纳米生物学及其应用.ppt

纳米生物学及其应用,亓立峰,浙江加州国际纳米技术研究院,前言：纳米微粒概述 Part I: 纳米药物与靶向设计 Part II: 肿瘤纳米分子影像与实时治疗 Part III: 纳米探针 前景展望,What is Nano?,Quantum dots, magnetic NP,Polymer NP,Fig1. A schemetic of nanoscale material,Phycoerythrin,前言,纳米粒子分类,某种物质或元素纳米级别粒子: nano Zinic; SiO2， nano gold, CdSe/ZnS, Fe2O3,Polymer NP Degradable,nanosphere,nanocapsule,Polymeric matrix,Polymeric membrane,Oily or aqueous core,前言,化学法： (1)沉淀法/透析法 把沉淀剂加入到盐溶液中反应后，（将沉淀热处理）得到纳米材料。其特点简单易行，但纯度低，颗粒半径大，聚合体纳米粒子、氧化物。 (2) 微乳液法/乳液扩散法 两种互不相溶的溶剂在表面活性剂的作用下形成乳液，在微泡中经成核、聚结、团聚、（热处理）后得纳米粒子。其特点粒子的单分散和界面性好，聚合体纳米粒子、族半导体纳米粒子多用此法制备,纳米微粒制备方法,前言,纳米粒子表征方法,These newly formed tumor vessels are usually abnormal in form and architecture. They are poorly-aligned defective endothelial cells with wide fenestrations, lacking a smooth muscle layer, or innervation with a wider lumen, and impaired functional receptors for angiotensin II. Furthermore, tumor tissues usually lack effective lymphatic drainage. All these factors will lead to abnormal molecular and fluid transport dynamics especially for macromolecular drugs. Namely, this phenomenon was coined “enhanced permeability and retention (EPR)-effect” of macromolecules and lipids in solid tumors. /wiki/Enhanced_Permeability_and_Retention_effect,Part I:纳米药物与肿瘤靶向治疗 EPR effects,Enhanced permeability and retention (EPR) effect. Long-circulating drug carriers (1), penetrate through the leaky pathological vasculature (2), into the tumor interstitium (3), and degrade there, releasing a free drug (4) and creating its high local concentration.,AAPS Journal. 2007;9(2):Article 15.DOI: 10.1208/aapsj0902015,Part I:纳米药物与肿瘤靶向治疗,Nanoparticles : clinical administration of anticancer drugs. Advantages: Controlled and targeted delivery of the drug Reduced systemic side effects Facilitated extravasation into the tumor cells (EPR effect: enhanced permeability and retention) High capability to cross various physiological barriers,Omic C.Farokhzad, and Robert Langer. ACS Nano, 2009,3:16,1.1 Chitosan NP for cancer therapy,Fig.3 Size and zeta potential of CNP and CNP-Cu,50 mV,96 mV,CNP-Cu,13 mV,Chitosan,CNP,CNP-Cu,L. Qi, et al, Colloids Surfaces A: Physicochem Eng Aspects 2004; 251: 183-190 ; Carbohydrate Research 2004; 2693-2700,CNP,Values are means of three experiments, standard deviation is given inparentheses (na = not active).,Table 1. Cytotoxic activity of chitosan, CNP (mean particle size = 40 nm), and CNP-Cu against different cell lines,Antitumor activity of CNP,L. Qi, et al, Bioorganic 15: 1357-1399,Fig.5 CNP-induced changes of MMP and zeta potential of membrane. A: Loss of MMP and zeta potential; Histogram of untreated cells (B) and cells treated with 100 g/mL CNP (C),CNP could neutralize the negative surface charge of BEL7402 cells so as to damage the cell membrane. The strong dissipation in MMP suggests a possible disruption of mitochondrial membrane after treated with CNP.,Loss of zeta potential and mitochondrial membrane potential (MMP) induced by CNP,L. Qi, et al, Eur. J. Cancer 2007; 43:184-93,aP0.01 vs Control,Table 2 Effects of CNP on fatty acid composition of membrane phosphoric lipid from BEL7402 cells,Fig. 6 Lipid peroxidation measured as thiobarbituric acid reactive substances (TBARs) production of BEL7402 cells non-treated or treated for 24 h with various doses of CNP.,Interaction with lipid bilayer of tumor cells,Increased ROS (Reactive oxygen species) production and LPO degree, decreased unsaturated fatty acid indicated that CNP exerted membrane penetration activity by induction of LPO,L. Qi, et al, Eur. J. Cancer 2007; 43:184-93,In vivo antitumor activity of CNP,Table 4 Effect of CNP with different particle size on BEL7402 tumor cell growth in nude mice by oral administration (1 mg/kg/day),Fig. 7 Effects of administration routes on antitumor efficacy of CNP against Sarcoma-180 subcutaneous tumor formation and growth in ICR mice,L. Qi, et al, , Bioorganic European Journal of Cancer, 2007.,Side effects of chemotherapy,Part II: 肿瘤纳米分子影像与实时治疗,Imaging: cancer diagnostics, staging, radiation planning, and evaluation of therapy. Standard clinical imaging modalities: CT, MRI, ultrasound, limitation in detection ( 0.5 cm), and distinguishing between benign and cancerous tumors. Molecular imaging: Multifunctional nanoparticles: MNP, QDs, gold NP, drug delivery (siRNA, DNA, drug), nanothermotherapy, photodynamic therapy.,What is Qdots,Size-tunable light emission Photostable Simultaneous excitation of QDs Large Stokes shift,Wu et al.Nat. Biotechnol. 21 416；Qi &Gao, ACS nano, 2008,2.1 Quantum dots for drug delivery and therapy,Qi and Gao, Expert Opin. Drug Deliv. 2008; 5:263-267,Figure 13. Schematic illustration of a multifunctional quantum dot coated with amphiphilic polymer.,Proton sponge coated QD for siRNA delivery and realtime imaging,Yezhelyev, Qi, ORegan, Nie, Gao. JACS, 2008,Fig. 14. Schematic diagram showing the steps of siRNA-QD in membrane binding, cellular entry, endosomal escape, capturing by RNA binding proteins, loading to RNA-induced silencing complexes (RISC), and target degradation.,Quantum Dot-Amphipol Nanocomplex for siRNA delivery,Fig. 16. Schematic drawing of the hybrid structure of QD and amphipol for siRNA delivery and real-time imaging in live cells.,Qi and Gao, ACS nano, 2008, 2(7), 14031410, 2008,Gel loading efficiency and protection against nuclease,Fig. 17 QD loading capacity and protection of siRNA molecules against nuclease degradation determined by gel electrophoresis.,Qi and Gao, ACS nano, 2008, 2(7), 14031410,Siliencing efficiency and cytotoxicity,Fig. 18 Gene silencing efficiency of siRNA targeting Her-2 using QDPMAL compared with the classic transfection agents, Lipofectamine and PEI.,Qi and Gao, ACS nano, 2008, 2(7), 14031410,纳米分子造影剂产品,1）3TP的癌症诊断技术，基于MRI的诊断乳腺癌、前列腺癌，区分恶性/良性，5mm瘤块。 2）主要以钆/氧化铁为组分核磁纳米造影剂 3）Palatin技术公司2007年获批准新型造影剂neutrospec，neutrospec含有一个以锝为标记的抗cd15单克隆抗体，可以选择性的结合于参与免疫应答的嗜中性粒细胞。neutrospec被注射入血后，可与感染部位存在的嗜中性粒细胞相结合，使这些细胞被放射性示踪剂所标记。,Part III: 纳米探针,Fig.22 Modulating signaling pathways and probing biological interactions with nanotopography,Oligo,3.1 Interaction of Nanotopography with Cells,Fig. 25. Cytotoxicity of CNC determined by detection of LDH release from BJ-5a cells cocultured with extracts of CNC for 72 h.,Fig. 26Actin localization in spreading cells with time,Control,CNC,Chitosan,3 h,3 d,11 d,L. Qi, et al, J. Biomed. Mater. Res. A. 2007 Dec. 18,3.2 纳米纤维转染DNA,Fig. 27. Human embryonic kidney cell line (HEK 293T) cultured on SiNWs (c) with and (d) without PEI treatment prior to GFP plasmid deposition. Yang et. al. J. AM. CHEM. SOC. 2007, 129, 7228-7229,3.3 靶分子监测纳米探针,Fig. 28. Well-dispersed antibody-nanoshell conjugates in the absence of analyte possess a well-defined extinction peak in the near-IR. In the presence of the complementary analyte, multiple nanoshells bind to the analyte, causing agglutination and acorresponding reduction in the extinction peak.,Methods in Molecular Biology 303NANOBIOTECHNOLOGYPROTOCOLS,Fig. 29. Signal detection of oligonucleotide-derivatized NBCs hybridized with complementary Cy5 oligonucleotide.,3.3 纳米金荧光检测核苷酸序列,Methods in Molecular Biology 303NANOBIOTECHNOLOGYPROTOCOLS,3.4 硅纳米纤维用于DNA监测,Fig. 30. Flow chamber setup for hybridization and visualization of fluorescence signals on the nanoarray. Optical microscope image of an optical fiber-bundle based nanoarray containing individual 300 nmdiameter fibers; (ii) magnified SEM image of a region of the array containing nanobeads; (iii) fluorescent nanobeads in the n