技术领域technical field
本发明属于复合纳米纤维及其制备领域，特别涉及一种载抗癌药物纳米纤维膜及其制备方法。The invention belongs to the field of composite nanofiber and its preparation, in particular to a nanofiber membrane loaded with anticancer drugs and its preparation method.
背景技术Background technique
肿瘤是严重威胁人类生命的常见病、多发病。据WHO统计，全球60亿人口中，平均每年死于恶性肿瘤者达690万人，新发病例870万，且数字还在逐年增长。目前我国肿瘤患者达180万人，死亡率在所有病因中占第二位。因此，世界各国政府、研究机构及制药公司一直对肿瘤治疗药物与治疗方法的研究给予高度重视与支持。目前，临床上治疗肿瘤常用的方法有化学疗法、放射疗法和手术切除疗法等。化学疗法由于副作用大，病人难以耐受长期治疗，使疗效受限制；放射疗法则存在部分肿瘤在提高放射剂量后仍不能被局部控制的问题，主要原因是许多肿瘤中存在对射线不敏感的乏氧细胞，为增强治疗效果常需增加放射剂量，但放射剂量超过一定限度就会损伤正常组织。Tumor is a common and frequently-occurring disease that seriously threatens human life. According to WHO statistics, among the 6 billion people in the world, an average of 6.9 million people die from malignant tumors each year, and 8.7 million new cases are diagnosed, and the number is still increasing year by year. At present, there are 1.8 million cancer patients in my country, and the mortality rate is the second among all causes. Therefore, governments, research institutes and pharmaceutical companies all over the world have been giving great importance and support to the research on tumor therapeutic drugs and methods. At present, the commonly used methods for clinical treatment of tumors include chemotherapy, radiotherapy and surgical resection. Due to the large side effects of chemotherapy, it is difficult for patients to tolerate long-term treatment, which limits the curative effect; while radiotherapy has the problem that some tumors cannot be locally controlled after increasing the radiation dose, the main reason is that many tumors have insufficient radiation sensitivity. Oxygen cells, in order to enhance the therapeutic effect, it is often necessary to increase the radiation dose, but if the radiation dose exceeds a certain limit, normal tissues will be damaged.
手术切除疗法是最常用的方法，但手术后常存在残留的肿瘤细胞，从而造成癌症的复发，严重制约了该疗法的实际疗效。目前临床上用来解决这个问题的主要方法有重复手术和化疗。这两种方法存在着如下问题：Surgical resection is the most commonly used method, but there are often residual tumor cells after surgery, resulting in recurrence of cancer, which seriously restricts the actual curative effect of this therapy. At present, the main methods used to solve this problem clinically are repeated surgery and chemotherapy. These two methods have the following problems:
1、重复手术费用高，对病人的伤害大。由于癌症患者在经过手术治疗后处于身心俱疲的状态，短期内再次进行手术治疗会给患者的身心带来极大的创伤和痛苦，重复手术费用高，对病人家庭与国家都是沉重经济负担。1. The cost of repeat operation is high and the harm to the patient is great. Because cancer patients are in a state of physical and mental exhaustion after surgery, re-operation in a short period of time will bring great trauma and pain to the patient's body and mind, and the cost of repeated operations is high, which is a heavy economic burden on the patient's family and the country. .
2、临床医生在手术后在肿瘤部位敷上抗肿瘤药物用来消灭残余肿瘤细胞，这种方法极其粗放，无法精确药量。大多数抗肿瘤药物对正常细胞都有着较大杀伤作用，所以药量的选择极其重要，否则药物浓度太大则会引起病人中毒而太少则无效；此外，采用这种方法药物在肿瘤部位无法缓释，药物的暴释或者会引起病人的中毒，或者会令药物的有效时间很短，当肿瘤复发时药物浓度已经远远低于其有效浓度。而肿瘤组织中存在着对放化疗不敏感的乏氧细胞，这就需要在手术部位通过缓释技术长期保持一定的药物浓度以便随时可以消除复发的肿瘤细胞。2. Clinicians apply anti-tumor drugs to the tumor site after surgery to eliminate residual tumor cells. This method is extremely extensive and cannot be accurately dosed. Most antineoplastic drugs have a relatively large killing effect on normal cells, so the choice of drug dosage is extremely important, otherwise the drug concentration will cause patient poisoning if the drug concentration is too large, and it will be ineffective if it is too small; Sustained release, the burst release of the drug may cause poisoning of the patient, or the effective time of the drug is very short, and the drug concentration is far lower than its effective concentration when the tumor recurs. However, there are hypoxic cells that are not sensitive to radiotherapy and chemotherapy in the tumor tissue, which requires a long-term maintenance of a certain drug concentration at the surgical site through slow-release technology so that recurrent tumor cells can be eliminated at any time.
因此，研究一种副作用小、选择性高、疗效好的微创方法来进行靶向示踪与治疗、消灭残留的肿瘤细胞，降低肿瘤的复发和转移，是当前国际医药学界面临的一项重要而又迫切的前沿课题。Therefore, researching a minimally invasive method with less side effects, high selectivity, and good curative effect to carry out targeted tracing and treatment, eliminate residual tumor cells, and reduce tumor recurrence and metastasis is an important task facing the current international medical community. and urgent frontier issues.
静电纺丝法即聚合物喷射静电拉伸纺丝法，将聚合物溶液或熔体带上几千至上万伏高压静电，带电的聚合物液滴在电场力的作用下在毛细管的Taylor锥顶点被加速。当电场力足够大时，聚合物液滴克服表面张力形成喷射细流；细流在喷射过程中溶剂蒸发或固化，最终落在接收装置上，形成类似非织造布状的纤维毡。用电纺丝技术制得的纤维，其直径可达纳米级，比表面积大、纤维粗细程度与均一性高、长径比大。The electrospinning method is the polymer jet electrostatic stretching spinning method. The polymer solution or melt is charged with thousands to tens of thousands of volts of high-voltage static electricity. was accelerated. When the electric field force is strong enough, the polymer droplets overcome the surface tension to form a jet stream; the solvent evaporates or solidifies during the spraying process, and finally falls on the receiving device to form a fiber mat similar to a non-woven fabric. The fiber prepared by electrospinning technology has a diameter of nanometer level, large specific surface area, high fiber thickness and uniformity, and large aspect ratio.
2002年美国专利（US20020181640），Ignatious和Baldoni两人用电纺丝纳米纤维设计出分别具有快速、即时、延时、缓慢、持续及阶段性等不同释药特性的复合药剂。2004年，Daniel J．Smith和Darrell H．Reneker两人公布了一项专利（US20040595329），他们用电纺丝制得了直链聚(乙撑亚胺)亚硝羟胺的纳米纤维，作为一种新型的医疗器材的外在包覆膜。这种覆膜可以缓慢而均衡地释放出一氧化氮(NO)，促进医疗器材周边的组织康复，并防止硬物对组织周边造成进一步伤害。这一方法给电纺纤维载药开辟了一个新的应用领域。In the US patent (US20020181640) in 2002, Ignatious and Baldoni used electrospun nanofibers to design composite agents with different drug release characteristics such as fast, instant, delayed, slow, sustained and staged. In 2004, Daniel J. Smith and Darrell H. The two of Reneker announced a patent (US20040595329), they used electrospinning to prepare nanofibers of linear poly(ethyleneimine) nitrosamines, which were used as the external coating of a new type of medical equipment. The coating slowly and evenly releases nitric oxide (NO), which promotes tissue healing around the medical device and prevents further damage to surrounding tissue from hard objects. This approach opens up a new application field for drug loading on electrospun fibers.
发明内容Contents of the invention
本发明所要解决的技术问题是提供一种载抗癌药物纳米纤维膜及其制备方法，该纳米纤维膜可以作为手术创口处的创伤敷料用来防止感染、止血，促进手术创口的愈合；在手术后，首先通过抗癌化疗药物杀死癌细胞；然后，进一步通过光纤定向照射，使光敏剂发生药效，从而无毒高效、简便地去除残余肿瘤细胞，防止肿瘤的复发，达到根治癌症的目的。The technical problem to be solved by the present invention is to provide a nanofiber membrane loaded with anticancer drugs and a preparation method thereof. The nanofiber membrane can be used as a wound dressing at the surgical wound to prevent infection, stop bleeding, and promote the healing of the surgical wound; Finally, the cancer cells are first killed by anti-cancer chemotherapy drugs; then, the photosensitizer is further irradiated through the optical fiber to make the photosensitizer effective, thereby removing residual tumor cells in a non-toxic, efficient and simple way, preventing tumor recurrence, and achieving the goal of curing cancer .
本发明的一种载抗癌药物纳米纤维膜，所述纳米纤维膜由载药和基体组成，其中，以摩尔比为1:1~2的光敏剂和化疗药物作为纳米纤维膜载药，以生物可降解高分子材料作为纳米纤维膜的基体，药物总质量为基体质量的4%。A nanofiber membrane loaded with anticancer drugs of the present invention, the nanofiber membrane is composed of drug loading and a matrix, wherein the photosensitizer and chemotherapeutic drug are used as the nanofiber membrane drug loading with a molar ratio of 1:1~2, and The biodegradable polymer material is used as the matrix of the nanofiber membrane, and the total mass of the drug is 4% of the mass of the matrix.
所述光敏剂为维替泊芬或四甲基卟吩。The photosensitizer is verteporfin or tetramethylporphine.
所述化疗药物为5-氟脲嘧啶或甲氨蝶呤。The chemotherapeutic drug is 5-fluorouracil or methotrexate.
所述生物可降解高分子材料为聚乳酸（PLLA）、聚对苯二甲酸乙二醇酯（PET）、聚羟基乙酸（PGA）或聚己内酯（PCL），分子量为8W-30W。The biodegradable polymer material is polylactic acid (PLLA), polyethylene terephthalate (PET), polyglycolic acid (PGA) or polycaprolactone (PCL), with a molecular weight of 8W-30W.
本发明的一种载抗癌药物纳米纤维膜的制备方法，包括：A method for preparing a nanofiber membrane loaded with anticancer drugs of the present invention, comprising:
（1）将化疗药物加入到冰醋酸中，加热使药物溶解，得到化疗药物溶液；将光敏剂和生物可降解高分子材料加入到有机溶剂中，搅拌均匀，然后加入化疗药物溶液，配制成高聚物溶液；(1) Add chemotherapeutic drugs to glacial acetic acid, heat to dissolve the drugs, and obtain a chemotherapeutic drug solution; add photosensitizers and biodegradable polymer materials into organic solvents, stir evenly, and then add chemotherapeutic drug solutions to prepare high polymer solution;
（2）将上述高聚物溶液进行静电纺丝，挥发有机溶剂和冰醋酸，制得载抗癌药物纳米纤维膜。(2) The above polymer solution was subjected to electrospinning, and the organic solvent and glacial acetic acid were volatilized to prepare the anticancer drug-loaded nanofiber membrane.
所述步骤（1）中化疗药物与冰醋酸的质量比为1:30~1:50；光敏剂和生物可降解高分子的总质量与有机溶剂的质量比为1:19。In the step (1), the mass ratio of the chemotherapeutic drug to glacial acetic acid is 1:30-1:50; the mass ratio of the total mass of the photosensitizer and biodegradable polymer to the organic solvent is 1:19.
所述步骤（1）中当生物可降解高分子材料为聚乳酸时，有机溶剂为体积比7:3的三氯甲烷与丙酮的混合液；当生物可降解高分子材料为聚对苯二甲酸乙二醇酯时，有机溶剂为体积比4:1的三氟乙酸与二氯甲烷的混合液；当生物可降解高分子材料为聚羟基乙酸时，有机溶剂为六氟异丙醇；当生物可降解高分子材料为聚己内酯时，有机溶剂为体积比4:1的三氟乙醇与水的混合液。In the step (1), when the biodegradable polymer material is polylactic acid, the organic solvent is a mixture of chloroform and acetone with a volume ratio of 7:3; when the biodegradable polymer material is polyterephthalic acid In the case of ethylene glycol ester, the organic solvent is a mixture of trifluoroacetic acid and methylene chloride at a volume ratio of 4:1; when the biodegradable polymer material is polyglycolic acid, the organic solvent is hexafluoroisopropanol; When the degradable polymer material is polycaprolactone, the organic solvent is a mixture of trifluoroethanol and water at a volume ratio of 4:1.
所述步骤（2）中的静电纺丝的工艺参数为：施加电压15KV～25KV，接收距离6cm～12cm，纺丝推进速度0.3ml/h～1.50ml/h，喷丝口直径0.8mm。The process parameters of the electrospinning in the step (2) are: applied voltage 15KV-25KV, receiving distance 6cm-12cm, spinning speed 0.3ml/h-1.50ml/h, spinneret diameter 0.8mm.
所述步骤（2）中的高聚物的均匀溶液中，有机溶剂、生物可降解高分子材料、抗肿瘤光敏剂、化疗药物之间混溶良好，无化学反应。In the homogeneous polymer solution in the step (2), organic solvents, biodegradable polymer materials, anti-tumor photosensitizers, and chemotherapeutic drugs are well miscible without chemical reactions.
所述步骤（2）所要求的载抗癌药物纳米纤维膜，化疗药物在手术后初期发生药效，光敏剂使用时要经过特定波长的激光照射才能发挥药效。In the anticancer drug-loaded nanofibrous membrane required in the step (2), the chemotherapeutic drug becomes effective in the early stage after operation, and the photosensitizer needs to be irradiated with a specific wavelength of laser light before it can exert its drug effect.
有益效果Beneficial effect
（1）本发明载光动力药物纳米纤维膜作为一种全新的抗肿瘤药物载体，可在手术切除肿瘤后，将载药纳米纤维膜置于肿瘤术后残腔，化疗药物从缓释载体中逐渐释放出来，从而维持稳定而有效的浓度；在需要的时候通过一定波长的激光的激活光敏剂药物，可无毒高效、简便、彻底的去处残余肿瘤细胞；具有生物相容性好、药效持久、可以控制等优点，达到防止肿瘤复发、根治癌症的目的；该膜植入后治疗简便；载药材料会自然降解，无毒副作用；(1) The photodynamic drug-loaded nanofiber membrane of the present invention is a brand-new anti-tumor drug carrier. After surgical resection of the tumor, the drug-loaded nanofiber membrane can be placed in the postoperative residual cavity of the tumor, and the chemotherapy drugs can be released from the slow-release carrier. It is gradually released to maintain a stable and effective concentration; when needed, the photosensitizer drug can be activated by a certain wavelength of laser light, which can remove residual tumor cells in a non-toxic, efficient, simple and thorough manner; it has good biocompatibility and efficacy Durable, controllable and other advantages to achieve the purpose of preventing tumor recurrence and curing cancer; the membrane is easy to treat after implantation; the drug-loaded material will degrade naturally, without toxic and side effects;
（2）本发明纳米纤维的多孔性结构有利于组织内外营养物质和氧的交换，促进手术创伤部位组织细胞的修复，恢复正常功能；纳米纤维高的比表面积和孔隙结构也有利于细胞的黏附、延展、增殖和分化；光敏剂产生的活性氧等小分子自由基可以通过纳米纤维的多孔结构扩散，这样就可以在长时间内根据需要随时进行光动力治疗，及时将复发的肿瘤细胞杀死：(2) The porous structure of the nanofibers of the present invention is conducive to the exchange of nutrients and oxygen inside and outside the tissue, and promotes the repair of tissue cells at surgical wound sites and restores normal functions; the high specific surface area and pore structure of the nanofibers are also conducive to cell adhesion , extension, proliferation and differentiation; small molecular free radicals such as reactive oxygen species produced by photosensitizers can diffuse through the porous structure of nanofibers, so that photodynamic therapy can be performed at any time for a long time as needed to kill recurrent tumor cells in time :
（3）本发明制备方法操作简单，成本低，具有良好的药物缓释性能，应用前景广阔。(3) The preparation method of the present invention is simple in operation, low in cost, has good drug sustained-release performance, and has broad application prospects.
具体实施方式Detailed ways
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
聚乳酸（PLLA）具有无毒、生物相容性好、机械性能好并且在生物体中可以完全降解等优点。因此，下面的实例中，选用此种材料作为基体，相应的有机溶剂选用氯仿与丙酮混合。化疗药物选择5-氟脲嘧啶，光敏剂选择四甲基卟吩，两种药物摩尔比为1：1，药物的质量是高分子基体的4%；静电纺的参数直接影响纳米纤维的形貌，从而影响其载药性能。Polylactic acid (PLLA) has the advantages of non-toxicity, good biocompatibility, good mechanical properties and can be completely degraded in organisms. Therefore, in the following examples, this material is selected as the matrix, and the corresponding organic solvent is selected from a mixture of chloroform and acetone. The chemotherapeutic drug is 5-fluorouracil, the photosensitizer is tetramethylporphine, the molar ratio of the two drugs is 1:1, and the quality of the drug is 4% of the polymer matrix; the electrospinning parameters directly affect the morphology of the nanofibers , thereby affecting its drug-loading performance.
实施例1Example 1
用天平称取0.0133g 5-氟脲嘧啶溶于0.5ml冰醋酸中，并且稍微加热使之全部溶解，用天平称取0.66g聚乳酸PLLA和0.0133g四甲基卟吩，将其溶解于7ml氯仿中，并封好瓶口，防止溶剂挥发，在磁力搅拌器上搅拌至完全溶解。然后加入上面配制好的5-氟脲嘧啶的冰醋酸溶液，继续在磁力搅拌器上搅拌；将混合均匀的溶液静置5小时后，将瓶口打开，再加入3ml丙酮，搅拌均匀，把即得静电纺高聚物溶液。对得到的溶液进行静电纺丝，施加电压为15KV，接收距离为12cm，纺丝推进速度为0.7ml/h，喷丝口直径为0.8mm。所得纳米纤维直径为350nm-1000nm之间。Weigh 0.0133g 5-fluorouracil with a balance and dissolve it in 0.5ml glacial acetic acid, and heat it slightly to dissolve it completely. Weigh 0.66g polylactic acid PLLA and 0.0133g tetramethylporphine with a balance, and dissolve them in 7ml In chloroform, and seal the bottle to prevent solvent volatilization, stir on a magnetic stirrer until completely dissolved. Then add the glacial acetic acid solution of 5-fluorouracil prepared above, and continue to stir on the magnetic stirrer; after the uniformly mixed solution was left to stand for 5 hours, the bottle mouth was opened, and then 3ml of acetone was added, stirred evenly, and the instant Obtain electrospinning polymer solution. Electrospinning was performed on the obtained solution, the applied voltage was 15KV, the receiving distance was 12cm, the spinning speed was 0.7ml/h, and the diameter of the spinneret was 0.8mm. The diameter of the obtained nanofiber is between 350nm and 1000nm.
实施例2Example 2
用天平称取0.0133g 5-氟脲嘧啶溶于0.5ml冰醋酸中，并且稍微加热使之全部溶解，用天平称取0.66g聚乳酸PLLA和0.0133g四甲基卟吩，将其溶解于7ml氯仿中，并封好瓶口，防止溶剂挥发，在磁力搅拌器上搅拌至完全溶解。然后加入上面配制好的5-氟脲嘧啶的冰醋酸溶液，继续在磁力搅拌器上搅拌；将混合均匀的溶液静置5小时，将瓶口打开，再加入3ml丙酮，搅拌均匀，即得静电纺高聚物溶液。对得到的溶液进行静电纺丝，施加电压为20KV，接收距离为12cm，纺丝推进速度为0.8ml/h，喷丝口直径为0.8mm。所得纳米纤维直径为500nm-1500nm之间。Weigh 0.0133g 5-fluorouracil with a balance and dissolve it in 0.5ml glacial acetic acid, and heat it slightly to dissolve it completely. Weigh 0.66g polylactic acid PLLA and 0.0133g tetramethylporphine with a balance, and dissolve them in 7ml In chloroform, and seal the bottle to prevent solvent volatilization, stir on a magnetic stirrer until completely dissolved. Then add the glacial acetic acid solution of 5-fluorouracil prepared above, and continue to stir on the magnetic stirrer; let the uniformly mixed solution stand for 5 hours, open the bottle mouth, then add 3ml of acetone, stir evenly, and obtain static electricity Spun polymer solution. The obtained solution was electrospun with an applied voltage of 20KV, a receiving distance of 12cm, a spinning speed of 0.8ml/h, and a spinneret diameter of 0.8mm. The diameter of the obtained nanofiber is between 500nm and 1500nm.
实施例3Example 3
用天平称取0.0133g 5-氟脲嘧啶溶于0.5ml冰醋酸中，并且稍微加热使之全部溶解，用天平称取0.66g聚乳酸PLLA和0.0133g四甲基卟吩，将其溶解于7ml氯仿中，并封好瓶口，防止溶剂挥发，在磁力搅拌器上搅拌至完全溶解。然后加入上面配制好的5-氟脲嘧啶的冰醋酸溶液，继续在磁力搅拌器上搅拌；将混合均匀的溶液静置5小时后，将瓶口打开，再加入3ml丙酮，搅拌均匀，即得静电纺高聚物溶液。对得到的溶液进行静电纺丝，施加电压为15KV，接收距离为12cm，纺丝推进速度为0.8ml/h，喷丝口直径为0.8mm。所得纳米纤维直径为400nm-1200nm之间。Weigh 0.0133g 5-fluorouracil with a balance and dissolve it in 0.5ml glacial acetic acid, and heat it slightly to dissolve it completely. Weigh 0.66g polylactic acid PLLA and 0.0133g tetramethylporphine with a balance, and dissolve them in 7ml In chloroform, and seal the bottle to prevent solvent volatilization, stir on a magnetic stirrer until completely dissolved. Then add the glacial acetic acid solution of 5-fluorouracil prepared above, and continue to stir on the magnetic stirrer; after the uniformly mixed solution is left to stand for 5 hours, open the bottle mouth, add 3ml of acetone, and stir evenly to obtain Electrospinning polymer solutions. Electrospinning was performed on the obtained solution, the applied voltage was 15KV, the receiving distance was 12cm, the spinning speed was 0.8ml/h, and the diameter of the spinneret was 0.8mm. The diameter of the obtained nanofiber is between 400nm and 1200nm.
实施例4Example 4
用天平称取0.0133g 5-氟脲嘧啶溶于0.5ml冰醋酸中，并且稍微加热使之全部溶解，用天平称取0.66g聚乳酸PLLA和0.0133g四甲基卟吩，将其溶解于7ml氯仿中，并封好瓶口，防止溶剂挥发。在磁力搅拌器上搅拌至完全溶解。然后加入上面配制好的5-氟脲嘧啶的冰醋酸溶液，继续在磁力搅拌器上搅拌；将混合均匀的溶液静置5小时，将瓶口打开，再加入3ml丙酮，搅拌均匀，即得静电纺高聚物溶液。对得到的溶液进行静电纺丝，施加电压为20KV，接收距离为12cm，纺丝推进速度为0.7ml/h，喷丝口直径为0.8mm。所得纳米纤维直径为500nm-1500nm之间。Weigh 0.0133g 5-fluorouracil with a balance and dissolve it in 0.5ml glacial acetic acid, and heat it slightly to dissolve it completely. Weigh 0.66g polylactic acid PLLA and 0.0133g tetramethylporphine with a balance, and dissolve them in 7ml Chloroform, and seal the bottle to prevent solvent volatilization. Stir on a magnetic stirrer until completely dissolved. Then add the glacial acetic acid solution of 5-fluorouracil prepared above, and continue to stir on the magnetic stirrer; let the uniformly mixed solution stand for 5 hours, open the bottle mouth, then add 3ml of acetone, stir evenly, and obtain static electricity Spun polymer solution. Electrospinning was performed on the obtained solution, the applied voltage was 20KV, the receiving distance was 12cm, the spinning speed was 0.7ml/h, and the diameter of the spinneret was 0.8mm. The diameter of the obtained nanofiber is between 500nm and 1500nm.
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