Treatment+of+Epithelial+Carcinoma+Using+anti-EGFR+Antibody+Conjugated+Gold+Particles

By: Blake Galeazzi and Dustyn Uchiyama

toc = = = = = = =Introduction=

Our project aims to evaluate the emerging cancer therapy known as photothermal therapy (PTT). This therapy takes advantage of the absorption and light scattering properties of gold nanoparticles. The use of nanoparticles in medicine has been advancing for quite some time, begin ning in the 1950s. In the past the particles have been used for cancer cell imaging using simple dark-field microscopy though now there is convincing evidence that they can be utilized for cancer therapy. This project will examine the application of nanoparticles in photothermal therapy and several proof-of-theory experiments. We have also included an outlook for this therapy and possible approaches to overcoming its limitations.

=What You Should Know=

Here we define and explain several key concepts that are needed to truly appreciate photothermal therapy.

**Nanoparticles**: small chemically engineered particles several nanometers in diameter, i.e. 10^-9 m. media type="youtube" key="jC8CUIID2HA" height="282" width="317" align="right" Gold and silver nanoparticles have strong optical resonances and support coherent oscillations of their valence electrons (surface plasmons) when illuminated by laser. Nanoparticles are generally either nanospheres, nano rods, or nanoshells (below). (2)

**How nanoparticles cause cell death**: The absorbed radiation is transferred into heat within picoseconds due to electron-phonon and phonon-phonon processes (2). The intense increase in heat causes cell death via denaturation of proteins, coagulation, deterioration of cell membrane, and bubble formation (microscopic version of underwater explosions) (3).

**How "tuning" of nanoparticles is achieved**: Size, shape, type of meta l and local environment of the metal nanoparticle are the main factors that determine the plasmon resonance wavelength. When the laser light is of the same wavelength as the plasmon resonance wavelength, maximum absorption by the nanoparticle is achieved. Smaller plasmonic nanoparticles are better absorbers, this property enables nanoparticles to kill malignant cells via highly localized photothermal heating when targeted by a laser (1). For one type of nanoparticle, nanoshells, the tuning of the optical resonance wavelength is achieved by specifically engineering the dielectric (silica) core and a metallic shell layer (1). Using gold nanoparticles that are active in the NIR-region is beneficial because this avoids the light extinction by intrinsic chromophores in native tissue. At wavelegths of 700-1000 nm water absorption is minimal and blood and tissue are maximally transmissive. Figure 2 (below) shows the differing absorption wavelengths of a) nanospheres, b) nanorods, and c) nanoshells (3).

**Targeting nanoparticles for cancerous cells**: There are two methods for targeting nanoparticles for cancerous cells/tumors. The first is passive targeting, which relies on the leaky vasculature of tumors to result in the accumulation of nanoparticles. Gaps within the newly formed, irregular blood vessels enable the nanoparticles, as well as large molecules, to be absorbed by the tumor; this is known as the enhanced permeability and retention (EPR) effect. The ability of the nanoparticles to be absorbed by the tumor is determined to an extent by the amount of time the nanoparticles can remain in circulation. The circulation of nanoparticles can be extended by attaching poly(ethylene glycol) (PEG) to the nanoparticle surface. When using PEG conjugated nanoparticles, nano shells have been shown to circulate for approximately one day and accumulate in the liver and spleen. Additional studies have shown nanoshells accumulate in the tumor at a maximum concentration 24 hours after injection.

This method is not really efficient and thus a new method, called antibody-targeting, was developed by scientists. Antibodies bind to cell receptors with much more specificity and high affinity, resulting in a much more efficient targeting method. One particular strategy for this is PEG linker molecules. Scientists have utilized the anti-EGFR (epidermal growth factor receptor) antibody in the majority of the proof-of-concept experiments, as we will discuss.

Having outlined the general parameters of nanoparticles, we can now proceed to understanding their unique application to photothermal therapy.

=Photothermal Therapy with Nanoparticles=

The first photothermal therapy experiments relied on irradiating cancerous cells with a laser which killed both malignant and benign cells. In this respect, photothermal therapy offered no novel advantages over traditional chemo and radiation therapies because there were still adverse side effects. However, with the emergence of nanoparticles, specifically targeting only malignant cells became a reality in photothermal therapy. The proof-of concept experiments primarily utilize anti-EGFR conjugated nanoparticles to target epithelial carcinom as. media type="youtube" key="9PJHa1NDuaI" height="226" width="402" align="left"



=An Overview of Carcinoma=

Carcinoma is the most common type of cancer. In fact, around 80 percent of cancers are of epithelial origin. In simple terms, it is a cancer that is found in the epithelial cells of the body. These tissues line the surfaces of the body, either inner (internal organs or glands) or outer (like the skin), and stem from the ectodermal or endodermal layers. Carcinomas in situ are localized carcinoma cells which have not yet invaded through the basal lamina, which would make them malignant, or invasive (7). In this case the cells will first spread to other local sites, but eventual ly will spread to other sites throughout the body. This act is called metastasis and occurs through the use of the blood and lymph nodes. Usually carcinomas are diagnosed through biopsies, in which doctors remove a portion of the tumor to examine whether or not the cells are cancerous. There are four main stages of carcinoma: Stages I and II in which the tumor has spread to local areas; Stage III in which they have spread to regional lymph nodes, tissues, or organ structures; State IV in which tumors have already metastasized [through the blood] to sites more distant fr om the original site (8).

There are many different treatments for carcinoma, and scientists and doctors are getting better at developing innovative new ways to treat the disease. Of course, mechanisms like radiation and chemotherapy are the most common, but throughout our project we will examine an emerging therapy -- photothermal therapy using gold nanoparticles and anti-EGFR antibodies -- which seems like it is a promising new way to target cancer cells.

=What is EGFR?=

Epidermal growth factor receptor (EGFR) is the cell-surface receptor for members of the epidermal growth factor family of extracellular protein ligands. The receptor is a special type of receptor tyrosine kinase, of which there are four closely related ones: EGFR, HER2, Her 3, and Her 4. EGFR exists on the cell surface and is activated by the binding of its specific ligands. EGFR is activated by its growth factor ligands and undergoes a transformation from an inactive monomeric form to an active homodimer. EGFR stimulates an intracellular protein-tyrosine kinase activity. Several signal transduction cascades occur with the activation of EGFR which are involved with things like DNA synthesis and cell proliferation. These proteins modulate phenotypes such as cell migration, adhesion, and proliferation. media type="youtube" key="zE4BkAw_lL4" height="315" width="420" align="right" One can see how the mutations related to EGFR can lead to cancer. The mutations will cause the cell to lose its ability to regulate its growth (of course inducing cancer). EGFR overexpression can result from mutations and have been associated with various epithelial cancers (carcinomas). In fact, problems with EGFR -- for example, mutations, amplifications, or misregulations -- could result in uncontrolled cell division and are implicated in about 30% of all epithelial cancers.

Many therapies have been developed which focus on EGFR, we are only discussi ng one of these in detail. Other therapeutic approaches have targeted EGFR directly, trying to use small molecules to inhibit EGFR tyrosine kinase. This would make it unable to activate itself (stopping signal cascades in the cell). The therapy we are discussing also targets epithelial growth factor receptor, just using photothermal therapy to target the cells specifically as opposed to other methods.

=Photothermal Therapy: Behind the Scenes=

In order to target specific tumors and tumor markers, antibody based targeting is used with photothermal therapy for carcinomas. The antibodies are selected to target a specific tumor marker. In this case, of course, EGFR antibodies can be used to target overexpressed EGFR in certain cancer cells. The antibodies are bound to negatively charged gold nanoparticles. Gold nanorods are capped with cetyltrimethyl-ammonium bromide (CTAB) molecules, which are positively charged. Antibodies can be absorbed onto gold nanorods by various molecular interactions. Cells are immersed into anti-EGFR-conjugated nanorods solution in order to transfer these particles into the cells. The cells are rinsed with a buffer and coated with glycerol.

EGFR is a good target for this therapy because, as mentioned, cancer cells have an overexpression of EGFR on their cell surfaces. In photothermal therapy as well as other cancer therapies, EGFR is exploited to selectively target cancerous cells.

**Synthesis of Nanoparticles (Nanorods)**: Gold nanorods are synthesized using 0.0005 M auric acid, 0.2 M CTAB, and 0.01 M of sodium borohydride. A 100 mL growth solution is prepared by the reduction of 0.001 M auric acid in a solution of 0.2 M CTAB, 0.15 M BDAC (Benzyldimethylhexadecyl-ammonium chloride), and 0.004 M silver salt, with ascorbic acid as well.

As mentioned, the nanorods are capped with CTAB. After, the positively charged surface of the nanorods is changed to a negatively charged one by exposing the nanoparticles to poly(styrenesulfonate) (PSS). These are then mixed with an antibody solution. The antibodies are bound to PSS-coated nanorods by electrostatic physisorption interactions (Figure 3, below from Huang et al.). The nanorods conjugated with anti-EGFR monoclonal antibodies are then centrifuged, in which they become stable for several days.

**Preparing Cell Lines for Experimentation**: A nonmalignant epithelial cell line and two malignant epithelial cell lines are cultured on 18 mm glass coverslips in a 12-well tissue culture plate and are allowed to grow for three days. The coverslips are coated with collagen type I, a protein which helps with cell growth. The monolayer is removed and rinsed with PBS buffer, and then immersed into the anti-EGFR-conjugated nanorods solution for about a half hour. It is then rinsed with PBS buffer again, and coated with glycerol, and sealed with a coverslip.

**Use of Nanoparticles in Photothermal Therapy**: The aim of PTT is to have the nanoparticles absorbed by the tumor and to then ablate the malignant cells via laser. In order for this therapy to become a reality, the nanoparticles must selectively bind and only be absorbed by malignant cells and the laser induction must only cause tissue damage to the cells associated with the nanoparticles. The anti-EGFR-conjugated nanoparticles are first injected into the body (in most experiments involving mice the injection is done through the tail vein). The cancerous cell surfaces become heavily saturated with the gold nanoparticles due to the specific binding of the anti-EGFR antibodies to the overexpressed EGFR on the surface of the cancer cells.

Once inside/attached to the desired cells, they can play multiple roles based on two distinguishing properties. Photothermal heating is the result of resonant absorption and bioimaging is possible due to the nanoparticles' light scattering properties. PTT is therefore primarily concerned with resonant absorption. The wavelength of resonant absorption is therefore extremely critical because it must be specific to the cells containing the nanoparticles, thereby reducing unnecessary tissue damage. Nanoshell optical resonance can be tuned from 700-1100 nm in wavelength, where there is minimal water absorption and blood and tissue are transmissive. The nanoparticle absorbs the light from the laser and quickly transforms the energy into heat. The heat released is enough to cause cell death by denaturation, and in some cases, the formation of bubbles from this reaction creates enough mechanical stress within the cell to kill it. This mechanism of cell death is termed photolysis, or the breaking of a cell using energy from photons.

=Parameters of Experimentation=


 * 1. Heating depends on both the laser power and the concentration of the nanoshells. **

Elliot et al. demonstrated that the size concentration of the nanoparticles influences the temperature distribution. In this experiment, the temperature distribution in tissue phantoms (110 nm diameter) was compared to that of nanoshell-laden tissue phantoms (180 nm in diameter, right). The two gels were targeted by a continuous wave (CW) laser at 808 nm wavelength. (1)


 * 2. The size and shape of nanoparticles affects their absorption and scattering wavelength. **

In regards to nanorods, the aspect ratio of length to width affects the sensitivity of the strong longitudinal band. Nanorods also enable the change of the absorption wavelength from visible to NIR region and an increase in their absorption (2). Nanorods have "a strong long-wavelength band due to the longitudinal oscillation of electrons and a weak short-wavelength band around 520 nm due to the transverse electronic oscillation" (below) (2).

In regards to nanoshells, the absorption wavelength is "fine-tuned" by adjusting the thickness of the gold shell and diameter of the silica core (2). Nanospheres have a visible absorption band around 520 nm.


 * 3. The length of the laser pulse is also important. **

When the laser is pulsed for a relatively long time (>100 ns), the heat dissipates and is lost to the surrounding particles. However, when the laser is pulsed for a short period of time (<20 ns) there is essentially no heat loss and all the heat is involved in heating the cell. One study calculated a theoretical temperature increase of 2500 C for a single cell with a bound 40 nm gold sphere irradiated with a laser at 520 nm.


 * 4. Significant cell death occurs after a temperature increase of 30-35 C. **

= Proof-of-Theory Experiments =

In one study by Hirsch et al., tumors 1 cm in diameter were grown in mice, and in another group PEGylated nanoshells were injected. In the control group only saline was injected. After the mice were euthanized, the tumors underwent histological evaluation and were observed to have suffered tissue damage only in the areas exposed to laser irradiation (Figure 2 below, 1).

Temperature maps showed an average temperature increase of 37.4 ± 6.6 °C, causing irreversible tissue damage after 4-6 minutes of laser irradiation in the tissue exposed to the nanoshells. The control samples only showed an average temperature rise of 9.1 ± 4.7 °C, which is safe for cell viability (1). Tumor destruction is readily observable by the human eye (Figure 4 below, 1). Other experiments (1) have shown the therapeutic efficacy and animal survival times over a 90 day period. The PEGylated nano shells were injected via the tail vein and were observed to accumulate in the tumor after 6 hours. The laser used was 808 nm at a power of 4 W/sq. cm for 3 minutes. In this experiment, one group of mice received the nanoshell injection and laser treatment; another received a saline injection and laser treatment; and another received no treatment. Ten days after treatment the group that received the nanoshell injection and laser treatment showed complete resorption of the tumor (Figure 3, right). The same group showed 100% survival after 60 days.

In an experiment by Huang et al., antibody conjugated nanospheres were used to kill malignant cells with only half the laser energy necessary to kill normal cells (2). The laser wavelength was 514 nm which overlaps with the strong wavelength band of nanospheres. The laser was only able to penetrate about 500 um, which would have little to no beneficial clinical impact.

Huang et al. later demonstrated that light in the NIR region can penetrate up to 10 cm due to low scattering and absorption by intrinsic tissue chromophores (2). This approach to PTT incorporates nanorods which can absorb light at 800 nm. This experiment also shows that cell death occurs at and above a laser power of 80 mW. The destruction of nonmalignant cells can be achieved at a laser power less than 120 mW (2). (Figure 3, below, Huang et al.) The blue spots show cell death.

In another experiment by Halas et al., anti-Her2-nanoshells were incubated with breast cancer cells. Only the anti-Her2-nanoshell-cancer cell conjugates were killed by the laser at 820 nm (Figure 6, below, Huang et al.). This experiment showed that an antibody other than EGFR could be incorporated into PTT and still achieve the same results. Also, the experiment demonstrated that nano shells are only effective when bound to the cancerous cells (3).



=Current Limitations=

As Lal et al. outlined, there are currently no quantification methods to calculate the exact concentration of nanoparticle accumulation in tissues. Currently, scientists are unsure as to whether or not nanoparticles tend to accumulate in other tissues, besides the targeted tumor. Additionally, it is important to be able to determine the amount of nanoparticles in tissues to understand the limitations in regards to minimum effective dosage and cytotoxicity. Understanding concentration profiles would also be beneficial because scientists need to characterize the resultant temperature elevation. In other words, it would be reckless to over-saturate a region which could lead to irreparable tissue damage by the transmission of heat alone.

The long term cytotoxicity of nanoparticles is still being investigated. With this being said, gold nanoparticles have traditionally been shown to have very little or no cytotoxic effects, though their use in increasing concentrations may change such information. In one study, it was discovered that carbon nanotubes, an associated type of nanoparticle, were shown to result in mesothelioma, a rare cancer which usually appears in the outer lining of the lungs (1).

In relation, there still needs to be research into the exact depth of penetration possible in PTT. The in vivo studies to date have all pertained to subcutaneous tumors which only require a few inches of NIR light penetration (1).

Lal also describes the mode of injection as a possible limitation. The studies utilize ectopic injection of malignant cells while most human tumors are orthotopic. In other words, the tumors in animal models may allow a net flow of nanoparticles, but this still needs to be determined in human tumors (this is pertinent to the delivery of nanoparticles to cancerous cells).

=Outlook of Photothermal Therapy=

Photothermal therapy provides a novel therapeutic approach to treating cancer. However, the use of nanoparticles and treatment by laser is not limited to "killing" cancer cells. In fact, several discoveries and developments in PTT may be applied to other realms of cancer therapy. Here is a discussion considering the potential of PTT and novel applications of this technology.

**Combining PTT with conventional therapies**: Multiple studies have shown that mild hyperthermia prevents hypoxia (1). This finding is significant because scientists believe that the hypoxic regions of tumors contain the most resistant and dangerous cancer cells. Due to the decreased b lood flow to hypoxic regions, drugs often have a hard time accessing these cancer cells, and thus they maintain constant proliferation. Hypoxic regions of tumors are also the least affected by radiation, chemotherapies, and immunotherapies (1). The use of nanoparticles is not necessarily to "kill" the cell, but merely "heat up" the cell (by 10 ** ° ** C) has been shown to prevent hypoxia. Diagaradjane et al. have used nanoshell-mediated hyperthermia in conjunction with radiation therapy (1). The study showed that the radiation therapy was more effective after general heating by use of nanoparticles and laser.

**Targeting hard to reach places (Using Nanoparticles)**: Hypoxic regions of tumors (believed to be responsible for recurrence and metastasis) are resistant to chemotherapy, and nanoparticles have a difficult time accumulating there. However, it has been observed that peripheral blood monocytes enter the hypoxic regions of a tumor along a chemoattractive gradient. The monocytes enter the tumor and differentiate into macrophages. Choi et al devised a “Trojan Horse” method of targeting nanoparticles for the hypoxic regions. They loaded monocytes with nanoparticles in which they proceeded to enter the tumor and differentiate into nanoshell-loaded macrophages. An in vitro tumor was developed using malignant breast epithelial cells and targeted by nanoshell-loaded macrophages. After laser irradiation tumor ablation was observed.

**Targeting hard to reach places (Using Laser)**: The use of fiber optic probes has been discussed to circumvent the weak penetrating power of lasers (1). By incorporating fiber optic probes, scientists are able to bypass tissues and blood and directly insert the laser into the tumor. Currently there are no data available regarding PTT and use of laser via fiber optic probes.

**Potential of PTT (ideas formulated by Blake and Dustyn)**: Whether or not these ideas are feasible is not our main focus, but we have brainstormed some ideas, that if applicable, could provide huge breakthroughs in cancer therapy.

1) Combining PTT with tumor resection: We hypothesized that the penetrating power of lasers could be circumvented if PTT was applied immediately after tumor resection (and the tissue was cut open). We believe that after the tumor is removed, nanoparticles could be injected and a laser could essentially "sweep" the area to kill any remaining cancer cells. We would hope that this "after surgery technique" could complement additional chemo and radiation therapies.

2) Laser induced nanoparticle-conjugated drugs: Nanoparticles conjugated to antibodies show the ability to target only malignant cells. We hypothesized that using this specificity of nanoparticles and conjugating them to drugs would enable specific drug delivery. There is currently research demonstrating specific drug delivery using conjugated drugs to antibodies, but perhaps also conjugating nanoparticles would add another layer of specificity to these drugs. In this scenario, the drugs would only be activated by the laser.

We leave you with an amazing development in PTT: media type="youtube" key="kMjRBXi2DIY" height="254" width="453" align="center"

Thank you for taking the time to read our Wiki!

-Blake Galeazzi and Dustyn Uchiyama

=References=

1. Lal, Surbhi, Susan E. Clare, and Naomi J. Halas. "Nanoshell-Enabled Photothermal Cancer Therapy: Impending Clinical Impact". __Accounts of Chemical Research__. //41//, 1842-1851. December 2008.

2. Huang, Xiaohua, Ivan H. El-Sayed, Wei Quan, and Mostafa A. El-Sayed. "Cancer Cell Imaging and Photothermal Therapy in the Near-Infrared Region by Using Gold Nanorods". __Journal of the American Chemical Society (JACS)__. //128//, 2115-2120. 26 January 2006.

3. Huang, Xiaohua, Prashant K. Jain, Ivan H. El-Sayed, and Mostafa El-Sayed. "Plasmonic photothermal therapy (PPTT) using gold nanoparticles". __Lasers Medical Science__. //23//, 217-228. 3 August 2007.

4. El-Sayed, Ivan, Xiaohua Huang, and Mostafa El-Sayed. "Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles". __Cancer Letters__. //239//, 129-135. (2006).

5. Dickerson, Erin B., Erik C. Dreaden, Xiaohua Huang, Ivan H. El-Sayed, Hunghao Chu, Sujatha Pushpanketh, John F. McDonald, and Mostafa El-Sayed. "Gold nanorod assisted near-infrared plasmonic photothermal therapy (PPTT) of squamous cell carcinoma in mice." __Cancer Letters__. //269//, 57-66 (2008).

6. Khlebtsov, Boris, Vladimir Zharov, Andrei Melnikov, Valery Tuchin, and Nikolai Khlebtsov. "Optical amplification of photothermal therapy with gold nanoparticles and nanoclusters". __Nanotechnology__. //17//, 5167-5179 (2006).

7. Islas, Angel. //BIOL 179: Cancer Biology Lecture//. Santa Clara University, Santa Clara, CA. 9 April 2012.

8. "Carcinoma." //Wikipedia//. 21 May 2012.