研究背景與目標
腦癌可說是現今棘手的癌症，其難以早期診斷以及治療的困難造成了極低的存活率與極高的治療副作用，而近期腦癌基因治療相關的可能性也被許多研究提出。有效的基因治療取決於如何有效的遞送干擾 RNA 進入細胞，目前常見利用奈米載體或者腺相關病毒載體 (adeno-associated viral vector, AAV或者JC-virus) 輸送系統來增強轉染效率，其最大缺點為製備成本過高及程序繁雜。本研究之主要目的即為利用類病毒奈米粒子為載體，改善上述於載體或者治療手段中的弱勢，開發具有多功能的RNAi載體平台。並透過降低細胞遷移活性以及抗藥性證明其功能。
研究方法
本研究提出利用基因工程技術一步合成同時含有螢光蛋白及干擾 RNA 之VLPs (Virus-like particles, VLPs)，此VLPs不僅具螢光追蹤功能且可降低腦癌細胞 c-MET的表現而抑制癌細胞生長遷徙並同時可增強 Temozolomide (TMZ) 對癌細胞毒殺反應性。經由進一步於表面修飾 transactivator of transcription (TAT) 以及 Apolipoprotein E (ApoE) 之功能多肽能夠提升細胞吞噬與穿越血腦屏障的能力，此輸送系統不僅可利用 E. coli. 快速、方便、大量生產且不具細胞毒性，大幅降低基因藥物製造成本。
研究結果
細胞實驗證實本研究採用之系統能有效降低c-MET表現量並抑制U87癌細胞株遷徙生長，對於TMZ之抗藥性也得以降低。動物實驗中藉由靜脈注射修飾有ApoE peptide與包覆紅色mCherry螢光蛋白的VLPs可於鼠腦部發現來自VLPs的紅色螢光，證明修飾擴充具有通過血腦屏障的能力。綜合以上結果，此自組裝且易於擴充之VLPs載體系統可望減低治療成本、減少抗藥性及提升輸送效率，作為具前瞻性的多功能RNAi治療平台。

Introducion
Brain tumour is one of the most lethal cancers due to difficulties of delivering therapeutic agents across blood-brain barrier (BBB) and serious side effect. Here, we describe a virus-like particles (VLPs) platform as multifunctional nanocarriers to deliver RNA molecules and fluorescence proteins for RNA interference and imaging tracking.

Methods
We have designed an RNA scaffold and co-expressed with Qβ coat protein and green fluorescent protein (GFP) or mCherry protein simultaneously through a two-plasmid system. Coat proteins and cargos will be produced and self assembled in E. coli. The TAT and ApoE peptide, were then incorporated to the exterior surface of VLPs via a linker (sulfo-SMCC) to enhance cell uptake and promote BBB penetration.

Results
The functional VLPs have been assessed by cell examination. VLPs do not show cytotoxicity and can be internalized insides cells. VLPs containing RNA molecules and GFP modulate gene expression and serve as fluorescent trackers within U87 cells. VLPs containing RNAi scaffold for c-MET silencing and GFP (c-MET@t-gVLPs) successfully down regulated the c-MET protein expression level of U87 cell. c-MET@t-gVLPs treated cells also showed lower Temozolomide (TMZ) drug resistance and limited cell migration.
ApoE conjugated VLPs have the ability to cross the BBB in vivo. Current findings showed that our VLPs could be a good RNAi delivery plateform for gene down-regulation therapy.
Conclusion
We believe this multifunctional VLP platform has potential to overcome impediments as mentioned earlier and well suited for RNAi-based therapeutic tools for brain tumour therapy.

目錄
審定書 i
誌謝 ii
中文摘要 iii
Abstract iv
目錄 vi
圖次 ix
表次 x
第一章、 緒論 1
1.1. 腦癌 1
1.1.1. 概況 1
1.1.2. 腦癌分類 2
(1) 依細胞型態分類 2
(2) 依來源分類 3
1.1.3. 腦癌治療 6
(1) 手術 6
(2) 放射治療 6
(3) 化學治療 7
(4) 其他療法 7
(5) 治療面臨困境 8
1.1.4. Temozolomide 抗藥性與c-MET 10
1.2. RNA干擾治療 15
1.2.1. RNA干擾的發現 15
1.2.2. RNA干擾原理 15
1.2.3. RNAi治療因子輸送途徑 18
1.3. 類病毒奈米粒子 (Virus-Like Particles, VLPs) 21
1.3.1. VLPs簡介 21
1.3.2. VLPs製作方法 22
1.3.3. VLPs現今應用範圍 23
1.3.4. 功能性多肽 25
(1) TAT peptide 25
(2) ApoE peptide 26
1.4. 研究動機與目的 28
第二章、 材料與實驗方法 29
2.1. 實驗材料 29
2.1.1. 藥品與耗材 29
(1) 質體、核酸與酶 29
(2) 化學藥品與實驗套裝 30
(3) 耗材用品 33
2.1.2. 細胞實驗 33
2.2. 實驗儀器 34
2.3. 研究方法 36
2.3.1. VLPs製作 36
(1) RNA干擾骨架設計 36
(2) 質體合成與轉殖 37
(3) 蛋白表現與粹取純化 39
(4) 多肽修飾 40
2.3.2. VLPs鑑定分析 41
(1) 粒徑、表面電位分析 41
(2) SDS page 蛋白質電泳分析 41
(3) 穿透式電子顯微鏡影像 42
(4) VLPs細胞毒性 42
(5) 細胞吞噬螢光影像 43
(6) 流式細胞儀 43
2.3.3. RNAi抑制能力評估與效果 44
(1) 西方墨點法 44
(2) 細胞遷移刮痕測試 45
(3) c-MET RNAi 與TMZ共同作用治療實驗 46
2.3.4. ApoE-VLPs 血腦屏障穿越實驗 46
第三章、 結果與討論 47
3.1. VLPs製作與鑑定 47
3.1.1. 基礎性質分析 47
3.1.2. VLPs表面修飾功能擴充 51
3.2. 細胞實驗 52
3.2.1. VLPs基本細胞毒性 52
3.2.2. 細胞吞噬能力比較 54
3.2.3. 細胞c-MET蛋白抑制之RNAi效果 59
(1) 西方墨點法蛋白表現抑制分析 59
(2) 刮痕癒合實驗c-MET抑制對細胞遷移能力影響 61
3.2.4. 細胞c-MET抑制與TMZ藥物抗藥性之交互影響 66
3.2.5. 修飾ApoE peptide之VLPs通過血腦屏障 70
3.3. 討論 71
第四章、 結論 80
參考文獻 81

 
圖次
圖1 1：衛福部國人十大死因統計圖。 1
圖1 2：血腦屏障 (BBB) 示意圖。 9
圖1 3：腦癌化療藥物TMZ之運作機構簡圖。 11
圖1 4：c-MET蛋白結構圖。 12
圖1 5：腦癌細胞內皮間質轉化示意圖。 13
圖1 6：RNAi作用機制圖。 17
圖1 7：本研究採用之Qβ噬菌體殼蛋白外型3D模擬圖。 22
圖1 8：TAT peptide 修飾之粒子於細胞吞噬作用之共軛交顯微攝影。 26
圖1 9：修飾Apo 之HSA奈米粒子通過SV 129鼠腦血腦屏障之TEM攝影。 27
圖2 1：RNAi干擾股架設計。 36
圖2 2：質體載體設計圖。 37
圖2 3：VLPs製作流程圖。 40
圖3 1：螢光VLPs (fVLPs) 螢光影像與TEM電子顯微影像。 49
圖3 2：VLPs DLS粒徑分析以及SDS page膠體電泳。 50
圖3 3：VLPs細胞毒性之XTT測試結果。 53
圖3 4：細胞吞噬螢光顯微鏡影像。 55
圖3 5：流式細胞術結果資料。 58
圖3 6：c-MET@t-gVLPs之功能初探西方墨點法影像。 60
圖3 7：U87細胞作用48小時之西方墨點詳細資料。 61
圖3 8：U87細胞刮痕癒合實驗。 63
圖3 9：刮痕癒合實驗之再現實驗。 65
圖3 10：U87細胞RNAi與TMZ共同作用之抗藥性評估XTT實驗結果。 67
圖3 11：不同TMZ濃度對不同材料施加時間點之細胞存活率作圖。 69
圖3 12：a-rVLPs穿越血腦屏障實驗。 70

表次
表1 1：常見原發性腦腫瘤分類。 5
表3 1：不同合成階段VLPs之DLS粒徑數據資料。 50
表3 2：流式細胞術訊號中具有GFP訊號之細胞比例。 56
表3 3：刮痕癒合實驗殘餘刮痕面積完整定量數據。 64

1. Ahmed J. Awad,Terry C. Burns,Ying Zhang et al. Targeting MET for glioma therapy. Neurosurgical Focus 37, E10, doi:10.3171/2014.9.focus14520 (2014).
2. 國家衛生研究院 癌症研究組 T. 腦癌之診斷與治療共識. (中華民國國家衛生研究院, 2004).
3. Wei K-C,Chu P-C,Wang H-Y J et al. Focused Ultrasound-Induced Blood–Brain Barrier Opening to Enhance Temozolomide Delivery for Glioblastoma Treatment: A Preclinical Study. PloS one 8, e58995, doi:10.1371/journal.pone.0058995 (2013).
4. Lalatsa A, Schatzlein A G & Uchegbu I F. Strategies to deliver peptide drugs to the brain. Mol Pharm 11, 1081-1093, doi:10.1021/mp400680d (2014).
5. Li M Y,Yang P,Liu Y W et al. Low c-Met expression levels are prognostic for and predict the benefits of temozolomide chemotherapy in malignant gliomas. Sci Rep 6, 21141, doi:10.1038/srep21141 (2016).
6. Huang M G,Liu T R,Ma P H et al. c-Met-mediated endothelial plasticity drives aberrant vascularization and chemoresistance in glioblastoma. J Clin Invest 126, 1801-1814, doi:10.1172/Jci84876 (2016).
7. Huang M,Liu T,Ma P et al. c-Met–mediated endothelial plasticity drives aberrant vascularization and chemoresistance in glioblastoma. The Journal of Clinical Investigation 126, 1801-1814, doi:10.1172/JCI84876 (2016).
8. Organ S L & Tsao M-S. An overview of the c-MET signaling pathway. Therapeutic Advances in Medical Oncology 3, S7-S19, doi:10.1177/1758834011422556 (2011).
9. Rai R,Banerjee M,Wong D H et al. Temozolomide analogs with improved brain/plasma ratios – Exploring the possibility of enhancing the therapeutic index of temozolomide. Bioorganic & Medicinal Chemistry Letters 26, 5103-5109, doi:https://doi.org/10.1016/j.bmcl.2016.08.064 (2016).
10. Donson A M,Addo-Yobo S O,Handler M H et al. MGMT promoter methylation correlates with survival benefit and sensitivity to temozolomide in pediatric glioblastoma. Pediatric Blood & Cancer 48, 403-407, doi:10.1002/pbc.20803 (2007).
11. Hegi M E,Diserens A-C,Gorlia T et al. MGMT Gene Silencing and Benefit from Temozolomide in Glioblastoma. New England Journal of Medicine 352, 997-1003, doi:doi:10.1056/NEJMoa043331 (2005).
12. Riganti C,Salaroglio I C,Pinzon-Daza M L et al. Temozolomide down-regulates P-glycoprotein in human blood-brain barrier cells by disrupting Wnt3 signaling. Cellular and molecular life sciences : CMLS 71, 499-516, doi:10.1007/s00018-013-1397-y (2014).
13. Ramirez Y P,Weatherbee J L,Wheelhouse R T et al. Glioblastoma Multiforme Therapy and Mechanisms of Resistance. Pharmaceuticals 6, 1475-1506, doi:10.3390/ph6121475 (2013).
14. Zhang J, Stevens M F & Bradshaw T D. Temozolomide: mechanisms of action, repair and resistance. Current molecular pharmacology 5, 102-114 (2012).
15. Bolós V,Gasent J M,López-Tarruella S et al. The dual kinase complex FAK-Src as a promising therapeutic target in cancer. OncoTargets and therapy 3, 83-97 (2010).
16. Iwadate Y. Epithelial-mesenchymal transition in glioblastoma progression. Oncol Lett 11, 1615-1620, doi:10.3892/ol.2016.4113 (2016).
17. Koochekpour S,Jeffers M,Rulong S et al. Met and hepatocyte growth factor/scatter factor expression in human gliomas. Cancer research 57, 5391-5398 (1997).
18. Joo K M,Jin J,Kim E et al. MET signaling regulates glioblastoma stem cells. Cancer research 72, 3828-3838, doi:10.1158/0008-5472.can-11-3760 (2012).
19. Li Y,Li A,Glas M et al. c-Met signaling induces a reprogramming network and supports the glioblastoma stem-like phenotype. Proceedings of the National Academy of Sciences of the United States of America 108, 9951-9956, doi:10.1073/pnas.1016912108 (2011).
20. Kohsaka S,Wang L,Yachi K et al. STAT3 Inhibition Overcomes Temozolomide Resistance in Glioblastoma by Downregulating MGMT Expression. Molecular Cancer Therapeutics 11, 1289-1299, doi:10.1158/1535-7163.mct-11-0801 (2012).
21. Lee E-S,Ko K-K,Joe Y A et al. Inhibition of STAT3 reverses drug resistance acquired in temozolomide-resistant human glioma cells. Oncol Lett 2, 115-121, doi:10.3892/ol.2010.210 (2011).
22. Zhang X,Liang H,Tan Y A N et al. A U87-EGFRvIII cell-specific aptamer mediates small interfering RNA delivery. Biomedical reports 2, 495-499, doi:10.3892/br.2014.276 (2014).
23. Chu S-H,Zhang H,Ma Y-B et al. c-Met Antisense Oligodeoxynucleotides as a Novel Therapeutic Agent for Glioma: In Vitro and In Vivo Studies of Uptake, Effects, and Toxicity. Journal of Surgical Research 141, 284-288, doi:http://dx.doi.org/10.1016/j.jss.2006.11.011 (2007).
24. Castanotto D & Rossi J J. The promises and pitfalls of RNA-interference-based therapeutics. Nature 457, 426-433, doi:10.1038/nature07758 (2009).
25. Fire A,Xu S Q,Montgomery M K et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811, doi:10.1038/35888 (1998).
26. Hasuwa H,Kaseda K,Einarsdottir T et al. Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Lett 532, 227-230 (2002).
27. Novobrantseva T I,Borodovsky A,Wong J et al. Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells. Mol Ther Nucleic Acids 1, e4, doi:10.1038/mtna.2011.3 (2012).
28. Davis M E,Zuckerman J E,Choi C H J et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464, 1067-U1140, doi:10.1038/nature08956 (2010).
29. Hannon G J & Rossi J J. Unlocking the potential of the human genome with RNA interference. Nature 431, 371-378 (2004).
30. Heidel J D,Liu J Y C,Yen Y et al. Potent siRNA inhibitors of ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro and in vivo. Clinical Cancer Research 13, 2207-2215, doi:10.1158/1078-0432.ccr-06-2218 (2007).
31. Burnett J C & Rossi J J. RNA-Based Therapeutics: Current Progress and Future Prospects. Chemistry & Biology 19, 60-71, doi:10.1016/j.chembiol.2011.12.008 (2012).
32. Bartel D P. MicroRNAs: Target Recognition and Regulatory Functions. Cell 136, 215-233, doi:http://dx.doi.org/10.1016/j.cell.2009.01.002 (2009).
33. Ambros V. microRNAs: Tiny Regulators with Great Potential. Cell 107, 823-826, doi:http://dx.doi.org/10.1016/S0092-8674(01)00616-X (2001).
34. Moss E G & Poethig R S. MicroRNAs: Something New Under the Sun. Current Biology 12, R688-R690, doi:http://dx.doi.org/10.1016/S0960-9822(02)01206-X (2002).
35. McNamara J O,Andrechek E R,Wang Y et al. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat Biotech 24, 1005-1015, doi:http://www.nature.com/nbt/journal/v24/n8/suppinfo/nbt1223_S1.html (2006).
36. Pan D W & Davis M E. Cationic Mucic Acid Polymer-Based siRNA Delivery Systems. Bioconjugate Chemistry 26, 1791-1803, doi:10.1021/acs.bioconjchem.5b00324 (2015).
37. Kim W J,Chang C-W,Lee M et al. Efficient siRNA delivery using water soluble lipopolymer for anti-angiogenic gene therapy. Journal of Controlled Release 118, 357-363, doi:http://dx.doi.org/10.1016/j.jconrel.2006.12.026 (2007).
38. Song W J,Du J Z,Sun T M et al. Gold nanoparticles capped with polyethyleneimine for enhanced siRNA delivery. Small 6, 239-246, doi:10.1002/smll.200901513 (2010).
39. Han J F,Cai J,Borjihan W et al. Preparation of novel curdlan nanoparticles for intracellular siRNA delivery. Carbohydrate Polymers 117, 324-330, doi:10.1016/j.carbpol.2014.09.069 (2015).
40. Destito G, Schneemann A & Manchester M. Biomedical nanotechnology using virus-based nanoparticles. Curr Top Microbiol Immunol 327, 95-122 (2009).
41. Galaway F A & Stockley P G. MS2 viruslike particles: a robust, semisynthetic targeted drug delivery platform. Mol Pharm 10, 59-68, doi:10.1021/mp3003368 (2013).
42. Fang P-Y,Gómez Ramos Lizzette M,Holguin Stefany Y et al. Functional RNAs: combined assembly and packaging in VLPs. Nucleic Acids Research, doi:10.1093/nar/gkw1154 (2016).
43. Kaczmarczyk S J,Sitaraman K,Young H A et al. Protein delivery using engineered virus-like particles. Proceedings of the National Academy of Sciences of the United States of America 108, 16998-17003, doi:10.1073/pnas.1101874108 (2011).
44. Ponchon L,Catala M,Seijo B et al. Co-expression of RNA-protein complexes in Escherichia coli and applications to RNA biology. Nucleic Acids Research 41, 13, doi:10.1093/nar/gkt576 (2013).
45. Golmohammadi R,Fridborg K,Bundule M et al. The crystal structure of bacteriophage Q beta at 3.5 A resolution. Structure (London, England : 1993) 4, 543-554 (1996).
46. Brown S D, Fiedler J D & Finn M G. Assembly of hybrid bacteriophage Qbeta virus-like particles. Biochemistry 48, 11155-11157, doi:10.1021/bi901306p (2009).
47. Aksyuk A A & Rossmann M G. Bacteriophage Assembly. Viruses 3, 172-203, doi:10.3390/v3030172 (2011).
48. Rhee J K,Hovlid M,Fiedler J D et al. Colorful virus-like particles: fluorescent protein packaging by the Qbeta capsid. Biomacromolecules 12, 3977-3981, doi:10.1021/bm200983k (2011).
49. Liu F,Ge S,Li L et al. Virus-like particles: potential veterinary vaccine immunogens. Res Vet Sci 93, 553-559, doi:10.1016/j.rvsc.2011.10.018 (2012).
50. Rhee J K,Baksh M,Nycholat C et al. Glycan-Targeted Virus-like Nanoparticles for Photodynamic Therapy. Biomacromolecules 13, 2333-2338, doi:10.1021/bm300578p (2012).
51. Patterson D P,Rynda-Apple A,Harmsen A L et al. Biomimetic antigenic nanoparticles elicit controlled protective immune response to influenza. ACS Nano 7, 3036-3044, doi:10.1021/nn4006544 (2013).
52. Brun A,Bárcena J,Blanco E et al. Current strategies for subunit and genetic viral veterinary vaccine development. Virus Research 157, 1-12, doi:http://dx.doi.org/10.1016/j.virusres.2011.02.006 (2011).
53. Aanei I L,ElSohly A M,Farkas M E et al. Biodistribution of Antibody-MS2 Viral Capsid Conjugates in Breast Cancer Models. Molecular Pharmaceutics 13, 3764-3772, doi:10.1021/acs.molpharmaceut.6b00566 (2016).
54. Ashley C E,Carnes E C,Phillips G K et al. Cell-Specific Delivery of Diverse Cargos by Bacteriophage MS2 Virus-Like Particles. ACS Nano 5, 5729-5745, doi:10.1021/nn201397z (2011).
55. Chen L S,Wang M,Ou W C et al. Efficient gene transfer using the human JC virus-like particle that inhibits human colon adenocarcinoma growth in a nude mouse model. Gene therapy 17, 1033-1041, doi:10.1038/gt.2010.50 (2010).
56. Chang L,Wang G,Jia T et al. Armored long non-coding RNA MEG3 targeting EGFR based on recombinant MS2 bacteriophage virus-like particles against hepatocellular carcinoma. Oncotarget 7, 23988-24004, doi:10.18632/oncotarget.8115 (2016).
57. Hovlid M L,Lau J L,Breitenkamp K et al. Encapsidated atom-transfer radical polymerization in Qβ virus-like nanoparticles. ACS Nano 8, 8003-8014, doi:10.1021/nn502043d (2014).
58. Anand P,O'Neil A,Lin E et al. Tailored delivery of analgesic ziconotide across a blood brain barrier model using viral nanocontainers. Sci Rep 5, 12497, doi:10.1038/srep12497 (2015).
59. Wadia J S, Stan R V & Dowdy S F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nature Medicine 10, 310-315, doi:10.1038/nm996 (2004).
60. Richard J P,Melikov K,Vives E et al. Cell-penetrating peptides - A reevaluation of the mechanism of cellular uptake. Journal of Biological Chemistry 278, 585-590, doi:10.1074/jbc.M209548200 (2003).
61. Trabulo S,Cardoso A L,Mano M et al. Cell-Penetrating Peptides—Mechanisms of Cellular Uptake and Generation of Delivery Systems. Pharmaceuticals 3, 961 (2010).
62. Debaisieux S,Rayne F,Yezid H et al. The Ins and Outs of HIV-1 Tat. Traffic 13, 355-363, doi:10.1111/j.1600-0854.2011.01286.x (2012).
63. Brooks H, Lebleu B & Vivès E. Tat peptide-mediated cellular delivery: back to basics. Advanced drug delivery reviews 57, 559-577, doi:http://dx.doi.org/10.1016/j.addr.2004.12.001 (2005).
64. Vives E, Brodin P & Lebleu B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. Journal of Biological Chemistry 272, 16010-16017, doi:10.1074/jbc.272.25.16010 (1997).
65. Banks W A, Robinson S M & Nath A. Permeability of the blood–brain barrier to HIV-1 Tat. Experimental Neurology 193, 218-227, doi:https://doi.org/10.1016/j.expneurol.2004.11.019 (2005).
66. Liu L,Guo K,Lu J et al. Biologically active core/shell nanoparticles self-assembled from cholesterol-terminated PEG–TAT for drug delivery across the blood–brain barrier. Biomaterials 29, 1509-1517, doi:http://dx.doi.org/10.1016/j.biomaterials.2007.11.014 (2008).
67. Zensi A,Begley D,Pontikis C et al. Albumin nanoparticles targeted with Apo E enter the CNS by transcytosis and are delivered to neurones. Journal of controlled release : official journal of the Controlled Release Society 137, 78-86, doi:10.1016/j.jconrel.2009.03.002 (2009).
68. Sarkar G,Curran G L,Sarkaria J N et al. Peptide carrier-mediated non-covalent delivery of unmodified cisplatin, methotrexate and other agents via intravenous route to the brain. PloS one 9, e97655, doi:10.1371/journal.pone.0097655 (2014).
69. Wagner S,Zensi A,Wien S L et al. Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model. PloS one 7, e32568, doi:10.1371/journal.pone.0032568 (2012).
70. Lim F, Spingola M & Peabody D S. The RNA-binding site of bacteriophage Qbeta coat protein. The Journal of biological chemistry 271, 31839-31845 (1996).
71. Witherell G W & Uhlenbeck O C. Specific RNA binding by Q.beta. coat protein. Biochemistry 28, 71-76, doi:10.1021/bi00427a011 (1989).
72. Roldao A,Mellado M C,Castilho L R et al. Virus-like particles in vaccine development. Expert review of vaccines 9, 1149-1176, doi:10.1586/erv.10.115 (2010).
73. Schiller J T & Lowy D R. Papillomavirus-like particle based vaccines: cervical cancer and beyond. Expert opinion on biological therapy 1, 571-581, doi:10.1517/14712598.1.4.571 (2001).
74. Wang J W & Roden R B S. Virus-like particles for the prevention of human papillomavirus-associated malignancies. Expert review of vaccines 12, 10.1586/erv.1512.1151, doi:10.1586/erv.12.151 (2013).
75. Boden D,Pusch O,Silbermann R et al. Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acids Research 32, 1154-1158, doi:10.1093/nar/gkh278 (2004).
76. Yan H,Wang L,Wang J et al. Two-order targeted brain tumor imaging by using an optical/paramagnetic nanoprobe across the blood brain barrier. ACS Nano 6, 410-420, doi:10.1021/nn203749v (2012).
77. Li J,Cai P,Shalviri A et al. A multifunctional polymeric nanotheranostic system delivers doxorubicin and imaging agents across the blood-brain barrier targeting brain metastases of breast cancer. ACS Nano 8, 9925-9940, doi:10.1021/nn501069c (2014).