Development of lipid-polymer hybrid nanoparticles for tendon regeneration

Jun 30, 2022, 12:20 PM
10m
Room: S3 B

Room: S3 B

Speaker

López Cerdá, Sandra (University of Helsinki)

Description

"Introduction: Tendinopathies are one of the most common musculoskeletal conditions. Unsatisfactory healing has a significant impact on the life of patients and imposes a remarkable socioeconomic burden. The recovery from tendon injuries is slow and requires extensive rehabilitation. The resulting scar tissue lacks the mechanical integrity of the original tissue, and therefore complete recovery is rarely achieved1 2. Several biological therapeutics have been proposed so far, such as the delivery of growth factors, stem cells and recently the application of gene therapy3, but limited success has been achieved. In this project, lipid-polymer hybrid nanoparticles (LPNs) are proposed for the co-loading of two biological drugs with the aim to achieve tendon regeneration. Interleukin-4 (Il-4) is an anti-inflammatory cytokine extensively used in macrophage polarization towards the anti-inflammatory M2 phenotype. Co-loading of IL-4 with an siRNA against one of the genes involved in fibrosis could potentially render a dual therapeutic effect: immunomodulation and fibrosis prevention 4,5.

Methodology: LPNs, consisting of a PLGA core and a lipid shell, were prepared using a newly developed method based on nanoprecipitation using a glass-capillary microfluidics technique. Empty LPNs were also analyzed by transmission electron microscopy (TEM) doing negative staining with phosphotungstic acid (PTA). The toxicity of the empty particles was assessed in RAW 264.7 cells with a luminescent CellTiter Glo® assay. The loading of a model siRNA (i.e., eGFP siRNA) in the lipid shell was quantified using the fluorescent Ribogreen assay. The loading of IL-4 in the PLGA core was determined using an ELISA assay. The transfection efficiency of the siRNA-loaded LPNs, measured as eGFP expression inhibition, was evaluated in RAW 264.7 cells expressing eGFP by flow cytometry. The ability of the IL-4-loaded LPNs to polarize macrophages was assessed in the same cell line by quantification of an increased expression of markers of M2 macrophages as compared to M1.

Results: Homogenous PLGA cores with sizes smaller than 300 nm were obtained by a glass-capillary microfluidics technique upon optimization of the process and formulation parameters. LPNs were prepared by two different microfluidics methods involving one and two steps, respectively, rendering homogenous particles of approximately 350 nm. The TEM images confirmed the results obtained by DLS and unraveled a spherical shape for the PLGA cores and a spherical/elongated shape for the hybrid NPs. LPNs were loaded with eGFP siRNA in the lipid shell and IL-4 in the PLGA core and a lack of toxicity was proved up to concentrations of 200 µg/mL.

Conclusion: This work demonstrates the potential of hybrid nanoparticles to load biological drugs with different physicochemical properties and therapeutic effects, allowing for the development of novel nanosystems with dual effects. The developed platform could potentially be used in tendinopathies to promote scarless tissue repair and recovery of the biomechanical function of the tissue."
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