Resumen de Nanotheranostics with microRNAs: applications in radiotherapy
Prostate cancer (PCa) is the second most common cancer in males, with an estimated 1.4 million new cases in 2020. Screening for PCa is an important tool for early detection of the disease, which can lead to a significant reduction in mortality, treatment costs, improvement in quality of life, and increased treatment success rates. Initial diagnosis is based on patient symptoms and usually involves a rectal exam, analysis of prostate-specific antigen (PSA) levels, urine analysis, and transrectal ultrasound. More recently, some additional biomarkers have been identified, but PSA levels remain the best biomarker, which correlates positively with the diagnosis of PCa. If initial tests reveal abnormal PSA levels, further tests such as biopsy, computed tomography (CT), magnetic resonance imaging, PET, are conducted to confirm diagnosis, staging, aggressiveness (presence of lymph nodes or tumor metastasis), and prognosis of the disease.
Once the disease is diagnosed and staged, the therapeutic strategy is carefully considered on a case-by-case basis and selected by a multidisciplinary team consisting of urologists, oncologists, radiation therapists, among others, who together decide on the most appropriate therapeutic strategy for the patient. There are various therapeutic modalities that can be selected, including surgical, radiation therapy (RT), hormonal therapy, and chemotherapy.
Regarding RT, it can be used at different stages of the disease, as the primary treatment or in combination with other previously mentioned therapeutic approaches. RT is one of the most used therapeutic approaches in the treatment of PCa, responsible for treating over 50% of patients. This technique uses high-energy ionizing radiation to destroy tumor cells, with the aim of reducing tumor size and preventing its spread. Although this therapeutic approach is effective in treating PCa, RT also has several advantages over other therapeutic approaches, as it is less invasive than surgery, the treatment is performed in a few minutes, and it can be used in patients for whom surgery is a risk, particularly in the elderly or patients with other associated comorbidities.
Despite the technical advancements in this therapeutic approach, radiation therapy (RT) has some limitations. Among the existing restrictions, non-selectivity, side effects, and radioresistance are the most relevant. With regards to radioresistance, tumor cells may be resistant to ionizing radiation and continue to grow even after treatment because of mutations and disturbances in gene expression. When a tumor cell is exposed to ionizing radiation, a complex series of molecular alterations is triggered that can induce cell death or allow the cell to survive and become resistant to additional treatments. It is known that miRNAs are small non-coding RNAs that play a crucial role in post-transcriptional gene regulation and modulate tumorigenesis through various processes, promoting or suppressing cell death depending on the specific miRNA involved.
Over the last few years, miRNAs have become promising therapeutic targets for cancer, as they are involved in many biological processes, including cell differentiation, proliferation, angiogenesis, and apoptosis. miRNAs can function as oncogenes, promoting tumor growth and progression, or as tumor suppressors, inhibiting cell growth. Mimic therapy with miRNAs and anti-miRNA therapy are two potential approaches for cancer treatment. Mimic therapy with miRNAs involves the delivery of exogenous miRNAs to tumor cells, where miRNA mimics are designed to imitate the action of tumor suppressor miRNAs and restore their lost function. Consequently, this therapy can induce tumor growth reduction and metastasis inhibition. On the other hand, anti-miRNA therapy involves inhibiting oncogenic miRNAs (oncomiRNAs) using antisense oligonucleotides or small molecules that target specific miRNAs. By reducing oncomiRNAs, which are frequently expressed in human tumors, it is possible to silence miRNAs that may contribute to tumor growth reduction and metastasis inhibition.
However, the clinical application of miRNAs as therapeutic agents is still limited by several factors, including dosage, specificity, and delivery. With regards to specificity, miRNAs must be directed and delivered to a specific tumor tissue depending on the target, to minimize side effects on the surrounding healthy tissues. In addition, bioavailability is also an important issue, as most miRNAs are rapidly degraded by the immune system and do not reach the site of action. Furthermore, new studies need to be developed to understand the possible unwanted side effects associated with miRNAs that could negatively affect other biological processes.
Nanotechnology is becoming increasingly important in miRNA therapy and RT treatments, as it allows overcoming some of the inherent limitations of the therapies mentioned. Nanotechnology can be defined as the observation, measurement, manipulation, assembly, control, and production of matter at dimensions between 1 and 100 nm, being one of the most promising technologies of the 21st century. By manipulating matter at the nanoscale level, researchers can create nanoscale materials and devices that interact with cancer cells in unique ways. Nanotechnology has shown potential to revolutionize cancer treatment, enabling early detection, efficient delivery of drugs/DNA/RNA, molecular imaging, personalized therapies, and real-time treatment monitoring. Several nanomaterials have been explored in cancer therapy, each with its own characteristics and applications. Furthermore, the development of these nanomaterials is a rapidly evolving field, with new materials and strategies constantly being developed. `Nanocarriers are a class of nanomaterials used to transport substances (drugs, miRNAs, DNA) that were previously identified as promising in the treatment of cancer. They are broadly categorized into two main groups: organic and inorganic. The organic group comprises of micelles, dendrimers, liposomes, hybrid, and compact polymeric nanoparticles, whereas the inorganic group comprises of fullerenes, quantum dots, silica, and metal nanoparticles.
In recent years, gold nanoparticles (AuNPs) have gained attention in miRNA therapy and RT as a promising tool to improve the efficacy and safety of ionizing radiation treatment and as a delivery platform for miRNA therapy due to their unique properties. AuNPs are relatively inert metallic nanoparticles and present several advantages over conventional therapeutic agents. The high atomic number of gold is an important factor that contributes to radiosensitization and increased contrast effect in CT. Thus, AuNPs can absorb a significant amount of ionizing radiation, allowing for an increase in the radiation dose in tumors, which is particularly useful in radiation-resistant tumors where higher doses are required to destroy tumor cells. Furthermore, the synthesis process of AuNPs is simple, and it is possible to synthesize AuNPs of different sizes and shapes, and they can be functionalized with specific ligands that will contribute to a more specific and targeted treatment, which will consequently reduce the exposure of surrounding healthy cells to ionizing radiation.
In addition, AuNPs have also become an attractive option as a delivery system capable of transporting miRNAs across the cell membrane into target cells. AuNPs offer several advantages over other miRNA delivery systems, including increased stability, protection against degradation, efficient cellular internalization, functionalization of the nanosystem, and low toxicity. AuNPs are highly stable and provide additional protection against miRNA degradation by the extracellular or intracellular environment, which significantly increases the half-life of miRNAs in bodily fluids and improves their delivery efficacy.
Currently, research has been expanding and investing in nanomedicine with the development of new nanocarriers for cancer treatment. Therefore, the aim of this project was to develop a nanosystem with AuNPs and incorporate miRNA on its surface in order to improve the efficacy of RT treatments and simultaneously be used as a contrast agent in computed tomography diagnosis.
In this sense, the objectives of this thesis were: (1) to synthesize and analyze the effect of different conformations of AuNPs (spherical gold nanoparticles, AuNPsp; gold nanostars, AuNPst; and gold nanorods, AuNPr) in RT treatments to understand the potential of different conformations of AuNPs as radiosensitizers of PCa cells; (2) optimize the miRNA binding process with AuNPs; and (3) evaluate variations in the expression level of four miRNAs (miRNA-95, -106b-5p, -145-5p, and -541-3p) in PCa cells with and without RT treatments and PEGylated gold nanorods (AuNPr-PEG).
Initially, AuNPs with different morphologies were developed, which were later functionalized with PEG. Functionalization of AuNPs with PEG (AuNPsp-PEG) has been a widely used strategy to improve stability under physiological conditions, bioavailability, and selectivity in biomedical applications. PEG molecules enhance the dispersion of AuNPs in aqueous solutions and increase their colloidal stability. Additionally, coating with PEG prevents the adsorption of proteins and opsonization of AuNPs by cells of the immune system, which can contribute to increasing the circulation time of AuNPs in the blood and their bioavailability. After functionalization, the AuNPs were characterized by various techniques, including UV-Visible spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), and zeta potential measurement.
After optimizing the production of AuNPs-PEG, the biocompatibility of the produced nanomaterial was evaluated, as well as the influence of AuNPs-PEG on metabolic activity pathways (gluconeogenesis, glycolysis, and beta-oxidation) in three PCa cell lines (PC3, DU145, and LNCaP).
In vitro metabolic activity assays are a valuable tool for medical and scientific research as they allow for the evaluation of cell metabolic rate under controlled laboratory conditions, which can lead to significant advances in the development of personalized treatments. In this case, cells were treated with different concentrations (0, 0.001, 0.01, 0.1, and 1 mM) of AuNPs-PEG for 24 hours. The results showed that the toxicity of AuNPs is concentration-dependent and cell-type-dependent. For PC3 and DU145 cells, AuNPs-PEG were the least toxic, followed by AuNPst-PEG and AuNPr-PEG. For LNCaP cells, AuNPst-PEG were the least toxic, followed by AuNPr-PEG and AuNPsp-PEG. Additionally, LNCaP cells were the most resistant to treatment with AuNPs-PEG, with only slight reductions in metabolic activity observed. Overall, AuNPr-PEG were found to be dose-dependent and the most efficient form of destroying both types of tumor cells (PC3 and DU145). However, further studies will need to be conducted to adequately quantify the cellular uptake efficiency of AuNPs and to understand the real effect of size and shape independently.
Regarding the effect of AuNPs on cellular metabolism, researchers have discovered that AuNPs can affect the expression of intracellular metabolites and, consequently, alter the functional genome, transcriptome, and proteome. After the Warburg effect, where oxidative phosphorylation in proliferative cells was switched to glycolysis even under aerobic conditions, metabolic alterations in tumor cells began to be explored. Tumor cells exhibit different sensitivities to various molecules related to the gluconeogenesis pathway, glycolysis, or fatty acid synthesis. Although some studies have explored the effect of AuNPs on the metabolism of tumor cells, much remains to be discovered. Based on our results, it has been shown that AuNPs-PEG altered the expression of intracellular enzymes involved in metabolic pathways in different PCa cells, but the level of expression was dependent on the cell lineage. AuNPsp-PEG and AuNPr-PEG tend to increase the expression of enzymes involved in glycolysis, such as hexokinase 2 (HK2) and pyruvate kinase (PKM) in PC3 and LNCaP cells, suggesting that they play a role in supporting tumor cell survival. In addition, AuNPs slightly increased glucose 6-phosphatase (G6Pase) in the PC3 cell line. Overall, the effect of AuNPs on the expression of metabolic enzymes is complex and context dependent. Although AuNPs can disrupt energy production and biosynthesis pathways in tumor cells, they can also promote NADPH production and support tumor cell survival. Further studies are needed to fully understand the mechanisms behind the effects of AuNPs on metabolic enzymes and their possible implications in cancer therapy.
The effect of AuNPs as a contrast agent in CT has also been explored. CT is an imaging technique that uses X-rays to create detailed images of the body, allowing doctors to visualize internal body structures with greater clarity. AuNPs have been widely used as contrast agents in medical imaging, including in CT. It is the ability of AuNPs to absorb X-rays that makes them an ideal contrast agent for CT imaging. In our study, AuNPs-PEG diluted in PBS at different concentrations of Au or iodine (reference group) from 0 to 4 mM were tested, and iomeprol was chosen as the clinical practice reference compound. Previously, studies have reported conflicting results on the influence of AuNP size as a contrast agent, but no information was found on the shape of AuNPs. Our results demonstrated that the attenuation factor of AuNPs was directly proportional to the concentration of AuNPs used. Among the tested AuNPs-PEG, AuNPr contributed to a higher attenuation compared to AuNPsp and AuNPst.
Next, the potential of different conformations of AuNPs as radiosensitizers for PCa cells was analyzed. The effect of AuNPs on PCa cells in the presence of ionizing radiation was evaluated through several in vitro assays, including viability, migration, colony formation, apoptosis, and reactive oxygen species (ROS). It was demonstrated that AuNPs are potential enhancers of ionizing radiation, and the responses are variable depending on the cell lines. Overall, AuNPst-PEG and AuNPr-PEG decreased cell viability in a dose-dependent manner. Additionally, AuNPst-PEG and AuNPr-PEG showed a tendency to reduce migration and colony formation with and without RT treatments. AuNPr-PEG were the most effective AuNPs-PEG in damaging PCa cells. However, further in vitro studies are needed to better understand the cellular mechanisms and the mechanism of AuNPs for radiosensitization.
Another approach to enhance the effect of ionizing radiation is to conjugate miRNAs on the surface of AuNPs. There are several approaches described in the literature that allow miRNAs to be linked to AuNPs, including covalent binding and electrostatic adsorption. In covalent binding, the miRNA is chemically modified to contain a reactive group that can be covalently linked to a functional group on the surface of AuNPs. Common reactive groups include thiol, amine, and carboxyl groups. Covalent conjugation provides stable and permanent binding between miRNA and AuNPs, but miRNA modification can affect its biological activity and specificity. In electrostatic adsorption, there is an electrostatic attraction between the negatively charged phosphate backbone of miRNA and the positively charged surface of AuNPs. Electrostatic adsorption is a simple and straightforward approach that does not require chemical modification of miRNA, but it can result in weak and unstable binding, leading to miRNA release under physiological conditions. The choice of approach for linking miRNAs to AuNPs depends on their specific application and the desired properties of the resulting complex. Throughout this work, a simple strategy for conjugating miRNAs to the surface of AuNPs has been optimized based on the protocol described in the literature. Over at least three days, the process of linking AuNPs with miRNA can be completed, in addition to stabilizing the nanosystem created with PEG.
We also evaluated the expression of miRNAs in in vitro assays using PCa cells subjected to AuNPr-PEG and RT treatments. After a literature review on the effect of miRNAs in RT treatment for PCa, four potential miRNAs were selected as potential radiosensitizers: miRNA-95, -106b-5p, -145-5p, and -541-3p. Overall, miRNA expression plays an important role in the response of tumor cells to RT. Our results showed that different cell types have different levels of miRNA expression, and that miRNA expression can be modulated by ionizing radiation and AuNPr-PEG. Among the studied miRNAs, it was suggested that miRNA-106-5p and miRNA-541-3p have the potential to be used as radiosensitizers in PC3 cells, miRNA-95 and miRNA-106-3p as radiosensitizers in DU145 and LNCAP cells, and as radioprotectors in HPrEpiC, a non-tumoral cell line. However, further studies should be done to investigate the molecular pathways involved in regulating radiation-induced cellular responses, such as cell cycle arrest, inhibition of cell proliferation, or cell death. Understanding how miRNA expression is affected by radiation may help develop new strategies to improve the effectiveness of RT while reducing side effects on normal cells.
Overall, the data presented in this thesis provided evidence that AuNPs can be used as a nanotheranostic agent, acting as a contrast agent and a radiosensitizer in metastatic PCa cell lines. Additionally, the analyzed miRNAs were shown to be capable of modulating the response to ionizing radiation in PCa cell lines, supporting the therapeutic strategy based on miRNAs to promote radiosensitization of PCa cells. However, further studies are needed, and we suggest conducting in vitro transfection assays with AuNPs conjugated with miRNAs to understand the true potential of this nanosystem in RT treatment.
Simultaneously, it would be interesting to develop a similar nanosystem capable of improving PCa radiosensitization through anti-angiogenic therapy. It has been demonstrated that the tumor microenvironment is essential for increasing the effectiveness of RT. Anti-angiogenic therapy remains an option in tumor growth, and AuNPs with miRNAs may be capable of modulating angiogenesis.
Furthermore, it would be interesting to replicate these results in a 3D in vitro model and evaluate the impact of AuNP-miRNA on PCa cell tumoroids and reassess survival, migration, and angiogenesis with and without RT before starting in vivo experiments. Currently, reducing animal testing is important, primarily for ethical reasons, and animal testing should only commence when results are consolidated. Therefore, tumoroids are multicellular three-dimensional culture systems used for the development of anticancer drugs, a promising technique because it mimics many characteristics of real solid tumors.
Once results in the 3D model are consolidated and validated, animal experiments can be considered as they offer several advantages over in vitro experiments and can provide information on how treatments are absorbed, distributed, metabolized, and excreted by the body and how they affect the body's biology before being tested in humans.
Furthermore, it would be interesting to explore the effect of AuNPs on FLASH RT, as radiotherapy has progressed, and new techniques have emerged. FLASH RT is a new technology that delivers unique ultra-high dose rates (¿ 40 Gy/s) in a very short period, usually in milliseconds. This technique has several advantages over traditional radiotherapy, being 400 times faster than conventional irradiation. By delivering the radiation dose rapidly, it reduces the amount of time that normal cells in the treatment area are exposed to radiation, contributing to the reduction of the risk of long-term side effects and damage to healthy tissues. Additionally, FLASH therapy has been shown to be more effective at destroying tumor cells than traditional radiotherapy and can treat deep lesions and/or larger areas of tissue in one go. We have reason to predict that FLASH RT will become a prevalent technique in clinical practice in the future. However, it is still in the early stages of development and more research is needed to fully understand its potential benefits and limitations.
