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Plasmonic photothermal therapy (PPTT) is a minimally-invasive oncological treatment strategy in which photon energy is selectively administered and converted into heat sufficient to induce cellular hyperthermia. The present work demonstrates the feasibility of in vivo PPTT treatment of deep-tissue malignancies using easily-prepared plasmonic gold nanorods and a small, portable, inexpensive near-infrared (NIR) laser. Dramatic size decreases in squamous cell carcinoma xenografts were observed for direct (P<0.0001) and intravenous (P<0.0008) administration of pegylated gold nanorods in nu/nu mice. Inhibition of average tumor growth for both delivery methods was observed over a 13-day period, with resorption of >57% of the directly-injected tumors and 25% of the intravenously-treated tumors.
The application of nanotechnology in medicine has been a rapidly growing field in recent years [1–8]. A variety of structures with unique structural [9, 10], optical [11, 12], electronic , magnetic , and catalytic  properties have been exploited in the areas of cancer imaging [2, 16–19], diagnostics [6, 20, 21], and treatment [22–30]. Noble metal nanoparticles provide remarkable opportunities in these applications due to their inherently low toxicity [31–33] and strongly enhanced optical properties associated with localized surface plasmon resonance [34–36]. The enhanced electromagnetic field surrounding such particles gives rise to large absorption, Rayleigh (Mie) scattering, raman scattering, and two-photon luminescence cross-sections, properties which have been utilized in photothermal cancer therapy [24–30] (PTT), surface enhanced Raman detection [37–39] (SERS), and diagnostic imaging [17–20] applications.
While surgical excision of tumors is a highly effective method of cancer treatment, curative strategies for primary tumors located in vital or poorly accessible tissues remains a challenge. In cases of recurrent tumors or those with ill-defined margins, alternative and multimodal oncological approaches are employed. The primary [40–42] and adjunctive [43–46] treatment of cancers by induced hyperthermia is a well established but burgeoning field of medical research. Here, temperatures in tumor-loaded tissues are elevated to 40–43°C  and above by selective or non-selective application of microwave, radio, ultrasound, alternating magnetic, infrared, or visible radiation. At temperatures greater than 43°C, protein denaturation and disruption of the cellular membrane is known to occur and ablation of tumor tissues has been shown in numerous cases [42, 48, 49]. Under mild temperature increases, clinical studies indicate an acceleration in both perfusion and reoxygenation [50, 51] of tumor tissues, thereby increasing the efficacy of cytostatic drug delivery (chemosensitization) and radiotherapy (radiosensitization), respectively. In all cases, clinical studies indicate statistically significant benefits to local tumor control and overall survival rates for primary [40–42] and conjunctive hyperthermia [52–56]. Although promising, conventional non-invasive hyperthermic strategies are often less selective than those based-on or used in combination with thermal contrast agents, in many cases, causing damage to surrounding healthy tissues, as well as significant discomfort. Moreover, hyperthermic treatments using commercially available instruments are often limited to shallow penetration depths  (<3 cm), lower treatment temperatures, and regions of the body with regular surface composition. Invasive approaches using microwave antennas are highly susceptible to interference, while magnetic particle treatments require large doses.
Photothermal therapy [49, 57–59] is a minimally-invasive treatment method in which photon energy is converted to thermal energy sufficient to induce cellular hyperthermia. Selectivity is achieved by focused directional control or invasive [40–42] (fiber optic) positioning of the incident radiation, often pulsed [28–30] or continuous wave [24–28, 30, 48] (cw) laser, and is typically accompanied by preferential administration of photoactive molecules [60–62] or nano-scale particles. Photoexcitation of the latter two results in non-radiative relaxation by local heat transfer to the surrounding tumor environment. In contrast, photodynamic therapy [63–65] (PDT), relies on non-radiative relaxation through local formation of cytotoxic singlet oxygen species. While PTT and PDT treatments have garnered significant attention, such methods are inherently limited by photobleaching effects and absorption cross sections several times weaker than those of noble metal nanoparticles.
Recent advances in the field of plasmonics present new opportunities for both primary and multimodal PTT strategies using noble metal nanoparticles. By photo-exciting conduction electrons which oscillate at the surfaces of such structures (surface plasmons), highly efficient local heating can be achieved by non-radiative relaxation through electron-phonon and subsequent phonon-phonon coupling processes . While several materials and spherical nanoparticles exhibit surface plasmon resonance in the visible region, opportunities for in vivo plasmonic photothermal therapy  (PPTT) are restricted due to a high degree of absorption by tissues at visible wavelengths. Such ablative treatments are therefore limited to shallow depths . In contrast, PPTT of deep tissue malignancies may be accomplished by laser exposure and plasmon absorption in the near-infrared region (NIR). Due to minimal attenuation by water and hemoglobin at these wavelengths, NIR transmission  in soft tissues may be achieved at depths exceeding 10 cm. By chemically varying the shape or composition of noble metal nanoparticles [9, 21, 24, 67–69], surface plasmon absorption can be tuned from ultraviolet (UV) to infrared (IR) wavelengths. The enhanced nonlinear optical properties of spherical metal nanoparticles have also been used by our group in in vitro near-infrared pulsed laser PPTT by second harmonic generation [29, 70, 71].
The potential uses of gold nanoparticles in near-infrared PPTT have been published using a variety of noble metal nanostructures, including gold nanoshells [26, 48], gold nanorods [8, 27, 72], and recently, gold nanocages . Studies using nanoshell-mediated PPTT indicate significantly improved local tumor control and survival times in animal models, while surface plasmon absorption of gold nanocages have been used in diagnostic imaging and in vitro therapy .
One of the simplest and widely used methods to obtain plasmonic nanoparticles involves the seed-mediated growth of colloidal gold nanorods . The use of such particles in near-infrared PPTT is highly attractive due to their rapid synthesis, facile bioconjugation, strong absorption cross-section, and tunable optical extinction. Recent calculations by discrete dipole approximation (DDA) show the absorption cross section of nanorod structures to be nominally larger than that of nanocages and more than twice that of nanoshell structures at their NIR plasmon resonance . By synthetically varying the aspect ratio of the nanorods, longitudinal plasmon absorption can be shifted throughout the visible, NIR, and IR regions [68, 74–76].
Our previous work  showed that gold nanorods conjugated to epithelial growth factor receptor antibodies (anti-EGFR) can serve as contrast agents for in vitro biodiagnostics. Moreover, due to overexpression of the EGF receptor on cancer cell surfaces and the specificity of antibody binding, malignant cells were found to require half the energy necessary to destroy normal cells when both were incubated with the same concentration of nanorod bioconjugates, a key feature of selective PPTT.
In the present work, the feasibility of in vivo near-infrared PPTT is demonstrated using colloidal gold nanorods in an animal model. Subcutaneous squamous cell carcinoma xenografts were grown in nude (nu/nu) mice and particles were selectively delivered to tumors by both direct and intravenous injection. Thiolated poly (ethylene) glycol (PEG5000) was covalently bound to the gold nanorod surface to increase biocompatibility [77–80], suppress immunogenic responses, and to decrease adsorption to the negatively charge luminal surface of blood vessels. Near-infrared PPTT was performed extracorporally using a small, portable, inexpensive, continuous wave diode laser. Making use of the enhanced permeability and retention (EPR) effect [81, 82], preferential accumulation of peylgated gold nanorods in tumor tissues was achieved due to the high density, extensive extravasation, and inherently defective architecture of the tumor vasculature, as well the diminished lymphatic clearance from associated interstitial spaces. Significant decreases in tumor growth were observed for both direct tumor injection (P<0.0001) and intravenous (P<0.0008) treatments. Inhibition of average tumor growth for both delivery methods was observed over a 13-day period, with resorption of >57% of the directly-injected tumors and 25% of the intravenously-treated tumors.
Seed-mediated growth was performed at 25 °C from freshly prepared aqueous solutions (18 MΩ) following methods of Nikoobakht and El-Sayed . Briefly, 2.50 mL of 1.00 mM HAuCl4 (Aldrich, 24459-7) was added to 5.00 mL of 0.200 M cetyltrimethylammonium bromide (CTAB, Aldrich). 600 μL of ice-cold 10 mM NaBH4 (Aldrich, 480886) was added to the stirred solution and allowed to react for several minutes, forming the pale brown gold seed solution. Next, 100.0 mL of 1.00 mM HAuCl4 was added to 100.0 mL of 0.200 M CTAB and 4.50 mL of 4.00 mM AgNO3 (Fischer). 1.40 mL of 78.8 mM ascorbic acid (Aldrich, A-7506) was added, followed by gentle mixing to form the transparent growth solution. 160 μL of the seed solution was added to the unstirred growth solution and allowed to react for 2 hours. Nanorods synthesized by this method are approximately 12 nm in width and 50 nm length (4.0 aspect ratio), with a longitudinal plasmon absorption maximum at 800 nm.
Gold nanorods solutions were centrifuged twice at 20,000 × g for 15 min and re-dispersed in deionized water to remove excess CTAB molecules. mPEG-SH (Nektar Therapeutics, MW5000) was added to the ~1 nM colloidal nanorod solution at a final concentration of 10 mM. Rods were sonicated overnight and centrifuged at 20,000 × g for 15 min and redispersed in deionized water to remove non-specifically bound PEG molecules. The pegylated gold nanorods were again centrifuged at 20,000 × g for 15 min, sterile filtered, and re-dispersed in 10 mM phosphate-buffered saline (PBS, Mediatech) to the desired optical density at 800 nm. Extinction spectra of the pegylated nanorod saline suspensions showed no peak shift, broadening, or reduction over a 1 week period prior to injection.
HSC-3 human squamous carcinoma cells were cultured in DMEM (Mediatech) supplemented with 10% v/v heat-inactivated fetal bovine serum (Invitrogen), 2 mM L-glutamine (Sigma), penicillin (100 U/ml) (Sigma), and streptomycin (100 μg/ml) (Sigma) in a 5% CO2 humidified atmosphere. Female nu/nu mice, 7–8 weeks of age, were obtained from Taconic (Hudson, NY). Mice were injected subcutaneously in the flank with 100 μL (3 × 106) HSC-3 cells suspended in 10 mM PBS. Near-infrared PPTT began once tumor burden reached 3 mm in diameter (7–9 days). All experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of the Georgia Institute of Technology (Atlanta, GA).
Tumor bearing mice were injected with gold nanorods (ODλ=800 = 120, 100 μl) via the tail vein, and euthanized at specified time points. Tumor tissues was excised, fixed in 10% formalin for 24 hours, and embedded in paraffin blocks. Blocks were sectioned (5 microns) and stained using the Silver Enhancer Kit SE-100 (Sigma-Aldrich) according to the manufacturer’s instructions. Incubation time for optimal visualization was determined to be 10 minutes. Silver staining of the tissue sections were examined using a BX60 Olympus microscope, and photographed using an Olympus Camedia digital camera.
Thermal transient measurements of HSC-3 tumor interstitia were obtained using a 33 gauge hypodermic thermocouple (Omega). The tip of the thermistor was positioned at the tumor center-of-mass and temperatures were recorded in 15 second intervals prior to, during, and following NIR exposure for direct (15 μL, ODλ=800=40, 10 min, 0.9–1.1 W/cm2, 6 mm dia) and intravenously (100 μL, ODλ=800=120, 10 min, 1.7–1.9 W/cm2, 6 mm dia) administered gold nanorods, as well as for comparably exposed sham/NIR treatments (15 μL direct intratumoral injection of 10 mM PBS).
In vivo imaging of pegylated gold nanorod accumulation was monitored by attenuation of near-infrared transmission (808 nm diode laser, Power Technologies) using a custom-built CCD device array. Control measurements were taken from images obtained by 15 μL direct intratumoral injection of 10 mM PBS, while directly and intravenously administered measurements were obtained using previously mentioned dosages.
Mice were anesthetized with ketamine/xylazine/acepromazine. 15 μL of pegylated gold nanorods (ODλ=800 = 40) were directly injected into the tumor interstitium or 100 μL of pegylated gold nanorods (ODλ=800 = 120) were intravenously (tail) injected. Control tumor sites were injected with 15 μL of 10 mM PBS with no NIR exposure. For direct administration, mouse tumors were extracorporeally exposed to NIR radiation (0.9–1.1 W/cm2, 6 mm dia, 10 min) within 2 minutes of injection to limit particle diffusion beyond the tumor boundaries. For intravenous administration, nanorods were allowed 24 hour circulation to maximize intratumoral particle accumulation prior to NIR exposure (1.7–1.9 W/cm2, 6 mm dia,10 min). Ellipsoidal tumor volume was calculated as V = (d)2(D)(π/6). Statistical hypothesis testing was performed using Welch’s t test and non-parametric analysis of variance was performed by the Kruskal-Wallis test. Due to the unusually rapid growth rates observed in the HSC-3 xenograft model, tumors and vital organs were harvested at day 14 for use in separate, ongoing toxicological investigations.
Pegylated gold nanorods were intravenously injected (tail vein) to assess optimal intratumoral particle accumulation. Following injection, HSC-3 tumors were excised at varying time intervals, fixed, sectioned, and stained with silver to visualize the extent of particle loading. Nanorods directly injected into tumors were used for as a positive control (data not shown). Fig. 1a shows a typical histological section from a HSC-3 tumor injected with 15 μL of 10 mM PBS (control). Fig. 1b and 1c illustrate representative tumor sections following 2 and 6 hours of accumulation, respectively. At these time points, no appreciable accumulation of particles was observed. In contrast, high particle loading was observed following 24 hours of circulation (Fig. 1d). Because the highest accumulation, and therefore PPTT selectivity, was observed at 24 hours, this time point was used for subsequent intravenous near-infrared PPTT treatments.
Transient particle accumulation following direct and intravenous administration was monitored by NIR transmission imaging (Fig. 2). Intensity line-scans of NIR extinction showed marginal diffusion of directly injected particles over 3 min, with no subsequent change observed over several hours. Intensity line-scans from NIR transmission images of HSC-3 tumor sites directly injected with 15 μL of 10 mM PBS show nominal extinction due to increased tissue density, while line-scans obtained following intravenous nanorod delivery at 24 hr accumulation showed extinction 2.00 times that observed for control sites. Directly injected tumor sites showed NIR extinction at 2 min nanorod diffusion more 2.18 times that observed by intravenous administration and 4.35 times that observed at control sites.
Thermal transient measurements for direct (Fig. 3a) and intravenous (Fig. 3b) near-infrared PPTT treatments show thermal equilibrium conditions prior to NIR irradiation. Rapid heating was observed upon exposure, followed by steady-state equilibrium. Note that >90% of the observed temperature increase occurred within the first 3 minutes. Upon removal of NIR exposure, tissues displayed expected Newtonian cooling behavior.
Heating efficiencies of PPTT treatments (the ratio of steady-state temperature change in the presence of plasmonic particles to that NIR exposed in their absence) were found to be 3.59±0.5 for direct-injection and 1.90±0.4 for intravenous injection of pegylated gold nanorods. The former value is remarkably similar to that observed during in vivo near-infrared PPTT treatments reported by Hirsch et al. by direct injection of gold nanoshells. Observed increases in temperature change for sham/NIR treatments using comparable exposure times and power densities as direct and intravenous administration conditions correlate well with increases in power density. Disparity of direct and intravenous PPTT heating efficiency scales proportionately with observed increases in NIR extinction (4.35 and 2.00 times greater than control extinction, respectively) and is attributed to decreased particle loading by intravenous delivery. Although particle volume and concentration was significantly higher for intravenous injections, accumulation is likely limited by the extent of tumor angiogenesis and uptake by the reticuloendothelial system (RES). Treatment selectivity and efficacy was most apparent for direct injections; however, both methods showed significantly improved local tumor control.
Groups of four to six mice were initially used to establish optimal conditions for near-infrared PPTT treatment of HSC-3 tumor xenografts. 15 μl of pegylated gold nanorods (ODλ=800=40) were directly administered at three sites within the tumor interstitium or 100 μL (ODλ=800=120) were intravenously injected (tail). After two minutes, tumors directly injected with nanorods were subjected to extracorporeal NIR exposure (808 nm, 6 mm dia) and it was determined that 10–15 minutes of irradiation at 0.9–1.1 W/cm2 was necessary for maximal tumor control and minimal damage to surrounding tissues. After 24 hours, tumors intravenously administered with nanorods were also subjected to extracorporeal NIR exposure (808 nm, 6 mm dia) and it was determined that 10–15 minutes of irradiation at 1.7–1.9 W/cm2 was necessary for maximal tumor control and minimal damage to surrounding tissues. In addition, no statistically significant differences in tumor growth were observed for direct nanorod injections without NIR exposure, sham/NIR exposure alone, and PBS intratumoral injections alone (P=0.427 at day 9) (S1). S2 illustrates typically observed tumor resorption and growth inhibition following direct injection of pegylated gold nanorods and near-infrared PPTT versus sham/NIR treatments.
Using previously established treatment conditions, change in tumor volume was recorded over a 13 day period for control mice, as well as those treated by intravenous and direct nanorod injections followed by PPTT (S3). Here, control mice were subjected to 15 μL direct injection of 10 mM PBS to the tumor interstitium, with no NIR exposure. Average change in tumor volume for each group was plotted (Fig. 4) and statistical hypothesis testing for differences in average tumor growth was performed (Table I). Figure 4 shows a >96% decrease in average tumor growth for directly treated HSC-3 xenografts and a >74% decrease in average tumor growth for intravenously treated HSC-3 xenografts at day 13 (relative to control tumors). Moreover, resorption of >57% of the directly treated tumors and 25% of the intravenously treated tumors was observed over the monitoring period.
Average tumor growth at day 13 for directly and intravenously treated tumors was significantly less than that observed in untreated control groups (P<0.0001 and P<0.0008, respectively). Differences in observed efficacy for direct and intravenous treatments gradually increased during the experiment, reaching statistical significance at the 8% level on day 13. Non-parametric analysis of variance for the treated and untreated groups (Table II) found statistically significant differences at the 2% level and below for the duration of the experiment. These results clearly indicate both the selectivity and specificity of near-infrared PPTT.
The dramatic changes in observed HSC-3 tumor growth are attributed to selective hyperemia of malignant tissues treated with pegylated gold nanorods by near-infrared PPTT. Preferential accumulation of pegylated gold nanorods within the tumor interstitium occurs due to the EPR effect . Because of their rapid metabolic rates, tumor cells are regarded as increasingly vulnerable to hyperthermic effects [44, 72] such as disruption of metabolic signaling processes, protein denaturation, and the onset of acidosis or apoptosis caused by the production of heat-shock proteins  and other immunostimulants. Small increases in local temperature are known to result in disruption of nuclear and cytoskeletal assemblies and indeed, previous [44, 84] and recent reports [29, 72] indicate significant membrane blebbing [85–87] under hyperthermic conditions. Under severe conditions, hyperthermic damage can result in impaired vasculature supply, endothelial swelling , and mictothrombosis associated with homeostatic disruption . In vitro, mild hyperthermia has been shown to impede the function of cell surface receptors, membrane transport, and RNA- and DNA-polymerization during protein synthesis. Repair of sublethal cell damage by DNA-polymerase-α and -β, such as that by incurred during radiotherapy, has also been shown to be inhibited by hyperthermia. While tumor growth suppression and resorption is likely a cumulative result of the previously mentioned effects, it is presumed here that ablation of the tumor vasculature and localized membrane disruption predominates.
Although the mechanism of cellular response in the present case is yet to be determined, the specificity of hyperthermic effects on tumor growth from both direct and intravenous near-infrared PPTT treatments is unmistakable (P<0.0001 and P<0.0008, respectively). Inhibition of average tumor growth and minimal damage to surrounding tissues is observed for both methods. Resorption of >57% of the directly-injected tumors and 25% of the intravenously-treated tumors clearly indicates the potential curative and adjunctive applications of NIR plasmonic photothermal therapy (PPTT) in pre-clinical settings.
The authors graciously thank Mariam Akhtar (GT Dept of Biology) for her assistance with silver staining studies. Generous support from the Georgia Cancer Coalition (EBD; Distinguished Cancer Scientist Award), the National Cancer Institute (MAE, ECD, & XH; Center of Cancer Nanotechnology Excellence Award U54CA119338), the Robinson Family Foundation (JFM), Ovarian Cycle (JFM), Golfers Against Cancer (JFM), and the US Dept of Energy (MAE, ECD, & XH; NO:DE-FG02-97 ER14799) is acknowledged.
Observed changes in HSC-3 xenograft growth for intratumoral nanorod injections in female nu/nu mice without NIR exposure, NIR exposure alone, and PBS intratumoral injections alone (P=0.427 at day 9) (S1). S2 illustrates representative tumor resorption and growth inhibition following direct injection of pegylated gold nanorods and near-infrared PPTT versus sham/NIR treatments. Change in tumor volume for individual mice following established conditions for near-infrared PPTT treatment of HSC-3 xenografts by control (a), intravenous (b), and direct (c) injections of pegylated gold nanorods (S3).
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