One-pot photoreduction to prepare NIR-absorbing plasmonic gold nanoparticles tethered by amphiphilic polypeptide copolymer for synergistic photothermal-chemotherapy

Siqi Yng,Linzhu Zhou,Yue Su,Rong Zhng*,Chng-Ming Dong,*

a School of Chemistry and Chemical Engineering,Shanghai Key Laboratory of Electrical Insulation and Thermal Aging,Shanghai Jiao Tong University,Shanghai 200240,China

b Joint Research Center for Precision Medicine,Shanghai Jiao Tong University Af fi liated Sixth People's Hospital South Campus,Shanghai Fengxian Central Hospital,Shanghai 201400,China

Key words:Near infrared Plasmonic gold nanoparticles Polypeptide Photothermal therapy Combination therapy

ABSTRACT We developed one-pot photoreduction strategy to prepare near infrared light(NIR)-absorbing plasmonic gold nanoparticles(Au NPs)tethered by amphiphilic polypeptide copolymer poly(L-cysteine)-b-poly(ethylene oxide)(PLC-b-PEO).The PLC-b-PEO@Au NPs possessed strong NIRabsorption at 700–1100 nm and ultrahigh photothermal conversion ef fi ciency of 62.1%.Upon the NIRirradiation(808 nm,2 W/cm 2,5 min),the PLC-b-PEO@Au NPs(1 mg/m L)sharply attained an elevation of 30.8?Cand the hyperthermia effect could efficiently kill cancer cells in vitro.As for the PT-CTtreatment,the doxorubicin(DOX)-loaded nanoparticles of DOX-PLC-b-PEO@Au NPs gave a combination index of 0.9 compared to single chemotherapy(CT)or photothermal therapy(PT),demonstrating a synergistic effect.

In the past decades,a myriad of gold nanoparticles(Au NPs)with excellent optical,catalytic and bio-physicochemical properties have attracted great attention in nanotechnology and nanomedicine as they intrinsically possess the localized surface plasmon resonance[1–8]Especially,some kinds of Au NPs with complicated architectures including gold nanoshells,nanorods,nanocages,and nanostars can absorb near-infrared light(NIR I,650?950 nm;NIRII,1000–1500 nm)and efficiently convert it into heat,making them great potentials for noninvasive photothermal therapy(PT)of cancers,bioimagings,and theranostics[3–8].Importantly,the hyperthermia effect might enhance NPs accumulation and penetration in tumor site,the cellular permeability and uptake,and the antitumor immunity that w ould further enable the tumor ablation of single PTor the synergistic therapeutic ef fi cacy of the combined photothermal-chemotherapy(PT-CT)[9–13].On the other hand,synthetic polymers are generally utilized not only to tune the plasmonic w avelength,but to enhance the stability and the biocompatibility of Au NPs[14].However,obvious draw backs including complicated fabrication procedures,toxic reducing agents,and non-biodegradable polymeric templates are urgent to be overcome,which w ill otherw ise hinder the plasmonic Au NPs from biomedical applications and clinical transitions[15–18].Therefore we are interested in how to fabricate NIR-absorbing plasmonic Au NPs in a simple and green manner.

Ow ing to good biocompatibility and biodegradability,synthetic polypeptides(i.e.,poly(amino acid)s)and their amphiphilic poly(ethylene oxide)(PEO)copolymers have been w idely investigated for various drug delivery vesicles,also providing opportunities to construct biocompatible plasmonic Au NPs[19–22].To addressthe above-mentioned problems,herein we develop one-pot photoreduction strategy for the preparation of plasmonic Au NPs with the NIR-absorption at 700–1100 nm by using the thiols-pendant poly(L-cysteine)-b-PEO (PLC-b-PEO)copolymer as a reducing and stabilizing agent(Fig.1A).The as-prepared polypeptide copolymer-tethered Au NPs(i.e.,PLC-b-PEO@Au NPs)have been thoroughly characterized by means of vis-NIR spectroscopy,XPS,DLS,TEM,and high resolution FE-TEM with tw o-dimensional element mapping,which convincingly demonstrated that the PLC-b-PEO@Au NPshasa thin layer of PLC-b-PEOcopolymer dangled on the surface of Au NPs.The PLC-b-PEO@Au NPs possess strong NIR absorption at 700–1100 nm and excellent photothermal conversion properties including ultrahigh photothermal conversion ef fi ciency of 62.1%and good photostability,enabling them promising for both PT and PT-CT treatments of cancers.

Fig.1.(A)One-pot fabrication of the NIR-absorbing plasmonic PLC-b-PEO@Au NPs;DLS(B)and TEM data(C)for the plasmonic PLC-b-PEO@Au NPs.

Inspired by UV photoreduction of Au3+to generate plasmonic Au NPs with visible light absorption[17],we tested the 365 nm UV irradiation(using a LED lamp)of HAuCl4in the presence of amphiphilic thiols-pendant polypeptide copolymer(PLC-b-PEO)in DMF/H2O solution.When the mixed solution of PLC-b-PEO(5 m L,1 mg/m L)with HAu Cl4(2.5 m L,5 mg/m L,Au/S=1 mol/mol)was irradiated at 60?C and then stirred at room temperature for different times,the resulting solution became pink red and then turned purple-black.This phenomenon indicates the formation of plasmonic Au NPs that were probably tethered and stabilized by amphiphilic PLC-b-PEO[9–12,14–15].The vis-NIR spectra show increasing broad NIR absorption at 700–1100 nm besides a maximal one at about 565 nm(Fig.S1Ain Supporting information).This evidences increasing amounts of plasmonic Au NPs was produced within 12 h UV irradiation in DMF/H2O solution and at 60?C,while black precipitate w ould occur for a longer time of irradiation.After 12 h UV irradiation and continuous stirring for different times at room temperature,the NIRabsorbance ranging at 700–1100 nm gradually increased over the time,suggesting the continuous grow th and/or aggregation of Au NPs in the presence of PLC-b-PEO(Fig.S1B)[12,14–15].As control,weak NIR absorption was observed without the polypeptide copolymer/UV irradiation or with the UV irradiation at room temperature(Figs.S2-S4 in Supporting information).As show n in Figs.1Band C,both dynamic light scattering(DLS)and transmission electron microscopy(TEM)showed that the resultant PLC-b-PEO@Au NPs had an irregular polyhedron morphology with a hydrodynamic diameter(62?3)nm(Fig.S5 in Supporting information),which was slightly bigger than(52?2)nm for the self-assembled PLC-b-PEOmicelles(Fig.S6 in Supporting information).Note that the morphology control of the PLC-b-PEO@Au NPs deserves to be further studied in the ongoing work.As can be discerned by TEM,the PLC-b-PEO@Au NPs have a thin copolymer layer of about 3 nm(also see the follow ing high resolution FE-TEM analysis).Note that the TEM size of the PLC-b-PEO@Au NPs is basically in agreement with that determined by DLS.As the irregular polyhedron morphology and the refractive index difference between Au NPs and the thin polypeptide copolymer layer w ould enhance a localized surface plasmon resonance(LSPR)coupling,which greatly shifted the LSPR peak to the red region that become broader with increasing NPs diameter[10].

Then we optimized the NIR-absorbing property of the PLC-b-PEO@Au NPs by changing reaction time and various molar ratios of Au to S element(Au/S=0.6–1.2,mol/mol).As found by vis-NIR spectroscopy(Fig.2A),the plasmonic PLC-b-PEO@Au NPs that were prepared under optimal ratio of Au:S=1:1 presented the strongest NIR absorption compared to the control.FT-IR spectra showed new vibrational peak at 1050 cm?1for sul fi nic or sulfonic acids,which were produced from the oxidization of thiols on PLC-b-PEO with concurrent gold reduction(Fig.S7 in Supporting information).The XPS of PLC-b-PEO@Au NPs gave the doublet peaks at 162.0 eV(S 2p3/2)and 163.5 eV(S 2p1/2),which were assigned to the coordinate bonding of sulfur-gold (S-Au).Moreover,the peak centered at 166.8 eV was assigned to oxidized sulfur species(Fig.2B).In all,these results persuasively evidenced that the thiols-pendant polypeptide copolymer PLC-b-PEO could reduce HAuCl4species under UVirradiation at 60?Cto produce the NIR-absorbing plasmonic PLC-b-PEO@Au NPs that were tethered by PLC-b-PEO via multivalent Au-S bondings[12,14–15].In addition,the gold weight percentage in PLC-b-PEO@Au NPs was determined to be 90.2 w t%by TGA analysis(Fig.S8 in Supporting information).

Fig.2.(A)Vis-NIRspectra of the PLC-b-PEOmicelles(1 mg/m L)and the plasmonic PLC-b-PEO@Au NPs with various ratiosof Au/S;XPSspectra(B);(C1)FE-TEM photograph of PLC-b-PEO@Au NPs;(C2)Corresponding TEM–EDXpro fi le recorded from one position(blue square)in the NPs;(C3)The magni fied FE-TEM image taken from the blue square;(C4-C7)The tw o-dimensional(2D)element mappings for the detected three elements Au,S,O,and Au+S.

Both FE-TEM and energy-dispersive X-ray spectroscopy(EDX)were further used to investigate the microstructure and elemental composition of the PLC-b-PEO@Au NPs.Fig.2-C1 is a typical FETEM image of a representative sample,which clearly showed irregular polyhedron morphology and the hybrid NPs surface was coated by the PLC-b-PEO copolymer layer.The EDX spectra in Fig.2-C2 con fi rmed that the elements Au,S,C and O were distributed in w hole NPs(note that the detected Cu element originates from the copper grid).The lattice fringes are clearly observed from Fig.2-C3 and the d-spacing value is 0.24 nm and 0.20 nm,corresponding to the{111}and{200}lattice planes,respectively[23].The tw o-dimensional(2D)elemental mapping analyses verify that PLC-b-PEO was homogeneously tethered on the gold nanocrystal surface(Fig.2C4-C7).Taken together,these experimental fi ndings convincingly demonstrated that the thiolspendant polypeptide copolymer PLC-b-PEO acted not only as a reducing agent for the oxidized gold species of HAu Cl4,but as a stabilizer for the reduced gold nanocrystal via forming multivalent S-Au bonds.

As the PLC-b-PEO@Au NPs have intrinsic NIR-absorbing capacity,we irradiated their solution using a continuous-w ave 808 nm laser to test the photothermal effect.As show n in Fig.3A,the solution temperature of the PLC-b-PEO@Au NPs(1 mg/m L)increased sharply over the irradiation time and a maximum of 30.8?Cwas attained upon 5 min laser irradiation at a power intensity of 2 W/cm2,however,the control was elevated only by 2.4?C.Moreover,both laser-on and laser-off cycle curves were repeated three times without any change,demonstrating good photostability.The magnitude of temperature elevation increased with the concentration of PLC-b-PEO@Au NPs,also representing intrinsic NIR-absorbing characteristics(Fig.3B).Similarly the temperature rise can be tuned by the laser power intensity(Fig.S9 in Supporting information).Furthermore,this temperature-elevating process was recorded by a thermoimaging camera,during which the NPs solution temperature doubled from 26.1?C to 52.0?C within initial 3 min irradiation and then slow ly increased to 56.6?C at 5 min(Fig.3C).Notably,the PLC-b-PEO@Au NPs gave higher photothermal conversion ef fi ciency(h)of 62.1%that is largely more than that(13%?22%)of the well-know n gold nanorods and nanoshells[3–8].This is attributed to the irregular polyhedral gold nanocrystals core stabilized by a thin copolymer layer,which enhanced the LSPR coupling and induced a “l(fā)ightning rod”effect upon an NIR laser irradiation [10].Meanwhile,there is a linear relationship between h and the concentration of PLC-b-PEO@Au NPs or with the laser power extinction(Figs.S10 and S11 in Supporting information),which is consistent with the previous report on the hyperthermia effect of the Au NPs[16].Collectively,these experimental results evidence that the PLC-b-PEO@Au NPs can act as an excellent photothermal nanoagent with strong NIR absorption,ultrahigh photothermal conversion ef fi ciency,and good photostability.In addition,because the plasmonic PLC-b-PEO@Au NPs has strong NIR-II absorbance,its NIR-II mediated PTT deserves to be further investigated in the future work.

The hyperthermia effect for killing cancer cells was directly observed by using a double fluorescent staining technique.After the NIR irradiation(5 min,808 nm,2 W/cm2)and incubation for 12 h,the HeLa cellswere co-stained with AOand EBto differentiate live(green)and dead(red)cells,respectively.The HeLa cells vividly kept alive upon with the NIR irradiation or PLC-b-PEO@Au NPs(256 m g/m L);however,they underwent nearly full death upon with same dose of PLC-b-PEO@Au NPs plus the NIR irradiation(Fig.4A).These experiments further veri fied that the hyperthermia effect mediated by PLC-b-PEO@Au NPs plus the NIR irradiation could efficiently kill the cancer cells in vitro,enabling them promising for the PT treatment of cancers[3–8].

Fig.3.(A)The temperature change curves of PLC-b-PEO@Au NPs solution(1 mg/m L)for three on-off laser irradiation cycles(808 nm,2 W/cm2);(B)The concentration dependent heating curves of the PLC-b-PEO@Au NPs solution upon 5 min NIRirradiation;(C)The IRthermal images of the PLC-b-PEO@Au NPs solution(1 mg/m L)upon NIR irradiation at different times.

Fig.4.(A)Fluorescence microscope imagesof HeLa cellstreated with PLC-b-PEO@Au NPs(256 m g/m L)or the NIRirradiation(5 min,808 nm,2 W/cm 2);(B)Cytotoxicity of the PLC-b-PEO@Au NPs incubated with L929 or HeLa cells;(C)Cytotoxicity of the PLC-b-PEO@Au NPs+NIRirradiation or the DOX-PLC-b-PEO@Au NPs or the DOX-PLC-b-PEO@Au NPs+NIR irradiation after incubated with HeLa cells.The data are presented as average?standard error(n=6).

The anticancer drug doxorubicin(DOX)was stirred with the PLC-b-PEO@ Au NPs solution to produce the drug-loaded counterparts(i.e.,DOX-PLC-b-PEO@Au NPs),which gave a high drug-loading capacity of about 27 w t%and a bigger hydrodynamic diameter of(85?3)nm than that((62?3)nm)of the blank ones(Fig.S12 in Supporting information).Ow ing to the complex interactions including hydrogen-bonding,van der Walls,and hydrophobic interactions among DOX,the tethered polypeptide copolymer chains,and the Au NPs surfaces,the loaded DOX was probably absorbed onto the interface layer between the copolymer and Au NPs during the self-assembly process[9–12].

With same dose of the NIR irradiation to the blank ones,the DOX-PLC-b-PEO@Au NPs solution(1 mg/m L)increased to 51.0?C from 25.0?C(Fig.S13 in Supporting information).Taking account of the DOX fraction in the DOX-PLC-b-PEO@Au NPs,the drug hardly had effect on the photothermal conversion ability of the drugloaded nanoparticles.The NIR-triggered cumulative DOX release was greatly enhanced to 44.5 w t%at 12 h compared to 26.0 w t%for non-triggered sample as the photo-converted heat accelerated the DOX diffusion in aqueous solution(Fig.S14 in Supporting information).This result illustrates that the built-in hyperthermia function of the drug-loaded DOX-PLC-b-PEO@Au NPs might improve the DOX-induced chemotherapy ef fi cacy[12].The cytotoxicity of PT,CT,and PT-CT of the PLC-b-PEO@Au NPs and/or the drug-loaded counterparts was evaluated by a standard MTT assay,respectively.As control,the same dose of NIRirradiation and the PLC-b-PEO@Au NPs themselves induced less cytotoxicity with cell viability>90%(Fig.4B).However,the cells viabilities sharply decreased over the drug-loaded NPs concentration(with or without NIRirradiation)and/or the blank ones plus NIRirradiation,and the half-maximal inhibitory concentrations(IC50)of cells could be calculated from Fig.4C.Based on the blank nanoparticles or DOX concentration,an IC50value of 54.78 m g/m Lor 13.53 m g/m L can be calculated for single PT.and single CT,respectively.Based on the concentration of the DOX-loaded PLC-b-PEO@Au NPs,a combined IC50of 27.02 m g/m L was given under PT-CT treatment,which consisted of 7.29 m g/m L for the combination CT and 19.73 m g/m L for the combination PT,respectively.As for the combination therapy,the combination index(CI)is generally used to evaluate the synergistic effect between different treatments,and the CI values of<1,1,and>1 indicate synergism,additive effect,and antagonism,respectively[24].Herein,the DOX-loaded PLC-b-PEO@Au NPs gave a CI of 0.9,demonstrating a synergistic effect of the combination PT-CT on the HeLa cell line.

In summary,we developed one-pot photoreduction strategy to fabricate the NIR-absorbing plasmonic PLC-b-PEO@Au NPs,during which the thiols-pendant polypeptide copolymer PLC-b-PEOacted not only as a reducing agent for HAu Cl4,but as a stabilizer for the reduced gold nanocrystalvia forming multivalent S-Au bonds.The PLC-b-PEO@Au NPs possessed strong NIR absorption at 700–1100 nm,an ultrahigh photothermal conversion ef fi ciency of 62.1%,and good photostability.The PLC-2IF]b-PEO@Au NPs mediated hyperthermia effect could efficiently kill the HeLa cells and the PT-CTtreatment produced a synergistic effect in vitro.Importantly,thiswork establishesa versatile platform for one-pot fabrication of the NIR-absorbing plasmonic Au NPsin a simple and green manner.

Acknow ledgm ents

The National Natural Science Foundation of China(No.21474061)and The Innovation Fund(No.IFPM2016B004)of Shanghai Jiao Tong University&Af fi liated Sixth People's Hospital South Campus are appreciated.

Appendix A.Supplem entary data

Supplementary data associated with thisarticle can be found,in the online version,at https://doi.org/10.1016/j.cclet.2018.02.015.