Novel Approach of Using Near-Infrared Responsive PEGylated Gold

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A Novel Approach of Using Near-Infrared Responsive PEGylated Gold Nanorod Coated Poly (L-Lactide) Microneedles to Enhance the Antitumor Efficiency of Docetaxel Loaded MPEG-PDLLA Micelles for Treating A431 Tumor Ying Hao, MingLing Dong, TaoYe Zhang, JinRong Peng, YanPeng Jia, YiPing Cao, and ZhiYong Qian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 18 Apr 2017 Downloaded from http://pubs.acs.org on April 19, 2017

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ACS Applied Materials & Interfaces

A Novel Approach of Using Near-Infrared Responsive PEGylated Gold Nanorod Coated Poly (L-Lactide) Microneedles to Enhance the Antitumor Efficiency of Docetaxel Loaded MPEG-PDLLA Micelles for Treating A431 Tumor Ying Hao1, MingLing Dong1, TaoYe Zhang2, JinRong Peng1, YanPeng Jia1, YiPing Cao2, ZhiYong Qian1,* 1

State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West

China Hospital, West China Medical School, Sichuan University, Chengdu, 610041, PR China 2

Key Laboratory of Optoelectronic Chemical Materials and Devices of Ministry of

Education, Jianghan University, Wuhan, 430056, PR China

*

To whom should be corresponded, [email protected] (Qian Z).

Tel/Fax:

ACS Paragon Plus Environment

+86-28-85501986,

E-mail:

ACS Applied Materials & Interfaces 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

ABSTRACT The combination of chemotherapy and photothermal therapy (PTT) plays a significant role in synergistic tumor therapy. However, high dosage of chemotherapy drugs or photothermal agents may cause series side effects. To overcome these challenges, we designed a near-infrared (NIR) responsive PEGylated gold nanorod (GNR-PEG) coated poly (L-lactide) microneedles (PLLA MNs) system (GNR-PEG@MNs) to enhance antitumor efficiency of docetaxel loaded MPEG-PDLLA (MPEG-PDLLA-DTX) micelles for treating A431 tumor. The as-made GNR-PEG@MNs contained only 31.83 ± 1.22 µg of GNR-PEG per patch and exhibited excellent heating efficacy both in vitro and in vivo. Meanwhile, GNR-PEG@MNs with the height of 480 µm had good skin insertion ability and was harmless to the skin. On the other hand, GNR-PEG@MNs had good heating transfer ability in vivo and the tumor sites could reach 50 oC within 5 min. In comparison with chemotherapy and PTT alone, the combination of low dosage MPEG-PDLLA-DTX micelles (5 mg/kg) and GNR-PEG@MNs completely eradicated the A431 tumor without recurrence in vivo, demonstrating a remarkable synergetic effect. Hence, GNR-PEG@MNs could be a promising carrier to enhance the antitumor effect of MPEG-PDLLA-DTX micelles for treating superficial tumors, and is expected to have a great potential in clinical translation for human epidermoid cancer therapy.

KEYWORDS: photothermal therapy (PTT); GNR-PEG@MNs; MPEG-PDLLA-DTX micelles; A431 tumor; synergetic effect.


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■ INTRODUCTION In recent decades, cancer is the most common life-threatening illness to people's health and has an upward trend in its morbidity and mortality.1 Among them, human epidermoid cancer is a public health problem which has a growing trend especially in Caucasian.2 Traditional therapies, such as photodynamic therapy,3 may cause skin DNA damage and other skin cancer.4,5 Great effort needed to be devoted to improve the therapy effect and reduce side effects. Docetaxel (DTX) is a broad-spectrum anti-tumor drug which has good antitumor effect.6,7 It is reported that the half-inhibitory concentration (IC 50) value of DTX was 6 nM in human epidermoid cancer cells (A431).8 And DTX loaded MPEG-PDLLA (MPEG-PDLLA-DTX) micelles has entered the clinical stage in South Korea,9 which could improve the efficacy of chemotherapy via enhanced permeability and retention (EPR) effect.10-13 However, it is reported that only fewer drugs could arrive at tumor site through the EPR effect.14,15 In order to increase the efficiency in chemotherapy, several synergetic strategies must be designed to further enhance the antitumor efficiency. Near-infrared (NIR) responsive photothermal therapy (PTT) is an attractive alternative to combine with chemotherapy as it could cause membrane damage, cell injury and protein denaturation as a systemic effect.16-19 What’s more, PTT could induce tumor thermal and improve the accumulation of nano-drug in tumor sites.20 Chen et al. in Soochow University have used an imagable and photothermal HSA-ICG-PTX nanoparticles to treat subcutaneous and metastatic breast tumors, the combination therapy achieved excellent synergistic therapeutic efficacy.21 To date, a variety of NIR-responsive nanostructures were used for photothermal therapy, such as gold nanostar,22 nanocage,23 nanocube,24 nanocluster,25 nanoshell26 and nanorod.27 Among them, gold nanorod (GNR) was chosen as an ideal photothermal agent, which has several advantages, including easy synthesis, small size and adjustable aspect ratio to achieve NIR light absorption.28-30 However, in traditional chemotherapy and PTT, chemotherapy drugs or photothermal agents were delivered to tumor sites either intratumorally or intravenously, which not only needed high dosage of chemotherapy drugs or photothermal agents to achieve better therapeutic efficacy, but also cause series side effects and reduce patients’ quality of life.31 Hence, it is urgent to develop



a novel synergetic system of chemotherapy and PTT to enhance the antitumor efficiency. Nowadays, microneedles delivery system was widely used to deliver drugs,32,33 DNA,34 RNA35 and vaccines36 as the microinjection is a rapid, cost-effective, painless and direct way for molecule delivery.37,38 Compared with other drug delivery way, the solid microneedles could pass through the cutin layer of skin and deliver drug to dermis directly, which could improve the local drug concentration and reduce systemic side effects. Both Gu39-42 et al. in North Carolina State University and Chen43-46 et al. in National Cheng Kung University have made great contribution in the field of microneedle delivery system, which proved that microneedles are ideal vehicles for molecule delivery. Herein, we developed a novel synergetic system of chemotherapy and PTT to treat A431 tumor by the combination of a NIR responsive PEGylated gold nanorod (GNR-PEG) coated poly (L-lactide) microneedles (GNR-PEG@MNs) and DTX loaded MPEG-PDLLA micelles shown in Figure 1. This system was composed of biodegradable poly (L-lactide) microneedles (PLLA MNs), photothermal agent GNR-PEG and antitumor nano-drug MPEG-PDLLA-DTX micelles. PLLA has been approved by the Food and Drug Administration (FDA) for clinical use which was suitable as a needle material. GNR-PEG was a harmless photothermal agent that efficiently converted the absorbed light energy into heat and made tumor site thermal.29,30 Meanwhile, MPEG-PDLLA was a safe nano-carrier, and DTX loaded MPEG-PDLLA-DTX micelles was sensitive to A431 cells.8,9 Last but not the least, the above-mentioned systems have not been reported for the combination of chemotherapy and PTT before. We aimed to use this novel NIR responsive GNR-PEG@MNs which contained only 31.83 ± 1.22 µg of GNR-PEG per patch and exhibited excellent heating efficacy to enhance the antitumor efficiency of low dosage MPEG-PDLLA-DTX micelles (5 mg/kg). In this study, we prepared MPEG-PDLLA-DTX micelles and GNR-PEG via described methods. And the GNR-PEG@MNs with the height of 480 µm was prepared by using NIR responsive GNR-PEG adsorbed on PLLA MNs which contained only 31.83 ± 1.22 µg of GNR-PEG per patch. In addition, the GNR-PEG@MNs was characterized in terms of scanning electron microscope (SEM), energy spectrum (EDS), skin insertion test, heating transfer experiment and NIR thermal imaging study. Finally, in vivo antitumor efficacy of ACS Paragon Plus Environment

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combined

chemotherapy

and

PTT

was

carried

on,

the

combination

of

MPEG-PDLLA-DTX micelles and GNR-PEG@MNs were compared with that of chemotherapy or PTT alone in mice bearing A431 tumor. The histopathological and cells proliferation of the tumors were also observed.

■ RESULTS AND DISCUSSION Characterization of MPEG-PDLLA The MPEG-PDLLA copolymer (Mn = 3765, 2000-1765) was synthesized via ring-opening method47 by MPEG 2000 and D, L-lactide as described before.48 (Figure S1A in the supporting information (SI) showed the procedure). The characterization of MPEG-PDLLA by 1H NMR spectra (Varian 400 spectrometer, Varian, USA), Fourier transform infrared spectroscopy (FTIR, NICOLET 200SXV, Nicolet, USA) and gel permeation chromatography (GPC, Agilent 110 HPLC, USA)49,50 confirmed that the MPEG-PDLLA copolymer was synthesized successfully and the molecular weight was 3821 with narrow distribution of 1.08. (Figure S1B-D in the SI) The DTX loaded MPEG-PDLLA micelles was prepared via a thin-film rehydration method.6,9 Dynamic light scattering (DLS) and transmission electron microscope (TEM) was used to character the DTX loaded MPEG-PDLLA-DTX micelles (Figure 2A). The particle size of DTX loaded micelles was about 22.07 ± 0.22 nm, and polydispersity index (PDI) was 0.200 ± 0.002. The drug loading (DL) and encapsulation efficiency (EE) of DTX loaded MPEG-PDLLA micelles were 4.98 ± 0.10% and 99.68 ± 1.96% respectively, which were determined by high performance liquid chromatography (HPLC) method as reported before.9 In the morphology study, MPEG-PDLLA-DTX micelles had uniform particle size and distributed as homogenous in the TEM image (inserted into Figure 2A). Meanwhile, we also study the in vitro cellular uptake efficiency and cytotoxicity assay of MPEG-PDLLA-DTX micelles (Figure S2). The cellular uptake efficiency of the micelles was time-dependent from 0.5 h to 4 h (Figure S2A, B), implying that the micelles could be internalized into the cytoplasm of A431 cells. Compared with DTX, the DTX loaded micelles strongly inhibited the growth of A431 cells with a dose-dependent manner. The IC 50 value was 3.53 ± 0.14 ng/mL after 48 h incubation which was in accordance with the



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previous study (Figure S2C, D).8 These results demonstrated that DTX was the suitable drug for treating A431 tumor. Characterization of GNR-PEG The GNR-PEG was prepared via a ligand exchange method from GNR. In detail, thiol ethylene glycol (PEG-SH) was used to replace the cetyltrimethyl ammonium bromide (CTAB) surfactant on GNR surface overnight, which could reduce the toxicity of CTAB.51 We used FTIR and EDS to ensure whether PEG-SH was modified on GNR. The IR spectra demonstrated that GNR had strong absorption peaks at 2917.85 cm-1 and 2849.31 cm-1 which belong to C-H stretch vibration, while after PEG-SH modification, the peaks at 2914.95 cm-1 and 1854.32 cm-1 became weaker sharply, confirming the CTAB surfactant was replaced by PEG-SH successfully (Figure S3A). Seen from the results of EDS in Figure S3B, the weight of Br element was decreased obviously from 17.81% to 0.98% when the PEG-SH was modified on the surface of GNR (Figure S3C), which suggested the PEG-SH was modified on GNR. Figure 2B was the UV-vis absorption spectra of GNR and GNR-PEG. Compared with GNR, the maximum absorption of GNR-PEG red shifted 10 nm, but it was still about 800 nm and the absorption intensity did not changes which will not affect the photothermal effect. What’s more, from GNR to GNR-PEG, the zeta potential range from 35.30 ± 1.80 mV to -7.65 ± 0.68 mV, which was suitable for further study as the zeta potential shifted closer to negative (Figure 2C, D) and further proved that PEG-SH was modified on GNR successfully. The TEM image of GNR (inserted into Figure 2C) and GNR-PEG (inserted into Figure 2D) revealed that GNR and GNR-PEG had uniform length about 50 nm and good dispersion. Characterization of GNR-PEG@MNs The NIR responsive GNR-PEG@MNs was obtained from the modification of PLLA MNs.

Firstly,

the

PLLA MNs

was fabricated by

melt-moulding

PLLA on

polydimethylsiloxane (PDMS) template and press-molded to obtain arrays of microneedles.34,52 And the NIR responsive GNR-PEG adsorbed on PLLA MNs via layer by layer method,53,54 then we used scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), FTIR, UV-vis transmittance spectroscopy and X-ray photoelectron spectroscopy (XPS) to character the microneedles. The size of PLLA MNs (left) was 1 cm ACS Paragon Plus Environment

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× 1 cm which consisted of 400 (20 × 20) microneedle tips. When the GNR-PEG absorbed on PLLA MNs to get GNR-PEG@MNs (right), the color of microneedles turned white to purple (Figure 3A). What’s more, the micrographs of PLLA MNs and GNR-PEG@MNs showed in Figure S4 presented the bright-field micrographs of microneedles structures. Figure 3B1 and Figure 3C1 were the SEM image of PLLA MNs and GNR-PEG@MNs, the microneedle tips was lined up in order. The base width and height of the microneedles was 300 µm and 480 µm respectively. The surface of PLLA MNs were clean and smooth (Figure 3B2), while the surface of GNR-PEG@MNs had a lot of rod-shaped object that were GNR-PEG (Figure 3C2). We used EDS to analyze the elemental content of PLLA MNs and GNR-PEG@MNs (Figure 3B3, C3), the results showed that only C, O elemental were on the surface of PLLA MNs, while the surface of GNR-PEG@MNs had C, O and Au elemental, demonstrating that the GNR-PEG absorbed on the surface of PLLA MNs successfully. This is further supported by IR spectra, UV-vis transmittance spectra and XPS results (Figure S5, S6). In details, when polyethylenimine (PEI) modified PLLA MNs, the absorption peak at 1591.21 cm-1 in the IR spectra was attributed to ⱱNH stretch vibration and the peak disappeared after GNR-PEG absorbed on PEI modified PLLA MNs (Figure S5A), revealing the GNR-PEG was absorbed on PLLA MNs successfully. What’s more, the transmittance measured via UV-vis transmittance spectroscopy declined from 81% to 64% as the modification carried on (Figure S5B), which also demonstrated we obtained GNR-PEG@MNs via layer by layer method. The surface atomic composition of GNR-PEG@MNs was identified by XPS analysis (Figure S6). Compared with PLLA MNs, GNR-PEG@MNs had an Au signal, indicating that the GNR-PEG was successfully absorbed on PLLA MNs. In vitro skin insertion test Skin insertion ability is the key factor to overcome the skin resistance for drug delivery.43,44 Trypan blue staining method was used to confirm whether the GNR-PEG@MNs could insert into the skin completely. After GNR-PEG@MNs inserted into mice skin for 5 min, the skin treated with trypan blue showed consistent microneedles insertion (Figure 4A). The surface of the skin was revealed rows of blue spots which were corresponded to the GNR-PEG@MNs puncture sites (Figure 4B), showing that ACS Paragon Plus Environment


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GNR-PEG@MNs had good skin insertion ability. Histological section (Figure 4C) further clearly demonstrated that GNR-PEG@MNs completely inserted into the skin. We used the H&E staining method to investigate whether the GNR-PEG@MNs was harmless to the skin that could recover rapidly to avoid wound infection. From results in Figure 4D, E, the skin that treated with GNR-PEG@MNs could be recovery 12 h later after the insertion, indicating that GNR-PEG@MNs was harmless to the skin and the damage induced by the GNR-PEG@MNs was reversible. Near-infrared thermal imaging of GNR-PEG@MNs GNR-PEG has been proved as a harmless and outstanding photothermal agent for PTT.28-30 To ensure whether the GNR-PEG@MNs still had good heating efficacy as GNR-PEG for PTT, we used an NIR thermal camera (Fluke Ti32, USA) to investigate the in vitro heating efficacy of PLLA MNs and GNR-PEG@MNs within 5 min (Figure 5A). As the time went on, the temperature of GNR-PEG@MNs quickly reached 55 oC, while the

temperature

of

PLLA

MNs

still

at

room

temperature,

indicating

that

GNR-PEG@MNs still had the heating efficacy as well as GNR-PEG. Heating curves and the temperature changes of PLLA MNs and GNR-PEG@MNs after irradiation with NIR light for 3 cycles were shown in Figure 5B, C. Once the irradiation was switched on, the temperature of GNR-PEG@MNs quickly rose to 55 oC, when the irradiation was switched off, the temperature of GNR-PEG@MNs rapidly cooled to room temperature, demonstrating that the temperature of GNR-PEG@MNs could be controlled by NIR light. Meanwhile the temperature of PLLA MNs was still at room temperature. Above all, the GNR-PEG@MNs that only contained 31.83 ± 1.22 µg of GNR-PEG per patch still had good heating efficacy and was a novel NIR responsive photothermal agent for the combination with chemotherapy. In vivo near-infrared thermal imaging and heating transfer efficacy study An ideal NIR responsive photothermal agent not only had good heating efficacy in vitro, but in vivo. As the tumor tissue could be damaged irreversibly when the temperature of the tumor sites reached 50 oC.55 In vivo thermal imaging study was carried on female A431 tumor-bearing balb/cA-nu mice (3 mice per group) (Figure 6). To evaluate whether transdermal drug delivery systems had advantages of other drug delivery systems to deliver ACS Paragon Plus Environment

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photothermal agent GNR-PEG for PTT, the group that smeared GNR-PEG and injected GNR-PEG intratumorly were also investigated. The temperature in the tumor sites in control and PLLA MNs (transcutaneous) group did not change a lot. The temperature of the group that smeared GNR-PEG at tumor sites raised to 45 oC with ∆T 8.9 oC within 5 min as the GNR-PEG solution was easier to dry when the 808 laser on which could decrease the heating efficiency of GNR-PEG. The temperature of GNR-PEG (intratumor) could reach 50 oC with ∆T 13 oC as GNR-PEG could not be evenly distributed in the tumor tissues which also reduce the heating efficiency at the tumor sites. The tumor treated with GNR-PEG@MNs transcutaneously increased quickly and reached 50 oC within 1 min with ∆T 23.8 oC, which were high enough for tumor tissue damaged irreversibly. These results demonstrated that the tumor treated with GNR-PEG@MNs had the best heating efficacy among other two groups. To confirm whether the heating could be transferred to the center of the tumor sites which could induce progressive necrosis of tumors. We further investigated the heating transfer efficacy in pork with the height, length and width of 2.5 cm, 3 cm and 3 cm respectively. (Figure 7). Compared with the group of GNR-PEG (smear) and GNR-PEG (subcutaneous), the group treated with GNR-PEG@MN (transcutaneous) had the best heating transfer efficacy and the depth of heating almost reached 1.5 cm. Additionally, the heating could be conductive to the middle position of the pork and reached to 55 oC within 5 min, revealing that the heating effect of the GNR-PEG@MNs could be transmitted to the center of tumor tissue in vivo. No significant temperature change was observed in the pork treated with PLLA MNs (transcutaneous) and control group. In summary, compared with other drug delivery system, the NIR responsive GNR-PEG@MNs only contained 31.83 ± 1.22 µg GNR-PEG had a big contact area for heating transfer to the center of the tumor sites evenly and was a promising carrier to deliver photothermal agent for PTT. In vivo antitumor efficacy To further study the synergetic effect of chemotherapy and PTT in vivo in comparison with chemotherapy and PTT alone, the tumor-bearing A431 mice were divided into 8 groups randomly (5 mice per group): (1) the saline group, (2) the GNR-PEG@MNs + laser group, (3) the DTX group (5 mg/kg), (4) the DTX group (10 mg/kg), (5) the DTX (5 ACS Paragon Plus Environment


mg/kg) + GNR-PEG@MNs + laser group, (6) MPEG-PDLLA-DTX group (5 mg/kg), (7) MPEG-PDLLA-DTX group (10 mg/kg) and (8) MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser group. According to photographs and growth curves of the tumors in Figure 8B, C, mice treated with (1) saline had a rapid tumor growth, then was the group of (2) GNR-PEG@MNs + laser, indicating that GNR-PEG@MNs + laser group could inhibit tumor slightly which was due to PTT. Additionally, the (5) DTX (5 mg/kg) + GNR-PEG@MNs + laser group had better inhibit ability than (3) DTX (5 mg/kg) and (4) DTX (10 mg/kg) group in the early time, while the tumors were recurrence at day 25 after the treatment, implying that the GNR-PEG@MNs could enhance the antitumor ability of DTX, but the low dosage of DTX could not inhibit tumor growth for long time. Compared the DTX group with DTX loaded MPEG-PDLLA micelles group at the same dosage, MPEG-PDLLA-DTX micelles group had better antitumor ability than the DTX group, which was assistant with the previously reported study.56,57 No matter DTX group or DTX loaded MPEG-PDLLA micelles group, the antitumor ability of high dose (10 mg/kg) was better than the low dose (5 mg/kg) group. It was noted that (8) MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser group had the rapider tumor volume reduction than group of (7) MPEG-PDLLA-DTX (10 mg/kg) and all mice were cured within 14 days after the treatment. In detail, after the combined treatment, the tumors were reduced rapidly and gradually replaced by a scab, then the scab was faded, and the skin was cured at day 20. Notably, no tumor recurrence was observed up to now, demonstrating that the GNR-PEG@MNs could enhance the antitumor ability of MPEG-PDLLA-DTX micelles and reduce the dosage of DTX. Moreover, the photograph of tumor in the mice (Figure 8E) consisted with Figure 8B, C. As shown in Figure 8D, the body weight of the mice did not change a lot in the treatment groups. The statistic data of the tumor weight was presented in Figure 8F. What’s more, the major tissues were collected for histopathology study (Figure S7), no lesions and inflammatory were found in the hearts, livers, spleens and kidneys tissues. For lung tissues, it was obvious to see congestion, necrosis and alveolar deformation in (1) saline and (2) GNR-PEG@MNs + laser group. These results demonstrated that the GNR-PEG@MNs was a safer carrier for PTT and could improve the antitumor ability of MPEG-PDLLA-DTX micelles. ACS Paragon Plus Environment

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In short, the synergetic effect of the combination of low dosage MPEG-PDLLA-DTX micelles (5 mg/kg) and GNR-PEG@MNs completely eradicated the A431 tumor without recurrence in vivo. The photothermal agent GNR-PEG@MNs was inserted into the tumor site transcutaneously which was harmless to the skin and minimal the uncomfortable feeling. With the combination of GNR-PEG@MNs, the dosage of MPEG-PDLLA-DTX micelles reduced to 5 mg/kg, which could reduce the occurrence of serious side effects. Compared with other chemo-photothermal therapy system,12,58 this synergetic system is expected to have a great potential in clinical translation for human epidermoid cancer therapy. Tumor cells histopathological and proliferation study Inspired by the results of in vivo antitumor efficacy, we explored the tumor cells histopathological and proliferation (3 mice per group). Histopathological study of tumor tissues (Figure 9A) revealed that although apoptosis and necroptosis occurred in the tumor site of the (2) GNR-PEG@MNs + laser group, there were still some survived tumor cells in the tumor site. Compared with the group of (3) DTX (5 mg/kg), (4) DTX (10 mg/kg) and (5) DTX (5 mg/kg) + GNR-PEG@MNs + laser, more tumor cells disappeared in the tumor site in (7) MPEG-PDLLA-DTX (10 mg/kg) micelles group and (8) MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser group, which further indicated that micelles has good antitumor ability and the GNR-PEG@MNs played an important role in enhancing the antitumor efficacy and reducing the dosage of drug for cancer therapy. The proliferation of tumor cell was investigated by Ki-67 immune histochemical staining (Figure 9B). The saline group has the most Ki-67 immunoreactivity than the treated groups. Compared with the DTX and the MPEG-PDLLA-DTX group, the micelles group had less Ki-67 positive cells in tumor tissues at the same dose, indicating that the micelles could significantly inhibit tumor cell proliferation. What’s more, the group of (5) DTX (5 mg/kg) + GNR-PEG@MNs + laser and (8) MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser group had better ability of inhibiting tumor cell proliferation than (3) DTX (5 mg/kg) and (6) MPEG-PDLLA-DTX (5 mg/kg) micelles group, demonstrating that the GNR-PEG@MNs could enhance the antitumor ability of MPEG-PDLLA-DTX ACS Paragon Plus Environment


under the irradiation of 808 nm laser and reduce the dosage of the drug. The quantitative data of the proliferation of tumor cells showed in Figure 9C.

■ CONCLUSIONS In summary, we successfully prepared MPEG-PDLLA-DTX micelles, GNR-PEG and PLLA MNs. Then we used layer by layer method to achieve GNR-PEG@MNs. The GNR-PEG@MNs had good skin insert ability and was harmless to the skin. In addition, the GNR-PEG@MNs had the heating efficacy as well as GNR-PEG and the temperature of GNR-PEG@MNs could be controlled by NIR light. The in vivo NIR thermal imaging and heating transfer efficacy study proved that the heating effect of the GNR-PEG@MNs could be transmitted to the middle of the tumor tissue and reach to 50 oC. The combination of GNR-PEG@MNs and low dosage of MPEG-PDLLA-DTX micelles (5 mg/kg) efficiently inhibited the tumor growth and cured all mice without recurrence. Therefore, this novel NIR responsive GNR-PEG@MNs could be a promising strategy to enhance the antitumor effect of the chemotherapy, and is expected to have a great potential in clinical translation for human epidermoid cancer therapy.

■ MATERIALS AND METHODS Preparation and characterization of DTX loaded micelles In this study, we prepared DTX loaded MPEG-PDLLA micelles according to a previously published method.6,9 In detail, 5 mg DTX and 95 mg MPEG-PDLLA were dissolved together in appropriate anhydrous ethanol. Then the ethanol was removed in vacuum at 37 oC by a rotary evaporator. Finally, the MPEG-PDLLA-DTX micelles were prepared by adding 5 mL deionized water at 60 oC and filtered with a 220 nm syringe filter. The DL and EE of DTX loaded MPEG-PDLLA micelles were determined by HPLC (Agilent 1260 HPLC, USA) method as reported before.9 DLS (Nano-ZS90, UK) and TEM (H-6009IV, Japan) were used to character the DTX loaded micelles. The cellular uptake efficiency and in vitro cytotoxicity of DTX and MPEG-PDLLA-DTX micelles were carried on A431 cell lines. Preparation and characterization of GNR-PEG The GNR was synthesized through a seed-mediated method described before.19 The


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GNR-PEG was synthesized via a ligand exchange method.51 Briefly, 1 mL GNR (1 mg/mL) reacted with 20 mg of PEG-SH dissolved in 19 mL deionized water and stirred at 25 oC. After reaction for the night, the GNR-PEG was centrifuged 12000 rpm for 15 min, then collected the precipitates and dispersed in deionized water. FTIR and EDS (JSM-7500F, JEOL, Japan) were used to conform whether PEG-SH was modified on GNR successfully. The morphology, zeta potential distribution and absorption spectra of GNR and GNR-PEG were measured by TEM, DLS and UV-vis absorption spectrometer (PE, USA) respectively. Preparation of PLLA MNs The microneedle master structure which was made from polydimethylsiloxane (PDMS) template was supplied by Jianghan University. The PLLA MNs was prepared as follows. Firstly, the dried PLLA was placed on a PDMS mold, then press-molded at 170 oC for 15 min on the as-prepared PDMS mold to obtain arrays of PLLA MNs. Preparation and Characterization of GNR-PEG@MNs Layer by layer method53,54 was used to prepare GNR-PEG@MNs. Briefly, the PLLA MNs was put into 50 mL PEI solution (0.5 mg/mL) for 2 h to get positive charge. Then pure water was used to wash the PLLA MNs and blew dry by nitrogen. In addition, PLLA MNs with positive charge was put into GNR-PEG solution (0.5 mg/mL) which had negative charge for 2 h. At last, pure water was used to wash them and blew dry by nitrogen to get GNR-PEG@MNs which the gold content was 31.83 ± 1.22 µg per patch measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES). FTIR and UV-vis transmittance spectroscopy (PE, USA) were used to ensure whether GNR-PEG absorbed on PLLA MNs to achieve GNR-PEG@MNs successfully. The morphology of GNR-PEG@MNs was measured by German ZEISS stereo microscope, SEM (JSM-7500F, JEOL, Japan) and the elemental content of GNR-PEG@MNs surface was measured by EDS and XPS (AXIS Ultra DLD, Kratos, UK). In vitro skin insertion test In this part, GNR-PEG@MNs was inserted into mice skin for 5 min, and then stained with trypan blue for 5 min and used normal saline to wash the exceeded trypan blue. In addition, skin section was processed for histological evaluation. Briefly, the skin was embedded with tissue freezing medium and sliced via a cryotome. The skin insertion ACS Paragon Plus Environment


ability was study via a fluorescence microscope (OLYMPUS, Germany). In order to observe whether the GNR-PEG@MNs was harmless to the skin that could recover rapidly to avoid wound infection.59 The GNR-PEG@MNs was inserted into mice skin for 5 min, then the mice of one group were euthanized and the skin was used for H&E staining. 12 h later, the mice of the other group were euthanized and used for H&E staining to study the recovery of the skin. Near-infrared thermal imaging of GNR-PEG@MNs The photothermal performance of GNR-PEG@MNs was measured by NIR thermal camera (Fluke Ti32, USA). To evaluate whether the temperature changes of GNR-PEG@MNs could be controlled by irradiating with NIR light (808 nm) at power density of 2 W/cm2 for 5 min. GNR-PEG@MNs was irradiated with NIR light for 3 cycles. During each cycle, the GNR-PEG@MNs was under NIR light for 5 min, and then switched off the NIR light for 5 min to cool GNR-PEG@MNs to room temperature. In vivo near-infrared thermal imaging and heating transfer efficacy study In vivo NIR thermal imaging study was carried on female A431 tumor-bearing balb/cA-nu mice. Briefly, a mixture of 5 × 106 A431 cells was injected subcutaneously at mice legs. When the tumor volume was about 200 mm3, the mice were divided into 5 group (3 mice per group): (1) the control group, in which the tumor were under 2 W/cm2 irradiation by 808 nm laser for 5 min; (2) the GNR-PEG (smear) group, in which the mice were smeared the GNR-PEG (32 µg) at the tumor site and under 2 W/cm2 irradiation by 808 nm laser for 5 min; (3) the GNR-PEG (intratumor) group, in which the mice were injected with GNR-PEG (32 µg) intratumorly and under 2 W/cm2 irradiation by 808 nm laser for 5 min; (4) PLLA MNs (transcutaneous) group, in which the mice were underwent insertion of PLLA MNs into the tumor sites from the skin surface and under 2 W/cm2 irradiation by 808 nm laser for 5 min; (5) GNR-PEG@MNs (transcutaneous) group, in which the GNR-PEG@MNs was penetrated into the tumor sites and under 2 W/cm2 irradiation by 808 nm laser for 5 min. Then the mice were imaged at appropriate time by NIR thermal camera (Fluke Ti32, USA). To study whether the heating could be transfer to the center of the tumor, the heating transfer efficacy study were carried on pork (Video 1, Figure S8A in the SI), the pork was ACS Paragon Plus Environment

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treated with above group under 2 W/cm2 irradiation by 808 nm laser for 5 min and imaged via NIR thermal camera (Fluke Ti32, USA). In vivo antitumor efficacy The in vivo antitumor efficacy of the combination of GNR-PEG@MNs and MPEG-PDLLA-DTX micelles was studied on A431 tumor-bearing balb/cA-nu mice (Figure 8A). In short, the right flank of the mice was injected with 100 µL of 5 × 106 A431 cells subcutaneously. When the volume of subcutaneous tumors were palpable, the mice were divided into 8 groups randomly (5 mice per group): (1) the saline group, in which the mice were intravenously injected 100 µL saline; (2) the GNR-PEG@MNs + laser group, in which the mice were underwent insertion of GNR-PEG@MNs into the tumor sites transcutaneously and under 2 W/cm2 irradiation by 808 nm laser for 5 min; (3) the DTX group (5 mg/kg), in which the mice were intravenously injected 100 µL DTX (5 mg/kg); (4) the DTX group (10 mg/kg), in which the mice were intravenously injected 100 µL DTX (10 mg/kg); (5) the DTX (5 mg/kg) + GNR-PEG@MNs + laser group, in which the mice were intravenously injected 100 µL DTX (5 mg/kg) and underwent insertion of GNR-PEG @MNs into the tumor sites with 2 W/cm2 irradiation by 808 nm laser for 5 min; (6) MPEG-PDLLA-DTX group (5 mg/kg), in which the mice were intravenously injected 100 µL MPEG-PDLLA-DTX micelles (5 mg/kg); (7) MPEG-PDLLA-DTX group (10 mg/kg), in which the mice were intravenously injected 100 µL MPEG-PDLLA-DTX micelles (10 mg/kg); (8) MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser group, in which the mice were intravenously injected 100 µL MPEG-PDLLA-DTX micelles (5 mg/kg) and treated with GNR-PEG@MNs transcutaneously at the tumor sites with 2 W/cm2 irradiation by 808 nm laser for 5 min (Video 2, Figure S8B in the SI). On days 8, 9 and 10, the mice were treated with the drugs mentioned above. It was noted that the GNR-PEG@MNs was took away from the tumor sites after 5 min treatment. The tumor size and body weight were measured every two days. On day 38, the mice in each group were killed and the major tissues were collected, fixed and sectioned, followed by hematoxylin and eosin (H&E) staining60-62. Tumor cells histopathological and proliferation study The mice of all groups (3 mice per group) were sacrificed 10 days after the treatment. ACS Paragon Plus Environment


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And the tumor tissues were harvested and used for histopathological and immune histochemical study which was detected by H&E staining60-62 and Ki-67 staining63,64 respectively. Statistical analysis All data were expressed as the mean value ± SD in this study. One-way analysis of variance (ANOVA) or a Student’s t-test was used for statistical analysis. P < 0.05 or P < 0.01 marked with “” or “” were considered to be statistically significant. ■ ASSOCIATED CONTENT Supporting Information The synthesis route, 1H NMR spectra, IR spectra and retention time of MPEG-PDLLA， the cellular uptake efficiency and in vitro cytotoxicity of MPEG-PDLLA micelles, the IR spectra and EDS of GNR and GNR-PEG, the bright-field micrographs of PLLA MNs and GNR-PEG@MNs, the IR spectra, UV-vis transmittance spectroscopy and X-ray photoelectron

spectroscopy

of

PLLA MNs,

PEI

modified

PLLA MNs

and

GNR-PEG@MNs, H&E stained images of major tissues, a brief look of video 1 and video 2, materials and some results and methods have been listed in Supporting information. ■ AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS This work was financially supported by The National Natural Science Fund for Distinguished Young Scholars (NSFC31525009), Sichuan Innovative Research Team Program for Young Scientists (2016TD0004), and Distinguished Young Scholars of Sichuan University (2011SCU04B18).


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Figure Captions: Figure 1. The schematic illustration of (A) the preparation of PLLA MNs and GNR-PEG@MNs, (B) the novel synergetic system of chemotherapy and photothermal therapy to treat A431 tumors by the combination of near-infrared responsive GNR-PEG@MNs and MPEG-PDLLA-DTX micelles. (Step 1: Injected the DTX loaded micelles; Step 2: After the injection, pressed the GNR-PEG@MNs at the tumor sites and under 2 W/cm2 irradiation by 808 nm laser within 5 min) Figure 2. (A) The particle size of MPEG-PDLLA-DTX micelles (the TEM image insert), (B) UV-vis absorption spectra of GNR and GNR-PEG, the zeta potential of (C) GNR and (D) GNR-PEG (the TEM image was inserted respectively). Figure 3. (A) Photograph of PLLA MNs (left) and GNR-PEG@MNs (right), SEM image of (B1-B2) PLLA MNs and (C1-C2) GNR-PEG@MNs (the black square represents the location of GNR-PEG), the surface elemental content of (B3) PLLA MNs and (C3) GNR-PEG@MNs. (B1,C1, scale bar, 100 µm; B2,C2, scale bar, 10 µm). Figure 4. The (A) photograph, (B) optical micrograph (scale bar, 200 µm) and (C) histological sections (scale bar, 20 µm) of mice skin stained with trypan blue after GNR-PEG@MNs application (the black arrow represents the microneedles puncture sites), H&E-stained mice skin sections (D) after GNR-PEG@MNs application (scale bar, 20 µm) (the black dotted line represents the microneedles puncture sites), (E) 12 h after GNR-PEG@MNs application (scale bar, 20 µm). Figure 5. (A) Near-infrared thermal imaging of PLLA MNs and GNR-PEG@MNs under 2 W/cm2 irradiation by 808 nm laser at different time (the black square represents the location of microneedles). (B) Heating curves of PLLA MNs and GNR-PEG@MNs. (C) Temperature changes of PLLA MNs and GNR-PEG@MNs after irradiation with NIR light for 3 cycles. Near Figure 6. (A) In vivo near-infrared thermal imaging of A431 tumor-bearing mice under 2 W/cm2 irradiation by 808 nm within 5 min (the black circle represents the location of tumors). (B) Heating curves in the tumor sites. “” means the P < 0.01. Figure 7. (A) The heating transfer efficacy in pork under 2 W/cm2 irradiation by 808 nm laser within 5 min (the black circle represents the location of microneedles, scale bar, 1 ACS Paragon Plus Environment


cm). (B) Heating curves of the center temperature of the pork. “” means the P < 0.01. Figure 8. (A) Schematic illustration of in vivo antitumor efficacy of the combination of GNR-PEG@MNs and MPEG-PDLLA-DTX micelles. (B) Representative photos of mice bearing A431 tumors after treatment for 7, 14, 21, 28 and 35 days (the black circle represents the location of tumors). (C) Growth curves and (D) body weight of the mice in each group. (E) Photograph and (F) weight of subcutaneous tumors in each group (the black circle represents the location of cured tumors). Data is represented as the mean ± standard deviation (n = 5). “” and “” means the P < 0.01 and P < 0.05. (1. Saline, 2. GNR-PEG@MNs + laser, 3. DTX (5 mg/kg), 4. DTX (10 mg/kg), 5. DTX (5 mg/kg) + GNR-PEG@MNs + laser, 6. MPEG-PDLLA-DTX (5 mg/kg), 7. MPEG-PDLLA-DTX (10 mg/kg), 8. MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser). Figure 9. (A) Representative H&E stained images (scale bar, 20 µm), (B) Ki-67 immune histochemical images (scale bar, 20 µm) and (C) Ki-67 LI of tumors in each group. “” means the P < 0.01. (1. Saline, 2. GNR-PEG@MNs + laser, 3. DTX (5 mg/kg), 4. DTX (10 mg/kg), 5. DTX (5 mg/kg) + GNR-PEG@MNs + laser, 6. MPEG-PDLLA-DTX (5 mg/kg), 7. MPEG-PDLLA-DTX (10 mg/kg), 8. MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser).


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Figure 1. The schematic illustration of (A) the preparation of PLLA MNs and GNR-PEG@MNs, (B) the novel synergetic system of chemotherapy and photothermal therapy to treat A431 tumors by the combination of near-infrared responsive GNR-PEG@MNs and MPEG-PDLLA-DTX micelles. (Step 1: Injected the DTX loaded micelles; Step 2: After the injection, pressed the GNR-PEG@MNs at the tumor sites and under 2 W/cm2 irradiation by 808 nm laser within 5 min)



Figure 2. (A) The particle size of MPEG-PDLLA-DTX micelles (the TEM image insert), (B) UV-vis absorption spectra of GNR and GNR-PEG, the zeta potential of (C) GNR and (D) GNR-PEG (the TEM image was inserted respectively).


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Figure 3. (A) Photograph of PLLA MNs (left) and GNR-PEG@MNs (right), SEM image of (B1-B2) PLLA MNs and (C1-C2) GNR-PEG@MNs (the black square represents the location of GNR-PEG), the surface elemental content of (B3) PLLA MNs and (C3) GNR-PEG@MNs. (B1,C1, scale bar, 100 µm; B2,C2, scale bar, 10 µm).



Figure 4. The (A) photograph, (B) optical micrograph (scale bar, 200 µm) and (C) histological sections (scale bar, 20 µm) of mice skin stained with trypan blue after GNR-PEG@MNs application (the black arrow represents the microneedles puncture sites), H&E-stained mice skin sections (D) after GNR-PEG@MNs application (scale bar, 20 µm) (the black dotted line represents the microneedles puncture sites), (E) 12 h after GNR-PEG@MNs application (scale bar, 20 µm).


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Figure 5. (A) Near-infrared thermal imaging of PLLA MNs and GNR-PEG@MNs under 2 W/cm2 irradiation by 808 nm laser at different time (the black square represents the location of microneedles). (B) Heating curves of PLLA MNs and GNR-PEG@MNs. (C) Temperature changes of PLLA MNs and GNR-PEG@MNs after irradiation with NIR light for 3 cycles.



Figure 6. (A) In vivo near-infrared thermal imaging of A431 tumor-bearing mice under 2 W/cm2 irradiation by 808 nm within 5 min (the black circle represents the location of tumors). (B) Heating curves in the tumor sites. “” means the P < 0.01.


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Figure 7. (A) The heating transfer efficacy in pork under 2 W/cm2 irradiation by 808 nm laser within 5 min (the black circle represents the location of microneedles, scale bar, 1 cm). (B) Heating curves of the center temperature of the pork. “” means the P < 0.01.



Figure 8. (A) Schematic illustration of in vivo antitumor efficacy of the combination of GNR-PEG@MNs and MPEG-PDLLA-DTX micelles. (B) Representative photos of mice bearing A431 tumors after treatment for 7, 14, 21, 28 and 35 days (the black circle represents the location of tumors). (C) Growth curves and (D) body weight of the mice in each group. (E) Photograph and (F) weight of subcutaneous tumors in each group (the black circle represents the location of cured tumors). Data is represented as the mean ± standard deviation (n = 5). “” and “” means the P < 0.01 and P < 0.05. (1. Saline, 2. GNR-PEG@MNs + laser, 3. DTX (5 mg/kg), 4. DTX (10 mg/kg), 5. DTX (5 mg/kg) + GNR-PEG@MNs + laser, 6. MPEG-PDLLA-DTX (5 mg/kg), 7. MPEG-PDLLA-DTX (10 mg/kg), 8. MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser).


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Figure 9. (A) Representative H&E stained images (scale bar, 20 µm), (B) Ki-67 immune histochemical images (scale bar, 20 µm) and (C) Ki-67 LI of tumors in each group. “” means the P < 0.01. (1. Saline, 2. GNR-PEG@MNs + laser, 3. DTX (5 mg/kg), 4. DTX (10 mg/kg), 5. DTX (5 mg/kg) + GNR-PEG@MNs + laser, 6. MPEG-PDLLA-DTX (5 mg/kg), 7. MPEG-PDLLA-DTX (10 mg/kg), 8. MPEG-PDLLA-DTX (5 mg/kg) + GNR-PEG@MNs + laser).



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Novel Approach of Using Near-Infrared Responsive PEGylated Gold

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