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The Formulation Of Tanshinol-loaded Bone-targeting Liposome And The Research Of Its Preventive And Therapeutic Effects On Fracture Delayed Union Of Mice

Posted on:2017-09-18Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y Z LiuFull Text:PDF
GTID:1364330488484854Subject:Pharmacy
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BackgroundBone fracture is one of the most common clinical musculoskeletal diseases,which approximately affects 2%people yearly in the world,and 5-10%of all the fracture patients would occur bone fracture union disorders.With the aging society and the increasing cases of fracture union disorder caused by different reasons,which create substantial economic burden to individuals,families and social medical resources.The treatment of bone fracture union disorders nowadays,no matter autologous bone grafts or BMP protein intervention,were both difficult to meet the actual clinical needs because of the limitation of their own.Moreover,the widespread application of these complex,economically expensive and even potentially harmful strategies in clinic are not clear.Therefore,a simple,safe and effective non-invasive strategy is strongly needed for the clinical treatment of bone fracture union disorders.Previous research of our group demonstrated tanshinol,a water-soluble ingredients monomer of traditional Chinese medicine named salviae miltiorrhizae,was effective for the treatment of osteoporosis.And due to previous traditional Chinese medicine treatment and researches had applied the effective ingredients of salviae miltiorrhizae in the treatment of bone fracture,we assume that tanshinol may be applied in the treatment of bone fracture union disorders.However,tanshinol has its limitation for easy oxidation,short half-life and also because of bone tissue has high hardness,low blood flow and poor permeability physiology issues,the prototype drug of tanshinol is not suitable directly applied for the treatment of fracture related disorders.Thus,the realistic requires to develop an appropriate drug formulation for tanshinol,which makes tanshinol stable and increases the retention time in the fracture site.To date,the development of nanoparticle target delivery strategy has been widely considered to be a potential promising strategy,which can provide several unique advantages for fracture union disorders,including deliver drug to its destination meanwhile maintain its concentration;attain maximum benefit from the drug;reduce side effects of the drug,protect drug from the dispersing and degradation in body fluids;increase the circulation time or retention time in vivo et al.In the nanoparticle target delivery system,liposome is an idea nanoparticle carrier for its simple preparation,non-toxic,no immunogenicity,easy loading and releasing agents and can deliver agents to specific destination by simple modifying its bilayer.Therefore,our group chose tanshinol as a basic drug molecule and liposome as a drug carrier with bone-targeting ability by modifying its surface,to develop a tanshinol-loaded bone-targeting liposome(Tan-BTL)for the treatment of fracture union disorders.Our research may provide new ideas and experimental basis for the further investigation of small molecule drugs of traditional Chinese medicine in the treatment of musculoskeletal diseases,and also may provide reference data for the development of modern creative small molecular natural drugs.Objectives1.To establish a standard formulation method for tanshinol-loaded bone-targeting liposome and characterize its basic properties.2.To investigate the bone-targeting properties of bone-targeting liposome in vitro and in vivo.3.To investigate the effects of tanshinol-loaded bone-targeting liposome on glucocorticoid-induced fracture delayed union of mice.Contents and methods1.The formulation and characterization of tanshinol-loaded bone-targeting liposomeTo establish a standard method of formulation,purification and detection for tanshinol-loaded bone-targeting liposome and characterize its basic properties.1.1 A certain proportion of natural lecithin and cholesterol were used to formulate liposome by reverse evaporation.In the liposome preparation process,bone-targeting molecule and tanshinol solution were added in the formulation.The bone-targeting molecule would insert into the bilayer of liposome and the bilayer would encapsulate tanshinol to form tanshinol-loaded bone-targeting liposome.Avanti mini-extruder was used to acquire desire size liposome and the liposome was purified by column chromatography.1.2 The size and the Zeta potential of tanshinol-loaded bone-targeting liposome were detected by dynamic light scattering system(DLS)for characterizing its physical properties.Transmission electron microscopy(TEM)was used to observed the bilayer structure of tansinol bone-targeting liposome.1.3 According to linear relationship between tanshinol concentration and the ultraviolet absorption peak area of the high performance liquid chromatography(HPLC),a tanshinol standard curve of HPLC was established by determining a series of tanshinol standard solution.And then fast protein liquid chromatography(FPLC)was used to separate the liposome and the free tanshinol.The free tanshinol was collected and determined by HPLC to acquire the concentration,then the encapsulation efficiency of tanshinol-loaded bone-targeting liposome was calculated.On the other hand,a different method was also used to calculate tanshinol encapsulation efficiency.Tanshinol standard curve of FPLC was also established by determining a series of tanshinol standard solution.FPLC was used to separate the liposome and the free tanshinol,and the concentration of free tanshinol was detected at the same time for the encapsulation efficiency calculation of tanshinol-loaded bone-targeting liposome.1.4 The size of tanshinol-loaded bone-targeting liposome was monitoring by DLS at the 1th,3th,8th,15th,30th and 45th day for stability test.2.To investigate the bone targeting properties of bone-targeting liposome(BTL)in vitro and in vivo.2.1 The liquid core of BTL was labeled by rhodamine B and the liposome was used for hydroxyapatite adhesion test.After the hydroxyapatite(HA)adhesion test,the adhered florescence liposome on HA powder was observed under a microscope and the florescence intensity of supernatant was determined by a microplate reader,which directly and indirectly characterized the HA affinity of BTL.On the other hand,the bilayer of BTL was labeled by FITC and the labeled liposome was also used for the HA adhesion test follow the same procedure.2.2 The BTL was labeled with rhodamine B and local injected on the femur surface of mice(Calcein was injected subcutaneously three days in advance the liposome injection for labeling the bone formation in vivo).The femur and tibia were harvested for frozen section.The distribution and bone affinity of florescence labeled BTL were investigated by microscope.On the other hand,thebone-targeting liposome was labeled with FITC and repeated the same experiment above(xylenol orange was subcutaneously injected 3 days in advance the liposome injection for labeling the bone formation florescence in vivo)2.3 The BTL and non-targeting lipsome(NTL)was labeled with IRdye 800CW and local injected in the femur closed fracture site of mice.The distribution and retention time of BTL were monitoring by LI-COR small animal florescence imaging system.On the other hand,bone-targeting liposomes with different ratios of bone-targeting agent were formulated for comparing the distribution and the retention time of these liposomes,which would profile the effects of bone-targeting agent ratio on the bone affinity.3.To investigate the effects of tanshinol-loaded bone-targeting liposome(Tan-BTL)on glucocorticoid-induced bone fracture delay of mice3.1 Mice were administered prednisone with low,medium and high dose(6 mg/kg,9 mg/kg and 12 mg/kg)by oral gavage for 12 weeks and were performed femur closed fracture on the 3th week,to investigate the effects of glucocorticoid-induced bone fracture delayed union on mice.Bone fracture healing process of mice were monitoring by a small animal X ray image system for selecting an optimal dose of prednisone to induce bone fracture delayed union on mice.3.2 Different doses of Tan-BTL(equivalent tanshinol dose 1.25 mg/kg,2.5 mg/kg and 5 mg/kg)were formulated and injected in the femur fracture site of mice,to investigate the effect of Tan-BTL on the treatment of bone fracture.The X ray image system and micro CT were used to monitor and analyze the fracture healing process in order to select an optimal dose of Tan-BTL for fracture treatment.3.3 Optimal dose of prednisone and Tan-BTL were selected to investigate the effects of Tan-BTL on glucocorticoid-induced bone fracture delayed union of mice.The small animal X ray image system and micro CT were used to monitor the fracture healing process and analyze the callus formation.Nine weeks after femur closed fracture surgery,three bending test was used to analyze the bone mechanical properties of mice.Results1.DLS data demonstrated the average size of Tan-BTL is 153nm,the PDI is 0.230 and the Zeta potential is-33.1mv.2.TEM images demonstrated Tan-BTL is large unilamellar vesicles with round shape,uniform size and nice polydispersity.The particle size of several liposomes in several field of images were measured to acquire the average particle size of Tan-BTL(166nm),which is similar to the data determined by DLS.3.The size of Tan-BTL was monitoring by DLS at the 1th,3th,8th,15th,30th and 45 th days demonstrated that the size,PDI and appearance did not change obviously during 45 days,which indicates that Tan-BTL is stable and not easy to aggregate or degenerate.4.Two methods were applied to calculate the encapsulation efficiency of Tan-BTL(1.HPLC combine with FLPC;2.FPLC).Method 1 results demonstrated the average encapsulation efficiency of Tan-BTL is 31.45%,the encapsulation efficiency of Tan-BTL determined by method 2 is 33.15%.Similar results were obtained from the two methods,which indicated that the formulation process and the encapsulation efficiency of Tan-BTL is stable.5.After the 6 hours HA adhesion test of rhodamine B labeled BTL,the supernatant data demonstrated that florescence intensity of rhodamine B labeled BTL decreased significantly compared to control and non-targeting liposome,which indirectly indicates the HA affinity of BTL.The microscope images demonstrated that rhodamine B labeled BTL showed bright red florescence on HA powders while non-targeting liposome and rhodamine B solution did not show any florescence.FITC labeled BTL also demonstrated a similar result.HA adhesion tests of rhodamine B and FITC labeled BTL both indicated that BTL has nice HA affinity.6.Femur frozen section images demonstrated that rhodamine B labeled BTL could distribute at different places in femur of mice,including the growth plate,the trabecular bone,the cortical bone and the bone lacuna.The red florescence of rhodamine B labeled BTL partly merge with the green florescence of calcein.FITC labeled BTL also repeated the same experiment.The images demonstrated FITC labeled BTL could distribute at the growth plate and the cortical bone of femur.The green florescence of FITC labeled BTL partly merge with the orange florescence of xylenol orange.The in vivo distribution and bone affinity test of BTL indicates that BTL could adhere to bone surface and distribute to different sites in bone tissue,but not limit to bone formation site.7.The IRdye 800 CW labeled BTL was locally injected in the fracture site of mice.The LI-COR small animal florescence imaging system demonstrated that BTL could concentrate on fracture site and the retention time of BTL at fracture lasted 10 to 20 days,including the BTL contains 2×10-6M targeted agent retain at the fracture site for 10 days,the BTL contains 4×10-6M targeted agent retain at the fracture site for 20 days and the BTL contains 8×10-6 M targeted agent retain at the fracture site for 14 days.The non-targeting liposome distributed throughout the body immediately after the local injection and a small amount of NTL retain at the fracture site until the 8th day.The IRdye 800CW solution degenerated 3 days after the local injection.These results indicate that the BTL has excellent bone targeting properties in vivo so that concentrate at fracture site and retain for up to 20 days.The retention time of BTL is related to the content of bone targeting agent in BTL,but is not a linear relationship.8.The prednisone dose selection test for femur fracture delayed union of mice,X ray images demonstrated that the fracture healing process of healthy control mice had completed in 30 days after the fracture surgery,while lots of callus still existed around the fracture site in GC-treated mice.The images indicated that GC induced fracture delayed union in mice.The fracture union time of mice treated with GC low dose mice was 12 days later than the healthy control mice,which the fracture union time increased 40%;The fracture union time of mice treated with GC medium and high dose(9 mg/kg/d or 12 mg/kg/d)were at least 34 days later than healthy control mice,which the fracture union time increased 113%at lowest.The data indicates that the fracture healing process is related to GC dose.9.X ray images demonstrated high dose Tan-BTL treatment significantly increased callus formation in femur closed fracture of mice compared to the healthy control mice.Micro CT analysis on callus demonstrated BV/TV in high dose Tan-BTL treated mice significantly increased 17%compared to the healthy control mice.The BV/TV in low and medium dose Tan-BTL treated mice showed no statistic significant difference compared to the healthy control mice.Micro CT data also suggest BV/TV of tibia trabecular increased with the dose of Tan-BTL.10.The investigation of the effects of Tan-BTL on glucocorticoid-induced fracture delayed healing mice.X ray images demonstrated that 18 days after fracture surgery,bone callus formation significantly decreased in GC-treated mice compared to the healthy control mice,while Tan-BTL treatment to GC-treated mice increased bone callus formation compared to GC-treated mice.Micro CT analysis on callus provided more details demonstrated that the callus BV/TV significantly decreased in GC-treated mice and Tan-BTL treatment to GC-treated mice obviously rescue BV/TV up to 57%compared to GC-treated mice,which indicates Tan-BTL treatment significantly stimulate callus formation in GC-treated mice.Tan-BTL treatment to GC-treated mice also could shorten the fracture union time at least 22 days(from>64 days to 42 days)and significantly improved bone biomechanical properties compared with GC-treated mice(>64 days).ConclusionThis study successfully formulated a tanshinol-loaded bone-targeting liposome with a standard procedure and its characterization had been established.The results demonstrated the property and encapsulation efficiency of Tan-BTL is stable and bone-targeting liposome had excellent bone affinity.Tan-BTL significantly increased bone callus formation in healthy fracture mice or GC-treated fracture mice,shortened fracture union time and significantly improved bone biomechanical properties in GC-treated mice.Tanshinol-loaded bone-targeting liposome could be developed as a potential promising natural small molecule creative drug for the treatment of fracture union disorders.
Keywords/Search Tags:Fracture, Delay union, Tanshinol, Bone targeting liposome, Mice
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