A poly(amidoamine)-based polymeric nanoparticle platform for efficient in vivo delivery of mRNA.

The successful use of mRNA vaccines enabled and accelerated the development of several new vaccine candidates and therapeutics based on the delivery of mRNA. In this study, we developed bioreducible poly(amidoamine)-based polymeric nanoparticles (PAA PNPs) for the delivery of mRNA with improved transfection efficiency. The polymers were functionalized with chloroquinoline (Q) moieties for improved endosomal escape and further stabilization of the mRNA-polymer construct. Moreover, these PAAQ polymers were covalently assembled around a core of multi-armed ethylenediamine (Mw 800, 2 % w/w) to form a pre-organized polymeric scaffolded PAAQ (ps-PAAQ) as a precursor for the formation of the mRNA-loaded nanoparticles. Transfection of mammalian cell lines with EGFP mRNA loaded into these PNPs showed a favorable effect of the Q incorporation on GFP protein expression. Additionally, these ps-PAAQ NPs were co-formulated with PEG-polymer coatings to shield the positive surface charge for increased stability and better in vivo applicability. The ps-PAAQ NPs coated with PEG-polymer displayed smaller particle size, electroneutral surface charge, and higher thermal stability. Importantly, these nanoparticles with both Q and PEG-polymer coating induced significantly higher luciferase activity in mice muscle than uncoated ps-PAAQ NPs, following intramuscular injection of PNPs loaded with luciferase mRNA. The developed technology is broadly applicable and holds promise for the development of new nucleotide-based vaccines and therapeutics in a range of infectious and chronic diseases.

Polymeric Nanoparticles Enable mRNA Transfection and Its Translation in Intervertebral Disc and Human Joint Cells, Except for M1 Macrophages.

Chronic lower back pain caused by intervertebral disc degeneration and osteoarthritis (OA) are highly prevalent chronic diseases. Although pain management and surgery can alleviate symptoms, no disease-modifying treatments are available. mRNA delivery could halt inflammation and degeneration and induce regeneration by overexpressing anti-inflammatory cytokines or growth factors involved in cartilage regeneration. Here, we investigated poly(amidoamine)-based polymeric nanoparticles to deliver mRNA to human joint and intervertebral disc cells. Human OA chondrocytes, human nucleus pulposus (NP) cells, human annulus fibrosus (AF) cells, fibroblast-like synoviocytes (FLS) and M1-like macrophages were cultured and transfected with uncoated or PGA-PEG-coated nanoparticles loaded with EGFP-encoding mRNA. Cell viability and transfection efficiency were analyzed for all cell types. Nanoparticle internalization was investigated in FLS and M1-like macrophages. No significant decrease in cell viability was observed in most conditions. Only macrophages showed a dose-dependent reduction of viability. Transfection with either nanoparticle version resulted in EGFP expression in NP cells, AF cells, OA chondrocytes and FLS. Macrophages showed internalization of nanoparticles by particle-cell co-localization, but no detectable expression of EGFP. Taken together, our data show that poly (amidoamine)-based nanoparticles can be used for mRNA delivery into cells of the human joint and intervertebral disc, indicating its potential future use as an mRNA delivery system in OA and IVDD, except for macrophages.

Cell uptake and intracellular trafficking of bioreducible poly(amidoamine) nanoparticles for efficient mRNA translation in chondrocytes.

Disulfide-containing poly(amidoamine) (PAA) is a cationic and bioreducible polymer, with potential use as a nanocarrier for mRNA delivery in the treatment of several diseases including osteoarthritis (OA). Successful transfection of joint cells with PAA-based nanoparticles (NPs) was shown previously, but cell uptake, endosomal escape and nanoparticle biodegradation were not studied in detail. In this study, C28/I2 human chondrocytes were transfected with NPs co-formulated with a PEG-polymer coating and loaded with EGFP mRNA for confocal imaging of intracellular trafficking and evaluation of transfection efficiency. Compared with uncoated NPs, PEG-coated NPs showed smaller particle size, neutral surface charge, higher colloidal stability and superior transfection efficiency. Furthermore, endosomal entrapment of these PEG-coated NPs decreased over time and mRNA release could be visualized both in vitro and in live cells. Importantly, cell treatment with modulators of the intracellular reducing environment showed that glutathione (GSH) concentrations affect translation of the mRNA payload. Finally, we applied a D-optimal experimental design to test different polymer-to-RNA loading ratios and dosages, thus obtaining an optimal formulation with up to ≈80% of GFP-positive cells and without toxic effects. Together, the biocompatibility and high transfection efficiency of this system may be a promising tool for intra-articular delivery of therapeutical mRNA in OA treatment.

Polymeric Nanoparticles for Drug Delivery in Osteoarthritis.

Osteoarthritis (OA) is a degenerative musculoskeletal disorder affecting the whole synovial joint and globally impacts more than one in five individuals aged 40 and over, representing a huge socioeconomic burden. Drug penetration into and retention within the joints are major challenges in the development of regenerative therapies for OA. During the recent years, polymeric nanoparticles (PNPs) have emerged as promising drug carrier candidates due to their biodegradable properties, nanoscale structure, functional versatility, and reproducible manufacturing, which makes them particularly attractive for cartilage penetration and joint retention. In this review, we discuss the current development state of natural and synthetic PNPs for drug delivery and OA treatment. Evidence from in vitro and pre-clinical in vivo studies is used to show how disease pathology and key cellular pathways of joint inflammation are modulated by these nanoparticle-based therapies. Furthermore, we compare the biodegradability and surface modification of these nanocarriers in relation to the drug release profile and tissue targeting. Finally, the main challenges for nanoparticle delivery to the cartilage are discussed, as a function of disease state and physicochemical properties of PNPs such as size and surface charge.