G.S. Terentyuk, D.S. Chumakov, I.L. Maksimova Saratov State University M.V. Basko

1. Evaluation of photothermal effects induced by laser heating of gold nanorods in suspensions and inoculated tumors G.S. Terentyuk, D.S. Chumakov, I.L. Maksimova Saratov State University M.V. Basko Saratov State Medical University A.V. Ivanov N.N. Blokchin Russian Cancer Research Centre B.N. Khlebtsov, N.G. Khlebtsov IBPPM RAS, Saratov Russia SFM'13 September 25-28, 2013, Saratov, Russia

2. Background Thermal ablation of cancer is gaining increasing attention as an alternative to standard surgical therapies , especially for patients with contraindications. Potential benefits of thermal ablation include reduced morbidity and mortality in comparison with standard surgical resection and the ability to treat nonsurgical patients. There is a wide range of ablation techniques that include : cryoablation, radiofrequency ablation, microwave ablation, ultrasound ablation and laser ablation.

3. Plasmonic photothermal therapy (PPTT) is a technique of cancer thermal therapy based on the laser heating of gold nanoparticles. One of the main advantages of this therapeutic technology is its high spatial selectivity that prevents surrounding healthy tissues from thermal damage. Advantages of gold nanoparticles as antitumor photothermal agents: • Unique optical properties • Photostability • Low toxicity • Well-known synthesis protocols Manthe R.L. et al . Mol . Pharm (2010) Dickerson et al. Chem. Soc Rev (2011)

4. Goal of the research To study photothermal effects under laser irradiation of aqueous suspensions of gold nanorods (in vitro experiments) and mice -inoculated Erlich carcinoma after intravenous injections of gold nanorods (in vivo experiments)

5. Gold nanorods (GNR) were synthesized by a seed-mediated approach and functionalized with PEG Geometrical parameters: L= 41 ± 8 nm d= 10,2 ± 2 nm r = 4,03 ± 0,7 Plasmon resonance at the wavelength820 nm Characteristics of nanoparticles Transmission electron microscopic image of gold nanorods

6. Scheme of in vitro experiments Suspension volume – 1,5 ml Duration of irraditaion– 5 min Power density - 1 W/сm2 Wavelength- 810 nm IRYSYS thermal imager was placed at the distance 37 cm of the test tube

7. Dependence of the temperature increment ∆T on the concentration of gold nanorods in the suspension after irradiation with laser light (810 nm, 1 W/сm2 ) during 5 min.

8. Distribution of temperature T along the axis x of test tubes under the laser heating of gold nanorod suspensions with concentrations 8 (а) and 100 (b) mg/ml. (B) (А)

9. Laboratory animals and tumour model Laboratory animals Female linear mice BALB/с Age: 3 months Body mass : 20-22 g Tumour model: Ehrlich’s ascites carcinoma The mean volume of the tumour was 1,7 ± 0,3 см2

10. Evaluation of tumor growth dynamic (7 animals) 7 animals (ААS) Scheme of in vivo experiments Experimental group № 2 Experimental group № 1 14 animals 14 animals Laser irradiation Wavelength - 810 nm Duration - 5 min Power Density- 1 W/cm2

11. Scheme of in vivo experiments Control group № 1 Control group № 2 6 animals 6 animals Laser irradiation Wavelength - 810 nm Duration - 5 min Power Density- 1 W/cm2 No irradiation Evaluation of tumor growth dynamic (4animals) Evaluation of tumor growth dynamic (4animals) 2 animals (AAS) 2 animals (AAS)

12. before laser irradiation1 min 5 min 2 D distribution of temperature over the surface of mice skin before the laser irradiation, in 1 min and in 5 min.

13. Kinetics of laser heating of tumor (1) and healthy (2) tissue in the vicinity of it maximum in 24 houres after intravenous injection of gold nanorods in the dose 2 (а) and 8 (b) mg /kg. Curve 3 shows the kinetics of the maximal heating of healthy muscle tissue without injecting the nanoparticles.

14. Biodistribution of gold nanorods in tumour and muscle (a) in liver and spleen (b) in 24 houres after intravenous injection of nanoparticles in doses 2 (1) and 8 (2) mg/kg. Curve 3 shows the corresponding distribution when injecting saline instead of nanoparticles.

15. Dependences of the tumour mean volume on the number of the days passed after perfoming a PPTT in the experimental groups with the dose of injected gold nanorods 8 (1) and 2 mg/kg (2) and for control groups with laser irradiation without injecting nanoparticles (3) and without laser irradiation (4).

16. Conclusion 1) PEG-coated nanorods with the size 40 × 10 nm in 24 houres after systemic injection to mice with inoculated Ehrlich carcinoma are accumulated in the tumor in the amount 4 mkg/g. In 24 houres after injection the concentration of nanoparticles is maximal in the spleen and liver. 2) Too high concentrations of gold nanoparticles in tumour may lead to undesirable effect of light absorption in surface layers and extremely nonuniform heat release 3) Even in the case of significant photothermal action on the tumour tissue , its complete resorption was not achieved and in 12 days after PPTT the growth of tumours recommenced. These  results  again  emphasise  the  necessity  to  use photothermotherapy in combination with chemotherapy and  radiotherapy in  order to provide high efficiency of the complex  therapy.