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The spontaneous healing of critical-sized bone defects is often limited, posing an increased risk of complications and suboptimal outcomes. Osteogenesis, a complex process central to bone formation, relies significantly on the pivotal role of osteoblasts. Despite the well-established osteogenic properties of vitamin D 3 (VD 3), its lipophilic nature confines administration to oral or muscle injection routes. Therefore, a strategic therapeutic approach involves designing a multifunctional carrier to enhance efficacy, potentially incorporating it into the delivery system. Here, we introduce an innovative sterosome-based delivery system, utilizing palmitic acid (PA) and VD 3 , aimed at promoting osteogenic differentiation and facilitating post-defect bone regeneration. The delivery system exhibited robust physical characteristics, including excellent stability, loading efficiency, sustained drug release and high cellular uptake efficiency. Furthermore, comprehensive investigations demonstrated outstanding biocompatibility and osteogenic potential in both 2D and 3D in vitro settings. A critical-sized calvarial defect model in mice recapitulated the notable osteogenic effects of the sterosomes in vivo. Collectively, our research proposes a clinically applicable strategy for bone healing, leveraging PA/VD 3 sterosomes as an efficient carrier to deliver VD 3 and enhance bone regenerative effects. [Display omitted] [ABSTRACT FROM AUTHOR]

Open fractures and internal fixation implants are often accompanied by bacterial infection, leading to osteomyelitis, characterized by intractable bone infection and sequestrum formation, and can result in lifelong disability or fatal sepsis. As common clinical treatment strategies, high-dose antibiotic application and autologous bone transplantation face the risk of recurrence and donor site injury. Herein, we designed and prepared a novel drug delivery system by rational selection of the antibacterial single-chain amphiphile (cetylpyridinium chloride, CPC) and osteoinductive sterol (20S-hydroxycholesterol, Oxy) to formulate CPC/Oxy sterosomes. We demonstrate their excellent biocompatibility and antibacterial ability through 2D and 3D settings in vitro. In addition, the osteogenic differentiation of bone marrow mesenchymal stem cells was investigated in cell monolayers and a hydrogel environment. Moreover, a rat infected critical-sized calvarial defect model was employed to illustrate the effects of antibacterial and osteogenic CPC/Oxy sterosomes in vivo. Our results showed that CPC/Oxy sterosomes not only exterminated bacterial infections, but also enhanced calvarial healing without additional antibiotics, bone formation promoters or exogenous cells. This research provides a promising and effective multifunctional sterosomal platform for the treatment of infected bone defects, with the potential to be combined with therapeutic genes, and small molecule drugs. [Display omitted] [ABSTRACT FROM AUTHOR]

The use of small molecular drugs with gene manipulation offers synergistic therapeutic efficacy by targeting multiple signaling pathways for combined treatment. Stimulation of mesenchymal stem cells (MSCs) with osteoinductive small molecule phenamil combined with suppression of noggin is a promising therapeutic strategy that increases bone morphogenetic protein (BMP) signaling and bone repair. Our cationic Sterosome formulated with stearylamine (SA) and cholesterol (Chol) is an attractive co-delivery system that not only forms stable complexes with small interfering RNA (siRNA) molecules but also solubilizes hydrophobic small molecules in a single vehicle, for directing stem cell differentiation. Herein, we demonstrate the ability of SA/Chol Sterosomes to simultaneously deliver hydrophobic small molecule phenamil and noggin-directed siRNA to enhance osteogenic differentiation of MSCs both in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. These results suggest a novel liposomal platform to simultaneously deliver therapeutic genes and small molecules for combined therapy.Statement of significanceApplication of phenamil, a small molecular bone morphogenetic protein (BMP) stimulator, combined with suppression of natural BMP antagonists such as noggin is a promising therapeutic strategy to enhance bone regeneration. Here, we present a novel strategy to co-deliver hydrophobic small molecule phenamil and noggin-targeted siRNA via cationic Sterosomes formed with stearylamine (SA) and high content of cholesterol (Chol) to enhance osteogenesis and bone repair. SA/Chol Sterosomes demonstrated high phenamil encapsulation efficiency, supported sustained release of encapsulated drugs, and significantly reduced drug dose requirements to induce osteogenic differentiation of mesenchymal stem cells (MSCs). Simultaneous deliver of phenamil and noggin siRNA in a single vehicle synergistically enhanced MSC osteogenesis and calvarial bone repair. This study suggests a new non-phospholipid liposomal formulation to simultaneously deliver small molecules and therapeutic genes for combined treatment.

Noggin is a specific antagonist of bone morphogenetic proteins (BMPs) that can prevent the interaction of BMPs with their receptors. RNA interfering molecules have been used to downregulate noggin expression and thereby stimulate BMP signaling and osteogenesis. Cationic liposomes are considered one of the most efficient non-viral systems for gene delivery. In the past decade, non-phospholipid liposomes (Sterosomes) formulated with single-chain amphiphiles and high content of sterols have been developed. In particular, Sterosomes composed of stearylamine (SA) and cholesterol (Chol) display distinct properties compared with traditional phospholipid liposomes, including increased positive surface charges and enhanced particle stability. Herein, we report SA/Chol Sterosome and small interfering RNA (siRNA) complexes that significantly enhanced cellular uptake and gene knockdown efficiencies in adipose derived mesenchymal stem cells with minimal cytotoxicity compared with commercially available lipofectamine 2000. Furthermore, we confirmed osteogenic efficacy of these Sterosomes loaded with noggin siRNA in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. The delivery of siRNA via novel SA/Chol Sterosomes presents a powerful method for efficient gene knockdown. These distinct nanoparticles may present a promising alternative approach for gene delivery.

The hedgehog signaling pathway plays a critical role in bone development and regeneration. Applications of hedgehog morphogens or small molecular agonists are of interest in bone repair but constrained by low stability, high dose requirement, and nonspecific targeting in vivo. Herein, a nanoparticulate agonist as a new type of hedgehog signaling activator is developed for efficacious bone healing. The shell of nanoparticulate agonist consists of palmitic acid and oxysterol, which could modify hedgehog function and bind with the smoothened receptor to positively modulate hedgehog signaling. Meanwhile, the core is assembled with the sonic hedgehog gene/polyethyleneimine complex, which could synergistically enhance hedgehog signaling with oxysterol constituents. Moreover, alendronate is introduced into the nanoparticulate agonist to bind with hydroxyapatite for potential bone tissue targeting. Lastly, the nanoparticulate agonist surface is decorated with the guanidine group to overcome cell membrane barriers. The created multifunctional nanoparticulate agonist is successfully integrated onto apatite‐coated 3D scaffolds and demonstrates greatly improved osteogenesis in vitro and calvarial bone healing. This work suggests a novel biomaterial design to specifically promote hedgehog signaling for the treatment of bone defects. [ABSTRACT FROM AUTHOR]

Sterosomes (STEs), a new and promising non-phospholipidic liposome platform based on palmitic acid (PA) and cholesterol (Chol) mixtures, need to have polyethylene glycol (PEG) chains grafted to their surface in order to obtain long-circulating nanocarriers in the blood stream. A post-insertion method was chosen to achieve this modification. The post-insertion process of PEG-modified distearoylphosphoethanolamine (DSPE-PEG) was monitored using the zeta potential value of STEs. Various conditions including PEG chain length and the DSPE-PEG/PA-Chol ratio, were explored. Zeta potential of STEs changed from about −40 mV for non-modified STEs to values close to 0 mV by the end of the process, i.e. for PEG-modified STEs. The kinetics of DSPE-PEG insertion and the stability of the resulting PEG-modified STEs were not considerably influenced, within the investigated range, by changes in PEG chain lengths and in DSPE-PEG/PA-Chol proportion. The post-insertion of PEG chains reduced in vitro complement activation as well as in vitro macrophage uptake compared to the non-modified STEs. Moreover, longer blood circulation time in mice was established for PEG-modified STEs intravenously injected compared to non-modified STEs. These results establish that post-insertion process of PEG chains to STEs is a promising strategy for developing long-term circulating drug delivery nanocarriers. [ABSTRACT FROM AUTHOR]

Sterosomes are recently developed types of non-phospholipid liposomes formed from single-chain amphiphiles and high content of sterols. Although sterosomes presented significantly increased stability compared to conventional phospholipid liposomes, current sterosome biomaterials are not truly bioactive and have no intrinsic therapeutic effects. The purpose of this study was to develop a sterosome formulation with osteoinductive properties by an effective selection of sterol, one of the sterosome components. Oxysterols are oxidized derivatives of cholesterol and are known to stimulate osteogenesis and bone formation. Thus, 20S-hydroxycholesterol (Oxy), one of the most potent oxysterols for bone regeneration, was examined as a promising candidate molecule to form fluid lamellar phases with a single-chain amphiphile, namely, stearylamine (SA). First, the optimal composition was identified by investigating the phase behavior of SA/Oxy mixtures. Next, the capacity of the optimized SA/Oxy sterosomes to promote osteogenic differentiation of bone marrow stromal cells was assessed in vitro in a hydrogel environment. Furthermore, we explored the effects of osteogenic oxysterol sterosomes in vivo with the mouse critical-sized calvarial defect model. Our results showed that SA/Oxy sterosomes induced osteogenic differentiation in vitro and enhanced calvarial healing without delivery of additional therapeutic agents, indicating their intrinsic bone-forming potential. This study suggests a promising non-phospholipid liposomal platform with osteoinductive properties for delivery of small molecular drugs and/or other therapeutic genes for enhanced bone formation.