The molecular markers in blood serum and transplanted liver tumor of rats as

indicators of effectiveness of plasmonic resonance photothermal therapy  

  

Alla B. Bucharskaya, Saratov State Medical University , RussiaGalina N. Maslyakova, Saratov State Medical University, RussiaNikita A. Navolokin, Saratov State Medical University, RussiaNatalya B. Zakharova, Saratov State Medical University, RussiaGeorgy S. Terentyuk, Saratov State Medical University, Saratov State University, RussiaAlexey N. Bashkatov, Saratov State University, RussiaElina A. Genina, Saratov State University, RussiaValery V. Tuchin, Saratov State University, RussiaBoris N. Khlebtsov, Institute of Biochemistry and Physiology of Plants and Microorganisms RAS, RussiaNikolai G. Khlebtsov, Institute of Biochemistry and Physiology of Plants and Microorganisms RAS, Saratov State University, Russia

to evaluate the molecular markers in blood and transplanted liver tumor of rats for control of effectiveness of plasmonic resonance photothermal therapy.

PURPOSE OF THE RESEARCH

TEM imaging of gold nanorods

Materials and methods30 mature outbred male rats with transplanted cholangiocarcinoma line PC-1

Gold nanorods (GN) coated silica and functionalized hematoporphyrin. Synthesized in IBPPM RAS, Saratov 

(L= 102 ±12 nm, d= 75 ± 6  nm, r= 1,4 ± 0,2)Max  extinction – 810 nm, 632 nm, optical density 20 

4 experimental groups and comparison group of white outbred rats (6 animals per group)Intratumor or intravenous injection of GN

The infrared 808-nm CW laser LS-2-N-808-10000 (Russia) at a power density 2.3 W/cm2 was used for plasmonic resonance photothermal therapy (PRPT) – 15 min. 

ELISA test (TNF-α, IGF-1, VEGF-C)Morphological and immunohistochemical studies   with Ki-67, p53, FAS-L, CD95 (Abcam, UK)

Animals with PC-1 tumors were divided into groups:• Comparison group - without any exposure • Experimental group 1. Intravenous administration of a

suspension of nanorods. • Experimental group 2. Laser irradiation of tumors.• Experimental group 3. Intravenous administration of a

suspension of nanorods. PRPT.• Experimental group 4. Intratumoral administration of a

suspension of nanorods. PRPT.The withdrawal of the animal from the experiment was performed in 24 hours after laser exposure. The blood serum for ELISA test and tissue samples of tumors for morphological studies were collected. The evaluation of TNF-α (Bioscience), VEGF-C (Bioscience), IGF-1 (Novozymes) was conducted using special ELISA kits.

Design of experiment

Results: effects of PRPT

Comparison group. PC-1 (x246,4)

Group 2. Irradiation of infrared laser (x246,4)

The tumor in group 2 (only laser treatment) kept lobed structure. There were small foci of necrosis (5-10% of the total area of tissue), the tumor cells with necrotic changes were noted. The vessels were full-blooded, thickening of the connective tissue septa, small foci of hemorrhages and infiltration of leukocytes were noted. At combined action of laser radiation and intravenous administration of gold nanoparticles the apparent necrotic changes in the tumor tissue were revealed - the tumor cells with degenerative changes persisted only in subcapsular zone, tumor necrosis occupied up to 80-90% of the area of the slice.

Group 3. PC-1 after PRPT(x246,4)

Immunohistochemical studies of PC-1 tumor biopsies

The expression decreasing of proliferation marker Ki-67 and the increasing of expression of marker p53 (suppressor of tumor growth) were noted after PRPT.

a) b)

c) d)

a)b) - expression of proliferation marker Ki-67 before and after PRPT (x774).c)d) - expression of marker p53 before and after PRPT (x246,4)

Immunohistochemical studies of PC-1 tumor biopsies

a)b) - expression of apoptosis marker FAS-L before and after PRPT (x246,4).c)d) - expression of apoptosis marker CD95 before and after PRPT (x246,4)

a) b)

c) d)

The increasing of expression of apoptosis markers FAS-L and CD95 after PRPT.

ELISA test: results

The increasing of amount of TNF-α and VEGF-C decreasing of IGF-1 were revealed in rat serum PC-1 after intravenous administration of nanorods and PRPT.

The decreasing of IGF-1 was revealed in rat serum PC-1, most pronounced in the group with intravenous administration of nanorods and PRPT.

ConclusionsThe results indicate that PRPT with gold nanorods has a significant damaging effect, which manifests in severe necrotic and degenerative changes in the tumor cells. The increasing of amount of inflammation markers in rat serum after PRPT may be due to the formation of the systemic inflammatory response, accompanied by the activation of the release of proinflammatory cytokines and angiogenesis factors. We noted the activation of apoptotic processes in the surviving tumor cells after PRPT. The levels of molecular markers in serum and transplanted tumors may serve as indicators of effectiveness of plasmonic resonance photothermal therapy.