Biodegradable Poly(amino acid)âGoldâMagnetic Complex with

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Biodegradable Poly(amino acid)-Gold-Magnetic Complex with Efficient Endocytosis for Multimodal Imaging-Guided Chemo-Photothermal Therapy Junjie Ma, Pengju Li, Weiwei Wang, Shunhao Wang, Xueting Pan, Fengrong Zhang, Shanshan Li, Shuang Liu, Hongyu Wang, Gan Gao, Bolong Xu, Qipeng Yuan, Heyun Shen, and Huiyu Liu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02750 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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Biodegradable Poly(amino acid)-Gold-Magnetic Complex with Efficient Endocytosis for Multimodal Imaging-Guided Chemo-Photothermal Therapy Junjie Ma,# Pengju Li,# Weiwei Wang, Shunhao Wang, Xueting Pan, Fengrong Zhang, Shanshan Li, Shuang Liu, Hongyu Wang, Gan Gao, Bolong Xu, Qipeng Yuan, Heyun Shen,* Huiyu Liu* Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Bionanomaterials & Translational Engineering Laboratory, State Key Laboratory of Organic-Inorganic Composites, Beijing Key Laboratory of Bioprocess, Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, China E-mail: Heyun Shen: [email protected], Huiyu Liu: [email protected]

ABSTRACT

The gold complex can serve as efficient photothermal converters for cancer therapy, but their non-biodegradability hinders the clinical bioapplications. Although enormous efforts have been devoted, the conventionally adopted synthetic methods of biodegradation are characterized by high cost and complicated procedure, which delay the process of further clinical translation of

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gold complex. Here, we report a multifunctional poly(amino acid)-gold-magnetic complex with self-degradation property for synergistic chemo-photothermal therapy via simple and green chemistry method. Nanoparticles with ~3 nm in the biodegradation product were observed in simulated body fluid in 4 days. The biodegradability mainly benefits from the weakened internal electrostatic interaction of the poly(amino acid) by the salt ion in simulated body fluid. It is demonstrated that the poly(amino acid)-gold-magnetic complex has great cellular endocytosis by taking advantage of the guanidine group in arginine. And the poly(amino acid)-gold-magnetic complex possesses the ability of multimodal imaging and efficient tumor ablation (94%). This study reports a possibility for gold-magnetic complex composed of poly(amino acid) may serve as a biodegradable nanotherapeutic for clinical applications.

KEYWORDS: biodegradable, gold-magnetic complex, poly(amino acid), chemo-photothermal therapy, endocytosis Photothermal therapy (PTT), which leads to cancer cells death via heat by photo-absorbing agents, has recently attracted widespread interests owing to its minimal invasiveness and specific spatial/temporal selectivity. Many nanomaterials can act as efficient photothermal converters, such as noble metal nanoparticles (NPs) (e.g., gold nanoshells,1 gold nanocages2 and gold naorods3), carbon nanomaterials (e.g., carbon spheres4 and carbon nanotubes5), semiconductors (e.g., copper sulfide6 and bismuth sulfide7 NPs) and some emerging two-dimensional nanostructures (e.g., graphene8 and black phosphorus9). But not all nanomaterials are adaptable for clinical translation. An ideal photothermal convert candidate should satisfy three basic requirements: good biocompatibility and biodegradability, high absorption in the near-infrared (NIR) region, and reasonable photothermal conversion performance. In this case, the plasmonic


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gold complex is one of the most promising PTT converters due to its good biocompatibility and excellent photothermal performance caused by radiative damping or Landau damping effect.10 Some reports, including ours, have demonstrated gold complexes-based PTT holds strong promise in cancer treatment.11–19 Encouragingly, Nanospectra Bioscience, Inc. has carried out the first clinical trial of gold nanoshell under the trademarked name of AuroShell particles for the selective and precise thermal destruction of head and neck.20 It undoubtedly provides perspectives of gold complex-based PTT in the clinic. Despite a good candidate for PTT, gold complex still has some limitations because of their non-biodegradability. In general, only NPs smaller than 5.5 nm could be excreted from the kidney.14 However, most gold complexes used in PTT, whose diameters are larger than 20 nm for better inhomogeneous polarization, are unsuitable for renal clearance.21 To solve this problem, considerable efforts have been devoted to synthesizing gold-based organic/inorganic hybrid NPs for better metabolism. For instance, Au NPs or nanorods modified with amphiphilic polymer could self-assemble into biodegradable gold vesicles. These gold vesicles can be dissociated by NIR light heating and/or enzymatic degradation.11–13 A comb-like amphipathic polymer Au NPs for rapid clearance was fabricated.14 But for this strategy, the interparticle distance between Au NPs need to be adjusted rationally to obtain a reasonable NIR wavelength according to Mie theory.22,23 Also, almost all methods involved in the synthesis of these nanomaterials need the organic solvent or harsh synthesis conditions, which may have a potential risk in the future clinical applications. Therefore, it remains a big challenge to fabricate biodegradable gold complex through a green method.


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Scheme 1. Schematic illustration of multimodal imaging-guided synergistic chemophotothermal therapy of tumor. Here, a green synthetic method for multifunctional biodegradable poly(amino acid)-goldmagnetic complex loading doxorubicin (DOX) with high endocytosis ability has been developed by introducing the magnetic Fe3O4, poly(L-arginine) (PA) and poly(γ-glutamic acid) (γ-PGA). As Scheme 1 indicated, this complex holds the following attractive advantages: 1) the guanidine group in PA facilitates the endocytosis and lysosomal escape of complex in cancer cells, through the formation of bidentate hydrogen bonds with cell membrane constituents such as sulfate, phosphate, and carboxylate.24–26 2) the poly(amino acid)-gold-magnetic complex could be degraded into around 3 nm Au/Fe3O4 NPs mixture in body fluid in 4 days after poly(amino acid) complex destruction. 3) The well-defined poly(amino acid)-gold-magnetic complex exhibited efficient in vivo multimodal imaging ability (photoacoustic tomography (PAT), magnetic


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resonance imaging (MRI), and computed tomography (CT)), precisely guiding synergistic chemo-photothermal

therapy

of

tumor.

And

DOX

released

effectively

under

the

stimulation/induction of NIR, presenting excellent synergistic chemo-photothermal therapy with an anti-tumor rate of 94%. Moreover, this design includes a self-assembly step and in situ reduction method without any harsh organic solvents. So it provides a simple, cost-effective, and green method for tailoring the biodegradability and biocompatibility of poly(amino acid)-goldmagnetic complex in a well-controlled manner. We believe that it can promote the clinical translation of gold-magnetic complex for synergistic chemo-photothermal cancer therapy.

RESULTS AND DISCUSSION


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Figure 1. Characterization of PF, PFDR, and PFDR-Au. a) Scheme of the preparation procedure for PFDR-Au. TEM images of b) PF and c) PFDR-Au. d) SEM image of PFDR-Au. e) FT-IR spectra of PF, PFDR, and PFDR-Au. f) XRD patterns of PF, PFDR, and PFDR-Au. g) UV-visNIR absorbance spectra of PF, PFDR, and PFDR-Au. h) Temperature increasing curves for PBS (control) and PFDR-Au (0.125, 0.25, 0.5, 1.0 and 2.0 mg mL−1) under 808 nm laser irradiation (2.0 W cm−2). The schematic preparation process of poly(amino acid)-gold-magnetic complex was shown in Figure 1a. (γ-PGA-Fe3O4) (PF) was prepared according to the previous study with a slight modification.27,28 First, we employed γ-PGA as a stabilizer to synthesize water-dispersible PF by a coprecipitation method. Then the DOX and PA were sequentially self-assembled to fabricate magnetic nanocluster (γ-PGA-Fe3O4-DOX-PA) (PFDR). Poly(amino acid)-gold-magnetic complex (PFDR-Au) was finally formed via in situ reduction of HAuCl4 on the surface of PFDR. As shown in Figure 1b, PF with the size of ~10 nm was observed using transmission electron microscopy (TEM). PFDR-Au complex is about 170 nm (Figure 1c, d) and the hydrodynamic size of PF, PFDR, and PFDR-Au increased stepwise (Figure S1a, b). The zeta potential of PF, PFDR, and PFDR-Au were −30.0, +36.8 and +6.8 mV, respectively (Figure S1c). The chemical composition of PF, PFDR, and PFDR-Au was confirmed by the Fourier transform infrared (FTIR) spectra (Figure 1e). The Fe−O vibration of PF had a strong band at 566 cm−1, then it shifted to 533 cm−1 and became weakened for PFDR-Au. The peak at 1622 cm−1 belonged to stretching vibration of C=O, the stretching vibrations of −CH2− groups were at 2852 and 2933 cm−1, respectively, and the peak at 3378 cm−1 belonged to the −NH groups, indicating the existence of γ-PGA and PA, and these peaks became weaker after the gold nanoshell was coated. The crystalline phases and structure of prepared samples were analyzed through the X-ray diffraction


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(XRD) patterns. As shown in Figure 1f, PF with the diffractions from the face-centered cubic (fcc) Fe3O4 at 2θ = 30.1°, 35.4°, 43.1° and 62.5° were attributed to the (220), (311), (400) and (440) planes (JCPDS 19-0629), respectively. For PFDR-Au, the (111), (200), (220) and (311) planes (JCPDS 04-0784) represented the diffractions from face-centered cubic (fcc) Au at 2θ = 38.2°, 44.4°, 64.6°, and 77.5°, respectively. After Au coating, the diffraction of Fe3O4 became weak and could not be detected due to the heavy atom effect of Au.29 As the localized surface plasmon resonance of PFDR-Au appeared in NIR region (Figure 1g), we investigated its photothermal performance and photothermal conversion efficiency. (Figure 1h, Figure S2). Results showed there was a significant temperature increase when the concentration of PFDR-Au varied from 0.125 to 2 mg mL−1. A 10 min NIR laser irradiation would make its final temperature reach 64 °C, while the phosphate buffer solution (PBS) showed negligible temperature changes. The photothermal conversion efficiency of PFDR-Au was 5.2% after calculation. Hence, the PFDR-Au presented feasible photothermal property under NIR laser irradiation, which can be a proper candidate for PTT of cancer.


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Figure 2. In vitro biodegradation of PFDR-Au. a) TEM images of PFDR-Au in water exposed to NIR laser for 5, 10, 15 and 20 min. Scale bar is 100 nm. TEM images of PFDR-Au cultured with b) simulated body fluid (SBF) and c) simulated lysosomal fluid (SLF) without NIR for different time durations. Scale bar is 100 nm. d) Size distribution and e) TEM image of NPs after incubation for 4 days in SBF. Scale bar is 10 nm. Bio-TEM was used to observe the structural evolution of PFDR-Au in 4T1 cancer cells after f) 2 days, g) 4 days and h) 8 days incubation with cells. i) enlarged view of h). To study the biodegradation property of PFDR-Au, we investigated it's in vitro biodegrade rate under different physiological conditions. First, PFDR-Au aqueous dispersion was exposed to NIR laser for various time. We observed the tightly packed PFDR-Au tended to collapse after 10


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min laser irradiation (2.0 W cm−2), and PFDR-Au disassembled to smaller particles in 20 min (Figure 2a). This phenomenon has been reported in some reports on polymersomes conjugated with gold nanoparticles.12–14 And these complexes could be used to release DOX on demand under the NIR laser irradiation. In our study, the electrostatic interaction between PFDR-Au and DOX was also disrupted by the laser-induced heat from PFDR-Au. Meanwhile, the increase of temperature promoted DOX diffusion, which induced almost complete DOX release from PFDR-Au at pH 5.0 (Figure S3). Then we directly observed the morphology evolution of PFDRAu incubated in simulated body fluid (SBF) and simulated lysosomal fluid (SLF) at 37 °C, respectively. The partially biodegraded PFDR-Au was observed by TEM (Figure 2b, c). After incubation for 2 days, most of the NPs of PFDR-Au were detached in SBF (salt concentration (SC) = 0.15 M, pH = 7.4), but the morphology of PFDR-Au had no obvious change in SLF (SC = 0.20 M, pH = 4.5). When the incubation time was prolonged to 4 days, no PFDR-Au could be observed in SBF, because PFDR-Au completely collapsed to 3 nm NPs (Figure 2b, d and e). Moreover, the energy-dispersive X-ray spectroscopy (EDX) analysis proved the existence of Au and Fe elements in the degradation products, which indicated that PFDR-Au could degrade into 3 nm Au/Fe3O4 NPs mixture (Figure S4). While in the SLF, most of PFDR-Au destructed and NPs with the size of around 3 nm were observed in TEM image after 8 days of incubation. Compared with SBF, there were less Fe3O4 NPs observed after 8 days of incubation with the SLF, this was because the low pH in SLF would accelerate the degradation of Fe3O4.17 In addition, we found the structure of PFDR-Au collapsed in simulated gastric fluid (SGF, SC = 0.03 M, pH = 1.2) after incubation for 8 days, and part destruction of the PFDR-Au was found in 1640 media (SC = 0.13 M, pH = 7.4) after 12 days. However, there were no obvious morphological change of PFDR-Au in simulated intestinal fluid (SIF, SC = 0.06 M, pH = 6.8)


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after 12 days of incubation (Figure S5). The presence of salt in the SBF shield charged the group of polyelectrolyte which reduced the electrostatic interaction between γ-PGA and PA,30 leading to a destruction of PFDR-Au and disassembling to NPs smaller than 5.5 nm in SBF, then it could be eliminated from the body through glomerular filtration.31,32 In case of the complex in SLF, acidic pH solution could induce partial γ-PGA protonation, thus reducing the electrostatic interaction between γ-PGA and PA as well as facilitating hydrogen bonding formation between PA and γ-PGA, resulting in slower (8 days) degradation of PFDR-Au than that in SBF (4 days). Although the acidic SGF solution contained enzyme molecules, the PFDR-Au was degraded slower than that in SBF. This indicated that ionic strength would be the main driving force for bioelimination of PFDR-Au and the pepsin in SGF was not a requirement factor in the degradation of PFDR-Au. In addition, in spite of similar ion strength between the SBF and 1640 media, a lot of glucose and the various amino acid could take effect on stabilization of PFDRAu. We further evaluated the intracellular biodegradation behavior of PFDR-Au by bio-TEM. After 4T1 cells incubation with PFDR-Au for 1 and 2 days, we found that the NPs could be endocytosed into cells, as shown in the yellow boxed regions (Figure 2f, Figure S6). Importantly, the PFDR-Au was slightly biodegraded in 4 days (in the yellow boxed region) (Figure 2g), and a complete intracellular biodegradation had been found after 8 days of incubation (Figure 2h, i). As indicated by the black arrow in the enlarged image, PFDR-Au was completely degraded into around 3 nm NPs. Above results suggested that obvious biodegradation of PFDR-Au was carried out by normal body fluid and cellular content. Hence, PFDR-Au would be a great candidate to fabricate carrier with complete bioelimination for the tumor theranostic applications.


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Figure 3. Endocytosis and pH dependence of red blood cells hemolysis. a) Cellular uptake of DOX, PFD, PFDK, PFDR, PFDK-Au, and PFDR-Au in 4T1 cells after 6 h incubation. Scale bar is 20 µm. b) The hemolysis evaluation of PFDK-Au and PFDR-Au. Red blood cells incubated with 0.2 mg mL−1 PFDK-Au and PFDR-Au in PBS with different pH value (pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.4), respectively. c) Photographs of red blood cells solution in water and pH 5.0 of PBS after 1 h incubation with 0.2 mg mL−1 PFDK-Au and PFDR-Au. d) Confocal images of 4T1 cells incubated with PFDK-Au and PFDR-Au for a different time to visualize the colocalization with lysosomes. Red: lysosomes, green: DOX of PFDK-Au and PFDR-Au, blue: nucleus. Scale bar is 10 µm. Moreover, cellular uptake of PFDR-Au in 4T1 cells was observed by confocal laser scanning microscopy (CLSM). The red fluorescence of DOX indicated the amount of NPs inside the cells (Figure 3a). To prove whether the amount of amino has a certain impact on endocytosis, we


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selected cationic poly(ε-lysine) (PL) to modify PF along with DOX loading (γ-PGA-Fe3O4DOX-PL) (PFDK) and gold nanoshell coating (PFDK-Au) by in situ reductions as positive controls. And PF along with DOX loading (PFD) as a negative control. Compared to the control groups, PA modified NPs (PFDR, PFDR-Au) showed more significant red fluorescent signals in 4T1 cells. This result demonstrated that the arginine of PFDR had higher cellular uptake ability due to the high membrane affinity of arginine. The guanidine of arginine can induce electrostatic interactions and bidentate hydrogen bonds with phosphate, carboxylate and sulfate moieties on cell surface composition, including phospholipids, sialic acids, proteoglycans, and so on.24–26,33 Because there is a relationship between endosomal disruption and hemolytic activity, red blood cells were used as a model for cells and lysosomal membranes to investigate the potential of the PFDR-Au to disrupt lipid bilayer membranes.34,35 The result in Figure 3b suggested the hemolytic activity of PFDR-Au and PFDK-Au was a function of pH. Neither PFDR-Au nor PFDK-Au had hemolytic activity at physiological pH values and tumor microenvironment (pH = 6.8), indicating that both PFDR-Au and PFDK-Au own great biocompatibility. However, the low pH led to a peak hemolytic activity of the PFDR-Au and PFDK-Au at pH 5.0. Because low pH values induced the protonation of carboxyl groups of γ-PGA and amino groups of arginine and lysine, thus increasing the zeta potential of NPs and improving the membrane permeability. Moreover, hemolysis value of PFDR-Au was about 1.7 times higher than that of PFDK-Au at pH 5.0. It was directly observed in naked eye (Figure 3c), indicating that guanidine group of arginine had better membrane permeability than amino groups of lysine at lysosomal acidic pH. On the other hand, the colocalization analysis was used to evaluate the escape of PFDR-Au or PFDKAu from the lysosomes. Figure 3d are the fluorescence confocal images of 4T1 cells treated by PFDR-Au or PFDK-Au for 3, 6, 9 and 12 h, the red fluorescence corresponds to lysosomes


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stained with LysoTracker Red, the green fluorescence corresponds to the DOX of PFDK-Au and PFDR-Au, and the blue fluorescence is from the 4′,6-diamidino-2-phenylindole (DAPI). As shown in Figure 3d, the red fluorescence overlapped well with the green fluorescence at 3 h, indicating that PFDR-Au and PFDK-Au could entry into lysosomes. And after 12 h incubation, PFDR-Au could effectively escape from lysosomes, but in the case of PFDK-Au, a lot of NPs still remained in lysosomes. Above results strongly demonstrated PFDR-Au had more excellent lysosomal escape ability than that of PFDK-Au in acidic pH. It should be noted that the hemolysis value of PFDR-Au was lower than 10% in whole acidic pH ranges and physiological pH, indicating that PFDR-Au had excellent biocompatibility for biomedical applications. Meanwhile, we investigated the number of terminal primary amino groups of PFDK-Au and PFDR-Au by trinitrobenzene sulfonic acid (TNBS) assay (Figure S7). When the weight of PFDR-Au was same with PFDK-Au, the number of PFDR-Au terminal primary amino groups was lower than that of PFDK-Au, which was also a strong evidence that the guanidine group of PFDR-Au had higher cell membrane affinity than amino group of PFDK-Au. Therefore, PFDRAu could induce effective endocytosis and lysosomal escape, which could help to improve synergistic chemo-photothermal cancer therapy.


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Figure 4. In vitro cell experiments. a) Cell viability of 4T1 cells incubated with PFR-Au for 24 and 48 h. b) The impact of synergistic chemo-photothermal therapy on 4T1 cells with free DOX, PFR-Au, and PFDR-Au. An equivalent DOX concentration was used in free DOX group and the PFDR-Au group. Data were presented as mean ± SD (n = 5), **P < 0.01, ***P < 0.001. c) Live/dead cell staining assays were observed by CLSM. Red: dead cells, green: live cells. Scale bar is 250 µm. Safety of nanomaterials should be the first consideration for future bioapplications. Therefore, we investigated the toxicity of PFDR-Au without DOX (PFR-Au) to 4T1 cells by a standard


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methyl thiazolyl tetrazolium (MTT) assay. The cell viability remained above 80% after 4T1 cells incubation with 200 µg mL−1 PFR-Au for 48 h (Figure 4a), demonstrating a high biocompatibility of the PFR-Au. Then, we investigated the effect of synergistic chemophotothermal therapy on 4T1 cells (Figure 4b). After incubated with PFDR-Au or PFR-Au for 12 h, the cells were exposed to NIR laser (2.0 W cm−2, 5 min). In PFDR-Au + NIR group, when NPs concentrations increased to 200 µg mL−1, the cell viability decreased to 21% and realized a high rate of apoptosis. No obvious cell death was found in PTT (PFR-Au + NIR) or chemotherapy (PFDR-Au) groups, and the apoptosis rates were 31% and 35% at the concentration of 200 µg mL−1, respectively. Moreover, the cell-killing ability of synergistic therapy was directly observed via propidium iodide (PI) and calcein acetoxymethyl ester (calcein-AM) double staining dead and live cells (Figure 4c). Compared with the PFDR-Au or PFR-Au + NIR, PFDR-Au + NIR group showed sufficient cell killing ability under NIR laser irradiation (200 µg mL−1, 2.0 W cm−2, 5 min). Under NIR stimulation, DOX released effectively and led to complete apoptosis of cancer cells. Above results strongly suggested that PFDR-Au owned excellent ability of synergistic chemo-photothermal therapy. In addition, we determined the synergistic effect of chemotherapy and PTT by the combination index (CI) using a ChouTalalay

method,

which

was

widely

used

to

evaluate

the

drug

interactions

in

pharmacodynamics.36,37 The formula and evaluation criterion of CI were added in supporting information. Based on Figure 4b, the CI value of PFDR-Au was calculated to be 0.179, indicating a strong synergistic effect after laser irradiation on 4T1 cells. Therefore, the theoretical data also can effectively demonstrate the strong synergistic chemo-photothermal therapy mediated by PFDR-Au.


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Figure 5. Biodistribution and IR thermal imaging of 4T1 breast tumor-bearing mice. a) In vivo DOX fluorescence imaging after i.v. injected with PFDR-Au (1 mg mL−1, 200 µL). b) Ex vivo fluorescence imaging and c) fluorescence intensity of tumor and major organs at 24 h postinjection. d) IR thermal images of mice i.v. injected with PFDR-Au (10 mg kg−1) and PBS. e) The heating curves of tumor upon NIR laser (2.0 W cm−2, 5 min) irradiation in PFDR-Au and PBS treated groups.


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Based on the strong in vitro synergistic therapeutic effects, we investigated the distribution of PFDR-Au in vivo. PFDR-Au could circulate in the blood for a long time and accumulate in the tumor region through the enhanced permeation and retention (EPR) effect. To investigate the fate of PFDR-Au in vivo, PFDR-Au and free DOX were intravenously (i.v.) injected into mice with the same dose of DOX. An in vivo imaging systems (IVIS) was used to track the distribution of PFDR-Au, and the fluorescence images were obtained at various time after injection. The maximum accumulation amount of PFDR-Au in tumor region was obtained at 24 h after injection, while there was no obviously DOX fluorescence signal at tumor region at all time points after injection in the free DOX group (Figure 5a, Figure S8). Moreover, ex vivo tissue analysis showed stronger fluorescence signals in tumor compared with that of other organs (Figure 5b, c). Above results suggested that PFDR-Au could effectively accumulate in tumor region by EPR effect and that DOX has efficiently released in the tumor microenvironment. In addition, in vivo photothermal effect of PFDR-Au was also investigated (Figure 5d). The local tumor temperature reached 38 °C in PBS group, indicating that the NIR laser was safe to normal tissue. In contrast, after PFDR-Au injection, the temperature at the tumor site reached 47 °C under laser irradiation for 5 min, and kept 47 °C during the treatment, inducing obvious cancer cell apoptosis (Figure 5d, e). In order to evaluate the pharmacokinetics of PFDR-Au, we i.v. injected the PFDR-Au into BALB/c mice and detected the Au content in blood by inductively coupled plasma mass spectrometry (ICP-MS). The first (t1/2α) and second (t1/2β) elimination halflife time for the PFDR-Au were 0.72 ± 0.11 and 4.33 ± 0.17 h, respectively. Meanwhile, the value of its area under the curve (AUC) was 393.99 ± 50.21 µg mL−1 h. Therefore, PFDR-Au displayed a long-circulation behavior (Figure S9). Moreover, to determine whether small gold nanoparticles can be effectively excreted from the body, we further determined the gold content


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in urine and feces at different time points after injection by ICP-MS (Figure S10). Au content in urine and feces reached the maximum at 36 h post-injection, which indicated that PFDR-Au could be degraded into small Au NPs and excreted from kidney, and the elimination of Au NPs through feces might be performed by hepatic processing and biliary excretion.38

Figure 6. In vivo multimodal imaging. a) PAT imaging before and after i.v. injection of 10 mg kg−1 PFDR-Au, yellow dotted circles are the location of the tumor. Red, blue and green represent oxyhemoglobin (HbO2), hemoglobin (Hb) and PFDR-Au, respectively. b) CT imaging before (left) and after (right) 10 min intratumoral injection of 30 mg kg−1 PFDR-Au. c) T2-weighted MRI before and after i.v. injection of 10 mg kg−1 PFDR-Au. Top and bottom row are the transverse and sagittal section, respectively. Red dotted circles are the location of the tumor. d) The corresponding T2-weighted MRI-signal intensity with the prolonging of duration after the injection. We defined the T2-weighted MRI-signal intensity of tumor before i.v. injection as 100%.


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Based on the physicochemical characteristics of PFDR-Au, we deduced that the PFDR-Au could serve as an excellent chemo-photothermal agent with multimodal diagnostic imaging properties, including PAT and CT imaging endowed by gold nanoshell, and MRI by superparamagnetic iron oxide. The multimodal imaging was used to detect the distribution of PFDR-Au and determine the tumor region for therapy guiding and therapeutic efficacy monitoring. First, the cross-sectional PAT images were obtained after PFDR-Au were i.v. injected into mice at different time intervals (Figure 6a). The result showed that there was more Hb and fewer HbO2 in the tumor region, and at 24 h post-injection the average PAT signal intensity of PFDR-Au had a 2.5-fold increase from 142 to 497, then declined. These results indicated that the tumor microenvironments were hypoxic and PFDR-Au could accumulate in tumor. As a high atomic number element (Z = 79), gold could produce a strong photoelectric effect to X-ray attenuation, suggesting a promising agent for CT imaging. As expected, an obvious contrast enhancement was found in the tumor region (red dot circles) after 10 min intratumoral injection of PFDR-Au (Figure 6b). In addition, if Fe3O4 NPs is larger than 5 nm it could enhance T2-weighted MRI, otherwise, it should be T1-weighted MRI contrast agents.39,40 Therefore, we evaluated the in vivo T2-weighted MRI performance of PFDR-Au. As Figure 6c, d showed, significant negative contrast increase was found in the tumor region at 24 h, and T2weighted signal intensity reduced to 60%. So MRI and PAT imaging both proved that the maximum accumulation amount of PFDR-Au in the tumor was obtained at 24 h after injection and then decreased. In addition, in vivo biodistribution of PFDR-Au determined the in vivo fate of PFDR-Au, which was also consistent with the above results (Figure 5a).


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Figure 7. In vivo synergistic chemo-photothermal therapy of PFDR-Au. a) Tumor volumes and b) body weights of mice with various treatments. Red arrows represent the initial injection time. Data were presented as mean ± SD (n = 5), ***P < 0.001. c) Photographs of mice at different times after various treatments. d) Photographs of the dissected tumors after 18-day therapy in each treated group. e) H&E stained results of the tumor for various treated groups. Scale bar is 50 µm. f) H&E stained results of liver, spleen, and lung after treatment (PFDR-Au + NIR). Scale bar is 50 µm. To prove the synergistic chemo-photothermal therapeutic effect in vivo, PFDR-Au was i.v. injected into the 4T1 tumor-bearing mice when the tumor volume reached about 50 mm3. We investigated the curative effect of free DOX, NIR, PFR-Au + NIR, PFDR-Au, and PFDR-Au +


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NIR, respectively. And tumor volumes were recorded (Figure 7a). The results showed the tumor size in the NIR irradiation, DOX, PFR-Au + NIR, PFDR-Au groups had a little decrease compared with the control group, but could not obtain desired anti-tumor effect (Figure 7a, c, and d). Moreover, the slight dermal damage of mice had completely recovered after 18 days treatment with PFDR-Au + NIR (Figure 7c). The body weight decreased in control group, but PFDR-Au group had slightly increased during the therapy course, which suggested that the health and living quality of mice were fine during the treatment by PFDR-Au (Figure 7b). At the same time, the synergistic chemo-photothermal therapy carried out by PFDR-Au under NIR irradiation induced an anti-tumor rate of 94% in all mice. Then all the animals were sacrificed at 18 days, and tumor together with main organs (heart, liver, spleen, lung, kidney) was collected for pathological analysis (histological examination (H&E stain)) (Figure 7e, f). In PFDR-Au + NIR group, extensive nuclear pyknosis and cancer necrosis occurred in the tumor as indicated by the black arrows, demonstrating the severe cancer necrosis. In PFR-Au + NIR group, the tendency of tumor tissue necrosis was observed from hemorrhagic necrosis and nuclear pyknosis as shown by the black arrow. At the same time, in groups of NIR, DOX, and PFDR-Au, only negligible necrosis was found as same as the control group. Besides, neither inflammation nor obvious damage was observed in other main organs because of the low cytotoxicity and immunogenicity of PFDR-Au (Figure 7f, Figure S11). These results indicated that PFDR-Au could achieve efficient synergistic chemo-photothermal therapy of cancer.

CONCLUSIONS

In this study, a biodegradable poly(amino acid)-gold-magnetic complex was fabricated by a green and facile synthesis method. Multimodal imaging-guided synergistic chemo-photothermal


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therapy was achieved by a dual response of intracellular acidic pH and NIR light, inducing effective tumor ablation with the anti-tumor rate of 94%. Importantly, the PFDR-Au completely degraded into around 3 nm NPs in the body fluid after 4 days induced by destruction of poly(amino acid) complex. Moreover, the guanidine group of arginine efficiently enhanced endocytosis and lysosomal escape ability of the PFDR-Au, leading to an excellent tumor elimination effect. The biodegradable PFDR-Au complex could facilitate the clinical translation of gold-magnetic complex for cancer treatment.

EXPERIMENTAL SECTION

Materials. Poly(γ-glutamic acid) (γ-PGA, Mw = 480 kDa) was purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. 4′,6-diamidino-2-phenylindole (DAPI), poly(L-arginine) (PA) were purchased from Sigma, St Louis, MO, USA. Poly(ε-lysine) (PL) was purchased from Shanghai yuanye Bio-Technology Co., Ltd. Hydroxylamine hydrochloride (NH2OH·HCl), ferric chloride hexahydrate (FeCl3·6H2O > 99%), sodium sulfite (Na2SO3) and ammonia (25-28% NH3 in water solution) were obtained from Sinopharm Chemical Reagent Co., Ltd., Beijing, China. Lysotracker Red DND-99 was purchased from Invitrogen, Carlsbad, California, USA. 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Doxorubicin hydrochloride (DOX·HCl) were purchased from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China. Chloroauric acid (HAuCl4) was purchased from Shenyang Jinke Reagent Factory, Shenyang, China. All chemicals were used as received. RPMI-1640 medium, PBS, fetal bovine serum (FBS), trypsin-EDTA solution, penicillin, and streptomycin were purchased from Corning (New York, USA). The dialysis membrane bag (MD34-5M, molecular weight cutoff (MWCO) =


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3500) was from MYM Biological Technology Co., Ltd., USA. Ultrapure water was purified by a Molresearch Water Purification System (Molecular, ChongQing, China). Synthesis of PFDR-Au Complex. The preparation of γ-PGA-Fe3O4 (PF) was according to the previous report with slight modification.28 First, FeCl3·6H2O (0.24 g) was placed in a threenecked flask (100 mL) containing ultrapure water (4 mL). N2 was bubbled for 5 min under stirring. Next, γ-PGA (48 mg) dissolved in 0.05 M NaHCO3 aqueous solution (8 mL) was added under stirring, whereafter dropwise added the Na2SO3 solution (40 mg, 2 mL). The reaction solution was heated to 60 °C when the color of the solution turned into yellow and ammonia (0.8 mL) was rapidly placed in the flask with stirring (350 rpm, 30 min). The obtained product was centrifuged (12000 rpm, 20 min) 3 times. DOX loading onto PF (PFD) was achieved by blending 2 mg DOX and 4 mg PF in 20 mL water under stirring in the dark (25 °C, 24 h). After centrifugation, PFD was gently washed with ultrapure water, and DOX loading content was measured via UV-vis measurement. Next, the positively charged PA (10 mL, 0.4 mg mL−1) was placed in a suspension of the negatively charged PFD (10 mL, 0.1 mg mL−1) incubation for 12 h to prepare PFDR. For Au growth on the surface of PFDR, HAuCl4·4H2O solution (212.4 µL, 48 mM) was added into an aqueous solution of PFDR (4 mL) under stirring in the dark for 4 h. Next, NH2OH·HCl (1148 µL, 445 mM) was added into the above reaction solution was stirred for another 40 min in the dark. The resultant product of PFDR-Au was obtained by centrifuging and washing. We have characterized the loading ratio of arginine, DOX, Au and Fe3O4 inside the final PFDR-Au product by TNBS method, UV-vis spectrophotometry, and ICP-MS, respectively. And the loading ratio of arginine, DOX, Au, and Fe3O4 are 27.5%, 0.6%, 27.2%, and 25.9%, respectively.


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Characterization. The morphology of PFDR-Au was investigated by TEM (JEM-2100, JEOL, Japan). Thermal camera (FLIRE64501, USA) was used to record the photothermal images. The temperature was acquired by TES-1315 K-type thermocouple thermometer (TES, China). TU-1901 spectrometer (Persee, China) was used to record the UV-vis-NIR spectrum. FT-IR spectra were acquired through Nicolet 6700 spectrometer (Thermo Fisher, USA) using the KBr pellet method. Powder XRD measurements were obtained by XRD-6000 X-ray diffractometer (Shimadzu, Japan). Leica TCS SP8 confocal fluorescence microscope (Leica Microsystems, Germany) was used to collect confocal fluorescence microscope images. Drug release was detected by PerkinElmer EnSpire (EnSpire, USA). In Vitro Synergistic Chemo-Photothermal Therapy of PFDR-Au. 4T1 murine breast cancer cells were initially purchased from American Type Culture Collection (ATCC). Frist, 4T1 cells were seeded in 96-well plate and cultivated in 1640 medium (containing FBS, penicillin, and streptomycin) with 5% CO2 at 37 °C. Then, 4T1 cells were incubated with PFR-Au (24 h and 48 h, 37 °C). And the cell viability was detected by MTT assay. To investigate the synergistic chemo-photothermal therapy, 1 × 104 4T1 cells were cultured with PFDR-Au for 12 h. Next, the cells were irradiated with NIR laser (2.0 W cm−2, 5 min), cultured for another 12 h, and tested the cell cytotoxicity by MTT assay. PI and calcein-AM double staining live and dead cells were also used to detect the cell activity. Frist, 5 × 105 cells were incubated with PFDR-Au, PFR-Au or DOX for 12 h, the cells were irradiated with NIR laser (2.0 W cm−2, 5 min) and incubated for another 12 h. Then, the cells were incubated with calcein-AM and PI co-staining (30 min, 37 °C), and then observed by CLSM. Evaluation of PFDR-Au Biodegradability in Vitro. To investigate the biodegradation behavior of PFDR-Au. PFDR-Au was incubated in the simulated lysosomal fluid, simulated


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body fluid, simulated gastric fluid, simulated intestinal fluid and 1640 media at 37 °C. The partially biodegraded PFDR-Au was observed by TEM. To evaluate the behavior of intracellular biodegradation, PFDR-Au (200 µg mL−1 in 1640 medium) was incubated with 4T1 cells and after different time intervals (1, 2, 4 and 8 days), the cells were sectioned for bio-TEM observation. Composition of SIF, SBF, SGF, and SLF. Simulated intestinal fluid (SIF): 6.8 g KH2PO4 was added to 250 mL water to dissolve, then NaOH solution (77 mL, 0.2 mol L−1) and 500 mL ultrapure water was added to above solution, after 10 g trypticase was dissolved, and the pH was adjusted to 6.8 ± 0.1 by using NaOH and HCl solution. Finally, diluted with water to 1000 mL. Simulated body fluid (SBF): pure 0.355 g NaHCO3, 8.35 g NaCl, 0.225 g KCl, 0.292 g CaCl2, 0.311 g MgCl2·6H2O, 0.072 g Na2SO4 and 0.231 g K2HPO4·3H2O were dissolved in ultrapure water. Pure 6.118 g NH2C(CH2OH)3, Tris and HCl (1.0 mol L−1, 39 mL) was used to adjust the pH to 7.40, and the final volume is 1000 mL. Simulated gastric fluid (SGF): 3.2 g pepsin (800~2500 active unit per mg) and 2.0 g NaCl are dissolved by adding HCl (7.0 mL) and water. The final pH of the SGF should be 1.2 and the final volume is 1000 mL. Simulated lysosomal fluid (SLF): 0.05 g MgCl2, 3.21 g NaCl, 0.071 g Na2HPO4, 0.039 g Na2SO4, 6.00 g NaOH, 0.128 g CaCl2·2H2O, 0.077 g sodium citrate, 0.059 g glycine, 20.8 g citric acid, 0.09 g sodium tartrate, 0.085 g sodium lactate, 0.086 g sodium pyruvate are added to 1000 mL water and the final pH is 4.5. Endocytosis and Colocalization with Lysosomes. To investigate the internalization of the PFDR-Au, 4T1 cells were incubated (12 h, 37 °C) with borosilicate chambered cover glass (5 × 105 cells per well). Then, the medium was removed and DOX, PF, PFD, PFDR, PFDK, PFDK-


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Au and PFDR-Au 1640 medium solution with a same concentration of DOX (except PF) was added. After incubation for 6 h and then observed by CLSM. To evaluate the colocalization of PFDK-Au or PFDR-Au with lysosomes, 4T1 cells were incubated with PFDK-Au or PFDR-Au for 3, 6, 9 and 12 h at 37 °C. Then, the 4T1 cells were stained by LysoTracker Red and DAPI. Leica TCS SP8 microscope was used to collect all of the confocal images. In Vivo Biodistribution of PFDR-Au. All animal experiments were performed adhering to the guidelines of the Institutional Animal Care and Use Committee of Peking University (Permit Number: 2011-0039). Female BALB/c mice with 4–6 weeks old were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). To develop the 4T1 tumor model, 50 µL suspension containing 1 × 104 4T1 cells were injected subcutaneously in the abdomen region of BALB/c mice. Tumor growth of the mice was monitored after intraperitoneal injection of D-luciferin (15 mg mL-1; AnaSpec, Inc., Fremont, CA, USA) at 8 min prior to imaging. To study biodistribution, 1 mg mL−1 PFDR-Au (200 µL, three mice per group) was i.v. injected into mice bearing 4T1 tumors. Moreover, tumor and major organs were obtained, and IVIS Spectrum Imaging System (PerkinElmer, Waltham, MA, USA) was used to investigate in vivo the distribution of PFDR-Au post-injection at each time point. Pharmacokinetics and Clearance of PFDR-Au. PFDR-Au (10 mg kg−1) was i.v. injected into Female BALB/c mice. After 0.083, 0.5, 1, 2, 4, 8, 12 and 24 h of injection, blood was collected from the mouse (n = 3) and then dissolved in chloroazotic acid to obtain the total amount of Au by using ICP-MS. To investigate the excretion of PFDR-Au, feces and urine were collected after mice (n = 3) i.v. injection with PFDR-Au (10 mg kg−1) after 2, 4, 8, 12, 24, 36, 48 and 96 h. And then the collected feces and urine were dissolved by chloroazotic acid and detected by ICP-MS.


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In Vivo Multimodal Imaging. The mice with tumor size of 200 mm3 were i.v. injected with PFDR-Au (10 mg kg−1). After 3, 6, 9, 12, 24, 36 and 48 h of i.v. injection, they were anesthetized for PAT imaging and MRI. T2-weighted MR images were obtained by a 1.5 T magnetic resonance system (Aspect Imaging, M3TM, Israel) at room temperature (TE = 50 ms, TR = 3000 ms). The PAT images were obtained from real-time multispectral optoacoustic tomography (MSOT, inVision 128, iThera Medical, Germany) of live mice. CT imaging was measured by a Micro-CT system (Caliper, Quantum FX, USA) before and after 10 min intratumoral injection of PFDR-Au (30 mg kg−1). In Vivo Synergistic Chemo-Photothermal Therapy of PFDR-Au. The 4T1 tumor-bearing mice with tumor size of 50 mm3 were randomly divided into six groups with various treatments: 1) without any treatment (control), 2) laser irradiation (NIR), 3) DOX only (DOX), 4) PFDR-Au only (PFDR-Au), 5) PFR-Au with laser irradiation (PFR-Au + NIR), and 6) PFDR-Au with laser irradiation (PFDR-Au + NIR). The whole tumor region was irradiated with NIR laser (2.0 W cm−2, 5 min) after i.v. injection of the sample (10 mg kg−1) for 24 h. The average temperature in the tumor region was measured by a thermal camera. Tumor volume (V) = ab2/2, where a and b is the length and width, respectively. All the mice were sacrificed on the 18 days of therapy and main organs and tumor were obtained for hematoxylin and eosin staining. Hemolysis Assay. The hematotoxicity of PFDR-Au was evaluated by using red blood cell (RBC). We added 200 µL of PBS solution (0.2 mg mL−1) of PFDR-Au and PFDK-Au (pH = 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.4) into 0.8 mL of freshly isolated blood. The different pH of PBS solution was adjusted by NaOH and HCl solution and was incubated (37 °C, 1 h). NaCl (0.9%) solution and ultrapure water acted as negative and positive controls, respectively. The mixtures were centrifuged and the supernatant was collected and then measured absorbance at 540 nm.


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Hemolysis ratio: hemolysis % = (sample absorbance − negative control absorbance) / (positive control absorbance − negative control absorbance) × 100%. Statistical Analysis. All data were presented as mean ± SD unless otherwise stated. And all the experiments were performed at least in triplicates. The statistical significance was determined using a two-tailed student′s test (*P < 0.05, **P < 0.01 and ***P < 0.001) unless otherwise stated.

AUTHOR INFORMATION

Author Contributions Liu. H. and Shen. H. planned the research, Ma. J. performed the materials synthesis, in vitro and in vivo experiments, most of the structural characterizations. Li. P. performed the cell experiments. Wang. W. performed the TEM measurement and analyzed the XRD and FT-IR data. Pan. X., Zhang. F., Li. S. and Wang. S. performed the in vivo treatment experiments. Yuan. Q. discussed experiment of drug release. The paper was co-written by Ma. J., Li. P., Liu. S., Wang. H., Gao. G. and Xu. B. All authors discussed the results and commented on the manuscript. Ma. J. and Li. P. contributed equally. Funding Sources This work was supported by National Basic Research Program of China (973 Program) under Grant No. 2016YFA0201500, the National Natural Science Foundation of China (NSFC, Grant No. 21304006, No. 51572271 and No. 5177021361) and Fundamental Research Funds for the Central Universities (buctrc201610, PYBZ1705).

ACKNOWLEDGMENT


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The authors would like to thank Drs Takami Akagi and Mitsuru Akashi from Osaka University for their helpful discussions and advice.

ASSOCIATED CONTENT

Supporting Information Supporting data includes DLS, bio-TEM and TEM measurements and properties analysis of poly(amino acid)-gold-magnetic complex. Pharmacokinetics studies and the clearance of PFDRAu in vivo. TNBS assay was used to measured primary amine group of complex. And H&E stained of organs and tumor was also presented. The following files are available free of charge via the internet at http://pubs.acs.org.

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Our study reports a biodegradable poly(amino acid)-gold-magnetic complex was fabricated through a green and facile method for multimodal imaging-guided synergistic chemophotothermal therapy.


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Biodegradable Poly(amino acid)âGoldâMagnetic Complex with

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