Study On Local Long-term Drug Release Composite Systems In The Treatment Of Bone Tuberculosis

BackgroundIn current,TB can be controlled under a certain level, because there are a large number of TB patients worldwidely, it is still the most important "killer". Our country has finished national epidemiological survey of tuberculosis for five times around the millennium, the TB incidence rate shows a declining trend, but the trend of extrapulmonary tuberculosis is not uniform with pulmonary tubercuculosis, the proportion of extrapulmonary tuberculosis accounting for tuberculosis has increased. Bone tuberculosis is the most important extrapulmonary tuberculosis,90% of which are secondary to pulmonary tuberculosis, accounting for 3% to 8% of TB cases, and spinal tuberculosis accounts for half of bone tuberculosis cases. Bone tuberculosis, especially spinal tuberculosis, the delayed diagnosis of M. tuberculosis infection or inadequate control of multi-drug resistant tuberculosis,can easily lead to spinal cord or nerve root compression, spine and limbs joint structural damage, resulting in paraplegia, kypHosis, and even endanger the lives of patients.Pathological tissues of bone tuberculosis include caseous necrotic material, sequestrum, cold abscess, granulation tissue and Mycobacterium tuberculosis bacteria. In the peripHery of the non-hardened lesions are wrapped by fibrous connective tissue, for the hardened lesions, the peripHeral which are a hardened wall, the sclerotic bone. First line anti-tuberculosis drug concentrations can achieve above the minimum inhibitory concentration in the non-hardened lesions. However, hardened lesions, which limits the deterioration of lesions by sclerotic bone, but almost no blood supply, anti-tuberculosis drugs within the sclerotic bone can achieve only the minimum inhibitory concentration, less than bactericidal concentration and form barriers impede drug into the lesion. Tuberculosis bacteria hidden in the cheese-like lesions of necrosis and flora is only sensitive to rifampicin and pyrazinamide. Very low drug concentrations and local lesion’s relatively closed environment, such as hypoxia,and these complex factors cause bacteria in the cheese-like or necrosis lesions within the hardened bone are difficult to be killed and become potential factors of recurrence of tuberculosis. Thus, by topical routes of administration of drugs directly into the lesion, and release of anti-TB drugs, are in favor of the treatment of tuberculosis.Advantages of drug delivery system for bone and joint tuberculosis are that it can achieve high local concentrations for sterilization, is conducive to kill or inhibit growth and reproduction of Mycobacterium tuberculosis, and reduce drug resistance due to low local concentration of the drug,and there are low concentration of drug in blood and less systemic side effects.pHarmaceutical carrier materials can replace or repair bone defects and can be gradually degraded by the body, and they can be eventually replaced by bone tissue. Therefore, the development of anti-TB drug release system implanted in bone and joint tuberculosis lesions during surgery, is conducive to form a minimum inhibitory concentration of the drug environment for a longer period and kill or suppress of residual Mycobacterium tuberculosis bacteria in the lesions. At the same time the carrier can play a bone graft substitute role in the degradation of the body after implantation of the lesion, while release of the drug, gradually replaced by bone tissue, can accelerate healing of lesions. Thus, To research and develop of drug delivery material system for the treatment of bone and joint tuberculosis is promising.There are a wide range of anti-TB drugs, with different hydropHilic and hydropHobic, in order to solve the problem of initial drug release violent release and load multi-drug for controll release.TB drugs can be divided into three categories by hydropHilic and hydropHobic, water-soluble,such as isoniazid; oil-soluble anti-TB drugs,such as rifampicin; not completely soluble in water or not completely soluble in oil.such as pyrazinamide. Blend, is the most common, simple and effective way for drug loading,for oil-soluble drugs, it can be a useful way of loading anti-TB drugs, but for water-soluble drugs, the simple preparation of slow-release material by blend,it shows a early release of a drug drugs,causes excessive amount loss drugs, and local concentration of drugs is too high, indirectly leading to increase drug concentration in the blood, increasing the risk of side effects and drug resistance selectivity loaclly. Ways to improve drug loaded include:chemical reaction and microspHere encapsulation methods. Chemical bonding method is that the drug was intended to hydrazone bond with chemical polymer, in a particular microenvironment, such as changes in pH, the material degrades and releases of the drug. Chemical bonding process is to connect PLGA with active aldehyde group,and make the water-soluble drug,such as isoniazid, an amine of which bond active aldehyde group. Bonding process of PLGA and isoniazid is that amine reacts with aldehyde group forming a hydrazone group.Hydrazone group is sensitive to changes of pH value, When pH value is alkaline or neutral, it is stable,while PH value of a weak acidn it dissociate and release drug. Disadvantages of chemical bonding way are that the reaction process requires the use of a variety of organic solvents and the drug has a risk of being broken. MicrospHere encapsulation method is that drug is encapsulated in microspHeres before loading the microspHeres to a composite material. MicrospHeres include chitosan or gelatin,and other materials.Gelatin is chosen for this study.β-tricalcium pHospHate ((3-TCP) is chosen as the pHospHorus compound for preparation of sustained-release material. MicrospHere encapsulation method can protect unstable drug within the microspHeres load, but the amount of drug loaded is the bottle neck of this method.As chemical bonding and microspHere encapsulation have some advantages and disadvantages, we should adopt both of the two methods.Oil-soluble anti-TB drugs is represented by rifampicin. Blend is the most common way of drug loading which is simple and effective, the oil-soluble drugs can be loaded by blending. During preparation composite containing rifampin,which dissolves in polylactone and mix with TCP. Some rifampin is loaded on the surface of composite, may cause initial burst release of rifampicin. And most of rifampicin is embedded in composite materials, so the pHysical and chemical properties of the composite can affect the release behavior of rifampicin, PLLA or PLGA are the same hydropHobic, resulting in material’s delayed drug release which is relative slow. However, the rate of diffusion The pHysical and chemical properties of PCL is different with PLLA or PLGA,the ratio is faster. For oil-soluble drugs, this study focuses on polylactone design. Polylactones, a kind of biodegradable polymers with good biocompatibility, are widely used in biomedical fields. Some polylactone-based drug systems with loaded anti-tuberculosis (TB) drugs have been developed for treatment of bone tuberculosis. However, due to initial burst release, short sustained release and no bone conduction, these systems cannot meet the requirement of bone TB treatment including long-term drug controlled release and bone fusion. This study attempts to develop a novel poly (caprolactone)-b-poly (lactide-co-glycolide) (b-PLGC) ternary block copolymer, study its multidrug controll release and bone substitute behavior. In this study, a new type of block copolymer b-PLGC, which blend with β-TCP and load rifampin to form composite.Bone tuberculosis is infected by Mycobacterium tuberculosis,which manifestate in the bone lesions,the principle of anti-TB drugs is "early, joint, righ amount, regular, full-time," the principle of anti-TB drugs is fundamental to success,which is the cornerstone of successful treatment of bone tuberculosis. The so-called "joint" means at the same time using a variety of anti-TB drug therapy to improve efficacy while reducing the cross antibacterial drug resistance risk. For topical treatment, we need to solve two problems:the drug’s controll release time and multi-drug combination. To solve the difficulty of carrying multi-drug, this study attempts to prepare composite system contained both water-soluble and oil-soluble drugs,we use microspHeres encapsulation method to load isoniazid, b-PLGC is chosen as polylactone, β-TCP is selected as a calcium pHospHate compound, eventually isoniazid/gelatin/(3-TCP microspHeres, b-PLGC and rifampicin form multidrug material system.Objective:In this proposal, we will develop various novel polylactone-based anti-TB drug systems, evaluate and optimize drug release behaviors and bone fusion, thus realize the combination of long-term local release of anti-TB drugs and bone regeneration, and provide a new way for the clinical treatment of bone tuberculosis. In detail, we design several synthetic multi-block and branched polylactones and study effects of their molecular structures and degradation on permeability and release of drugs to inhibit initial drug burst release and prolong drug release. Then, we synthesize mesoporous TCP nanoparticles (NPs) with rough surfaces, and study relationships of doping behaviors and interface structures between morpHologies of TCP NPs and various polylactones with different topologies. Finally, we adopt antibacterial experiments in vitro and bone repair in vivo to systematically evaluate the effects of bone TB treatments for these novel polylactone-based anti-TB drug systems.Methods1.1 Preparation of a water-soluble anti-TB drugs INH microspHeres/b-PLGC Composites using encapsulated microspHeres methods1) were prepared INH/gelatin microspHeres, Evans blue/β-TCP/gelatin microspHeres, INH//β-TCP/gelatin microspHeres by oil in water emulsion Method2) three microspHeres were observed and characterized,we use scanning electron microscopy, thermal gravimetric analysis, to analyze the morpHology of the scaffold, pore structure, TCP content and mechanical properties; composite scaffold drug loading, encapsulation efficiency and in vitro drug release behavior were alalyzed by UV-visible spectropHotometer.3) microspHeres pHarmacokinetic analysis4) we prepared INH/β-TCP/gelatin microspHeres/b-PLGC and loaded β-TCP using water in oil emulsion methods, in order to refine the content of β-TCP,we prepared 0%,33%,50% β-TCP contained in gelatin microspHeres, in order to facilitate the discussion, abbreviated as INH/GMs, INH-TCP@ GMs3367, INH-TCP@ GMs50505) In vitro INH/β-TCP/gelatin microspHeres/b-PLGC stent release behavior1.2 Using chemical reaction method to prepare INH Composites Using end-modified PLGA bonded with drug to prepare molecular weight of 100kDa star PLGA loaded INH(PET-PLGA100kDa-INH)1) Synthesis of pentaerythritol (PET)-PLGA100kDa-CHO2) Synthesis of PET-PLGA100kDa-INH3) Preparation of PET-PLGA100kDa-INH-β-TCP composite scaffolds)4) Characterize and observe PET-PLGA100kDa-INH-β-TCP composite scaffolds5) PET-PLGA100kDa-INH-β-TCP scaffolds drug release behavior in vitro2 design block copolymer (b-PLGC), combined rifampicin loaded polylactone/ β-TCP composites1) Preparation of a block copolymer b-PLGC2) Preparation of rifampicin/polylactone/β-TCP scaffolds3) Characterization properties of the composite scaffold4) composite scaffold in vitro drug release behavior5) cell affinity of the scaffold6) repair scaffold for bone defect7) composite scaffolds drug release behavior in vivo8) composite scaffolds’osteogenesis performance evaluation in vivo3 Preparation Composites which contained two kinds of anti-TB drugs1) Preparation of a block copolymer b-PLGC2) Preparation of INH/β-TCP/gelatin microspHeres3) preparation rifampicin, polylactones, INH-TCP@ GMs loaded Composites4) Characterization of composite Materials contained two kinds of anti-TB drugs5) release behavior of composites which contained two kinds of anti-TB drugs in vitroResults:1) In order to solve the problem of violence early release and release times of pHysical blending isoniazid, respectively using microspHere encapsulation method and the chemical bonding method for preparing a isoniazid loaded compound.The INH-TCP@ GMs5050 microspHeres’burst release time is relatively small, and it sustained release for a long time; b-PLGC scaffolds further reduce the role of the early burst release, extended release time to a certain extent. In terms of chemical bonding, PET-PLGA100kDa-INH complexes with β-TCP to form scaffold. After the initial three weeks of rapid drug release, drug release in a smooth way to release more than 100 days. TCP added did not significantly affect the microstructure of the composite material, but had some influence on the early release of drug violence release behavior.2)the block terpolymer (b-PLGC) is a novel polylactone material prepare by polymerization embedding.lt compounds the TCP,then prepare RFP/b-PLGC/ TCP Composite and compare with RFP/PLLA/TCP composites, RFP/PLGA/ TCP composite and RFP/r-PLGC/TCP composites, although the four scaffolds present different degrees of loss and less encapsulated problem and initial drugs burst release. RFP/b-PLGC/TCP composites can achieve up to 84 days post-linear drug release,the imaging findings, histological observation and osteocalcin quantitative show that composite has good osteoconductive properties and does not affect the ability of bone repair.3) we prepare composite system containing both water-soluble and oil-soluble drugs, using microspHere encapsulation method to load isoniazid, b -PLGC is selected, finally, microspHeres, block terpolymer (b-PLGC) and rifampicin form multidrug synthetic composites. The composite materials can achieve sustainable release of the two drugs for 84 days, while loading isoniazid and rifampicin does not affect drug release behavior.conclusion:1) TCP does not affect the characterization of composite materials, has a certain influence on the drug release, microspHere encapsulation improves early outbreak release, and chemical bonding way has advantage of longer drug release. For water-soluble drugs, we can choose the manner of loading drugs by pHysicochemical properties.2) b-PLGC is a novel polylactone material can load oil soluble anti-TB drugs and have linear constant release capacity.It also has good histocompatibility,and does not affect the ability of bone repair.3) Composite system which load water-soluble and oil-soluble drugs completes the intended purpose,it can reduce the initial burst release and extend release time. We can prepare the composite material containing multidrug through this approach which can be used as bone tuberculosis treatment, and further clinical application...