Nanoparticle Delivery of siRNA in Lung Cancer Treatments
Introduction
Lung cancer accounts for the highest mortality rate out of all cancer types (Siegel et al., 2021) due to late diagnosis and poor prognosis. Lung cancer can be classified into non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). NSCLC accounts for 80-85% of lung cancers and is subdivided into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma (Osmani et al., 2018). Common treatments for cancer include chemotherapy or radiotherapy. However, these treatments have limitations as they indiscriminately target both healthy and tumor cells, causing serious side effects. Multi-drug resistance (MDR) can also be an issue during treatment as the tumor cells have innate resistance or develop resistance to drugs through gene mutations. (Zhao et al., 2023). A way to overcome these limitations is through treatments using RNA interference.
RNA interference (RNAi) is a process in which RNA molecules inhibit sequence-specific gene expression. siRNA, or short interfering RNA, is a type of RNA molecule that suppresses target genes by binding with their complementary mRNA, inhibiting protein expression. The potential of specific gene silencing in siRNA makes it a promising tool for the treatment of various diseases, including cancer. siRNA therapies can address issues found in current cancer treatments as they selectively silence cancer-related genes and regulators (Krishnaswami & Kandasamy, 2022) and resensitize cells to chemotherapy through the co-delivery of siRNA and chemotherapeutic drugs (Zhao et al., 2023). For effective treatment, effective carriers for siRNA are essential, as naked siRNAs are unstable in physiological conditions. Therefore, nanoparticle carriers are used for siRNA protection and targeted delivery to specific sites. This review will introduce siRNA as a potential treatment for lung cancer and elaborate on different nanoparticle systems as a way to successfully achieve siRNA delivery.
Mechanism of siRNA
RNA interference is an eukaryotic defense mechanism for degrading exogenous nucleic acids, as well as a regulator of gene expression. When double-stranded RNAs (dsRNAs), likely from viral sources, are found present in the cell, they are cut up into short 21’ nucleotide RNAs called short interfering RNAs(siRNAs) by a ribonuclease Dicer. These dsRNAs are cleaved into guide and passenger single-stranded RNAs. The passenger strand is degraded while the guide strand is integrated into the RNA-induced silencing complex (RISC), a protein complex key to gene silencing. RISC uses this guide strand to bind complementary mRNA molecules present. Once the guide strand is base-paired to the mRNA, the mRNA is cleaved by the Argonaute protein, an endonuclease component of RISC. The unstable mRNA is then degraded, preventing gene expression.
Researchers have taken advantage of this degrading mechanism by engineering exact siRNAs complementary to the target mRNA, enabling targeted silencing of specific genes (Dykxhoorn & Lieberman, 2006). Cancers typically emerge from a build-up of genetic mutations, in particular those that activate oncogenes, impair tumor repressor genes, and facilitate overexpression of genes that aid tumor progression. These genes are therefore potential targets for siRNA designs when combating cancer.
Therapeutic use of siRNA in lung cancer
Common mutations that lead to lung cancer include the endothelial growth factor receptor (EGFR), KRAS, ALK, p53, and cMyc mutations (Zarredar et al., 2018). These gene mutations have been successfully targeted with siRNA resulting in tumor inhibitions. For example, Yu et al. (2021) demonstrated that EGFR-targeted siRNA delivered using gold nanoparticles effectively reduced tumor weight by 70% for mice models. In another study, siRNA was used to target the undruggable cMyc gene, which promotes cell growth, proliferation, and angiogenesis. It resulted in induced cell downregulation of cMyc, which inhibited tumor proliferation (Zhang et al. 2013). Other siRNA targets include vascular endothelial growth factor (VEGF) pathways to inhibit angiogenesis (Crintea et al., 2021) and over-activated pathways such as the PI3K-AKT pathway that affect various stages of cell growth, proliferation, and survival (Zarredar et al., 2018; Krishnawami & Kandasamy, 2022).
In addition to being effective in tumor reduction on its own, siRNA can also reinforce the efficacy of chemotherapy. Like bacterial resistance to antibiotics, cancerous cells can acquire resistance to chemotherapy drugs through adaptive development of more membrane proteins such as P-glycoprotein and MDR proteins, which pump drugs out of the cell (Gottesman & Pastan, 2015; Song et al., 2020); or by developing better defense mechanisms against cell apoptosis pathways that such drugs may activate (Chen et al., 2018). siRNAs can be designed to target genes powering these resistance mechanisms such that it, when co-delivered with chemotherapy, effectively disables the cancerous cells’ ability to resist the drugs. This was successfully demonstrated by Zhao et al. (2013) in their administration of the anticancer drug cisplatin, which disrupts mitosis by binding to and forming adducts with mitotic DNA, with USP14 siRNA to mice carrying cisplatin-resistant lung cancer (A549/DDP) cells. The resistance to cisplatin arose from overexpression of the protein USP14, which is reported to play a role in cisplatin resistance through deubiquitinating EGFR and enhancing its stability. The increased EGFR leads to downstream activation of the EGFR/PI3K/AKT pathway, inhibiting apoptosis even with DNA damage by cisplatin. By suppressing USP14 expression with siRNA, a significantly stronger antitumor response was achieved compared to treatments using cisplatin alone.
Combination therapy with siRNA has also been effective in restoring sensitivity to MDR cells for other common drugs against NSCLC including gemcitabine (Khatri et al., 2015; Zhang et al. 2013). Specifically, Zhang et al. (2013) demonstrated that co-delivery of cMyc targeting siRNA with gemcitabine monophosphate in a single nanoparticle produced enhanced treatment effects than when administered separately.
Barriers to siRNA delivery
While siRNA treatments show great promise in their high specificity, there are still several hurdles to cross before actual clinical use. In particular is systemic delivery. In general, RNA molecules are unstable in physiological conditions and are degraded by nucleases when injected into the bloodstream or consumed orally. If not degraded, they can be filtered by the kidney or taken up by the reticuloendothelial system (RES) (Guo et al., 2011).
Therefore, siRNA must escape the vascular endothelium to reach the target tissues. This is made easier in tumor tissues due to the enhanced permeability and retention (EPR) effect. The EPR effect is created by the irregular vascular structure of tumor cells due to angiogenesis. Drugs can easily slip into these tissues and accumulate 10-50 times more in 1-2 days than normal tissues (Lyer et al., 2006). When the molecule reaches the target tissue, it must now overcome cellular barriers such as cellular uptake via endocytosis. However, RNAs are negatively charged, making it difficult to passively diffuse across the negatively charged cell membrane. Therefore, it needs to be packaged into vesicles to enter the cell. Once it is inside the membrane via endocytosis, it must escape the endosome before it is degraded by lysosomes.
siRNAs also activate an innate immune response by binding with receptors capable of recognizing foreign genetic materials (Judge et al., 2005). It stimulates innate interferon and cytokine responses that create toxicity issues for the cell. Off-target effects also present a problem for siRNA treatments.
For siRNA delivery to the lungs, pulmonary delivery such as intratracheal, intranasal, and inhalational delivery methods can be more effective than systemic delivery (Ahn et al., 2023; Krishnaswami & Kandasamy, 2022). Delivery through the lungs presents new obstacles. First of all, physiological barriers arise from the reticular pulmonary structure from the trachea to the alveoli. Processes to clear up foreign materials in the lungs such as cough reflex, mucociliary clearance, and immune responses pose another challenge. Respiratory mucus and airway surface liquid can inhibit drug penetration (Sanders et al., 2009).
Another obstacle to treating lung cancer with siRNA is that it is difficult to apply animal studies to humans as their lung structures have significant differences (Hofmann et al., 1989) Therefore, it is difficult to gauge the efficacy of a treatment based on animal trials, so a human lung delivery trial must be conducted for accurate assessment (Ahn et al., 2023).
A successful carrier must overcome all of these barriers to deliver stable siRNA to the target. It must also be biodegradable, nonimmunogenic, and biocompatible (Xu & Wang, 2015).
siRNA Nanoparticle Delivery Systems
Nanoparticles encapsulate siRNA, protecting it from degradation and delivering it to the targeted site. Modification of chemical and physical properties promotes different factors such as gene transfection, targeted delivery, and toxicity. The most well-known modification is the polyethylene glycol (PEG) surface modification, which increases carrier stability and blood circulation time (Iversen et al., 2013). Though some nanoparticles reach the target site passively through the EPR effect, active targeting is preferred for its specificity. For active targeting of cells or organs, various ligands such as aptamers (Zhao et al., 2023), peptides (Khatri et al., 2015; Nascimento et al., 2017), antibodies, and folate (Song et al., 2020) are used to guide the nanoparticles to cancer cells. These ligands are recognized by cancerous cells as they are often overexpressed in tumors.
1. Organic-based Nanoparticles
1.1: Lipid-based Nanoparticles
Lipid-based nanoparticles have many advantages. As cell membranes are also composed of lipids, lipid nanoparticles have increased cellular uptake and lower immunogenic reactions than most polymeric materials (Chen et al., 2018). One of the most commonly used lipid nanoparticles is the liposome. A liposome is a vesicle made of bilayer lipids like the cell membrane. It has a hydrophilic, aqueous core and a hydrophobic membrane, allowing it to carry both hydrophilic and hydrophobic agents. Cationic liposomes spontaneously form lipoplexes with anionic RNAs, making it a good carrier for siRNAs (Hayes et al., 2006). Liposomes can also be modified in many different ways. PEGylated liposomes are of particular interest as they exhibit stealth behavior that increases the siRNA circulation half-time (Nag et al., 2013). Many liposomes also utilize calcium phosphate cores to form complexes with nucleic acids (Khatri et al., 2015; Zhang et al., 2013). Other lipid-based nanoparticles include micelles, stabilized nucleic acid lipid particles (SNALP), and solid lipid nanoparticles (Chen et al., 2018).
1.2: Polymer-based nanoparticles
The main polymers used for siRNA delivery are cationic polymers that form complexes with negative nucleic acids from natural electrostatic interactions. They are wide in variety from synthetic polymers (e.g. polyethylenimine (PEI)) to natural polymers (e.g. chitosan, cyclodextrin). As with other nanoparticles, conjugation of PEG chains improves serum stability, and chemical modifications for targeting improve cell uptake. Polyethylenimine (PEI) is a commonly used cationic polymeric nanoparticle conjugated with various modifications for different purposes. Xu et al. (2015) reported using PEI conjugated with doxorubicin, pH linker cis-aconitic anhydride (CA), and Bcl2 targeting siRNA for pulmonary co-delivery. The PEI-CA-DOX/Bcl2 siRNA complex indicated aggregation in tumor tissues and inhibited tumor growth in metastatic lung cancer models in vivo. Chitosans are natural polysaccharides that are made of β linked glucosamine and N-acetylglucosamine from deacetylated chitin (Liu et al., 2007). They are easy to make, biodegradable, and non-toxic, making them a good choice for drug carriers (Kazmi et al., 2023). For example, a 2017 study by Nascimento et al. (2017) utilized EGFR-targeted chitosan nanoparticles for combination treatment with siRNA and cisplatin in vivo. The targeted delivery inhibited tumor growth and validated EGFR-targeted chitosan as an effective siRNA carrier. For pulmonary delivery, chitosan presents another major advantage as these particles bind to mucosal surfaces and enhance absorption (Kazmi et al., 2023).
2. Inorganic-based Nanoparticles
Inorganic materials such as gold, silver, silica, and platinum can function as siRNA carriers. Gold nanoparticles (AuNP) are frequently used metal nanoparticles due to their non-toxicity and versatility (Ghosh et al., 2008). Yu et al. (2021) conducted in vitro and in vivo experiments with EGFR siRNA carrying collagen AuNP. This resulted in a successful knockdown of EGFR genes and tumor suppression in lung cancer cells with minimal immunogenicity. Metal oxides, such as iron oxides and zinc oxides are also used with various modifications (Zhao et al., 2023). Using magnetic carriers such as iron oxide can open new doors to targeted delivery as they can be actively directed to tumor sites with an external magnetic field (Price et al., 2018). Mesoporous silica nanoparticles (MSN) are also common inorganic nanocarriers due to their adjustable pore size, large surface area, and wide range of functionalities (Li et al., 2012). For example, Song et al. (2020) loaded MRP-1 siRNA into folic acid-conjugated MSN to target NSCLC cells. A co-delivery of these siRNAs with myricetin inhibited tumor growth, inducing significant apoptosis.
Conclusion
This review discussed the mechanism, barriers, and potential delivery systems of siRNA along with therapeutic applications to lung cancer. siRNA therapeutics via nanoparticle delivery inhibited tumors through silencing cancer-related genes and co-delivery with chemotherapeutic agents. Currently, there are active clinical trials for lung cancer treatments using siRNA therapies. NBF-006 is an ongoing siRNA treatment for non-small cell lung cancer that is currently in stage I of clinical trials (Narasipura et al., 2023). The siRNA targets Glutathione S-Transferase-P (GSTP) which is overexpressed in KRAS-mutated NSCLC. NBF-006 utilizes lipid nanoparticles for siRNA delivery, successfully targeting lung tumor tissues for siRNA uptake and resulting in significant tumor repression (O’Brien et al., 2018). Such clinical trials are underway not just for NSCLC but for different kinds of cancers and diseases as well, demonstrating that precise and targeted treatments using siRNA-nanoparticles hold great potential for treatments in all sectors of medicine.
Author’s Note
I wrote this review for my UWP 104E class in the summer of 2023. Unfortunately, many of my friends were struggling with cancer during that time, especially lung cancer. That led me to explore various novel treatments available for cancer and, ultimately, to this topic of siRNA treatments. It was fascinating to see how much careful planning goes into developing this one branch of cancer therapy. There are so many options to choose from and so many aspects to consider. I hope this article provides insight into the complexities of siRNA-based treatments and highlights their potential in the future of cancer therapy.
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