1. Imaging in vitro

2. Quantum dots as tools
for cellular imaging

3. What are quantum dots ?
•
•
•
•
Crystalline fluorophores
CdSe semiconductor core
ZnS Shell (surface passivation)
Hydrophobic crystals

4. What are quantum dots
• Unique Spectral properties
Broad absorption
Narrow emission
Wavelength depends on size
CdSe/ZnS
QD
samples
Annu. Rev. Anal. Chem. 2013. 6:143–62, Michalet , X. et al. Science. 2005, 307 , 538 – 44

5. Making hydrophobic quantum dots biocompatible
• Various methods for
making them watersoluble
– 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

6. Conjugating quantum dots to biomolecules
• Avidin or protein-G with
positively charged tail conjugated
to negatively charged DHLA coat
of quantum dots
Goldman E.R. et al.( 2002 ) JACS,124 , 6378 – 82 ;
Analytical Chemistry , 74 , 841 – 7 .
Avidin
protein G

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

8. Quantum dots for multiplexing

9. Quantum dots for multiplexing
benign prostate
gland
Multiplex
Immunostaining
gland with a single
malignant cell
Malignant
HRS cells
To differentiate
Hodgkin’s from
non-Hodgkin’s
lymphoma
B cells
T cells
Liu J et al. Anal. Chem. 2010, 82, 6237–6243; Am. Chem. Soc. Nano 4:2755–65

10. Quantum dots v/s other fluorescent
probes
Photostability (quantum dots do not photobleach)
3T3 cells
Scale bar 10 µm.
Red: qdot 605 Conjugate
Green: Alexa488 Conjugate
Wu et al. Nat. Biotechnol. 2003;21(1):41-62

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

12. Quantum dots and imaging
In vivo visualization of capillaries
Quantum dots
FITC-Dextran
Larson et al. 2003, Science 300:1434-1436

13. Quantum dots and imaging
anti-α-tubulin
antibody
anti-Her2
antibody
Cancer cell surface marker red & green Microfilaments
biotinylated
phalloidin
Actin filaments
Nuclear antigens
Wu et al. Nat. Biotechnol. 2003;21(1):41-62

14. Quantum dots and imaging
Glycine Receptors
Diffusion of single
Qdot-GlyRs in
synaptic boutons
Primary antibody ↔ secondary Antibody – biotin ↔ QD Streptavidin
Dahan et al. Science. 2003, 302:442-445

15. Quantum dots and imaging
EGF receptor
EGF-QD
Live imaging of receptor mediated endocytosis
Lidke et al. Nat. Biotechnol. 2004, 22:198

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

17. Quantum dots and imaging: FRET

18. 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

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. Multifunctional NP for Imaging
24

25. Imaging techniques and related contrast agents
X-ray  Iodinated contrast materials
Au
MRI  Gadolinium-based
Fe-based
19
F-based
PET  Radioactively labelled agents (11C, 18F)

26. 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

27. nanocage
nanorod
nanoshell
nanosphere
Size 4-40 nm

28. 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

29. 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 )

30. 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
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
.
• 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.
B
32

33. 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).

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

35. 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
36

37. 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

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

39. Formation of MCL
• magnetite NP dispersed in distilled water
• N-(α-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

40. 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
42

43. 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
43

44. Effect of Hyperthermia
Treated
Tumor
Before Treatment
Untreated
Tumor
Rectum
antitumor immune response
After Treatment

45. 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
46

47. Magnetic Nanoparticles/Carriers
M
• Magnetite Core
• Starch Polymer Coating
M
M
M
• Bioavailable
• Phosphate in coating for functionalization
• Chemo Drug attached to Coating
M
Magnetite
Core
Starch Polymer
M
M
• Mitoxantrone
• Drug Delivered to Rabbit with Carcinoma
47

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)
48

49. 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.

51. 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
52
vivo

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

54. MICROBUBBLES
• Small (1-7 µm) 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.

55. 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

56. 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

57. Shell
Air or High Molecular
Weight Gases
1-7µm

58. Preparation of microbubbles
- Homogeneization
- Sonication
- microfluidic T-devices

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

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