The authors have declared that no competing interests exist.
Conceived and designed the experiments: AMA Mostafa A. El-Sayed AAS OAE NAY SSM SME. Performed the experiments: AMA EA WHE NMF Marwa A. El-Shaer NS. Analyzed the data: AMA NAY SSM SME. Contributed reagents/materials/analysis tools: EA NAY SSM SME. Wrote the manuscript: AMA NAY SSM SME. Data revision and fund management: Mostafa A. El-Sayed AAS OAE.
Gold nanorods (GNR) within tumor microregions are characterized by their ability to absorb near IR light and emit heat in what is called photoplasmonic effect. Yet, the efficacy of nanoparticles is limited due to intratumoral tissue distribution reasons. In addition, distribution of GNRs to normal tissue might result in non specific toxicity. In the current study, we are assessing the intratumoral and tissue distribution of PEGylated GNRs on the top of its antitumor characteristics when given intravenously or intratumoral to solid tumor bearing mice and coupled with laser photoplasmonic sessions. PEGylated GNRs with a longitudinal size of less than 100 nm were prepared with aspect ratio of 4.6 showing strong surface plasmon absorption at wavelength 800 nm. Pharmacokinetics of GNR after single I.V. administration (0.1 mg/kg) showed very short systemic circulating time (less than 3 h). On the other hand, tissue distribution of I.V. GNR (0.1 mg/kg) to normal animals showed preferential deposition in spleen tissue. Repeated administration of I.V. GNR resulted in preferential accumulation in both liver and spleen tissues. In addition, I.V. administration of GNR to Ehrlich carcinoma tumor bearing mice resulted in similar tissue distribution; tumor accumulation and anti-tumor effect compared to intratumoral administration. In conclusion, the concentration of GNR achieved within tumors microregions after I.V. administration was comparable to I.T. administration and sufficient to elicit tumoral growth arrest when coupled with laser-aided photoplasmonic treatment.
Cancer is so far a national and international health problem [
Nanomaterials have diverse effects that draw the attention of scientists from different specialties. Attributed to their unique properties, nanomaterials have been involved in numerous applications including biomedical uses [
Gold nanoparticles are subclass of nanomaterials intensively investigated for biomedical uses which is attributed to their excellent biocompatibility with human tissues. Moreover, gold nanoparticles is currently approved by the United States Food and Drug Administration for the treatment of rheumatoid arthritis [
Herein, we have evaluated the tumor and normal tissue distribution of gold nanorods injected systemically in normal and tumor bearing animals. In addition, we assessed the antitumor effect of systemically administered gold nanorods coupled with near IR laser plasmon photothermal therapy in subcutaneously transplanted Ehrlich carcinoma solid tumor model.
Transmission electron microscope (TEM) image of the prepared GNRs were shown in
TEM image of GNRs with Plasmon band energies at 800 nm (A) and UV- Visible NIR absorption spectra of the GNRs (B) prepared using single surfactant mixtures. Scale bar = 100 nm.
The optical properties of metallic nanoparticles depend on shape. This is due to the absorption of visible light both along the length of the nanorod (the longitudinal plasmon band) and along the width of the nanorod (the transverse plasmon band). The ultraviolet visible (UV–VIS) spectra of the GNRs in deionized water as a solvent were shown in
After single I.V. administration of GNRs to normal male and female animals, preliminary distribution into different major organs (liver, spleen and kidney) was evaluated after two weeks of exposure (
GNRs were administered by I.V. injection (0.1 mg/kg) and assayed after two weeks in major excretory organs (liver, spleen and kidney). Concentration of GNRs (A) and the percent residual amount of the total administered dose (B) are presented. Data are presented as mean ± SEM (n=6).
In clinical setting, chemotherapy and radiotherapy are usually prescribed in the form of treatment cycles to achieve maximum tumor killing effect. In this context, the accumulation of GNRs has been studied after repeated I.V. administration (0.1 mg/kg) into normal animals with special emphasis on liver, spleen, kidney and brain tissues (
GNRs were administered by I.V. injection (0.1 mg/kg for five consecutive days of each month and repeated for 6 months). Three week after the last injection major target organs (liver, spleen, kidney and brain) were assayed for tissue concentration of GNRs (A) and the percent residual amount of the total administered dose (B). Data are presented as mean ± SEM (n=6).
To assess tumor/normal tissue distribution of GNRs in tumor bearing animals, a dose of 1.5 mg/kg was administered intratumoral (I.T.) or intravenous (I.V.) and tissue concentration of GNRs was determined in tumor, liver, spleen, and kidney tissues at different time intervals (
GNRs were administered by I.V. (◌) or I.T. (●) injection (1.5 mg/kg) to tumor bearing mice. Tissue concentration of gold in tumor (A), liver (B), spleen (C), and kidney (D) tissues were assayed for Gold conetnt at different time intervals until two weeks. Data are presented as mean ± SEM (n=3).
Cmax |
AUC0-last |
AUC0-inf |
Elimination half life time (days) |
Cmax ratio | Tissue exposure ratio (TER) |
Tissue Exposure Index (TEI) |
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---|---|---|---|---|---|---|---|---|---|---|---|---|
(µg/g tissue) |
(day. µg/g tissue) |
(day. µg/g tissue) |
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I.V. | I.T. | I.V. | I.T. | I.V. | I.T. | I.V. | I.T. | I.V. | I.T. | |||
7.3 | 15.14 | 75.7 | 121.4 | 101.2 | 162 | 11.6 | 13.8 | 2.1 | 1.6 | 100 | 100 | |
2.0 | 1.5 | 24.6 | 18.2 | 34.1 | 26.6 | 23.1 | 69.3 | 0.75 | 0.74 | 32.5 | 15.0 | |
16.8 | 19.8 | 157.1 | 145.6 | 185.8 | 180.4 | 4.1 | 6.3 | 1.2 | 0.93 | 207.5 | 119.9 | |
4.2 | 5.8 | 49.8 | 54.8 | 64.8 | 71.3 | 11.6 | 11.6 | 1.4 | 1.1 | 65.8 | 45.1 |
Tissue exposure ratio is calculated as ratio between AUCI.T /AUCI.V and indicates the relative degree of exposure of each tissue to gold nanorods when administered locally or systemically.
Tissue Exposure Index is calculated as ratio between AUC of gold nanorods in normal tissue relative to tumor tissue and indicates the relative degree of tumor tissue targeting.
Tissue exposure index (TEI) represents the relative normal organ exposure to GNRs to tumor tissue after I.T. or I.V. administration. Liver and kidney tissues showed TEI indicative of lower organ/tumor exposure characteristics after either I.T. or I.V. administration (TEI ranged from 14.99-65.79%). TEI of spleen indicated higher organ/tumor exposure and was higher in I.V. than I.T. administration showing 207.5 and 119.9%, respectively (
Relative tissue exposure ratio (TER) is a parameter to present the relative overall organ exposure to GNR after I.T. administration relative to I.V. administration (
To assess the anti-tumor effect of GNRs coupled with PPT, 1.5 mg/kg GNRs was injected I.T. and I.V. every three weeks to EAC tumor bearing mice, exposed to near IR laser beam (50 W/cm2 for 2 min) and tumor growth rate was measured for up to 47 days. Immediately after the session, tumor tissue was overheated to about 79 °C in the center of laser beam exposure. The temperature of the tumor tissue declined outward to reach about 41 °C at tumor boundaries (
EACC tumor bearing mice were given gold NRs (1.5 mg/kg every three weeks) by I.V. (▲) and I.T. (■) administration compared to PBS treated animals (●). Animals were exposed to laser plasmonic beam (50 W/cm2 for 2 min) every week. Tumor size was measured every three days and plotted (A). Representative tumors are shown in panel (B). Data are presented as mean ± SEM (n=10).
To assess the pathological changes in solid tumor after treatment with GNRs coupled with laser plasmonic therapy, the cellular, vascular and stromal compartments were examined in H&E stained tumor sections. Solid Erlich carcinoma in the control group appeared as compact subcutaneous masses of tumor cells which invade the subjacent connective tissue. Tumor cells were highly cellular anaplastic, atypical pleomorphic, polygonal, with abundant eosinophilic cytoplasm and prominent central nuclei. The surrounding stroma was reduced with some inflammatory cell infiltration (
EACC tumors of control group (A); gold NRs IT treated group (B); and IV treated group (C) were stained by H&E regular stain. GNR coupled with PTT showed massive tissue destruction appeared as non-cellular debris eosinophilic areas (arrows) Scale bar = 20 µm.
In tumors treated with direct I.T. GNR coupled with laser plasmonic therapy, remarkable cellular debris (indicated by arrows) was observed in addition to profound desmoplastic stroma in the form of fibrocollagenous bundles and peri-vascular hyalinosis. These findings might explain the regression of tumor growth and further tumor invasion to the surrounding connective tissues. In addition, the marked lymphocytic infiltration was noticed in the surrounding stroma (
Intravenous GNRs coupled with laser plasmonic therapy showed abundant tumoral cell debris, more oesinophilic tumor cells (indicated by arrows) and peri-vascular hyalinosis indicative of nuclear damage. Marker stromal desmoplastic fibrotic bundles were observed which might explain the limitation of tumor invasion (
Multifunctional nanoparticles, nonetheless, gold nanorods are designed to take over various functions in the field of oncology, such as tumor targeting, imaging and selective therapy, which offer great promise for the future of cancer prevention, diagnosis, imaging and treatment [
Apart from photonic physical properties, biocompatibility and potential toxicity of GNRs constitutes obstacle in front of using nanometalic structures as a therapeutic remedy. Not only the metal itself, but also chemical materials used in the synthesis such as CTAB, might carry some toxic effect
In tumor bearing animals, GNRs accumulated in tumor tissue comparably after I.V. and I.T. administration, as well as, in the rest of all examined tissues (liver, spleen, kidney and brain). Surprisingly in the current work, PEGylated GNRs disappeared from plasma only after 3 h of I.V. administration (data not shown). In general, prolonging the circulating time of GNR’s via PEGylation was considered as a tool to achieve better tumor tissue accumulation [
In our previous work, systemically administered GNRs coupled with near IR laser plasmon therapy efficiently diminished the growth of squamous cell carcinoma [
In conclusion, GNRs administered systemically was equally distributed to Ehrlich carcinoma solid tumor tissues compared to intra-tumoral administration of GNRs. In addition, systemically administered GNRs was equipotent to local intra-tumoral administered GNRs when coupled with laser plasmon thermal therapy in inducing tumor growth arrest. Finally, accumulation of GNRs in vital organs such as liver and spleen apparently was non-toxic; however, might warrant further toxicological detailed studies. Further formulations of better surface plasmon characteristics and less accumulation kinetics in vital organs might pave promise in the field of laser-induced photo plasmon thermal therapy of solid tumors. It is recommend for the current GNRs formulation to be considered for clinical assessment in solid tumor treatment coupled with laser photoplasmonic therapy.
Cetyltrimethylammonium bromide (CTAB) and Sodium borohydride (99%) were purchased from Mercy and LOBA chemic respectively. Silver nitrate was purchased from Sigma-Aldrich, L-ascorbic acid. All the reagents were analytical grade, and used without further purification. Deionized water (18 MΩ) was used in all the experiments.
. The nanorods were synthesized according to the seed-mediated growth method [
CTAB solution (5 mL, 0.20 M) was mixed with HAuCl4 (5 mL, 0.0005 M) under vigorous stirring. Next, 0.6 mL of ice-cold 0.01 M NaBH4 was added to the solution. The solution turned brownish yellow immediately after adding NaBH4, indicating particle formation. The particles in this solution were used as seeds. Vigorous stirring of the seed solution was continued for 2 min. After the solution was stirred, it was kept at 25°C.
In a clean test tube, 10 mL of gentle mixing growth solution, containing (5 mL, 0.20 M) of CTAB and (5 mL, 0.001M) of HAuCl4, was mixed with 0.35 mL of 0.004 M AgNO3 solution at 25°C. To this solution, 5 mL of 1M HCl was added, and after gentle mixing of the solution 70µL of 0.0788 M ascorbic acid was added. Ascorbic acid as a mild reducing agent changes the growth solution from dark yellow to colorless. It is worth noting that the growth solutions above are identical except for their silver ion content. The final step was the addition of 12µL of the seed solution to the growth solution at 27-30°C. The color of the solution gradually changed within
10-20 min. For longer NRs, the color change takes place more slowly. The temperature of the growth medium was kept constant at 27-30°C in all the experiments. This pathway produces pure NR solutions with aspect ratios 4.6.
GNRs colloidal solutions were centrifuged twice (20,000g for 15 min) then re-dispersed in deionized water to get rid of excess CTAB. A final concentration of 10 mM mPEG-SH (MW5000, Sigma-Aldrich) and 1 nM colloidal GNRs were mixed. GNR were sonicated overnight and then centrifuged (20,000g for 15 min) and re-dispersed in deionized water to remove non-specifically bound PEG molecules. The PEGylated GNRs were centrifuged (20,000g for 15 min), sterile by filteration (0.22 µm pore size filter), and re-dispersed in sterile 10 mM phosphate-buffered saline (PBS, Gibco) 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.
Absorption spectra of the prepared solutions were measured in the range of 1000–200nm using Jasco 570 UV–VIS-NIR spectrophotometer. The morphology of gold NRs was studied by Transmission Electron Microscope (JEOL-JEM 2010) operated at 200 kV accelerating voltage. The preparation of TEM grid, the TEM image was taken after separating the surfactant from the metal particles by centrifugation. Typically 1 mL of the sample was centrifuged for 10 min at a speed of 14000 rpm. The upper part of the colorless solution was removed and the solid portion was redispersed in 1 mL of water. 2 µL of this redispersed particle suspension was placed on a carbon coated copper grid and dried at room temperature.
Male and female BalbC mice (weight 20-25 g) and Sprauge Dawly rats (weight 120-150 g) were maintained in the pathogen free area of the National Research Center animal house facility (Dokki, Giza, Egypt). Animals had an access to food and water
Murine Ehrlich Ascitis Carcinoma cell line, EACC was obtained from National Cancer Institute (Cairo, Egypt), and maintained in RPMI-1640 media supplemented with 100 µg/ml streptomycin, 100 units/ml penicillin and 10% heat-inactivated fetal bovine serum in a humidified chamber at 37°C supplied with 5% (v/v) CO2.
For tumor induction, cells were collected and washed 3 times with serum free media, and 1x107 viable cells were injected s.c. into the flank region of each mice.
To determine the tissue distribution and potential targeting of NRs to major organ of normal animals, rats were given NRs preparation (0.1 mg/kg) intravenousely in PBS solution. Two weeks later, animals were euthanized by cervical dislocation, and these organs (liver, spleen, and kidney) were harvested within 20 min and stored at -80°C to be assayed.
To determine the tissue accumulation of NRs in major organs of normal animals, rats were given NR preparation intravenousely in PBS solution (0.1 mg/kg every day for the first 5 days of each month and repeated for 6 months). Three weeks after the last administration, animals were euthanized by cervical dislocation, and their organs (liver, spleen, kidney and brain) were harvested within 20 min and stored at -80°C to be assayed.
When tumor size reached 300 mm3, mice were given NR preparation (1.5 mg/kg) either intratumorally or intravenousely in PBS solution. At predetermined time points, mice were euthanized by cervical dislocation, and their organs (tumor, liver, spleen, kidney, brain) were harvested within 20 min and stored at -80°C to be assayed.
Tissues samples were incinerated for 48 h at 550°C. Ashes were dissolved in concentrated nitric acid and gold content were measured by atomic absorption. Standard curve of HAuCl4 were plotted and measured by atomic absorption.
To evaluate anti tumor efficacy; when tumor size reached 300 mm3, mice were given GNR preparation (1.5 mg/kg) either intratumorally or intravenousely in PBS solution every three weeks. Animals were exposed weekly to laser beam (50 W/cm2 for 2 min). Tumor tissue temperature was scanned using surface thermometer probe (Ugo Basile, Comerio, Italy); and temperature distribution was plotted against the radius of tumor tissue. Tumor size and overall survival were monitored for total of 7 weeks. Tumor size was determined using the formula: Volume = (Width2 x length)/2
Any animal showed massive weight loss, ascitis, change in motility or any other sign of morbidity was immediately sacrificed.
Histological examination for tumor tissues were performed according the lab routine protocol. Briefly, paraformaldhyde fixed tissues were embedded in paraffin wax. Cross vertical sections (5 µm) were obtained and after dewaxing and rehydration sections were stained with H&E.
Data are presented as mean ± SEM. Analysis of variance (ANOVA) with LSD post hoc test was used for testing the significance using SPSS® for windows, version 17.0.0. p<0.05 was taken as a cut off value for significance.
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We are greatly thankful to Dr Hany E. El-Nazer for his keen and careful administrative support to the project team. We would like to thank Dr Ashraf B. Abdel-Naim, Tissue Culture Unit, Pharmacology Department, Faculty of Pharmacy, Ain Shams University for using their technical facilities.