Retracted Article: CuS nanocrystal@microgel nanocomposites for light-regulated release of dual-drugs and chemo-photothermal synergistic therapy in vitro

Abstract

In this article, we described the synthesis of novel light-sensitive inorganic@organic core/shell nanocomposites that consisted of CuS nanocrystals as the core and a poly(N-isopropylacrylamide)-graft-chitosan (PNIPAM-g-CS) microgel as the shell. The CuS@PNIPAM-g-CS nanocomposites were synthesized by temperature-tunable copolymerization of NIPAM and CS in the presence of CuS nanocrystals (∼5.4 nm). The nanocomposites showed an average diameter of ∼56 nm and a strong longitudinal surface plasmon band in the near-infrared (NIR) region. Due to the photothermal effect of CuS under NIR light (980 nm) irradiation, the nanocomposites presented photothermal-sensitive volume shrinkage of the PNIPAM-g-CS microgel. After loading of doxorubicin (Dox), the nanocomposites were utilized as versatile nanocarriers for photothermal-induced release of Dox. After loading of Dox, nitric oxide (NO) photodonors (RBS) were then loaded into nanocomposites to fabricate Dox/RBS dual-loading CuS@PNIPAM-g-CS nanocarriers. Upon visible light (365 nm) irradiation, the nanocarriers could release NO due to the photolysis of RBS. Experimental results implied that NIR and visible light, respectively, triggered the release of Dox and NO from the nanocarriers. Together with the photothermal effect of CuS, the nanocarriers simultaneously realized the light-triggered release of dual-drugs and synergistic chem-photothermal therapy to cancer cells in vitro.

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1. Introduction

As a well-known p-type semiconductor material, recently copper sulfide (CuS) has generated much attention due to its potential application in photothermal therapy.¹ CuS nanocrystals (NCs) possess a series of advantages such as low cost, low cytotoxicity and intrinsic near-infrared (NIR) absorption originating from energy band transition instead of surface plasmon resonance.² Upon irradiation with NIR-light, the high photothermal conversion efficiency and photothermal ablation therapy of CuS NCs have been extensively reported in recent years.³ NIR-light (700–1100 nm) is considered as an effective mode in photothermal therapy, when compared to other external stimuli (such as pH, ultrasound, redox, glucose, enzyme, magnetic field, etc.) that have been exploited for controlled drug release.⁴ As another advantage, NIR-light irradiation enables a deep penetration but minimal absorbance in tissues, promoting the use of CuS NCs in theranostic nanomedicine.⁵

The combination of CuS NCs and drug delivery vehicles together could produce dual-effects of photothermal ablation and chemo-therapy, which thus provides an efficient synergistic therapeutic treatment to cancer cells. In previous reports, CuS@mesoporous silica nanocomposites have been developed to perform NIR-induced photothermal therapy from CuS NCs and pH-regulated drug release from mesoporous silica.⁶ Poly(ethylene glycol)–graphene oxide/CuS nanocomposites were used for NIR chemo-photothermal therapy.⁷ The NIR-light triggered both drug release and CuS-mediated photothermal ablation. CuS nanoparticles with DNA sequences were conjugated with mesoporous silica to obtain a new drug delivery platform.^5b This platform enabled NIR-responsive drug release from mesoporous silica and photothermal therapy of CuS. To further improve chemo-photothermal therapy, additional light-regulated modes of drug release (e.g., drug release triggered by low-energy visible light) are attractive for the nanocomposites consisting of CuS NCs and drug delivery vehicles. Accompanied with photothermal therapy of CuS, the introduction of NIR- and visible-light triggered drug release into one system would realize light-regulated release of dual-drugs and improve chemo-photothermal synergistic therapy.

Herein, we described light-sensitive nanocomposites consisting of CuS NCs as a core and poly (N-isopropylacrylamide)-graft-chitosan (abbreviated as PNIPAM-g-CS) microgels as a shell. The nanocomposites (abbreviated as CuS@PNIPAM-g-CS) were synthesized by temperature-tunable copolymerization of NIPAM and CS in the presence of CuS NCs. Due to the photothermal effect of CuS under NIR light (980 nm), the nanocomposites showed photothermal-sensitive volume shrink of PNIPAM-g-CS. After loading of doxorubicin (Dox), the nanocomposites were utilized as smart nanocarriers for photothermal-induced Dox release. Then, nitric oxide (NO) photodonors (Fe₄S₃(NO)₇⁻, or abbreviated as RBS that could photo-sensitively release NO under visible-light irradiation)⁸ were subsequently loaded into the nanocomposites to form Dox and RBS dual-loaded CuS@PNIPAM-g-CS nanocarriers. Upon visible light (365 nm), the nanocarriers could release NO due to RBS photolysis. NIR and visible light respectively triggered release of Dox and NO from the nanocarriers (Scheme 1). Together with the photothermal effect of CuS, the nanocarriers simultaneously realized light-triggered release of dual-drugs and synergistic chem-photothermal therapy to cancer cells.

Scheme 1 Schematic illustration of the fabrication and light-regulated drug release of Dox/RBS loaded CuS@PNIPAM-g-CS nanocarriers.

2. Experimental

2.1. Materials and apparatus

CuCl₂ (99%), Na₂S (98%), sodium citrate (99%), NIPAM (99%), CS (deacetylation degree 90%, M_v = 3.8 × 10⁵), ammonium persulphate (APS, 99%), N,N′-methylenebisacrylamide (MBA, 99%) were bought from Sigma-Aldrich. Roussin's Black Salt (RBS, Fe₄S₃(NO)₇⁻) was prepared based on the report from Seyferth et al.⁸ RBS in the solid form is stored in dark and inert atmosphere. Dox (hydrochloride salts) was obtained from Beijing Huafeng Corp. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and other biological reagents were bought from Invitrogen Corp. HeLa cells were provided by cell bank of Shanghai Science Academe of China. Other chemicals with analytical grade were obtained from Shanghai Chemical Reagent Corp. All chemicals can be utilized directly as received without any purification. Distilled water was used in experiments throughout. Phosphate buffer solutions (PBS, 1 mM) were prepared by adjusting the ratio of Na₂HPO₄ and NaH₂PO₄.

Transmission electron microscope (TEM, Jeol) images were acquired with a JEOL JEM-1400 TEM operating at 120 kV of acceleration voltage. UV-vis-NIR absorption spectra were recorded on a UV-2450 spectrophotometer (Shimadzu). Fourier transform infrared (FTIR) spectra (Nicolet) were recorded with a Nicolet 6700 FTIR spectrometer. Dynamic light scattering (DLS, Malven Instruments) was utilized to record hydrodynamic diameter. For the analysis of TEM and DLS, the concentration of relevant samples was fixed to be 1.0 mg mL⁻¹.

2.2. Preparation of CuS@PNIPAM-g-CS nanocomposites

Citrate-stabilized CuS NCs were prepared by a slightly modified method reported previously (details are available in ESI, Part S1†).⁹ CuS@PNIPAM-g-CS nanocomposites were prepared via a temperature-regulated copolymerization of NIPAM monomer and CS in the presence of CuS NCs.¹⁰ Typically, 1.5 g of NIPAM and 0.1 g of MBA dissolved in 50 mL of distilled water were loaded into a 250 mL three-necked flask, which was bubbled with N₂ inlet for 30 min to remove dissolved oxygen. In the mixture solution, 10 mL of aqueous suspension of CuS NCs (2 mg mL⁻¹) was slowly injected under ultrasonic and stirring to form the reaction solution. Then, 0.06 g of APS dissolved in 5 mL distilled water was added in the reaction solution to initiate polymerization for 2 h at room temperature (25 °C). Afterward, 0.04 g of APS, 0.2 g of CS, 0.2 mL of acetic acid dissolved in 10 mL distilled water was added in the reaction solution. The reaction temperature was raised to 70 °C for a further polymerization, with continuous stirring of 300 rpm and N₂ flow of 200 mL min⁻¹. After 6 h of polymerization reaction, the crude products were centrifuged. The as-obtained precipitates were dispersed in distilled water and then dialyzed (Spectra/Por 7 dialysis membrane, MWCO 5 × 10⁴) against water frequently, lasting for 5 days. The aqueous suspension of CuS@PNIPAM-g-CS nanocomposites were condensed by rotary evaporators and dispersed in PBS (pH 7.4) for subsequent experiments. PNIPAM-g-CS microgels were prepared by using the method of CuS@PNIPAM-g-CS in the absence of CuS NCs.

2.3. Drug loading and release assay

CuS@PNIPAM-g-CS nanocomposites (20 mg) were ultrasonically dispersed into 25 mL of Dox solution (1 mg mL⁻¹, 1% NaCl in PBS at pH 7.4) to form mixture solution that was continuously stirred at 25 °C for 24 h until the Dox concentration [Dox] in solution stabilized. The suspension was centrifuged and washed with water twice to remove unbound and surface-absorbed Dox. The mass of Dox loaded in nanocomposites was calculated by subtracting the Dox in supernatant from the total Dox in initial solution. Free Dox in solution was detected by UV-vis spectroscopy at 480 nm using Lambert–Beer law. The loading efficiency (LE) and loading capacity (LC) of Dox were calculated by the equations as below;^10b–d

Dox-LE (%) = 100 × (M_total-Dox − M_free-Dox)/M_total-Dox (1)

Dox-LC (%) = 100 × (M_total-Dox − M_free-Dox)/M_{total-nanocomposites} (2)

Under slight stirring at 25 °C, 25 mL of RBS solution (1 mg mL⁻¹) was added into the above Dox-loaded nanocomposites, keeping for 24 h incubation. The treatments and calculations of RBS loading were similar to the case of Dox loading, and the mass of RBS in solution was determined by UV-vis spectroscopy at 375 nm.^8b

To evaluate NIR light-triggered Dox release, 20 mg of Dox-loaded nanocomposites dispersed in 20 mL of PBS (10 mM, pH 7.4, 6.5 or 5.5) were transferred into a dialysis tube (MWCO 5 × 10⁴) that was shaken slightly in water bath at room temperature. As a fixed release time (0–12 h), 1 mL of each sample was withdrawn and analyzed by UV-vis spectroscopy at 480 nm to determine the [Dox] from nanocomposites. Dox release was studied without and with 0–5 min light irradiation at 980 nm (0.5 W), respectively. Upon 365 nm light irradiation at 0.5 W, RBS release from Dox/RBS dual-loaded nanocomposites was evaluated during 0–30 min. NO concentration [NO] from RBS photolysis was calculated using the colorimetric Griess reaction (in ESI, Part S2†).¹¹

2.4. Photothermal and chemo-cytotoxicity assay

The cytotoxicities of CuS@PNIPAM-g-CS nanocomposites, free Dox, Dox-loaded or Dox/RBS dual-loaded CuS@PNIPAM-g-CS nanocarriers were studied before and after light irradiation. HeLa cells were cultured as the sub-confluent monolayers on 25 cm² cell culture plates with vent caps in 1× minimum essential and a medium supplemented with 10% of fetal bovine serum in a humidified incubator with 5% of CO₂. Upon the growth to sub-confluence, the HeLa cells were dissociated from the surface with trypsin solution (0.25%) and the aliquots (100 mL) were seeded (1 × 10⁴ cells) into a 96-well plate. After incubation for 24 h, the medium was replaced with 10 mL of serum-free Dulbecco modified Eagle essential medium containing 0–1 mg mL⁻¹ of different samples. These treated cells were incubated for 24 h in dark. Afterward, these cells were irradiated with 980 nm light (0.5 W) for 5 min alone, and were also irradiated with 365 nm light (0.5 W) for 25 min during the drug release time of 0–30 min. The viabilities of HeLa cells were quantitated by using a standard MTT assay.¹⁰

3. Results and discussion

3.1. Synthesis and characterization

According to the monomer polymerization, the encapsulation of CuS NCs with PNIPAM-g-CS microgels to prepare CuS@PNIPAM-g-CS nanocomposites is a simple route for rapid entrapment of inorganic materials within a polymer matrix. This route needs to conquer the phase separation between NCs and microgels, and the aggregation of NCs in polymerization. Above the low critical solution temperature (LCST, ∼32 °C) of PNIPAM,¹⁰ NCs and PNIPAM are incompatible. The functionalized amphiphilic polymers are added as polymerizable groups onto the surface of NCs, allowing to copolymerizing with NIPAM.¹² Incompatibility of hydrophilic NCs and hydrophobic PNIPAM above LCST could result in a low percentage of NCs–PNIPAM hybrids, making them difficult to be separated from polymerization products. Herein, aqueous citrate-stabilized CuS NCs directly copolymerized with NIPAM to form hybrids by tuning polymerization temperature. Below the LCST (25 °C), CuS NCs and PNIPAM networks are well compatible. Above the LCST (70 °C), PNIPAM networks collapse to form nanospheres, leading to the entrapment of NCs into nanospheres. Then, PNIPAM becomes compatible with NCs above LCST, and the entrapped NCs could hardly any expelled outside of the nanospheres.¹³ The temperature-tunable copolymerization of NIPAM in the presence of CuS NCs is feasible, facile and effective, without any modification on the surface of NCs. Furthermore, it facilitates the following graft polymerization of CS with PNIPAM to fabricate PNIPAM-g-CS microgels¹⁰ and CuS@PNIPAM-g-CS nanocomposites.

In Fig. 1a, the peak at 1645 cm⁻¹ is assigned to the stretching vibration of [double bond splayed left]C=O from PNIPAM. The characteristic absorption bands at 2970 and 1390 cm⁻¹ also revealed the existence of PNIPAM chains in CuS@PNIPAM-g-CS.¹⁰ The peak at 1560 cm⁻¹ is assigned to the bending vibration of –NH₂ from CS. Below 600 cm⁻¹, a sharp band indicated the characteristic absorption of inorganic substances (CuS NCs, based on the synthesis procedures).¹⁴ The ¹H-NMR spectrum (in ESI, Fig. S1†) further indicates the achievement of PNIPAM-g-CS microgels. These results revealed the copolymerization of PNIPAM and CS, and the integration of PNIPAM-g-CS microgels and CuS NCs in nanocomposites. The absorption spectrum of CuS@PNIPAM-g-CS nanocomposites (Fig. 1b) displayed a weak transverse plasmon band at ∼650 nm and a strong longitudinal surface plasmon band at ∼1400 nm, attributed to the presence of CuS NCs.¹⁵ Thus, the photothermal effect of CuS@PNIPAM-g-CS can be simply conducted by a NIR laser. Compared with the use of an 808 nm laser to trigger photothermal ablation of CuS NCs,^1a the 980 nm laser enables a higher absorbance of CuS NCs, deeper tissue penetration and higher conservative limit for human skin.⁹ NIR light with a wavelength over 1000 nm will produce water absorption in biomedical systems, and so reduces penetration depth and spatial resolution.^5c By contrast, a 980 nm laser was chosen as the NIR light to trigger drug release in subsequent experiments. TEM images of CuS NCs (Fig. 1c) exhibited uniform and discrete nanoparticles with an average diameter of 5.4 ± 0.4 nm. After copolymerization, core/shelled nanoparticles were clearly observed with an average size of 56.0 ± 1.5 nm (Fig. 1d). The black cores were coated by grey shells. Based on the synthesis procedures, these results implied that CuS NCs were coated by PNIPAM-g-CS microgels and therefore had been embedded within nanocomposites favorably.

Fig. 1 (a) FTIR spectra of PNIPAM-g-CS microgels and CuS@PNIPAM-g-CS nanocomposites. (b) UV-vis-NIR absorption spectrum of CuS@PNIPAM-g-CS nanocomposites. TEM images of (c) CuS NCs and (d) CuS@PNIPAM-g-CS nanocomposites.

3.2. Photothermal sensitivity of nanocomposites

The continuous exposure of CuS@PNIPAM-g-CS nanocomposites to NIR light induced a rapid temperature elevation, which is a key feature of CuS NCs-based nanomaterials for controlled drug release and photothermal ablation therapy to tumors.^1,2 Under a 980 nm light irradiation at 0.5 W, the aqueous suspension of nanocomposites yielded a marked temperature elevation (Fig. 2a). After 120, 240 and 300 s of irradiation, the temperature of aqueous suspension was elevated to 34.9, 41.5 and 43.0 °C, respectively. In contrast, no marked temperature increase (∼0.5 °C) was detected when PBS as the control was exposed to the NIR light irradiation, which thus suggested the good photothermal efficiency of nanocomposites. After 5 min irradiation, a temperature above 42 °C could be obtained, which is sufficient for hyperthermia treatment to kill tumor cells.¹⁶ Hence, the nanocomposites could be used as an efficient NIR-light absorber for photothermal tumor therapy. During the light irradiation of 0–5 min, the hydrodynamic diameter of nanocomposites exhibited a regular decrease due to the photothermal effect of CuS NCs, as shown in Fig. 2b. Above the LCST, PNIPAM networks (from PNIPAM-g-CS microgels in CuS@PNIPAM-g-CS) will undergo an abrupt collapse to form hydrophobic spheres, exhibiting a coil–globule volume phase transition.¹⁰ Upon the NIR irradiation below 120 s, a negligible (<5%) volume shrink of nanocomposites was observed. Between 120 and 210 s of NIR irradiation, a marked volume shrink occurred and the hydrophobic diameter of nanocomposites decreased from 91.5 to 39.9 nm (determined by DLS in ESI, Fig. S2 and S3†). At a longer irradiation above 210 s, there is almost no volume shrink that implies the achievement of highly compact nanocomposites. As a comparison, we performed relevant experiments to confirm the volume shrink of nanocomposites that was attributed to the increased temperature in a water bath (in ESI, Fig. S4†). With the temperature increase of a water bath (from 25 to 45 °C), the hydrodynamic diameter of CuS@PNIPAM-g-CS nanocomposites exhibited a similar change trend to the case of light irradiation treatment at 980 nm (in Fig. 2b). Hence, the dramatic volume shrink of CuS@PNIPAM-g-CS nanocomposites was produced from the promising NIR light-triggered photothermal effect of CuS NCs. When drugs were loaded into nanocomposites, the photothermal effect-induced volume shrink would squeeze loaded drugs out of nanocomposites, causing a controllable drug release. These characters of nanocomposites could be efficiently applied to the NIR light-regulated chemo-photothermal synergistic therapy.

Fig. 2 (a) Temperature elevation of aqueous suspension of CuS@PNIPAM-g-CS nanocomposites (1.0 mg mL⁻¹) as a function of irradiation time with a 980 nm light (0.5 W) in 10 mM of PBS at 7.4. (b) Effect of irradiation time on hydrodynamic diameters of the nanocomposites.

3.3. Drug loading and light-induced drug release

The CuS@PNIPAM-g-CS nanocomposites were loaded with Dox at different concentrations for 24 h at 25 °C (Fig. 3a). Both the loading capacity (LC) and loading efficiency (LE) were distinctly affected by Dox, and gradually increased with [Dox] increasing. When [Dox] increased from 0.05 to 1.0 mg mL⁻¹, the LC increased from 2.6 to 48.0%, while the LE increased from 14.9 to 62.3%. These results revealed that the Dox loading into nanocomposites was [Dox]-dependent. Generally, Dox molecule consists of acidic phenolic hydroxyl and alkaline amino groups that could interact with amino and hydroxyl groups of CS from PNIPAM-g-CS microgels in nanocomposites to form intermolecular complexes by hydrogen bonding,¹⁷ which will cause a high LE. In comparison with other drugs with a small molecule size (e.g. 5-aminosalicylic) and macromolecular drugs (e.g. bovine serum albumin/BSA), Dox has a much larger molecular size than 5-aminosalicylic, but is much smaller than BSA. Thus, the encapsulation of Dox into nanocomposites was much efficient since the nanocomposites had no adsorption interaction with Dox. Otherwise, a reduced trend for LE would be observed upon the [Dox] increase.¹⁸ Dox release from Dox-loaded nanocomposites in PBS at different pHs was studied. In Fig. 3b, the release of Dox from the nanocomposites is slower at neutral than mildly acidic pH as Dox is less soluble in water at neutral pH and would therefore prefer to remain in the nanocomposites. As early reported, the solubility of Dox at pH 4.0–5.5 was optimal.¹⁷ Nevertheless, these release processes were relatively slow. At pH 6.5 (a potential acidic condition for tumor tissues), only less than 20% of Dox release was obtained at the initial stage (0–2 h).

Fig. 3 (a) Effects of Dox concentration on the LC and LE of Dox into CuS@PNIPAM-g-CS nanocomposites. Cumulative Dox release profiles from Dox-loaded nanocomposites: (b) incubated in PBS at 25 °C, (c) incubated in PBS (pH 6.5) without or with NIR-light irradiation (980 nm, 0.5 W), and (d) incubated in PBS before and after the NIR irradiation.

A rapid and controllable drug release toward targeted tumor sites is necessary and improves the therapy efficiency of serious tumors. For Dox-loaded nanocomposites incubated in PBS at 6.5, the release of Dox was triggered by 980 nm light irradiation (0.5 W). Upon the increase of irradiation time (0–5 min), an obviously accelerated Dox release was found in Fig. 3c. After 5 min irradiation, the cumulative amount of Dox reached over 62%. Without NIR-light irradiation, the released Dox was close to only 20% in the release time of 0–2 h, revealing that the NIR-light irradiation could dramatically accelerate the release of Dox. As well established, the NIR-light irradiation induced photothermal effect of CuS@PNIPAM-g-CS nanocomposites (from CuS NCs) that so resulted in the volume shrink of PNIPAM-g-CS microgels. Thus, the loaded Dox into PNIPAM-g-CS was rapidly squeezed out, leading to a rapid release of Dox. Except for the case of pH 6.5, similar Dox-release trends were also found at the cases of pH 7.4 and pH 5.5 (Fig. 3d). In other words, the NIR-light irradiation could obviously accelerate Dox release at different pH conditions. Compared to the case without irradiation (0 min), the accelerated degree of irradiation-induced Dox release was more significant at a higher pH. At different pHs, the cumulative released Dox even reached an almost equal amount after NIR-light irradiation for 5 min, which thus further confirmed the high feasibility of the NIR-light irradiation to efficiently trigger Dox release.

For Dox/RBS dual-loaded CuS@PNIPAM-g-CS nanocomposites, NO release was investigated by irradiation with visible light.^8b In Fig. 4, 365 nm of light irradiation triggered dramatic release of NO at an increasing rate (0–25 min). After 25 min, a slight decrease in the instant content of released NO was observed due to the oxidation of NO by oxygen in solution.¹⁹ By contrast, hardly any NO release could be detected when the nanocomposites were incubated in the dark (with no irradiation). The maximum NO release reached a dose of ∼0.86 μM at 25 min. Although more NO can be released under a high-power light irradiation (>0.5 W) or an extended release time (>30 min),¹¹ a shorter excitation time and a lower irradiation power are especially favorable for their biomedical applications. These conditions can maximally reduce the potential damage from light irradiation itself to normal physiological systems.²⁰

Fig. 4 (a) NO release from Dox/RBS dual-loaded CuS@PNIPAM-g-CS nanocomposites incubated in PBS (at pH 6.5) without and with NIR-light irradiation (365 nm, 0.5 W).

3.4. Chemo-photothermal therapy to tumor cells

To further investigate the cytotoxicities of CuS@PNIPAM-g-CS, free Dox and Dox-loaded CuS@PNIPAM-g-CS nanocomposites, HeLa cells were incubated with them for 24 h. As shown in Fig. 5a, high cell viabilities were determined upon incubation with blank CuS@PNIPAM-g-CS. At 1 mg mL⁻¹, 84.0% of cell viability was maintained, indicating low cytotoxicity and favorable biocompatibility. As a reference, the cytotoxicity of CuS NCs was also evaluated under the same conditions. Consequently, after 24 h incubation with 1 mg mL⁻¹ of CuS NCs, the viability of HeLa cells reached 81.0% (in ESI, Fig. S5†), implying the low cytotoxicity of CuS NCs. The slightly enhanced cell viability (from 81% to 84%) further suggested a higher biocompatibility of CuS NCs after the surface coating of PNIPAM-g-CS microgels as the shell. A distinct decrease in cell viability was observed when incubated with Dox-loaded CuS@PNIPAM-g-CS. At 0.1–1.0 mg mL⁻¹, the viability of treated cells reduced from 83.5 to 42.0%. The cytotoxicity of Dox-loaded CuS@PNIPAM-g-CS (with the same Dox concentration with free Dox) was less than that of free Dox. These results revealed that the Dox released from Dox-loaded CuS@PNIPAM-g-CS still retained a high inhibition to cancer cells or a high anticancer activity. In fact, the concentration of nanocomposites was too low to produce toxicity for cancer cells.¹⁰

Fig. 5 (a) In vitro cell viabilities after incubation with different dosages (0–1 mg mL⁻¹) of CuS@PNIPAM-g-CS, free Dox and Dox-loaded CuS@PNIPAM-g-CS nanocomposites with the same Dox concentration with free Dox. (b) In vitro cell viabilities after incubation with 0.1 mg mL⁻¹ of Dox/RBS-loaded CuS@PNIPAM-g-CS nanocomposites incubated for 0–30 min in dark, irradiated by NIR light (980 nm, 0.5 W) for 5 min and irradiated by visible light (365 nm, 0.5 W) for 25 min.

At a low concentration of the Dox/RBS-loaded CuS@PNIPAM-g-CS nanocomposites (0.1 mg mL⁻¹), the cytotoxicities of nanocomposites were investigated under different conditions of light irradiation. In Fig. 5b, the cells treated with nanocomposites showed higher cell viability in dark. During the incubation time from 0 to 30 min, more than 78.5% of cell viability was maintained. In contrast, 5 min NIR-light irradiation (980 nm) produced a significant and gradual decrease of cell viability. After an additional incubation for 25 min, the viability of HeLa cells reduced to less than 23.5%. The significantly decreased viability of HeLa cells treated with nanocomposites should be due to the photothermal effect of CuS NCs and photothermal-induced Dox release. After 980 nm light irradiation for 5 min (0.5 W), the system temperature rose to over 42 °C, which is sufficient for hyperthermia treatment to kill HeLa cells. Meanwhile, the photothermal-induced temperature rise could trigger dramatic volume shrink of PNIPAM-g-CS microgels and thus it would squeeze loaded Dox out of nanocomposites, resulting in the dramatic release of anticancer agent Dox. The results illustrated the successful achievement of chemo-photothermal synergistic therapy to HeLa cells. Besides 980 nm of light irradiation for 5 min, an additional treatment with 365 nm of light irradiation for 25 min was also conducted to improve the therapy efficiency to cancer cells. As a result, the light irradiation (365 nm) further accelerated the cell death. Compared to the case of only 980 nm light irradiation (∼23.5% of cell viability), the additional 365 nm light irradiation induced a dramatic decrease in cell viability (reduced to ∼1%) at the experiment time of 0–30 min. As established, visible (365 nm) light irradiation triggered a dramatic NO release due to RBS photolysis, while the released NO could produce an appreciable level of cytotoxicity in cancer cell lines.²¹ Hence, the dramatic cell death triggered by the additional 365 nm light irradiation should be attributed to NO-induced cytotoxicity.

Without incubation with the CuS@PNIPAM-g-CS, HeLa cells were directly treated with light irradiation at 980 nm or 365 nm. Consequently, 980 nm light irradiation (0.5 W) for 30 min only induced a negligible temperature elevation in cell microenvironment (<1 °C) (in ESI, Fig. S6a†).⁹ Although the light energy of 365 nm is higher than that of 980 nm, the low power (0.5 W) is not enough to produced a remarkably elevated temperature for cell microenvironment (<3 °C) (in ESI, Fig. S6b†). Negligible temperature elevation further verified that the marked death of HeLa cells was from the light-triggered released anticancer agents (Dox, RBS) and the photothermal effect of CuS NCs, rather than the light irradiation itself. Based upon these experimental results, Dox/RBS-loaded CuS@PNIPAM-g-CS nanocomposites could achieve light irradiation-regulated release of dual-drugs and chemo-photothermal synergistic therapy to cancer cells.

4. Conclusions

In summary, light-sensitive core@shell CuS@PNIPAM-g-CS nanocomposites were synthesized by temperature-tunable copolymerization of NIPAM and CS in the presence of CuS NCs. Due to the photothermal effect of CuS under NIR light irradiation, the nanocomposites demonstrated the photothermal-induced volume shrinkage of Dox-loaded PNIPAM-g-CS microgels, leading to the release of Dox. Dox and RBS dual-loaded CuS@PNIPAM-g-CS nanocomposites were developed. Upon NIR- and visible-light irradiation, the nanocomposites could respectively release Dox (be squeezed out due to volume shrinkage of PNIPAM-g-CS) and NO (RBS photolysis). Experimental results revealed that the nanocomposites simultaneously realized light-triggered release of dual-drugs and improved chem-photothermal synergistic therapy to cancer cells in vitro. The versatile nanocomposites would be further utilized towards the high efficient therapy of tumor-bearing living small animals in vivo, and facilitate the fabrication of novel multifunctional nanocarrier systems for biomedical applications.

Acknowledgements

This work was financially supported by the Shandong Provincial Natural Science Foundation of China (ZR2015YL041 and ZR2014BQ001), the Project of Shandong Province Higher Educational Science and Technology Program (J14LM02), the Science and Technology Research Program of Shangdong Academy of Medical Sciences (2014-40) and the Postdoctoral Applied Research Project of Qingdao (2015136).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra25870g

‡ These authors have the equivalent contribution to this work.

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