Energy Pumping by Surface Collectors on Upconversion Nanoparticles for Extended Transfer and Efficient Self-Evaluable Photodynamic Therapy

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Apr 2022Energy Pumping by Surface Collectors on Upconversion Nanoparticles for Extended Transfer and Efficient Self-Evaluable Photodynamic Therapy Xiaobo Zhang†, Weiwei Chen†, Xiaoyu Xie, Yue Zhang, Zhicong Chao, Haibo Ma, Ying Liu and Huangxian Ju Xiaobo Zhang† State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 , Weiwei Chen† State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 , Xiaoyu Xie Institute of Theoretical and Computational Chemistry, Nanjing University, Nanjing 210023 , Yue Zhang State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023 , Zhicong Chao State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 , Haibo Ma Institute of Theoretical and Computational Chemistry, Nanjing University, Nanjing 210023 , Ying Liu *Corresponding author: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 Chemistry and Biomedicine Innovation Center, Nanjing University, Nanjing 210023 and Huangxian Ju *Corresponding author: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023 https://doi.org/10.31635/ccschem.021.202100951 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Although upconversion nanoparticle (UCNP)-based luminescence resonance energy transfer (LRET) has attracted intensive attention, its application in sensing strategies for biomacromolecule detection is still limited due to the low energy-transfer efficiency. Here, we develop an energy pumping strategy to enhance the upconversion LRET efficiency of UCNP from 3.6% to 70.3%. Surface collector dye cyanine-3 (Cy3) is modified on the UCNP surface to concentrate the scattered emission energy from interior UCNP to particle surface and then further pumped to the surface extended acceptor. Using caspase-3 as a target, its recognition and cleavage of QSY7-labeled peptide co-assembled on UCNP-Cy3 leads to the release of QSY7 to recover Cy3 fluorescence. Owing to the multiple emissions of UCNP, this system can be integrated with another surface collector to enhance reactive oxygen species (ROS) generation for photodynamic therapy (PDT). Caspase-3, as a PDT product, is imaged in situ with an internal standard to self-evaluate the PDT effect. Download figure Download PowerPoint Introduction Recently, the multiplex upconversion emissions and relatively low background have made upconversion nanoparticles (UCNPs) a popular nanomaterial for designing bioprobes in bioanalysis and cancer therapy.1–6 Taking UCNPs as energy donors and the dye attached on its surface as an energy acceptor, different “off–on” upconversion bioprobes have been designed for sensing of small molecules such as metal ions,7,8 adenosine 5′-triphosphate (ATP),9 free radicals,10,11 pH,12,13 sulfur compounds,14 and drug metabolites.15,16 In these sensing strategies, the energy acceptors act as the target recognition domain, and are needed to be maintained as close as possible to the emitter inside UCNPs to obtain satisfactory luminescence resonance energy transfer (LRET) efficiency. However, the size of the UCNPs, often larger than 20 nm in diameter, limits the approach of the energy acceptors to randomly distributed emitters inside the UCNP, which correspondingly reduces the LRET efficiency from the emitters to surface extended acceptors. Thus, the bioprobes prepared with UCNP and large molecule recognition domains, such as DNA strands or peptides, exhibit low LRET efficiency due to the long outward transmission path.17–19 To improve the LRET efficiency of UCNP, some nanomaterials with strong energy absorption capacity, such as gold20 and carbon nanoparticles,21 have been utilized as acceptors of UCNP emission energy to construct the “off–on” bioprobes. Unfortunately, the broad absorption spectra of these acceptors seriously influence the multiple UCNP emissions, and their nanometer size increase the energy transmission path and steric hindrance for target recognition, which greatly limits the full utilization of UCNP multiemissions. Therefore, our previous study22 developed an energy concentration zone strategy with multishell-structured UCNP to confine the emitter ions Er3+ in a 2.7-nm thin-layer proximity to the UCNP surface, which achieved 3600-fold enhancement of upconversion emission intensity and also boosted the energy-transfer efficiency to tightly attached surface acceptors. Theoretically, the upconversion energy concentration via UCNP structure manipulation is still incapable of transferring energy to surface stretched acceptors spaced 4 nm away from UCNP interior emitters, because the Förster distance between rare earth emitters as donors and dye acceptors is reduced to below 4 nm.23–26 Therefore, a strategy promoting the outward energy-transfer efficiency is needed to extend the multiplex functionalities of UCNPs in biosensing, bioimaging, and biomedicine. Instead of the structure confinement, herein we design an energy collecting system to pump the scattered upconversion emission energy to remote functional energy acceptors. The energy pumping strategy is achieved by immobilizing surface collectors on UCNPs to concentrate the scattered upconversion emissions at different wavelengths, respectively, and subsequently transfers to their corresponding acceptors extended from the particle surface (Figure 1). The strategy of upconversion energy concentration and continuous transfer can solve the bottleneck that limits the application of UCNPs in biomacromolecular detection. As a proof-of-concept, small molecule dye cyanine-3 (Cy3), whose absorptions and emission wavelengths match with the UCNP emission at 540 nm and QSY7 absorption, respectively, was chosen as the surface collector. Cy3 was immobilized on the NaYF4:Gd,Yb,[email protected]4 UCNP surface, and QSY7 was labeled on the terminus of peptide KADEVDAC that immobilized on the UCNP as a surface extended acceptor to demonstrate the energy pumping and long-distance transmission for biosensing of biomacromolecule caspase-3. The efficient energy collection of 540 nm emission from dispersed Er3+ in UCNP, in accordance with subsequent transmission to QSY7 located at extended distance from UCNP surface, result in the complete quenching of Cy3 luminance to perform the “off–on” detection of target caspase-3. In addition, the operation of collector Cy3 does not influence the emission of Er3+ located in the center of UCNP, which is beyond the effective collection distance. This leads to a steady emission at 540 nm regardless of target concentration change, thus providing an internal standard for target quantification. Figure 1 | Schematic illustration of energy pumping from Er3+ emitters in UCNP to surface extended energy acceptors with surface co-immobilized collectors Cy3 and Ppa for performing “off–on”-typed biosensing and self-evaluable highly efficient PDT. Download figure Download PowerPoint Photosensitizer pyropheophorbide-a (Ppa) is co-immobilized on UCNP to act as another surface collector. Another scattered UCNP emission at 654 nm from dispersed Er3+ in UCNP is collected by Ppa27 with matching absorption and further pumped to dissolved oxygen as the energy acceptor to generate reactive oxygen species (ROS) to perform enhanced photodynamic therapy (PDT). Taking advantage of the multiemissions of UCNP with dual surface collectors, PDT with in situ, efficient self-evaluation of therapeutic effects is achieved by detecting the PDT product, caspase-3. The energy pumping strategy with extended transfer distance can broaden the application of UCNPs in precision medicine. Experimental Methods Materials and apparatus Yttrium chloride (YCl3) (99.9%), ytterbium chloride (YbCl3) (99.9%), erbium chloride (ErCl3) (99.9%), gadolinium chloride (GdCl3) (99.9%), oleic acid (OA), 1-octadecene, matrix metalloproteinase-2 (MMP-2), cathepsin-B, and Ppa were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Sodium hydroxide (NaOH), ammonium fluoride (NH4F), cyclohexene, alendronic acid (ADA), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), 1,3-diphenylisobenzofuran (DPBF), dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), trichloromethane (CHCl3), and methanol (MeOH) were purchased from Aladin Co. Ltd. (Shanghai, China). 4-Dimethylaminopyridine (DMAP) was purchased from Adamas Reagent Co. Ltd. (Shanghai, China). Cy-3 NHS ester (NHS-Cy3) and Cy-3.5 C5-maleimide (Mal-Cy3.5) were purchased from Fanbo Biochemicals Co. Ltd. (Beijing, China). QSY-7 C5-maleimide (Mal-QSY7) was purchased from Invitrogen (USA). NHS-PEG4, NHS-PEG4-NHS, and NH2-PEG-FA (MW 2000, PEG-FA; PEG = polyethylene glycol, FA = folic acid) were purchased from ToYong Bio (Shanghai, China). Human cervix carcinoma (HeLa) cell lines, Annexin V-FITC/PI apoptosis detection kit (FITC = Fluorescein Isothiocyanate, PI = Propidium Iodide), apoptosis detection kit, trypsin, dihydrorhodamine (DHR), and 3-(4,5-dimethylthiazol-2-yl)-2-diphenyltetrazolium bromide (MTT) were purchased from KeyGen Biotech. Co. Ltd. (Nanjing, China). Human caspase-3 and caspase-9 recombinant proteins along with assay buffer were purchased from EMD Millipore (Billerica, MA). Peptide KADEVDAC was obtained from GL Biochem Co. Ltd. (Shanghai, China). Phosphate-buffered saline (PBS; pH 7.4) contained 136.7 mM NaCl, 2.7 mM KCl, 8.72 mM Na2HPO4, and 1.41 mM KH2PO4. All other chemical reagents were of analytical grade and used without further purification. DNAs oligonucleotides were synthesized and purified by Sangon Biotech Co. Ltd. (Shanghai, China) with sequences listed as follows: Capture DNA: 5’-COOH-TATCTTGTATTTAAGGTTTA-3’ [email protected]: 5’-TAAACCTTAAATACAAGATA-Cy3-3’ [email protected]: 5’-TAAACCTTAAATACA/iCy3/AGATA-3’ [email protected]: 5’-TAAACCTTAA/iCy3/ATACAAGATA-3’ [email protected]: 5’-TAAAC/iCy3/CTTAAATACAAGATA-3’ [email protected]: 5’-Cy3-TAAACCTTAAATACAAGATA-3’ Transmission electron microscopy (TEM) images were obtained on JEM-2800 transmission electron microscope (JEOL, Tokyo, Japan). Dynamic light scattering (DLS) was conducted on ZetaPlus 90 Plus/BI-MAS (Brook haven, Holtsville, USA). Zeta potential analysis was conducted on Nano-Z Zetasizer (Malvern, Malvern, UK). Absorption spectra were recorded on Maya 2000 Pro spectrophotometer (Ocean Optics, Shanghai, China). Flow cytometric analysis was conducted on Coulter FC-500 flow cytometer (Beckman-Coulter, Brea, CA). The fluorescence spectra were acquired on FluoroMax-4 spectrofluorophotometer (HITACHI, Tokyo, Japan) with an external continuous-wave laser (980 nm) as the excitation source. Fluorescence lifetimes were detected with a F980 spectrophotometer (Edinburgh Instruments, Livingston, UK). Cell images were obtained on TCS SP5 confocal laser scanning microscope (CLSM) (Leica, Wetzlar, Germany). MTT assays were conducted on Hitachi/Roche System Cobas 6000 (Bio-Rad, Hercules, CA). Synthesis of NaYF4:Gd,Yb,[email protected]4 (UCNP) Two mmol of Ln(OA)3 (Y:Gd:Yb:Er = 0.68:0.10:0.20:0.02) was initially added to the mixture of OA and 1-octadecene (40 mL, v:v = 1:3), degassed in vacuum at 150 °C for 1 h, and cooled to 45 °C. Ten mL MeOH solution of NH4F (8.0 mmol) and NaOH (5 mmol) was then dropwise added in the obtained solution in 30 min under stirring. The reaction mixture was heated to 110 °C to completely remove MeOH and continued to react at 300 °C for 90 min under nitrogen atmosphere to obtain NaYF4:Gd,Yb,Er (UCNP core). The outer layer precursor was prepared by adding 0.3 mmol of Y(OA)3 in the mixture of OA and 1-octadecene (8 mL, v:v = 1:3) and degassed in vacuum at 150 °C for 1 h. After cooling to 45 °C, 10 mL MeOH solution of NH4F (1.2 mmol) and NaOH (0.75 mmol) was added dropwise into the obtained solution in 30 min under stirring. After heating to 110 °C to completely remove MeOH, the obtained outer layer precursor was injected into the above prepared UCNP core and stirred for 30 min to generate NaYF4:Gd:Yb,[email protected]4 (UCNP). The reaction mixture was then cooled to room temperature, precipitated with acetone, repeatedly washed with cyclohexane, and re-dispersed in 20 mL CHCl3 or cyclohexane for further use. Synthesis of UCNP-NH2 To functionalize UCNP with amine groups, UCNP (200 mg) was mixed with ADA (50 mg), CHCl3 (10 mL), EtOH (4 mL), and water (6 mL), and the solution pH was adjusted to 2–3 with HCl (1 M) to react for 30 min under stirring (880 rpm). The upper liquid was transferred to a new centrifuge tube, washed with water to obtain UCNP-NH2, which was then stored in aqueous solution. Synthesis of UCNP-Cy3/peptide-QSY7/Ppa/FA To synthesize photosensitizer Ppa and FA-conjugated UCNP-Cy3/peptide-QSY7 as self-evaluation upconversion nanoprobe (SEUCNP), the carboxyl group of Ppa (1 mM in 1 mL DMF) was initially activated with 0.1 mg/mL DCC and 0.1 mg/mL NHS in 0.1 mL DMF. After 2.5 mL of 2.0 mg/mL UCNP-NH2 in DMF was mixed with 1.5 mM NHS-Cy3 in 0.1 mL DMF containing 0.1 mg/mL DMAP, 0.1 mL of activated Ppa in DMF was added in the mixture to stir for 6 h at room temperature, which was centrifuged and washed with DMF to get UCNP-Cy3/Ppa. 2.5 mL of 2.0 mg/mL UCNP-Cy3/Ppa in DMF was then mixed with 0.1 mL of 0.1 M NHS-PEG4-NHS in DMF containing 0.1 mg/mL DMAP to react for 6 h under stirring at room temperature to get UCNP-Cy3/Ppa/NHS after centrifugation and washed with DMF. 2 mg UCNP-Cy3/Ppa/NHS in 2.5 mL DMF was finally mixed with the mixture of 0.2 mL peptide-QSY7 and 0.1 mL 0.15 mM NH2-PEG-FA in DMF to react under stirring for 6 h at room temperature to get SEUCNP after centrifugation and washed with DMF. In vitro measurements of caspase-3 activity and fluorescence lifetime After UCNP-Cy3/peptide-QSY7 (10 μL 5 mg/mL) was incubated with caspase-3 recombinant protein in 90 μL assay buffer at 37 °C for 100 min, 100 μL DMSO was added to stop the reaction, and the fluorescence spectrum was collected from 530 to 610 nm with excitation at 980 nm (10 W/cm2). The fluorescence lifetimes of UCNP, UCNP-Cy3, UCNP-peptide-Cy3.5, and [email protected] to 20 were measured at 540 nm to study the resonance energy-transfer efficiency of UCNP with an excitation wavelength of 980 nm. The fluorescence spectra for caspase activity detection were acquired on FluoroMax-4 spectrofluorophotometer (HITACHI) with an external continuous-wave laser (980 nm). CLSM imaging After 1 × 104 HeLa cells were seeded in a confocal dish and incubated in culture medium for 24 h at 37 °C, the medium was replaced with 200 μL fresh culture medium containing 400 μg/mL SEUCNP to incubate at 37 °C for 6 h. Afterward, the cells were washed twice with PBS, and CLSM imaging was performed at an excitation wavelength of 980 nm to collect the fluorescence signals from 560 to 620 nm for caspase detection, and from 500 to 550 nm as internal standard. All images were digitized and analyzed with the Leica Application Suite Advanced Fluorescence (LAS-AF) software package. Detection of ROS 0.8 mg/mL SEUCNP (100 μL) was mixed with 4 mM DPBF (100 μL) to irradiate with 980 nm light-emitting diode (LED) light at 2.0 W/cm2, during which the absorbance of DPBF was measured at 412 nm every 5 min to monitor the formation of ROS. To monitor the generation of intracellular ROS, HeLa cells were initially incubated with SEUCNP for 6 h and 50 mM DHR for another 30 min at 37 °C, followed with exposure under 980 nm LED light at 2.0 W/cm2 for 30 min with 5-min break at 15 min. CLSM fluorescence images were then collected from 510 to 550 nm with an excitation wavelength of 488 nm. PDT All experiments were performed according to the Institutional Animal Use and Care Regulations approved by Nanjing University of Chinese Medicine (202104A027). After HeLa cells were seeded into 96-well plates (1 × 104 cells/chamber) and incubated in 200 μL culture medium for 24 h at 37 °C, the cells were washed with PBS and incubated with 400 μg/mL SEUCNP for 6 h. Upon washing with PBS twice, the cells were exposed under 980-nm LED light with a power density of 2.0 W/cm2 for different times with 5-min breaks after each 15-min irradiation to perform MTT assay and self-evaluation of the therapeutic effect with CLSM imaging. Results and Discussion Distance-dependent upconversion energy-transfer efficiency NaYF4:Gd,Yb,[email protected]4 UCNP was synthesized according to the previous report.23,28 Gd was doped in a UCNP core NaYF4:Gd,Yb,Er to control size at around 20.0 nm ( Supporting Information Figure S1a) and guarantee the subsequent uniform coating of NaYF4 shell.23 The coating of NaYF4 shell increased the size of NaYF4:Gd,Yb,[email protected]4 to 21.4 nm (Figure 2a). It could avoid the surface quenching of upconversion emission by water molecules28 and greatly enhanced the emission of UCNP at 520, 540, and 654 nm under 980 nm excitation ( Supporting Information Figure S1b). The as-obtained UCNP was then functionalized with ADA, and the obtained UCNP-NH2 showed slightly increased hydrodynamic diameter from 24.4 to 25.5 nm, while the zeta potential increased from 25.6 to 43.3 mV ( Supporting Information Figure S1c). Ninhydrin, which reacts with free amino groups of ADA to produce blue-purple compounds, was used to titrate the stoichiometry of ADA on UCNP surface ( Supporting Information Figure S2) and resulted in 2376 ± 243 ADA per nanoparticle. Figure 2 | (a) TEM image of NaYF4:Gd,Yb,[email protected]4. (b) Schematic illustration of UCNP-Cy3 LRET pairs with donor–acceptor distances from 1 to 20 bp. (c) Corresponding luminescence spectra compared with 0.25 mg/mL UCNP-DNA under 980-nm excitation (10 W/cm2). (d) Theoretical counts of photons emitted at different distances to the UCNP center. (e) Theoretical Cy3-accepting percentages of photons emitted at different distances to the UCNP center. Download figure Download PowerPoint Considering the wide absorption overlapped with the emission of UCNP at both 520 and 540 nm, Cy3 was chosen as energy acceptor to demonstrate the dependence of upconversion outward energy-transfer efficiency on distance ( Supporting Information Figure S3a). A series of duplex DNA strands with Cy3 labeled at different positions was conjugated to UCNP-NH2, and the distances of Cy3 to UCNP surface were arranged from 1 to 20 bp (Figure 2b). Only [email protected] with a donor–acceptor distance of 1 bp demonstrated strong Cy3 fluorescence at 580 nm, which suppressed the upconversion emission of UCNP at 520 and 540 to a certain extent (Figure 2c). The Cy3 fluorescence sharply decreased with extended distance, indicating the quick decrease of energy outward transfer efficiency. Using the fluorescence lifetimes of [email protected] at 540 nm (89, 173, 196, 210, and 215 μs respectively) ( Supporting Information Figure S3b), the energy outward transfer efficiency η for upconversion emission was estimated according to eq. 1: η = 1 − τ complex τ donor (1)where τdonor and τcomplex are the lifetimes of UCNP in the absence and presence of Cy3. The [email protected] showed the maximum energy-transfer efficiency of 60.4%, while the efficiency of [email protected] quickly decreased to 21.0%. The outward transmission efficiency for rare earth emitters substantially decreased with increasing distance away from the UCNP surface. Since each Er3+ contributes to the emission of UCNP NaYF4:Gd,Yb,[email protected]4, the emission energy accepted by acceptor from different donor ions located inside UCNP is spatially Thus, was to the in structure of UCNP ( Supporting Information Figure and generate the counts emitted at different distances to UCNP center (Figure The percentages of these photons by that the energy-transfer efficiency at different distances were followed Figure and η = 6 6 6 is the distance between UCNP interior emitter Er3+ and Cy3 labeled on DNA and the Förster distance of donor–acceptor which is to be nm with the fluorescence of [email close to [email protected] demonstrated the energy-transfer efficiency of while the energy-transfer efficiency for [email protected] was below and [email protected] and showed with the of fluorescence intensity and lifetime (Figure and Supporting Information Figure It is that the emitters Er3+ located in the center of UCNP were away from the surface collector Cy3 for effective collection of upconversion energy and thus the Cy3 showed a steady emission at 540 nm an internal standard for Upconversion energy pumping with surface collector Cy3 to extended distance Cy3 attached on the UCNP surface showed an energy-transfer efficiency (Figure The concentration of Cy3 was and the Cy3 emission intensity was achieved with Cy3 concentration of 30 which to ± 15 Cy3 per nanoparticle ( Supporting Information Figure to the as energy acceptor, the attached Cy3 could act as a surface collector to scattered upconversion emission from Er3+ dispersed in UCNP (Figure which to the increase of the Cy3 emission at 580 nm and the decrease of UCNP emission at 540 nm (Figure Figure | (a) Schematic illustration of long-distance LRET and energy pumping for biosensing of caspase-3. Upconversion emission spectra of UCNP and in presence and absence of surface collector Cy3 UCNP, UCNP-Cy3, and UCNP-Cy3/peptide-QSY7 (c) and UCNP-Cy3/peptide-QSY7 in to caspase-3 from to 6 and caspase-3 (e) of in (d) caspase-3 in caspase-3. All experiments were performed under 980-nm excitation (10 with 0.25 mg/mL Download figure Download PowerPoint To demonstrate that the energy collected by surface collector Cy3 could be to extended distance, peptide KADEVDAC was co-immobilized on UCNP-Cy3, which ( Supporting Information Figure with increasing size from to nm and zeta potential from to mV ( Supporting Information Figure due to the presence of the peptide The absorption of at 580 nm overlapped with Cy3 thus was used as the extended acceptor to generate an emission at 610 nm ( Supporting Information and while the showed greatly decreased Cy3 which was from both UCNP and under 980 nm excitation (Figure the long-distance energy transfer from the emitter Er3+ dispersed in UCNP to peptide terminus which demonstrated the efficient upconversion energy pumping of collector Cy3. The extended energy transmission was also by the luminance lifetime of UCNP at 540 nm, which decreased from μs for UCNP to μs for UCNP-peptide-Cy3.5, and μs for ( Supporting Information Figure The lifetime the of surface collector Cy3 to extended energy which increased transfer efficiency from 3.6% to 70.3%. It was that collector Cy3 not the UCNP emission at 654 nm due to the absorption as by the luminance lifetime around μs for UCNP, UCNP-peptide-Cy3.5, and ( Supporting Information Figure detection of biomacromolecule with extended energy transmission To “off–on”-typed biosensing the designed pumping the peptide of was labeled with QSY7 for of nanoprobe UCNP-Cy3/peptide-QSY7 (Figure QSY7 showed a wide absorption around 580 nm ( Supporting Information Figure which Cy3 of QSY7 were and complete of Cy3 luminance was achieved with QSY7 ( Supporting Information Figure indicating the efficient energy transfer from collector Cy3 to surface extended The surface of QSY7 was by its absorbance at nm and resulted in 104 ± 4 QSY7 per nanoparticle ( Supporting Information Figure Upon of caspase-3 to the of the KADEVDAC was to release QSY7 from UCNP surface, which the extended energy transfer from Cy3 to and thus Cy3 luminance at 580 nm with the increasing caspase-3 concentration (Figure the a steady emission at 540 nm from Er3+ located in the center of UCNP was (Figure regardless of the of caspase-3 concentration (Figure Therefore, the emission of nanoprobe at 540 nm could be used as an internal standard for the of caspase-3 with a luminance intensity of Cy3 emission at 580 nm to UCNP emission at 540 nm As in Figure the of caspase-3 concentration a from to 6 with a in to 2 which a detection of at a of 10 ( Supporting Information Figure In with the of caspase-3 at 2 the nanoprobe showed to such as cathepsin-B, and ( Supporting Information and indicating of the detection PDT with extended energy transmission Owing to the absorption of both Cy3 and QSY7 at 654 nm ( Supporting Information and the emission of the designed nanoprobe at 654 nm could be further used for PDT by another energy collector photosensitizer Ppa on UCNP surface for ROS was also conjugated to the UCNP surface for cancer cells to complete a SEUCNP (Figure and Supporting Information Figure with UCNP-NH2 and the obtained SEUCNP showed larger size and zeta potential due to the presence of peptide and PEG (Figure The surface of Ppa was and as ± 4 Ppa per nanoparticle ( Supporting Information Figure The MTT assay demonstrated low of SEUCNP and under irradiation ( Supporting Information Figure further the of SEUCNP ( Supporting Information Figure In addition, SEUCNP demonstrated hydrodynamic diameter with of Cy3 fluorescence in indicating ( Supporting Information Figure The ROS generation of SEUCNP irradiation at 980 nm was also ROS sensing which demonstrated decrease of the absorbance at 412 nm min due to the by ROS (Figure The generation efficiency was than previous due to the efficient energy pumping of 654 nm

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