Quantum Sensing for Real-Time Monitoring of Drug Efficacy in Synovial Fluid from Arthritis Patients

Diamond-based T1 relaxometry is a new technique that allows nanoscale magnetic resonance measurements. Here we present its first application in patient samples. More specifically, we demonstrate that relaxometry can determine the free radical load in samples from arthritis patients. We found that we can clearly differentiate between osteoarthritis and rheumatoid arthritis patients in both the synovial fluid itself and cells derived from it. Furthermore, we tested how synovial fluid and its cells respond to piroxicam, a common nonsteroidal anti-inflammatory drug (NSAID). It is known that this drug leads to a reduction in reactive oxygen species production in fibroblast-like synoviocytes (FLS). Here, we investigated the formation of free radicals specifically. While FLS from osteoarthritis patients showed a drastic decrease in the free radical load, cells from rheumatoid arthritis retained a similar radical load after treatment. This offers a possible explanation for why piroxicam is more beneficial for patients with osteoarthritis than those with rheumatoid arthritis.

A part of the cultured cells was fixed with 4% formaldehyde and stained with phalloidin-TRITC to visualize cell morphology (F-actin cytoskeleton).Images were taken with TissueFAXS microscope (63x/1.40oil objective).
Samples from both OA and RA patients were negative for the expression of CD33, CD3, CD11b, CD14, and CD86, as indicated by the full overlap between the signal from these markers and the one from the isotype control (see supplementary Figure S2).Conversely, samples from RA and OA patients were highly positive for CD90 (see Figure 4 a, b).Even when there was some overlap between the CD90 signal and the one from the isotype control.
Fibroblast-like synoviocytes are important cellular components of the inner layer of the joint capsule.They can be found in both layers of the synovial membrane and contribute to normal joint function by producing extracellular matrix components.However, under inflammatory conditions, they may start to proliferate, undergo phenotypical changes, and become central elements of OA or RA.

Materials:
The fluorescent nanodiamonds (FNDs) in this work were commercially available particles from Adamas Nanotechnology (USA).These particles have a hydrodynamic diameter of 70 nm and are produced by grinding HPHT diamonds and irradiation with 3MeV electrons at a fluence of 5*10 19 e/cm 2 .After irradiation, they undergo high-temperature annealing.They contain an average of 500 NV centers (Nitrogen-Vacancy centers) per diamond (determined by EPR by the manufacturer) [ 1 ].The final step of the production is a cleaning in oxidizing acids, which removes non-diamond carbon residues.As a result, the surface chemistry of these FNDs is oxygen-terminated.Previous literature has already characterized these particles extensively [ 2 , 3 ].Dulbecco's Modified Eagle's Medium (DMEM-HG) was provided by Sigma Aldrich (The Netherlands), Fetal Bovine Serum (FBS), penicillin and streptomycin trypsin/EDTA from Life Technologies (The Netherlands), and phosphate-buffered saline (PBS) from Invitrogen (The Netherlands).Piroxicam and phalloidin-TRITC was bought from Sigma Aldrich (The Netherlands).2',7'-dichlorodihydrofluorescein diacetate (DCFDA) was purchased from Millipore Corp. (USA).

Ethical considerations:
The University Medical Center Groningen (UMCG) review board approved this study (14.07.2021METc 2021/419 Magnetometry as a new tool to study joint inflammation), and all participants signed an informed consent.

Detection of reactive oxygen species
In order to evaluate the overall level of reactive oxygen species in collected samples, we have performed a DCFDA assay according to the manufacturer's recommendation and as shown earlier [ 4 ].After entering the cell, DCFDA is deacetylated and later oxidized by ROS to 2,7dichlorodihydrofluorescein (DCF), which is highly fluorescent.These fluorescence intensity changes were measured using a plate reader (excitation at 485 nm and emission at 535 nm) to determine cellular ROS production directly in the synovial fluid and in cultured cells with and without piroxicam treatment.The positive control was prepared by treating synovial fluid or cells with 25 µM H2O2.

Intracellular T1 measurements
The laser (532 nm, CNI, Changchun, China) was attenuated to 50 μW (2 × 105 W cm −2 ) at the location of the sample (at continuous illumination).To collect the fluorescence, we used an Olympus UPLanSApo40× NA = 0.95 air objective.A 550 nm long-pass filter and a confocal pinhole are used to separate scattered laser light from fluorescence.For detection, we used an avalanche photodiode (SPCM-AQRF-15-FC) in single-photon counting mode.The NV centers were polarized with 5 μs long laser pulses intermitted by dark times (from 0.2 μs to 10 ms).To determine the polarisation state of the NV centers, we observed the fluorescence within the first 0.6 μs of each laser pulse.Since particles within cells are moving (and for drift correction in stationary particles), we also track the particle.To this end, the measurement is automatically paused every 5 seconds to perform a quick scan to find the particle and move the laser to the new location of the particle.While each individual measurement takes several microseconds, we usually repeat the measuring sequence about 10,000 times to obtain an excellent signal-tonoise ratio.
The collected synovial fluid (SF) was used to establish patient-specific cell cultures.For that purpose, the SF was centrifuged for 5 min at 1000 rpm.The pellet of cells was resuspended in a DMEM high glucose medium containing 10% FBS and 1% of penicillin-streptomycin.Cells were plated on glass-bottom Petri dishes.After one and seven days in culture, cells were incubated for 4 hours with 2 µg/ml of 70 nm FNDs resuspended in the medium.Afterwards, the samples were rinsed with fresh medium, and the initial T1 relaxometry measurements were performed.Next, the same cells were treated with Piroxicam at a final 2 µg/ml concentration.Then T1 measurements were performed using the same FNDs as in the initial measurements.
Supplementary Figure S1.Results of T1 relaxometry control measurements without cells or synovial fluid.The experiments were repeated 21 times for each step (dry, PBS, and Piroxicam) in the sequence, and error bars represent standard deviations.
Cell Differentiation Molecules (HCDM) → BioLegend Supplementary Figure S2.Markers (CD3, CD118, CD14, CD33, CD86) with no detectable expression in cell cultures from synovial fluid of OA and RA patients.Samples from OA patients (A, C, E, G, I) and RA patients (B, D, F, H, J).Histograms were normalized to the number of cells.

Table 1
Interrogated cell markers and corresponding cell types.