Dual-Stimuli-Responsive, Polymer-Microsphere-Encapsulated CuS

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Dual-stimuli Responsive Polymer Microspheres Encapsulated CuS Nanoparticles for Magnetic Resonance ImagingGuided Synergistic Chemo-Photothermal Therapy Li Zhang, Zhe Yang, Wei Zhu, Zhilan Ye, Yiming Yu, Zushun Xu, Jinghua Ren, and Penghui Li ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00204 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 27, 2017

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ACS Biomaterials Science & Engineering

Dual-stimuli Responsive Polymer Microspheres Encapsulated CuS Nanoparticles for Magnetic Resonance Imaging-Guided Synergistic Chemo-Photothermal Therapy Li Zhang,1 Zhe Yang,1 Wei Zhu,1 Zhilan Ye,2 Yiming Yu,1 Zushun Xu,1, * Jinghua Ren,2, * and Penghui Li3, *

1

Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials; Ministry of Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei University, Wuhan, Hubei 430062, China

2

Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430022, China 3

Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China

*

To whom correspondence should be addressed.

Prof. Zushun Xu, School of materials science and engineering, Hubei University. 368 Youyi Road, Wuchang

District, Wuhan, Hubei 430062, China. E-mail: [email protected]. Tel: +86 27 88661897; Fax: +86 27

8866561092

Dr. Jinghua Ren, Huazhong University of Science & Technology, Cancer Center, Union Hospital. 109 Machang

Road, Jianghan District, Wuhan, Hubei 430062, China. E-mail: [email protected].

Dr. Penghui Li, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese

Academy of Sciences. 1068 Xueyuan Road, Nanshan District, Shenzhen 518055, China. E-mail: [email protected].

ACS Paragon Plus Environment


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ABSTRACT: Integrating biomedical imaging and multimodal therapies into one platform for enhanced anti-cancer efficacy is of great significance. Herein, a core/shell structured nano-theranostic

(CuS@copolymer)

for

magnetic

resonance

imaging

(MRI)-guided

chemo-photothermal therapy was simply prepared via emulsifier-free emulsion polymerization with the full participation of hydrophilic CuS NPs, styrene (St), N-isopropylacrylamide (NIPAm), methacrylic acid (MAA), and polymerizable rare earth complex (Gd(AA)3phen). The synthesized multifunctional microspheres with excellent biocompatibility exhibited high loading capacity (15.3 wt%) for DOX·HCl and excellent drug release under low pH and high temperature. The photosensitive CuS cores which can simultaneously efficiently absorb near infrared (NIR) light and convert NIR light to fatal heat, leading to a synergistic therapeutic effect combined photothermal therapy (PTT) with chemotherapy. Moreover, the temperature sensitive copolymer attached onto the CuS nanoparticles was able to be productively infected by the thermal effect and give rise to a highly controllable DOX release. Furthermore, the CuS@copolymer/DOX showed an enhanced therapeutic efficacy against 4T1 cells than separate photothermal therapy or chemotherapy. Additionally, the drug delivery procedure could be visualized by in vivo MR images and the longitudinal relaxivity (r1) was calculated to be 10.72 mM-1 s-1. These results suggest the CuS@copolymer microspheres highly attractive candidates for biomedical applications. KEYWORDS: MRI, stimuli-responsive, CuS nanoparticles, NIR, synergistic therapeutic.

1. INTRODUCTION Cancer is still a major devastating disease for human beings and the incidence rate is increasing


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year by year. Over the past decades, chemotherapy has been one of the most commonly used therapeutic approaches in clinical cancer therapy. However, the available chemotherapeutic drugs used clinically are known to suffer from the drawbacks of systemic side effects and insufficient dosage to the pathologic tissues.1-2 In order to solve this problems, various strategies have been exploited for drug delivery systems (DDSs) aiming to regulate drug release, improve the anticancer efficacy, and reduce side-effects.3-7 In particular, stimuli-responsive DDSs using “smart” nanocarriers are considered effective tools, which can release drugs quickly in response to local tumor circumstance, such as pH,8 temperature,9 redox,10 enzyme,11 and light,12 etc. Among them, methylacrylic acid (MAA) and N-isopropylacrylamide (NIPAm) as the representatives of pH and temperature sensitive materials have been the intense focus of research for years. Moreover, the combination of pH and temperature sensitive polymers would produce a significant synergistic effect and resulted in accelerated drug release.13-14 Unfortunately, although great progress has been made, the primary tumor would still metastasize and invade other organs, and drug resistance in solid tumors remain formidable problems with chemotherapy.15 PTT is a newly developed treatment modality which commonly utilizes photosensitizing agents to strongly absorb light from near-infrared (NIR, λ = 700-1100 nm) light energy sources and to convert luminous energy into heat for in situ cancerous cell killing or ablation.16 Compared to conventional chemotherapy, PTT possesses the advantages including improved selectivity, precise and minimally invasion, little harm to healthy tissues, and strong antitumor effect.17-18 More importantly, PTT can directly eradicate the cancer cells in primary tumor or local metastasis in lymph node nearby to combat the initial stage of cancer metastasis.19 Nowadays, an increasing number of nanomaterials have been widely investigated as efficient NIR-absorbing candidates



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comprising both inorganic nanoparticles (gold nanostructures,20 palladium nanosheets,21 MoS2 nanosheets,22 and copper chalcogenide,23 etc.) and organic compounds (carbon nanotubes,24 graphene,25 polypyrrole,26 polydopamine,27 and indocyanine green (ICG),28 etc.). Of those, copper sulfide (CuS), a well-known p-type semiconductor material has emerged as a new class of photothermal agents attributing to their simplicity of synthesis, good biocompatibility, low cost, high photostability and outstanding photothermal conversion efficiency.29 Additionally, unlike the gold nanostructures, the broad absorption in the NIR region originates from the d-d energy band transition of Cu2+ ions instead of the surface plasmon phenomenon, which is hardly altered by the particle size, shape and surrounding medium, resulting in better stability of photothermal conversion.18 On account of the unsatisfactory results acquired from single treatment modality, a synergistic strategy binding different drugs and mechanisms with enhanced efficacy for cancer therapy is extremely appealing. Some studies have focused on the combination therapy systems, such as chemo-photothermal therapy,21 chemo-photodynamic therapy,30 photothermal-photodynamic therapy,31 photodynamic-radiation therapy,32 etc. Peculiarly, the integration of chemotherapy and photothermal therapy into one drug delivery vehicle has been demonstrated to provide a strong synergetic effect to maximize therapeutic efficacy, overcome treatment resistance, and diminish adverse effects.15 Drug delivery vehicles for NIR-triggered release are mainly composed of NIR absorption agents and a drug-containing moiety. Numerous good researches have suggested that the incorporation photothermal therapy with chemotherapy played better role in destroying tumor than each separate treatment.33 In some examples, Lu et al. developed a facile one-pot approach to synthesis of colloidal stable, monodisperse, highly PEGylated mesoporous silica coated copper


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sulfide nanocomposites for the combination of photothermal therapy and chemotherapy.34 Bi et al. presented a novel and multifunctional doxorubicin (DOX) conjugated copper sulfide nanoparticle (CuS-DOX NP) drug delivery system using hydrazone bonds to conjugate carboxyl-functionalized copper sulfide nanoparticles (CuS NPs) and DOX for efficient synergistic therapy triggered by near-infrared light.29 Su et al. designed mesoporous silica-coated prussian blue nanocubes with PEGyltation

to

construct

multifunctional

PB@mSiO2-PEG

nanocubes

for

combined

photothermal-chemotherapy of tumor.35 Importantly, the NIR light responsive system for drug delivery combines chemotherapy and photothermal therapy, exhibiting synergistic effects for cancer therapy due to enhanced cytotoxicity of the anticancer drug doxorubicin hydrochloride (DOX) at elevated temperatures and higher heat sensitivity for the cells exposed to the DOX. Nevertheless, most of the synthetic procedure suffer from complicated surface modification steps and involve in numerous organic solvents, which is not favorable for further biological applications. Molecular imaging has been widely used in the early diagnosis of cancer. However, most cancer diagnosis and treatment are relatively independent process of each other currently, which is far from the requirement of medical treatment.36 Imaging guidance could be utilized to identify the tumor sites before treatment or visualize the procedure during treatment in real time, providing valuable information to enhance anti-cancer efficacy and optimize therapy with greater safety, accuracy and sensitivity.37 Therefore, the design and construction of a proper theranostic platform with diagnostic capabilities and simultaneously release drugs attracted considerable research interest.38 As an evolving new field, theranostic probes which embrace numerous advanced capabilities in an all-in-one single platform to provide a comprehensive diagnosis and an



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individualized therapy at earliest stage, have better advantages over traditional imaging and therapeutics, consist of multimodality imaging as well as sustained/controlled/targeted delivery.21,39 Among multitudinous imaging modalities, MRI has become the most commonly used diagnostic tool because of its excellent high spatial and temporal resolutions, especially in soft-tissue imaging. As one of the most powerful non-invasive medical imaging techniques, MRI can offer superior three dimensional details and tissue-depth-independent images. Gadolinium (III) based complexes are testified to be the most frequently used clinically for T1-weighted MR imaging as a consequence of their unique spin paramagnetism.40-41 Therefore, it would be particularly significant for us to investigate such MR imaging-guided theranostics. Herein, we designed a core/shell structured CuS@copolymer nano-theranostic for MRI-guided chemo-photothermal therapy by emulsifier-free emulsion polymerization. The reaction was conducted only in the aqueous phase with a mild temperature and microspheres with clean surface were obtained in a short time. The synthesized CuS@copolymer microspheres possess good biocompatibility and exhibit high loading capacity for DOX·HCl. The escape of the loaded drug molecules was sped up not just at lower pH or higher temperature in a pH/temperature-responsive manner but also by NIR light. Further experiments demonstrated that the synergistic therapeutic effects combined photothermal therapy with enhanced chemo-therapy was highly efficacious for cancer therapy. Additionally, the microspheres showed a long circulation in blood vessels and considerable accumulation in tumor, which was visualized by MRI (Scheme 1). 2. EXPERIMENTAL SECTIONS 2.1. Preparation of hydrophilic CuS nanoparticles


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The CuS NPs were prepared on the basis of reported method without further modification as followed.42 CuS NPs (2 mmol) was dispersed in a 500 mL four-necked flask with 200 mL ethanol-water mixed solvents (volume ratio = 1:1). Then OA (2 mmol) was added dropwise at with a thermostatic water-bath at 80 °C. The desired OA/CuS NPs were obtained via centrifugation to get rid of unreacted OA after 2 h. Subsequently, the OA/CuS were diverted into a beaker containing 50 mL NaUA (0.5 g) aqueous solution. Finally, hydrophilic (OA/NaUA)/CuS NPs were synthesised by ultrasound with a solid content of 0.36 %. 2.2. Preparation of CuS@copolymer and DOX-loaded CuS@copolymer microspheres The synthesis of NaUA and Gd(AA)3Phen were according to our previous paper.43 CuS@copolymer composite microspheres with a core-shell structure were prepared via one-pot emulsifier-free emulsion polymerization. Briefly, 0.5 g of hydrophilic CuS fluid, 0.6 g of St, 1.0 g of NIPAm, 0.25 g of MAA, and 0.12 g of KPS were dissolved in 50 mL of deionized water by ultrasound dispersion in an ice bath for 20 min. Then the mixture was transferred to a 250 mL four-necked flask bubbled with flowing N2 to get rid of dissolved oxygen and under stirring to form homogeneous solution. The polymerization was carried out at 78 °C via a thermostatic water-bath and the stirring rate was maintained at 300 rpm. Half an hour later, 0.08 g of Gd(AA)3Phen dissolved in 15 mL deionized water was added in the mixture. After another 3 hours of polymerization reaction, the dark green products were dealt with overnight dialysis (cut-off Mw = 14000 Da) in deionized water to purify for seven days. DOX loaded process was carried out via direct mingling DOX·HCl and CuS@copolymer to obtain theranostic probes. Typically, 50 mg of refined CuS@copolymer was dissolved in a 50 mL two-necked flask with 15 mL of deionized water, then 10 mg of DOX·HCl dissolved in 15 mL



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deionized water was added with moderate stirring of 200 rpm for overnight in the dark. Subsequently, a dialysis tube was used to get rid of the free DOX via dialysis method (cut-off Mw = 14000 Da). The resulting products were used to measure the concentration of DOX·HCl according to previous method.44 The drug loading content (DLC) and the drug loading efficiency (DLE) according to the following equations: DLC (%) = weight of drug encapsulated in CuS@copolymer/DOX / weight of CuS@copolymer × 100%

(1)

DLE (%) = weight of drug encapsulated in CuS@copolymer/DOX / weight of drug in feed × 100%

(2)

3. RESULTS AND DISCUSSION 3.1. FT-IR characterization FT-IR was later performed to confirm the chemical structures of products. The peaks at 565 cm-1 and 620 cm-1 were found in both Figure 1(a) and Figure 1(c) (red circle), which are respectively associated with the Cu-O and Cu-S vibration. Moreover, the absorption at 1629 cm-1 in Figure 1(a) corresponds to carbonyl stretching vibration of OA and no peaks are observed between 1690 cm-1 and 1720 cm-1, testifying that carboxylate oxygen and copper were bonding with coordinated bond. As shown in Figure 1(b), the sharp peak at 3420 cm-1 belongs to typical carboxylic O-H vibration and the peak around 1721 cm-1 is identified as C=O stretching of carboxyl groups in PMAA. The peaks at 1638 cm-1 and 1552 cm-1 are attributed to C=O stretching vibration and N-H bending vibration of PNIPAm and peaks at 1382 cm-1 and 1368 cm-1 are associated with the isopropyl groups.45 The peaks at 815 cm-1, 768 cm-1 and 696 cm-1 can be ascribed to flexural vibration of


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C-H in benzene ring. Compared with the Figure 1(a) and Figure 1(b), the weak absorption peak at 450 cm-1 is derived from Gd-O vibration in Gd(AA)3Phen.43 All these observations support the monomers have been successfully participated in the polymerization reaction. 3.2. Morphology and size TEM was used to investigate the morphology and size distribution of the as-synthesized samples. As shown in Figure 2(a), some aggregates was observed because of the high surface energy of CuS NPs and their diameter was measured to be 8~9 nm. And in Figure 2(b), it is clear that the pure copolymer microspheres are respectively uniform with a same size around 235 nm. In addition, as shown in Figure 2(c), the CuS@copolymer microspheres are monodisperse and spherical in shape, the average size was about 295 nm. The detailed structure of the CuS NPs within the latex was visualized and the CuS nanocrystals were completely encapsulated into the polymer microspheres. The diameters of the pure copolymer and CuS@copolymer in water were also measured by DLS. As displayed in Figure 2(d) and Figure 2(e), the average sizes are 262 nm and 312 nm, respectively, which are slight larger than that of TEM results due to their swelling in water. In addition, the polydispersity indexes (PDI) are 0.059 and 0.070, suggesting the narrow size distribution of the prepared microspheres. 3.3. XRD characterization The as-obtained CuS NPs and CuS@copolymer were performed by XRD patterns to get insight into the phase structures. As shown in Figure S1, the location and strength of all diffraction peaks of as-prepared CuS NPs are very consistent with the standard diffraction peaks of CuS (JCPDS No. 79-2321). No obvious impure peaks were observed, suggesting the synthesized CuS NPs were



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highly pure. The peaks at 2θ = 27.6°, 29.3°, 31.7°, 32.7°, 48°, 52.6°, 59.2° are ascribed separately to the (101), (102), (103), (006), (110), (108) and (116) planes of CuS, and the wide diffraction peak around 13° belongs to the crystalline nature of the copolymer. The XRD pattern peaks of CuS@copolymer are consistent with that in as-prepared CuS NPs, although the intensities of the diffraction peaks were greatly weakened. These consequences further indicate that the CuS NPs maintained phase structures well after polymers coating. 3.4. Stabilities characterization Physiological stability is a key parameter for potential biomedical applications of synthesized products. The sizes of CuS@copolymer microspheres before and after 7 day’s dialysis at deionized water and PBS buffer (pH 7.4) are 312 nm and 315 nm, 328 nm and 336 nm, respectively, and the zeta potentials of CuS@copolymer microspheres before and after 7 day’s dialysis at deionized water and PBS buffer (pH 7.4) are -28.9 mv and -29.6 mv, -31.8 mv and -33.4 mv, respectively, suggesting that the CuS@copolymer microspheres are stable in this pH range (Figure S2 and Figure S3). Additionally, there are negligible free Gd ions released from the polymeric microspheres after dialyzed for 7 days, which can also verify the good physiological stability of synthesized CuS@copolymer microspheres. The thermostability of as-synthesized sample was measured by TGA in N2 atmosphere. As shown in Figure S4(a), the initial weight loss of 0.83% is owing to the volatilization of surface absorption water. With continuous increasing in temperature, the long carbon chains of OA begin to decompose and the total weight loss percentage is about 30.3%. In the pure copolymer, a sharp curve between 250 °C and 420°C occurred due to removal of the Poly(St-NIPAm-MAA) and the residual weight was almost 0. Compared with Figure S4(b), the remnant of 0.94% in Figure S4(c)


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should be that of the pure CuS nanoparticles and tiny amounts of gadolinium. The investigation results demonstrate that CuS@copolymer possess good thermostability with an onset degradation temperature of 250 °C. 3.5. Stimuli-responsive properties The pH and temperature properties of the as-synthesized CuS@copolymer were performed by DLS measurement at various pH and temperatures values, respectively. As shown in Figure 3(a), with the increase of the pH value from 2 to 9, the hydrodynamic diameter (Dh) of the CuS@copolymer raised from 291 nm to 350 nm, indicating that the CuS@copolymer were prominently pH-responsive. When pH is less than 6, more hydrogen bonds were formed among MMA (pKa ~6)46 units due to the protonation of carboxyl groups, resulting in shrinkage of the microspheres. On the contrary, the diameter increased because the carboxyl groups are ionized and hydrogen bonds decreased under high pH values. Figure 3(b) shows the changes in Dh of the CuS@copolymer as a function of temperature. It was found that Dh of the CuS@copolymer had slightly decrease at lower temperatures and significantly dropped to 278 nm when temperature past 33 °C, revealing a lower critical solution temperature (LCST) of the CuS@copolymer at 33 °C. These results might be explained by the intermolecular and intramolecular hydrogen bonding of temperature-responsive PNIPAm, leading to a hydrophilic state at below LCST and a hydrophobic state at above LCST. 3.6. Photothermal performance The optical properties of as-obtained CuS@copolymer were characterized by UV-vis-NIR absorption spectra. As shown in Figure 4(a), the CuS@copolymer presented a wide absorption in the wavelength from 300 to 1100 nm, which may result from the d-d intra-band transitions of Cu2+



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rather than the localized surface plasmon resonance. Moreover, the UV-vis-NIR absorption spectra of CuS@copolymer almost in line with the overlap of pure copolymer and CuS patterns, indicating the successful coating of CuS in the latex particles. In addition, the typical absorption peak of DOX at 480 nm appeared after DOX loading, confirming the successful preparation of CuS@copolymer/DOX. The strong optical absorption of CuS@copolymer at the NIR area indicates the possibility for photothermal conversion. In order to investigate the photothermal conversion effect, a digital thermometer with a thermocouple probe was used to record the real-time temperatures of samples under 980 nm laser irradiation for 10 min (2.0 W/cm2). As Figure 4(b) shows, the temperature of CuS@copolymer aqueous dispersions increased strongly when concentration and irradiation time increased. Compared to control experiment of pure water, the temperature of (NaUA/OA)/CuS NPs rose 33.8 °C from 25.5 to 59.3 °C within 10 min at 100 µg/mL CuS while pure water only increased to 31.2 °C. In short, the photothermal heating effect at such a low concentration shows that the CuS@copolymer could be applied to photothermal therapy as a promising photosensitizer. 3.7. DOX loading and in vitro DOX release profile In the process of DOX loading onto CuS@copolymer, we maintained the pH value of reaction system at 7.0. Due to the protonation of DOX·HCl (pKa ~8.25)47 and the ionization of carboxyl groups in PMAA (pKa ~6) at neutral environment, the -NH3+ of DOX·HCl and -COO- of PMAA generates electrostatic interactions and promote the procedure. What's more, the DOX loading ability is also affected by the hydrogen bonding between DOX·HCl and CuS@copolymer. The DLC was calculated to be 15.3 % and the DLE to be about 76.4 wt%, the final concentration of DOX was measured to be 305.6 µg/mL. Based on pH and temperature-responsive swelling


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behaviors of CuS@polymer, the drug release profile under various pH and temperature conditions were investigated using a dialysis membrane against PBS-buffer solution. Two pH values at pH = 7.4 and pH = 5.0 were selected to simulate a normal physiological environment and a cellular lysosome environment, respectively. Similarly, the DOX release was studied at two different temperatures, resulting in following four different media: (1) pH = 5.0 and T = 25 °C, (2) pH = 5.0 and T = 43 °C, (3) pH = 7.4 and T = 25 °C, (4) pH = 7.4 and T = 43 °C. As depicted in Figure 5(a), the CuS@copolymer displayed an obvious pH-dependent DOX release profile. The cumulative release of DOX was significantly accelerated with 66% (pH = 5.0 and T = 25 °C) of drug release after dialysis for 60 h while the DOX release was restricted and could only reach up to 14% (pH = 7.4 and T = 25 °C) otherwise the same condition. This pH-sensitive drug releasing property may be ascribed to the destruction of the electrostatic interaction between PMAA segments and DOX originated from the protonation of the side carboxyl groups under lower pH value. Meanwhile, the temperature-responsive DOX release was also assessed at different temperature points ranging from 25 to 43 °C in PBS. The cumulative DOX release increased from 14% (25 °C) to 31% (43 °C) with 60 h at pH 7.4, which suggested that the release behavior could also be triggered by higher temperature. These results can be ascribed to the shrinkage of the polymer brushes when the temperature reached up to the LCST. Note that, the CuS@copolymer exhibited a synergistically DOX release derived from the pH and temperature double stimulus response and the respective release efficiency is 14% (pH = 7.4 and T = 25 °C) and 87% (pH = 5.0 and T = 43 °C) within 60 h dialysis. Additionally, whether the NIR light could trigger DOX release was carried out on account of above temperature-responsive results. Figure 5(b) revealed the cumulative DOX releasing curves



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of pH = 7.4 at 37 °C without and with 980 nm NIR laser for 10 min (2.0 W/cm2) at particular releasing time points of 1 h, 3 h, 5 h, 7 h, 9 h, respectively. It was apparent that the amount of DOX release was activated in presence of 980 nm laser light irradiation and the DOX release reached about 36% after 10 h, which is much higher than that control group (11%). The considerable increased release amount of DOX indicated NIR irradiation can also accelerate the drug release due to PTT-induced heating. In general, the as-prepared CuS@copolymer/DOX microspheres possess the potential as multi-functional drug carrier and offer the controlled drug release by pH, temperature and NIR light. 3.8. Cellular internalization Prior to investigation of as-synthesized nanocomposites for in vitro and in vivo application, DOX-loaded CuS@copolymer microspheres were incubated with HeLa cells to evaluate the cell uptake behavior. After incubation for several time points, the cells were collected and cellular red fluorescence of DOX was visualized by CLSM. Herein, the longest incubation time point was chosen at 2 h to assess the fluorescence intensity due to considering the highly toxic of DOX after longer incubation time. As shown in Figure 6(b), higher red fluorescence signals appeared in the cytoplasm and showed a measurable dependence over time, indicating the efficient intracellular of the CuS@copolymer microspheres. What's more, CuS@copolymer microspheres showed a faster cellular internalization compared with free DOX in Figure 6(a). 3.9. Cell viability assays In vitro cell viability against 4T1 cells containing CuS@copolymer at various concentrations were evaluated using standard MTT assay (Figure 7). The results showed that the CuS@copolymer were practically non-toxic to 4T1 cells (cell viabilities > 80%), although up to 1000 µg/mL,


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revealing low cytotoxicity of CuS@copolymer as drug carriers for biomedical application. In order to study the in vitro therapy efficacy of CuS@copolymer as a synergistic platform for photothermal therapy and anticancer drug nanocarrier, four groups of free DOX, CuS@copolymer + Laser, CuS@copolymer/DOX and CuS@copolymer/DOX + Laser were treated with 4T1 cells in detail. The cells were irradiated upon a 980 nm NIR laser for 10 min after incubation for 4 h for sufficient uptake of microspheres by 4T1 cells. As controls, the NIR laser irradiation itself causes no significant cell death when cells were treated without the material. Figure 8(a) depicted decreased cell viability against 4T1 cells as the concentration increased in both cases. DOX as a typical model drug is clearly able to play a partial role in enhancing anticancer effect. Moreover, the cell viability of CuS@copolymer/DOX (with the equivalent DOX concentration as free DOX) was demonstrated slightly lower than free DOX and nearly 50% cell death was observed at DOX concentration of 20 µg/mL. This is because DOX-loaded microspheres could be taken up more easily through endocytosis while the delivery of free DOX into cells is mainly caused by passive diffusion or active transportation.48-49 In comparison with chemotherapeutic group, there was a gradual and substantial cell death when the cells were treated with CuS@copolymer in the presence of 10 min NIR irradiation. And less than 52% 4T1 cells could survive with the involvement of CuS@copolymer with a concentration of CuS at 50 µg/mL, which should be owing to the excellent heat-induced cell ablation by the inside CuS nanoparticles. Notably, in the case of CuS@copolymer/DOX under NIR irradiation, the CuS@copolymer/DOX showed a drastically increasing cytotoxicity with only 18% cell survival, which demonstrated a remarkably cell-killing efficacy comparing with the separate chemotherapy (CuS@copolymer/DOX without NIR irradiation) and photothermal therapy (CuS@copolymer with NIR irradiation). The



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significantly inhibition ratio of 4T1 cells might be ascribed to several possible reasons. First, the pH/temperature-responsive characteristics of CuS@copolymer/DOX would be facilitated in the microenvironment of cancer cells due to the protonation and hydrogen-bond interaction between DOX and CuS@copolymer, leading to a sustained release of DOX. Second, the embedded CuS nanoparticles could be used as a high-efficiency NIR photosensitizer to absorb light and produce localized heat to kill the cancer cells via hyperthermia effect. Third, the elevated temperature during NIR irradiation could further promote the DOX release owing to the shrinkage of polymer chains, resulting in enhanced therapeutic treatment. These observations suggest the as-prepared CuS@copolymer microspheres could be used as an efficient drug carrier and achieve a synergistic effect for chemo-photothermal therapy. To further intuitively visualize the therapeutic effects, 4T1 cells were stained by calcein-AM and PI after laser irradiation to distinguish live (green) and dead (red) cells by CLSM. As shown in Figure 8(b), cells without any treatment were used as control and displayed no red fluorescence. Conversely, vast cells were killed within the laser spot after being incubated with CuS@copolymer microspheres, indicating that the CuS@copolymer microspheres could mediate the photothermal ablation of cancer cells by converting the NIR into the heat. It is worth noting that the cells experienced substantial death when exposed to laser with the participation of CuS@copolymer/DOX microspheres. The red cells appeared not only within but also out of the laser irradiation spot mainly because of the cooperative efficacy of photothermal therapy and chemotherapy. More importantly, the DOX release would be prominent accelerated in response to near-infrared stimulus. 3.10. Assessment of in vitro and in vivo MRI The feasibility of CuS@copolymer as potential T1-weighted MRI contrast agent was assessed on


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clinical MRI scanner. Gadolinium-containing contrast agents possess excellent paramagnetism properties because their seven unpaired inner 4f electrons could promote the longitudinal (T1) relaxation of surrounding water protons, thus exhibiting a positive contrast enhancement. As illustrated in Figure 9(a), favorably brighter of MR signals were noticed by responsive in vivo T1 phantom images of CuS@copolymer compared to pure water. Meanwhile, the representative T1-weighted images became gradually lighter with the increased Gd concentration. Subsequently, as Figure 9(b) shows, the longitudinal relaxivity value (r1) was computed to be 10.72 mM-1 s-1 from the slope of longitudinal relaxation rates (1/T1) versus molar concentration of Gd ranging from 0 to 0.5 mM, which was more than twice the r1 value of Magnevist®. Furthermore, to validate the potential capability of the nanocomposites for in vivo MR imaging, CuS@copolymer aqueous dispersion at a dosage of 0.05 mmol Gd/kg of body weight was administrated on a 4T1 tumor-bearing mice by tail intravenous injection. In vivo MR images of the tumor at scheduled time points Pre- and Post-injection were collected and shown in Figure 9(c). A gradually enhanced signal emerged apparently in tumor site due to the enhanced permeability and retention (EPR) effect over time.50 Meanwhile, the signal intensity was quantitatively studied using region-of-interest (ROI) quantification. As shown in Figure 9(d), we found that the signal intensity displayed a remarkable rise with 130% at 3 h post-injection when compared with that of pre-injection, which significantly enhanced the positive contrast effect. Moreover, obvious contrast signal enhancement effect of the tumor occurred at 24 h post-injection and the signal intensity increased up to 165%, indicating greater nonacomposites accumulation after 24 h injection. Overall, these results proved that the as-prepared nonacomposites could be a promising contrast agent.



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3.11. In vivo photothermal properties Encouraged by the superior photothermal therapy capacity in vitro, the feasibility of CuS@copolymer microspheres for in vivo photothermal therapy was conducted on 4T1 tumor-bearing mice. The 4T1 tumors were irradiated with a 980 nm laser (2 W/cm2) for 10 min at the time points of 3, 6, 12 and 24 h after injection. The temperature change of tumor was monitored in real-time by an infrared thermal camera to assess the photothermal effect (Figure 10). The local temperature of the tumor treated with CuS@copolymer microspheres showed a sharp increase upon laser treatment and the temperature increased as a function of exposure time. Expressly, it can be found that for the CuS@copolymer-injected group, the maximum temperature occurred after 24 h post injection which revealed a gradually increasing tumor uptake over time in agreement with in vivo MR images. The temperature was enhanced from 36.2 °C to 51.5 °C while the PBS-injected mice only add up to 40.8 °C with equal exposure period, which was sufficient for effective photothermal ablation of tumors in vivo. These results revealed that the CuS@copolymer microspheres indeed reach the tumor sites and conduct in vivo PTT efficiently. 3.12. Therapeutic effect and tissue analysis To further verify the therapeutic effect, four groups of PBS, free DOX, CuS@copolymer + Laser, and CuS@copolymer/DOX + Laser were treated with 4T1 mice (Laser groups were irradiated upon a 980 nm NIR laser for 10 min at specific time points), and then tumor tissues were collected for histological analysis through haematoxylin and eosin (H&E) stained images. As shown in Figure 11(a), the PBS treated mice was set as control, no obvious cell damage was observed and the cells maintained normal morphology at the most extent in this group. In contrast, the CuS@copolymer/DOX with laser irradiation group showed a large number of cell membrane


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destruction and nuclear contraction, which are obvious pathological features. Additionally, the tissue necrosis of CuS@copolymer/DOX + Laser group is far more than that of pure DOX group and CuS@copolymer + Laser group, revealing the prominent therapeutic characteristics of CuS@copolymer/DOX for in vivo synergistic chemotherapy and photothermal therapy. The body weight of mice treated with different groups was shown in Figure 11(b). The body weight of the mice did not show a significant decrease, indicating no adverse drug reactions in all groups. At the same time, the tumor volumes of different groups were selected after treatment for intuitive observation of the therapeutic effect. As shown in Figure 11(c), we can see the rapid growth in the control group due to no treatment. Moreover, the tumor size among pure DOX group and CuS@copolymer + Laser group were inhibited to a certain extent, respectively, due to chemotherapy and photothermal therapy. Furthermore, tumor growth was significantly inhibited in the CuS@copolymer/DOX + Laser group, and the tumor volume was much lower than the initial size after 14 days, confirming the synergistic effect of chemo/photothermal therapy. Additionally, the representative images of 4T1 tumors collected from the mice after treatment were shown in Figure S5. At the same time, to determine whether the agents give rise to any possible tissue damage, H&E staining of the representative organs containing heart, liver, spleen, lung, and kidney in different groups were also performed (Figure 12). As we can see, no noticeable tissue lesion took place and the results confirmed the hypotoxicity and safety of CuS@copolymer microspheres for potential clinical application. 4. CONCLUSION To summary, a facile and moderate procedure for the synthesis of multifunctional



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CuS@copolymer microspheres was developed via emulsifier-free polymerization. The prepared intelligent nanoplatform exhibited low cytotoxicity and high drug loading efficiency. It was found that the drug release could not only be triggered by low pH and high temperature, but also could be accelerated when exposing with NIR light. Moreover, the combined photothermal with enhanced chemo-therapy for high therapeutic efficacy due to the inner CuS NPs was also investigated. Furthermore, the microspheres possess good T1-MRI contrast enhancing effect, which are able to monitor the drug delivery procedure. Such a core/shell nanostructured polymer microspheres incorporate all these attractive features into one single system, providing a universal approach for constructing multifunctional theranostics to achieve simultaneous imaging diagnostics and multiple treatment modalities.

SUPPORTING INFORMATION The following files are available free of charge. The materials, measurement of photothermal performance, cell viability measurements, cellular uptake assay, animals and tumor models, in vitro and in vivo MRI, characterization, and other supplementary figures, including XRD patterns of CuS NPs and CuS@copolymer, the sizes of CuS@copolymer microspheres before and after 7 day’s dialysis at deionized water and PBS buffer (pH 7.4), the zeta potentials of CuS@copolymer microspheres before and after 7 day’s dialysis at deionized water and PBS buffer (pH 7.4), TG curves of OA/CuS NPs, pure copolymer, and CuS@copolymer, representative images of 4T1 tumors collected from the mice at the end of experiment. AUTHOR INFORMATION

Corresponding Author


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*

E-mail addresses: [email protected]; [email protected]; [email protected].

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We acknowledge the funding support from the National Natural Science Foundation of China (Grant NO. 51573039, 51273058, 81372712), and Science and Technology Research Funding of Shenzhen (NO. JCYJ20150401150223643). REFERENCES (1) Andre Luis Branco de Barros and Daniel Cristian Ferreira Soares. Theranostic nanoparticles: imaging and therapy combined. Molecular Pharmaceutics & Organic Process Research 2014, 2, 113-114. (2) Cabal, C.; Darias, D.; González, E.; Musacchio, A. Theranostics and Molecular Imaging: new concepts and technologies for drug development. Biotecnología Aplicada 2013, 30, 172-177. (3) Kalluru, P.; Vankayala, R.; Chiang, C. S.; Hwang, K. C. Nano-graphene oxide-mediated In vivo fluorescence imaging and bimodal photodynamic and photothermal destruction of tumors. Biomaterials 2016, 95, 1-10. (4) Chen, L.; Feng, W.; Zhou, X.; Qiu, K.; Miao, K.; Zhang, Q.; Qin, M.; Li, L.; Zhang, Y. and He, C. Facile synthesis of novel albumin-functionalized flower-like MoS2 nanoparticles for in vitro chemo-photothermal synergistic therapy. RSC Adv. 2016, 6, 13040-13049. (5) Floyd, J. A.; Galperin, A.; Ratner, B. D. Drug encapsulated polymeric microspheres for intracranial tumor therapy: A review of the literature. Adv. Drug Delivery Rev. 2015, 91, 23-37. (6) Yang, G.; Gong, H.; Qian, X.; Tan, P.; Li, Z.; Liu, T.; Liu, J.; Li, Y.; Liu, Z. Mesoporous silica nanorods intrinsically doped with photosensitizers as a multifunctional drug carrier for combination therapy of cancer. Nano Research 2014, 8, 751-764. (7) Agudelo, D.; Berube, G.; Tajmir-Riahi, H. A. An overview on the delivery of antitumor drug doxorubicin by carrier proteins. Int. J. Biol. Macromol. 2016, 88, 354-360. (8) Gothwal, A.; Khan, I.; Gupta, U. Polymeric micelles: recent advancements in the delivery of anticancer drugs. Pharm. Res. 2016, 33, 18-39. (9) Kesharwani, P.; Iyer, A. K. Recent advances in dendrimer-based nanovectors for tumor-targeted drug and gene delivery. Drug Discov Today 2015, 20, 536-547.



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Figure Captions: Scheme 1. Illustration of the preparation of CuS@copolymer/DOX microspheres and the combined chemo-photothermal therapy upon a 980 nm near-infrared laser irradiation. Figure 1. FT-IR spectra of (a) OA/CuS NPs, (b) pure copolymer, and (c) CuS@copolymer. Figure 2. TEM images of (a) OA/CuS NPs, (b) pure copolymer, (c) CuS@copolymer, (d) and, (e) Hydrodynamic diameter and size distribution of pure copolymer and CuS@copolymer, respectively. Figure 3. Hydrodynamic diameters of CuS@copolymer as a function of (a) pH and (b) temperature. Figure 4. (a) UV-Vis-NIR absorbance spectra of pure copolymer, (NaUA/OA)/CuS NPs, pure DOX, CuS@copolymer, CuS@copolymer/DOX in deionized water and (b) The temperature increase of PBS containing different concentrations of CuS@copolymer and (NaUA/OA)/CuS NPs as a function of irradiation time. Figure 5. (a) Cumulative drug release profiles of DOX-loaded CuS@copolymer in PBS buffer (0.01 M) varying different temperature or pH conditions and (b) without or with a 980 nm NIR laser at a power density of 2.0 W/cm2 at different time points (red column, each time point radiated for 10 min) under 37 °C at pH 7.4. Figure 6. CLSM images of HeLa cells incubated with (a) free DOX and (b) CuS@copolymer/DOX for 0.5 h, 1 h and 2 h at 37 °C. Each series can be classified into the nuclei of the cells (being dyed in blue by DAPI for visualization), the free DOX or CuS@copolymer/DOX and overlay of the above, respectively. Scale bar = 100 µm. Figure 7. Cell viability test for cytotoxicity of CuS@copolymer microspheres in 4T1 cell for 24 h by MTT assay. Figure 8. (a) Viabilities of 4T1 cells after incubation with different concentrations of

free DOX, 2

CuS@copolymer and CuS@copolymer/DOX with or without 10 min of NIR irradiation (2.0 W/cm , 980 nm), and 4T1 cells (incubated with only medium) with 10 min of NIR irradiation. At the each equivalent DOX point, CuS@copolymer has the same Cu concentration as CuS@copolymer/DOX and (b) Photothermal/chemotherapy of CuS@copolymer/DOX in vitro against 4T1 tumor cells (Equivalent CuS concentration at 50 µg/mL and equivalent DOX concentration at 20 µg/mL). Viable cells were stained green with calcein-AM, and dead cells were stained red with PI. Inside the dashed curve was irradiation zone. Scale Bar = 50 µm. Figure 9. (a) T1-weighted MR images of pure water, CuS@copolymer with different Gd concentrations. (b) Relaxation rate (1/T1) as a function of Gd concentration (mM) of CuS@copolymer solution. (c) T1-weighted MR images of 4T1 breast tumor acquired before and at different time points after injection of CuS@copolymer. (d) Relative signal intensity of 4T1 tumor collected before and at different time points after administration of CuS@copolymer. Figure 10. In vivo infrared thermographic images of Balb/c mice with 4T1 breast tumors exposed to a 980 nm laser at 2.0 W/cm2 for 10 min at different time points after intravenous injection of CuS@copolymer. Figure 11. (a) Typical images of H&E stained tumor slices obtained after 14 days of treatments, the scale bar = 50 µm. (b) The normalized body weight of mice in different groups after treatments. (c) The tumor growth curves of mice after different treatments. Figure 12. H&E stained images of major organs from pure DOX treated mice, CuS@copolymer treated mice with laser, CuS@copolymer/DOX treated mice with laser, and PBS-injected group as control.



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Scheme 1.


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Figure 1.



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Figure 2.


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Figure 3.



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Figure 4.


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Figure 5.



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Figure 6.


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Figure 7.



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Figure 8.


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Figure 9.



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Figure 10.


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Figure 11.



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Figure 12.


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TABLE OF CONTENTS GRAPHIC

Dual-stimuli Responsive Polymer Microspheres Encapsulated CuS Nanoparticles for Magnetic Resonance Imaging-Guided Synergistic Chemo-Photothermal Therapy Li Zhang,1 Zhe Yang,1 Wei Zhu,1 Zhilan Ye,2 Yiming Yu,1 Zushun Xu,1, * Jinghua Ren,2, * and Penghui Li3, *


Dual-Stimuli-Responsive, Polymer-Microsphere-Encapsulated CuS

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