Abstract
Radiation therapy, while a functional means of cancer treatment is flawed for its ineffectiveness in the hypoxic environment of solid tumor cells. Current research seeks to design a more effective and selective means of cancer cell targeting in hypoxic environments through nanoparticle aided radiotherapy. This study examines the effects of infusing gold nanoparticles less than 2 nm in diameter into lung cancer cell lines as a potential radiosensitizer in the absence of oxygen. It also examines the effectiveness of gold nanoparticles in providing increased treatment specificity by limiting collateral cell damage during radiotherapy. By exploiting the hypervascularity of solid tumors and the Enhanced Permeability and Retention Effect, nanoparticles can passively accumulate inside the nucleus of tumor cells. When exposed to sufficient doses of external radiation, these nanoparticles emit Photoelectric and Auger electrons, which mimic the DNA damaging effects of reactive oxygen species, leading to tumor cell apoptosis. A549 and LLC1 cancer cells were examined using clonogenic survival assay techniques, with and without 100 µM of GNP, in single point radiation doses of 0, 2, 6, 10 and 20 gray. Combinational treatment was found to be more effective than isolated radiation with a viable colony count of zero after normalization compared to the 43% viability observed with RT alone. A clonogenic toxicity study was conducted to determine threshold dose levels of GNP in HUVEC cells which proved to be cytotoxic at doses greater than 1 mg/ml. The use of GNP as a radiosensitizer can be an effective means of solid tumor cell treatment in both hypoxic and normoxic environments. By acting as both a reactive oxygen species generator and a radiosensitization enhancer, it can elicit increased tumor cell apoptosis leading to reduced external beam radiation treatment doses.
Keywords: Hypoxia, External Beam Radiotherapy, Enhanced Permeability and Retention Effect, Gold Nanoparticles
Introduction
Cancer—a family of diseases characterized by uncontrolled proliferation of malignant abnormal cells, is caused by genomic mutations arising from environmental and hereditary influences. Exposure to carcinogenic elements such as lead, arsenic, and inorganic fluoride, and implementation of detrimental lifestyle behaviors such as tobacco intake, consumption of processed foods and lack of exercise can lead to the formation of mutations and subsequently carcinogenesis. Cancer results in millions of deaths each year worldwide, with a staggering mortality rate of 8.2 million in 20121. Current projections estimate the number of new cancer diagnosis within the United States to reach 1.7M in 20182. Due to its prevalence and its often fatal nature, medical facilities, research institutions and governmental organizations globally have pooled together their resources to improve upon the detection, diagnosis, and treatment of many forms of cancers. Currently, treatment options include chemotherapy, surgery, immunotherapy, molecular targeted therapy, stem cell transplant, and radiation therapy.
While each treatment has been designed to most effectively combat specific cancer types at specific stages for increased efficacy, clinicians and patients must often confront the limitations of these therapies. Many cancer therapies—mainly those dealing with personalized forms of cancer treatment, are based on recent novel discoveries and are presently not feasible for clinical use as they require further analysis. As such, the main focus in cancer treatment continues to be on the improvement of surgical, radiotherapeutic and chemotherapeutic means of cancer treatment, with an emphasis in radiotherapy due to its ease of use, specificity and efficacy in combinational therapies. Much of the research being conducted in the field of oncology attempts to devise mechanisms for understanding and improving the efficacy of these cancer therapy.
Chemotherapy
Chemotherapy, a form of cancer treatment that utilizes cytostatics (cytotoxic substances that act as cell growth inhibitors) as a mode of inducing cellular destruction, forms one of the three pillars of cancer therapy. Due to the high proliferative nature of cancer cells, cytostatics are used as a means of disrupting critical cell-growth regulating mechanisms, leading to inhibition of cell division and prevention of subsequent proliferation. Chemotherapy is an effective means of dealing with late-stage metastatic malignancies due to its ability to target large areas. However, because cytostatics are extremely nonspecific and equally affect the growth regulating mechanisms of cancerous and non-cancerous cells, patients are often faced with severe side effects after treatment such as; anemia, lymphedema, neutropenia, hair loss and bleeding. Current research in this field seeks to increase the specificity and effectivity of these cytotoxic agents while drastically reducing their side effects3,4. Advances in chemotherapeutic techniques such as the development of doxorubicin hydrochloride (Adriamycin) for the treatment of soft tissue and skeletal sarcoma, and hyperthermia for treating deep-seated tumors by localized magnetic-loop induction show promise in increasing specificity5. Chemotherapy is often avoided whenever possible as the first step of cancer treatment due to its poisonous nature and is usually utilized in small doses and in combination with other cancer treatments types for increased efficacy.
Surgery
Surgery, the second pillar of cancer treatment serves as a highly specific alternative to chemotherapy. It allows for the precise removal of large localized tumors while minimizing collateral damage to surrounding tissue. However, its effectivity is limited only to single masses and localized tumors and cannot be utilized for metastatic forms. It also lacks the ability to remove microscopic tumor cells thus increasing the likelihood of recurrence. Is also a highly invasive form of treatment often requiring significant amounts of time for proper recovery as well as having the opportunity for infection. Due to these limitations, surgery is often used in early-stage cancer before metastasis and in combination with other forms of treatment.
Personalized Cancer Therapy
The field of personalized cancer therapy is a relatively new field of precision medicine. Its premise lies in understanding and sequencing the biological markers of patient cancers and using the generated information to determine a personalized and precise form of treatment. The field of personalized cancer therapy shows promise in reducing autoimmune responses that may result from specific cancer types, while also increasing treatment specificity.
Immunotherapy
Immunotherapy—a relatively new branch of personalized cancer therapy, attempts to harness the power of the immune system as an anti-tumor agent. Early stage research details a phenomenon where the localized treatment of a primary tumor using external beam radiation causes both shrinking of the primary tumor in addition to shrinking of distal tumors outside the localized zone of treatment. This phenomenon, now known as the Abscopal effect was first referenced by Mole in 1953 on his work with whole body irradiation6. It has been attributed to the enhancement of endogenous anti-tumor and adaptive immune response systems7, the induction of antigen-specific immune response by T cells via cross-presentation by antigen-presenting cells (APC) using radiation-based antigens, and the release of inflammatory cytokines such as Tumor Necrosis Factor alpha (TNF-a), Interleukin 1 (IL-1) and Interleukin 6 (IL-6)8. Ionizing radiation produces up-regulation of tumor antigens and antigen presenting machinery leading to enhanced tumor recognition and killing and induction of positive immunomodulatory pathways9. As a result, the immune system is primed to selectively find and destroy cancerous cells.
Molecular Targeted Therapy
Molecular Targeted Therapy attempts to block the proliferation of cancer cells through the inhibition of molecules known to promote tumor growth and carcinogenesis. This therapy relies on understanding the molecular cancer cell characteristics for each individual patient and has proven difficult due to the fact that individual patients have variations in the molecular composition of their cancer cells making molecular targeting difficult and time-consuming.
Stem Cell Transplant
Stem cell transplant, often a secondary treatment for patients initially treated with particularly high doses of radiation and chemotherapy, is used to contain the large collateral cell damage often associated with these doses. High dose treatment causes significant destruction of noncancerous cells—including immune and stem cell populations. To counteract this, stem cells from donors are transplanted into host patients to regenerate lost stem cell populations. However, in some instances, the host immune system can reject the stem cell transplant, and treat it as a foreign invader. The use of nanoparticles in external beam radiation therapy proves to be promising in reducing the dose of radiation required while still providing enough localized damage for dealing with the resistant malignant cancer cells characteristic of late-stage cancer.
Radiation Therapy
Radiation therapy—one of the most common forms of cancer treatment utilizes the generation of high energy particles to destroy cancer cell DNA and induce apoptosis. Alpha particles, beta particles, and x-rays have the capacity to ionize surrounding molecules through the removal of tightly bound electrons from their orbital shells10. These ions are extremely reactive with high velocities that can create breaks in the sugar-phosphate backbone of the DNA strands and can disrupt the hydrogen bond base pairing of DNA strands upon contact. Alpha particles are known to have heavier charges than beta particles and X-rays and as a result, they have the capacity to do more damage when in contact with cellular DNA11. A field of radiation therapy known as radioimmunotherapy has been developed around this concept. Through Targeted Alpha particle Therapy (TAT), molecular substances undergoing alpha decay can be inserted into tumor cells through monoclonal antibodies and then irradiated to generate alpha particles and damage DNA11. DNA damage generated by radiation therapy is often repaired by cellular DNA repair mechanisms such as base excision repair, homologous recombination, nucleotide excision repair, mismatch repair and non-homologous end joining (NHEJ). However, in the case of catastrophic DNA damage—mainly double-strand breaks, DNA may undergo apoptosis due to genomic instability12.
Current research in the field of oncology seeks to increase our understanding of personalized cancer therapy due to its promise of being an effective tool in cancer treatment. However, this field is still in its infancy and not much is known about the mechanisms of cancer biology. Thus, other forms of cancer treatment must be utilized and improved upon to serve in the meantime. Radiation therapy is often chosen as the best form of treatment for cancer patients due to its high specificity (when compared to chemotherapy) as well as its decreased invasiveness and ability to deal with metastasis (when compared with surgery). Improving the efficacy of radiation therapy—namely its ineffectiveness in the oxygen-deprived environment of solid tumor cells, as well as its precision is a highly active field of research. The use of nanoparticles shows promise for increasing RT efficacy in hypoxic and normoxic conditions
Tumor Hypoxia
During the increased volume progression of solid tumor cells, angiogenesis is initiated. Production of new blood vessels serves to supply tumor cells with blood and oxygen levels necessary for cell survival. This hypervascularity aids in the rapid growth and proliferation of tumor cells by increasing blood and oxygen delivery but proves unsustainable. Over time, oxygen concentrations required for by the solid tumor mass greatly outweighs the capacity of the blood vessels to supply. This leads to subsections of the solid tumor highly lacking in oxygen—a phenomenon known as hypoxia.
Radiation therapy relies extensively on the ability to generate radiosensitizers from molecular oxygen (O2). Following the absorption of high energy radiation, molecular oxygen can be transformed into reactive oxygen species (free radicals) with the capacity of producing DNA damage such as single and double strand breaks—the latter being extremely catastrophic13. Without the presence of molecular oxygen in the tumor microenvironment, the efficacy of radiation treatment is drastically reduced. Radiation therapy in an anoxic environment shows that cells are ~3x more resistant to radiation than cells with normal oxygen levels14 highlighting the importance of environmental molecular oxygen in radiation treatment. It is therefore integral to find a substitute for ROS species in hypoxic environments.
Gold Nanoparticles as radiosensitizers
Metallic nanoparticles are nanoscale molecules composed of metallic alloys that can be utilized in a variety of forms for the treatment of cancer. Recently, these molecules have begun to play an increasingly important role in the field of nanotechnology and cancer biology due to their ability to generate free radicals through the photoelectric and Auger effect. The Auger effect is a phenomenon first observed independently by Austrian physicist Lise Meitner in 1922 on her work with nuclear beta electrons15 and by French physicist Pierre Auger in 1923 on his work with Secondary β Rays16. It details a phenomenon where the displacement of an electron from the lowest valence shell of an atom generates a vacancy causing atomic instability. To compensate for this, an electron from the highest valence shell of the atom (with the highest energy level) will drop down to fill the vacancy and upon doing so, will release energy equal to the difference between its initial orbital energy and its final orbital energy (ΔE)17. This transition energy can be transferred into another electron and if it is higher than the orbital binding energy, it will cause the receiving electron to be ejected from the atom at an extremely high velocity. This electron is known as an Auger electron and due to its high velocity, it can be extremely destructive if it comes into contact with cellular DNA—causing lesions, single strand breaks, and double-strand breaks.
The displacement of the first electron is achieved through the use of ionizing radiation. By ionizing the area surrounding an atom, it is possible to excite its electrons to such a state that the highest valence electrons (auger electrons) are displaced. The velocity of the auger electron, when ejected from the atom, determines how catastrophic it will be when in contact with cellular DNA and is directly proportional to the transition energy it receives. In order to maximize this velocity, an atom must be chosen with the largest possible valence shells to create a large difference in transition energy from the highest valence shell to the lowest valence shell. Due to the large valence shell numbers present in transition metals, they serve as perfect candidates to generate high-velocity auger electrons.
The photoelectric effect is a secondary phenomenon activated by the use of radiation therapy on metallic nanoparticles. When metallic particles are exposed to electromagnetic radiation of specific wavelengths (known as the threshold frequency), light particles (photons) are absorbed by an electron from the metal atom, generating a current18. The energy absorbed by the electron ionizes it leading to its displacement and subsequent damaging of DNA
When a metallic nanoparticle is injected into a solid tumor tissue and is exposed to external beam radiation, it is possible to generate both auger and photoelectric electrons both of which can cause significant projectile damage when localized near tissue and increase the efficacy of radiation treatment. Metallic nanoparticles are efficient because they can be conjugated with antibodies, ligands, and spacers for drug delivery systems.
Enhanced Permeability and Retention Effect
The Enhanced Permeability and Retention Effect is a highly debated premise in the field of cancer biology first referenced by Dr. Hiroshi Maeda in his work describing the Conjugation of poly(styrene-co-maleic acid) derivatives to the anti-tumor protein neocarzinostatin (SMANCS) in murine tumor models19. It defines a unique phenomenon where molecules of specific sizes (mainly nanoparticles), have a tendency to accumulate in tumor tissue to a higher degree than they do to normal tissue due to their hypervascular nature. Tumor cells are also lacking in lymphatics—thin channels that carry lymph fluids (containing lymphocytes) from the body to the bloodstream. Unable to conduct lymphatic drainage, certain molecules such as nanoparticles are not properly filtered out leading to their retention and accumulation over time. Inversely, due to the extremely tight endothelial junctions of normal tissue, nanoparticles do not have the capacity to penetrate their junctions and thus cannot enter20. Over time, certain molecules are found in much higher concentrations in tumor tissue than normal cell tissue.
Tumor cells develop abnormalities in their perivascular cells and the basement membrane or smooth-muscle layer. This leads to the creation of a much wider lumen and less control of the permeability of certain molecules21. The vascular endothelial growth factor is an extremely potent angiogenic factor necessary for the growth of vascular endothelial cells and is significantly upregulated in tumors leading to an incredibly rapid rate of blood vessel formation22. It is a leading cause of the rampant angiogenesis observed in tumor cells. This rapid rate of blood vessel formation leads to the establishment of aberrant leaky vascular architecture due to the fact that the blood vessels are grown in a rushed, misguided manner. This is compounded by the fact that tumor tissues produce an extensive amount of vascular permeability factors which lead to extravasation within tumor tissues23. Tumor cells develop abnormal molecule transport dynamics when compared to normal tissue forming the basis of which the enhanced permeability and retention effect occurs.
The Enhanced Permeability and Retention Effect has been replicated by multiple studies and is now considered to be one of the cornerstones in tumor targeting chemotherapy and nanoparticle-based radiation therapy24. By taking advantage of the leaky vasculature of solid tumor cells, and the enhanced permeability and retention effect, it is possible to inject gold nanoparticles into a treatment site and in do so, create an accumulation in the tissue of solid tumor cells. Subsequent irradiation will then cause generation of Auger electrons and thus DNA damage.
Benefits of GNP for Reducing Collateral Cell Damage
Due to the fact that gold nanoparticles accumulate in cancer cells to a much higher degree than they do in normal epithelial cells, it is possible to utilize their ROS generating capabilities to act in reducing collateral cell damage in normoxic environments. In normoxic conditions with the presence of GNP, radiation therapy can generate ROS species using molecular oxygen as well as free radicals through the Auger effect. Therefore in normoxic conditions, cancer cells can be bombarded with both ROS species and Auger electrons, leading to twice the amount of DNA damage for the same radiation dose. This acts to limit the actual dose of radiation required to destroy cancerous cells, limiting the harm that normal cells are exposed to.
Methods
Plating Efficiency
To determine the plating efficiency of LLC1 and A549 cells, these cell lines were seeded at different densities (300, 400, 500 cells/well for A549, 150, 250, 350 cells/well for LLC1) and allowed to grow for 7 days with subsequent media change every 48 hours.
Clonogenic Assay using GNP
To determine the effect of GNP on radiation treatment, mouse Lewis lung carcinoma LL/2 (LLC1) cells were plated at 200 cells per well in 6 well plates. 24 hours after plating, 100 uM GNP was pipetted into each well. For control groups, 100 uM of culture medium was pipetted into each well. 24 hours after treatment, cells were irradiated using 0, 2, 6, 10 and 20gy doses at 100kVP. The plates were allowed to grow for 7 days with regular media change until 80% confluence. They were subsequently stained and counted to determine viable cell count and generate a survival curve.
Adenocarcinomic human alveolar basal epithelial cells (A549) were plated at 350 cells per well in 6 well plates. 24 hours after plating, 100 uM GNP was pipetted into each well. For control groups, 100 uM of culture medium was pipetted into each well. 24 hours after treatment, cells were irradiated using 0, 2, 6, 10 and 20gy doses at 100kVP. The plates were allowed to grow for 5 days with regular media change until 80% confluence. They were subsequently stained and counted to determine viable cell count and generate a survival curve.
GNP Diffusion Pattern
To determine the rate and pattern of diffusion, we created varied concentrations of agar gel in 6-well plates and injected 0.1% of gold nanoparticles with less than 2nm diameter into the gels and observed speed and pattern of diffusion over 6 hours.
Injection of Gold Nanoparticles
In delivering gold nanoparticles into solid tumors, intravenous drug delivery methods will be extremely hindered due to the leaky vasculature of cancerous cells25. The rate of diffusion and dispersal pattern of gold nanoparticles must be taken into account to determine the mode of drug delivery. This will determine whether single site delivery or multiple tumor site injections would be necessary during treatment. In the case where gold nanoparticles aggregate at a single point within the cell lumen, multiple sites of injection would be necessary during treatment. However, if the gold nanoparticles disperse evenly across the solid tumor cell, then a single point dose would be sufficient. We delivered nanoparticles at single and multi-point doses using needles.
Testing for Auger electrons
N-acetyl-L-cysteine (NAC) is a derivative of the amino acid L-cystine that can be utilized as an antioxidant26. It is often used in cancer biology to test radical oxygen species inducers as well as to act as a free radical scavenger26. To evaluate the assumption that gold nanoparticles injected inside cancer cells and exposed to ionizing radiation under hypoxic conditions will develop auger electrons (free radicals) species usually present in normoxic environments, we utilized the ROS scavenging properties of N-acetyl-L-cysteine. In the presence of N-acetyl-L-cysteine, we should see no effects of combinational treatment using gold nanoparticle and radiation therapy on solid tumor cells because all generated auger and photoelectric electrons will be removed. In the absence of N-acetyl-L-cysteine, we should see the characteristic drop in tumor survival rate.
Results
LLC1 Survival Curve
Treatment of LLC1 cell lines with 100 uM dose of GNP and 4gy radiation dose at 100kVp. Control groups (NT) were given equal volumes of media instead of GNP. There was significant tumor killing observed in combinational treatment with GNP and radiation therapy compared to just radiation alone (figure 1). 120 hours after treatment (5 days) we see 0% cell viability with combinational treatment and 43%viability with radiation therapy alone (normalized data). We see a larger increase in cell death with combinational therapy (figure 2)
A549 Survival Curve
Treatment of A549 cell lines with 100 uM dose of GNP and 4gy dose of radiation at 100kVp. Control groups (NT) were given an equal volume of media instead of GNP. There was significant tumor killing observed in combinational treatment with GNP and radiation therapy than using radiation therapy in isolation (figure 3). 168 hours after treatment (7 days) we see 0% cell viability with combinational treatment and 52% viability with radiation therapy alone (normalized data). Again we see a larger increase in cell death with combinational therapy (figure 4)
GNP Diffusion
To understand the rate and pattern of diffusion for GNP studies, we used agar gels at different concentrations to mimic the tumor microenvironment. In figure 5, We evaluated the dispersal of GNP at 0.2%, o.5%, and 1% agar concentrations over a period of 3 hours and we saw that GNP disperses evenly and does not localize to a specific spot. This means that for treatment of cancer patients, it is sufficient to inject GNP at a single injection site.
Metal Nanoparticles Cytotoxicity
While many metals may be harmless in trace levels, they have the capacity to become toxic at certain concentrations. It is thus important to selectively choose which metallic nanoparticles to use for treatment. We decided to evaluate the toxicity of gold nanoparticles. To determine its cytotoxic effects on normal endothelial cells, we conducted a clonogenic toxicity study using HUVEC cells. At doses equal to or greater than 1 mg/ml, GNP showed cellular toxicity suggesting that doses higher than these should not be used in clinical studies (figure 6).
Discussion
The efficacy of radiation therapy is drastically reduced in the hypoxic microenvironment of solid tumor cells. Due to a lack of molecular oxygen (O2), ionizing radiation is unable to create reactive oxygen species and as a result, the amount of catastrophic DNA damage and apoptosis that occurs is drastically reduced. This often leads to an increase in radiation treatment doses to compensate for the low treatment efficacy and as a result, patients often have greater collateral tissue damage. The use of metal nanoparticles has the potential to reduce treatment dose by enhancing efficacy. By utilizing gold nanoparticles, ionizing radiation can generate high velocity photoelectric and Auger electrons that can mimic ROS species and lead to DNA damage. Because the auger effect is independent of the oxygen levels in the environment, it can serve as a viable replacement in the hypoxic solid tumor microenvironment.
The relatively large error bars presents in figures 1 and 3 might signify variations in environmental conditions and/or errors in technique that results in inconsistent viability of LLC1 and A549 cells.To account for this, future replicative studies are currently being conducted to generate a mean that tends closely to the true mean as well as to generate standard error of the mean (SEM) calculations to account for experimental variables.
Current research in our lab also attempts to create a more accurate tumor microenvironment to gain a better understanding of how combinational treatment using gold nanoparticles and radiation therapy will behave in vivo. We are also attempting to induce hypoxia in vitro through clonogenic assay inside a hypoxia chamber. Future studies hope to determine its effects in an in-vivo hypoxic tumor microenvironment through the use of mice models. We will also be looking into combinational clonogenic assays where two cancer cell line types are grown in the same environment to better mimic the tumor microenvironment.
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Appendix
- Figure 1: Survival Assay of LLC1 cells treated with combinational treatment of 4 Gy Radiation and 100 μM GNP and isolated treatment of 4 gy radiation.
- Figure 2: Clonogenic survival assay of LLC1 cells treated with 100 μM GNP and 4gy radiation at 100kvP. Cells were observed for 72 hours after irradiation.
- Figure 3: Survival Assay of A549 cells treated with combinational treatment of 4 Gy Radiation and 100 μM GNP and isolated treatment of 4 gy radiation.
- Figure 4: Clonogenic survival assay of LLC1 cells treated with 100 μM GNP and 4gy radiation at 100kvP. Cells were observed for 72 hours after irradiation.
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