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CN114533952B - 一种负载地舒单抗的人工骨及其制备方法 - Google Patents

一种负载地舒单抗的人工骨及其制备方法 Download PDF

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CN114533952B
CN114533952B CN202210131151.5A CN202210131151A CN114533952B CN 114533952 B CN114533952 B CN 114533952B CN 202210131151 A CN202210131151 A CN 202210131151A CN 114533952 B CN114533952 B CN 114533952B
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denosumab
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邓幼文
谭伟
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Third Xiangya Hospital of Central South University
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Abstract

本发明公开了一种负载地舒单抗的人工骨及其制备方法，所述人工骨由羧甲基壳聚糖粉末、纳米羟基磷灰石粉末、2‑甲基咪唑锌盐、地舒单抗组成，所述羧甲基壳聚糖粉末、纳米羟基磷灰石粉末、2‑甲基咪唑锌盐、地舒单抗四种组分的质量比为（2‑4）:（2‑4）:（0.05‑0.2）:（0.12‑0.72）。该人工骨可以治疗溶骨性骨肿瘤的用途，其通过抑制溶骨型骨肿瘤术后骨缺损局部肿瘤复发及转移发挥生物学效应。

Description

一种负载地舒单抗的人工骨及其制备方法
技术领域
本发明属于生物医药材料领域，涉及一种负载地舒单抗的人工骨及其制备方法。
背景技术
肿瘤性骨缺损是良、恶性骨肿瘤等疾病手术所致的巨大骨缺损，面临着修复困难及肿瘤复发的巨大挑战。目前常用骨修复材料有自体骨、同种异体骨及人工骨。其中自体骨来源有限，且造成取骨部位二次创伤，同种异体骨有感染疾病风险，因此人工骨是一个重要选择。
目前，不管自体骨、同种异体骨，还是常规人工骨均不具备抑制肿瘤作用。常用抗肿瘤化疗药物及局部放疗毒副作用巨大，开发抗肿瘤人工骨修复肿瘤性骨缺损局部抗肿瘤，可以有效减少化疗药物剂量，减轻化疗药物耐药性。
发明内容
本发明旨在克服现有技术的不足，提供一种负载地舒单抗的人工骨及其制备方法。
为了达到上述目的，本发明提供的技术方案为：
所述负载地舒单抗的人工骨由羧甲基壳聚糖粉末(CMCS)、纳米羟基磷灰石粉末(nHA)、2-甲基咪唑锌盐(ZIF-8)、地舒单抗(Denosumab)组成，所述羧甲基壳聚糖粉末、纳米羟基磷灰石粉末、2-甲基咪唑锌盐、地舒单抗四种组分的质量比为(2-4):(2-4):(0.05-0.2):(0.12-0.72)。
优选地，所述羧甲基壳聚糖粉末、纳米羟基磷灰石粉末、2-甲基咪唑锌盐、地舒单抗四种组分的质量比为2:4:0.1:0.72。
优选地，所述羧甲基壳聚糖粉末的羧化度≥90％，pH值为6-8，黏度为10m-80mPa.s；所述羟基磷灰石粉末的粒径＜200nm；所述地舒单抗为生物医药级的地舒单抗。
上述负载地舒单抗的人工骨的制备方法包括如下步骤：
(1)按前述比例准备工骨由羧甲基壳聚糖粉末、纳米羟基磷灰石粉末、2-甲基咪唑锌盐、地舒单抗；将地舒单抗加入超纯水中，然后加入2-甲基咪唑锌盐进行物理混合获得Denosumab@ZIF-8混悬液，再加入羧甲基壳聚糖粉末，充分物理混合后获取CMCS/Denosumab@ZIF-8混合凝胶，最后加入纳米羟基磷灰石粉末，混合后得CMCS/nHA/Denosumab@ZIF-8混合凝胶，于30-37℃条件下搅拌30-60min；所述CMCS/nHA/Denosumab@ZIF-8混合凝胶中羧甲基壳聚糖粉末的质量百分比含量为30.3-65.4％，纳米羟基磷灰石粉末的质量百分比含量为30.3—65.4％，2-甲基咪唑锌盐的质量百分比含量为0.57-4.63％，地舒单抗的质量百分比含量为1.5-13％；
(2)将步骤(1)所得的CMCS/nHA/Denosumab@ZIF-8混合凝胶注入无菌模具板后置于真空冷冻干燥机冷阱，在-20℃条件下预冻1-2h，继而抽吸冷阱至真空，最后冷冻干燥12-24h，即得负载地舒单抗的人工骨，于无菌袋封装后在-20℃保存备用。
优选地，所述步骤(2)中冷冻干燥的温度为-20℃，功率为850W，真空度＜10pa，补水能力4kg/24h。
本发明所述的负载地舒单抗的人工骨可用于制备治疗溶骨性骨肿瘤人工骨材料，也可以用于制备骨修复增强性人工骨材料。
下面对本发明作进一步说明：
本发明旨在解决肿瘤性骨缺损修复困难这一难题。纳米羟基磷灰石(nHA)抗压强度良好、骨传导性能优异，是正常人体骨质主要成分，羧甲基壳聚糖(CMCS)的生物相容性、降解性能、药物负载性能优异，2-甲基咪唑锌盐(ZIF-8)具有高效吸附药物及pH响应药物缓释功能，肿瘤微环境呈酸性，在酸性环境可以加速ZIF-8的降解，进而释放药物，地舒单抗(Denosumab)是人源单克隆抗体，具有竞争性抑制RANKL的作用，进而影响破骨细胞RANKL/RANK/OPG信号轴发挥作用导致破骨作用受到抑制，进而直接发挥抗肿瘤作用，且其特异性强，相对细胞毒性抗肿瘤化疗药物，其副作用更小；因机体成骨效应跟破骨效应处于动态平衡，当破骨作用受到抑制，成骨效应相应增强，因此该药可以间接促进成骨，又同时具有抑制骨肿瘤细胞的双重功效，三者结合制备复合人工骨实现优势互补，可用于临床肿瘤性骨缺损的修复或替换。
本发明将材料CMCS、nHA、ZIF-8、Denosumab三种组分复合，采用冷冻干燥方法，在低温环境中，将CMCS、nHA组分添加进入含Denosumab@ZIF-8的溶液中，在-20℃低温下冻干制备的人工骨力学强度好，药物分布均匀，生物活性好，ZIF-8吸附药物后缓慢稳定释放。Denosumab为单克隆抗体，生物靶向性高，较常规化疗药物毒副作用小，除具有强烈抑制骨肿瘤细胞溶骨作用外，兼具促骨修复、改善骨质的功效，非常适合用于溶骨性骨肿瘤术后骨缺损的修复过程，既能促进骨确实修复，又能抑制肿瘤细胞。同时该人工骨亦可用于骨科手术的植骨融合。
本发明将RANKL抑制剂Denosumab与常用骨修复材料复合；Denosumab是RANKL的竞争性拮抗剂，能够抑制破骨细胞RANKL/RANK/OPG细胞信号轴的表达，具有抑制骨吸收、抗溶骨性骨肿瘤细胞的作用，其对具有溶骨特性的骨肿瘤均有抑制作用，特别适用骨巨细胞瘤、骨转移瘤；Denosumab还能够抑制破骨细胞RANKL/RANK/OPG细胞信号轴的表达，间接增强支架成骨能力，适用植骨融合(椎板间、横突间、椎间隙等)、骨缺损填充治疗，尤其是伴有骨质疏松症的骨缺损。
本发明的价值在于：
(1)本发明制备的骨人工骨兼具抗肿瘤及诱导成骨功能，能够解决骨肿瘤术后局部残留肿瘤细胞复发的问题；同时增强骨缺损的修复速度及质量。其中CMCS优良的生物相容性、可降解性、药物吸附性，支架的三维多孔结构，nHA优异的骨传导功能、Denosumab抑制破骨细胞作用，ZIF-8的是药物Denosumab的良好载体，并高效吸附并缓慢释放，四种组分优势互补，有利于骨缺损部位修复。
(2)本发明制备的人工骨最适用于肿瘤性骨缺损部位的填充治疗，制备方法简单，生物力学性能接近正常松质骨，降解速率跟骨修复速率基本匹配，药物稳定释放，符合临床使用的需求，具有广阔的应用前景。
(3)本发明制备的人工骨间接增强支架成骨能力，提高植骨融合率，适用植骨融合(椎板间、横突间、椎间隙等)、骨缺损填充治疗，尤其是伴有骨质疏松症的骨缺损。
总之，本发明所述的人工骨具有强度高、力学性质接近人骨，具有良好骨传导性、生物相容性、骨修复、抗肿瘤作用以及稳定的药物释放能力。本发明首次提出利用药物地舒单抗赋予人工骨抗肿瘤功效及诱导成骨能力，既能达到抑制肿瘤细胞增殖复发的效果，又解决目前肿瘤性骨缺损局部易复发的缺陷，同时又增强骨缺损修复速度骨质量，从而可能为肿瘤性骨缺损的治疗提供一种新的治疗选择。该人工骨治疗肿瘤性骨缺损的治疗主要针对溶骨性骨肿瘤，如骨巨细胞瘤、溶骨性骨转移瘤。
附图说明
图1为骨支架电镜观察图；
图2为骨支架的傅里叶红外光谱图(FTIR)；
图3为骨支架的X线衍射图(XRD)；
图4为骨支架的热重分析图(TGA)。
具体实施方式
空白对照组
1.复合凝胶制备
(1)混料：将适量0.1g 2-甲基咪唑锌盐(ZIF-8)加入100ml超纯水中，继而加入适量2g羧甲基壳聚糖，充分物理混合后获取CMCS/ZIF-8混合凝胶，继而加入适量4g纳米羟基磷灰石，经充分物理混合获得CMCS/nHA/ZIF-8混合凝胶；
(2)搅拌：使用恒温磁力搅拌器搅拌均匀，搅拌时间30-60min，温度20-30℃；悬浮液中羧甲基壳聚糖的质量百分比含量为32.8％，羟基磷灰石的质量百分比含量为65.6％％,ZIF-8的质量百分比含量为1.6％。
2.冷冻干燥成形
(1)分装：将步骤(1)所得的凝胶用注射器抽取后分别注入48孔无菌模具板，每孔加入1ml混合凝胶，全程遵循无菌操作原则；
(2)冷冻干燥成形：将无菌模具板置于真空冷冻干燥机冷阱，在-20℃环境预冻1-2h，继而抽吸冷阱至真空，最后冷冻干燥12h-24h；
(3)收集样品：收集样品至无菌袋中，密封后置于-20℃备用。
3.性能评估：
(1)物相结构：骨支架经X射线衍射(XRD)、傅里叶红外光谱(FTIR)、热重分析(TGA)等物相检测分析均能检测到其特异性波峰存在，提示支架由明胶、纳米羟基磷灰石构成；
(2)弹性模量及细胞毒性：抗压缩实验测得复合骨支架的弹性模量为12MPa，CCK—8细胞毒性试验显示：相对于未接种至支架的细胞组，该支架对破骨细胞系RAW264.7细胞及骨髓间充质干细胞细胞活力无明显影响(p＞0.05)。
实施例1
1.复合凝胶制备
(1)混料：将Denosumab(120mg)加入100ml去离子后，继而加入适量0.1g ZIF-8粉末进行物理混合获得Denosumab@ZIF-8混悬液，继而加入适量2g羧甲基壳聚糖，充分物理混合后获取CMCS/Denosumab@ZIF-8混合凝胶，继而加入适量4g纳米羟基磷灰石，经充分物理混合获得CMCS/nHA/Denosumab@ZIF-8混合凝胶；
(2)搅拌：使用恒温磁力搅拌器搅拌均匀，搅拌时间30-60min，温度20-30℃；悬浮液中羧甲基壳聚糖的质量百分比含量为32.2％，纳米羟基磷灰石的质量百分比含量为64.3％,地舒单抗的质量百分比含量为1.9％，ZIF-8的质量百分比含量为1.6％。
2.冷冻干燥成形
(1)分装：将步骤(1)所得的凝胶用注射器抽取后分别注入48孔无菌模具板，每孔加入1ml混合凝胶，全程遵循无菌操作原则；
(2)冷冻干燥成形：将无菌模具板置于真空冷冻干燥机冷阱，在-20℃环境预冻1-2h，继而抽吸冷阱至真空，最后冷冻干燥12h-24h；
(3)收集样品：收集样品至无菌袋中，密封后置于-20℃备用。
3.性能评估：
(1)物相结构：骨支架经X射线衍射(XRD)、傅里叶红外光谱(FTIR)、热重分析(TGA)等物相检测分析发现支架的主要组分为羧甲基壳聚糖、纳米羟基磷灰石、且能检测到地舒单抗的存在；且药物波峰高度跟支架中地舒单抗剂量成正相关；
(2)弹性模量及细胞毒性：抗压缩实验测得复合人工骨支架的弹性模量为11.2MPa，CCK—8细胞毒性试验显示：相对于空白对照组，该支架抑制破骨细胞系RAW264.7细胞活力，对骨髓间充质干细胞细胞活力无显著差异(p＞0.05)。
该骨支架与空白组力学强度接近(p＜0.05)。骨支架电镜观察图见图1；骨支架的傅里叶红外光谱图(FTIR)见图2；骨支架的X线衍射图(XRD)见图3；骨支架的热重分析图(TGA)见图4。
实施例2
1.复合凝胶制备
(1)混料：将Denosumab(360mg)加入100ml去离子后，继而加入适量0.1g 2-甲基咪唑锌盐(ZIF-8)进行物理混合获得Denosumab@ZIF-8混悬液，继而加入适量2g羧甲基壳聚糖，充分物理混合后获取CMCS/Denosumab@ZIF-8混合凝胶，继而加入适量4g纳米羟基磷灰石，经充分物理混合获得CMCS/nHA/Denosumab@ZIF-8混合凝胶；
(2)搅拌：使用恒温磁力搅拌器搅拌均匀，搅拌时间30-60min，温度20-30℃；悬浮液中羧甲基壳聚糖的质量百分比含量为31％，纳米羟基磷灰石的质量百分比含量为61.9％,地舒单抗的质量百分比含量为5.6％，ZIF-8的质量百分比含量为1.5％。
2.冷冻干燥成形
(1)分装：将步骤(1)所得的凝胶用注射器抽取后分别注入48孔无菌模具板，每孔加入1ml混合凝胶，全程遵循无菌操作原则；
(2)冷冻干燥成形：将无菌模具板置于真空冷冻干燥机冷阱，在-20℃环境预冻1-2h，继而抽吸冷阱至真空，最后冷冻干燥12h-24h；
(3)收集样品：收集样品至无菌袋中，密封后置于-20℃备用。
3.性能评估：
(1)物相结构：骨支架经XRD、FTIR、TGA等物相检测分析发现支架的主要组分为明胶、且均能检测到纳米羟基磷灰石、地舒单抗的存在，且药物波峰高度跟支架中地舒单抗含量成正相关；
(2)弹性模量及细胞毒性：抗压缩实验测得复合骨支架的弹性模量为10.9MPa，CCK—8细胞毒性试验显示：相对于空白对照组，该支架抑制破骨细胞系RAW264.7细胞活力，同时对骨髓间充质干细胞细胞活力无显著差异。
相对于空白对照组，该支架力学强度稍低，跟药物的掺入导致羧甲基壳聚糖/纳米羟基磷灰石比例下降有关。
实施例3
1.复合凝胶制备
(1)混料：将Denosumab(720mg)加入100ml去离子后，继而加入适量0.1g 2-甲基咪唑锌盐(ZIF-8)进行物理混合获得Denosumab@ZIF-8混悬液，继而加入适量2g羧甲基壳聚糖，充分物理混合后获取CMCS/Denosumab@ZIF-8混合凝胶，继而加入适量4g纳米羟基磷灰石，经充分物理混合获得CMCS/nHA/Denosumab@ZIF-8混合凝胶；
(2)搅拌：使用恒温磁力搅拌器搅拌均匀，搅拌时间30-60min，温度20-30℃；悬浮液中羧甲基壳聚糖的质量百分比含量为29.3％，纳米羟基磷灰石的质量百分比含量为58.7％,地舒单抗的质量百分比含量为10.5％，ZIF-8的质量百分比含量为1.5％。
2.冷冻干燥成形
(1)分装：将步骤(1)所得的凝胶用注射器抽取后分别注入48孔无菌模具板，每孔加入1ml混合凝胶，全程遵循无菌操作原则；
(2)冷冻干燥成形：将无菌模具板置于真空冷冻干燥机冷阱，在-20℃环境预冻1-2h，继而抽吸冷阱至真空，最后冷冻干燥12h-24h；
(3)收集样品：收集样品至无菌袋中，密封后置于-20℃备用。
3.性能评估：
(1)物相结构：骨支架经XRD、FTIR、TGA等物相检测分析发现支架的主要组分为明胶、且均能检测到纳米羟基磷灰石、地舒单抗的存在，且药物波峰高度跟支架中地舒单抗含量成正相关；
(2)弹性模量及细胞毒性：抗压缩实验测得复合骨支架的弹性模量为9.1MPa，CCK-8细胞毒性试验显示：相对于空白对照组，该支架抑制破骨细胞系RAW264.7细胞活力，对骨髓间充质干细胞细胞活力无显著差异。
相对于空白组及实施组1、2，该支架力学强度进一步降低，跟药物的掺入导致羧甲基壳聚糖/纳米羟基磷灰石比例进一步下降有关。
实施例4
1.复合凝胶制备
(1)混料：将Denosumab(840mg)加入100ml去离子后，继而加入适量0.1g 2-甲基咪唑锌盐(ZIF-8)进行物理混合获得Denosumab@ZIF-8混悬液，继而加入适量2g羧甲基壳聚糖，充分物理混合后获取CMCS/Denosumab@ZIF-8混合凝胶，继而加入适量4g纳米羟基磷灰石，经充分物理混合获得CMCS/nHA/Denosumab@ZIF-8混合凝胶；
(2)搅拌：使用恒温磁力搅拌器搅拌均匀，搅拌时间30-60min，温度20-30℃；悬浮液中羧甲基壳聚糖的质量百分比含量为28.8％，纳米羟基磷灰石的质量百分比含量为57.6％,地舒单抗的质量百分比含量为12.1％，ZIF-8的质量百分比含量为1.44％。
2.冷冻干燥成形
(1)分装：将步骤(1)所得的凝胶用注射器抽取后分别注入48孔无菌模具板，每孔加入1ml混合凝胶，全程遵循无菌操作原则；
(2)冷冻干燥成形：将无菌模具板置于真空冷冻干燥机冷阱，在-20℃环境预冻1-2h，继而抽吸冷阱至真空，最后冷冻干燥12h-24h；
(3)收集样品：收集样品至无菌袋中，密封后置于-20℃备用。
3.性能评估：
(1)物相结构：骨支架经XRD、FTIR、TGA等物相检测分析发现支架的主要组分为明胶、且均能检测到纳米羟基磷灰石、地舒单抗的存在，且药物波峰高度跟支架中地舒单抗含量成正相关；
(2)弹性模量及细胞毒性：抗压缩实验测得复合骨支架的弹性模量为6.8MPa，CCK-8细胞毒性试验显示：相对于空白对照组，该支架抑制破骨细胞系RAW264.7细胞活力，对骨髓间充质干细胞细胞活力有显著影响。

Claims (4)

1.一种用于抗溶骨性骨肿瘤的负载地舒单抗的人工骨，其特征在于，所述人工骨由羧甲基壳聚糖粉末、纳米羟基磷灰石粉末、2-甲基咪唑锌盐、地舒单抗组成，所述羧甲基壳聚糖粉末、纳米羟基磷灰石粉末、2-甲基咪唑锌盐、地舒单抗四种组分的质量比为2:4:0.1:0.72。
2.如权利要求1所述用于抗溶骨性骨肿瘤的负载地舒单抗的人工骨，其特征在于，所述羧甲基壳聚糖粉末的羧化度≥90%，pH值为6-8，黏度为10m-80mPa.s；所述羟基磷灰石粉末的粒径＜200nm；所述地舒单抗为生物医药级的地舒单抗。
3.如权利要求1或2所述用于抗溶骨性骨肿瘤的负载地舒单抗的人工骨的制备方法，其特征在于，所述方法包括如下步骤：
（1）按权利要求1所述的比例准备羧甲基壳聚糖粉末、纳米羟基磷灰石粉末、2-甲基咪唑锌盐、地舒单抗；将地舒单抗加入超纯水中，然后加入2-甲基咪唑锌盐进行物理混合获得Denosumab@ZIF-8混悬液，再加入羧甲基壳聚糖粉末，充分物理混合后获取CMCS/Denosumab@ZIF-8混合凝胶，最后加入纳米羟基磷灰石粉末，混合后得CMCS/nHA/Denosumab@ZIF-8混合凝胶，于30-37℃条件下搅拌30-60min；所述CMCS/nHA/Denosumab@ZIF-8混合凝胶中羧甲基壳聚糖粉末的质量百分比含量为30.3-65.4%，纳米羟基磷灰石粉末的质量百分比含量为30.3—65.4%，2-甲基咪唑锌盐的质量百分比含量为0.57-4.63%，地舒单抗的质量百分比含量为1.5-13%；
（2）将步骤（1）所得的CMCS/nHA/Denosumab@ZIF-8混合凝胶注入无菌模具板后置于真空冷冻干燥机冷阱，在-20℃条件下预冻1-2h，继而抽吸冷阱至真空，最后冷冻干燥12-24h，即得负载地舒单抗的人工骨，于无菌袋封装后在-20℃保存备用。
4.如权利要求3所述的方法，其特征在于，所述步骤（2）中冷冻干燥的温度为-20℃，功率为850W，真空度＜10pa，补水能力4kg/24h。
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