 1,550 cm⁻¹ (δ(N-H), amide II). Hence, the FTIR results could provide evidence for the surface modification and polymerization on AuNRs.

Figure 7 and Table 1 showed the effect of the molar ratios of NIPAAm/PEGMA on the LCSTs of the Au_rod@pNIPAAm-PEGMA nanogel. The Au_rod@pNIPAAm (the molar ratio of NIPAAm/PEGMA, 1:0) exhibited an LCST of approximately 32°C, which was consistent with pure pNIPAAm 13 . It is clearly shown in Table 1 that the LCSTs of the Au_rod@pNIPAAm-PEGMA nanogel could be tuned by changing the molar ratio of NIPAAm/PEGMA. Namely, as the molar ratio of NIPAAm/PEGMA decreased, the LCST of the nanogel increased. For example, when the molar ratio of NIPAAm/PEGMA was set at 18:1, the LCST of Au_rod@pNIPAAm-PEGMA nanogels could be up to 36°C. The addition of hydrophilic PEGMA increased the hydrophilicity of pNIPAAm due to the strong interactions between water and hydrophilic groups on the polymer, which led to an increased LCST 29 . It is thus expected that this attractive property of tunable LCST might make Au_rod@pNIPAAm-PEGMA nanogels more promising in drug delivery application.

NIR-mediated release of ZnPc₄ loaded in Aurod@pNIPAAm-PEGMA nanogels was investigated with the irradiation of a NIR laser (808 nm). When the sample was irradiated at 200 mW/cm², the release efficiency was about 23.5% in the initial 20 min. As the irradiated time was prolonged, the cumulative release efficiency was up to 37.4% within 1 h (Figure 8A). This can be explained by the AuNRs of Au_rod@pNIPAAm-PEGMA nanogels absorbing a certain SPR wavelength light and converting it into heat 30 . The heat diffused into the polymer shell and caused the shrinkage of the pNIPAAm-PEGMA nanogels and the release of ZnPc₄.

The effect of laser power density on drug release was studied (Figure 8B). Exposure of Au_rod@pNIPAAm-PEGMA nanogels to an 808-nm laser with the power of 100 mW/ cm² for 15 min caused 20% of the loaded ZnPc₄ released. More loaded ZnPc₄ (43.7%) in Au_rod@pNIPAAm-PEGMA nanogels could be released upon the irradiation power of 800 mW/ cm². This is because when irradiated with a low-power NIR laser, small shrinkage of nanogels occurred, whereas a laser at high power might make nanogels shrink considerably and rapidly 31 , consequently more ZnPc₄ could be released. It is thus speculated that the NIR-responsive Au_rod@pNIPAAm-PEGMA nanogel, acting as drug delivery carriers, could offer specific drug delivery to the targeted site, such as a tumor zone.

In PDT, the photosensitizing drugs should preferentially accumulate in target tissues and subsequently be activated by light with a matching wavelength to generate reactive singlet oxygen 32 . The singlet oxygen will cause the destruction of target cells by a complex cascade of chemical, biological, and physiological reactions 33 . The Au_rod@pNIPAAm-PEGMA nanogels served as ZnPc₄ carrier in PDT; besides the excellent properties of drug loading and release, its effect on the capability of loaded ZnPc₄ to generate singlet oxygen was also investigated.

Photo-induced ¹O₂ of ZnPc₄ was examined by a chemical method by using DMA, which could react with ¹O₂ to form an endoperoxide. The decrease in amount of DMA can be recorded by measuring the absorption at 377 nm. As shown in Figure 9, when ZnPc₄-loaded Au_rod@pNIPAAm-PEGMA nanogels or pure ZnPc₄ was irradiated by an 808-nm laser, the absorption of DMA remained unchanged with the increase of exposure time to light, whereas the absorption of DMA continuously decreased as the ZnPc₄-loaded Au_rod@pNIPAAm-PEGMA nanogels or pure ZnPc₄ was irradiated by a 680-nm light. This decrease indicated the production of ¹O₂, which can irreversibly react with DMA. Moreover, the generation curve of ZnPc₄-loaded Au_rod@pNIPAAm-PEGMA nanogels was similar with that of pure ZnPc₄, demonstrating that the capacity of generating ¹O₂ of ZnPc₄ was hardly affected after being loaded in Au_rod@pNIPAAm-PEGMA nanogels. It is thus suggested that the Au_rod@pNIPAAm-PEGMA nanogel might be a promising drug carrier for photodynamic therapy in the future.

The in vitro PDT study, represented in Figure 10, showed the percentage of cell viability after treatment of Hela cells with the ZnPc₄-loaded Au_rod@pNIPAAm-PEGMA nanogel (300 μg/mL) at different irradiated conditions. Compared with the cells’ group with no light treatment, no significant difference of the cell viability was found in the 808-nm laser-treated group. However, for the 680-nm light-treated group, the cell viability decreased to 40%. It is interesting to note that when irradiated by the two lights, the cell viability decreased to 10%. This is because the 808-nm laser treatment might result in the release of ZnPc₄ from nanogels, which could improve the efficiency of the generation of singlet oxygen after the 680-nm irradiation and thus enhance the PDT effect on Hela cells.