Construction Of Bone Targeting Drug Delivery System Based On Lipid Nanocarriers And Applications In The Treatment Of Osteoporosis

Osteoporosis is a disease of the skeletal system characterized by the degeneration of bone tissue microstructure and bone mass reduction,having been ranked by the World Health Organization as the second most harmful disease to human health after cardiovascular disease.At present,most of the drugs used for the treatment of osteoporosis are to inhibit osteoclasts and thereby reduce the loss of bone mass in the human body,but they cannot reverse the bone mass and repair the damaged bone tissue.Simvastatin(SIM)is one of the commonly used lipid-lowering drugs in clinic,but recent studies have found that SIM can promote osteoblast differentiation and mineralization through the BMP-Smad signaling pathway,thereby promoting bone regeneration,which opened up a new way for osteoporosis treatment.However,as a poorly water-soluble drug,SIM has low bioavailability in the human body and less bone tissue distribution.To achieve the therapeutic effect of osteoporosis,it is necessary to increase the dose of administration,but this will inevitably cause toxicity and side effect of other tissues and organs.Therefore,there is an urgent need to develop a SIM-loaded bone targeting transporter to increase the distribution of SIM in bone tissue and improve the efficacy of the drug.Lipid nanoparticles play an important role in drug delivery due to their good biocompatibility,high encapsulation rate of fat-soluble drugs,and drug sustained-release effect.Therefore,in this study,aspartate hexapeptide(ASP₆)was used as the bone-targeting group,and the bone-targeting lipid nanoparticles(SIM/ASP₆-LNPs)with a particle size of about 100 nm were prepared by using an aqueous solvent diffusion method.The encapsulation rate of SIM/ASP₆-LNPs to SIM can reach more than 97%,and it has a slow-release effect on SIM,and the cumulative release amount in 48h exceeds 70%.Taking mouse precranial cranial bone cell subclone 14 cell line(MC3T3-E1 subclone 14)as a model cell,bone-targeting lipid nanoparticles can be well taken up by MC3T3-E1 cells,and compared to free drug SIM,it can effectively reduce the cytotoxicity of SIM.Alizarin red staining experiments and alkaline phosphatase activity experiments have shown that SIM-loaded bone targeting lipid nanoparticles can achieve a drug effect similar to free SIM,that is,promote the differentiation and mineralization of osteoblasts,thereby promoting bone repair and regeneration.ASP₆ modified bone targeting lipid nanoparticles can be distributed more in bone tissue and effectively reduce the distribution in non-targeting tissue.Female SD rats were used as model animals,and bilateral ovaries were removed to establish an osteoporosis model.Micro-CT results show that different treatment groups have different degrees of bone mass increase,of which the bone targeting lipid nanoparticles modified with ASP₆(SIM/ASP₆-LNPs)are the most obvious(bone density is0.176g/cm²),basically reaching the bone mass of the sham operation group(Sham)(the bone density is 0.186g/cm²).In addition,the expression of enzymes and proteins related to the osteoblast differentiation and mineralization period was the largest in the SIM/ASP₆-LNPs group,indicating that the bone targeting lipid nanoparticles constructed in this study can transport more SIM to bone tissue to exert medicinal effects.At the same time,the therapeutic effect of soybean lecithin(PL)modified calcium carbonate nanoparticles on osteoporosis was also studied.Amorphous calcium carbonate nanoparticles(ACC)with a particle size of about 80 nm were prepared by gas diffusion method,and the surface of ACC was modified with soybean lecithin(PL)and another bone targeting group,tetracycline(TC),to prepare bone targeting complex lipids nanoparticles(TC/ACC).Then we prepared SIM-loaded bone targeting composite lipid nanoparticles(TC/ACC/SIM)with SIM as a model drug to achieve the synergistic treatment of osteoblast promoter SIM and calcium supplement(TC/ACC).The drug loading of SIM in bone targeting composite lipid nanoparticles can reach 8%.TC-modified bone targeting composite lipid nanoparticles have a higher affinity for hydroxyapatite.Rat primary osteoblasts were used as model cells.Bone targeting composite lipid nanoparticles can be well taken up by osteoblasts,and compared with the free drug SIM,it can effectively reduce the cytotoxicity of SIM.The alizarin red staining experiment and the alkaline phosphatase activity experiment show that both SIM and TC/ACC can increase the ALP activity during the differentiation period and increase the calcium salt deposition during the mineralization period,and the TC/ACC/SIM group can achieve synergistic treatment effect.Micro-CT results show that the TC/ACC/SIM group has the most obvious reversal of bone mass loss compared to the single-treatment TC/ACC and SIM,and achieves the synergistic treatment of calcium and SIM.Finally,considering that the treatment of osteoporosis is a long-term process,in order to improve patient compliance,we continue to explore nanocarriers available for oral administration based on the previous synergistic treatment.In this study,PL-modified ACC was packed into glyceryl monostearate(MS),which is relatively stable in gastrointestinal tract,and TC was modified to obtain oral bone targeting composite lipid nanoparticles TC/MS/ACC.When the ratio of MS to ACC is 1:1,TC/MS/ACC transports best through the intestinal mucosa.Using SIM as a model drug,oral drug-loaded bone targeting composite lipid nanoparticles(TC/MS/SIM)with a particle size of about 100 nm were prepared.Micro-CT results show that the TC/MS/SIM constructed in this study,compared with commercially available calcium carbonate tablets(bone density of 0.098g/cm²),due to the bone targeting effect of TC and the inclusion on SIM of composite lipid nanoparticles,protecting which from gastrointestinal tract damage,it has the strongest effect of reversing bone mass loss(bone density of 0.138g/cm²),basically reaching the bone mass of sham operation group(sham)of 0.139g/cm².In addition,due to the slow-release effect of TC/MS/SIM on SIM,it can significantly reduce the hepatotoxicity of SIM and has a good vivo safety.The bone targeting lipid nanoparticles and bone targeting composite lipid nanoparticles constructed in this study can effectively promote the differentiation and mineralization of osteoblasts,thereby promoting bone repair and regeneration,providing a new strategy for the treatment of osteoporosis.