Hydrogen‐bonded organic framework core–shell composite for synergistic antimicrobial therapy

Hydrogen‐bonded organic frameworks (HOFs) are crystalline porous materials with permanent voids formed via self‐assembly of organic molecules through hydrogen bonding and intermolecular forces. Further combination of HOFs with functional material would broaden their application horizon but were less explored in existing literature. Herein, a highly porous and photosensitive HOF was successfully coated onto upconversion nanoparticles (UCNPs) to construct a core–shell structure named UNCPs@PFC‐73‐Ni. To enhance spectral overlap and maximize energy conversion efficiency, this study utilized the Er and Tm co‐doped UCNPs, which can effectively convert infrared light into visible light emission thereby exciting the porphyrin shell. Subsequent investigation reveals that the composite exhibits significant photodynamic and photothermal effects under infrared light. Encouraged by its noticeable photoactivity, UCNPs@PFC‐73‐Ni was evaluated as an antibacterial agent against Escherichia coli. Notably, significant antibacterial efficacy was observed, highlighting the potential of UCNPs@PFC‐73‐Ni as an effective antibacterial agent under infrared light irradiation.


INTRODUCTION
As a developing porous crystalline material, hydrogenbonded organic framework materials (HOFs) [1,2] have gained wide usage in diverse fields, including gas adsorption and separation, [3,4] catalysis, [5] fluorescence sensing, [6][7][8][9][10] and biomedicine, [11][12][13] owing to their attributes of effortless synthesis, permanent pore, and various organic ligands.Nevertheless, customizing HOFs to satisfy specific functional needs has proved to be quite a challenging task.Simply modifying the constituent building blocks of HOFs is often insufficient in addressing complex functional requirements.A more effective alternative approach involves integrating materials with desired functionalities into HOFs.This integration enables the realization of functions that are otherwise unattainable with stand-alone HOFs.However, it is a great challenge to integrate a variety of materials with different functions into a composite with HOFs.In previous reports, molecular recognition, [14] ion exchange, [15] and "ship-in-a-bottle" strategies [16] have been exploited to introduce small active molecules or inorganic nanoparticles into the cavities of HOFs for fabricating multifunctional composites.Additionally, constructing composite materials through the reverse approach of creating a core-shell structure has been found to be effective.
Porphyrins are effective organic photosensitizers that play crucial roles in photodynamic and photothermal bacteriostasis. [17,18]Among these, PFC-73-Ni, [19] derived from [5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin]-Ni(TCPP-Ni) and formed through the self-assembly of HOFs with a square-layer network topology, demonstrated positive environmental stability and exhibited a wide light absorption range in the ultraviolet-visible (UV-vis) band.These attributes position PFC-73-Ni as a promising contender for applications in photodynamic and photothermal bacteriostasis.As is widely understood, infrared light is commonly employed as a light source for biotherapy and sensing due to its excellent tissue penetration and biocompatibility.
However, the infrared part's absorption of PFC-73-Ni is feeble, thereby confining its application in photochemistry.To address this issue, we decided to combine PFC-73-Ni with materials that have the ability to produce upconversion luminescence.
Upconversion luminescence [20] is a phenomenon wherein two or more photons of low energy and long wavelengths are successively absorbed to emit photons of higher energy with shorter wavelengths.Among the various types of upconversion nanoparticles (UCNPs), NaYF 4 -based nanocrystals [21][22] are commonly utilized, which can emit short-wavelength, high-energy light in the UV-vis band by adjusting the doped rare earth ions and their content under 980 nm infrared light excitation.Herein, to enhance the overlap between the upconversion luminescence spectrum and the absorption range of PFC-73-Ni, multi-shell UCNPs co-doped with Er and Tm were employed, which emits both blue and green light simultaneously (Figure S5).This is in contrast to the more frequently used single Er-doped UCNPs emitting only green light.
In this study, core-shell nanoparticle complexes were synthesized by combining Er-Tm co-doped UCNPs with PFC-73-Ni to achieve photodynamic and photothermal.The UCNPs were activated by infrared light, which upconverted into high-energy photons and was absorbed by the outer PFC-73-Ni layer to achieve the therapeutic effects of photodynamic and photothermal bacteriostasis, showing the application potential in the field of antimicrobial therapy.

Synthesis of UCNPs
The synthesis of the materials was based on the literature that has been reported. [23]In general, 1.0 mmol of rare-earth chloride (RECl 3 ) (RE = 0.79Y + 0.2Yb + 0.01Tm) was mixed with 6 mL of oleic acid (OA) and 15 mL of 1-octadecene (ODE) in a three-orifice flask.The solution was heated to 100 • C under a vacuum to remove impurities from the reaction vessel.The solution was then heated to 156 • C to obtain a homogeneous solution in an N 2 environment and then cooled to room temperature.Ten milliliters of methanol solution containing 0.100 g NaOH and 0.148 g NH 4 F was slowly added to the flask and stirred for 30 min.The methanol in the system was then removed by slow heating and vacuumed at 100 • C for 10 min, followed by a final rapid heating to 300 • C and reaction for 1 h under the protection of N 2 .After the reaction system was cooled, 30 mL of ethanol was added to precipitate the product, which was washed several times with ethanol and cyclohexane.The final NaYF 4 :Yb, Tm UCNPs were dispersed in 5 mL cyclohexane for subsequent use.An amount of 2.5 mL of purified NaYF 4 :20%Yb, 2% Er nanoparticle solution was mixed with 10 mL of OA and 10 mL of ODE.The resulting mixed solution was evacuated at 110 • C for 30 min to remove cyclohexane and then heated to 310 • C under N 2 atmosphere.Furthermore, 4 mL of a mixed solution of OA and ODE (v/v = 1:1) containing 0.25 mmol of CF 3 COONa and 0.25 mmol of Y(CF 3 COO) 3 was injected into the reaction system.One hour later, the mixed solution of 4 mL of OA, ODE (v/v = 1:1) containing 1 mmol of CF 3 COONa, 0.4 mmol of Yb(CF 3 COO) 3 , 0.05 mmol of Tm(CF 3 COO) 3 , and 0.595 mmol of Y(CF 3 COO) 3 was injected into the reaction system.After 1 h, 4 mL of a mixed solution of OA, ODE (v/v = 1:1) containing 0.5 mmol of CF 3 COONa, and 0.5 mmol of Y(CF 3 COO) 3 was injected into the reaction system, and the reaction was continued for 1 h.Finally, the reaction mixture was cooled to room temperature, and the product was collected by centrifugation and dispersed in cyclohexane for further use.

Synthesis of PFC-73-Ni
PFC-73-Ni was synthesized following a slightly modified pathway reported previously. [19]The synthesis method of TCPP-Ni is shown in Figure S1.

Synthesis of UCNPs@PFC-73-Ni
TCPP-Ni (10 mg) was dissolved in DMF (5 mL) before the UCNPs (5 mg, 0.2 mL) were dispersed.The mixture was sealed and stirred at 100 • C for 24 h.The red powder was collected via centrifugation at 12,000 rpm for 5 min and washed with dichloromethane and DMF.After vacuum drying at 80 • C, red samples of TCPP-Ni ligandanchored UCNPs (abbreviated as UCNPs-L) were collected.The UCNPs-L obtained above was added to a 15 mL pressure-resistant tube together with 10 mg TCPP-Ni, and 1 mL DMF was added to 2 mL TCB, which was dissolved and dispersed by sonication.The solution was placed in an open oil bath at 90 • C and stirred for volatilization.With the volatilization of DMF, TCPP-Ni undergoes heterogeneous nucleation on the surface of UCNPs-L.The resulting red powder (UCNPs@PFC-73-Ni) was washed several times with dichloromethane.

Characterization
The building block and crystal structure model of PFC-73-Ni are shown in Figure 1A.The core-shell UCNPs@PFC-73-Ni nanocomposites were synthesized using the fractionation method (Figure 1B).Initially, UCNPs with oil solubility were synthesized, followed by the mixing of UCNPs with TCPP-Ni in DMF.The strong affinity of the carboxyl group for metal ions allowed for the replacement of OA molecules on the surface of UCNPs at 100 • C heating, resulting in UCNPs-L with TCPP-Ni anchors. [20]Subsequently, PFC-73-Ni grows on the surface of UCNPs-L by heterogeneous nucleation, leading to the final formation of UCNPs@PFC-73-Ni with a core-shell structure.The synthesis process was characterized using transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR).TEM analysis revealed successful synthesis of UCNPs with OA, and the OA layer disappeared after ligand substitution.The UCNPs@PFC-73-Ni had a PFC-73-Ni layer with a thickness of approximately 5-6 nm (Figure 2B).FTIR analysis showed the disappearance of the OA absorption peak (2920 cm −1 for methylene [─CH 2 ─] stretching vibration) [24] after ligand substitution, and the appearance of the TCPP-Ni-related infrared absorption peak (1680 cm −1 for the carboxylic group [─C═O] stretching vibration and 1398 cm −1 for imine [─C─N─] bending vibration) (Figure S6). [25]Additionally, powder X-ray diffraction (PXRD) analysis confirmed the successful preparation of UCNPs@PFC-73-Ni, showing good agreement with PFC-73-Ni and UCNPs, as the diffraction peaks of the first 30 • closely matched those of PFC-73-Ni (Figure 2A).The upconversion emission spectra of UCNPs and UCNPs@PFC-73-Ni were measured and normalized with emission at 660 nm.A comparison of the two groups of spectra showed that the emission intensity of UCNPs@PFC-73-Ni at 300-600 nm was significantly decreased compared to that of unwrapped UCNPs (compared with that at 660 nm), indicating that the emission light of the corresponding wavelength (250-600 nm) emitted by the core UCNPs was absorbed by the shell PFC-73-Ni.This result was consistent with the strong absorption capacity of PFC-73-Ni in the UV-vis absorption spectrum (Figure 2C).The UV-vis-near-infrared (NIR) absorption spectra of UCNPs, PFC-73-Ni and UCNPs@PFC-73-Ni are shown in Figure S7.When contrasted with the individual UCNPs sample, UCNPs@PFC-73-Ni showed shorter lifetimes, supporting the existence of Förster resonance energy transfer (FRET) between two components. [26]Meanwhile, the FRET efficiencies were calculated according to the shortened luminescence lifetimes at the overlapped wavelength (540 nm) using Equation (S1), resulting in a FRET efficiency of 61.4% for UCNPs@PFC-73-Ni (Figure 2D).

Photodynamic therapy
Using 2,2,6,6-tetramethyl-1-piperidinyloxy as a 1 O 2 capture agent, [27] the generation of triplet peaks in the electron paramagnetic resonance (EPR) spectrum under NIR irradiation verified that the photoinduced reactive oxygen species was 1 O 2 .To evaluate the photodynamic activity at 980 nm, we conducted an EPR analysis, which revealed that after 15 min of 980 nm illumination, the EPR spectrum of the UCNPs@PFC-73-Ni solution exhibited a clear triplet peak, which was significantly higher than its intensity under dark conditions (Figure 3A).This confirmed that UCNPs@PFC-73-Ni could effectively produce a photodynamic effect under infrared conditions.
To evaluate singlet oxygen production under NIR light irradiation, we used 1,3-diphenylisobenzofuran (DPBF) [28] as a singlet oxygen indicator and a 980 nm (1.5 W/cm 2 ) laser as the light source to illuminate the sample.We regularly detected the attenuation of the absorbance of the suspension at 300-500 nm over time using a UV-vis spectrophotometer to evaluate the singlet oxygen production of the material.Meanwhile, the DPBF solution was used as the blank control group without any treatment.At 980 nm, the degradation of the DPBF solution in the blank group was almost nonexistent, and the degradation ability of the DPBF solution in the pure PFC-73-Ni condition was almost negligible.However, UCNPs@PFC-73-Ni achieved about 15% degradation of the DPBF solution in 20 min under the irradiation of 980 nm (Figures 3B and S8).This confirmed that UCNPs@PFC-73-Ni could produce singlet oxygen under infrared light with an excellent photodynamic effect.

Photothermal therapy
To observe the photothermal properties of the material more directly, the material was laid on a quartz sheet, and the temperature change process of the material was observed and recorded under infrared light irradiation. [29]Upon irradiation with a 980 nm infrared laser (1.5 W/cm 2 ), the temperature of the composite rapidly reached about 170 • C, while PFC-73-Ni alone only reached about 110 • C (Figure 3C).As organic materials have a low specific heat capacity and the sample is porous, it tends to absorb water vapor from the air, leading to significant changes in its specific heat capacity, making the photothermal precision of the solid powder less accurate.Therefore, a 0.5 mg/mL water dispersion was used to measure the photothermal conversion efficiency of the material. [30]he data from the above experiment showed that the aqueous solution of UCNPs@PFC-73-Ni reached 61.4 • C when exposed to infrared light, whereas the aqueous solution of PFC-73-Ni only reached 52.6 • C (Figure 3D).By calculating the relevant formula for photothermal conversion efficiency, it was determined that UCNPs@PFC-73-Ni has a conversion efficiency of 14.83%, which was higher than the 8.36% conversion efficiency of PFC-73-Ni alone (Figure S9 and Table S1).In addition, we present complementary PXRD patterns of UCNPs@PFC-73-Ni before and after 48 h in aqueous solution.The photothermal stability of UCNPs@PFC-73-Ni was also investigated (Figure S10).These results indicate that the photothermal effect of the composite material is superior to that of its components.

Bacteriostatic experiment
The outstanding photothermal and photodynamic effects of UCNPs@PFC-73-Ni inspired us to investigate its antimicrobial performance.E. coli was chosen as the model microorganism to microorganism to evaluate the antibacterial activity. [31]E. coli was cultured using a plate coating method; and then incubated in a blank solution, PFC-73-Ni, and UCNPs@PFC-73-Ni (0.5 mg/mL aqueous solution, 2 mL) for 20 min on the ultra-clean table while being irradiated with a 980 nm laser.The bacterial solution was diluted 10 6 times with ultrapure water, coated, and cultured on solid Luria-Bertani agar plate for 20 h.The amount of colony growth was compared to evaluate the antibacterial activity of the material.Additionally, a group of material solution cultures was cultured without exposure to light.The antibacterial activity rate of the material was calculated by dividing the number of colonies after treatment by the number of colonies in the dark condition without any treatment.Through calculation, it can be seen that the inhibition rate of the control group was approximately 9.9% under infrared light irradiation.The inhibition rate of PFC-73-Ni was 56.5% in the dark condition and 82% under infrared light.UCNPs@PFC-73-Ni exhibited an inhibition rate of 64.3% in dark conditions and 97.4% under infrared light (Figure 4).In general, the construc-tion of a core-shell nanostructure has led to the successful realization of a synergistic photothermal and photodynamic antimicrobial effect in response to NIR light.

CONCLUSION
In this work, we achieved the successful synthesis of hexagonal upconverted nanoparticles with a co-doped structure of Er and Tm, which exhibited impressive luminescence effects by efficiently converting infrared excitation light into visible wavelengths.Building upon this, we employed a heterogeneous nucleation and ligand substitution process to grow hydrogen-bonded organic frameworks on the surfaces of these nanoparticles.The formation of nanocomposites with core-shell structures leads to UCNPs@PFC-73-Ni.This composite maintains the unique qualities of its parent materials and establishes an effective FRET pathway between the two components.As a result, superior performance is achieved by the HOF composites, exceeding that of the individual components.Moreover, the UCNPs@PFC-73-Ni exhibited excellent photothermal and photodynamic effects when exposed to infrared light.The material's photoactivity results in remarkable bacteriostatic efficiency, achieving 97.4% in the E. coli inhibition test under infrared light irradiation.These findings not only provide a new way for functionalizing HOFs but also demonstrate its promising potential in the field of biology.

A C K N O W L E D G M E N T S
The authors gratefully acknowledge the National Natural Science Foundation of China (grant numbers: 22071246, 22033008 and 22272178), the CAS-Iranian Vice Presidency for Science and Technology Joint Research Project (grant number: 121835KYSB20200034), and the CAS Youth Interdisciplinary Team (grant number: JCTD-2022-12).

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 they have no conflicts of interest.

F I G U R E 4
(A) Inhibition ratios corresponding to different treatments.(B) Escherichia coli medium after treatment (980 nm, 0.5 W/cm 2 ) (D: dark, L: light).
The 1 H NMR spectrums of TCPPCOOMe, TCPPCOOMe-Ni, and TCPP-Ni are shown in FiguresS2-S4.TCPP-Ni was dissolved in 2 mL of N,Ndimethylformamide (DMF) in a 15 mL uncapped glass bottle.Subsequently, 4 mL of 1,2,4-trichlorobenzene (TCB) was added to the bottle.The uncapped glass bottle was heated and stirred at 100 • C for 2 days, and the powdered samples of PFC-73-Ni were harvested.The samples were soaked in CH 2 Cl 2 for 3 days and then activated under vacuum at 100 • C for 8 h.