Quantum dots have unique spectral properties that make them useful fluorescent probes for cellular imaging. They can be made water-soluble and conjugated to biomolecules for targeting specific cells and structures. Quantum dots have advantages over traditional fluorescent probes like greater photostability and the ability to multiplex imaging. They have been used for in vitro and in vivo imaging applications like labeling cancer cells, visualizing capillaries and receptors, and observing subcellular structures in real-time. While useful imaging tools, quantum dots have limitations like potential toxicity that must be addressed for in vivo use.

1. Imaging in vitro

2. Quantum dots as tools
for cellular imaging

3. What are quantum dots ?
• Crystalline fluorophores
• CdSe semiconductor core
• ZnS Shell

4. What are quantum dots
• Unique Spectral properties
– Broad absorption
– Narrow emission
– Wavelength depends on size
• Hydrophobic crystals
3 nm

5. 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

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

8. Quantum dots v/s other
fluorescent probes
Photostability (quantum dots do not photobleach)
Wu et al. 2003
Red: qdot 605 Conjugate Green: Alexa488 Conjugate

9. 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

10. Quantum dots v/s other
fluorescent probes
• Brighter than other fluorophores
Quantum dots
Fluorescein
Larson et al. 2003

11. Quantum dots and imaging
Quantum dots FITC-Dextran
In vivo visualization of capillaries
Larson et al. 2002

12. Quantum dots and imaging
Cancer cell surface marker red & green Microfilaments
Actin filaments Nuclear antigens Wu et al. 2003

13. Quantum dots and imaging
Diffusion of single
Qdot-GlyRs in
synaptic boutons
Glycine Receptors
Dahan et al. 2003

14. Quantum dots and imaging
EGF-QD
EGF receptor
Live imaging of receptor mediated endocytosis
Lidke et al. 2004

15. Quantum dots and imaging
Individual vesicles - Dendritic cell processes
Temperature variations
Observing high resolution structure in dendritic cells

16. Quantum dots and imaging
1 m
200 nm
200 nm
Single quantum dot crystals can be observed in electron micrographs

17. Quantum dots and imaging
• Quantum dots have been used in FRET
• In conjunction with Texas Red
• In conjunction with fluorescent quenchers
Willard et al. 2003

19. QDOTS IN VIVO

20. 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

21. 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

22. Imaging in vivo

24. X-raydense,
fluorescent,
metallic,
or magnetic cores

25. Multifunctional Nanoparticles
For imaging
25

27. 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

28. nanocage
nanorod
nanosphere
nanoshell
Size 4-40 nm

29. MRI
several 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

30. 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.

31. Magnetic Nanoparticles
31

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.
32

33. 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.

34. 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).

36. 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

37. 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
37

38. Variation of Formation
• Change Coating Material
– Various other starches, Sulfated Dextran (for
functionalization)
• Crosslinking coating material
– Increases plasma half-life
– Same Particle Size
38

39. Magnetite Cationic Liposomes (MCL)
Fe3O4
• Why Cationic?
– Interaction between + liposome and – cell
– membrane results in 10x uptake.

40. 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

41. 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

44. 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.
44
Ito A., Honda H., Kobayashi T. Cancer Immunol Immunother Res 2006 55; 320-328

45. Delivery Magnetic nanoparticles
• 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 applied
Alternating Magnetic Field
Ito A., Honda H., Kobayashi T. Cancer Immunol Immunother Res 2006 55; 320-328 45

46. Effect of Hyperthermia
Treated
Tumor
Before Treatment
Untreated
Tumor
Rectum
• Non-local heating in body is the
result of eddy-currents
– The currents resulting from the After Treatment
magnetic field produce heat

47. Uses of Nano Magnets
• 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

50. 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.

51. 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
51

52. Magnetic Nanoparticles/Carriers
M
M
• Magnetite Core M
• Starch Polymer Coating M
Magnetite
Core
M
• Bioavailable
Starch Polymer
• Phosphate in coating for functionalization
M
• Chemo Drug attached to Coating M
• Mitoxantrone
• Drug Delivered to Rabbit with Carcinoma
52

53. Results of Drug Delivery
• External magnetic
field (dark)
• deliver more
nanoparticles to tumor
• No magnetic field
(white)
• most nanoparticles in
non tumor regions
53

54. 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…).

55. 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
55
Yang, etal. Angew. Chem. 2007, 119, 8992 –8995.

56. 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.

57. 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 57

58. MICROBUBBLES
• Used with ultrasound echocardiography and
magnetic resonance imaging (MRI)
• Diagnostic imaging - Traces blood flow and
outlines images
• Drug Delivery and Cancer Therapy

59. • 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.

60. 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

61. 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

62. Shell
Air or High Molecular
Weight Gases
1- m

63. 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

64. O2 microbubbles coated with PAAs
Cationic PAA PAA-cholesterol
Diameter = 549.5 ± 94.7 nm Diameter = 491.4 ± 38.2 nm
PZ = 8.54±1.21 PZ = 6.22±1.17
pH = 3.28 pH = 6.50

65. Application of microbubble technology for ultrasound
imaging of the heart