Bedingfield, S.

Colazo, J.

Decuzzi, P

Di Francesco, M.

Di Francesco, V.

Di Mascolo, D.

Duvall, C.

Ferreira, M.

Yu, F.

• Fragassi, Agnese.

English

Archivio istituzionale della ricerca - Università di Genova

Squared PLGA microPlates as injectable depot for the management of Post-Traumatic OsteoarthritisAgnese Fragassia,b, Martina Di Francescoa, Sean K. Bedingfieldc, Juan M. Colazoc , Valentina Di Francescoa, Fang Yuc, Miguel Ferreiraa, Daniele Di Mascoloa, Craig Duvallc, Paolo Decuzziaa  Laboratory of Nanotechnology for Precision Medicine, Fondazione Istituto Italiano di Tecnologia (IIT), via Morego 30, Genova, 16163, Italyb Department of Chemistry and Industrial Chemistry, University of Genova, 16146 Genova, ItalycDepartment of Biomedical Engineering,Vanderbilt University, Nashville, Tennessee 37212, United StatesOsteoarthritis (OA) is a chronic inflammatory joint disease that affects population across all ages. It is caused by progressive breakdown ofjoint cartilage and underlying bone, resulting in joint pain, and, eventually, permanent disability. Currently, there is no disease-modifying drugavailable to reverse the progression of the OA and conventional therapies are palliative, providing only a temporary relief from the symptoms1.INTRODUCTIONSYNTHESIS AND PHYSICAL-CHEMICAL CHARACTERIZATION OF µPLsWithin this context, a top-down strategy was used for synthetizing squared-poly (D,L-lactide-co-glycolide) (PLGA) microPlates (μPLs) 2,3 forprolonged release of Dexamethasone (DEX)2 and matrix metalloproteinase 13 (MMP-13) RNA interference nanoparticles (siMMP13-NPs)3. Afull characterization of the physical-chemical and mechanical properties of both formulations was performed. μPLs exhibited an edge lengthof 20 µm and a height of 10 µm and a stiffness value of about 3.1 ± 0.9 MPa (Fig. 1)..In addition, the bio-pharmacological properties of µPLs were investigated (Fig. 2). Under the sink conditions, 80% of DEX was releasedfrom µPLs within the first 10 days (Fig. 2a). Conversely, under confined conditions, DEX µPLs provided a sustained drug release for severalmonths, with ~20% of DEX released in 1 month (Fig. 2b)2. Equally, in vitro siMMP13-NP release profile from μPLs in PBS (0.75 mg of μPLsin 1.5 mL) showed prolonged release over the 5-week experiment (Fig 2c and d) 3.BIOPHARMACOLOGICAL CHARACTERIZATION OF µPLSFig 1. a. SEM images of the empty µPLs released from the PVA template; b. Confocal microscopy image of Cy5-siNP loaded Curcumin-µPLs; c. Force-displacement curvefor a flat punch indentation experiment on an ensemble of μPLs (average curve and standard deviation).; d. Energy dissipation ability of µPL upon cyclic mechanical loading(frequency 5 Hz) as a function of the force oscillation amplitude.Fig 2. a. DEX release profile from µPLs under sink conditions (4 L, red line) and fit to the Weibull empirical drug release model (red line; 95% confidence margin: redmargin); b. DEX release profile from µPLs under confined conditions, mimicking the synovial volume (500 µL, Weibull: green line with 95% confident band); c. siMMP13-NP release profile from μPLs as measured by Quant-iT Ribogreen assay; d. Cy5-siNP release kinetics from μPLs as measured by Cy5 fluorescence.IN VIVO PHARMACOKINETIC STUDY OF µPLSTo evaluate in vivo μPLs intra-articular retention time, Cy5 was conjugated on particles surface. After a single intra-articular injection of Cy5-μPLs into a cohort of mice with mechanically induced osteoarthritis (PTOA), a time course of intravital imaging, ex vivo imaging, and confocalmicroscopy analyses was performed. Fluorescent μPLs were detected in the joint, for up to 30 days (Fig. 3a) and a surface erosion evident byloss of fluorescence (heterogenous) was observed in a significant number of individual Cy5-μPLs at days 25 and 30 as compared to days 1 and5, as showed in Fig. 3b 2,3.The therapeutic efficacy of an intra-articular injection of DEX-µPLs in the PTOA mouse model. In particular, a single injection of DEX-µPLsdecreased the expression of IL-1β, TNF-α, IL-6 and MMP-13 by approximately half compared to free DEX at 4 weeks post-treatment (Fig4a)2. In the same animal model, gene silencing efficiency was obtained by siMMP13-NPs released from µPLs for the whole duration of thestudy (5 weeks), preserving ~50% silencing after 4 weeks (Fig 4b and c).3.In conclusion, top-down fabrication approach allowed to synthetize shaped-defined μPLs that can act an effective depot system for the localtreatment of OA. They provided a sustained drug release for several weeks, reducing inflammation and pain and improving mechanicalproperties of OA-affected joint.CONCLUSIONSFig 3 a. Representative pharmacokinetic time course intravital images (skin on) and ex vivo knee images (skin off) of Cy5-μPLs injected intra-articularly into PTOA mouseknee joints (D-#, where # represents days after intra-articular injection); b. Magnified images of Cy5-μPLs within the joint at multiple time points; c. Quantification of relativefluorescence unites (RFU) of individual Cy5-siNP-μPLs in histological sections over time plotted as mean + SEM.IN VIVO THERAPEUTIC EFFICACY OF µPLSFig 4. a. In vivo expression of IL-1β, TNF-α, IL-6, and MMP-13 measured by TaqMan qPCR (for each treatment groups n = 6, while for the healthy group n = 4); b. in vivolongitudinal inhibition of MMP13 and reduction of inflammatory gene expression in the total joint and c. synovium.REFERENCES 1. Martel-Pelletier et al., Nat Rev Dis Primers. 2016, 2, 16072.2. Di Francesco et al., ACS applied materials & interfaces, 2021, 13, 31379.3. Bedingfield et al., ACS nano, 2021, 15, 9, 14475–14491.

Squared PLGA microPlates as injectable depot for the management of Post-Traumatic Osteoarthritis

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Squared PLGA microPlates as injectable depot for the management of Post-Traumatic Osteoarthritis

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Archivio istituzionale della ricerca - Università di Genova