Nano Labs (Lec7 cell nanoparticle)

Nano Labs (Lec7 cell nanoparticle) - Interactions Between...

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Unformatted text preview: Interactions Between Living Cells and Nanoparticles Nano Labs, MAE C187L/C287L Nano Lab MAE C187L/C287L Nano Can Engineers Understand Can Engineers Understand Cell Biology? • Cell anatomy – Lipid membrane and vesicles – Nucleus – Cytoskeleton – Internalization • Internalization and application of nanoparticles – Gene delivery – Drug delivery – Actuation Nano Lab MAE C187L/C287L Cell Consists of Organelles Cell Consists of Organelles There are many different types, sizes, and shapes of cells in the body. A cell consists of three parts: the cell membrane, the nucleus, and between the two, the cytoplasm. Cell is Contained Cell is Contained by a Plasma Membrane Every cell in the body is enclosed by a cell membrane. The cell membrane separates the material outside the cell, extracellular, from the material inside the cell, intracellular. It maintains the integrity of a cell and controls passage of materials into and out of the cell. All materials within a cell must have access to the cell membrane for the needed exchange. The cell membrane is a double layer of phospholipid molecules. Proteins in the cell membrane provide structural support, form channels for passage of materials, act as receptor sites, function as carrier molecules, and provide identification markers. Nucleus is the Control Center Nucleus is the Control Center The nucleus, formed by a nuclear membrane around a fluid nucleoplasm, is the control center of the cell. The nucleus contains deoxyribonucleic acid (DNA), the genetic material of the cell. The nucleolus is a dense region of ribonucleic acid (RNA) in the nucleus and is the site of ribosome formation. The nucleus determines how the cell will function, as well as the basic structure of that cell. Cytoplasm is the Matrix Cytoplasm is the Matrix for Other Organelles The cytoplasm is the gel-like fluid inside the cell. It is the medium for chemical reaction. It provides a platform upon which other organelles can operate within the cell. All of the functions for cell expansion, growth and replication are carried out in the cytoplasm of a cell. Within the cytoplasm, materials move by diffusion, a physical process that can work only for short distances. Schematic showing the cytoplasm, with major components of a typical animal cell. (1) nucleolus (2) nucleus (3) ribosome (4) vesicle (5) rough endoplasmic reticulum (6) Golgi apparatus (7) cytoskeleton (8) smooth endoplasmic reticulum (9) mitochondria (10) vacuole (11) cytosol (12) lysosome (13) centriole. Vesicles Act as Transport Shuttles Vesicles Act as Transport Shuttles • Intracellular transport vesicles – Continual budding and fusing – Carry proteins, lipids, etc. between cellular compartments – Assembly of clathrin coating • Budding is driven by proteins Nano Lab MAE C187L/C287L Cytoskeleton Enables Cytoskeleton Enables Shape and Movement • Actin actin nucleus • Microtubules – Structure – Movement – Transport – Shape and motility – Muscle contraction microtubules Nano Lab MAE C187L/C287L Motility of 3T3 Fibroblasts Motility of 3T3 Fibroblasts Nano Lab MAE C187L/C287L Mechanisms of Internalization Mechanisms of Internalization • Endocytosis is an active process – Pinocytosis “cellular drinking” • Vesicle <150 nm – Phagocytosis “cellular eating” • Vesicle >250 nm Nano Lab MAE C187L/C287L Pinocytosis is continual in Pinocytosis is continual in eucaryotic cells • Continual cycling of plasma membrane • Mediated by clathrin – Intake of fluid, small particles – Macrophage recycles membrane in 30 min • Can be selective for a macromolecule – “Receptor­mediated endocytosis” Nano Lab MAE C187L/C287L Specialized phagocytes ingest Specialized phagocytes ingest large particles • Phagocytes defend against foreign particles – Macrophages – Neutrophils Triggered by coating on particle • Neutrophil phagocytosis, Marks and Maxfield • Neutrophil chases bacterium Nano Lab MAE C187L/C287L Inner Life of a Cell Inner Life of a Cell • Inner Life, Harvard University – Macrophage cell rolling – Fluid lipid membrane, lipid rafts – Actin filaments – Assembly and dissociation of microtubules – Vesicle transport along microtubule – Ribosomal translation of RNA – Exocytosis Nano Lab MAE C187L/C287L Neuronal Cell Structure Neuronal Cell Structure • The neuron has a cell body, called soma, which contains the nucleus, ribosomes, and other subcellular organelles, and is responsible for routine metabolic “housekeeping” functions. Their sizes are a few µ m to a hundred µ m. The neuron has a long cylindrical process, called axon, with diameters ranging from 0.2 to 20 µ m, and lengths ranging from µ m up to m. The neuron also has branched extensions, called dendrites, with lengths from µ m up to mm. • • Neuronal Membrane Structure Neuronal Membrane Structure 6 nm Neuronal membrane contains a phospholipid bilayer, which is very good isolation layer. Ion channels are embedded in the member which allows specific ions to pass through. The sodium-potassium ion pump embedded in the membrane pumps the ions across the membrane against their concentration gradients. Voltage Gated Na+ Channel Voltage Gated Na A model to explain how the pulse is generated: (1) The channel is closed at rest potential; (2) The channel is opened when the membrane is depolarized; (3) The channel is blocked by the potion of protein after sodium ions passed through; (4) It will back to standby status after the depolarized voltage is removed. How is the Pulse Generated? How is the Pulse Generated? (A) At standby status, only K channels are permeable; (B) A depolarized potential opens the Na channels, the Na ions rush into the neuron; (C) After a while, the Na channels are closed, the K ions are driven out by the positive potential. (D) The ion pumps bring ions back to standby concentrations, and ion channels to standby status. Signal Propagation in Neuron Signal Propagation in Neuron Propagation of the pulse signal along the axon is similar to the propagation of a fire. When a axon is depolarized at a point, the Na ion channels open. The influx of the Na ions will also diffuse to its neighbor area, until the change of the ion concentrations there reaches a threshold value, it will also induce a pulse there, and the pulse will propagate in this way further. Synapse Type II: Synapse Type II: Chemical Synapse • • • Most of the synapses are chemical synapses. They are separated by synaptic cleft of 20-50 nm wide. The terminal typically contains dozens of small spheres, about 50 nm in diameter, called synaptic vesicles. The vesicles contains chemicals called neurotransmitter. The postsyneptic membrane contains neurotransmitter receptors. • Neurotransmitter Neurotransmitter There are many different kinds of neurotransmitters. Most of the neurotransmitters fall into one of the three categories: (a) Amino acids (b) Amines (c) Peptides Release of Neurotransmitter Release of Neurotransmitter 1. 2. 3. 4. 5. The release of neurotransmitter: A synaptic vesicle is loaded with neurotransmitters A signal pulse from the presynaptic neuron opens the voltage gated Ca+ ion channel in the presynaptic membrane. The influx of Ca+ ions induce the release of neurotransmitters from the vesicle. The vesicle is recycled. The time for the release of neurotransmitters may take less than 1 ms. Learning Process Learning Process by Modifying Synapses During associate learning, we form association between events, such as classic conditioning, e.g. Before conditioning, the sound of bell, the conditioned stimulus (CS), get no response from the dog, in sharp contrast with to its response to a piece of meat, the unconditioned stimulus. After conditioning the dog by pairing the bell and meat, the dog learns to respond to the bell. Ligand Coating Ligand Coating can Promote Internalization Nanoparticle carrier Payload Internalization Release Interaction between nanoparticles and cell involves attaching molecules, known collectively as ligands, to the nanoparticle surface. These ligands specifically bind to complementary molecules, or receptors, found on the surface of cells, allowing the nanoparticles to enter the cell with high efficiency. Packaging can Promote Nanoparticle Packaging can Promote Nanoparticle Internalization Liposomes are a form of vesicles that consist either of many, few or just one phospholipid bilayers. The polar character of the liposomal core enables polar drug molecules to be encapsulated. Amphiphilic and lipophilic molecules are solubilized within the phospholipid bilayer according to their affinity towards the phospholipids. Participation of nonionic surfactants instead of phospholipids in the bilayer formation results in niosomes. Channel proteins can be incorporated without loss of their activity within the hydrophobic domain of vesicle membranes, acting as a sizeselective filter, only allowing passive diffusion of small solutes such as ions, nutrients and antibiotics. Thus, drugs that are encapsulated in a nanocage-functionalized with channel proteins are effectively protected from premature degradation by proteolytic enzymes. Drug encapsulation in liposomes. Biomedical Applications of Biomedical Applications of Nanoparticles • Advantages of versatility • Key aspects of examples: – Nanoparticle carrier – Biochemical payload – Mechanism of internalization – Actuation and transduction – Verification of delivery Nano Lab MAE C187L/C287L Nanoparticles for Gene Delivery Nanoparticles for Gene Delivery On encountering a cell, the cell engulfs the nanoparticles in a little piece of cell membrane that is pinched off inside the cell. It turns out that the solution inside these endosomes is significantly more acidic than the surroundings, and this triggers the polymersome nanoparticles to fall apart, releasing its DNA. This, in turn, generates an osmotic pressure sufficient to burst open the endosome, releasing the DNA into the cell interior, where it is available for translation. Carbon Nanotubes for Carbon Nanotubes for Gene Delivery • Carbon Nanotube – Single/multi wall – Ammonium – functionalized (+) Pristine nanotube • Plasmid DNA After DNA loading After DNA loading – Phosphate groups (­) – β­galactosidase marker gene – Plasmid condenses Pantarotto, 2004 Nanotubes continued Nanotubes continued • Electrostatic adsorption • Internalization Whole cell Internalized NT – Not endocytosis – Spontaneous insertion into membrane? Partially inserted NT Pantarotto, 2004 Nanotubes … Nanotubes … • • Transduction – Intracellular translocation – Gene expression Verification of expression – Chemiluminescent reporter kit Gene expression ND ND+DOX Pantarotto, 2004 Incubation time Charge ratio Nanoparticles for Drug Delivery Nanoparticles for Drug Delivery A difficulty of current cancer therapy is that the injected drug goes everywhere throughout the body, causing the patient to significant side effects and lose their lives. Besides causing these side-effects, non-targeted drug pills have to face the acidic stomach, the cell membranes, and the various cleavage enzymes that are ever-ready to break them down. The drug loses most of its efficacy before it even reaches the target site. The ligands attached to the nanopartciles can recognize and bind to complementary receptors on the surface of cancer cells. When such targeting molecules are added to a drug delivery nanoparticle, more of the anticancer drug finds and enters the tumor cell, increasing the efficiency of the treatment and reducing toxic effects on surrounding normal tissue. Nanoparticles releasing the contained drug at the exact site of tumor. Active Nanodiamonds for Active Nanodiamonds for Chemotherapy • Nanodiamond – 2 to 10 nm – Hydrophilic functional groups Pristine nanodiamond After DOX loading 10 nm • Doxorubicin hydrochloride (DOX) – Apoptosis drug used in chemotherapy – 2 to 10 nm coating Huang, 2007 Nanodiamonds continued Nanodiamonds continued • Salt mediates ad/desorption • Internalization – Mechanism not stated Immediately after ND exposure 1 hr after ND exposure Nucleic stain Huang, 2007 Nanodiamonds … Nanodiamonds … • Transduction • Verification of cellular effects – Viability – DNA electrophoresis ND+DOX DOX ND Normal – DOX induces apoptosis Normal DOX ND ND+DOX Huang, 2007 Magnetic Nanoparticles Enable Magnetic Nanoparticles Enable Mechanical Actuation • Application of magnetic force on cells – Transportation of cells – Induced cell death Nano Lab MAE C187L/C287L Choi, 2006 Nickel Nanowire for Cell Sorting Nickel Nanowire for Cell Sorting • Ni Nanowire After antibody stain – 100 nm diameter – 5 to 35 μm long • Coating with mouse antibody – Allows fluorescent staining later Hultgren, 2005 Nanowires continued Nanowires continued • Non­specific protein adsorption Paxillin focal adhesion • Internalization – Focal adhesions – Phagocytosis 30 min: External nanowire 24 hr: Internal nanowire Hultgren, 2005 Nanowires … Nanowires … • Transduction Heterogeneous Sorted – Application of external magnetic field • Visualization of cell sorting – Yield as function of nanowire length Hultgren, 2005 Structure Carbon magnetic Nanoparticles Dendrimers Ceramics Nanoparticles Chitosan Nanoparticles Liposomes Low Density Lipoprotein Nanoemulsions Nanolipispheres Nanoparticles composites Nanoparticles Nanopill/Micelle Nanospheres Nanovesicles Polymer Nanocapsules Size 40-50 nm 1-20 nm ~35 nm 110-180 nm 20-25 nm 20-25 nm 20-25 nm 25-50 nm ~40 nm 25-200 nm 20-45 nm 50-500 nm 25-3000 nm 50-200 nm Role in drug delivery For drug delivery and targeted cell destruction Holding therapeutics substances such as DNA in their cavities Accumulate exclusively in the tumor tissue and allow the drug to act as sensitizer for photodynamics therapy without being released High encapsulation efficiency .In vitro release studies show a burst effect flowed by a slow and continuous release. A new generation of liposomes that incorporate fullerenes to deliver drug that are not water soluble, that tend to have large molecules Drug solublized in the lipid core or attached to the surface Drug in oil/or in liquid phases to improve absorption Carrier incorporation of lipophilic and hydrophilic drugs Attached to guiding molecules such as Mabs for targeted drug delivery Act as continuous matrices containing dispersed or dissolved drug Made for two polymer molecules-one water -repellent and the other hydrophobic-that self assemble into a sphere called a micelle that can deliver drugs to specific structures within the cell Hollow ceramic nanospheres created by ultrasound Single or multilamellar bilayer spheres containing the drugs in lipids Used for enclosing drugs Lab instructor: Andrew Fung, [email protected] Time: 5/16 May 9, Friday. Session I, 9 am-12 noon; Session II, 1 pm-4 pm. Lab: Bldg. CMISE cell culture room in Boelter 1538 & CNSI Building, Room 4537 Objective: This lab will combine concepts and skills from previous sessions to observe the interaction of live cells with nanoparticles. Fluorescent particles with different functionalities will be dispersed into a culture of fibroblasts. The time-dependent interaction of the cells with the nanoparticles will be examined by confocal microscope imaging. Reminder: Experiment starts at 9 am and 1 pm in Boelter 1538. Please bring your flash drives for image recording. Nano Lab MAE C187L/C287L Nano References • MAE298 Mechanics of Cells, Prof. Bill Klug • Biofunctionalization of Nanomaterials, Edited by Challa Kumar, Wiley-VCH Edited • Nanodevices for the life sciences, Edited by Challa Kumar, Wiley-VCH Challa • Nanoscale technology in biological systems, by R. S. Greco, F. B. Prinz, R. L. Smith by Nano Lab MAE C187L/C287L Nano ...
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This note was uploaded on 03/03/2009 for the course MECH&AE 187L taught by Professor Yongchen during the Spring '08 term at UCLA.

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