Study of the Synergistic Immunomodulatory and Antifibrotic Effects of Dual-Loaded Budesonide and Serpine1 siRNA Lipid–Polymer Nanoparticles Targeting Macrophage Dysregulation in Tendinopathy

Musculoskeletal diseases involving tissue injury comprise tendon, ligament, and muscle injury. Recently, macrophages have been identified as key players in the tendon repair process, but no therapeutic strategy involving dual drug delivery and gene delivery to macrophages has been developed for targeting the two main dysregulated aspects of macrophages in tendinopathy, i.e., inflammation and fibrosis. Herein, the anti-inflammatory and antifibrotic effects of dual-loaded budesonide and serpine1 siRNA lipid–polymer hybrid nanoparticles (LPNs) are evaluated in murine and human macrophage cells. The modulation of the gene and protein expression of factors associated with inflammation and fibrosis in tendinopathy is demonstrated by real time polymerase chain reaction and Western blot. Macrophage polarization to the M2 phenotype and a decrease in the production of pro-inflammatory cytokines are confirmed in macrophage cell lines and primary cells. The increase in the activity of a matrix metalloproteinase involved in tissue remodelling is proven, and studies evaluating the interactions of LPNs with T cells proved that dual-loaded LPNs act specifically on macrophages and do not induce any collateral effects on T cells. Overall, these dual-loaded LPNs are a promising combinatorial therapeutic strategy with immunomodulatory and antifibrotic effects in dysregulated macrophages in the context of tendinopathy.


Preparation and characterization of empty and co-loaded LPNs.
The hybrid nanoparticles without drug (LPNs) and loaded with the drugs were prepared using an in-house manufactured glass-capillary microfluidic device following a two-steps microfluidics method, as described elsewhere 1 .Briefly, PLGA cores were formed in a first microfluidics step in which 5 mg/mL PLGA and budesonide dissolved in acetone were mixed with 1% w/v PVA.Budesonide was added in the organic phase using a 5:2.5 weight ratio of PLGA:budesonide.After purification of the PLGA cores by ultracentrifugation, the PLGA cores were resuspended in ethanol and the lipid mixture was dissolved in this organic phase.
In a second microfluidics step, this organic phase was mixed at high speed with the aqueous phase of serpine1 siRNA dissolved in 1% w/v PVA, rendering budesonide and serpine1 siRNA dual-loaded LPNs.Upon purification by ultracentrifugation, the final formulation was resuspended in RNase-free milli-Q water.The size and size homogeneity (polydispersity index) of the empty and dual loaded-LPNs was controlled by dynamic light scattering (DLS) using the Zetasizer Nano ZS instrument (Malvern Panalytical Ltd., UK), as previously described. 1The loading degree of BUD and the encapsulation efficiency of siRNA were quantified by a previously described HPLC method and Ribogreen assay, respectively. 1Scheme S1.Representation of the gating strategy of the negative control (cells with no antibody staining) used for the macrophage polarization studies in Figures 4 and 5 Pro-inflammatory cytokines analysis from supernatants of human primary macrophages.

Figure S1 .
Scheme S2.Schematic representation of the reporter T cells system used to identify a potential activation of TLRs signalling by LPNs and its components using flow cytometry analysis.The reporter T cells and the reporter THP-1 monocytes were previously established elsewhere.2,3

Figure S2 .
Figure S2.Analysis of the pro-inflammatory cytokines in the supernatants of the samples collected for the macrophage polarization study with human primary macrophages Data are presented as the mean ± SD (n = 4 biological replicates).A one-way ANOVA followed by a Dunnett post-hoc test was used for the statistical analysis.The significance levels of the differences were set at the probabilities of **p < 0.01 for comparing the treatment samples with the M1 positive control *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure S3 .
Figure S3.Activation of toll-like receptors (TLRs) in Jurkat reporter T cells by single-loaded LPNs, dual-loaded LPNs and the drugs (BUD and serpine1 siRNA alone).The cells were incubated for 48 h with the corresponding nanoparticles/drugs and cells were harvested to analyse the expression of GFP as indicator of TLR signalling activation. 2 Phorbol myristate acetate (PMA) and PMA + ionomycin were used as positive controls for (A) TLR4, (B) TLR2/1, (C) TLR2/6 and (D) TLR2/1/6 reporter T cell lines, and LPS was used as positive control for (E) reporter THP-1 monocytes.Data represents the mean fluorescence intensity (MFI) ± s.d.(n=3).A one-way ANOVA followed by a Dunnett post-hoc test was used for the statistical analysis to compare the positive controls to the treatment samples.The significance levels of the differences were set at the probabilities of *p < 0.05, **p < 0.01, and ***p <0.001, to compare the negative control (only cells) with the treatment samples.