納米生物學及其應用ppt課件

1、亓立峰亓立峰 浙江加州國際納米技術(shù)研究院浙江加州國際納米技術(shù)研究院12022-1-28v前言：納米微粒概述vPart I: 納米藥物與靶向設(shè)計vPart II: 腫瘤納米分子影像與實時治療vPart III: 納米探針v前景展望22022-1-28What is Nano?Quantum dots, magnetic NPPolymer NPFig1. A schemetic of nanoscale materialPhycoerythrin前言32022-1-28某種物質(zhì)或元素納米級別粒子: nano Zinic; SiO2， nano gold, CdSe/ZnS, Fe2O3,Poly

2、mer NPDegradablenanospherenanocapsulePolymeric matrixPolymeric membraneOily or aqueous core前言42022-1-28化學法： (1)沉淀法/透析法把沉淀劑加入到鹽溶液中反應后，（將沉淀熱處理）得到納米材料。其特點簡單易行，但純度低，顆粒半徑大，聚合體納米粒子、氧化物。(2) 微乳液法/乳液擴散法兩種互不相溶的溶劑在表面活性劑的作用下形成乳液，在微泡中經(jīng)成核、聚結(jié)、團聚、（熱處理）后得納米粒子。其特點粒子的單分散和界面性好，聚合體納米粒子、族半導體納米粒子多用此法制備納米微粒制備方法前言52022-1-28

3、納米粒子表征方法測 量 方 法測 量 功 能適用的尺寸范圍使用的主要儀器離心沉降法等效直徑25nm高速離心機，分光光度計或暗場法光學系統(tǒng)氣體吸附法（容量法或重量法）比表面積尺寸：約110nm比表面積：0.11000m2/gBET吸附裝置或重量法裝置光散射法平均直徑約3nm喇曼光譜儀X射線衍射峰寬法晶粒平均尺寸約500nm常用于50nmX射線衍射儀小角度X射線散射線晶粒平均尺寸約2nm透射電子顯微鏡掃描隧道顯微鏡法（STM）形貌與尺寸寬范圍掃描隧道顯微鏡粒徑分析粒徑分布 粒徑分析儀熒光光譜法熒光圖譜 熒光光譜儀62022-1-28These newly formed tumor vessels

4、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 lympha

5、tic 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_P

6、ermeability_and_Retention_effectEnhanced 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

7、 Journal. 2007;9(2):Article 15.DOI: 10.1208/aapsj09020157v Nanoparticles : clinical administration of anticancer drugs. v Advantages: Controlled and targeted delivery of the drug Reduced systemic side effects Facilitated extravasation into the tumor cells (EPR effect: enhanced permeability and reten

8、tion) High capability to cross various physiological barriersOmic C.Farokhzad, and Robert Langer. ACS Nano, 2009,3:1682022-1-28 74.94nm 0.001.00 um2.00 x 2.00 um020000.40000.60000.80000.1.e+51.2e+5-100.0100.200.Intensity (kcps)Zeta Potential (mV)Zeta Potential Distribution01.e+52.e+53.e+54.e+5100.In

9、tensity (kcps)Zeta Potential (mV)Zeta Potential DistributionFig.3 Size and zeta potential of CNP and CNP-Cu50 mV96 mVCNP-Cu050000.1.e+51.5e+52.e+52.5e+5-200.-100.0100.200.Intensity (kcps)Zeta Potential (mV)Zeta Potential Distribution13 mVChitosanCNPCNP-CuL. Qi, et al, Colloids Surfaces A: Physicoche

10、m Eng Aspects 2004; 251: 183-190 ; Carbohydrate Research 2004; 2693-2700CNP92022-1-28Panel of cell lineCell line Cytotoxicity (IC50,g/mL) ChitosanCNPCNP-CuLiverL-02 na na na Colon cancer Calo320 1200( (27) 28(3)3) 14(2)2) Gastric cancerBGC823 1200(35)35) 21(2)2) 8 (1)1) Liver cancerBEL7402 1200(2222

11、) 15(2)2) 6 (1)1) 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 linesL. Qi, et al, Bioorganic & Medicinal Chemistry Letters 2005; 15:

12、1357-1399 102022-1-2802468101201234Time (h) potential (-mV)051015202530354045%Cells with loss of MMPZeta potentialMMPRh123+M1:7.30%Rh123+M1:74.34%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 wit

13、h 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.L. Qi, et al, Eur. J. Cancer 2007; 43:184-93112022-1-28(g/mg) (g/mg) a

14、aP0.01 vs Control 00.811.21.40255075100CNP (g/mL)TBARs (nmol/10 6 cells)Table 2 Effects of CNP on fatty acid composition of membrane phosphoric lipid from BEL7402 cellsFig. 6 Lipid peroxidation measured as thiobarbituric acid reactive substances (TBARs) production of BEL7402 cells non-treat

15、ed or treated for 24 h with various doses of CNP.Increased ROS (Reactive oxygen species) production and LPO degree, decreased unsaturated fatty acid indicated that CNP exerted membrane penetration activity by induction of LPOL. Qi, et al, Eur. J. Cancer 2007; 43:184-93122022-1-28Table 4 Effect of CN

16、P 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 miceL. Qi, et al, , Bioorganic & Medicinal Chemistr

17、y Letters 2006; European Journal of Cancer, 2007.Side effects of chemotherapy132022-1-28 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 benig

18、n and cancerous tumors. Molecular imaging: Multifunctional nanoparticles: MNP, QDs, gold NP, drug delivery (siRNA, DNA, drug), nanothermotherapy, photodynamic therapy.142022-1-28 Size-tunable light emission Photostable Simultaneous excitation of QDs Large Stokes shiftWu et al.Nat. Biotechnol. 21 416

19、；Qi &Gao, ACS nano, 2008152022-1-28Qi and Gao, Expert Opin. Drug Deliv. 2008; 5:263-267Figure 13. Schematic illustration of a multifunctional quantum dot coated with amphiphilic polymer.162022-1-28Yezhelyev, Qi, ORegan, Nie, Gao. JACS, 2008Fig. 14. Schematic diagram showing the steps of siRNA-QD

20、 in membrane binding, cellular entry, endosomal escape, capturing by RNA binding proteins, loading to RNA-induced silencing complexes (RISC), and target degradation.172022-1-28Fig. 16. Schematic drawing of the hybrid structure of QD and amphipol for siRNA delivery and real-time imaging in live cells

21、. Qi and Gao, ACS nano, 2008, 2(7), 14031410, 2008182022-1-28Fig. 17 QD loading capacity and protection of siRNA moleculesagainst nuclease degradation determined by gel electrophoresis.Qi and Gao, ACS nano, 2008, 2(7), 14031410192022-1-28Fig. 18 Gene silencing efficiency of siRNA targeting Her-2 usi

22、ng QDPMAL compared with the classic transfection agents, Lipofectamine and PEI.Qi and Gao, ACS nano, 2008, 2(7), 14031410202022-1-28納米分子造影劑產(chǎn)品1）3TP的癌癥診斷技術(shù)，基于MRI的診斷乳腺癌、前列腺癌，區(qū)分惡性/良性，5mm瘤塊。2）主要以釓/氧化鐵為組分核磁納米造影劑3）Palatin技術(shù)公司2007年獲批準新型造影劑neutrospec，neutrospec含有一個以锝為標記的抗cd15單克隆抗體，可以選擇性的結(jié)合于參與免疫應答的嗜中性粒細胞。neut

23、rospec被注射入血后，可與感染部位存在的嗜中性粒細胞相結(jié)合，使這些細胞被放射性示蹤劑所標記。212022-1-28Part III: 納米探針Fig.22 Modulating signaling pathways and probing biological interactions with nanotopographyOligo222022-1-2801020304050abcdeCSContrCNCCytotoxicity%* * * * * * * Fig. 25. Cytotoxicity of CNC determined by detection of LDH releas

24、e from BJ-5a cells cocultured with extracts of CNC for 72 h. Fig. 26Actin localization in spreading cells with timeControlCNCChitosan3 h3 d11 dL. Qi, et al, J. Biomed. Mater. Res. A. 2007 Dec. 18232022-1-283.2 納米纖維轉(zhuǎn)染納米纖維轉(zhuǎn)染DNAFig. 27. Human embryonic kidney cell line (HEK 293T) cultured on SiNWs (c)

25、with and (d) without PEI treatment prior to GFP plasmid deposition. Yang et. al. J. AM. CHEM. SOC. 2007, 129, 7228-7229242022-1-283.3 靶分子監(jiān)測納米探針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

26、 complementary analyte, multiple nanoshells bind to the analyte, causing agglutination and acorresponding reduction in the extinction peak.Methods in Molecular Biology 303NANOBIOTECHNOLOGYPROTOCOLS252022-1-28Fig. 29. Signal detection of oligonucleotide-derivatized NBCs hybridized with complementary

27、Cy5 oligonucleotide.3.3 納米金熒光檢測核苷酸序列Methods in Molecular Biology 303NANOBIOTECHNOLOGYPROTOCOLS262022-1-28Fig. 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) m