The morpho-functional assessment of plasmonic photothermal therapy e ® ects on transplanted liver tumor

A. B. Bucharskaya*||, G. N. Maslyakova*, G. A. Afanasyeva*, G. S. Terentyuk*†, N. A. Navolokin*, O. V. Zlobina*, D. S. Chumakov, A. N. Bashkatov, E. A. Genina, N. G. Khlebtsov†‡, B. N. Khlebtsov and V. V. Tuchin†¶ *Saratov State Medical University n.a. V.I. Razumovsky, Russia Saratov State University n.a. N.G. Chernyshevsky, Russia Institute of Biochemistry and Physiology of Plants and Microorganisms RAS, Saratov, Russia Institute of Precise Mechanics and Control RAS, Saratov, Russia ¶Optoelectronics and Measurement Techniques Laboratory University of Oulu, Oulu, Finland ||allaalla_72@mail.ru


Introduction
Currently the development of novel medical technologies that can improve the results of treatment of malignant tumors is the urgent area of research.
One of the well established approaches for cancer treatment is laser-induced interstitial thermotherapy (LIIT). 1,2The major drawback of LIIT is its low spatial selectivity at tissue heating, a®ecting both tumor and surrounding healthy tissue.The selectivity can be signi¯cantly increased at staining a target tissue by indocyanine green dye.This photothermal therapy technology was pioneered by Chen and nowadays it is used in combination with the immunoadjuvant that helps to enhance therapeutic e±ciency of cancer treatment. 3][6][7][8][9][10][11][12][13][14] For applications in medicine, the resonant optical properties of nanostructures should be con¯gured to ¯t the wavelength range of transparency window of biological tissues. 15Spectral tuning of plasmon resonance of nanoparticles and the relation between their absorption and scattering was implemented by changing the size, shape, form and structure of the nanoparticles. 14][9][10] Such technology has been developed over the last decade and it was named a plasmonic photothermal therapy (PPTT). 16he possibility of spectral tuning of plasmon resonance of nanoparticles in the \therapeutic window of transparency" and achieving an acceptable ratio between their absorption and scattering e±ciencies was reported. 14,17old nanorods have a high brightness and increased sensitivity of resonant scattering to the dielectric environment as compared with equivolume spheres.The main optical resonance of nanorods is easily adjusted by changing their axial ratio. 18It causes promising prospects of their use for optical photothermolysis of tumor cells.
A number of authors [19][20][21] have shown that excessive formation of free radicals and inactivation of antioxidant enzyme on the background of antitumor laser therapy ensures the accumulation of toxic products of oxidative stress intensifying the severity of endogenous tumor intoxication.Secondary metabolic disorders are accompanied by toxic damage of cell membranes of various internal organs and tissues, which is often the limiting factor for further treatment.Therefore, it is necessary to assess the e®ectiveness and safety of new method of selective laser hyperthermia of malignant tumors.
The objectives of our work were to identify the morphological changes in the internal organs and transplanted liver tumors and to assess the degree of autointoxication of laboratory animals exposed to PPTT.

Methods and Materials
Animal experiments were performed in accordance with the guidance hInternational Guiding principles for Biomedical Research Involving Animalsi. 22 total of 30 male outbred albino rats weighing 160 AE 20 g were used to study the e®ect of laser hyperthermia with a single intratumoral injection of gold nanorods.0.5 mL of 25% tumor cell suspension of alveolar liver cancercholangiocarcinoma PC-1, obtained from the bank of tumor strains of Russian Cancer Research Center n.a.N.N.Blokhin (Moscow, Russia), was implanted subcutaneously in rats.When the tumor reached a diameter of 1:0 AE 0:3 cm, the animals were randomly divided into three groups (10 rats in each group): group 1 without exposure (comparison group), group 2 with the laser irradiation of the tumor, group 3 with intratumoral administration of gold nanorods and laser irradiation of the tumor.As control group 10 nontumor bearing animals were used.
Gold nanorods were synthesized by previously established method. 18To prevent nanoparticles aggregation in biological tissue and enhance biocompatibility, nanoparticles were functionalized with thiolated polyethyleneglycol (Mw ¼ 5000, Nektar, USA) by previously established method. 23eometrical parameters of gold nanorods were determined from analysis of transmission electron microscopy (TEM) images (Libra-120, Carl Zeiss, Germany) by using standard graphic editor.
Extinction spectrum of the sample was measured with a Specord 250 BU spectrophotometer (Analytik, Jena, Germany).Before measurements, the nanorods suspension was diluted to 1:10 and was placed in 2 mm quartz cuvette.
Nanorods zeta-potential measurements were made with a Zetasizer Nano-ZS instrument (Malvern, UK).In a good agreement with previously reported data, 23 pegilated nanorods have slightly negative zeta-potential (À14 mV).
The infrared 808-nm CW laser LS-2-N-808-10000 (Laser Systems, Ltd., St. Petersburg, Russia) at a power density of 2.3 W/cm 2 was used for PPTT.The area of the laser spot on the skin surface was about 0.5 cm 2 .An hour before laser irradiation, the animals were injected intratumorally with the solution of gold nanorods in the amount of 30% of the tumor volume.
The injection of the gold nanoparticles directly into the tumor was conducted according to the paper of Xie and co-workers, 24 where the authors demonstrated that using such a modi¯cation of intratumoral injection leads to prolonged retention of nanoparticles in tumor tissue.
Size of the nanorods was 41 AE 8 nm long and 10 AE 2 nm wide and concentration of nanorods in the suspension was 400 pg/mL, which corresponds to optical density of 20 at this wavelength.Irradiation was carried out percutaneously within the area of a tumor during 15 min.Temperature control of the tumor heating was provided by IR imager IRI4010, Infrared Integrated System (IRYSYS, UK).
Prior to any medical procedure or treatment, the rats were anaesthetized with Zoletil 50 (Virbac, France) in dose of 0.05 mg/kg.The removal of animal from the experiment was performed 24 h after laser exposure.The tissue samples of internal organs and tumors for morphological studies were collected.The standard histological techniques with hematoxylin and eosin staining were used.
The study of lipid peroxidation activity was carried out using measuring of intermediate products of lipid peroxidationmalondialdehyde (MDA) and lipid hydroperoxide (GPL)in the blood serum of experimental animals with standard biochemical methods on spectro°uorometer RF-5301 PC (Shimadzu Corp., Japan).MDA was determined by reaction with 2-thiobarbituric acid.The absorbance of samples was measured by spectrophotometric recording at ¼ 532 nm, the concentration of MDA was expressed in M/mL.The method of hydroperoxide determination was based on absorption of ultraviolet radiation by lipid hydroperoxide at the wavelength of 233 nm.The results were expressed in relative optical density units per 1 mL of plasma.The severity of autointoxication was assessed by integrative indicatorthe level of average mass molecules (AMM) in the blood serum of rats.The coarse serum proteins were precipitated by adding 10% trichloroacetic acid solution which was centrifuged for 20 min at 3000 rev/min.The supernatant was then examined in a spectrophotometer at the wavelength of 254 nm.The results were expressed in relative optical density units per 1 mL of plasma. 25

Results and Discussion
As shown in Fig. 1(a), the average particle shape corresponds to cylinders with hemispherical ends.The number of particles with another shape, such as with °at ends and lesser particle diameter for the nanorod middle was very small.Length, diameter and the axial ratio of each particle were determined for an ensemble of about 500 particles.Average values for the length and diameter of rods were 41 AE 8 nm and 10:2 AE 2 nm, respectively.Such geometrical parameters lead to strong plasmon resonance in NIR region (810-820 nm).In agreement with this speculation, the longitudinal resonances are located near 810 nm [Fig.1(b)].The ratio of major to minor resonance maximum equals indicating that the percentage of impurity particles (sphere, cubes, etc.) is small. 18On the other hand, The morpho-functional assessment of PPTT e®ects 1541004-3 the full width at half maximum (FWHM) is about 200 nm, which is typical of the moderate polydispersity of Au NRs over length and width.
The inset in Fig. 1(a) shows the histogram of the numerical distribution of the particles ÁN i =Ár i on the axial ratio r i ¼ L i =d i with the average value r ¼ 4:03 AE 0:7.
Morphologically tumors in the comparison group of rats had a lobed structure; segments were separated by thin layers of connective tissue.Tumor cells had oval-rounded shape with eccentrically located nuclei.A signi¯cant portion of cytoplasm was occupied by large vacuoles containing mucus.There were clusters of mucous masses in the intercellular spaces.
Simultaneously, a signi¯cant increase in the level of MDA (p < 0:001), GPL (p < 0:001) and AMM (p < 0:001) was found in the serum as compared with those in the group of nontumor bearing animals.This indicates the activation of lipid peroxidation and the development of autointoxication in experimental animals with cholangiocarcinoma (Table 1).
In morphological studies of animals with cholangiocarcinoma, there were no changes in the liver, kidney and spleen in comparison with the control group.The study of liver tissue demonstrated that architectonic structure was intact, the moderate congestion in small capillaries and necrosis of individual hepatocytes were observed after laser hyperthermia.The moderate congestion in large vessels and necrosis of individual hepatocytes were detected in the liver after PPTT with gold nanorods (Fig. 2).
In kidney, moderate congestion in the glomeruli and foci of hemorrhage between the tubules were observed after laser hyperthermia.Foci of hemorrhage, the swelling between the capsule of glomerulus, large full-blooded vessels were observed after PPTT (Fig. 3).
The morphological changes were not pronounced in the organs of detoxi¯cation because of the short time between the start of exposure and deducing animals from the experiment.
In spleen, after laser hyperthermia, the signs of lymphoid cell activation were not observed in the  white pulp.Increasing of follicles with germinal center and density of lymphoid cells in periarterial zone (Fig. 4), as well as immunoblast predominance were observed in spleen after PPTT with gold nanoparticles.These changes in spleen are a sign of the immune system activation.At only laser exposure, the tumor temperature increased up to 42 C.The morphologic study of tumors in group 2 (only laser treatment) proved that the tumor maintained a lobed structure.A small foci of necrosis were noted (5-10% of the total area of tissue); the necrotic changes in the tumor cells occurred; the single mitosis was identi¯ed; the vessels were full blooded.Thickening of the connective tissue septa, in¯ltration of leukocytes and focal hemorrhages were observed [Figs.5(c) and 5(d)].
At the same time MDA, GPL and AMM levels were still signi¯cantly higher than those control indicators.The absence of signi¯cant morphological changes in the tumor in this series of experiments was accompanied by preservation of the activity of lipid peroxidation processes.However, the level of AMM grew progressively (Table 1).
We noted a signi¯cant increase in tumor temperature (up to 60 C) in experiment with PPTT of tumorsusing laser irradiation and gold nanorods as nanosensitizers.The pronounced necrotic changes developed in the tumor center, which occupied up to 80-90% of the slice.The tumor cells with marked degenerative changes were located in subcapsular tumor zone [Figs.5(e) and 5(f)].
The formation of local necrosis in organs and tissues was accompanied by the development of systemic toxicity and metabolic disorders in the body.In a series of experiments on animals with cholangiocarcinoma, the excessive accumulation of MDA (p2 < 0:001) was revealed in the serum of rats after PPTT exposure compared with group of animals exposed to laser irradiation without the use of nanoparticles.
The growth of cholangiocellular cancer is accompanied by increase of the lipid peroxidation activity and the development of severe autointoxication compared to intact animals.This is con¯rmed by an increase in the content of the intermediate products of lipid peroxidation -MDA and the GPLand the accumulation of AMM excess in serum.The obtained results are consistent with literature data, indicating that tumor growth in the body violates oxidative balance and causes activation of free radical oxidation. 17,21These shifts are due to changes occurring on the stages of transformation of tumor cells, tumor progression and promotion at the local and systemic levels, as well as associated with the manifestations of functional, biochemical and morphological atypia of tumor cells.
In previous experiments, we found there were no signi¯cant di®erences of morphofunctional features in group of tumor bearing animals after single intratumoral injection of gold nanorods.
Our data on the activation of free radical oxidation in the blood serum of animals after using PPTT are consistent with the results of the authors, [19][20][21] who discovered the intensi¯cation of lipid peroxidation, a progressive increase in lipid peroxidation products in the serum of patients with malignant neoplasms after complex therapy including surgery, radiation therapy and adjuvant combination chemotherapy.Activation of free lipid accumulation in animal serum lipid peroxidation products as a result of exposure to laser radiation on the tumor may have an ambiguous interpretation.Free radical destabilization of biological membranes of cells exposed to laser hyperthermia shows e®ect on tumor cells and con¯rms the e®ectiveness of the treatment with using nanosensitizers according to Wang. 26t the same time, the radiation e®ect along with the cytotoxic e®ect on malignant cells may provide a pronounced cytotoxic e®ect on cells of various tissues and organs outside the tumor.Formation system paraneoplastic metabolic disorders in the form of accumulation of lipid peroxidation products in the systemic circulation as a result of laser hyperthermia can signi¯cantly intensify the severity of the disease and contribute to the development of secondary dysfunctions of organs and body systems on the background of autointoxication.
In this regard, it seems promising to study the application of antihypoxants, antioxidants, membrane protectors and immunoadjuvant to improve the e®ectiveness of PPTT therapy for malignant tumors.

Conclusion
The most pronounced necrotic processes in the tumor tissue were detected after PPTT with gold nanorods as nanosensitizers.The accumulation of intermediate products of lipid peroxidation and the development of autointoxication were revealed after PPTT with gold nanorods that may be the leading pathogenetic factors that can result in damage of membranes of tumor cells at PPTT.The results not only demonstrate the antitumor e±cacy of the proposed therapeutic technology but also reveal side e®ects in the presence of peroxidation products in systemic circulation.

Fig. 1 .
Fig. 1.(a) TEM image of gold nanorods.The insert in Fig. 1(a) shows the histogram of the numerical distribution of the particles ÁN i =Ár i on the axial ratio r i ¼ L i =d i with the average value r ¼ 4:03 AE 0:7.(b) Extinction spectrum of gold nanorod suspension.

Fig. 2 .
Fig. 2. Histological sections of liver: (a) Comparison group (rats with PC-1).The moderate congestion of the capillaries.(b) Group with laser hyperthermia.The necrosis of individual hepatocytes.(c) Group with PPTT.The hepatocyte necrosis.Extension of vein of portal tract.The blood separation phenomenon -Staining with hematoxylin and eosin.(Â400).

Fig. 3 .Fig. 4 .
Fig. 3. Histological sections of kidney: (a) Comparison group (rat with PC-1).The moderate congestion in the glomeruli.(b) Group with laser hyperthermia.The moderate congestion in the glomeruli, foci of hemorrhage between the tubules.(c) Group with PPTT.The swelling between the capsule of glomerulus.Staining with hematoxylin and eosin.(Â774).

Fig. 5 .
Fig. 5. Histological sections of a liver tumor: (a), (b) Comparison group (rats with PC-1).Tumor lobe with intact tumor cells.(c), (d) Group with laser hyperthermia.(c) Tumor lobe with degenerative changes of cells.(d) The leukocyte in¯ltration of the connective tissue septa, congestion of vessels.(e), (f) Group with PPTT.(e) Pronounced necrosis in tumor center.(f ) The dystrophic changes in the tumor cells of subcapsular zone.Hematoxylin and eosin.(Â774).

Table 1 .
Indicators of lipid peroxidation and autointoxication of rats.Note: pwas counted compared with control group; p1was counted compared with liver tumor group; p2was counted compared with liver tumor group after laser hyperthermia; Maverage value; mstandard deviation.