NANOPARTICLES AS THERAGNOSTIC
AGENTS:APPLICATIONS IN CANCER NANOTECHNOLOGY（
和訳：癌診断・治療剤としての多機能・生体整合磁 性ナノ粒子の開発：癌ナノテクノロジーへの応用）
著者 SIVAKUMAR BALASUBRAMANIAN
学位授与大学 東洋大学
取得学位 博士
学位の分野 生命科学
報告番号 32663甲第350号
学位授与年月日 2013‑09‑25
URL http://id.nii.ac.jp/1060/00006458/
Creative Commons : 表示 ‑ 非営利 ‑ 改変禁止 http://creativecommons.org/licenses/by‑nc‑nd/3.0/deed.ja
氏 名（ 本 籍 地 ） 学 位 の 種 類 報 告・ 学 位 記 番 号 学 位 記 授 与 の 日 付 学 位 記 授 与 の 要 件 学 位 論 文 題 目
論 文 審 査 委 員
SIVAKUMARBALASUBRAMANIAN（インド）
博士（生命科学）
甲第350号（甲生第29号）
平成25年９月25日
本学学位規則第３条第１項該当
MULTIFUNCTIONALBIOCOMPATIBLEMAGNETIC NANOPARTICLESASTHERAGNOSTICAGENTS:
APPLICATIONSINCANCERNANOTECHNOLOGY
（和訳：癌診断・治療剤としての多機能・生体整合磁性ナノ 粒子の開発：癌ナノテクノロジーへの応用）
主査 教授 Ph.D. D.SakthiKumar
副査 教授 工学博士 前 川 透
副査 教授 博士（工学） 花 尻 達 郎
副査 准教授 博士（工学） 森 本 久 雄
副査 特任准教授 博士（工学） 水 木 徹
【論文審査】Review of the thesis
Magneticnanoparticleshavegarneredalotofattentioninbiomedicalandindustrialappli- cationsowingtotheirbiocompatibility,easeofbiofunctionalizationanditsinherentmag- netic properties. The development of multifunctional magnetic nanoparticles with biocompatibilitycanenhancetheinteractionandcanbeexploitedforbothactiveandpas- siveimaging,drugdeliveryandmagnetichyperthermiaforthedestructionofcancercells.
ThethesisentitledsubmittedbyMr.B.Sivakumar“Multifunctionalbiocompatiblemagnetic nanoparticles as theragnostic agents: applications in cancer nanotechnology” has been dividedinto9chapters,whichmainlydealswiththedevelopmentofmultifunctionalmag- neticnanoparticlesexploitingthenaturallyoccurringbiopolymersforin vitrocellularimag- ing,efficientdrugdeliveryandhyperthermiastudiesincancercells.
Inthe1stchapterofthethesis,Mr.Sivaprovidedanintroductiontomultifunctionalmag- netic nanoparticles and their theragnostics applications for simultaneous diagnosis and therapeutics(deliveringtargeteddrugstocancerspecificcells).Thedevelopmentofmag- neticnanoparticles,thedesignofmultifunctionalmagneticnanoparticles,factorsrendering multifunctionalityandsomeofthemajorbiomedicalapplicationswithspecialreferenceto drug targeting (passive, active and magnetic) and magnetic hyperthermia are provided in
thischapter.
Inthe2ndchapterMr.Sivasummarizedthetheoreticaldetailsoftoolsandinstrumentsthat areusedforthecharacterizationofnano-sizedstructures.
Inthe3rdchapter,Mr.SivamentionedaboutthedevelopmentofMultifunctionalcarboxym- ethylcellulose-basedmagneticnanovectorasatheragnosticsystemforfolatereceptortar- geted chemotherapy, imaging, and hyperthermia against cancer. A multifunctional biocompatible nanovector based on magnetic nanoparticle and carboxymethyl cellulose (CMC) wasdeveloped.TheresultsindicatethatthemultifunctionalCMCMNPspossessa highdrugloadingefficiencyandhighbiocompatibilityandwithlowcellcytotoxicityandcan beconsideredtobepromisingcandidatesforCMC-basedtargeteddrugdelivery,cellular imaging,andmagnetichyperthermia(MHT).
The4thchapterdealswiththedevelopmentofbacterialexopolysaccharidecoatedandsta- bilizedmagneticnanoparticles.Microbialexopolysaccharides(EPSs)arehighlyheteroge- neouspolymersproducedbyfungiandbacteriathathavegarneredconsiderableattention andhaveremarkablepotentialinvariousfields,includingbiomedicalresearch.Inthisstudy Mr.Sivahascoatedmagneticnanoparticles(MNPs)withtwobacterialEPS-mauran(MR) andgellangum(GG).ThebiocompatibilityofEPScoatedMNPswasenhancedandhepro- videdthemulti-functionalitybyattachingtargetingmoiety,incorporatingimagingmoieties and encapsulated with potent anticancerous drug. The biocompatibility of EPS coated MNPsandsynergisticeffectofmagnetichyperthermiaanddrugforenhancedantiprolifera- tion of cancer cells was evaluated. The results suggests that MR and GG coated MNPs exhibitedexcellentbiocompatibilitywithlowcellcytotoxicity,hightherapeuticpotential, andsuperparamagneticbehaviorthatcanbeemployedasprospectivecandidatesforbacte- rialEPSbasedtargeteddrugdelivery,cancercellimagingandformagnetichyperthermia (MHT)forkillingcancercellswithinshortperiodoftime.
In the 5th chapter, Mr. Siva discusses the synthesis of curcumin encapsulated PLGA- magnetic nanoparticles and their augmented cellular uptake and antiproliferation against pancreatic cancer cells induced by nanoformulation. He has developed a multifunctional nanoconjugatebasedonPLGAthathasbeenprovedtobecompletelysafebyFDAtolabel, specificallytargetandefficientlytreattumorcells.Superparamagneticironoxidenanopar- ticles(SPIONs)areusedformagneticallytargeteddrugdelivery,magnetichyperthermia andasanimagecontrastagent.Inthepresentstudy,thepotentialityofSPIONsandcur- cumin, a natural antitumor agent, encapsulated in PLGA nanoparticles (Cur-SPION- PLGA-NPs) against pancreatic cancer cells was investigated. Specificity to the
nanoconjugate was imparted by conjugating HER2 to Cur-SPION-PLGA that confirmed enhanced uptake by the cancer cells. HER2-Cur-SPION-PLGA NPs establishes a mag- neticdrugdeliverysystemasobservedbytheenhanceduptakeofnanoparticlesbycancer cellsundertheactionofexternalmagneticfield.HER2-Cur-SPION-PLGAdemonstrated greaterandsustainedtherapeuticefficiency,however,uponexposuretoexternalmagnetic fieldtheanti-proliferativeactivityofHER2-Cur-SPION-PLGAwasaugmentedowingto superior cellular uptake that resulted in enhanced cytotoxicity. In vitro results support potentialclinicalapplicationsofHER2-Cur-SPION-PLGANPsbyactivatingthelossof mitochondrialmembranepotentialtherebytriggeringapoptosisincancercells.
Inthe6thchapter,Mr.Sivademonstratesthedevelopmentofdoubletargetedanddouble drugloadedPLGAmagneticnanoparticles.Theefficienttargetingandtherapeuticefficacy ofcombinationofdrug(curcuminand5FU)andmagneticnanoparticlesencapsulatedPLGA nanoparticles, functionalized with two targeting ligands specific towards cancer are dis- cussedinthework.Thismultifunctional,highlyspecificnanoconjugateresultedinsuperior uptakeofnanoparticlesbycancercells.Byusingmagnetichyperthermia;duetotheadded advantage of the incorporation of magnetic nanoparticles that synergistically acting with thedrugs,destroyedcancercellswithinaveryshortperiodoftime.Theremarkablemulti- modalefficacyattainedbythistherapeuticnanoformulationoffersthepotentialfortarget- ing,imagingandtreatmentofthecancerwithinashortperiodoftimebyinitiatingearly andlateapoptosis.
Inthe7thChapterthedevelopmentofaptamertargetedtheragnosticmultifunctionalmag- neticnanoparticlesasnanoplatformforpancreaticcancertherapyhasbeendiscussed.The developmentofdesignermultifunctionaltherapeuticswithmagneticnanoparticles(MNP)is a promising approach to selectively deliver therapeutics and image them in cancer cells.
Thisstudyexploredthepreparation,characterizationandapplicationofatargetednano- system represented by aptamer conjugated dual drug (Curcumin and Gemcitabine-loaded) andMNPencapsulatedPLGAnanoparticles(Apt-Cur-Gem-PLGA-MNPs)forpancreatic cancer therapy. The aptamer targeted NPs exhibited excellent antiproliferative activity leadingtoapoptosisowingtoenhancedcellularuptake.ThepresenceofMNPinnanoconju- gate was exploited for magnetic hyperthermia and magnetic ablation of cancer cells by rotationalmagneticfield.Thus,Apt-Cur-Gem-PLGA-MNPsisabletodeliveraneffectual andtargeteddeliveryoftherapeuticsforpancreaticcancertreatment.
The8thchapterdiscussesthedevelopmentofmultifunctionalhybridmagneticnanoparticles as theragnostic agents. Gold-coated magnetic nanoparticles was developed and hybrid
nanoparticlewasincorporateintoPLGAnanoparticlesalongwithtwodrugs(curcuminand gemcitabine).Thenanoconjugatewasmadespecifictopancreaticandbreastcancercellsby attaching three specific targeting ligands (folate, transferrin and AS1411 aptamer). He investigated the three different modes of cancer cell destruction with the nanoconjugate.
Thusan‘allinone’approachforsimultaneouschemotherapeutic,photothermalandmag- netichyperthermiamediateddestructionofcancercellswasinvestigated.
Theconcludingremarksofthethesisarediscussedinthefinal9thchapter.
【審査結果】 Summary and decision
The thesis entitled“Multifunctional biocompatible magnetic nanoparticles as theragnostic agents: applications in cancer nanotechnology”focuses on the synthesis of theragnostic agents(TherapyandDiagnosis)bycouplingmagneticnanoparticles,drugsandimagingmoi- eties. Aptamer and folate targeting ligands have been used to direct these theragnostic agentstocancercells.Thistargetingwashighlysuccessfulinsparingthenormalhealthy cells.Theresultsshowninthethesisareoutstandingininternationalpointofviewandthe salientfeaturesofthisworkaresummarizedbelow.
• A multifunctional biocompatible nanovector based on magnetic nanoparticle and car- boxymethylcellulose(CMC)hasbeendemonstrated.Theresultsindicatethatthemulti- functionalCMCMNPspossessahighdrugloadingefficiencyandhighbiocompatibility and with low cell cytotoxicity and can be considered to be promising candidates for CMC-basedtargeteddrugdelivery,cellularimaging,andmagnetichyperthermia(MHT).
• MauranandGellangumcoatedMNPsexhibitedexcellentbiocompatibilitywithlowcell cytotoxicity, high therapeutic potential, and superparamagnetic behavior that can be employed as prospective candidates for bacterial EPS based targeted drug delivery, cancercellimagingandformagnetichyperthermia(MHT)forkillingcancercellswithin shortperiodoftime.
• HER2-Cur-SPION-PLGA demonstrated greater and sustained therapeutic efficiency, however, upon exposure to external magnetic field; the anti-proliferative activity of HER2-Cur-SPION-PLGA was augmented owing to superior cellular uptake that resultedinenhancedcytotoxicity.Invitroresultssupportpotentialclinicalapplications of HER2-Cur-SPION-PLGA NPs by activating the loss of mitochondrial membrane potentialtherebytriggeringapoptosisincancercells.
• Aptamer conjugated dual drug (Curcumin and Gemcitabine-loaded) and MNP
encapsulated PLGA nanoparticles (Apt-Cur-Gem-PLGA-MNPs) used for pancreatic cancertherapy.TheaptamertargetedNPsexhibitedexcellentantiproliferativeactivity leadingtoapoptosisowingtoenhancedcellularuptake.ThepresenceofMNPinnano- conjugatewasexploitedformagnetichyperthermiaandmagneticablationofcancercells by rotational magnetic field. Thus, Apt-Cur-Gem-PLGA-MNPs is able to deliver an effectualandtargeteddeliveryoftherapeuticsforpancreaticcancertreatment.
Based on the work, 2 first-authoring and 1 co-authoring papers have been published in internationaljournalssuchasLangmuirandInternational Journal of Nanomedicine.
Judgingbytheresultsshowninthethesisandthenumberofinternationalpaperspublished sofar,thelevelofthepresentresearchresultsisdefinitelyhighbyinternationalstandards andthepresentresultsmaywellmakeagreatcontributiontothenanodrugdeliveryagainst cancer.Inconclusion,thethesisisconsideredasahighquality,highstandardonebyinter- nationalstandards.
