重组人干扰素纳米磁性脂质体的构建及靶向治疗人原发性肝癌的研究
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摘要
肿瘤靶向治疗是利用特定的载体将药物在特定的导向机制作用下选择性地定位于肿瘤组织,提高肿瘤组织内的药物浓度,达到提高疗效、降低毒副反应、增加病人顺从性的目的。在本系列实验中,采用逆相蒸发法研制了纳米级磁性干扰素脂质体,考察了磁性干扰素纳米脂质体的体外稳定性、体内外磁场响应性和在原发性肝癌移植瘤裸鼠体内的生物分布,我们也进行了MIL静注后动物的溶血性和急毒性实验,以及用双抗夹心ELISA法检测各实验组和对照组在外加磁场和不加磁场两种状态下,药物干预后动物的血药浓度和组织浓度方面的药动学研究。分别用MTT法、细胞计数法、吖啶橙染色法和DNA ladder法进行细胞生长抑制和凋亡检测。还进行了MIL靶向治疗原发性肝癌移植瘤裸鼠后的分子生物学方面的机理探讨。
主要研究结果如下:
1、磁性干扰素纳米脂质体粒径平均170nm,干扰素包封率62.71%,磁性颗粒均匀分布于脂质体中。不同的类脂比例、药脂比、有机溶剂种类和油水相体积比影响磁性干扰素纳米脂质体粒径和干扰素包封率。制备后4℃和-20℃保存,8h内渗漏率较低。结果提示磁性纳米脂质体有可能作为干扰素的新型载体应用于恶性肿瘤的靶向治疗中。
2、磁性干扰素纳米脂质体体外、体内具有良好的磁场响应性。应用磁性纳米脂质体加磁场的方式给药,干扰素可以有效地聚集到靶部位,显著提高靶部位干扰素的浓度,降低其它组织和器官中的药物浓度。作为干扰素的有效载体,磁性纳米脂质体加磁场的给药方式有望成为肿瘤靶向治疗的新途径。
3、通过溶血实验和急毒性实验检测发现MIL在动物体内用药是安全的;采用双抗体夹心ELISA法检测IFN组、MIL组(加磁场)和MIL组(不加磁场)在小鼠体内i.v.后血清和组织中的药物浓度,结果发现:在MIL组(加磁场),药物在肝和肾内浓度明显升高(p＜0.01),且滞留时间延长,肝和肾外各组织药物浓度低。说明:MIL在外加磁场作用下,药物在靶区的靶向选择性最好,滞留时间延长;与普通IFN相比,在外加磁场的作用下,MIL改变了IFN在小鼠体内的分布特性,延长了IFN的半衰期,提高了IFN的生物利用度,具有很强的靶向性和缓释性。
4、分别用游离IFN、ML、IL和MIL对体外培养的Bel-7402肿瘤细胞进行药物干预,然后再分别用MTT法、细胞计数法、吖啶橙染色法和DNA ladder法进行细胞生长抑制和凋亡检测。实验发现:各组Bel-7402肿瘤细胞的生长受到抑制,并且出现细胞凋亡现象。各实验组和对照组比较显示:MIL组和IL组明显高于游离IFN组,而游离IFN组明显高于ML组和对照组(仅含培养基)。以上结果均具有统计学意义p＜0.05)。以上结果说明MIL能够显著抑制Bel-7402细胞的生长,其作用机制是导致细胞凋亡的发生。
5、在原发性肝癌移植瘤裸鼠肿瘤组织外固定永久磁铁后,静脉注射磁性干扰素纳米脂质体可以显著抑制肿瘤组织的生长,抑瘤率为62.50%,高于IFN(27.61%)和IL(28.17%);RT-PCR显示,MIL的VEGFmRNA表达明显低于对照组和其它实验组(p＜0.01),而MIL的Caspase-3mRNA表达明显高于对照组和其它实验组(p＜0.01);Western-blot显示,MIL的VEGF蛋白表达明显低于对照组和其它实验组(p＜0.01),而MIL的Caspase-3蛋白表达明显高于对照组和其它实验组(p＜0.01)。
本研究提示作为化疗药物的新型载体磁性纳米脂质体在肿瘤部位磁场的作用下可以有效提高肿瘤组织内化疗药物浓度减低化疗药物毒副反应显著抑制肿瘤组织的生长。本研究为磁性纳米脂质体作为化疗药物载体进一步应用于临床肿瘤治疗奠定了基础靶向治疗有可能成为肿瘤治疗的新方法。
Targeting therapy for solid tumors, using specific carriers to locate anticancer drugs selectively to solid tumors under specific guiding mechanism, may increase the drug concentration in the required sites. Such an approach would dramatically increase the anticancer drugs' therapeutic efficacy, reduce the drugs' toxic side effects and increase the patient's compliance.
The current study investigated the magnetic nano-liposome designed to act as interferon -α2b(IFN-α2b) carrier, which could be effectively delivered to solid tumors via intravenous administration. Magnetic interferon-α2b nano-liposome(MIL) was prepared by the reverse-phase evaporation method. We studied the response of MIL to the magnetite both out of and within the body, investigated the biological distribution of the MIL in the nude mice bearing colon cancer. The therapeutic effect of MIL to nude mice bearing colon cancer was also intensively studied. We also studied the aeutetoxieity and haemolysis of MIL by i.v. Double antibody sandwich ELISA analysis was used for testing drug concentration in serum and tissues after i.v. of IFN、MIL (in the presence of magnetic field) and MIL(in the absence of magnetic field) in mice.To study the growth inhibitory effect and apoptosis inducing effect of MIL on tumor Bel- 7402 cells by MTT, Cell counting, Acridine Orange and DNA ladder test. And to study treatment with MIL via intravenous administration under magnetic force fixed in the surface of the tumor by analyse of the mRNA expression and Western blot.
The main results of this study are as follows:
1. The particle size of MIL was 170nm, the concentration of IFN-α2b in MIL was 5000 IU IFN/ml, and the entrapment rate of IFN-α2b was about 62.71%. Different lipid ratio, drug lipid ratio, organic solvent and ratio of water/oil phase may influence the particle size of MIL and the entrapment rate of IFN-α2b. The leakage rate of IFN-α2b in MIL was very low within the preservative 8 hours under 4℃and -20℃after preparation.
2. MIL had a good response to the magnetite both out of and within the body. Administration of MIL under magnetic field could be used to deliver IFN-α2b effectively to the targeted site, increasing IFN-α2b concentration in the tumor and decreasing IFN-α2b concentration in other tissues and organs.
3. The experiment of aeutetoxieity and haemolysis of MIL showed that the MIL was safe if it was inject by i.v. Double antibody sandwich ELISA analysis was used for testing drug concentration in serum and tissues after i.v. of IFN、MIL (in the presence of magnetic field) and MIL(in the absence of magnetic field) in mice. The result showed: the drug concentration of liver and kidney of MIL with magnetic field group was remarkable higher than those of free IFN group and MIL without magnetic field group, but that of normal liver and tissues outside liver was lower. The drug selective index of liver and kidney tumor of the with magnetic field group was significantly higher than those of other groups, and the drug selective index of liver and kidney tumor of MIL without magnetic field group was also higher than that of free IFN group. All of them had statistical meaning (P<0.01).
4. Tumor cell Bel-7402 treated by the free IFN, ML, IL, MIL, which had high level of inhibitory action, was detected by MTT, Cell counting, Acridine Orange and DNA ladder test. It was found that the growth of Bel-7402 was inhibited and the apoptosis was happened. And the tumor cell Bel-7402 inhibition rate and apoptosis rate of MIL group and IL group was remarkably higher than those of the IFN group. And the tumor cell Bel-7402 inhibition rate and apoptosis rate of free IFN group was remarkably higher than those of the ML group and Control group. All of them had statistical meaning (p<0.05). The result shows that the MIL had inhibitory action for tumor cell Bel-7402. The mechanism was making the apoptosis happen.
5. Treatment with MIL via intravenous administration under magnetic force fixed in the surface of the tumor showed significant greater anticancer activity than any other modality. The tumor inhibition rate of MIL group was 62.50% , which was remarkably higher than those of the IFN group (27.61%) and IL group (28.17%). RT-PCR showed that the mRNA expression of VEGF of MIL group is the lowest of all groups (P<0.01) and the mRNA expression of Caspase-3 of MIL group is the highest of all groups (P<0.01) . Western blot showed that the VEGF165 protein (monomer, P21) expression is the lowest of all groups (P<0.01) and the cleaved Caspase-3 protein (P17) expression is the highest of all groups (P<0.01) .
The present results suggested that as a new carrier of IFN-α2b, MIL could be used to deliver IFN-α2b effectively to the tumor fixed in the surface of its with a permanent magnet, increasing IFN-α2b concentration in the tumor and decreasing the side effects of other tissues and organs. This new treatment approach involving a combination of magnet implantion in the center of the tumor and intravenous administration of MIL could effectively control the tumor growth.
In summary, this study indicated that magnetic nano-liposome, as an carrier of anticancer drug, had a wide window of opportunity to achieve even anticancer effects in clinical settings. Targeting therapy could be a novel approach to the cancer treatment.
主要研究结果如下:
1、磁性干扰素纳米脂质体粒径平均170nm,干扰素包封率62.71%,磁性颗粒均匀分布于脂质体中。不同的类脂比例、药脂比、有机溶剂种类和油水相体积比影响磁性干扰素纳米脂质体粒径和干扰素包封率。制备后4℃和-20℃保存,8h内渗漏率较低。结果提示磁性纳米脂质体有可能作为干扰素的新型载体应用于恶性肿瘤的靶向治疗中。
2、磁性干扰素纳米脂质体体外、体内具有良好的磁场响应性。应用磁性纳米脂质体加磁场的方式给药,干扰素可以有效地聚集到靶部位,显著提高靶部位干扰素的浓度,降低其它组织和器官中的药物浓度。作为干扰素的有效载体,磁性纳米脂质体加磁场的给药方式有望成为肿瘤靶向治疗的新途径。
3、通过溶血实验和急毒性实验检测发现MIL在动物体内用药是安全的;采用双抗体夹心ELISA法检测IFN组、MIL组(加磁场)和MIL组(不加磁场)在小鼠体内i.v.后血清和组织中的药物浓度,结果发现:在MIL组(加磁场),药物在肝和肾内浓度明显升高(p＜0.01),且滞留时间延长,肝和肾外各组织药物浓度低。说明:MIL在外加磁场作用下,药物在靶区的靶向选择性最好,滞留时间延长;与普通IFN相比,在外加磁场的作用下,MIL改变了IFN在小鼠体内的分布特性,延长了IFN的半衰期,提高了IFN的生物利用度,具有很强的靶向性和缓释性。
4、分别用游离IFN、ML、IL和MIL对体外培养的Bel-7402肿瘤细胞进行药物干预,然后再分别用MTT法、细胞计数法、吖啶橙染色法和DNA ladder法进行细胞生长抑制和凋亡检测。实验发现:各组Bel-7402肿瘤细胞的生长受到抑制,并且出现细胞凋亡现象。各实验组和对照组比较显示:MIL组和IL组明显高于游离IFN组,而游离IFN组明显高于ML组和对照组(仅含培养基)。以上结果均具有统计学意义p＜0.05)。以上结果说明MIL能够显著抑制Bel-7402细胞的生长,其作用机制是导致细胞凋亡的发生。
5、在原发性肝癌移植瘤裸鼠肿瘤组织外固定永久磁铁后,静脉注射磁性干扰素纳米脂质体可以显著抑制肿瘤组织的生长,抑瘤率为62.50%,高于IFN(27.61%)和IL(28.17%);RT-PCR显示,MIL的VEGFmRNA表达明显低于对照组和其它实验组(p＜0.01),而MIL的Caspase-3mRNA表达明显高于对照组和其它实验组(p＜0.01);Western-blot显示,MIL的VEGF蛋白表达明显低于对照组和其它实验组(p＜0.01),而MIL的Caspase-3蛋白表达明显高于对照组和其它实验组(p＜0.01)。
本研究提示作为化疗药物的新型载体磁性纳米脂质体在肿瘤部位磁场的作用下可以有效提高肿瘤组织内化疗药物浓度减低化疗药物毒副反应显著抑制肿瘤组织的生长。本研究为磁性纳米脂质体作为化疗药物载体进一步应用于临床肿瘤治疗奠定了基础靶向治疗有可能成为肿瘤治疗的新方法。
Targeting therapy for solid tumors, using specific carriers to locate anticancer drugs selectively to solid tumors under specific guiding mechanism, may increase the drug concentration in the required sites. Such an approach would dramatically increase the anticancer drugs' therapeutic efficacy, reduce the drugs' toxic side effects and increase the patient's compliance.
The current study investigated the magnetic nano-liposome designed to act as interferon -α2b(IFN-α2b) carrier, which could be effectively delivered to solid tumors via intravenous administration. Magnetic interferon-α2b nano-liposome(MIL) was prepared by the reverse-phase evaporation method. We studied the response of MIL to the magnetite both out of and within the body, investigated the biological distribution of the MIL in the nude mice bearing colon cancer. The therapeutic effect of MIL to nude mice bearing colon cancer was also intensively studied. We also studied the aeutetoxieity and haemolysis of MIL by i.v. Double antibody sandwich ELISA analysis was used for testing drug concentration in serum and tissues after i.v. of IFN、MIL (in the presence of magnetic field) and MIL(in the absence of magnetic field) in mice.To study the growth inhibitory effect and apoptosis inducing effect of MIL on tumor Bel- 7402 cells by MTT, Cell counting, Acridine Orange and DNA ladder test. And to study treatment with MIL via intravenous administration under magnetic force fixed in the surface of the tumor by analyse of the mRNA expression and Western blot.
The main results of this study are as follows:
1. The particle size of MIL was 170nm, the concentration of IFN-α2b in MIL was 5000 IU IFN/ml, and the entrapment rate of IFN-α2b was about 62.71%. Different lipid ratio, drug lipid ratio, organic solvent and ratio of water/oil phase may influence the particle size of MIL and the entrapment rate of IFN-α2b. The leakage rate of IFN-α2b in MIL was very low within the preservative 8 hours under 4℃and -20℃after preparation.
2. MIL had a good response to the magnetite both out of and within the body. Administration of MIL under magnetic field could be used to deliver IFN-α2b effectively to the targeted site, increasing IFN-α2b concentration in the tumor and decreasing IFN-α2b concentration in other tissues and organs.
3. The experiment of aeutetoxieity and haemolysis of MIL showed that the MIL was safe if it was inject by i.v. Double antibody sandwich ELISA analysis was used for testing drug concentration in serum and tissues after i.v. of IFN、MIL (in the presence of magnetic field) and MIL(in the absence of magnetic field) in mice. The result showed: the drug concentration of liver and kidney of MIL with magnetic field group was remarkable higher than those of free IFN group and MIL without magnetic field group, but that of normal liver and tissues outside liver was lower. The drug selective index of liver and kidney tumor of the with magnetic field group was significantly higher than those of other groups, and the drug selective index of liver and kidney tumor of MIL without magnetic field group was also higher than that of free IFN group. All of them had statistical meaning (P<0.01).
4. Tumor cell Bel-7402 treated by the free IFN, ML, IL, MIL, which had high level of inhibitory action, was detected by MTT, Cell counting, Acridine Orange and DNA ladder test. It was found that the growth of Bel-7402 was inhibited and the apoptosis was happened. And the tumor cell Bel-7402 inhibition rate and apoptosis rate of MIL group and IL group was remarkably higher than those of the IFN group. And the tumor cell Bel-7402 inhibition rate and apoptosis rate of free IFN group was remarkably higher than those of the ML group and Control group. All of them had statistical meaning (p<0.05). The result shows that the MIL had inhibitory action for tumor cell Bel-7402. The mechanism was making the apoptosis happen.
5. Treatment with MIL via intravenous administration under magnetic force fixed in the surface of the tumor showed significant greater anticancer activity than any other modality. The tumor inhibition rate of MIL group was 62.50% , which was remarkably higher than those of the IFN group (27.61%) and IL group (28.17%). RT-PCR showed that the mRNA expression of VEGF of MIL group is the lowest of all groups (P<0.01) and the mRNA expression of Caspase-3 of MIL group is the highest of all groups (P<0.01) . Western blot showed that the VEGF165 protein (monomer, P21) expression is the lowest of all groups (P<0.01) and the cleaved Caspase-3 protein (P17) expression is the highest of all groups (P<0.01) .
The present results suggested that as a new carrier of IFN-α2b, MIL could be used to deliver IFN-α2b effectively to the tumor fixed in the surface of its with a permanent magnet, increasing IFN-α2b concentration in the tumor and decreasing the side effects of other tissues and organs. This new treatment approach involving a combination of magnet implantion in the center of the tumor and intravenous administration of MIL could effectively control the tumor growth.
In summary, this study indicated that magnetic nano-liposome, as an carrier of anticancer drug, had a wide window of opportunity to achieve even anticancer effects in clinical settings. Targeting therapy could be a novel approach to the cancer treatment.
引文
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