SLIDE 1
GOLD NANORODS AS NEW NANOCHROMOPHORES FOR PHOTOTHERMAL THERAPIES
Roberto Pini, Fulvio Ratto, Paolo Matteini, Francesca Rossi Istituto di Fisica Applicata “Nello Carrara”, Consiglio Nazionale delle Ricerche, Sesto Fiorentino (Italy)
Saratov Fall Meeting - SFM'09
September 21 - 24, 2009, Saratov, Russia
SLIDE 2 Motivations of this study Motivations of this study
The biomedical use of gold nanoparticles activated by near The biomedical use of gold nanoparticles activated by near infrared (NIR) light is an intriguing perspective, which takes infrared (NIR) light is an intriguing perspective, which takes advantage of the good transmittance of biological tissues advantage of the good transmittance of biological tissues in a window between ~ 700 and 1300 nm in a window between ~ 700 and 1300 nm These nanoparticles may be delivered to selected tissues, These nanoparticles may be delivered to selected tissues, and then triggered from a remote NIR laser to perform and then triggered from a remote NIR laser to perform minimally invasive diagnostics, therapeutics and sensing minimally invasive diagnostics, therapeutics and sensing As a model example of photothermal therapy, we tested the As a model example of photothermal therapy, we tested the use of aqueous colloids of gold nanorods in the laser use of aqueous colloids of gold nanorods in the laser welding of eye tissues and arteries welding of eye tissues and arteries Perspectives on the use of gold nanoparticles in tumor Perspectives on the use of gold nanoparticles in tumor diagnostics and therapy will be also discussed diagnostics and therapy will be also discussed
IFAC IFAC-
CNR
SLIDE 3 Background
Combination of near-infrared (NIR) lasers and NIR chromophores → Minimally invasive photothermal therapies Model therapy: NIR laser welding of connective tissues (already in the clinical phase in Ophthalmology) Standard NIR chromophores:
applied topically
SLIDE 4 Introducing laser welding of ocular tissues Introducing laser welding of ocular tissues Our approach: NIR laser + ICG Our approach: NIR laser + ICG
Utilization of diode laser radiation at 810 nm (in both CW and Pulsed emissions) in association with the topical application of ICG (indocyanine green) as the photo-enhancing chromophore (optical absorption peak ~ 800 nm). Advantages in comparison with
Lower laser doses (CW: ~ 100 mW, 12 W/cm2) (Pulsed: ~ 50 mJ, 100 ms) localized welding only in presence
Absorption spectrum
Diode laser emission line IFAC IFAC-
CNR
SLIDE 5 Our clinical experience in laser corneal Our clinical experience in laser corneal welding in penetrating, lamellar and welding in penetrating, lamellar and endothelial transplants endothelial transplants
substitution or as a support to conventional suturing
femtosecond laser corneal sculpturing
transplants in patients Advantages:
- Lesser inflammation
- Stable post-op astigmatism
- Faster healing time
Disadvantages:
prepared at time of surgery IFAC IFAC-
CNR
F.Rossi, P.Matteini, F.Ratto,L.Menabuoni, I.Lenzetti, R. Pini Laser tissue welding in ophthalmic surgery Journal of Biophotonics, 1 (4), pp. 331-342 (2008).
SLIDE 6 Motivations for new chromophores
ICG proved effective in many welding applications The range of biomedical applications of ICG is limited: Organic dyes suffer from:
- Limited extinction efficiency;
- Limited stability in the body;
- Limited biochemical flexibility
Objective: To replace organic dyes by innovative solutions → To extend their use to relevant applications, such as photothermal
- r photoacoustic treatment of
cancer.
SLIDE 7 E
+
Pros:
- Extremely efficient;
- Well stable;
- Chemically flexible (e.g. for drug delivery)
- Wavelength absorption at around 520 nm
Light irradiation excites localised plasmon resonances → Near field enhancement (~800-fold
{Cubukcu, Appl. Phys. Lett. 89, 093120 (2006)});
Rayleigh scattering; Optical absorption: Luminescence (e.g. e-h recombination) (TPL
~60 times brighter than rhodamine {Wang, Proc. Nat.
- Acad. Sci. 102, 15752 (2005)});
Electron-phonon coupling…
(Molar extinction higher by ~5 orders of magnitude than ICG {Jain, J. Phys. Chem. B 110, 7238 (2006)})
SLIDE 8 E
+
40 – 80 nm
Gold nanorods
NIR radiation excites localised plasmon resonances → Near field enhancement (~800-fold
{Cubukcu, Appl. Phys. Lett. 89, 093120 (2006)});
Rayleigh scattering; Optical absorption: Luminescence (e.g. e-h recombination) (TPL
~60 times brighter than rhodamine {Wang, Proc. Nat.
- Acad. Sci. 102, 15752 (2005)});
Electron-phonon coupling…
(Molar extinction higher by ~5 orders of magnitude than ICG {Jain, J. Phys. Chem. B 110, 7238 (2006)})
Pros:
- Extremely efficient;
- Well stable;
- Chemically flexible (e.g. for drug delivery)
- Wavelength tunable (in the range 700 nm – 1000 nm)
11.4 nm r; 3.1 A.R. 11.4 nm r; 3.9 A.R. Extinction Scattering Absorption
P.K. Jain et al., J. Phys. Chem. B 110, 7238 (2006)
SLIDE 9 Introducing the new Introducing the new nanostructured nanostructured chromphores chromphores for NIR lasers: Gold Nanorods for NIR lasers: Gold Nanorods
Colloidal gold nanorods have been synthesized in a seed-mediated approach from reduction of gold ions from HAuCl4 Typical sizes: 40-80 nm in length, 10-20 nm in width Exceptional optical absorption in the NIR is due to excitation of longitudinal surface plasmon resonances
IFAC IFAC-
CNR
SLIDE 10 The Power of Gold Nanorods The Power of Gold Nanorods as novel NIR as novel NIR chromphores chromphores
Comparison with organic chromophores:
- Tuning of the NIR optical absorption
peak, which depends on the ratio between length and width of the nanorod;
- Greater efficiency: optical absorption
coefficient ~ 5 orders of magnitude larger than ICG!!!;
- Improved chemical and thermal stability;
- Improved photo-bleaching threshold;
- Possibility to target specific tissues via
functionalization; IFAC IFAC-
CNR
11.4 nm r; 3.1 A.R. 11.4 nm r; 3.9 A.R. 21.9 nm r; 3.9 A.R. Extinction Scattering Absorption
P.K. Jain et al., J. Phys. Chem. B 110, 7238 (2006)
SLIDE 11 Biomedical applications of gold nanorods
Therapeutics: Welding; Hyperthermia; Disruption
Selectivity at the single-cell level!
Diagnostics: Scattering; Luminescence (incl. FLIM); Photoacoustics
Resolution at the single-cell level!
Biosensing: Enhancement of Raman scattering
(by up to 14 orders of magnitude {Arya, Phys. Rev. B 74, 195438 (2006)});
Enhancement of luminescence And a variety of non biomedical applications…
SLIDE 12 Biomedical applications of gold nanorods: therapeutics
The group of El-Sayed demonstrated the efficient and selective photothermal therapy
- f cancer cells irradiated by a
CW near infrared laser. Gold nanorods conjugated with suitable antibodies are retained by the cancer cells selectively. As a consequence, much lower NIR laser fluences are required to induce a photothermal damage in the cancer cells (which retain the gold nanorods) than in the healthy cells (which do not retain the gold nanorods).
- X. Huang, I.H. El-Sayed, M.A. El-Sayed, J. Am. Chem. Soc. 128, 2115 (2006)
Photothermal
SLIDE 13 Biomedical applications of gold nanorods: therapeutics
Gold nanorods were conjugated with folate and therefore adhered to the membranes
- f the cancer cells selectively.
Then cavitation micro-bubbles were produced under FS laser irradiation. These micro-bubbles disrupted the membrane of cancer cells, which causes immediate cell death or induced apoptosis.
T.B. Huff, M.N. Hansen, A. Wei, J.X. Cheng, Adv.
(2007) Photoacoustic The group of Wei demonstrated an even better efficiency and selectivity by the use
laser pulses, which induce a photoacoustic effect.
SLIDE 14 Biomedical applications of gold nanorods: diagnostics
The group of Wei demonstrated the possibility to image individual gold nanorods in a blood flow by two photon luminescence. The group of Niidome (Japan) used gold nanorods conjugated with suitable antibodies as an efficient and selective contrast agent to image cancer cells using darkfield microscopy (which exploits the intense Rayleigh scattering from the gold nanorods).
- H. Takahashi, T. Niidome, T. Kawano, S. Yamada,
- Y. Niidome, J. Nanopart. Res. 10, 221 (2008)
Back scattering
- H. Wang, T.B. Huff, D.A. Zweifel, W. He, P.S.
Low, A. Wei, J.X. Cheng, PNAS 102, 15752 (2005) TPL
SLIDE 15 Biomedical applications of gold nanorods: diagnostics
Optoacoustic signal generated by Au-NRs detected through a 4 cm thick scattering media. Excitation induced by a ns NIR laser. The x-axis represents the time following triggering of laser pulse. Au-NRs were detectable at a concentration of 7.5·108 NRs per ml (1.25 pM). The group of Alexander Oraevsky demonstrated the possibility to detect a photoacoustic signal from gold nanorods at a concentration as low as ~ 1 pM. This allowed to map the distribution of the gold nanorods injected in a mouse.
- M. Eghtedari, A. Oraevsky, J.A. Copland, N.A. Kotov, A. Conjusteau, M. Motamedi,
Nano Lett. 7, 1914 (2007) Photoacoustic
SLIDE 16 Synthesis of gold nanorods
Self-assembly of colloids of gold nanorods → Anisotropic
nanoseeds Reduction of HAuCl4 by ascorbic acid in the presence
sustainable process CTAB drives the shape anisotropy and stabilises the suspension
SLIDE 17 Synthesis of gold nanorods
Self-assembly of colloids of gold nanorods → Anisotropic
nanoseeds Reduction of HAuCl4 by ascorbic acid in the presence
sustainable process CTAB drives the shape anisotropy and stabilises the suspension
10-10 M gold nanorods (760 nm)
SLIDE 18 Synthesis of gold nanorods
Self-assembly of colloids of gold nanorods → Anisotropic
nanoseeds Reduction of HAuCl4 by ascorbic acid in the presence
sustainable process CTAB drives the shape anisotropy and stabilises the suspension
SLIDE 19 Synthesis of gold nanorods
Self-assembly of colloids of gold nanorods → Anisotropic
nanoseeds Reduction of HAuCl4 by ascorbic acid in the presence
sustainable process Control over amount of HAuCL4 reduced → Control
- ver size of gold nanorods
600 × 400 nm2
A C D B A B C D
SLIDE 20 Synthesis of gold nanorods
Self-assembly of colloids of gold nanorods → Anisotropic
nanoseeds Reduction of HAuCl4 by ascorbic acid in the presence
sustainable process Control over kinetics of reduction of HAuCL4 → Control over shape of gold nanorods
This flexibility in the definition of the size and shape of the gold nanorods may be important for their functionalization and their interaction with cells (e.g. cell uptake), as well as for the response to near infrared laser light
Ratto, F., Matteini, P., Rossi, F., Pini, R. Size and shape control in the overgrowth of gold nanorods J Nanoparticle Research, pp. 1-8. DOI 10.1007/s11051-009-9712-0 (2009).
SLIDE 21 Materials and Methods Materials and Methods
About 10-2 M atomic gold (10-6 M gold nanorods) in an aqueous solution with 0 – 20% collagen Absorption spectrum
Diode laser line IFAC IFAC-
CNR
SLIDE 22 Laser welding of eye tissue with gold nanorods
Transplant of patches of porcine lens capsules (ex vivo)
Surgical procedure
capsule from a donor;
- 2. A few drops of a colloid of
gold nanorods deposited;
- 3. The stained capsule laid
face down onto the lens capsule from a recipient;
- 4. 40 ms, ~ (70 – 100) mJ,
810 nm diode laser spots delivered through a fiberoptic (200 μm Ø) laid onto the specimen.
Donor capsule Recipient capsule Stain Light
SLIDE 23 Application of the laser welding in lens refilling procedures: creation
- f a flap-valve on the recipent capsule for refilling operations
(already tested ex vivo with ICG, US patent)
Opening of a small capsulorhexis Phacoemulsification
Application of the stained patch Partial welding of the patch perimenter (flap valve) Lens refilling Final closure of the capsulorhexis
SLIDE 24 Mat&Meth Mat&Meth
Capsular patches from freshly enucleated porcine eyes Surgical procedure: 1 Upon excision, the capsule from the donors was sprinkled with a droplet of gold nanorod colloid; 2 The droplet was dried in air, and then the capsule was rinsed with water; 3 The stained capsule was laid face down onto the recipient’s capsule, following removal of the cornea;
IFAC IFAC-
CNR
SLIDE 25 Results Results
: welding effectiveness
The adhesion of the welded patch was by qualitatively evaluated by exerting mechanical traction Successful and reproducible pulsed welding was achieved in with 40 ms laser pulses in the range of fluences (80 - 110) J·cm-2, with satisfactory mechanical strength. These fluences did not produce any detectible effects in specimens with no gold nanorods. IFAC IFAC-
CNR
SLIDE 26 Successful spots at (100 – 140) J·cm-2 diode laser fluence Histological sections (toluidine blue stain): Collagen locally denatured and shrunk → Local temperatures well above 50 °C, up to beyond 60 μm from the interface
- F. Ratto, P. Matteini, F. Rossi, L. Menabuoni, N. Tiwari, S.K. Kulkarni, R. Pini,
Photothermal effects in connective tissues mediated by laser-activated gold nanorods, Nanomedicine: Nanotechnology, Biology, and Medicine, 5 (2), pp. 143-151 (2009).
Transplant of patches of porcine lens capsules (ex vivo)
Laser welding of eye tissue with gold nanorods
SLIDE 27
Laser welding with gold nanorods - simulations
Solution of the bioheat equation by the finite element method: → local temperature rise up to 80 ° C at the interface of the welded layers → photothermal effect well confined within the spots
25° C 80° C
SLIDE 28
Laser welding with gold nanorods: preliminary tests on arteries
Solution of GNRs dispersed in a collagen gel Welding of 1-2 mm long longitudinal cuts CW welding (0.7 W) Test of effective blood flux with no leaks Follow up observations up to 15 days Closure of cuts in the carotid artery of rabbits (in vivo)
SLIDE 29
SLIDE 30 Work in progress – functionalisation
CTAB coated gold nanorods are less than ideal:
- Partially cytotoxic;
- Environmentally labile;
- Chemically rigid
Further advances: To replace CTAB by something better (e.g. silica, PEG…) →
- Biocompatible;
- Shielded from the
environment;
functional molecules
SLIDE 31 CTAB coated gold nanorods are less than ideal:
- Partially cytotoxic;
- Environmentally labile;
- Chemically rigid
Further advances: To replace CTAB by something better (e.g. silica, PEG…) →
- Biocompatible;
- Shielded from the
environment;
functional molecules
Work in progress – functionalisation
SLIDE 32 CTAB coated gold nanorods are less than ideal:
- Partially cytotoxic;
- Environmentally labile;
- Chemically rigid
Further advances: To replace CTAB by something better (e.g. silica, PEG…) →
- Biocompatible;
- Shielded from the
environment;
(e.g. for drug delivery…)
(180 × 120) nm2 TEM micrograph of gold nanorods coated by a silica shell
Work in progress – functionalisation
SLIDE 33
We are carrying of studies on the photoacoustic effects induced by the excitation of the gold nanorods with pulses of near infrared laser light in the ns regime, in order to set up minimally invasive applications such as photoacoustic diagnosis, imaging and microsurgery of cancer.
Hot electron – cold electron coupling (some 100 fs); Electron – NP phonon coupling (a few ps); NP phonon – bio phonon coupling (several 100 ps)… V.P. Zharov et al., J. Phys. D 38, 2571 (2005)
Under ns pulse irradiation these dynamics develop simultaneously
The process depends on a delicate balance of properties of the nanoparticles and the nano–bio interfaces, which may dynamically change over time →
A thorough experimental exploration of pulse parameters such as power and duration is most interesting
Work in progress – photoacoustic effects mediated by gold NPs
SLIDE 34 Perspectives – Applications in cancer diagnosis and therapy
Upon systemic delivery, the functionalised gold nanordods will target the cancer cells selectively. Then they will be irradiated by a near-infrared laser, to generate diagnosis, imaging and therapy
1) At low laser power, the photoacoustic effects will result into the generation of ultrasound, which will be used for the molecular diagnosis and imaging of the cancer cells, even in small metastases; 2) At higher laser power, the photothermal and photoacoustic response will lead to the formation of cavitation micro- bubbles, which will damage the membranes and induce the apoptosis of the cancer cells with high efficiency, selectivity and non-invasiveness.
SLIDE 35 10-9 M gold nanorods (760 nm) in PVA irradiated (left) for 10 s at 0.5 W (810 nm diode laser, 300 μm fiberoptic); and (right) for 700 s with 2*104 W, 15 ns, 10 Hz pulses (760 nm Ti : sapphire laser, 200 μm fiberoptic).
We are exploring these concepts from colloids of gold nanorods dispersed into biologically relevant media Example 2: Studies in cell cultures and small animals are underway in cooperation with the Dept of Pathologic Clinics at the Univ. of Florence . Example 1: Investigation on the stability of the gold nanorods (immobilized in PVA phantoms)
Work in progress – photoacoustic effects
SLIDE 36 CONCLUSIONS
NIR excited gold nanorods hold the promise of manifold biomedical applications The synthesis of functional gold nanorods is highly sustainable and offers novel strategies to control different parameters, such as their average size and shape (which may give a substantial flexibility in the definition of their interaction with cells and tissues) We demonstrated in vitro and in vivo that gold nanorods are suitable exogenous chromophores in welding
In tumor therapy, gold nanorods may provide cancer cells apoptosis by photothermal and photoacoustic effects
SLIDE 37
Thank Thank you you for for your your time time
ACKNOWLEDGEMENTS: Dr Luca Menabuoni, Dr. Ivo Lenzetti Unità Operativa Oculistica Azienda USL 4 Prato (Italy) Dr Alfredo Puca, Dr Giuseppe Esposito Istituto di Neurochirurgia, Policlinco Gemelli, Roma Dr Guido Toci IFAC-CNR, Sesto Fiorentino (Italy) Dr Neha Tiwari, Prof Sulabha K. Kulkarni Department of Physics, University of Pune, Pune (India)
