# BMP-2 Delivery through Liposomes in Bone Regeneration
## 3 Liposomes  
Liposomes are biocompatible, self-assembled, spheric vesicles composed of concentric phospholipid bilayers wrapped around an aqueous compartment. Phospholipids are composed of a hydrophilic polar head and a hydrophobic non-polar tail. They were first described in the 1960s [85] and have been used as delivery systems since the 1970s [86]. As their structure is similar to cell membranes, they can easily merge with cells, penetrating them, and discarding their cargo. They can incorporate many types of molecules depending on the structure of the liposome and the hydrophilicity of the entrapped molecule. The hydrophilic drugs are entrapped into their inner compartment and the hydrophobic ones are linked to them directly or indirectly, either on the surface or between the two lipid layers (Figure 1). They are extremely versatile. Although there is little researched on their role in bone regeneration, their role in other areas is well documented. Liposomes are used as a vector in more than 20% of approved clinical trials in controlled drug delivery [87]. There are numerous FDA-approved clinical applications of liposomes in vaccine development, as well as antibiotic and analgesic delivery [88]. In cancer therapy, antibody conjugated liposomes (immunoliposomes) have been used in clinical trials to eliminate circulating cancer cells, preventing metastasis [89]. In the COVID-19 pandemic, the first approved vaccines were using liposomal transfection of messenger RNA [90]. By conjugating with magnetic nanoparticles and imaging agents, in vivo traceability of liposomes has been achieved. Targeted release has been obtained by exposure to ultrasound [91] or magnetic actuation [92; 93]. Using a combination of radioactive molecules bonded to liposomes, diagnostics, targeting, and treatment have been obtained, which has been called theranostics [94]. Attempts were made to immobilize liposomal BMP-2 onto different biocompatible scaffolds for a sustained, long-term release. Electrospun poly(L-lactic acid) fibers, functionalized with hydroxyapatite (HA) can be a good carrier for BMP-2 loaded liposomes. Adipose tissue-derived MSCs were seeded onto the scaffolds. The levels of ALP and calcium ions were significantly higher in the liposome and HA containing scaffolds than in the control group (HA-coated scaffolds with free BMP-2). The expression level of the genes related to osteogenesis were three-fold as compared with the control group. The osteoconductivity of the constructs was tested in vivo by subcutaneous implantation into rats. At the site of the implants, MSCs aggregated and primary ossification centers appeared [95]. Magnetic liposomes have also been used to carry BMP-2 and in combination with magnets and have had good results in maintaining the proteins at the injury site for a prolonged period of time. Entrapment efficiency was approximated to be the same with fluorescein isothiamate-conjugated dextran (FD-40) which has the same molecular weight as BMP-2. The magnetic liposomes had entrapped a lower quantity of FD-40 than the conventional ones. A critical size bone defect was created in the animal's femur and  
Figure 1: Structure of liposomes and the molecules that can be delivered through them. a magnet was inserted. Different magnetic and non-magnetic liposomes were injected at the injury site, at different timepoints. Only the animals injected with magnetic liposomes immediately after the surgery, presented complete bone bridge formation [96]. In situ gels are a good alternative for drug delivery systems. They are liquid ex vivo and turn into gels in the organism depending on several factors. Growth factors included in liposomes can be entrapped in gels for a prolonged and controlled release, resulting in longer and more stable plasma levels of the protein and significantly more bone formation when injected into critical size bone defects [97]. Hydroxapatite is found only in bone tissue and designing systems that can link to it is an important goal in osteogenic research.