Imparting multi-functionality to covalent organic framework nanoparticles by the dual-ligand assistant encapsulation strategy

The potential applications of covalent organic frameworks (COFs) can be further developed by encapsulating functional nanoparticles within the frameworks. However, the synthesis of monodispersed core@shell structured COF nanocomposites without agglomeration remains a significant challenge. Herein, we present a versatile dual-ligand assistant strategy for interfacial growth of COFs on the functional nanoparticles with abundant physicochemical properties. Regardless of the composition, geometry or surface properties of the core, the obtained core@shell structured nanocomposites with controllable shell-thickness are very uniform without agglomeration. The derived bowl-shape, yolk@shell, core@satellites@shell nanostructures can also be fabricated delicately. As a promising type of photosensitizer for photodynamic therapy (PDT), the porphyrin-based COFs were grown onto upconversion nanoparticles (UCNPs). With the assistance of the near-infrared (NIR) to visible optical property of UCNPs core and the intrinsic porosity of COF shell, the core@shell nanocomposites can be applied as a nanoplatform for NIR-activated PDT with deep tissue penetration and chemotherapeutic drug delivery.


Synthesis of core@shell structured nanocrystals:
The core@shell nanoparticles were fabricated by using the one-pot successive layer-by-layer (SLBL) protocol, which was developed by our group. [6] 2.5 mL of the purified NaYF4:Yb/Er core nanoparticles solution (~ 0.25 mmol) were mixed with 4.0 mL of OA and 6.0 mL of ODE. The flask was pumped down at 70 °C for 30 min to remove cyclohexane. After that, the system was switched to Ar flow and the reaction mixture was further heated to 280 °C at a rate of ~ 20 °C/min. Then pairs of Gd-OA (0.10 M, 1.0 mL) and Na-TFA-OA (0.40 M, 0.50 mL) precursors were alternately introduced by dropwise addition at 280 °C and the time interval between each injection was 15 min. Finally, the obtained S4 NaYF4:Yb/Er@NaGdF4 core@shell nanoparticles were precipitated and washed in the same way as the core nanoparticles, and dispersed in cyclohexane.
The core-shell NaGdF4:5%Nd@NaGdF4 nanoparticles were also synthesized by the similar protocols described above except that the precursors were correspondingly replaced.

Surface modification with PEI
The as-prepared seeds (e.g. SiO2 nanospheres) were dispersed in water at a concentration of 20 mg/mL, then 1.0 mL of PEI aqueous solution (100 mg/mL) was slowly added into the dispersion.
The mixture was stirred at room temperature for 1 h. Then, the PEI modified nanoparticles were collected and washed by high-speed centrifugation.

Selectively etching of SiO2 from the core@shell structured SiO2@COF
To obtain bowl-shape COF, the SiO2@COF prepared with 2.0 mg of DMTP was dispersed in 2 M NaOH aqueous solution and stirred overnight. The products were obtained and repeatedly washed by centrifugation. Similarly, the SiO2@COF prepared with 4.0 mg DMTP can transform to hollow structured COF after the etching process.

Synthesis of functional UCNPs@SiO2
To synthesize UC-COF, the oleic acid-stabilized upconversion NaYF4:Yb/Er@NaGdF4 nanocrystals were transferred to hydrophilic by coating a silica shell. Inverse microemulsion method was used to synthesize NaYF4:Yb/Er@NaGdF4@SiO2 according to our previous report. [7] Then the NaYF4:Yb/Er@NaGdF4@SiO2 nanoparticles were modified with PEI as described above.

Synthesis of SiO2@LZU-1 and porphyrin-based COF coated SiO2
For the synthesis of SiO2@LZU-1, 6 mg of PEI modified SiO2 nanospheres and 40.0 mg of PVP were added into 10.0 mL mixed solution of o-dichlorobenzene/n-butyl alcohol (v/v = 1:1). The dispersion was treated with ultrasonic for more than 0.5 h. Then, 3.5 mg of triformylbenzene and 3.5 mg of p-phenylenediamine were added. After stirring for 5 min, 100 μL of glacial acetic acid was added. The reaction was maintained at room temperature for 4 h. Afterward, 400 μL of acetic acid and 480 μL of DI water was added. The mixture was degassed by three freeze-pump-thaw cycles, sealed under vacuum, and kept at 120 °C for 5 days. The products were obtained and washed with acetone by centrifugation.
For the growth of porphyrin-based COF, the PEI modified SiO2 nanospheres were used as core S5 and the synthetic procedures were identical with the synthesis of UC-COF.

Characterization
The morphology of prepared nanoparticles was observed by transmission electron microscopy (TEM) under accelerated voltage of 200 kV (FEI, American). Scanning electron microscopy (SEM) images were captured using field emission scanning electron microscopy (FESEM, Hitachi S-4800, Japan). X-ray diffractions (XRD) of samples were recorded on a D/max-2550 PC X-ray diffractometer (XRD; Rigaku, Japan). UV-vis-NIR absorption spectra were measured on a Shimadz spectrophotometer (UV-3150) (Japan). Size distribution and Zeta potential of the samples were recorded by using Zetasizer Nano ZS apparatus (Malvern, UK). The concentrations of Gd element were analyzed by a Leeman Prodigy inductively coupled plasma-atomic emission spectroscopy (ICP-AES) system (Hudson, NH03051, USA). Fourier transform infrared (FTIR) spectra were recorded using a NICOLET MX-1E FTIR spectrometer.

In vitro cytotoxicity evaluation
The cells were cultured in standard Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% (v/v) FBS, 100 mg mL −1 streptomycin and 100 U mL −1 penicillin at 37 °C in a humidified incubator with 5% CO2.
The cytotoxicity of the prepared UC-COF was assessed using standard CCK-8 assay. 4T1 or HUVECs were seeded in 96-well plates (10 4 cells/well) for 24 h. After that, the cells were incubated with fresh medium containing different concentrations of UC-COF (400, 200, 100, 50, 25, 12.5, 6.25 μg/mL) for another 24 h. Then, the medium was discarded and the cells were slightly washed by PBS, followed by the standard CCK-8 protocol according to the manufacturer's instruments.
Cells treated with pure medium without UC-COF was used as control group. Four parallel experiments were performed for each group.

In Vitro Cell Uptake
The cell uptake of UC-COF was first investigated by confocal laser scanning microscopy (CLSM, Olympus FV900). 4T1 cells were seeded in glass-bottom culture dish (3.0 × 10 5 cells per dish) and incubated with UC-COF at two different pH conditions (7.4 and 6.5) for 4 h. Subsequently, the cells were slightly rinsed with PBS, fixed with 4% paraformaldehyde, and stained with DAPI for direct observation.

In vitro ROS detection
To evaluate the ROS generation ability, 200 μL of UC-COF in DMSO was mixed with 10 μL of DPBF solution (6 mM). The mixed solution was then irradiated by 980 nm laser (1 W/cm 2 ). The change of absorption of DPBF at 420 nm was recorded by a UV-vis spectrophotometer.

Tissue depth-dependent PDT
Further, the impact of tissue depth on killing effect of NIR light-triggered and visible lighttriggered PDT was also investigated. The cells were subjected to the same treatment as described above, except that the cell plates were placed under a cylindrical culture dish with 4-mm thickness 1% Intralipid®, which was applied as a simulated tissue due to its similar scattering characteristics. [8] The cell viability of different groups was evaluated by CCK-8 assay, and the cells were stained with Calcein-AM/PI after different treatments for CLSM observation.

Statistical analysis
At least three independent groups were performed throughout experiment. Significant difference between different groups was analyzed by one-way analysis of variance (ANOVA) and Turkey's post hoc test. The criterion was expressed as *P < 0.05 and **P < 0.01.    In addition, the COF shell become thicker after the thermal crystallization, indicating that the crystallization process is accompanied with the reconstruction of the polymer shell to form the highly crystalline COF shell.   In the absence of PEI and PVP, the COF shell is not encapsulated on SiO2 and only phase-separated flake-like COF is observed, implying that the interaction between COF monomers and SiO2 nanospheres is very weak. When only PEI is modified on the SiO2 surface, the core@shell structured SiO2@COF nanocomposites with serious agglomeration is obtained, and the products are settled down at the bottom of the flask after the reaction, indicating the poor colloidal dispersity of the obtained samples. In S25 the case of PVP modified SiO2, although the core@shell structured can be formed, the phase-separation is not avoided, and the thickness of the COF shell is obviously thinner than that of on the PEI-modified SiO2. with the increased amount of PVP, further confirming that PVP can regulate the growth of COF shell. Cooperated with PEI, the growth of COF shell can be well manipulated to form monodispersed core@shell structure.