Journal of Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Materials Chemistry B View Article Online
PAPER
Cite this: DOI: 10.1039/d0tb01915a
View Journal
A tumor-targeted theranostic nanomedicine with strong absorption in the NIR-II biowindow for image-guided multi-gradient therapy† Qi Chen,‡a Ziliang Zheng,‡a Xiaojing He,‡a Shuo Rong,a Yufei Qin,a Xiaoyang Penga and Ruiping Zhang *b Developing new strategies to enhance drug accumulation in the tumor and therapeutic efficacy is of great importance in the field of tumor therapy. Herein, a peanut-like multifunctional nanomedicine (CuS-PGH NMs) made of CuS nanoparticles encapsulated in poly(L-lysine)(PLL)/glucose oxidase (GOx)hyaluronic acid (HA) shells has been constructed via layer-by-layer (LbL) assembly, and shows good biocompatibility and effective multi-gradient therapy. Because of the enhanced permeability and retention (EPR) effect, the CuS-PGH NMs could significantly enhance the cellular uptake by tumors overexpressing CD44 receptors, which respond to hyaluronidase (HAase)-triggered surface charge conversion. Once internalized by the tumor, GOx was the first to be exposed and could effectively deplete endogenous glucose for starvation therapy, and the excess H2O2 was then converted into highly toxic hydroxyl radicals ( OH) via a Cu+-mediated Fenton-like reaction for chemodynamic therapy (CDT). Meanwhile, the as-obtained Cu+ ions accompanied the regenerated less-active Cu2+ ions. Interestingly, the high content of H2O2 could, in turn, accelerate Cu2+/Cu+ conversion to promote the Cu+–H2O2 reaction for enhanced chemodynamic therapy (CDT), thereby achieving efficient tumor growth suppression via synergistic starvation/CDT therapy. Subsequently, owing to the strong NIR-II absorption
Received 8th August 2020, Accepted 15th September 2020
capability of CuS-PGH NMs, effective photothermal tumor ablation of the weakened tumor cells could
DOI: 10.1039/d0tb01915a
be realized with the precise guidance of NIR-II PAI. This multi-gradient therapeutic strategy has been demonstrated to have excellent antitumor activity with minimal nonspecific damages, and offers a new
rsc.li/materials-b
avenue to precise tumor therapy.
1. Introduction Breast cancer is associated with relatively high mortality rates among women worldwide. Especially triple-negative breast cancer (TNBC), which is one of the breast cancer subtypes, presents the worst survival rate due to its highly aggressive and metastatic behavior.1–3 Current treatments for TNBC patients mainly depend on traditional surgical operation, radiotherapy or chemotherapy, and often involve issues of non-specific distribution, drug resistance and systemic side effects.4,5 For these reasons, the development of targeted diagnostic and therapeutic platforms is an urgent need for effective TNBC therapy. a
Department of biochemistry and molecular biology, Shanxi Medical University, Taiyuan 030001, China b Department of Radiology, Third hospital of Shanxi Medical University, Taiyuan 030032, China. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/ d0tb01915a ‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2020
Photothermal therapy (PTT), a new type of physiotherapy, has attracted increasing interest in the field of cancer treatment.6–9 PTT, utilizing photothermal nanoagents, exhibits good tumor-specific localization by converting the absorbed near-infrared (NIR) laser energy into heat and therefore, efficiently ablates cancer cells and tissues with minimal invasiveness.10–12 Notably, severe hypoxia and low pH in the tumor microenvironment (TME) make the cancer cells more sensitive to heat than normal cells.13,14 Second near-infrared (NIR-II, 900–1700 nm) light has many advantages compared with traditional first near-infrared light, such as deeper penetration of biological tissues, less tissue scattering or absorption, higher signal-to-noise ratio and lesser interference of fluorescent proteins.15,16 Furthermore, nanoagents that absorb light in the NIR-II window combine well with photoacoustic imaging (PAI) and result in high-performance photothermal treatment outcomes.17–20 Therefore, NIR-II PAI could achieve high-spatialresolution diagnosis to guide tumor-depth PTT. It is well-known that diagnosis and treatment effects can be achieved only with a certain deposition amount due to the
J. Mater. Chem. B
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Paper
Journal of Materials Chemistry B
dynamic equilibrium between the uptake and excretion rates of nanomedicines at the tumor site.21 Therefore, the prerequisite for accurate tumor imaging and therapeutic effects is the efficient delivery of a multifunctional agent into the cancer cells.22,23 One of the feasible delivery approaches is the construction of nanomedicines, which can be significantly enriched at the tumor sites through the acknowledged enhanced permeability and retention (EPR) effect.24 However, when the delivery relies solely on this intrinsic characteristic of nanomedicine through the EPR effect, it exhibits insufficient accumulation and inadequate tumor retention, which inevitably leads to a suboptimal therapeutic effect.25,26 As a desirable strategy to solve this issue, stimuli-responsive nanomedicines, which perform the theranostics function only after specific activation at the tumor sites and remain in a ‘‘silent state’’ in non-neoplastic lesions, have been exploited to achieve precision anti-tumor efficacy and decrease the adverse effects.27,28 Bao and co-workers29 constructed a nanolongan delivery system, which could reverse the negative surface charge to positive at the tumor sites by dimethylmaleic anhydride (DMMA) decoration, resulting in ferroptosis-apoptosis combined anticancer therapy. Chen et al.30 obtained ICG/TPZ@HSA dNMs that could efficiently eradicate the tumors through a cascade of synergistic events triggered by sequential laser irradiation in the tumor area. Hence, the rational design of stimuli-responsive nanomedicines is crucial for enhancing efficient uptake by the tumor cells and thus achieving excellent antitumor performance. Based on the above principles, we have developed a novel multifunctional nanomedicine (CuS-PGH NMs), which shows excellent efficacy in tumor eradication through a multi-gradient therapy (two-step treatment, including synergistic starvation/ CDT therapy and NIR-II PAI-guided PTT) without detectable
Scheme 1
side effects. By adopting the layer-by-layer (LBL) technique, CuS-BSA nanoparticles were encapsulated in poly(L-lysine) (PLL) through electrostatic attraction and further decorated with glucose oxidase (GOx) and a hyaluronic acid (HA) shell (Scheme 1). In detail, the shielding effect of the HA shell would ensure prolonged circulation time and provide more opportunities for CuS-PGH NMs to reach the tumor region via the EPR effect. Meanwhile, the active-targeting effect would enhance the retention and internalization of CuS-PGH via the specific interaction of HA with its primary receptor CD44, which is over-expressed in TNBC cells. In the tumor regions, the HA shell of CuS-PGH NMs decomposed, reversing the surface charge and exposing GOx. GOx then consumed the endogenous glucose for starvation therapy by catalyzing its oxidation to generate gluconic acid and H2O2 (eqn (1)). The elevated H2O2 levels led to the reduction of Cu2+ to Cu+ ions, due to which the CuS-PGH NMs exhibited excellent Fenton-like reaction ability, generating highly toxic OH from excess H2O2 (Eqn (2) and (3)). Therefore, simultaneously producing OH and accelerating Cu2+/Cu+ conversion in response to the high H2O2 levels in the TME endows CuS-PGH NMs with the ability for CDT, thereby leading to efficient tumor growth suppression in combination with starvation therapy. Based on these, the strong NIR-II absorption capability of CuS-PGH NMs offers complete tumor ablation and enables the use of NIR-II PAI for diagnosis and PTT guidance. We believe this work provides a novel multi-gradient therapeutic strategy to achieve highly efficient tumor treatment. GOx
Glusoce þ H2 O þ O2 ! Glucinicacid þ H2 O2
(1)
Cu2+ + H2O2 - Cu+ + H+ + HO2
(2)
Cu + H2O2 - Cu + OH + OH
(3)
+
+
Schematic illustration of the construction of CuS-PGH NMs for multi-gradient antitumor therapy.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2020
View Article Online
Journal of Materials Chemistry B
2. Materials and methods Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
2.1.
Materials
Bovine serum albumin (BSA) was obtained from Acmec Biochemical (Shanghai, China). Copper nitrate trihydrate (Cu (NO3)23H2O), sodium sulfide nonahydrate (Na2S9H2O), polyL-lysine (Mw 2000–5000), glucose oxidase (GOx), and hyaluronic acid (HA) were acquired from Aladdin (Shanghai, China). Sodium hydroxide (NaOH), propidium iodide (PI) and calcein acetoxymethyl ester (Calcein–AM) were purchased from SigmaAldrich. Reagent-grade water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA) for all the experiments. All chemicals used here were of analytical grade and used without further purification. 2.2.
Synthesis of CuS@BSA (CuS)
The CuS@BSA nanoparticles were prepared in an aqueous solution at 90 1C according to the biomineralization strategy. Typically, aqueous Cu(NO3)23H2O solution (0.025 mmol, 8 mL) and BSA solution (30 mg mL1, 8 mL) were mixed in a onenecked flask (25 mL) with magnetic stirring. After rapid mixing, the mixture instantly showed white turbidity, but immediately turned into a light-yellow solution. Five minutes later, the pH of the system was adjusted to 12 using a 2 M NaOH solution, and the mixture turned transparent and deep purple. Afterwards, 400 mL of Na2S9H2O (242.16 mg mL1) was quickly injected into the above system, and the solution turned deep brown. The reactor was placed in a constant-temperature oil bath with a magnetic stirrer and maintain at a temperature of 90 1C for 30 minutes. Then, the CuS synthesis experiment ended when the solution changed black in colour. The solution was transferred into an ultrafiltration centrifuge tube and centrifuged at 3500 rpm for 15 minutes. After repeating the above operation 3 to 5 times with pure water, the solution was freeze-dried, sealed, and stored. It is also feasible to dissolve the lyophilized powder of CuS in pure water. 2.3.
Preparation of CuS@BSA-PLL/GOx/HA (CuS-PGH NMs)
The CuS nanoparticles obtained in the previous step were dissolved in pure water at a concentration of 1 mg mL1. Then, 5 mL of the above solution was added dropwise to a PLL solution (1 mg mL1, 5 mL) at a constant rate. Excess PLL was removed by centrifugation at 3000 rpm for 10 min and re-dissolved in 5 mL pure water. An aqueous GOx solution (1 mg mL1) and a HA solution (1 mg mL1) were then mixed in the ratio of 1 : 1. This mixed solution was very slowly added dropwise to the re-dissolved CuS@BSA-PLL solution in the ratio of 3 : 5. After centrifuging again at 3000 rpm for 10 min to remove excess HA and GOx, CuS-PGH NMs were obtained. It is able to dissolve the lyophilized powder in pure water for later experiments. 2.4.
Material characterization
The TEM images, element mapping images and EDS linescanning of the CuS-PGH NMs were obtained using a Tecnai G2 F20 S-twin transmission electron microscope (FEI). X-ray photoelectron spectroscopy (XPS) was performed with a V.G.
This journal is © The Royal Society of Chemistry 2020
Paper Scientific ESCALAB250 spectrometer using Al Ka radiation (1486.6 eV, 150 W). Dynamic light scattering (Nano ZS, Malvern) was used to record the hydrodynamic size distribution and z-potential of the CuS-PGH NMs. Fourier-transform infrared (FT-IR) spectroscopy was performed on a Vertex PerkinElmer 580BIR spectrophotometer (Bruker) using the KBr pellet technique to identify the bonds and quantify their relative abundance. The UV-vis absorption of the samples was obtained using a TU-1901 dual-beam UV-vis spectrophotometer (PerkinElmer). 2.5. Measurement of photothermal effect of CuS-PGH NM in aqueous solution A 1064 nm laser was used for the photothermal effect measurements and the following in vitro/in vivo PTT experiments. Aqueous CuS-PGH NM solutions (200 mL) at a series of concentrations (0.0625, 0.125, 0.25, 0.5, and 1 mg mL1) were placed in Eppendorf tubes and exposed to the NIR laser (1064 nm, 1.0 W cm2) for 5 min. Simultaneously, an infrared camera (Fluke Ti400) was used to take pictures and record the real-time temperature of the liquid at 15 s intervals. Photothermal stability cycling tests were also conducted repeatedly to observe the temperature increase induced by the laser with a 0.25 mg mL1 CuS-PGH NM aqueous solution. Chicken breast meat slices of different thicknesses (4 mm, 6 mm, 8 mm) were used to verify the penetration effect and photothermal effect when the CuS-PGH NMs aqueous solution (0.25 mg mL1) was irradiated by the 1064 nm laser. 2.6.
In vitro degradation and response test
The pH values of the solution after the addition of HAase and glucose were measured over time since GOx can catalyze the oxidation of glucose to gluconic acid. Glucose was added to 5 mL of the CuS-PGH NM solution or the CuS-PH NM solution (0.25 mg mL1) to a final glucose concentration of 0.5 mg mL1. Then, the pH value of each solution was measured over time. Considering that H2O2 is produced when GOx reacts with glucose, Micro Hydrogen Peroxide (H2O2) Assay Kit (Solarbio, Beijing, China) was used to quantify the amount of H2O2. We used methylene blue (MB) to verify the production of OH. CuSPGH NMs (0.1 mg mL1), H2O2 (1 mM) and MB (0.01 mg mL1) were prepared in a 25 mM NaHCO3 solution to a total volume of 1 mL. Then, the reaction system was shaken in darkness at 37 1C. At different time points, the UV-vis absorption curves of the mixture were tested and recorded. 2.7. In vitro cytotoxicity of CuS-PGH NMs and photothermal therapy assays A standard Cell Counting Kit (CCK8) (Sigma-Aldrich, St. Louis, MO, USA) assay was conducted using a breast cancer cell line (4T1) to evaluate the in vitro cytotoxicity of CuS-PGH NMs. Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% (v/v) fetal bovine serum (FBS) and 1% antibiotics (penicillin–streptomycin) (Corning) at 37 1C under a 5% CO2 atmosphere. Typically, 4T1 cells (8 103/well) were seeded into 96-well plates, and then, the cells were incubated in the culture medium for 24 h at 37 1C. The culture
J. Mater. Chem. B
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Paper medium was then removed, and the cells were incubated with 100 mL of fresh medium containing CuS-PGH NMs at different concentrations (0.0625, 0.125, 0.25, 0.5 and 1 mg mL1) for 24 h. The CCK-8 solution (10 mL, 5 mg mL1) was added into the plates along with the culture medium, and the cells were incubated for another 2 h. Finally, the OD450 value (abs.) of each well was measured using a microplate reader (BioTek Epoch 2). In another set of experiments, after incubating with different concentrations of CuS-PGH NMs, the 4T1 cells were exposed to a 1064 nm laser at an irradiation density of 1 W cm2 for 5 min. The above CCK-8 method was used to observe the in vitro phototherapeutic efficacy of the nanoparticles and cell viability at different glucose concentrations (0.125, 0.25, 0.5, 1 and 2 mg mL1) for verifying GOx function. 2.8.
Animal model
All animal experimental procedures were performed in compliance with the criteria of the National Regulation of China for Care and Use of Laboratory Animals and Animal Use Committee of Shanxi Medical University (Approval No. 2016LL141, Taiyuan,
Journal of Materials Chemistry B China). Tumor-bearing mouse models were established by injecting 4T1 cells (1 106) subcutaneously into the right thigh of female nude mice (6–8 weeks old, bodyweight 18–22 g), which were purchased from Weitong Lihua Experiment Animal Technology Co. Ltd (Beijing) and acclimatized to the animal facility for at least 7 days. Tumor-bearing nude mice were used in experiments when the tumor volume approached 50–100 mm3. 2.9.
PAI imaging
The in vitro PAI property was examined using a real-time multispectral optoacoustic tomographic (MSOT) imaging system. The phantom was filled with the CuS-PGH NMs aqueous solution at different concentrations (0, 0.0625, 0.125, 0.25, 0.5 and 1 mg mL1) and suspended inside a water tank and imaged under a continuous Nd:YAG laser. For in vivo PAI, the tumor-bearing nude mice were anesthetized by isoflurane inhalation and administered CuS-PGH NMs dispersed in 200 mL of PBS via the tail vein. PAI signals at different time points (after injection for 1, 4, 6, 8, 10, and 12 h) were acquired using a real-time multispectral optoacoustic tomographic (MSOT) imaging system.
Fig. 1 (a and b)The TEM images, (c) the corresponding elemental mapping images and the (d) Cu 2p and (e) S 2p XPS spectra of CuS-PGH NMs. The (f) DLS distribution and zeta potential trend, (g) the FT-IR spectra, and (h) the absorption spectra (in the wavelength range of 200–1000 nm and 1000–1400 nm) of CuS, CuS-P and CuS-PGH NMs.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2020
View Article Online
Journal of Materials Chemistry B
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
2.10.
In vivo photothermal imaging and PTT
When the tumor volume reached B50 mm3, the 4T1 tumorbearing nude mice were randomly divided into four groups (n = 5, in each group) (dose = 15 mg kg1): (1) PBS + laser, (2) CuS-PGH, (3) CuS + laser, and (4) CuS-PGH + laser. After 6 h of intravenous injection, the tumor areas of the (1), (3) and (4) groups were exposed to a 1064 nm laser (1 W cm2, 5 min). An infrared thermal camera (Fluke Ti400) was used to monitor the temperature changes at the tumor sites during NIR-II irradiation.
3. Results and discussion A peanut-like structure with multiple CuS nanoparticles (about 6–8 nm in diameter) uniformly embedded in HA shells could be observed visually in the transmission electron microscopy (TEM) images of CuS-PGH NMs (Fig. 1a and b). Moreover, it clearly revealed the lattice fringes of d103 = 0.29 nm and d107 = 0.18 nm, which were in agreement with those of the hexagonal CuS phase.31,32 The elemental mapping images of CuS-PGH NMs in Fig. 1c indicate the presence of the corresponding elements (Cu and S) in the sample. As seen in Fig. 1d and e, X-ray photoelectron spectroscopic (XPS) analyses were used to determine the components and chemical states of Cu and S in CuS-PGH NMs. The intense peaks at the binding energies of 932.5 (Cu 2p3/2) and 952.4 eV (Cu 2p1/2) could be assigned to
Paper Cu2+ from CuS.28 In the XPS spectrum of S element, different valence states of S (peaks at 163.7 eV and 168.2 eV) were identified in the NMs, which could be attributed to the S2 state of CuS and the disulfide bonds (–S–S–) of BSA, respectively.33 Moreover, CuS-PGH NMs were characterized by X-ray diffraction (XRD) to confirm the crystal-structure (Fig. S1, ESI†). Although the diffraction peaks of XRD were relatively weak, they could be well indexed to the CuS standard peaks (JCPDS No. 06-0464), further indicating the successful formation of CuS in CuS-PGH NMs. To analyze the structural properties of CuS-PGH NMs, dynamic light scattering (DLS) and zeta potentials were recorded to track the assembly process (LBL technique) (Fig. 1f). The average hydrodynamic diameter gradually increased from 19.8 nm to 45.7 nm with the systematic modification with PLL(+) and GOx/HA(), confirming the successful assembly of the CuS-PGH NMs. The zeta potential of the nanoparticles decreased from 27.1 mV to 10.6 mV after the modification with the negatively charged GOx and HA. Although the peak of the point was in the positive charge region, it had already crossed the point of electrical neutrality, which indicated that the electrostatic force was so weak that only a small amount of negatively charged proteins in the body can attach to the surface of the nanoparticles.34 In addition, after soaking CuS-PGH NMs in various solutions, including water, PBS, saline and the cell growth medium, for 24 h, the solutions were still uniform and translucent, indicating good
Fig. 2 (a) Photothermal heating curves of CuS-PGH NMs at different concentrations under laser irradiation (1064 nm, 1 W cm2) as a function of time (0–300 s). (b) Photothermal stability of CuS-PGH NMs (0.25 mg mL1). (c) The heating curves and (d) the infrared thermal images of CuS-PGH NMs (0.25 mg mL1) placed below chicken meat of varying thicknesses under laser irradiation (1064 nm, 1 W cm2).
This journal is © The Royal Society of Chemistry 2020
J. Mater. Chem. B
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Paper dispersion and stability of CuS-PGH NMs (Fig. S2, ESI†). The TEM images demonstrated the difference between CuS and CuS-PGH NMs (Fig. S3, ESI†) as the morphology and size of the samples changed during the LbL assembly process. Moreover, FT-IR spectroscopy was carried out to monitor the change in material configuration during the formation of CuS, CuS-P and CuS-PGH NMs (Fig. 1g). It could be distinctly observed that the bands for N–H (bends) at 1397 cm1 underwent remarkable attenuation since PLL and GOx/HA wrapped the nanoparticle core, and the peaks of BSA were less prominent than those in the initial state.35 In brief, the positively charged PLL layer and the negatively charged GOx/HA layer sequentially encapsulated the CuS nanoparticles via layer-by-layer assembly (LbL) to form CuS-PGH NMs. The optical property of CuS-PGH NMs and its two predecessor nanoparticles was evaluated based on their UV absorption spectra. As displayed in Fig. 1h, the absorption curve (600– 1000 nm) exhibited a good upward trend, and we chose another near-infrared scanner with a longer wavelength range, which
Journal of Materials Chemistry B could scan absorption in the NIR-II wavelength range. The CuS-PGH NMs showed a broad absorption band in the wavelength region of 1000–1400 nm (inset Fig. 1h), which is very beneficial for PAI and PTT in the NIR-II region. Encouraged by the absorption characteristics of CuS-PGH NMs, a 1064 nm laser was chosen to investigate the PTT properties of solutions at different concentrations (0.0625, 0.125, 0.25, 0.5, and 1 mg mL1). As expected, after irradiation (1.0 W cm2) for 5 min (Fig. 2a), the temperature of the CuSPGH NMs solution increased rapidly even at a rather low concentration (0.125 mg mL1) in a concentration-dependent manner. Comparatively, only a negligible increment was observed when pure water was used in parallel. Moreover, CuS-PGH NMs possessed high photothermal stability, and no attenuation of photothermal efficiency was observed even after five cycles of switching (Fig. 2b). It is widely believed that a NIR-II wavelength laser can provide deeper penetration and higher contrast compared with a NIR-I laser. During the measurements, we further exploited the photothermal effect
Fig. 3 (a) The pH values as a function of reaction time and (b) H2O2 production arising from the CuS-PGH NM-catalyzed decomposition reaction of glucose. UV/vis spectra of the MB aqueous solution after degradation by (c) CuS-PGH NM-mediated Fenton-like reaction and (d) by CuS-PGH NMs and glucose at different time points; [CuS-PGH NMs] = 0.1 mg mL1; [H2O2] = 1 mM, [glucose] = 0.5 mg mL1. (e) 4T1 cell viability after treatment with various concentrations of glucose or glucose along with CuS-PGH NMs (0.25 mg mL1) for 12 h. (f) Cell viability of 4T1 cells after incubation with gradient concentrations of CuS-PGH NMs and CuS-PGH NMs + Laser in DMEM medium containing 0.5 mg mL1 glucose (1064 nm, 1 W cm2, 5 min). (g) Micrographs of 4T1 cells incubated with CuS-PGH NMs (0.25 mg mL1) before and after irradiation with a 1064 nm laser (1 W cm2); dead cells are stained with trypan blue. ***P o 0.001.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2020
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Journal of Materials Chemistry B of CuS-PGH NMs in deep tissues by using the chicken breast tissues of different thicknesses (4, 6, and 8 mm) as models of biological tissues. The chicken breast tissue was placed between the laser source and the CuS-PGH NMs solution (Fig. S4 ESI†). As illustrated in Fig. 2c, the temperatures of all the CuS-PGH NMs solutions increased to around 60 1C within 600 s of 1064 nm laser irradiation and reached over 43.8 1C after a rather short irradiation time of less than 160 s. Considering the threshold temperature for PTT (43 1C), the observed temperature increment exceeded the temperature rise required (13 1C) to ablate tumor cells in living mice.36,37 More importantly, no obvious temperature change was recorded in the tissue after irradiation (Fig. 2d). After verifying the encapsulation of GOx in the CuS-PGH NMs, the catalytic activity of the immobilized GOx was evaluated by detecting pH variation and H2O2 generation since GOx can catalyze the oxidation of glucose to gluconic acid and H2O2. As shown in Fig. 3a, a significant drop in the pH of the CuS-PGH NMs solution (from 6.7 to 3.4) was observed after treatment with glucose (0.5 mg mL1) for 1.5 h. However, the pH change in the sample solution without GOx was much smaller (from 6.9 to 6.5) than that of the GOx-loaded sample under the same circumstance. Titanium sulfate was selected to evaluate the efficiency of H2O2 production by measuring the fluorescent titanium peroxide complex (Fig. 3b).38 The generated H2O2 quickly reached the maximum in 1 h, indicating that CuS-PGH NMs possessed high GOx catalytic activity. Remarkably, the production of H2O2 gradually decreased after 1 h, which indicates the possible conversion of H2O2. As reported previously, the Cu+ ion could react with H2O2 to produce OH
Paper through a Fenton-like reaction, which could effectively induce cancer cell apoptosis.39,40 Hence, the methylene blue (MB) degradation experiment was performed to detect the generation of hydroxyl radicals ( OH). When CuS-PGH NMs and excess H2O2 were added, the absorbance of MB solution at 650 nm showed an obvious decrease with increasing reaction duration (Fig. 3c), manifesting that H2O2 was converted to OH due to the Fenton-like catalytic ability of Cu+.41,42 Moreover, MB was partly degraded in the presence of CuS-PGH NMs and glucose (Fig. 3d) due to glucose depletion and the Fenton-like reaction. These results indicated that the CuS-PGH NMs could mediate a cascade of reactions, including GOx-triggered glucose oxidation and then the Cu+-mediated Fenton-like reaction. Based on the above results, 4T1 cells were used to test the cascaded multi-gradient therapeutic performance of CuS-PGH NMs in vitro. As shown in Fig. 3e, glucose was added as an energy source for cell growth and proliferation. However, the cell activity dramatically decreased with the addition of CuSPGH NMs due to GOx-triggered glucose-depletion for cell starvation therapy and the generation of Cu+-mediated OH for CDT. This result demonstrated that the cell starvation as the first stage in the cascade of multi-gradient therapy could undoubtedly lead to a reduction in cell viability. However, CuS-PGH NMs exhibited negligible cytotoxicity against the tumor cells over the range of experimental concentrations in DMEM medium containing 0.5 mg mL1 glucose, indicating that the multi-gradient synergistic therapeutic effect was indeed weak at low concentrations of glucose (Fig. 3f). In comparison, significant cell apoptosis and death were caused by the increased concentration of CuS-PGH NMs. Notably, CuS-PGH
Fig. 4 (a) The PAI mapping of CuS-PGH NMs at different concentrations. (b) The photoacoustic spectrum of the CuS-PGH NMs solution between 660 and 1150 nm wavelengths. (c) Signal intensity changes and (d) PAI images of 4T1 tumor-bearing mice after CuS or CuS-PGH NMs injection up to 12 h under 1064 nm laser irradiation. ***P o 0.001.
This journal is © The Royal Society of Chemistry 2020
J. Mater. Chem. B
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Paper NMs concentration of 0.25 mg mL1 resulted in low cell viability, demonstrating the potential minimum effective concentration of CuS-PGH NMs for the tumor cell killing. To further demonstrate the capability of CuS-PGH NMs in generating ROS in vitro, cellular ROS was determined with DCFH-DA dyes as they are converted to fluorescent DCF via ROS oxidation (Fig. S5, ESI†). The intercellular ROS levels increased dramatically in CuS-PGH NM-stimulated cells compared with control cells. Further, the fluorescence of the CuS-PGH group was 2 times as bright as the CuS group. This indicated an increase in cellular oxidative stress and that the total ROS level increased sharply, which further promoted CDT. Moreover, the cells incubated with 0.25 mg mL1 CuS-PGH NMs were almost entirely destroyed after laser irradiation, as seen in Fig. 3g, demonstrating that the multi-gradient ablation of tumor cells could be achieved by the combined utilization of CuS-PGH NMs and laser for further anti-tumor applications. The PAI signals of CuS-PGH NMs were monitored to evaluate their photoacoustic properties in vitro. As shown in Fig. 4a, the PAI signals exhibited a downward trend as the concentration of the solution decreased. This feature is also seen in the upper right corner of the PAI images. Moreover, the PAI data of the
Journal of Materials Chemistry B medium density condition (0.25 mg mL1) was selected to process the photoacoustic spectrum of CuS-PGH NMs (Fig. 4b), and the overall trend was roughly the same as the UV absorption curve at the wavelength of 1000 nm (Fig. 2a). Based on the above results, the in vivo PAI capability of CuSPGH NMs was tested in a subcutaneous 4T1 tumor model, and the PAI images of the tumors were longitudinally recorded and quantified at 0 h, 1 h, 4 h, 6 h, 10 h and 12 h post-injection. As shown in Fig. 4c and d, the PAI intensities of CuS and CuS-PGH NMs gradually enhanced over time and displayed maximal retention at 6 h post-injection. Noticeably, CuS only showed low accumulation in a limited region and failed to cover the entire tumor site. Meanwhile, after intravenous injection with CuS-PGH NMs, the PAI signals did not only accumulated efficiently in the tumor, but also exhibited a much higher contrast throughout the tumor than the surrounding tissues. This phenomenon confirmed that the increased PAI signals from CuS-PGH NMs at the tumor site may arise from HAmediated recognition and HAase-responsive reverse charging to facilitate tumor uptake. Moreover, the semiquantitative analysis of PAI further revealed that CuS-PGH NMs acted as an excellent contrast agent and were able to penetrate deep into
Fig. 5 In vivo antitumor study. (a) IRT images and (b) corresponding temperature curves of tumor-bearing mice injected with PBS, CuS or CuS-PGH NMs against time under NIR-laser irradiation (1064 nm, 1 W cm2). (c) Representative photographs of the mice in the different groups after treatment. (d) Relative body weight of mice in the different groups after their treatment. (e) H&E staining of the tumor tissues collected from mice at the end of treatment. Scale bar: 100 mm. n.s. = not significant.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2020
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Journal of Materials Chemistry B the tumor cells, which helps identify the tumor region for guided diagnosis and treatment. The highly efficient PTT effect and the superb targeting ability of CuS-PGH NMs with PAI guidance enabled the evaluation of the in vivo antitumor performance on tumor-bearing nude mice. The 4T1 tumor-bearing nude mice were intravenously administered PBS, CuS and CuS-PGH NMs (15 mg kg1) and subjected to a 1064 nm NIR laser (1 W cm2) for 5 min at 6 h post-injection. Based on the real-time thermal images recorded using an IR thermal camera (Fig. 5a and b), the temperature in the tumor region had increased by 20 1C in the presence of CuS-PGH NMs upon laser irradiation, which was much higher than those of the CuS (11.5 1C) and PBS (4 1C) groups. The difference in temperature variation was attributed to the high photothermal conversion efficiency of CuS and the efficient tumor accumulation of CuS-PGH NMs. The change in tumor volume is a direct indicator of the therapeutic effect, which is shown in Fig. 5c and Fig. S6 (ESI†). The tumor-bearing nude mice were randomly divided into four groups, including (1) PBS + laser, (2) CuS-PGH, (3) CuS + laser, and (4) CuS-PGH + laser. At 6 h after the intravenous injection, the tumor areas of the (1), (3) and (4) groups were exposed to a 1064 nm laser for 5 min (1 W cm2). The growth of tumors in the CuS-PGH group could be inhibited, revealing the high efficiency of the synergistic starvation/CDT therapy. Owing to higher accumulation in the tumor, the treatment effects in the CuS-PGH NMs + laser group were greater than that in the CuS + laser group, and the tumor even disappeared thoroughly on the 11th-day post-treatment. Such effective therapeutic efficacy of CuS-PGH NMs in the presence of laser could be attributed to the photothermal therapy that further promoted glucose depletion and the cascading production of OH due to the wellretained catalytic activities of both GOx and CuS. Therefore, the photothermal therapy with the assistance of starvation and CDT using highly concentrated CuS-PGH NMs at the tumor site was sufficient for tumor ablation. In addition, the bodyweight of all the tested groups remained stable throughout the experiment, suggesting no systemic side effects after the treatment (Fig. 5d). The superior antitumor efficacy of CuS-PGH NMs is attributed to the multistage therapy that leads to a cascade of reactions. In the process of the reactions, the exposed GOx accelerated the consumption of intratumoral glucose, achieving starvation therapy. Notably, the excess H2O2 generated by glucose oxidation was then converted into highly toxic OH via a Cu+-mediated Fenton-like reaction, thereby accomplishing CDT. This cascade reaction-enhanced synergistic starvation and CDT effect demonstrated high-efficiency suppression of tumor cell activity. Ultimately, the photothermal performance of CuS-PGH NMs induced apoptosis and necrosis of the tumor cells under 1064 nm laser irradiation, resulting in excellent antitumor effects. H&E staining at the tumor tissue level further verified the cancer cell killing capability of CuS-PGH NMs. Consistent with the photothermal results, the highest apoptosis of tumor cells was shown by the CuS-PGH NMs + laser group compared with other treatment groups (Fig. 5e). The histological observations suggested that increased apoptosis may account for
This journal is © The Royal Society of Chemistry 2020
Paper tumor growth inhibition, which is consistent with the variation in the tumor growth curve. Moreover, major organs (heart, liver, spleen, lung, and kidney) of the mice were collected after treatment and sliced for H&E staining and examination. As shown in Fig. S7 (ESI†), the H&E staining images indicated no noticeable signs of toxic side effects. The above experimental results indicated that CuS-PGH NMs exhibit good biosafety and excellent antitumor effects and can serve as a novel nano-theranostic agent for cancer therapy.
4. Conclusion In summary, a theranostic agent with a novel peanut-like structure, CuS-PGH NMs, was developed for efficient NIR-II PAI-guided multistage therapy through LBL assembly. The charge conversion and specific recognition of CD44 by the HA shell did not only provide long circulation time for CuS-PGH NMs and active tumortargeting ability, but also enabled its high accumulation and retention at the tumor site. After HA degradation in the tumor microenvironment, GOx consumed glucose and produced H2O2 for starvation therapy, and H2O2 further reduced Cu2+ to Cu+ via a Fenton-like reaction. Meanwhile, the Cu+ ions effectively catalyzed the decomposition of H2O2 into highly toxic OH, thus presenting a distinctive cascade reaction-enhanced synergistic starvation and CDT effect for tumor suppression. Subsequently, under the guidance of NIR-II PAI, the CuS core endowed the CuS-PGH NMs with NIR-II photothermal conversion ability, which raises the tumor temperature, ensuring the thorough killing of the tumor cells by PTT. Moreover, PTT could also highly enhance Fenton-like activity to accomplish CDT. Based on high tumor accumulation, biocompatibility and the stimuli-responsive behavior of CuS-PGH NMs, this work provides an innovative strategy for effective cancer treatment by a cascade effect, which might have promising value in future clinical practices.
Conflicts of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81571747, 81771907), Science and technology innovation team project of Shanxi Province (No. 201705D131026), Engineering Technology Research Center of Shanxi Province (No. 201805D121008), Scientific and technological achievements transformation project of Shanxi Province (No. 201704D131006), Laboratory Construction Project of Shanxi Province, The Projects for Local Science and Technology Development Guided by the Central Committee (YDZX20191400002537), Scientific and Technologial Innovation Programs of Higher Education Institutions in Shanxi (2019L0415), Shanxi Province Science Foundation for Youths (201901D211343).
J. Mater. Chem. B
View Article Online
Paper
Journal of Materials Chemistry B
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
References 1 R. L. Siegel, K. D. Miller and A. Jemal, Ca-Cancer J. Clin., 2016, 66, 7–30. 2 L. M. Heiser, G. B. Mills and J. W. Gray, Cancer Cell, 2019, 35, 341–343. 3 A. M. Brewster, M. Chavez-MacGregor and P. Brown, Lancet Oncol., 2014, 15, e625–e634. 4 W. Chen, C. A. Glackin, M. A. Horwitz and J. I. Zink, Acc. Chem. Res., 2019, 52, 1531–1542. 5 J. Park, Y. Choi, H. Chang, W. Um, J. H. Ryu and I. C. Kwon, Theranostics, 2019, 9, 8073–8090. 6 Y. Liu, P. Bhattarai, Z. Dai and X. Chen, Chem. Soc. Rev., 2019, 48, 2053–2108. 7 S. He, Y. Jiang, J. Li and K. Pu, Angew. Chem., Int. Ed., 2020, 59, 10633–10638. 8 J. Li, D. Cui, J. Huang, S. He, Z. Yang, Y. Zhang, Y. Luo and K. Pu, Angew. Chem. Int. Ed., 2019, 58, 12680–12687. 9 X. Zhen, P. Cheng and K. Pu, Small, 2019, 15, e1804105. 10 W. Fan, B. Yung, P. Huang and X. Chen, Chem. Rev., 2017, 117, 13566–13638. 11 X. Zhen, C. Xie and K. Pu, Angew. Chem., Int. Ed., 2018, 57, 3938–3942. 12 Z. Li, H. Xu, J. Shao, C. Jiang, F. Zhang, J. Lin, H. Zhang, J. Li and P. Huang, Appl. Mater. Today, 2019, 15, 297–304. 13 Q. Chen, X. Liu, J. Zeng, Z. Cheng and Z. Liu, Biomaterials, 2016, 98, 23–30. 14 W. Sun, X. Zhang, H. R. Jia, Y. X. Zhu, Y. Guo, G. Gao, Y. H. Li and F. G. Wu, Small, 2019, 15, e1804575. 15 X. Wang, X. Zhong, H. Lei, Y. Geng, Q. Zhao, F. Gong, Z. Yang, Z. Dong, Z. Liu and L. Cheng, Chem. Mater., 2019, 31, 6174–6186. 16 Y. Jiang, P. K. Upputuri, C. Xie, Y. Lyu, L. Zhang, Q. Xiong, M. Pramanik and K. Pu, Nano Lett., 2017, 17, 4964–4969. 17 Y. Jiang, J. Li, X. Zhen, C. Xie and K. Pu, Adv. Mater., 2018, 30, e1705980. 18 Y. Sun, F. Ding, Z. Chen, R. Zhang, C. Li, Y. Xu, Y. Zhang, R. Ni, X. Li, G. Yang, Y. Sun and P. J. Stang, Proc. Natl. Acad. Sci. U. S. A., 2019, 116, 16729–16735. 19 S. K. Hong, S. O. Kim and K. J. Lee, Adv. Mater., 2020, 32, e2000696. 20 J. Huang and K. Pu, Angew. Chem., Int. Ed., 2020, 59, 11717–11731. 21 T. Ji, Y. Zhao, Y. Ding, J. Wang, R. Zhao, J. Lang, H. Qin, X. Liu, J. Shi, N. Tao, Z. Qin, G. Nie and Y. Zhao, Angew. Chem., Int. Ed., 2016, 55, 1050–1055. 22 B. Yu, S. Goel, D. Ni, P. A. Ellison, C. M. Siamof, D. Jiang, L. Cheng, L. Kang, F. Yu, Z. Liu, T. E. Barnhart,
J. Mater. Chem. B
23
24 25
26 27 28
29 30
31 32
33 34 35 36 37 38
39 40 41 42
Q. He, H. Zhang and W. Cai, Adv. Mater., 2018, 30, e1704934. Z. Qin, T. Du, Y. Zheng, P. Luo, J. Zhang, M. Xie, Y. Zhang, Y. Du, L. Yin, D. Cui, Q. Lu, M. Lu, X. Wang and H. Jiang, Small, 2019, 15, e1902755. X. Hao, C. Li, Y. Zhang, H. Wang, G. Chen, M. Wang and Q. Wang, Adv. Mater., 2018, 30, e1804437. T. Ojha, V. Pathak, Y. Shi, W. E. Hennink, C. T. W. Moonen, G. Storm, F. Kiessling and T. Lammers, Adv. Drug Delivery Rev., 2017, 119, 44–60. X. Hu, F. Li, S. Wang, F. Xia and D. Ling, Adv. Healthcare Mater., 2018, 7, e1800359. F. Li, Y. Du, J. Liu, H. Sun, J. Wang, R. Li, D. Kim, T. Hyeon and D. Ling, Adv. Mater., 2018, 30, e1802808. Y. Hao, Z. Dong, M. Chen, Y. Chao, Z. Liu, L. Feng, Y. Hao, Z. L. Dong, M. C. Chen, Y. Chao, Z. Liu and L. Z. Feng, Biomaterials, 2020, 228, 119568. W. Bao, X. Liu, Y. Lv, G. H. Lu, F. Li, F. Zhang, B. Liu, D. Li, W. Wei and Y. Li, ACS Nano, 2019, 13, 260–273. J. Chen, L. Liu, S. M. Motevalli, X. Wu, X.-H. Yang, X. Li, L. Han, A. Magrini, W. Guo, J. Chang, M. Bottini and X.-J. Liang, Adv. Funct. Mater., 2018, 28, 1707291. J. Sheng, L. Wang, Y. Han, W. Chen, H. Liu, M. Zhang, L. Deng and Y. N. Liu, Small, 2018, 14, 1702529. T. Yang, Y. Wang, H. Ke, Q. Wang, X. Lv, H. Wu, Y. Tang, X. Yang, C. Chen, Y. Zhao and H. Chen, Adv. Mater., 2016, 28, 5923–5930. H. Yan, J. Chen, Y. Li, Y. Bai, Y. Wu, Z. Sheng, L. Song, C. Liu and H. Zhang, Biomater. Sci., 2018, 7, 92–103. Z. Yao, B. Zhang, T. Liang, J. Ding, Q. Min and J. J. Zhu, ACS Appl. Mater. Interfaces, 2019, 11, 18995–19005. Q. Zhao, Z.-X. Fang, M.-M. Chen, M. Li, Z.-M. Zhou, B. Du and Q.-Q. Zhang, Micro Nano Lett., 2018, 13, 835–837. J. Li, C. Xie, J. Huang, Y. Jiang, Q. Miao and K. Pu, Angew. Chem., Int. Ed., 2018, 57, 3995–3998. X. Zhen, J. Zhang, J. Huang, C. Xie, Q. Miao and K. Pu, Angew. Chem., Int. Ed., 2018, 57, 7804–7808. L. Wang, J. Hou, S. Liu, A. J. Carrier, T. Guo, Q. Liang, D. Oakley and X. Zhang, Sens. Actuators, B, 2019, 287, 180–184. L. H. Fu, Y. R. Hu, C. Qi, T. He, S. Jiang, C. Jiang, J. He, J. Qu, J. Lin and P. Huang, ACS Nano, 2019, 13, 13985–13994. Z. Zhao, W. Wang, C. Li, Y. Zhang, T. Yu, R. Wu, J. Zhao, Z. Liu, J. Liu and H. Yu, Adv. Funct. Mater., 2019, 29, 1905013. Z. Yu, P. Zhou, W. Pan, N. Li and B. Tang, Nat. Commun., 2018, 9, 5044. Y. Sun, P. Tian, D. Ding, Z. Yang, W. Wang, H. Xin, J. Xu and Y.-F. Han, Appl. Catal., B, 2019, 258, 117985.
This journal is © The Royal Society of Chemistry 2020
Materials Chemistry B View Article Online
PAPER
Cite this: DOI: 10.1039/d0tb01915a
View Journal
A tumor-targeted theranostic nanomedicine with strong absorption in the NIR-II biowindow for image-guided multi-gradient therapy† Qi Chen,‡a Ziliang Zheng,‡a Xiaojing He,‡a Shuo Rong,a Yufei Qin,a Xiaoyang Penga and Ruiping Zhang *b Developing new strategies to enhance drug accumulation in the tumor and therapeutic efficacy is of great importance in the field of tumor therapy. Herein, a peanut-like multifunctional nanomedicine (CuS-PGH NMs) made of CuS nanoparticles encapsulated in poly(L-lysine)(PLL)/glucose oxidase (GOx)hyaluronic acid (HA) shells has been constructed via layer-by-layer (LbL) assembly, and shows good biocompatibility and effective multi-gradient therapy. Because of the enhanced permeability and retention (EPR) effect, the CuS-PGH NMs could significantly enhance the cellular uptake by tumors overexpressing CD44 receptors, which respond to hyaluronidase (HAase)-triggered surface charge conversion. Once internalized by the tumor, GOx was the first to be exposed and could effectively deplete endogenous glucose for starvation therapy, and the excess H2O2 was then converted into highly toxic hydroxyl radicals ( OH) via a Cu+-mediated Fenton-like reaction for chemodynamic therapy (CDT). Meanwhile, the as-obtained Cu+ ions accompanied the regenerated less-active Cu2+ ions. Interestingly, the high content of H2O2 could, in turn, accelerate Cu2+/Cu+ conversion to promote the Cu+–H2O2 reaction for enhanced chemodynamic therapy (CDT), thereby achieving efficient tumor growth suppression via synergistic starvation/CDT therapy. Subsequently, owing to the strong NIR-II absorption
Received 8th August 2020, Accepted 15th September 2020
capability of CuS-PGH NMs, effective photothermal tumor ablation of the weakened tumor cells could
DOI: 10.1039/d0tb01915a
be realized with the precise guidance of NIR-II PAI. This multi-gradient therapeutic strategy has been demonstrated to have excellent antitumor activity with minimal nonspecific damages, and offers a new
rsc.li/materials-b
avenue to precise tumor therapy.
1. Introduction Breast cancer is associated with relatively high mortality rates among women worldwide. Especially triple-negative breast cancer (TNBC), which is one of the breast cancer subtypes, presents the worst survival rate due to its highly aggressive and metastatic behavior.1–3 Current treatments for TNBC patients mainly depend on traditional surgical operation, radiotherapy or chemotherapy, and often involve issues of non-specific distribution, drug resistance and systemic side effects.4,5 For these reasons, the development of targeted diagnostic and therapeutic platforms is an urgent need for effective TNBC therapy. a
Department of biochemistry and molecular biology, Shanxi Medical University, Taiyuan 030001, China b Department of Radiology, Third hospital of Shanxi Medical University, Taiyuan 030032, China. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/ d0tb01915a ‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2020
Photothermal therapy (PTT), a new type of physiotherapy, has attracted increasing interest in the field of cancer treatment.6–9 PTT, utilizing photothermal nanoagents, exhibits good tumor-specific localization by converting the absorbed near-infrared (NIR) laser energy into heat and therefore, efficiently ablates cancer cells and tissues with minimal invasiveness.10–12 Notably, severe hypoxia and low pH in the tumor microenvironment (TME) make the cancer cells more sensitive to heat than normal cells.13,14 Second near-infrared (NIR-II, 900–1700 nm) light has many advantages compared with traditional first near-infrared light, such as deeper penetration of biological tissues, less tissue scattering or absorption, higher signal-to-noise ratio and lesser interference of fluorescent proteins.15,16 Furthermore, nanoagents that absorb light in the NIR-II window combine well with photoacoustic imaging (PAI) and result in high-performance photothermal treatment outcomes.17–20 Therefore, NIR-II PAI could achieve high-spatialresolution diagnosis to guide tumor-depth PTT. It is well-known that diagnosis and treatment effects can be achieved only with a certain deposition amount due to the
J. Mater. Chem. B
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Paper
Journal of Materials Chemistry B
dynamic equilibrium between the uptake and excretion rates of nanomedicines at the tumor site.21 Therefore, the prerequisite for accurate tumor imaging and therapeutic effects is the efficient delivery of a multifunctional agent into the cancer cells.22,23 One of the feasible delivery approaches is the construction of nanomedicines, which can be significantly enriched at the tumor sites through the acknowledged enhanced permeability and retention (EPR) effect.24 However, when the delivery relies solely on this intrinsic characteristic of nanomedicine through the EPR effect, it exhibits insufficient accumulation and inadequate tumor retention, which inevitably leads to a suboptimal therapeutic effect.25,26 As a desirable strategy to solve this issue, stimuli-responsive nanomedicines, which perform the theranostics function only after specific activation at the tumor sites and remain in a ‘‘silent state’’ in non-neoplastic lesions, have been exploited to achieve precision anti-tumor efficacy and decrease the adverse effects.27,28 Bao and co-workers29 constructed a nanolongan delivery system, which could reverse the negative surface charge to positive at the tumor sites by dimethylmaleic anhydride (DMMA) decoration, resulting in ferroptosis-apoptosis combined anticancer therapy. Chen et al.30 obtained ICG/TPZ@HSA dNMs that could efficiently eradicate the tumors through a cascade of synergistic events triggered by sequential laser irradiation in the tumor area. Hence, the rational design of stimuli-responsive nanomedicines is crucial for enhancing efficient uptake by the tumor cells and thus achieving excellent antitumor performance. Based on the above principles, we have developed a novel multifunctional nanomedicine (CuS-PGH NMs), which shows excellent efficacy in tumor eradication through a multi-gradient therapy (two-step treatment, including synergistic starvation/ CDT therapy and NIR-II PAI-guided PTT) without detectable
Scheme 1
side effects. By adopting the layer-by-layer (LBL) technique, CuS-BSA nanoparticles were encapsulated in poly(L-lysine) (PLL) through electrostatic attraction and further decorated with glucose oxidase (GOx) and a hyaluronic acid (HA) shell (Scheme 1). In detail, the shielding effect of the HA shell would ensure prolonged circulation time and provide more opportunities for CuS-PGH NMs to reach the tumor region via the EPR effect. Meanwhile, the active-targeting effect would enhance the retention and internalization of CuS-PGH via the specific interaction of HA with its primary receptor CD44, which is over-expressed in TNBC cells. In the tumor regions, the HA shell of CuS-PGH NMs decomposed, reversing the surface charge and exposing GOx. GOx then consumed the endogenous glucose for starvation therapy by catalyzing its oxidation to generate gluconic acid and H2O2 (eqn (1)). The elevated H2O2 levels led to the reduction of Cu2+ to Cu+ ions, due to which the CuS-PGH NMs exhibited excellent Fenton-like reaction ability, generating highly toxic OH from excess H2O2 (Eqn (2) and (3)). Therefore, simultaneously producing OH and accelerating Cu2+/Cu+ conversion in response to the high H2O2 levels in the TME endows CuS-PGH NMs with the ability for CDT, thereby leading to efficient tumor growth suppression in combination with starvation therapy. Based on these, the strong NIR-II absorption capability of CuS-PGH NMs offers complete tumor ablation and enables the use of NIR-II PAI for diagnosis and PTT guidance. We believe this work provides a novel multi-gradient therapeutic strategy to achieve highly efficient tumor treatment. GOx
Glusoce þ H2 O þ O2 ! Glucinicacid þ H2 O2
(1)
Cu2+ + H2O2 - Cu+ + H+ + HO2
(2)
Cu + H2O2 - Cu + OH + OH
(3)
+
+
Schematic illustration of the construction of CuS-PGH NMs for multi-gradient antitumor therapy.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2020
View Article Online
Journal of Materials Chemistry B
2. Materials and methods Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
2.1.
Materials
Bovine serum albumin (BSA) was obtained from Acmec Biochemical (Shanghai, China). Copper nitrate trihydrate (Cu (NO3)23H2O), sodium sulfide nonahydrate (Na2S9H2O), polyL-lysine (Mw 2000–5000), glucose oxidase (GOx), and hyaluronic acid (HA) were acquired from Aladdin (Shanghai, China). Sodium hydroxide (NaOH), propidium iodide (PI) and calcein acetoxymethyl ester (Calcein–AM) were purchased from SigmaAldrich. Reagent-grade water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA) for all the experiments. All chemicals used here were of analytical grade and used without further purification. 2.2.
Synthesis of CuS@BSA (CuS)
The CuS@BSA nanoparticles were prepared in an aqueous solution at 90 1C according to the biomineralization strategy. Typically, aqueous Cu(NO3)23H2O solution (0.025 mmol, 8 mL) and BSA solution (30 mg mL1, 8 mL) were mixed in a onenecked flask (25 mL) with magnetic stirring. After rapid mixing, the mixture instantly showed white turbidity, but immediately turned into a light-yellow solution. Five minutes later, the pH of the system was adjusted to 12 using a 2 M NaOH solution, and the mixture turned transparent and deep purple. Afterwards, 400 mL of Na2S9H2O (242.16 mg mL1) was quickly injected into the above system, and the solution turned deep brown. The reactor was placed in a constant-temperature oil bath with a magnetic stirrer and maintain at a temperature of 90 1C for 30 minutes. Then, the CuS synthesis experiment ended when the solution changed black in colour. The solution was transferred into an ultrafiltration centrifuge tube and centrifuged at 3500 rpm for 15 minutes. After repeating the above operation 3 to 5 times with pure water, the solution was freeze-dried, sealed, and stored. It is also feasible to dissolve the lyophilized powder of CuS in pure water. 2.3.
Preparation of CuS@BSA-PLL/GOx/HA (CuS-PGH NMs)
The CuS nanoparticles obtained in the previous step were dissolved in pure water at a concentration of 1 mg mL1. Then, 5 mL of the above solution was added dropwise to a PLL solution (1 mg mL1, 5 mL) at a constant rate. Excess PLL was removed by centrifugation at 3000 rpm for 10 min and re-dissolved in 5 mL pure water. An aqueous GOx solution (1 mg mL1) and a HA solution (1 mg mL1) were then mixed in the ratio of 1 : 1. This mixed solution was very slowly added dropwise to the re-dissolved CuS@BSA-PLL solution in the ratio of 3 : 5. After centrifuging again at 3000 rpm for 10 min to remove excess HA and GOx, CuS-PGH NMs were obtained. It is able to dissolve the lyophilized powder in pure water for later experiments. 2.4.
Material characterization
The TEM images, element mapping images and EDS linescanning of the CuS-PGH NMs were obtained using a Tecnai G2 F20 S-twin transmission electron microscope (FEI). X-ray photoelectron spectroscopy (XPS) was performed with a V.G.
This journal is © The Royal Society of Chemistry 2020
Paper Scientific ESCALAB250 spectrometer using Al Ka radiation (1486.6 eV, 150 W). Dynamic light scattering (Nano ZS, Malvern) was used to record the hydrodynamic size distribution and z-potential of the CuS-PGH NMs. Fourier-transform infrared (FT-IR) spectroscopy was performed on a Vertex PerkinElmer 580BIR spectrophotometer (Bruker) using the KBr pellet technique to identify the bonds and quantify their relative abundance. The UV-vis absorption of the samples was obtained using a TU-1901 dual-beam UV-vis spectrophotometer (PerkinElmer). 2.5. Measurement of photothermal effect of CuS-PGH NM in aqueous solution A 1064 nm laser was used for the photothermal effect measurements and the following in vitro/in vivo PTT experiments. Aqueous CuS-PGH NM solutions (200 mL) at a series of concentrations (0.0625, 0.125, 0.25, 0.5, and 1 mg mL1) were placed in Eppendorf tubes and exposed to the NIR laser (1064 nm, 1.0 W cm2) for 5 min. Simultaneously, an infrared camera (Fluke Ti400) was used to take pictures and record the real-time temperature of the liquid at 15 s intervals. Photothermal stability cycling tests were also conducted repeatedly to observe the temperature increase induced by the laser with a 0.25 mg mL1 CuS-PGH NM aqueous solution. Chicken breast meat slices of different thicknesses (4 mm, 6 mm, 8 mm) were used to verify the penetration effect and photothermal effect when the CuS-PGH NMs aqueous solution (0.25 mg mL1) was irradiated by the 1064 nm laser. 2.6.
In vitro degradation and response test
The pH values of the solution after the addition of HAase and glucose were measured over time since GOx can catalyze the oxidation of glucose to gluconic acid. Glucose was added to 5 mL of the CuS-PGH NM solution or the CuS-PH NM solution (0.25 mg mL1) to a final glucose concentration of 0.5 mg mL1. Then, the pH value of each solution was measured over time. Considering that H2O2 is produced when GOx reacts with glucose, Micro Hydrogen Peroxide (H2O2) Assay Kit (Solarbio, Beijing, China) was used to quantify the amount of H2O2. We used methylene blue (MB) to verify the production of OH. CuSPGH NMs (0.1 mg mL1), H2O2 (1 mM) and MB (0.01 mg mL1) were prepared in a 25 mM NaHCO3 solution to a total volume of 1 mL. Then, the reaction system was shaken in darkness at 37 1C. At different time points, the UV-vis absorption curves of the mixture were tested and recorded. 2.7. In vitro cytotoxicity of CuS-PGH NMs and photothermal therapy assays A standard Cell Counting Kit (CCK8) (Sigma-Aldrich, St. Louis, MO, USA) assay was conducted using a breast cancer cell line (4T1) to evaluate the in vitro cytotoxicity of CuS-PGH NMs. Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% (v/v) fetal bovine serum (FBS) and 1% antibiotics (penicillin–streptomycin) (Corning) at 37 1C under a 5% CO2 atmosphere. Typically, 4T1 cells (8 103/well) were seeded into 96-well plates, and then, the cells were incubated in the culture medium for 24 h at 37 1C. The culture
J. Mater. Chem. B
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Paper medium was then removed, and the cells were incubated with 100 mL of fresh medium containing CuS-PGH NMs at different concentrations (0.0625, 0.125, 0.25, 0.5 and 1 mg mL1) for 24 h. The CCK-8 solution (10 mL, 5 mg mL1) was added into the plates along with the culture medium, and the cells were incubated for another 2 h. Finally, the OD450 value (abs.) of each well was measured using a microplate reader (BioTek Epoch 2). In another set of experiments, after incubating with different concentrations of CuS-PGH NMs, the 4T1 cells were exposed to a 1064 nm laser at an irradiation density of 1 W cm2 for 5 min. The above CCK-8 method was used to observe the in vitro phototherapeutic efficacy of the nanoparticles and cell viability at different glucose concentrations (0.125, 0.25, 0.5, 1 and 2 mg mL1) for verifying GOx function. 2.8.
Animal model
All animal experimental procedures were performed in compliance with the criteria of the National Regulation of China for Care and Use of Laboratory Animals and Animal Use Committee of Shanxi Medical University (Approval No. 2016LL141, Taiyuan,
Journal of Materials Chemistry B China). Tumor-bearing mouse models were established by injecting 4T1 cells (1 106) subcutaneously into the right thigh of female nude mice (6–8 weeks old, bodyweight 18–22 g), which were purchased from Weitong Lihua Experiment Animal Technology Co. Ltd (Beijing) and acclimatized to the animal facility for at least 7 days. Tumor-bearing nude mice were used in experiments when the tumor volume approached 50–100 mm3. 2.9.
PAI imaging
The in vitro PAI property was examined using a real-time multispectral optoacoustic tomographic (MSOT) imaging system. The phantom was filled with the CuS-PGH NMs aqueous solution at different concentrations (0, 0.0625, 0.125, 0.25, 0.5 and 1 mg mL1) and suspended inside a water tank and imaged under a continuous Nd:YAG laser. For in vivo PAI, the tumor-bearing nude mice were anesthetized by isoflurane inhalation and administered CuS-PGH NMs dispersed in 200 mL of PBS via the tail vein. PAI signals at different time points (after injection for 1, 4, 6, 8, 10, and 12 h) were acquired using a real-time multispectral optoacoustic tomographic (MSOT) imaging system.
Fig. 1 (a and b)The TEM images, (c) the corresponding elemental mapping images and the (d) Cu 2p and (e) S 2p XPS spectra of CuS-PGH NMs. The (f) DLS distribution and zeta potential trend, (g) the FT-IR spectra, and (h) the absorption spectra (in the wavelength range of 200–1000 nm and 1000–1400 nm) of CuS, CuS-P and CuS-PGH NMs.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2020
View Article Online
Journal of Materials Chemistry B
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
2.10.
In vivo photothermal imaging and PTT
When the tumor volume reached B50 mm3, the 4T1 tumorbearing nude mice were randomly divided into four groups (n = 5, in each group) (dose = 15 mg kg1): (1) PBS + laser, (2) CuS-PGH, (3) CuS + laser, and (4) CuS-PGH + laser. After 6 h of intravenous injection, the tumor areas of the (1), (3) and (4) groups were exposed to a 1064 nm laser (1 W cm2, 5 min). An infrared thermal camera (Fluke Ti400) was used to monitor the temperature changes at the tumor sites during NIR-II irradiation.
3. Results and discussion A peanut-like structure with multiple CuS nanoparticles (about 6–8 nm in diameter) uniformly embedded in HA shells could be observed visually in the transmission electron microscopy (TEM) images of CuS-PGH NMs (Fig. 1a and b). Moreover, it clearly revealed the lattice fringes of d103 = 0.29 nm and d107 = 0.18 nm, which were in agreement with those of the hexagonal CuS phase.31,32 The elemental mapping images of CuS-PGH NMs in Fig. 1c indicate the presence of the corresponding elements (Cu and S) in the sample. As seen in Fig. 1d and e, X-ray photoelectron spectroscopic (XPS) analyses were used to determine the components and chemical states of Cu and S in CuS-PGH NMs. The intense peaks at the binding energies of 932.5 (Cu 2p3/2) and 952.4 eV (Cu 2p1/2) could be assigned to
Paper Cu2+ from CuS.28 In the XPS spectrum of S element, different valence states of S (peaks at 163.7 eV and 168.2 eV) were identified in the NMs, which could be attributed to the S2 state of CuS and the disulfide bonds (–S–S–) of BSA, respectively.33 Moreover, CuS-PGH NMs were characterized by X-ray diffraction (XRD) to confirm the crystal-structure (Fig. S1, ESI†). Although the diffraction peaks of XRD were relatively weak, they could be well indexed to the CuS standard peaks (JCPDS No. 06-0464), further indicating the successful formation of CuS in CuS-PGH NMs. To analyze the structural properties of CuS-PGH NMs, dynamic light scattering (DLS) and zeta potentials were recorded to track the assembly process (LBL technique) (Fig. 1f). The average hydrodynamic diameter gradually increased from 19.8 nm to 45.7 nm with the systematic modification with PLL(+) and GOx/HA(), confirming the successful assembly of the CuS-PGH NMs. The zeta potential of the nanoparticles decreased from 27.1 mV to 10.6 mV after the modification with the negatively charged GOx and HA. Although the peak of the point was in the positive charge region, it had already crossed the point of electrical neutrality, which indicated that the electrostatic force was so weak that only a small amount of negatively charged proteins in the body can attach to the surface of the nanoparticles.34 In addition, after soaking CuS-PGH NMs in various solutions, including water, PBS, saline and the cell growth medium, for 24 h, the solutions were still uniform and translucent, indicating good
Fig. 2 (a) Photothermal heating curves of CuS-PGH NMs at different concentrations under laser irradiation (1064 nm, 1 W cm2) as a function of time (0–300 s). (b) Photothermal stability of CuS-PGH NMs (0.25 mg mL1). (c) The heating curves and (d) the infrared thermal images of CuS-PGH NMs (0.25 mg mL1) placed below chicken meat of varying thicknesses under laser irradiation (1064 nm, 1 W cm2).
This journal is © The Royal Society of Chemistry 2020
J. Mater. Chem. B
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Paper dispersion and stability of CuS-PGH NMs (Fig. S2, ESI†). The TEM images demonstrated the difference between CuS and CuS-PGH NMs (Fig. S3, ESI†) as the morphology and size of the samples changed during the LbL assembly process. Moreover, FT-IR spectroscopy was carried out to monitor the change in material configuration during the formation of CuS, CuS-P and CuS-PGH NMs (Fig. 1g). It could be distinctly observed that the bands for N–H (bends) at 1397 cm1 underwent remarkable attenuation since PLL and GOx/HA wrapped the nanoparticle core, and the peaks of BSA were less prominent than those in the initial state.35 In brief, the positively charged PLL layer and the negatively charged GOx/HA layer sequentially encapsulated the CuS nanoparticles via layer-by-layer assembly (LbL) to form CuS-PGH NMs. The optical property of CuS-PGH NMs and its two predecessor nanoparticles was evaluated based on their UV absorption spectra. As displayed in Fig. 1h, the absorption curve (600– 1000 nm) exhibited a good upward trend, and we chose another near-infrared scanner with a longer wavelength range, which
Journal of Materials Chemistry B could scan absorption in the NIR-II wavelength range. The CuS-PGH NMs showed a broad absorption band in the wavelength region of 1000–1400 nm (inset Fig. 1h), which is very beneficial for PAI and PTT in the NIR-II region. Encouraged by the absorption characteristics of CuS-PGH NMs, a 1064 nm laser was chosen to investigate the PTT properties of solutions at different concentrations (0.0625, 0.125, 0.25, 0.5, and 1 mg mL1). As expected, after irradiation (1.0 W cm2) for 5 min (Fig. 2a), the temperature of the CuSPGH NMs solution increased rapidly even at a rather low concentration (0.125 mg mL1) in a concentration-dependent manner. Comparatively, only a negligible increment was observed when pure water was used in parallel. Moreover, CuS-PGH NMs possessed high photothermal stability, and no attenuation of photothermal efficiency was observed even after five cycles of switching (Fig. 2b). It is widely believed that a NIR-II wavelength laser can provide deeper penetration and higher contrast compared with a NIR-I laser. During the measurements, we further exploited the photothermal effect
Fig. 3 (a) The pH values as a function of reaction time and (b) H2O2 production arising from the CuS-PGH NM-catalyzed decomposition reaction of glucose. UV/vis spectra of the MB aqueous solution after degradation by (c) CuS-PGH NM-mediated Fenton-like reaction and (d) by CuS-PGH NMs and glucose at different time points; [CuS-PGH NMs] = 0.1 mg mL1; [H2O2] = 1 mM, [glucose] = 0.5 mg mL1. (e) 4T1 cell viability after treatment with various concentrations of glucose or glucose along with CuS-PGH NMs (0.25 mg mL1) for 12 h. (f) Cell viability of 4T1 cells after incubation with gradient concentrations of CuS-PGH NMs and CuS-PGH NMs + Laser in DMEM medium containing 0.5 mg mL1 glucose (1064 nm, 1 W cm2, 5 min). (g) Micrographs of 4T1 cells incubated with CuS-PGH NMs (0.25 mg mL1) before and after irradiation with a 1064 nm laser (1 W cm2); dead cells are stained with trypan blue. ***P o 0.001.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2020
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Journal of Materials Chemistry B of CuS-PGH NMs in deep tissues by using the chicken breast tissues of different thicknesses (4, 6, and 8 mm) as models of biological tissues. The chicken breast tissue was placed between the laser source and the CuS-PGH NMs solution (Fig. S4 ESI†). As illustrated in Fig. 2c, the temperatures of all the CuS-PGH NMs solutions increased to around 60 1C within 600 s of 1064 nm laser irradiation and reached over 43.8 1C after a rather short irradiation time of less than 160 s. Considering the threshold temperature for PTT (43 1C), the observed temperature increment exceeded the temperature rise required (13 1C) to ablate tumor cells in living mice.36,37 More importantly, no obvious temperature change was recorded in the tissue after irradiation (Fig. 2d). After verifying the encapsulation of GOx in the CuS-PGH NMs, the catalytic activity of the immobilized GOx was evaluated by detecting pH variation and H2O2 generation since GOx can catalyze the oxidation of glucose to gluconic acid and H2O2. As shown in Fig. 3a, a significant drop in the pH of the CuS-PGH NMs solution (from 6.7 to 3.4) was observed after treatment with glucose (0.5 mg mL1) for 1.5 h. However, the pH change in the sample solution without GOx was much smaller (from 6.9 to 6.5) than that of the GOx-loaded sample under the same circumstance. Titanium sulfate was selected to evaluate the efficiency of H2O2 production by measuring the fluorescent titanium peroxide complex (Fig. 3b).38 The generated H2O2 quickly reached the maximum in 1 h, indicating that CuS-PGH NMs possessed high GOx catalytic activity. Remarkably, the production of H2O2 gradually decreased after 1 h, which indicates the possible conversion of H2O2. As reported previously, the Cu+ ion could react with H2O2 to produce OH
Paper through a Fenton-like reaction, which could effectively induce cancer cell apoptosis.39,40 Hence, the methylene blue (MB) degradation experiment was performed to detect the generation of hydroxyl radicals ( OH). When CuS-PGH NMs and excess H2O2 were added, the absorbance of MB solution at 650 nm showed an obvious decrease with increasing reaction duration (Fig. 3c), manifesting that H2O2 was converted to OH due to the Fenton-like catalytic ability of Cu+.41,42 Moreover, MB was partly degraded in the presence of CuS-PGH NMs and glucose (Fig. 3d) due to glucose depletion and the Fenton-like reaction. These results indicated that the CuS-PGH NMs could mediate a cascade of reactions, including GOx-triggered glucose oxidation and then the Cu+-mediated Fenton-like reaction. Based on the above results, 4T1 cells were used to test the cascaded multi-gradient therapeutic performance of CuS-PGH NMs in vitro. As shown in Fig. 3e, glucose was added as an energy source for cell growth and proliferation. However, the cell activity dramatically decreased with the addition of CuSPGH NMs due to GOx-triggered glucose-depletion for cell starvation therapy and the generation of Cu+-mediated OH for CDT. This result demonstrated that the cell starvation as the first stage in the cascade of multi-gradient therapy could undoubtedly lead to a reduction in cell viability. However, CuS-PGH NMs exhibited negligible cytotoxicity against the tumor cells over the range of experimental concentrations in DMEM medium containing 0.5 mg mL1 glucose, indicating that the multi-gradient synergistic therapeutic effect was indeed weak at low concentrations of glucose (Fig. 3f). In comparison, significant cell apoptosis and death were caused by the increased concentration of CuS-PGH NMs. Notably, CuS-PGH
Fig. 4 (a) The PAI mapping of CuS-PGH NMs at different concentrations. (b) The photoacoustic spectrum of the CuS-PGH NMs solution between 660 and 1150 nm wavelengths. (c) Signal intensity changes and (d) PAI images of 4T1 tumor-bearing mice after CuS or CuS-PGH NMs injection up to 12 h under 1064 nm laser irradiation. ***P o 0.001.
This journal is © The Royal Society of Chemistry 2020
J. Mater. Chem. B
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Paper NMs concentration of 0.25 mg mL1 resulted in low cell viability, demonstrating the potential minimum effective concentration of CuS-PGH NMs for the tumor cell killing. To further demonstrate the capability of CuS-PGH NMs in generating ROS in vitro, cellular ROS was determined with DCFH-DA dyes as they are converted to fluorescent DCF via ROS oxidation (Fig. S5, ESI†). The intercellular ROS levels increased dramatically in CuS-PGH NM-stimulated cells compared with control cells. Further, the fluorescence of the CuS-PGH group was 2 times as bright as the CuS group. This indicated an increase in cellular oxidative stress and that the total ROS level increased sharply, which further promoted CDT. Moreover, the cells incubated with 0.25 mg mL1 CuS-PGH NMs were almost entirely destroyed after laser irradiation, as seen in Fig. 3g, demonstrating that the multi-gradient ablation of tumor cells could be achieved by the combined utilization of CuS-PGH NMs and laser for further anti-tumor applications. The PAI signals of CuS-PGH NMs were monitored to evaluate their photoacoustic properties in vitro. As shown in Fig. 4a, the PAI signals exhibited a downward trend as the concentration of the solution decreased. This feature is also seen in the upper right corner of the PAI images. Moreover, the PAI data of the
Journal of Materials Chemistry B medium density condition (0.25 mg mL1) was selected to process the photoacoustic spectrum of CuS-PGH NMs (Fig. 4b), and the overall trend was roughly the same as the UV absorption curve at the wavelength of 1000 nm (Fig. 2a). Based on the above results, the in vivo PAI capability of CuSPGH NMs was tested in a subcutaneous 4T1 tumor model, and the PAI images of the tumors were longitudinally recorded and quantified at 0 h, 1 h, 4 h, 6 h, 10 h and 12 h post-injection. As shown in Fig. 4c and d, the PAI intensities of CuS and CuS-PGH NMs gradually enhanced over time and displayed maximal retention at 6 h post-injection. Noticeably, CuS only showed low accumulation in a limited region and failed to cover the entire tumor site. Meanwhile, after intravenous injection with CuS-PGH NMs, the PAI signals did not only accumulated efficiently in the tumor, but also exhibited a much higher contrast throughout the tumor than the surrounding tissues. This phenomenon confirmed that the increased PAI signals from CuS-PGH NMs at the tumor site may arise from HAmediated recognition and HAase-responsive reverse charging to facilitate tumor uptake. Moreover, the semiquantitative analysis of PAI further revealed that CuS-PGH NMs acted as an excellent contrast agent and were able to penetrate deep into
Fig. 5 In vivo antitumor study. (a) IRT images and (b) corresponding temperature curves of tumor-bearing mice injected with PBS, CuS or CuS-PGH NMs against time under NIR-laser irradiation (1064 nm, 1 W cm2). (c) Representative photographs of the mice in the different groups after treatment. (d) Relative body weight of mice in the different groups after their treatment. (e) H&E staining of the tumor tissues collected from mice at the end of treatment. Scale bar: 100 mm. n.s. = not significant.
J. Mater. Chem. B
This journal is © The Royal Society of Chemistry 2020
View Article Online
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
Journal of Materials Chemistry B the tumor cells, which helps identify the tumor region for guided diagnosis and treatment. The highly efficient PTT effect and the superb targeting ability of CuS-PGH NMs with PAI guidance enabled the evaluation of the in vivo antitumor performance on tumor-bearing nude mice. The 4T1 tumor-bearing nude mice were intravenously administered PBS, CuS and CuS-PGH NMs (15 mg kg1) and subjected to a 1064 nm NIR laser (1 W cm2) for 5 min at 6 h post-injection. Based on the real-time thermal images recorded using an IR thermal camera (Fig. 5a and b), the temperature in the tumor region had increased by 20 1C in the presence of CuS-PGH NMs upon laser irradiation, which was much higher than those of the CuS (11.5 1C) and PBS (4 1C) groups. The difference in temperature variation was attributed to the high photothermal conversion efficiency of CuS and the efficient tumor accumulation of CuS-PGH NMs. The change in tumor volume is a direct indicator of the therapeutic effect, which is shown in Fig. 5c and Fig. S6 (ESI†). The tumor-bearing nude mice were randomly divided into four groups, including (1) PBS + laser, (2) CuS-PGH, (3) CuS + laser, and (4) CuS-PGH + laser. At 6 h after the intravenous injection, the tumor areas of the (1), (3) and (4) groups were exposed to a 1064 nm laser for 5 min (1 W cm2). The growth of tumors in the CuS-PGH group could be inhibited, revealing the high efficiency of the synergistic starvation/CDT therapy. Owing to higher accumulation in the tumor, the treatment effects in the CuS-PGH NMs + laser group were greater than that in the CuS + laser group, and the tumor even disappeared thoroughly on the 11th-day post-treatment. Such effective therapeutic efficacy of CuS-PGH NMs in the presence of laser could be attributed to the photothermal therapy that further promoted glucose depletion and the cascading production of OH due to the wellretained catalytic activities of both GOx and CuS. Therefore, the photothermal therapy with the assistance of starvation and CDT using highly concentrated CuS-PGH NMs at the tumor site was sufficient for tumor ablation. In addition, the bodyweight of all the tested groups remained stable throughout the experiment, suggesting no systemic side effects after the treatment (Fig. 5d). The superior antitumor efficacy of CuS-PGH NMs is attributed to the multistage therapy that leads to a cascade of reactions. In the process of the reactions, the exposed GOx accelerated the consumption of intratumoral glucose, achieving starvation therapy. Notably, the excess H2O2 generated by glucose oxidation was then converted into highly toxic OH via a Cu+-mediated Fenton-like reaction, thereby accomplishing CDT. This cascade reaction-enhanced synergistic starvation and CDT effect demonstrated high-efficiency suppression of tumor cell activity. Ultimately, the photothermal performance of CuS-PGH NMs induced apoptosis and necrosis of the tumor cells under 1064 nm laser irradiation, resulting in excellent antitumor effects. H&E staining at the tumor tissue level further verified the cancer cell killing capability of CuS-PGH NMs. Consistent with the photothermal results, the highest apoptosis of tumor cells was shown by the CuS-PGH NMs + laser group compared with other treatment groups (Fig. 5e). The histological observations suggested that increased apoptosis may account for
This journal is © The Royal Society of Chemistry 2020
Paper tumor growth inhibition, which is consistent with the variation in the tumor growth curve. Moreover, major organs (heart, liver, spleen, lung, and kidney) of the mice were collected after treatment and sliced for H&E staining and examination. As shown in Fig. S7 (ESI†), the H&E staining images indicated no noticeable signs of toxic side effects. The above experimental results indicated that CuS-PGH NMs exhibit good biosafety and excellent antitumor effects and can serve as a novel nano-theranostic agent for cancer therapy.
4. Conclusion In summary, a theranostic agent with a novel peanut-like structure, CuS-PGH NMs, was developed for efficient NIR-II PAI-guided multistage therapy through LBL assembly. The charge conversion and specific recognition of CD44 by the HA shell did not only provide long circulation time for CuS-PGH NMs and active tumortargeting ability, but also enabled its high accumulation and retention at the tumor site. After HA degradation in the tumor microenvironment, GOx consumed glucose and produced H2O2 for starvation therapy, and H2O2 further reduced Cu2+ to Cu+ via a Fenton-like reaction. Meanwhile, the Cu+ ions effectively catalyzed the decomposition of H2O2 into highly toxic OH, thus presenting a distinctive cascade reaction-enhanced synergistic starvation and CDT effect for tumor suppression. Subsequently, under the guidance of NIR-II PAI, the CuS core endowed the CuS-PGH NMs with NIR-II photothermal conversion ability, which raises the tumor temperature, ensuring the thorough killing of the tumor cells by PTT. Moreover, PTT could also highly enhance Fenton-like activity to accomplish CDT. Based on high tumor accumulation, biocompatibility and the stimuli-responsive behavior of CuS-PGH NMs, this work provides an innovative strategy for effective cancer treatment by a cascade effect, which might have promising value in future clinical practices.
Conflicts of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81571747, 81771907), Science and technology innovation team project of Shanxi Province (No. 201705D131026), Engineering Technology Research Center of Shanxi Province (No. 201805D121008), Scientific and technological achievements transformation project of Shanxi Province (No. 201704D131006), Laboratory Construction Project of Shanxi Province, The Projects for Local Science and Technology Development Guided by the Central Committee (YDZX20191400002537), Scientific and Technologial Innovation Programs of Higher Education Institutions in Shanxi (2019L0415), Shanxi Province Science Foundation for Youths (201901D211343).
J. Mater. Chem. B
View Article Online
Paper
Journal of Materials Chemistry B
Published on 17 September 2020. Downloaded by State University of New York at Stony Brook on 10/3/2020 12:45:14 PM.
References 1 R. L. Siegel, K. D. Miller and A. Jemal, Ca-Cancer J. Clin., 2016, 66, 7–30. 2 L. M. Heiser, G. B. Mills and J. W. Gray, Cancer Cell, 2019, 35, 341–343. 3 A. M. Brewster, M. Chavez-MacGregor and P. Brown, Lancet Oncol., 2014, 15, e625–e634. 4 W. Chen, C. A. Glackin, M. A. Horwitz and J. I. Zink, Acc. Chem. Res., 2019, 52, 1531–1542. 5 J. Park, Y. Choi, H. Chang, W. Um, J. H. Ryu and I. C. Kwon, Theranostics, 2019, 9, 8073–8090. 6 Y. Liu, P. Bhattarai, Z. Dai and X. Chen, Chem. Soc. Rev., 2019, 48, 2053–2108. 7 S. He, Y. Jiang, J. Li and K. Pu, Angew. Chem., Int. Ed., 2020, 59, 10633–10638. 8 J. Li, D. Cui, J. Huang, S. He, Z. Yang, Y. Zhang, Y. Luo and K. Pu, Angew. Chem. Int. Ed., 2019, 58, 12680–12687. 9 X. Zhen, P. Cheng and K. Pu, Small, 2019, 15, e1804105. 10 W. Fan, B. Yung, P. Huang and X. Chen, Chem. Rev., 2017, 117, 13566–13638. 11 X. Zhen, C. Xie and K. Pu, Angew. Chem., Int. Ed., 2018, 57, 3938–3942. 12 Z. Li, H. Xu, J. Shao, C. Jiang, F. Zhang, J. Lin, H. Zhang, J. Li and P. Huang, Appl. Mater. Today, 2019, 15, 297–304. 13 Q. Chen, X. Liu, J. Zeng, Z. Cheng and Z. Liu, Biomaterials, 2016, 98, 23–30. 14 W. Sun, X. Zhang, H. R. Jia, Y. X. Zhu, Y. Guo, G. Gao, Y. H. Li and F. G. Wu, Small, 2019, 15, e1804575. 15 X. Wang, X. Zhong, H. Lei, Y. Geng, Q. Zhao, F. Gong, Z. Yang, Z. Dong, Z. Liu and L. Cheng, Chem. Mater., 2019, 31, 6174–6186. 16 Y. Jiang, P. K. Upputuri, C. Xie, Y. Lyu, L. Zhang, Q. Xiong, M. Pramanik and K. Pu, Nano Lett., 2017, 17, 4964–4969. 17 Y. Jiang, J. Li, X. Zhen, C. Xie and K. Pu, Adv. Mater., 2018, 30, e1705980. 18 Y. Sun, F. Ding, Z. Chen, R. Zhang, C. Li, Y. Xu, Y. Zhang, R. Ni, X. Li, G. Yang, Y. Sun and P. J. Stang, Proc. Natl. Acad. Sci. U. S. A., 2019, 116, 16729–16735. 19 S. K. Hong, S. O. Kim and K. J. Lee, Adv. Mater., 2020, 32, e2000696. 20 J. Huang and K. Pu, Angew. Chem., Int. Ed., 2020, 59, 11717–11731. 21 T. Ji, Y. Zhao, Y. Ding, J. Wang, R. Zhao, J. Lang, H. Qin, X. Liu, J. Shi, N. Tao, Z. Qin, G. Nie and Y. Zhao, Angew. Chem., Int. Ed., 2016, 55, 1050–1055. 22 B. Yu, S. Goel, D. Ni, P. A. Ellison, C. M. Siamof, D. Jiang, L. Cheng, L. Kang, F. Yu, Z. Liu, T. E. Barnhart,
J. Mater. Chem. B
23
24 25
26 27 28
29 30
31 32
33 34 35 36 37 38
39 40 41 42
Q. He, H. Zhang and W. Cai, Adv. Mater., 2018, 30, e1704934. Z. Qin, T. Du, Y. Zheng, P. Luo, J. Zhang, M. Xie, Y. Zhang, Y. Du, L. Yin, D. Cui, Q. Lu, M. Lu, X. Wang and H. Jiang, Small, 2019, 15, e1902755. X. Hao, C. Li, Y. Zhang, H. Wang, G. Chen, M. Wang and Q. Wang, Adv. Mater., 2018, 30, e1804437. T. Ojha, V. Pathak, Y. Shi, W. E. Hennink, C. T. W. Moonen, G. Storm, F. Kiessling and T. Lammers, Adv. Drug Delivery Rev., 2017, 119, 44–60. X. Hu, F. Li, S. Wang, F. Xia and D. Ling, Adv. Healthcare Mater., 2018, 7, e1800359. F. Li, Y. Du, J. Liu, H. Sun, J. Wang, R. Li, D. Kim, T. Hyeon and D. Ling, Adv. Mater., 2018, 30, e1802808. Y. Hao, Z. Dong, M. Chen, Y. Chao, Z. Liu, L. Feng, Y. Hao, Z. L. Dong, M. C. Chen, Y. Chao, Z. Liu and L. Z. Feng, Biomaterials, 2020, 228, 119568. W. Bao, X. Liu, Y. Lv, G. H. Lu, F. Li, F. Zhang, B. Liu, D. Li, W. Wei and Y. Li, ACS Nano, 2019, 13, 260–273. J. Chen, L. Liu, S. M. Motevalli, X. Wu, X.-H. Yang, X. Li, L. Han, A. Magrini, W. Guo, J. Chang, M. Bottini and X.-J. Liang, Adv. Funct. Mater., 2018, 28, 1707291. J. Sheng, L. Wang, Y. Han, W. Chen, H. Liu, M. Zhang, L. Deng and Y. N. Liu, Small, 2018, 14, 1702529. T. Yang, Y. Wang, H. Ke, Q. Wang, X. Lv, H. Wu, Y. Tang, X. Yang, C. Chen, Y. Zhao and H. Chen, Adv. Mater., 2016, 28, 5923–5930. H. Yan, J. Chen, Y. Li, Y. Bai, Y. Wu, Z. Sheng, L. Song, C. Liu and H. Zhang, Biomater. Sci., 2018, 7, 92–103. Z. Yao, B. Zhang, T. Liang, J. Ding, Q. Min and J. J. Zhu, ACS Appl. Mater. Interfaces, 2019, 11, 18995–19005. Q. Zhao, Z.-X. Fang, M.-M. Chen, M. Li, Z.-M. Zhou, B. Du and Q.-Q. Zhang, Micro Nano Lett., 2018, 13, 835–837. J. Li, C. Xie, J. Huang, Y. Jiang, Q. Miao and K. Pu, Angew. Chem., Int. Ed., 2018, 57, 3995–3998. X. Zhen, J. Zhang, J. Huang, C. Xie, Q. Miao and K. Pu, Angew. Chem., Int. Ed., 2018, 57, 7804–7808. L. Wang, J. Hou, S. Liu, A. J. Carrier, T. Guo, Q. Liang, D. Oakley and X. Zhang, Sens. Actuators, B, 2019, 287, 180–184. L. H. Fu, Y. R. Hu, C. Qi, T. He, S. Jiang, C. Jiang, J. He, J. Qu, J. Lin and P. Huang, ACS Nano, 2019, 13, 13985–13994. Z. Zhao, W. Wang, C. Li, Y. Zhang, T. Yu, R. Wu, J. Zhao, Z. Liu, J. Liu and H. Yu, Adv. Funct. Mater., 2019, 29, 1905013. Z. Yu, P. Zhou, W. Pan, N. Li and B. Tang, Nat. Commun., 2018, 9, 5044. Y. Sun, P. Tian, D. Ding, Z. Yang, W. Wang, H. Xin, J. Xu and Y.-F. Han, Appl. Catal., B, 2019, 258, 117985.
This journal is © The Royal Society of Chemistry 2020
![[No title available]](/assets/img/hold.png)
