纳米生物技术用于药物输运与生物成像的初步研究
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摘要
纳米生物技术是纳米技术和生物技术相结合的产物,将它应用于疾病的治疗,诊断,监测及对生物系统的控制即为纳米医学。在众多的纳米医学研究中,纳米药物和纳米诊断性试剂(如纳米粒子作为造影剂进行成像)的合理化靶向性输运成为纳米医学研究领域的前沿。
本论文的研究内容包括两个方面:
一是纳米生物技术用于药物输运:即利用两亲性超支化共聚物HPAE -co-PLA在水相中形成的纳米胶束包载新型抗肿瘤药物NS1,形成以疏水性药物NS1和聚合物疏水端为核,以聚合物亲水端为壳的载药纳米粒子,实现该药物的水溶性,并进行载药纳米粒子的尺度和结构表征,测定包封率与载药量,进而以HepG2和H1299癌细胞为模型细胞,比较了游离药物和纳米药物在相同的剂量下,对肿瘤的杀伤作用,并考察了纳米药物的摄取、胞内滞留和胞内转运等过程。
二是纳米生物技术用于生物成像:即利用Apoferritin做为模板合成了贵金属M纳米粒子,形成了以Apoferritin为壳,以多个贵金属M纳米粒子为核的生物纳米结构,称之为Apo-M荧光探针;对合成的荧光探针进行了表征,并测定了其荧光性质;进而利用该荧光探针进行体内与体外生物成像的初步研究;同时测定其对细胞生长的影响及细胞对其吞噬的作用。结果表明,作为纳米粒子造影剂,该荧光探针有望用于相关疾病的诊断。
Nanotechnology, an interdisciplinary research field involving physics, chemistry, materialogy, informatics, biology, and more, is a new technology emerging in 1990s. Nanotechnology, information technology and biotechnology have been regarded as the most important technologies around the world in 21st century. Nanobiotechnology is combination of nanotechnology and biotechnology. The application of nanobiotechnology to disease treatment, diagnosis, monitoring, and to the control of biological systems has recently been referred to as nanomedicine. Research into the rational delivery and targeting of pharmaceutical, therapeutic, and diagnostic agents (for example, imaging with nanoparticle contrast materials) is at the forefront of projects in nanomedicine.
Nanoparticle drug delivery system in the context of nanomedicine should be viewed as science and technology of nanometer scale complex system (1-1000nm) consisting of drug and nanocale carrier. Several nanomaterials had been used for drug delivery, for example, hydrogels, copolymer micelles, liposomes, dendrimers. Copolymer micelles are spherical super-molecular assemblies of amphiphilic copolymer. The core of micelles can accommodate hydrophobic drugs, and the shell is a hydrophilic brush-like corona that makes the micelle water soluble, thereby allowing delivery of the poorly soluble drugs.
In this study, a novel amphiphilic copolymer with hyper-branched poly(amine-ester) (HPAE) and polylactide (PLA) was used to encapsulate the novel and potent anti-cancer agent, NS1( Fe chelators), for anti-tumor investigations. Hyper-branched polymers have structures and topologies similar to dendrimers and exhibit a higher solubility and a lower solution viscosity compared to linear analogues. In addition, the large number of functional groups offer the possibility for further modification and special applications. As one of the most prominent biodegradable polymers, poly ( D, L-lactic acid) ( PLA) is widely used in the fields of drug delivery and tissue engineering due to its non-toxic, biocompatible and bioabsorbable characteristics.
The HPAE-co-PLA NPs with or without loading of NS1 were produced using an emulsion/solvent evaporation technique. The spherical structure of NS1-loaded NPs formed was revealed by ESEM. Drug-loading content and encapsulation efficiency of the NS1-loaded NPs determined using a Visible-UV spectrophotometer were 8.12±0.04%, 59.3±3.27% respectively. The anti-tumor efficacy of NS1-loaded NPs was nearly equal to free NS1 at higher concentrations (1-10μM ) at 48 h treatment in H1299 and HepG2 and the mechanism in inhibiting tumor cell growth was different with DOX. The intrinsic fluorescence of NPs was used to monitor the cellular uptake of fluorescent NPs and cellular localization of NPs in cells. This observation indicated that cellular uptake of NPs was time-dependent and reached maximal levels between 15 and 24 h. To study the intracellular localization, the intrinsic fluorescence of NPs was recorded with a confocal microscope. It was found that there was a significant overlap between the fluorescence of NPs and the lysosomes. These results indicated that NPs were mainly localized in organelles consistent with lysosomes, uptake and distribution of NPs occured via the endosome-lysosome pathway.
Imaging with nanoparticle contrast materials is another important research field of nanomedicine. Several nanopaticles as contrast materials had been used for biomedical imaging, for example, metal oxides, quantum dots, metals.
Ferritin is a protein complex consisting of 24 subunits forming a sphere with 12 nm outer diameter and an inner cavity of 7.6 nm. The junctions of the subunits provide 14 channels, 3.4 ? in diameter, which perforate the protein shell and serve as pathways between the exterior and interior. As iron storage protein, ferritin is present in many organisms. Releasing the iron containing core from the cavity leads to a hollow sphere, the apoferritin, which has already been used as template for the preparation of various nanoparticles.
In this study, apoferritin was used as template to synthesize nobel metal M nanoparticles which located in the apoferritin cavity. This combination of apoferritin and M nanoparticles was called the Apo-M fluorescent probe because M nanoparticles had fluorescence. The core/shell structure of Apo-M was revealed by TEM. Excitation wavelength and emission wavelength of Apo-M determined using a spectrofluorophotometer were 475nm, 669nm respectively. Raw264.7 cells were imaged with Apo-M which mainly located in cytoplasm, furthermore, near-infrared whole-body imaging with Apo-M was realized in the nude mouse. Study about effects of Apo-M and apoferritinat to cell growth showed that they didn’t have significant toxicity to Raw264.7 at 12h treatment and had toxicity at 24h treatment, however, didn’t have significant toxicity to HepG2 at 12h or 24h treatment. The intrinsic fluorescence of Apo-M was used to monitor the cellular uptake of Apo-M, this observation indicated that cellular uptake of Apo-M was time-dependent.
In summary, our study makes hydrophobic NS1 water soluble, so it’s a promise that this novel and potent anti-cancer agent is used in clinic;Apo-M fluorescent probes as contrast materials are expected for disease diagnosis.
本论文的研究内容包括两个方面:
一是纳米生物技术用于药物输运:即利用两亲性超支化共聚物HPAE -co-PLA在水相中形成的纳米胶束包载新型抗肿瘤药物NS1,形成以疏水性药物NS1和聚合物疏水端为核,以聚合物亲水端为壳的载药纳米粒子,实现该药物的水溶性,并进行载药纳米粒子的尺度和结构表征,测定包封率与载药量,进而以HepG2和H1299癌细胞为模型细胞,比较了游离药物和纳米药物在相同的剂量下,对肿瘤的杀伤作用,并考察了纳米药物的摄取、胞内滞留和胞内转运等过程。
二是纳米生物技术用于生物成像:即利用Apoferritin做为模板合成了贵金属M纳米粒子,形成了以Apoferritin为壳,以多个贵金属M纳米粒子为核的生物纳米结构,称之为Apo-M荧光探针;对合成的荧光探针进行了表征,并测定了其荧光性质;进而利用该荧光探针进行体内与体外生物成像的初步研究;同时测定其对细胞生长的影响及细胞对其吞噬的作用。结果表明,作为纳米粒子造影剂,该荧光探针有望用于相关疾病的诊断。
Nanotechnology, an interdisciplinary research field involving physics, chemistry, materialogy, informatics, biology, and more, is a new technology emerging in 1990s. Nanotechnology, information technology and biotechnology have been regarded as the most important technologies around the world in 21st century. Nanobiotechnology is combination of nanotechnology and biotechnology. The application of nanobiotechnology to disease treatment, diagnosis, monitoring, and to the control of biological systems has recently been referred to as nanomedicine. Research into the rational delivery and targeting of pharmaceutical, therapeutic, and diagnostic agents (for example, imaging with nanoparticle contrast materials) is at the forefront of projects in nanomedicine.
Nanoparticle drug delivery system in the context of nanomedicine should be viewed as science and technology of nanometer scale complex system (1-1000nm) consisting of drug and nanocale carrier. Several nanomaterials had been used for drug delivery, for example, hydrogels, copolymer micelles, liposomes, dendrimers. Copolymer micelles are spherical super-molecular assemblies of amphiphilic copolymer. The core of micelles can accommodate hydrophobic drugs, and the shell is a hydrophilic brush-like corona that makes the micelle water soluble, thereby allowing delivery of the poorly soluble drugs.
In this study, a novel amphiphilic copolymer with hyper-branched poly(amine-ester) (HPAE) and polylactide (PLA) was used to encapsulate the novel and potent anti-cancer agent, NS1( Fe chelators), for anti-tumor investigations. Hyper-branched polymers have structures and topologies similar to dendrimers and exhibit a higher solubility and a lower solution viscosity compared to linear analogues. In addition, the large number of functional groups offer the possibility for further modification and special applications. As one of the most prominent biodegradable polymers, poly ( D, L-lactic acid) ( PLA) is widely used in the fields of drug delivery and tissue engineering due to its non-toxic, biocompatible and bioabsorbable characteristics.
The HPAE-co-PLA NPs with or without loading of NS1 were produced using an emulsion/solvent evaporation technique. The spherical structure of NS1-loaded NPs formed was revealed by ESEM. Drug-loading content and encapsulation efficiency of the NS1-loaded NPs determined using a Visible-UV spectrophotometer were 8.12±0.04%, 59.3±3.27% respectively. The anti-tumor efficacy of NS1-loaded NPs was nearly equal to free NS1 at higher concentrations (1-10μM ) at 48 h treatment in H1299 and HepG2 and the mechanism in inhibiting tumor cell growth was different with DOX. The intrinsic fluorescence of NPs was used to monitor the cellular uptake of fluorescent NPs and cellular localization of NPs in cells. This observation indicated that cellular uptake of NPs was time-dependent and reached maximal levels between 15 and 24 h. To study the intracellular localization, the intrinsic fluorescence of NPs was recorded with a confocal microscope. It was found that there was a significant overlap between the fluorescence of NPs and the lysosomes. These results indicated that NPs were mainly localized in organelles consistent with lysosomes, uptake and distribution of NPs occured via the endosome-lysosome pathway.
Imaging with nanoparticle contrast materials is another important research field of nanomedicine. Several nanopaticles as contrast materials had been used for biomedical imaging, for example, metal oxides, quantum dots, metals.
Ferritin is a protein complex consisting of 24 subunits forming a sphere with 12 nm outer diameter and an inner cavity of 7.6 nm. The junctions of the subunits provide 14 channels, 3.4 ? in diameter, which perforate the protein shell and serve as pathways between the exterior and interior. As iron storage protein, ferritin is present in many organisms. Releasing the iron containing core from the cavity leads to a hollow sphere, the apoferritin, which has already been used as template for the preparation of various nanoparticles.
In this study, apoferritin was used as template to synthesize nobel metal M nanoparticles which located in the apoferritin cavity. This combination of apoferritin and M nanoparticles was called the Apo-M fluorescent probe because M nanoparticles had fluorescence. The core/shell structure of Apo-M was revealed by TEM. Excitation wavelength and emission wavelength of Apo-M determined using a spectrofluorophotometer were 475nm, 669nm respectively. Raw264.7 cells were imaged with Apo-M which mainly located in cytoplasm, furthermore, near-infrared whole-body imaging with Apo-M was realized in the nude mouse. Study about effects of Apo-M and apoferritinat to cell growth showed that they didn’t have significant toxicity to Raw264.7 at 12h treatment and had toxicity at 24h treatment, however, didn’t have significant toxicity to HepG2 at 12h or 24h treatment. The intrinsic fluorescence of Apo-M was used to monitor the cellular uptake of Apo-M, this observation indicated that cellular uptake of Apo-M was time-dependent.
In summary, our study makes hydrophobic NS1 water soluble, so it’s a promise that this novel and potent anti-cancer agent is used in clinic;Apo-M fluorescent probes as contrast materials are expected for disease diagnosis.
引文
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