EcN共价PSMA/PELA载药微球的制备及其靶向肿瘤低氧环境的初步研究
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摘要
目的将Escherichia coli Nissle 1917(EcN)与聚苯乙烯马来酸酐(poly styrene-co-maleic anhydride, PSMA)/聚DL-乳酸-聚乙二醇共聚物[(poly (DL-Lactic acid) and poly ethyleneglycol block copolymer, PELA]聚合物载药微粒共价结合,研究其靶向肿瘤低氧环境的效果。方法溶剂挥发法制备不同混合比例(质量比为7∶3和5∶5)的PSMA和PELA携载阿霉素(Dox)的PSMA/PELA微粒;通过EDC和NHS催化反应使得EcN表面的氨基和PSMA/PELA载药微粒表面的酸酐发生共价结合,平板计数法表征接枝后EcN的生长活性;利用HepG2细胞体外构建3D肿瘤模型,研究EcN共价PSMA/PELA载药微粒在肿瘤低氧区域的渗透效果;小鼠肝癌细胞H22建立皮下肿瘤模型,体内验证其靶向肿瘤低氧区域的效果。结果成功制备载Dox的PSMA/PELA微粒(DMP_(7/3)和DMP_(5/5)),两种微粒球形规则,粒径700～800 nm;DMP_(7/3)和DMP_(5/5)载药量分别为(4.27±0.19)%和(3.94±0.11)%,包裹效率分别为(85.4±2.6)%和(78.8±3.5)%,载药量和包裹效率差异无统计学意义;CLSM结果显示,EcN成功接枝在DMP_(7/3)表面,且接枝后的细菌具有生长活力;体外渗透实验研究表明,与EcN未共价接枝的DMP比较,EcN修饰的聚合物载药微粒(EcN@DMP_(7/3))可较多地渗透至肿瘤内部;体内分布实验证实,静脉给药4 h和24 h后,EcN@DMP_(7/3)治疗组肿瘤组织内Dox浓度分别是DMP_(7/3)治疗组浓度的3倍和6倍(P<0.01)。结论通过EDC和NHS催化反应方法可使EcN表面的氨基和PSMA/PELA载药微粒表面的酸酐发生共价反应,且反应后的EcN依然维持其生长活性,能有效驱动PSMA/PELA载药微粒靶向肿瘤内部低氧区域。
Objective To conjugate Escherichia coli Nissle 1917(EcN) with drug-loaded poly styrene-co-maleic anhydride(PSMA)/poly(DL-lactic acid) and poly ethyleneglycol block copolymer(PELA) polymer micro-particles and investigate the ability of the micro-particles to target the hypoxic tumor environment. Methods PSMA and PELA were mixed at the ratio of 7∶3 or 5∶5(m/m) to prepare doxorubicin(Dox)-loaded PSMA/PELA micro-particles through solvent evaporation method. The covalent binding was achieved by EDC-NHS catalytic reaction of the carboxyl groups on the surface of EcN with the amino groups on the surface of PSMA/PELA drug-loaded micro-particles. In an in vitro 3 D tumor model constructed using HepG2 cells, we investigated the permeation of EcN-conjugated PSMA/PELA drug-loaded micro-particles into the hypoxic tumor area. We further tested the performance of the drug-loaded micro-particles for targeting the hypoxic tumor environment in a mouse model bearing subcutaneous hepatoma H22 cell xenograft. Results We successfully prepared Dox-loaded micro-particles with different PSMA/PELA ratios(DMP_(7/3) and DMP_(5/5)) using solvent evaporation method, and both of the micro-particles showed a regular spherical shape with an average size of 700 to 800 nm. DMP_(7/3) and DMP_(5/5) micro-particles had comparable Dox-loading efficiency [(4.27±0.19)% vs(3.94±0.11)%] and encapsulation efficiency [(85.4±2.6)% vs(78.8±3.5)%]. Confocal laser scanning microscopy showed that EcN was successfully conjugated on the surface DMP_(7/3) micro-particles. Compared with DMP microparticles without EcN conjugation, the EcN-conjugated micro-particles(EcN@DMP_(7/3)) permeated efficiently into the central area in the in vitro 3 D tumor model. In the tumor-bearing mice, bio-distribution analyses revealed that at 4 and 24 h after intravenous administration, EcN@DMP_(7/3) resulted in 3-folder and 6-folder increases in Dox accumulation in the tumors, respectively, as compared with the non-conjugated DMP_(7/3) micro-particles(P<0.01). Conclusion EDC-NHS catalytic reaction allows the amino groups on the outer surface of EcN to covalently bind to anhydride on the surface of drug-loaded PSMA/PELA particles without affecting the growth activity of EcN, which effectively drives the drug-loaded particles to target the hypoxic region inside the tumor.
Objective To conjugate Escherichia coli Nissle 1917(EcN) with drug-loaded poly styrene-co-maleic anhydride(PSMA)/poly(DL-lactic acid) and poly ethyleneglycol block copolymer(PELA) polymer micro-particles and investigate the ability of the micro-particles to target the hypoxic tumor environment. Methods PSMA and PELA were mixed at the ratio of 7∶3 or 5∶5(m/m) to prepare doxorubicin(Dox)-loaded PSMA/PELA micro-particles through solvent evaporation method. The covalent binding was achieved by EDC-NHS catalytic reaction of the carboxyl groups on the surface of EcN with the amino groups on the surface of PSMA/PELA drug-loaded micro-particles. In an in vitro 3 D tumor model constructed using HepG2 cells, we investigated the permeation of EcN-conjugated PSMA/PELA drug-loaded micro-particles into the hypoxic tumor area. We further tested the performance of the drug-loaded micro-particles for targeting the hypoxic tumor environment in a mouse model bearing subcutaneous hepatoma H22 cell xenograft. Results We successfully prepared Dox-loaded micro-particles with different PSMA/PELA ratios(DMP_(7/3) and DMP_(5/5)) using solvent evaporation method, and both of the micro-particles showed a regular spherical shape with an average size of 700 to 800 nm. DMP_(7/3) and DMP_(5/5) micro-particles had comparable Dox-loading efficiency [(4.27±0.19)% vs(3.94±0.11)%] and encapsulation efficiency [(85.4±2.6)% vs(78.8±3.5)%]. Confocal laser scanning microscopy showed that EcN was successfully conjugated on the surface DMP_(7/3) micro-particles. Compared with DMP microparticles without EcN conjugation, the EcN-conjugated micro-particles(EcN@DMP_(7/3)) permeated efficiently into the central area in the in vitro 3 D tumor model. In the tumor-bearing mice, bio-distribution analyses revealed that at 4 and 24 h after intravenous administration, EcN@DMP_(7/3) resulted in 3-folder and 6-folder increases in Dox accumulation in the tumors, respectively, as compared with the non-conjugated DMP_(7/3) micro-particles(P<0.01). Conclusion EDC-NHS catalytic reaction allows the amino groups on the outer surface of EcN to covalently bind to anhydride on the surface of drug-loaded PSMA/PELA particles without affecting the growth activity of EcN, which effectively drives the drug-loaded particles to target the hypoxic region inside the tumor.
引文
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[19] LOUISE MEYER R,ZHOU X F,TANG L,et al.Immobilisation of living bacteria for AFM imaging under physiological conditions[J].Ultramicroscopy,2010,110(11):1349-1357.DOI:10.1016/j.ultramic.2010.06.010.
[20] WANG C,TANG Z,ZHAO Y,et al.Three-dimensional in vitro cancer models:A short review[J].Biofabrication,2014,6(2):022001.DOI:10.1088/1758-5082/6/2/022001.
[21] WANG Y,MIRZA S,WU S,et al.3D hydrogel breast cancer models for studying the effects of hypoxia on epithelial to mesenchymal transition[J].Oncotarget,2018,9(63):32191-32203.DOI:10.18632/oncotarget.25891.
[22] LUO X M,JIA G Q,SONG H X,et al.Promoting antitumor activities of hydroxycamptothecin by encapsulation into acid-labile nanoparticles using electrospraying[J].Pharm Res,2014,31(1):46-59.DOI:10.1007/s11095-013-1130-4.
[23] LUO C H,HUANG C T,SU C H,et al.Bacteria-mediated hypoxia-specific delivery of nanoparticles for tumors imaging and therapy[J].Nano Lett,2016,16(6):3493-3499.DOI:10.1021/acs.nanolett.6b00262.
[2] PRABHU R H,PATRAVALE V B,JOSHI M D.Polymeric nanoparticles for targeted treatment in oncology:current insights[J].Int J Nanomedicine,2015,10:1001-1018.DOI:10.2147/IJN.S56932.
[3] SHA H,ZOU Z,XIN K,et al.Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy[J].J Control Release,2015,200:188-200.DOI:10.1016/j.jconrel.2014.12.039.
[4] DAS M,DUAN W,SAHOO S K.Multifunctional nanoparticle–EpCAM aptamer bioconjugates:A paradigm for targeted drug delivery and imaging in cancer therapy[J].Nanomedicine,2015,11(2):379-389.DOI:10.1016/j.nano.2014.09.002.
[5] DHANI N,FYLES A,HEDLEY D,et al.The clinical significance of hypoxia in human cancers[J].Semin Nucl Med,2015,45(2):110-121.DOI:10.1053/j.semnuclmed.2014.11.002.
[6] HONG M,ZHU S,JIANG Y,et al.Efficient tumor targeting of hydroxycamptothecin loaded PEGylated niosomes modified with transferrin[J].J Control Release,2009,133(2):96-102.DOI:10.1016/j.jconrel.2008.09.005.
[7] FELFOUL O,MOHAMMADI M,TAHERKHANI S,et al.Magneto-aerotactic bacteria deliver drug-containing nanoliposomes to tumour hypoxic regions[J].Nat Nanotechnol,2016,11(11):941-947.DOI:10.1038/nnano.2016.137.
[8] ZHOU S B,GRAVEKAMP C,BERMUDES D,et al.Tumour-targeting bacteria engineered to fight cancer[J].Nat Rev Cancer,2018,18(12):727-743.DOI:10.1038/s41568-018-0070-z.
[9] THAMM D H,KURZMAN I D,KING I,et al.Systemic administration of an attenuated,tumor-targeting Salmonella typhimurium to dogs with spontaneous neoplasia:phase I evaluation[J].Clin Cancer Res,2005,11(13):4827-4834.DOI:10.1158/1078-0432.CCR-04-2510.
[10] TOSO J F,GILL V J,HWU P,et al.Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma[J].J Clin Oncol,2002,20(1):142-152.DOI:10.1200/JCO.2002.20.1.142.
[11] SOUZA é L,ELIAN S D,PAULA L M,et al.Escherichia coli strain Nissle 1917 ameliorates experimental colitis by modulating intestinal permeability,the inflammatory response and clinical signs in a faecal transplantation model[J].J Med Microbiol,2016,65(3):201-210.DOI:10.1099/jmm.0.000222.
[12] STRITZKER J,WEIBEL S,HILL P J,et al.Tumor-specific colonization,tissue distribution,and gene induction by probiotic Escherichia coli Nissle 1917 in live mice[J].Int J Med Microbiol,2007,297(3):151-162.DOI:10.1016/j.ijmm.2007.01.008.
[13] ZHOU S N,ZHANG M X,WANG J S.Tumor-targeted delivery of TAT-Apoptin fusion gene using Escherichia coli Nissle 1917 to colorectal cancer[J].Med Hypotheses,2011,76(4):533-534.DOI:10.1016/j.mehy.2010.12.010.
[14] SHI C L,GUO X,QU Q Q,et al.Actively targeted delivery of anticancer drug to tumor cells by redox-responsive star-shaped micelles[J].Biomaterials,2014,35(30):8711-8722.DOI:10.1016/j.biomaterials.2014.06.036.
[15] SIMS L B,HUSS M K,FRIEBOES H B,et al.Distribution of PLGA-modified nanoparticles in 3D cell culture models of hypo-vascularized tumor tissue[J].J Nanobiotechnology,2017,15(1):67.DOI:10.1186/s12951-017-0298-x.
[16] KRAMER M G,MASNER M,FERREIRA F A,et al.Bacterial therapy of cancer:promises,limitations,and insights for future directions[J].Front Microbiol,2018,9:16.DOI:10.3389/fmicb.2018.00016.
[17] LIU K,CHEN W J,YANG T T,et al.Paclitaxel and quercetin nanoparticles co-loaded in microspheres to prolong retention time for pulmonary drug delivery[J].Int J Nanomed,2017,12:8239-8255.DOI:10.2147/IJN.S147028.
[18] KAMALY N,YAMEEN B,WU J,et al.Degradable controlled-release polymers and polymeric nanoparticles:mechanisms of controlling drug release[J].Chem Rev,2016,116(4):2602-2663.DOI:10.1021/acs.chemrev.5b00346.
[19] LOUISE MEYER R,ZHOU X F,TANG L,et al.Immobilisation of living bacteria for AFM imaging under physiological conditions[J].Ultramicroscopy,2010,110(11):1349-1357.DOI:10.1016/j.ultramic.2010.06.010.
[20] WANG C,TANG Z,ZHAO Y,et al.Three-dimensional in vitro cancer models:A short review[J].Biofabrication,2014,6(2):022001.DOI:10.1088/1758-5082/6/2/022001.
[21] WANG Y,MIRZA S,WU S,et al.3D hydrogel breast cancer models for studying the effects of hypoxia on epithelial to mesenchymal transition[J].Oncotarget,2018,9(63):32191-32203.DOI:10.18632/oncotarget.25891.
[22] LUO X M,JIA G Q,SONG H X,et al.Promoting antitumor activities of hydroxycamptothecin by encapsulation into acid-labile nanoparticles using electrospraying[J].Pharm Res,2014,31(1):46-59.DOI:10.1007/s11095-013-1130-4.
[23] LUO C H,HUANG C T,SU C H,et al.Bacteria-mediated hypoxia-specific delivery of nanoparticles for tumors imaging and therapy[J].Nano Lett,2016,16(6):3493-3499.DOI:10.1021/acs.nanolett.6b00262.