I. INTRODUCTION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTION <<II. EXPERIMENTIII. RESULTS AND DISCUSSI...IV. SUMMARY AND CONCLUSIO...REFERENCESDeoxyribose nucleic acid (DNA) nanostructure and nanoparticle hybrids have attracted considerable attention as smart drug delivery carriers in the cancer therapy community due to their high therapy efficacy and low side effects.1–31. K. Chen, T. Fu, W. Sun, Q. Huang, P. Zhang, Z. Zhao, X. Zhang, and W. Tan, Theranostics 9, 3262 (2019). https://doi.org/10.7150/thno.318852. Q. Jiang, S. Zhao, J. Liu, L. Song, Z. G. Wang, and B. Ding, Adv. Drug Delivery Rev. 147, 2 (2019). https://doi.org/10.1016/j.addr.2019.02.0033. J. Li, C. Fan, H. Pei, J. Shi, and Q. Huang, Adv. Mater. 25, 4386 (2013). https://doi.org/10.1002/adma.201300875 Inherited from the DNA nanostructure, the nanohybrid possesses good biocompatibility and stability, high drug loading and cellular uptake efficiency, tailorable nanostructure, and easy functionalization with targeting and smart response units.4,54. W. Sun, T. Jiang, Y. Lu, M. Reiff, R. Mo, and Z. Gu, J. Am. Chem. Soc. 136, 14722 (2014). https://doi.org/10.1021/ja50880245. M. Xiao, W. Lai, F. Wang, L. Li, C. Fan, and H. Pei, J. Am. Chem. Soc. 141, 20354 (2019). https://doi.org/10.1021/jacs.9b10765 Benefiting from the nanoparticles, the nanohybrid not only has a reinforced nanostructure, but also unique photonic, electronic, or thermal properties.6,76. J. Kim, D. Jang, H. Park, S. Jung, D. H. Kim, and W. J. Kim, Adv. Mater. 30, 1707351 (2018). https://doi.org/10.1002/adma.2017073517. P. Zhang, Z. He, C. Wang, J. Chen, J. Zhao, X. Zhu, C.-Z. Li, Q. Min, and J.-J. Zhu, ACS Nano 9, 789 (2015). https://doi.org/10.1021/nn506309d These physical properties, thus, regulate the kind of stimuli that can be used to release the drug under the desired sites, enhance the drug delivery in different parts of the body, and work synergistically with the released drug to further improve the therapy efficacy, and at the same time, largely decrease the side effects. For example, the smart nanocarrier with gold nanorods as cores and stimuli-responsive DNA Y motifs and temperature-sensitive polymers as shells showed excellent NIR-guided accumulation, massive drug release, efficient gene silence, and severe apoptosis in target HeLa cells.88. P. Zhang et al., ACS Nano 10, 3637 (2016). https://doi.org/10.1021/acsnano.5b08145 Rapid and efficient DOX release in target cancers is realized by the upconversion-luminescence-fueled DNA-azobenzene nanopump. Under NIR irradiation, the upconversion nanoparticle cores emit both UV and visible photons to fuel the continuous photoisomerization of azo, which acts as an impeller pump to trigger cyclic DNA hybridization and dehybridization for controllable DOX release in desired tumor sites.99. Y. Zhang, Y. Zhang, G. Song, Y. He, X. Zhang, Y. Liu, and H. Ju, Angew. Chem. Int. Ed. 58, 18207 (2019). https://doi.org/10.1002/anie.201909870 The triangle DNA nanostructure and gold nanorod nanohybrids showed enhanced therapy efficacy due to a synergistic interaction of the on-demand released antitumor drugs and the gold photothermal therapeutic agents.1010. Y. Du et al., Adv. Mater. 28, 10000 (2016). https://doi.org/10.1002/adma.201601710 Indeed, the integration of DNA nanostructure with nanoparticles endows the drug delivery nanocarriers with smart responses, enhanced therapy efficacy, and largely minimized side effects. However, most of these hybrid nanostructures are constructed on the basis of inorganic nanoparticles, which have attractive physicochemical properties but are hard to degrade in biosystems and have potential cytotoxicity. The exploration of DNA nanostructure and nanoparticle hybrids with more biocompatible and degradable properties is still desirable.Polydopamine (PDA) nanoparticles are the main components of melanin, a natural biopolymer widely distributed in almost all living organisms and have many distinct functions, including easy synthesis with tailorable size, free radical quenching, biodegradability, high photothermal conversion efficiency, and plenty of functional groups on their interface.11,1211. Y. Liu, K. Ai, J. Liu, M. Deng, Y. He, and L. Lu, Adv. Mater. 25, 1353 (2013). https://doi.org/10.1002/adma.20120468312. Y. Liu, K. Ai, and L. Lu, Chem. Rev. 114, 5057 (2014). https://doi.org/10.1021/cr400407a Owing to these features, PDA nanoparticles have been widely used as photothermal therapeutic agents and drug carriers.13–1513. W. Pan, X. Zhang, P. Gao, N. Li, and B. Tang, Chem. Commun. 55, 9645 (2019). https://doi.org/10.1039/C9CC04486H14. Y. Li, X. Liu, W. Pan, N. Li, and B. Tang, Chem. Commun. 56, 1389 (2020). https://doi.org/10.1039/C9CC08447A15. J. Cui, Y. Wang, A. Postma, J. Hao, L. Hosta-Rigau, and F. Caruso, Adv. Funct. Mater. 20, 1625 (2010). https://doi.org/10.1002/adfm.201000209 Photothermal therapy is a platform to fight cancer with high therapeutic efficacy for targeting cells but low damage to adjacent normal tissues.1616. Y. Cheng, F. Yang, G. Xiang, K. Zhang, Y. Cao, D. Wang, H. Dong, and X. Zhang, Nano Lett. 19, 1179 (2019). https://doi.org/10.1021/acs.nanolett.8b04618 Under NIR light, PDA nanoparticles can convert more than 99% of the absorbed photon energy nonradiatively into heat within 50 ps and greatly enhance the tumor’s local temperature to kill the cancer cells.1717. X. Wu, Q. Jiang, D. Ghim, S. Singamaneni, and Y.-S. Jun, J. Mater. Chem. A 6, 18799 (2018). https://doi.org/10.1039/C8TA05738A Recently, PDA-based nanoplatforms with dual pH and NIR light response properties have been used for on-demand drug release and cancer chemo-photothermal synergistic therapy.18–2118. Z. Li, X. Shan, Z. Chen, N. Gao, W. Zeng, X. Zeng, and L. Mei, Adv. Sci. 8, 2002589 (2021). https://doi.org/10.1002/advs.20200258919. W. Cheng, X. Zeng, H. Chen, Z. Li, W. Zeng, L. Mei, and Y. Zhao, ACS Nano 13, 8537 (2019). https://doi.org/10.1021/acsnano.9b0443620. Y. Peng et al., Biomater. Sci. 6, 1084 (2018). https://doi.org/10.1039/C7BM01206C21. H. Zhang, Y. Sun, R. Huang, H. Cang, Z. Cai, and B. Sun, Eur. J. Pharm. Biopharm. 128, 260 (2018). https://doi.org/10.1016/j.ejpb.2018.05.013 Besides, they also had a high median lethal dose but did not induce long-term toxicity during their retention in rats.2222. S. Wang et al., Adv. Mater. 29, 1701013 (2017). https://doi.org/10.1002/adma.201701013 Despite these impressive behaviors, the PDA nanoparticles still have the limitation of low drug loading capacity. Similar to most of the nanoparticle-based drug nanocarriers,23,2423. Y. Liu, G. Yang, T. Baby, Tengjisi, D. Chen, D. A. Weitz, and C.-X. Zhao, Angew. Chem. Int. Ed. 59, 4720 (2020). https://doi.org/10.1002/anie.20191353924. P. Couvreur, Adv. Drug Delivery Rev. 65, 21 (2013). https://doi.org/10.1016/j.addr.2012.04.010 the drug loading amount is generally less than 10%.To overcome these limitations, the [email protected] nanohybrids were prepared and used as smart nanocarriers for on-demand drug release and chemo-photothermal synergistic therapy of cancers. The [email protected] nanohybrids were designed with PDA nanospheres as cores and the S6 aptamer cross-linked nanogel as multifunctional smart shells. The DNA nanogel self-assembled by two DNA Y motifs and two DNA linkers integrated the functions of specific cancer cell recognization, fluorescence bioimaging, high drug loading, and on-demand release. The anticancer drug doxorubicin (Dox) intercalated into the GC base pair of DNA helix (Scheme 1), with a loading capacity of 50%. Upon specific recognition by the S6 aptamer to the A549 cancer cells (human lung adenocarcinoma epithelial cell line), the [email protected] nanohybrids were internalized into the cancer cells to release the drug under the control of NIR light and the guidance of the fluorescence image of FAM. The PDA cores could effectively convert the absorbed light into heat, increase the local temperature, dehybridize the DNA double-stranded nanostructure, and release the intercalated Dox. The synergistic interaction of the released drug and the local enhanced temperature effectively killed the cancer cells.

II. EXPERIMENT

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. EXPERIMENT <<III. RESULTS AND DISCUSSI...IV. SUMMARY AND CONCLUSIO...REFERENCES

A. Reagents

The HPLC-purified oligonucleotide sequences were purchased from Sangon Biotech. Co., Ltd. (Shanghai, China) and listed in Table S1 in the supplementary material. Dopamine hydrochloride and doxorubicin hydrochloride (Dox) were purchased for Aladdin Industrial Company. Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco (New York, USA). The ammonia solution, poly(ethylene glycol)2-thioethyl ether acetic acid (SH-PEG-COOH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich. Uranyl acetate solution (2%) was obtained from Shanghai Bairong Biotech Company. All chemicals were of analytical grade and used as-received without further purification. Double distilled water was used during the whole experiment.

B. Synthesis of PDA nanospheres

The synthesis method of PDA nanospheres was adapted from a previous study.2525. J. Li et al., J. Am. Chem. Soc. 137, 1412 (2015). https://doi.org/10.1021/ja512293f In a typical synthesis of PDA nanospheres with an average diameter of 70 nm, 0.5 g of dopamine hydrochloride was dissolved in 10 ml of water and mixed with a solution containing 3.5 ml of ammonia aqueous solution, 40 ml of ethanol, and 90 ml of de-ionized water. After stirring at 30 °C for 30 min, the solution turned to pale yellow and then gradually changed to dark brown. The reaction was allowed to proceed for 24h and the PDA nanoparticles were obtained.The [email protected] nanoparticles were synthesized according to the previous reports with slight modifications.1111. Y. Liu, K. Ai, J. Liu, M. Deng, Y. He, and L. Lu, Adv. Mater. 25, 1353 (2013). https://doi.org/10.1002/adma.201204683 At first, the stoichiometric quantities of the ssDNA strands for the Y-shaped monomer A (YMA), Y-shaped monomer B (YMB), and DNA linker (LK) were separately added to three tubes containing 20 mM of Tris buffer (pH 7.5) and 100 mM of NaCl. Then, each mixture was heated to 95 °C for 5 min and cooled down to room temperature to form the desired building units. Then, the PDA nanospheres were premodified with T15-ay single-stranded DNA and PEG to make them stable at Tris solutions. At last, the three building units of YMA, YMB, and LK were added to the above T15-ay and PEG-stabilized PDA nanospheres. The ideal molar ratio of YMA to the linker is 1:1.5 and that of YMB to the linker is 2:1. The mixture was heated at 50 °C for 5 min, cooled down to room temperature, centrifuged, and washed with Tris buffer. The product was the final [email protected] nanohybrids, which were stored at 4 °C for further use.

D. TEM measurements

For TEM measurements, 10 μl of the sample was deposited onto a carbon-coated grid (300 mesh) and left at room temperature for 10 min. The resulting grid was washed with distilled water to get rid of the salts. Then, 2% uranyl acetate solution was used to stain the sample. The grid was then washed with distilled water and dried at room temperature. Finally, the sample was analyzed by a JEOL JEM-2010 transmission electron microscope with an accelerating voltage of 100 kV.

To evaluate the photothermal conversion efficiency (η), different concentrations of [email protected] nanohybrids solutions (i.e., 0, 50, 100, and 200 μg/ml) were first irradiated by 808 nm NIR laser light (2 W/cm) for 10 min. Then, the laser light was shut off, the temperature was measured, and the photothermal conversion efficiency was determined by the following equations: η=hAΔTmax−QsI(1−10−Aλ),

In the two equations, h is the heat-transfer coefficient, S is the surface area of the container, m (0.5 g) is the mass of the solvent, C (4.2 J/g) is the heat capacity of the solvent, τs is the sample system time constant, Qs is the heat associated with light absorption by the solvent, I is the laser power, and Aλ is the absorbance of the solution at 808 nm.

For drug loading, [email protected] nanospheres were dispersed into different Dox aqueous solutions (Dox: [email protected] = 0:1, 1:4, 1:2, 2:3, and 1:1) and followed with gentle shaking for 12 h in the dark. After equilibrium, the Dox-loaded [email protected] nanoparticles were collected via centrifugation at 10000 rpm for 10 min and washed several times with pure water. The Dox loading capacity was determined by the UV–Vis spectrophotometer according to the maximal absorbance of Dox at 480 nm.The in vitro drug release performance of the [email protected] was evaluated at pH 5.0 and 7.4, with and without the 808 nm light irradiation for 10 min, respectively. In detail, the [email protected] nanospheres were dispersed in different release systems and followed with gentle shaking. During each 2 h interval, some supernatant was taken out for recording the UV–vis absorption spectrum. To keep the system stable, same volume of fresh buffer solution was added to the above drug release system. According to the maximal absorbance of Dox at 480 nm and the calibration curve of Dox, the drug release amount was measured.

G. Confocal fluorescence imaging analysis

Prior to confocal microscopy study, A549 cells and HeLa cells were plated at a density of 106 cells per dish on the glass bottom of cell culture dishes at 37 °C in an atmosphere containing 5% CO2 for 24 h. Then, the cells were incubated with 1 ml of DMEM culture medium containing 200 μg/ml of [email protected] nanoparticles for 4 and 8 h, respectively. For control, the cells were also incubated with a blank DMEM culture medium, at the same experimental conditions of 37 °C and 5% CO2. After the incubation was completed, the cells were washed with fresh culture medium and imaged by the confocal laser scanning fluorescence microscope.

H. Cytotoxicity assay and chemo-photothermal therapy

For evaluating the cytotoxicity and the chemo-photothermal therapy efficacy of the [email protected] nanoparticles, A549 cells were seeded in a 96-well plate at a density of 104 cells per well for 24 h at 37 °C and in 5% CO2. DMEM culture solutions without and with the addition of [email protected], free Dox, and [email protected], respectively, were used to incubate the A549 cells at 37 °C and in 5% CO2 for 8 h. Then, the incubation solutions were removed, the cells were washed three times with PBS, and 100 μl of MTT solutions (0.5 mg/ml) were added to incubate the cells for another 4 h. Finally, 100 μl of DMSO was transferred to the wells, and the absorbance at 540 nm was measured. To evaluate the chemo-photothermal therapy efficacy, another set of MTT experiments with 808 nm NIR light irradiation for 10 min were carried out.

III. RESULTS AND DISCUSSION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. EXPERIMENTIII. RESULTS AND DISCUSSI... <<IV. SUMMARY AND CONCLUSIO...REFERENCESThe synthesis of the [email protected] nanohybrids started with the preparation of PDA nanospheres.2626. Z. H. Miao, H. Wang, H. Yang, Z. L. Li, L. Zhen, and C. Y. Xu, ACS Appl. Mater. Interfaces 7, 16946 (2015). https://doi.org/10.1021/acsami.5b06265 By controlling the ammonia and dopamine concentrations in a mild ethanol and H2O mixture, the dopamine self-polymerized into PDA nanospheres. The TEM image shows that the nanospheres have an average diameter of 70 nm, and the dynamic light scattering experiment shows that the nanohybrids have a narrow size distribution [Figs. 1(a) and 1(b)]. The hydrodynamic diameter is slightly larger than the TEM diameter, which is attributed to the swollen corona around the PDA cores. For cellular uptake and drug delivery, nanocarriers in the range from 50 to 200 nm are preferred due to their high accumulation amount in the tumor sites.2727. J. Kim, Y. M. Lee, Y. Kang, and W. J. Kim, ACS Nano 8, 9358 (2014). https://doi.org/10.1021/nn503349g The PDA nanospheres exhibited a broad absorption ranging from ultraviolet to NIR wavelengths (Fig. S1 in the supplementary material),3535. See the supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002170 for information on the sequence used in the experiment, the UV-vis-NIR absorption spectra of [email protected] nanohybrids with different concentrations, photographs of the [email protected] nanohybrids after their dispersion in different solutions for 48 h, temperature elevation of water and [email protected] nanohybrids with different concentrations as a function of irradiation time, temperature evaluation of the [email protected] nanohybrids over three NIR laser on/off irradiation cycles, the photothermal response of the [email protected] nanohybrids (200 μg/ml) with a 808 nm NIR laser irradiation (2 W/cm) for 600 s, the linear time data versus –lnθ, the Vis-NIR absorption spectra of Dox and [email protected] in aqueous solution, the Dox loading efficiency at different Dox: [email protected] NPs ratios, and the in vitro drug-release profiles of [email protected] NPs in media with different pH values, and with and without NIR laser irradiations, respectively. endowing the biopolymer with strong light absorption capacity. The PDA nanospheres were full of hydroxyl groups and phenol groups and could participate in various interfacial interactions such as hydrogen bonding, metal coordination, π−π/cation-π interactions, and thiol reduction.1111. Y. Liu, K. Ai, J. Liu, M. Deng, Y. He, and L. Lu, Adv. Mater. 25, 1353 (2013). https://doi.org/10.1002/adma.201204683 Taking advantage of the π−π and hydrogen bonding interactions between DNA strands and PDA nanospheres, the DNA nanogel assembling units,2525. J. Li et al., J. Am. Chem. Soc. 137, 1412 (2015). https://doi.org/10.1021/ja512293f Y-motif A, Y-motif B, and the linker (Table S1 in the supplementary material)3535. See the supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002170 for information on the sequence used in the experiment, the UV-vis-NIR absorption spectra of [email protected] nanohybrids with different concentrations, photographs of the [email protected] nanohybrids after their dispersion in different solutions for 48 h, temperature elevation of water and [email protected] nanohybrids with different concentrations as a function of irradiation time, temperature evaluation of the [email protected] nanohybrids over three NIR laser on/off irradiation cycles, the photothermal response of the [email protected] nanohybrids (200 μg/ml) with a 808 nm NIR laser irradiation (2 W/cm) for 600 s, the linear time data versus –lnθ, the Vis-NIR absorption spectra of Dox and [email protected] in aqueous solution, the Dox loading efficiency at different Dox: [email protected] NPs ratios, and the in vitro drug-release profiles of [email protected] NPs in media with different pH values, and with and without NIR laser irradiations, respectively. were hybridized together to form DNA nanoshell on the PDA nanospheres. As shown in Figs. 1(c) and 1(e), the TEM images reveal that the resultant [email protected] nanoparticles were spherical in shape and with thick DNA layers around the PDA cores. Correspondingly, the hydrodynamic diameter of the [email protected] nanohybrids significantly increased from 100 [Fig. 1(b)] to 230 nm [Fig. 1(d)]. The thicker DNAnanogel shells guaranteed the nanohybrids with high drug loading amount. Meanwhile, the Zeta potential (ζ) varied with the coating as well, showing a negative charge of approximately −18 mV for the naked PDA and a negative charge of −25 mV when further assembled with negatively charged DNA nanogel [Fig. 1(f)]. All these features indicated the successful assembly of DNA nanogel on the PDA cores. To assess the colloid stability, the [email protected] nanohybrids were incubated with different media including H2O, PBS, and cell culture medium, respectively, and no agglomeration or sediment existed in the solution after 48 h incubation (Fig. S2 in the supplementary material),3535. See the supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002170 for information on the sequence used in the experiment, the UV-vis-NIR absorption spectra of [email protected] nanohybrids with different concentrations, photographs of the [email protected] nanohybrids after their dispersion in different solutions for 48 h, temperature elevation of water and [email protected] nanohybrids with different concentrations as a function of irradiation time, temperature evaluation of the [email protected] nanohybrids over three NIR laser on/off irradiation cycles, the photothermal response of the [email protected] nanohybrids (200 μg/ml) with a 808 nm NIR laser irradiation (2 W/cm) for 600 s, the linear time data versus –lnθ, the Vis-NIR absorption spectra of Dox and [email protected] in aqueous solution, the Dox loading efficiency at different Dox: [email protected] NPs ratios, and the in vitro drug-release profiles of [email protected] NPs in media with different pH values, and with and without NIR laser irradiations, respectively. indicating that nanocarriers were highly stable in physiological conditions.The photothermal conversion efficiency of the [email protected] nanohybrids was then systematically studied under the irradiation of 808 nm NIR light, which had a deep penetration distance and no damage to healthy cells.2828. D. Jaque, L. Martinez Maestro, B. del Rosal, P. Haro-Gonzalez, A. Benayas, J. L. Plaza, E. Martin Rodriguez, and J. Garcia Sole, Nanoscale 6, 9494 (2014). https://doi.org/10.1039/C4NR00708E As shown in Fig. S3(a) in the supplementary material, after irradiated by a power of 808 nm laser at 2 W/cm2 for 10 min, the temperature of the [email protected] solution (200 μg/ml) reached 55.4 °C, whereas the temperature of control water reached 27.2 °C. The temperature increased monotonically with the increase of the [email protected] concentration in the range of 50–200 μg/ml. It has been well demonstrated that the cancer cells can be killed after maintenance at 42 °C for 15–60 min, this duration can be shortened to 4–6 min for temperatures over 50 °C.29,3029. P. Vijayaraghavan, C. H. Liu, R. Vankayala, C. S. Chiang, and K. C. Hwang, Adv. Mater. 26, 6689 (2014). https://doi.org/10.1002/adma.20140070330. R. W. Habash, R. Bansal, D. Krewski, and H. T. Alhafid, Crit. Rev. Biomed. Eng. 34, 459 (2006). https://doi.org/10.1615/CritRevBiomedEng.v34.i6.20 Assuming that the in vivo temperature of the human body is 36 °C, after injection of [email protected] nanohybrids, the tumor tissues can be easily heated to over 50 °C within 10 min, thus efficiently killing the cancer cells. The [email protected] hybrids showed good photothermal stability, which has been kept stable for at least three on-off irradiation cycles [Fig. S3(b) in the supplementary material].3535. See the supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002170 for information on the sequence used in the experiment, the UV-vis-NIR absorption spectra of [email protected] nanohybrids with different concentrations, photographs of the [email protected] nanohybrids after their dispersion in different solutions for 48 h, temperature elevation of water and [email protected] nanohybrids with different concentrations as a function of irradiation time, temperature evaluation of the [email protected] nanohybrids over three NIR laser on/off irradiation cycles, the photothermal response of the [email protected] nanohybrids (200 μg/ml) with a 808 nm NIR laser irradiation (2 W/cm) for 600 s, the linear time data versus –lnθ, the Vis-NIR absorption spectra of Dox and [email protected] in aqueous solution, the Dox loading efficiency at different Dox: [email protected] NPs ratios, and the in vitro drug-release profiles of [email protected] NPs in media with different pH values, and with and without NIR laser irradiations, respectively. The photothermal conversion efficiency was calculated to be 38% following Liu's report [Figs. S3(c) and S3(d) in the supplementary material].11,3511. Y. Liu, K. Ai, J. Liu, M. Deng, Y. He, and L. Lu, Adv. Mater. 25, 1353 (2013). https://doi.org/10.1002/adma.20120468335. See the supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002170 for information on the sequence used in the experiment, the UV-vis-NIR absorption spectra of [email protected] nanohybrids with different concentrations, photographs of the [email protected] nanohybrids after their dispersion in different solutions for 48 h, temperature elevation of water and [email protected] nanohybrids with different concentrations as a function of irradiation time, temperature evaluation of the [email protected] nanohybrids over three NIR laser on/off irradiation cycles, the photothermal response of the [email protected] nanohybrids (200 μg/ml) with a 808 nm NIR laser irradiation (2 W/cm) for 600 s, the linear time data versus –lnθ, the Vis-NIR absorption spectra of Dox and [email protected] in aqueous solution, the Dox loading efficiency at different Dox: [email protected] NPs ratios, and the in vitro drug-release profiles of [email protected] NPs in media with different pH values, and with and without NIR laser irradiations, respectively. It is higher than those widely studied photothermal conversion agents such as gold nanorod (21%) and Cu9S5 (25.7%).31,3231. C. M. Hessel, V. P. Pattani, M. Rasch, M. G. Panthani, B. Koo, J. W. Tunnell, and B. A. Korgel, Nano Lett. 11, 2560 (2011). https://doi.org/10.1021/nl201400z32. Q. Tian et al., ACS Nano 5, 9761 (2011). https://doi.org/10.1021/nn203293t These results revealed that [email protected] nanohybrids were promising agents for photothermal therapy of cancer treatment.Dox, a clinically used chemotherapy drug, was loaded into the [email protected] via intercalation into the double-stranded GC or CG base pairs.3333. J. Cui, Y. Yan, G. K. Such, K. Liang, C. J. Ochs, A. Postma, and F. Caruso, Biomacromolecules 13, 2225 (2012). https://doi.org/10.1021/bm300835r After Dox loading [Fig. S4(a) in the supplementary material],3535. See the supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002170 for information on the sequence used in the experiment, the UV-vis-NIR absorption spectra of [email protected] nanohybrids with different concentrations, photographs of the [email protected] nanohybrids after their dispersion in different solutions for 48 h, temperature elevation of water and [email protected] nanohybrids with different concentrations as a function of irradiation time, temperature evaluation of the [email protected] nanohybrids over three NIR laser on/off irradiation cycles, the photothermal response of the [email protected] nanohybrids (200 μg/ml) with a 808 nm NIR laser irradiation (2 W/cm) for 600 s, the linear time data versus –lnθ, the Vis-NIR absorption spectra of Dox and [email protected] in aqueous solution, the Dox loading efficiency at different Dox: [email protected] NPs ratios, and the in vitro drug-release profiles of [email protected] NPs in media with different pH values, and with and without NIR laser irradiations, respectively. a typical peak at 480 nm was observed in the UV–vis absorption spectrum. A maximal drug loading amount of 50% was obtained when the concentration ratio of Dox to the nanohybrid got to 1:1 [Fig. S4(b) in the supplementary material].3535. See the supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002170 for information on the sequence used in the experiment, the UV-vis-NIR absorption spectra of [email protected] nanohybrids with different concentrations, photographs of the [email protected] nanohybrids after their dispersion in different solutions for 48 h, temperature elevation of water and [email protected] nanohybrids with different concentrations as a function of irradiation time, temperature evaluation of the [email protected] nanohybrids over three NIR laser on/off irradiation cycles, the photothermal response of the [email protected] nanohybrids (200 μg/ml) with a 808 nm NIR laser irradiation (2 W/cm) for 600 s, the linear time data versus –lnθ, the Vis-NIR absorption spectra of Dox and [email protected] in aqueous solution, the Dox loading efficiency at different Dox: [email protected] NPs ratios, and the in vitro drug-release profiles of [email protected] NPs in media with different pH values, and with and without NIR laser irradiations, respectively. It should be noted that the Dox loading amount was much higher than most of the nanoparticle-based nanocarriers, such as PDA nanocapusules3333. J. Cui, Y. Yan, G. K. Such, K. Liang, C. J. Ochs, A. Postma, and F. Caruso, Biomacromolecules 13, 2225 (2012). https://doi.org/10.1021/bm300835r and gold nanorod decorated triangle DNA nanostructure,1010. Y. Du et al., Adv. Mater. 28, 10000 (2016). https://doi.org/10.1002/adma.201601710 which was attributed to the thick DNA nanogel shell. The stimuli-responsive drug release behavior of the Dox-loaded [email protected] nanohybrids was then investigated with diverse stimuli including pH and NIR laser irradiation. As shown in Fig. S4(c) in the supplementary material,3535. See the supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002170 for information on the sequence used in the experiment, the UV-vis-NIR absorption spectra of [email protected] nanohybrids with different concentrations, photographs of the [email protected] nanohybrids after their dispersion in different solutions for 48 h, temperature elevation of water and [email protected] nanohybrids with different concentrations as a function of irradiation time, temperature evaluation of the [email protected] nanohybrids over three NIR laser on/off irradiation cycles, the photothermal response of the [email protected] nanohybrids (200 μg/ml) with a 808 nm NIR laser irradiation (2 W/cm) for 600 s, the linear time data versus –lnθ, the Vis-NIR absorption spectra of Dox and [email protected] in aqueous solution, the Dox loading efficiency at different Dox: [email protected] NPs ratios, and the in vitro drug-release profiles of [email protected] NPs in media with different pH values, and with and without NIR laser irradiations, respectively. at pH 7.4, only 14% of Dox was released from the [email protected] nanohybrids in 24 h. In contrast, about 32% of the Dox was released at pH 5.0 due to the increased solubility of protonated Dox. Interestingly, under the irradiation of 808 nm NIR light, a burst release of Dox to 50% was observed. This pH/NIR dual stimuli-responsive drug release behavior made them promising for on-demand drug release and chemotherapy.The targeted cellular uptake behavior of the [email protected] nanohybrids was investigated by confocal laser scanning microscopy (CLSM). FAM fluorophore was labeled on the nanohybrid to track the location of the smart nanocarrier. Aptamer S6 is the desired candidate with a specific affinity to A549 cancer cells but not HeLa cells.3434. H. Shi, X. Ye, X. He, K. Wang, W. Cui, D. He, D. Li, and X. Jia, Nanoscale 6, 8754 (2014). https://doi.org/10.1039/C4NR01927J The prepared [email protected] nanohybrids were incubated with A549 cells and HeLa cells at 37 °C for 4 and 8 h, respectively. After removing the unbound nanohybrids by PBS washing, the CLSM images were recorded (Fig. 2). An intensive fluorescence signal was observed in the cytoplasm of A549 cells. However, almost no fluorescence signal could be observed in HeLa cells under identical conditions. Thus, the [email protected] nanohybrids could be selectively internalized into the A549 cancer cells with the help of the S6 aptamer.The cell cytotoxicity and in vitro chemo-photothermal combination therapy of the [email protected] nanohybrids were evaluated on A549 cells by a MTT assay method. As shown in Fig. 3, the [email protected] nanohybrids exhibited negligible toxicity to cells even at a high concentration of 400 μg/ml after incubation for 8 h, indicating the good biocompatibility of the drug loading matrix. However, only 46% of cell viability was left when the NIR light was applied on the Dox-loaded [email protected] nanohybrids ([email protected]). In comparison, negligible toxicity was observed when the A549 cells were exposed to the NIR light alone, and the free Dox and [email protected] without Dox caused 23% and 32% of cell viability loss, respectively. Obviously, the improved therapy efficacy should be attributed to the synergistic interaction of chemo-photothermal therapy.