Study Of A Neutrophil-based Bone Marrow-targeted Drug Delivery System

Bone is one of the most important structures of the body and plays an important role in support and protection.Also,inside the bone is the bone marrow,the birthplace of numerous immune and hematopoietic cells.Bone-related diseases are common,and in addition to diseases such as fractures,which are encountered on a daily basis,there are more complex diseases such as bone cancer,osteomyelitis,osteoporosis and osteoarthritis.In particular,diseases that develop in the bone marrow,the inner part of the bone,are more difficult to treat.This is because the presence of the blood-bone marrow barrier,in addition to the low blood perfusion in the bone marrow,makes it difficult for general drugs to reach the bone marrow area,resulting in poorer therapeutic results.Neutrophils are one of the most common cells in the body,accounting for 50-70%of the white blood cells in the blood.Neutrophils play a major role in the body in finding and migrating to the site of inflammation,phagocytosis,and killing bacteria,and are one of the important component cells of the body’s immune system,as well as being bone marrow-derived cells.Neutrophils develop and mature in the bone marrow and migrate into the bloodstream.At this time,neutrophils are highly expressed in CXCR2 and low in CXCR4 and are recruited to function at sites of inflammation when stimulated by inflammatory signals(e.g.IL8).However,the half-life of neutrophils is only a dozen hours.Most of the neutrophils gradually go senescent during the blood circulation,during which the expression of CXCR2,which was originally highly expressed,is downregulated,while CXCR4 is up-regulated,and finally stimulated by the chemokine CXCL12(SDF-1α)secreted by bone marrow stromal cells,through the CXCR4/CXCL12 axis remigration back to the bone marrow and apoptosis.In this dissertation study,we designed a drug delivery system that can specifically target bone marrow for the treatment of bone-related diseases by targeting senescent neutrophils back to bone marrow.We first prepared poly(lactic-co-glycolic acid)(PLGA)nanoparticles encapsulating the antitumor drug cabazitaxel(CTX)by a single-emulsion method,and the cabazitaxel-loaded PLGA nanoparticles(CTX-NPs)could be released slowly in vitro for 10 days.Due to the superior phagocytic ability of neutrophils,CTXNPs-loaded neutrophils(CTX-NPs@NEs)can be obtained by simple incubation with neutrophils.Meanwhile,the responsiveness of blank neutrophils(NEs)and CTXNPs@NEs to CXCL12,which was absent in dead cells,was demonstrated in vitro by Transwell assay.Next,approximately 30% of labeled neutrophils that had ingested fluorodeoxyglucose(18F-FDG)were detected by PET/CT back to the bone marrow,and the ability of neutrophils to deliver free drug(18F-FDG)into the bone marrow was also demonstrated.After intravenous infusion of neutrophils ingesting fluorescently labeled nanoparticles into the body,both live imaging and immunofluorescence section images observed that nanoparticle-loaded neutrophils were more concentrated in the bone marrow and spleen than nanoparticles alone,whereas free nanoparticles not taken up by neutrophils were mainly trapped in the liver after injection.The above study demonstrated the feasibility of a neutrophil-based bone marrowtargeted drug delivery system.Next,we applied this system to bone metastases from breast cancer.Patients with advanced breast cancer are highly susceptible to bone metastases,when removing the tumor through surgery is impossible.After treatment with CTX-NPs@NEs,bone metastases were effectively controlled in mice compared to controls,reducing secondary metastases of bone metastatic tumor cells to the lung,while protecting the bone and slowing bone loss.In addition,we also validated the effectiveness of a neutrophil-based bone marrow-targeted drug delivery system in a post-ovariectomy mouse model of osteoporosis.We synthesized nanoparticles of teriparatide(PTH)and prepared PTH-NPs@NEs.In the anti-osteoporosis assay,relevant serum indices and bone parameters were largely restored and osteoporosis symptoms were alleviated in the PTHNPs@NEs group of mice.Finally,we systematically evaluated the safety of the drug delivery system because the drug concentration in the bone marrow would be dramatically increased under the action of the system.Toxicity analysis of major organs such as heart,liver,and kidney,as well as changes in the ratio of bone marrow cell fractions,revealed that the neutrophil-based drug delivery system itself is almost nontoxic,but encapsulation of CTX,a cytotoxic drug with myelosuppressive properties,amplifies the myelosuppressive effect.Therefore,the selection of target drugs and models for treatment is critical.In summary,this thesis designed a neutrophil-based drug system for bone marrow targeting from a biopharmacological perspective based on the apoptotic properties of senescent neutrophils returning to the bone marrow,and validated it on two models of bone metastases and osteoporosis,providing some ideas for early diagnosis and improving the therapeutic effects of bone-related diseases in clinical practice.