[Audio] BME 695N: Engineering Nanomedical Systems (Fall 2007)

With Deborah Knapp as guest lecturer.

Outline:Introduction and overview How does the FDA think about nanomedical systems? The 2006 Nanotechnology Task Force Some details of the Nanotechnology Task Force Report General findings of the report Some initial recommendations of the Task Force Where the FDA may need to meet EPA on nanoscale materials Will FDA re-visit GRAS products containing nanomaterials?How will the FDA consider nanomedical systems? Nanomedical systems are integrated nanoscale drug and drug delivery devices Either a drug or a device? How about a "Combination Product"? Drug-Biologic combination productsEPA and other regulatory agency issues Assessing environmental impact of emerging nanotechnologies Concept of life cycle assessment (LCA) Toxicity of nanomaterials Some recommendations of the 2006 International Conference on Nanotechnology and Life Cycle AssessmentNanotechnologies and the workplace NIOSH – Formulating workplace safety standards for nanotechnology Protecting workers in the workplace Assessing hazards in the workplace Establishing a Nanotechnology Safety SystemThe future of nano-healthcare products

Outline:Overview What does cGMP mean? Why GMP? Controlling processes means more predictable outcomes… Enforcement What can be learned from the semi-conductor industry clean-room and manufacturing? What doesn’t fit this paradigm?cGMP-level manufacturing Predictable methods lead to predictable products The CFR (Code of Federal Regulations) sections on GMPs What is covered under cGMP?Bionanomanufacturing So what is special about biomanufacturing? Nano-clean water necessary for nano-pharmaceuticals Contaminants at the nano-level Can you scale up the process?Some quality control issues – how to test Correctness of size – size matters! Composition – atomic level analyses Monodispersity versus agglomeration Order and correctness of layers Correctness of zeta potentials Does the nanomedical system contain the correct payload? Targeting (and mis-targeting) specificity and sensitivity

Outline:Overview – the in-vitro to ex-vivo to in-vivo paradigm In-vitro - importance of choosing suitable cell lines Ex-vivo – adding the complexity of in-vivo background while keeping the simplicity of in-vitro In-vivo - all the complexity of ex-vivo plus the “active” components of a real animalIn-vivo systems are open, “active” systems with multiple layers of complexity In-vitro and ex-vivo are mostly “closed” systems, but not absolutely What is an “open” system? Attempts to isolate open systemsLayers of complexity of in-vivo systems Human cells in nude mice – a mixture of in-vitro and in-vivo “Model” small animal systems Bbetter model larger animal systemsExamples of the in-vitro to in-vivo experimental pathway Kopelman group – multifunctional NPs for MRI and photodynamic therapy Langer group – aptamer-targeted NPs for cancer therapy in-vivo Leary group – peptide-guided NPs to human tumors in nude mice magnetic nanoparticles as MRI contrast agents in tissue phantoms

Outline:Outline – the need for single cell measures of nanotoxicity There is more than one way for a cell to die... Necrosis" vs. "Apoptosis" There are other forms of "toxicity" Some other challenges in measuring toxicity of nanomaterialsNecrosis vs. Apoptosis mechanisms Necrosis is unplanned "cell injury" Apoptosis is planned "programmed cell death" Why it is important to distinguish between necrosis and apoptosisSingle cell assays for necrosis and apoptosis Dye exclusion assays for necrosis TUNEL assays for late apoptosis Annexin V assays for early apoptosis COMET assays for DNA damage and repair Light scatter assaysNanotoxicity in vivo – some additional challenges Single cell nanotoxicity, plus.... Accumulations of nanoparticles can change toxicity locally to tissues and organs Filtration issues of nanoparticles – size matters – toxicity to liver and lung

Outline:Introduction and overviewNanomedical treatment at the single cell level requires evaluation at the single cell levelFor evaluation purposes, does structure reveal function?The difficulty of anything but simple functional assaysThe need for assays which at least show correlation to functional activityQuantitative single cell measurements of one or more proteins per cell by flow and image/confocal cytometryCell surface measures of protein expression on live, single cellsHigh-throughput flow cytometric screening of bioactive compoundsChallenges of measuring protein expression inside fixed, single cellsWhen location is important 2D or 3D imaging is required to get spatial location of proteinsinside cellsQuantitative multiparameter phospho-specific flow cytometryAttempts to measure "functional proteins" by detecting phosphorylationExample of phospho-specific, multiparameter flow cytometryExample of measuring single cell gene silencing by phospho-specific flow cytometryQuantitative measures of gene expression – the promises and the realitiesIs gene expression at the single cell level really possible?Is it even useful to measure a single gene's changes?Gene arrays of purified cell subpopulationsRNA amplification techniques to attempt to perform single cell gene arrays

Outline:Introduction and overviewSome of the advantages of therapeutic genesSome of the advantages of molecular biosensor feedback control systemsWhy a nanodelivery approach is appropriateThe therapeutic gene approachWhat constitutes a "therapeutic gene" ?Transient versus stable expression modesMolecular feedback control systemsDrug delivery has traditionally not used feedback controlsWhy feedback control might be a very good idea!Positive or negative feedback?Molecular Biosensors as a component of a nanomedicine feedback control systemWhat is a molecular biosensor?How a molecular biosensor functions as a therapeutic gene switchBuilding integrated molecular biosensor/gene delivery systems –some examplesRequired componentsExample of a ribozyme/antivirus systemExample of an ARE biosensor/DNA repair system

Outline:Overview of drug dosing problemProblems of scaling up doses from animal systemsBasing dosing on size, area, weight of recipientVast differences between adults in terms of genetics, metabolismDosing in children – children are NOT smaller adults!Pharmacokinetics – drug distribution, metabolism, excretion, breakdownConventional dosing assumes drug goes everywhere in the bodyTargeted therapies – a model for future nanomedical systems?From the animal dosing to human clinical trialsImportance of picking an appropriate animal model systemDoes drug dosing really scale?The human guinea pig in clinical trials and beyondSome drug dosing methodsAttempts to scale up on basis of areaAttempts to scale up on weight/volumeAttempts to use control engineering principlesGenetic responses to drug dosingAll humans are not genomically equivalent!Predicting on basis of family tree responsesSNPs, chips, and beyond…predicting individual drug responseAfter the $ 1000 individual genome scan… more closely tailored individual therapiesDosing in the era of directed therapies – a future model for nanomedical systems?How directed therapies change the dosing equationCurrent generation directed antibody therapies dosingSome typical side effects of directed therapiesNanomedical systems are the next generation of directed therapiesMost directed therapies are nonlinear processesCurrent and pending FDA approved directed therapiesSome examples of how a few directed therapies workComplement directed cytotoxicityADCC-mediated adaptive immunity switchAntibody-directed enzyme producing therapyOther ways of controlling dose locallyMagnetic field release of drugsLight-triggered release of drugs

Outline:Introduction – the importance of the zeta potentialNanoparticle-nanoparticle interactionsNanoparticle-cell interactionsPart of the initial nanomedical system-cell targeting processLow zeta potential leads to low serum protein binding and potentially longer circulationZeta potential basicsWhat is the zeta potential?How is it measured?Some factors affecting the zeta potentialpHIonic strengthSome zeta potential experiences

With Dmitry Zemlyanov as guest lecturer.

With Donald E. Bergstrom as guest lecturer.

What Helen McNally as guest lecturer.

Outline:Assessing nanomedical system (NMS) targeting at the single cell levelFluorescent labeling of NMSsFirst estimates of NMS binding by fluorescence microscopyInternal of external binding by confocal microscopySingle-cell image/confocal analysisFlow cytometric quantitation of NMS binding to specific cell typesImage confocal analysis of NMS binding to single cellsAbility to scan/locate cells of interestPhotobleaching challengesOptical sectioning for 3D location of NMSs on/within cellsA quick overview of flow cytometryBasic principlesCapabilities and limitationsUse for assessing specificity and sensitivityRare-event analysis of NMS targeting to desired cellsBasic concepts of rare-event analysisStrategies for rare cell detectionMore advanced flow cytometry for ultra-rare cell detectionExamples of rare cell detectionRare cell sampling statistics

Outline:IntroductionCore building blocksFunctional coresFunctionalizing the core surfaceFerric oxide coresParamagnetic coresSuperparamagnetic coresFerric nanorodsAdvantages and disadvantagesC60 and carbon nanotubesSize and structure of C60Elongation of C60 into carbon nanotubesAdvantages and disadvantagesGold coresGold nanoparticlesGold nanorodsOther shapes (e.g. "stars")Gold nanoshellsAdvantages and disadvantagesSilica coresSilica nanoparticlesAdvantages and disadvantagesHybrid materialsGold-ferrric oxide nanoparticles and nanorodsCobalt-Platinum magnetic nanoparticles

Outline:Introduction to measuring technologies for nanomedical system interaction with cellsThe importance of quantitative or at least semi-quantitative single cell measurementsto detect presence and location of nanomedical systemsBelow "optical limit" imagingRequirements on the NMS to have X-ray dense, fluorescent, metallic, or magnetic coresCan you study living cells?Technologies – Advantages and disadvantagesFlow cytometry – a "zero order" imaging deviceScanning and Transmission electron microscopyConfocal microscopy – one and two-photonSurface Plasmon Resonance (SPR) ImagingAtomic Force MicroscopyMagnetic Sorting/MRI contrast agents for in-vivo imaging

Outline:IntroductionThe general problem of cell entryChoosing modes of cell entryHow does Nature do it? (biomimetics)Non-specific uptake mechanismsPinocytosis by all cellsPhagocytosis by some cellsReceptor mediated uptakeReceptor mediated transport of desired moleculesExample- transferrin receptor transport of iron for metabolismNanoparticle uptakeSize mattersAgglomeration reduces uptakeDrug delivery by "shedding"Extracellular drug delivery by sheddingIntracellular drug release by shedding

Outline:Overview: targeting nanosystems to cellsAntibody targetingPeptide targetingAptamer targetingAntibodies – polyclonal and monoclonalWhere do antibodies come from – in nature?How do we make them in the laboratory?Monoclonal antibodiesTherapy problems with mouse monoclonal antibodies“Humanizing” monoclonal antibodies to reduce adverse host immune reactionsWhy antibodies may not be a good overall choices for targeting nanosystems to cellsPeptide targetingHow does a peptide target?Examples of peptide targetingCreating new peptides by random peptide phage display librariesHigh-throughput screening of those peptide librariesAdvantages and disadvantages of peptide targetingAptamer targetingWhat are aptamers and how do they target?Some different types of aptamersHow do you make aptamers?How do you screen for useful aptamers?

Outline:Bridging the gap between diagnostics and therapeuticsHow conventional medicine is practiced in terms of diagnostics and therapeuticsThe consequences of separating diagnostics and therapeuticsA new approach – "theragnostics" (or "theranostics")Examples of current theragnostic systemsExample: Rituxan ("Rituximab)(an example of not using diagnostics to guide the therapy)Example 1: Herceptin ("terastuzumab")Example 2: Iressa ("Gefitinib)How theragnostics relates to Molecular ImagingConventional imaging is not very specificTypes of In-vivo ImagingX-rays, CAT (Computed Axial Tomography) scansMRI (magnetic Resonance Imaging)PET (Positron Emission Tomography) scans"Molecular Imaging"Engineering nanomedical systems for simultaneous molecular imagingUsing nanomedical cores for MRI contrast agentsDifficulties in using PET probes for nanomedical devicesUsing cell-specific probes for molecular imaging of nanomedical devicesBreaking the "diffraction limit" – nano-level imagingTheragnostic nanomedical devicesUsing nanomedical devices to guide separate therapeutic deviceWhen might we want to combine diagnostics and therapeutics?

Outline:Nanomedical systems – levels of challengesEssential elements of a nanomedical systemRequirements for specific cell targetingConsequences of mis-targetingEngineering around the consequences of mis-targetingSome ways to lower mis-targeting to non-diseased cells

Outline: Features of NanomedicineBottoms up rather than top down approach to medicine Nano-tools on the scale of molecules Cell-by-cell repair approach – regenerative medicine Feedback control system to control drug dosing Elements of good engineering designWhenever possible, use a general design that has already been testedUse multiple specific molecules to do multi-step tasksControl the order of molecular assembly to control the order of eventsTherefore, perform the molecular assembly in reverse order to the desired order of events Building a nanodeviceChoice of core materialsAdd drug or therapeutic geneAdd molecular biosensors to control drug/gene deliveryAdd intracellular targeting moleculesResult is multi-component, multi-functional nanomedical deviceFor use, design to de-layer, one layer at a timeThe multi-step drug/gene delivery process in nanomedical systems The challenge of drug/gene dosing to single cellsPrecise targeting of drug delivery system while protecting non-targeted cells from exposure to the drugHow to minimize mis-targetingHow to deliver the right dose per cellOne possible solution – in situ manufacture of therapeutic genes