BioNanoSci. DOI 10.1007/s12668-016-0320-z
Plasmonic Photothermal Therapy of Transplanted Tumors in Rats at Multiple Intravenous Injection of Gold Nanorods A. B. Bucharskaya 1 & G. N. Maslyakova 1 & N. I. Dikht 1 & N. A. Navolokin 1 & G. S. Terentyuk 1,2 & A. N. Bashkatov 2,3 & E. A. Genina 2,3 & B. N. Khlebtsov 2,4 & N. G. Khlebtsov 2,4 & V. V. Tuchin 2,3,5
# Springer Science+Business Media New York 2016
Abstract The aim of this study was to evaluate the morphological changes in tumor tissue in rats with transplanted liver cancer PC-1 after repeated intravenous (IV) administration of gold nanorods (GNRs) and plasmonic photothermal therapy (PPT). GNRs with aspect ratio 4.1 and plasmonic peak at 810 nm were functionalized with thiolated polyethylene glycol and IV administered at a single dose 0.4 mg of Au and by repeated injection of the same dose for 2 and 3 days (the total doses were 0.8 and 1.2 mg of Au, respectively). One day after the last IV injection of GNRs, the tumors were irradiated by an 808-nm NIR diode laser at a power density 2.3 W/cm2 during 15 min. The withdrawal of the animals from the experiment and sampling of the tissues for morphological study and GNR distribution were performed 24 h after the PPT. Repeated IV administration of GNRs in tumorbearing rats resulted in the highest accumulation of nanoparticles in both liver and spleen tissues. After triplicate IV injection of GNRs, they accumulated in the tumor and related PPT effects were comparable with those observed after direct intratumoral injection of the single GNR dose. Keywords Gold nanorods . Plasmonic photothermal therapy . Transplanted tumors * A. B. Bucharskaya
[email protected]
1
Saratov State Medical University n.a. V.I. Razumovsky, Saratov, Russia
2
Saratov National Research State University, Saratov, Russia
3
National Research Tomsk State University, Tomsk, Russia
4
Institute of Biochemistry and Physiology of Plants and Microorganisms, RAS, Saratov, Russia
5
Institute of Precision Mechanics and Control, RAS, Saratov, Russia
1 Introduction In oncology, laser hyperthermia is applied for a long time as a treatment method based on laser heating and destruction of tumors. The effects of hyperthermia on mammalian cells are complex. In addition to multiple effects on cellular physiology, relatively short exposure to temperature in excess of 40– 41 °C inhibits cancer cell growth due to cytotoxicity. Extensive protein denaturation has been demonstrated to occur in mammalian cells during exposure to 40–45 °C for moderate periods of time (15∼60 min), and numerous cellular functions damaging or inactivation have been identified [1]. Currently, the thermosensitizers are widely used to increase the efficiency of laser hyperthermia; there are substances which can efficiently absorb the laser radiation and convert it to heat. One of the most effective and promising methods of laser therapy is a plasmonic photothermal therapy, which uses gold nanoparticles as thermosensitizers [2]. The relevance of gold nanoparticles application is determined by electrochemical and optical properties of the colloidal gold, in particular, their surface plasmon resonance. For practical purposes, it is preferable to use thermosensitizers for absorbing light in the near infrared (NIR) region (700–1000 nm), where the absorption of biological tissues themselves is minimal, in the therapeutic transparency window of tissues [3]. Thus, the plasmon resonance of gold nanoparticles for in vivo use should be tuned to the NIR range. Recently, several research groups reported the use of various gold nanoparticles such as nanoshells, nanorods, nanocages, etc. for the plasmon resonance hyperthermia [4–9]. The use of gold nanorods (GNRs) for photothermal therapy is preferred due to their colloidal stability and easy tuning of nanorod plasmon resonance to the laser wavelength by changing the nanorod aspect ratio [10]. To improve the biocompatibility of the nanoparticles and to enhance their
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stability, different biocompatible polymers are applied [11]. A longer circulation time and better accumulation in tumors show nanoparticles coated with neutrally charged polymers, including polyethylene glycol (PEG) [12]. We have previously tested the method of photothermal plasmon resonance therapy in tumor-bearing rats with alveolar liver cancer PC-1 at intratumoral administration of gold nanorods [13]. The solution of PEGylated gold nanorods with concentration of 400 μg/mL (length of 41 ± 8 nm and diameter of 10 ± 2 nm 6, absorption maximum at the wavelength of 808 nm) was administered intratumorally in a volume corresponding to 30 % of tumor volume. Laser exposure was done percutaneously over the tumor for 15 min in 1 h after nanoparticle injection using 808-nm laser LS-2-N-808-10000 (St.Petersburg, Russia) with a power density of 2.3 W/cm2. Temperature control of tumor heating was performed every 30 s using infrared thermograph IRI4010 (IRYSYS, UK). During the laser irradiation, a significant rise in temperature (up to 65 ± 2 °C) was found, most pronounced in the first 2 min of irradiation. Twenty-four hours after laser-induced hyperthermia, animals were withdrawn from the experiment, and the marked changes were revealed at morphological study of tumors. Survived tumor cells with degenerative changes were detected only in the subcapsular area of the tumors. Unfortunately, this technique has some limitations associated with the intratumoral injection of nanoparticles, which is possible only at a superficial tumor localization. In addition, intratumoral administration may result in localized damages in the tumor that can provoke its metastasis. Analyzing the data of existing methods for laser hyperthermia with IV administration of nanoparticles, we observed that the effectiveness of laser hyperthermia depends on a right choice of treatment time after nanoparticle administration. The accumulation of nanoparticles in tumor tissue increases dramatically the temperature gradient between the tumor and the surrounding healthy tissue, providing local heating of the tumor. This makes the laser-induced thermal imaging of a tumor and reduces the negative effects of laser irradiation on surrounding healthy tissues. However, the optimal doses of GNR suspension needed for effective gold accumulation in tumors and optimal protocols of plasmonic photothermal therapy (PPT) have not been reported until now. The aim of this study was to evaluate the morphological changes in transplanted liver tumors after multiple IV administration of a constant dose of gold nanorods (GNRs) and PPT.
2 Material and Methods 2.1 Preparation and Characterization of GNRs For photothermal experiments, the gold nanorods were synthesized in the Laboratory of Nanobiotechnology (Institute of
Biochemistry and Physiology of Plants and Microorganisms RAS, Saratov, Russia) by previously reported method [14] as described in detail elsewhere [15]. In brief, gold Bseeds^ were prepared by the addition of 0.1 mL of an ice-cold 10 mM sodium borohydride solution to 1 mL of aqueous CTAB followed by the addition of 0.025 mL of HAuCl4. Then, 2 mL of 4 mM AgNO3, 5 mL of 10 M HAuCl4, 1 mL of 100 mM ascorbic acid, 1 mL of 1 M HCl, and 1 mL of the gold Bseed^ solution were sequentially added to 90 mL of 0.1 M CTAB. The mixture was kept undisturbed overnight at 30 °C. To prevent nanoparticle aggregation in a tissue and enhance biocompatibility, nanoparticles were functionalized with thiolated polyethylene glycol (MW = 5000, Nektar, USA) as reported previously [15]. Geometrical parameters of gold nanorods were determined from analysis of transmission electron microscopy (TEM) images (Libra-120, Carl Zeiss, Germany) in Centre of Collective Use of IBPPM RAS (Fig. 1a). The nanorod dimensions were 41 ± 8 nm (length) and 10 ± 2 nm (diameter), and the concentration of nanorod suspension was 400 μg/mL, which corresponds to optical density of 20 at 810 nm. 2.2 In Vivo Experiments The experiments were performed with 30 healthy mature albino male rats (weight 180–220 g) according to the University’s Animal Ethics Committee and the relevant national agency regulating animal experiments in Centre of Collective Use of Saratov State Medical University. The International Guiding Principles for Biomedical Research Involving Animals of the Council for the International Organization of Medical Sciences and International Council for Laboratory Animal Science were followed during the animal experiments [16]. The experimental model of rat liver cancer (cholangiocarcinoma line PC-1) was reproduced by transplantation of tumor cell suspension, obtained from the bank of tumor strains of Russian Cancer Research Center n.a. N.N. Blokhin. 0.5 mL of 25 % tumor cell suspension in Hank’s solution was implanted subcutaneously in the shoulder area of rats. When the tumor reached a diameter of 3.0 ± 0.3 cm3, the animals were randomly divided into five groups (6 rats in each group): group 1—without treatment (blank control), group 2—after only laser treatment (laser effect cotrol), group 3—with a single injection of 1 mL of GNR suspension (total dose is 0.4 mg of Au) and PPT, group 4—with double injections of 1 mL of GNR suspension for each daily injection (total dose is 0.8 mg of Au) and PPT, group 5—with triple injections of 1 mL of GNR suspension for each daily injection (total dose is 1.2 mg of Au) and PPT. One day after the last injection of GNRs, the tumors were irradiated by an 808-nm NIR diode laser LS-2-N-808-10000 (Laser Systems, Ltd., St.Petersburg, Russia) during 15 min at a power density 2.3 W/ cm2. Temperature control of the tumor heating was provided by
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Fig. 1 TEM image of gold nanorods (a). Thermography of tumors (1) after only laser treatment; (2) after PPT with single IV administration of GNRs; (3) after PPT with double IV administration of GNRs; (4) after
PPT with triple IV administration of GNRs; (5) after PPT treatment with intratumoral administration of GNRs (5) (b)
IR imager IRI4010 (IRYSYS, UK). Prior to all medical procedures or treatments, the rats were anesthetized with Zoletil 50 (Virbac, France) in a dose of 0.05 mg/kg. For comparison, the data of our previous experiment with intratumoral administration of GNR and PTTT were used [13]. The withdrawal of the animals from the experiment and sampling of tissues for morphological study and gold distribution were performed 24 h after PPT. The morphological examination of tumor tissues was performed according the standard histological protocol. In brief, formalin fixed tissues were embedded in paraffin, the tissue sections of a 5-μm thickness were obtained and, after dewaxing, the sections were stained with H&E. The gold distribution in the liver, spleen, kidney, and tumor was evaluated by atomic absorption spectroscopy (AAS) with a Dual Atomizer Zeeman AA iCE 3500 spectrophotometer (Thermo Scientific Inc., USA). The process of tissue sample preparation was carried out in an automatic mode with constant control of the temperature in a microwave system «MARS Xprees» (USA).
IV GNR administration was insignificant (Table 1); thus, the temperature was increased due to only laser radiation hyperthermic action. In the group without treatment, the tumors had a lobed structure; tumor cells had oval-rounded shape with eccentrically located nuclei (Fig. 2a). After PPT therapy, the small foci of necrosis were noted in tumors, which take 20–30 % of slice area; the tumor cells with necrotibiotic changes were noted in a small amount (Fig. 2c). These morphological changes were comparable to similar changes in the tumor with only laser treatment (Fig. 2b). For rats with the double GNP injection, we observed the increase of tumor temperature up to 45.3 °C at PPT (Fig. 1, curve 3). The gold content in the tumor tissue increased almost 9 times (up to 1.24 ± 0.01 μg/g) compared to the group with a single injection (Table 1). The more pronounced necrotic changes were revealed in the tumor tissue after PPT; tumor necrosis occupied up to 30–50 % of slice area (Fig. 2d). Finally, for rats with 3-fold nanoparticle injection, we observed the increase of tumor temperature up to 68.2 °C at PPT (Fig. 1, curve 4), and the temperature rise was comparable with that achived for PPT after direct intratumoral GNR injection (Fig. 1, graph 5), as found previously for intratumoral GNR injection [12]. The gold content in the tumor tissue significantly increased (up to 10.67 ± 0.39 μg/g) compared to group with a single injection (Table 1). The most pronounced necrotic changes were revealed in tumor tissue in this group of rats after PPT, tumor necrosis occupied up to 70–80 % of slice area, the tumor cells with necrotibiotic changes were presented only in subcapsular zone (Fig. 2e). Damage effects were comparable to
3 Results and Discussion For rats with a single GNR injection, the tumor temperature increased from 35 up to 40 °C during PPT (Fig. 1, curve 2) and the temperature rise was comparable with that observed with only laser irradiation (Fig. 1, curve 1). The AAS data showed that gold accumulation in the tumor tissue after single
BioNanoSci. Table 1 The concentration of gold in tissue after IV administration of GNRs
Tissue
Group without
Single injection
Double injection
Triple injection
1.24 ± 0.01 2.91 ± 0.27 19.09 ± 1.20a 2.33 ± 0.28a
10.67 ± 0.39a 9.30 ± 0.94a 69.27 ± 7.58a 2.38 ± 0.28a
treatment Concentration of gold (μg/g) Tumor Liver Spleen Kidney
0.09 ± 0.01 0.27 ± 0.02 0.49 ± 0.03 0.03 ± 0.01
0.14 ± 0.02 1.72 ± 0.20 11.38 ± 1.40a 0.72 ± 0.06
Data are presented as mean ± standard deviation. p < 0.05was taken as a cutoff value for significance a
The significant differences with control group
similar changes in the tumor after PPT with direct intratumoral GNR-injection (Fig. 2f). The gold content was significantly increased in spleen tissue after single and, in particular, after multiple injections of GNRs; the marked gold accumulation was noted in the liver after triple IV injection of GNRs. The results indicate that triple IV administration of gold nanorods and follow-up by PPT caused the most pronounced necrotic changes in transplanted liver tumors. Currently, several approaches have been proposed to increase the nanoparticle accumulation in tumor, including the multiple dosing strategy [12, 17, 18]. Specifically, a recent study by Wang et al. [12] has demonstrated the maximal gold accumulation in the tumor and in the internal organs in 24 and 72 h, respectively, after single IV injection of 200 μL of GNRs (60 × 15 nm, length and diameter) at a concentration of 0.1 mg/mL to Balb/c mice inoculated with mammary carcinoma 4T1. In a work by Puvanakrishnan et al. [17], a repeated dosing strategy was used for IV injection of PEGylated 135nm gold nanoshells and 24 × 7 nm GNRs in Swiss nu/nu mice with subcutaneous CRL-155 tumors. The greatest accumulation of nanoparticles in tumors has been achieved for triplicate administration of GNRs with 24 h intervals. Our main finding is that the repeated IV injection of GNRs enhances the efficacy of PPT and inhibits the tumor growth similarly to the PPT inhibition after a direct intratumoral injection at a comparable single dose. This conclusion is in agreement with results by Fig. 2 Liver cancer PC-1 without treatment (a), after only laser treatment (b), after PPT with a single IV administration of GNRs (c), after PPT with a double IV administration of GNRs (d), after PPT with a triple IV administration of GNRs (e), and after PPT with intratumoral administration of GNRs (f). H&E staining, magnification ×246.6
Puvanakrishnan et al. [17] and El-Sayed et al. [18] revealing enhanced accumulation of GNRs in tumors after repeated IV injection. In the study by El-Sayed et al. [18], the solution of PEGylated gold nanorods (length of 60 ± 5 and aspect ratio of 4.6, absorption maximum at the wavelength of 800 nm) was administered intravenously to Balb/c mice inoculated with Ehrlich carcinoma at a dose of 1.5 mg/kg every three weeks. Previously, the evaluation of gold concentration in the tumor by atomic absorption spectroscopy showed that the maximum accumulation of gold in intertwined tumors observed 3 days after IV administration of GNRs. Seven days after GNR IV administration, mice were treated by a diode laser with a power of 50 W/cm2 for 2 min, i.e., fluence of 6.0 kJ/cm2. The tumors were heated up to 79 °C, the PPT effects were compared to intratumoral administration of GNRs. For protocol described in this paper, by providing a 3-fold less fluence, of only 2.1 kJ/cm2, tumors were heated up to 65 °C. Thus, we have a greater potential to control tumor temperature at elevation of laser power density which was 22 times less than that used in Ref. [18]. We would also like to stress that only 1.0– 1.5 min is needed to reach a saturation in an abrupt temperature increase at triplicate NP administration (Fig. 1b). That allows for much shorter laser exposures of 1.0–1.5 min to be used in order to reach needed temperatures of 70–80 °C by elevation of laser power density up to 5–10 W/cm2 (see also the related theoretical consideration in [19]).
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It is known that the toxicity of GNRs directly correlates to accumulation in internal organs, nonetheless, the reticuloendothelial system such as the liver and spleen [20]. Recent studies revealed application of different techniques to visualize the biodistribution of nanomaterials in biological tissues. In particular, Naumenko et al. [21] applied the enhanced dark-field microscopy to visualize the distribution of halloysite nanotubes in tissues. Currently, various optical approaches, including the elemental imaging with 3D capabilities, are used for visualization of nanomaterials at the whole-body scale [22, 23]. However, a common use of such techniques is limited by their cost and complexity. Thus, determination of the optimal dosage and time intervals of GNR-administration remains under further discuss i o n . U n f o r t u n a t e l y, s u r v i v e d c a n c e r c e l l s w i t h necrotibiotic changes may retain in subcapsular zone of tumor after PPT treatment, and then may be responsible for tumor regrowth. Further studies are needed for development of optimal PPT protocols. One of the ways is to assess the degree of tumor vascularization to select the optimal mode of GNR administration and follow-up PPT.
4 Conclusion In this work, we have shown that the multiple IV injection of gold nanorods and further PPT of transplanted liver tumors in rats result in significant damaging effect which was manifested in pronounced necrotic and degenerative changes of cancer cells. The antitumor effects of PPT after triple IV injection were comparable with those obtained at direct intratumoral administration of similar total dose of GNRs. Increasing of the effectiveness of treatment was due to a maximal accumulation of gold in tumors after multiple IV administration of gold nanoparticles. Further research should be focused on the development of criteria for evaluation of the effectiveness and safety of the proposed PPT protocol.
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15. Acknowledgments The work of BNK and NGK was supported by grant No. 14-13-01167 from the Russian Science Foundation. The experimental and morphological studies done by ABB, GNM, NID and NAN were conducted by the state assignment of Russian Ministry of Health. The work on laser treatment and light dose evaluation done by ANB, EAG, and VVT was supported by grant No. 14-15-00186 of the Russian Science Foundation. The authors thank Dr. S.V. Eremina (Department of English and Intercultural Communication of Saratov State University) for the help in manuscript translation to English.
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