*4.5. Layer-by-Layer Assembly*

Layer-by-layer assembly involves the electrostatic adsorption of oppositely charged anions or cations on the UCNP surface. Electrostatic attraction is one of the strongest and most stable interactions known in nature. Specific layer-by-layer modification usually contributes to the specific biological applications of UCNP. The advantage of this method is that it can prepare coated colloids of different shapes and sizes, with uniform layers of different compositions and controllable thickness [139]. Most importantly, this method can control the surface potential, size, and incorporated functional group ligands of UCNP, which is very important for cell internalization and biological targeting. Polyacrylic acid (PAA) coated with Yi groups on the surface of upconversion luminescent nanocrystals by the layer assembly method. PAA contains 25% octylamino groups and 40% isopropylamine groups, which can transfer oil-soluble upconversion luminescent nanocrystals to the water phase and further couple with biomolecules [140].

In addition to the improvement of biocompatibility, surface modification can also improve the optical properties of UCNPs, laying a foundation for the further application of UCNPs in bioimaging and other fields.

#### **5. Biological Applications of UCNPs**

Compared with traditional luminescent materials (including organic dyes and quantum dots), UCNPs have the advantages of high chemical stability, good optical stability, and narrow band gap emission. In addition, under the excitation of near-infrared light, it has strong biological tissue penetration, no damage to biological tissue, high signal-tonoise ratio, and has been widely used in the biological field. This paper also focuses on the application of up conversion luminescent materials in biological imaging, biological detection, and photodynamic therapy [141,142].

For highly infiltrative cancers, including glioblastoma multiform (GBM), it cannot be resected completely. Routinely, the conventional direct introduction to UCNPs suspension into the tissue will cause a series of biocompatibility problems. Thus, implantation of optical fiber has a heavy burden on the patient's body and a high risk of infection. Daniel et al. [143] reported the fabrication of an optical-guided UCNPs implant. Comparatively, the highly biocompatible UCNPs implant is placed into the brain via craniotomy and sealed. Then, the NIR light source passes through the healed scalp and points in the implant to stimulate the UCNPs implant, which will emit visible light to target the photosensitive metabolite protoporphyrin-IX (PpIX) in brain tumors. The flexible light guide with FEP coating approved by the FDA can also maintain NIR to visible light spectrum transduction when the implant is bent to 90◦, as shown in Figure 4a. The tumor size of PDT-treated mice was significantly smaller than untreated mice, as shown in Figure 4b. Implant-based UCNPs also allow them to recover from the tissue when they are no longer needed, which cannot be achieved by direct injection of UCNPs suspension into the tissue. A wide range of emission spectrum can be engineered into UCNPs so that the implant can activate multiple drugs simultaneously without cross-interference.

Wang et al. [144] reported a method that can change intracellular pH UCNP@ZIF + TPP + PA nanometer material. The intracellular pH is usually weakly alkaline, which is not conducive to the application of acid-responsible nanomaterials. The proposed new nanomaterials (UCNP@ZIF + TPP + PA) were designed with NaYF4: Yb: Tm (UCNPs) inner core coated by the framework of porous imidazolium zeolite (ZIF-8) for co-loading of photoacid (PA) and acid-responsive porphyrin (TPP). With 980 nm laser irradiation, the emission light of UCNPs activated PA to release H+. In the new acidic environment, TPP was protonated to increase its water solubility and reduce its aggregation. Simultaneously, transformed UV Vis also activates protonated TPP, which produces more singlet oxygen (1O2) to kill cancer

cells, to enhance the therapeutic effect of photodynamic therapy (PDT); the synthesis route and therapeutic principle are shown in Figure 5.

**Figure 4.** (**a**) UCNPs implant when not excited with NIR and emission intensities visualization at different angles of UCNPs implant bending, excited with 1583 mW cm<sup>−</sup><sup>2</sup> of NIR. (**b**) IVIS imaging indicates that PDT mouse tumors were regressing, as compared to other control groups. The normalized change of tumor burden in all experiment groups over time (*n* = 5 mice/group. \* *p* = 0.0327, \*\*\* *p* = 0.0002, \*\*\*\* *p* < 0.0001. Two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test), reprinted with permission from ref. [143]. Copyright 2020 John Wiley and Sons.

**Figure 5.** Synthetic route and anticancer mechanism of UCNP@ZIF + TPP + PA. Responding to weak acid tumor microenvironment, UCNP@ZIF + TPP + PA released TPP. When acid-responsive TPP entered the weak alkaline cell, it aggregated again. With 980 nm light irradiation, UCNP emitted UV-Vis light. Photoacid absorbed UV-Vis light and changed its structure to produce H+, which restructured the intracellular pH value. In the new weak pH, the aggregation of TPP was decreased by its protonation. Meanwhile, protonated TPP was activated by the transformed UV-Vis light and produced more 1O2 for enhancing PDT, reprinted with permission from ref. [144]. Copyright 2014 Royal Society of Chemistry.

One of the key challenges in the process of PDT therapy is the accurate killing of cancer cells without destroying normal cells to achieve the desired therapeutic effect. Li et al. [145] designed a type of upconversion nanoprobes (mUCNPs) for intracellular cathepsin B (CAB) reactive PDT, composed of multi-shell upconversion nanoparticlesNaYF4: Gd@NaYF4:Er, Yb@NaYF4: Nd, Yb), with the function of in situ self-tuning therapy effect prediction, as shown in Figure 6.

**Figure 6.** Schematic illustrations of (**a**) synthesis of the upconversion nanoprobe and (**b**) Intracellular CaB-Activated PDT with CaB imaging for Therapeutic Effect Prediction, reprinted with permission from ref. [145]. Copyright 2020 American Chemical Society.

Similarly, the Zhang group [146] designed an amplifier with multiple upconversion luminescence, composed of photo-caged DNA nano-combs and upconversion nanoparticles (UCNPs) sensitized with IRDye® 800CW, to realize the near-infrared light switch cascade reaction triggered by specific microRNA and accurate photodynamic therapy for early cancer. Under 808 nm light irradiation, the generated ultraviolet light cuts off the "photozipper" to induce the cascade hybridization reaction of the microRNA response. This activates the photosensitizer connected to different hairpins to produce reactive oxygen species (ROS) under the blue light emitted simultaneously, to carry out effective PDT, as shown in Figure 7. The amplifier showed desirable serum stability, excellent controllability of reactive oxygen species generation, high specificity for target cancer, and sensitivity to specific microRNA expression. In vivo and in vitro experiments showed strong inhibition on cell proliferation, strong ability to induce apoptosis of tumor cells, and distinct inhibition of tumor growth.

**Figure 7.** (**a**) Schematic illustration of NIR photo-switched miRNA amplifier for precise PDT. (**b**) Representative images at day 10 and (**c**) tumor volumes of early-stage breast cancer-bearing mice, treated with saline, CSUCNPs-DNC'Pc-PPa' and CSUCNPs-DNCPc-PPa' before and after 808 nm light irradiation at 1 W/cm2. Error bars indicate means ± SD (*n* = 5), reprinted with permission from ref. [146]. Copyright 2020 John Wiley and Sons.

Lin et al. [147] designed a spindle-like UCNPs nanoprobe, coated with a layer of gold nanoparticles to enhance upconversion luminescence (UCL), as shown in Figure 8. The results of biocompatibility, blood routine, bioimaging, and anti-cancer tests showed that it was easier for the spindle-like nanoprobes to enter biological tissues. In addition, the combination of SPS@Au and ZnPc (SPSZ) is a potential candidate for synergistic immune photodynamic therapy (PDT), with enhanced UCL effect and excellent biocompatibility.

**Figure 8.** TEM image of (**a**) spindle precursor and (**b**) SPS@Au (LBL1) and (**c**) SPS@Au (LBL2) and (**d**) SPS@Au (LBL3). Reprinted with permission from ref. [147]. Copyright 2020 American Chemistry Society.

The Li group [148] designed core-satellite metal-organic through electrostatic selfassembly framework@UCNP superstructures, composed of a single metal-organic framework (MOF) NP as the core, and Nd3+-sensitized UCNPs as the satellites. In vitro and in vivo experiments show that the double photosensitizer superstructure has a three-mode (magnetic resonance/UCL/ fluorescence) imaging function and excellent anti-tumor effect under the excitation of 808 nm near-infrared light, avoiding overheating caused by laser irradiation. After being exposed to an 808 nm laser for 5 min, the temperature of the irradiated area was lower than 42 ◦C, and without damage to mice. However, under the same conditions, a 980 nm laser can heat the irradiated area to above 50 ◦C and severely burn the skin. These findings indicate that 808 nm excitation has a much weaker tissue thermal effect and is more suitable for biological applications.

Sun et al. [149] designed and prepared one kind of lanthanide (Ln3+)-doped upconversion nanocomposites with multi-functions, which can not only provide temperature

feedback in PTT process, but also play the photodynamic therapy (PDT) function for the synergistic effect of tumor therapy. Based on NaYF4:Yb, Er upconversion nanoparticles (UCNPs), mesoporous SiO2 was modified on the surface combined with photosensitizer Chlorin e6 (Ce6) molecules, which could be excited by red emission of Er3+ under the 980 nm laser. Cit-CuS NPs were further linked on the surface of the composite as a photothermal conversion agent, therefore, the temperature of the PTT site can be monitored by recording the ratio of I525/I545 of green emissions, especially within the physiological range, as shown in Figure 9. Based on the guidance obtained from spectral experiments, they further investigated the dual-modal therapy effect both in vitro and in vivo, respectively, and acquired decent results.

In addition, these rare earth doped nanoparticles also have strong scintillation luminescence that can be used for X-ray induced photodynamic therapy, which is a very hot area, as this new therapy can be used for deep as well as skin cancer treatment [150–163].

**Figure 9.** (**a**) Schematic diagram of the detection of the temperature and emission spectrum of UCNPs-Ce6@mSiO2-CuS incubated with cells in physiological range; (**b**) UCL emission spectrum of UCNPs-Ce6@mSiO2-CuS incubated with cells at different temperatures by external heating. The peaks were normalized at 525 nm; (**c**) FIR of the green UC emissions for the 2H11/2/4S3/2 → 4I15/2 transitions relative to the temperature of UCNPs-Ce6@mSiO2-CuS incubated with cells. The inset picture is the AFM image of the cell after spectral detection; (**d**) A plot of ln(I525/I545) versus 1/T to calibrate the thermometric scale for UCNPs-Ce6@mSiO2-CuS incubated with cells. Reprinted with permission from ref. [149]. Copyright 2019 Elsevier.

#### **6. Summary and Perspective**

Over the last decades, UCNPs have made remarkable advances in the treatment of critical diseases, greatly promoting the application of modern precision medicine in the life system with its enhanced therapeutic effect, high space-time controllability, deep tissue penetration, and minimal invasion. However, despite the remarkable achievements, there are still some challenges of UCNPs. (1) The stability of luminous efficiency after surface modification: in the surface modification, the oil-soluble molecules will be modified to improve biocompatibility. Nevertheless, the dispersing ability of UCNPs in oil and water is different. After surface modification, it is easy to cause fluorescence quenching and reduce the upconversion efficiency. (2) The biological toxicity of UCNPs: many studies have shown that the reasonable optimization of chemical composition, particle size distribution, and surface modification can significantly improve the biocompatibility of UCNPs, which can be used in biomedical applications. However, there are no tests to evaluate its longterm toxicity, including potential immune response and mutagenic effect. (3) Technical gaps in clinical trials: so far, UCNPs-based phototherapy has not been applied to human beings, mainly due to biosafety or therapeutic effect. There is still a long way to go from laboratory animals to human-level technical standard updates. In summary, UCNPs offer a tremendous opportunity to practice precision medicine. We expect that stable surface modification, low toxicity, and clinical trials will make UCNPs more competitive in the biological field.

**Author Contributions:** Conceptualization, methodology, Q.L.; validation, formal analysis. and; investigation, resources, visualization; writing—original draft preparation, C.G.; data curation, P.Z. and S.H.; supervision, writing—review and editing, J.W., C.X., T.J., H.T. and W.C.; project administration and instruction, J.W.; funding acquisition, Y.W., D.L. and S.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Science Foundation of China No. 11904125, the Open Research Subject of Key Laboratory of Dielectric and Electrolyte Functional Material Hebei Province No. HKDE201902, BIGC Project No. 22150121034/026, No. 22150121003/050, No. Eb202105, and JENU No. XZD201802. W. C. would like to thank Solgro Inc. and UT Arlington distinguished award.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The study did not report any data.

**Conflicts of Interest:** The authors declare no conflict of interest.
