DNA Nanorobot Programmed to Target and Induce Apoptosis in Cancer Cells

By: Peter Lee, Daniel Howard, Evan Miller, and Trent Weiss

Abstract

Richard Feynman first seeded the conceptual foundation of nanotechnology in 1959 in

his talk “There’s Plenty of Room at the Bottom.” The field gained massive attention during the 1980s after the invention of the scanning tunneling microscope and atomic force microscope. These inventions allowed for visualization of individual atoms and molecules at the nanoscale, which remained impossible up until this time. Concurrently, Nadrian Seeman proposed the idea of DNA nanotechnology, which is centered on bottom-up molecular self-assembly. Taking a bottom-up molecular self-assembly approach is desirable due to the relatively inexpensive ability to create similar nanostructures to those produced under nonautonomous conditions. In DNA nanotechnology, the Watson-Crick base pairing tendencies of nucleotides are exploited to form highly specified and predictable structures. A specific selection of sequences will result in a recognizable structure because it is known how DNA will interact with itself to form three-dimensional structures based on the base sequences.

Until liposomes began to be used for targeted drug delivery in the early 2000s, targeted drug delivery was unheard of. In 2009, DNA nanorobots were proposed and patented as a new method of targeted drug delivery, specifically for cancer cells by scientists at the Wyss Institute for Biologically Inspired Engineering at Harvard University [2]. The DNA nanorobots offer an improvement upon the targeting of cancer cells and the quantity of drugs delivered. While the technology is encouraging, it is still in the early stages of development. Significant further research must be done to optimize the production of nanorobots. Due to the high prices of early prototypes, finding another model to construct the nanorobot entirely out of DNA could potentially provide a cost effective alternative that would bring this technology towards commercialization. Background

The DNA nanorobot is most applicable to diseases such as cancer where currently one of the only viable innocuous treatments lies in targeted drug delivery. Cancer is one of the most threatening health issues facing the modern world. To surmount this issue, four principal approaches have been taken: discovery, detection, delivery, and destruction. Our research specifically focuses on the targeted delivery of therapeutic drugs to the cancer cell to induce apoptosis. Some of the various drug delivery systems constructed are centered on magnetic nanoparticles (MNPs), liposomes, and micelles. The drug delivery system presented in this paper is constructed using DNA as the self-assembling building material. DNA was chosen as the

material as oppose to the others aforementioned due to a multiplicity of factors, including programmability, biocompatibility, and biodegradability.

It is easy to construct a DNA-based drug delivery system due to the programmability of DNA, which exploits traditional, complementary Watson-Crick base-pairing rules with adenine forming a double hydrogen bond with thymine and guanine forming a triple hydrogen bond with cytosine. Complementary base pairing allows for manipulation in the creation of the nanostructures as shown in Figure 1.

Moreover, DNA as a building material is proven to be biocompatible with the human body system as DNA is an inherent component of our genetic make-up. Furthermore, DNA is naturally a biodegradable object that leaves no potentially dangerous substance within the human body system.

Figure 1: DNA origami techniques used to create various structures

Method of Construction

There are two principal methods in structural DNA Nanotechnology to construct any arbitrary three-dimensional object out of DNA: tile-based structures that assemble from smaller components and DNA origami. The former was the initial methodology adopted by Nadrian Seeman when he first constructed 3D structures using DNA. To do so, he combined branched 4-arm Holliday junctions via sticky ended cohesion to form planar lattices that curve back upon themselves to create three- dimensional structures [4]. The latter, DNA origami, however, is the process by which our drug delivery system is constructed. Paul Rothemund invented DNA origami in 2006, and it essentially works by folding DNA at the nanoscale. A long chain single strand of viral DNA called a scaffold is folded with the aid of multiple short single strands of viral DNA called staples. These staples aid in the folding process by binding onto different areas of the scaffold under Watson-Crick base pairing rules. Images are then drawn with a raster fill of the scaffold. This design is then put into a computer software program called Cadnano, which computes the individual staple placement calculations. The DNA is then mixed and annealed. During the cooling portion of annealing, the staples pull the scaffold into the desired three-

dimensional shape as DNA has self-assembling properties. To observe the synthesized three-dimensional object, various microscopes such as the atomic force microscope, electron microscope, and fluorescence microscope can be used [9].

Our DNA nanorobot is primarily formed by using a scaffold of about 7,000 bases [2]. Aforementioned, in order to keep this structure robust, staples need to be binded to specific sites on the scaffold. Using Cadnano, electronic impulses will be sent to the DNA chain to directly influence its shape. Once the general structural outline is laid out, the staples are added in to allow the structure to fully form around the skeletal foundation. Subsequently, aptamer locks are then positioned and stapled to the structure. Method of Operation The way the nanorobot works is very simple yet extremely effective. On the corners of one side of the nanorobot, there are specific antigen aptamer locks as shown in red in Figure 2. These locks are protein-specific, meaning that each lock will only open once in contact with the specific protein. In the case of drug delivery, the specific protein selected would be an antigen on the membrane of the targeted cell, which is produced by a specific strain of cancer. The locks are made up of two strands of DNA, which bond together. However, it is important to note that one strand is more strongly attracted to target protein, so the lock will open when in contact with the specified protein. This is extremely advantageous, as the aptamer locks can be changed on the nanorobot to only open when in contact with many distinct types of proteins, and hence a certain type of cell. The locking mechanism significantly prevents the nanorobot from opening too early and releasing the payload accidentally, as there is only a 0.6% chance of off target binding. Also, the locks work as a logic AND gate, meaning both locks need to be in contact with the target antigens for the DNA nanorobot to open. This also prevents one side of the nanorobot opening and releasing the payload, but not on the target cells.

Figure 2: Structure of the DNA nanorobot

The payload itself can be bonded to the DNA nanorobot at 14 different points. When both the aptamer locks are opened, a signal is sent down the DNA nanorobot to release the payload

from its DNA strands: this is achieved by the DNA being released from the large structure. The payload is then able to land directly on the targeted cell.

Due to the programmable properties of the DNA nanorobot, many types of payloads can be used as very few will interact with the DNA itself. This is advantageous because it is feasible to add payloads to treat many different types of illnesses including various types of cancers and other elusive diseases.

Laboratory Trials

In the primary source used, A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads by Shawn M. Douglas, Ido Bachelet and George M. Church, two experimental trials were completed to test the efficiency, reliability, and initial application of the DNA nanorobots. In the first trial, eight different types of DNA nanorobots were constructed containing combinations of three different types of aptamer locks (41t, TE17 and sgc8c) as well as a positive and negative control (always open and unopenable respectively)[2]. The nanorobots were loaded with fluorescent markers, which would cause marked cells to be identified under flow cytometry. These nanorobots were then introduced into six cell cultures: Burkitt’s lymphoma (possessing no keys), acute myeloblastic leukemia (possessing keys for 41t, TE17 and sgc8c), aggressive NK leukemia (possessing keys for 41t and TE17), T-cell leukemia (possessing keys for TE17 and sgc8c), acute lymphoblastic leukemia (possessing keys for TE17 and sgc8c) and neuroblastoma (possessing the key for scg8c)[2]. The gathered results can be found in Figure 3.

Figure 3: Activation of nanorobot by different cancer cell types

From the results collected in this trial it can be determined that the nanorobot can accurately respond to many types of antigens by the manipulation of the aptamer locks attached to the barrel structure. This generality implies that the nanorobot can potentially be applied in many cases for which the disease-infected cells produce modified or extraneous surface proteins.

The trial also further verified that the logic gate would only activate in the presence of the correct antigen combinations and not without the activation of both locks.

In the second trial, two varieties of DNA nanorobots were prepared, both containing a combination of antibody to human CD33 and antibody to human CDw328 Fab′ fragments [2]. This payload had previously been experimentally determined as detrimental to the growth of leukemic cells. One variety of nanorobot was programmed with two sgc8c locks, while the other had no aptamer locks attached and was therefore open [2]. The target in this experiment was leukemic natural killer cells (NKLs). Each type of nanorobot was introduced to an environment of NKLs for five hours and a sample without nanorobots was also observed [2]. The gathered results can be observed in Figure 4.

Figure 4: Induction of growth arrest in cancerous NKL cells

The results from this set of experiments indicate that increased dosage of DNA

nanorobots containing apoptotic drugs directly correlated with arrested growth. While the positive control gave a slightly higher ratio, it is not applicable to in vivo use as the specificity of drug delivery is the primary benefit of the nanorobot. Advantages and Disadvantages

The DNA nanorobot has many advantages to it. Firstly, it can be easily programmed and into many different shapes or scaffolds, using Cadnano. It is also extremely biodegradable, as it is made up of molecules found in our body, and can easily be broken down by the liver and passed out of our body. Many nanoparticles that are currently being investigated as methods of targeted drug delivery are potentially toxic to the body. Alongside its biodegradability it is biocompatible. DNA nanorobots are not toxic to the body, especially as we all have DNA inside each of our cells. This means that there are almost no side effects to having the DNA nanorobot pass through our body. Also, nanorobots reduce the exposure of the body to the significant adverse side effects of conventional cancer medications (most significantly immunosuppression) such as chemotherapy. The combination of active and passive targeting allows for reduced dose

sizes and increased dose efficiency, so that ultimately smaller amounts of harmful substances need to be introduced into the body. However, there are several significant problems with the DNA nanorobot. Currently, DNA nanorobots are highly expensive to produce. Also, nuclease, a restriction enzyme found in the body, can often break down the DNA nanorobot before it has reached its target cells. The DNA nanorobot may also be brought to the liver by phagocytes to be destroyed.

There are very effective techniques at combatting the effect of the nuclease on the DNA nanorobot. The nanorobot can be coated with polyethylene glycol, which disguises the nanorobot and hides it from the nuclease. However, the addition of polyethylene glycol prevents the Nanorobot from passing through the blood-brain barrier. Comparison to Other Drug Delivery Systems There are many forms of drug delivery systems available. The first to have been certified by the FDA was the use of liposomes. Liposomes are spherical structures made up of a phospholipid bilayer that have an inner core, which is hydrophilic. Different types of drugs can be inserted into the liposomes, however they cannot by hydrophobic, as if they were they would be repelled. The liposomes, often in the form of large unilamellar vesicles (LUVs) would contain a semi-permeable core [6], which would allow for the slow release of the drugs. However, this means that the drugs cannot all be released at once and in a specific area, but rather a larger region. Magnetic nanoparticles can also be used in drug delivery. They are biocompatible and can easily travel around the body. The MNPs are easy to locate via MRI and are also able to pass through the blood-brain barrier. However, there is few treatments possible with the MNPs, such as induced hyperthermia, as you cannot add different types of drugs to the nanoparticles. This is a very big limitation to MNPs and is a huge advantage to using a DNA nanorobot. Both of these other forms of drug delivery are useful and are very good at what they do. However, overall, the DNA nanorobot has been proven to be more effective. Not only is it smaller in size than both of these, but it can also be programmed to become different shapes for different environments, and is able to target and destroy almost all kinds of cells, due to its programmable aptamer locks and the availability to attach many different payloads to it. Future Research DNA nanorobots are currently an inadequate and impractical method of targeted drug delivery because they are extremely costly and thereby cannot be commercially produced. Further research must be completed before the DNA nanorobot can become a useful and effective method of treating severe diseases such as cancer. Even though the nanorobot is only in preclinical trials and is still approximately a decade from becoming an available treatment, production must be increased enormously. An estimated 1000 fold increase in production is necessary for commercialization [2]. Furthermore, DNA is currently extremely expensive even

though there is ongoing research attempting to drastically reduce the prices of strands and bases of DNA. Instead of relying on external research to improve the efficiency of the nanorobot, research can be done on the nanorobot itself to increase its cost-efficiency and manufacturability. Current research is examining modified structures of the nanorobot, including a cage structure as seen in Figure 5 and a spherical structure. The cage structure would eliminate unnecessary DNA, or DNA that is not truly adding rigidity to the structure. It would also eliminate the need for bonding the payload to the nanorobot because the cage would be fully enclosed unlike the cylindrical structure as shown in Figure 2. The spherical structure would also be fully enclosed. In addition, it would maximize the volume to surface ratio, allowing for the greatest amount of payload to be inserted into the structure. Disintegration after delivery could also be looked into, as this would prevent the phagocytes from attempting to engulf the nanorobots after use, thereby partially compromising the immune system and requiring the body to expend energy to break them down. In addition, the construction of the nanorobot could be modified so that the loading step of the payload is eliminated. This could be accomplished by building the nanorobot around the payload in the spherical or cage structures in which the payload is not covalently bonded to the nanorobot. Finally and possibly most importantly, integrating artificial materials such as polymers like polyethylene glycol can reduce the cost of the nanorobot. By using the polymer chains in a similar way as the DNA staples, they can maintain the rigidity of the structure while keeping the nanorobot at a price that would allow for commercialization in years to come.

Figure 5: Image of DNA cage structure Conclusion

There is also some research being done on testing the DNA nanorobot in vivo to treat cancer in different pests, such as rats and cockroaches. This is currently being done in Europe, and human trials are predicted to start in the next few years [8]. Even so, the DNA nanorobot is still many years of trials from commercialization and use beyond the laboratory. If successfully mass-produced, targeted drug delivery using a DNA nanorobot could be a viable treatment for a broad spectrum of diseases including some cancers.

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