Degradable NIR-PTT Nanoagents with a Potential Cu@Cu2O

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Degradable NIR-PTT nanoagents with a potential Cu@Cu2O@polymer structure Yu-Wei Tai, Yi-Chun Chiu, Po-Ting Wu, Jiashing Yu, Yu-Cheng Chin, Shu-Pao Wu, Yu-Chun Chuang, Ho-Chen Hsieh, Ping-Shan Lai, Hsiu-Ping Yu, and Mei-Yi Liao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15109 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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ACS Applied Materials & Interfaces

Degradable NIR-PTT nanoagents with a potential Cu@Cu2O@polymer structure. Yu-Wei Tai,a† Yi-Chun Chiu,b† Po-Ting Wu,a Jiashing Yu,a* Yu-Cheng Chin,c Shu-Pao Wu,d YuChun Chuang,e Ho-Chen Hsieh,d Ping-Shan Lai,f Hsiu-Ping Yu,f and Mei-Yi Liaoc* a.

Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan. Email:

[email protected] b.

Division of Urology, Department of Surgery, Zhong Xiao Branch, Taipei City Hospital, Taipei,

Taiwan c.

Department of Applied Chemistry, National Pingtung University, Pingtung 90003, Taiwan.

Email: [email protected] d. Department of Applied Chemistry, National Chiao Tung University, Hsinchu, Taiwan e. National Synchrotron Radiation Research Center, Hsinchu, 300, Taiwan f. Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan † These authors contributed equally.

Keywords: Cu nanoparticle, core-shell structure, extraction, biodegradable, near-infrared absorption, photothermal ablation, photothermal-chemotherapy 0 ACS Paragon Plus Environment

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Abstract:

Cu@Cu2O@PSMA polymer nanoparticles (Cu@Cu2O@polymer NPs) with near-infrared (NIR) absorption were successfully synthesized in a single-step oxidation reaction of Cu@PSMA polymer NPs at 100 °C for 20 min. The shape, structure, and optical properties of the Cu@Cu2O@polymer NPs were tailorable by controlling the reaction parameters, e.g., using the initial Cu@PSMA polymer NP as a template and varying the halide ion content, heating temperature, and reaction time. The Cu@Cu2O@polymer NPs exhibited robust NIR absorption between 650 nm and 710 nm and possessed superior oxidation resistance in water and culture media. In vitro assays demonstrated the low cytotoxicity of the Cu@Cu2O@PSMA polymer NPs to HeLa cells through an improved cell viability, high IC50, low injury incidence from the supernatant of the partly dissociated Cu@Cu2O@PSMA polymer NPs, and minor generation of reactive oxygen species (ROS). More importantly, we demonstrated the inorganic Cu-based nanocomposite (+0.34V vs NHE) was degradable in an endogenous H2O2 (+1.78V vs NHE) environment. Cu ions were detected in the urine of the mice, which illustrates the possibility of extraction after the degradation of the Cu-based particles. After an examination of the HeLa cells received the Cu@Cu2O@polymer NPs and a 660 nm LED, the photoablation of 50 % and 90 % cells was observed at NP doses of 20 ppm and 50 ppm, respectively. These results demonstrate that NIR-functional and moderate redoxactive Cu@Cu2O@polymer NPs are potential next-generation photothermal therapy (PTT) nanoagents because of combined features of degradation resistance in the physiological 1 ACS Paragon Plus Environment

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environment, enabling the delivery of efficient PTT; a possibly improved ability to selectively harm cancer cells by releasing Cu ions under high-H2O2 and/or low-pH conditions; and an ability to be extracted from the body after biodegradation.

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1. Introduction

In recent years, transition metal, metal oxide, metal sulfide nanoparticles (NPs) have found potential application in many fields, including catalysis, energy, and biomedical applications.1-3 The small size of NPs not only endows these materials with new physicochemical features of quantum confinement, surface plasmon resonance (SPR), and interface reactions owing to their high surface-to-volume ratio but also provokes specific biological responses, e.g., internalization, recognition, and enhanced permeability and retention (EPR) effects.4-8 Many pioneers as well as our group have reported that the passivation of NP surfaces increases their half-life in the circulatory system9-12 and facilitates passive and active targeting for disease treatment and diagnosis.9-12,13

Recently, the development of noble metal-based nanostructures has been widely studied for use as intense near-infrared (NIR) photoabsorption photothermal agents via the plasmonic effect.14-15 Different from inorganic semiconductors16-17 and conjugated polymers18 in terms of the nonirradiated transition of excited electrons back to the ground state, plasmonic NPs have large extinction coefficients, enabling possible treatment with less photons and/or a lower sample loading for efficient photothermal therapy (PTT) of cancer cells. Interest in designing Au nanocages, Au nanorods, Au@polymer nanomaterials, Pd plate-related nanostructures, and Ag-hybrids to successfully tailor the localized SPR (LSPR) in the NIR optical window (NRs) has increased.1, 1920

Even though researchers have claimed that Au and Pd nanomaterials are nontoxic to biological 3

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tissues during the delivery-therapeutic period, their stable and robust structure makes these NPs non-degradable and thus limits their practical applications in vivo. Long-term retention in the human body can pose unknown injury risks to organs.

Among the plasmonic nanomaterials,1, 19-21 copper (Cu) NPs are a logical choice for nextgeneration NIR nanoagents because of several advantages determined in previous investigations: i) biodegradability,22-23 ii) moderate tolerance in living cells,24 and iii) intended SPR interband transition at approximately 550-600 nm25-27 near the edge of the NIR-I wavelength (660-950 nm). The IC50 of Cu-based NPs was measured at 1.71 μg/mL,28 2.55-6.5 μg/mL,29-30 and 6.6 μg/mL31 when incubated with A-375 cells, HeLa cells, and THP-1 cells, respectively, and the LD50 of CuNPs in mice was 413 mg/kg in in vivo experiments.32 Recently, Hu and co-workers reported the clinical use of Cu-related NPs in cancer therapy had less adverse effects than other noble metal NPs as well as other types of anti-cancer drugs.33 Although excess unbound Cu ions are harmful to living systems, trace levels of Cu ions play important roles in organisms, e.g., in cellular energy production, the regulation of central nervous transmission, blood clotting, and oxygen transport. For example, the Cu ion within superoxide dismutase is able to drive a decrease in the activity of reactive oxygen species (ROS).34 Strategically controlling the shape geometry of Cu NPs to a size regime of < 100 nm to red-shift the SPR band has not been possible until now.26, 35-37 Achieving geometrical coverage of a Cu2O shell structure onto a noble metal has provided a new route to large red-shifts of the SPR band to 4 ACS Paragon Plus Environment


wavelengths of over 700 nm because of the high refractive index of Cu2O (∼2.7) at wavelengths above 600 nm.38 Susman used UV-visible measurements to study the kinetic oxidation of Cu NPs in a solid-state reaction39 and observed a nanoscale Kirkendall effect in the formation of holes accompanied by the growth of the copper oxide shell, which affected the wavelength position of the SPR band. Compared to the formation of Au nanostructures through a seed-growth reaction,38, 40-41

the formation of copper oxide nanoshells through the oxidation of Cu nanocolloids is highly

dependent on the ligand42 and solvent system37, 43 in the solution-phase reaction. Such a postoxidation process used to form core-shell Cu-Cu2O NPs might incur continuous damage because of oxidation after purification in air, leading to time-dependent changes in the SPR band position. The complete corrosion of the plasmonic Cu nanocore and/or disintegration of the Cu2O shell structure can occur in H2O/O2 conditions after long-term storage. In this work, our approach was to prepare Cu@Cu2O@polymer NPs (sub-100 nm) by directly oxidizing PSMA-encapsulated, sub-30 nm Cu NPs as a template, as shown in Scheme 1. The decoration of the Cu NPs (~20 nm) with a PSMA-polymer-combined oxide shell (~15 nm) shifted the original SPR band at 588 nm toward the NIR wavelength region at 650-710 nm. We found that the addition of halide ions in the synthesis altered the Cu nanocore structure but accelerated the dissolution of plasmonic Cu NPs, which lowered the extinction coefficient as the aging due to oxidation increased. The PSMA-Cu2O composite utilizes the surface structure of the hydrophobic polystyrene block of the PSMA polymer to protect the as-synthesized Cu-Cu2O core-shell structure 5 ACS Paragon Plus Environment

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from oxidation, thus preserving the NIR-I absorbance of the plasmonic Cu NPs. The carboxylic acid moieties in the PSMA polymer can chelate metal ions to prevent the burst release of high Cu ion concentrations that can harm living cells. The as-prepared Cu@Cu2O@polymer NPs demonstrated less cell damage, i.e., low ROS generation and an improved IC50 value, and superior photothermal conversion than Cu@polymer NPs. After a 660 nm light-triggered photothermal treatment using an LED light source, being the region closest in NIR wavelength, cellular examination revealed that a low dose of the Cu@Cu2O@polymer NPs enhanced the elimination of HeLa cancer cells up to 90 %. Relying on the electrochemical reactions inspired the work described herein, the oxidation of metallic copper is susceptible to halide ions, H2O/O2, and H2O2 oxidant. The exploration on the Cu@Cu2O@polymer NPs showed medium-dependent degradation of < 5 % in water, ~30 % in culture medium, and 100 % in an H2O2 oxidant solution, compared with the recently developed transition metal oxide, metal sulfide, and Au NPs.1-3, 14-21 In an in vivo study, Cu ions were detected in urine at 4-24 h post-injection after metabolism. Cancer cells reportedly contain high levels of ROS,44-45 such as H2O2 and superoxide anions, and have a low pH environment46 compared with normal cells. Finally, we exemplified the ROS enhanced the release of Cu ions from redox-active Cu@Cu2O@polymer NPs in metastatic cancer cells and induced by toxic lipopolysaccharides (LPS). Similar to the intercalation of Pt drugs into the DNA double helix,47-50 we hypothesized that the additional response of endogenous H2O2 oxidant and acids would stimulate the burst release of Cu ions, which could be used for a site6 ACS Paragon Plus Environment


specific cancer treatment modality involving the synergetic effect of combined photothermalchemotherapy over a relatively long time period.

2. Results and discussion

2.1. Synthesis of Cu@polymer NPs

Synthesis of the Cu@polymer NPs involved a one-pot hydrothermal reaction of CuCl2, PSMA, HCl, and N2H4.51 A TEM image (Figure 1a) was utilized to directly observe the microstructure of the product, which consisted of a dark region related to the Cu nanocore decorated with light regions corresponding to the PSMA polymer shell in the surrounding. The overall diameter of the spherical Cu@polymer NPs was approximately 50 ± 8 nm, and the diameter of the spherical Cu core was approximately 18 ± 6 nm. The peak of the diameter distribution determined from dynamic light scattering (DLS) measurements was ~93 nm (Figure S1). The higher particle size determined from DLS compared to that determined from the TEM image could be attributed to the interaction of the PSMA nanoshell with water molecules in aqueous solution. Furthermore, the high-resolution TEM image in Figure 1b shows a polycrystalline-like structure with a crystal lattice spacing of 2.1 Å and 1.8 Å, which matches the {111} and {200} planes, respectively, of the crystalline Cu. XRD was also used to characterize the structure of the resulting product, as shown in Figure 1c. The peaks of the crystal planes include (111), (200) and (220), in agreement with the planes of crystalline Cu based on a face-centered cubic structure. The UV-visible absorption spectrum (Figure 1d) of the 7 ACS Paragon Plus Environment

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Cu@polymer NPs showed strong absorbance at 588 nm, representing the SPR of the Cu@polymer NPs. 2.2. Effects of Halides in the Synthesis of the Cu@polymer NPs Many studies have shown that the nuclei-growth reaction of metallic nanostructures is significantly affected by halide interactions.52-53 In the present approach, the structures of the Cu@polymer NPs in the presence of 125 μL of NaI (Figure 2a) and 125-500 μL of NaBr (Figure S2a-c) were similar to the structure of Cu@polymer colloids without halide addition (Figure 1a). We found hardly any alterations to the optical and particle structure of the product when I- ions were replaced with Br- ions (125-500 μL) (Figure S2d). However, when the halide addition was increased to 375 μL, some of Cu core structure generated multiple core structures (Figure 2b), which indicates that the halide ions interfered with the crystallization of the Cu@polymer NPs by modifying the nucleation and growth process. Figure 2c illustrates that several of the Cu NPs converted to core-free and rod-like structures upon the addition of 500 μL of I- ions (Figure 2c). The UV-visible spectrum in Figure 2d shows that a slight red-shift of the SPR band from 584 nm to 600 nm with peak broadening was observed in the synthesis of the Cu@polymer NPs when the addition of I- ions was increased from 125 μL to 500 μL.

2.3. Generation of a Cu2O interface layer on Cu@polymer NPs



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Several groups have demonstrated that the oxide layer formed on the surface of Cu NPs was capable of shifting the SPR peak toward longer wavelengths.37,

39, 42-43, 54

Therefore, we next

examined a novel fabrication method for the Cu@Cu2O@polymer structure in aqueous solution, enabling a green, cost-efficient, and fast-reaction approach to controlling SPR bands. A facile single-step oxidation reaction of Cu@polymer NPs was investigated in DI water at 25-100 °C. In Figure 3a, the TEM image shows that the structure of the Cu@polymer NPs did not significantly change after aging at 25 °C for 7 days. The UV-visible spectra (Figure 3b) were additionally employed to monitor the oxidation process, showing only a slight red-shift (~600 nm) in the absorption peak of the Cu@polymer NPs. The SPR band was present after 7 days of aging, demonstrating that complete oxidation was prevented. The surface passivation with PSMA stabilized the Cu nanocores. When heating temperatures of 40 °C and 60 °C were implemented, the spectrum profile of the optical absorption bands between 0 min and 30 min of the oxidized Cu@polymer NPs did not remarkably change (Figure S3). At 80 °C, the optical bands of the Cu@polymer NPs were red-shifted from 588 nm (5 min) to 610 nm (30 min), which suggests progressive growth of a dielectric oxide layer on the surface of the Cu core (Figure 3c). A similar trend was observed for the oxidation of the Cu@polymer NPs at 100 °C. Compared to the reaction at 80 °C, the shift of the band toward 610 nm required one-third of the heating time. An SPR band spanning 570-800 nm appeared at 20 min of heating (Figure 3d).


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This single-step heating approach is very easy to carry out to oxidize Cu@polymer NPs and subsequently generate a Cu2O interlayer in a wet chemistry reaction. For example, Cu@Cu2O@polymer NPs produced intense NIR absorbance at 650-710 nm compared with the SPR peak at 600 nm produced by hydrocarbon chain-coated Cu@CuxO NPs prepared by organic-phase synthesis.37, 42-43 After 30 min of oxidation, the absorption band of the Cu@polymer NPs had almost completely disappeared, indicating the dramatic decomposition of the Cu nanocore. Notably, the Cu@polymer NPs oxidized for 20 min (at 100 °C) exhibited a simultaneous decrease in the extinction of the overall absorbance due to a lower scattering effect from the decreased density of the solid Cu core. However, a ratio of 1.10 at 660 nm/450 nm is comparable to a value of 1.24 at 588 nm/450 nm for the Cu@polymer NPs after and before oxidation at 100 °C for 20 min. Based on the shift in the absorbance of the product toward longer wavelengths, we speculated that the growth of the Cu2O layer under 100 oC of refluxing time was thicker on the surface of the Cu nanocore.38-39 Figure 4a shows the corresponding size distribution histogram of Cu@polymer core-shell structure almost no changes with the refluxing time from 5 min to 30 min in accordance with the time-dependent TEM image measurements (data not shown). X-ray absorption spectroscopy (XAS) analysis was employed to determine the electronic transition change in the chemical state and environment of the Cu-related samples.55 As shown in Figure 4b, around 80% Cu and 20% Cu2O in the starting materials were measured. A refluxing process was implemented at 100 oC to oxidize the Cu nanocore leading the metallic Cu fraction declined from 73% at 5 min, 10 ACS Paragon Plus Environment


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60% at 10 min, 52% at 20 min, to 42% at 30 min, while the electronic transitions related to Cu2O material absorption edge has subsequently increased. As shown in Figure 4a, the oxide layer decorated on the Cu NP grew thicker as Cu core size decrease, which could be attributed to the gradual oxidation process for Cu2O structure formation. To confirm the generation of a copper oxide layer, XRD (Figure 4c) measurement was used to characterize the Cu@polymer NPs oxidized for 20 min (at 100 °C). The appearance of new peak at 37° was assigned to the (111) crystal plane of the Cu2O structure (JCPDS 65-3288) in addition to the (111), (200) and (220) reflection planes from the Cu (JCPDS 04-0836) nanocore. An evident broad peak at 37°likely indicates the appearance of amorphous copper oxide structure, which the result was further confirmed with the assignment of the diffused ring structure in selected area electron diffraction (Figure S4a). Intriguingly, TEM image with an acceleration voltage at 200 eV showed some holes embedded within the interior of the Cu NPs and the generation of a lighter region at the boundary between the Cu NPs and the polymer shell (Figure 4d). The observation of the porous structure in the interior of the particles and the medium electron scattering at the Cu2O interlayer is difficult to be observed by a high magnetization TEM image with 80 eV (Figure 4e). The lattice spacing was approximately 3.0 Å and 2.1 Å, which is consistent with the {111} and {100} crystal planes, respectively, of the Cu2O crystal structure (Figure 4f) around the metallic Cu core.


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FT-IR spectra verified the vibrational structures of the PSMA polymer shell kept intact before and after the oxidation process at 100 oC (20 min) (Figure S4b). Specifically, the carboxylate groups of the PSMA shell could bond to the Cu ions and preferentially generate copper oxide in the confined area of the polymer nanocapsule once the oxygen atoms diffused into the polymer shell. The formation of pores in the interior accompanied by the growth of the Cu2O shell could be explained by the faster outward diffusion of Cu cations relative to the inward diffusion of oxygen anions. This oxidation process is known as the “Kirkendall effect”, which describes the generation of vacancies during concomitant inward diffusion.39, 56-59 2.4. Synthesis of Cu@Cu2O@polymer NPs from I--assisted Cu@polymer NPs Optical broadening was immediately observed in the I--assisted preparation of the Cu@polymer after the post-injection of the sample into water at 80 °C (Figure 5a). One can observe rapid depletion of the SPR band with the reaction time. Although the shift of the SPR peak toward 660 nm was observed at 10 min, the extinction coefficient (ε) of Cu at 660 nm was 870.4±10.5 L mol-1 cm-1, which is lower than the coefficient of the Cu@Cu2O@polymer NPs of 2261.0±38.8 L molcm-1. Taken together with the TEM image (Figure S5), the inferior absorbance was probably attributed to the removal of a large portion of the plasmonic Cu NPs in the interior. This ε value (2261±38.8 L mol-cm-1) is calculated based on the atom molar concentration, which is independent of the particle geometry, particle concentration, and size. The result is compatible with the spherical Au nanoparticles (~20 nm)60 and higher than NIR iron oxide,61 as shown in Table 1. 12 ACS Paragon Plus Environment


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Among the different oxidation treatments, the Cu@polymer NPs prepared using halide-free conditions with heating at 100 °C for 20 min had the optimal spectra region with a high ε value at 660 nm. Applying illumination at 660 nm, which is close to the boundary between the red and NIR regions in the biological window, is advantageous for the Cu@Cu2O@polymer NP-meditated PTT of cancer cells. 2.5. Examination of the physicochemical properties of the Cu@Cu2O@polymer NPs The facilitation of electron transfer from NaBH4 to 4-nitrophenol by the Cu@Cu2O@polymer NPs was investigated. Optical analysis revealed a decrease in absorbance at 400 nm followed by an increase in absorbance at 310 nm within 30 min (Figure S6), indicating an efficient conversion from 4-nitrophenol to 4-aminophenol. This result provided direct evidence that (1) the metallic Cu nanocore could act as catalyst within the polymer nanocapsule and (2) molecules could inwardly/outwardly diffuse across the polymer shell and Cu2O layer before/after interacting with the Cu nanocatalyst. 2.6. Examination of the stability of the Cu@Cu2O@polymer NPs To determine the particle stability, the morphology and SPR band position of the Cu@Cu2O@polymer NPs were analyzed after storage at 25 °C for 7 days (Figures 5b and S7). The aged colloid solution barely changed compared with fresh samples. Neither the Cu@polymer nor Cu@Cu2O@polymer NPs underwent obvious weight loss after aging in the water, as determined from AA measurements (Figure 5c). The PSMA coating served as a protective barrier to prevent 13 ACS Paragon Plus Environment

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the decomposition of metallic Cu by the H2O/O2 molecules. However, the Cu@polymer NPs degraded rapidly within 4 h in culture medium that contained L-glutamine, glucose, sodium pyruvate, sodium bicarbonate, fetal bovine serum and an antibiotic-antimycotic solution. The biological environment, e.g., the extracellular and intracellular matrix, has a complex composition that includes salts, proteins, H2O2, and other biological molecules, which can interact with Cu NPs and cause the release of Cu ions and damage to living cells.24,

28-32, 62-63

By contrast, the

Cu@Cu2O@polymer NPs retained approximately 70 % of mass after dispersion in a culture medium for 48 h because the composition of the polymer-Cu2O shell structure enhanced the stability of the Cu nanocore against severe etching by biomolecules. Cancer cells have higher levels of endogenous H2O2 than normal cells.44,45 To study whether the Cu@Cu2O@polymer NPs were degradable in the presence of H2O2 as a strong oxidizer (+1.78V vs NHE), UV-visible spectra of the Cu@Cu2O@polymer NPs were recorded after exposure to different H2O2 concentrations for 1 h (Figure 5d). The absorbance intensity at 650-710 nm decreased with the increase in H2O2 oxidant from 0.03 mM to 3 mM. AAS measurements determined the degradation of the metallic Cu core with respect to different H2O2 concentrations as ~30 % at 0.3 mM H2O2 and over 90 % at 3 mM H2O2. 2.7. Cytotoxicity of the Cu-based NPs To evaluate the cytotoxicity of the NPs, HeLa cells incubated with different concentrations of Cu@polymer NPs and Cu@Cu2O@polymer NPs were examined. After removing the Cu-based 14 ACS Paragon Plus Environment


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sample solution, washing with a PBS solution, and replacing the medium with fresh culture medium at 4 h and 24 h, the NP-treated cells were cultured for another 24 h. The results of an MTT assay performed to determine the cell viability are shown in Figure 6a. Remarkably, neither material caused cell death at 10 ppmCu for 4-24 h. Upon the addition of 20 ppmCu, the cytotoxicity of the Cu@polymer NPs and Cu@Cu2O@polymer NPs slightly decreased to ~82 % cell viability. Compared with the Cu@polymer NPs at 20 ppm (below 60 % cell viability), the Cu@Cu2O@polymer NPs showed low toxicity, resulting in approximately ~80 % cell viability after 24 h. Such improved biocompatibility could be attributed to the superior protective layer of the PSMA-Cu2O shell structures that retarded the degradation of the Cu nanocore. As the added particle concentration was increased to 50 ppm, the Cu@Cu2O@polymer NPs were still less toxicity to cells at 4 h (71 %) than at 24 h (49 %). The results indicated the increase of cell death with sample dose and long co-culture time were associate with the gradually release of Cu ions from the Cu@Cu2O@polymer NPs. The IC50 was estimated to be 20 ppm for the Cu@polymer NPs and 50 ppm for the Cu@Cu2O@polymer NPs after 24 h of incubation. These IC50 values were 7fold higher than those determined in a previous study.29-31 The potential cytotoxicity of Cu-based materials is mainly attributed to the generation of intramolecular ROS30, 63 and the release of Cu ions that can damage DNA.64-65 AAS measurements were employed to determine the Cu ion concentration after 4 h of co-culturing. No dramatic distinct effect was observed for the cells incubated with the Cu@polymer and Cu@Cu2O@polymer NPs at 15 ACS Paragon Plus Environment

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10 ppm (~0.8 %) or 20 ppm (~3.6 %) (Figure 6b). At 50 ppm, the Cu concentration released within the cells was ~8 % from the Cu@polymer NPs and 5.8 % from the Cu@Cu2O@polymer NPs. Based on the above dissolution experiment (Figure 5c), we suggest that the additional increase in the Cu content from Cu@polymer NPs could be attributed to the internalization of particles combined with the diffusion of decomposed Cu species more. To analyze the intracellular response of the Cu-based materials inside cells after 4 h of incubation, the ROS generation triggered by the Cu@polymer and Cu@Cu2O@polymer NPs was investigated using a DCFH-DA assay. Figure 6c shows that brightener fluorescence was obtained with the Cu@polymer NPs (20 ppm) over 4-24 h, indicating that these cells experienced high oxidative stress compared with the cells treated with particle-free and Cu@Cu2O@polymer NPs (20 ppm). Shi et al. examined ROS generation in the presence of similar Cu-Cu2O core-shell structures in the solution phase.63 These authors found that the formation of a Cu2O shell stabilized Cu-based NPs and reduced the activity of Cu with oxygen to yield less ROS. Based on this result63 and our intracellular analysis, we propose that the oxidative stress level of cells induced by Cu@Cu2O@polymer NPs decreased, and the cell viability subsequently improved. Considering 30 % decomposition of the Cu@Cu2O@polymer NPs in the medium during 4 h of culturing (Figure 5c), the potential cytotoxicity of these species was carefully studied. The supernatant of the Cu@Cu2O@polymer NPs aged for 4 h was collected and subjected to coculturing with cells again. We observed no additional injury to these cells after 4 h and 24 of 16 ACS Paragon Plus Environment


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incubation (Figure S8), further illustrating the high tolerance of cells for the Cu@Cu2O@polymer NPs and their decomposition products. The cell death pathway could be further identified by analyzing these particle-treated cells with annexin V and PI staining. Both green and red fluorescence of cells incubated with the Cu@polymer NPs were highly expressed at 4 h (Figure S9). Cells in late apoptosis and already dead cells appeared after this incubation. By contrast, red fluorescence was much less detectable in cells after incubation with the Cu@Cu2O@polymer NPs. Very little green fluorescence was observed in a merged image, consistent with a lower cytotoxicity to cells. The red fluorescence from PI and green fluorescence from annexin V-FITC were almost not observed in the control group, which indicates that most of the cells were alive after 4 h of incubation. According to the stability and in vitro analyses, the Cu@Cu2O@polymer NPs posed little risk to cells in terms of ROS and presented acceptable biocompatibility at 20-50 ppm within 4 h of treatment, which serve as proof-of-concept for subsequently demonstrating their applicability to PTT.

2.8. Temperature Elevation by Cu-based NPs

The temperature elevation of the medium by the Cu-based NPs under light exposure was observed, and the results are shown in Figure 7a. After illumination at 660 nm of the Cu@polymer and Cu@Cu2O@polymer NPs at the same Cu concentration, the temperature of the surrounding


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medium increased to 23 °C and 28 °C, respectively, compared with the temperature increase of the particle-free medium (~4.2 °C). Although the mass of the plasmonic core of the Cu NPs occupied a lower proportion in the Cu@Cu2O@polymer than in the Cu@polymer NPs, the local SPR effect and heat generation of the Cu@Cu2O@polymer NPs were promoted because the absorption band matched closely with the incident wavelength at 660 nm.

2.9. Cytotoxicity to particle-treated HeLa cells with and without photothermal killing

The performance and efficiency of the Cu@polymer and Cu@Cu2O@polymer NPs in the in vitro PTT of HeLa cells was analyzed using the MTT assay, and the results are shown in Figure 7b. The cell viability of light-irradiated cells was not significantly different from that of the cells in the light-free treatment. The viability of cells incubated with Cu@Cu2O@polymer NPs at 20 ppm decreased by approximately 50 % after 660 nm light exposure for 4 min, whereas for the cells treated with particles alone only drop by ~20 %. The further decrease in the cell viability by treating 660 nm light was ascribed to the synchronized effects of the acute PTT with continuous Cu ion release, displaying an efficient nanoparticle-based combination therapy for cancer treatment. Efficient phototoxicity was obtained using 50 ppm Cu@Cu2O@polymer NPs, resulting in HeLa cell death of over 90 %. In the same photothermal experiment, only moderate photothermal destruction of cells was observed when using the Cu@polymer NPs. The degradation of the Cu@polymer NPs after 4 h of incubation in the culture medium likely resulted in the lower



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photothermal effect under illumination at 660 nm. On the other hand, the photothermal performance of the Cu@Cu2O@polymer NPs was related to the dose-dependent evolution of temperature in terms of the stronger absorbance at 650-710 nm and better stability in the culture medium. The introduction of light to excite the Cu@Cu2O@polymer NPs did not exhibit an energy transfer pathway from the singlet state to the excited energy of p-type Cu2O to generate single oxygen species (Figure S10), and mainly contributed to photon energy conversion to release heat, causing photothermal injury to HeLa cells. In this proof of concept experiment, we hypothesize the endogenous H2O2 oxidant accelerate the Cu ion release through oxidation of Cu@Cu2O@polymer NPs. The HeLa cells were first co-culture with Cu@Cu2O@polymer NPs for 4 h then followed by LPS endotoxin treatment,66 leading to provoke ROS levels, for another 1 h. DCFHDA dye was utilized to determine the intracellular ROS levels within the cell body (Figure 8a). The result showed strong green fluorescence in the Cu@Cu2O@polymer with LPS-treated group (1 h). To analyze the DCF signals in the cells, semiquantification measurements were measured by ImageJ and shown in Figure 8b. Cu@Cu2O@polymer/LPS-treated cells represent a at least 4.7-fold increase in fluorescence intensity, signifying higher ROS levels, from the treatments by Cu@Cu2O@PSMA or LPS only. Subsequently, (Z)-2-(2-(pyren-1-ylmethylene)hydrazinyl) pyridine67 was utilized to detect the release of Cu ions from Cu@Cu2O@polymer NPs within the HeLa cells (Figure 8c). This pyridinebased dye will generate blue fluorescence when Cu ions are present. Figure 8d provided a direct 19 ACS Paragon Plus Environment

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comparison that cells with Cu@Cu2O@polymer+LPS treatment exhibited 7.2-fold more strong blue fluorescence than the particle-treated alone. Blue emission obtained from the cells reacted with LPS was insignificant (1h). MTT assay showed that the HeLa cell number decreased after the LPSstimulated ROS generation (Figure 8e) in particle-treated group. We observed that the Cu ion release (the middle row in Figure 8c) from the Cu@Cu2O@polymer NPs assisted with LPS stimulation presented an increase of the ROS levels within the cell body (the middle row in Figure 8a). It is probability that the increase of ROS amount was favorable dissolution of more Cu ions within cells from Cu@Cu2O@polymer NPs and thus cause more cells death in the progress. Our preliminary results showed that metastatic cancer cells (cervical HeLa and bladder T24 cells) possessed larger ROS amounts in comparison to the 3T3 fibroblast normal cells (Figure S11). MTT assay examined on Cu@Cu2O@polymer NPs resulted enhanced cytotoxicity of malignant HeLa and grade-III T24 cells in contrast to 3T3 cells in LPS-free groups (Figure 8e). Although this phenomenon relied on intracellular ROS expression, the specific lysis toward cancer cells was possibly induced which would be related to dissolution and release of Cu ions within cell body under high ROS concentrations. Again, direct observation with LPS-induced ROS biostimulation within HeLa and T24 cells showed enhanced cytotoxicity with Cu@Cu2O@polymer NPs (Figure 8f). It is worthy to mentioned that grade-III T24 cells generate highest levels of ROS, which might activate the selectivity of lysis toward T24 cells. 2.10 Survival rate of mice administered Cu@Cu2O@polymer NPs 20 ACS Paragon Plus Environment


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Because excretion is an important safety consideration, the metabolism of potentially toxic Cu ions released from the Cu@Cu2O@polymer NPs in vivo was further evaluated. Eight male BALB/c mice (weight range: 28-30 g) were administered a 20 mg kg-1 sample. Figure 9a shows that the viability rates in the experimental groups were 100 % during two weeks post-injection. Body weight change of mice from the Cu@Cu2O@polymer NPs-treated groups is consistent with no-injection mice (Figure 9b), indicating no acute toxicological impact. We further collected urine from mice at 1 day, 2 days, 3 days post-injection. The samples were analyzed by AAS to determine the levels of Cu ions in the urine, which revealed an accumulated release of Cu ions (Figure 9c). These results suggested that the Cu@Cu2O@polymer is metabolizable, and thus the particle is more appropriate for practical application as an inorganic NIR nanoreagent to avoid the possible long-term retention of non-degradable NPs in the body. The release of higher Cu ions in the urine from the mice group after Cu@polymer NPs treatment was observed (Figure 9c). However, a 75% death rate of mice was found after 3 day of post-injection time when a 20 mg kg-1 of Cu@polymer NPs was administration via i.v. injection (Figure 9a). This result pointed out that a passivation layer onto the Cu-based NPs could significantly decrease the Cu burst release and improve its biocompatibility. 3. Conclusions We successfully fabricated NIR-activated Cu@Cu2O@polymer NPs in a single-step oxidation of Cu@polymer NPs with heat assistance. The absorption band position of the Cu@polymer NPs shifted toward NIR wavelengths during the oxidation process upon 21 ACS Paragon Plus Environment

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variation of the heating temperature and time. The resulting nanoreagent retained its absorbance within the NIR region because the PSMA-Cu2O shell structure stabilized the surface of the plasmonic Cu nanocores. After exposure to biological environments, the moderate redoxactive Cu@Cu2O@polymer NPs did not cause injury to mice and could degrade in the presence of biological molecules and H2O2. Our experiments revealed that the Cu@Cu2O@polymer NPs produced sufficient phototoxicity to kill cancer cells via photothermal effects combined with a low sample dosage (20-50 ppm) and laser power density (610 mW/cm2). LPS endotoxin led to additional endogenous H2O2 oxidant readily increased the release of Cu ions, enabling a more accessible lysis of cancer cells. Even though their toxicity still needs to be addressed in detail, our investigation produced preliminary results demonstrating that the IC50 increases 7-fold using the Cu@Cu2O@polymer NPs, and these particles are degradable and extractable. Xia et al. reported that a metallic Pt nanocluster assembly exhibited the pH-sensitive release of Pt and induces DNA damage.47 In their work, the nanocapsules entered cells, and the Pt release successfully overcame the cisplatin resistance of hepatocellular carcinoma. Xu and co-workers presented the anticancer effects of FePt-CoS2 yolk-shell NPs after cellular uptake due to the release of Pt atoms from the core.49-50 Inspired by Pt-based nanotherapeutics that have led to responsive chemotherapy, we demonstrated that the disintegration of redox-active Cu@Cu2O@polymer NPs into Cu ions would occur after internalization into cancer cells due to their the high level of 22 ACS Paragon Plus Environment


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endogenous H2O245 and/or the intracellular acidic environment (pH < 5.5) in malignant cancerous cells. If the Cu@Cu2O@polymer NPs combined with targeting ligands can be delivered locally to a tumor area, the selective generation of ROS activated by Cu species at elevated H2O2 concentrations, in addition to the photothermal treatment, could harm the nucleus and mitochondria. This could be a useful strategy for synchronous chemo- and photothermal therapy to injure cancer cells more efficiently.

4. Experimental method

4.1. Materials

Copper (II) chloride dehydrate (CuCl2ꞏ2H2O) (Riedel-de Haën 31286), poly(styrene-alt-maleic acid) sodium salt solution (PSMA) (Aldrich 662631), hydrazine hydrate (N2H4ꞏH2O) (Acros Organics 196711000), phosphate buffered saline (PBS) tablet (Sigma P4417), sodium hydroxide (NaOH) (Mallinckrodt 7708-10), hydrochloric acid (HCl) (Fish Scientific 231-595-7), dimethyl sulfoxide (DMSO) (Scharlau SU0155), Dulbecco's modified Eagle's medium - high glucose (DMEM/HG)

(Thermo

Hyclone

SH30003.02),

antibiotic

antimycotic

Solution

(penicillin/streptomycin/amphotericin) (Biological 03-033-1B), fetal bovine serum (FBS) (Biological 04-001-1A), trypsin-EDTA (Biological 03-051-5B), sodium bicarbonate (NaHCO3) (Sigma S7277), trypan blue solution (0.4%) (Sigma T8154), sodium pyruvate (C3H3NaO3) (ACROS 132150250), thiazolyl blue tetrazolium bromide (MTT) (Alfa Aesar L11939), 2’, 7’23 ACS Paragon Plus Environment

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dichlorofluorescin diacetate (DCFH-DA) (Sigma-aldrich D6833), annexin V-FITC fluorescence microscopy kit (BD Pharmingen 550911), propidium iodide staining solution (BD Pharmingen 556463), hydrogen peroxide solution (H2O2) (Sigma H0904), 4-nitrophenol (Sigma-Aldrich 73560), sodium borohydride (NaBH4) (Alfa-Aesar 13432), N,N-dimethyl-4-nitrosoaniline (RNO) (Sigma D172405), Imidazole (Sigma I0250), sodium bromide (NaBr) (J.T.Baker 3588-01), and sodium iodide (NaI) (Alfa-Aesar A15480). All chemicals were used as received.

4.2. Synthesis of Cu@polymer NPs

Cu@polymer NPs were synthesized using a procedure that has been reported previously.51 First, 1 mL of CuCl2 aqueous solution (5 mM) was prepared and mixed with 7.5 mL of deionized water, 2.5 mL of poly(styrene-alt-maleic acid) sodium salt (PSMA) solution (24 mg/mL), 18 μL of 2 M HCl, and 0.1 mL of N2H4 hydrate in a 23 mL Teflon-lined hydrothermal synthesis autoclave reactor. Then, the reactor was placed in a circulator oven to conduct hydrothermal treatment at 158 oC for 3 hr. Next, the reaction product was centrifuged at 11000 rpm for 10 min to separate the NPs from the dissolved reactants. The supernatant of the product was removed, and the NPs were resuspended with deionized water. After washing three times, the NP suspension was purified and collected.

4.3. Synthesis of Cu@polymer NPs with halide addition

250 mM of sodium halide (NaBr and NaI) in a water solution was prepared before use. The desired halide solution volumes of 125 μL, 375 μL, and 500 μL were added to the same CuCl224 ACS Paragon Plus Environment


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PSMA-HCl-N2H4 solution. After 3 hr of hydrothermal reaction, a repeated process including centrifugation and washing was carried out to purify the resulting nanoproducts.

4.4. The preparation of Cu@Cu2O@polymer NPs

10 mL of Cu@polymer NPs (0.8 mM) were added to a round-bottom flask. Thereafter, the flask was heated at 40/60/80/100 oC for 5/10/20/30 min, respectively, to investigate the generation of Cu@Cu2O@polymer NPs with NIR absorbance. The resulting product was collected and centrifuged at 8600 rpm for 10 min, followed by a repeated process including centrifugation and washing with DI water.

4.5. Characterization of Cu-based NPs

0.4 mL of 4.5 M NaOH and 1.5 mL of 4.5 M HCl were sequentially added to 0.1 mL of Cubased NPs for digestion of the polymer and metal core. The concentration of Cu ions was quantified using an atomic absorption analyzer (AA) (AAnalyst200, Perkin Elmer). The structures, compositions, and optical properties of the synthesized NPs were characterized by transmission microscopy (TEM) (JEM-1400 operating at 120 keV and JEM-2100F operating at 200 keV for high-resolution analysis), scanning electron microscopy (SEM) with an energy dispersive X-ray spectrometer (EDS) (NanoSEM 230, Nova), a multipurpose X-ray thin-film diffractometer (XRD) (D/MAX250, Rigaku), a UV-visible spectrophotometer (8452A, Hewlett Packard), dynamic light scattering, and Zeta potential measurement (Zetasizer Nano Z, Malvern), respectively. 25 ACS Paragon Plus Environment

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4.6. Stability and degradability of Cu-based NPs

The stability of Cu-based NPs (167 ppm) in DI water and medium solution for 7 days was analyzed by using TEM images and UV-visible spectra combined with AA measurements to determine the dissolution of Cu ions from the mother solution. The same analyses were performed to evaluate the degradability of 210 μL of Cu-based NPs (42 ppm) after adding 0.72/7.2/72 μL of an H2O2 solution.

4.7. Physicochemical properties of Cu-based NPs

The catalytic reduction property was examined by mixing 20 μL of 0.1 mM Cu-based NPs with 1 mL of 0.175 mM 4-nitrophenol and 1 mL of 0.5 M NaBH4. Then UV-visible spectrum was employed to analyze the absorbance at 400 and 310 nm for the mixed solution at different time intervals. The photochemical reaction of the singlet oxygen generation of Cu-based NPs was demonstrated by using an RNO/ imidazole test. 1μL of a 25 mM RNO solution and 1 μL of a 25 mM imidazole solution were added into 300 μL of 300 ppm Cu-based NPs. Then, the mixed solution was excited with light under 660 nm laser irradiation at a power intensity of 32 mW/cm2. After 0/4/8/16 min of exposure, the absorption peak at 440 nm was monitored with a UV-visible spectrum.

4.8. Cell culture



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HeLa cells were cultured in a DMEM-HG culture medium supplemented with 10% fetal bovine serum and a 1% antibiotic-antimycotic solution for in vitro testing. First, the cells were seeded on a 10 cm culture dish at 37 oC in a humidified incubator under 5% CO2. When the cells were attached on the dish and had proliferated to an appropriate amount, the medium was removed, and the cells were washed with PBS. Then, trypsin-EDTA was added and incubated with the cells for 4 min at 37 oC to detach the cells from the culture plate. To stop the reaction between the trypsin-EDTA and the cells, the culture medium was added to the dish. The medium with the suspended cells was moved to a centrifuge tube and centrifuged at 900 rpm for 5 min. Then, the supernatant was removed to eliminate the trypsin-EDTA, and the cells were resuspended in the culture medium. To estimate the number of cells, trypan blue was used to mark the dead cells, and a hemocytometer was used to evaluate the amount of viable cells. The suspended cells were prepared for use in the subsequent experiments.

4.9. Cytotoxicity of Cu-based NPs

For the cytotoxicity test, HeLa cells were incubated in a 96-well plate at a density of 5000 cells per well for 1 day. Then, the Cu-based NPs were diluted to the desired concentration by centrifugation and redispersion in a culture medium for use as a cell culture. Next, after the PBS wash, the incubated cell medium was replaced with 100 μL of a culture medium with a different concentration of NPs. To measure the cytotoxicity of the Cu-based NPs after a short incubation


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time, the cells were cultured with an NP-containing medium for 4 hr. Then, the medium was removed, and pure medium was added for another 20 hr of incubation. The cytotoxicity for 1 day was also measured by incubating the cells with an NP-containing medium for 24 hr. These two groups were then tested using an MTT assay. The medium was replaced with a new medium containing 10% MTT solution (5 mg/mL) by volume. Then, the cells were incubated for 3.5 hr, and DMSO was used to dissolve purple formazan produced by viable cells. The absorbance at 570 nm was measured with a microplate reader (Awareness Technology), and cell viability was defined as the ratio between the treated groups and the control group that was not exposed to the Cu-based NPs. Similar experimental processes were performed for examining the in vitro cytotoxicity of the supernatant collected from the 4h-aged Cu-based NP solution.

4.11. Cellular uptake of the copper element

To determine the amount of Cu within the cells, HeLa cells were co-cultured with the desired concentration of Cu-based NPs for 1 day. After 4 and 24 hr of incubation, the medium was removed and washed with a PBS solution. The particle-treated cells were lysed with 100 μL of DMSO. Finally, NaOH and HCl were respectively reacted with the cells to digest the Cu-based NPs. An AA measurement was used to analyze the concentration of Cu ions.

4.12. ROS generation test of Cu-based NPs 28 ACS Paragon Plus Environment


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HeLa cells were incubated in a 24-well plate at a density of 50000 cells per well for 1 day. The culture medium was removed, and the cells were washed with PBS. Then, a DCFH-DA stock solution (1 mM) was diluted to 40 μM with PBS, and the cells were incubated with 1 ml of the diluent for 30 min to uptake the DCFH-DA. Next, the cells were washed with PBS to remove any excess DCFH-DA, and 1 mL of medium with 50 ppm Cu-based NPs was added and incubated with the cells. After 4/24 hr of incubation, the medium was removed, and the cells were washed twice and rinsed with PBS. Finally, the fluorescence emission was observed with a fluorescence microscope (Nikon Eclipse 80i) to estimate the ROS concentration level.

4.13. Identification of cell death pathway

1 mL of medium with 50 ppm of Cu-based NPs was added and incubated with 50000 cells per well. After 4 hr of incubation, the culture medium was removed, and each group was washed twice with cold PBS. Also, an annexin V binding buffer was prepared by diluting 10X annexin V binding buffer in an annexin V-FITC fluorescence microscopy kit to 1X with deionized water, and the working solution was prepared by mixing annexin V-FITC, PI and an annexin V binding buffer at a ratio of 5:1:45 by volume. The cells were rinsed with the annexin V binding buffer and stained with 1 mL of the working solution at room temperature for 15 min. Finally, the cells were washed twice and rinsed with the annexin V binding buffer, and fluorescence images of the cells were observed with a fluorescence microscope.


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4.14. Temperature elevation of Cu-based NPs under irradiation

A 0.1 mL medium solution including Cu-based NPs (50 ppm) was added into a 96-well plate and subjected to irradiation conditions under an LED board with a wavelength of 660 nm and a power of 610 mW/cm2 in an enclosed environment by using a Styrofoam box to cover the entire plate. The temperature of the solution was monitored every 30 s by using a thermocouple probe with a plastic insulation-encapsulated copper wire linked with a digital thermometer.

4.15. In vitro photothermal therapy

HeLa cells were incubated in a 96-well plate at a density of 5000 cells per well for 1 day. Then, the Cu-based NPs were diluted to the desired concentration by centrifugation and redispersion in a culture medium for use as a cell culture. Next, after a PBS wash, the incubated cell medium was replaced with 100 μL of the culture medium with different concentrations of NPs. The cells were cultured with an NP-containing medium for 4 hr in a culture plate and then removed from the incubator, which was enclosed with a 15.5 cm/15.5 cm/18 cm L/W/H Styrofoam box and irradiated on the bottom with an LED board with a wavelength of 660 nm and a power of 610 mW/cm2 for 4 min. Thereafter, the Cu-based NP-containing medium was replaced with pure medium for another 20 hr of incubation. Another examination was performed for the cells incubated with the Cu-based NPs without 660 nm-light illumination as a control experiment. The cell viability with light-excited



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and light-free cells were measured using an MTT assay to investigate the effect of photothermal therapy.

4.16. Pyrene-based chemosensor to assess the dissolution of Cu-based NPs.

The HeLa and 3T3 cells cultured in DMEM and T24 cultured in McCoy's 5A medium were treated with 20 and 50 ppm[Cu] solutions of Cu-based NP and incubated at 37 °C for 4 h. The treated cells were washed with PBS solution to remove free Cu-based NP. Afterwards, 50μg/ml of LPS endotoxin in medium was added to the particle-treated cells to induce the ROS generation after 60 min of incubation time. After washing excess LPS reagents with PBS solution, mediums including (Z)-2-(2-(pyren-1-ylmethylene)hydrazinyl) pyridine chemosensor (10 μM) and DCFHDA (20 μM) were added to these particle-treated cells to determine the Cu ions and ROS levels, respectively, within cells. The staining time is 30 min, followed by a purification process with PBS before observation. We employed a fluorescence microscopy combined with DAPI and FITC channels to detect pyridine chemosensor- and DCFHDA-stained cells, respectively. 4.17. Collection of Cu ions from the mice urine after intravenous injection 4-5 weeks old male ICR mice were housed in metabolic cages with food and water. Mice were acclimatized for 3 days before injection. After tail vein injection of Cu@Cu2O@polymer NPs at a dosage of 20 mg Cu/kg body weight, urine samples were collected at 24 h, 48 h, and 72 h post injection. The concentrations of Cu ions were quantified by AA and the volumes of urine were


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recorded. The body weights were measured by using an electronic balance electronic balance (ATY224, SHIMADZU) every day for 14 days after injection. X-ray absorption spectroscopy was collected at NSRRC Beam Line 17C1. The Si (111) crystals were used as a monochromator for energy selection. The spectrum of target element foil was concomitantly recorded for internal energy calibration through the first inflection point. The Cu K-edge spectra was scanned over the energy 8.77-9.67 KeV on the fluorescence mode by using a Lytle detector, with a step size in the near-edge region of 0.4 eV.

4.17. Statistical analysis

All data are expressed as means ± standard deviation. Comparison of different groups was determined using a Student’s t-test and a significant difference was assumed to be a p-value ≤ 0.05. The statistical data was analyzed using SigmaPlot 10.0. ASSOCIATED CONTENT DLS measurement and TEM images of Cu@polymer NPs prepared with NaBr (125 μL – 500 μL) and NaI (500 μL) agent with refluxing reaction at 40 oC and 60 oC. SAED pattern and FT-IR spectra of Cu@Cu2O@polymer NPs. UV-visible analysis of the reduction of 4-nitrophenol and the RNO/imidazole indicator with Cu@Cu2O@polymer NPs. TEM image for the stability of Cu@Cu2O@polymer NPs. MTT assay, fluorescent image (stained with annexin V-FITC (green)/PI (red)), and the integrated DCFH-DA fluorescence intensity of cells treated with Cubased NP using 660 nm LED or under dark condition. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author:

*Prof. Jiashing Yu E-mail: [email protected]. 32 ACS Paragon Plus Environment


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*Prof. Mei-Yi Liao E-mail: [email protected]

ORCID Jiashing Yu: 0000-0002-0782-2328. Mei-Yi Liao: 0000-0003-3872-0049. Author Contributions † Yu-Wei Tai and Yi-Chun Chiu contributed equally.

Notes The authors declare no competing financial interest.

Acknowledgments Thanks to Prof. Chi-Shen Lee for help on the XAS experiments. This work was supported in part by grants from the National Science Council, Taiwan (MOST 104-2113-M-153-001MY2 and 104-2221-E-002 -124 -MY3 and 106-2113-M-153-001), and a grant from the Taipei City Hospital (TCH) and the Department of Health, Taipei City Government (TCH No. 10401-62-058 and 10601-62-015).

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Table 1. Summary of the extinction coefficient (ε) by spherical Au NPs,60 Cu@Cu2O@polymer NPs, NIR iron oxide material61 with element molar concentration (M) and mass concentration (ppb). Samples

Au NPs(peak maximum shifted)

Cu NPs (@660 nm)

NIR iron oxide (@808 nm)

5 nm

20 nm

50 nm

Cu@Cu2O@polymer

Fe2O3/Fe3O4

Unit: L∙mol-1∙cm-1

1536

2561

4728

2261 ± 38.8

620

Unit60: ppb-1∙cm-1

7.8 ± 0.04 × 10-6

1.3 ± 0.002 × 10-5

2.4 ± 0.01 × 10-5

3.6± 0.06 × 10-4

1.1 × 10-4


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Scheme 1. Flow chart of the synthesis of the Cu@Cu2O@polymer NPs through the oxidation of Cu@polymer NPs. These Cu-based NIR nanoagents enabled the relaxation of NIR photons for the thermal destruction of cancer cells and were able to be degraded and excreted.



Figure 1. a) TEM image, b) HR-TEM image, c) XRD pattern, and d) UV-visible spectrum of the as-prepared Cu@polymer NPs. The photograph inserted in d) shows a colloidal Cu@polymer solution.


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Figure 2. a-c) TEM images and d) UV-visible spectra of the Cu@polymer NPs synthesized in the presence of (a) 125 μL (b) 375 μL (c) 500 μL of a NaI (250 mM) solution.



Figure 3. a) TEM images of the Cu@polymer NPs after 7 days of aging. b) UV-visible spectra of the Cu@polymer NPs in water after 7 days of aging. UV-visible spectra of the Cu@polymer NPs after heating at c) 80 °C and d) 100 °C for 0-30 min.


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Figure 4. a) Histogram of the size distribution characterizations and b) XAS analysis for the Cu@polymer NPs through different refluxing reaction time. c) XRD pattern, d) 200 keV TEM, e) high-magnification TEM (120 keV), and f) 200keV HRTEM images of the Cu@Cu2O@polymer NPs prepared at 100 °C after 20 min.



Day 0 Day 1 Day 4 Day 7

b) Normalized Absorbance

Absorbance (a.u.)

a)

0 min 5 min 10 min 15 min 20 min

400

c) 200

500

600 Wavelength (nm)

700

500

d)

Cu@polymer Cu@polymer NPs in DI water Cu@polymer Cu@polymer NPs in medium medium Cu@Cu Cu@Cu2O@polymer NPsin inDI DIwater water 2O@polymer NPs Cu@Cu2O@polymer NPsin inmedium medium Cu@Cu 2O@polymer NPs

150

400

800

600 Wavelength (nm)

100

50

700

800

0 mM 0.03 mM 0.3 mM 3 mM

Absorbance (a.u.)

Remained percentage (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 0

10

20 30 Time (hr)

40

50

400

500

600 700 800 Wavelength (nm)

900

1000

Figure 5. UV-visible spectra monitoring the oxidation of a) the I--assisted synthesized Cu@polymer NPs at 80 °C and b) the stability of the as-prepared Cu@Cu2O@polymer NPs at 25 °C for 7 days. c) AA measurements of the percentage of Cu material remaining in the precipitate after dispersing the Cu@polymer and Cu@Cu2O@polymer NPs in DI water and culture medium for 0-48 h. d) UV-visible absorption spectra of the Cu@Cu2O@polymer NPs dispersed in an H2O2 solution (0.03-3 mM) for 1 h.


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Figure 6. a) MTT assay with HeLa cells treated with Cu@polymer and Cu@Cu2O@polymer NPs. The excess materials were removed after 4 h and 24 h of incubation, followed by another incubation for 24 h. b) AA analysis of the cellular uptake of the Cu materials after 4 h of incubation. c) Fluorescence images of HeLa cells stained with DCFH-DA and then treated with Cu-based NPs for 4 h and 24 h of incubation. Scale bar: 50 μm



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Figure 7. a) Temperature difference between the Cu@polymer NPs and Cu@Cu2O@polymer NPs at 50 ppm dispersed in medium upon irradiation with 660 nm light at 610 mW/cm2. b) MTT assay to evaluate the cell viability of HeLa cells treated with Cu@polymer and Cu@Cu2O@polymer NPs for 4 h under two conditions of excitation with 660 nm light for 4 min and dark aging (4 min), followed by further incubation for 24 h.


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Figure 8. Intracellular ROS production of HeLa cells co-cultured with Cu@Cu2O@PSMA, Cu@Cu2O@PSMA with LPS, and LPS only. Microscopic image was taken under different treatment (a, c) and fluorescence intensity per area was analyzed in each group (b, d). The left, middle, and right lane represent the bright field, fluorescence image, and merged image. MTT result showed e) Cu@Cu2O@PSMA treatment only and f) cell cytotoxicity enhancement when LPS is co-cultured with Cu@Cu2O@PSMA. All data are presented as mean ± standard deviation (n = 4)



100 No injection Ctrl Cu@polymer Cu

Survival percentage (%)

a)

75

Cu@Cu2O@polymer Cu2O

50 25 0 0

2

b) 160

Relative Body Weight (%)

4

6 8 10 Days after injection

12

14

No Injection NoNPs injection

140

Cu@Cu2O@polymer Cu2O

120 100 80 60 40 20 0 0

c)

1

2

3

4

5 6 7 8 9 10 11 12 13 14 Days after injetion

30

Noinjection injection No 25

Copper accumulation (μg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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20

Cu@polymer Cu Cu2O Cu@Cu2O@polymer

15 10 5 0 24

48 Hours after injection

72

Figure 9. a) Survival analysis of mice under different treatments. Sample dose is at 20 mg/kg (n=8). b) Body weight changes for mice treated with Cu@Cu2O@polymerNPs at doses of 20 mg/kg and without NPs (n=8). c) Cumulative excretion of Cu ions from the whole urine of the mice injected with Cu@Cu2O@polymer NPs (20 mg/kg (n=8)) and Cu@polymer NPs (20 mg/kg (n=8)) and without NPs. 49 ACS Paragon Plus Environment

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TOC


Degradable NIR-PTT Nanoagents with a Potential Cu@Cu2O

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