骨粉内纳米银缓释及抗菌性能研究

  本文关键词：骨粉内纳米银缓释及抗菌性能研究，由笔耕文化传播整理发布。

        背景：口腔种植中，多种原因（拔牙后牙槽骨萎缩、创伤、感染、肿瘤、先天畸形等）可引起牙种植体植入区骨量不足从而影响种植体的植入及远期效果。骨组织替代材料的植入成为解决这一问题的必要手段，而如何控制术后感染成为种植治疗中的难点及热点。目前广泛研究的抗生素如万古霉素、四环素、左氧氟沙星等负载到骨粉上技术因细菌耐药性、抗生素释放持续时间短暂等，而未能达到理想的控制骨粉感染的目的。银、锌、铜等无机抗菌剂掺杂入骨粉材料内可有效抑制细菌感染，但其对哺乳动物细胞的毒性成为应用中的隐忧，且如何使其持续有效的发挥作用还未很好解决。此外，如何有效利用纳米技术来延缓抗菌剂的释放还需进一步研讨。研究目的：本研究尝试用环式糊精（CD）及壳聚糖（CS）作为配体合成纳米银，再将不同配体的纳米银（NP）负载到人工骨粉内，结合乳化技术，外包裹聚乳酸羟基乙酸（PLGA），制成能延缓释放无机抗菌剂纳米银的骨粉生物材料。本实验的特点是利用环糊精、壳聚糖对纳米银的覆盖并不同程度增强其抗菌性，及负载纳米银的羟基磷灰石外包裹聚乳酸羟基乙酸，制成骨粉材料控制其感染，延缓纳米银的释放。通过本实验，希望获得能持续有效释放纳米银，直到自体骨形成，达到理想的控制骨粉感染的目的。方法与结果：1、环式糊精配体纳米银的制备及性能借鉴文献[1]中纳米银的合成方法合成环式糊精配体纳米银，方法是：不同浓度的β-CD水溶液作为配体，硼氢化钠溶液还原硝酸银水溶液制得CD-NP。通过扫描电镜、透射电镜观察制得的CD-NP的表面形貌、粒子直径大小及粒子的聚集情况，观察其稳定性，检测不同浓度纳米银的抗菌性。紫外分光光度计观测CD-NP的吸收峰值。结果显示：据文献[1]报道,随环式糊精浓度增加，制得纳米银抗菌性增强，因此选用20mM的环式糊精作为配体，与2mM的硝酸银溶液持续搅拌20分钟后，加入20mM的硼氢化钠水溶液，变黑30s后形成黄褐色溶液，搅拌24小时后可制得稳定性良好的纳米银。电镜结果显示：纳米银颗粒成圆形，粒径在24-33nm之间，分布较均匀，无明显聚集。紫外吸收光谱显示：CD-NP的吸收峰值在410nm。细菌实验表明：壳聚糖配体纳米银有明显抗菌性，且随纳米银的浓度增高，抗菌性增强。2、壳聚糖配体纳米银的制备及性能借鉴文献[2]中纳米银的合成方法合成壳聚糖配体纳米银，方法是利用壳聚糖作为配体和还原剂，氢氧化钠和乙酸调节PH值，制得CS-NP。将CS-NP溶于0.1%乙酸溶液中通过扫描电镜、透射电镜观察制得的CD-NP的表面形貌、粒子直径大小及粒子的聚集情况，观察其稳定性，检测不同浓度纳米银的抗菌性。紫外分光光度计观测CS-NP的吸收峰值。结果显示：脱乙酰度>75%的壳聚糖溶于1%的乙酸溶液中，制得1mg/ml的壳聚糖溶液，加热至45℃并稳定搅拌至壳聚糖完全溶解形成均质溶液，24ml的壳聚糖均质液与1ml100mM的硝酸银溶液混合后，将0.1M的氢氧化钠溶液75ml缓慢加入上述混合液中，得黄褐色溶液。电镜结果显示：CS-NP成大小不等的卵圆形，粒径在5-25nm之间，有聚集现象，粒子大小分布不太均匀。紫外吸收光谱显示：CS-NP的吸收峰值在400nm。细菌实验表明：壳聚糖配体纳米银有明显抗菌性，且随纳米银的浓度增高，抗菌性增强。3、羟基磷灰石负载纳米银的制备及性能方法：利用冻干法将不同浓度的环式糊精和壳聚糖配体纳米银负载到羟基磷灰石里，通过扫描电镜、透射电镜观察制得的CD-NP-HA/CS-NP-HA的表面形貌、粒子直径大小及粒子的聚集情况，观察其稳定性，并测试负载不同配体不同浓度纳米银的羟基磷灰石的抗菌性。将纳米银羟基磷灰石浸泡于模拟体液中，检测其体外缓释性能。结果显示：将0.1umol、0.2umol、0.5umol、1.0umol、2.0umol、4.0umol、10.0umol的CD-NP加入到100mg羟基磷灰石水溶液中（10mg/ml）搅拌4小时后冻干。将0.1umol、0.2umol、0.5umol、1.0umol、2.0umol、4.0umol、10.0umol的CS-NP加入到100mg羟基磷灰石水溶液中（10mg/ml）搅拌4小时后冻干。冻干后的负载纳米银的羟基磷灰石呈白色粉末状。电镜结果显示：CD-NP-HA/CS-NP-HA成大小不等的颗粒状，，粒径在9nm--60nm，颗粒聚集，稳定性好。细菌实验结果表明：负载0.1umol及以上浓度的CD-NP-HA/CS-NP-HA均有抗菌性。缓释实验结果表明：在第14天检测时释放达高峰。4、包裹聚乳酸羟基乙酸的羟基磷灰石制备及其性能方法：将聚乳酸羟基乙酸（PLGA）溶于氯仿中至完全溶解，负载纳米银的羟基磷灰石冻干粉溶于上述溶液中，剧烈搅拌至冻干粉溶解，将上述溶液迅速倒入含0.4%甲基纤维素的蒸馏水中，剧烈搅拌直至氯仿完全挥发至尽。离心、水洗后，将得到的固体冻干。通过扫描电镜、透射电镜观察制得的CD-NP-HA-PLGA/CS-NP-HAPLGA的表面形貌、粒子直径大小及粒子的聚集情况，观察其稳定性，并测试负载不同配体不同浓度纳米银的羟基磷灰石的抗菌性。将包裹PLGA的纳米银羟基磷灰石浸泡于模拟体液中，检测其体外缓释性能。结果：将200mg的PLGA溶于20ml氯仿，50mg负载不同浓度纳米银的羟基磷灰石溶于上述溶液中，溶解后倒入200ml含0.4%甲基纤维素的蒸馏水中，得到粉末状的缓释纳米银的骨粉材料。电镜结果显示：冻干粉成大小不等的球状，直径相差叫悬殊。最大直径可达100多nm，最小可至10nm以下。细菌实验结果显示：含0.1umolCD-NP和0.1umolCS-NP及以上浓度的缓释微球均具有明显抗菌性。缓释实验结果显示：至第42天时，其释放纳米银仍呈上升趋势，未见明显下降。结论：1、通过不同方法合成环式糊精与壳聚糖配体的纳米银，再通过冻干法将纳米银负载到羟基磷灰石里，再将其表面包裹聚乳酸聚羟基乙酸，冻干后得目标骨粉。含0.1umol纳米银的羟基磷灰石及含0.1umol纳米银的羟基磷灰石再包裹聚乳酸聚羟基乙酸均有明显的抗菌性，体外缓释实验结果显示，纳米银羟基磷灰石在第14天时释放纳米银达高峰，包裹聚乳酸聚羟基乙酸后，在第42天时释放纳米银仍呈上升趋势，未见明显下降。2、还需细胞相关实验检测负载不同浓度纳米银的羟基磷灰石及包裹聚乳酸聚羟基乙酸的羟基磷灰石对细胞的影响，及动物实验检测其在种植体周围炎模型中是否可以正常成骨且可以抗感染，有望成为好的缓释载体材料。

    【Background】In implantation,a variety of reasons (alveolar bone atrophy after exodontia,trauma, infection, cancer, birth defects, etc.) can cause lack of bone mass in dentalimplants implanted area,which will affect the implant and long-term effect.Implanted bone substitute materials become the essential means to solve this problem,but how to control for postoperative infection is a growing difficulty andhotspot in the treatment of implantation. Currently,widely research of antibioticssuch as vancomycin, tetracycline, levofloxacin due to load onto the bone technologiessuch as bacterial drug resistance and antibiotic release duration is short, but failed toachieve the goal of the ideal control bone infection. Silver, zinc, copper and otherinorganic antibacterial agent doping into bone powder materials can effectively inhibitbacterial infections, but its toxicity to mammalian cells become concerns in theapplication, and how to make its sustained and effective work is not very good solve.In addition, how to effectively use nanotechnology to delay the release ofantimicrobial agent still need further discussion.【Aim】This study try to use beta-cyclodextrin (β-CD) and chitosan (CS) as ligandsynthesis of silver nanoparticles, then different ligands of silver nanoparticles (NP)load in the artificial bone, combined the technology of emulsification, Polylacticacid hydroxy acetic acid(PLGA), PLGA outside the package made antibacterial bonebiomaterial to delay the release of inorganic nano silver. This experiment use thecharacteristics of cyclodextrin, chitosan to cover silver nanoparticles and enhance itsantimicrobial properties in different levels, and nano silver hydroxyapatite wrappedby Polylactic acid hydroxy acetic acid(PLGA),made into bone powder materialcontrol the infection, and slow release of silver nanoparticles. Through theexperiment, hopely can get sustained release of nano silver, until autologous boneformation, achieve the objective of the ideal control bone infection.【Methods and results】1.Preparation and properties of CD-NP Reference literature[1]in the synthesis of silver nanoparticles synthesis nano silverof β-CD ligand, the method is: different concentrations of beta-CD aqueoussolution as a ligand, sodium borohydride as reducts, reduct silver nitrate aqueoussolution to CD-NP. By scanning electron microscopy (sem), transmission electronmicroscopy (sem) observate CD-NPs’ surface morphology, particle diameter andparticle size of aggregate, the stability of the observation period.Spectrophotometerobservate CD-NPs’ absorption peak.Results：As reported in literature[1], with cyclodextrin concentration increased, theobtained nano-silver antibacterial properties enhanced.so that20mM β-CD as ligands,and2mM silver nitrate solution, stirring after20minutes, add sodium borohydridesolution of20mM, get dark solution，30s later, brown solution was formed，after24hs’ stiring, can obtain good stable nano-silver. Electron microscopy (sem)results showed that nano-silver particles into a round shape, particle size, between24to33nm are uniformly distributed and no obvious aggregation and test antimicrobialproperties of different concentrations of silver nanoparticles.. Ultraviolet absorptionspectra showed that CD-NP absorption peak at410nm. Bacteria experiment showedthat chitosan ligands nano-silver have obvious antimicrobial properties, and alongwith the increased concentration of silver nanoparticles, antimicrobial propertiesenhanced.2.Preparation and properties of CS-NPReference literature[2],chitosan as ligand in the synthesis of silver nanoparticles.The method is using chitosan as a ligand and reductant, Sodium hydroxide andacetic acid as adjusts to adjust PH value, obtained CS-NP. CS-NP soluble in0.1%acetic acid solution by scanning electron microscopy (sem) and transmission electronmicroscopy (sem) observate of CS-NP surface morphology, particle diameter andparticle size of aggregate, the stability of CS-NP and test antimicrobial propertiesof different concentrations of silver nanoparticles. Spectrophotometer observateCS-NPs’ absorption peak.Results showed that chitosan(degree>75%) is soluble in1%acetic acidsolution, the chitosan solution was1mg/ml, heating up to45℃and stir until completely dissolved to form chitosan homogeneous solution,24ml chitosanhomogeneous fluid mixed with1ml100mM silver nitrate solution,and add75ml of0.1M sodium hydroxide solution slowly to the mixture, obtaining brown solution.Electron microscopy (sem) results show that CS-NP size ovoid, particle size between5to25nm, have gathered phenomenon, particle size distribution is not uniform.Ultraviolet absorption spectra showed that CS-NP absorption peak at400nm.Bacteria experiment showed that chitosan ligands nano-silver have obviousantimicrobial properties, and along with the increased concentration of silvernanoparticles, antimicrobial properties enhanced.3.Preparation and properties of CD-NP-HA/CS-NP-HAMethods:Different concentration of CD-NP/CS-NP load into the hydroxyapatite bylyophilization.By scanning electron microscopy (sem) and transmission electronmicroscopy (sem) observate CD-NP-HA/CS-NP-HA ‘surface morphology,particle diameter and particle size of aggregate, stability of the observation period,and test antimicrobial properties of hydroxyapatite loading silver nanoparticles indifferent ligand and different concentration.The nano-silver hydroxyapatiteimmersing in simulated body fluid, test slow-release performancein vitro.Results showed that:0.1umol,0.2umol,0.5umol,1.0umol,2.0umol,4.0umol,10.0umol CD-NP to join100mg hydroxyapatite (10mg/ml) in aqueous solution,lyophilization after stirring4hours.0.1umol,0.2umol,0.5umol,1.0umol,2.0umol,4.0umol,10.0umol CS-NP to100mg of hydroxy apatite (10mg/ml) inaqueous solution, lyophilization after stirring4hours. After lyophilizationhydroxyapatite loading nano-silver is into white powder. Electron microscopy(sem) results showed that CD-NP-HA/CS-NP-HA into granular size, particle sizein9nm-60nm, particle aggregation, stability is good. Bacteria experiment resultsshow that the load0.1umol CD-NP-HA/CS-NP-HA have antimicrobial properties.Prolonged-release experimental results show that: on the14th day,releasing is intothe peak.4.Preparation and properties of CD-NP-HA-PLGA/CS-NP-HA-PLGAMethods: Polylactic acid hydroxy acetic acid (PLGA) dissolved in trichloromethane until completely dissolved, CD-NP-HA/CS-NP-HA dissolved in the solution above,violent stir until completely dissolved, above solution quickly into the distilledwater containing0.4%methyl cellulose, stir until chloroform volatilize completely todo. After centrifugal, washing, the solid freeze-dried.By scanning electronmicroscopy (sem), transmission electron microscopy (sem) observation of CD-NPHAPLGA/-CS-NP-HA PLGA surface morphology, particle diameter and particlesize of aggregate, observe its stability, and test load different ligand concentration ofsilver nanoparticles hydroxyapatite antimicrobial properties. PLGA nano-silverhydroxyapatite immersing in simulated body fluid, test slow-release performanceinvitro.Results:200mg of PLGA will dissolve in20ml chloroform,50mg hydroxyapatiteloading nano-silver in different concentration，dissolved in above solution，then allpoured into200ml of distilled water containing0.4%methyl cellulose, getting thebone powder that slowly release silver nanoparticles. Electron microscopy (sem)results showed that the product is into the ball size, diameter is disparity. Maximumcan reach more than100nm in diameter, minimum can be below10nm. Bacteriaexperiment results show that:0.1umolCD-NP and0.1umolCS-NPslow-releasemicrospheres have obvious antimicrobial properties. Slow release experiment resultsshow that:To42days, the release of nano-silver is still on the rise, without significantdecline.Conclusion:1.Through different methods for synthesis of CD-NP、 CS-NP bylyophilization,which load into the nano-hydroxyapatite and wrapedd bypoly(lactic-co-glycolic) acid (PLGA) after freeze-dried,getting target bone meal.Hydroxyapatite containing0.1umol NP and NP-HA-PLGA containing0.1umol NPhave obvious antimicrobial properties.Slow-releasing experimental in vitro resultsshow that the nano-silver hydroxyapatite in14days to release up to peak, afterwraped by PLGA，until42days release the nano-silver is still on the rise,withoutsignificant decline.2.It still need to cell-related experiment testing different concentrations of silver nanoparticles hydroxyapatite and NP-HA-PLGA effects on cells, and model of theinflammation around the implant in animal experiments test whether it can benormal osteogenesis and anti-infection,it is expected to become a good slow-releasecarrier material.

骨粉内纳米银缓释及抗菌性能研究

中文摘要4-8Abstract8-12第1章 绪论16-26    1.1 前言16-17    1.2 相关材料的研究现状17-26        1.2.1 银17-19        1.2.2 壳聚糖19-20        1.2.3 环式糊精20-23        1.2.4 羟基磷灰石23-24        1.2.5 聚乳酸聚羟基乙酸24-26第2章 实验部分26-52    2.1 材料与设备26-27    2.2 制备方法27-28        2.2.1 环式糊精、壳聚糖配体纳米银的制备27        2.2.2 负载纳米银的羟基磷灰石（NP-HA）的制备27        2.2.3 聚乳酸聚羟基乙酸包裹负载纳米银的羟基磷灰石（NP-HA- PLGA）的制备27-28    2.3 性质检测28-30        2.3.1 电镜观察28        2.3.2 紫外吸收光谱分析28        2.3.3 傅里叶红外光谱分析28        2.3.4 抑菌性检测28-29        2.3.5 缓释效果检测29-30        2.3.6 数据分析30    2.4 结果与分析30-47        2.4.1 电镜结果30-36        2.4.2 紫外吸收光谱分析结果36-37        2.4.3 红外吸收光谱分析结果37-40        2.4.4 抑菌性检测结果40-41        2.4.5 缓释效果检测41-47    2.5 结论47-48    2.6 讨论48-52        2.6.1 缓释系统48-49        2.6.2 纳米银的合成49        2.6.3 PLGA 与羟基磷灰石的结合状态49-50        2.6.4 银的有效杀菌浓度50-52参考文献52-58作者简介及学期间取得的科研成果58-59致谢59-60

  本文关键词：骨粉内纳米银缓释及抗菌性能研究，由笔耕文化传播整理发布。