All rare earth reagents used in this study were 5N (99.999%). In a beaker, a solution containing 1.2 mmol NaCl, 0.48 mmol YCl₃, 0.108 mmol YbCl₃, 0.012 mmol HoCl₃ (Aladdin Chemicals Co., Ltd., Shanghai, China), 0.15 g of PEI was prepared in 15 ml of ethylene glycol. After stirring on a magnetic stirrer, 0.4 g of NaF was added, the solution was stirred for 10 min. The reaction kettle was sealed into a muffle furnace and heated for 2 h at 200°C. After slow cooling to room temperature, the precipitate was collected by centrifugation. The pellet was washed three times with water and ethanol. After drying, the nanoparticles were dispersed in water. The particle size of the UCPs was determined by dynamic light scattering and transmission electron microscopy (TEM) on a JEOL/JEM-2010F TEM (JEOL, Tokyo, Japan). Infrared spectroscopy was used to analyze the surface of the UCPs. The luminescence spectra of the particles were determined by fluorescence spectrometer using an external 980-nm light source.

The pHLIP polypeptide was synthesized by the solid phase method and using the following sequence: Ala Cys Glu Gln Asn Pro Ile Tyr Trp Ala Arg Tyr Ala Asp Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Val Asp Ala Asp Glu Gly Thr Gly.

Aqueous phase transfer is the first prerequisite for biomedical applications of UCPs, but ifyou need to use UCPs to achieve specific functions, for example, targeted imaging or light detection for specific objects, it is necessary to carry out surface modification of UCP, which is usually achieved through coupling antibodies or other targeting molecules. The silane treatment makes the particles hydrophilic, its surface mainly presenting hydroxyl groups, which cannot directly link proteins. Therefore, the silane shell surface was modified to present amino or carboxyl groups by the crosslinking agent 1-ethyl-3-(3-aminopropyl-methyl-2) carbon imine (EDC) and two N- hydroxysuccinimide (NHS) reactions.

The linker (SMCC; Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for the coupling of the polypeptide to the particles through the Cysthiol group and the particle surface amino group at the N terminal side of the peptide. Cy5-labeled pHLIP was used as positive control, and unlabeled UCP was used as negative control. Anurinary tract epithelial cancer cell line cultured in vitro was used as a model. The cell binding reaction of pHLIP-UCP was observed at pH 5–7. The experiments were repeated to determine the optimal pHLIP-UCP coupling conditions.

For linking proteins to nanoparticles using SMCC, 50 mg of SMCC was dissolved in 5 ml of DMF to obtain a solution at 10 mg/ml. High-brightness purified UCPs were added to obtain a solution at about 0.015 mmol and 5 ml of phosphate-buffered saline were added and mixed quickly. The solution was left to react for 30 min at 37°C. Excess SMCC was removed by PBS dialysis, 1 ml of 0.15 M of pHLIP polypeptide was added, and the reaction was continued at 37°C for 30 min. After a period of silane reaction, the clear solution gradually became milky white, indicating that the silane carried on the coupling reaction on the surface of the particles. The coated particles gradually precipitated the organic phase. In order to observe the continuous process of silane modification to determine the optimal modification time, samples were observed after 12, 24, 36, 42, and 48 h by TEM. The nanoparticles were obtained after centrifugal precipitation and repeated washing. Nanoparticles were kept at 4°C.

An animal model of subcutaneous tumor was established in 4-week old nude mice using UTUC cells. All applicable institutional and national guidelines for the care and use of animals were followed. The tumors were observed from the first day after inoculation. pHLIP-Cy5.5 was used as the positive control, prepared as previously described (19). Uncoupled UCPs were used as the negative control. A foreign body was implanted at the side of the inoculation site to cause local inflammation and to observe the effect of inflammation on the sensitivity of the probe.

Cultured urinary tract epithelial cancer cell line T₂₄ was cultured. About 0.5×10⁶ cells in 0.1 ml were injected in the diaphragm below the heart. PHLIP-UCP solution (1 ml) was injected in the abdominal cavity of each animal.

Optical detection was performed using a small animal living body multi spectral imaging system (Maestro; CRi, Inc., Woburn, MA, USA). The spectral detection range was 400–950 nm. The excitation source (infrared laser diode) was set at 980 nm and the power was 0.2 W (Changchun Mingwan Optics Co., Changchun, China) (20). The image was captured by a CCD camera (Andor Clara with Sony ICX285 CCD; Sony, Connecticut, NE, USA). Images were analyzed using the Maestro software analysis system 2.4 (CRi, Inc.). Fluorescence intensity was determined to represent the enrichment degree and sensitivity of the probe to the tumor cells.

Fig. 1 shows that the UCPs dispersed in chloroform emitted no light under natural light (center vial), while they emitted a green light when excited with a 980-nm laser (left vial).

The spectra presented in Fig. 2 were captured using green and red filters. The graph shows that the emission of the excitation channel was uniform and transparent and the intensity was high. The maximum emission wavelength of Ho³⁺-doped UCPs was about 550 nm and the red emission region was around 650 nm. When the doping was 2%, the green luminescence dominates and the proportion of red emission was increased when the proportion of active ions was increased.

The diameter of the UCPs was 10.9±1.7 nm. They had a round shape. The NaYbF₄ particles had a crystal structure of about 0.38 nm composed of a planar hexagon. The surface of NaYF₄: Yb, Ho³⁺UPCs was coated with a uniform layer of silica. Fig. 3 shows that the UCPs dispersed evenly in aqueous solution and that the surface of the particles was coated with peptides to achieve the function of the UPCs. A continuous process of silane reaction is shown in Fig. 3. It can be seen that the UCPs were uniformly coated with a layer of silane and that the shell became gradually thicker with elapsing time.

As shown in Fig. 4, the solution is transparent in aqueous solution. Because the coated UCPs had a tendency to agglomerate and precipitate, the yield of the UCPs in the aqueous phase was reduced and the overall fluorescence intensity of the aqueous phase particles was reduced. Compared with the corresponding organic phase solution, the fluorescence intensity was decreased.

The nanoparticles were injected into the frontal chest and diaphragm (below the heart) of the mice. Fig. 5A shows the mice and tumor imaging after UCP injection. Fig. 5B shows that the tumor is visible with a visible 550 nm upconversion fluorescence emission. By analyzing the spectral slices at 550 nm, as shown in Fig. 5C, the fluorescence intensity measurement is carried out by selecting the fluorescence emission region and the surrounding background region. The average counts signal is obtained and the average intensity signal is plotted over the corresponding region. Regions 3 and 4 represent the upconversion fluorescence region while regions 1 and 2 represent the background region. Taken from the ventral side, the image shows a signal-to-noise ratio of 18.5. Fig. 5D shows that the angle of view is important. Indeed, when imaged from the side, the ribs interfere with the laser and the emitted light, resulting in weaker signal. This can be overcome by increasing exposure time, but the background noise is increased. Spectral analysis of this region, as shown in Fig. 5E, shows that the 550-nm fluorescence emission is very close to the background. By analyzing the fluorescence intensity of the 550 nm spectra, as shown in Fig. 5F, the signal-to-noise ratio is 2.08, showing that the signal is still positive. Fig. 5G shows the relative location map of the conversion fluorescence emission for deep injection, which is expressed in the form of intensity.