壳-核结构多柔比星脂质磷酸钙纳米粒的制备及体外性能评价
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摘要
脂质体作为一种重要的药物载体,具有生物相容性高、免疫原性低等优点,已被广泛应用于药物传递领域,尤其是肿瘤的靶向治疗。然而传统脂质体由流动的动态磷脂膜构成,极易发生相互融合从而导致聚集和药物泄露。此外,较低的聚乙二醇(polyethylene glycol,PEG)修饰程度也限制了该载体在体内的靶向递药性能。鉴于传统脂质体存在的问题,本文设计了一种将无机载体磷酸钙与脂质体相结合的纳米粒靶向药物递送系统,即脂质磷酸钙(lipid coated calcium phosphate,LCP)。以多柔比星(doxorubicin,DOX)为模型药物,采用反相微乳液法制备载药脂质磷酸钙纳米粒(DOX/LCP),并对制备条件进行考察。采用红外光谱、能量色散光谱和透射电镜对磷酸钙内核进行结构表征和形态观察,并对DOX/LCP的粒径、包封率、载药量、稳定性及体外释放行为进行了考察。在此基础上,采用共聚焦显微镜和流式细胞仪分别对LCP介导DOX在耐药肿瘤细胞株MCF-7/DOX中的摄取进行了定性和定量评价,并采用噻唑蓝比色法考察了其细胞毒性作用。结果表明:制得的LCP具有典型的核-壳结构,且尺寸均一、分散性良好,粒径为(48.6±3.9)nm,zeta电位为(-12.1±1.2)m V,包封率>80%,在模拟血浆中具有良好的粒径稳定性。体外释放具有明显的p H依赖性,当环境p H为7.4时,24 h累计释放度低于20%;随着释放介质p H值的降低,DOX/LCP释放速度逐渐加快,在p H为4.5介质中,24 h累计释放量超过90%。LCP可以显著促进耐药细胞对DOX的摄取和蓄积,且体外对耐药肿瘤的抑制率显著提高,DOX/LCP组和游离DOX组的半数抑制浓度(half maximal inhibitory concentration,IC50)分别为4.6和11.8μg·m L-1,两者相比具有显著性差异(P<0.05)。综上,本研究制备的LCP粒径小、包封率高、稳定性好,具有环境响应性及潜在的逆转肿瘤耐药性能,有应用于临床研究的潜力。
As an important drug carrier,liposome has the advantages of high biocompatibility and low immunogenicity.It has been widely used in the field of drug delivery,especially the targeted treatment of tumors.However,traditional liposomes are composed of flowing dynamic phospholipid membranes,which are easy to fuse together,resulting in aggregation and drug leakage.In addition,the lower degree of polyethylene glycol(PEG) modification also limits the targeted delivery performance of the vector in vivo.In view of the problems,a nanoparticle-targeted drug delivery system combining the inorganic carrier calcium phosphate with liposomes was designed,namely lipid calcium phosphate(LCP).Using doxorubicin(DOX) as a model drug,doxorubicin-loaded lipid calcium phosphate nanoparticles(DOX/LCP) were prepared by reverse microemulsion method,and the preparation conditions were investigated.The structure and morphology of calcium phosphate cores were observed by infrared spectroscopy,EDS spectroscopy,and transmission electron microscopy.The particle size,encapsulation efficiency,drug loading,stability and release behavior in vitro of DOX/LCP were investigated.Confocal microscopy and flow cytometry were used to qualitatively and quantitatively evaluate the uptake of DOX in drug-resistant tumor cell line MCF-7/DOX by LCP,respectively,and the thiazolium MTT colorimetric method was used to examine its cytotoxicity.LCP exhibited a typical core-shell structure with good size uniformity and dispersibility.The particle size was in(48.6 ± 3.9) nm,the potential was in(-12.1 ± 1.2) m V,and the encapsulation efficiency was above 80%.Moreover,it has a good stability in simulated plasma.In vitro release of LCP had a significant p H dependence.When the p H of the environment was 7.4,the cumulative release within 24 hours was less than 20%; as the p H of the release medium decreases,the release rate of DOX/LCP was accelerated gradually.Accumulated release over 24 hours exceeded 90% in the p H 4.5 medium.LCP significantly promoted the uptake and accumulation of DOX by drug-resistant cells,and the inhibition rate of drug-resistant tumors was significantly increased in vitro.The half maximal inhibitory concentrations(IC50) of LCP/DOX and free DOX were 4.6 and 11.8 μg·m L-1,respectively,and there was a significant difference between the two groups(P < 0.05).In summary,the LCP prepared in this study had a small particle size,high encapsulation efficiency and good stability.It had environmental responsiveness and potential inhibition of tumor drug resistance,which suggests a potential in the clinical application.
As an important drug carrier,liposome has the advantages of high biocompatibility and low immunogenicity.It has been widely used in the field of drug delivery,especially the targeted treatment of tumors.However,traditional liposomes are composed of flowing dynamic phospholipid membranes,which are easy to fuse together,resulting in aggregation and drug leakage.In addition,the lower degree of polyethylene glycol(PEG) modification also limits the targeted delivery performance of the vector in vivo.In view of the problems,a nanoparticle-targeted drug delivery system combining the inorganic carrier calcium phosphate with liposomes was designed,namely lipid calcium phosphate(LCP).Using doxorubicin(DOX) as a model drug,doxorubicin-loaded lipid calcium phosphate nanoparticles(DOX/LCP) were prepared by reverse microemulsion method,and the preparation conditions were investigated.The structure and morphology of calcium phosphate cores were observed by infrared spectroscopy,EDS spectroscopy,and transmission electron microscopy.The particle size,encapsulation efficiency,drug loading,stability and release behavior in vitro of DOX/LCP were investigated.Confocal microscopy and flow cytometry were used to qualitatively and quantitatively evaluate the uptake of DOX in drug-resistant tumor cell line MCF-7/DOX by LCP,respectively,and the thiazolium MTT colorimetric method was used to examine its cytotoxicity.LCP exhibited a typical core-shell structure with good size uniformity and dispersibility.The particle size was in(48.6 ± 3.9) nm,the potential was in(-12.1 ± 1.2) m V,and the encapsulation efficiency was above 80%.Moreover,it has a good stability in simulated plasma.In vitro release of LCP had a significant p H dependence.When the p H of the environment was 7.4,the cumulative release within 24 hours was less than 20%; as the p H of the release medium decreases,the release rate of DOX/LCP was accelerated gradually.Accumulated release over 24 hours exceeded 90% in the p H 4.5 medium.LCP significantly promoted the uptake and accumulation of DOX by drug-resistant cells,and the inhibition rate of drug-resistant tumors was significantly increased in vitro.The half maximal inhibitory concentrations(IC50) of LCP/DOX and free DOX were 4.6 and 11.8 μg·m L-1,respectively,and there was a significant difference between the two groups(P < 0.05).In summary,the LCP prepared in this study had a small particle size,high encapsulation efficiency and good stability.It had environmental responsiveness and potential inhibition of tumor drug resistance,which suggests a potential in the clinical application.
引文
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[20]Tang J,Li L,Howard CB,et al.Preparation of optimized lipid-coated calcium phosphate nanoparticles for enhanced in vitro gene delivery to breast cancer cells[J].J Mater Chem B,2015,3:6805-6812.
[21]Zhang L,Wang Y,Gao H,et al.The construction of cellpenetrating peptide R8 and p H sensitive cleavable polyethylene glycols co-modified liposomes[J].Acta Pharm Sin(药学学报),2015,50:760-766.
[22]Huang L,Liu Y.In vivo delivery of RNAi with lipid-based nanoparticles[J].Annu Rev Biomed Eng,2011,13:507-530.
[23]Suk JS,Xu Q,Kim N,et al.PEGylation as a strategy for improving nanoparticle-based drug and gene delivery[J].Adv Drug Deliv Rev,2016,99:28-51.
[24]Ma P,Mumper RJ.Anthracycline nano-delivery systems to overcome multiple drug resistance:a comprehensive review[J].Nano Today,2013,8:313-331.
[25]Markman JL,Rekechenetskiy A,Holler E,et al.Nanomedicine therapeutic approaches to overcome cancer drug resistance[J].Adv Drug Deliv Rev,2013,65:1866-1879.
[26]Hu Y,Haynes MT,Wang Y,et al.A highly efficient synthetic vector:nonhydrodynamic delivery of DNA to hepatocyte nuclei in vivo[J].ACS Nano,2013,7:5376-5384.
[2]Yin H,Kanasty RL,Eltoukhy AA,et al.Non-viral vectors for gene-based therapy[J].Nat Rev Genet,2014,15:541-555.
[3]Choi AH,Ben-Nissan B.Calcium phosphate nanocoatings and nanocomposites,part I:recent developments and advancements in tissue engineering and bioimaging[J].Nanomedicine(Lond),2015,10:2249-2261.
[4]Tabakovi?A,Kester M,Adair JH.Calcium phosphate-based composite nanoparticles in bioimaging and therapeutic delivery applications[J].Wiley Interdiscip Rev Nanomed Nanobiotechnol,2012,4:96-112.
[5]Xu HH,Wang P,Wang L,et al.Calcium phosphate cements for bone engineering and their biological properties[J].Bone Res,2017,5:17056.
[6]Parent M,Baradari H,Champion E,et al.Design of calcium phosphate ceramics for drug delivery applications in bone diseases:a review of the parameters affecting the loading and release of the therapeutic substance[J].J Control Release,2017,252:1-17.
[7]Li J,Yang Y,Huang L.Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for si RNA delivery to the tumor[J].J Control Release,2012,158:108-114.
[8]Ginebra MP,Canal C,Espanol M,et al.Calcium phosphate cements as drug delivery materials[J].Adv Drug Deliv Rev,2012,64:1090-1110.
[9]Elzoghby AO,Hemasa AL,Freag MS.Hybrid proteininorganic nanoparticles:from tumor-targeted drug delivery to cancer imaging[J].J Control Release,2016,243:303-322.
[10]Deshpande PP,Biswas S,Torchilin VP.Current trends in the use of liposomes for tumor targeting[J].Nanomedicine(Lond),2013,8:1509-1528.
[11]Yang Y,Xie X,Yang Y,et al.A review on the influences of size and surface charge of liposome on its targeted drug delivery in vivo[J].Acta Pharm Sin(药学学报),2013,48:1644-1650.
[12]Fang Y,Xue J,Gao S,et al.Cleavable PEGylation:a strategy for overcoming the“PEG dilemma”in efficient drug delivery[J].Drug Deliv,2017,24:22-32.
[13]Zhang D,Xu H,Hu M,et al.“PEG dilemma”for liposomes and its solving approaches[J].Acta Pharm Sin(药学学报),2015,50:252-260.
[14]Haynes MT,Huang L.Lipid-coated calcium phosphate nanoparticles for nonviral gene therapy[J].Adv Genet,2014,88:205-229.
[15]Satterlee AB,Huang L.Current and future theranostic applications of the lipid-calcium-phosphate nanoparticle platform[J].Theranostics,2016,6:918-929.
[16]Haynes MT,Huang L.Maximizing the supported bilayer phenomenon:liposomes comprised exclusively of PEGylated phospholipids for enhanced systemic and lymphatic delivery[J].ACS Appl Mater Interfaces,2016,8:24361-24367.
[17]Li J,Chen YC,Tseng YC,et al.Biodegradable calcium phosphate nanoparticle with lipid coating for systemic si RNA delivery[J].J Control Release,2010,142:416-421.
[18]Uskokovi?V,Desai TA.Phase composition control of calcium phosphate nanoparticles for tunable drug delivery kinetics and treatment of osteomyelitis.I.Preparation and drug release[J].J Biomed Mater Res A,2013,101:1416-1426.
[19]Guo S,Miao L,Huang L.Lipid coated calcium phosphate nanoparticles for drug delivery[J].Acta Biophy Sin(生物物理学报),2013,29:823-830.
[20]Tang J,Li L,Howard CB,et al.Preparation of optimized lipid-coated calcium phosphate nanoparticles for enhanced in vitro gene delivery to breast cancer cells[J].J Mater Chem B,2015,3:6805-6812.
[21]Zhang L,Wang Y,Gao H,et al.The construction of cellpenetrating peptide R8 and p H sensitive cleavable polyethylene glycols co-modified liposomes[J].Acta Pharm Sin(药学学报),2015,50:760-766.
[22]Huang L,Liu Y.In vivo delivery of RNAi with lipid-based nanoparticles[J].Annu Rev Biomed Eng,2011,13:507-530.
[23]Suk JS,Xu Q,Kim N,et al.PEGylation as a strategy for improving nanoparticle-based drug and gene delivery[J].Adv Drug Deliv Rev,2016,99:28-51.
[24]Ma P,Mumper RJ.Anthracycline nano-delivery systems to overcome multiple drug resistance:a comprehensive review[J].Nano Today,2013,8:313-331.
[25]Markman JL,Rekechenetskiy A,Holler E,et al.Nanomedicine therapeutic approaches to overcome cancer drug resistance[J].Adv Drug Deliv Rev,2013,65:1866-1879.
[26]Hu Y,Haynes MT,Wang Y,et al.A highly efficient synthetic vector:nonhydrodynamic delivery of DNA to hepatocyte nuclei in vivo[J].ACS Nano,2013,7:5376-5384.