D‐type neuropeptide decorated AIEgen/RENP hybrid nanoprobes with light‐driven ROS generation ability for NIR‐II fluorescence imaging‐guided through‐skull photodynamic therapy of gliomas

Glioma is one of the most common malignant tumors of the central nervous system, leading high mortality rates in human. Aggregation‐induced emission (AIE) photosensitizers‐based photodynamic therapy (PDT) has emerged as a promising therapeutic strategy for least‐invasive treatment of glioma, which involves local irradiation of the tumor using an external near‐infrared (NIR) laser. Unfortunately, most AIE photosensitizers suffered from poorly penetration of the visible light excitation, bad spatiotemporal resolution in deep tissues and low efficient blood‐brain barrier (BBB) crossing ability, which greatly limited the clinical practice of AIE photosensitizers for especially deep‐seated brain tumor treatment. In this work, we developed a multifunctional NIR‐driven theranostic agent through hybrid of AIE photosensitizers TIND with rare‐earth doping nanoparticles (RENPs) NaGdF4:Nd/Yb/Tm with up/down dual‐mode conversion luminescence. The theranostic agent was further decorated with D‐type neuropeptide DNPY for crossing BBB and targeting glioma. Under the 808‐nm light irradiation, the down‐conversion NIR‐II luminescence could indicate the position glioma and the upconversion NIR‐I luminescence could trigger the AIE photosensitizers producing reactive oxygen species to inhibit orthotopic glioma tumor growth in situ. These results demonstrate that the integration of D‐type neuropeptide, AIE photosensitizers and RENPs could be promising candidates for in vivo NIR‐II fluorescence image‐guided through‐skull PDT treatments of brain tumors.


INTRODUCTION
6] Despite the tremendous efforts have been devoted, the effects of treatment and diagnosis of glioma was still limited in the past few years. [7,8]Taking advantage of their localized treatment with reduced side effects, easy operation, minimal invasiveness, and high spatial accuracy, photodynamic therapy (PDT) are alternative promising modalities for glioma treatment.PDT involves strong light absorption by a photosensitizer to efficiently generate cytotoxic reactive oxygen species (ROS). [9]However, because of the lack of deep tissue-excitable photosensitizer and appropriate strategies, the utility of PDT for glioma treatment is challenging. [10,11]Furthermore, conventional photosensitizers (porphyrin, phenylthiazinium derivatives, etc.) usually possess planar and extended π-conjugated structure with hydrophobic nature, leading to aggregation-caused quenching (ACQ) in aggregates in water medium with weaker fluorescence and reduced ROS generation.Aggregationinduced emission (AIE), an opposite property to ACQ, has attracted increasing attention to develop new generation image-guided PDT agents.[14] Unfortunately, most of highly efficient AIE photosensitizers are excited by ultraviolet or visible light and not in the near-infrared (NIR) region, which limits their therapeutic effect in glioma.[20] Ling et al. constructed a near infrared responsive photodynamic nanoagents based on α-NaYF 4 :Yb(80%),Er(2%)@CaF 2 core@shell nanoparticles, achieving the high-efficiency PDT with deep penetration by combining the photosensitizer Ce6. [21]Under the irradiation of 980-nm laser, the RENPs could convert visible light to activate Ce6 to generate ROS in tumor acidic microenvironment.As NIR-to-visible emitters, the developed RENPs could only serve as NIR light "transducer" to active photosensitizer under NIR light-regulated hybrid photosensitizer to solve the issue of limited tissuepenetration depth in PDT.Jin et al. developed a monodisperse nanoplatform by direct encapsulation of NaYF 4 :Yb/Er with a spectrally matchable AIE photosensitizer, which offered targeted cell imaging and PDT under single NIR laser illumination. [22][33][34] However, there were no report on integration of up/down dual-mode conversion nanoparticles and AIE photosensitizer to develop multifunctional nanoprobes for glioma diagnosis and therapy.
Besides the limitation of light penetration required for of AIE photosensitizer photonactivation, the existence of blood-brain barrier (BBB) can be regarded as another major impediment, which exclude over 98% of therapeutic drugs or contrast agents to the brain. [35,36]Even when BBB are partially disrupted so that the drug can cross the BBB, the drugs at the tumor site are insufficient for treatment, eventually to death.Therefore, the development of more efficient and accurate method for diagnosis and treatment of gliomas is critical and challenging.Endogenous receptor-mediated transcytosis is one of the most effective ways to cross the BBB.Furthermore, natural peptides composed of L-type amino acids have been widely used for BBB crossing through specific receptor, but they can be recognized and degraded by various proteolytic enzymes and loss of bioactivity.However, synthetic chiral peptides composed of D-type amino acids show better enzyme stability and BBB permeability due to the resistance to proteolysis. [37]Notably, our previous work confirmed that D-type neuropeptide Y (NPY) Y 1 R ligand D [Asn 28 , Pro 30 , Trp 32 ]-NPY(25-36) ( D NPY) modified nanomicelles could bind selectively to Y 1 R-expressed human brain endothelial cells and glioma cells, and improve the BBB permeability and glioma targeting. [38]Therefore, the decoration of nanocomposites based on D-type NPY Y 1 R ligands will be favorable for the diagnosis and treatment of glioma.Herein, we designed a photo-theranostic nanoplatform RENPs@AIE-D NPY based on dual-mode conversion luminescence RENPs integrating with AIE photosensitizer and D-type neuropeptide.Under the 808-nm light irradiation, the down-conversion NIR-II luminescence could indicate the position glioma and the upconversion NIR-I luminescence could trigger the AIE photosensitizers producing ROS to inhibit orthotopic glioma tumor growth in situ.Both In vitro and in vivo results demonstrated that the integration of D-type neuropeptide, AIE photosensitizers and RENPs could be promising candidates for in vivo NIR-II fluorescence image-guided through-skull PDT treatments of brain tumors.
Then, the optical properties of RENPs were further investigated.As shown in Figure 2D, the RENPs in cyclohexane have multiple absorption peaks in the range 500-1000 nm, of which 808-nm can be considered as the optimal excitation wavelength with low autofluorescence for biological tissue.Under 808-nm laser excitation, the emission spectra of RENPs clearly show down-conversion (DC) and upconversion (UC) photoluminescence from visible to NIR-II regions (Figure 2E and Figure 2F).Among the UC spectrum, there are two bands centered at 500-520 nm and 600-650 nm, which could be applied for PDT.Apparently, the DC spectrum exhibits high fluorescence intensity at 980-and 1060-nm wavelength in the NIR-II window, achieving lower tissue autofluorescence and deeper biological tissue penetration.Consistent with the DC spectrum, the RENPs exhibit bright NIR-II fluorescence under 808-nm laser irradiation, which was positively correlated with the concentration.Since photostability is important for long-term tumor imaging, the RENPs were dispersed in the phosphate buffered saline (PBS) for photostability test (Figure 2G).After being continuously irradiated for 30 min, the fluorescence images and intensity of RENPs showed no obvious change.Furthermore, in order to explore the penetration depth, the pork tissues were used with different thicknesses (0-12 mm) to shield the RENPs.As shown in Figure 2H, the NIR-II fluorescence intensity of the RENPs decreases with the increase of thickness, and its maximum penetrating depth is 10 mm.In contrast, the penetration depth of visible light is only 1-3 mm. [40]Therefore, the RENPs exhibit excellent tissue penetration depth and have the potential for in vivo NIR-II fluorescence imaging.

Synthesis and characterization of RENPs@AIE-D NPY
Based on afore-mentioned properties, we selected the dualmode conversion luminescence RENPs to undergo further modification for the bioimaging and treatment of glioma.Briefly, we used amphiphilic DSPE-PEG 2000 and DSPE-PEG-D NPY to encapsulate the hydrophobic RENPs and AIE photosensitizer TIND, which hydrophilic PEG chains spread out into the aqueous phase to improve the dispersity of nanocomposites in water.The resultant nanocomposites are referred as RENPs@AIE and RENPs@AIE-D NPY, and the morphology is uniform and monodisperse (Figure 3A and Figure S2).Moreover, their zeta potential and hydrodynamic size are shown in Figure 3B and C, respectively.Additionally, the zeta potential decreased with the loading of AIE or D NPY, which may be due to the introduction of negatively charged groups, like acetate or carboxyl group.The synthesis route and characterization of TIND is shown in Figure S3 to S7, which proves the successful synthesis of this AIE photosensitizer.To verify the loading of TIND into nanocomposites, the ultraviolet-visible (UV-vis) absorption spectra of different nanocomposites were characterized, respectively.As shown in Figure 3D and Figure S8, the nanocomposites exhibit the absorption at 584-nm, which indicates the successful loading of TIND and the loading rate reaches 10.4%.
In order to verify whether the nanocomposite composed of RENPs and AIE photosensitizer can have photodynamic effects, the UC spectra was characterized under the excitation of 808-nm laser.As shown in Figure 3F, the UC band at 500-520 nm and 650-700 nm for RENPs@AIE was strongly quenched due to AIE photosensitizer loading, indicating the efficient energy transfer between RENPs and AIE photosensitizer.To further verify the generation of singlet oxygen, Figure 3G and Figure S9 showed the absorbance spectra (at 410-nm) of DPBF by reacting with RENPs@AIE or TIND under the irradiation of 808-nm laser with different time.Additionally, theoretical calculations were performed using time-dependent density functional theory.The calculated energy gaps between LUMO and HOMO for TIND was 2.41 eV (Figure S10).Furthermore, theoretical calculations also indicate that TIND have an ΔE ST ≈ 0.5 eV (Figure S11).The results of the final theoretical calculations show that TIND's superior ROS generating capability is attributable to its tiny ΔE ST . [41]Therefore, it could be concluded that singlet oxygen is generated between photosensitizer and RENPs in short distance, and the RENPs can serve as the lighttransducers to activate the photosensitizer for effective PDT in deep tissues.
In addition to the PDT performance, the NIR-II fluorescence is also a spotlight under the irradiation of 808-nm laser.As shown in Figure 3E and Figure S12, the fluorescence intensity of RENPs@AIE had a slight decrease intensity when comparing with RENPs at 980-and 1060-nm wavelength.Even so, under the irradiation of 808-nm laser, RENPs@AIE-D NPY showed bright fluorescence images and the brightness is almost comparable to that of RENPs (Figure 3H).Moreover, RENPs@AIE-D NPY keep great photostability within 30 min of continuous irradiation and the penetration depth of nanocomposite is still much higher than that of visible light (Figure 3I and Figures S13-S15).

In vitro cytotoxicity and in vivo biocompatibility
Good biocompatibility of nanocomposites is the premise to ensure their biomedical application.Herein, we verified the cytotoxicity of different nanocomposites based on RENPs to U87-MG cell lines without laser irradiation (Figure 4A and B).The cell viability was above 85% at the concentration of 60 μg mL −1 , indicating the appreciable biocompatibility of RENPs@AIE-D NPY.And RENPs also kept a low cytotoxicity even at a high concentration of 400 μg mL −1 .Then, the in vivo biocompatibility of different nanocomposites was evaluated by hematoxylin and eosin (H&E) staining, blood routine, and biochemical analysis.As shown in Figure S16, major organs had no apparent damage or inflammation in all groups.Besides, the blood routine and biochemical analysis results of RENPs@AIE-D NPY group were similar to those in the PBS group, all within the range of healthy indexes (Figures S17 and S18).It suggested that at the dose tested of RENPs@AIE-D NPY, the liver and kidney function of the mice remained normal, and no obvious blood changes occurred.

In vitro cellular uptake and photodynamic properties
The cellular uptake of RENPs@AIE-D NPY by U87-MG cells was measured using the flow cytometry analysis.As shown in Figure 4C, the mean fluorescence intensity (MFI) of the cells incubated with RENPs@AIE-D NPY was 4.5-fold stronger than that of the RENPs@AIE group after 4 h of incubation.Alternatively, the images of confocal laser scanning microscope also intuitively showed that the RENPs@AIE-D NPY group could be taken up by U87-MG cells (Figure 4G).Besides, in vitro BBB crossing efficiency of RENPs@AIE-D NPY group was about 3-fold higher than that of the RENPs@AIE group (Figure S19).Therefore, these results all indicated that the ligand-mediated active targeting not only helped nanoparticles selectively target the glioma but also promoted cellular endocytosis of the nanoparticles.
The photodynamic performance of U87-MG cells by RENPs@AIE-D NPY under the irradiation of 808-nm laser was evaluated.For the cell treated with RENPs@AIE-D NPY, the cell viability significantly decreased as the concentration of TIND (AIEgen) and irradiation time as well as power density increased, suggesting that RENPs@AIE-D NPY had a significant PDT effect (Figure 4D to F).More intuitively, the images of confocal laser scanning microscope further confirmed the presence of ROS when using the 2,7dichlorodihydrofluorescein diacetate (DCFH-DA) molecular indicator (Figure 4H).That is, RENPs@AIE-D NPY could generate the cytotoxic ROS to induce cell death under 808-nm laser irradiation.As shown in Figure S20, the fluorescence images of live (green) and dead (red) showed that RENPs@AIE-D NPY could generate selective photodynamic cytotoxicity to the U87-MG cancer cells compared with bEnd.3 normal cells.As a reslut, the great PDT performance of RENPs@AIE-D NPY was demonstrated through above in vitro experiments.

In vivo NIR-II fluorescence imaging of orthotopic gliomas
Fluorescence-guided therapy has proven to be of great clinical value, in which imaging probes with excellent photostability and good permeability into the brain are key elements of brain tumor imaging.To evaluate the in vivo performance of RENPs@AIE-D NPY nanocomposites in NIR-II fluorescence imaging, we respectively intravenously injected 100 μL of RENPs, RENPs@AIE, and RENPs@AIE-D NPY solutions (the dose set at 25 mg kg −1 ) into U87-MG orthotopic glioma.Afterward, the NIR-II fluorescence images of orthotopic glioma mice under excitation by an 808-nm laser were collected at different time points postinjection.As shown in Figure 5A, compared with the images collected with the control group (PBS), RENPs@AIE-D NPY nanocomposites could efficiently cross the BBB facilitated by the receptor-mediated transcytosis and enrich at the glioma tumor site, which correspond to bioluminescence images.Evidently, the fluorescence intensity of glioma gradually increased with time, reached the highest at 8 h postinjection and then still maintained at 24 h.At this time point, the through skull TBR of RENPs@AIE-D NPY is as high as 5.08, achieved accurate localization of glioma.In contrast, the passive-targeted nanocomposites (RENPs and RENPs@AIE) had less accumulation in the orthotopic glioma and achieved relatively low TBR values.Moreover, the fluorescence intensity of the RENPs and RENPs@AIE groups in the mouse brain were comparable, which indicated that the addition of AIE photosensitizer did not have a significant effect on the fluorescence of NIR-II region in vivo (Figure 5B).To further evaluate the biodistribution of nanocomposites, the ex vivo fluorescence signals of main organs were also collected at 12 h postadministration (Figure 5C).Compared to other groups, RENPs@AIE-D NPY had significantly increased accumulation in the brain, which was consistent with the in vivo NIR-II fluorescence imaging.Together, these results confirmed that our prepared RENPs@AIE-D NPY have great potential for NIR-II fluorescence imaging nanoprobes, laid the foundation of PDT for deep glioma.

In vivo PDT of orthotopic gliomas
Inspired by the as-proved good photodynamic effect of RENPs@AIE-D NPY in vitro, the therapeutic effect on orthotopic glioma models was further investigated (Figure S21).The orthotopic glioma nude mice were divided into three groups: PBS + laser, RENPs@AIE + laser, and RENPs@AIE-D NPY + laser.The orthotopic glioma nude mice were treated after 14 days of tumor cell inoculation, which was regarded as the 0th day of treatment (Figure 6A).Guided by NIR-II imaging in vivo, the optimal time for treatment was at 8 h postinjection.Bioluminescence imaging was used to monitor the growth of glioma in mice over 15 days.As shown in Figure 6B and C, the bioluminescence signals in glioma of mice treated with RENPs@AIE-D NPY + laser group were suppressed within 15 days, obviously weaker than that of all other groups.Besides, the in vivo anti-glioma effect was further evaluated by Kaplan-Meier survival analysis (Figure 6D).Consistent with the bioluminescence results, almost the mice treated with PBS + laser or RENPs@AIE + laser eventually died due to excessive glioma growth.The result suggested that RENPs@AIE-D NPY could effectively inhibit the growth of glioma and prolong the lifetime.

CONCLUSION
In conclusion, we have successfully designed a D-type neuropeptide modified nanocomposite RENPs@AIE-D NPY for glioma NIR-II imaging guided PDT.The nanocomposite mainly takes the advantage of rare earth doped nanoparticles, which exhibit great dual-mode conversion (up and down conversion) photoluminescence under the excitation of a single wavelength.When further combined with the AIE photosensitizers, the RENPs with up-conversion luminescence provide efficient photodynamic effects, overcoming the problem of insufficient penetration depth.Then, the RENPs@AIE-D NPY nanocomposite induces significant human U87-MG glioma death in vitro and inhibits orthotopic glioma tumors in vivo.More importantly, the nanocomposite can provide a precise NIR-II fluorescence imaging for glioma with high contrast collectively, the results suggested a great promising of RENPs@AIE-D NPY for efficient photo-theranostics of glioma, and new insights into the integration of diagnosis and treatment of brain tumors.

Synthesis of RENPs@AIE nanocomposites
RENPs@AIE nanocomposite was prepared via a solvent evaporation method.Chloroform (5 mL) containing 1.5 mg TIND and 10 mg RENPs was added dropwise into 5 mL chloroform containing 20 mg DSPE-mPEG 2000 while magnetically stirring for 5 min.The organic solvent was removed by evaporation under vacuum at 45 • C. The obtained dry film was hydrated with water, and then magnetically stirred for 5 min.The unencapsulated free TIND was removed by ultrafiltration (MW = 3500 Da).Finally, the resultant RENPs@AIE nanocomposites were kept at 4 • C for further use.

Synthesis of RENPs@AIE-D NPY nanocomposites
Y 1 R ligand D NPY-modified RENPs@AIE nanocomposite was prepared via a solvent evaporation method.Chloroform (5 mL) containing 1.5 mg TIND and 10 mg RENPs was added dropwise into 5-mL chloroform containing DSPE-mPEG:DSPE-PEG-NPY (20:1, w/w) with magnetic stirring for 5 min.The organic solvent was removed by evaporation under vacuum at 45 • C. The obtained dry film of RENPs@AIE-D NPY nanocomposites was hydrated with water, and then magnetic stirred for 5 min.The unencapsulated free TIND was removed by ultrafiltration (MW = 3500 Da).Finally, the resultant nanocomposites were kept at 4 • C for further use.

Singlet oxygen detection
DPBF was used as a chemical probe to determine the generation of 1 O 2 .DPBF (20 μL, 2.5 mg mL −1 in dimethyl sulfoxide) was added into the RENPs@AIE nanocomposites (2 mL, 2.0 mg mL −1 in water), kept in the dark and continuously irradiated with 808-nm laser for 20 min (0.6 W cm −2 ).Then the absorbance intensity of DPBF at 410-nm was collected per 5 min.

NIR-II fluorescence imaging
Different concentration gradients of RENPs, RENPs@AIE and RENPs@AIE-D NPY were prepared to acquire corresponding fluorescence images by NIR II imaging system (Suzhou NIR-Optics Co., Ltd.China).Then, the RENPs, RENPs@AIE, and RENPs@AIE-D NPY nanocomposites were continuously irradiated with 808-nm laser for 0, 5, 10, 15, 20, 25, and 30 min.The fluorescence images were obtained at different times.To evaluate the tissue penetration depth, the RENPs, RENPs@AIE, and RENPs@AIE-D NPY nanocomposites was added to a 24-well plate, and pork tissues of a certain thickness were added in turn.During this period, the NIR II imaging system was used to obtain fluorescence images and corresponding fluorescence intensity values.The parameters are set as: 808-nm excitation and LP1000 filter (to filter fluorescence below 1000-nm), and the exposure time is 100 ms.

Cellular uptake
U87-MG cells were cultured with fresh medium, RENPs@AIE and RENPs@AIE-D NPY (10 μg mL −1 ).After a further 4 h of incubation, the cells were washed with PBS to remove any absorbed free nanocomposites.For flow cytometry analysis, the MFI of cells (1 × 10 4 counts) was analyzed by flow cytometer (FACS Calibur, BD Biosciences, USA), all experiments were conducted in triplicate and data are presented as the means ± SD.For laser scanning confocal microscopy (LSCM) analysis, the cells were then fixed with 4% formaldehyde for 30 min, treated with 0.1% triton for 5 min, and then treated with 1.0% Bovine Albumin (BSA) for 30 min at 25 • C. Stained with FITC phalloidin and Hoechst for 30 min, finally the samples were observed with CLSM (TCS SP5 II, Leica, Germany).

Intracellular ROS generation
U87-MG cells were treated with RENPs@AIE or RENPs@AIE-D NPY in fresh medium for 4 h (10 μg mL −1 ).Afterward, the cells were incubated with 1-mL fresh serum-free medium containing 10 μM DCFH-DA and Hoechst at 37 • C for 20 min.After washing, the cells were exposed to 808-nm laser irradiation for 10 min, followed by CLSM imaging.

In vivo toxicity evaluation
Healthy BALB/c mice were intravenously injected with PBS, RENPs, RENPs@AIE, or RENPs@AIE-D NPY nanoparticles (25 mg kg −1 ), respectively.After 14 days postinjection, the blood samples of treated mice were collected for blood biochemistry and blood routine examination, then mice were euthanized and major organs (heart, liver, spleen, lung, and kidney tissues) were harvested for H&E staining.

In vivo NIR-II fluorescence imaging of orthotopic gliomas
NIR-II fluorescence imaging was performed on the orthotopic glioma mice by using a small animal NIR-II imaging system.The system parameters were set as: 808-nm excitation and LP1000 filter, and the exposure time is 200 ms.The mice were intravenously injected with PBS, RENPs, RENPs@AIE, and RENPs@AIE-D NPY (100 μL, 25 mg kg −1 ), respectively.Fluorescence images were obtained at different time points (0, 2, 4, 6, 8, 10, 12, 24, and 48 h) with the in vivo fluorescence imaging system (n = 3 for each imaging probe).At 12 h postinjection, mice were euthanized to collect the main organs (heart, liver, spleen, lung, kidney, and brain) for ex vivo fluorescence imaging.The images were processed by using ImageJ software.

4.12
In vivo PDT of orthotopic gliomas Photodynamic treatment was carried out on the 14th day of the establishment of orthotopic glioblastoma mice model (regarded as the 0th day of treatment).The mice were randomly assigned into three groups (n = 5 mice per group).They were control (PBS + Laser), negative (RENPs@AIE + Laser), positive (RENPs@AIE-D NPY + Laser).All mice groups were intravenously injected with solution of PBS, RENPs@AIE, and RENPs@AIE-D NPY.The laser treatment was performed using an 808-nm laser (0.6 W cm −2 , 10 min) on the first and seventh days of treatment, respectively.The therapeutic efficiency from 0 to 15 days was monitored by the bioluminescence imaging.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

F I G U R E 1
The synthesis route and mechanism of rare-earth doping nanoparticles (RENPs)@aggregation-induced emission (AIE)-D NPY.A D-type neuropeptide D NPY decorated AIEgen/RENP hybrid nanoprobes can cross blood brain barrier and specifically target glioma sites.Under the 808-nm light irradiation, the down-conversion near-infrared (NIR)-II luminescence could indicate the position glioma, and the up-conversion NIR-I luminescence could trigger the AIE photosensitizers producing reactive oxygen species to inhibit orthotopic glioma tumor growth in situ.

F I G U R E 3
The physicochemical characterization of as-prepared rare-earth doping nanoparticles (RENPs)@aggregation-induced emission (AIE)-D NPY nanocomposites.(A) TEM image of RENPs@AIE-D NPY nanocomposite.(B) DLS curves of different nanocomposites.(C) Zeta potential of different nanocomposites.(D) UV-vis absorption spectra of TIND (AIE photosensitizer), RENPs and RENPs@AIE.(E) The up-conversion photoluminescence spectra of different nanocomposites under the excitation of 808-nm laser.(F) The down-conversion photoluminescence spectra of different nanocomposites under the excitation of 808-nm laser.(G) UV-vis absorption spectra of RENPs@AIE after adding DPBF (irradiated with 808-nm laser).(H) Near-infrared (NIR) II fluorescence images of different nanocomposites under the excitation of 808-nm laser.(I) The fluorescence intensity curve of RENPs@AIE-D NPY nanocomposite at 1060-nm vary with irradiation time.

F I G U R E 4
In vitro performance evaluation of U87-MG cells incubated with different nanocomposites.(A) The cell viability of U87-MG cells incubated with rare-earth doping nanoparticles (RENPs) for 24 h.(B) The cell viability of U87-MG cells incubated with TIND (aggregation-induced emission [AIE] photosensitizer), RENPs@AIE and RENPs@AIE-D NPY for 24 h.(C) The mean fluorescence intensity (MFI) values of flow cytometry analysis for U87-MG cells incubated with different nanocomposites at 4 h.(D) The cell viability under the irradiation of 808-nm laser (10 min) by changing the laser power, Mean ± SD (n = 3), ***p < 0.005.(E) The cell viability under the irradiation of 808-nm laser (0.6 W/cm 2 ) by changing the irradiation time, Mean ± SD (n = 3), ****p < 0.0001.(F) The cell viability of RENPs@AIE and RENPs@AIE-D NPY nanocomposites under the irradiation of 808-nm laser (0.6 W/cm 2 , 10 min) by changing the concentration, Mean ± SD (n = 3), **p < 0.01.(G) The confocal laser scanning microscopy (CLSM) images of U87-MG cells incubated with different nanocomposites for 4 h.Blue, green, and red represent the nucleus (Hoechst), cell membrane (FITC) and TIND (AIE photosensitizer) respectively (scale bar = 50 μm).(H) The detection of reactive oxygen species by CLSM images of U87-MG cells incubated with different nanocomposites for 4 h.Blue and green represent the nucleus and reactive oxygen species respectively (scale bar = 50 μm).

F I G U R E 5
The Near-infrared (NIR)-II fluorescence imaging performance evaluation of rare-earth doping nanoparticles (RENPs)@aggregation-induced emission (AIE)-D NPY nanocomposite in orthotopic glioma mice.(A) In vivo NIR-II fluorescence imaging of orthotopic glioma mice at different time periods after injection of PBS, RENPs, RENPs@AIE, and RENPs@AIE-D NPY via tail vein, respectively, and the bioluminescence images of the mouse on the right.(B) Tumor signal-to-background ratio (TBR) of orthotopic glioma mice after injection of material through tail vein.Mean ± SD (n = 3).(C) Ex vivo NIR II fluorescence images of orthotopic glioma mice at 12 h postinjection of different groups via tail vein.Main organs are heart, liver, spleen, lung, kidney, and brain.

F I G U R E 6
In vivo anticancer efficacy of rare-earth doping nanoparticles (RENPs)@aggregation-induced emission (AIE)-D NPY nanocomposites using an orthotopic glioma model.(A) Schematic illustration of establishment of orthotopic glioma model and treatment with RENPs@AIE-D NPY nanocomposites under 808-nm laser irradiation at the maximum RENPs@AIE-D NPY concentration, guided by near-infrared (NIR)-II fluorescence imaging.(B) The representative bioluminescence images of orthotopic U87-MG tumor mice from each group, recorded every 5 days.(C) The semi-quantitative bioluminescence signal intensity in the brain, recorded every 5 days.Mean ± SD (n = 5) (D) Kaplan-Meier survival curves of mice with orthotopic glioma in each group.
This work was financially supported by Natural Science Foundation of China (grant numbers: T2222021, 32011530115, and 32025021), National Key R&D Programs (grant number: 2019YFE0198700), Science and Technology Bureau of Ningbo City (grant numbers: 2020Z094 and 2021Z072), Excellent Member of Youth Innovation Promotion Association Foundation of CAS (grant number: Y2021079), and the Innovation and Technology Commission (grant number: MHP/047/19).All animal procedures were carried out by the Ningbo University Guidelines for the Care and Use of Laboratory Animals and were approved by the Ningbo University Animal Ethics Committee (permit number: SYXK (Zhe) 2019-0005).