Novel Intrapolymerization Doped Manganese‐Eumelanin Coordination Nanocomposites with Ultrahigh Relaxivity and Their Application in Tumor Theranostics

Abstract While magnetic resonance imaging contrast agents have potential in noninvasive image‐guided tumor treatment, further developments are needed to increase contrast, biodegradability, and safety. Here, novel engineered manganese‐eumelanin coordination nanocomposites (MnEMNPs) are developed via a facile one‐pot intrapolymerization doping (IPD) approach in aqueous solution, through simple chemical oxidation–polymerization of the 3,4‐dihydroxy‐DL‐phenylalanine precursor with potassium permanganate serving as the Mn source and an oxidant. The resulting MnEMNPs possess ultrahigh longitudinal relaxivity (r 1 value up to 60.8 mM−1 s−1 at 1.5 T) attributed to the high manganese doping efficiency (>10%) and geometrically confined conformation. Due to their high manganese chelation stability, excellent biocompatibility, and strong near‐infrared absorption, high‐performance longitudinal‐transverse (T 1 ‐T 2) dual‐modal magnetic resonance/photoacoustic imaging and photothermal tumor ablation are achieved. Furthermore, the hydrogen peroxide‐triggered decomposition behavior of MnEMNPs circumvents the poor biodegradation issue of many nanomaterials. This facile, convenient, economical, and efficient IPD strategy will open up new avenues for the development of high‐performance multifunctional theranostic nanoplatforms in bionanomedicine.

S3 proceed under vigorous stirring for 6 h. Then, the products were retrieved by several sequential centrifugation/redispersion processes in deionized water (17500 rpm, 15 min), to remove excess precursors and reactants. Finally, the as-obtained MnEMNPs were redispersed in deionized water. The yields of MnEMNPs were determined by weighing after lyophilization and the Mn content was quantified by inductively coupled plasma mass spectrometry (ICP-MS) following digestion by aqua regia overnight.
To optimize the reaction conditions, the effect of reaction time and DL-DOPA/KMnO 4 feeding molar ratio on the nanostructure formation were systematically investigated. Detailed reaction parameters for the synthesis of MnEMNPs are summarized in Table S1. The same synthetic procedure were  were irradiated by 808 nm laser (2 W cm -2 , 3 min). Deionized water was set as control.
The solution temperature was recorded using an infrared thermal camera. MnEMNPs (200 μg mL -1 ) aqueous solution were irradiated by 808 nm laser at various power densities (0.5-2.5 W cm -2 ) for 3 min. The photothermal stability of MnEMNPs was assessed at 100 μg mL -1 via cyclic-irradiation assay (2 W cm -2 , laser irradiation for 2 min and then shut off for 7 min). To compare the photothermal stability of MnEMNPs and Au nanorods. MnEMNPs and Au nanorods (100 μg mL -1 ) were irradiated by 808 nm laser (2 W cm -2 , 30 min), the changes of optical absorption and morphology were measured by UV-vis spectrometer and TEM, respectively. MRI relaxivity measurement. Magnetic property measurement was performed using a vibrating sample magnetometer (VSM) (Quantum, USA) at 300 K. Proton 1/T 1 and 1/T 2 NMRD profiles at various magnetic fields in the range of 0.09-1.45 T (corresponding to 4-62 MHz proton Larmor frequencies) were measured on a fast field-cycling relaxometer (Stelar, Italy) at room temperature. To measure the longitudinal (r 1 ) and transverse (r 2 ) relaxivity, MnEMNPs were dispersed in ultrapure water with various metal molar concentrations (0-0.5 mM), using Gd-DTPA and Mn ion standards as control. Phantom images were acquired using a 7.0 T small animal cm. The r 1 and r 2 relaxivity were obtained using 1/relaxation time (s -1 ) plotted against metal concentrations (mM) and calculated by a linear fit, respectively. The MRI relaxivity under various magnetic fields was also measured using a 9. For in vitro cell imaging, 2x10 6 U87MG cells were seeded in cell culture dishes and allowed to 85% confluence. Then, various concentrations of PMnEMNPs were incubated with cells for 6 h. After washed three times with PBS, the cells were digested by trypsin, collected and suspended in 1% low melting agarose (200 μL). In vitro cell MR and cell PA images were acquired using a 7.0 T small animal MRI scanner and NEXUS 128 PAI system, respectively.
For in vitro photothermal cytotoxicity, U87MG cells were seeded in a 12-well plate with 5x10 4 cells per well and incubated overnight at 37 ℃. PMnEMNPs (50 μg mL -1 ) were incubated with cells for 6 h. Then, cells were washed with PBS for three times and replaced with fresh medium, following by 808 nm laser irradiation (2 W cm -2 , 5 min). After incubation for another 4 h, cells were co-stained with Calcein AM and PI S9 for 30 min. The cells were visualized using a fluorescence microscope, which live and dead cells exhibited green and red fluorescence, respectively. To quantify the photothermal cytotoxicity of PMnEMNPs, 5x10 3 U87MG cells were seeded in a 96-well plate. PMnEMNPs with various concentrations were incubated with cells for 6 h. Then, cells were washed with PBS and exposed to 808 nm laser (2 W cm -2 , 5 min), and then incubated with fresh media for another 24 h. Cell viability was determined by standard MTT assay. Statistical analysis. All data were presented as mean ± standard deviation.
Statistical significance was determined by a two-tailed Student's t test assuming equal variance using SPSS 19.0. P value < 0.05 was considered statistically significant.

Supplementary Results
The PEGylation of MnEMNPs was confirmed by FT-IR spectra (Figure 3c).
Characteristic peaks from 3300 cm -1 to 3500 cm -1 were attributed to N-H and O-H stretching vibrations. The peak at 1617 cm -1 was assigned to aromatic C=C bonds.         , where T, T surr , and T max is the solution temperature, ambient temperature of the surroundings, and the equilibrium temperature, respectively. Here, , the time constant for heat transfer from the sample system and was determined to be , by applying linear time data from the cooling period vs negative natural logarithm of (-Ln ) ( Figure S14e).
Here, hS is a dimensionless driving force temperature and was determined according to Equation 2: , where m s (0.2 g) and C p (4.2 J g -1 ) are the mass and heat capacity of pure water, respectively. Substituting value into Equation 2, hS was S23 determined to be 7.11 mJ. The η of MnEMNPs at 808 nm was calculated according to Equation 3 as previously described: [2] , where h is the heat transfer coefficient, S is the surface area of the container, ΔT max is the temperature change of MnEMNPs solution at the maximal steady-state temperature (40.69 o C), Qs is the heat associated with the NIR light absorbance of the pure water (measured to be 35.73 mW), I is the laser power density (2 W cm -2 ), and A 808 is the absorbance of MnEMNPs at 808 nm (0.82). Substituting all values into Equation 3, the η value of MnEMNPs was calculated to be 23.5%. The PTCE of MnEMNPs was remarkably higher than 13.1% for polydopamine-coated magnetic composite particle (Fe 3 O 4 @PDA-5), [3] 19.5% for CuS nanocrystals, and 22% for Au nanorods. [4] Supplementary Figure  The as-prepared MnEMNPs could be dispersed in water without noticeable aggregation for more than six months indicating prominent water dispersion.
However, they partially precipitated from other biological media including PBS, 0.9% NaCl, 5% BSA, and DMEM after 24 h incubation ( Figure S17). In comparison, the PMnEMNPs exhibited consistent dispersion stability in these media without observable agglomeration.