Abstract

Cancer, the uncontrolled proliferation of malignant cells caused by genetic mutations, affects 1.8 million people in the United States annually. Current cancer treatments are imprecise and often ineffective in preventing cancer recurrence. Likewise, long treatment durations and adverse effects carried by such are strenuous on the body. Our solution is Trojan Horse, a membrane-bound container modified with a sperm flagellum and peripheral antibodies which allow it to infiltrate the cellular membrane and kill cancer from the inside. The liposome carries VEGF-targeted CRISPR Cas-9 gene-editing proteins and Cytochrome C, which are released inside cancer cells, inducing cell death. After the first injection, a second dose containing location-tracking chips is administered to monitor tumor location within the body through the transmission of electromagnetic signals via a nanochip to external machinery and scanners. Trojan Horse streamlines cancer treatment by reducing adverse side effects, maximizing efficiency, and utilizing a simple yet potent design.

 

 

Present Technology

Cancer Treatment

Local cancer treatment methods include surgery and radiation therapy, which operate on specific organs or areas of the body. Surgery involves removing cancerous tissue using several methods, including cutting, burning, or freezing. While surgery carries dangers like infection and bleeding, it can be a very successful treatment for some cancers. Infection, bleeding, and long recovery drawbacks are avoided with Trojan Horse because tissue is not cut into or exposed to environmental factors. The other local treatment, radiation therapy, uses X-rays and other high-energy beams to kill cancer cells. The beams are aimed at the tumor, damaging the cancer cells' DNA to prevent them from proliferating. However, radiation therapy can also have serious adverse effects, such as exhaustion and harm to healthy cells.
 

Systemic cancer treatment methods include chemotherapy, hormone therapy, and targeted therapy, and work by targeting the body. Chemotherapy uses medications to kill cancer cells. Typically administered intravenously, the medications reach cancer cells across the body through blood circulation. Chemotherapy may treat blood cancer extremely effectively, but it can also cause substantial side effects including nausea and hair loss. Like radiation therapy, these side effects are a result of the treatment’s lack of precision, often harming nearby healthy cells along with cancerous ones. Trojan Horse will address this issue by using tumor-specific antibodies to bind cancerous cells only more precisely and leave healthy cells unscathed. Next, hormone therapy is used to both treat cancer and ease cancer symptoms. Hormone therapy works by either inhibiting or increasing hormone production to eliminate a tumor. A significant drawback is that hormone therapy is not versatile and usually only addresses prostate and breast cancer since these tumors use the hormones estrogen and testosterone as “fuel” to grow. Lastly, targeted therapy uses drugs that target proteins involved in cancer cell growth and division. One method uses small molecules that bypass the cellular membrane to deliver an inhibitor to certain proteins within the cancer cell. Another method utilizes synthetic antibodies to target antigens located on the surface of the cellular membrane. Tumor-Specific Antigens (TSA) are only found on cancer cells, so lab-grown proteins called monoclonal antibodies can be engineered to target and attach to these TSAs. Targeted therapy is less harsh on the human body than chemotherapy and radiation therapy because it is designed to avoid attacking healthy cells.

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Nanomedicine

Nanomedicine is a branch of medicine that uses nanotechnology to treat disease. Two cancer-related examples are Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and liposomes. CRISPR is a gene editing technology that allows for the precise modification of DNA sequences. It works by using an enzyme called Cas9, which acts as a "molecular scissors" to cut specific regions of DNA, and a guide RNA (gRNA) that binds to a specific target sequence. The gRNA directs the Cas9 enzyme to the precise location in the DNA where the cut needs to be made. Once the DNA is cut, the cell will naturally try to repair the break. This is where the true power of CRISPR lies, as researchers can use this repair process to add, delete, or replace specific genes with precision. CRISPR has been used to edit T cells to recognize and attack cancer, but the treatment is still unprecise. The technology is administered in a variety of ways, including injection, inhalation, or oral delivery, depending on the type of cells or tissues being targeted.­­­­­­­­

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Liposomes are microscopic, membrane-bound vesicles used to administer drugs to cells. They are composed of phospholipids and cholesterol.  The two hydrophobic phospholipid bilayers of liposomes are used to protect drugs from degradation in vivo, as well as in controlling drug release, modifying biodistribution, targeting drug delivery to a specific site, and enhancing biological compatibility. The vesicles may encapsulate water-soluble drugs in their aqueous spaces and lipid-soluble drugs within the membrane itself. Liposomes are manufactured via solvent evaporation, solvent dispersion, and reverse-phase evaporation. These processes entail the dissolution of phospholipids in an organic solvent which is then removed. Liposomes may be administered orally or via injection. Liposomes float freely in their environment until they undergo endocytosis (fusion with a cell’s membrane) and release their contents into the accepting cell. However, liposomes can often not penetrate deep enough into tumors and rely on a passive flow. Trojan Horse Liposomes (THLs) have a motile tail, allowing them to move through tumor cells without the use of blood flow.

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History

Cancer results from the abnormal growth and proliferation of cells resulting from factors that damage genetic information and alter cancer-regulating proteins such as proto-oncogenes or tumor suppressors. When these proteins are altered, cells may bypass important checkpoints during cell division, becoming cancerous. Tumors can be benign, constricted to a localized area, or malignant, metastatically invasive. Each year, approximately 600 thousand people of 1.8 million cancer cases die in the United States alone.

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Evidence of cancer cells can be traced back to dinosaur fossils dating 70-80 million years old. The reported case of human cancer dates to 3000 BCE, recorded in ancient Egypt. However, no major breakthroughs in modern cancer research were made until the 18th century. In 1846, with the invention of anesthesia, surgery became a safer and more commonplace procedure, allowing surgical removal to become standard in cancer treatment.

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In 1895, the first X-ray machine was built, quickly giving way to the first practice of radiation therapy in 1896. The Golden Age of Medicine, beginning in the mid-20th century, led to significant leaps in the field of medicine and molecular biology. Closely following the invention of hormone therapy, chemotherapy was first practiced in 1942 and involved the chemical agent nitrogen mustard to temporarily slow the spread of lymphosarcoma. Other breakthroughs in this era of research include the first complete cure of tumor via chemotherapy in 1953, the first combination of chemotherapeutic drugs in 1958, the discovery of the commonly mutated tumor suppressor, P53, in 1979, the invention of CRISPR in 2012, and the recent development of more targeted forms of liposomes.

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Future Technology

While there have been several major milestones in detecting and combating cancer, Trojan Horse would propel the medical field into a new era of cancer treatment. Utilizing a host of liposomal, motile, genetic, and immunological techniques, Trojan Horse not only prevents cancer growth but also eradicates cancerous tissues entirely. An overview design of the Trojan Horse involves the attachment of a liposome to cryopreserved sperm flagellum to produce a system that mimics sperm-like motility. The human body’s smallest capillaries measure 5000 nanometers in diameter, allowing 4500 nanometer Trojan Horse Liposomes (THLs) to reach any cancer cell that blood flows to. The THLs’ phospholipid bilayers would measure 4 nanometers in width, the same as a human’s lipid bilayer. The flagellum, coming from cryopreserved sperm flagellum, would consist of an axoneme (11 microtubules collectively), a thin cell membrane encapsulating the axoneme, and mitochondria arranged around the axoneme. The motile flagellum would measure a length of approximately 5000 nanometers to provide sufficient thrust. The diameter would measure 10 nanometers, encompassing the mitochondrial, membrane, and axoneme layers.

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Attached to the extracellular portion of the first dose THLs would be our monoclonal antibodies. These antibodies would be tailored to the type of tumor a patient has. For instance, cells with lung cancer may harbor different TSAs compared to cells with breast cancer, requiring different types of antibodies on the respective THL treatment. After recognition of TSAs, the THLs would undergo endocytosis to release the “soldiers” hidden inside. The interior of the THL would consist of two major components: VEGF-targeting CRISPR-cas9 systems and free-floating Cytochrome C. VEGF is an angiogenesis-promoting gene that when expressed, propagates a signal to create blood vessels to feed the cancerous tissue nutrients and oxygen. Without it, no more blood vessels can be created for cancer to grow. The free-floating Cytochrome Cs released during liposomal endocytosis would bind to Apaf-1 to form apoptosomes, recruiting caspases that cleave protein structures and trigger a cascade of events leading to apoptosis. The Trojan Horse’s VEGF-cutting CRISPR-cas9 system acts as a fallback to prevent further cancer growth in case ­­­Cytochrome C induced apoptosis fails. The Trojan Horse would be administered via intravenous injection. After undergoing all the processes required to kill a cell, the material would be engulfed by a phagocyte and broken down. If a Trojan Horse does not bind to TSAs, it would be easily filtered by the kidney since it is composed of organic material. First dose THLs would also include a coating of synthetic antigens which will become relevant in the second dosage.

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Future technological advancements may entail a second dosage of THLs. However, liposomes in this dose would have distinct modifications from the first dose of THLs; the second dose would be dedicated to monitoring the targeted tumor. The second dose THLs are coated with a second type of monoclonal antibody that binds to the synthetic antigens placed on the surface of the first dose THLs. To detect endocytosis, the microchip within the liposome would use a pH indicator. The liposome has an internal pH of 7, while the cytoplasm within a cell is slightly basic at 7.4 pH. After sensing a rise in pH, which indicates that the contents of the liposome have entered a cell, the microchip sends a simple electromagnetic signal to be received and located by an external device such as a smartphone or smartwatch. From there, patients and doctors can monitor the size and location of tumors using signals from Trojan Horse microchips.

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Ultimately, Trojan Horse would become the keystone of cancer treatment. Its cutting-edge, personalized, and precise technology would take the war against cancer to a new level, delivering the hidden soldiers that are CRISPR and Cytochrome C into cells. Easy monitoring with a second dose would allow for a new and unprecedented level of awareness to effectively eradicate cancerous tissue. Welcome to the future of cancer treatment.

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Breakthroughs

In order for Trojan Horse to become a reality, technological advancements in various areas are imperative.  First, breakthroughs in Cytochrome C viability are needed for the Trojan Horse to effectively kill cancerous tissues. To test for viability, techniques for isolating Cytochrome C such as chromatography on synthetic ion exchange resin have been shown to be very effective in many organisms (dogs, cows, salmon, horses, etc.). Likewise, aqueous solutions of ammonium sulfate and other solvent solutions have been successful in storing Cytochrome C, ranging from below freezing to ambient temperatures (-20°C to 25°C). However, maintaining enzymatic viability and protein structure throughout the process of Cytochrome C insertion into liposomes and intravenous injection remains a hurdle. To test Cytochrome C viability, we will be conducting a laboratory experiment where Cytochrome C is placed into aqueous environments for a certain time at a certain temperature and transferred to stem cells. Researchers would carry out the procedures as follows:

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1. Isolate Cytochrome C in 10 Petri dishes, each at different temperatures ranging from -20 degrees Celsius to 70 degrees Celsius.

2. After an hour of storage, the Cytochromes would be placed in 10 different Petri dishes given the respective temperatures they were held at, each containing human stem cells.

3. Observe changes in free-floating Cytochromes to determine whether apoptosomes have been formed.

4. All previous steps may be repeated by changing the independent variable from temperature of storage to amount of time stored before insertion into cells.

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Since apoptosomes form when free-floating Apaf-1 and Cytochrome Cs combine, we can measure the amount of apoptosomes formed by subtracting the original number of cytochromes put in and the number of cytochromes left free-floating. These differences can be quantified using Western Blot and fluorescence microscopy. The former involves cell fractionation, isolation of mitochondria, and gel electrophoresis. The latter involves analysis of immunolabeled cells via an optical microscope that uses scattering, reflection, attenuation, and absorption of light to observe changes in organic substances.

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Another obstacle in making Trojan Horse a reality includes flagellum attachment to liposomes. Current technology is not capable of effectively programming flagellum to move the liposome to the target site. Brownian motion and the effect of entropy exponentially make the task of movement within cellular environments challenging.

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Manufacturing microscopic circuits to be used in a THL requires technology that is currently beyond reach. In 2022, IBM showed it is possible to build two-nanometer microchips, which are small enough to fit within a THL. However, this microchip requires a power source to function, especially when our goal is to emit a high-power radio signal, and with today’s technology, no battery would both fit inside a THL and supply sufficient power.

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Today, liposomes have only reached a maximum diameter of 2500 nanometers, 2000 less than THLs. Manufacturing larger liposomes is key for increasing the storage capabilities of nanomedicine such as liposomes. Overall, Trojan Horse requires advancements in biomolecule viability, biomolecule movement mechanics, and microtechnology to become a reality.

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Design Process

Three alternative features we considered for Trojan Horse included an internal “factory” to manufacture liposomes, shape-shifting antibodies for more diverse recognition of cancerous tissue, and the combination of medication and a chip into one liposome dosage instead of two.

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We rejected the idea of making a large non-organic “factory” for liposomes. This nanobot would only require one dose, as it would produce motile liposomes by using resources absorbed by the digestive system. However, the size of the “factory” is too unrealistic and would entail hundreds if not thousands of enzymes to create liposomes with medication inside. If produced, such advanced technology would have an exorbitant cost, making the treatment inaccessible to most patients. The final concept of Trojan Horse solves this issue by administering two doses of pre-manufactured organic biomolecules which can be broken down and filtered within the body itself, be it phagocytes eating dead cells or kidneys filtering out waste.

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The next rejected idea for Trojan Horse was shape-shifting surface antibodies for more diverse tumor recognition. This concept would entail having custom antibodies measuring 10 nanometers in length that have a mechanism to change shapes to detect all forms of cancer. Again, this technology would be more than decades away and cost a myriad of money for patients to consume. The final Trojan Horse addresses these issues by having customized antibodies for each patient. Instead of having shape-shifting antibodies, the liposomes have one specific type of antibody to bind to cancerous tissue more precisely.

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We rejected the last idea of putting both the microchip and the medication in the same liposome because THLs are too small to fit current location-tracking microchips and the medication would interfere with the chip’s functionality. Trojan Horse solves this issue by separating the medication and tracking technology into two doses to allow for more precise location of tumors.

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Consequences

The benefits of Trojan Horse for society are innumerable. Its non-invasive and highly targeted specific approach to eradicating cancer prevents adverse effects of radiation therapy, chemotherapy, and surgical operations. The great degree of personalization in switching antibodies on the surface of THLs bypasses the issues of restricted treatment such as in hormone therapy. Thus, the economic toll in producing THLs would not burden and possibly harbor socioeconomic divisions within society. Rather, its administration through vaccination makes the treatment very accessible to all and quick acting. Trojan Horse is organic, minimizing harmful side effects and allowing easy filtration by the kidney. Its second dose allows for an unprecedented level of awareness for patients and physicians to assess the size and location of cancerous tissue for treatment.

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Although Trojan Horse provides many alleviations for cancer treatment, there are a few drawbacks that may negatively impact patients and society. First, left-over Trojan Horses that may have been unable to recognize cancerous tissue may accumulate in the kidney, where they could affect its osmotic gradient. When the Trojan Horses are filtered into the descending loop of Henle, osmolarity could be disrupted, inhibiting the release of necessary solvent (water) to carry the filtered waste to the bladder. However, this is unlikely because of Trojan Horse’s organic structure and microscopic size. Second, malfunctions with any part of the biomolecule could have several adverse repercussions. For example, malfunctions with antibody recognition could lead to the Trojan Horse attacking non-cancerous cells, mimicking the negative effects of chemotherapy on healthy cells. Finally, doctors may become too dependent on the second dose of Trojan Horse to identify where tumor cells are. Over-reliance on Trojan Horse’s tumor-tracking second dose could be harmful to doctors in society: if there is an error in the recognition of tumor cells and doctors do not question it, procedures could significantly harm the patient being treated.

 

 

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