Imaging Rheumatoid Arthritis in Mice Using Combined Near Infrared and 19F Magnetic Resonance Modalities

Rheumatoid arthritis (RA) is an autoimmune disease that causes pain and tissue destruction in people worldwide. An accurate diagnosis is paramount in order to develop an effective treatment plan. This study demonstrates that combining near infrared (NIR) imaging and 19F MRI with the injection of labelled nanoparticles provides high diagnostic specificity for RA. The nanoparticles were made from poly(ethylene glycol)-block-poly(lactic-co-glycolic acid) (NP) or PLGA-PEG-Folate (Folate-NP), loaded with perfluorooctyl bromide (PFOB) and indocyanine green (ICG) and evaluated in vitro and in a collagen-induced arthritic (CIA) mouse model. The different particles had a similar size and a spherical shape according to dynamic light scattering (DLS) and transmission electron microscopy (TEM). Based on flow cytometry and 19F MRI analysis, Folate-NP yielded a higher uptake than NP in activated macrophages in vitro. The potential RA-targeting ability of the particles was studied in CIA mice using NIR and 19F MRI analysis. Both NP and Folate-NP accumulated in the RA tissues, where they were visible in NIR and 19F MRI for up to 24 hours. The presence of folate as a targeting ligand significantly improved the NIR signal from inflamed tissue at the early time point (2 hours), but not at later time points. Overall, these results suggest that our nanoparticles can be applied for combined NIR and 19F MRI imaging for improved RA diagnosis.


PLGA-PEG-Folate synthesis
The PLGA-PEG-Folate polymer was synthesized as described by Esmaeili et. al, 2008 with minor modifications 1 . Briefly, the carboxylic group of PLGA resomer RG 504H (Evonik industries, Germay) was converted into PLGA-NHS using N-hydroxysucconimide (EDC)/1-ethyl-3-(3-dimethylamino) carbodimide (NHS) with molar ratio 1:10:10 for 24 h in dry DCM. The polymer was precipitated in ice-cold methanol, centrifuged and washed 3 times to remove unreacted agent and dried using vacuum. The PLGA-NHS was then reacted with bis-amine 6 kDa PEG with molar ratio (1:5) in dry DCM for a 24 hours. The reaction was precipitated in a cold mixture of methanol and diethyl ether, centrifuged and washed 3 times and dried under low pressure. Folic acid (13 mg) was activated with N,N'-dicyclohexylcarbodiimide DCC (13 mg) in dry Dimethyl sulfoxide (DMSO) overnight. Next, PLGA-PEG-NH 2 was added to react with folic acid overnight and slowly dropped into 50 ml mixture of methanol/water before transfer to a 14 kDa dialysis membrane to remove unconjugated product for 2 days (water were changed twice a day).
The final product was freeze dried and kept at -20°C. The conjugation of folic acid to PLGA-PEG was confirmed by NMR Bruker 500 MHz (Bruker, Billeria, MA, USA). The concentration of folic acid in the polymer was optimized using UV-Vis at wavelength (280 nm to 285 nm)

Weight of nanoparticles
One hundred microliters of purified nanoparticles (n =3) were put in an Eppendorf tube and freeze dried for 6 hours. The net weights of nanoparticles were measured using the scale.

Measuring concentration of PFOB and ICG loaded nanoparticle
Concentration of PFOB in the nanoparticles was estimated using 19 F MRI. In brief, 100 µl of purified nanoparticles were placed in an NMR tube together with various concentration of PFOB in CDCl 3 . The multi-slice multi-echo MSME pulse sequence was used with an echo time (TE) of 3.36 ms, a repetition time (TR) of 12 s, a field of view (FOV) of 22x22 mm 2 , slice thickness 10 mm, an image matrix of 32x32, 10 averaging scans (NA), and an excitation frequency of -83 ppm. After the MRI acquisition, the 19 F SNRs of each NMR tube were measured and standard curves were built. The amounts of PFOB in nanoparticles were then calculated using the standard curve.
The encapsulation efficiency of PFOB in nanoparticle was calculated by comparing the ratio of amount PFOB/weight of nanoparticle before formulation and after purification.
The concentration of ICG in nanoparticles was measured by DMSO extraction. Briefly, ICG-loaded nanoparticles (after freeze drying) were dissolved in 500 μl of DMSO and shaken for 3 h. The solution was then centrifuged at 17,000 g for 10 min, to remove all precipitants. The concentration of ICG was measured using a Fluorometer (Horiba-Yvon, Japan) at an excitation wavelength of 765 nm and the concentration was calculated using a standard curve from a various ICG solutions.
After 8 hours of incubation, the media was renewed and cells were grown overnight. On the next day, a standard MTT assay was performed according to the manufacturer's protocol (Sigma) in order to estimate cell viability. Data was subject to the student t-test and P<0.05 was considered statistically significant.

Rheumatoid arthritis scoring
RA developed in mice at 4 to 8 weeks after induction. The following rubric score was used to access the extent of the RA: 0 No evidence of erythema and swelling 1 Erythema and mild swelling confined to the tarsals or ankle joint 2 Erythema and mild swelling extending from the ankle to tarsals 3 Erythema and moderate swelling extending from the ankle to metatarsal joints