2.1.

Reagents

The following reagents were used to provide protocol of gold nanoparticle synthesis: tetraethyl orthosilicate (TEOS, Aldrich, Saint Louis, Missouri) tetrakis (hydroxymethyl) phosphonium chloride (THPC, Fluka, Saint Louis, Missouri), 3-aminopropyltrimethoxysilane (APTMS, Aldrich), PEG-thiol (Nektar, San Carlos, California), absolute ethanol, tetrachloroauric acid (TCAA, Aldrich), potash (Reachim Company, Russia), and formaldehyde (Serva, Heidelberg, Germany). All chemicals were of research grade, and 25% aqua ammonia was of analytical grade. Ethanol was purified by additional distillation.

2.2.

Gold Nanoparticle Characteristics and Quality Control

For experiments described in this work, the plasmonic nanoparticles (gold nanospheres and nanoshells) were fabricated in the Laboratory of Nanobiotechnology at the Institute of Biochemistry and Physiology of Plants and Microorganisms (IBPPM) RAS. The poly (ethylene glycol) molecules have been attached (using end thiol groups) to the surface of nano-particles to provide their defense from reticuloendothelial systems of the organisms under study.

Silica/gold nanoshells were fabricated as described in paper Ref. 37, with minor modifications related to reagent concentrations. First, silica nanoparticles were grown by reducing TEOS with $\mathrm{N}{\mathrm{H}}_4\mathrm{O}\mathrm{H}$ in absolute ethanol. To do this, we mixed $10\mathrm{ml}$ of absolute ethanol with $0.6\text{-}\mathrm{ml}$ 25% aqua ammonia and $0.3\text{-}\mathrm{ml}$ TEOS. For the preparation of gold seeds, $220\mu \mathrm{l}$ of $1\text{-}\mathrm{M}$ aqueous NaOH and $6\mu \mathrm{l}$ of 80% THPC were added to $20\mathrm{ml}$ of triply distilled water. The solution was vigorously agitated on a magnetic stirrer at $1000\mathrm{rpm}$ , and after that $880\mu \mathrm{l}$ of a 1% TCAA solution were added. Next, aminated silica particles were added to the gold-seed dispersion. Gold particles adsorb to the amine groups on the silica surface, resulting in silica nanoparticle covering by the gold colloid. Gold nanoshells were then grown by reacting $\mathrm{H}\mathrm{Au}{\mathrm{Cl}}_4$ with the silica/gold colloid particles in the presence of formaldehyde at room temperature. In this process, additional gold reduces on the adsorbed metal particles, which act as nucleation sites. The nanoshells were centrifuged and sonicated in HPLC-grade water before use.

The following PEGylation protocol was used for silica/gold nanoshells. The nanoshells’ solution was centrifuged at $7000\mathrm{g}$ for $20\min$ to pellet the particles, decanted, and then nanoshells were resuspended in the same amount of water. One hundred microliters of $2\text{-}\mathrm{mM}$ potassium carbonate and $10\mu \mathrm{l}$ of $1\text{-}\mathrm{mM}$ PEG-thiol solution were added per $1\mathrm{ml}$ of the nanoparticles’ solution. The mixture was stored overnight at room temperature, then the centrifuged, decanted, and prepared PEGylated nanoshells were resuspended in water several times to remove the PEG-thiol excess.

Nanoshells have silica cores of $140\mathrm{nm}$ in diameter with $20\text{-}\mathrm{nm}$ -thick gold shells. These parameters have been chosen to provide plasmon resonance in the IR region of the spectrum where diode lasers radiate.

Parameters of fabricated gold nanoparticles were tested by spectral analysis (measuring of optical density) and by transmission electron microscopy (TEM). The average diameter of nanoshells of $160\mathrm{nm}$ was evaluated using TEM micrograms of nanoshell suspensions (Fig. 1 ). Synthesized nanoshell suspensions had a sufficient narrow size distribution.

Fig. 1

TEM microgram of suspensions of silica/gold nanoshells, $140∕20\mathrm{nm}$ .

Spectral characteristics of gold nanoparticles calculated using the Mie theory are presented in Fig. 2a . Solid gold nanospheres of 15 and $50\mathrm{nm}$ in diameter have extinction maximums at the wavelengths near $520\mathrm{nm}$ . Optical properties of such particles are characterized by strong absorption and weak scattering. For providing tissue hyperthermia by $810\text{-}\mathrm{nm}$ laser diode, the best is using gold nanoshells with the ratio of core diameter to shell thickness as $140∕20\mathrm{nm}$ , because these particles have extinction maximums at this particular wavelength, which fits well with the optical transparency window of the tissue. The measured spectrum of optical density of fabricated silica/gold nanoshells is presented in Fig. 2b. There is a good correlation between calculated and measured spectra; however, the experimental curve is smoother due to particle size heterogeneity.

Fig. 2

(a) Theoretical spectral curves of extinction cross section for gold nanoparticles of 15 and $50\mathrm{nm}$ in diameter, and silica/gold nanoshells $(140∕20\mathrm{nm})$ . (b) Experimental spectral curve of optical density for silica/gold nanoshells $(140∕20\mathrm{nm})$ .

2.3.

Animals

For in vivo experiments, the white laboratory rats, $8\text{to}10\text{weeks}$ of age (weight $180\text{to}200\mathrm{g}$ ), were used. Animals were bred under SPF conditions and a barrier was maintained for animals involved in experiments. Drinking water and conventional food were provided ad libitum. Husbandry conditions were maintained according to all applicable provisions of the national laws. The experimental protocol was approved by an independent ethical committee prior to the study.

2.4.

Experimental Design

Investigation of thermal effects and alterations of tissue morphology induced by laser irradiation at subcutaneous and/or intramuscular injection of $0.1\text{-}\mathrm{ml}$ silica/gold nanoshell suspensions was carried out using white laboratory rats. In solution of designed silica/gold nanoshells $(140∕20\mathrm{nm})$ , gold content was of $150\mu \mathrm{g}∕\mathrm{ml}$ . Similar concentration expressed in the number of particles, $N=5\times {10}^9{\mathrm{cm}}^{-3}$ , was used in in vitro experiments with tissue phantoms. In in vivo studies, 48 animals were divided into two groups, 24 rats in each; rats from the first and the second groups were irradiated by continuous wave (cw) and pulse laser light, accordingly. Each of the 24 animals in each group was injected with a solution of gold nanoparticle suspension once subcutaneously and then in $20\min$ intramuscularly. The first injection was done subcutaneously in front of the abdominal wall, the second intramuscular one was done into the hip far enough from the first injection area. After two minutes elapsed after each injection, the skin area around the injection puncture was exposure to laser light to produce photothermal effects mediated by the inserted nanoparticles within the skin or within the muscle tissue. A $20\text{-}\min$ delay and different locations of treated areas allowed us to consider tissue photothermal responses at subcutaneous and intramuscular gold nanoparticle delivery for the particular animal as independent ones. We used the diode laser from Opto Power Corporation (Tusca, Aricona), providing a mean wavelength of $810\mathrm{nm}$ , optical-fiber output, and cw and pulse regimes. The cw laser output power was $2\mathrm{W}$ . In pulse mode, peak power was $8\mathrm{W}$ , with an on-off time ratio of 0.25, and pulse duration of $1\mathrm{msec}$ . The preliminary estimation of heating kinetics and size of the heated area was carried out at different concentrations of nanoparticles in a test tube. For noninvasive measurement of spatial distribution of surface temperature of objects under investigation, the IR Imager IRISYS 4010, Infrared Integrated System Limited (Northampton, United Kingdom) was used. General anesthesia was used for providing experiments in vivo. Tissue samples for histological examination were collected after injection of nanoparticles before, after, and $24\text{-}\mathrm{hr}$ past laser heating. Heating duration was from $10\text{to}360\sec$ . To prove the evidence of nanoparticle delivery into the tissue and to evaluate qualitatively their spatial distribution within the tissue, TEM and dark-field optical microscopy were used.

2.5.

Imaging of Gold Nanoparticles

The dark-field optical microscopy was used for imaging of gold nanoparticles in tissue slices [Biolam-M microscope (LOMO, Saint Petersburg, Russia)].

For electron microscopic examinations, the cut-out strips of tissue of size $3\times 5\mathrm{mm}$ were rapidly ground by blade to pieces not more than $1\times 1\mathrm{mm}$ . Further fixation, dehydration, and subsequent impregnation with resin via standard procedures were carried out. Tissue thin slices were obtained by the ultratome Reichert (Austria), and thickness was from $40\text{to}80\mathrm{nm}$ . Each slice was mounted to the grids without the base layers and contrasted by the saturated alcoholic solution of uranyl acetate at a temperature of $56°\mathrm{C}$ during $10\min$ . Specimens were investigated using a Hitachi Hu-1a electron microscope (Japan) in transmission microscopy (TEM) mode.

Diffuse backscattering spectroscopy at the application of fiber optic spectrometer LESA-01 was used for noninvasive in vivo detecting of subcutaneously injected gold nanoparticles. The sample was illuminated with the source fiber (fiber core diameter of $200\mu \mathrm{m}$ ), and diffuse reflected light was collected with six detector optical fibers $\left(200\mu \mathrm{m}\right)$ , which were $\sim 0.3\mathrm{mm}$ apart from the source fiber. Distance between the fiber probe tip and skin was $0.5\mathrm{mm}$ . The detector fibers were coupled to a spectrometer, and a diffuse reflectance spectrum was collected over a wavelength range of $450\text{to}1000\mathrm{nm}$ . Prior to spectral analysis, recorded signals were corrected for system response.

2.6.

Thermography

An IR thermograph was used for noninvasive monitoring of surface temperatures. The thermal imaging system IRISYS 4010 is based on the uncooled bolometric matrix providing temperature recording in the range from $-10\text{to}+250°\mathrm{C}$ , with sensitivity of $0.15°\mathrm{C}$ . The system is sufficiently fast $\left(8\text{frames}\text{per}\text{second}\right)$ to provide animal studies in vivo. The memory flash of the thermal imager makes it possible to record up to 6000 thermograms with $120\times 160\text{pixel}$ size. Range of focus is from $30\mathrm{cm}$ to infinity. The minimal size of the object, whose temperature can be measured, depends on the distance between the IR imager and the object. The wavelength range of the thermal imager comprises $8\text{to}14\mu \mathrm{m}$ , thus the scattered laser radiation $\left(810\mathrm{nm}\right)$ does not affect thermal-vision measurements.

3.1.

Model Experiments on Near Infrared Laser Heating Mediated by Gold Nanoparticles

A preliminary investigation using phantom studies and computer simulations are necessary to solve the inverse problem of reconstruction of in-depth temperature distribution on the basis of surface thermal imaging for the specified experimental conditions. For optimization of laser heating technology, in-depth temperature distributions were investigated using $2\text{-}\mathrm{ml}$ test tubes ( $1\mathrm{cm}$ diam) containing solutions of silica/gold nanoshells $(140∕20\mathrm{nm})$ with different concentrations (Fig. 3 ). Heating was provided at irradiating by a cw NIR diode laser $\left(810\mathrm{nm}\right)$ for $2\min$ (total dose of $120\mathrm{J}$ , power density $4\mathrm{W}∕{\mathrm{cm}}^2$ ). From the measurements, it follows that at high concentrations of particles the whole phantom volume is not heated directly by laser light, but only the part which is closer to the incident laser beam. The rest volume of nanoparticle solution is heated up indirectly due to a thermal diffusion process. Thus, inhomogeneity of heating could be expected. With lesser concentration of particles, the whole volume is heated directly by laser light, but this heating could be insufficient. Evident inhomogeneity of in-depth heating of nanoparticle solutions is followed from data presented in Fig. 3. The effective heating depth for highly concentrated solutions $(N=5\times {10}^9{\mathrm{cm}}^{-3})$ does not exceed $5\text{to}7\mathrm{mm}$ . This puts restrictions on nanoparticle concentration for clinical applications for tumors with sizes bigger than $1{\mathrm{cm}}^3$ . If the concentration decreases four times, the effective heating depth is increased two times. Uniform temperature distribution in the test tube is recorded only at the thinning of the nanoparticle solution 16 times. In this case, maximal temperature alterations do not exceed $18°\mathrm{C}$ , while at a concentration of $5\times {10}^9{\mathrm{cm}}^{-3}$ , temperature rise is $40°\mathrm{C}$ . It is necessary to note that $810\text{-}\mathrm{nm}$ laser radiation is also absorbed by water, thus in control measurements with the test tube filled up by physiological solution, background heating was about $6°\mathrm{C}$ .

Fig. 3

Thermograms of the test tube containing solutions of silica/gold nanoshells $(140∕20\mathrm{nm})$ and physiological solution of heating by diode laser $\left(810\mathrm{nm}\right)$ . Maximal particle concentration is $N=5\times {10}^9{\mathrm{cm}}^{-3}$ .

The temperature measurements using a test tube allowed us to determine the upper limit of the temperature increase in tissues. The concentration of gold nanoparticles in tissues is conceivably lower than in the initial injected solution. To estimate temperature rise in tissues, experimental dependence of temperature rise on concentration of gold nanoparticles in a phantom can be used (see Fig. 4 ). Temperature kinetics for the solution of gold nanoparticles with a concentration of $N=5\times {10}^9{\mathrm{cm}}^{-3}$ is shown in Fig. 5 . The observed temporal dependence of temperature rise is nonlinear. The most heating occurs over the first $100\sec$ of laser irradiation, then temperature increase is saturated with time. After laser switch off, the temperature decreases more slowly than it increased at laser heating. Even after $5\min$ elapsed since the laser was switched off, the temperature of nanoparticle solution was about $10°\mathrm{C}$ above the reference one. Evidently, the cooling rate depends on the thermal diffusivity of material and sample volume. The characteristic thermal time response of an object is defined by its dimension $R_o$ (the radius for a cylinder form) and the thermal diffusivity of its material $a_T$ :⁴

Fig. 4

Maximal temperature of gold nanoparticle solution with different concentrations measured at the end of cw laser irradiation within $2\min$ (power $2\mathrm{W}$ , total energy $120\mathrm{J}$ , power density $4\mathrm{W}∕{\mathrm{cm}}^2$ ).

Fig. 5

Temporal dependence of temperature in the center of the laser spot of the cw $(4\mathrm{W}∕{\mathrm{cm}}^2)$ diode laser $\left(810\mathrm{nm}\right)$ used for heating the solution of gold nanoparticles with a concentration of $N=5\times {10}^9{\mathrm{cm}}^{-3}$ in the test tube.

Experimental values for the thermal diffusivity $a_t$ of many soft tissues lie within the rather narrow range from $1.03\times {10}^{-7}{\mathrm{m}}^2∕\mathrm{s}$ (hydrated collagen containing 50% of water) to $1.46\times {10}^{-7}{\mathrm{m}}^2∕\mathrm{s}$ (pure water). Therefore, the characteristic thermal time response for our experiments with phantoms and tissues according to expression 1 is around $200\text{to}300\sec$ . This value is in good agreement with our experimental results presented in Fig 5. Therefore, the real time of object overheating exceeds the laser exposure time. This finding is necessary to take into account to assign a duration of local laser hyperthermia mediated by gold nanoparticles.

3.2.

In Vivo Near-Infrared Laser Hyperthermia Mediated by Gold Nanoshells

These experimental studies aim the in vivo determination of the spatial distributions of temperature corresponding to dif-ferent depths and concentrations of nanoparticles in tissues of white laboratory rats. The solution of silica/gold nanoshells ( $140∕20\mathrm{nm}$ , $150\mu \mathrm{g}∕\mathrm{ml}$ of gold) was injected $\left(0.1\mathrm{ml}\right)$ subcutaneously or intramuscularly. The diffuse backscattering spectrum of the skin as a scattering medium is influenced by inserted gold nanoparticles due to the specific absorption spectrum of the particles (see Fig. 2). Evidently, the effect of nanoparticles on the backscattering spectrum should be lower as the depth of the layer of the inserted gold nanoparticles increases. Experimental diffuse reflectance spectra measured for rat skin with injected gold nanoparticles (Fig. 6 ) validate the possibility to detect gold nanoparticles within some depth in tissue. These spectra are different from the absorption spectrum of gold nanoparticle solution. The maximum of diffuse reflectance of the skin with gold nanoparticles is mismatched with the absorbance peak of gold colloid. The rise of backscattering intensity (curve 3) nearly $530\mathrm{nm}$ for tissue with nanoshells is due to the backscattering of these particles. The absorption peaks [ $\lambda =530\mathrm{nm}$ for particles with $15\text{-}\mathrm{nm}$ size and $\lambda =810\mathrm{nm}$ for nanoshells with $140∕20\text{-}\mathrm{nm}$ size (Fig. 2)] correspond to the minimal values (dips) of diffuse backscattering spectra. This dips are formed because of nanoparticle absorption of scattered radiation in inhomogeneous medium.

Fig. 6

Diffuse reflectance spectra of rat skin: 1—skin without nanoparticles; 2—subcutaneously injected gold nanopartilces of $15\mathrm{nm}$ in diameter; and 3—subcutaneously injected silica/gold nanoshells $(140∕20\mathrm{nm})$ .

In vivo temperature spatial distributions were measured for cw and pulsed laser irradiation and different injection depths of nanoparticles into tissues (Fig. 7 ). It is seen that heating is happening more rapidly and temperature rise is substantially higher at laser irradiation of the skin with inserted nanoparticles than for skin free of particles. The rate of heating is very important for photothermal therapy of cancer. Temperature rise to $46\text{to}50°\mathrm{C}$ is accepted as optimal for this purpose.³⁸ Such temperatures admittedly stimulate apoptosis in tumor tissue, in contrast to heating in the range $37\text{to}43°\mathrm{C}$ , which is very undesirable.³⁹ In our experiments, by using nanoshells the time interval for tissue heating up to $46°\mathrm{C}$ was $15\text{to}20\sec$ , and in their absence it was more than tenfold prolonged, up to $3\text{to}4\min$ .

Fig. 7

In vivo measured temporal dependences of rat skin temperature at cw (●,○) and pulse (△,Δ) laser heating, 1, 3—without nanoparticles, 2, 4—with silica/gold nanoshells $(140∕20\mathrm{nm})$ , subcutaneous injection.

It is interesting to note that both temperature kinetic curves for cw and pulsed irradiation of tissue with nanoparticles have a local maximum at $60\sec$ (Fig. 7). The origin of this maximum may be explained by the manifestation of compensatory reaction of organisms to temperature rise, which has a characteristic time of about $100\sec$ . With the fast heating caused by nanoparticles, the inertial mechanism of heat regulation could not provide any immediate temperature compensation. In contrast, essentially good compensation is realized for laser heating without inserted particles, and some compensation is seen in a larger time scale for heating with inserted nanoparticles. Also important to note is that laser pulse heating is more controllable in a large time scale and has similar short time kinetics as cw. In that sense, pulsed irradiation could be preferable because it gives fast and self-limited temperature rise. The comparison of curves 2 and 4 (Fig. 7) shows that the increase of effectiveness of hyperthermia is possible by a combination of different heating modes. It is reasonable to use the prime cw mode with a higher power density of laser radiation, and switch over to pulse mode or cw mode with lower power.

At $24\mathrm{h}$ after laser action, biopsy samples of irradiated tissues were taken for histological investigation. Dark-field microscopy was used for imaging of nanoparticles in muscle tissue (Fig. 8 ).

Fig. 8

Histological study of muscle tissue $(200\times )$ . Staining by hematoxylin/eosin: (a) dark-field image and (b) optical transmission image. The white arrows show the gold particle clusters.

In vivo thermal measurements and histological studies of the tissue before and after laser irradiation were compared. Figure 9 shows the thermorgams and structure of the skin and muscle tissue before and after laser action on tissue with the administrated nanoshells.

Fig. 9

Comparison of local temperature rise and corresponding alterations in tissue morphology: (a) histology of rat skin before laser action, (b) thermogram at laser heating with subcutaneous administration of silica/gold nanoshells $(140∕20\mathrm{nm})$ , (c) histology of rat skin after $30\sec$ of laser action, (d) histology of rat muscle tissue before laser action, (e) thermogram at laser heating with intramuscular administration of silica/gold nanoshells $(140∕20\mathrm{nm})$ , and (f) histology of rat muscle tissue after $30\sec$ of laser action.

In the control samples, we did not observe any pathological changes [Figs. 9a and 9d]. For subcutaneously administrated nanoshells after laser irradiation, epidermis of the skin is partly absent with the formation of vesicles filled up by serous fluid. In the derma, we found severe edema with disorientation of collagen fibers [Fig. 9c]. In the muscle tissue we observed edema, hyperemia, and inflammatory infiltration by leukocytes. The control of the spatial distribution of the temperature of the skin surface using the IR imager showed that after $30\sec$ of laser action, the maximal temperature in the center of the laser spot is about $65°\mathrm{C}$ [Fig. 9b].

At intramuscular administration of the nanoshells, we revealed moderate edema with defibrillation of collagen fibers in muscle tissue [Fig. 9f]. In the connective tissue, we observed inflammatory leukocyte infiltration and edema. In the case of intramuscular particle introduction, we did not observe significant changes in color or structure of tissue surface during the irradiation time. However, the surface temperature was approximately $7°\mathrm{C}$ higher than in the control measurements without nanoparticles. Thus, the laser thermolysis can be achieved in the region of nanoparticle localization without damage of the surface tissues. This conclusion is confirmed by histological examinations.

Figure 10 shows aggregates (clusters) of nanoshells (marked by white arrows) injected immediately in tissue before laser action. There are evident signs of cell damage: the nucleus contains vacuoles (V), the cytoplasmic membrane (CM) is ruinous, and cytoplasm is pouring out into extracellular space. Near the cell there are unstructured necrotic masses (NM), maybe remains of destroyed cells.

Fig. 10

TEM image of skin after subcutaneous injection of nanoparticles and laser irradiation.