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Dual-triggered drug-release vehicles for synergistic cancer therapy
Colloids and Surfaces B: Biointerfaces 173 (2019) 788–797
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Dual-triggered drug-release vehicles for synergistic cancer therapy a,1
a,b,c,1
a
b,c
a
Ting-Yu Tu , Shu-Jyuan Yang , Ming-Hsien Tsai , Chung-Hao Wang , Shin-Yu Lee , ⁎ Tai-Horng Younga, Ming-Jium Shieha,d,e,
T
a
Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, 100, Taiwan Gene’e Tech Co. Ltd. 2F., No.661, Bannan Rd., Zhonghe Dist., New Taipei City 235, Taiwan Apius Bio Inc. 1F., No.92, Daxin St., Yonghe Dist., New Taipei City 234, Taiwan d Department of Oncology, National Taiwan University Hospital, #7, Chung-Shan South Road, Taipei, 100, Taiwan e Department of Biomedical Engineering, National Taiwan University Hospital, #7, Chung-Shan South Road, Taipei, 100, Taiwan b c
ARTICLE INFO
ABSTRACT
Keywords: Gold nanorod Human serum albumin Chemotherapy Photothermal therapy Controlled drug release
Cancer is a complex and tenacious disease. Drug-delivery systems in combination with multimodal therapy strategies are very promising candidates for cancer theranostic applications. In this study, a new drug-delivery vehicle that combine human serum albumin (HSA)- and poly(sodium 4-styrenesulfonate) (PSS)-coated gold nanorod nanoparticles(GNR/PSS/HSA NPs) was developed for synergistic cancer therapy. Doxorubicin (DOX) was loaded onto GNR/PSS/HSA NPs, by electrostatic and hydrophobic forces, to create multimodal DOX@GNR/ PSS/HSA NPs. DOX@GNR/PSS/HSA NPs were found to be highly biocompatible and stable in physiological solutions. Furthermore, GNR/PSS/HSA NPs with or without DOX were designed to exhibit strong absorbance in the near-infrared region and high photothermal conversion efficiency. Therefore, bimodal DOX release from DOX@GNR/PSS/HSA NPs could be triggered by an acidic pH and by near-infrared irradiation after NPs preferentially accumulated at tumor sites, leading to a significant chemotherapeutic effect. Moreover, DOX@GNR/ PSS/HSA NPs were designed to be applied during chemo- and photo-thermal combination therapy and exhibited a synergistic anticancer effect that was superior to the effect of monotherapy, from both in vitro and in vivo results. These results suggest that DOX@GNR/PSS/HSA NPs are a strong candidate for a nanoplatform for future antitumor therapeutic strategies.
1. Introduction Cancer remains one of the most devastating diseases. Chemotherapy is an integral component of most cancer treatments. It regulates the abnormal proliferation of cancer cells and blocks the supply of nutrients required for growth. Although chemotherapy is a powerful tool and can help to avoid cancer metastasis, a major issue of chemotherapy is its non-specificity. To improve the efficacy of chemotherapy, nanodrug carriers with sizes of 100–200 nm have been developed. These nanodrug carriers passively target the tumor site, through the enhanced permeability and retention (EPR) effect and result in high tumor accumulation [1,2]. In addition, various internally or externally triggered drug-release approaches, such as pH, temperature, ultrasound, magnetic field, and light, have been explored, to achieve better treatment outcomes [3–8]. Near-infrared (NIR) light with a wavelength of 650–900 nm is widely used as a trigger, because it penetrates up to 10 cm into living tissue and induces minimal damage to normal tissues
[9,10]. NIR-light resonance gold nanomaterials, such as gold nanorods (GNRs), have many advantages for biomedical applications [11–14]. The NIR-light absorption and scattering characteristics of GNRs can be manipulated, by adjusting the type or proportion of the reducing agent, the size, or the aspect ratio (length-to-diameter ratio) of the GNR. Upon exposure to proper NIR light, GNRs convert light energy to thermal energy, through the surface plasmon resonance (SPR) effect [15]. The resultant local increase of temperature induces significant thermal and mechanical stress within the system, and results in the bursting off of the carrier and the release of the payload [16,17]; furthermore, upon NIR-laser stimulation, GNRs can induce hyperthermia within the tumor tissue (known as photothermal therapy [PTT]), and the extent of hyperthermia depends on the thermal dosage and exposure time of NIRlaser stimulation [15,18]. In addition, these SPR-induced tunable absorption and scattering features allow the use of GNRs as a contrast agents for bioimaging applications [19,20].
Corresponding author. E-mail addresses: [email protected], [email protected] (M.-J. Shieh). 1 The first two authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.colsurfb.2018.10.043 Received 25 June 2018; Received in revised form 20 September 2018; Accepted 16 October 2018 Available online 19 October 2018 0927-7765/ © 2018 Published by Elsevier B.V.
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GNRs can been easily synthesized by using a cationic surfactant hexadecyltrimethylammonium bromide (CTAB)-templated growth strategy [21]. However, the toxicity of CTAB on GNRs must be reduced to allow biological applications [22]. The layer-by-layer (LbL) coating technique is used widely for a safe surface coating because of its versatility, simplicity, and applicability in aqueous conditions [23]. Other advantages of this technique are that the deposited multilayer films can be designed to have high biocompatibility and that payload drugs can be loaded into separate layers for sequential, controlled drug release [24,25]. Overall, LbL coating techique is a relatively easy method which can add active substances to GNRs and thus enhance biocompatibility. Human serum albumin (HSA) is an endogenous protein and the most abundant protein in human blood. HSA binds to and transports several metabolic compounds and therapeutic drugs in the blood circulation [26]. Moreover, HSA has high affinity for tumor tissue [27,28]; it actively accumulates at tumor sites via gp60 receptor-mediated transcytosis as a result of its high binding affinity for the specific glycoprotein, secreted protein acidic and rich in cysteine(SPARC), which is secreted by several cancer cells and is abundant in tumor interstitial tissue [29]. HSA possesses numerous and different functional groups that can be employed for chemical modification; this characteristic makes HSA an ideal carrier for cancer-drug delivery [30,31]. In this study, we developed a thermo- and pH-triggered drug-delivery vehicle, poly(sodium 4-styrenesulfonate) (PSS)/HSA-coated GNRs nanoparticles (GNR/PSS/HSA NPs), for cancer PTT and chemotherapy. GNR/PSS/HSA NPs were created from GNRs, which were synthesized by using a CTAB-templated growth strategy and then undergoing LbL coating with PSS and HSA, by electrostatic force. Consequently, the GNR/PSS/HSA NPs would exhibit good biocompatibility and not be detected by the reticuloendothelial system, leading to a longer circulation time. The GNR/PSS/HSA NPs were loaded with doxorubicin (DOX) (DOX@GNR/PSS/HSA NPs), by using electrostatic and hydrophobic forces. DOX has been widely used for cancer chemotherapy. However, unspecific accumulation of DOX brings about serious side effects, such as severe cardiac toxicity. These side effects limit the use of DOX for cancer treatment [32–34]. However, GNR/PSS/ HSA NPs can transport DOX to the tumor site passively (via the EPR effect) and actively (via gp60 receptor-mediated transcytosis). Thus, DOX leakage into the blood circulation could be reduced compared to that of conventional chemotherapy. Importantly, DOX on DOX@GNR/ PSS/HSA NPs accumulated at the tumor site can be triggered and released by the acidic pH within endosomes/lysosomes and by NIR-laser irradiation during PTT. Simultaneously, PTT can induce cancer-cell death via apoptosis or necrosis (Scheme 1). Therefore, we expect that a
combination of PTT and chemotherapy involving DOX@GNR/PSS/HSA NPs will be a promising tool in cancer therapy, to kill cancer cells and decrease the probability of tumor recurrence. 2. Materials and methods 2.1. Materials GNRs were kindly supplied by Gene’e Tech Co., Ltd. (Taiwan). PSS, HSA, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (USA). Doxorubicin, hydrochloride salt (> 99%) was purchased from LC Laboratories (USA). Calcein acetoxymethyl ester (Calcein-AM) and ethidium homodimer-1 (EthD-1) were purchased from Invitrogen (USA). Matrigel matrix was purchased from Discovery Labware, Inc (USA). 2.2. Preparation of GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs were prepared by using the LbL technique. First, 10 mg/mL of PSS stock solution was prepared by dissolving 10 mg PSS powder in 1 ml of 1 mM NaCl solution. Then, 1 mL of GNR/CTAB solution (OD = 2) was mixed gently with 1 mL of PSS stock solution for 1 h, to obtain GNR/PSS NPs. GNR/ PSS NPs were washed three times and were centrifuged (at 16,000 g for 10 min) to remove excess PSS. Then, the pH value of the GNR/PSS solution was adjusted to 4.2, and the GNR/PSS solution was mixed with 1 mL of HSA in aqueous solution (10 mg/mL, pH = 4.2) for 2 h at room temperature. GNR/PSS/HSA NPs were collected, after centrifugation at 16,000 g for 10 min to remove free HSA. DOX@GNR/PSS/HSA NPs were prepared by gently mixing the GNR/PSS solution (pH = 4.2) with 150 μL of 1 mg/mL DOX･HCl solution. After 1 h of gentle stirring, 1.15 mL of the DOX@GNR/PSS NP solution was incubated with 1 mL of HSA in aqueous solution (10 mg/mL, pH = 4.2) for 2 h at room temperature. DOX@GNR/PSS NPs were then centrifuged at 16,000 g for 10 min, to remove free DOX and free HSA. Finally, the GNR/PSS/HSA and DOX@GNR/PSS/HSA precipitates were separately re-dispersed in DI water and stored at 4 °C until use. 2.3. Characterization of GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs The chemical compositions of GNR/CTAB, GNR/PSS, and GNR/ PSS/HSA NPs were evaluated by using an ATR-FTIR spectrometer (Thermo Scientific Nicolet 6700; Waltham, MA, USA), to determine the characteristics of the LbL deposition. The morphology and the particle
Scheme 1. The EPR effect and gp60 receptor-mediated transcytosis enhance the uptake of DOX@GNR/PSS/HSA NPs into the tumor and triggering of DOX drug release. The synergistic effect of combined DOX chemotherapy and photothermal therapy that includes NIR-laser irradiation is also shown. 789
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size of GNR/CTAB, GNR/PSS, and GNR/PSS/HSA NPs were examined by transmission electron microscopy (TEM, Hitachi H-7500, Tokyo, Japan) with and without 1% phosphotungstic acid (PTA) staining. The absorption properties of GNR/CTAB, GNR/PSS, and GNR/PSS/HSA NPs were evaluated, based on the absorption spectra obtained by using an ELISA microplate reader (SpectraMax M2 Multi-Mode Microplate Readers, Molecular Devices, USA). The zeta potentials of the prepared NPs were measured by using a Zetasizer Nano-ZS90 (Malvern Instruments Ltd, UK). The fluorescence intensity of the unloaded DOX in the NPs was determined by using high-performance liquid chromatography (HPLC, Waters e2696), and the loading efficiency of DOX was calculated. The drug encapsulation efficiency (E.E.) was calculated by using the following equation: Encapsulation efficiency (%) = (Weight of the feeding DOX – Weight of the unloaded DOX)/ Weight of the feeding DOX × 100%
Au/mL) or GNR/PSS/HSA NPs (0, 3.125, 6.25, 12.5, 25, or 50 μg of Au/ mL) for 2 h. After another wash with PBS, the cells were cultured in fresh medium for another 24 h before being subjected to the MTT assay, involving the use of a scanning multiwell ELISA reader (Microplate Autoreader EL311, BioTek Instruments Inc., Winooski, VT, USA). Furthermore, the cytotoxicities of free DOX and that of DOX@GNR/PSS/ HSA NPs at DOX concentrations of 0, 0.3125, 0.625, 1.25, 2.5, 5, and 10 μg/mL were evaluated. The MTT assay results were used to determine the chemotherapy effect. For combined chemotherapy with PTT, the HeLa cells were treated with GNR/PSS/HSA NPs or DOX@GNR/PSS/HSA NPs at Au concentrations of 0, 5, 10, 25, or 50 μg/mL for 2 h. During the 2-h treatment, the cells were exposed to laser irradiation (808 nm, 1 W/cm²) for 2 min. After the 2-h treatment, the cells were washed and cultured in fresh medium for another 24 h and then subjected to the MTT assay. In addition, the groups of cells treated with GNR/PSS/HSA NPs in combination with NIR-light irradiation were immediately stained with Calcein-AM (Ex/Em: 494/517 nm) and EthD-1 (Ex/Em: 495/635 nm) and visualized under a fluorescence microscope (X51 Olympus Optical Co., Tokyo, Japan) to observe live and dead cells.
2.4. In vitro photothermal measurement The GNR/PSS/HSA NPs, the DOX@GNR/PSS/HSA NPs, and the HSA, PSS, and DOX aqueous solutions were irradiated with an 808-nm laser (1 W/cm²) for 2 min. During the irradiation, the change in temperature was recorded every 10 s by using a thermocouple device. In addition, 150 μL of DOX@GNR/PSS/HSA NP solution was dispersed in DMEM (Gibco BRL) at various GNR concentrations (20, 40, 80, 100, and 150 μg/mL, respectively), and the resulting mixtures were then irradiated with an 808-nm laser (1 W/cm²) for 5 min. During the irradiation, the change in temperature was recorded every 10 s, to determine the relationship between the concentration of DOX@GNR/PSS/ HSA NPs and the rate of heat generation. To further investigate the photothermal properties of the NPs, DOX@GNR/PSS/HSA NPs (100 μg/ ml) were irradiated with an 808-nm NIR laser (1.0 W/cm2) for three cycles of a 2-min NIR-light exposure period followed by a period of cooling to room temperature. The thermocouple device was used to record all of the temperature changes.
2.8. In vivo animal model Female BALB/cAnN.Cg-Foxnlnu/CrlNarl nude mice with 4 weeks age were purchased from the National Laboratory Animal Center (Taipei, Taiwan). The in vivo experimental protocols were approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee. To develop the in vivo tumor model, a 100-μL aliquot containing 5 × 106 HeLa cells was subcutaneously injected into the flank region of each mouse. The tumor volume was calculated as 1/2 × a × b², where a is the length and b is the width of the tumor. 2.9. In vivo temperature measurement and photothermal imaging When the tumor sizes reached 100–200 mm3, the tumor-bearing mice were divided into four different groups that were to be administered PBS (control), free DOX (10 mg of DOX/kg), GNR/PSS/HSA NPs (Au: 55 mg of Au/kg), or DOX@GNR/PSS/HSA NPs (10 mg of DOX/kg, 55 mg of Au/kg), respectively, through the tail vein of each mouse. A total of 24 h after the mice underwent administration of the experimental preparations, the tumors were exposed to an 808-nm laser at a power density of 1.5 W/cm2 for 5 min. The temperature at each tumor site was measured by using an infrared thermal imaging camera (Thermo Shot F30, Nippon Avionics Co., Ltd, Japan). The thermal images were captured every 1 min, and the temperature at each tumor site was determined.
2.5. In vitro thermo- and pH-controlled drug release The DOX@GNR/PSS/HSA NPs with or without proteinase K (BioShop, Canada) were dispersed in a dialysis bag with a 3.5-kDa molecular-weight cut-off that was then immersed in 40 mL of PBS at 37 °C [35]. After shaking for 1, 3, 6, 8, and 24 h, 400 μL of the release medium was withdrawn from the exterior solution. The fluorescence intensity of the released DOX at each time point was determined by HPLC. In order to analyze the photothermal effect on trigging drug release, DOX@GNR/PSS/HSA NPs in PBS buffer (pH 7.4) were first irradiated with an 808-nm laser (1.4 W/cm²) for 0, 2, 5, 10, or 15 min, followed by incubation at 37 °C for 8 h. Then, 400 μL of the particlecontaining solution was centrifuged at 18,000 g for 10 min, and the fluorescence intensity of the released DOX in the supernatant was measured by HPLC.
2.10. In vivo antitumor efficacy Treatments were started when the tumor sizes reached 100–200 mm³. Five groups of tumor-bearing mice (n = 3 in each group) were respectively treated with (a) PBS, (b) free DOX (10 mg of DOX/kg), (c) GNR/PSS/HSA NPs (Au: 55 mg of Au/kg) with laser irradiation, (d) DOX@GNR/PSS/HSA NPs (10 mg of DOX/kg, 55 mg of Au/kg) without laser irradiation, or (e) DOX@GNR/PSS/HSA NPs (10 mg of DOX/kg, 55 mg of Au/kg) with laser irradiation. The mice were intravenously injected only once at day 0. For the laser irradiation-treated groups, the tumor was irradiated for 5 min by using a 808-nm laser (1.5 W/cm²) at 24 h post injection. The tumor sizes and the body weights of the mice were measured every 3 days until 30 days had passed.
2.6. Cell culture Human uterine cervix carcinoma cell line (HeLa) cells were cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillinstreptomycin-amphotericin B antibiotic–antimycotic solutions at 37 °C under a humidified atmosphere containing 5% CO2. 2.7. In vitro cytotoxicity of the prepared NPs with or without laser irradiation In vitro cytotoxicity was evaluated by using the MTT assay and the EthD-1 assay [36]. HeLa cells were seeded into a 96-well plate at a density of 1.6 × 104 cells/well and incubated for 24 h. The cells were then washed with PBS and incubated in media containing different concentrations of GNR/CTAB NPs (0, 3.125, 6.25, 12.5, 25, or 50 μg of
2.11. Statistical analysis The mean ± standard deviation and graphs were used to describe the data. Student’s t-test was used to determine statistically significant 790
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differences between groups. * indicates p < 0.05.
30 °C, respectively, during irradiation with an 808-nm laser (1 W/cm²) for 2 min (Fig. 2A). This reflects the great photothermal effect of the GNRs. Moreover, the temperature of the solution rapidly increased with the increase of GNR concentration (Fig. 2B), indicating a concentrationdependent photothermal effect. Further cycle-heating experiments (2 min of irradiation followed by cooling to room temperature) showed that the photothermal performance of DOX@GNR/PSS/HSA NPs was not affected if NIR light irradiation was repeatedly administrated (Fig. 2C).
3. Results and discussion 3.1. Characterization of GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs The GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs were prepared by using an LbL coating method (Fig. 1A). The FTIR spectra of GNR/CTAB, GNR/PSS, and GNR/PSS/HSA NPs are shown in Fig. 1B. The absorption peak at 1477 cm−1 was assigned to CeH bending of CTAB [37]. Characteristic FTIR absorption peaks at 1008, 1030, 1114, and 1174 cm−1 were observed in the obtained spectrum for the GNR/ PSS NPs. Those were assigned to the symmetric and antisymmetric stretching vibrations of the O]S]O groups of SO3− in PSS [38]. The absorption peaks at 1640 cm−1 and 1535 cm−1 were ascribed to the stretching of the C]O bond and to the bending of NeH of the CONH group [39,40], respectively, indicating the successful coating the surface of GNR/PSS NPs with HSA. Fig. 2C and D show the particle size and the zeta potential of GNR/ CTAB, GNR/PSS, and GNR/PSS/HSA NPs, as determined by TEM and by the Zetasizer, respectively. The particle size of GNR/CTAB NPs was 58.1 nm (the aspect ratio was about 3:1), with a zeta potential of 62 mV. The particle size of GNR/PSS NPs was about 65.4 nm, with a zeta potential of −43 mV. The changes in the size and the zeta potential indicate that the coating of GNR/CTAB NPs with PSS was successful. After coating with HSA, GNR/PSS/HSA NPs exhibited a positive surface charge (a zeta potential of 5 mV), a particle size of 67.3 nm at pH 4.2, and a negative surface charge (a zeta potential of −15 mV) at pH 7.4. This change occurred because the isoelectric point of HSA lies at pH 4.7. Thus, the net charge of the protein changes from positive at pH 4.2 to negative at pH 7.4 [41]. The TEM image of GNR/PSS/HSA NPs revealed a surrounding layer with a strong contrast against the background after negative staining with PTA. Taken together, the increase in particle size and the reversed zeta potential indicate that the LbL coating of GNR/CTAB NPs with PSS and HSA was successful. The UV–vis-IR absorption spectra of the designed NPs and that of free DOX are shown in Fig. 1E. The longitudinal SPR absorption of the GNR/CTAB NPs was centered at 810 nm and was slightly blue-shifted (about 10 nm) after the NPs were coated with PSS and HSA. This might have resulted from the slight aggregation of GNR/PSS/HSA NPs. This aggregation may have been caused by the neutralization of the positive charges of CTAB by the negative charges of PSS, which removed the stabilizing repulsive force between the GNRs [42]. After GNR/PSS/HSA NPs were loaded with DOX, the longitudinal plasmon resonance maximum red-shifted back toward the original position, indicating that the positive charges of DOX contributed to a stabilizing repulsive force between the DOX@GNR/PSS/HSA NPs. Moreover, a significant absorption peak appeared at 400–550 nm. This indicated the successful loading of the DOX molecules. In addition, we found that 1.0 μg of GNRs could maximally be loaded with 0.43 μg of DOX (Table 1) and that the E.E. of DOX was 40.7%. Moreover, DOX@GNR/PSS/HSA NPs did not aggregate in aqueous solution over a period of 1 month, showing excellent stability, without broadening or tailing of the SPR band (Fig. S1). Furthermore, DOX@GNR/PSS/HSA NPs were stable in culture medium with and without FBS for over 24 h, indicating that the HSA-coated NPs minimized undesired protein absorption, which is important because such undesired protein interaction may cause severe aggregation of NPs in the blood circulation (Fig. S2) [43].
3.3. In vitro thermo- and pH-controlled drug release DOX release from DOX@GNR/PSS/HSA NPs in PBS was analyzed under various conditions. The DOX release rate was much higher in acidic environments (pH 5.5) than that in neutral environments (pH 7.4) (Fig. 3A). After 24 h, the proportion of released DOX at pH 7.4 (25.0%) was lower than that at pH 5.5 (44.8%). The accelerated drugrelease rate at pH 5.5 might be a result of weaker interactions between DOX and HSA due to more protonated HSA groups in the acidic environment compared with those in the neutral environments. In addition, attributed to two phenols and one amine moiety in DOX molecules, the degree of protonation and polarity of DOX could be manipulated by changing pH [44]. Therefore, in the acidic environment, the hydrophilicity and the solubility of protonated-DOX molecules were increased, leading to a higher diffusion rate of DOX from the NPs to the aqueous phase. Notably, in the presence of proteinases at pH 5.5, up to 69.1% of DOX was released after 24 h. This indicates that the drug was selectively released in the proteinase-rich environment of the endosomes/lysosomes. These results indicate that DOX@GNR/PSS/ HSA NPs may not only reduce undesirable DOX leakage into the blood circulation, avoiding potential damage to normal tissues, but also facilitate site-specific drug delivery to the tumor environment and within the cellular endosomes/lysosomes. To investigate the effect of the temperature increase resulting from NIR irradiation on drug release, DOX@GNR/PSS/HSA NPs were irradiated with the 808-nm laser for different time periods. As shown in Fig. 3B, longer laser exposure times yielded greater drug release. Compared with the group without NIR irradiation, exposure to NIR irradiation for 5 min and 15 min resulted in 2.4- and 3.4-times greater DOX release, respectively. Thus, DOX release could be triggered by laser irradiation. The drug release probably occurred because of the elevation of temperature under NIR-irradiation. When the temperature was increased to a higher level than the first melting temperature of HSA (55 °C), parts of the broken helical structure of HSA might expose to the solvent and result in morphological changes and an increase in hydration [45]. Therefore, the outermost HSA coating on the DOX@GNR/PSS/HSA NPs would become more swell and get a loose structure after exposure to NIR irradiation, resulting in the entrapped DOX be released at a faster rate. 3.4. In vitro single- and dual-modality therapy with the prepared NPs Cellular uptake into HeLa cells was observed using fluorescence microscopy, after incubation with DOX@GNR/PSS/HSA NPs (Au conc = 50 μg/mL) for 0.5, 2, and 4 h. As shown in Fig. S3, the red fluorescence signal of DOX was detected in the nuclei of HeLa cells after incubation with DOX@GNR/PSS/HSA NPs, indicating that the DOX@GNR/PSS/HSA NPs had been internalized. The intensity of the DOX fluorescence signal in the nuclei significantly increased with incubation time, indicating that, after internalization, DOX immediately dissociated from the DOX@GNR/PSS/HSA NPs in the endosomes/lysosomes and then diffused into the nuclei. To evaluate in vitro single- and dual-modality therapy effects, the viability of HeLa cells was determined using the MTT assay after treatment of the cells with GNR/CTAB NPs, GNR/PSS/HSA NPs, free DOX, and DOX@GNR/PSS/HSA NPs. The viability of the HeLa cells
3.2. In vitro photothermal measurement Compared with ddH2O, DMEM, PSS, HSA, and DOX solutions, the temperature of the solutions containing GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs increased markedly from 0 °C to 31.6 °C and 791
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Fig. 1. (A) Fabrication of GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs. (B) FTIR spectra of GNR, GNR/PSS, and GNR/PSS/HSA NPs. (C) TEM images of GNR/ CTAB, GNR/PSS, and GNR/PSS/HSA NPs. The scale bar in the images is 50 nm. (D) Zeta potentials at the different adsorption steps for the preparation of GNR/PSS/ HSA NPs. Inset: zeta potentials of GNR/PSS/HSA NPs at pH values of 4.2 and 7.4. (E) UV–vis-NIR spectra of the prepared NPs before and after PSS, HSA, and DOX coating.
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Fig. 2. (A) Photothermal effects of GNR/PSS/HSA NPs, DOX@GNR/PSS/HSA NPs, and HSA, PSS, and DOX aqueous solutions upon NIR irradiation (λ = 808 nm; 1 W/cm² for 5 min). (B) Photothermal effects of DOX@GNR/PSS/HSA NP aqueous dispersions at different GNR concentrations (20, 40, 80, 100, and 150 μg/mL) under NIR-laser irradiation (λ = 808 nm; 1 W/cm²) for 5 min. (C) Real-time temperature measurement of a DOX@GNR/PSS/HSA NP aqueous dispersion (100 μg/ml) under 808-nm NIR-light irradiation with a power density of 1.0 W/cm2 for three cycles. Each cycle consisted of 2 min of irradiation followed by cooling to room temperature.
DOX at equivalent DOX concentrations. This indicates that the efficacies of DOX@GNR/PSS/HSA NPs and free DOX for chemotherapy are the same. To evaluate the photothermal effect of GNR/PSS/HSA NPs on HeLa cells, cells were incubated with GNR/PSS/HSA NPs (100 μg/mL) for 2 h, irradiated with an 808-nm laser for 2 min, and stained with Calcein-AM and EthD-1 to visualize the live and dead cells. Live and dead cells showed green and red fluorescence, respectively. As shown in Fig. 4C, no obvious red fluorescence, for dead cells, was detected after the cells had been treated with only laser irradiation. In contrast, a significant portion of dead cells was observed when the cells have been treated with GNR/PSS/HSA NPs followed by laser irradiation. To investigate the in vitro effect of the combined chemotherapy and PTT, the viabilities of the HeLa cells after treatment with GNR/PSS/ HSA NPs and DOX@GNR/PSS/HSA NPs with or without NIR-laser irradiation were determined using an MTT assay. As shown in Fig. 4D, GNR/PSS/HSA NPs without laser irradiation exhibited no cytotoxicity. In contrast, DOX@GNR/PSS/HSA NPs without laser irradiation showed dose-dependent cytotoxicity. After NIR irradiation for 2 min, the GNR/ PSS/HSA NP-treated cells exhibited significant cell death, especially at
Table 1 The loading capacity of DOX in the prepared DOX@GNR/PSS/HSA NPs. Designed DOX/Au (w/w)
200/280 (0.71)
150/280 (0.54)
100/280 (0.36)
E.E. (%) Final DOX/Au (w/w)
40.7 ± 2 0.43
27.5 ± 3.11 0.22
8.3 ± 0.25 0.04
significantly decreased with increasing GNR/CTAB NPs concentration, which resulted from the toxicity of CTAB (Fig. 4A). Compared with the cell viabilities of the GNR/CTAB NP-treated groups, the cell viability was much higher (> 80%) for the GNR/PSS/HSA NP-treated group. This can be attributed to the fact that, after LbL coating of GNR/CTAB NPs with PSS and HSA, the level of cytotoxicity of the cationic CTAB was significantly decreased. Moreover, a dose-dependent cytotoxicity was observed after incubation with free DOX and DOX@GNR/PSS/HSA NPs for 2 h (Fig. 4B). Notably, the viability of cells treated with DOX@GNR/PSS/HSA NPs was as high as that of cells treated with free
Fig. 3. (A) DOX-release profiles of DOX@GNR/PSS/HSA NPs in buffers of pH 7.4 and 5.5 with or without proteinase K. (B) Percentage of DOX released from DOX@GNR/PSS/HSA NPs under NIR-laser irradiation (λ = 808 nm; 1.4 W/cm²) at different time points. 793
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Fig. 4. (A) Viability of HeLa cells after incubation with GNR/CTAB NPs and GNR/PSS/HSA NPs at different Au concentrations. (B) Viability of HeLa cells after treatment with free DOX and DOX@GNR/PSS/HSA NPs at different DOX concentrations. (C) Fluorescence images of HeLa cells co-stained with ethidium homodimer1 (red, dead cells) and Calcein-AM (green, live cells) and pretreated with DMEM (control) or GNR/PSS/HSA NPs followed by laser irradiation (λ = 808 nm; 1 W/cm² for 2 min). The scale bar in the images is 100 μm. (D) Viability of HeLa cells after treatment with GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs at different Au concentrations with or without laser irradiation (λ = 808 nm; 1 W/cm² for 2 min). *: p < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 794
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Fig. 5. (A) Tumor temperature and (B) infrared thermographic maps at different time points of nude mice bearing HeLa tumors. The mice were injected with GNR/ PSS/HSA NPs and DOX@GNR/PSS/HSA NPs after laser irradiation (808 nm, 1.5 W/cm² for 5 min).
an Au concentration of 25–50 μg/mL, indicating a strong photothermal effect of the prepared NPs on cytotoxicity. When DOX@GNR/PSS/HSA NP-treated cells were irradiated with an NIR laser for 2 min, significant cytotoxicity was observed. This cytotoxicity was not only induced by DOX but also by the localized heat generated by the GNRs upon NIRlaser irradiation. In addition to the heat-induced ablation of cancer cells, heat also could accelerate the release of DOX from the NPs. This may result in an enhanced chemotherapeutic effect.
prognostic factor that is associated with a survival and a response to treatment [47]. Therefore, DOX@GNR/PSS/HSA NPs was shown to be well-tolerated at the given dose and has moderate biocompatibility. For the mice in the group treated with GNR/PSS/HSA NPs followed by NIR irradiation, a significant suppression of tumor growth by day 30 was observed, compared to that for the mice of the PBS-, free DOX-, and DOX@GNR/PSS/HSA NP-treated groups. Interestingly, similar levels of tumor-growth suppression were observed for the group treated with DOX@GNR/PSS/HSA NPs and NIR-laser irradiation and that treated with GNR/PSS/HSA NPs and NIR-laser irradiation during the first 15 days. By day 30, however, a remarkable suppression of tumor growth was observed for the DOX@GNR/PSS/HSA NPs and NIR-laser irradiation treated group. Taken together, our results suggest a synergistic antitumor effect of PTT and chemotherapy including DOX@GNR/PSS/ HSA NPs. Thus, DOX@GNR/PSS/HSA NPs have a high potential of being implemented in future cancer therapeutic strategies.
3.5. In vivo photothermal effect To evaluate the in vivo photothermal effect, the temperatures of tumors with the DOX@GNR/PSS/HSA NP-injected via the tail vein upon 808-nm laser irradiation were measured by using the infrared thermal imaging camera. As shown in Fig. 5, the tumor temperature increased with increased NIR-laser exposure time. After irradiation with a 808-nm laser at a power density of 1.5 W/cm² for 5 min, GNR/PSS/ HSA NP-treated and DOX@GNR/PSS/HSA NP-treated tumors both showed a significant temperature increase from 35 °C to about 52 °C, and the temperature increase was higher than that of tumors treated with free DOX or PBS. If the tumor temperature was maintained at 50 °C – 52 °C for about 5 min, irreversible cellular damage occurred [46]. Therefore, we suggest that GNR/PSS/HSA NPs and DOX@GNR/ PSS/HSA NPs that are prepared by following our protocol can be applied for in vivo PTT for cancer treatment.
4. Conclusions We successfully produced multilayer-coated and DOX-loaded GNR Nanoparticles (DOX@GNR/PSS/HSA NPs) that seem to be a promising platform for in vivo combined cancer PTT and chemotherapy. Compared with unmodified GNRs, the PSS- and HSA-modified GNRs exhibited negligible toxicity and excellent colloidal stability. Besides improving biocompatibility, surface coating of the GNRs with HSA also promoted endocytosis of the NPs and provided a suitable layer for the loading of DOX. In addition, DOX@GNR/PSS/HSA NPs exhibited heat- and pHtriggered and site-specific drug-release behavior, which led to an enhanced antitumor effect at the cellular level. Moreover, DOX@GNR/ PSS/HSA NPs showed synergistic photothermal and chemotherapeutic effects in vivo in a HeLa-tumor-bearing mouse model. This was due to the high photothermal conversion efficiency of DOX@GNR/PSS/HSA NPs under irradiation with NIR light that penetrated deep into the tissue. This study demonstrates the potential of DOX@GNR/PSS/HSA NPs to be used in combined chemotherapy and photothermal therapy anticancer applications.
3.6. In vivo antitumor efficacy Tumor growth was measured in order to examine the in vivo antitumor efficacy of PBS, free DOX, GNR/PSS/HSA NPs, and DOX@GNR/ PSS/HSA NPs with and without NIR-laser irradiation for HeLa tumorbearing mice. The tumor size was normalized to the tumor size at day 0. Fig. 6A showed that DOX@GNR/PSS/HSA NPs only and free DOX only exhibited similar effects on the suppression of tumor growth, which was consistent with the in vitro cytotoxicity results shown in Fig. 6B. However, significant loss of body weight was observed for the administration of free DOX only and not for the administration of DOX@GNR/ PSS/HSA NPs (Fig. 6B). Significant Loss of body weight is a major
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Fig. 6. (A) Tumor volumes and (B) body weights of nude mice bearing HeLa tumors that were treated with PBS, free DOX, GNR/PSS/HSA NPs, or DOX@GNR/PSS/ HSA NPs with or without laser irradiation (808 nm, 1.5 W/cm², 5 min). *: p < 0.05.
Funding sources
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Dual-triggered drug-release vehicles for synergistic cancer therapy a,1
a,b,c,1
a
b,c
a
Ting-Yu Tu , Shu-Jyuan Yang , Ming-Hsien Tsai , Chung-Hao Wang , Shin-Yu Lee , ⁎ Tai-Horng Younga, Ming-Jium Shieha,d,e,
T
a
Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, 100, Taiwan Gene’e Tech Co. Ltd. 2F., No.661, Bannan Rd., Zhonghe Dist., New Taipei City 235, Taiwan Apius Bio Inc. 1F., No.92, Daxin St., Yonghe Dist., New Taipei City 234, Taiwan d Department of Oncology, National Taiwan University Hospital, #7, Chung-Shan South Road, Taipei, 100, Taiwan e Department of Biomedical Engineering, National Taiwan University Hospital, #7, Chung-Shan South Road, Taipei, 100, Taiwan b c
ARTICLE INFO
ABSTRACT
Keywords: Gold nanorod Human serum albumin Chemotherapy Photothermal therapy Controlled drug release
Cancer is a complex and tenacious disease. Drug-delivery systems in combination with multimodal therapy strategies are very promising candidates for cancer theranostic applications. In this study, a new drug-delivery vehicle that combine human serum albumin (HSA)- and poly(sodium 4-styrenesulfonate) (PSS)-coated gold nanorod nanoparticles(GNR/PSS/HSA NPs) was developed for synergistic cancer therapy. Doxorubicin (DOX) was loaded onto GNR/PSS/HSA NPs, by electrostatic and hydrophobic forces, to create multimodal DOX@GNR/ PSS/HSA NPs. DOX@GNR/PSS/HSA NPs were found to be highly biocompatible and stable in physiological solutions. Furthermore, GNR/PSS/HSA NPs with or without DOX were designed to exhibit strong absorbance in the near-infrared region and high photothermal conversion efficiency. Therefore, bimodal DOX release from DOX@GNR/PSS/HSA NPs could be triggered by an acidic pH and by near-infrared irradiation after NPs preferentially accumulated at tumor sites, leading to a significant chemotherapeutic effect. Moreover, DOX@GNR/ PSS/HSA NPs were designed to be applied during chemo- and photo-thermal combination therapy and exhibited a synergistic anticancer effect that was superior to the effect of monotherapy, from both in vitro and in vivo results. These results suggest that DOX@GNR/PSS/HSA NPs are a strong candidate for a nanoplatform for future antitumor therapeutic strategies.
1. Introduction Cancer remains one of the most devastating diseases. Chemotherapy is an integral component of most cancer treatments. It regulates the abnormal proliferation of cancer cells and blocks the supply of nutrients required for growth. Although chemotherapy is a powerful tool and can help to avoid cancer metastasis, a major issue of chemotherapy is its non-specificity. To improve the efficacy of chemotherapy, nanodrug carriers with sizes of 100–200 nm have been developed. These nanodrug carriers passively target the tumor site, through the enhanced permeability and retention (EPR) effect and result in high tumor accumulation [1,2]. In addition, various internally or externally triggered drug-release approaches, such as pH, temperature, ultrasound, magnetic field, and light, have been explored, to achieve better treatment outcomes [3–8]. Near-infrared (NIR) light with a wavelength of 650–900 nm is widely used as a trigger, because it penetrates up to 10 cm into living tissue and induces minimal damage to normal tissues
[9,10]. NIR-light resonance gold nanomaterials, such as gold nanorods (GNRs), have many advantages for biomedical applications [11–14]. The NIR-light absorption and scattering characteristics of GNRs can be manipulated, by adjusting the type or proportion of the reducing agent, the size, or the aspect ratio (length-to-diameter ratio) of the GNR. Upon exposure to proper NIR light, GNRs convert light energy to thermal energy, through the surface plasmon resonance (SPR) effect [15]. The resultant local increase of temperature induces significant thermal and mechanical stress within the system, and results in the bursting off of the carrier and the release of the payload [16,17]; furthermore, upon NIR-laser stimulation, GNRs can induce hyperthermia within the tumor tissue (known as photothermal therapy [PTT]), and the extent of hyperthermia depends on the thermal dosage and exposure time of NIRlaser stimulation [15,18]. In addition, these SPR-induced tunable absorption and scattering features allow the use of GNRs as a contrast agents for bioimaging applications [19,20].
Corresponding author. E-mail addresses: [email protected], [email protected] (M.-J. Shieh). 1 The first two authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.colsurfb.2018.10.043 Received 25 June 2018; Received in revised form 20 September 2018; Accepted 16 October 2018 Available online 19 October 2018 0927-7765/ © 2018 Published by Elsevier B.V.
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GNRs can been easily synthesized by using a cationic surfactant hexadecyltrimethylammonium bromide (CTAB)-templated growth strategy [21]. However, the toxicity of CTAB on GNRs must be reduced to allow biological applications [22]. The layer-by-layer (LbL) coating technique is used widely for a safe surface coating because of its versatility, simplicity, and applicability in aqueous conditions [23]. Other advantages of this technique are that the deposited multilayer films can be designed to have high biocompatibility and that payload drugs can be loaded into separate layers for sequential, controlled drug release [24,25]. Overall, LbL coating techique is a relatively easy method which can add active substances to GNRs and thus enhance biocompatibility. Human serum albumin (HSA) is an endogenous protein and the most abundant protein in human blood. HSA binds to and transports several metabolic compounds and therapeutic drugs in the blood circulation [26]. Moreover, HSA has high affinity for tumor tissue [27,28]; it actively accumulates at tumor sites via gp60 receptor-mediated transcytosis as a result of its high binding affinity for the specific glycoprotein, secreted protein acidic and rich in cysteine(SPARC), which is secreted by several cancer cells and is abundant in tumor interstitial tissue [29]. HSA possesses numerous and different functional groups that can be employed for chemical modification; this characteristic makes HSA an ideal carrier for cancer-drug delivery [30,31]. In this study, we developed a thermo- and pH-triggered drug-delivery vehicle, poly(sodium 4-styrenesulfonate) (PSS)/HSA-coated GNRs nanoparticles (GNR/PSS/HSA NPs), for cancer PTT and chemotherapy. GNR/PSS/HSA NPs were created from GNRs, which were synthesized by using a CTAB-templated growth strategy and then undergoing LbL coating with PSS and HSA, by electrostatic force. Consequently, the GNR/PSS/HSA NPs would exhibit good biocompatibility and not be detected by the reticuloendothelial system, leading to a longer circulation time. The GNR/PSS/HSA NPs were loaded with doxorubicin (DOX) (DOX@GNR/PSS/HSA NPs), by using electrostatic and hydrophobic forces. DOX has been widely used for cancer chemotherapy. However, unspecific accumulation of DOX brings about serious side effects, such as severe cardiac toxicity. These side effects limit the use of DOX for cancer treatment [32–34]. However, GNR/PSS/ HSA NPs can transport DOX to the tumor site passively (via the EPR effect) and actively (via gp60 receptor-mediated transcytosis). Thus, DOX leakage into the blood circulation could be reduced compared to that of conventional chemotherapy. Importantly, DOX on DOX@GNR/ PSS/HSA NPs accumulated at the tumor site can be triggered and released by the acidic pH within endosomes/lysosomes and by NIR-laser irradiation during PTT. Simultaneously, PTT can induce cancer-cell death via apoptosis or necrosis (Scheme 1). Therefore, we expect that a
combination of PTT and chemotherapy involving DOX@GNR/PSS/HSA NPs will be a promising tool in cancer therapy, to kill cancer cells and decrease the probability of tumor recurrence. 2. Materials and methods 2.1. Materials GNRs were kindly supplied by Gene’e Tech Co., Ltd. (Taiwan). PSS, HSA, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (USA). Doxorubicin, hydrochloride salt (> 99%) was purchased from LC Laboratories (USA). Calcein acetoxymethyl ester (Calcein-AM) and ethidium homodimer-1 (EthD-1) were purchased from Invitrogen (USA). Matrigel matrix was purchased from Discovery Labware, Inc (USA). 2.2. Preparation of GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs were prepared by using the LbL technique. First, 10 mg/mL of PSS stock solution was prepared by dissolving 10 mg PSS powder in 1 ml of 1 mM NaCl solution. Then, 1 mL of GNR/CTAB solution (OD = 2) was mixed gently with 1 mL of PSS stock solution for 1 h, to obtain GNR/PSS NPs. GNR/ PSS NPs were washed three times and were centrifuged (at 16,000 g for 10 min) to remove excess PSS. Then, the pH value of the GNR/PSS solution was adjusted to 4.2, and the GNR/PSS solution was mixed with 1 mL of HSA in aqueous solution (10 mg/mL, pH = 4.2) for 2 h at room temperature. GNR/PSS/HSA NPs were collected, after centrifugation at 16,000 g for 10 min to remove free HSA. DOX@GNR/PSS/HSA NPs were prepared by gently mixing the GNR/PSS solution (pH = 4.2) with 150 μL of 1 mg/mL DOX･HCl solution. After 1 h of gentle stirring, 1.15 mL of the DOX@GNR/PSS NP solution was incubated with 1 mL of HSA in aqueous solution (10 mg/mL, pH = 4.2) for 2 h at room temperature. DOX@GNR/PSS NPs were then centrifuged at 16,000 g for 10 min, to remove free DOX and free HSA. Finally, the GNR/PSS/HSA and DOX@GNR/PSS/HSA precipitates were separately re-dispersed in DI water and stored at 4 °C until use. 2.3. Characterization of GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs The chemical compositions of GNR/CTAB, GNR/PSS, and GNR/ PSS/HSA NPs were evaluated by using an ATR-FTIR spectrometer (Thermo Scientific Nicolet 6700; Waltham, MA, USA), to determine the characteristics of the LbL deposition. The morphology and the particle
Scheme 1. The EPR effect and gp60 receptor-mediated transcytosis enhance the uptake of DOX@GNR/PSS/HSA NPs into the tumor and triggering of DOX drug release. The synergistic effect of combined DOX chemotherapy and photothermal therapy that includes NIR-laser irradiation is also shown. 789
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size of GNR/CTAB, GNR/PSS, and GNR/PSS/HSA NPs were examined by transmission electron microscopy (TEM, Hitachi H-7500, Tokyo, Japan) with and without 1% phosphotungstic acid (PTA) staining. The absorption properties of GNR/CTAB, GNR/PSS, and GNR/PSS/HSA NPs were evaluated, based on the absorption spectra obtained by using an ELISA microplate reader (SpectraMax M2 Multi-Mode Microplate Readers, Molecular Devices, USA). The zeta potentials of the prepared NPs were measured by using a Zetasizer Nano-ZS90 (Malvern Instruments Ltd, UK). The fluorescence intensity of the unloaded DOX in the NPs was determined by using high-performance liquid chromatography (HPLC, Waters e2696), and the loading efficiency of DOX was calculated. The drug encapsulation efficiency (E.E.) was calculated by using the following equation: Encapsulation efficiency (%) = (Weight of the feeding DOX – Weight of the unloaded DOX)/ Weight of the feeding DOX × 100%
Au/mL) or GNR/PSS/HSA NPs (0, 3.125, 6.25, 12.5, 25, or 50 μg of Au/ mL) for 2 h. After another wash with PBS, the cells were cultured in fresh medium for another 24 h before being subjected to the MTT assay, involving the use of a scanning multiwell ELISA reader (Microplate Autoreader EL311, BioTek Instruments Inc., Winooski, VT, USA). Furthermore, the cytotoxicities of free DOX and that of DOX@GNR/PSS/ HSA NPs at DOX concentrations of 0, 0.3125, 0.625, 1.25, 2.5, 5, and 10 μg/mL were evaluated. The MTT assay results were used to determine the chemotherapy effect. For combined chemotherapy with PTT, the HeLa cells were treated with GNR/PSS/HSA NPs or DOX@GNR/PSS/HSA NPs at Au concentrations of 0, 5, 10, 25, or 50 μg/mL for 2 h. During the 2-h treatment, the cells were exposed to laser irradiation (808 nm, 1 W/cm²) for 2 min. After the 2-h treatment, the cells were washed and cultured in fresh medium for another 24 h and then subjected to the MTT assay. In addition, the groups of cells treated with GNR/PSS/HSA NPs in combination with NIR-light irradiation were immediately stained with Calcein-AM (Ex/Em: 494/517 nm) and EthD-1 (Ex/Em: 495/635 nm) and visualized under a fluorescence microscope (X51 Olympus Optical Co., Tokyo, Japan) to observe live and dead cells.
2.4. In vitro photothermal measurement The GNR/PSS/HSA NPs, the DOX@GNR/PSS/HSA NPs, and the HSA, PSS, and DOX aqueous solutions were irradiated with an 808-nm laser (1 W/cm²) for 2 min. During the irradiation, the change in temperature was recorded every 10 s by using a thermocouple device. In addition, 150 μL of DOX@GNR/PSS/HSA NP solution was dispersed in DMEM (Gibco BRL) at various GNR concentrations (20, 40, 80, 100, and 150 μg/mL, respectively), and the resulting mixtures were then irradiated with an 808-nm laser (1 W/cm²) for 5 min. During the irradiation, the change in temperature was recorded every 10 s, to determine the relationship between the concentration of DOX@GNR/PSS/ HSA NPs and the rate of heat generation. To further investigate the photothermal properties of the NPs, DOX@GNR/PSS/HSA NPs (100 μg/ ml) were irradiated with an 808-nm NIR laser (1.0 W/cm2) for three cycles of a 2-min NIR-light exposure period followed by a period of cooling to room temperature. The thermocouple device was used to record all of the temperature changes.
2.8. In vivo animal model Female BALB/cAnN.Cg-Foxnlnu/CrlNarl nude mice with 4 weeks age were purchased from the National Laboratory Animal Center (Taipei, Taiwan). The in vivo experimental protocols were approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee. To develop the in vivo tumor model, a 100-μL aliquot containing 5 × 106 HeLa cells was subcutaneously injected into the flank region of each mouse. The tumor volume was calculated as 1/2 × a × b², where a is the length and b is the width of the tumor. 2.9. In vivo temperature measurement and photothermal imaging When the tumor sizes reached 100–200 mm3, the tumor-bearing mice were divided into four different groups that were to be administered PBS (control), free DOX (10 mg of DOX/kg), GNR/PSS/HSA NPs (Au: 55 mg of Au/kg), or DOX@GNR/PSS/HSA NPs (10 mg of DOX/kg, 55 mg of Au/kg), respectively, through the tail vein of each mouse. A total of 24 h after the mice underwent administration of the experimental preparations, the tumors were exposed to an 808-nm laser at a power density of 1.5 W/cm2 for 5 min. The temperature at each tumor site was measured by using an infrared thermal imaging camera (Thermo Shot F30, Nippon Avionics Co., Ltd, Japan). The thermal images were captured every 1 min, and the temperature at each tumor site was determined.
2.5. In vitro thermo- and pH-controlled drug release The DOX@GNR/PSS/HSA NPs with or without proteinase K (BioShop, Canada) were dispersed in a dialysis bag with a 3.5-kDa molecular-weight cut-off that was then immersed in 40 mL of PBS at 37 °C [35]. After shaking for 1, 3, 6, 8, and 24 h, 400 μL of the release medium was withdrawn from the exterior solution. The fluorescence intensity of the released DOX at each time point was determined by HPLC. In order to analyze the photothermal effect on trigging drug release, DOX@GNR/PSS/HSA NPs in PBS buffer (pH 7.4) were first irradiated with an 808-nm laser (1.4 W/cm²) for 0, 2, 5, 10, or 15 min, followed by incubation at 37 °C for 8 h. Then, 400 μL of the particlecontaining solution was centrifuged at 18,000 g for 10 min, and the fluorescence intensity of the released DOX in the supernatant was measured by HPLC.
2.10. In vivo antitumor efficacy Treatments were started when the tumor sizes reached 100–200 mm³. Five groups of tumor-bearing mice (n = 3 in each group) were respectively treated with (a) PBS, (b) free DOX (10 mg of DOX/kg), (c) GNR/PSS/HSA NPs (Au: 55 mg of Au/kg) with laser irradiation, (d) DOX@GNR/PSS/HSA NPs (10 mg of DOX/kg, 55 mg of Au/kg) without laser irradiation, or (e) DOX@GNR/PSS/HSA NPs (10 mg of DOX/kg, 55 mg of Au/kg) with laser irradiation. The mice were intravenously injected only once at day 0. For the laser irradiation-treated groups, the tumor was irradiated for 5 min by using a 808-nm laser (1.5 W/cm²) at 24 h post injection. The tumor sizes and the body weights of the mice were measured every 3 days until 30 days had passed.
2.6. Cell culture Human uterine cervix carcinoma cell line (HeLa) cells were cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillinstreptomycin-amphotericin B antibiotic–antimycotic solutions at 37 °C under a humidified atmosphere containing 5% CO2. 2.7. In vitro cytotoxicity of the prepared NPs with or without laser irradiation In vitro cytotoxicity was evaluated by using the MTT assay and the EthD-1 assay [36]. HeLa cells were seeded into a 96-well plate at a density of 1.6 × 104 cells/well and incubated for 24 h. The cells were then washed with PBS and incubated in media containing different concentrations of GNR/CTAB NPs (0, 3.125, 6.25, 12.5, 25, or 50 μg of
2.11. Statistical analysis The mean ± standard deviation and graphs were used to describe the data. Student’s t-test was used to determine statistically significant 790
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differences between groups. * indicates p < 0.05.
30 °C, respectively, during irradiation with an 808-nm laser (1 W/cm²) for 2 min (Fig. 2A). This reflects the great photothermal effect of the GNRs. Moreover, the temperature of the solution rapidly increased with the increase of GNR concentration (Fig. 2B), indicating a concentrationdependent photothermal effect. Further cycle-heating experiments (2 min of irradiation followed by cooling to room temperature) showed that the photothermal performance of DOX@GNR/PSS/HSA NPs was not affected if NIR light irradiation was repeatedly administrated (Fig. 2C).
3. Results and discussion 3.1. Characterization of GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs The GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs were prepared by using an LbL coating method (Fig. 1A). The FTIR spectra of GNR/CTAB, GNR/PSS, and GNR/PSS/HSA NPs are shown in Fig. 1B. The absorption peak at 1477 cm−1 was assigned to CeH bending of CTAB [37]. Characteristic FTIR absorption peaks at 1008, 1030, 1114, and 1174 cm−1 were observed in the obtained spectrum for the GNR/ PSS NPs. Those were assigned to the symmetric and antisymmetric stretching vibrations of the O]S]O groups of SO3− in PSS [38]. The absorption peaks at 1640 cm−1 and 1535 cm−1 were ascribed to the stretching of the C]O bond and to the bending of NeH of the CONH group [39,40], respectively, indicating the successful coating the surface of GNR/PSS NPs with HSA. Fig. 2C and D show the particle size and the zeta potential of GNR/ CTAB, GNR/PSS, and GNR/PSS/HSA NPs, as determined by TEM and by the Zetasizer, respectively. The particle size of GNR/CTAB NPs was 58.1 nm (the aspect ratio was about 3:1), with a zeta potential of 62 mV. The particle size of GNR/PSS NPs was about 65.4 nm, with a zeta potential of −43 mV. The changes in the size and the zeta potential indicate that the coating of GNR/CTAB NPs with PSS was successful. After coating with HSA, GNR/PSS/HSA NPs exhibited a positive surface charge (a zeta potential of 5 mV), a particle size of 67.3 nm at pH 4.2, and a negative surface charge (a zeta potential of −15 mV) at pH 7.4. This change occurred because the isoelectric point of HSA lies at pH 4.7. Thus, the net charge of the protein changes from positive at pH 4.2 to negative at pH 7.4 [41]. The TEM image of GNR/PSS/HSA NPs revealed a surrounding layer with a strong contrast against the background after negative staining with PTA. Taken together, the increase in particle size and the reversed zeta potential indicate that the LbL coating of GNR/CTAB NPs with PSS and HSA was successful. The UV–vis-IR absorption spectra of the designed NPs and that of free DOX are shown in Fig. 1E. The longitudinal SPR absorption of the GNR/CTAB NPs was centered at 810 nm and was slightly blue-shifted (about 10 nm) after the NPs were coated with PSS and HSA. This might have resulted from the slight aggregation of GNR/PSS/HSA NPs. This aggregation may have been caused by the neutralization of the positive charges of CTAB by the negative charges of PSS, which removed the stabilizing repulsive force between the GNRs [42]. After GNR/PSS/HSA NPs were loaded with DOX, the longitudinal plasmon resonance maximum red-shifted back toward the original position, indicating that the positive charges of DOX contributed to a stabilizing repulsive force between the DOX@GNR/PSS/HSA NPs. Moreover, a significant absorption peak appeared at 400–550 nm. This indicated the successful loading of the DOX molecules. In addition, we found that 1.0 μg of GNRs could maximally be loaded with 0.43 μg of DOX (Table 1) and that the E.E. of DOX was 40.7%. Moreover, DOX@GNR/PSS/HSA NPs did not aggregate in aqueous solution over a period of 1 month, showing excellent stability, without broadening or tailing of the SPR band (Fig. S1). Furthermore, DOX@GNR/PSS/HSA NPs were stable in culture medium with and without FBS for over 24 h, indicating that the HSA-coated NPs minimized undesired protein absorption, which is important because such undesired protein interaction may cause severe aggregation of NPs in the blood circulation (Fig. S2) [43].
3.3. In vitro thermo- and pH-controlled drug release DOX release from DOX@GNR/PSS/HSA NPs in PBS was analyzed under various conditions. The DOX release rate was much higher in acidic environments (pH 5.5) than that in neutral environments (pH 7.4) (Fig. 3A). After 24 h, the proportion of released DOX at pH 7.4 (25.0%) was lower than that at pH 5.5 (44.8%). The accelerated drugrelease rate at pH 5.5 might be a result of weaker interactions between DOX and HSA due to more protonated HSA groups in the acidic environment compared with those in the neutral environments. In addition, attributed to two phenols and one amine moiety in DOX molecules, the degree of protonation and polarity of DOX could be manipulated by changing pH [44]. Therefore, in the acidic environment, the hydrophilicity and the solubility of protonated-DOX molecules were increased, leading to a higher diffusion rate of DOX from the NPs to the aqueous phase. Notably, in the presence of proteinases at pH 5.5, up to 69.1% of DOX was released after 24 h. This indicates that the drug was selectively released in the proteinase-rich environment of the endosomes/lysosomes. These results indicate that DOX@GNR/PSS/ HSA NPs may not only reduce undesirable DOX leakage into the blood circulation, avoiding potential damage to normal tissues, but also facilitate site-specific drug delivery to the tumor environment and within the cellular endosomes/lysosomes. To investigate the effect of the temperature increase resulting from NIR irradiation on drug release, DOX@GNR/PSS/HSA NPs were irradiated with the 808-nm laser for different time periods. As shown in Fig. 3B, longer laser exposure times yielded greater drug release. Compared with the group without NIR irradiation, exposure to NIR irradiation for 5 min and 15 min resulted in 2.4- and 3.4-times greater DOX release, respectively. Thus, DOX release could be triggered by laser irradiation. The drug release probably occurred because of the elevation of temperature under NIR-irradiation. When the temperature was increased to a higher level than the first melting temperature of HSA (55 °C), parts of the broken helical structure of HSA might expose to the solvent and result in morphological changes and an increase in hydration [45]. Therefore, the outermost HSA coating on the DOX@GNR/PSS/HSA NPs would become more swell and get a loose structure after exposure to NIR irradiation, resulting in the entrapped DOX be released at a faster rate. 3.4. In vitro single- and dual-modality therapy with the prepared NPs Cellular uptake into HeLa cells was observed using fluorescence microscopy, after incubation with DOX@GNR/PSS/HSA NPs (Au conc = 50 μg/mL) for 0.5, 2, and 4 h. As shown in Fig. S3, the red fluorescence signal of DOX was detected in the nuclei of HeLa cells after incubation with DOX@GNR/PSS/HSA NPs, indicating that the DOX@GNR/PSS/HSA NPs had been internalized. The intensity of the DOX fluorescence signal in the nuclei significantly increased with incubation time, indicating that, after internalization, DOX immediately dissociated from the DOX@GNR/PSS/HSA NPs in the endosomes/lysosomes and then diffused into the nuclei. To evaluate in vitro single- and dual-modality therapy effects, the viability of HeLa cells was determined using the MTT assay after treatment of the cells with GNR/CTAB NPs, GNR/PSS/HSA NPs, free DOX, and DOX@GNR/PSS/HSA NPs. The viability of the HeLa cells
3.2. In vitro photothermal measurement Compared with ddH2O, DMEM, PSS, HSA, and DOX solutions, the temperature of the solutions containing GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs increased markedly from 0 °C to 31.6 °C and 791
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Fig. 1. (A) Fabrication of GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs. (B) FTIR spectra of GNR, GNR/PSS, and GNR/PSS/HSA NPs. (C) TEM images of GNR/ CTAB, GNR/PSS, and GNR/PSS/HSA NPs. The scale bar in the images is 50 nm. (D) Zeta potentials at the different adsorption steps for the preparation of GNR/PSS/ HSA NPs. Inset: zeta potentials of GNR/PSS/HSA NPs at pH values of 4.2 and 7.4. (E) UV–vis-NIR spectra of the prepared NPs before and after PSS, HSA, and DOX coating.
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Fig. 2. (A) Photothermal effects of GNR/PSS/HSA NPs, DOX@GNR/PSS/HSA NPs, and HSA, PSS, and DOX aqueous solutions upon NIR irradiation (λ = 808 nm; 1 W/cm² for 5 min). (B) Photothermal effects of DOX@GNR/PSS/HSA NP aqueous dispersions at different GNR concentrations (20, 40, 80, 100, and 150 μg/mL) under NIR-laser irradiation (λ = 808 nm; 1 W/cm²) for 5 min. (C) Real-time temperature measurement of a DOX@GNR/PSS/HSA NP aqueous dispersion (100 μg/ml) under 808-nm NIR-light irradiation with a power density of 1.0 W/cm2 for three cycles. Each cycle consisted of 2 min of irradiation followed by cooling to room temperature.
DOX at equivalent DOX concentrations. This indicates that the efficacies of DOX@GNR/PSS/HSA NPs and free DOX for chemotherapy are the same. To evaluate the photothermal effect of GNR/PSS/HSA NPs on HeLa cells, cells were incubated with GNR/PSS/HSA NPs (100 μg/mL) for 2 h, irradiated with an 808-nm laser for 2 min, and stained with Calcein-AM and EthD-1 to visualize the live and dead cells. Live and dead cells showed green and red fluorescence, respectively. As shown in Fig. 4C, no obvious red fluorescence, for dead cells, was detected after the cells had been treated with only laser irradiation. In contrast, a significant portion of dead cells was observed when the cells have been treated with GNR/PSS/HSA NPs followed by laser irradiation. To investigate the in vitro effect of the combined chemotherapy and PTT, the viabilities of the HeLa cells after treatment with GNR/PSS/ HSA NPs and DOX@GNR/PSS/HSA NPs with or without NIR-laser irradiation were determined using an MTT assay. As shown in Fig. 4D, GNR/PSS/HSA NPs without laser irradiation exhibited no cytotoxicity. In contrast, DOX@GNR/PSS/HSA NPs without laser irradiation showed dose-dependent cytotoxicity. After NIR irradiation for 2 min, the GNR/ PSS/HSA NP-treated cells exhibited significant cell death, especially at
Table 1 The loading capacity of DOX in the prepared DOX@GNR/PSS/HSA NPs. Designed DOX/Au (w/w)
200/280 (0.71)
150/280 (0.54)
100/280 (0.36)
E.E. (%) Final DOX/Au (w/w)
40.7 ± 2 0.43
27.5 ± 3.11 0.22
8.3 ± 0.25 0.04
significantly decreased with increasing GNR/CTAB NPs concentration, which resulted from the toxicity of CTAB (Fig. 4A). Compared with the cell viabilities of the GNR/CTAB NP-treated groups, the cell viability was much higher (> 80%) for the GNR/PSS/HSA NP-treated group. This can be attributed to the fact that, after LbL coating of GNR/CTAB NPs with PSS and HSA, the level of cytotoxicity of the cationic CTAB was significantly decreased. Moreover, a dose-dependent cytotoxicity was observed after incubation with free DOX and DOX@GNR/PSS/HSA NPs for 2 h (Fig. 4B). Notably, the viability of cells treated with DOX@GNR/PSS/HSA NPs was as high as that of cells treated with free
Fig. 3. (A) DOX-release profiles of DOX@GNR/PSS/HSA NPs in buffers of pH 7.4 and 5.5 with or without proteinase K. (B) Percentage of DOX released from DOX@GNR/PSS/HSA NPs under NIR-laser irradiation (λ = 808 nm; 1.4 W/cm²) at different time points. 793
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Fig. 4. (A) Viability of HeLa cells after incubation with GNR/CTAB NPs and GNR/PSS/HSA NPs at different Au concentrations. (B) Viability of HeLa cells after treatment with free DOX and DOX@GNR/PSS/HSA NPs at different DOX concentrations. (C) Fluorescence images of HeLa cells co-stained with ethidium homodimer1 (red, dead cells) and Calcein-AM (green, live cells) and pretreated with DMEM (control) or GNR/PSS/HSA NPs followed by laser irradiation (λ = 808 nm; 1 W/cm² for 2 min). The scale bar in the images is 100 μm. (D) Viability of HeLa cells after treatment with GNR/PSS/HSA NPs and DOX@GNR/PSS/HSA NPs at different Au concentrations with or without laser irradiation (λ = 808 nm; 1 W/cm² for 2 min). *: p < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 794
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Fig. 5. (A) Tumor temperature and (B) infrared thermographic maps at different time points of nude mice bearing HeLa tumors. The mice were injected with GNR/ PSS/HSA NPs and DOX@GNR/PSS/HSA NPs after laser irradiation (808 nm, 1.5 W/cm² for 5 min).
an Au concentration of 25–50 μg/mL, indicating a strong photothermal effect of the prepared NPs on cytotoxicity. When DOX@GNR/PSS/HSA NP-treated cells were irradiated with an NIR laser for 2 min, significant cytotoxicity was observed. This cytotoxicity was not only induced by DOX but also by the localized heat generated by the GNRs upon NIRlaser irradiation. In addition to the heat-induced ablation of cancer cells, heat also could accelerate the release of DOX from the NPs. This may result in an enhanced chemotherapeutic effect.
prognostic factor that is associated with a survival and a response to treatment [47]. Therefore, DOX@GNR/PSS/HSA NPs was shown to be well-tolerated at the given dose and has moderate biocompatibility. For the mice in the group treated with GNR/PSS/HSA NPs followed by NIR irradiation, a significant suppression of tumor growth by day 30 was observed, compared to that for the mice of the PBS-, free DOX-, and DOX@GNR/PSS/HSA NP-treated groups. Interestingly, similar levels of tumor-growth suppression were observed for the group treated with DOX@GNR/PSS/HSA NPs and NIR-laser irradiation and that treated with GNR/PSS/HSA NPs and NIR-laser irradiation during the first 15 days. By day 30, however, a remarkable suppression of tumor growth was observed for the DOX@GNR/PSS/HSA NPs and NIR-laser irradiation treated group. Taken together, our results suggest a synergistic antitumor effect of PTT and chemotherapy including DOX@GNR/PSS/ HSA NPs. Thus, DOX@GNR/PSS/HSA NPs have a high potential of being implemented in future cancer therapeutic strategies.
3.5. In vivo photothermal effect To evaluate the in vivo photothermal effect, the temperatures of tumors with the DOX@GNR/PSS/HSA NP-injected via the tail vein upon 808-nm laser irradiation were measured by using the infrared thermal imaging camera. As shown in Fig. 5, the tumor temperature increased with increased NIR-laser exposure time. After irradiation with a 808-nm laser at a power density of 1.5 W/cm² for 5 min, GNR/PSS/ HSA NP-treated and DOX@GNR/PSS/HSA NP-treated tumors both showed a significant temperature increase from 35 °C to about 52 °C, and the temperature increase was higher than that of tumors treated with free DOX or PBS. If the tumor temperature was maintained at 50 °C – 52 °C for about 5 min, irreversible cellular damage occurred [46]. Therefore, we suggest that GNR/PSS/HSA NPs and DOX@GNR/ PSS/HSA NPs that are prepared by following our protocol can be applied for in vivo PTT for cancer treatment.
4. Conclusions We successfully produced multilayer-coated and DOX-loaded GNR Nanoparticles (DOX@GNR/PSS/HSA NPs) that seem to be a promising platform for in vivo combined cancer PTT and chemotherapy. Compared with unmodified GNRs, the PSS- and HSA-modified GNRs exhibited negligible toxicity and excellent colloidal stability. Besides improving biocompatibility, surface coating of the GNRs with HSA also promoted endocytosis of the NPs and provided a suitable layer for the loading of DOX. In addition, DOX@GNR/PSS/HSA NPs exhibited heat- and pHtriggered and site-specific drug-release behavior, which led to an enhanced antitumor effect at the cellular level. Moreover, DOX@GNR/ PSS/HSA NPs showed synergistic photothermal and chemotherapeutic effects in vivo in a HeLa-tumor-bearing mouse model. This was due to the high photothermal conversion efficiency of DOX@GNR/PSS/HSA NPs under irradiation with NIR light that penetrated deep into the tissue. This study demonstrates the potential of DOX@GNR/PSS/HSA NPs to be used in combined chemotherapy and photothermal therapy anticancer applications.
3.6. In vivo antitumor efficacy Tumor growth was measured in order to examine the in vivo antitumor efficacy of PBS, free DOX, GNR/PSS/HSA NPs, and DOX@GNR/ PSS/HSA NPs with and without NIR-laser irradiation for HeLa tumorbearing mice. The tumor size was normalized to the tumor size at day 0. Fig. 6A showed that DOX@GNR/PSS/HSA NPs only and free DOX only exhibited similar effects on the suppression of tumor growth, which was consistent with the in vitro cytotoxicity results shown in Fig. 6B. However, significant loss of body weight was observed for the administration of free DOX only and not for the administration of DOX@GNR/ PSS/HSA NPs (Fig. 6B). Significant Loss of body weight is a major
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Fig. 6. (A) Tumor volumes and (B) body weights of nude mice bearing HeLa tumors that were treated with PBS, free DOX, GNR/PSS/HSA NPs, or DOX@GNR/PSS/ HSA NPs with or without laser irradiation (808 nm, 1.5 W/cm², 5 min). *: p < 0.05.
Funding sources
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