7 imaging

Imaging in vitro

Quantum dots as tools for cellular imaging

What are quantum dots ?

• Crystalline fluorophores

• CdSe semiconductor core

• ZnS Shell

What are quantum dots

• Unique Spectral properties– Broad absorption– Narrow emission– Wavelength depends on size

• Hydrophobic crystals

3 nm

Making hydrophobic quantum dots bio-compatible

• Various methods for making them water-soluble– Derivatizing surface with

mercaptoacetic acid

– Encapsulating in phospholipid micelles or liposomes

– Coating them with amine-modified polyacrylic acid

Conjugating quantum dots to biomolecules

• Avidin or protein-G with positively charged tail conjugated to negatively charged DHLA coat of quantum dots

Avidin

protein G

Quantum dots v/s other fluorescent probes

Photostability (quantum dots do not photobleach)

Red: qdot 605 Conjugate Green: Alexa488 Conjugate

Wu et al. 2003


• Broader excitation spectrum and narrower gaussian emission spectrum

• No spectral overlap between dots of different size – better for multiplexing

Jaiswal & Simon 2004


• Brighter than other fluorophores

Larson et al. 2003

Quantum dots

Fluorescein

Quantum dots and imaging

In vivo visualization of capillaries

Larson et al. 2002

Quantum dots FITC-Dextran


Wu et al. 2003

Cancer cell surface marker red & green Microfilaments

Actin filaments Nuclear antigens


Dahan et al. 2003

Glycine Receptors

Diffusion of single Qdot-GlyRs in

synaptic boutons


Lidke et al. 2004

Live imaging of receptor mediated endocytosisEGF receptor

EGF-QD


Observing high resolution structure in dendritic cells

Individual vesicles -Temperature variations

Dendritic cell processes


Single quantum dot crystals can be observed in electron micrographs

1 m

200 nm

200 nm


• Quantum dots have been used in FRET• In conjunction with Texas Red• In conjunction with fluorescent quenchers

Willard et al. 2003

QDOTS IN VIVO

Advantages

• Specific labeling of cells and tissues• Useful for long-term imaging• Useful for multi-color multiplexing• Suitable for dynamic imaging of

subcellular structures• May be used for FRET-based analysis

Disadvantages

• Colloidal polymer-coated quantum dots can aggregate irreversibly

• Toxic in vivo• Quantum dots are bulkier than many organic

fluorophores– Accessibility issues

– Mobility issues

• Cannot diffuse through cell membrane– Use of invasive techniques may change physiology

Imaging in vivo

X-raydense, fluorescent, metallic,or magnetic cores

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Multifunctional NanoparticlesMultifunctional NanoparticlesFor imagingFor imaging

X-ray CT

CT is ubiquitous in the clinical setting as. The increasing use and development of micro-CT and hybrid systems that with PET, MRI.The most investigated NPs in this field are gold NPs, since they have large absorption coefficients against the x-ray source used for CT imaging and may increase the signal-to-noise ratio of the technique.To date, different types of gold NPs have been tested in a preclinical setting as contrast agents for molecular imaging: nanospheres, nanocages, nanorods and nanoshells. Gold NPs formulations as an injectable imaging agent have been utilized to study the distribution in rodent brain ex vivo

nanocage

nanosphere

nanorod

nanoshell

Size 4-40 nm

MRIseveral nanotechnological approaches have been devised, based on the idea of carrying a substantial payload of Gd chelates. Examples include liposomes micelles dendrimers fullerenes. However, this approach has not yet achieved clinical applications.

To this end, magnetic NPs (MNPs) are of considerable interest because they may behave either as contrast agents or carriers for drug delivery. Among these, the most promising and developed NP system is represented bysuperparamagnetic iron oxide agents

PET The strategy utilized is consisting in incorporating PET emitters within the components of the NP, or entrapping them within the core. Oku et al (2011) employed PET to image brain cancer using positron-emitting labelled liposomes in rats. Plotkin et al (2006) used PET radioisotopes for targeting the intra-tumourally injected magnetic NPs in patients with glioblastoma.

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Magnetic Nanoparticles

B

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Types of Magnets

• Ferromagnetic materials: the magnetic moments of

neighboring atoms align resulting in a net magnetic moment.

• Paramagnetic materials are randomly oriented due to Brownian

motion, except in the presence of external magnetic field. B

Superparamagnetic• Combination of paramagnetic and ferromagnetic

properties

– Made of nano-sized ferrous

magnetic particles, but affected by Brownian Motion.

• They will align in the presence of an external magnetic field.

The most promising and developed NP system is represented by superparamagnetic iron oxide agents, consisting of a magnetite (Fe3O4) and/or maghemite (Fe2O3) crystalline core surrounded by a low molecular weight carbohydrate (usually dextran or carboxydextran) or polymer coat.. Iron oxide NPs can be classified according to their core structure, such as Monocrystalline (MION; 10–30 nm diameter), or according to their size as ultra-small superparamagnetic (USPIO) (20–50 nm diameter), superparamagnetic (SPIO) (60–250 nm).

Dextran Coated Magnetite Nanoparticles

• Synthesis of polysaccharide covered superparamagnetic oxide colloids – For MRI imaging

• FDA max size for injectables = 220 nm.

• Smaller sizes (<100 nm) have longer plasma half-life. – Blood clearance by Reticuloendothelial system (RES)– Liver and Spleen

• Without coating, opsonin proteins deposit on Magnetite and mark for removal by RES

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Formation of Nanoparticles• Solution of Dextran and Ferric hexahydrate (acidic

solution)

– Less Dextran Larger Particles

• Drip in Ammonium hydroxide (basic) at ~2oC

• Stirred at 75oC for 75 min.

• Purified by washing and

ultra-centrifugation

• Resulting Size ~ 10-20 nm

• Plasma half-life: 200 min

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Variation of Formation

• Change Coating Material– Various other starches, Sulfated Dextran (for

functionalization)

• Crosslinking coating material– Increases plasma half-life– Same Particle Size

Magnetite Cationic Liposomes (MCL)

• Why Cationic?

– Interaction between + liposome and – cell

– membrane results in 10x uptake.

Fe3O4

Formation of MCL

• magnetite NP dispersed in distilled water

• N-(-trimethyl-amminoacetyl)-didodecyl-D-glutamate chloride (TMAG) Dilauroylphosphatidylcholine (DLPC) Dioleoylphosphatidyl-ethanolamine (DOPE) added to dispersion at ratio of 1:2:2

• Stirred and sonicated for 15 min

• pH raised to 7.4 by NaCl and Na phosphate buffered and then sonicated

Uses of Nano Magnets

• Hyperthermia

– An oscillating magnetic field on nanomagnets result in local heating by (1) hysteresis, (2) frictional losses (3) Neel or Brown relaxation

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Cancer Treatment

• Heating due to magnetic field results in two possibilities Death due to overheating

Increase in heat shock

proteins result in

anti-cancer immunity.

Ito A., Honda H., Kobayashi T. Cancer Immunol Immunother Res 2006 55; 320-328

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Delivery Magnetic nanoparticles

Ito A., Honda H., Kobayashi T. Cancer Immunol Immunother Res 2006 55; 320-328

• Magnetite nanoparticles encapsulated in liposomes– (1) Antibody conjugated

(AML)– (2) Positive Surface Charge

(MCL)• Sprague-Dawley rats injected with

two human tumors.– Liposomes injected into 1 tumor (black) and appliedAlternating Magnetic Field

Effect of Hyperthermia

TreatedTumor

UntreatedTumor

Rectum

After Treatment

Before Treatment

• Non-local heating in body is the result of eddy-currents

– The currents resulting from the magnetic field produce heat


• MRI imaging.Iron oxide agents shorten T2 and T2* relaxation times on T2-

and T2*-weighted MRI images, creating low signal or negative contrast. They can also be detected by MRI with T1, off resonance, and steady-state free precession sequences


• External Magnetic field for nanoparticle delivery

– Magnetic nanoparticles loaded with

drug can be directed to diseased site for

Drug Delivery or MRI imaging.

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Magnetic Drug Delivery System

• Using Magnetic Nanoparticles for Drug Delivery• Widder & others developed method in late 1970s• Drug loaded magnetic nanoparticles introduced through IV or IA

injection and directed with External Magnets • Requires smaller dosage because of targeting, resulting in fewer side

effects

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Magnetic Nanoparticles/Carriers

• Magnetite Core

• Starch Polymer Coating• Bioavailable

• Phosphate in coating for functionalization

• Chemo Drug attached to Coating • Mitoxantrone

• Drug Delivered to Rabbit with Carcinoma

Magnetite Core

Starch Polymer

M

M

MM

M

MM

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Results of Drug Delivery

• External magnetic field (dark)

• deliver more nanoparticles to tumor

• No magnetic field (white)

• most nanoparticles in non tumor regions

Magnetic nanoparticles in medicineThey consist of a metal or metallic oxide core, encapsulated in an inorganic or a polymeric coating, that renders the particles biocompatible, stable, and may serve as a support for biomolecules.

• Drug or therapeutic radionuclide is bound to a magnetic NP, introduced in the body, and then concentrated in the target area by means of a magnetic field.• Depending on the application, the particles release the drug or give rise to a local effect (hyperthermia). • Drug release can proceed by simple diffusion or take place through mechanisms requiring enzymatic activity or changes in physiological conditions (pH, osmolality, temperature, etc…).

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Multifunctional Magnetic NanoparticlesMultifunctional Magnetic Nanoparticles

• Magnetic nanocrystals as ultrasensitive MR contrast agents: MnFe2O4

• Anticancer drugs as chemotherapeutic agents: doxorubicin, DOX

• Amphiphilic block copolymers as stabilizers: PLGA-PEG

• Antibodies to target cancer cells: anti-HER antibody (HER, herceptin) conjugated by carboxyl group on the surface of the MMPNs

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Yang, etal. Angew. Chem. 2007, 119, 8992 –8995.

Magnetic nanoparticles

The application of magnetic nanoparticles in cancer therapy is one of the most successful biomedical exploitations of nanotechnology. The efficacy of the particles in the treatment depends upon the specific targeting capacity of the nanoparticles to the cancer cells. Efficient, surface-engineered magnetic nanoparticles open up new possibilities for their therapeutic potential.

… effective conjugation of folic acid on the surface of superparamagnetic iron oxide nanoparticles (SPION) enables their high intracellular uptake by cancer cells.

Such magnetic-folate conjugate nanoparticles are stable for a long time over a wide biological pH range: additionally, such particles show remarkably low phagocytosis as verified with peritoneal macrophages.

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Conclusions• Nanomagnets can be made bioavailable by

liposomal encapsulation with targeting

• Nanoparticles smaller than 20 nm can be useful for local heat generation

• Intracellular hyperthermia kills the cancer cell and releases heat shock proteins. These are used to target and kill other cancer cells.

• Results in reduction in growth of tumor size

• Nanomagnets can be used for MR Imaging in vivo

• Used with ultrasound echocardiography and magnetic resonance imaging (MRI)

• Diagnostic imaging - Traces blood flow and outlines images

• Drug Delivery and Cancer Therapy

MICROBUBBLES

• Small (1-7 m) bubbles of air (CO2, Helium) or high molecular weight gases (perfluorocarbon).

• Enveloped by a shell (proteins, fatty acid esters).• Exist - For a limited time only! 4 minutes-24

hours; gases diffuse into liquid medium after use.

• Size varies according to Ideal Gas Law (PV=nRT) and thickness of shell.

ultrasound

• Ultrasound uses high frequency sound waves to image internal structures

• The wave reflects off different density liquids and tissues at different rates and magnitudes

• It is harmless, but not very accurate

Ultrasound and Microbubbles

• Air in microbubbles in the blood stream have almost 0 density and have a distinct reflection in ultrasound

• The bubbles must be able to fit through all capillaries and remain stable

1-m

Shell

Air or High Molecular Weight Gases

Preparation of microbubbles

1. Water

2. Fluorinated hydrocarbon

3. Polymer solution

4. Ethanol palmitic acid solution with

Epikuron® 200

5. Homogeneization for 10 min at 12000 rpm

O2 microbubbles coated with PAAs

Diameter = 549.5 ± 94.7 nmPZ = 8.54±1.21pH = 3.28

Diameter = 491.4 ± 38.2 nmPZ = 6.22±1.17pH = 6.50

Cationic PAA PAA-cholesterol

Application of microbubble technology for ultrasound imaging of the heart

7 imaging

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