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'09September 21 - 24, 2009, Saratov, Russia

Motivations of this studyMotivations 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 t issues advantage of the good transmittance of biological t issues in a window between ~ 700 and 1300 nmin a window between ~ 700 and 1300 nm

These nanoparticles may be delivered to selected ti ssues, These nanoparticles may be delivered to selected ti ssues, and then triggered from a remote NIR laser to perfo rm and then triggered from a remote NIR laser to perfo rm minimally invasive diagnostics, therapeutics and se nsingminimally invasive diagnostics, therapeutics and se nsing

As a model example of photothermal therapy, we test ed the As a model example of photothermal therapy, we test ed the use of aqueous colloids of gold nanorods in the las er use of aqueous colloids of gold nanorods in the las er welding of eye tissues and arterieswelding of eye tissues and arteries

Perspectives on the use of gold nanoparticles in tu mor Perspectives on the use of gold nanoparticles in tu mor diagnostics and therapy will be also discusseddiagnostics and therapy will be also discussed

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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: organic dyes such as ICG, applied topically

Introducing laser welding of ocular tissuesIntroducing laser welding of ocular tissuesOur approach: NIR laser + ICGOur approach: NIR laser + ICG

Utilization of diode laser radiation at 810 nm (in both CW and Pulsed emissions) in association with the topical applicat ion of ICG(indocyanine green) as the photo-enhancing chromophore (optical absorption peak ~ 800 nm).

Advantages in comparison with other laser approaches:

� Lower laser doses (CW: ~ 100 mW, 12 W/cm 2)(Pulsed: ~ 50 mJ, 100 ms)

� localized welding only in presence of ICG

Absorption spectrum of ICG in corneal tissue

Diode laser emission line

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Our clinical experience in laser corneal Our clinical experience in laser corneal welding in penetrating, lamellar and welding in penetrating, lamellar and endothelial transplantsendothelial transplants

• Laser welding in substitution or as a support to conventional suturing

• Combination with femtosecond laser corneal sculpturing

• More than 100 corneal transplants in patients

Advantages :• Lesser inflammation• Stable post-op astigmatism• Faster healing timeDisadvantages :• ICG solution must be

prepared at time of surgery

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F.Rossi, P.Matteini, F.Ratto,L.Menabuoni, I.Lenzett i, R. PiniLaser tissue welding in ophthalmic surgery Journal of Biophotonics, 1 (4), pp. 331-342 (2008).

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 or photoacoustic treatment of cancer .

E

+

-

Gold nanorods

Pros:• Extremely efficient;• Well stable;• Chemically flexible (e.g. for drug delivery)• Wavelength absorption at around 520 nm

Light irradiation excites localisedplasmon 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)})

E

+

-

10 – 20 nm

40 –80 n

m

Gold nanorodsNIR radiation excites localisedplasmon 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.

ExtinctionScatteringAbsorption

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Introducing the new Introducing the new nanostructurednanostructuredchromphoreschromphores for NIR lasers: Gold Nanorodsfor 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

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The Power of Gold Nanorods The Power of Gold Nanorods as novel NIR as novel NIR chromphoreschromphores

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;

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11.4 nm r; 3.1 A.R.

11.4 nm r; 3.9 A.R.

21.9 nm r; 3.9 A.R.

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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…

Biomedical applications of gold nanorods: therapeut ics

The group of El-Sayeddemonstrated the efficient and selective photothermal therapy of 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 lowerNIR 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

Biomedical applications of gold nanorods: therapeut ics

Gold nanorods were conjugated with folate and therefore adhered to the membranes of 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.

L. Tong, Y. Zhao, T.B. Huff, M.N. Hansen, A. Wei, J.X. Cheng, Adv. Mater. 19, 3136 (2007) Photoacoustic

The group of Wei demonstrated an even better efficiency and selectivity by the use of ultrashort NIR laser pulses, which induce a photoacoustic effect.

Biomedical applications of gold nanorods: diagnosti cs

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

Biomedical applications of gold nanorods: diagnosti cs

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 Oraevskydemonstrated 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

Synthesis of gold nanorods

Self-assembly of colloids of gold nanorods → Anisotropic overgrowth of gold nanoseeds

Reduction of HAuCl4 by ascorbic acid in the presence of CTAB → Highly sustainable process

CTAB drives the shape anisotropy and stabilises the suspension

Synthesis of gold nanorods

Self-assembly of colloids of gold nanorods → Anisotropic overgrowth of gold nanoseeds

Reduction of HAuCl4 by ascorbic acid in the presence of CTAB → Highly sustainable process

CTAB drives the shape anisotropy and stabilises the suspension

10-10 M gold nanorods (760 nm)

Synthesis of gold nanorods

Self-assembly of colloids of gold nanorods → Anisotropic overgrowth of gold nanoseeds

Reduction of HAuCl4 by ascorbic acid in the presence of CTAB → Highly sustainable process

CTAB drives the shape anisotropy and stabilises the suspension

Synthesis of gold nanorods

Self-assembly of colloids of gold nanorods → Anisotropic overgrowth of gold nanoseeds

Reduction of HAuCl4 by ascorbic acid in the presence of CTAB → Highly sustainable process

Control over amount of HAuCL4 reduced → Control over size of gold nanorods

600

×40

0 nm

2

A

C D

B

A

B

C

D

Synthesis of gold nanorods

Self-assembly of colloids of gold nanorods → Anisotropic overgrowth of gold nanoseeds

Reduction of HAuCl4 by ascorbic acid in the presence of CTAB → Highly sustainable process

Control over kinetics of reduction of HAuCL4 →

Control over shape of gold nanorods

A: slow; B: fast200 × 200 nm2

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).

Materials and Methods Materials and Methods ��������

Chromophore preparation :

About 10 -2 M atomic gold (10-6 M gold nanorods)

in an aqueous solution

with 0 – 20% collagen

Absorption spectrum of Gold Nanorods in H2O

Diode laser line

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Laser welding of eye tissue with gold nanorodsTransplant of patches of porcine lens capsules (ex vivo)

Surgical procedure

1. Removal of the lens 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 capsuleRecipient capsule

Stain

Light

Application of the laser welding in lens refilling procedure s: creationof a flap-valve on the recipent capsule for refilling op erations

(already tested ex vivo with ICG, US patent)

Opening of a smallcapsulorhexis

Phacoemulsificationof the lens

Application of the stained patch

Partial welding of the patch perimenter (flap valve)

Lens refilling Final closure of the capsulorhexis

Mat&MethMat&Meth ��������

Animal model :

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;

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ResultsResults ��������: welding effectiveness : welding effectiveness

� The adhesion of the welded patch was by qualitatively evaluated by exerting mechanical traction

� Successful and reproducible pulsed welding was achiev ed 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 effec ts in specimens with no gold nanorods.

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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 μmfrom the interface

F. Ratto, P. Matteini, F. Rossi, L. Menabuoni, N. T iwari, 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

Laser welding with gold nanorods - simulations

Solution of the bioheatequation 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

Laser welding with gold nanorods: preliminary tests on arteries

Solution of GNRsdispersed 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)

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;• Better conjugation with functional molecules

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;• Better conjugation with functional molecules

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;• Further functionalizable (e.g. for drug delivery…) (180 × 120) nm2 TEM micrograph of

gold nanorods coated by a silica shell

Work in progress – functionalisation

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 simultaneouslyThe 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 paramete rs such as power and duration is most interesting

Work in progress – photoacoustic effects mediated by gold NPs

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 of cancer:

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.

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

CONCLUSIONS

NIR excited gold nanorods hold the promise of manif old biomedical applications

The synthesis of functional gold nanorods is highly sustainable and offers novel strategies to control different parameters, such as their average size an d shape (which may give a substantial flexibility in the definition of their interaction with cells and tiss ues)

We demonstrated in vitro and in vivo that gold nanorods are suitable exogenous chromophores in welding operations

In tumor therapy, gold nanorods may provide cancer c ells apoptosis by photothermal and photoacoustic effects

ThankThank youyou forfor youryour timetime

ACKNOWLEDGEMENTS:

Dr Luca Menabuoni, Dr. Ivo LenzettiUnità Operativa Oculistica Azienda USL 4 Prato (Italy)

Dr Alfredo Puca, Dr Giuseppe EspositoIstituto di Neurochirurgia, Policlinco Gemelli, Roma

Dr Guido TociIFAC-CNR, Sesto Fiorentino (Italy)

Dr Neha Tiwari, Prof Sulabha K. KulkarniDepartment of Physics, University of Pune, Pune (Ind ia)