Polypyrrole confined in dendrimer-like silica nanoparticles for combined photothermal and chemotherapy of cancer

Abstract

Multifunctional drug delivery systems that combine photothermal therapy and chemotherapy have become a potential approach for cancer treatment. Herein, polypyrrole (PPy), a near-infrared (NIR) light-absorbing polymer, is employed to be confined in dendrimer-like silica nanoparticles (DSNs). Then the obtained PPy@DSNs–NH₂ is further modified with biocompatible polyethylene glycol (PEG) through the reaction of –COOH and –NH₂ groups to improve its biocompatibility and stability in physiologic conditions. The as-prepared PPy@DSNs–PEG nanocomposite has been demonstrated to possess high photothermal conversion efficiency and high drug loading capacity as well as low cytotoxicity. The anticancer drug doxorubicin hydrochloride (DOX) loaded PPy@DSNs–PEG (DOX/PPy@DSNs–PEG) shows pH-responsive and heat-sensitive drug release properties. Furthermore, the in vitro cytotoxicity results display that DOX/PPy@DSNs–PEG under NIR irradiation shows the highest death rate on U251 and U87 MG cells, owing to PPy@DSNs–PEG mediated photothermal ablation and DOX-triggered cytotoxicity. Thus, the combined therapy of DOX/PPy@DSNs–PEG reveals enhanced therapeutic efficacy compared with single chemotherapy or photothermal therapy. These results indicate that the prepared multifunctional drug delivery system will be a potential candidate for biomedical applications.

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1. Introduction

Cancer is still a major public health problem in the world due to its high death rate.¹ Over the past decade, a single therapy of anticancer drugs has been frequently selected for cancer treatment. Due to the non-specificity of traditional anticancer drugs, collateral damage and adverse side effects to normal tissue cells during the chemotherapeutic process are almost inevitable. To address this formidable problem, controlled drug delivery systems (CDDS), such as hydrogels,² exosomes,³ polymeric microspheres⁴ and various inorganic nanoparticles-based nanomaterials^5,6 have been developed to achieve enhanced therapeutic efficacy and decreased side effects. Therefore, designing multifunctional CDDS that are responsive to external physical stimuli, has also attracted extensive interest in the area of nanomedicine.^7–9

In recent years, photothermal ablation (PTA) therapy, which can effectively kill cancer cells at a specific region by converting light energy into heat, has received significant interest as a potentially effective treatment on tumor necrosis.^10,11 Compared with traditional treatment, such as surgery and chemotherapy, PTA therapy is non-invasive and targeted, which make it an excellent alternative for cancer therapy. Currently, a large variety of nanomaterials have been widely explored as photothermal agents based on their high near-infrared (NIR) photothermal effect, including noble metal nanomaterials (e.g., gold nanorods), semiconductor nanomaterials (e.g., CuS nanoparticles), carbon-based nanomaterials (e.g., carbon nanotubes) and organic nanomaterials (e.g., polypyrrole nanoparticles).^10,12–14 Moreover, combined therapy, such as composed of chemotherapy and photothermal therapy, has recently emerged as a practical and efficient therapeutic strategy for cancer treatment due to its outstanding synergistic therapeutic efficacy.^8,15 In such system, photothermal therapy will not only be able to kill the cancer cells by photothermal agent-mediated hyperthermia, but also can increase the sensitivity of chemotherapy and synergistically enhance the therapeutic effects.¹⁶ Hence, it is highly desired that a drug delivery system with excellent biocompatibility possesses both the photothermal and chemotherapeutic functions.

Polypyrrole (PPy), because of its high biocompatibility and good electroconductive properties, has been widely investigated as a promising biomaterial for tissue engineering, such as nerve regeneration and bone repair.^17–19 Furthermore, with strong NIR absorbance, PPy-based materials have also been developed by several groups as a photothermal agent, showing excellent cancer ablation effect in vitro and in vivo.^10,20,21 In order to further improve the therapeutic effect, anticancer drugs are thus loaded into PPy-based nanocomposites to achieve chemo-photothermal synergistic therapy. For example, the anticancer drug doxorubicin (DOX) was loaded into the PPy shell of Fe₃O₄@PPy core–shell nanoparticles, showing an outstanding in vitro and in vivo synergistic antitumor effect.²² In a previous report, PPy was successfully confined in ordered mesoporous silica SBA-15 channels by an in situ polymerization technique for electrorheological investigation.²³ Therefore, with the good photothermal effect of PPy, it is encouraging to develop PPy-incorporated silica nanoparticles as a potential photothermal agent for cancer treatment.

In this work, PPy incorporated dendrimer-like silica nanoparticles are fabricated by in situ polymerization in the channels of silica nanoparticles and then further modified with polyethylene glycol (PEG) to improve the biocompatibility and stability in physiological conditions. Moreover, the DOX molecules can be loaded into the remaining room within mesopore channels of obtained PPy@DSNs–PEG nanocomposite. The resultant nanoparticles were characterized by various techniques. Also, the photothermal performance and in vitro drug release were investigated. Most importantly, we further evaluate the synergistic therapeutic efficacy of DOX/PPy@DSNs–PEG by in vitro MTT assays and confocal microscope observation. Our results suggest that the prepared DOX/PPy@DSNs–PEG may be a promising multifunctional drug delivery system for applications in biomedicine.

2. Experimental section

2.1. Materials

Tetraethyl orthosilicate (TEOS), 3-aminopropyl-triethoxysilane (APTES), cetyltrimethylammonium bromide (CTAB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), FeCl₃·6H₂O and 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazolium bromide (MTT) were purchased from Sigma Aldrich. Pyrrole, aqueous ammonia (NH₄OH, 25–28%), hydrochloric acid (HCl, 36–38%) and ethyl ether were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). PEG monomethyl ether with one end of carboxyl group (PEG–COOH, M_w = 2000) was from Shanghai Yanyi Biotechnology Corporation (Shanghai, China). Doxorubicin hydrochloride (DOX) was purchased from Beijing Huafeng United Technology Co., Ltd (Beijing, China). All the chemicals were analytical grade and used as received without further purification.

2.2. Synthesis of dendrimer-like silica nanoparticles

DSNs–NH₂ was prepared by one-pot sol–gel synthesis procedure based on previous report.²⁴ Briefly, 0.5 g of CTAB was dissolved in an emulsion system composed of 70 mL of H₂O, 0.8 mL of aqueous ammonia, 15 mL of ethyl ether and 5 mL of ethanol. After the mixture was vigorously stirred for 0.5 h at 15 °C, a mixture of 2.5 mL of TEOS and 0.1 mL of APTES was quickly added into the above mixture. The resulting mixture was kept to stir for 4 h. Then, 1 mL of HCl was added to stop the base-catalyzed reaction. The product was collected by centrifugation at 4000 rpm for 10 min, and then washed with ethanol and water for three times. The as-prepared product was purified by gradient centrifugation. First, the resulting product was redispersed into ethanol and treated by ultrasonic dispersion for 30 min. After that, different centrifugation rates (1500, 2000, 2500, 3000 and 3500 rpm) were performed. Finally, the above suspension containing nanoparticles was centrifuged at 4000 rpm for 10 min to obtain the purified nanoparticles. Subsequently, the CTAB template was removed by extraction in acidic ethanol (15 mL of HCl in 120 mL of ethanol) via stirring at 70 °C for 24 h. The surfactant-free DSNs–NH₂ were collected by centrifugation and washed with water for three times. DSNs–NH₂ were kept in water or dried under vacuum for analysis.

2.3. Synthesis of polypyrrole confined DSNs–NH₂ nanocomposite

PPy was confined in DSNs–NH₂ nanoparticles according to the literature.^11,23 25 mg of dried DSNs–NH₂ nanoparticles was firstly dispersed in 692 μL (1384 or 4152 μL) of pyrrole monomer. Then, the mixture was dried under vacuum at room temperature for 24 h to allow pyrrole driven into the pores. The pyrrole-containing DSNs–NH₂ was immersed in 5 mL of FeCl₃·6H₂O (3.73 g) aqueous solution and stirred at room temperature for another 24 h. The resulting PPy@DSNs–NH₂ were collected by centrifugation and washed several times with water, and then kept in water or DMSO for further use, or dried under vacuum for analysis. In addition, pure PPy was also prepared under the same conditions.

2.4. Synthesis of PEGylated PPy@DSNs–NH₂ nanocomposite

In brief, 5 mg of PEG–COOH was first activated with 23.96 mg of EDC and 27.14 mg of NHS in 10 mL of DMSO at room temperature for 3 h. Then 50 mg of PPy@DSNs–NH₂ dispersed in 20 mL of DMSO was added into above solution under vigorous magnetic stirring at room temperature for 48 h. Finally, the products were collected by centrifugation and washed several times with water.

2.5. Characterization

To observe the morphology and structure of nanoparticles, field emission scanning electron microscope (FESEM) was performed on a Hitachi S-4800 (Hitachi, Japan) at 10 kV and transmission electron microscope (TEM) was performed on a JEM 2100F (JEOL, Japan) at 200 kV. Fourier transform infrared (FTIR) spectra were recorded on a Nexus 670 (Thermo Nicolet, USA) spectrometer. Thermogravimetric analysis (TGA) was carried out using a TG 209 F1 (NETZSCH Instruments Co., Ltd., Germany) thermogravimetric analyzer. The particle size distributions of nanoparticles were determined by dynamic light scattering (DLS) using a BI-200SM multi-angle dynamic/static laser scattering instrument (Brookhaven, USA). The zeta potential measurements were performed on a Zetasizer Nano ZS instrument (Malvern, UK). The surface analysis was performed by nitrogen (N₂) adsorption–desorption measurements with a Micromeritics Tristar 3000 system. The specific surface area and the pore size distribution were calculated by the Barrett–Emmett–Teller (BET) and Barrett–Joyner–Halanda (BJH) methods, respectively. UV-visible absorption spectra were measured with a Lambda 35 UV-visible spectrophotometer (PerkinElmer, USA) at room temperature under ambient conditions.

2.6. Photothermal performance

To measure the photothermal performance of as-synthesized PPy@DSNs–PEG, 0.4 mL of the sample solution was added into the quartz cuvette and irradiated with an 808 nm laser (Xi'an Tours Radium Hirsh Laser Technology Co., Ltd. China) at a power density of 1 W cm⁻². PPy@DSNs–PEG nanoparticles with different concentrations (62.5, 125, 250, 500 and 1000 μg mL⁻¹) were exposed to the laser irradiation for 10 min, and the temperature of the solution was recorded by a thermometer every 20 s. Water was used as a control. To determine the impact of NIR power density, the PPy@DSNs–PEG solution with the concentration of 250 μg mL⁻¹ was irradiated under different power densities (1, 1.5 and 2 W cm⁻²). To detect the thermal stability of PPy@DSNs–PEG, the samples were cyclically irradiated.

2.7. DOX loading and in vitro release

For DOX loading, 10 mg of PPy@DSNs–PEG was mixed with 4 mL of 0.5 mg mL⁻¹ DOX solution in PBS (pH 7.4). After stirred for 24 h under dark condition, the DOX loaded PPy@DSNs–PEG (DOX/PPy@DSNs–PEG) was collected by centrifugation and washing several times with PBS. To evaluate the DOX loading capacity, the adsorption measurements of the original solution and the supernatant were recorded using a UV-visible spectrophotometer at the wavelength of 480 nm. The DOX loading efficiency (LE) and entrapment efficiency (EE) were all calculated.

For the in vitro DOX release experiment, 0.4 mL portions of DOX/PPy@DSNs–PEG solution (5 mg mL⁻¹) in PBS at different pHs (5.0 and 7.4) were loaded into dialysis tubes (molecular weight cut-off: 7000 Da), and then placed in tubes containing 2 mL of corresponding buffer. The tubes were shaken at 100 rpm and 37 °C. At determined time points, aliquots of each 0.5 mL dialysis solution were taken out for analysis, and the same volume of fresh corresponding buffer was added back. For investigating the photothermal-triggered drug release form DOX/PPy@DSNs–PEG, each DOX/PPy@DSNs–PEG solution was irradiated with an 808 nm NIR laser (1 W cm⁻²) for 10 min at determined time points. Aliquots of each 0.4 mL dialysis solution were taken out before and after NIR stimulation and equivalent volume of fresh buffer solution were added back. The amount of released DOX was determined using a UV-visible spectrophotometer at the wavelength of 480 nm.

2.8. Cell culture

The glioma cell lines U251 and U87 MG were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DMEM medium supplemented with 10% FBS, 1% penicillin and 1% streptomycin stock solution at 37 °C in a 5% CO₂ atmosphere. The culture medium was changed every two days, and cells were passaged by trypsinization before confluence.

2.9. Cell viability assay

The cell viability was measured by the standard MTT assay. First, U251 and U87 MG cells were cultured in 96-well plates with 100 μL of culture medium at a density of 1 × 10⁴ cells per well and cultured in 5% CO₂ at 37 °C for 24 h. After removing the original culture medium, the cells was exposed to fresh medium (blank control) and PPy@DSNs–PEG dispersed in the fresh culture medium at different concentrations (7.8, 15.6, 31.3, 62.5, 125, 250 and 500 μg mL⁻¹) for 24 h. Then the medium was replaced with 90 μL fresh culture medium, and followed by the addition of 10 μL MTT solution. After incubation for 4 h, the mixture was removed and 200 μL DMSO was added to each well to dissolve the insoluble formazan crystals. 100 μL solutions were taken out for test. The OD value at 492 nm was measured using a microplate reader (MK3, Thermo, USA). The cytotoxicity was expressed as the percentage of the cell viability as compared with the blank control, and the mean value was calculated from four parallel wells.

2.10. Hemolysis assay

For the hemolysis determination, the collected human red blood cells (RBCs) were diluted with sterile PBS solution up to a concentration of 10% (v/v). Then 300 μL of diluted RBCs was incubated with 1200 μL of PPy@DSNs–PEG solution with different concentrations (15.6, 31.3, 62.5, 125, 250 and 500 μg mL⁻¹). Ultrapure water and PBS were served as positive and negative controls, respectively. The mixture were gently shaken up and then kept at 37 °C for 3 h. Afterward, the samples were centrifuged for 10 min at 3000 rpm and the absorbance values of supernatant were measured using a UV-visible spectrophotometer at 541 nm. The hemolysis percentages of PPy@DSNs–PEG sample were calculated by the following formula:

Hemolysis (%) = [(OD_sample − OD_{negative control})/(OD_{positive control} − OD_{negative control})] × 100% (1)

2.11. In vitro photothermal ablation and DOX delivery

To study the photothermal ablation of PPy@DSNs–PEG, U251 cells were firstly seeded in a 48-well plate at a density of 2 × 10⁴ cells per well for 24 h prior treatment. After that, the cells were incubated with 125 μg mL⁻¹ of PPy@DSNs–PEG solution. After incubation for 2 h, the cells were irradiated with an 808 nm laser at a power density of 1 W cm⁻² for 10 min. The cells were then washed with PBS and stained with 0.4% trypan blue solution for 5 min. Finally, the cells were washed with PBS and the images of the stained cells were taken immediately using a light microscope (Olympus IX71).

For drug delivery observation, U251 cells were seeded in a 48-well plate at a density of 2 × 10⁴ cells per well for 24 h to allow the cells attachment. Then the medium was removed and the cells were incubated with DOX/PPy@DSNs–PEG at DOX concentration of 2 μg mL⁻¹ (the relative concentration of PPy@DSNs–PEG was 12.44 μg mL⁻¹) for specific time points (2, 4, 6 and 12 h). Finally, the cells were fixed with 4% paraformaldehyde and observed by using a fluorescence microscope (Olympus IX71). The DOX fluorescence signal was imaged at λ_ex = 480–490 nm, λ_em ≥ 515 nm with red filter.

2.12. Chemo-photothermal synergistic effect on cancer cells

For the chemo-photothermal therapy assay, U251 and U87 MG cells were firstly cultured in 96-well microplates with 100 μL of culture medium at a density of 1 × 10⁴ cells per well and cultured in 5% CO₂ at 37 °C for 24 h. PPy@DSNs–PEG and DOX/PPy@DSNs–PEG with various PPy@DSNs–PEG concentrations (15.6, 31.3, 62.5, 125 and 250 μg mL⁻¹) were added separately into wells and cultured for 2 h. After that, the cells of photothermal groups were irradiated with an 808 nm laser at a power density of 1 W cm⁻² for 10 min. The cells exposed to fresh medium without irradiation were used as blank control. The cells incubated with DOX/PPy@DSNs–PEG without irradiation were also performed. The culture medium was then replaced with fresh medium and cultured for 24 h. Subsequently, the MTT assay was carried out to determine the cell viability relative to the blank control.

2.13. Confocal fluorescence imaging analysis

To study the chemo-photothermal effect on cell cytoskeleton, U251 cells were seeded into 20 mm glass bottom culture dishes at the density of 1 × 10⁵ cells and incubated for 24 h. The cells were washed with PBS, followed by cultured with PPy@DSNs–PEG and DOX/PPy@DSNs–PEG at the PPy@DSNs–PEG concentration of 125 μg mL⁻¹. After incubation for 2 h, corresponding culture dishes were irradiated with an 808 nm NIR laser at a power density of 1 W cm⁻² for 10 min. The untreated cells and the cells treated with PPy@DSNs–PEG without irradiation were used as control. The cells were then incubated again at 37 °C for 12 h. After this treatment, the cells were rinsed with PBS and fixed with 4% paraformaldehyde for 20 min. The fixed cells were washed with PBS and then permeabilized with 0.1% Triton X-100 in PBS for 5 min, followed by blocking with 1% BSA in PBS for 30 min. After washing with PBS, F-actin of the cells was stained with Alexa Fluor 488 conjugated phalloidin for 30 min and then the nuclei were stained with DAPI for 5 min. Finally, all samples were washed several times with PBS and observed by confocal lasers scanning microscope (CLSM, Carl Zeiss LSM 700).

2.14. Statistical analysis

All values were presented as mean ± standard deviation. Statistical analysis was carried out by the one-way analysis of variance (one-way ANOVA) and Scheffe's post hoc test. The criteria for statistical significance were *P < 0.05 and **P < 0.01.

3. Results and discussion

3.1. Characterization of synthesized nanoparticles

In this study, DSNs–NH₂ was first fabricated using TEOS as silane precursor, APTES as co-condensation silane, NH₃·H₂O as basic catalyst, CTAB as mesopore template and emulsion stabilizer, ethyl ether as oil phase, and ethanol as co-solvent by a one-pot sol–gel synthesis procedure.²⁵ PPy chains were then incorporated in the channels of DSNs–NH₂ by an in situ chemical oxidative polymerization to obtain the PPy@DSNs–NH₂ nanoparticles, as shown in Fig. 1. The PEGylation of PPy@DSNs–NH₂ (PPy@DSNs–PEG) was achieved through the reaction of –COOH and –NH₂ group by using cross-linking reagents. Finally, the remaining room within the channels of PPy@DSNs–PEG was used to load the anti-cancer drugs.

Fig. 1 Schematic illustration of the preparation of DOX/PPy@DSNs–PEG for combined chemo-photothermal therapy.

The SEM and TEM images of as-synthesized nanoparticles are shown in Fig. 2. From the SEM images, it can be seen that the DSNs–NH₂ nanoparticles are spherical with an average diameter around 233 nm, and the wrinkled sheets on nanoparticles surface are arranged in three dimensions to form the large pores (Fig. 2A). The TEM images show that the pore structure is radially oriented, and pore size gradually increase from the center to the outer surface (Fig. 2D). When PPy is formed within the channels of DSNs–NH₂, the size of large pores decreased (Fig. 2B) and the dendrimer-like skeleton of DSNs–NH₂ turned to thicker (Fig. 2E). After PEGylation, there were no difference in morphology and structure between PPy@DSNs–NH₂ and PPy@DSNs–PEG (Fig. 2C and F). When two-fold of pyrrole monomer was added during the synthesis process, more PPy was formed within the channels of DSNs–NH₂ (Fig. S1A and B†). Furthermore, when the volume of added pyrrole monomer reached to six-fold, the channels of DSNs–NH₂ were filled with PPy and the outer surface was also coated with PPy (Fig. S1C†). These images indicate that PPy can be readily incorporated into the channels of DSNs–NH₂, and DSNs–NH₂ still has the remaining room to load other guests when prepared with the appropriate amount of pyrrole monomer. Thereby, DSNs–NH₂ prepared with the lowest amount of pyrrole monomer was used in the following experiments.

Fig. 2 SEM (A–C) and TEM (D–F) images of DSNs–NH₂ (A and D), PPy@DSNs–NH₂ (B and E) and PPy@DSNs–PEG (C and F).

The FTIR spectra of the samples before and after incorporation of PPy are displayed in Fig. 3A. For DSNs–NH₂, the bands at 1086, 798 and 953 cm⁻¹ are assigned to Si–O–Si asymmetric stretching vibration and symmetric stretching vibration and Si–OH vibration,²⁶ and the bands at 1491 and 1454 cm⁻¹ are attributed to N–H bending vibration,²⁴ respectively. After incorporation of PPy, the characteristic absorption peaks of DSNs–NH₂ are not disappeared. Moreover, the bands at 1554, 1456 and 1303 cm⁻¹ respectively assigned to pyrrole ring stretching, C–N stretching and =C–H in plane vibrations of pure PPy also appear in the PPy@DSNs–NH₂ (at 1560, 1465 and 1313 cm⁻¹).²⁷ However, the corresponding peaks in PPy@DSNs–NH₂ shift to higher wavenumber compared to pure PPy, suggesting the PPy chain formed within PPy@DSNs–NH₂ is shorter than that of pure PPy.²³ After PEGylation, slightly enhanced bands at 2924 and 1456 cm⁻¹ are appeared in PPy@DSNs–PEG sample, which can be respectively attributed to the stretching vibration and bending vibration of C–H bonds in PEG.²⁸ The data of FTIR spectra confirm that PPy was confined in the channels of DSNs–NH₂. Next, TGA measurement was used to estimate the amount of PPy in the channels. As shown in Fig. 3B, after heating to 900 °C, DSNs–NH₂, PPy@DSNs–NH₂ and PPy@DSNs–PEG display a weight loss of 17.2 wt%, 59.3 wt% and 62.9 wt%, respectively. Thus, the amount of PPy loaded in the channels of PPy@DSNs–PEG sample can be calculated to be about 42.1 wt%. This high PPy loading of PPy@DSNs–PEG brings the feasibility to serve as photothermal material.

Fig. 3 Characterization of prepared nanoparticles. (A) FTIR spectra of pure PPy, DSNs–NH₂, PPy@DSNs–NH₂ and PPy@DSNs–PEG. (B) TGA curves of DSNs–NH₂, PPy@DSNs–NH₂ and PPy@DSNs–PEG. (C) Zeta potential change of DSNs–NH₂, PPy@DSNs–NH₂ and PPy@DSNs–PEG. (D) Size distribution curves of DSNs–NH₂ and PPy@DSNs–PEG aqueous dispersions. (E) Photos of PPy@DSNs–NH₂ and PPy@DSNs–PEG in different solutions after 4 h.

The zeta potential measurements were conducted to further verify the conjugation of PEG on the PPy@DSNs–NH₂ (Fig. 3C). We observe that the zeta potential of DSNs–NH₂ (+27.3 mV) is positive charged due to the presence of amino group, and then increase to +32.0 mV after incorporation of PPy. After PEGylation, the zeta potential decrease to +24.5 mV, due to the reaction between carboxyl groups of the PEG–COOH and amino groups of DSNs–NH₂. This change of zeta potential demonstrates the successful conjugation of PEG on DSNs–NH₂ nanoparticles. In addition, the hydrodynamic diameter and size distribution of DSNs–NH₂ and PPy@DSNs–PEG were measured by DLS. As shown in Fig. 3D, the diameter of DSNs–NH₂ and PPy@DSNs–PEG are 335 and 386 nm, respectively, larger than that observed from TEM images because of the hydrate layer on nanoparticles in aqueous solution.⁷ However, the DSNs–NH₂ has a significant increase of hydrodynamic diameter when dispersed in cell medium, while the hydrodynamic diameter of PPy@DSNs–PEG almost remains constant (Fig. S2†). Fig. 3E shows PPy@DSNs–NH₂ and PPy@DSNs–PEG samples dispersed in different solutions, including water, PBS solution and cell medium. It can be seen that the PEGylated PPy@DSNs–NH₂ exhibits better stability in these solutions, allowing it to be further investigated for biological applications.

3.2. Photothermal performance of PPy@DSNs–PEG

The optical property of the PPy@DSNs–NH₂ and PPy@DSNs–PEG aqueous dispersion was examined by UV-visible spectroscopy. The UV-visible–NIR absorbance spectrum show that PPy@DSNs–PEG exhibit a high NIR absorption from 700 to 1100 nm (Fig. 4A), making it a potential photothermal agent. In order to further explore the potential use of PPy@DSNs–PEG as a photothermal agent, the PPy@DSNs–PEG aqueous solution with different concentrations were exposed using an 808 nm NIR laser at a power density of 1.0 W cm⁻² for 10 min. As seen in Fig. 4B and C, the photothermal heating effects of PPy@DSNs–PEG are concentration-dependent and laser power density-dependent. When the concentrations PPy@DSNs–PEG are 125 and 250 μg mL⁻¹, the temperature can reach to 44.5 and 50.9 °C with the temperature elevations of 25.6 and 31.3 °C (Fig. S3A†), respectively. As a control, the temperature of pure water has no obvious change. With the increase of measured power density (1–2 W cm⁻²), the temperature of sample solutions rapidly increase and can increase by 31.3, 43.6 and 54.6 °C (Fig. S3B†), respectively. To investigate the photostability of PPy@DSNs–PEG, the temperature was recorded by three repeated cycles of laser irradiation (Laser on/off). We do not observe any negative effect on the photothermal conversion efficiency (Fig. 4D). These results indicate the prepared PPy@DSNs–PEG has potential for thermal ablation in cancer treatment.

Fig. 4 (A) UV-visible absorption spectra of PPy@DSNs–NH₂ and PPy@DSNs–PEG. (B) Photothermal heating curves of pure water and PPy@DSNs–PEG aqueous dispersions at different concentrations under an 808 nm laser irradiation at the power density of 1 W cm⁻². (C) Photothermal heating curves of PPy@DSNs–PEG aqueous dispersions (250 μg mL⁻¹) at various power densities. (D) Temperature elevation of PPy@DSNs–PEG aqueous dispersions (250 μg mL⁻¹) over three lasers on/off cycles under an 808 nm laser irradiation at the power density of 1 W cm⁻².

3.3. In vitro DOX loading and release

Before drug loading, the surface analysis of PPy@DSNs–PEG was firstly investigated by N₂ adsorption–desorption measurement (Fig. 5A). The sample exhibits a type IV isotherm according to the IUPAC classification, which indicates the mesoporous structure of PPy@DSNs–PEG. The BET surface area, average pore size and pore volume were calculated to be 165.9 m² g⁻¹, 3.8 nm and 0.34 cm³ g⁻¹, respectively. Thus, the remaining mesopores of PPy@DSNs–PEG can be employed to load the drug molecules. To evaluate the drug loading capacity, the chemotherapeutic drug DOX was chosen to load into the channels of PPy@DSNs–PEG. It was measured that the entrapment efficiency (EE) and loading efficiency (LE) are 86.92% and 16.08% (Fig. 5B), respectively. The result reveals that PPy@DSNs–PEG still possesses high drug loading capacity, owing to that PPy can effectively bind DOX via π–π stacking and the pore adsorption.²²

Fig. 5 (A) N₂ adsorption–desorption isotherms and the inset pore size distribution curves of PPy@DSNs–PEG. (B) The DOX loading efficiency (LE) and entrapment efficiency (EE) of PPy@DSNs–PEG. The inset photo is the DOX solutions before and after loading. Cumulative release profiles of DOX from DOX/PPy@DSNs–PEG at different pHs (C) without NIR irradiation and (D) with 1 W cm⁻² NIR irradiation.

Next, the DOX release behaviors of DOX/PPy@DSNs–PEG under different pH values were investigated. As clearly shown in Fig. 5C, the DOX release is rapid within the first 6 h at pH 5.0, with the total amount of about 41.75 ± 1.21% at 48 h. But at pH 7.4, only 5.63 ± 0.44% was released within the 48 h. This result is mainly due to the protonation of the amino group in the DOX molecule under acidic pH which make DOX a positive charge, and thus facilitating the drug release.²² In addition, the photothermal effect of PPy@DSNs–PEG on the drug release behavior was also studied. The NIR irradiation can enhance the cumulative release of DOX from DOX/PPy@DSNs–PEG at different time points and pH values, as shown in Fig. 5D. The total amount of released DOX is improved to 34.98 ± 0.93% after 8 h at pH 5.0 under the irradiation, with 9.60% increase compared to that without NIR irradiation (25.38 ± 1.32%), whereas rather limited DOX release was enhanced at pH 7.4. The data show that DOX release from the DOX/PPy@DSNs–PEG can be triggered by an external NIR laser and the release rate was significantly increased under the acidic pH. This phenomenon can be attributed to the weakening binding between DOX and PPy and also the reduction of electrostatic interaction between DOX and silica by local temperature increase.^22,29 On the other hand, the release of DOX from DOX/PPy@DSNs–PEG exhibits a pH-responsive pattern (Fig. 5C and D). It has been reported that the tumor microenvironment can be stabilized at a mildly acidic with a pH range of 5.8–7.1 and the intracellular environment is more acidic with a pH of 5.0.^30,31 Thus, the undesirable leakage of DOX can be minimized when the DOX/PPy@DSNs–PEG nanoparticles are outside the tumor sites, while the DOX release can be efficient after entering cell endosomes and lysosomes, where the pH value is 5–6. As a result, we deduce that the pH-sensitive and NIR-triggered release of DOX can significantly improve the therapeutic effect.

3.4. In vitro cell viability and hemolysis assays of PPy@DSNs–PEG

Before confirming the therapeutic efficacy of DOX/PPy@DSNs–PEG, we first evaluate the in vitro cytotoxicity of PPy@DSNs–PEG via the standard cell viability MTT assay on U251 cells and U87 MG cells. As expected, we find that the PPy@DSNs–PEG exhibit negligible toxicity to both types of cells at the determined concentrations (Fig. 6A and B). On the other hand, we further investigate the impacts of PPy@DSNs–PEG on the hemolysis behavior of RBCs as blood compatibility is a critical concern to the application of nanoplatform. The hemolytic phenomena of PPy@DSNs–PEG are almost invisible by the direct observation from inset photographic image of Fig. 6C. It is also found that the hemolysis percentages of PPy@DSNs–PEG are negligible even at a high concentration of 500 μg mL⁻¹. Therefore, the as-prepared PPy@DSNs–PEG has low cytotoxicity and good blood compatibility, which is greatly favorable for the potential application as a photothermal agent.

Fig. 6 The in vitro cell viability of (A) U251 cells and (B) U87 MG cells incubated with PPy@DSNs–PEG at different concentrations. (C) Hemolysis percentage of RBCs incubated with PPy@DSNs–PEG at different concentrations for 3 h, using PBS as a negative control and pure water as a positive control. The inset photographic image is taken after centrifugation.

3.5. Photothermal ablation and drug delivery

In order to demonstrate the photothermal effect of PPy@DSNs–PEG at cellular level, the trypan blue staining experiments of U251 cells were conducted (Fig. 7A). The dead cells different from live ones can be stained with blue color by using trypan blue. When exposed to the laser at 1 W cm⁻², almost all the cells were killed. In contrast, the cells keep alive when they were treated with the PPy@DSNs–PEG nanoparticles without NIR irradiation or laser irradiation alone compared with the control. That means the photothermal ablation of PPy@DSNs–PEG on cancer cells is efficient.

Fig. 7 (A) Optical microscopy images of trypan blue staining of U251 cells incubated with cell medium (control), NIR irradiation alone, PPy@DSNs–PEG without NIR irradiation and PPy@DSNs–PEG with NIR irradiation. (B) Fluorescence microscopy images of U251 cells incubated with DOX/PPy@DSNs–PEG at DOX concentration of 2 μg mL⁻¹ for 2, 4, 6 and 12 h; the blue signal and red signal indicate the cell nuclei and DOX, respectively. All images, bar = 200 μm.

For the evaluation of drug delivery by PPy@DSNs–PEG, the U251 cells were incubated with DOX/PPy@DSNs–PEG and then observed via fluorescence microscope. As shown in Fig. 7B, the red fluorescence of DOX becomes more intense with increasing incubation time. This result reveal that the DOX/PPy@DSNs–PEG nanoparticles can be easily internalized by the cells, and more and more nanoparticles were endocytosed into the cells as the incubation time extended. We also observe that the uptake of DOX/PPy@DSNs–PEG nanoparticles was mainly distributed in the cytoplasm in the initial time. After 6 h incubation, the slight red signal was overlapped with blue signal in the nuclei, which means the little amount of released DOX from carrier can rapidly accumulate in the cell nuclei through diffusion. The result indicates that the DOX was delivered into the cells by the endocytosis of DOX/PPy@DSNs–PEG nanoparticles, followed by releasing from the carrier within the cytoplasm and then eventually diffusing into the nuclei. As a result, the constructed drug delivery system can be effectively taken up by cancer cells and release the loaded drug within the cells.

3.6. In vitro chemo-photothermal synergistic effect

To evaluate the therapeutic efficacy of DOX/PPy@DSNs–PEG in vitro, cell viability of U251 and U87 MG cells treated with different treatments was measured by the MTT assay (Fig. 8). As demonstrated in Fig. 8A and B, the quantitative results on U251 and U87 MG cells show a similar dose-dependent manner. After irradiation, the viability of U251 cells incubated with PPy@DSNs–PEG decreased from 72.7% to 24.6% with the particle concentration increasing from 62.5 to 250 μg mL⁻¹, while that of U87 MG cells decreased from 57.4% to 23.6%. When the cells incubated with DOX/PPy@DSNs–PEG without the laser exposure, it shows the cell viability less than 50% for U251 and U87 MG cells at the concentration of 250 μg mL⁻¹. Furthermore, we observe that the cytotoxicity of DOX/PPy@DSNs–PEG with irradiation dramatically increase, with the viability decreasing from 43.8% to 12.0% for U251 cells and 36.8% to 9.8% for U87 MG cells when the particle concentration from 62.5 to 250 μg mL⁻¹. Obviously, the DOX/PPy@DSNs–PEG nanoparticles with the laser irradiation exhibits the highest cytotoxicity compared with other groups (P < 0.01), which can be attributed to the photothermal effect and heating-triggered drug release.

Fig. 8 The in vitro cytotoxicity of (A) U251 and (B) U87 MG cells incubated with PPy@DSNs–PEG under NIR irradiation at 1 W cm⁻², and DOX/PPy@DSNs–PEG with and without NIR irradiation. **P < 0.01 vs. other groups.

Afterwards, we observe the photothermal heating effect on cytoskeleton and nucleus via confocal images (Fig. 9). Compared with the untreated cells, the cytoskeleton of the U251 cells treated with PPy@DSNs–PEG still shows homogenous and well-spread morphology, and the nuclei also keep normal morphology, suggesting the good compatibility of PPy@DSNs–PEG. However, when the cells treated with PPy@DSNs–PEG plus the NIR irradiation, the morphology of the cytoskeleton is atrophic and fragmented, and the nuclei turn into dense nuclear parts, indicating that the prepared PPy@DSNs–PEG with NIR irradiation can efficiently kill cancer cells. As expected, the DOX/PPy@DSNs–PEG with the laser irradiation group causes more obvious cell damage. It is clearly seen that the cell number is less, and the cell nuclei turn to be denser and some of them become severely fragmented, which demonstrate the synergistic effect of photothermal therapy and chemotherapy by DOX/PPy@DSNs–PEG. In addition to the photothermal ablation of PPy@DSNs–PEG, DOX also induce the irreversible damage to cells by DNA damage and topoisomerase II inhibition.³² These results collectively suggest an effective chemo-photothermal synergistic therapy based on DOX/PPy@DSNs–PEG nanocomposite.

Fig. 9 Confocal images of U251 cells under different treatments, including PPy@DSNs–PEG with and without NIR irradiation, and DOX/PPy@DSNs–PEG with NIR irradiation. The blue signal and red signal indicate the cell nuclei and actin, respectively. All images, bar = 20 μm.

PPy has been demonstrated to possess strong NIR absorbance and thus developed as a photothermal agent. Therefore, PPy-based materials were explored by several groups for photothermal treatment of cancer, including pure PPy nanoparticles and PPy-based nanocomposites.^33–35 Specially, the PPy-based nanocomposites endow themselves with multifunctional properties. However, they were always prepared by coating the PPy onto the matrix,^36–38 which may shield the functionality of the matrix and make the nanocomposites not easy for the further modification. In our design, PPy was formed within the channels of dendrimer-like silica nanoparticles by in situ polymerization. The surface amino groups will not be covered when PPy@DSNs–NH₂ prepared with the appropriate amounts of pyrrole monomer (Fig. 2 and S1†). In this case, the presence of amino groups on the nanocomposites ensures them easily to be modified. Although the PPy was hydrophilic,³⁹ the PPy incorporated nanocomposites were inevitably aggregated under the condition of PBS solution and cell medium, while the PEG-modified nanocomposites exhibit better stability in those condition (Fig. 3E), which demonstrated that the PEG modification played a significant role in improving the stability. In addition, the as-prepared PPy@DSNs–PEG still has mesopores after PPy incorporation (Fig. 5A), providing the room to load the DOX. The release of DOX from the carrier shows pH-responsive and heat-sensitive release profiles (Fig. 5C and D). This drug release property was beneficial to improve the therapeutic efficiency because of the acidic intracellular environment in cancer cells.^30,31 The PPy@DSNs–PEG nanocomposites have low cytotoxicity but exhibit high photothermal conversion efficiency and efficient photothermal ablation on cancer cells (Fig. 6 and 7A), meeting the demand to be used as a therapeutic agent. Importantly, the drug delivery experiment shows that the DOX can be easily delivered into the cancer cells and the released DOX eventually enter into the nuclei via diffusion (Fig. 7B). As expected, the in vitro cytotoxicity assays clearly reveal that the DOX/PPy@DSNs–PEG with the combination of photothermal therapy and chemotherapy shows the highest cytotoxicity on cancer cells (Fig. 8), implying the chemo-photothermal synergistic therapy, which is attributed to the photothermal effect and released drug. This outstanding killing efficacy to cancer cells was possibly dependent on the cytoskeleton and nucleus damage which caused by the photothermal ablation and drug induction, as evidenced by the confocal observation (Fig. 9).

4. Conclusions

In summary, a multifunctional drug delivery system DOX/PPy@DSNs–PEG for synergistic chemo-photothermal therapy has been successfully constructed by a facile approach. The as-prepared PPy@DSNs–PEG shows good physiological stability and high NIR absorbance as well as efficient heat transformation. Also, PPy@DSNs–PEG exhibits a high DOX loading capacity, due to the strong π–π stacking interactions between PPy and DOX. The DOX released from DOX/PPy@DSNs–PEG can be controlled by external stimulation, including pH and NIR light. In addition, the in vitro cell viability and hemolysis experiments demonstrate that PPy@DSNs–PEG has no visible cytotoxicity against U251 and U87 MG cells at the concentration of 7.8–500 μg mL⁻¹ and negligible hemolysis activity in a broad concentration range of 15.6–500 μg mL⁻¹. Meanwhile, the cells can be killed by photothermal conversion of PPy@DSNs–PEG under the NIR irradiation. Importantly, based on the in vitro results, the DOX/PPy@DSNs–PEG nanocomposite with the NIR irradiation could show better therapeutic efficacy compared with individual photothermal therapy or chemotherapy. Our results indicate that the constructed DOX/PPy@DSNs–PEG nanocomposite as a multifunctional drug delivery system has great potential for chemo-photothermal synergistic therapy of cancer.

Acknowledgements

This work was supported financially by the National Natural Science Foundation of China (81201990).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: TEM images of PPy@DSNs–NH₂ prepared with different volume of pyrrole monomer are shown in Fig. S1. Size distribution curves of DSNs–NH₂ and PPy@DSNs–PEG in cell culture medium are shown in Fig. S2. Temperature changes (ΔT) over a period of 10 min NIR irradiation under different particle concentrations and different power densities are displayed in Fig. S3. See DOI: 10.1039/c6ra03314h

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