Imaging in vitro

Quantum dots as tools for cellular imaging

What are quantum dots ?

• Crystalline fluorophores

• CdSe semiconductor core

• ZnS Shell (surface passivation)

• Hydrophobic crystals

What are quantum dots• Unique Spectral properties

Broad absorptionNarrow emissionWavelength depends on size

Annu. Rev. Anal. Chem. 2013. 6:143–62, Michalet , X. et al. Science. 2005, 307 , 538 – 44

CdSe/ZnS QD samples

Making hydrophobic quantum dots bio-compatible

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

with bifunctional ligands

– Encapsulating in phospholipid micelles or liposomes

– Polymer coating

Gao , X. et al. Adv.Experim.Med. Biol. 2007, 620,57-73

Conjugating quantum dots to biomolecules

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

Avidin

protein GGoldman E.R. et al.( 2002 ) JACS,124 , 6378 – 82 ; Analytical Chemistry , 74 , 841 – 7 .

Quantum dots v/s other fluorescent probes

• Broader excitation spectrum and narrower gaussian emission spectrum

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

Jaiswal & Simon 2004

Quantum dots for multiplexing

T cellsB cells

Malignant HRS cells

Liu J et al. Anal. Chem. 2010, 82, 6237–6243; Am. Chem. Soc. Nano 4:2755–65

benign prostategland

gland with a single malignant cell Multiplex

Immunostaining

To differentiate Hodgkin’s from non-Hodgkin’s lymphoma

Quantum dots for multiplexing

Quantum dots v/s other fluorescent probes

Photostability (quantum dots do not photobleach)

Red: qdot 605 Conjugate Green: Alexa488 Conjugate

Wu et al. Nat. Biotechnol. 2003;21(1):41-62

3T3 cells

Scale bar 10 µm.

Quantum dots v/s other fluorescent probes

• Brighter than other fluorophores

Larson et al. 2003

Quantum dots

Fluorescein

Quantum dots and imaging

In vivo visualization of capillaries

Larson et al. 2003, Science 300:1434-1436

Quantum dots FITC-Dextran

Quantum dots and imaging

Cancer cell surface marker red & green Microfilaments

Actin filaments Nuclear antigens

Wu et al. Nat. Biotechnol. 2003;21(1):41-62

anti-α-tubulin antibody

biotinylated phalloidin

anti-Her2antibody

Quantum dots and imaging

Dahan et al. Science. 2003, 302:442-445

Glycine Receptors

Diffusion of single Qdot-GlyRs in

synaptic boutons

Primary antibody ↔ secondary Antibody – biotin ↔ QD Streptavidin

Quantum dots and imaging

Lidke et al. Nat. Biotechnol. 2004, 22:198

Live imaging of receptor mediated endocytosis

EGF receptor EGF-QD

Quantum dots and imaging

Single quantum dot crystals can be observed in electron micrographs

1 m

200 nm

200 nm

Quantum dots and imaging: FRET

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

Willard et al. Nat Materials. 2003, 2:575

Quantum dots and imaging: FRET

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

24

Multifunctional NP for Imaging

Imaging techniques and related contrast agents

X-ray Iodinated contrast materials Au

MRI Gadolinium-based Fe-based 19F-based

PET Radioactively labelled agents (11C, 18F)

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

PET

The strategy utilized is consisting in incorporating PET emitters within the components of the NP, or entrapping them within the core.

brain cancer

Oku et al (2011), Int. J. Pharm. 403 :170–177

MRI

MRI relies upon the enhancement of local water proton relaxation in the presence of a contrast agent (CA). CA are compounds that catalytically shorten the relaxation times of bulk water protons.

T1 (longitudinal relaxation– in simple terms, the time taken for the protons to realign with the external magnetic field) Positive CA (Gd)

T2 (transverse relaxation –in simple terms, the time taken for the protons to exchange energy with other nuclei) Negative CA (Iron oxide agents )

Gd-based NP

several nanotechnological approaches have been devised, based on 2 ideas:-carrying many Gd chelates;-slow down the rotation of the complex

Examples include liposomes micelles dendrimers fullerenes.

However, this approach has not yet achieved clinical applications.

31

Magnetic Nanoparticles

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 by superparamagnetic iron oxide agents

32

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).

J Lodhia et al. Biomed Imaging Interv J 2010; 6(2):e12

Polymer Coated Magnetite Nanoparticles

Dextran, silane, poly-lactic acid, PEG, dextran, chitosan, gelatin, ethylcellulose…

36

Formation of Nanoparticles

• Solution of Dextran, FeCl3.6H2O and FeCl2.4H2O (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

Variation of Formation

• Change Coating Material

• Crosslinking coating material

– Increases plasma half-life

– Same Particle Size

Lee H et al. J. AM. CHEM. SOC. 2007,129, 1273-12745

• Why Cationic?

– Interaction between + liposome and – cell

– membrane results in 10x uptake.

Magnetite Cationic Liposomes (MCL)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

DLPC

DOPE

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

42

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

43

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

antitumor immune response

Uses of Nano Magnets

• External Magnetic field for nanoparticle delivery

– Magnetic nanoparticles loaded with drug can be directed to diseased site for Drug Delivery or MRI imaging.

46

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

47

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

48

Results of Drug Delivery

• External magnetic field (dark)

• deliver more nanoparticles to tumor

• No magnetic field (white)

• most nanoparticles in non tumor regions

Alexiou C et al ANTICANCER RES 27: 2019-2022 (2007)

Magnetic nanoparticles in medicine

They 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…).

50

Multifunctional 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

50

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.

52

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.

MICROBUBBLES

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

Soft Matter, 2008, 4, 2350–2359

1-m

Shell

Air or High Molecular Weight Gases

Preparation of microbubbles

- Homogeneization- Sonication- microfluidic T-devices

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