A Novel Design of Bone Targeting Through Bisphosphonate Lipid Nanoparticles

Introduction: Lipid nanoparticles (LNPs) for targeting the bone microenvironment offer significant potential for nucleic acid therapeutic applications in bone and bone-related tissues. However, biological barriers, such as low blood flow in bone, blood−bone marrow barriers, and low affinity between drugs and bone minerals, substantially hinder therapeutic delivery to bone, resulting in unfavorable dosage conditions in the bone microenvironment. To solve this problem, bisphosphonate (BP) ionizable lipids are introduced, which have a high affinity for bone minerals by enhancing LNP targeting and accumulation in the bone microenvironment. Here we formulate a series of bone-targeting ionizable lipids with a robust piperazine backbone linking bisphosphonate group and use them to make BP-LNPs with bone-target effect. A library of 56 BP-LNPs is constructed, and BP-LNPs with the highest transfection efficiency are selected by in vitro screening on BJ cells. Further, we build up a novel in vitro model mimicking the bone microenvironment to prove high bone mineral localization of BP-LNPs and their effective transfection to cells adhering to bone minerals. The bone microenvironment transfection model and in vivo results demonstrate a high affinity and localization of BP-LNPs in bone minerals compared to LNPs without BP groups. In conclusion, these results validate the potential of bone-target mRNA delivery by BP-LNPs. This study proposes a novel target-delivery approach to a bone-like/dental environment which solves the need for developing new therapeutic methods, potentially being utilized for a range of mRNA therapeutic applications including nucleotide delivery to bone marrow cells, bone regeneration, and craniofacial tissues.

Materials and

Methods: First, we synthesize 56 bisphosphonate ionizable lipids (BP-lipids) with a robust piperazine backbone and corresponding BP-LNPs are prepared at specific lipid formulation parameters (Ionizable lipid/DOPE/Cholesterol/C14-PEG2000 = 35/16/46.5/2.5%, molar ratios) encapsulating firefly luciferase mRNA via a microfluidic device. The particle size and zeta potential are measured by DLS and the encapsulation efficiency and mRNA concentration of BP-LNPs are measured by RiboGreen assay. BJ cells are used for in vitro screening, and firefly luciferase expression is quantified using the Luciferase Assay System after 24 hours of LNP transfection. BP-LNPs with the top performance are selected for the following experiments.
To experimentally demonstrate the interaction between LNPs and hydroxyapatite (HA), HA disc surfaces are coated with either negative control group LNPs based on 'C12-200' ionizable lipid or BP-LNPs. After 24 hours, the surfaces are thoroughly rinsed with deionized water to remove unbound LNPs. We use AFM to visually confirm whether bone-targeted LNPs effectively bind to the HA discs. Next, we investigate whether BP- LNPs bind to HA and transfect under in vitro experimental conditions. BJ cells are seeded on the surface of HA discs soaked in a mixture of DMEM media and BP-LNPs. After 48 hours, the cells are lysed and luciferase expression is measured using Luciferase Assay System.
In vivo transfection are performed with the optimal BP-LNPs. Mice receive a single intravenous injection of FLuc mRNA LNPs at a dosage of 0.5 mg/kg via the tail vein. Luciferase expression is assessed using an IVIS Spectrum imaging system 12 hours after injection.

Results, Conclusions, and Discussions: The synthesis of various forms of piperazine-based bisphosphonate-linked ionizable lipids, derived from economically viable starting materials is accomplished. Corresponding BP-LNPs are formulated with a fixed molar ratio produced by microfluidic devices. A library of 56 BP-LNPs is constructed and in vitro screening is performed on BJ cells. BP-LNPs exhibit very low toxicity and high mRNA transfection within cells. A heatmap is generated to evaluate the influence of various structural parameters on in vitro mRNA delivery. Among the BP-LNPs generated with the fixed molar ratios, LNPs based on '200-C12', '197-C12', and '488-C10' ionizable lipids exhibited the highest efficiencies. Transfection occurs more efficiently when a rigid backbone like piperazine is present compared to amine cores without piperazine structures. Considering the relationship between tail length and transfection, it can be inferred that shorter tails are advantageous for transfection when the molecular size of the capping ligand and amine core is large, facilitating interactions among adjacent lipids. Ionizable lipids tend to have a higher solubility in DMF solvent which also facilitates the formation and transfection of BP-LNPs.
Further, HA binding studies show that BP-LNPs can be immobilized on a bone mineral surface, which enables three-dimensional shape analysis using analytical equipment such as AFM. The morphology of the empty HA disc and the non-binding groups are similar while the BP-LNP group exhibits a completely different pattern. This difference is also evident in the roughness values, indicating that the hydroxyapatite disc surface with rough features is enveloped by BP-LNPs, showing its binding and accumulation on hydroxyapatite.
After demonstrating the in vitro efficacy of BP-LNPs for bone targeting, we further evaluate whether BP-LNPs enhance mRNA delivery in vivo. High luciferase expression is observed in the leg, particularly in the femur, patella, and tibia. The in vivo experiments are consistent with the binding studies demonstrating that the newly developed BP-LNPs can strongly bind to bones, highlighting their potential for bone-targeted RNA delivery.
In conclusion, the newly designed BP-LNPs show high efficiency in mRNA transfection with high localization and accumulation to the bone microenvironment, which holds immense potential in developing next-generation bone-target delivery systems.

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