Special Poster Session 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Lipid Nanoparticles as mRNA delivery platform for intervertebral disc regeneration (#SP-6d)

Aida Moreira 1 , Chantal Voskamp 1 , Daniele Corraini 1 , Raymond M. Schiffelers 2 , Marianna A. Tryfonidou 1
  1. Clinical Sciences, Utrecht University, Utrecht, Netherlands
  2. Central Diagnostic Laboratory Research, University Medical Centre Utrecht, Utrecht, Netherlands

 

Introduction

Non-surgical local treatments for intervertebral disc degeneration-related low back pain, including cell-based therapeutic approaches, come with relatively high costs and lack restoration of tissue structure. In this work we aim to develop a cell free nanomedicine based on Lipid Nanoparticles (LNPs) carrying rejuvenating biomolecular factors in RNA form. LNPs are the state-of-the art delivery system for nucleic acids [1, 2]. In this work, four LNP formulations (3 clinical approved) were prepared to evaluate their therapeutic potential as mRNA delivery platform to disc cells. We performed in vitro culture of dog Nucleus Pulposus Cells (NPCs) and intervertebral disc explants, to evaluate cell association with LNPS and mRNA translation.

Methods

LNPs were prepared by microfluidic technology (NanoAssemblr®) that allows the preparation of small nanoparticles with high reproducibility and scalability. After preparation, LNPs diameter and surface charge were characterized by Dynamic Light Scattering and Zeta-Potential, respectively. Cy5.5-labelled LNPs were prepared to allow the tracking (cellular association and internalization) of LNPs. Internalization (1h-4h) and transfection efficiency (24h) of eGFP mRNA-loaded LNPs in dog NPCs were determined by Flow Cytometry. Ex vivo disc culture was carried out to evaluate the spatial distribution of the Cy5.5-LNPs within the disc and the functional delivery of eGFP-encoding mRNA (eGFP protein expression). The four LNPs were injected intradiscally, and the internalization and mRNA translation evaluated at 4h, 24h and 48h after injection.

Results

The four LNP formulations prepared had a small diameter (60-80 nm) and were monodisperse (PdI of 0.05-0.2). Zeta Potential values were between 0 and -5 mV and indicated the surface of the LNPs had a neutral charge. When Cy5.5-LNPs were incubated with dog NPCs in a pro-degenerate environment (350 mOsm/L, pH 6.8), the internalization of LNPs increased 2- to 10-fold compared to a healthy disc environment (450 mOsm/L, pH 7.1) (figure1). From the four LNPs tested, we found that MC3 and SM-102 based formulations were the most efficient in mRNA delivery to the cytosol. These formulations showed the highest increase in eGFP cellular expression, measured by Flow Cytometry. Furthermore, we showed that we can culture intact discs ex vivo.

Discussion

Until today, there has been no effective treatment that targets IVD regeneration. Innovative approaches to regenerate the disc are key to restore tissue composition and structure. In our team we aim to develop an injectable cell free therapy based on mRNA for mild to moderate IVD degeneration. Local mRNA delivery to the disc is an interesting approach, since several mRNAs encoding for different rejuvenating biomolecules can be targeted at the same time. This work enabled us to optimize our delivery platform for future functional testing of mRNAs coding for rejuvenating molecules.

 6554c8b1000fe-Figure2_healthy+vs+degenerate+internalization.jpg

 Figure 1. Internalization of Cy5.5-LNPs by dog NPCs. NPCs were seeded either in healthy (450 mOsm/L, pH 7.1) or in a pro-degenerate media (350 mOsm/L, pH 6.8). Next day, cells were incubated with Cy5.5-LNPs for 1h, 2h and 4h. Cellular association was evaluated by Flow Cytometry, with untreated cells as control. Data presented as average ± SD of 1 donor.  

  1. Evers, M.J.W., et al., State-of-the-Art Design and Rapid-Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery. Small Methods, 2018. 2(9): p. 1700375.
  2. Akinc, A., et al., The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nature Nanotechnology, 2019. 14(12): p. 1084-1087.