# Targeted chondrogenic differentiation of human MSCs using niosomes for SOX9 gene delivery: comparison of minicircle and conventional plasmids

## TL;DR

This study explores using niosomes to deliver the SOX9 gene to human stem cells, promoting cartilage formation more effectively than traditional methods.

## Contribution

The study introduces niosomes as a non-viral delivery system for SOX9 gene transfer, showing enhanced chondrogenic differentiation in human MSCs.

## Key findings

- Niosome-delivered SOX9 significantly increased chondrogenic markers like SOX9, ACAN, and COLII.
- Lipofectamine-based transfection led to hypertrophic and fibrocartilaginous phenotypes.
- Parental plasmids provided more consistent chondrogenic outcomes compared to minicircle plasmids.

## Abstract

Niosomes represent a promising non-viral gene delivery system, offering an alternative to viral vectors for the genetic modification of hard-to-transfect cells, such as mesenchymal stem cells (MSCs), which are pivotal in regenerative medicine. Specifically, SOX9 gene transfer is a valuable strategy for cartilage tissue repair, as it promotes chondrocyte differentiation while repressing hypertrophic and osteogenic markers. In this study, we investigated the potential of niosomes to deliver SOX9, using both parental and minicircle plasmids, to induce chondrogenic differentiation in primary bone marrow-derived human MSCs (hMSCs).

Niosomes were synthesised using the thin-film hydration method and complexed with either parental or minicircle SOX9 plasmids to form nioplexes. Physicochemical properties of niosomes and nioplexes were studied in terms of size, zeta potential, complexation, and protection capacity. Primary hMSCs were transfected in a 2D monolayer and 3D aggregate cultures using Lipofectamine as a positive control of transfection. Chondrogenic differentiation was assessed by gene expression (SOX9, ACAN, COLII, COLI, COLX), histological and immunohistochemical staining (Toluidine blue, haematoxylin & eosin and SOX9, COLII, COLI, COLX, respectively), and biochemical (proteoglycans, DNA and protein contents) analyses of main cartilage markers.

SOX9 delivery via DP20CQ niosome systems significantly enhanced the expression of key chondrogenic markers (SOX9, ACAN, and COLII) and increased production of a characteristic hyaline-like cartilage matrix. In contrast, Lipofectamine-based complexes induced hypertrophic and fibrocartilaginous phenotypes, evidenced by increased expression of COLX and COLI. Quantification of proteoglycan production, along with proteins and DNA content, supported these findings. Both plasmid types promoted comparable chondrogenic outcomes, but parental plasmids yielded more consistent results than minicircles.

Delivery of SOX9 plasmids via niosomes promotes enhanced chondrogenic differentiation of primary hMSCs in a 3D aggregate culture system, leading to the formation of hyaline-like cartilage tissue. This non-viral strategy represents a promising gene delivery platform for cartilage reparative therapies.

The online version contains supplementary material available at 10.1186/s13287-025-04867-5.