Breast cancer is a heterogeneous disease distinguished by the growth of malignant tumors in mammary glands (Heer et al., 2020). Clinical particularities of this cancer comprise breast lumps, armpit/breast pain, breast skin redness, nipple bleeding, nipple rash, change in the morphology of the nipple, weight loss, and generalized fatigue, among other symptoms (Elshami et al., 2022). Unfortunately, this disease represents the most common cancer with over 2.3 million new cases worldwide and 685,000 deaths in 2020, which accounted for 16% of cancer deaths in women (Arnold et al., 2022). Given the elevated prevalence and mortality rates of breast cancer, novel genetic therapies are currently under development, intending to improve patients’ quality of life. Specifically, miRNA-loaded NPs have shown to be a promising treatment as they can precisely target and silence oncogenes and multiple breast-carcinogenic-related pathways.

In such regard, miR-21, responsible for promoting tumor growth and metastasis, has been reported to be overexpressed in triple-negative breast cancer (TNBC) tumors, as well as in other types of cancer (e.g., lung cancer and glioblastoma). In fact, miR-21 is a well-established oncomiR. On this basis, a miR-21 inhibitor (miR-21i) was conjugated with core-shell tecto dendrimers (CSTDs) to target this RNA molecule in TNBC cell lines (Figure 3B). Moreover, previous work has shown that the co-delivery of chemotherapeutic drugs along with miRNA inhibitors enhances cytotoxic response compared to the drug alone. Thus, the anticancer drug doxorubicin (DOX) was encapsulated inside the CSTDs/miR-21i polyplex for co-delivery. Polyplexes (polymeric systems electrostatically bound to nucleic acids) were successfully internalized by MDA-MB-231 human breast adenocarcinoma cells (Song et al., 2020). Furthermore, efficient in vitro transfection of miR-21i by the polyplexes induced apoptosis activation and strong anti-migratory effect in the treated cancer cells due to the downregulation of miR-21 expression and significant upregulation of miR-21 targets involved in the regulation of tumor proliferation, such as phosphatase and tensin homolog (PTEN), programmed cell death protein 4 (PDCD4), tumor protein p53 (p53), and caspase-3. Overall, this study highlighted the applicability of CSTDs for the synergistic administration of miRNAs and chemotherapeutic drugs for improved antitumor effects (Song et al., 2020).

Ahir et al. (2020) designed MSNs to deliver miR-34a-mimic and antisense-miR-10b as a novel therapeutic approach that could retard tumor metastasis in TNBC since miR-10b is upregulated in this type of cancer, while miR-34a is downregulated. This abnormal miRNA expression encourages tumor formation and dissemination. On that account, simultaneous overexpression of miR-34a and downregulation of miR-10b was pursued. A cationic basic side chain was attached to the NPs’ core to enhance the MSN interaction with cell membranes, providing it with the capability of transporting dual miRNAs with amplified efficiency. Moreover, the loaded MSNs were coated with a hyaluronic acid-poly (ethylene glycol)-poly (lactide-co-glycolide) polymer (HA-PEG-PLGA), as it provides protection against degradation and better affinity with the CD44 receptor (overexpressed in TNBC cells). Finally, the results of this therapeutic method yielded effective inhibition of tumor growth, cancer cell death, and delayed metastasis in TNBC in vitro and in vivo (Ahir et al., 2020) (Figure 3A).

The expression of miR-22-3p has been reported to be downregulated in TNBC cells. This miRNA regulates the eukaryotic elongation factor 2 kinase (eEF2K), which promotes the proliferation and invasion of cancer cells, enhances tumor formation, and triggers drug resistance when overexpressed. Thus, increased miR-22-3p levels are essential to inhibit eEF2K carcinogenic functions. Gorur et al. (2021) transfected MDA-MB-231 and MDA-MB-436 cell lines with miR-22-3p. Later, grafts of the same type of cell lines were put into the mammary fat of mice, which were then injected with single-lipid liposomal NPs (SLNPs) loaded with miR-22-3p. In both cases, expression levels of miR-22-3p were restored, silencing eEF2K, inducing apoptosis, and inhibiting cancer cell proliferation, colony formation, invasion, and metastasis (Gorur et al., 2021) (Figure 3E).

Subsequently, a targeted multilayer FA-decorated polyelectrolyte nanocarrier was designed by Motiei et al. (2021) to deliver LTX-315 (an oncolytic peptide with antitumor activity) and Melittin (Mel) peptides, as well as miR-34a into the MDA-MB-231 breast cancer cell line. MiR-34a, involved in tumor proliferation, invasion and metastasis, is commonly underexpressed in this type of cancer. Meanwhile, LTX-315 is a peptide that has been identified to trigger immune responses against cancer by causing mitophagy. On the other hand, Mel is a 26 amino acid peptide capable of downregulating B-cell lymphoma 2 (BCL2) antiapoptotic protein and upregulating p53 proapoptotic protein. Additionally, Mel can downregulate the expression of cell division-regulating genes cyclin D1 and CDK4, thereby inhibiting cancer cell proliferation (Motiei et al., 2021). On the whole, BCL2 and cyclin D1 gene downregulation, as well as low caspase-8 activity (triggered by the synergistic regulatory activity of miR-34a and Mel) intrinsically induced apoptosis and inhibited cell proliferation. Nonetheless, caspase-8 showed enhanced activity under the presence of LTX-315, suggesting apoptosis induction via the extrinsic way (Motiei et al., 2021) (Figure 3G).

It has been well established that miR-34a is downregulated in breast cancer and plays a key role in regulating genes involved in the cell cycle, such as cyclin-dependent kinase 4 (CDK4) and CDK6, as well as BCL2, which participates in apoptotic mechanisms. On the other hand, docetaxel (DTX) is a chemotherapeutic drug that promotes cell cycle arrest, impaired mitosis, and apoptosis. However, this agent possesses low solubility in water, a fast body clearance rate, non-specific biodistribution, and can generate drug resistance. In order to maximize the stability and the antitumoral effects of both miR-34a and DXT, they were co-delivered into 4T1 and MDA-MB-231 cell lines using folate-targeted hybrid lipo-polymeric nanoplexes composed mainly by a polycarbonate backbone and cholesterol (Sharma et al., 2021). The folate-targeted polycarbonate backbone from the nanoplexes facilitated endo-lysosomal fate escape, avoiding payload degradation. Besides, the folate moiety enhanced active cancer cell targeting due to the overexpressed folate receptors located on the surface of carcinogenic cells. Finally, the cholesterol grafted to the backbone allowed lipid-mediated endocytosis (Sharma et al., 2021).

This delivery method demonstrated an optimal encapsulation and delivery of the two therapeutic molecules of interest since BCL2 anti-apoptotic protein was found to be downregulated after the treatment. In this case, miR-34a worked in synergy with DTX to reduce BCL2 expression since the miRNA avoided protein synthesis via gene silencing, while the drug inhibited BCL2 phosphorylation (Sharma et al., 2021). Once the treatment was completed, BCL2-associated X (BAX) pro-apoptotic protein expression was enhanced, and Ki-67 antigen nuclear protein (responsible for tumor cell proliferation) was downregulated. Further, inhibition of angiogenesis, proliferation, metastasis, and cancer progression were detected, in addition to epithelial-mesenchymal transition (EMT) regression and enhanced apoptosis (Sharma et al., 2021) (Figure 3D).

In another recent study, antisense oligonucleotides (anti-miRNAs) were designed against the metastasis-associated and chemoresistance-associated miRNAs miR-155 and miR-21. To improve anti-miRNAs’ effectiveness, photodynamic therapy was also incorporated into the system at issue by adhering the chlorin E6 (Ce6) photosensitizer to anti-miR-21 and anti-miR-155 (Shang et al., 2022). In fact, Ce6 reacts to 660 nm wavelengths, resulting in energy liberation, which in turn reacts with oxygen molecules to produce reactive oxygen species (ROS), triggering oxidative stress that eventually leads to apoptosis. Importantly, superhydrophobic zeolitic imidazolate framework NP (ZIF-90) was employed to deliver Ce6-anti-miR-21 and Ce6-anti-miR-155 into human breast cancer cell line MDA-MB-231. The pH responsiveness of ZIF-90 allowed it to self-decompose and release its nucleic acid content upon exposure to the acidic environment in tumor cells. Consequently, it was found that the combination of photodynamic therapy and anti-miRNA-controlled release suppresses metastasis and tumor cell proliferation (Shang et al., 2022) (Figure 3F).

Proper conjugation of AuNPs with miRNAs represents a challenging drawback in the field of NP-mediated miRNA delivery. Nonetheless, said issue was overcome by Chaudhari et al. (2022), who developed a simpler method for the fabrication of miRNA-loaded AuNPs with the aim of treating luminal-A type of breast cancer with miR-206, which has been reported to be downregulated in this type of breast cancer and involved in metastasis. These authors developed a complex consisting of AuNPs with limited toxicity inferred by the citrate capping method, further modified with a polyethylene glycol (PEG) moiety to provide a positive charge that simplifies miRNA attachment and stabilizes the platform. After several in vitro assays, it was evidenced that the engineered system reduced the cell viability of MCF-7 breast cancer cells, induced G0-G1 cell arrest, and activated mitochondria-mediated apoptosis. Remarkably, even under nanomolar (nM) concentrations of the miR-206, Au-PEG-miRNA NPs triggered cancer cell death by hindering the expression of a well-known target of miR-206, neurogenic locus notch receptor 3 (NOTCH3, responsible for cell proliferation and tumorigenesis). Furthermore, the nanoplatform downregulated BCL2 and increased BAX expression (Chaudhari et al., 2022) (Figure 3H).

Current research has shown that the downregulation of miR-27b is linked with chemoresistance. Under that premise, expression induction of this miRNA was carried out with the purpose of reversing paclitaxel (PTX) resistance in breast cancer cell lines (MCF-7/Adr) and breast cancer-bearing mice. To carry out this experiment, single-stranded miR-27b was conjugated with fullerene C₆₀ (also known as buckminsterfullerene) to synthesize a fullerene nanospherical miRNA. This nanocarrier was then phagocytized by the cells, where miR-27b is being released (Xu et al., 2022). Subsequently, this miRNA triggered the downregulation of cyclic adenosine monophosphate responsive element binding protein 1 (CREB1) and cytochrome P450 family 1 subfamily B member 1 (CYP1B1) proteins at the transcriptional level. CREB1 regulates the proliferation, survival, and differentiation of cancerous cells, while the latter is involved in drug detoxification. On the other hand, fullerene nanospheres stimulated ROS production under 365 nm irradiation. Altogether, ROS output, along with CREB1 and CYP1B1 downregulation, reverted PTX recalcitrance, allowing apoptosis and suppression of metastasis in resistant breast cancer cells in both in vivo and in vitro assays (Xu et al., 2022) (Figure 3C).

Although promising, the current studies investigating the delivery of miRNAs via NPs for the treatment of breast cancer have certain limitations that need to be addressed. One of the major concerns in this matter corresponds to the unknown adverse effects of the nanocarriers employed in the delivery of miRNA-based drugs. A comprehensive safety profile evaluation is necessary to assess potential side effects and mitigate any risks before advancing to clinical trials. As well, understanding the biodistribution and potential accumulation of NPs in various organs and tissues is essential for assessing their systemic impact and ensuring patient safety.

Besides, determining the optimal dosage, frequency of administration, and duration of treatment is crucial for achieving maximum therapeutic efficacy while minimizing off-target effects. Accordingly, the interactions between miRNAs and NP, their pharmacokinetics, pharmacodynamics, and specific mechanisms of action in breast cancer cells need to be elucidated. More importantly, conducting well-designed pre-clinical and clinical trials is essential to validate the safety, efficacy, and overall therapeutic potential of miRNA-loaded NPs in breast cancer.

Lung cancer is characterized by the presence of thymic, lymph-node, vascular, connective, or neurogenic tumors in the lung, pleura, or mediastinum, which are caused by mutations that commonly affect epidermal growth factor receptor (EGRF), kristen rat sarcoma viral oncogene (KRAS), phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha (PIK3A), and PTEN (Bironzo et al., 2022). Although the estimated heritability of these mutations is 18% (Rifkin et al., 2023), most lung cancer cases are triggered by smoking (Rustagi et al., 2023). Individuals affected by this disease present a cluster of symptoms encompassed by chest pain, fatigue, sleep deprivation, cough, dyspnea, hemoptysis, and weight loss (Ruano-Raviña et al., 2020; Luo et al., 2023). Thereupon, given the escalated incidence and severity of this type of cancer, miRNA-loaded NPs have received significant attention in the last years due to their clinical significance in the modulation of oncogenic protein expression.

As a matter of fact, the lungs are one of the most common sites for cancer metastasis since the presence of collagen in this organ facilitates the dissemination of tumor cells. Yan et al. (2022b) studied for the first time the therapeutic efficacy of miR-29 on inhibiting collagen expression in the lung. Data analysis showed that miR-29a-3p negatively regulates the expression of collagen I (Col1a1), and the upregulated expression of said miRNA is associated with better prognosis in patients. Therefore, in order to deliver miR-29 into mouse lung fibroblasts, they designed two nanovesicle systems. The first one consisted of naturally elicited exosomes upon cisplatin chemotherapeutic treatment (CPT-Exo), which successfully downregulated Col1a1 both in vitro and in vivo, therefore decreasing tumor cell metastasis. The second one comprised a liposome-based nano-delivery system referred to as lipoplex (LPX), which turned out to be less biocompatible than CPT-Exo as they are easily cleared by immune cells. Nonetheless, LPX had enriched tissue-specific delivery attributed to the previously optimized (Kranz et al., 2016) charge ratio adjustment of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)/cholesterol to negatively-charged miRNA (4:1). This proportion facilitated the targeted delivery of the miR-29a-3p-LPX nanosystem specifically to lung tissues. Even so, in both cases, the ability shown by the efficient delivery of miR-29a-3p alleviated the establishment of a pro-metastatic environment for circulating lung tumor cells (Yan et al., 2022b) (Figure 4A).

Notably, folic acid (FA) receptors are widely overexpressed on the surface of cancerous lung cells. Accordingly, FA can be used as a targeting ligand that would covalently bind to these receptors and induce endocytosis of the conjugated drug delivery material, ultimately enhancing cellular uptake. On the other hand, chitosan (CS) is a highly abundant polysaccharide with several advantages as a drug delivery material, including non-toxicity, non-immunogenicity, stability, and the fact that its amino-functional surface groups can be easily modified and functionalized. In this context, Golafzani et al. (2022) designed an FA-decorated CS (FA-CS) NP platform to effectively deliver miR-126 to A549 lung cancer cells. Quantitative Real-Time PCR (qRT-PCR) revealed the improved ability of miR-126 delivered through the FA-CS nano-complex to downregulate the expression of epidermal growth factor-like domain 7 (EGFL7), an angiogenic promoter. Besides, an increased expression of the apoptosis inductors caspase-9 and BAX genes was observed. As well, miR-126 enhanced the expression of genes with an important role in autophagy induction, such as autophagy-related 5 (ATG5) and beclin 1 (BECN1). Altogether, the FA-CS-miR-126 delivery system demonstrated cancer inhibition in vitro (Golafzani et al., 2022) (Figure 4C).

MiR-148b has been reported to suppress a class of proteins in non-small cell lung cancer (NSCLC) called cell adhesion molecules (CAMs), which are responsible for intercellular attachment, adhesion to surfaces, and cell migration. In fact, expression levels of miR-148b have been found to be inversely proportional to A549 NSCLC cells’ survivability. In this regard, Arrizabalaga et al. (2022) designed IONPs with chemical attachment sites on their surface as ideal spots for miRNA junctions. It is noteworthy to emphasize that the oligonucleotide was coupled with the NP using a Diels–Alder linker, which permitted the spatiotemporally controlled miRNA delivery into Human Caucasian Lung Carcinoma cells (A549) when stimulated by alternating magnetic field radiofrequency (AMR-RF). Confocal microscopy demonstrated the efficient and selective uptake of the nano-complex by A549 cells. Meanwhile, viability assays confirmed the subsequent induction of cytotoxicity, with significantly enhanced results in the groups that included AMR-RF compared to those in which AMR-RF was not applied. Viability tests also evidenced that AMR-RF-exposed A549 cells displayed less survival rates (Arrizabalaga et al., 2022) (Figure 4B).

According to the results of the studies herein, the delivery of miRNAs via NPs holds promise as a potential treatment strategy for lung cancer. However, several constraints persist that hinder their successful translation into clinical practice. One key limitation is the lack of knowledge regarding the adverse effects of NP-mediated miRNA delivery in lung cancer patients. While in vitro and animal studies have shown positive outcomes, the long-term safety and potential side effects in humans remain uncertain. Hence, thorough evaluation and monitoring of potential toxicities are essential to ensure patient safety and effectively mitigate any risks before progressing to human trials.

Moreover, the specific biological and physiological characteristics of lung cancer pose additional challenges in the nanocarrier-mediated delivery of miRNA-focused drugs. In this sense, the complex tumor microenvironment, heterogeneous cell populations, and variations in gene expression patterns necessitate a deeper understanding of the pharmacodynamics and pharmacokinetics of these nanoformulations. To overcome these limitations, it is crucial to employ appropriate biological models that closely mimic the specific conditions of lung cancer in humans (e.g., patient-derived xenografts, lung cancer organoids, and 3D cell cultures). By utilizing relevant models that faithfully replicate the characteristics of lung tumors and the surrounding microenvironment, researchers could obtain more accurate insights into treatment responses, potential adverse effects, and overall efficacy, thereby improving the chances of successful clinical translation.

Glioblastoma is a type of cancer characterized by glioma development within the central nervous system, particularly in the brain (Tołpa et al., 2023). This type of brain malignancy represents the most lethal and aggressive primary neoplasm, given that it has a median survival of 14 months and only 5% of the patients survive beyond 5 years after being diagnosed (Zhong et al., 2020; Huan et al., 2023). Clinical indicators of glioblastoma commonly include headaches, seizures, nausea, vomiting, alterations in personality, imbalance when walking, urinary incontinence, and visual difficulties (Hansen et al., 2023). Additionally, the prognosis remains unfavorable despite the remarkable advances in neuro-oncology and biomedicine. In the last years, nanomedicine has shown important potential for NP-mediated local targeted delivery of miRNAs, which are able to simultaneously regulate mutated tumor genes that promote proliferation, differentiation, and motility in glioblastoma.

In an intriguing investigation, a polymeric NP was developed to deliver anti-miR-21 and miR-124 into glioblastoma multiforme (GBM) tissue. Interestingly, oligopeptide angiopep-2 was incorporated to ensure specific binding of the NP to the highly expressed lipoprotein receptor-related protein 1 (LRP-1) receptors in GBM tumor cells. This promoted enhanced GBM uptake and easy traverse of the blood-brain barrier. Moreover, angiopep-2 was able to encapsulate and protect aforesaid miRNAs from degradation via electrostatic, hydrogen-bond, and hydrophobic interactions. Significantly, these interactions became neutralized when the NP encountered the ROS stimuli present in tumor cells, which enabled precise miRNA release (Liu et al., 2021). The delivery of anti-miR-21 via polymeric NPs repressed the expression of miR-21, permitting the tumor suppression activity of PTEN. On the other hand, miR-124 has been found to be downregulated in GBM; this miRNA is capable of inhibiting tumor growth and invasion by targeting the rat sarcoma (RAS) family, which is responsible for controlling cell division (Liu et al., 2021). Consequently, the delivery of miR-124 into cancerous brain cells provoked tumor growth deceleration. On the whole, anti-miR-21 and miR-124 synergic activity in orthotopic GBM xenograft models demonstrated a strong antitumor effect since diminished proliferation, invasion, angiogenesis, and migration of tumor cells was detected, leading to higher survival rates (Liu et al., 2021) (Figure 5A).

It has been elucidated that miR-7 is able to sensitize TRAIL to effectively repress tumors. Likewise, the X-linked inhibitor of apoptosis (XIAP) is a target gene of miR-7 and is responsible for the regulation of cell death sensitivity (Kang et al., 2021b). In such a regard, miR-7 was loaded into a near-infrared (NIR) absorbing semiconducting polymer-based/miRNA nanocarrier (SP/miNP). The primary advantage that this NP possess relies on the fact that it is capable of generating strong photoacoustic radiation (PA) force when a NIR-pulse laser is applied toward the target tissues and vessels. The SP then absorbs light, allowing it to smoothly overcome the endothelial barriers so that rapid and intact delivery of miR-7 into U87-GM-bearing mice could be pursued. Furthermore, this gene therapy was improved with a TRAIL-inducing compound (TIC), which worked synergistically with miR-7 to trigger TRAIL-induced apoptosis. Assays demonstrated that this treatment achieved miR-7 intact delivery in 20 min, resulting in the inhibition of tumor growth, apoptosis induction, and angiogenesis reduction by downregulating the expression of XIAP and upregulating TRAIL expression (Kang et al., 2021b) (Figure 5B).

Later, anti-miR-21 was loaded onto a spermine-modified acetalated dextran NP system (SpAcDex), whose highly positive charges allowed a miRNA encapsulation efficiency of 90% (Zheng et al., 2022). Additionally, the nanomaterial was conjugated with bradykinin peptide, which exhibits great affinity for the B1 receptor, highly expressed in brain tumor microvascular systems. When the association between the bradykinin ligand and its B1 receptor counterpart is established, the permeability of vessels in the blood-tumor barrier (BTB) is adjusted, facilitating SpAcDex penetration and uptake by GBM tumor cells, allowing an efficient cargo release. In vivo assays performed in orthotopic U87MG glioma-bearing mice, as well as in vitro trials in human (U87MG) and rat (C6) glioma cells displayed continuous anti-miR-21 release, cell apoptosis, tumor growth deceleration, and blood vessel normalization (Zheng et al., 2022) (Figure 5D).

MiR-219 is known to be downregulated in GBM. In fact, data indicates that miR-219 overexpression triggers the downregulation of both roundabout guidance receptor 1 (ROBO1) and spalt-like transcription factor 4 (SALL4), which are linked with prolonged patient survival. To investigate their gene delivery potential, chitosan NPs (CNPs) loaded with miR-219 were constructed using an ionic gelation method, achieving an entrapment efficiency of 95% (Alswailem et al., 2022). Subsequently, sustained release patterns of this miRNA from the complex reduced tumor proliferation, migration, and invasion of U87 MG human brain cancer cells without affecting fibroblasts in vitro (representing healthy cells), which suggests that this NP-mediated treatment is safe and specific. Said effects were proved to be mediated by the downregulation of miR-219 target genes epidermal growth factor receptor (EGFR), ROBO1 and SALL4, inducing apoptosis (Alswailem et al., 2022) (Figure 5C).

In spite of the fact that some reports have shown encouraging outcomes, the use of miRNA nanoformulations for glioblastoma treatment still faces several challenges that need to be addressed. For instance, the current models used in glioblastoma research do not fully mimic the complexity of human disease. Traditional cell culture models or animal models may not adequately replicate the specific microenvironment of glioblastoma, including the interactions with surrounding cells and the blood-brain barrier. Therefore, it is essential to develop advanced biological models that better capture the unique characteristics of glioblastoma, such as patient-derived xenografts or three-dimensional organoids, to improve the translatability of miRNA nanoformulations into clinical trials.

On the other hand, the lack of comprehensive long-term studies in animal models or clinical trials hinders our understanding of potential toxicities and off-target effects of these therapeutic approaches. Consequently, a thorough evaluation of the toxicity profiles, as well as the biodistribution and accumulation of NPs in healthy brain tissues, is crucial before advancing into clinical applications for glioblastoma patients. To overcome these limitations, comprehensive studies evaluating the pharmacodynamics and pharmacokinetics of miRNA nanoformulations in reliable animal models and eventually in human patients are necessary to optimize their therapeutic potential and ensure safe and effective treatment strategies for glioblastoma.

Liver cancer is a type of carcinoma that grows in hepatic cells (Wu et al., 2023). It is the fourth leading cancer-related cause of mortality, and approximately 800,000 cases are diagnosed each year (Bakrania et al., 2023). Certain associated risk factors that could lead to liver cancer development are chronic viral hepatitis, alcohol abuse, excess body fat, and constant smoking (Arafa et al., 2023). The main clinical particularities of this cancer involve white stools, hepatitis B, hepatitis C, jaundice, considerable weight loss, vomiting, and abdominal pain (Mehmood et al., 2022). Nonetheless, symptoms are insidious at early disease stages, and most patients are diagnosed only when cancer reaches advanced levels (Jiang et al., 2023). Overall, given the elevated number of liver cancer-related deaths, incidence, and severity, chemo-gene combinatorial therapies have been devised to provide an alternative treatment against hepatic cancer (Wang et al., 2021c). Particularly, miRNAs have caught special attention for the diagnosis and therapy of liver cancer as they regulate the progression of oncogenes.

One of the key target miRNAs involved in hepatocarcinogenesis is miR-122 owing to the fact that it can suppress tumorigenic proteins in liver cells and downregulate permeable glycoproteins (P-gp), which work as channels that mediate the expulsion of drugs from the cell. Considering this, miR-122, the anticancer drug PTX, and hepatocyte targeting ligands were co-delivered into HepG2 cells using multivalent 6-way junction (6WJ) RNA NPs (Wang et al., 2021a). This nanoscale delivery system possesses a rubber-like property, which allows it to have amplified permeability and retention when entering the tumor’s vasculature, thus increasing its efficiency to target cancer cells. Similarly, conjugated hepatocyte targeting ligands function as an endocytosis-mediator receptor, providing 6WJ RNA NPs with higher tumor specificity (Wang et al., 2021a). In vitro and in vivo assessments demonstrated successful NP-tumor cell binding, cellular uptake, PTX intracellular retention, and induction of severe cytotoxicity; showing the potential synergistic effect of miR-122 and PTX to treat hepatocellular carcinoma (HCC). Importantly, expression levels of miR-122 target genes such as oncogenic disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and multi-drug resistance 1 (MDR1) were diminished, preventing tumorigenesis and drug efflux, respectively (Wang et al., 2021a). Successful drug retention, NP tumor specificity, correct biodistribution, low toxicity, as well as suppression of tumorigenic proteins contributed to the growth reduction of liver tumors (Wang et al., 2021a).

Hyperbranched polyamidoamine (PAMAM) functionalized with lactobionic acid (LA) was formulated in another investigation to selectively administer a miR-218 expressing DNA plasmid (pmiR-218) into HepG2 cell lines. Concretely, LA displays significant binding affinity towards asialoglycoprotein receptor 1 (ASGPR1), overexpressed in HCC. This provided PAMAM with enhanced biocompatibility and better cellular uptake via mediated-endocytosis activation. On the other hand, miR-218, expressed limitedly in HCC tissue, serves a critical function in inhibiting tumor proliferation and promoting apoptosis (Elfiky et al., 2021). In vitro and in vivo experiments demonstrated effective miR-218 restoration, as well as downregulation of the miR-218 target Homeobox A1 (HOXA1), responsible for tumor progression. Consequently, diminished tumor proliferation was detected in addition to improved liver histological features (Elfiky et al., 2021).

Remarkably, N-acetylgalactosamine (GalNac)-decorated NPs represent a noteworthy approach in the realm of liver cancer research. This moiety displays a great affinity for asialoglycoprotein receptors, which are widely expressed on the surface of hepatocytes. Conjugation of the GalNac ligand to RNA strands enhances selectivity and uptake by hepatocellular carcinoma-positive cells (Debacker et al., 2020; Brown et al., 2021). Under such premise, GalNac-coated exosomes have been proved to effectively deliver miR-122 and PTX into liver tumors, avoiding the need for endosomal trapping and achieving increased cancer inhibition (Ellipilli et al., 2023). Nonetheless, the use of GalNac has not yet been widely applied in miRNA nano-complexes for liver cancer. Hence, exploring the incorporation of GalNac into NPs designed to deliver artificial miRNAs represents a promising avenue to harness the full potential of miRNA-based therapeutics. Moreover, a deeper inquiry into the oral administration of GalNac-decorated nano-complexes is encouraged, as it will represent an important advancement for use in chronically-ill patients (Debacker et al., 2020).

Notwithstanding the above, the use of miRNA-based drugs delivered via NPs in treating liver cancer is currently accompanied by several restraints that must be solved to reach the clinical panorama. For example, relying solely on cell cultures and mouse models for studying the efficacy of these treatments is inadequate. While convenient for initial screening, cell cultures fail to accurately represent the complexity and heterogeneity of liver cancer. Similarly, although mouse models provide some insights into tumor behavior, they do not fully mimic the intricate interactions between liver cancer cells and the human microenvironment. Thus, the translational potential of miRNA-based nanoformulations to clinical settings remains uncertain without rigorous evaluation in more sophisticated and relevant biological systems that better mimic the human liver cancer microenvironment.

Additionally, while short-term studies may demonstrate favorable results, these therapies’ long-term effects and potential adverse reactions require thorough investigation. As a vital organ responsible for numerous metabolic processes, the liver must carefully evaluate any potential hepatotoxicity or systemic effects resulting from miRNA NP treatments. Comprehensive long-term studies, including biodistribution analyses, monitoring of treatment response over extended periods, and assessment of potential off-target effects, are crucial to validate the safety and efficacy of miRNA-centered drugs delivered via nanocarriers in liver cancer treatment.

Former studies have confirmed that miR-451 regulates the expression of P-gp in several multi-drug resistant ovarian and gastric cancer cell lines. As previously stated, silencing P-gp expression is clinically important since this protein transports chemotherapeutics out of the cell. Taking this into account, calcium carbonate NPs coated with cancer cell membranes (designed with the aim of rising the tumor-localization capacity of the drug delivery system) were loaded with miR-451 and Adriamycin (Adr), and delivered into bladder cancer cells (Wei et al., 2020). Calcium carbonate mediated an effective miRNA binding process and proper biocompatibility, while the cancer cell membrane made possible NP retention within the tumor tissue and the overcoming of extracellular barriers. Finally, in vivo and in vitro studies showed successful delivery of miR-451, which significantly downregulated P-gp levels and maintained high intracellular levels of Adr due to the avoidance of drug efflux. Moreover, elevated expression of apoptotic genes caspase-3 and cytochrome-3 was found; while the level of BCL2 apoptosis suppressor gene was lower compared to the control group, leading to mitochondria-mediated apoptosis. On the whole, the synergistic effect of Adr and miR-451 contributed to overcoming multidrug resistance in bladder cancer cells (Wei et al., 2020).

Omentum fibroblasts are known to secrete abundant exosomes, which are ideal gene delivery candidates due to their stability in blood circulation and efficacy in transporting nucleic acids. Since the majority of ovarian cancer (OC) patients undergo omentectomy, Kobayashi et al. (2020) proposed using omentum-derived exosomes to carry miR-199a-3p, a representative tumor suppressor miRNA, as a cancer replacement therapy. Remarkably, the nano-complex was successfully accumulated in cancer cells, inhibiting tumor proliferation and invasion. In vitro and in vivo experiments demonstrated an increase in miR-199a-3p expression and suppression of its direct target tyrosine-protein kinase Met (c-Met), as well as downstream target molecules, such as matrix metalloproteinase-2 (MMP2) and the phosphorylation of extracellular signal-regulated kinase (ERK). Reduced tumor burden and inhibition of metastasis were also reported in the groups treated with the nanodevice. However, more studies are required to properly determine the biodistribution of exosomes in animal models (Kobayashi et al., 2020).

Later, the attention was centered on cervical cancer treatment by synthesizing SiO₂-polyethyleneimine NPs loaded with miR-let-7c-5p. When delivered, this miRNA was successfully transferred to epithelial carcinoma (HeLa) cells, inhibiting the development of cancer cells under shallow cytotoxicity conditions. As a result, miR-let-7c-5p/insulin-like growth factor 1 receptor/phosphatidylinositol-3 kinase/AKT serine/threonine kinase 1 (miR-let-7c-5p/IGF-1R/PI3K/AKT) and beta-catenin/snail family transcriptional repressor 2 (β-catenin/SLUG) were highlighted as potential targets against cervical cancer (Shao et al., 2020).

MiRNA miR-200c has been identified to stimulate the entrance of CD8⁺ T cells into tumors, elevate mutated cells’ sensitivity to apoptotic signals, suppress vascular formation around tumors, and downregulate programmed death-ligand 1 (PD-L1) expression, thus limiting its immunosuppressive action. More importantly, dabrafenib (Dab), a v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitor is a drug capable of slowing tumor growth, and its combination with miR-200c turned out to enhance cancer cells’ sensitivity to such medicine by inhibiting the expression of PD-L1 (Nguyen et al., 2021). These molecules were co-delivered into murine colon carcinoma cells MC-38 using chemokine receptor type 4 (CXCR4)-targeted polymeric NPs coated with poly-L-glutamic acid and a CXCR-4 antagonist peptide, mediating better binding with chemokine receptors, enhanced cytotoxicity and drug release, as well as content stability. In vivo and in vitro experiments showed that this cancer treatment method effectively inhibits PD-L1 expression and triggers a powerful immune response against accelerated cell differentiation. Remarkably, only a low dose of the drug was required to observe notable tumor suppression (Nguyen et al., 2021).

In another study, nona-arginine (R9) peptide labeled with ¹²⁵I radioactive particles were combined with arginine-glycine-aspartate (RGD) short cyclic peptide, and Ce6 photosensitizer to build a self-assembling nano-complex to deliver miR-139-5p into cancer cells. This nano-complex was used in conjunction with Ce6-mediated photodynamic-radio cancer therapy to increase its therapeutic potential in vitro and in vivo. ¹²⁵I radioactive particles produced a large number of free radicals, which caused DNA damage, thus propitiating proapoptotic p53 expression and, consequently, apoptosis, as well as tumor growth retardation, metastasis inhibition, and proliferation repression of HeLa cells (Wang et al., 2021b). On the other hand, miR-139-5p was critical for the regulation of DNA maintenance and repair genes DNA polymerase theta (POLQ), DNA topoisomerase 1 (TOP1), DNA topoisomerase II alpha (TOP2A), RAD54 like (RAD54L), and X-ray repair cross-complementing 5 (XRCC5), as well as the ROS defense-related gene methionine adenosyltransferase 2A (MAT2A). When delivered into the HeLa cells, the intracellular level of miR-139-5p became upregulated and promoted apoptosis of cancer cells under radiotherapy. Overall, the synergistic action of all nano-complex components in combination with photodynamic therapy and radiotherapy displayed destruction of neoplasms (Wang et al., 2021b).

Chen et al. (2021) developed a miRNA-conjugated NP treatment for gastric cancer (GC). It is widely known that disturbing mitochondrial-related pathways are an efficient method for inducing apoptosis in gastric tumor cells since GC is considered a mitochondrial metabolic disease. Therefore, to trigger apoptosis via mitochondrion pathways, miR-532-3p was delivered using poly d,l-lactide-co-glycolide (PLGA)-PEG NPs labeled with vitamin B12 (VB12), which selectively target the transcobalamin II receptor CD320, overexpressed in GC cells. MiR-532-3p works as a therapeutic oligonucleotide since it inhibits the expression of the apoptosis repressor with the caspase recruitment domain (ARC). Consequently, once the ARC restraint effect has been suppressed, the pro-apoptotic protein BAX enters the mitochondria, decreasing mitochondrial membrane potentials, activating ROS production, and promoting mitochondrial permeability transition pores opening (Chen et al., 2021). On the whole, all these events result in mitochondrial damage that promotes the activation of caspase-dependent cell apoptosis. As expected, in vivo and in vitro tests displayed inhibition of GC cells proliferation after the miRNA treatment, suggesting that miR-532-3p delivered by PLGA-PEG-VB12 NPs regulates the ARC/Bax/mitochondria signaling pathway to cause apoptosis (Chen et al., 2021).

One of the principal disadvantages of radiotherapy relies on the fact that this method becomes less efficient due to radioresistance triggered by DNA damage repair and hypoxia. To avoid a limited response from cells to radiotherapy, radiosensitizers such as miR-181a were delivered into cancerous esophageal squamous cells using 2D graphdiyne-cerium oxide (GDY-CeO₂) nanozymes. As a result, miR-181a directly bound to the transcripts of its target gene RAD17 checkpoint clamp loader component (RAD17), responsible for regulating checkpoint kinase 2 (Chk2) pathway, causing RAD17 downregulation in radioresistant cells and the overexpression of Chk2 protein (Zhou et al., 2021). Importantly, the downregulation of RAD17 protein prevented DNA repair, therefore enhancing DNA-damage-induced apoptosis, while the accumulation of Chk2 inhibited cancer cell proliferation. Particularly, GDY-CeO₂ exhibited a better catalase activity by transforming H₂O₂ into O₂ when surrounded by a weakly acidic tumor microenvironment, which caused the mitigation of tumor hypoxia (therefore reversing radioresistance), promoted radiation-induced DNA damage, inhibited tumor growth, and enhanced radiotherapy sensitivity in esophageal squamous cell carcinoma (ESCC) (Zhou et al., 2021).

Ghaffari et al. (2021) reported a PEGylated niosomal formulation capable of simultaneously loading miR-15a and miR-16–1 which was subsequently delivered into the prostate cancer cell line PC3. Niosomes are biodegradable cationic lipids made of synthetic non-ionic surfactants with advantages such as low immunogenicity, cost-effective production, stable structure, and no cytotoxicity. Further, the cationic charges found on their lipid bilayer allow strong electrostatic interactions with miRNAs, resulting in a high loading efficiency. Therefore, the miR-15a and miR-16-1-loaded nioplexes significantly decreased the expression of BCL2 augmenting the degree of cell death in PC3 cells. As well, nioplexes loaded with both miRNAs showed a higher cellular uptake compared to the NPs loaded with one miRNA alone (Ghaffari et al., 2021).

Yan et al. (2022a) designed a multifunctional chemical/gene-therapeutic-loaded nanomaterial that restrains epithelial ovarian cancer cell (SKOV₃) development by detecting and reversing drug resistance, along with treating cancer in a chemical-photothermal manner. Concretely, these researchers embedded Cyanine 5 (Cy5)-modified miRNA let-7i and platinum (IV) onto a nano-graphene oxide platform (NGO), which was transported into the cisplatin-resistant ovarian cancer cell line SKOV3DDP. Let-7i was employed owing to its well-known capacity to negatively regulate cyclin D1, a protein involved in cell proliferation. Besides, the attached fluorophore Cy5 was employed as a probe to detect mRNAs related to drug resistance when miRNAs were bound to them. On the other hand, platinum (IV) was included in the formulation since it gets converted into the chemotherapeutic particle platinum (II) under the stimuli of glutathione (GSH) in the acidic and reductive tumor microenvironment. Moreover, folate was included in the nanocomposite to achieve targeted delivery to drug-resistant cancer cells. NGO photothermal properties were activated via NIR laser radiation, which significantly enhanced the therapeutic efficiency of this bio-responsive nanocomposite. Ultimately, Cy5-modified miRNA, folate and platinum-loaded PEGylated nano-graphene oxide complex (^Cy5−miRNGO-PEG-FA-Pt) showed outstanding stability, remarkable drug uptake capacity, and great proficiency to induce cytotoxic effects in cisplatin-resistant carcinoma cells (Yan et al., 2022a).

Soon after, miR-320 was proposed as a promising miRNA to treat head and neck cancer (HNC). This miRNA is naturally downregulated in HNC cells and stimulates apoptosis, represses angiogenesis, counteracts drug resistance, and inhibits cancer progression. However, its delivery into HNC cells has been challenging due to its poor cellular penetration, degradation, and inefficient endosomal escape. Thereby, to overcome such drawbacks, Lo et al. (2022) delivered miR-320 into human tongue squamous carcinoma cell lines (SAS) and SAS-bearing mice using cationic solid lipid NPs (SLNs) coated with a combination of trans-activator (TAT) and peptides containing the asparagine-glycine-arginine motif (NGR). This peptide combination, characterized by cysteine-rich transcriptional activation domains and arginine-rich RNA binding motifs was named T-peptide for efficiency purposes. Accordingly, T-peptide provided SLNs with tumor-homing moiety and mediated cell penetration; this initial miRNA-containing NP activated diverse signaling pathways, leading to the inhibition of drug resistance-related mechanisms. As a result, it sensitized HNC cells for the subsequent NP. Further, with the purpose of enhancing miR-320 therapeutic impact, chemotherapy drug oxaliplatin (Oxa) was delivered through liposomes covered with T-peptide and a nuclear-localization-sequence containing peptide (R-peptide) to provide the nanocarrier with cell- and nucleus-penetrating capability. Oxa is a platinum-related compound that impedes DNA replication, transcription, and repair in cancer cells by triggering a DNA cross-linkage at the N₇ positions of adenine and guanine (Lo et al., 2022). SLNs and liposomes were sheathed with polyglutamic acid and polyethylene glycol (PGA-PEG) to make the nanocarriers pH-responsive. In this way, the NPs’ cargoes were optimally released when exposed to the acidic tumor environment. Overall, in vivo and in vitro assays demonstrated satisfactory uptake of both Oxa and miR-320, decreased Oxa-associated toxicities (i.e., neutropenia and thrombocytopenia), and antitumor activity (Lo et al., 2022).

As previously stated, radiotherapy has been associated with low oxygen concentrations in the tumor microenvironment. To overcome this issue in rectal cancer, zeolitic imidazolate framework-8 (ZIF-8) nano-complexes were built to deliver nano-MnO₂ particles and miR-181a mimics together into mouse colon cancer cell line MC38 and MC38-bearing mice. ZIF-8 was chosen as the nanocarrier because of its controlled component release, excellent biodegradability, and negligible toxicity (Hao et al., 2022). Findings showed that MnO₂ NPs reacted with endogenous H₂O₂ to form O₂, which led to a reduction in hypoxia and subsequent sensitivity to radiotherapy. Further, MnO₂ considerably blocked the scavenging activity of glutathione on hydroxyl groups by transforming this compound to glutathione disulfide, therefore magnifying the apoptotic efficacy of chemodynamic therapy. Additionally, induced miR-181a overexpression triggered DNA damage after radiotherapy via double-strand breaks, hence working as a potential inhibitor of angiogenesis and tumor growth. Moreover, the expression of HIF1-α was found to be significantly reduced. On the whole, tumors showed increased radiation sensibility, inhibited proliferation, decreased migration, and apoptosis improvement (Hao et al., 2022).

Oncogenic miR-30a-5p has received particular attention for the treatment of ocular melanoma. Concretely, this miRNA downregulates E2F7, responsible for promoting the proliferation, migration, and apoptosis of carcinogenic cells. Under such premises, this nucleic acid was released into MUM2B, CRMM2, and CM 2005.1 ocular melanoma cell lines, as well as into MUM2B-bearing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice (Ma et al., 2023). To achieve optimal transportation, the chosen delivery vehicles were GSH-responsive quasi-mesoporous magnetic nanospheres (MMNs), since they protect the miRNA from nuclease degradation and promote M1-like macrophages to secrete pro-inflammatory cytokines that induce apoptosis. Furthermore, NP-derived Fe²⁺ reacted with H₂O₂ produced by M1 macrophages to generate Fe³⁺, OH-, and free OH radicals, promoting apoptosis. Overall, the outcomes of this inquiry suggest that miR-30a-5p can be efficiently delivered via GSH-responsive MMNs, blocking E2F7 and caspase-3 expression, thus facilitating the inhibition of ocular melanoma and induced apoptosis both in vivo and in vitro (Ma et al., 2023). The main findings regarding the application of nanotechnology-mediated delivery of miRNA-based drugs in these other types of cancer are listed in Table 1.