LnNP@ZIF8 Smart System for In Situ NIR-II Ratiometric Imaging-Based Tumor Drug Resistance Evaluation

Just-in-time evaluation of drug resistance in situ will greatly facilitate the achievement of precision cancer therapy. The rapid elevation of reactive oxygen species (ROS) is the key to chemotherapy. Hence, suppressed ROS production is an important marker for chemotherapy drug resistance. Herein, a NIR-II emission smart nanoprobe (LnNP@ZIF8, consisting of a lanthanide-doped nanoparticle (LnNP) core and metal-organic framework shell (ZIF8)) is constructed for drug delivery and in vivo NIR-II ratiometric imaging of ROS for tumor drug resistance evaluation. The drug-loaded nanoprobes release therapeutic substances for chemotherapy in the acidic tumor tissue. As the level of ROS increases, the LnNPs shows responsively descending fluorescence intensity at 1550 nm excited by 980 nm (F1550, 980Ex), while the fluorescence of the LnNPs at 1060 nm excited by 808 nm (F1060, 808Ex) is stable. Due to the ratiometric F1550, 980Ex/F1060, 808Ex value exhibiting a linear relationship with ROS concentration, NIR-II imaging results of ROS change based on this ratio can be an important basis for determining tumor drug resistance. As the chemotherapy and resistance evaluation are explored continuously in situ, the ratiometric imaging identifies drug resistance successfully within 24 h, which can greatly improve the timeliness of accurate treatment.


Introduction
Despite tremendous advances in targeted therapy and immunotherapy [1][2][3], cytotoxic chemotherapies remain the backbone for cancer treatment at different stages [4,5]. The commonly used chemotherapeutic drugs for breast cancer include doxorubicin (DOX), paclitaxel, and cyclophosphamide [6,7]. Studies have shown that one of the main mechanisms of doxorubicin in tumor treatment is to stimulate an intracellular oxidative stress state to produce excessive intracellular reactive oxygen species (ROS) levels, causing death signals such as DNA damage and activation of apoptosis [8]. However, to compensate for this and maintain redox homeostasis, tumor cells can develop a set of complex resistant mechanisms during treatment, such as enhancing antioxidant capacity [9,10]. Recently, more attention has been focused on the antioxidant system in tumors [11,12]. Unfortunately, there is no technology available for detecting in situ drug resistance to chemotherapy. Hence, the ability to relieve oxidative stress and elevated antioxidant capacity are potential hallmarks of drug-resistant cancer [13]. Based on this principle, in situ ROS monitoring during chemotherapy will be used as a basis for the evaluation of tumor drug resistance. first successfully performed in vitro validation of the ROS response using H2O2.
Once the DOX-loaded probe was captured by cancer cells, the outer ZIF8 woul gradually degrade under acidic conditions (pH ≈ 5.5) and release the drug (Figure 1). Sub sequently, the exposed LnNPs would be monitored for in situ ROS levels. To avoid signa fluctuations due to differences in probe uptake, the stable emission of 1060 nm light from the LnNP core at 808 nm excitation (F1060, 808Ex) can be used as an internal index fo signal correction. At the same time, the outer layer of the LnNP core emits intense 155 nm light (980 nm excitation) (F1550, 980Ex) at low levels of ROS. However, this phenom enon indicates that ROS production had been inhibited and was in a drug-resistant stat ( Figure 1, lower left pink area). In contrast, there was a rapid rise in ROS in drug-sensitiv cells and a trend toward quenching of F1550, 980Ex (Figure 1, lower right blue area). Th final ratio of F1550, 980Ex to F1060, 808Ex is a quantitative assessment of in situ tumo drug resistance.
To our knowledge, this is the first work to report a spatiotemporal real-time rat ometric infrared monitoring evaluation of tumor ROS levels in vivo during chemother apy. This redox imaging probe has the potential to inform therapeutic response and dru resistance. Ultimately, this would facilitate early intervention, allowing the clinician t adapt the treatment regimen, with the potential to achieve a better prognosis.  Once the DOX-loaded probe was captured by cancer cells, the outer ZIF8 would gradually degrade under acidic conditions (pH ≈ 5.5) and release the drug ( Figure 1). Subsequently, the exposed LnNPs would be monitored for in situ ROS levels. To avoid signal fluctuations due to differences in probe uptake, the stable emission of 1060 nm light from the LnNP core at 808 nm excitation (F1060, 808Ex) can be used as an internal index for signal correction. At the same time, the outer layer of the LnNP core emits intense 1550 nm light (980 nm excitation) (F1550, 980Ex) at low levels of ROS. However, this phenomenon indicates that ROS production had been inhibited and was in a drug-resistant state (Figure 1, lower left pink area). In contrast, there was a rapid rise in ROS in drug-sensitive cells and a trend toward quenching of F1550, 980Ex ( Figure 1, lower right blue area). The final ratio of F1550, 980Ex to F1060, 808Ex is a quantitative assessment of in situ tumor drug resistance.
To our knowledge, this is the first work to report a spatiotemporal real-time ratiometric infrared monitoring evaluation of tumor ROS levels in vivo during chemotherapy. This redox imaging probe has the potential to inform therapeutic response and drug resistance. Ultimately, this would facilitate early intervention, allowing the clinician to adapt the treatment regimen, with the potential to achieve a better prognosis.
Transmission electron microscopic (TEM) images were captured on a JEM-2100 transmission electron microscope (JEOL Ltd., Tokyo, Japan). Formvar/Thick Carbon Film Coated grids (BZ11024a) were purchased from Beijing zhongjingkeyi Technology Co. (Beijing, China). and the accelerating voltage was 200 kV. Dynamic light scattering (DLS) was conducted on a ZetaPlus 90 Plus/BI-MAS particle size analyzer (Brook haven, Atlanta, GA, USA). The crystal structures of the obtained materials were characterized by X-ray diffraction (XRD) performed on an Ultima IV 285 X-ray powder diffractometer (Rigaku Co., Tokyo, Japan). NIR-II images were captured by an IVIS Lumina XR III in vivo imaging system (PerkinElmer, Boston, MA, USA). 4 :Nd,Gd@NaYF 4 :Gd@NaYF 4 :Gd,Yb,Er@ NaYF 4 :Ce LnNP-OA Synthesis of multilayer structured LnNP-OA: UCNP-OA was synthesized through seed-mediated growth. The LnNP core was firstly synthesized by adding 1 mmol of Ln(OA) 3 (Y:Nd:Gd = 0.9:0.05:0.05) to the mixture of OA and 1-octadecene (20 mL, v:v = 1:3). After being degassed in vacuum at 150 • C for 1 h, this mixture was cooled down to 45 • C, and added with 10 mL methanol solution of NH 4 F (4.0 mmol) and NaOH (2.5 mmol) dropwise under stirring for 30 min. After the reaction mixture was heated to 110 • C to completely remove methanol, and 300 • C under a nitrogen atmosphere for 90 min, LnNPs (NaYF 4 :Nd,Gd) with a diameter of 18 nm were obtained.

Preparation of NaYF
Various amounts of outer layer precursor were injected into the above reaction mixture of the UCNP core and stirred for 30 min to grow the emitting layer on the core (NaYF 4 :Nd,Gd@NaYF 4 :Gd). The third layer was then grown by injecting its precursor in the mixture and stirring for 30 min to get multilayer structured UCNP (NaYF 4 :Nd,Gd@NaYF 4 :Gd@NaYF 4 :Gd,Yb,Er). The four-layer structure of NaYF 4 :Nd, Gd@NaYF 4 :Gd@NaYF 4 :Gd,Yb,Er@NaYF 4 :Ce LnNP-OA can be obtained in the same way. The mixture was then cooled down to room temperature, precipitated with acetone, repeatedly washed with cyclohexane, and re-dispersed in 10 mL CHCl 3 or cyclohexane for further use.

Preparation of PVP-Capped LnNP (LnNP-PVP)
To create a suitable surface for growing the ZIF8, OA ligands on the surface of the LnNPs were removed and then coated with a PVP layer. Briefly, LnNPs (0.4 mmol) were dispersed in 10 mL (0.1 M) of dilute HCl solution and sonicated for 1 h. Then they were washed three times with distilled water and dispersed in 5 mL of ethanol. Then, 15 mL of chloroform containing 0.96 g of PVP (Mw = 24,000) was dropwise added into the solution with vigorous stirring. After stirring for 24 h, PVP-capped LnNPs were obtained. Finally, the products were collected from the solution by centrifugation and washed with ethanol to remove excess PVP molecules.

Synthesis of the LnNP@ZIF8 Nanocomposites
Typically, 0.4 mL of PVP-LnNP in methanol (0.1 M) was added into 5 mL of 2methylimidzole (0.13 g/mL) methanol solution, and stirred for about 30 min, then mixed with 5 mL of Zn(NO 3 ) 2 ·6H 2 O (0.059 g/mL) methanol solution. The mixture was stirred at room temperature for 24 h. The resulting white solid was collected by centrifugation, washed with methanol three times, and re-dispersed in methanol for further use.

Preparation of LnNP@ZIF8-DOX Nanoprobes
The dried precipitate of LnNP@ZIF8 (4 mM) was mixed with 0.5 mg, 1 mg, and 2 mg of DOX into 2 mL of distilled water for ultrasonic dispersion, respectively. The mixture was vibrated for 24 h, allowing the DOX to be adsorbed into the pores of LnNP@ZIF8. The LnNP@ZIF8-DOX nanoprobes were then obtained by centrifugation at 10,000 r/min (4480× g) for 5 min and washed with water three times.

Quantitative Detection of ROS
The LnNP@ZIF8-DOX was reacted with H 2 O 2 with concentrations from 0 to 80 µM, and the emission spectra excited by 808 nm and 980 nm laser light were measured respectively. The fluorescence intensity ratio at 1060 nm and 1550 nm (F1550,980Ex/F1060,808Ex) was calculated. Then, 500 µL of the above mixture solution was added to a 24-well plate, and images were taken by the NIR-II fluorescence imaging setup.

In Vitro Selectivity Studies of LnNP
LnNPs (0.5 mg·ml −1 ) in PBS solution were reacted with other ROS (80 µM) and various amino acids and ions (GSH, Gly, Glu, glucose, CaCl 2 ) (1 mM) for 30 min, and the emission spectra excited by 808 nm and 980 nm laser light were measured, respectively. The F1550,980Ex/F1060,808Ex ratio was calculated and a student two-sided t-test was performed.

Cell Uptake and Imaging
MCF-7 cells and 4T1 (5 × 10 5 mL −1 ) were seeded into 12-well plates. After 8 h, these were treated with LnNP@ZIF8 or LnNP@ZIF8-DOX (100 µg/mL) and incubated for 6 h, then washed with PBS. The cells were then fixed with 1 mL of 4% paraformaldehyde for 10 min and washed with PBS. All cell samples were photographed under a NIR microscope. The NIR-II images were taken by a NIRVana HS camera (Teledyne Princeton Instruments, Trenton, NJ, USA) and processed by Lightfield software.

Construction of 4T1 Orthotopic Mouse Model
Ten six-to-eight-week-old female Balb/c mice were purchased from the Model Animal Research Center of Nanjing University (MARC, Nanjing, China) and maintained under specific pathogen-free conditions in the Animal Core Facility of Nanjing Medical University. The mice were anesthetized using inhaled isoflurane (RWD Life Science, Shenzhen, China). In total, 1 × 10 5 4T1 cells in 100 µL of PBS were injected subcutaneously into the right inguinal mammary fat pads of each animal.

Construction of MCF-7 Human Breast Tissue-Derived Orthotopic Mouse Model
Ten five-to-seven-week-old female severe combined immunodeficiency (SCID) mice were purchased from the Model Animal Research Center of Nanjing University (MARC, Nanjing, China). The SCID mice were kept under specific pathogen-free (SPF), temperaturecontrolled conditions. Cages, bedding, and drinking water were autoclaved and changed regularly. The mice were maintained in a daily cycle of 12 h periods of light and darkness. Normal human breast tissues were obtained from freshly discarded material of elective reduction mammoplasty surgery according to the ethical guidelines of the Declaration of Helsinki and approved by the ethics and research committee of the First Affiliated Hospital of Nanjing Medical University. The breast tissues were stripped of excess fat and sliced under sterile conditions into pieces of approximately 4 × 4 × 4 mm. Three pieces were selected randomly for histological examination and other pieces were placed in phosphatebuffered saline (PBS) at 0 • C until implanted in the SCID mice. Before implantation, mice were anesthetized using inhaled isoflurane (RWD Life Science, Shenzhen, China). Surgical procedures were modified according to the previous description. Briefly, 5-6 mm incisions were made by a scalpel in the skins of the left and right mid-dorsal flanks. Implantation was finished within 6 h of the mammoplasty surgery. The mice received gentamicin in their drinking water (800,000 U/L) up to 1 week following the implantation. Ten SCID tissue-derived orthotopic mice were divided into the MCF-7 WT group and the MCF-7 ADR group randomly and equally. Cells were harvested with 0.25% trypsin and 0.02% disodium edetate, washed in the medium, counted, and resuspended in PBS. One week after the implantation of human breast tissues, the MCF-7 WT cells and MCF-7 ADR cells (5 × 10 5 ) in 0.2 mL PBS were inoculated into the left implanted breast tissues.

Mouse Treatment Procedures
Subcutaneous injection of murine 4T1 cells into Balb/c mice led to the formation of primary tumors. At 21 days after 4T1 cell injection, when the tumor diameter was >7 mm (range, 7-10 mm), ten mice were divided into two groups: treated with LnNP@ZIF8 (LnNP@ZIF8) or treated with LnNP@ZIF8-DOX (LnNP@ZIF8-DOX). The mice were anesthetized using inhaled isoflurane (RWD Life Science, Shenzhen, China). The tumor site was sanitized with a 75% ethanol tincture before treatment. All the procedures were performed aseptically. Then, a 100µL mixture of the LnNPs in PBS (5 mg/mL) was injected subcutaneously into the 4T1 tumors.
Eight of ten SCID tissue-derived orthotopic mice models constructed successfully. Then, a 100 µL mixture of the LnNP@ZIF8-DOX (5 mg/mL) in PBS was injected subcutaneously into the MCF-7 WT and MCF-7 ADR tumors.
NIR-II fluorescent images were recorded at 24 h after probe injection using an in vivo imaging system equipped with an IVIS Lumina XR III in vivo imaging system (PerkinElmer, Waltham, MA, USA).
All mice were sacrificed by cervical dislocation after CO 2 euthanasia.

Declarations
All mice studies were conducted according to the Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Nanjing

Statistical Analysis
Origin 2018 64 Bit and GraphPad Prism (Version 8.0) were used to analyze any differences present between groups in the data. Significance was analyzed by the Student t-test. All experiments were independently repeated three times, and p < 0.05 was used for statistical significance.

Preparation and Characterization
We designed lanthanide-doped nanoparticles (LnNPs) with multilayer core shells (Figure 2A, 44.1 ± 2.1 nm) with NIR orthogonal excitation characteristics. Firstly, hydrophobic NaYF 4 :Nd,Gd nanoparticles were synthesized as the core of the nanoprobe via a facile high-temperature co-precipitation method [24,25]. The core is wrapped in inert Shell 1 (S1) and emits 1060 nm light stably under 808 nm excitation, which acts as an internal standard during the detection process ( Figure S1). Shell 2-3 forms a sensor responsive to ROS, with its 1550 nm emission decreasing as ROS rises under 980 nm excitation ( Figure S2). To create a suitable surface for growing the ZIF8, hydrochloric acid was used to remove the OA ligands. Then, polyvinylpyrrolidone (PVP) was coated onto the bare LnNPs. After stirring for 24 h, PVP-capped LnNPs were obtained. Finally, a ZIF8 layer mixing DOX aqueous solution was coated onto the LnNP nanodisks to form the hydrophilic LnNP@ZIF8-DOX core−shell nanostructure. Transmission electron microscopy (TEM) images clearly showed the obvious core-shell geometry (57.5 ± 4.1 nm) ( Figure 2B), indicating the successful synthesis of LnNP-OA structures ( Figure S3 and Figure 2A). The DLS data likewise confirm this parcel trend (average diameter increased by 16.2 nm). ( Figure 2C). X-ray diffraction (XRD) was employed to characterize the structural evolution of the LnNPs and LnNP@ZIF8-DOX ( Figures 2D and S4).

Response of LnNP to ROS
Since Ce 3+ ions in the host matrix were exposed on the surface of the ligand-free NPs after the acid treatment, H2O2 or other ROS species can directly oxidize Ce 3+ to Ce 4+ through redox reactions, resulting in the quenching of the NIR-II emission of Er 3+ upon

Response of LnNP to ROS
Since Ce 3+ ions in the host matrix were exposed on the surface of the ligand-free NPs after the acid treatment, H 2 O 2 or other ROS species can directly oxidize Ce 3+ to Ce 4+ through redox reactions, resulting in the quenching of the NIR-II emission of Er 3+ upon excitation at 980 nm [26,27]. Benefiting from such redox reactions, LnNPs can be explored as an effective probe for the detection of ROS. Next, to investigate the quenching effect of ROS on the NIR-II emission of the LnNPs, ta ligand-free probe (0.5 mg/mL) was applied to react with different concentrations of H 2 O 2 in the range of 0-80 µM. After the reaction, NIR-II fluorescent images and the intensity of the nanoprobe were acquired upon 808 or 980 nm excitation, respectively. It was observed that the fluorescent signal at 1550 nm of the nanoprobe gradually decreased with increasing H 2 O 2 concentration upon irradiation with a 980 nm laser, due to the redox reaction between the H 2 O 2 and Ce 3+ ions ( Figure 3A). However, the intensity of the NIR-II fluorescent signal at 1060 nm irradiated with an 808 nm laser remained stable ( Figure 3B). As a result, the nanoprobe exhibited a ratiometric response for H 2 O 2 in the range of 1 to 80 µM ( Figure 3C) and the limit of detection (LOD) of H 2 O 2 (calculated according to the following formula: LOD = 3 SD/K (SD: standard deviation of the response, K: the slope of the standard curve)) was determined to be 0.190 µM. NIR-II fluorescent imaging of the solution also showed that the signal at 1550 nm gradually became darker with increasing ROS concentration under irradiation with a 980 nm laser, whereas the signal at 1060 nm was largely unchanged after irradiation with an 808 nm laser. The ratiometric NIR-II image showed gradual dimming with increasing ROS concentration ( Figure 3D). The selectivity of this probe was also tested. In the presence of various ROS species, the fluorescent signal at 1550 nm excited by a 980 nm laser (F1550,980Ex) was quenched to the same level, while the ratio of signal (F1550,980Ex/F1060,808Ex) remains stable under the interference of other amino acids and ions ( Figure 3E). This suggested that the LnNP@ZIF8 probe had adequate sensitivity and universality for ROS species.

Drug Releasing of LnNP@ZIF8-DOX and Cellular Uptake
Firstly, we examined the drug-loading ability of freshly synthesized LnNP@ZIF8-DOX NPs. Different concentration of DOX stock solution was mixed with LnNP-PVP (5mg/mL), during the final step of the synthesis. The drug loading percentages of the nanoparticles were 17.8 wt%, 37.3 wt%, and 64.7 wt%, respectively ( Figure 4A), determined by the UV/Vis analysis of unloaded drug in the supernatant following centrifuge treatment.
The successful encapsulation of DOX motivated us to study the degradation behavior of LnNP@ZIF8-DOX further. Owing to its acid instability, ZIF8 can be degraded under acid conditions, resulting in the release of embedded DOX. The responsive experiment under different pH conditions was carried out to confirm this specific degradation of ZIF8. It can be seen from Figure 4B that the ZIF-shell of the nano-assemblies collapsed under acetate buffer solution at pH 5.5, with a rapid release in the first 24 h and over 80% release after 72 h, whereas no obvious changes in the release amount of DOX were observed in bovine serum solution at pH 7.4 under 37 • C (below 10% in 3 days). Subsequently, we studied the cellular uptake of our nanoparticles. The near-infrared microscope images ( Figure 4C) showed that the cell uptake of LnNP@ZIF8 and LnNP@ZIF8-DOX was efficient, which led to efficient drug delivery in wildtype MCF-7 cells without using a toxic organic solvent in the cell culture.
The ability of LnNP@ZIF8 and LnNP@ZIF8-DOX against cancer cells was evaluated by measuring the cell viability reduction after introducing the NPs to MCF-7 WT cells. The experiments were carried out by CCK-8 assay. The result showed that LnNP@ZIF8-DOX has a significant cytotoxic effect on MCF-7 wildtype cells, whereas LnNP@ZIF8 without DOX did not affect cell survival rate ( Figure S5). ions ( Figure 3E). This suggested that the LnNP@ZIF8 probe had adequate sensitivity and universality for ROS species.

Drug Releasing of LnNP@ZIF8-DOX and Cellular Uptake
Firstly, we examined the drug-loading ability of freshly synthesized LnNP@ZIF8-DOX NPs. Different concentration of DOX stock solution was mixed with LnNP-PVP (5mg/mL), during the final step of the synthesis. The drug loading percentages of the nanoparticles were 17.8 wt%, 37.3 wt%, and 64.7 wt%, respectively ( Figure 4A), determined by the UV/Vis analysis of unloaded drug in the supernatant following centrifuge treatment.
The successful encapsulation of DOX motivated us to study the degradation behavior of LnNP@ZIF8-DOX further. Owing to its acid instability, ZIF8 can be degraded under acid conditions, resulting in the release of embedded DOX. The responsive experiment under different pH conditions was carried out to confirm this specific degradation of ZIF8. It can be seen from Figure 4B that the ZIF-shell of the nano-assemblies collapsed under acetate buffer solution at pH 5.5, with a rapid release in the first 24 h and over 80% release after 72 h, whereas no obvious changes in the release amount of DOX were observed in bovine serum solution at pH 7.4 under 37 °C (below 10% in 3 days). Subsequently, we studied the cellular uptake of our nanoparticles. The near-infrared microscope images ( Figure 4C) showed that the cell uptake of LnNP@ZIF8 and LnNP@ZIF8-DOX was efficient, which led to efficient drug delivery in wildtype MCF-7 cells without using a toxic organic solvent in the cell culture. The ability of LnNP@ZIF8 and LnNP@ZIF8-DOX against cancer cells was evaluated by measuring the cell viability reduction after introducing the NPs to MCF-7 WT cells. The experiments were carried out by CCK-8 assay. The result showed that LnNP@ZIF8-DOX has a significant cytotoxic effect on MCF-7 wildtype cells, whereas LnNP@ZIF8 without DOX did not affect cell survival rate ( Figure S5).

ROS Generation and Detection of LnNP@ZIF8-DOX In Vitro and In Vivo
The main anticancer action of DOX is believed to be due to topoisomerase II inhibition and ROS generation [22]. Next, we tried to explore whether the nanoprobe could be applied to detect changes in ROS during LnNP@ZIF8-DOX co-culture with breast cancer cells. The MCF-7 cell line incubated in 12-well plates was divided into two groups for the following experiments. In the first group, MCF-7 cells were incubated with LnNP@ZIF8,

ROS Generation and Detection of LnNP@ZIF8-DOX In Vitro and In Vivo
The main anticancer action of DOX is believed to be due to topoisomerase II inhibition and ROS generation [22]. Next, we tried to explore whether the nanoprobe could be applied to detect changes in ROS during LnNP@ZIF8-DOX co-culture with breast cancer cells. The MCF-7 cell line incubated in 12-well plates was divided into two groups for the following experiments. In the first group, MCF-7 cells were incubated with LnNP@ZIF8, which encapsulated no drugs, whereas cells of the other group were co-cultured with LnNP@ZIF8-DOX. After incubation for 24 h, NIR-II images irradiated with 808 or 980 nm laser light were recorded. The results showed that the cells treated with LnNP@ZIF8 alone were not able to quench the NIR-II emission of nanoprobes upon 980 nm excitation, since no ROS was generated. In the second group, MCF-7 cells treated with LnNP@ZIF8-DOX for 24 h showed relatively weak F1550,980Ex signals, and weak ratiometric signals were observed, indicating that some extent of ROS was generated in cells by DOX released from the nano-assemblies ( Figure 5A). In the first group, the ratio was 0.824 ± 0.026, and ROS mean concentration in the MCF-7 cells was 1.618 µM compared with the standard curve. In the second group, the ratio was 0.406 ± 0.065, and the calculated ROS mean concentration in the MCF-7 cells was 43.787 µM ( Figure 5B). These data illustrated the feasibility of the ratiometric nanoprobe for intracellular ROS detection.
Given the excellent performance of the nanoprobe for the generation and detection of ROS in vitro, we further explored its applicability for ROS detection in vivo by using the 4T1 orthotopic breast cancer mouse model. The nanoprobes were injected into the 4T1 tumors. After 24 h, NIR-II imaging of the whole mice was recorded with 808 or 980 nm laser light. Consistent with the in vitro results, the orthotopic tumors treated with LnNP@ZIF8-DOX emitted relatively weaker fluorescence at 1550 nm irradiated with a 980 nm laser compared with tumors treated with LnNP@ZIF8, while emitting stable fluorescence at 1060 nm irradiated with a 980 nm laser ( Figure 5C). In addition, uptake, and distribution of DOX in tumor tissues after the injection of LnNP@ZIF8-DOX were shown by immunofluorescence images ( Figure S6). The fluorescence changes of the nanoprobes in tumors over time were studied ( Figure S7). At the same time, nanoprobe safety was also demonstrated by H&E staining ( Figure S8). Based on the requirement for timeliness and comparative imaging results, subsequent studies using 24 h imaging are discussed. In the LnNP@ZIF8 group, the ratio at 24 h was 0.729 ± 0.025, and the tested mean ROS level (converted to H 2 O 2 ) in 4T1 tumors of mice was 3.426 µM compared with the standard curve ( Figure 5D). In the LnNP@ZIF8-DOX group, the ratio was 0.389 ± 0.041, and the calculated mean ROS concentration (converted to H 2 O 2 ) in 4T1 tumor tissues was 50.061 µM ( Figure 5D). This result further proved that the nanoprobe could accurately quantify ROS in mice and possess excellent performance in tumor imaging.

Capability of Identifying DOX Resistance
ROS generated by DOX might exceed a certain ROS threshold in DOX-sensitive cells, resulting in detrimental oxidative stress and ultimately apoptosis or necrosis. In contrast, DOX-resistant cells are able to counteract increased ROS levels through their endogenous antioxidant systems. Thus, monitoring the ROS level is a hopeful strategy for predicting doxorubicin resistance, which inspired us to apply our ROS-sensing probe to identifying DOX resistance [28]. Experiments were carried out on wildtype MCF-7 (MCF-7-WT) and MCF-7-ADR, a breast cancer cell line and its DOX-resistant counterpart, to explore the nanoprobe's identification ability. Cells were incubated with LnNP@ZIF8-DOX for 24 h, and then NIR-II images were recorded. As we can see from Figure 6A, the emission at 1550 nm irradiated with a 980 nm laser was distinctly quenched by ROS generated by LnNP@ZIF8-DOX, which is consistent with the results given above. It is noteworthy that, in MCF-7-ADR cells, emission at 1550 nm irradiated with a 980 nm laser maintains a certain intensity, and the ratio diagrams replaced with the pseudo-colored version still showed brightly. The calculated NIR-II FL ratio of MCF-7-WT and MCF-7-ADR cells at 24 h was 0.424 ± 0.060 and 0.661 ± 0.049, respectively ( Figure 6B). Compared with the standard curve, the mean level of ROS (converted to H 2 O 2 ) was only 5.905 µM, while the concentration in the MCF-7-WT group was 37.985 µM ( Figure 6B). To confirm that DOX kept its therapeutic potential and the MCF-7-ADR cells acquired DOX resistance, we explored the IC 50 of LnNP@ZIF8-DOX for the MCF-7-WT cells and MCF-7-ADR cells ( Figure S9). Furthermore, we tested the nanoprobe's ability to identify DOX resistance in vivo. In this study, we developed a human breast tissue-derived orthotopic mouse model of breast cancer, in which the normal human breast tissues were implanted subcutaneously to create a normal human mammary microenvironment, after which MCF-7-WT and MCF-7-ADR cells were inoculated into the implants [29]. After 24 h of intratumor injection of the probe, a significantly decreased signal at 1550 nm irradiated with a 980 nm laser was observed in the MCF-7-WT cells, indicating an intense response of nanoprobes to ROS ( Figure 6C, left). However, the MCF-7-ADR tumor tissues treated with LnNP@ZIF8-DOX emitted obvious NIR-II signals in both irradiation wavelengths and showed a bright ratiometric signal ( Figure 6C, right). According to the data of the MCF-7-WT group, the NIR-II fluorescence ratio of MCF-7-WT and MCF-7-ADR tissues at 24 h was 0.440 ± 0.042 and 0.708 ±0.045, respectively ( Figure 6D). Based on the standard curve, the mean ROS concentration (converted to H 2 O 2 ) of MCF-7-WT tumor tissues in situ was 33.481 µM, while the level in the MCF-7-ADR group was 4.075 µM (Figures 3C and 6D). In the second group, the ratio was 0.406 ± 0.065, and the calculated ROS mean concentration in the MCF-7 cells was 43.787 μM ( Figure 5B). These data illustrated the feasibility of the ratiometric nanoprobe for intracellular ROS detection. Given the excellent performance of the nanoprobe for the generation and detection of ROS in vitro, we further explored its applicability for ROS detection in vivo by using the 4T1 orthotopic breast cancer mouse model. The nanoprobes were injected into the 4T1 tumors. After 24 h, NIR-II imaging of the whole mice was recorded with 808 or 980 nm laser light. Consistent with the in vitro results, the orthotopic tumors treated with LnNP@ZIF8-DOX emitted relatively weaker fluorescence at 1550 nm irradiated with a 980 nm laser compared with tumors treated with LnNP@ZIF8, while emitting stable fluorescence at 1060 nm irradiated with a 980 nm laser ( Figure 5C). In addition, uptake, and distribution of DOX in tumor tissues after the injection of LnNP@ZIF8-DOX were shown by immunofluorescence images (Figure S6). The fluorescence changes of the nanoprobes in tumors over time were studied ( Figure S7). At the same time, nanoprobe safety was also demonstrated by H&E staining ( Figure S8). Based on the requirement for timeliness and comparative imaging results, subsequent studies using 24 h imaging are discussed. In the LnNP@ZIF8 group, the ratio at 24 h was 0.729 ± 0.025, and the tested mean ROS level (converted to H2O2) in 4T1 tumors of mice was 3.426 μM compared with the standard curve ( Figure 5D). In the LnNP@ZIF8-DOX group, the ratio was 0.389 ± 0.041, and the calculated mean ROS concentration (converted to H2O2) in 4T1 tumor tissues was 50.061 μM ( Figure 5D). This result further proved that the nanoprobe could accurately quantify ROS in mice and possess excellent performance in tumor imaging.  Here, these data show that LnNP@ZIF8-DOX is uniquely sensitive to the upregulated antioxidant pathways present in drug-resistant human breast tumors. In the drug-resistant cell lines and tumor tissues, lower ratio intensity corresponded with decreased ROS concentrations in comparison to drug-sensitive tumors. Therefore, our redox imaging agent enables the identification of patients with breast cancer that are refractory to the standard of care, as well as monitoring their response posttreatment.

Conclusions
In conjunction with DNA damage, chemotherapies generate high levels of oxidative stress in tumors and lead to the apoptosis of cells sensitive to treatment [30]. However, Here, these data show that LnNP@ZIF8-DOX is uniquely sensitive to the upregulated antioxidant pathways present in drug-resistant human breast tumors. In the drug-resistant cell lines and tumor tissues, lower ratio intensity corresponded with decreased ROS concentrations in comparison to drug-sensitive tumors. Therefore, our redox imaging agent enables the identification of patients with breast cancer that are refractory to the standard of care, as well as monitoring their response posttreatment.

Conclusions
In conjunction with DNA damage, chemotherapies generate high levels of oxidative stress in tumors and lead to the apoptosis of cells sensitive to treatment [30]. However, drug resistance has become a major obstacle to the clinical use of doxorubicin [31,32]. In addition, there is no satisfactory way to identify patients who become refractory to the standard of care. DOX-resistant cancer cells can maintain redox homeostasis to protect themselves from the harmful effects of oxidative stress, thereby conferring treatment resistance. Consequently, elevated antioxidant capacity is an ideal hallmark of doxorubicin insensitivity in breast cancer cells [33]. ROS-sensing probes have the potential to assess the efficacy of chemotherapeutics that converge with the induction of oxidative stress. In a recent study, the 18 F-FSPG imaging system, reflecting the tumor antioxidant response to doxorubicin, was shown to be a promising biomarker of drug resistance in animal models of ovarian cancer [28]. Among the optical sensing methods currently being explored, ratiometric fluorescence sensing has received particular attention as a technique with the potential to provide precise and quantitative analyses [34][35][36].
In our study, the acidic tumor microenvironment was exploited to respond to pHsensitive doxorubicin-loaded LnNP@ZIF8, resulting in the rapid release of doxorubicin at low pH and an increase in ROS levels. Furthermore, the core-shell LnNPs showed ultrasensitive performance for ROS detection. The findings demonstrate that ZIFs are prime candidates as drug carriers for monitoring drug efficacy. Carrying out matched drugsensitive and drug-resistant breast cancer cells, we confirmed that resistance to doxorubicin corresponded with an increase in tumor antioxidant capacity, defined through the decrease in ROS [37]. lower ROS corresponded to a decreased emission signal at 1550 nm in drugresistant versus drug-sensitive cells.
Together, we envisage this nano-assembled platform has the potential to predict drug resistance prior to invalid chemotherapy. Ultimately, this would result in fewer ineffective cures and a better prognosis.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/nano12244478/s1, Figure S1: NIR-II emission spectra of LnNP treated with the addition of ROS or not upon excitation at 808 nm; Figure S2: NIR-II emission spectra of LnNP treated with the addition of ROS or not upon excitation at 980 nm; Figure S3: TEM images of core of LnNP core, LnNP CS1 and LnNP CS1S2; Figure S4: XRD patterns of LnNP-OA; Figure S5: cell viability of MCF-7-WT cells with different treatments; Figure S6: uptake and distribution of DOX in tumor tissues; Figure S7. NIR-II FL imaging of MCF-7 tumor mice at different time points; Figure S8. H&E staining images of Vital organs. Figure S9. IC50 of the LnNP@ZIF8-DOX in the MCF-7-WT and MCF-7-ADR cells.