Albumin-mediated “Unlocking” of supramolecular prodrug-like nanozymes toward selective imaging-guided phototherapy

Construction of an activatable photosensitizer and integration into an adaptive nanozyme during phototherapy without producing off-target toxicity remains a challenge. Herein, we have fabricated a prodrug-like supramolecular nanozyme based on a metallic-curcumin and cyanine co-assembly. The albumin-mediated phenol AOH group transformation of nanozyme changes its adjustable oxygen stress from negative superoxide dismutase-like activity of ROS-scavenging to positive photo oxidase activity with an ROS-amplifying capacity. It further increases the depth penetration of a nanozyme in a tumor spheroid, selectively targeting tumorous phototherapy. It also triggers a signal in targeted tumor cells and helps increase cancer cell ablation. This work suggests new options for development of activatable supramolecular nanozymes and provides a synergetic prodrug-like nanozyme strategy for early diagnosis and preclinical phototherapeutics.


Characterization
Transmission electron microscopy (TEM) analysis was conducted on a JEM-2100F (JEOL) microscope at 100 kV. UV-visible absorption spectra were recorded with an Evolution 201 UV/vis spectrometer (Thermo Fisher Scientific) using a quartz cuvette.
The particle size distribution in solution was measured via dynamic light scattering (DLS) using a Nanotrac Wave. Fluorescence spectra were recorded by a spectrofluorometer (Edinburgh FL900/FS900). Fourier transform infrared (FTIR) spectroscopy was performed using a VERTEX 80/80v FTIR spectrometer (BRUKER).
The 1 H-NMR spectra were produced with a Bruker AM 300. Confocal laser scanning microscopy images were obtained using an Olympus Fluoview FV1200 confocal laser scanning microscope. A Bruker EMX EPR spectrometer equipped with a halogen lamp was used to investigate the ROS scavenging and ROS generation of samples.
High-resolution mass spectrometry was performed on a Synapt G2-HDMS mass spectrometer (Waters, Manchester, U.K.), which was operated using MassLynx 4.1 software at KBSI (Korea Basic Science Institue, Ochang, Center of Research Equipment).

Synthesis of Mn-curcumin
The complex was synthesized by mixing curcumin with manganese (II) chloride at a molar ratio of 1:1 in methanol. Curcumin (0.74 g, 2 mmol) was dissolved in 50 mL pure methanol and heated at 60℃ under nitrogen. Manganese (II) chloride (0.406 g, 2 mmol) was added to the curcumin methanol solution, and the mixture was refluxed for 2 hours under nitrogen. The yellow solid product was filtered and washed with cold methanol and water to remove residual reactants; the product was dried under vacuum overnight.  Figure S2. Synthesis of compound IRCOOH.

Molecular dynamics simulation
The molecular dynamics (MD) simulation was performed using Gromacs (Version 5.1.4) package. [1] The force field of Mn-curcumin complex and IRCOOH was generated by antechamber program in Ambertools18 package [2] and acpype.py program [3] , among which the bonded force field of metal-ligand was constructed by VFFDT program [4] . The force field of protein was amber03. The atomic charges of the two molecules were fitted by DFT calculation under the restrained electrostatic potential (RESP) formalism and the resp program in Ambertools18. Water molecule was modeled using the tip3p potential. All solution models were firstly minimized utilizing the conjugate-gradient algorithm, and then equilibrated through running for 500 ps NVT simulations followed by 500ps NPT simulations. Production runs in the NPT ensemble were then run for 150 ns at 298 K and 1 bar, employing the leapfrog algorithm with a time step of 2 fs to integrate the equations of motion. The electrostatic forces were treated with the particle-mesh Ewald approach. Both the cutoff values of van der Waals forces and electrostatic forces were set to be 1.2 nm.
The LINCS algorithm was utilized to preserve bonds.

Molecular docking computations
The molecular docking computations were performed by Autodock 4.2.6 package. [5] The albumin structure 4OR0 was employed from Protein Data Bank. [6] Briefly

Selectivity of nanomaterials in cancer cells and normal cells
HeLa cells (1×10 4 cells· well -1 ) and L929 cells ( Following these treatments, we allow the HeLa cells to grow up for another 24 hours.
Finally, standard MTT assays were conducted.

Reduced mouse model
We coated inner plate wells with 60 μL of 1.5% agarose. After cooling the agarose solution to room temperature in 20 minutes, 3D multicellular tumor spheroids were prepared with HeLa cells in the aforementioned ultra-low attachment 96-well plate (Corning) at a density of 5000 cells per well in 100 μL of culture medium. 3D cell spheroids were incubated at 37 °C under a 5% CO 2 atmosphere. After the spheroids formed over 3 days, they were processed and incubated with Mn-curcumin/IRCOOH or Mn-curcumin/IRCOOH/Albumin for 24 hours at the desired concentration (50 μg·mL -1 ) and washed twice with PBS. They were subsequently transferred to a confocal dish and imaged at different depths (z-stacking) with a laser scanning confocal fluorescence microscope.