Background

In recent years, it has been shown that the tumor microenvironment (TME) comprises different types of cell subpopulations. Cancer stem cells (CSCs) are rare populations in the TME, which is considered the origin of the cancer. Hence, they have also been called tumor-initiating cells (TICs) [1]. Despite having a minor population, CSCs play a significant role in cancer cell invasion, tumor development and progression, resistance to chemical drugs and stress conditions within the TME (such as hypoxia or nutrient deprivation), colony formation capacity, epithelial-to-mesenchymal transition (EMT), tumor dormancy, and long-term reproliferating potential [2,3,4,5]. Furthermore, it has been shown that CSCs are the leading cause of cancer relapse after therapies by repopulating new cancer cells and are responsible for the metastasis of cancer [6, 7].

CSCs also display “stemness,” which means they hold properties similar to normal stem cells, including enhanced self-renewal capacity and the potential to regrow and differentiate into heterogeneous lineages of cancer cells and reproduce cells with metastasis behavior that ultimately causes tumor recurrence following conventional therapies [8]. It has been shown that stemness is fundamentally related to malignant properties of cancer cells, such as resistance to therapeutic agents and tumor progression and relapse [9, 10]. Therefore, CSCs drive tumor progression, conventional therapy resistance, relapse, and metastasis [11, 12]. Overall, the clinical and biological significance of CSCs appears to have a positive correlation between cancer stemness and enhanced metastatic propensity [13]. Many recent studies focused on targeting CSCs through chemical drugs and RNA-based therapeutics such as microRNAs (miRNAs) to reduce the malignancy of cancer and enhance therapeutic efficiency. In this regard, different types of nanoparticles (NPs) have been used to deliver these therapeutic agents into CSCs. Nature-based nanoparticles, such as chitosan nanoparticles (CSNPs), as natural polymer-based nanoparticles and bioinspired nanoparticles, such as exosomes, have been widely used in drug delivery. In the present study, we review the therapeutic application of CSNPs and exosomes in the delivery of miRNAs into CSCs and compare their properties.

Identification of cancer stem cells

Like normal stem cells, CSCs are regulated by similar regulatory signaling pathways, including Wnt, Notch, JAK/STAT3 (Janus kinase/signal transducers and activators of transcription), PI3K/AKT/mTOR (Phosphoinositide 3-kinase/AKT/mammalian target of rapamycin), NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells), and the Hedgehog pathway. Activation of these pathways is associated with tumorigenesis and metastasis and maintains CSCs’ stemness [14].

CSC Biomarkers are the most effective way to characterize and isolate this cell population in tumors. CSCs can be characterized by several specific cell-surface markers, such as CD44, also known as Homing Cell Adhesion Molecule (HCAM), CD24, also known as Heat Stable Antigen (HSA), CD133 (Prominin-1), ALDH, and EpCAM (CD326) [15, 16]. CD44+/CD24− cells present CSCs in breast cancer, prostate cancer, head and neck squamous cell carcinoma, and ovarian cancer [17]. Transcription factors, as intracellular markers, also play a key role in regulating CSC development and maintenance. These pluripotent transcription factors, including OCT4, SOX2, Nanog, Klf4, and MYC (c-myc), can be overexpressed in many CSCs [18].

Herein, therapeutic approaches based on targeting these markers are currently being developed. CSCs are frequently identified as a contributing factor to multi-drug resistance (MDR). Although conventional anticancer agents can kill non-CSCs in the tumor mass, they are not efficient in eradicating CSCs because these therapeutic agents affect only proliferating cells. Therefore, CSCs escape conventional treatment methods because they undergo a quiescent state. Furthermore, due to their advanced resistance mechanisms, such as expressing MDR transporters and enhanced DNA repair capacity, CSCs are highly resistant to multiple therapeutic agents [19, 20]. Additionally, CSCs highly express chemotherapeutic resistance-related genes, such as anti-apoptotic genes and the Human ATP-binding cassette (ABC) transporter family, which contribute to escape from apoptotic signaling pathways and survival. Activation of DNA damage checkpoints facilitates repair mechanisms that enhance cell survival [21].

To enhance the efficacy of cancer treatment and overcome therapy-resistant CSCs, the design of new drugs and their delivery routes should be prioritized to eliminate these highly resistant subpopulations from tumor masses [22].

Targeting CSCs in new cancer therapy approaches

In recent years, targeting CSCs has been an attractive therapeutic strategy in cancer treatment. Combining conventional non-CSC-targeted therapies and CSC-targeted treatments will be a novel approach to overcoming drug resistance and tumor recurrence. In developing new anticancer drugs, researchers have focused on signaling pathways, drug transporters, apoptosis pathways, DNA damage machinery, and biomarkers of CSCs that underlie stemness and malignant properties Although anti-CSC new therapeutic strategies, ranging from monoclonal antibodies (mAb) and small molecules, including cytotoxic anticancer drugs, have emerged, cancer resistance continues to develop [23, 24].

In recent years, novel approaches based on therapeutic nucleic acids aimed at combating unique drug resistance pathways have emerged rapidly [25]. The technology of RNA interference (RNAi), including small interfering RNA (siRNA), miRNA, and long noncoding RNA (lncRNA), has emerged as an alternative to the conventional chemical drug-based treatments that were developed to target CSCs (Fig. 1).

Fig. 1
figure 1

Cancer stem cell-targeted therapy compared to conventional anticancer therapies. In contrast to traditional anticancer therapies, specific CSC-targeted therapies are more effective in eradicating tumor cells. Specific targeting of CSCs is considered to be essential for complete tumor regression. Created in BioRender. Valizadeh, M. (2025) https://BioRender.com/j93u291

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MiRNAs and CSCs

miRNAs, with a length of 21–25 nucleotides, are a class of endogenous small noncoding RNA (ncRNAs) that regulate their target gene expression at the post-transcriptional level through partial binding to seed sequences in the 3′-untranslated region (3′-UTR) of target mRNAs, which occurs in RNA-induced silencing complex (RISC) [26]. As a single miRNA regulates the expression of several genes associated with various biological processes such as cancer development, proliferation, invasion, and migration, it plays a fundamental role in cancer development and progression. miRNAs regulate the malignant properties of cancer cells, such as migration, angiogenesis, metastasis, drug resistance, and tumorigenicity [27]. Various findings revealed that miRNA expression is dysregulated in different types of cancers, and they can be categorized into two classes: upregulated miRNAs in cancer cells, which act as oncogenic miRNAs (oncomiRs), while downregulated miRNAs, which prevent cancer initiation, are considered tumor suppressor miRNAs (tsmiRs). Dysregulated expression of miRNAs affects signaling pathways and molecular machinery underlying CSCs, which are associated with cancer progression [28]. Furthermore, it has been documented that CSC-related genes are also regulated by miRNAs, which contribute to regulating cancer cell stemness and CSC populations in an in-vitro model (Fig. 2) [29, 30].

Fig. 2
figure 2

miRNAs regulating cancer stem cells. miRNAs play key roles in regulating CSC-specific properties, including differentiation, self-renewal capacity, metastasis, tumor recurrence, drug resistance, and regulation of CSC marker expression. Created in BioRender. Valizadeh, M. (2025) https://BioRender.com/j93u291

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Hence, silencing oncomiRs with specific miRNA inhibitors or antagonists or upregulating tsmiRs using miRNA mimics has been demonstrated as a promising approach for targeting CSCs. Recently, miRNA-based therapeutics have gained attention in cancer treatment, especially CSC targeting. Nevertheless, these nucleic acid-based agents have faced some challenges, such as low cellular uptake, negatively charged nature, immune system activation, short circulation time, instability, nuclease degradation, and undesirable off-targets [31]. Nanomedicines emerged as a novel and promising therapeutic approach to increase the effectiveness of miRNA delivery and address existing limitations.

Nanotechnology-based therapy for targeting CSCs

NPs have advantages in the delivery of therapeutics into CSCs, such as improving the bioavailability of therapeutic agents, enhancing RNAi stability, increasing the efficiency of drug delivery, and providing targeted delivery into CSCs. NP surfaces can be modified with proper targeting ligands to reduce undesirable side effects, enhance the uptake of therapeutic agents, and increase the distribution of nanomaterials in the target cells. These ligands can bind to overexpressed and specific CSC antigens or receptors. For example, CD44 is overexpressed on CSCs, and hyaluronic acid (HA), as a natural polymer, has an affinity to bind CD44. Coating NPs with HA can selectively target and deliver anticancer agents specifically to CSCs [32].

Various nanomaterials, such as natural or synthetic polymers, lipids, inorganic polymers, and exosomes, as bio-inspired nanocarriers, are applied to deliver therapeutic materials such as RNAi molecules and chemical drugs into CSCs (Fig. 3) [33]. As shown in Table 1, various NPs have been investigated to deliver tsmiRs or oncomiRs into different CSCs. This review discusses the significant findings of recent advances in exosome-mediated and chitosan-mediated miRNA delivery systems for targeting CSCs in representative cancer types. Although both NPs are natural, chitosan is derived from a natural substance, whereas exosomes are extracted from natural components such as a wide range of cells.

Fig. 3
figure 3

Nanocarriers in targeting Cancer Stem Cell Therapy. Nanocarriers in different formulations deliver miRNAs and anticancer drugs or, in combination, into different cancer stem cells. Created in BioRender. Valizadeh, M. (2025) https://BioRender.com/j93u291

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Table 1 The role of oncogenic miRNAs( OncomiRs) and tumor suppressor miRNAs TsmiRs) in CSC propagation and Inhibition via different delivery systems
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Exosomes-mediated MiRNA delivery in CSC therapy

Extracellular vesicles (EVs) are vesicles derived from cells and released into the extracellular space. Based on their biogenesis, dimensions, and membrane characteristics, they are classified into three primary categories: microvesicles (MVs) (50–1000 nm), apoptotic bodies (ApoBs) (50–5000 nm), and exosomes (30–100 nm) [54]. Exosomes are characterized based on their size and the expression of surface antigens [55].

Exosomes are membrane-covered bio-vesicles that reflect their parent cells’ characteristics, enclosing a variety of cargo such as transcription factors, receptors, proteins, lipids, and various nucleic acids, including DNA, mRNA, miRNA, and ncRNAs (Fig. 4) [56, 57]. Some studies have shown that exosomes perform their roles by exchanging their cargo between different cells or tissues [58]. Exosomes are secreted by different cell types and present in various biological fluids, including blood, serum, saliva, and urine, playing a crucial role in mediating communication between different types of cells [59].

As shown in Fig. 4, the typical process of exosome biogenesis contains the following steps: First, the cytoplasmic membrane invaginates, forming early endosomes. Next, intraluminal vesicles (ILVs) formed and contained within the endosome are termed multivesicular bodies (MVBs). Finally, the MVB membrane fuses with the cell membrane, releasing the ILVs as exosomes into the extracellular space. Target cells can take up exosomes through several forms of endocytosis, including clathrin-based endocytosis, caveolin-based endocytosis, macropinocytosis, and phagocytosis [60], and their functions are influenced by exosome cargo. Exosomes are appreciated as essential mediators of cell-cell communication [61].

Exosomes have been shown to participate in physiological and pathological processes such as cancer development, migration, metastasis, formation of the TME, and chemoresistance. Therefore, they gained considerable attention in mediating intracellular communication by playing a vital role in delivering therapeutics into specific target cells. Upon reaching their target cells, exosomes can be internalized through endocytosis, directly merge with the cell membrane, or connect with surface antigens, activating intracellular signaling mechanisms. Endocytosis refers to the mechanism through which cells ingest external substances by enveloping them with their plasma membrane [57, 62].

To date, exosomes have been employed as vehicles for the delivery of various therapeutics in numerous studies due to excellent advantages, including similar structure to the cell membrane, potential targeting capabilities, efficient cell uptake rate, capacity to penetrate through physiological barriers, unique biocompatibility, low immunogenicity, high stability in blood circulation, and lower toxicity compared to nanoparticles in in-vivo models [63]. In recent years, a vast number of studies, including our studies, revealed that exosomes have been introduced as promising nanocarriers to deliver RNA therapeutics, including miRNA, into cancer cells and specifically CSCs [64–69].

Fig. 4
figure 4

Biogenesis of exosome and its contents. Exosomes derived from MVBs deliver many biomolecules such as proteins, lipids, and nucleic acids. Created in BioRender. Valizadeh M (2025) https://BioRender.com/j93u291

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In a TME, exosomes also play an essential role in the interaction between non-CSCs and CSCs and in maintaining the homeostasis of CSCs and stemness through regulating CSC-specific signaling pathways, which are crucial for regulating numerous biological processes, including self-renewal ability, drug resistance, and tumorigenesis of CSCs. Furthermore, exosomes are significant in the chemoresistance mechanisms of CSCs, which encompass various strategies, such as augmented DNA repair capabilities, enhanced anti-apoptotic responses, decelerated cell cycle progression, and drug efflux. Selectively targeting CSCs through the abovementioned pathways utilizing exosome-loaded agents, such as miRNA, is a promising strategy for eradicating CSCs [70]. In fact, many recent studies have revealed that exosomes could be regarded as attractive biological carriers for efficiently delivering miRNAs to target CSC and cancer stemness.

Exosomes come from various sources, such as mesenchymal stem cells (MSCs), immune cells, and cancer cells, each with unique biological traits. MSCs-derived exosomes are known for low immune response, high compatibility, and stability as carriers. They effectively deliver drugs by specifically transporting therapeutic agents to target sites, avoiding immune detection and degradation, and controlling the release of combined treatments at the correct location [71].

In contrast to MSCs, Adipose-derived stem cells (ADSCs) have a longer lifespan, higher proliferative capacity, shorter doubling time, and show delayed aging in vitro. Additionally, harvesting ADSCs is simpler and less invasive, resulting in higher yields. However, their clinical use is limited due to potential tumor-promoting effects [72].

Immune cell-derived exosomes can activate immune cells to fight pathogens, viral infections, and cancer. The therapeutic potential of immune cell-derived exosomes is a significant focus in nanobiomedicine research, including diagnostic platforms, treatments, and vaccines [73].

Tumor cell-derived exosomes contain many molecules from cancer cells and are recognized and taken up by immune cells, facilitating communication between tumors and the immune system. They can also suppress immune cell functions, helping tumors evade immune surveillance within the tumor microenvironment [74].

In a study by Naseri et al., MSC-derived exosomes delivered antimiR-142-3p into breast cancer stem cells (BCSCs). The results revealed that anti-miR142-3p-loaded exosomes suppressed the colony formation capabilities in MCF-7 cells-derived CSCs by inhibiting miR-142-3p. Unloaded exosomes did not affect the colony formation in these CSC populations [48].

Based on evidence, the EMT process is a complex cellular process that changes cancer cell phenotype from epithelial-like morphology into mesenchymal form. Cancer cells that undergo EMT acquire invasive properties, including migratory capacity, enhanced resistance to apoptosis, immune escape capabilities, therapy resistance, and stemness [75, 76]. Inhibition of EMT through the delivery of miRNA in exosomes has been evaluated in many studies. A study by our team showed that MSC-exosomes loaded with miR-218 inhibit EMT in the MDA-MB-231 breast cancer cell line [67]. To evaluate the effect of exosomes enriched with miRNA on cancer stemness, Rezaei et al. purified exosomes from HT-29 and SW480, two human colon cancer (CRC) cell lines, and delivered miR-375 mimic to CRC cell lines for reversing the EMT process. The EMT process upregulates the cell surface expression of CD133 and CD44, which are CSC markers related to cancer metastasis and poor prognosis. miR-375 is negatively associated with EMT and suppresses the migratory phenotypes of CRC cells. miR-375 loaded exosomes significantly downregulated the expression of Vimentin, snail, ZEB-1, and β-catenin, mesenchymal markers, and upregulated epithelial markers, E-cadherin. Furthermore, the CD44+ and CD133+ CRC-CSC population percentages significantly decreased after exosome treatment [44]. In another study, Lee et al. used MSCs-exosomes to deliver miR-124 or miR-145 mimics to CD133 + glioma stem cells (GSCs). The results showed that overexpression of miR-124 mimic decreased the expression of Sox2 mRNA and self-renewal capacity. Also, transfected GSCs with MSCs-exosomes containing miR-124 and miR-145 suppressed neurosphere formation compared to GSCs transfected with exosomes expressing control miRNA [45]. Chang et al. used M2 macrophage-derived exosomes to deliver miR-21-5p and anti-miR-21-5p to CD24+CD44+EpCAM+ pancreatic CSCs. miR-21-5p exerts an oncogenic role in various solid tumors. Factor Krüppel-like factor-3 (KLF3), a member of a subset of transcriptional Factor Krüppel-like factors (KLFs), has been a target gene of miR-21. Pancreatic CSCs treated with miR-21 loaded exosomes showed higher expression of Nanog and Oct4. Also, exosomes promote cell sphere-forming and colony-forming ability in pancreatic CSCs. In order to downregulate the expression of miR-21, M2 macrophage-secreted exosomes were treated with miR-21-5p inhibitor and transferred into pancreatic CSCs. The results showed that the expression of Nanog and Oct4 downregulated after introducing exosomes containing anti-miR-21-5p. Also, antimiR-21-5p in M2 macrophage-derived exosomes inhibited the pancreatic CSCs’ sphere formation ability [47]. Seo et al. investigated the roles of ADSC-derived exosomes containing miR-503-3p on CD44+ BCSCs and breast cancer stemness. The results revealed that Nanog was downregulated in BCSCs transfected with exosomes containing miR-503-3p. Also, exosomes containing miR-503-3p significantly inhibited sphere formation in BCSCs, which indicated the regulatory role in breast cancer stemness [46]. In a study performed by Li et al., BCSC-derived exosomes were applied to deliver miR-197 inhibitors into breast cancer cells. BCSCs used in this study were isolated from primary tumors. miR-197 has an oncogenic role in breast cancer. The results showed that exosomes containing miR-197 inhibitor significantly enhanced colony formation ability in breast cancer cells [77].

Concerning the advantages of exosomes, some limitations, such as time-consuming production, potential for toxicity, storage conditions, and the unknown nature of exosomes, limit the use of this carrier. In conclusion, although exosomes present considerable potential as promising drug delivery systems, extensive research and development efforts are imperative to overcome their existing constraints. Consequently, natural NPs are applied as a safe alternative for gene delivery in cancer.

Chitosan role in CSC research

Chitosan (CS) is a naturally occurring polymer that originates from chitin and is the second most prevalent naturally occurring polymer after cellulose. CS constitutes a linear polysaccharide formed by the random repetition of N-acetyl-D-glucosamine and D-glucosamine units, which are interconnected through β (1→4) glycosidic bonds, ultimately decomposing into harmless and easily assimilated sugars. CS has many biomedical applications, such as acting as an anticancer and antimicrobial agent, cell culture scaffold in tissue engineering, wound healing agent, and drug delivery vehicle. According to drug delivery applications, CS comes in many forms, such as nanoparticles, hydrogels, and nanofibers, to deliver various drugs into different cell types [78]. One of the most well-known areas where CS is used for drug delivery in cancer. CSNPs act as natural polymer-based NPs to deliver anticancer agents such as chemical drugs and RNAi to different cancer cells [79].

CSNPs are known to be taken up by cancer cell membranes through the endocytosis pathway, which involves two primary mechanisms: phagocytosis and pinocytosis. Moreover, the pinocytosis of CSNPs can be categorized into caveolin-dependent, cadherin-dependent, and clathrin-dependent mechanisms. Phagocytosis pertains to the internalization of particles exceeding 250 nm in diameter. At the same time, pinocytosis encompasses the cellular uptake pathways of CSNPs, which rely on factors such as size and surface modifications [80]. Due to the presence of amine groups, CS is a positively charged polysaccharide. Simultaneously, the cellular membranes with negatively charged groups enable their interaction with positively charged CSNPs and cell-membrane adsorption, resulting in the cellular uptake of CSNPs and internalization of nucleic acid payloads. Also, when CS is exposed to mildly acidic conditions, the primary amines (-NH2) within the CS structure acquire a positive charge (-NH3+), facilitating CSNPs binding to negatively charged miRNA through electrostatic interactions. This interaction, driven by the charge disparity between CS and miRNA, leads to the spontaneous formation of NPs in an aqueous environment, leading to the condensation and protection of DNA or RNAi, such as siRNA, shRNA, and miRNA, from nuclease degradation, which is called the ionic gelation method [81, 82]. CS has favorable features such as non-toxicity properties, antibacterial activity, biocompatibility, and biodegradability [83, 84]. An added advantage of CSNPs is a functional surface that can be conjugated with synthetic or natural materials and functional moieties to enhance therapeutic efficacy in targeting delivery approaches [85]. Another advantage of CSNPs is their ability to escape from the proton sponge effect, which prevents hydrolysis inside acidic lysosomal compartments inside the tumor cells. Therefore, CSNPs are promising tools to overcome miRNA limitations by enhancing low stability and bioavailability [86].

In several studies, our team investigated the effects of CSNPs containing different miRNAs, such as miR-340 and miR-155, on breast cancer cell proliferation and immune response. The results showed that CSNPs showed good stability, high transfection rate, good drug release profile, and high protection against enzyme degradation [87–89].

Fig. 5
figure 5

Applications of chitosan nanoparticles. Chitosan is an attractive natural polymer that has a wide range of applications, such as delivery systems in the field of cancer research and treatment. Created in BioRender. Valizadeh, M. (2025) https://BioRender.com/j93u291

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In addition to drug delivery in cancer research, previous studies have shown that CS has been used to culture CSCs (Fig. 5). CS-based scaffolds were also used as promising devices to mimic CSC niches to study the stemness of cancer cells. CS scaffolds can enhance stemness properties and increase cell migration, quiescent cancer cell population, chemoresistance, self-renewal capacity, and the expression levels of CSC markers, including Oct4, Nanog, CD133, CD44, and EpCAM in colon and Hepatocellular carcinoma (HCC) cells [90].

Another study by F.M. Kievit et al. reported that three-dimensional (3D) CS-alginate (CA) scaffolds enhanced the proliferation of CD133+ glioma cell population and also increased the expression of stemness and EMT markers, such as CD44 and CD133 [91]. In another study, Sims-Mourtada et al. showed that breast cancer cells cultured on 3D scaffolds of Polycaprolactone (PCL)-CS nanofibers showed increases in CD44+/CD24− BCSCs population and significantly enhanced mammosphere-forming ability compared with cells grown on the polystyrene scaffold [92]. A study by Huang et al. has revealed that non-small cell lung carcinoma (NSCLC) cells cultured on CS-HA scaffolds mimic the TME of human NSCLC tumors and enhanced CSC-like properties, promoted tumor spheroids formation, and showed higher levels of NSCLC stem cell markers [93]. Also, it has been reported that CS-hyaluronan membranes can improve the stemness of MSCs by sustaining the expression of stemness markers and enhancing sphere formation ability [94].

Numerous studies have focused on CS NPs’ role in delivering drugs and RNA therapeutics into cancer cells and CSCs. In one of the studies that investigated the effect of CSNPs on CSCs, Lazer et al. used CS-folic acid-Herceptin (CFH) NPs functionalized with Double cortin-like kinase 1 (DCLK1) antibody to target colon CSCs selectively. DCLK1 acts as a CSC biomarker and maintains cancer progression, EMT, and stemness. The results showed that these NPs can inhibit colonosphere formation and downregulate the expression of DCLK1 as a specific colon CSC [95]. In another study, Rao et al. developed CS-decorated NPs composed of Pluronic F127 polymer encapsulating doxorubicin (DOX) to target CD44+/CD133+ mammospheres. The mammosphere is an in vitro 3D tumor model for studying the BCSCs. It represents an assembly of cells containing the CSC niche so that the CSCs can be enriched and survive. The results showed that these NPs inhibit the formation of mammosphere cells in 3D model cell culture. Compared to free DOX used in this study, cellular uptake of nano-formulated DOX is higher in MCF-7 mammosphere in 3D cell culture [96].

A study by Chattopadhyay et al. explored the effect of CS NPs on delivering miRNA for gene therapy of NSCLC in 3D cell culture. They formulated biodegradable NPs composed of CS and poly (lactic-co-glycolic acid) (PLGA) to provide a caspase 8 (CASP8) inhibitor and two tsmiRs, miR-29a/b1 and miR-34a, into NSCLC cell lines, both in 2D and 3D in vitro models. The results showed that the CS-PLGA NPs effectively penetrated lung tumor spheroids and delivered miR-29a/b1 and miR-34a, which induced apoptosis in the spheroids’ core, where the expression of stemness markers was not measured [97]. Table 2 summarizes the delivery systems for miRNA using two prominent carriers: exosomes and CSNPs for CSC targeting. Despite extensive research on using CSNPs to deliver anticancer drugs to CSCs and considering the importance of miRNAs in regulating the stemness of cancer cells, the delivery of miRNAs to cancer cells by CSNPs has not been extensively studied. Since exosomes provide a biomimetic, efficient delivery route with minimal immunogenicity, CSNPs offer better versatility in formulation and the potential for large-scale production, making them attractive for miRNA-based therapeutic applications.

Table 2 Delivery system of MiRNA using exosomes and CSNPs for CSCs
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Advantages and limitations of Chitosan versus exosomes

Despite the promising potential of using exosomes as advanced drug delivery tools for cancer therapy, many hurdles have impeded their progress in clinical trials. Exosomes are considered superior candidates because of their inherent properties from structural components like proteins, nucleic acids, and lipids. They possess several beneficial features for therapeutic delivery, including lower immunogenicity, higher biocompatibility, increased circulation stability when derived from the same individual, endogenous homing ability that allows them to cross biological membranes and target specific cells, and greater efficiency in delivering contents to the cytosol [98].

Some challenges include the lack of a standardized protocol for exosome isolation, low purity of the exosomes obtained, poor characterization of exosomes, and low exosome cell production. The composition of exosomes varies depending on cell type and physiological state, which may present challenges for standardization and reproducibility. Tackling these concerns is essential for transforming exosomes from experimental research into practical and dependable therapeutic options [99].

One key focus area is improving production and scalability. Creating standardized protocols for isolating, purifying, and characterizing exosomes is essential for maintaining consistency and quality across different production batches. Extensive research and development are necessary to overcome existing challenges. Critical focus areas include adopting scalable production technologies like bioreactor systems and high-throughput isolation methods, stability, storage, in vivo performance, drug loading and release mechanisms, and regulatory and ethical issues [100]. Chitosan is well-known for its biocompatibility, biodegradability, and non-toxicity, making it attractive for drug delivery. It is soluble primarily in acidic conditions, but not very useful in neutral or basic pH environments. To address this, extensive research has been conducted to chemically modify chitosan and improve its solubility. Developing surface modifications may enhance their therapeutic effectiveness. Additionally, investigating targeting mechanisms is essential to optimize exosome delivery to CSCs, thereby improving therapeutic accuracy [101]. Considering that exosomes and chitosan have certain advantages and disadvantages as listed in Table 3, an alternative approach would be to develop hybrid delivery systems to overcome the limitations of each method. Ongoing research into hybrid exosome systems, which combine natural exosomes with nanoparticles, may overcome issues like stability and drug loading.

Song et al.‘s study used CS hydrogel incorporated with bone marrow MSC-derived exosomes (BMSC-Exo) enriched with endogenous tissue inhibitor of metalloproteinase 2 (TIMP2) to treat cholangiocarcinoma (CCA). CS can overcome the short half-life of exosomes in the systemic circulation and prolong their bioavailability. This innovative system also enhances targeted delivery to CCA cells. Further studies are needed to explore using a combination delivery system in miRNA-based CSC therapy. This will be the subject of future research [102].

Table 3 Some advantages and limitations of exosomes and Chitosan nanoparticles as a drug delivery system
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Conclusion and future perspective

CSCs are a rare population of cancer cells within the tumor mass with a strong self-renewal ability and differentiation potential into different cell types. CSCs play a key role in tumor heterogeneity, tumor relapse, metastasis, and therapy resistance. Recent advances in cancer treatments include both CSC-targeting strategies and non-CSC approaches that have emerged to target all cancer cells efficiently. However, multiple barriers need to be overcome to kill CSCs efficiently. CSCs exploit their inherent stemness characteristics to sustain their lineage against stress conditions and therapy. Most CSC-targeted drugs can only kill a small proportion of CSCs by targeting surface markers or CSC pathways. Therefore, effective strategies focusing on the specific properties of CSC, such as their metabolic pathways, should be designed to deliver anti-CSC drugs to CSCs efficiently.

Emerging evidence suggests that noncoding RNAs, such as miRNAs, have essential roles as therapeutic molecules in regulating tumor cell growth, invasion, drug resistance, metastasis, and stemness of CSCs. Compared with traditional cancer treatment strategies, miRNA-based targeting of CSCs will provide a novel and more effective therapeutic approach for targeting and killing CSCs. This will lead to the inhibition of tumor aggressiveness and decrease recurrence and therapeutic resistance.

Several problems exist with miRNA-based therapeutics, including low cellular uptake due to the size and negative charge of the miRNAs. The application of NPs possesses significant promise in cancer treatment. NPs are very attractive vehicles for encapsulating drugs and delivering them into tumor tissue through the leaky tumor vasculature. Different novel and efficient nano-scale vehicles have been developed to eliminate obstacles. Natural NP-based approaches are gaining attention as a safe delivery vehicle for miRNAs by protecting miRNAs from degradation and enhancing their therapeutic efficiency in targeting cancer cells, especially CSCs. Several studies showed that NPs can improve the bioavailability of therapeutics in CSC populations in 3D spheroid models.

Exosomes are bio-inspired nanovesicles that, owing to their natural potential to carry nucleic acids such as DNA and RNA to the targeted cells, have attracted increased interest as delivery vehicles. While many studies have revealed the role of exosome-mediated delivery of miRNAs into several cancer cell lines, side effects caused by the unknown content of exosomes remain challenging. More efforts are still needed to overcome these challenges. Exosome engineering by conjugating ligands against surface markers and key proteins associated with CSC signaling pathways helps deliver miRNAs to CSCs efficiently and precisely with fewer treatment-related adverse effects.

As another natural carrier, CSNPs have great potential to provide a cheap, sustainable, biodegradable, biocompatible, naturally available vehicle for miRNA delivery. Despite being a promising miRNA delivery system for targeting cancer cells, its role in the delivery of miRNA into CSCs has not been well elucidated. Much focus should be put on CSCs’ biology, CSNPs properties, and miRNAs targeting CSC key genes, which is highly recommended to intensify our knowledge. CS NPs and other ligands have been modified to evade the immune system, increase circulation time, and increase cellular uptake.

It is posited that through persistent investigation, we can entirely leverage the benefits of exosomes and CS NPs as innate vectors while circumventing their limitations. In light of this, we may formulate innovative approaches that utilize engineered NPs to transport tumor-suppressive proteins, nucleic acid elements, or targeted therapeutics that serve as personalized medicine. Additional inquiry is required to assess the implications of administering existing therapeutic miRNAs to CSCs to enhance the effectiveness of anti-CSC interventions.