Injectable 2D MoS2‐Integrated Drug Delivering Implant for Highly Efficient NIR‐Triggered Synergistic Tumor Hyperthermia

MoS2 nanosheets and a doxorubicin (DOX)-containing poly (lactic-co-glycolic acid) (PLGA)/MoS2/DOX composite implant are successfully constructed based on the unique phase-changing behavior of PLGA/MoS2/DOX oleosol within tumors. The fast phase transformation can firmly restrict MoS2 and DOX within tumors, and the integrated MoS2 and DOX can endow the implant with high synergistic photothermal and chemotherapeutic efficiency against tumors. As a minimally or noninvasively therapeutic method, photothermal therapy (PTT) of tumors induced by near-infrared (NIR) laser has attracted great attentions in recent years.1-3 NIR laser (λ = 808 nm in this study) can efficiently penetrate through tissues with concurrent high depth of several centimeters and less tissues absorption.2, 4 The introduced photo absorbing agents (PTA) can absorb and convert the penetrated NIR laser into heat, raise the tumor temperature, and finally ablate the tumor.5 Recently, 2D transition metal dichalcogenide MoS2 nanosheets has been explored as PTA, computed tomography imaging and drug delivery material.6-8 However, during tumor PTT, only a trace amount of the intratumorally or intravenously injected MoS2 nanosheets was accumulated in tumor tissue.9 A myriad of nanosheets unfortunately penetrate into the bloodstream and are captured by liver, spleen, and lung tissues, which will induce potential safety threats to normal organ.9 Chemotherapy is one of the most frequently used therapeutic modalities for cancer treatment.10 However, chemotherapy suffers from several therapeutic bottlenecks, such as limited drug accumulation in tumor tissue, drug loss due to the enhanced permeability and retention (EPR) effect of tumor vasculature, multi-drug resistance, and side effects toward normal tissues resulted from frequent drug administrations.1, 11 In addition, traditional single tumor PTT or chemotherapy is difficult to obtain satisfactory therapeutic efficacy. On this ground, it is highly desirable to construct a system that can deliver high amount of PTA and drug into tumor site for efficient multi-modal tumor synergistic therapy with significantly mitigated side effects. Poly(lactic-co-glycolic acid) (PLGA), as one of the US Food and Drug Administration (FDA) proved biocompatible polymers, has been extensively used in biomedical applications.12 PLGA chains can be readily dissolved into organic solvent such as N-methylpyrrolidone (NMP) to form an oleosol. It was found that MoS2 nanosheets and doxorubicin (DOX) can also be uniformly dispersed or dissolved into NMP. Therefore, a multifunctional PLGA/MoS2/DOX (PMD) oleosol can be obtained simply by homogenizing PLGA, MoS2, and DOX together into NMP. The PMD oleosol with a good syringeability could be in situ administrated into tumor via a minimally invasive manner. As a super hydrophobic polymer, PLGA chains in PMD oleosol will undergo an immediate liquid–solid phase transformation upon contacting water in vitro and in vivo (Scheme 1a,c). MoS2 and DOX can be encapsulated within the formed solid after the transient phase transforming process (Scheme 1b), forming a 2D MoS2-based composite PMD implant within tumor. To the best of our knowledge, this is the first conceptual breakthrough on the phase-changing organic–inorganic composite oleosol for localized tumor-synergistic therapy by the integration with 2D inorganic MoS2 nanosheest and organic PLGA. Unlike traditional drug/carrier "dispersed-in-suspension" manner,8 such a unique localized therapeutic implant is featured with the following advantages: first, the fast formed PMD implant enables combined in vivo tumor photothermal and chemotherapy at a very low PTA and drug dosage (DOX: 30 μg; MoS2: 75 μg); second, MoS2 nanosheets and DOX packaged within the PLGA matrix will not enter the bloodstream, thus ensuring excellent in vivo biosafety; third, MoS2 nanosheets with high NIR laser absorbance act as a high performance contrast agents for photoacoustic (PA) imaging,6, 13 thus the accurate location of solid PMD implant can be monitored by PA. Last but not least, heat transformed from NIR irradiation can not only cause the efficient tumor hyperthermia and coagulation necrosis but also effectively trigger the drug release from the implant matrix, to realize on-demand drug release and thus an enhanced chemotherapeutic outcome. MoS2 nanosheets with lateral diameter of ≈100 nm (Figure 1a) were facilely prepared in large scale via a bottom-up approach as developed by our previous study.9 It was found that PLGA, MoS2, and DOX could readily dissolve/disperse into NMP to form homogeneous dispersions (Figure S1a–c, Supporting Information). The syringeability of the oleosol is a dominate factor for the minimally invasive administration.14 The PLGA concentration (0.5 g mL−1) is competent in maintaining good syringeability of the PMD oleosol. PMD oleosol could be readily filled into a standard 1 mL syringe with a 21 gauge pinpoint (Figure S1d, Supporting Information) and smoothly pumped out through the needle pinpoint (Figure S1e,f and Movies 1 and 2, Supporting Information). Upon contacting with water, PLGA, PLGA/MoS2 (PM) or PMD oleosol solidified immediately with the quick diffusion of NMP solvent (Scheme 1a, and Figure S1g and Movies 1 and 2, Supporting Information), forming a visible solid (called PLGA, PM or PMD implant). The microstructure and composition of implants were visualized by electron microscopic observation. All the PLGA (Figure 1b), PM (Figure 1c), and PMD (Figure 1d) implants show a rough surface with irregular holes. These holes facilitate the diffusion of encapsulated drugs out of the implant. Polyethylene glycol (PEG) modified MoS2 (MoS2-PEG) nanosheets incorporated into PM/PMD implants matrix showed an aggregated microstructure (Figure 1c,d) while pure PLGA did not show such a microstructure (Figure 1b). The corresponding energy dispersive X-ray spectroscopy (EDX) analysis confirms the existence of Mo and S elements within the organic framework (Figure S2, Supporting Information). Obvious characteristic peaks relating to signals of MoS2 nanosheets at 232.1 eV (Mo 3d 3/2) and 228.2 eV (Mo 3d 5/2) corresponding to Mo4+ can be detected from the X-ray photoelectron spectroscopy (XPS) spectrum of the formed PMD implant (Figure 1e). Compared to the X-ray diffraction (XRD) patterns of PLGA and MoS2 nanosheets (Figure S3, Supporting Information), the PM implant shows the sharp characteristic peaks at 2θ = 8.5° and 32.4°, which can be attributed to the (002) and (100 + 101) crystal planes of MoS2 nanosheets (Figure 1f, JCPDS Card No. 17-0744), respectively. These characterizations strongly support the successful incorporation of MoS2 nanosheets within the PM implant. Owing to the high photothermal conversion efficiency of MoS2 nanosheets,6, 9 the formed PMD implant shows a high performance in tumor PTT. Under the NIR irradiation, the PMD implant presents a laser power density and MoS2 mass ratio dependent temperature increase (Figure 2a; Figure S4a, Supporting Information). The highest temperature increment of up to ≈21 and ≈50 °C was achieved after 30 s and 5 min irradiations (power density = 1.0 W cm−2), respectively. A maximum of ≈10 °C was still observed at an extremely lower power density (0.2 W cm−2). Comparatively, the photothermal effect is negligible for pure PLGA implant, of which only ≈2 °C increase at the same power density. The photothermal images of NIR irradiated PMD implant (Figure 2b; Figure S4b,c, Supporting Information) corresponding to Figure 2a and Figure S4a (Supporting Information) further illustrate the excellent photothermal conversion efficiency. The photothermal performance of PMD implant was further evaluated in a physiological simulated environment, where the implant was immersed into saline. As shown in Figure 2c,d, a similar laser power density and MoS2 mass ratio dependent temperature elevation of saline was recorded. A temperature increasing of 30.2, 22.5, 15.4, 12.1, and 7.9 °C could be realized after 5 min irradiations at a power density of 1.0, 0.8, 0.6, 0.4, and 0.2 W cm−2, respectively. Such a high in vitro photothermal conversion efficiency is expected to highly favor the further in vivo photothermal ablation of tumor. Cytocompatibility and hemocompatibility are the two predominate factors15 determining the clinical translation of PMD implant. The cytocompatibility was studied via CCK-8 assay and cell morphology observation. Compared with untreated L929 cells (Figure S5b, Supporting Information), in vitro viability and cell morphology of PMD implant treated cell were not affected (Figure S5c, Supporting Information), indicating the excellent cytocompatibility of PMD implant. PMD implant also shows an excellent in vitro hemocompatibility. Similar to the human red blood cells (HRBCs) exposed to phosphate buffer solusion (PBS, negative control), no visible in vitro hemolytic phenomenon was observed from HRBCs treated with PMD implant with different MoS2-PEG contents (hemolysis percentages of less than 5%, Figure S6, Supporting Information). In contrast, HRBCs exposed to water were totally damaged (inset of Figure S6, Supporting Information). The in vitro blood coagulation investigation (Figure S7a, Supporting Information) confirmed that the prothrombin time (PT), activated partial thromboplastin time (APTT), and (fibrinogen) FIB of PMD implant treated plasma showed no obvious differences with the untreated plasma, indicating that PMD implant did not cause any blood coagulation. PMD implant with such a high cyto- and hemocompatibility indicates its high biocompatibility for potential clinical translation. Different from traditional protocols for DOX loading via noncovalent "π–π" stacking or physical absorbing characterized by repetitive centrifuging operation,16, 17 DOX can be simply packaged within the matrix of PMD implant during the phase transforming procedure. The DOX-loading efficiency is controllable, which is dependent on PLGA and DOX concentrations. Up to 95% of DOX could be incorporated when PLGA concentration of 1 g mL−1 was used (Figure S8, Supporting Information). Importantly, the encapsulated DOX could be released upon external triggers. As shown in Figure 3, the release of DOX from PMD implant follows a controlled pH- and NIR-responsive manner. DOX releasing rate was much slower at physiological pH (pH = 7.4) and without NIR irradiation. About 18.9% of DOX was released after 96 h incubation at acidic pH condition (pH = 5.4, simulates the acidic tumor microenvironment), while only 9.4% of DOX was released at physiological pH at the same time point. DOX molecules will transform into a soluble DOX.HCl salt form under an acidic pH (pH = 5.4),18 thus, the acidic tumor extracellular environment and organelles (e.g., endosomes and lysosomes: pH = 5.0–5.5) can trigger the fast release rate of DOX.17, 19 Due to the hydrophobic nature of PLGA, the encapsulated DOX molecules have less chance to contact and release into the surrounded water, thus, the release of encapsulated DOX is relatively slow even at acidic pH condition. After 5 min continuous NIR irradiation, the percentage of released DOX increases from 8.7% to 31.8% in an acidic releasing medium (Figure 3c). The NIR light triggered drug release can be attributed to the following reasons: first, under the external heat activation, the PMD implant will expand and the binding effect toward the DOX molecules was reduced; second, the generated heat also accelerates DOX molecular motion, thus increasing the drug releasing rate. The photothermal activation cannot make the solid PMD implant flow because the contacting with the aqueous surrounding will further restrict the implant into solid form. The PMD implant can efficiently encapsulate not only DOX but also other NMP-soluble antibiotics (such as amoxicillin (AMX), Figure S9, Supporting Information) with a high loading amount and efficiency, indicating the versatility of the durg loading precedure of this PLGA-based composite implant. Such a dual stimuli (pH and NIR light) responsive drug releasing performance of PMD implant is expected to significantly enhance the therapeutic efficiency and mitigate the side effects of therapeutic agents. The PMD implant with high in vitro photothermal conversion efficiency and pH/NIR light dual stimuli responsive drug release could realize the synergistic photothermal and chemotherapy of tumor. As shown in Figure 3, cells treated with saline or PM implant still kept healthy, indicative of excellent cytocompatibility of PM implant (Figure 3a). Due to the cell killing effect of the released DOX, viability of PMD implant treated cells was significantly lower than PM implant treated cells (p < 0.01). Moreover, viability of PM implant treated cells was significantly suppressed after irradiation (p < 0.05, versus PLGA + NIR treated group, Figure 3a), implying the high in vitro photothermal therapeutic efficiency of the integrated MoS2 nanosheets. Due to the NIR-triggered MoS2-based photothermal ablation and DNA damage by released DOX, cancer cells treated by PMD implant and NIR suffered from a synergistic killing effect. Cells presented completely morphology damage and death with a viability of less than 5%, significantly lower than cell treatments by PM + NIR (p < 0.01) and PMD implant (p < 0.001). The microscopic observation of cell morphology (Figure 3b1–b6) further demonstrates the synergistic photothermal and chemotherapy efficiency. Comparatively, morphologies of cells treated with saline (Figure 3b1), PM implant (Figure 3b2), and PLGA implant + NIR (Figure 3b3) kept unchanged. Cell morphologies treated with PMD implant (Figure 3b4) and PM implant + NIR (Figure 3b5) were partially destroyed, while the cell morphology treated by PMD implant + NIR was totally damaged (Figure 3b6). The cytotoxicity of NMP solvent should be taken into consideration. As shown in Figure S10a (Supporting Information), NMP can kill L929 cells at a relatively high concentration (cell viability: 41.9% ± 1.6%, NMP 50 μL mL−1; 28.1% ± 3.9%, NMP 100 μL mL−1). While, at a low NMP concentrations (no higher than 50 μL mL−1), the cell survival rate is higher than 80% and cell morphology (Figure S10c,d, Supporting Information) is unaffected compared with control cells (Figure S10b, Supporting Information) after 24 h incubation. Thus, the NMP dosage is a key point for the in vivo application of PMD implant. The injected 30 μL PMD oleosol transforms into solid state immediately after the injection without leakage (Figure S11, Supporting Information). Under the irradiation of NIR, a fast tumor temperature elevation was recorded (Figure 4a; Figure S10a, Supporting Information), which reached 43.8 and 56.5 °C after 30 s and 5 min irradiation, respectively. The high temperature area spread quickly and covered almost the whole tumor site within 150 s. The PMD implant could readily elevate the tumor temperature higher than the temperature threshold (≈42 °C)20 required for cancer cell death and tumor coagulation necrosis, thus caused restriction of tumor malignant proliferation in vivo. In contrast, the photothermal effect of control mice group was not obvious (only ≈1.7 °C; Figure S12, Supporting Information). In vivo anticancer efficiency of PMD implant after single NIR laser irradiation was evaluated by recording the tumor volume within given duration (Figure 4b). Photothermal or chemotherapy alone could only partly inhibit the tumor malignant proliferation. After 28 d feeding, tumor volume of control, PM implant with NIR, and PMD implant without NIR expanded 1050.5%, 250.5%, and 507.1%, respectively (Figure 4b; Figure S13, Supporting Information). Comparatively, PMD implanted tumor treated with NIR shrank significantly after NIR irradiation where tumor almost disappeared after 7 d feeding. The left scar was completely healed after 4 weeks feeding (Figure 4d4) and no tumor reccurrence was observed in the following days (within 2 months, Figure S14, Supporting Information). The in vivo microscopic therapeutic outcomes of tumor cells were further evaluated by the typical CD31 immunohistochemical staining (Figure S15a,b, Supporting Information) and hexatoxylin & eosin (H&E) staining (Figure S15c,d, Supporting Information). All tumor cells received hyperthermia were remarkably destructed (Figure S15d, Supporting Information), and no CD31 positive tumor microvessels was observed (Figure S15b, Supporting Information). Comparatively, no obvious cell death was found from control tumor (Figure S15a,c, Supporting Information). Tumor histological examination strongly demonstrates the high antitumor efficiency of PMD implant. The life span of mice in PMD implant + NIR group were obviously prolonged (no death and tumor reccurrence within 2 months, Figure S14, Supporting Information), while mice death occur continuously in other groups. In vivo anticancer outcome strongly demonstrates the synergistic effect of tumor photothermal and chemotherapy efficiency (Figure 4c). Under laser irradiation, the generated heat from the implanted PMD implant can cause the thermal expansion of tumor tissue and transmit/distinct PA signal (Figure S14, Supporting Information). Thus, the in vivo location of PMD implant can be accurately tracked via PA imaging. Additionally, MoS2 nanosheets and DOX will be restricted within tumor during the phase changing process of PLGA. It is noted that the subcutaneous formed PMD implant could be in vivo biodegraded, as shown in Figure S17 (Supporting Information). These features endow the multifunctional PMD implanting system a high hemo- and histocompatibility. It was found that mice under several different treatments show no distinct weight variations over the feeding time (Figure S18a, Supporting Information). Only trace amount of the injected MoS2 nanosheets (less than 1% of the injected dosage per gram (ID% g–1) tissue) could be detected in major organs including the heart, liver, spleen, lung, and kidney at 6, 24, 72, 120, and 168 h post in situ administration (Figure S18b, Supporting Information), which is much superior to traditional drug administration mode where nanoparticles are mostly captured by liver and spleen. The longer term in vivo safety analysis from H&E staining of the major organs on day 14 and day 28 (Figure S18c, Supporting Information) shows no obvious pathological tissue damage/abnormality within major organs of mice, further indicating that the administrated NMP dosage will not cause side effects to normal tissues. During the fast liquid–solid phase changing process, the NMP solvent may diffuse into the blood circulation owing to the enhanced permeability and retention effect of tumor blood vessel.21 Thus, the in vivo hemocompatibility should be considered. As shown in Figure S7b (Supporting Information), blood plasma PT, APTT, and FIB values of PMD implant treated mice show no obvious differences compared to untreated mice, indicating that PMD implant could not cause in vivo blood coagulation. No significant difference in blood routine parameters and serum biochemistry parameters among experimental group and control group was found (Figure S19a–f, Supporting Information), indicating that the injected NMP dosage could not affect the normal function of blood and metabolism of main organs. Combining the high anticancer efficiency, such high in vivo hemo- and histocompatibility renders the PMD implant a promising clinical translation potential in combating the cancer. In summary, a multifunctional PLGA/MoS2/DOX composite implant has been constructed for highly efficient and versatile synergistic tumor therapy based on the phase transforming behavior of FDA approved PLGA polymer. The fast solidifying of PLGA/MoS2/DOX oleosol upon contacting body fluid can concurrently encapsulate MoS2 nanosheets and anticancer drug (DOX) inside the implant matrix in vivo, forming a solid implant with high potential in synergistic tumor photothermal and chemotherapy. The generated heat upon NIR laser irradiation not only causes the significant tumor coagulation necrosis but also enhances the tumor chemotherapeutic efficiency via triggering the fast release of encapsulated DOX molecules. Tumor could be completely erased without reccurrence by this therapeutic protocol. Importantly, MoS2 nanosheets and anticancer agents do not diffuse into the body fluid circulation, and the implant could also be in vivo biodegraded. Therefore, the potential long term side effects to normal issue and organs are significantly mitigated. In addition to antitumor DOX agent, other antibiotic drug such as AMX was also demonstrated to be successfully incorporated into the implant matrix, showing the versatility of this drug loading strategy in biomedicine. The prepared PMD implant with convenient handling and administrating procedure, low required dosage, multiple stimuli-responsive drug release, high in vivo hemo/histocompatibility and enhanced synergistic tumor therapeutic outcome is believed to provide a promising clinical translation potential for efficient localized tumor therapy. This work was supported by China National Funds for Distinguished Young Scientists (51225202), National Natural Science Foundation of China (Grant No. 51072212), Shanghai Excellent Academic Leaders Program (Grant No. 14XD1403800), and Shanghai technical platform for testing and characterization on inorganic materials (14DZ2292900). The authors also thank the support of China Postdoctoral Science Foundation (2014M551463). All animal experiments were performed under the guideline approved by the Chongqing Medical University and were in accordance with the policies of National Ministry of Health. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.