Author: Layla Adeli

Layla Adeli is a student at Palisades Charter High School in Pacific Palisades, California. She is a cross country runner and a violinist and loves spending her free time reading or listening to music. She is interested in the fields of cancer biology, nanotechnology, and genetics and hopes to become an oncologist in the future!

Categories
Biomedical Research

Nanotechnology In Cancer Therapeutics and Imaging

By Layla Adeli

Published 9:56 EST, Sat October 16th, 2021

Abstract:

Cancer is a disease caused by abnormal cells that rapidly divide, evade cell death, and can metastasize across an organism’s body. There are many hallmarks of cancer, such as avoiding immune destruction, replicating indefinitely, and destabilization of the genome, all of which pose as possible targets for cancer therapies. Due to the unstable nature of cancer, therapy and imaging are often difficult to perfect. For instance, chemotherapy, hormonal therapy, and radiotherapy––the three most common forms of cancer therapy––all can suppress cancer growth to an extent. However, these therapies can be toxic to healthy tissue by spreading harmful chemicals or radiation throughout the body. To combat this, new nanoparticle-based technologies, including immunotherapy and fluorescent imaging, have been used to specifically target cancer cells and encapsulate harmful drugs prior to release. This novel approach lowers toxicity and risk of off-target effects while enhancing therapeutic efficacy. In this paper, I review multiple types of nanoparticle-based therapies that use nanotechnology to conjugate and combine cancer therapies. Among these methods are chemotherapy drugs, immunotherapy, fluorescent imaging agents, and radiation in combination with nanoparticles to create more efficient and effective cancer therapies and imaging techniques.

Keywords: Cancer, Chemotherapy, Nanoparticles, Fluorescent Imaging, Immunotherapy

Introduction:

Types of Cancer:

Cancer comes in many forms, proving to be difficult to image and treat. Cancer can arise from nearly every tissue in the body and is named by the type of cell that causes the tumor, the phenotype of those cells, and how advanced the disease is within the patient; this is known as staging and is influenced by size and metastasis. [1] Early detection and effective therapies are two critical components required to reduce the mortality rate of cancer worldwide. For instance, gastric cancer is the 14th most common cancer in the world and one of the most common causes of cancer-related deaths in the world due to a high percentage of patients who are not diagnosed until they present with an advanced or metastatic form of the disease.[2] This makes it important to focus on improved imaging to attempt to catch the cancer earlier, reducing the fatality of the disease. Furthermore, breast cancer is one of the most frequently diagnosed diseases in females and is a heterogeneous disease that is broken up into 4 groups: luminal A, luminal B, HER2 positive and triple negative breast cancer (TNBC).Current treatments for luminal A and luminal B (ER/PR +) include Tamoxifen, while specific antibodies like Trastuzumab are used to target HER2+.[3] However, due to the lack of active hormone receptors for TNBC, there is currently a need for adequate treatment options for TNBC patients, giving way to a need for nanoparticle-based cancer therapy for TNBC. Lastly, colon cancer is difficult to image as well, and has proved to be a challenge regarding early detection. [4] It is currently the second leading cause of death from cancer, but more efficient screening can allow for polypectomy, finding early-stage colon cancer with less toxicity.

Imaging:

There are a variety of imaging techniques for cancer. For instance, magnetic resonance imaging, or MRI, is a popular form of imaging. MRI is an extremely powerful and non-invasive imaging technique for high quality and multiplanar images of soft tissue in vivo. [5] This can still be improved however, with the addition of fluorescent nanoparticles, as they have the potential to increase the contrast between the imaging agent and the patient’s non-cancerous tissue. For instance, near infrared (NIR) dyes such as Indocyanine Green (ICG) and other cyanine dyes with similar structures can be incorporated in albumin-based nanoparticles that also contain tumor targeting ligands. These have been extremely helpful regarding colon cancer due to the resulting enhancement of detection ability during early imaging of colonic polyp. [6]

Immunotherapy and Gene Therapy:

         Immunotherapy has changed the world of cancer therapy by incorporating targeted therapies, and by blocking inhibitory tumor ligands. Cytokines for effective cancer therapies have been a difficult task to manage due to toxicity. However, there is more evidence now that strategies based on ligand delivery (conjugating a cytokine to an antibody or peptide) of lower dose cytokines can aid in the enhancement of immunotherapies by making the cytokine more stable and targeting it specifically to the desired location. [7]

Furthermore, PD-L1 is an immune checkpoint in tumors that will bind to the PD-1 receptor in T cells, causing the downregulation of T cell anti-tumor activity, allowing the tumor to continue growing and avoiding a full immune response. By including a nanoparticle-based immunotherapy, PD-L1 could be inhibited, allowing a full immune response against the tumor, and giving a higher chance of survival for the patient. [8] Gene therapy has been a very important form of immunotherapy, with drugs based on a viral vector like a glybera. [9] In cancer, gene therapy can enhance tumor suppressor genes, such as p53. Furthermore, p53 can also be used along with chemotherapeutics by enhancing sensitivity of drug-resistant tumors to the drugs; however, the complex nature of this has led to some obstacles in finding the most efficient and effective combinations of p53 and chemotherapeutics. [10] Because DNA is addressable and programmable, one modification of gene therapy using nanotechnology has been using DNA nanostructures, or DNA origami, to codeliver drugs without any increase in cytotoxicity or immunogenicity.

Chemotherapy and Radiotherapy:

Chemotherapy is used to aid anti-tumor effects by stopping mitotic processes. There are many types of chemotherapy drugs; for instance, the chemotherapy drug paclitaxel (PTX) has been used various times for many types of cancers; however, it is highly toxic, and has a low bioavailability. [11]  While there have been formulations of paclitaxel, such as Abraxane, which uses albumin bound nanoparticles and paclitaxel, other forms of nanoparticles are being considered to lower the off-target toxicity of paclitaxel, while continuing its benefits against cancer. The use of immune cells as a delivery agent is a popular approach to this issue since they have a natural affinity to gather near tumors, and to carry chemotherapeutic agents. Nanoparticles can also be conjugated to the surface of the immune cells and can enhance the anti-tumor activity initiated by the cells. Furthermore, doxorubicin is another chemotherapeutic drug that binds with DNA to prevent replication and is very popular in its use to combat breast cancer, yet it is also highly toxic and can lead to nausea, fatigue, and heart failure. [12]

Radiotherapy is used to either eradicate a tumor or control tumor growth. Radiotherapy works by destroying cancer cells and damaging DNA through an excessive amount of radiation. Unfortunately, this therapy comes with many negative side effects because healthy cells are also susceptible to radiation damage; this is a severe consequence that researchers are trying to combat. Additionally, many types of cancer have a disappointing recurrence rate in patients after radiation therapy. By incorporating nanotechnology, radiotherapy could hopefully become less toxic, allowing the benefits to outweigh the negative effects.

Nanoparticles:

Nanoparticles and nanotechnology are complex but can be helpful when containing elements or therapeutic drugs. They have been an interest in cancer therapeutics for many reasons, but most notably due to their enhanced permeability and retention (EPR) effect which relies on specific defects in the tumor microenvironment such as leaky vasculature to cause an accumulation of nanoparticles. For instance, Gadolinium (Gd) contributes to the overall signal intensity of T1-weighted images. However, Gd3+ ions, when free in vivo, are toxic and can pose  a threat to the organism, so a new generation of Gd-containing nanoprobes like the Gd encapsulated carbon dots (CDs), have been made to suppress the toxicity. Carbon dots require a simple preparation, have many unique structures, are low in toxicity, and have a superb cell membrane permeability, [13] thus making them excellent candidates for MRI probing and cancer therapy drug carriers. Furthermore, albumin-bound NPs use the endogenous albumin pathways to carry hydrophobic molecules in the bloodstream. Albumin naturally binds to the hydrophobic molecules, and so it has been adapted and used as drug delivery vehicles. [14]   There is an increase in the interest regarding albumin nanoparticles due to factors such as biocompatibility and biodegradability. Albumin is also one of the most used proteins in the pharmaceutical field, and albumin nanoparticles are currently used in chemotherapy as drug carriers resulting in drugs such as Abraxane; this provides insight to the increase in nanotechnology in modern cancer therapies. Furthermore, gold or silver NPs can emit radiation, and liposomes and BSA. Gold NPs or AuNps have a high X-ray absorption coefficient and are also very easy to synthesize. After the ionizing radiation is applied to the organism/system, the radiosensitization of Gold NPs can aid in cancer DNA damage and cell death and can enhance the dose at megavoltage radiation energy range. [14]   

Discussion:

Chemotherapy:

Gd@CDs for Triple Negative Breast Cancer:

A promising study investigated near infrared triggered Gd@CDs for MRI guided photothermal chemotherapeutic drug delivery specifically for triple negative breast cancer. Gd@CDs were synthesized, leading to the development of the nanoplatform, Dox@IR825@Gd@CDs, which delivers the chemotherapy drug Dox and photothermal agent IR825 for an MRI-guided photothermal chemotherapy for triple negative breast cancer (Figure 1). [15]

The Gd@CDs were tested for their absorption band and showed an absorption peak at 340 nm in the carboxyl group, and an emission peak at 437 nm (very similar to regular carbon dots). Furthermore, the in vivo toxicity analysis showed that there was no damage in the Gd@CDs treated groups (in the heart, liver, and kidney) after 24 hours of Gd@CDs solution injection. Then, IR825 and an anti-tumor drug, Dox, were loaded onto the surface of the Gd@CDs and absorbed onto the carbon dots.

In the combined photothermal chemotherapy aspect of the study in vitro, 4T1 cells, a line of breast cancer cells, were incubated with the Dox@IR825@Gd@CDs and DAPI, a fluorescent dye, for 4 hours. The red fluorescent Dox was present in the 4T1 cytosol, while the blue DAPI was present in many cell nuclei; this provides evidence for an easy Dox@IR825@Gd@CDs entrance into the cell. Furthermore, the 4T1 cells had over 95% viability, indicating a much lower toxicity. However, when using NIR radiation, the cellular viability of 4T1 cells dropped to a mere 26%, proving that the anticancer efficacy of Dox@IR825@Gd@CDs was drastically improved due to only 5 minutes of NIR irradiation, destroying nearly all cancer cells. Moreover, when the concentration of Dox@IR825@Gd@CDs was 0.5 mg/mL, over 97% of 4T1 cells in the groups including combined treatment died in comparison to single chemotherapy or phototherapy groups.

Due to the immense success of the in vitro experiments using Dox@IR825@Gd@CDs, the next study focused on combined therapeutic efficacy in vivo. Female nude mice that had 4T1 tumors were randomly split into four groups. The mice were injected with the Dox@IR825@Gd@CDs solution, the temperature changes on the surface of the tumor were tracked using thermal imaging, and the tumor volumes of the mice were measured daily. The temperatures were quickly increased under irradiation in IR825@Gd@CDs and Dox@IR825@Gd@CDs groups, thus causing irreversible damage to the tumor tissue due to the high temperature. When compared with the group injected with saline, the tumors treated with chemotherapy alone and photothermal therapy alone grew very slowly, and the tumor growth was inhibited to a small extent. But the mice treated with Dox@IR825@Gd@CDs with NIR irradiation showed the best tumor growth inhibition due to the synergistic nature and efficiency of the combined photothermal chemotherapy.

Lastly, with regards to imaging, Gd@CDs acted as T1 contrast agents and improved the MRI effect due to the increasing concentration of Gd@CDs and under the same Gd dose, the MRI of Gd@CDs was brighter and clearer than that of Gd-DTPA, the regular MRI contrast agent. Overall, Gd-CDs were shown to exhibit great biocompatibility and showed that they could be used to create a Dox@IR825@Gd@CDs nanoplatform to further favorable MRI results, improve combination therapy (photothermal and chemotherapy), and to lower toxicity of treatment regarding triple negative breast cancer. These superb results further show the success of nanotechnology in cancer research, by improving efficacy and lowering toxicity of a once toxic chemotherapeutic drug.

Figure 1: Gd@CDs were loaded with Dox and IR 825 and injected into mice, along with NIR irradiation for MRI Guided Photothermal Chemotherapy

DNA Origami Nanostructures:

A 2018 study showed that a triangle DNA origami (TO) can efficiently load doxorubicin (DOX), which can interlace into the base pairs of DNA while also assembling the tumor suppressor gene, p53. [16] This results in TODP, or a “nano-kite” which is finished with the addition of a glycoprotein, MUC1, aptamer. The TO was designed to contain two capture strands, A16-LAS20 and A45-LAS20 on one side. This was for the assembly of the LS20-p53-Cap gene. The TO was then loaded with DOX, and the nanostructure containing both DOX and the p53 gene (TODP) was assembled by adding the LS20-p53-Cap gene to the DOX DNA origami (Figure 2)

In order to see how efficient the co-delivery nanostructure was in regards to cellular uptake and penetration, DOX-resistant MCF-7 breast cancer cells were tested with TODP; the results showed that the targeted DNA origami could pass through the cell membrane, and that the collection of the TODP and TOD encased DOX was higher than free DOX. Regarding the efficiency of gene delivery, there was a higher expression of p53 in the TOP and TODP groups in comparison to the control group. The nanostructure was then further observed in vitro, to determine the ability to prevent the spread of MCF-7 dox-resistant breast cancer cells. First, it was found that there was no inhibition with free Dox, p53 plasmid, or the unstructured DNA strands; however, the TODP showed phenomenal inhibition of MCF-7 cell division, while demonstrating a great anti-tumor effect with roughly 40% cell viability after 24 hours.

Due to the success of the in vitro experiments, in vivo effects were thus investigated using MCF-7 tumor-bearing nude mice, who were injected with a fluorescent DNA origami (TOP-cy5.5 with MUC1 aptamer), and then sacrificed after 24 hours. The administration of the nanostructures took place as followed: saline, TO, TOP, DOX, TOD or TODP by tail vein injection. Remarkable results were demonstrated, one being that there was a much higher accumulation of the MUC1 aptamer assembled DNA nanostructures in the tumor tissue when compared to the non-targeted group. Furthermore, while there was no inhibition from the free saline carrier (TO), there was a consistent inhibition by the TODP treatment and a moderate inhibition by the TOP and TOD groups; this further shows the immense increase in efficacy by the combination of chemotherapeutics and gene therapy, when compared to the therapies alone. While there was a large amount of destruction to the heart and liver in the free DOX group (high toxicity), there were no obvious differences in the organs of the TODP group when compared to the saline group.

In conclusion, this novel form of cancer therapy perfectly combines gene therapy and chemotherapeutics to effectively deliver p53 and doxorubicin to a targeted tumor site, while enhancing the anti-tumor effect of both the gene and the drug. It is further impressive in its self-assembly and provides a glimpse of the future of cancer therapy through the remarkable results of biocompatible and efficient nano-kite platforms to co-deliver tumor-suppressor genes and chemotherapeutics.

Figure 2: p53 and Dox were interlaced onto DNA origami figures to create a TODP with a MUC1 aptamer. This was then injected into a MDR tumor (known to have MUC1 receptors).

Fluorescent Imaging:

Albumin NPs For Colon Cancer:

In a study regarding the synthesis of NIR fluorescent albumin nanoparticles to detect colon cancer, nanoparticle based NIR fluorescence has been shown to have enhanced photostability, greater biocompatibility, and a simple conjugation with the biomolecules on the nanoparticle surface. [17] In this experiment, the high affinity of albumin and cyanine dyes prepared NIR fluorescent albumin nanoparticles, using a synthesized carboxylic acid byproduct of IR-783, known as CANIR, as well as the NIR fluorescent human serum albumin (HSA) nanoparticles. HSA nanoparticles were activated through a covalent conjugation of targeting agents like PNA and anti-CEA to the nanoparticle surface and were used to detect colon cancer tumors in mice in vivo. Different concentrations of CANIR were added to a 4% HSA solution, which was followed with the formation of the nanoparticles. Then, photobleaching experiments were followed through for both CANIR that was encapsulated in HSA nanoparticles, and for “free” CANIR dye, with an illumination at 800 nm; it was found that the intensity of the NIR HSA nanoparticles were unaltered, while free CANIR decreased by a large amount, showing that HSA prevents photobleaching of CANIR to a very large extent. Furthermore, for optimal tumor detection peptide nucleic acid (PNA) which binds to sugar on the Thomsen-Friedenreich antigen, was conjugated to the nanoparticles.

The PNA-conjugated nanoparticles in phosphate-buffered saline (PBS) were then injected into the colon wall of the mice and the colons were washed 20 minutes later; half (3) of the mice were able to rest for 1.5 hours while the other half rested for 4 hours. Other mouse trials were performed with 4-hour recovery time but used monoclonal antibodies against CEA and TAG-72, which are expressed highly in human carcinomas; these NIR HSA nanoparticles were tested in LS174t and HT29 cancer cell lines, with HT29 being the negative control in the experiment due to low expression of CEA and TAG-72.

It was found that while non-conjugated nanoparticles did not detect tumors on either line, the anti-CEA and anti-TAg-72 nanoparticles labeled the LS174t tumor with a high signal to background ratio. Due to a high SBR, it can be suggested that NIR fluorescent nanoparticle imaging has an advantage over standard colonoscopy in terms of an accurate type of imaging and detection. Lastly, in order to prove that the NIR HSA nanoparticles are responsible for the fluorescence, tumor sections were analyzed to show the difference between the untreated tumor without nanoparticles and the treated tumor with nanoparticles. 

The conclusions of this study were positive and supported the potential of NIR fluorescent HSA nanoparticles in the diagnosis and detection of colon cancer. The results proved that nanoparticles can not only detect cancer efficiently, but by conjugating the nanoparticles with other targeting agents, could enhance a fluorescent signal, thus improving efficiency and specificity of cancer detection in the future.

BRCAA1 Monoclonal Antibody NP Conjugation:

Furthermore, in a recent study concerning gastric cancer imaging, a BRCAA1 monoclonal antibody was conjugated with a fluorescent nanoparticle for in vivo targeted imaging of gastric cancer. [18] BRCAA1 is an antigen gene that is most associated with breast cancer but is also heavily overexpressed in gastric cancer as well, making it a significant player in advancing gastric imaging. The BRCAA1 antibodies were prepared against a “purified fusion protein” BRCAA1. Male nude mice were then injected with the protein four times, and three days after the final injection, the spleen cells were harvested and fused with the Sp 2/0 myeloma cell that was then coated in the BRCAA1 protein. The monoclonal antibodies were chosen to bind with the protein and purified. Furthermore, fluorescent magnetic nanoparticles (FMNPs)–specifically Fe3O4 nanoparticles–were prepared, and a two-step process was used to create a stable conjugation of the anti-BRCAA1-FMNPs.

The mice were injected with MGC-803 gastric cancer cells; the tumors were allowed to grow for 4-5 weeks. The BRCAA1-FMNPs nanoprobes were then injected into the tail vein, and the mice were monitored at different hours in order to obtain fluorescent images.

The results of this study were overall enlightening, showing that there was a strong fluorescence signal at the tumor site 6 hours post-injection, proving that the nanoprobes were integrated into the tumor tissue perfectly. Furthermore 12 hours post-injection, the fluorescence was still strong, yet there was no strong sign of the particles in non-cancerous organs, which demonstrated that the nanoprobes can target the tumor tissue extremely efficiently. Moreover, the nanoprobes exited the body from the cholecystic system after 12 hours post-injection, which indicates beneficial biosafety by not staying in the bodies of the nude mice for too long.

In conclusion, this study showed a phenomenal example of the use of nanoparticles for cancer imaging by harnessing biomarkers and magnetic nanoparticles to create an effective targeting imaging tool that can be used to spot gastric cancer before it becomes fatal. It showed that the conjugation of anti-BRCAA1-FMNPs yielded positive results, with no evident repercussions to the mice’s health in the study, and had a high intensity fluorescence, making it a large factor in the direction that future imaging studies will follow.

Immunotherapy:

pH Triggered Met/siFGL1 NP:

In a recent study, a pH-responsive platform made for metformin (Met) and siRNA (short interfering RNA) was shown to aid in the immense inhibition of cancer growth. [19] Met was used in this as it activates the adenosine monphosphate-acvtivated protein kinase (AMPK), which is important in cancer progression, but also decreases glucose levels, thus suppressing cancer cell proliferation. The platform was used to target a fibrinogen-like protein 1 mRNA (siFGL1) using biomimetic membrane camouflaged poly (PLGA) nanoparticles.

A pH triggered CO2 generating nanoplatform was made using the guanidine Met group, and the release of Met aided in the degradation of PD-L1 by blocking the inhibitory signals of PD-L1 while also aiding in the release of the siRNA. Then, siFBL1 delivery through the camouflage nanoparticles silenced the FGL1 gene, thus increasing anti-tumor immunity and aiding in T-cell immune responses. FGL1 is the main ligand of LAG3, one of the main inhibitory receptors on depleted T lymphocytes, and thus the blocking of FGL1 is crucial in antitumor immunity.

In this study, MET-Co2 was incorporated along with siFGL1 onto PLGA-Met-Co2/siFGL1 NPs. Next, the cell membrane was isolated from RAW264.7 macrophage-like cells (M) and 4T1 cancer cells (C), the RAW-4T1 hybrid membrane was fused, and then either the RAW264.7, 4T1, or RAW-4T1 membranes were coated on the cores of PLGA-Met-Co2/siFGL1 NPs. The drugs were then injected into the tail vein of mice with 4T1 breast cancer- bearing mice (broken into 6 groups) every 4 days.

It was shown that the tumors in the saline group and siFGL1 only group grew extremely quickly. Furthermore, the siFGL1-treated was inhibited by 2.66%, while the MC-PLGA-siFGL1 NP-treated group was inhibited by 56.1% and the MC-PLGA-Met-Co2/siFGL1 NP group was inhibited by 97.3%, showing the strength of the MC hybrid camouflaged NP method. Furthermore, the MC-PLGA-Met-Co2/siFGL1 NPs increased the survival period of the mice by just under 40 days. In conclusion, this study had an immense impact on the course of immunotherapy by showing how the usage of PLGA/Met-Co2 nanoparticles to target siFGL on LAG3 is extremely beneficial in the inhibition of cancer growth. Its results will help pave the way for future ligand-based immunotherapies that could not only prevent cancer growth, but also potentially co-deliver chemotherapeutic drugs.

Fab’-anti-PD-L1 Doxorubicin Liposomes:

Furthermore, a new study used the chemotherapy drug Doxorubicin (Dox) combined with immune checkpoint inhibitors (ICI) to inhibit PD-L1. [20] The Dox was administered into liposomal nanoparticles in order to lower cardiotoxicity and to improve efficacy by aiding in active targeting through selective binding (Figure 3). The α-PD-L1-Fab’(antigen-binding fragment) Dox liposomes were made through targeted micelles and incubation time, resulting in 3 liposomal formulations: empty liposomes (LP), non-targeted Dox liposomes (LPD) and Fab’-anti-PD-L1 Dox liposomes (LPF).

The conjugation of Fab’ and incorporation of Dox did not greatly influence the physiochemical characteristics of the liposomes. The subjects in this study were female mice that were injected with B16OVA melanoma cells and had their tumor growth monitored twice a week; after the tumor reached about 5-6 mm, they were then divided into multiple groups: control, free Dox, LPD, LPD plus free α-PD-L1, and LPF. The results were outstanding and indicated that the intensity and accumulation were higher for the targeted liposomes when compared to the non-targeted liposomes. This further proves the selective nature of PD-L1 and explains that targeting provides a higher intensity and a higher chance for the formulation to reach the cytoplasm of the cells.

Furthermore, the cytotoxicity by the Dox drug was analyzed 72 hours post-exposure, and it was found that the highest cytotoxic activity was in the free Dox, followed by the LPF and finally the LPD. Thus, the targeted liposomes, or the LPF, had a higher impact than regular liposomes in terms of cytotoxicity in cancer cells, making them the more impactful cancer therapy. Moreover, the immunoliposomes proved to be less toxic to the non-tumor cells in the body by quickly degrading when they completed infiltrating cancer cells. Furthermore, the immunoliposomes triggered an immune response, as the targeted liposomes promoted a large change in the number of tumor-infiltrating T cells (TILs) while also activating tumor specific lymphocytes (CD8+), which was not activated by the combination of the free monoclonal antibodies (α-PD-L1) and non-targeted liposomes. Lastly, the immunoliposomes were able to control and suppress tumor growth, as the encapsulated Dox not only shrank tumors more than the free Dox, but the immunoliposomes were able to control growth entirely for over 40 days; in comparison to the non-targeted group, the targeted group had an increased life span of nearly a month.

Overall, this study was a prime example of the increase in immunotherapy for cancer therapies and provides a new combination of Dox and liposomes, through the advantageous use of targeting-based therapies. It not only showed the increase in tumor suppression, but also the efficient clearing of immunoliposomes and increased intensity of cell/cytoplasm invasion, while also having an increase in the efficacy of the anti-tumor effects. In conclusion, it is the first study to succeed in the chemo-immunotherapy approach using immunoliposomes and remains an important landmark in the field of nanoparticle-based cancer therapy.  

Figure 3: Doxorubicin was added onto targeted liposomes along with anit-PD-L1 micelles as a form of targeted immune-chemotherapeutics.

Radiotherapy:

In a recent radiotherapy study, using a 4T1 breast tumor model, there was a large reduction in the tumor interstitial fluid pressure 24 hours after using a polyethylene glycol-TNF-coated gold nanoparticle (CYT-6091) alone and combined with radiation. [21] In contrast to the combined therapy, radiation alone had a small effect on the interstitial fluid pressure (IFP). Furthermore, in this study there was greater than 2 times the delay in tumor growth when CYT-6091 was combined with a single radiation dose. Also, when hypo-fractionated radiation was applied with CYT-6091 treatment, more than five times growth delay was observed. [22]

Radiotherapy also uses other elements besides gold including Hafnium which is another man-made compound. It is a high-Z material that can develop into many different shapes and sizes and evolve to become optimal for any tumor cells. For example, in a soft tissue sarcoma (STS) phase I study hafnium oxide NPs (NBTXR3) in addition to an external beam radiation therapy (EBRT) was shown to shrink tumors up to 40% median tumor shrinkage rate and caused 26% residual malignant cells rate. Even more recently, there was another study in the I/II clinical trial phase which also uses STS, NBTXR3 and RT. [21] In this study, the patients were broken up into 2 groups, one of which received EBRT and the other getting NBTXR3 mediated EBRT. The results were mainly positive, with the NBTXR3 group having a 16% response rate compared to an 8% response rate by the EBRT group. While the percentages are not high, the NBTXR3 group shows improvement by doubling response rates, and thus providing a hopeful look toward the future for Hf nanoparticle mediated RT.

Lastly, radiation therapy can also utilize Bi NPs to maximize radiation absorption efficiency and sensitivity. [21] Recently they have been modified with folate red blood cells (F-RBC) for breast cancer RT. In this study it was found that after a radiation treatment for mice with a 4T1 tumor, the F-RBC Bi NPs aided in tumor regression and increased survival. Overall, adding nanoparticles such as gold, hafnium, and bismuth to radiotherapy have shown to be extremely effective due to their ability to repress toxicity and increase survival rate, while enhancing the effects of radiotherapy by increasing the anti-tumor efficacy. In the future, it would be essential to improve radiotherapy as currently it is very toxic for cancer patients and not extremely efficient in its anti-tumor behavior; nanoparticles and nanotechnology provide a clear window as to how to further the effects of radiotherapy, while improving quality of life for patients and eradicating cancer faster than before.

Conclusion and Future Directions:

Nanotechnology has improved cancer therapy to a large extent, as seen through the overwhelmingly positive results in in vivo trials. Nanoparticles, such as carbon dots and albumin bound chemotherapeutic drugs have been shown to decrease toxicity of common cancer therapies, while enhancing the effects of chemotherapeutic drugs, fluorescent imaging, and immunotherapies. Nanotechnology can incorporate DNA origami approaches for gene therapy and targeted chemotherapeutic uses, while also co-delivering NIR, opening the doors to safer therapy for cancer patients soon.

Nanotechnology has much potential for furthering cancer therapies to an even larger extent as it can also enhance the use of specialized biomarkers of cancer, including neutrophil gelatinase-associated lipocalin (NGAL). NGAL has been shown to be upregulated in ovarian cancer cells, and thus expression of NGALs can be used to help in early diagnosis and screening of ovarian cancer. It is not present in healthy ovaries but can be present in benign ovarian tumors and in early grade malignant ovarian tumors. In a 2006 study done to determine the feasibility of using immunoreactive NGAL as a biomarker for ovarian cancer, blood from patients who were divided into 6 groups was tested; the groups were as follows: control, benign, borderline, grade 1, grade 2 and grade 3.[23] NGAL is upregulated in the blood of ovarian cancer patients, and it increases with grade. Grade 2 and 3 tumors had less expression than grade 1, borderline, or benign; however, this is still more NGAL expression than normal ovarian stroma, which had no expression of NGAL. Furthermore, the mean expression of irNGAL was much higher in the benign and grade 1 samples than all other groups. This study is integral in the future of ovarian cancer therapies, as NGAL is a protein that can be used to specifically target ovarian tumor cells. By harnessing a cell that is often recruited by the tumor microenvironment, one could further the uses of targeted chemotherapeutics, with the possibility of it working alongside MRI imaging.

Therefore, I propose the use of a NGAL-ligand binder (MMP9, hydrophobic molecules, or FeEnt), as well as platelet-derived growth factor (PDGF), which would attach to a Gd@CD. PDGF binds to PDGFR-a, which is prevalent on ovarian cancer cells. This novel nanotechnology would be used for two separate functions: imaging for ovarian cancer, and chemotherapeutic delivery once the patient has been diagnosed. First, the carrier would be used to carry a photothermal agent (IR) to work alongside NIR and further the destruction of cancer cells.[15] If a patient was then diagnosed with ovarian cancer, the nanoparticle could be modified through the addition of a chemotherapeutic drug, such as doxorubicin (Dox), and be a form of targeted chemotherapy. The targeting of chemotherapy using NGALs would also lower cytotoxicity, as chemotherapeutics would only be sent to the tumors and not to other cells in the body.

Using PDGF in addition to a NGAL binder ensures that the chemotherapeutics will only be released once the carrier encounters both NGAL and PDGF; this furthers the specificity of the drug distribution, and in theory would lower toxicity by only allowing drugs to enter ovarian cancer cells and not neutrophils or other cells. Furthermore, Gd@CDs suppress toxicity of Gd, and CDs have a phenomenal cell membrane permeability, which makes them good candidates for MRI probing and drug carriers.[15] Using targeted imaging agents in MRI would also aid in diagnosis accuracy, as you can use the fluorescence to better the results of the MRI. Lastly, using IR within the nanoparticle would also be beneficial because NIR has been shown to destroy cancer cells. Using targeted photo thermal therapy and chemotherapy together can improve results and lower cytotoxicity. [15] This therapy can be revolutionary due to its modularity and ability to be easily modified for ovarian cancer imaging and therapeutics.

This proposed ovarian cancer dual-targeted imaging and therapeutic agent is just one of countless examples of the progressing cancer therapies. While it is superb in its targeting abilities, there are many more questions to be answered in the next couple years of cancer research. Among these questions are: How to make DNA origami more reliable, how to provide better funding for these expensive therapies, and how to better the reliability of existing cancer therapies such as radiation? Thus, while the increasing prevalence of nanotechnology in cancer research is greatly aiding the progression of this field, in order to further progress in the field of cancer therapy these questions must be tackled first.   

Acknowledgments:

 I would like to thank and acknowledge my mentor, Nina Horowitz. A PhD candidate at Stanford University, she took time out of her own research-filled schedule to guide me in the complex world of cancer therapeutics and nanotechnology, and without her I would not have been able to complete this project.

Layla Adeli Youth Medical Journal 2021

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Figure References: 

  1. Jiang, Qunjiao, Li Liu, Qiuying Li, Yi Cao, Dong Chen, Qishi Du, Xiaobo Yang, et al. “NIR-Laser-Triggered Gadolinium-Doped Carbon Dots for Magnetic Resonance Imaging, Drug Delivery and Combined Photothermal Chemotherapy for Triple Negative Breast Cancer.” Journal of Nanobiotechnology 19, no. 1 (2021). https://doi.org/10.1186/s12951-021-00811-w.
  2. Liu, Jianbing, Linlin Song, Shaoli Liu, Qiao Jiang, Qing Liu, Na Li, Zhen-Gang Wang, and Baoquan Ding. “A DNA-Based Nanocarrier for Efficient Gene Delivery and Combined Cancer Therapy.” Nano Letters 18, no. 6 (2018): 3328–34. https://doi.org/10.1021/acs.nanolett.7b04812.
  3. Merino, María, Teresa Lozano, Noelia Casares, Hugo Lana, Iñaki F. Troconiz, Timo L. ten Hagen, Grazyna Kochan, Pedro Berraondo, Sara Zalba, and María J. Garrido. “Dual Activity of PD-L1 Targeted Doxorubicin Immunoliposomes Promoted an Enhanced Efficacy of the Antitumor Immune Response in Melanoma Murine Model.” Journal of Nanobiotechnology 19, no. 1 (2021). https://doi.org/10.1186/s12951-021-00846-z.

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