Porous silicon nanoparticles for cancer photothermotherapy
© Hong et al; licensee Springer. 2011
Received: 9 September 2010
Accepted: 11 April 2011
Published: 11 April 2011
The in vitro cell tests and in vivo animal tests were performed to investigate the feasibility of the photothermal therapy based on porous silicon (PSi) in combination with near-infrared (NIR) laser. According to the Annexin V- fluorescein isothiocyanate Apoptosis assay test results, the untreated cells and the cells exposed to NIR laser without PSi treatment had a cell viability of 95.6 and 91.3%, respectively. Likewise, the cells treated with PSi but not with NIR irradiation also had a cell viability of 74.4%. Combination of these two techniques, however, showed a cell viability of 6.7%. Also, the cell deaths were mostly due to necrosis but partly due to late apoptosis. The in vivo animal test results showed that the Murine colon carcinoma (CT-26) tumors were completely resorbed without nearly giving damage to surrounding healthy tissue within 5 days of PSi and NIR laser treatment. Tumors have not recurred at all in the PSi/NIR treatment groups thereafter. Both the in vitro cell test and in vivo animal test results suggest that thermotherapy based on PSi in combination with NIR laser irradiation is an efficient technique to selectively destroy cancer cells without damaging the surrounding healthy cells.
In recent years, photothermotherapy (PTT) techniques based on inorganic nanomaterials and near-infrared (NIR) light have attracted significant attention owing to their advantages over conventional surgical treatments. The advantages of PTT include the anticipated reduction in morbidity and mortality, low cost, suitability for real-time imaging guidance, and the ability to perform ablative procedures on outpatients because of its non-invasive nature . In the conventional PTTs based on simple heating, i.e., hyperthermia [, and references therein], most treatment failures result from insufficient temperature rises in the tumor tissues. Therefore, it is essential to use a thermal coupling agent with a good photothermal property to secure irreversible destruction of tumor cells in a short time without damaging adjacent healthy cells in thermotherapy.
The inorganic nanomaterials currently demonstrated as thermal coupling agents in PTTs are gold nanoparticles (Au NPs) [3–12], gold nanorods [13–16], gold nanoshells [1, 17–19], gold nanocages [20, 21], gold nanocrystals [22, 23], single wall carbon nanotubes (SWCNTs) [24–26], and porous silicon (PSi) [27, 28]. Of these nanomaterials, we are particularly interested in PSi because it is known to have many important properties such as biocompatibility , biodegradability [30, 31], and a readily functionalized surface  which a therapeutic agent should have desirably as well as an excellent photothermal property . In addition, PSi has a merit that it can be easily prepared by the simple electrochemical anodization of silicon. As regards biomedical applications of PSi, their use in drug delivery [34–39] and photodynamic therapy [40–42] applications have been reported before. We previously reported on the excellent heat generation ability of PSi and the ability of PSi to irreversibly destroy cancer cells under NIR laser irradiation by using temperature rise measurement and MTT assay results, respectively [27, 28]. In this paper, we report the Annexin V-fluorescein isothiocyanate (FITC) apoptosis assay test and in vivo animal test results of PSi in combination with NIR laser to investigate the ability of PSi to kill cancer cells as well as the death modes of cancer cells and the ability of PSi to inhibit the growth of tumors, respectively.
Preparation of PSi/EtOH:PEG drug solutions
First, meso-PSi layers were prepared on 2.5 cm × 2.5 cm × 0.05 cm pieces of p-type Si(100) with a resistivity of 1-5 mΩcm by anodic etching in an 3:1 (by volume) solution of 46% HF and 95% C2H5OH at a current density of 200 mA/cm2 for 150 s. PSi is generally classified into three different types in terms of the pore size: macro-PSi (d > 50 nm), meso-PSi (2 nm < d < 50 nm), micro-PSi (d < 2 nm). According to our experience, meso-PSi is the most suitable for photothermotherapy since it shows the highest photothermal effect and can be easily to be fractured into nanoparticles with proper sizes. The porosity and thickness of the PSi layers determined by weight measurements  were about 73% and 55 μm, respectively. The details of the anodization process are described elsewhere . The PSi layers formed on Si(100) were then lifted off by anodic etching in an 1:15 (by volume) solution of 46% HF and 95% C2H5OH at a current density of 4 mA/cm2 for 250 s. Next, the free-standing PSi layers were fractured by ultrasonicating in 10 mL of ethanol for 24 h. The PSi nanoparticles were subsequently filtered twice by using a 450 nm membrane first and then by using a 220 nm membrane. PSi/EtOH:PEG drug solutions were prepared by dispersing the resulting PSi particles in 10 mL of ethanol mixed with 10 mL of thiolated polyethyleneglycol (PEG-SH) and centrifuged for 24 h until all the PSi particles were dispersed.
Measurement of heating of the PSi/EtOH:PEG solution by NIR irradiation
Heterochromatic NIR light irradiation was performed on six different samples by using NIR laser. The samples include a PSi/EtOH:PEG solution and a EtOH:PEG solution as well as a solid PSi (a free standing PSi layer). The three different kinds of samples were irradiated continuously for 20 min by the NIR laser at 1.5 W/cm2. The distance between the laser source and each sample was fixed to be 2 cm. Change in the temperature of the samples with the NIR exposure time was measured at 30-s intervals by using an IR thermometer (model: AZ 8859, max. output: 1 mW, wavelength: 670 nm, measurement range: -20 to 420°C).
Cells and materials
The Murine colon cancer cell lines (CT-26) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). CT-26 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. FBS, cell culture media, penicillin-streptomycin and all other agents used in cell culture studies were purchased from Invitrogen™ (GIBCO, NY, USA). Cultures were maintained at 37°C in a CO2 incubator with a controlled humidified atmosphere composed of 95% air and 5% CO2. Trypan blue dyes were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Annexin V- FITC apoptosis assays
CT-26 cells were cultured in DMEM. The incubations of 1 × 106 CT-26 cells were carried out at 37°C and in 5% CO2 atmosphere for approximately 24 h in 24-well plates, with the cells having been seeded in a 100 nm dish for approximately 18 h before incubation. After incubation, the cell media were removed from the wells, and the cells were washed using PBS and then incomplete DMEM was added to each well. Then, the PSi/EtOH:PEG solution was added to each well. Annexin V- FITC apoptosis assays were performed on four different mouse CT-26 cell sample groups to see the ability of our technique to irreversibly destroy cells and the modes of cell deaths: the CT-26 cell control group given neither PSi nor laser treatment, the CT-26 cell group not treated with PSi but with laser, the group not treated with laser but with PSi, the group treated with both PSi and laser. For the preparation of the last sample group, CT-26 cells were treated with the PSi/EtOH:PEG drug solution (0.7 g/L) first and then NIR laser at 600 mW/cm2 for 20 min. Next, the 2 × 106 cells were removed from the culture, washed twice with cold PBS, and double-stained with Annexin V-FITC and propidium iodide (PI) (BD Biosciences, San Jose, CA, USA) in Annexin-binding buffer, followed by analysis on a FAC-Scalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 488-nm argon laser. To avoid nonspecific fluorescence from dead cells, live cells were gated using forward and side scatter.
Trypan blue cell death assays
Cell viability was performed by the trypan blue cell death assay. Briefly, CT-26 cells were plated at a density of 3 × 105 cells in 60 mm culture dishes for 24 h. Then, the medium was removed, and the cells were treated with a PSi/EtOH:PEG solution to each plate. The final concentration of ethanol in the medium was ≤0.5% (v/v). Detached CT-26 cells treated with a PSi/EtOH:PEG solution were exposed to NIR laser at 600 mW/cm2 for 20 min. After NIR irradiation, a collection of supernatants and adherent cells obtained by trypsinization was incubated in 0.4% trypan blue and pipetted onto a hemocytometer and manually counted under a microscope at ×100 magnification. The percentage of cells admitting trypan blue dye to the total number of cells was determined by counting three different fields for each experimental condition, which was done in triplicates .
In vivo animal tests
Animal care and all experimental procedures were conducted in accordance with the Guide for Animal Experiments edited by the Korean Academy of Medical Sciences. The CT-26 cells (1 × 106 cells) were suspended in 100 μL PBS, and subcutaneously injected into the back of male mice of each group (n = 5, 5- to 6-week-old, Balb/c). When the tumors were grown up to a volume of 65-70 mm3, mice were randomized into four groups: (a) mice were simply monitored without any other treatment; (b) mice were intratumorally injected with 100 mL of PBS and then irradiated NIR laser 4 times at 1.5 W/cm2 for 2 min each time with a time interval of 2 min under the ether anesthesia; (c) a PSi/EtOH:PEG solution (0.7 g/L, 100 μL) was intratumorally injected without NIR laser irradiation; (d) a PSi/EtOH:PEG (0.7 g/L, 100 μL) was injected into tumor, then NIR laser was immediately irradiated on the tumor region in the same manner as in (a). The mice were anesthetized by injecting 40 μL of a 9:1 solution of ketamine (100 mg/mL) and rompun (100 mg/mL). Tumor volumes, animal body weights, and tumor conditions were recorded weekly for the duration of the study . The tumor size of each group was measured using a skinfold caliper, and tumor volumes were calculated using the following equation: tumor volume = ab 2/2, where a is the maximum diameter of tumor and b is the minimum diameter of tumor . All the procedures for in vivo experiments were performed in accordance with Inha University of Biomedical Science guidelines on animal care and use.
Results and discussion
Photothermal properties of the PSi/EtOH:PEG solution
Annexin V- FITC apoptosis assay tests
We previously reported as a result of the in vitro cell test based on MTT assay that the cell viabilities of the mouse groups untreated, treated only with PSi, treated only with laser, and treated with PSi followed by laser treatment were 99.8, 95.2, 98.1, and 2.6%, respectively . The present Annexin V-FITC Apoptosis assay result is somewhat worse than the previous MTT assay result. In the previous test, the PSi/NaCl suspension was used instead of the PSi/EtOH:PEG solution as a PSi drug solution. Another difference is that the PSi concentration in the PSi/NaCl suspension (~10.0 g/L) was far higher than that in PSi/EtOH:PEG solution (0.7 g/L) since the PSi was not filtered by using a 220-nm membrane in the former suspension whereas PSi was filtered prior to the in vitro cell test in the latter solution. Therefore, the higher cell viability, i.e., the lower cell death rate of the group treated with both PSi and laser in the present test may be mainly attributed to the lower PSi concentration in the PSi drug solution.
Trypan blue cell death assay tests
In vivo animal tests
Tumor size after PSi or laser treatment (mm)
5.5 × 5.4
12 × 12
12 × 11
12 × 15
21 × 27
18 × 22
18 × 24
Weight change after laser treatment
The in vitro cell test and in vivo animal test results were performed to investigate the feasibility of the photothermal therapy based on PSi in combination with 808 nm NIR laser. Combination of PSi and NIR laser treatment techniques shows a substantially higher cell death rate than only one of these two techniques. The Murine colon carcinoma (CT-26) tumors were completely resorbed without nearly giving damage to surrounding healthy tissue within 5 days of PSi and NIR laser treatment. All the mice given both treatments remained healthy and free of tumors and side effects for more than 3 months. The preliminary results in this work shows the feasibility of photothermotherapy based on PSi in combination with NIR laser irradiation in selectively destroying cancer cells without damaging the surrounding healthy cells. However, the systematic administration of cancers still remains as a challenge in this therapeutic approach. The experiments on this issue are under way using tumor targeting techniques such as functionalization of PSi with specific antibodies.
- Au NPs:
Dulbecco's modified Eagle's medium
fluorescent-activated cell sorter
fetal bovine serum
single wall carbon nanotubes
This work was supported by the Korea Engineering and Science Foundation (KOSEF) through 'the 2007 National Research Lab Program'.
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