Traumatic musculoskeletal injuries require advanced therapeutic intervention to heal effectively. Regenerative medicine research has aimed to address this by using biomaterials to deliver gene therapeutic nanoparticles (NPs) to the injury site to direct healing. However, clinical translation has proven challenging due to the short shelf-life of NPs and requirements for cold storage conditions. Thus, this study aimed to investigate lyophilisation as a process to formulate 'off-the-shelf' NPs that can be incorporated into biomaterial scaffolds at the point of use and can be stored and transported at ambient temperatures. To this end, NPs consisting of a non-viral delivery vector, glycosaminoglycan-enhanced transduction (GET) peptide, complexed with plasmid DNA (pDNA), were formulated at three charge ratios (CRs - 6, 9, 12) and lyophilised. Firstly, the effects of lyophilisation on NP physicochemical properties were investigated; it did not affect NP size, polydispersity or charge. Next, the ability of the lyophilised NPs to express the pDNA cargo in mesenchymal stem cell (MSC) 2D monolayer culture was assessed. Transfection with lyophilised NPs at each CR promoted stable transgene expression and furthermore, once lyophilised, transgene expression could be maintained following long-term storage at room temperature. Transfection with lyophilised GET-pSOX9 NPs also significantly increased MSC-mediated articular cartilage matrix deposition in methacrylated hyaluronic acid (MeHA)-collagen type II (Col II) injectable hydrogel scaffolds, highlighting the therapeutic potential of this NP formulation. In conclusion, this study outlines an effective method for formulating 'off-the-shelf' NPs for regenerative medicine applications that could be applied to the musculoskeletal system as well as other tissues.