Fluorescent nanodiamonds encapsulated by Cowpea Chlorotic Mottle Virus (CCMV) proteins for intracellular 3D-trajectory analysis

Long-term tracking of nanoparticles to resolve intracellular structures and motions is essential to elucidate fundamental parameters as well as transport processes within living cells. Fluorescent nanodiamond (ND) emitters provide cell compatibility and very high photostability. However, high stability, biocompatibility, and cellular uptake of these fluorescent NDs under physiological conditions are required for intracellular applications. Herein, highly stable NDs encapsulated with Cowpea chlorotic mottle virus capsid proteins (ND-CP) are prepared. A thin capsid protein layer is obtained around the NDs, which imparts reactive groups and high colloidal stability, while retaining the opto-magnetic properties of the coated NDs as well as the secondary structure of CPs adsorbed on the surface of NDs. In addition, the ND-CP shows excellent biocompatibility both in vitro and in vivo. Long-term 3D trajectories of the ND-CP with fine spatiotemporal resolutions are recorded; their intracellular motions are analyzed by different models, and the diffusion coefficients are calculated. The ND-CP with its brilliant optical properties and stability under physiological conditions provides us with a new tool to advance the understanding of cell biology, e.g., endocytosis, exocytosis, and active transport processes in living cells as well as intracellular dynamic parameters.


Experimental section
Centrifuge Device with Omega Membrane were used for particle preparation.

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The homogenate is filtrated to remove the larger plant debris. The homogenate is kept at 1 T=4 °C for an hour to allow the proteins to precipitate. 3 The homogenate is subjected to 2 low-speed centrifugation to precipitate the leaf tissue. The pellet is discarded and the 3 supernatant is added to 10 % (w/v) solid PEG (MW=6000 g/mol). The mixture is stirred 4 for 1 h at T=4 °C. The precipitate is pelleted by low-speed centrifugation. The supernatant 5 is discarded and the bottles are drip-dried thoroughly to remove the PEG solution (Hebert, The pellet is suspended in cold virus buffer (0.1 M NaAc, 1 mM Na2EDTA, 1 mM NaN3) 8 with the help of a glass stick or a pipette. The resuspension is cleared of undissolved 9 material by low-speed centrifugation. 10 The pellet is discarded and the supernatant is mixed with cesium chloride. Once completely 11 dissolved, the mixture is subjected to a density gradient centrifugation. The obtained 12 solution is dialyzed against virus buffer at T= 4°C, and then stored at T=4 °C. The presence 13 and purity is checked by SDS-PAGE and FPLC. Typical yields of CCMV are 200-300 mg 14 per kg of cowpea tissue. During the whole process, the virus solution is kept cold either in 15 an ice bath or in the cold room (T= 4°C).   3 In a typical experiment, NDs solution (400 μL, 0.2 mg/mL; H 2 O) is added to a solution of 4 CCMV coat protein (100 μL, 15 mg/mL; pH 7.2; 250 mM Tris, 500 mM NaCl) and allowed 5 to incubate overnight at 4 ⁰C. The reaction mixture is subsequently resulting CP-NDs are 6 purified using preparative FPLC.  Injection of 500 µL pre-filtered samples which are injected on a 24 mL superpose-6 12 column. Compound elution is monitored using a UV-vis spectrometer at 260 nm, 280 nm. 13 Fractionation are collected separately 14 15 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 16 SDS-PAGE samples are prepared by mixing 10 µL of sample with 9 µL of sample buffer 17 (125 mM Tris-HCl, 20% (v/v) glycerol, 5% (w/v) sodium dodecyl sulfate, 0.02% (w/v) 18 bromophenol blue, pH 6.8) and 1 µL 2-mercaptoethanol. The mixture was heated at 99 °C 19 for 5 minutes to denature the protein, after which the mixture was used to fill the wells of   Transmission Electron Microscopy (TEM) 10 4 µL 0.1 mg/mL solution of ND-CPs in MilliQ was placed onto an oxygen treated carbon 11 coated copper grid. After 10 minutes the solution was removed using a filter paper and 12 grids were stained with uranyl acetate 4% for 1.5 minutes. The grids were washed three 13 times with MilliQ water and dried before measuring. A Jeol 1400 transmissions electron 14 microscope was used to obtain bright field images. And Image J software was used to 15 process the data. was removed and 300 µL MilliQ water was applied onto the mica surface, forming a 2 droplet for measuring in liquid. Samples were scanned with scan rates between 1 and 2 Hz 3 and scan sizes between 0.5 and 2 µm. Images were processed with NanoScope Analysis 4 1.8.   Here, mth = mean time to haemorrhage (in seconds), mtl = mean time to start vascular lysis 7 (in seconds), mtc = mean time to start coagulation (in seconds). When no irritation 8 component (such as hemorrhage, lysis, or coagulation) was observed within 5 minutes after 9 sample application, the contribution of respective irritation component was considered zero 10 to calculate the total irritation score (IS).  Table S1. Classification of irritant based on the irritation score (IS) 8

Classification
Irritation Score (IS)

Custom-built confocal microscopy for bioimaging and Intracellular tracking 17
The confocal setup is a conventional custom-built setup, driven by the software Qudi, 18 which could perform a variety of basic measurement functionalities.

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The key hardware of the setup consists of an oil-immersion objective lens (Olympus, 1.35, 20 60×), a 532 nm continuous-wave laser, a spectrometer (Princeton Instruments, Acton SP  1 Another way to distinguish normal and anomalous diffusion (either confined diffusion or directed 2 motion) is by calculating the cumulative distribution function (CDF) of the square displacements 3 (Δr 2 ) at a particular t lag . 9, 14 Here we focused on "Trajectory 1", the longest trajectory recorded in 4 this work, and t lag = 2Δt = 10 s. The probability distribution function (PDF) of the square 5 displacements were first calculated ( Figure 4B, top panel), based on which the CDF was computed 6 ( Figure 4B, bottom panel). We further fitted the CDF data with single and double exponential 7 functions as follows, 8 where r 0 2 is the MSD at t lag , and 9 where r 1 2 and r 2 2 are the MSDs at t lag , corresponding to the fast and slow mobility components, 10 respectively. The contributions of these two components to the CDF are weighted with factors w 11 and (1-w) respectively. Whereas Equation S3 implies normal diffusion, Equation S4 covers both 12 normal (w = 0 or 1) and anomalous (w → 0.5) diffusion. The r i 2 (i = 0, 1, 2) are related to the 13 diffusion coefficient D i as r i 2 = 6D i t lag . From the fitting, we obtained r 0 2 = 1.2540 × 10 -2 μm 2 , r 1 2 14 = 1.7715 × 10 -3 μm 2 , r 2 2 = 1.7590 × 10 -2 μm 2 , and w = 0.2616. As a result we obtained the following  Figure 4A), we obtained the following three diffusion coefficients, 1.25 × 10 -4 , 5 3.07 × 10 -4 , and 1.39 × 10 -3 μm 2 /s ( Figure 4C), which are comparable to the values obtained from 6 the single and double exponential fits to the CDF data at t lag = 10 s. The nominal diffusion 7 coefficient of the ND-CP in the HeLa cell was then determined to be the average of these three 8 values, that is, 6.07 × 10 -4 μm 2 /s.