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BioNanoScience ISSN 2191-1630Volume 7Number 1 BioNanoSci. (2017) 7:216-221DOI 10.1007/s12668-016-0320-z

Plasmonic Photothermal Therapy ofTransplanted Tumors in Rats at MultipleIntravenous Injection of Gold Nanorods

A. B. Bucharskaya, G. N. Maslyakova,N. I. Dikht, N. A. Navolokin,G. S. Terentyuk, A. N. Bashkatov,E. A. Genina, B. N. Khlebtsov, et al.

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Plasmonic Photothermal Therapy of Transplanted Tumorsin Rats at Multiple Intravenous Injection of Gold Nanorods

A. B. Bucharskaya1 & G. N. Maslyakova1 & N. I. Dikht1 & N. A. Navolokin1&

G. S. Terentyuk1,2& A. N. Bashkatov2,3 & E. A. Genina2,3 & B. N. Khlebtsov2,4 &

N. G. Khlebtsov2,4 & V. V. Tuchin2,3,5

Published online: 17 October 2016# Springer Science+Business Media New York 2016

Abstract The aim of this study was to evaluate the morpholog-ical changes in tumor tissue in rats with transplanted liver cancerPC-1 after repeated intravenous (IV) administration of goldnanorods (GNRs) and plasmonic photothermal therapy (PPT).GNRs with aspect ratio 4.1 and plasmonic peak at 810 nm werefunctionalized with thiolated polyethylene glycol and IVadmin-istered at a single dose 0.4 mg of Au and by repeated injection ofthe same dose for 2 and 3 days (the total doses were 0.8 and1.2 mg of Au, respectively). One day after the last IV injection ofGNRs, the tumors were irradiated by an 808-nmNIR diode laserat a power density 2.3 W/cm2 during 15 min. The withdrawal ofthe animals from the experiment and sampling of the tissues formorphological study and GNR distribution were performed 24 hafter the PPT. Repeated IV administration of GNRs in tumor-bearing rats resulted in the highest accumulation of nanoparticlesin both liver and spleen tissues. After triplicate IV injection ofGNRs, they accumulated in the tumor and related PPT effectswere comparable with those observed after direct intratumoralinjection of the single GNR dose.

Keywords Goldnanorods .Plasmonicphotothermal therapy .

Transplanted tumors

1 Introduction

In oncology, laser hyperthermia is applied for a long time as atreatment method based on laser heating and destruction oftumors. The effects of hyperthermia on mammalian cells arecomplex. In addition to multiple effects on cellular physiolo-gy, 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 oc-cur in mammalian cells during exposure to 40–45 °C for mod-erate periods of time (15∼60 min), and numerous cellularfunctions damaging or inactivation have been identified [1].

Currently, the thermosensitizers are widely used to increasethe efficiency of laser hyperthermia; there are substanceswhich can efficiently absorb the laser radiation and convertit to heat. One of the most effective and promising methods oflaser therapy is a plasmonic photothermal therapy, which usesgold nanoparticles as thermosensitizers [2].

The relevance of gold nanoparticles application is deter-mined by electrochemical and optical properties of the colloidalgold, in particular, their surface plasmon resonance. For practi-cal purposes, it is preferable to use thermosensitizers for absorb-ing light in the near infrared (NIR) region (700–1000 nm),where the absorption of biological tissues themselves is mini-mal, in the therapeutic transparency window of tissues [3].Thus, the plasmon resonance of gold nanoparticles for in vivouse should be tuned to the NIR range.

Recently, several research groups reported the use of vari-ous gold nanoparticles such as nanoshells, nanorods,nanocages, etc. for the plasmon resonance hyperthermia[4–9]. The use of gold nanorods (GNRs) for photothermaltherapy is preferred due to their colloidal stability and easytuning of nanorod plasmon resonance to the laser wavelengthby changing the nanorod aspect ratio [10]. To improve thebiocompatibility of the nanoparticles and to enhance their

* A. B. Bucharskayaallaalla_72@mail.ru

1 Saratov State Medical University n.a. V.I. Razumovsky,Saratov, Russia

2 Saratov National Research State University, Saratov, Russia3 National Research Tomsk State University, Tomsk, Russia4 Institute of Biochemistry and Physiology of Plants and

Microorganisms, RAS, Saratov, Russia5 Institute of Precision Mechanics and Control, RAS, Saratov, Russia

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stability, different biocompatible polymers are applied [11]. Alonger circulation time and better accumulation in tumorsshow nanoparticles coated with neutrally charged polymers,including polyethylene glycol (PEG) [12].

We have previously tested the method of photothermalplasmon resonance therapy in tumor-bearing rats with alveolarliver cancer PC-1 at intratumoral administration of gold nano-rods [13]. The solution of PEGylated gold nanorods with con-centration of 400 μg/mL (length of 41 ± 8 nm and diameter of10 ± 2 nm 6, absorption maximum at the wavelength of808 nm) was administered intratumorally in a volume corre-sponding to 30 % of tumor volume. Laser exposure was donepercutaneously over the tumor for 15 min in 1 h after nano-particle 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 every30 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 first2 min of irradiation. Twenty-four hours after laser-inducedhyperthermia, animals were withdrawn from the experiment,and the marked changes were revealed at morphological studyof tumors. Survived tumor cells with degenerative changeswere detected only in the subcapsular area of the tumors.

Unfortunately, this technique has some limitations associ-ated with the intratumoral injection of nanoparticles, which ispossible only at a superficial tumor localization. In addition,intratumoral administrationmay result in localized damages inthe tumor that can provoke its metastasis.

Analyzing the data of existing methods for laser hyperther-mia with IVadministration of nanoparticles, we observed thatthe effectiveness of laser hyperthermia depends on a rightchoice of treatment time after nanoparticle administration.The accumulation of nanoparticles in tumor tissue increasesdramatically the temperature gradient between the tumor andthe surrounding healthy tissue, providing local heating of thetumor. This makes the laser-induced thermal imaging of atumor and reduces the negative effects of laser irradiation onsurrounding healthy tissues. However, the optimal doses ofGNR suspension needed for effective gold accumulation intumors and optimal protocols of plasmonic photothermaltherapy (PPT) have not been reported until now.

The aim of this study was to evaluate the morphologicalchanges in transplanted liver tumors after multiple IV adminis-tration 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 syn-thesized in the Laboratory of Nanobiotechnology (Institute of

Biochemistry and Physiology of Plants and MicroorganismsRAS, Saratov, Russia) by previously reported method [14]as described in detail elsewhere [15]. In brief, goldBseeds^ were prepared by the addition of 0.1 mL of anice-cold 10 mM sodium borohydride solution to 1 mL ofaqueous CTAB followed by the addition of 0.025 mL ofHAuCl4. Then, 2 mL of 4 mM AgNO3, 5 mL of 10 MHAuCl4, 1 mL of 100 mM ascorbic acid, 1 mL of 1 MHCl, and 1 mL of the gold Bseed^ solution were sequen-tially added to 90 mL of 0.1 M CTAB. The mixture waskept undisturbed overnight at 30 °C. To prevent nanopar-ticle aggregation in a tissue and enhance biocompatibility,nanoparticles were functionalized with thiolated polyeth-ylene glycol (MW = 5000, Nektar, USA) as reported pre-viously [15]. Geometrical parameters of gold nanorodswere determined from analysis of transmission electronmicroscopy (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 concentrationof nanorod suspension was 400 μg/mL, which corre-sponds to optical density of 20 at 810 nm.

2.2 In Vivo Experiments

The experiments were performedwith 30 healthymature albinomale rats (weight 180–220 g) according to the University’sAnimal Ethics Committee and the relevant national agencyregulating animal experiments in Centre of Collective Use ofSaratov State Medical University. The International GuidingPrinciples for Biomedical Research Involving Animals of theCouncil for the International Organization of Medical Sciencesand International Council for Laboratory Animal Science werefollowed during the animal experiments [16]. The experimentalmodel of rat liver cancer (cholangiocarcinoma line PC-1) wasreproduced by transplantation of tumor cell suspension, obtain-ed from the bank of tumor strains of Russian Cancer ResearchCenter n.a. N.N. Blokhin. 0.5 mL of 25 % tumor cell suspen-sion in Hank’s solution was implanted subcutaneously in theshoulder 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 con-trol), 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 doubleinjections of 1 mL of GNR suspension for each daily injection(total dose is 0.8 mg of Au) and PPT, group 5—with tripleinjections of 1 mL of GNR suspension for each daily injection(total dose is 1.2 mg of Au) and PPT. One day after the lastinjection of GNRs, the tumors were irradiated by an 808-nmNIR 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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IR imager IRI4010 (IRYSYS, UK). Prior to all medicalprocedures or treatments, the rats were anesthetized withZoletil 50 (Virbac, France) in a dose of 0.05 mg/kg. Forcomparison, the data of our previous experiment withintratumoral administration of GNR and PTTT wereused [13]. The withdrawal of the animals from the ex-periment and sampling of tissues for morphologicalstudy and gold distribution were performed 24 h afterPPT. The morphological examination of tumor tissueswas performed according the standard histological pro-tocol. In brief, formalin fixed tissues were embedded inparaffin, the tissue sections of a 5-μm thickness wereobtained and, after dewaxing, the sections were stainedwith H&E. The gold distribution in the liver, spleen,kidney, and tumor was evaluated by atomic absorptionspectroscopy (AAS) with a Dual Atomizer Zeeman AAiCE 3500 spectrophotometer (Thermo Scientific Inc.,USA). The process of tissue sample preparation wascarried out in an automatic mode with constant controlof the temperature in a microwave system «MARSXprees» (USA).

3 Results and Discussion

For rats with a single GNR injection, the tumor temperatureincreased from 35 up to 40 °C during PPT (Fig. 1, curve 2)and the temperature rise was comparable with that observedwith only laser irradiation (Fig. 1, curve 1). The AAS datashowed that gold accumulation in the tumor tissue after single

IV GNR administration was insignificant (Table 1); thus, thetemperature was increased due to only laser radiation hyper-thermic action.

In the group without treatment, the tumors had a lobedstructure; tumor cells had oval-rounded shape with eccentri-cally located nuclei (Fig. 2a). After PPT therapy, the small fociof necrosis were noted in tumors, which take 20–30 % of slicearea; the tumor cells with necrotibiotic changes were noted ina small amount (Fig. 2c). These morphological changes werecomparable to similar changes in the tumor with only lasertreatment (Fig. 2b).

For rats with the double GNP injection, we observed theincrease of tumor temperature up to 45.3 °C at PPT (Fig. 1,curve 3). The gold content in the tumor tissue increased almost9 times (up to 1.24 ± 0.01 μg/g) compared to the group with asingle injection (Table 1). The more pronounced necroticchanges were revealed in the tumor tissue after PPT; tumornecrosis occupied up to 30–50 % of slice area (Fig. 2d).

Finally, for rats with 3-fold nanoparticle injection, we ob-served the increase of tumor temperature up to 68.2 °C at PPT(Fig. 1, curve 4), and the temperature rise was comparable withthat achived for PPT after direct intratumoral GNR injection(Fig. 1, graph 5), as found previously for intratumoral GNRinjection [12]. The gold content in the tumor tissue significantlyincreased (up to 10.67 ± 0.39 μg/g) compared to group with asingle injection (Table 1). The most pronounced necroticchanges were revealed in tumor tissue in this group of rats afterPPT, tumor necrosis occupied up to 70–80 % of slice area, thetumor cells with necrotibiotic changes were presented only insubcapsular zone (Fig. 2e). Damage effects were comparable to

Fig. 1 TEM image of gold nanorods (a). Thermography of tumors (1)after only laser treatment; (2) after PPT with single IV administration ofGNRs; (3) after PPT with double IV administration of GNRs; (4) after

PPT with triple IV administration of GNRs; (5) after PPT treatment withintratumoral administration of GNRs (5) (b)

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similar changes in the tumor after PPTwith direct intratumoralGNR-injection (Fig. 2f). The gold content was significantlyincreased in spleen tissue after single and, in particular, aftermultiple injections of GNRs; the marked gold accumulationwas noted in the liver after triple IV injection of GNRs.

The results indicate that triple IV administration of goldnanorods and follow-up by PPT caused the most pronouncednecrotic changes in transplanted liver tumors.

Currently, several approaches have been proposed to in-crease the nanoparticle accumulation in tumor, including themultiple dosing strategy [12, 17, 18]. Specifically, a recentstudy by Wang et al. [12] has demonstrated the maximal goldaccumulation in the tumor and in the internal organs in 24 and72 h, respectively, after single IV injection of 200 μL of GNRs(60 × 15 nm, length and diameter) at a concentration of0.1 mg/mL to Balb/c mice inoculated with mammary carcino-ma 4T1. In a work by Puvanakrishnan et al. [17], a repeateddosing strategy was used for IV injection of PEGylated 135-nm gold nanoshells and 24 × 7 nmGNRs in Swiss nu/nu micewith subcutaneous CRL-155 tumors. The greatest accumula-tion of nanoparticles in tumors has been achieved for triplicateadministration of GNRs with 24 h intervals. Our main findingis that the repeated IV injection of GNRs enhances the efficacyof PPT and inhibits the tumor growth similarly to the PPTinhibition after a direct intratumoral injection at a comparablesingle dose. This conclusion is in agreement with results by

Puvanakrishnan et al. [17] and El-Sayed et al. [18] revealingenhanced accumulation of GNRs in tumors after repeated IVinjection. In the study by El-Sayed et al. [18], the solution ofPEGylated gold nanorods (length of 60 ± 5 and aspect ratio of4.6, absorption maximum at the wavelength of 800 nm) wasadministered intravenously to Balb/c mice inoculated withEhrlich carcinoma at a dose of 1.5 mg/kg every three weeks.Previously, the evaluation of gold concentration in the tumorby atomic absorption spectroscopy showed that the maximumaccumulation of gold in intertwined tumors observed 3 daysafter IV administration of GNRs. Seven days after GNR IVadministration, mice were treated by a diode laser with a pow-er of 50 W/cm2 for 2 min, i.e., fluence of 6.0 kJ/cm2. Thetumors were heated up to 79 °C, the PPT effects were com-pared to intratumoral administration of GNRs. For protocoldescribed in this paper, by providing a 3-fold less fluence, ofonly 2.1 kJ/cm2, tumors were heated up to 65 °C. Thus, wehave a greater potential to control tumor temperature at eleva-tion of laser power density which was 22 times less than thatused 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 tempera-ture increase at triplicate NP administration (Fig. 1b). Thatallows for much shorter laser exposures of 1.0–1.5 min to beused in order to reach needed temperatures of 70–80 °C byelevation of laser power density up to 5–10 W/cm2 (see alsothe related theoretical consideration in [19]).

Fig. 2 Liver cancer PC-1 withouttreatment (a), after only lasertreatment (b), after PPT with asingle IV administration of GNRs(c), after PPTwith a double IVadministration of GNRs (d), afterPPT with a triple IV administra-tion of GNRs (e), and after PPTwith intratumoral administrationof GNRs (f). H&E staining, mag-nification ×246.6

Table 1 The concentration ofgold in tissue after IVadministration of GNRs

Tissue Group without

treatment

Single injection Double injection Triple injection

Concentration of gold (μg/g)

Tumor 0.09 ± 0.01 0.14 ± 0.02 1.24 ± 0.01 10.67 ± 0.39a

Liver 0.27 ± 0.02 1.72 ± 0.20 2.91 ± 0.27 9.30 ± 0.94a

Spleen 0.49 ± 0.03 11.38 ± 1.40a 19.09 ± 1.20a 69.27 ± 7.58a

Kidney 0.03 ± 0.01 0.72 ± 0.06 2.33 ± 0.28a 2.38 ± 0.28a

Data are presented as mean ± standard deviation. p < 0.05was taken as a cutoff value for significancea The significant differences with control group

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It is known that the toxicity of GNRs directly correlatesto accumulation in internal organs, nonetheless, the reticu-loendothelial system such as the liver and spleen [20].Recent studies revealed application of different techniquesto visualize the biodistribution of nanomaterials in biolog-ical tissues. In particular, Naumenko et al. [21] applied theenhanced dark-field microscopy to visualize the distribu-tion of halloysite nanotubes in tissues. Currently, variousoptical approaches, including the elemental imaging with3D capabilities, are used for visualization of nanomaterialsat the whole-body scale [22, 23]. However, a common useof such techniques is limited by their cost and complexity.Thus, determination of the optimal dosage and time inter-vals of GNR-administration remains under further discus-s ion. Unfor tunate ly, survived cancer cel ls wi thnecrotibiotic changes may retain in subcapsular zone oftumor after PPT treatment, and then may be responsiblefor tumor regrowth. Further studies are needed for devel-opment of optimal PPT protocols. One of the ways is toassess the degree of tumor vascularization to select theoptimal mode of GNR administration and follow-up PPT.

4 Conclusion

In this work, we have shown that the multiple IV injec-tion of gold nanorods and further PPT of transplantedliver tumors in rats result in significant damaging effectwhich was manifested in pronounced necrotic and degen-erative changes of cancer cells. The antitumor effects ofPPT after triple IV injection were comparable with thoseobtained at direct intratumoral administration of similartotal dose of GNRs.

Increasing of the effectiveness of treatment was due toa maximal accumulation of gold in tumors after multipleIV administration of gold nanoparticles. Further researchshould be focused on the development of criteria for eval-uation of the effectiveness and safety of the proposedPPT protocol.

Acknowledgments The work of BNK and NGK was supported bygrant No. 14-13-01167 from the Russian Science Foundation. Theexperimental and morphological studies done by ABB, GNM, NIDand NAN were conducted by the state assignment of RussianMinistry of Health. The work on laser treatment and light doseevaluation done by ANB, EAG, and VVT was supported by grantNo. 14-15-00186 of the Russian Science Foundation. The authors thankDr. S.V. Eremina (Department of English and InterculturalCommunication of Saratov State University) for the help in manuscripttranslation to English.

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