Nanoscale

PAPER

Cite this: DOI: 10.1039/c8nr08269c

Received 21st November 2018,Accepted 22nd December 2018

DOI: 10.1039/c8nr08269c

rsc.li/nanoscale

Surface-engineered vanadium nitride nanosheetsfor an imaging-guided photothermal/photodynamic platform of cancer treatment†

Chunyu Yang, ‡a Huahai Yu, ‡a Yan Gao, a Wei Guo, b Zizuo Li, c

Yaodong Chen, c Qinmin Pan, a Mingxing Ren, *b Xiaojun Han *a andChongshen Guo *a,b

Of the many strategies for precise tumor treatment, near-infrared (NIR) light-activated “one-for-all” thera-

nostic modality with real-time diagnosis and therapy has attracted extensive attention from researchers.

Herein, a brand-new theranostic nanoplatform was established on versatile vanadium nitride (VN)

nanosheets, which show significant NIR optical absorption, and resultant photothermal effect and reactive

oxygen species activity under NIR excitation, thereby realizing the synergistic action of photothermal/

photodynamic co-therapy. As expected, systematic in vitro and in vivo antitumor evaluations demon-

strated efficient cancer cell killing and solid tumor removal without recurrence. Meanwhile, the surface

modification of VN nanosheets with poly(allylamine hydrochloride) and bovine serum albumin enhanced

the biocompatibility of VN and made it more suitable for in vivo delivery. Moreover, VN has been ascer-

tained as a potential photoacoustic imaging contrast for in vivo tumor depiction. Thus, this work high-

lights the potential of VN nanosheets as a single-component theranostic nanoplatform.

Introduction

Surgery is the most universal treatment for cancer, but its useis limited for diffuse tumors and it suffers from high recur-rence and surgical risk as well.1,2 Chemotherapy and radiother-apy as adjuvant therapies to surgery can inhibit tumor develop-ment in some cases; however, they have disadvantages likeside effects from drugs or short-wave rays and serious post-operative complications.3,4 Therefore, it is urgent to developnovel therapeutic techniques to surmount the above issues.5,6

Phototherapy, including photothermal therapy (PTT) andphotodynamic therapy (PDT), has attracted widespread interestowing to the merits of high selectivity, minimal damage, lowcomplications and deeper penetration with near-infrared (NIR)

light.7–11 For phototherapy, a photothermal agent (PAT) forPTT implementation and a photosensitizer (PS) for the PDTprocesses could be triggered by NIR light to generate on-sitehyperthermia or reactive oxygen species (ROS) to damage solidtumors.12–15 Instead of X-rays in radiotherapy, NIR light of650–1400 nm has been intensively used for phototherapy invirtue of its biosafety and deeper penetration depth,16–18

especially for the second NIR (NIR-II) window of1000–1350 nm, offering deeper tissue penetration than thefirst NIR (NIR-I) window of 650–950 nm.16,19–21

Although great advances have been made in PTT and PDTresearches, single role of PTT or PDT has some unavoidableflaws, typically as oxygen-depletion induced ROS cessation forPDT and the heat-shock effect for PTT.22,23 Combination ofPDT and PTT in one system greatly enhances the overall photo-therapuetic outcome, and previous research realized thisusually by making PAT and PS into a complicated system.24

Nevertheless, drawbacks emerge with the multicomponentsystem, such as systematic complexity, interference andabsorption discordance between PAT and PS.25–27 To overcomethe above problems, the development of multifunctional nano-materials combining both photothermal effect and photo-induced ROS production is urgently needed for cancer treat-ment.28,29 Theranostic agents with the light-activated photo-therapy (PAT and PS) effect and real-time imaging modalityhave currently attracted tremendous attention for clinicalapplications.30–33 Photoacoustic (PA) imaging, which collects

†Electronic supplementary information (ESI) available: The calculation processof photothermal conversion efficiency, zeta potential data, dispersed photo-graphs, plots of temperature change, absorption spectra, ESR spectra, mitochon-drial membrane potential data, the relative body weights of mice, and hemato-logical analyses. See DOI: 10.1039/c8nr08269c‡Equal contribution from Chunyu Yang and Huahai Yu.

aSchool of Chemistry and Chemical Engineering, Harbin Institute of Technology,

Harbin, 150001, China. E-mail: hanxiaojun@hit.edu.cn, chongshenguo@hit.edu.cnbKey Laboratory of Micro-systems and Micro-structures Manufacturing

(Ministry of Education), Academy of Fundamental and Interdisciplinary Sciences,

Harbin Institute of Technology, Harbin, 150080, China. E-mail: renmx@aliyun.comcDepartment of Abdominal Ultrasonography, The First Affiliated Hospital of Harbin

Medical University, Harbin, 150001, China

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the reconstruction information of an ultrasound signal result-ing from thermoelastic expansion under pulse lightirradiation, can provide deep tissue penetration and highspatial resolution.34–38 Meanwhile, PA imaging is used tolocate lesion tissue and monitor the treatment process,improving the diagnostic accuracy and therapeutic effect. It isreported that the PA effect is closely related to the photother-mal property of nanomaterial contrasts.39 Therefore, a “one-for-all” theranostic system could be achieved by combiningdiagnosis and therapy with one species,40,41 avoiding theshortcomings of a multicomponent theranostic system.

In this work, vanadium nitride (VN) nanosheets with strongphotoabsorption in the NIR region were employed as theranos-tic agent for the first time. Poly(allylamine hydrochloride)(PAH) and bovine serum albumin (BSA) were successivelymodified onto the surface of the VN nanosheets via electro-static interaction (abbreviated as VNPs for VN@PAH andVNPBs for VN@PAH@BSA) to improve biocompatibility. Moreattractively, VNPBs nanosheets exhibit photothermal effect,ROS generation and PA imaging feature under NIR excitationto achieve imaging-mediated complementary PTT/PDT syner-gistic phototherapy owing to the outstanding NIR-drivenphotothermal and photosensitive properties (Scheme 1).Resultantly, in vivo phototherapeutic results demonstrated thatVNPBs nanosheets exhibited admirable tumor inhibitionefficiency, which could thoroughly destroy solid tumors andprevent their recurrence.

Results and discussionCharacterization of VNPBs nanosheets

In this study, the surface of negatively charged VN nanosheets(−50.7 ± 4.42 mV, Fig. S1†) was alternately modified with posi-tively charged PAH and negatively charged BSA by electrostaticinteraction for improving the biocompatibility. The zeta poten-tials of the as-prepared VNPs and VNPBs are 26.1 ± 1.19 mVand −16.7 ± 3.22 mV, respectively, confirming the successful

modification of PAH (positive charge) and BSA (negativecharge) onto the VN nanosheets (Fig. S1†). The correspondingtransmission electron microscopy (TEM) images are presentedin Fig. 1a–c. It is clearly seen that the bare VN has an irregular2D sheet-like morphology. It shows a darkened contrast assurface modification proceeded, indicating the successfulpreparation of VNPBs nanosheets. Moreover, the averagehydrodynamic diameter increased from 200.5 ± 7.5 nm for VN,to 264.5 ± 5.3 nm for VNPs, and finally being 317.8 ± 7.8 nmfor VNPBs, as revealed by dynamic light scattering (DLS;Fig. 1d) results. The polydispersity index values correspondingto the DLS results in deionized water are 0.005, 0.005 and0.002, suggesting the excellent monodispersity of VN, VNPs,and VNPBs nanosheets, respectively. Then, X-ray diffraction(XRD) was employed to investigate the crystal nature ofsample. Obviously, all the characteristic Bragg peaks can beindexed to the cubic structure of standard VN (JCPDS no. 73-0528; Fig. 1e) without any impurities. Additionally, X-rayphotoelectron spectroscopy (XPS) was performed to investigatethe chemical composition and valence. It reveals that VNnanosheets are made up of V, N, O, C elements (Fig. 1f). Thepresence of characteristic bands of carbon and oxygen may beattributed to the absorption of carbon dioxide molecules andinevitable slight surface oxidation of VN nanosheets in theatmospheric conditions, respectively. As displayed in the highresolution XPS spectra, spin–orbit doublets at 514.3 eV (V 2p3/2)and 521.8 eV (V 2p1/2) belong to the V3+ ions, while character-istic peaks located at 516.9 eV (V 2p3/2) and 524.4 eV (V 2p1/2)are assigned to the V5+ ions (Fig. 1 g). Additionally, in the N 1sspectra, binding energies at 397.6 eV and 399.6 eV corres-pond to V–N and V–O–N, respectively (Fig. 1h). All of the aboveXPS results are consistent with typical characteristic peaks ofreported VN.42–44 Finally, the surface modification process ofVN nanosheets was monitored stepwise by Fourier transforminfrared (FT-IR) spectra. As presented in Fig. 1i, threeadditional bands emerged after modification with PAH. Thedistinct bands at 3195 and 3413 cm−1 belong to the symmetricand asymmetric stretching vibrations of N–H, while theabsorption band at 1640 cm−1 is assigned to the N–H bendingvibration mode of PAH. Furthermore, VNPBs present one morecharacteristic peak of asymmetric stretching vibration of COO−

after the electrostatic interaction with BSA, thus confirmingthe successful modification of PAH and BSA onto the surfaceof VN nanosheets.

Optical and photothermal properties of VNPBs nanosheets

An optical response in the NIR region and light-activatedphotothermal properties are prerequisites for a phototherapeu-tic agent. We firstly explored the optical properties of VNpowder. As shown in the Fig. 2a, VN powder exhibits a typicalbroad and strong absorption band spanning the visible andNIR regions, including the NIR-II window (1000–1350 nm) ofinterest. Then, the absorbance of VNPBs dispersion in de-ionized water was determined. It was found that the absor-bance value is proportional to the concentration of VNPBs(Fig. 2b). Moreover, the VNPBs nanosheets exhibit remarkable

Scheme 1 Schematic illustration of the synthetic process and theapplication of VNPBs nanosheets as theranostic agents.

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dispersibility in each of deionized water, Dulbecco’s modifiedEagle medium (DMEM), phosphate buffered saline (PBS) andfetal bovine serum (FBS), and no obvious macroscopic aggre-gates are discerned (Fig. S2†). Considering that VNPBs haveoutstanding absorption in the whole of the NIR region, theymay show a prominent photothermal performance. Gradientconcentrations of VNPBs dispersion were exposed to a NIRlaser (1064 nm, 2 W cm−2) for 10 min and the photothermalproperties were monitored by an FL-IR System i7 infraredcamera. As illustrated in Fig. 2c and d, the solution tempera-ture increased as a function of concentration and irradiationtime. With a higher concentration (0.5 mg mL−1), the tempera-ture increased by 27.4 °C after irradiation for 10 min(Fig. S3†). In contrast, a subtle increase of 7.4 °C occurred forthe control (deionized water), indicating that the VNPBsnanosheets could efficiently convert NIR light energy intothermal energy. To further assess the photothermal perform-ance of VNPBs, the photothermal conversion efficiency (η) wasquantified according to a previously established method.45,46

VNPBs nanosheets were continuously irradiated for 10 min,followed by natural cooling to room temperature for 10 min.In this way, the time constant (τs) was calculated to be 91.1 saccording to the cooling curve (inset of Fig. 2e). Thus, the η

Fig. 1 Characterization of VN nanosheets. (a-c) TEM images of VN, VNPs and VNPBs (scale bar = 200 nm) and corresponding DLS results (d). (e)XRD pattern of VN nanosheets. (f ) Full-range XPS survey spectrum of VN nanosheets; (g) V 2p XPS spectra; (h) N 1s XPS spectra. (i) FT-IR spectra ofVN, VNPs and VNPBs.

Fig. 2 Optical and photothermal properties of VN nanosheets. (a)Powder absorption spectrum of VN nanosheets. (b) Optical absorptionspectra of VNPBs aqueous dispersion with various concentrations. (c)Photothermal curves and (d) thermographic images of VNPBs dispersionof different concentrations under NIR laser irradiation. (e) Temperaturechange of VNPBs aqueous dispersion for 10 min laser irradiation, fol-lowed by natural cooling (inset shows the curve of −Ln(θ) versus time).(f ) Temperature curve of VNPBs aqueous dispersion with repeatedirradiation on/off cycles (1064 nm, 2 W cm−2).

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value was determined to be 29.0% (Fig. 2e). Furthermore,VNPBs nanosheets maintained excellent photostability duringeight laser on/off cycles, as shown in Fig. 2f. These findingsconfirm that VNPBs are an excellent photothermal agent.

Detection of ROS generation

Next, we tested the feasibility of VNPBs nanosheets as a photo-sensitizer. The ROS generation mediated by VNPBs nanosheetsin aqueous solution was detected with 1,3-diphenylisobenzo-furan (DPBF), which has a characteristic absorption at 420 nm.DPBF could be decomposed by ROS and results in a photo-absorption loss accordingly. As shown in Fig. S4,† photo-excited VNPBs nanosheets led to a marked absorptiondecrease of DPBF as compared to deionized water, suggestingthat VNPBs nanosheets possess excellent PDT efficiency forROS generation. Further, the intracellular ROS generationin HepG2 cells (human liver cancer cell line) was detectedthrough the ROS-sensitive nonfluorescent probe 2′,7′-dichloro-dihydrofluorescein diacetate (H2DCFDA), which could presentstrong green fluorescence after reacting with ROS to form2′,7′-dichloroflorescein. Herein, this well-known ROS scavengerof sodium azide (NaN3)

47,48 was used to annihilate ROS duringNIR (1064 nm) laser irradiation for comparison. Meanwhile,untreated cells and cells treated with H2O2 (50 mM, 200 μL)were used as negative and positive controls, respectively. Ascan be seen in Fig. 3a–c, the green fluorescence is undetect-able for the untreated cells (negative control group), and1064 nm NIR laser irradiation and VNPBs nanosheets treatedgroups. In sharp contrast, the positive control group (H2O2)and the cells treated with VNPBs nanosheets and 1064 nm NIRlaser irradiation give rise to intense green fluorescence, asshown in the Fig. 3d and e. In Fig. 3f, addition of NaN3 annihi-lated ROS produced by VNPBs as proved by non-fluorescentimage under 1064 nm laser irradiation. These findings indi-cate that VNPBs nanosheets are an outstanding PS for PDT.

In this study, the VNPBs nanosheets for the first time wereemployed as a PS for PDT and the underlying mechanism isunclear so far. However, a possible mechanism of ROS gene-

ration under laser irradiation may be as follows according tosome related works:49,50

V3þ þ hv ! V4þ þ e� ð1Þ

e� þ O2 ! •O2� þHþ ! •OOH ! •OH ð2Þ

•O2� þ •OH ! 1O2 þ OH� ð3Þ

V4þ þ OH� ! V3þ þ •OH ð4ÞThe NIR irradiation could induce free electron production

with a charge variation from V3+ to V4+, which is similar to pre-viously reported WO3−x. Then, the free electrons are capturedby dissolved oxygen to form various ROS via a series of photo-chemical reactions, and then singlet oxygen as stable andtypical ROS is formed. Finally, V4+ would react with OH− andrevert back to V3+, realizing a full catalytic cycle.

Moreover, the photothermal effects could also induce ROSgeneration. Hyperthermia or hot electrons could convert thedissolved oxygen of the solution to ROS (i.e. •OH, •O2

−, H2O2

and 1O2) when the solution temperature is above 37 °C accord-ing to the literature.51,52 Consequentially, the amount of ROSgenerated is proportional to temperature. In view of the out-standing photothermal properties, the VNPBs nanosheets ofthis study may generate desirable amounts of ROS in this wayas well. Meanwhile, the generation of 1O2 was also monitoredby electron spin resonance (ESR) spectrum. We used 2,2,6,6-tetramethylpiperidine (TEMP) as spin trap agent for the detec-tion of 1O2. After irradiation (1064 nm laser, 2 W cm−2) for10 min, the appearance of a typical equal-intensity three-linesignal in the ESR spectrum of VNPBs nanosheets solution canbe attributed to the characteristic of TEMPO adduct (Fig. S5†),suggesting the production of 1O2.

53

In vitro cell cytotoxicity and phototherapeutic treatment

Motivated by the excellent photothermal properties and ROSgeneration ability of VNPBs nanosheets under NIR irradiation,we then investigated the phototherapeutic effect in vitro usingHepG2 cells. Prior to the phototherapy investigation, 3-(4,5-di-methylthialzol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay was employed to assess the potential cytotoxicity ofVNPBs nanosheets. Nearly no cytotoxicity was observed afterthe co-incubation of HepG2 cells with VNPBs nanosheets for24 h (Fig. 4a), verifying the good biocompatibility of VNPBsnanosheets. The in vitro phototherapeutic effects of VNPBs-mediated PTT, PDT and synergetic PTT/PDT were further deter-mined. Untreated cells were used as control, and HepG2 cellswere incubated with 250 µg mL−1 of VNPBs nanosheets for24 h, followed by 1064 nm NIR irradiation for 10 min (2 Wcm−2). Meanwhile, we introduced NaN3 (10 μM, 50 μL) and anice-bath (≈4 °C) to quench ROS for PTT investigation andremove thermal destruction for PDT inspection, respectively.As expected, PTT and PDT groups (77.1% and 65.7% celldeath) show a lower cell killing efficiency as compared with thesynergetic PTT/PDT group (84.4% cell death) under 1064 nmNIR laser irradiation, confirming that both ROS and hyperther-

Fig. 3 Fluorescence microscope images of cells with H2DCFDA stain-ing. (a) Cells without any treatment; (b) cells treated with NIR laserirradiation; (c) cells incubated with VNPBs nanosheets; (d) H2O2 treatedcells; (e) cells treated with VNPBs nanosheets and NIR irradiation; (f )cells treated with VNPBs nanosheets + NaN3 + NIR irradiation (NIR laser:1064 nm, 2 W cm−2, 10 min; scale bar = 200 µm).

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mia produced by VNPBs nanosheets contribute to the cancercell killing (Fig. 4b). Then, the living/dead cell staining assaywas visualized using the green fluorescence of calcein acetoxy-methyl ester (Calcein-AM) and the red fluorescence of propi-dium iodide (PI), respectively. As shown in the fluorescencemicroscope images, HepG2 cells treated with both VNPBsnanosheets and NIR laser irradiation present a remarkable redfluorescence, which is not surprising owing to the generationof ROS and hyperthermia in such cases. Moreover, there is atime-dependent death circle (Fig. 4f–h); that is, the longer theirradiation duration applied, the greater the number of deadcells. In marked contrast, the untreated cells (negative controlgroup), NIR laser irradiation or VNPBs nanosheets treatedgroups exhibit only widespread green fluorescence signals(Fig. 4c–e), indicating negligible cell apoptosis. Takentogether, these results indicate that VNPBs nanosheets are anefficient NIR-triggered phototherapy agent (synergetic PTT/PDT) for cancer treatment. It is well known that mitochondriaplay a key role in inducing cell apoptosis. Thus, we hypoth-esized that cancer cell death is closely related to disruption ofmitochondrial membrane potential (MMP) after phototherapy.The MMP changes were monitored in the presence/absence ofVNPBs nanosheets with/without NIR laser irradiation with5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylimidacarbocyanineiodide (JC-1) indicator, which is a probe to assess MMPchanges. JC-1 shows red fluorescence of aggregates at highmembrane potentials in normal and healthy mitochondria,whereas it forms green fluorescent monomers at low mem-brane potentials after mitochondrial damage. Therefore, theratio of green to red fluorescence reflects the degree of MMPloss. As shown in Fig. S6,† the control (untreated cells) andNIR laser irradiation and VNPBs nanosheets treated groupsdisplay only red fluorescence signals. In sharp contrast, thecells treated with VNPBs nanosheets and 1064 nm NIR laserirradiation exhibit striking green fluorescence signals. These

findings indicate that VNPBs nanosheet-mediated photother-apy caused cell apoptosis through dysfunction of mitochon-dria with MMP loss.

In vivo phototherapeutic investigation

The in vitro synergistic PTT/PDT outcomes of VNPBs nanosheetsmotivated us to investigate their phototherapeutic efficacyin vivo. Phototherapy experiments in vivo were conducted withHepG2-tumor-bearing nude mice as typical model animals.Twenty-five mice were randomly assigned to five groups:“control (group I)”, “mice treated with 1064 nm NIRirradiation (group II)”, “mice treated with VNPBs injection(group III)”, “mice treated with VNPBs + Vc + 1064 nm NIRirradiation (group IV)”, “mice received with VNPBs + 1064 nmNIR irradiation (group V)”. It is well known that vitamin C (Vc)as a ROS scavenger can annihilate ROS in vitro and in vivo.54,55

In this work, we also employed DPBF as a probe to verify thescavenging of ROS by Vc under NIR laser irradiation. As shownin Fig. S4,† photoabsorption loss of DPBF by ROS was sup-pressed obviously with addition of Vc, suggesting that Vc canbe used as a ROS quenching agent. An infrared (IR) thermalcamera was applied to monitor the temperature change oftumor site during 1064 nm NIR laser irradiation (2 W cm−2).The tumor region with VNPBs injection (groups IV and V)exhibited much higher temperature than that free of VNPBsunder NIR irradiation (group II), as illustrated in the photo-thermal images (Fig. 5a). The temperature of tumor siterapidly increased to about 58.8 °C within 10 min of irradiationin the presence of VNPBs, regardless of Vc presence or not. Incomparison, the NIR-treated group displayed a slight tempera-ture change (from 36.9 to 43.8 °C) due to the absence ofVNPBs, as shown in Fig. 5b. After the treatments, the tumorvolume changes and weight fluctuations of mice in groups I–Vwere recorded by a vernier caliper and electronic balance.Simultaneously, photographs of mice in all groups were takenat 0, 3rd, 7th and 14th day post-treatment. As shown in Fig. 5c,compared with groups I–III, the tumor volumes of groups IVand V display lower growth rate, especially for group V, inwhich tumors gradually shrank until almost complete dis-appearance at the 14th day, confirming an effective tumor inhi-bition ability by NIR-triggered PTT/PDT. The antitumorefficiency difference between groups IV and V is due to theabsence of PDT role. Group IV is free of ROS owing to theintroduction of Vc, resulting in PTT effect only in this case.While in group V, the synergetic PDT/PTT functions were rea-lized. Consequently, this confirms that ROS and hyperthermiado play a role in solid tumor suppression to some extent bycomparing the antitumor effect between groups IV andV. Meanwhile, the body weight changes of mice as a functionof time for groups I–V are plotted in Fig. S7.† The relative bodyweight of each group increased slightly in the following twoweeks post-treatment, indicating no significant toxicity effectof both VNPBs delivery and NIR laser irradiation. Moreover,the representative photographs and tumors of mice fromgroups I–V also clearly indicted the ablation of tumor by VNPBs-mediated phototherapy (Fig. 5d). To evaluate in vivo toxicity of

Fig. 4 In vitro cell cytotoxicity assay and phototherapeutic effect ofVNPBs nanosheets toward HepG2 cells. (a) Relative cell viability ofHepG2 cells treated with different concentrations of VNPBs nanosheetsfor 24 h. (b) Relative cell viability for various treatment groups. (c–h)Living/dead cell staining images of HepG2 cells after receiving differenttreatments (scale bars = 500 µm, NIR laser: 1064 nm, 2 W cm−2).

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VNPBs nanosheets, the major organs (heart, liver, spleen, lungand kidney) and the tumors of mice in groups I–V wereextracted at the 14th day. Hematoxylin and eosin (H&E) stain-ing was employed to investigate the histological changes. Asshown in Fig. 5e, the tumor slices of groups IV and V exhibit alarger region dead cells and cell shrinkage, as well as a greatercollapse of the extracellular matrix, as compared with groupsI–III (cell death was scarcely detectable), especially in group V. Inaddition, histological examination for main organs and hemato-logical analyses were also conducted. No obvious pathologicaland blood abnormalities were observed in groups I–V (Fig. 6and Fig. S8†), suggesting that VNPBs nanosheets are a potentialand biosafe phototheraputic agent for tumor treatment.

In vitro and in vivo PA imaging

PA imaging is a kind of bio-imaging modality with high spatialresolution, short scan time and noninvasiveness, which canprovide 3D structural and biological information of targetedtissue. It is reported that the PA effect is closely related to thephotothermal property of endogenous tissue or exogenouscontrast. Inspired by the outstanding in vitro and in vivo photo-thermal property, VNPBs nanosheets were developed as a PAcontrast agent for tumor depiction. We firstly evaluated the PAsignal intensity of VNPBs aqueous dispersions. As shown inthe Fig. 7a and b, strong PA signal intensity and bright PAimages were observed at high concentration of VNPBsnanosheets (1 mg mL−1). On the whole, the PA signal intensityenhancement increased almost linearly with concentration of

VNPBs nanosheets at 880 nm (Fig. 7c), suggesting a satisfac-tory PA contrast potency in vitro. Further, in vivo PA imagingwas conducted on HepG2-tumor-bearing mice. VNPBsnanosheets were injected into the mice intratumorally or intra-venously and then the tumor area was imaged. Fig. 7d showsdistinct PA signal at tumor site after intratumoral injection. Inthis way, PA signal intensity at tumor site was remarkablystronger than that of control (0 h) without VNPBs delivery. As a

Fig. 5 In vivo phototherapy study. (a) Photothermal images and corresponding (b) temperature variation at tumor sites under NIR irradiation withand without VNPBs injection. (c) Variation of relative tumor volume after different treatments during 14 days. (d) Representative photographs of miceand tumors. (e) H&E stained images of tumor slices after different treatments at 14th day (scale bars = 50 µm).

Fig. 6 H&E histological images of major organ slices harvested fromdifferent groups at 14th day (scale bars = 50 µm).

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comparison, delivery of VNPBs intravenously was performed.Accumulation of VNPBs at tumor was maximum at 6 h post-injection attributed to the enhanced permeability and reten-tion effect as presented in Fig. 7e. These results demonstratethat VNPBs nanosheets can serve as an effective and satisfac-tory PA contrast for cancer diagnosis.

Conclusions

In summary, PAH- and BSA-coated VN nanosheets were fabri-cated and used as multimodal theranostic agents for cancerphototherapy and PA imaging for the first time. VNPBsnanosheets as a brand-new photoactive substance gave rise toexcellent hyperthermia and ROS performance under NIR-IIwindow irradiation, resultantly leading to the synergisticphototherapy efficiency of PTT/PDT. The single roles ofhyperthermia and ROS produced by VNPBs have been verifiedto play a role in cancer cell killing, while the combination ofthem contributed to a better antitumor outcome. The in vivostudies confirmed that NIR-mediated VNPBs nanosheets ablateda solid tumor without recurrence within 14 days, while causingno impairment to normal cells or tissues. All in all, VNPBsnanosheets as a novel theranostic nanoplatform displayedpotential in cancer treatment for further clinical application.

ExperimentalMaterials

The VN nanocrystals were obtained from commercial supplier(Shanghai Chaowei Nano Technology Co. Ltd) and stored in a

desiccator. BSA was purchased from Aladdin. PAH (Mw =17 500), H2DCFDA, DPBF, PI, Calcein-AM and MTT were pur-chased from Sigma-Aldrich.

Surface modification process of VN nanocrystals

Prior to the modification, the purchased VN nanocrystals wereheated in NH3 atmosphere at 850 °C for 2 h for full nitridation.Then, the powder was dispersed in water and underwent ultra-sonic dispersion for 20 min. The suspension was centrifugedat 4000 rpm for 5 min to remove bulk particles. The VNPBswere prepared via electrostatic interaction according to our pre-viously published method with minor modifications.51,53 Inbrief, the VN nanocrystals and PAH with a mass ratio of 1 : 4were dissolved in NaCl (0.5 M) aqueous solution and subjectedto ultrasonication for 20 min. Then, the sample was collectedby centrifugation (12 000 rpm, 5 min) and rinsed three timeswith NaCl solution (0.1 M) to obtain VNPs. After that, VNPsand BSA with a mass ratio of 1 : 3 were dissolved in deionizedwater and continuously stirred for 4 h in an ice bath. After cen-trifugation and washing, VNPBs were obtained. Finally, VNPBsnanosheets were re-dispersed into the deionized water forfurther use.

Characterization

The morphology of products was characterized using TEM(JEM-1400). The hydrodynamic size of samples was measuredby DLS (Zeta PALS BI-90 Plus, Brookhaven Instruments). XPS(Thermo Fisher Scientific ESCALAB 250Xi) and XRD (X’PERTPRO MPD) were used for analyzing the chemical valence andcrystal phase of the sample. Optical absorption was recordedby a Hitachi spectrometer (Japan). FT-IR spectra were

Fig. 7 PA properties of VNPBs nanosheets. (a) In vitro PA signal intensity of VNPBs aqueous dispersion with different concentrations. (b) PA imagesof VNPBs aqueous dispersion as a function of concentration. (c) Linear relationship between PA signal intensity and concentration of VNPBsnanosheets. In vivo PA imaging of tumor site for HepG2-tumor-bearing mice before and after (d) intratumoral and (e) intravenous injection of VNPBsnanosheets (2 mg mL−1, 100 μL) at different time points.

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measured using an AVATAR360 spectrometer. The temperaturechange was acquired by an FL-IR System i7 infrared camera. APA computed tomography scanner (MOST invision 128) wasused for monitoring PA signals.

Photothermal evaluation of VNPBs nanosheets

VNPBs suspensions of various concentrations (0, 0.0625,0.125, 0.25, 0.5 mg mL−1) in a 0.5 mL quartz tube were irra-diated by a NIR laser (1064 nm, 2 W cm−2) for 10 min. Thetemperature change was detected every 30 s by an FL-IRSystem i7 infrared camera.

Cell culture

HepG2 human liver cancer cells were incubated in highglucose DMEM (Corning) containing 1% (v/v) antibiotics(penicillin–streptomycin, Corning) and 10% (v/v) FBS (Gibco)at 37 °C under 5% CO2 atmosphere.

Detection of ROS generation

Extracellular ROS generation was detected with DPBF as probe.Briefly, N,N-dimethylformamide (20 μL) solution containingDPBF (1 mg mL−1) was added to 3 mL deionized water orVNPBs suspension, which was irradiated by a NIR laser(1064 nm, 2 W cm−2). After centrifugation of VNPBs suspen-sions, the absorbance of the supernatant was measured by aspectrophotometer. The intracellular ROS generation wasdetermined through the ROS-sensitive probe H2DCFDA.HepG2 cells seeded in a 35 mm quartz cuvette were treatedwith VNPBs solution (250 µg mL−1) for 4 h. Then, the cellswere replaced with fresh medium and washed with PBS. Afterthe treatments, the cells were stained with H2DCFDA (10 mM,50 μL) for 1 h. Meanwhile, the cells were randomly dividedinto six groups for receiving various treatments: group I(untreated cells as negative control); group II (1064 nm laserirradiation for 10 min); group III (incubation with VNPBs);group IV (incubated with 200 μL of 50 mM H2O2 for 1 h aspositive control); group V (VNPBs + 1064 nm laser irradiationfor 10 min); group VI (VNPBs + 500 μL of 10 μM sodium azide+ 1064 nm laser irradiation for 10 min). Finally, the fluo-rescence visualization images were acquired by an OlympusBX53 microscope.

Evaluation of cytotoxicity in vitro

The standard MTT assay was used to evaluate the cytotoxicityof VNPBs in vitro. HepG2 cells were seeded into a 96-well platewith 1 × 104 cells per well. After being cultured for 12 h, thecells were incubated in 200 μL of DMEM containing VNPBs ofdifferent concentrations for 24 h. Then, 20 μL of 5 mg mL−1

MTT solution in PBS was added into each well, followed byincubation for another 4 h. After that, DMEM was replaced by150 μL of dimethyl sulfoxide for 30 min. Finally, the absor-bance was recorded at fixed wavelength of 490 nm by a micro-plate reader (SynergyTM HT, BioTek Instruments Inc., USA).Untreated cells were used as a control. The in vitro photothera-peutic effects of PTT, PDT and synergetic PTT/PDT were esti-mated by the MTT method as well. For synergetic PTT/PDT

group, HepG2 cells were seeded into 96-well plates in the pres-ence of VNPBs (250 µg mL−1) for 24 h and then irradiated infresh medium by a NIR laser (1064 nm, 2 W cm−2) for 10 min.Then, the cell viability was determined by MTT assay. It isworth noting that phototherapy processes are similar for PTTand PDT groups, except for introducing sodium azide (10 μM,50 μL) and an ice bath to remove PDT and PTT effects,respectively.

In vitro phototherapeutic investigation

HepG2 cells were cultured with VNPBs (250 µg mL−1) in a35 mm quartz cuvette at a density of 3 × 105 cells per dish for6 h. After replacing with fresh medium and washing with PBS,the cells were illuminated by a NIR laser (1064 nm, 2 W cm−2)for 2, 6, and 10 min. Untreated cells, cells only irradiated bythe NIR laser or incubated with VNPBs were used as controls.Then, PI and Calcein-AM were used to stain dead and livingcells, respectively. Finally, the fluorescence visualizationimages were observed with an Olympus BX53 microscope.

In vivo phototherapeutic investigation

Female BALB/C nude mice (4–5 weeks old) were purchasedfrom Beijing Vital River Laboratory Animal Technology Co.Ltd. All the animal experiments were implemented accordingto the criteria of the National Regulation of China for Care andUse of Laboratory Animals and approved by the EthicsResearch Committee of the School of Chemistry and ChemicalEngineering of Harbin Institute of Technology. Animal oper-ations were carried out strictly by the standard of the NationalRegulations on the Administration of Laboratory Animals ofthe State Council of the People’s Republic of China (http://www.gov.cn/gongbao/content/2017/content_5219148.htm) andGuidelines for the Care and Use of Laboratory Animals by theHeilongjiang Provincial People’s Congress (http://www.hljkjt.gov.cn/html/ZWGK/ZCFG/heilongjiang/show-18086.html). In vivophototherapy was carried out when the tumor volume reachedabout 150 mm3. The antitumor efficiency was judged by thetumor volume, which was calculated as V = length × width2/2.The HepG2-tumor-bearing mice were randomly divided intofive groups (n = 5 for each group): group I (PBS as control);group II (1064 nm NIR laser irradiation for 10 min); group III(VNPBs injection); group IV (VNPBs + Vc (25 μmol kg−1) injec-tion + 1064 nm NIR laser irradiation for 10 min); group V(VNPBs + 1064 nm NIR laser irradiation for 10 min). GroupsIII–IV were intratumorally injected with 100 µL VNPBs suspen-sions (1 mg mL−1). The mice (in groups II, IV, V) were irra-diated by a 1064 nm laser at a power intensity of 2 W cm−2 for10 min at 2 h post-intratumoral injection of VNPBs (in groupsIV, V), and the temperature change was monitored every 30 sby an FL-IR System i7 infrared camera.

Histology staining

Histology staining of tumors and major organs (includingspleen, heart, liver, kidney, lung) were analyzed at 14 dayspost-phototherapy. The tissues were preserved in 4% parafor-

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maldehyde solution and stained with H&E. Finally, images ofthe tissues were observed by an Olympus BX53 microscope.

Hematological analysis

An automatic blood analyzer (HF-3800) was employed tomeasure blood (20 µL) of mice at 14 days post-treatment.

PA imaging

For PA imaging in vitro, VNPBs were dispersed in deionizedwater with various concentrations (0, 0.125, 0.25, 0.5 and 1 mgmL−1), and then measured with a MOST invision 128 system.As to PA imaging in vivo, HepG2-tumor-bearing mice wereintratumorally or intravenously injected with VNPBs (100 µL,2 mg mL−1) and then scanned at tumor sites with a MOST invi-sion 128 system to obtain in vivo PA images. Then, the PAsignals of tumor sites were reconstructed at different timepoints (0, 1, 3, 6, 12, 24 h).

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (No. 51572059).

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