Functional-Protein-Assisted Fabrication of Fe−Gallic AcidCoordination Polymer Nanonetworks for Localized PhotothermalTherapyYaqiong Wang,† Jing Zhang,† Chuyu Zhang,† Bingjie Li,† Jiaojiao Wang,† Xuejun Zhang,† Dong Li,*,‡

and Shao-Kai Sun*,†

†School of Medical Imaging, Tianjin Medical University, Tianjin 300203, China‡Department of Radiology, Tianjin Medical University General Hospital, Tianjin 300052, China

*S Supporting Information

ABSTRACT: Fe−polyphenols coordination polymers have emergedas a versatile theranostic nanoplatform for biological applicationsowing to the appealing biocompatibility of precursors from nature.Incorporating bioactive molecules with Fe−polyphenols coordina-tion polymers is greatly significant to take full advantages of theirsuperiorities for advanced application. Herein, we show functional-protein-assisted fabrication of Fe−gallic acid (GA) nanonetworks viaa mild and facile biomineralization for photothermal therapy. Mildalkaline condition is crucial to obtain protein−Fe−GA nanonetworkswith intense near-infrared absorption and their unique networkstructure allows reducing the leakage to the surrounding normaltissues, benefiting high photothermal therapeutic efficacy andminimal side effects. The proposed bovine serum albumin−Fe−GA nanonetworks are successfully used to eradicate tumorin vivo. In addition, this universal method can be extended to synthesize other protein-involved nanonetworks, such as humanserum albumin−Fe−GA and ovalbumin−Fe−GA. More importantly, the intrinsic bioactivity of protein can be retained in thenanonetworks, and the ovalbumin−Fe−GA nanonetworks enable inducing the maturation of immune cells, showing thesuccessful fusion of immune activity of ovalbumin into the nanonetworks. The proposed biomineralization strategy shows abright prospect in incorporating various functional proteins, such as enzymes and antibodies, to form protein−Fe−GAnanonetworks with good biocompatibility, favorable photothermal effect, and specific biological function.

KEYWORDS: Polyphenols, Coordination polymer, Biocompatibility, Biomineralization, Immune activation, Photothermal therapy

■ INTRODUCTION

Traditional cancer treatments, such as surgery excision,radiotherapy, and chemotherapy, have been widely appliedfor clinical cancer therapy, but these suffer from the risk of sideeffects such as destroying the immune system and increasingthe incidence of cancer recurrence. Photothermal therapy(PTT) with the merits of high selectivity and minimalinvasiveness is emerging as an alternative method for cancertherapy in recent years. PTT utilizes near-infrared (NIR) light-absorbing agents to convert the NIR light energy into heat forthermal ablation of malignant tumors, and spatially controlledlight illumination and localized accumulation of photothermalagents endow PTT with high selectivity and minimalinvasiveness.1,2 Besides, PTT not only owns the ability todirectly “burn” tumors but also can assist the efficacy ofchemotherapy, radiotherapy, and immunotherapy, effectivelyinhibiting tumor relapse and metastasis.3−5 Up to now, variousphotothermal agents such as carbon nanostructures,6,7 noblemetal nanomaterials,8,9 metal chalcogenides and oxide,10−17

semimetal nanoparticles,18 black-phosphorus nanomateri-als,19,20 photosensitizer-containing polymers and lipo-

some,21−26 organic nanomaterials,27−29 and dyes30,31 havebeen extensively explored for PTT. Despite outstandingtherapeutic efficacy, great concerns regarding the long-termsafety of current photothermal agents seriously hinder theirclinical applications.Coordination polymer nanostructures, also known as

nanoscale metal−organic frameworks, are formed by the self-assembly between metal ions and organic polydentate bridgingligands, and show great potential as a versatile nanoplatformfor diverse biomedical applications due to their structural andchemical diversity and high loading capacity.32−38 Never-theless, the majority of coordination polymers inevitably sufferfrom tedious synthesis process and use of toxic metal elementsand organic ligands.29 The rational choice of metal center andligands with special function is the key to developing high-performance polymers for biological applications. Fe is anessential component of hemoglobin, a substance in red blood

Received: September 13, 2018Revised: November 1, 2018Published: November 22, 2018

Research Article

pubs.acs.org/journal/ascecgCite This: ACS Sustainable Chem. Eng. 2019, 7, 994−1005

© 2018 American Chemical Society 994 DOI: 10.1021/acssuschemeng.8b04656ACS Sustainable Chem. Eng. 2019, 7, 994−1005

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cells that carries oxygen around the body, and plays a crucialrole in extensive biological processes.39 The definite biologicalfunction and biosafety of Fe element make it an excellent metalion candidate for building coordination polymers. In light oforganic ligands, some precursors derived from naturalresources have attracted increasing attention and have beenapplied to fabricate coordination polymers instead of toxicones such as imidazole and terephthalic acid. Gallic acid (GA)is a type of organic tea polyphenol with low-molecular weightderived from plant sources40 and has been extensively used inthe food and medicine industries.41,42 The strong bidentatecoordination interaction between Fe3+ and the two phenolichydroxyl groups in ortho position in GA makes it possible forthe fabrication of Fe−GA coordination polymer. Moreover, theFe−GA complex possesses intense NIR absorption derivedfrom the strong delocalization in the π-electron structure andhas been employed as an excellent PTT agent with favorablebiocompatibility.43 Several hydrophilic polymers, such aspoly(vinyl alcohol) (PVA),44 polyethylene glycol (PEG),45

and poly(vinylpyrrolidone) (PVP)46 have been employed assurface stabilizers for Fe−GA coordination polymers. How-ever, previous surface modifications merely employed inactivemolecules to enhance water solubility, so it is greatly significantbut challenging to incorporate bioactive molecules to take fulladvantages of the superiorities of Fe−GA coordinationpolymer for advanced applications.47 Proteins, such asantibodies, enzymes, structural proteins, signaling proteins,and transport proteins, fulfill many functions as one type ofmost significant biomolecules in biology.48 Proteins are rich infunctional groups such as amino, carboxyl, and thiol groups,thus exhibiting strong coordination capacities with transitionmetal ions like Fe3+. Moreover, the unique spatial structure andmolecular chain flexibility make protein an expansive hollownanoreactor suitable for biomineralization.49 Up until now,plenty of bioactive proteins, such as bovine serum albumin(BSA), human serum albumin (HSA), ovalbumin (OVA),catalase, lactoferrin, transferrin, ferritin, pepsin, trypsin, papain,horseradish peroxidase (HRP), ribonuclease, lysozyme, andinsulin, have been employed for the fabrication of diversenanostructures with fascinating properties for biologicalapplications.50,51 For instance, noble nanoclusters (Pt, Au,Cu),52−55 metal sulfide (PbS, Bi2S3, CuS, FeS, ZnS:Mn),56−58

and metal oxide (Gd2O3, Au/Gd2O3, and MnO2)59−63 have

been synthesized via biomineralization for theranostics ofvarious diseases. Therefore, bioactive proteins as excellenttemplates show great potential in the fusion of diversebiological functions with the unique merits of Fe−GAcoordination polymer.64−68

Herein, we showed functional-protein-assisted fabrication ofFe−GA nanonetworks via a mild and facile biomineralizationway for localized photothermal therapy. Mild alkalinecondition permitted the formation of Fe−GA nanonetworkswith favorable NIR absorbance and impressive photothermalperformance. The BSA−Fe−GA nanonetworks exhibited aunique network structure with an appropriate size of 193.5 nm,facilitating their retention in the tumor site and minimizing theleakage to surrounding normal tissues after localizedadministration. The BSA−Fe−GA nanonetworks with out-standing biocompatibility and excellent photothermal perform-ance were successfully applied in localized photothermaltherapy in vitro and in vivo against tumors. To testify theuniversality of the protein-assisted fabrication strategy ofcoordination polymers, other proteins like HSA and OVAwere also employed to construct Fe−GA coordinationpolymers. Both HSA and OVA enabled the formation ofprotein−Fe−GA coordination polymers with similar networkstructure and excellent photothermal effect to those of BSA−Fe−GA nanonetworks (Scheme 1). Moreover, the proposedOVA−Fe−GA nanonetworks successfully induced the matura-tion of dendritic cells (DC2.4) and macrophage cells(RAW264.7). The immune activity of OVA in OVA−Fe−GA nanonetworks was retained efficiently via the mildsynthesis approach, showing great potential in the develop-ment of multifunctional nanovaccine.69 The proposedbiomineralization strategy shows a bright prospect in theincorporation of functional proteins, such as enzymes andantibodies, with Fe−GA coordination polymers for variousbiological applications.

■ RESULTS AND DISCUSSIONSynthesis and Characterization of BSA−Fe−GA,

OVA−Fe−GA, and HSA−Fe−GA Nanonetworks. BSA−Fe−GA nanonetworks were prepared through a facile and one-step biomineralization approach. Briefly, 60 mg of BSA and 80

Scheme 1. Schematic Illustration of Functional-Protein-Assisted Fabrication of Fe−GA Coordination Polymer Nanonetworksfor Localized Photothermal Therapy

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mg of FeCl3·6H2O were dissolved in 15 mL of water withmagnetically stirring. Then, 1 mL of NaOH solution (1 M)was rapidly added to the mixture liquid with pH changing from3 to 9. GA aqueous solution (4 mL, 10 mg/mL) was thenintroduced into the orange complex mixture, immediatelygiving rise to a dark solution due to rapidly self-cross-linking ofFe3+ and GA along with the existence of protein in water.Alkaline aqueous environment induced loose and unfoldedstructure of protein, contributing a stable network of BSA−Fe−GA coordination polymer.70 Moreover, a mild basiccondition is crucial to obtain BSA−Fe−GA nanonetworkswith maximum NIR absorption, since the slightly high pH canstrengthen the bidentate coordination interaction betweenFe3+ and the two phenolic OH groups in ortho position in GA(Figure S1 and Table S1).43 Besides, mild synthesis conditionalso ensured the minimal impairment to the function ofprotein. The resulting BSA−Fe−GA nanonetworks werepurified by dialysis against deionized water for 24 h andstored at 4 °C for further use. OVA−Fe−GA and HSA−Fe−

GA nanonetworks were also synthesized under optimalsynthetic conditions for BSA−Fe−GA nanonetworks.The composition, size, and morphology of the as-prepared

protein−Fe−GA nanonetworks were characterized by trans-mission electron microscopy (TEM), dynamic light scattering(DLS), Fourier transform-infrared spectroscopy (FT-IR), andinductively coupled plasma-atomic emission spectrometry(ICP-AES). The TEM images indicated BSA−Fe−GAcoordination polymer owned a network structure (Figures 1aand S2a), and analogous morphologies were observed forOVA−Fe−GA and HSA−Fe−GA nanonetworks (FigureS2b,c). The hydrodynamic sizes of BSA−Fe−GA, OVA−Fe−GA, and HSA−Fe−GA nanonetworks in water weredetermined to be 193.5, 233.1, and 196.5 nm, respectively(Figure 1b). Protein−Fe−GA nanonetworks were dispersed indifferent buffer solution (10 mM, pH 4.0, 5.0, 7.0, 7.4, and 8.0)for the zeta potential test. The zeta potential values of BSA−Fe−GA, OVA−Fe−GA, and HSA−Fe−GA nanonetworkswere around −20 to −30 mV in neutral and weak alkalineenvironment due to abundant carboxyl groups in protein and

Figure 1. Characterization of the protein−Fe−GA nanonetworks. (a) TEM image of BSA−Fe−GA nanonetworks. Scale bar: 200 nm. (b)Hydrodynamic sizes of BSA−Fe−GA, OVA−Fe−GA, and HSA−Fe−GA nanonetworks dispersed in pure water. (c) Zeta potential of 0.1 mg/mLBSA−Fe−GA, OVA−Fe−GA, and HSA−Fe−GA nanonetworks dispersed in PBS (pH 7.4, 10 mM). BFG, OFG, and HFG mean the abbreviationof BSA−Fe−GA, OVA−Fe−GA, and HSA−Fe−GA, respectively. (d) FT-IR spectra of BSA, GA, and BSA−Fe−GA nanonetworks. (e) UV−vis−NIR absorption spectra of BSA−Fe−GA nanonetworks solution with different concentrations (0.1, 0.2, 0.3, and 0.4 mg/mL). (f) UV−vis−NIRabsorbance at 808 nm as function of gradient concentrations of BSA−Fe−GA nanonetworks and ultrasmall BSA−Fe−GA.

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GA (Figures 1c and S3). The presence of BSA and GA in theprepared protein−Fe−GA nanonetworks was verified by theircharacteristic bands in FT-IR spectra. −CH2− symmetricalvibrations at 2932 cm−1

, amide I (mainly CO stretchingvibrations) at 1654 cm−1, and amide II (coupling of bendingvibrations of N−H and stretching vibrations of C−N) bands at1534 cm−1 all correspond to BSA in BSA−Fe−GA nanonet-works. The characteristic peak at 1265 cm−1 (C−O stretch)and 1026 cm−1 (O−H in-plane deformation) of GA confirmedthe incorporation of GA in BSA−Fe−GA nanonetworks.Besides, the disappearance of characteristic peak at 3283 cm−1

(O−H stretching) of GA indicated the coordinationinteraction between the phenolic groups and carboxylic groupsin GA and Fe3+ in BSA−Fe−GA nanonetworks (Figure 1d).71

FT-IR spectra of GA, HSA, OVA, OVA−Fe−GA, and HSA−Fe−GA nanonetworks also proved successful incorporation ofHSA and OVA into Fe−GA coordination polymers (FigureS4a,b). BSA−Fe−GA nanonetworks had a strong NIRabsorbance resulting from the strong delocalization in the π-electron structure, benefiting the high photothermal efficacyunder an 808 nm light irradiation.43 Even at a lowconcentration at 0.1 mg/mL, the absorbance of BSA−Fe−GA nanonetworks at 808 nm could reach around 0.3 (Figure1e). The extinction coefficient of the proposed BSA−Fe−GAnanonetworks was 2.902 L·g−1·cm−1, which was 7.5 timeslarger than that of ultrasmall BSA−Fe−GA nanoparticles(0.388 L·g−1·cm−1) at 808 nm wavelength (Figures 1f andS5).67,68,72 The UV−vis−NIR absorption spectra and ex-tinction coefficient of OVA−Fe−GA and HSA−Fe−GAnanonetworks both revealed similar NIR absorbance to thatof BSA−Fe−GA nanonetworks, testifying the universality ofproposed method (Figure S6a−c). The content of Fe in theproposed BSA−Fe−GA, OVA−Fe−GA, and HSA−Fe−GAnanonetworks were determined to be 16.6, 17.7, and 18.8%(w/w), respectively. The high loading of Fe−GA complexensured the intense NIR absorption of the proposed BSA−Fe−GA nanonetworks.Long-Term Colloidal Stability of Protein−Fe−GA

Nanonetworks. The as-prepared BSA−Fe−GA nanonet-works can be easily dissolved in various media includingwater, PBS (10 mM, pH 7.4), normal saline, 1640 culturemedium, DMEM culture medium, and fetal bovine serum(FBS), and no apparent precipitation was observed for 0.1 mg/mL BSA−Fe−GA nanonetworks for 20 days. The hydro-dynamic size of BSA−Fe−GA nanonetworks exhibited noobvious change during 20 days, further confirming their long-term colloidal stability (Figure 2a,b). We further evaluated thecolloidal stability of protein−Fe−GA nanonetworks in buffersolution with different pH values. Owing to these threeproteins isoelectric point at around 4.8, precipitate appeared inthe protein−Fe−GA solution when pH value was 4.0 or 5.0.However, no apparent precipitation and obvious hydro-dynamic diameter change were observed for BSA−Fe−GAand OVA−Fe−GA nanonetworks dispersed in phosphatebuffer solution (10 mM, pH 7.0, 7.4, and 8.0) for 20 days,further indicating their long-term colloidal stability in thesimulated physiological environment (Figure S7a−c). HSA−Fe−GA nanonetworks showed a relatively large hydrodynamicsize and poor colloidal stability, which is probably attributed tothe small chemical structure difference between BSA and HSA(Figure S7d).Cytotoxicity of BSA−Fe−GA Nanonetworks. The

cytotoxicity of BSA−Fe−GA nanonetworks was investigated

by a standard cellular methyl thiazolyl tetrazolium (MTT)assay. Three cell lines (4T1, DC2.4 and RAW264.7) wereincubated with different concentrations of BSA−Fe−GAnanonetworks ranging from 0 to 400 μg/mL. High cellviability (over 70%) was observed after the treatment ofdifferent concentrations of BSA−Fe−GA nanonetworks,demonstrating the low cytotoxicity of BSA−Fe−GA nanonet-works (Figures 2c and S8a).

In Vitro Cellular Uptake Test. To investigate the in vitrocellular distribution of protein−Fe−GA nanonetworks, the Fecontents in cells were determined after different treatments,and the results revealed that in vitro DC2.4 and RAW264.7cells can internalize OVA−Fe−GA and BSA−Fe−GA nano-networks in a concentration and time dependent manner(Figure S8b,c). Such an effective phagocytosis of protein−Fe−GA nanonetworks in vitro ensured their potential for theimmune activation.

Immune Activation of OVA−Fe−GA Nanonetworks inVitro. OVA as model antigen has been widely applied to elicitimmune response.73−75 To verify the function of OVA in thefabricated nanonetworks, we typically investigated the immuneactivation effect of OVA−Fe−GA nanonetworks via costimu-latory molecules expression and cytokines secretion of DC2.4and RAW264.7 cells.76 Before flow cytometry quantificationand ELISA analysis were performed, the cytotoxicity of OVA−Fe−GA nanonetworks toward immune cells was alsoinvestigated by the standard cellular MTT assay. DC2.4 andRAW264.7 cells were incubated with different concentrationsof OVA−Fe−GA nanonetworks ranging from 0 to 400 μg/mL,and the MTT results clearly showed the low cytotoxicity ofOVA−Fe−GA nanonetworks (Figure S8d). The capability ofOVA−Fe−GA nanonetworks to induce DCs and macrophagesmaturation was evaluated by upregulation of costimulatorymolecules CD80 and CD83 on their membrane. CD80 canrecognize and bind to B7 molecules on the T cells, providingthe “second signal” for T cell activation. CD83 can modulate

Figure 2. (a) Monitoring the hydrodynamic size change of BSA−Fe−GA nanonetworks during 20 days. (b) Photos of 0.1 mg/mL BSA−Fe−GA nanonetworks dispersed in different media (from left to right:water, PBS, normal saline, 1640 culture media, DMEM culture media,and FBS) for 20 days. (c) MTT assay of 4T1 cells after incubationwith different concentrations (0 to 400 μg/mL) of BSA−Fe−GAnanonetworks.

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the maturity of DC and macrophage and further activate T andB cells. Taken together, increased expression of CD80 andCD83 molecules could be recognized as important markers forimmune cells maturation.77 The percentages of mature DCsexpressing CD80 increased from 1.37 to 18.4%, and CD83increased from 1.36 to 13.1% after incubation with 200 μg/mLOVA−Fe−GA nanonetworks, respectively (Figures 3a,b andS9). We further carried out the same immune activationexperiments on RAW264.7 cells, and the percentages ofmature RAW264.7 cells with costimulatory molecules ex-pression were determined to be 4.91% (CD80) and 11.7%(CD83) after incubation with 200 μg/mL OVA−Fe−GAnanonetworks (Figures 3c,d and S10). The CD80 and CD83

expression levels induced by OVA−Fe−GA nanonetworks inDC2.4 and RAW264.7 cells were both superior to those re-sulted from pure OVA at the same concentration. ELISAquantification of inflammatory cytokines (IL-6 and TNF-α)secretion of DC2.4 and RAW264.7 cells incubated with pureOVA, OVA−Fe−GA, and LPS for 24 h demonstrated thatOVA−Fe−GA nanonetworks could induce higher levels ofcytokines secretion compared with pure OVA (Figure 3e−h).Such evident immune responses in vitro manifested thesufficient immune activation capability of OVA−Fe−GAnanonetworks, which showed great potential in nanovaccine-based immunotherapy. In addition, BSA−Fe−GA nanonet-works also exhibited a stronger immunoactivation effect than

Figure 3. Immune stimulation effect of OVA−Fe−GA nanonetworks in vitro. Expressions of the costimulatory molecules of CD80 (a) and CD83(b) in DC2.4 and CD80 (c) and CD83 (d) in RAW264.7 cells incubated with OVA, OVA−Fe−GA, and LPS for 24 h. IL-6 (e) and TNF-α (f)inflammatory cytokines secreted by DC2.4, and IL-6 (g) and TNF-α (h) inflammatory cytokines secreted by RAW264.7 cells cocultured withOVA, OVA−Fe−GA, and LPS for 24 h, respectively.

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free BSA, which serves as an exogenous antigen for DC2.4 andRAW264.7 cells (Figures S9−S11).78Photothermal Performance of Protein−Fe−GA Nano-

networks. To evaluate the photothermal performance of thedesigned nanonetworks, pure water and BSA−Fe−GA nano-networks were both irradiated by an 808 nm laser (2 W/cm2)for 10 min. The concentration-dependent temperatureelevation was observed. The 0.4 mg/mL BSA−Fe−GAnanonetworks can raise temperature by 23.6 °C, while purewater only showed a slight temperature change of 4.7 °C(Figure 4a). Besides, BSA−Fe−GA nanonetworks exhibitedexcellent photothermal stability, and maximum temperatureincreased over 20 °C during four LASER ON/OFF cycles withan 808 nm laser irradiation (Figure 4b). The excellentphotothermal heating effect of HSA−Fe−GA and OVA−Fe−GA nanonetworks were also confirmed by the fact ofremarkable temperature increase, and the temperature of 0.4mg/mL HSA−Fe−GA and OVA−Fe−GA nanonetworks canincrease by 22.2 and 27.6 °C, respectively (Figure S12a,b).The infrared thermal photos further intuitively proved theoutstanding photothermal effect of BSA−Fe−GA, HSA−Fe−GA, and OVA−Fe−GA nanonetworks (Figure S12c). Thephotothermal conversion efficiency (η) was measured

according to the precious work.79 The 808 nm laser heatconversion efficiencies (η) of the as-synthesized BSA−Fe−GA,OVA−Fe−GA, and HSA−Fe−GA nanonetworks are furthercalculated to be 20.33, 18.76, and 14.66%, respectively (FigureS13). Therefore, the as-prepared protein−Fe−GA nanonet-works with high photothermal efficiency could serve aseffective photothermal agents to ablate tumors.

In Vitro Photothermal Therapy of BSA−Fe−GANanonetworks toward 4T1 Tumor Cells. The photo-thermal therapy in vitro was assessed using 4T1 cells whichwere incubated with BSA−Fe−GA nanonetworks andirradiated by an 808 nm laser for 10 min. MTT assay revealedthat the cell viability with the treatment of the nanonetworksor laser irradiation alone was similar to the negative controlgroup. However, the cell viability showed an obvious reductionwith the combined treatments of BSA−Fe−GA nanonetworksand laser irradiation. Majority of 4T1 cells were destroyed afterthe incubation with 400 μg/mL BSA−Fe−GA nanonetworksunder an 808 nm laser irradiation at a power density of 2 W/cm2 (Figure 4c). Ultimately, the temperature of 4T1 cells withdifferent treatments after 10 min were measured by an infraredthermometer camera, indicating distinct temperature rise afterPTT (Figure 4d,e). Moreover, live/dead cell dual staining was

Figure 4. Photothermal performance of BSA−Fe−GA nanonetworks in vitro. (a) Photothermal heating curves of pure water and differentconcentrations of BSA−Fe−GA nanonetworks (0.1, 0.2, and 0.4 mg/mL) under an 808 nm laser irradiation (2 W/cm2) for 10 min. (b)Temperature elevation of BSA−Fe−GA nanonetworks over four LASER ON/OFF cycles with an 808 nm laser irradiation at a power density of 2W/cm2 (LASER ON time: 10 min; LASER OFF time: 15 min). (c) 4T1 cell viability after incubation with 0, 200, and 400 μg/mL BSA−Fe−GAnanonetworks under an 808 nm laser irradiation at power densities of 0, 0.5, 1, and 2 W/cm2; n = 3; *, p < 0.05. Infrared thermal photos (d) andterminal temperature (e) of 4T1 cells in a 96-well plate incubated with 0, 200, and 400 μg/mL BSA−Fe−GA nanonetworks under an 808 nm laserirradiation at power densities of 0, 0.5, 1, and 2 W/cm2 for 10 min. (f) 4T1 cells were exposed to 0, 200, and 400 μg/mL BSA−Fe−GAnanonetworks and an 808 nm laser irradiation at power densities of 0, 0.5, 1, and 2 W/cm2 for 10 min, and then live and dead cells were stainedwith calcein AM and PI to be green and red, respectively.

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carried out to intuitively assess the photothermal therapyefficacy. The fluorescence images of cells costained by calcineacetoxymethyl ester (Calcine AM) and propidium iodide (PI),which differentiated the live (shown in green) and dead cells(shown in red), also proved the remarkable photothermaltherapy effect of BSA−Fe−GA nanonetworks (Figure 4f).In Vivo Photothermal Therapy of BSA−Fe−GA Nano-

networks. The BALB/c mice with xenografts 4T1 cells wererandomly divided into 3 groups (n = 3) for differenttreatments, and BSA−Fe−GA nanonetworks or PBS wereintratumorally injected to the mice. The temperature of tumor

surface could increase by 37.2 °C and reach to 65.8 °C afterthe treatment of BSA−Fe−GA nanonetworks plus laserirradiation for 10 min, which was high enough to kill tumorcells. In contrast, the tumor in mice treated with PBS (10 mM,pH 7.4) plus laser irradiation only exhibited a slighttemperature increase of 8.4 °C (Figures 5a and S14). Thedigital photographs of 4T1 tumor-bearing mice at differenttime points (0, 3, 6, 21, and 30 d) intuitively confirmed thecomplete eradication of tumor by BSA−Fe−GA nanonetworksplus irradiation (Figure 5d). The average relative tumorvolume (V/V0) change of different treatments during 30 days

Figure 5. Photothermal therapy effects of BSA−Fe−GA nanonetworks in mice. (a) Infrared thermal images of mice bearing 4T1 tumors afterintratumoral injection of PBS (10 mM, pH 7.4) and BSA−Fe−GA nanonetworks (13.2 mg/mL) under an 808 nm laser irradiation at a powerdensity of 0.5 W cm/2 for 10 min. (b) Average relative tumor volume (V/V0) change after diverse treatments, shown as means ± SD; *, p < 0.05.(c) Ratio of tumors weight to body weight of mice in each group, shown as means ± SD; *, p < 0.05. (d) Digital photographs of 4T1 tumor-bearingmice at different time points (0, 3, 6, 21, and 30 d) after different treatments with PBS + L, BFG alone, and BFG + L. L means laser irradiation (808nm, 0.5 W/cm2, 10 min). BFG: BSA−Fe−GA. TW: tumor weight; BW: body weight.

Figure 6. Toxicity of BSA−Fe−GA nanonetworks in vivo. Representative hematoxylin and eosin (H&E) stained tissue sections of major organsincluding heart, liver, spleen, kidney, and tumor of BALB/c mice after different treatments. Scale bar: 100 μm. L: laser irradiation (808 nm, 0.5 W/cm2, 10 min); BFG: BSA−Fe−GA.

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manifested the extraordinary photothermal tumor ablationeffect of BSA−Fe−GA nanonetworks (Figure 5b). The largesttumors growth occurred in mice treated with BSA−Fe−GAnanonetworks alone, and the mice treated with PBS plus laserirradiation displayed insufficient thermal therapeutic effecttoward tumors. Complete tumors ablation and no moretumors recurrence were observed with the treatment of BSA−Fe−GA nanonetworks plus laser irradiation. The ratio changeof tumor weight to body weight of the mice in each group alsoindicated the remarkable photothermal therapeutic effect ofBSA−Fe−GA nanonetworks (Figure 5c). These results provedthe admirable photothermal therapy efficacy of BSA−Fe−GAnanonetworks in vivo.The BSA−Fe−GA nanonetworks-based therapy did not

cause notable body weight decline, showing their goodbiocompatibility in vivo (Figure S15). To further study thepotential toxicity of the BSA−Fe−GA nanonetworks in vivo,hematoxylin and eosin staining (H&E staining) of differentorgans including heart, liver, spleen, and kidney and tumor ofmice were carried out after diverse treatments (Figure 6). Nomorphological changes, inflammation, cell apoptosis, ornecrosis was observed in the heart, liver, spleen, and kidneyof mice in each group. No tumor tissue was found in micetreated with BSA−Fe−GA nanonetworks plus laser irradiation.On the contrary, the tumor tissue slices of mice treated withPBS plus laser irradiation and BSA−Fe−GA nanonetworksalone both illustrated a large quantity of tumor cells withoutobvious necrosis. These data indicated that BSA−Fe−GAnanonetworks not only possessed a powerful photothermaltherapeutic effect in vivo but also exhibited good biocompat-ibility.

■ CONCLUSIONSIn summary, we reported functional-protein-assisted fabrica-tion of Fe−GA nanonetworks for localized photothermaltherapy. The proposed BSA−Fe−GA nanonetworks not onlypossessed a unique network structure with an appropriate sizearound 200 nm minimizing the leakage to surrounding normaltissues after localized administration but also exhibitedfavorable NIR absorbance and impressive photothermalperformance. Moreover, the proposed BSA−Fe−GA nanonet-works showed excellent biocompatibility due to intrinsicbiosafety of the precursors and were successfully applied inlocalized photothermal therapy in vitro and in vivo. Thisuniversal design can be readily extended to fabricatecoordination polymers using other functional proteins, suchas HSA and OVA. The formed HSA−Fe−GA and OVA−Fe−GA nanonetworks also exhibited excellent photothermal effectdue to their intense NIR absorption. Moreover, the immuneactivity of OVA in OVA−Fe−GA nanonetworks was retainedefficiently due to the mild synthesis condition, and the OVA−Fe−GA nanonetworks can induce the maturation of DC2.4and RAW264.7 cells efficiently, showing great potential in thedevelopment of multifunctional nanovaccine. We believe theproposed functional protein assisted-synthesis strategy laysdown a novel and universal way for the integration offunctional proteins with coordination polymers for advancedapplications.

■ EXPERIMENTAL SECTIONReagents and Materials. The chemical materials were all at least

analysis-grade. Bovine serum albumin (BSA) and human serumalbumin (HSA) were purchased from Beijing Dingguo Biotechnology

Co., Ltd. (Beijing, China). Ovalbumin (OVA) was obtained fromSigma-Aldrich Corporation (Shanghai, China). FeCl3·6H2O, sodiumhydroxide (NaOH), methyl thiazolyl tetrazolium (MTT), and gallicacid (GA) were provided by Aladdin Reagent Co., Ltd. (Shanghai,China). Ultrapure water (Hangzhou Wahaha Group Co., Ltd.) wasused throughout the synthesis procedure. Dimethyl sulfoxide(DMSO) and formaldehyde were purchased from ConcordTechnology (Tianjin, China). Calcein acetoxymethyl ester (CalceinAM) and propidium iodide (PI) were obtained from dojindolaboratories (Shanghai, China). Anti-mouse CD80 FITC-conjugatedand CD83 PE-conjugated antibodies were purchased from eBio-science. Mouse IL-6 and TNF-α ELISA kits were obtained fromMultiSciences (Lianke) Biotech Co., Ltd. (Hangzhou, China).

Characterization. The Fe element content was quantified byinductively coupled plasma-atomic emission spectrometry (ICP-AES,Thermo IRIS Advantage). The size and zeta potential of protein−Fe−GA nanonetworks were determined on a Malvern Zetasizer (Nanoseries ZS, UK). The size and morphology of the preparednanonetworks were characterized by a Hitachi HT7700 transmissionelectron microscopy (TEM) (Hitachi HT7700, Japan). Fouriertransform-infrared (FT-IR) spectra (of 400−4000 cm−1) of thenanonetworks were recorded with pure KBr as the background via aNicolet IR AVATAR-360 spectrometer (Nicolet, USA). The infraredthermal photos were taken by infrared thermometer camera (FLIRE50 seires, USA). The absorption spectra of protein−Fe−GAnanonetworks were determined by a UV-3600 plus spectrophotom-eter (Hitachi, Japan).

Preparation of Protein−Fe−GA Nanonetworks. First, 60 mgof protein (BSA, OVA, or HSA) and 80 mg of FeCl3·6H2O weredispersed in 15 mL of ultrapure water under gentle magnetic stirringfor 3 min, and then 1 mL of NaOH (1 M) was added to adjust pH to9. After stirring for 0.5 h, GA aqueous solution (4 mL, 10 mg/mL)was introduced to the mixture and reacted for another 10 h. Theobtained protein−Fe−GA nanonetworks were purified by dialysis(molecular weight cutoff is 8−14 kDa). Then, the purifiednanonetworks were stored at 4 °C for following use.

Long-Term Colloidal Stability. First, 0.1 mg/mL BSA−Fe−GAnanonetworks were dispersed in ultrapure water, PBS (10 mM, pH7.4), normal saline, Gibco 1640 culture medium, Gibco DMEMculture medium, and fetal bovine serum (FBS), and their photos werethen taken for 20 days. The hydrodynamic sizes of BSA−Fe−GAnanonetworks dissolved in water at different time points were alsorecorded.

To further investigate the influence of pH on the colloidal stabilityof different protein−Fe−GA nanonetworks, 0.1 mg/mL BSA−Fe−GA, OVA−Fe−GA, and HSA−Fe−GA nanonetworks were dispersedin different buffer solution including sodium acetate−acetic acidbuffer solution (10 mM, pH 4.0 and 5.0) and phosphate buffersolution (10 mM, pH 7.0, 7.4, and 8.0), and their photos were thentaken during 20 days. The hydrodynamic sizes of BSA−Fe−GA,OVA−Fe−GA, and HSA−Fe−GA nanonetworks in different buffersolution at different time points were also recorded.

Measurement of the Extinction Coefficient at 808 nm ofProtein−Fe−GA Nanonetworks. The NIR absorbance in protein−Fe−GA nanonetworks with different concentrations were determinedby a UV-3600 plus spectrophotometer. The extinction coefficient at808 nm was then calculated according to the Lambert−Beer law (A/L= αC, where A is the absorption intensity, L is the length of thecuvette, C is the concentration, and α is the extinction coefficient).

Assessment of Photothermal Performance. Different concen-trations (0.1, 0.2, and 0.4 mg/mL) of BSA−Fe−GA nanonetworksand ultrapure water were irradiated with an 808 nm laser irradiation(2 W/cm2) for 10 min. HSA−Fe−GA and OVA−Fe−GA nanonet-works solution were also irradiated with the same parameters. Thetemperature increase of pure water and protein−Fe−GA nanonet-works (0.1, 0.2, 0.4 mg/mL) was recorded by an infraredthermometer camera during the photothermal heating process.Magnetic stirring was necessary to ensure the homogeneity of heatdistribution.

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To calculate the photothermal conversion efficiency, protein−Fe−GA solutions (0.4 mg/mL) were irradiated with an 808 nm laser (2W/cm2) until the solution temperature reached a steady state. Then,the solution naturally cooled down to the ambient temperature afterthe stoppage of laser irradiation. The photothermal conversionefficiency (η) of protein−Fe−GA nanonetworks were calculated asfollow equation:

hs T T Q

I

( )

(1 10 )Amax surr 0η =

− −− − (1)

where h is the heat transfer coefficient and s is the surface area of thecontainer. (Tmax − Tsurr) is the temperature change of the protein−Fe−GA solution at the maximum steady-ambient temperature. Q0represents heat dissipated from light absorbed by the solvent and thecontainer. I is the power density of the laser, and A is the absorbanceof protein−Fe−GA at 808 nm.In Vitro Cytotoxicity Assay. The cytotoxicities of BSA−Fe−GA

and OVA−Fe−GA nanonetworks were evaluated by 4T1 cells andimmune cells (DC2.4 and RAW264.7 cells), respectively. 4T1 cellswere cultured in Gibco DMEM with 10% FBS and 1% streptomycin−penicillin in an atmosphere of 5% CO2 at 37 °C. DC2.4 andRAW264.7 cells were cultured in Gibco DMEM with 10% FBS, 1%streptomycin−penicillin, and 1% L-glutamine in an atmosphere of 5%CO2 at 37 °C. The cells were seeded in a 96-well plate at density of 1× 104 per well. After 24 h, the cells were washed by PBS (10 mM, pH7.4) to remove dead cells and incubated with by fresh mediacontaining BSA−Fe−GA and OVA−Fe−GA nanonetworks withdifferent concentrations for another 24-h incubation. After beingwashed with PBS again, the cells were treated with fresh mediumcontaining MTT (10 μL, 5 mg/mL), and then incubated for 4 h in anatmosphere of 5% CO2 at 37 °C. Then, 120 μL of DMSO wasrespectively added into each well to replace the media and dissolvedthe purple formazan crystals. After 10 min of gentle shaking, theabsorbance of each well at 490 nm was recorded by a microplatereader (Biotek, USA). The cell viabilities were calculated according tothe following formula.

Cell viability OD /OD 100%exp con= × (2)

where ODexp and ODcon are the optical density (OD) for cells treatedwith different concentrations of nanonetworks and cells in controlgroup, respectively.In Vitro Cellular Uptake Test. The cellular uptake of protein−

Fe−GA by DC2.4 and RAW264.7 cells was investigated by ICP-AESmeasurement of Fe content in cells. Briefly, DC2.4 and RAW264.7cells were seeded in 12-well plates at a density of 5 × 105 cells perwell. After 24 h, the cells were washed by PBS (10 mM, pH 7.4) toremove dead cells and incubated with by fresh media containingBSA−Fe−GA and OVA−Fe−GA nanonetworks with differentconcentrations (0, 100, 200, and 400 mg/L) for different incubationtime (8, 12, and 24 h). After that, each group of cells was washedthree times with PBS. The cells in each well were processed with aquaregia for 2 days and then diluted with water to 4 mL for ICP-AES test.In Vitro Activation and Maturation of Immune Cells

Induced by OVA−Fe−GA and BSA−Fe−GA Nanonetworks.Immature immune cells including DC2.4 and RAW264.7 were seededin a 12-well plate at a density of 5 × 105 per well and cultured for 24h. Then, cells were washed with PBS and treated with fresh culturemedia containing OVA, BSA, OVA−Fe−GA, BSA−Fe−GA, and LPSfor another 24 h, respectively. The cell culture supernatants werecollected together and then purified by centrifugation for 3 min at1000 rpm. The obtained supernatants were stored at −80 °C forfollowing analysis. The amounts of pro-inflammatory cytokines IL-6and TNF-α released by cells after different treatments were analyzedby ELISA according to the manufacturer’s directions. Then, cells weresubsequently washed with PBS and stained for 1 h at 4 °C with FITC-conjugated anti-CD80 and PE-conjugated anti-CD83 antibodies.After again washing 3 times with PBS and dispersing with 200 μL ofPBS, these labeled cells were then collected for flow cytometry.

In Vitro Photothermal Therapy of 4T1 Cells with BSA−Fe−GA Nanonetworks. 4T1 cells were seeded in a 96-well plate at adensity of 1 × 104 per well and cultured in an atmosphere of 5% CO2at 37 °C for 24 h. Then, the cells were incubated with differentconcentrations of BSA−Fe−GA nanonetworks (0, 200, and 400 μg/mL) for 1 h, following by an 808 nm laser irradiation at a powerdensity of 0, 0.5, 1, and 2 W/cm2 for 10 min. The infrared thermalphotos of cells with different treatments were recorded by an infraredthermometer camera. The cell viabilities were investigated by astandard MTT assay as aforementioned. Calcein acetoxymethyl ester(Calcein AM) and propidium iodide (PI) were used to stain live anddead cells after different treatments. After incubation for 15 min, eachwell was washed twice with PBS carefully to remove dyes completely.The fluorescence images were obtained on an inverted fluorescencemicroscope.

Animal Model. The tumor models were established by thesubcutaneous planting of 4T1 cells onto the back of the BALB/c mice(15−18 g, HFK Bioscience Co., Ltd., Beijing, China). When thetumor size reached 5−7 mm in diameter, in vivo photothermaltherapy was carried out on the mice.

In Vivo Photothermal Therapy of BSA−Fe−GA Nanonet-works. The tumor-bearing mice were randomly divided into 3 groups(A−C) with 3 mice in each group. The mice in different groups weretreated as follows: A, intratumor injection with 50 μL of PBS (10 mM,pH 7.4) + 808 nm laser irradiation (0.5 W/cm2 for 10 min); B,intratumorally injection with 50 μL of BSA−Fe−GA nanonetworkssolution (13.2 mg/mL); C, intratumor injection with 50 μL of BSA−Fe−GA nanonetworks solution (13.2 mg/mL) + 808 nm laserirradiation (0.5 W/cm2 for 10 min). The photothermal heating effecton 4T1 tumor-bearing mice in groups A and C were recorded by aninfrared thermometer camera during laser irradiation. Body weight,tumor sizes, and digital photos of 4T1 tumor-bearing mice afterdifferent treatments were recorded for 30 days. Heart, liver, spleen,kidney, and tumor were dissected from the sacrificial mice and thenprocessed with 4% formaldehyde on day 30 and disposed with atypical H&E staining procedure to study pathologic changes.

Statistical Analysis. The data are presented as the means ±standard deviation (SD). The comparison between groups wasanalyzed with two-tailed t tests or one-way ANOVA. Differences wereconsidered statistically significant when the p values were less than0.05 (p < 0.05).

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acssusche-meng.8b04656.

Optimization of experimental conditions, UV−vis−NIRabsorbance, TEM, FT-IR, zeta potential, hydrodynamicsize, photothermal heating curves, extinction coefficientsfitting lines, time constants, infrared thermal photos,MTT assay, flow cytometry quantification, ELISAquantification, and average body weight (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: dr_lidong@163.com (D.L.).*E-mail: shaokaisun@tmu.edu.cn (S.-K.S.).ORCIDShao-Kai Sun: 0000-0001-6136-9969NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSY.Q.W. and J.Z. contributed equally to this work. This workwas supported by the financial support from the National

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Natural Science Foundation of China (Grant Nos. 21435001and 21874101) and Natural Science Foundation of TianjinCity (No. 18JCYBJC20800).

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