Targeting drug resistant colorectal cancer with apigenin nanoarchitectures

Highlights

• •Apigenin-loaded nanoparticles increased the drug delivery and bioavailability in CRC.
• •Apigenin induces apoptosis in drug-resistant CRC through mitochondrial disruption.
• •Apigenin-loaded liposomes display synergy with 5-FU in drug-resistant CRC cells.
• •Nanocarriers enable sustained release and increased cellular uptake of apigenin.
• •Combining apigenin with chemo drugs sensitizes resistant CRC cells to therapy.

Abstract

Drug resistance remains a critical obstacle in the treatment of colorectal cancer (CRC), contributing to high mortality rates, particularly in advanced stages. Conventional therapies, including surgery, chemotherapy, and targeted drugs, often face limitations such as systemic toxicity, poor drug selectivity and the development of multi-drug resistance (MDR). Recent studies have explored the use of apigenin, a naturally occurring flavonoid, as a promising therapeutic agent against drug-resistant CRC. This review aims to summarize and critically evaluate current research on the use of apigenin-loaded nanoarchitectures in overcoming drug resistance mechanisms in CRC. Specifically, it examines the efficacy of apigenin when delivered via various nanocarriers, including PLGA nanoparticles, lipid-polymer hybrid nanoparticles and liposomal formulations. These nanoarchitectures enhance the bioavailability, targeted delivery and sustained release of apigenin, improving its therapeutic efficacy. Apigenin-loaded nanoparticles effectively increase cellular uptake, circumvent drug efflux pumps and induce apoptosis in drug-resistant CRC cells. They also inhibit cancer cell proliferation by arresting the cell cycle and suppressing oncogenic pathways, such as PI3K/Akt/mTOR. Furthermore, apigenin disrupts angiogenesis and inflammation, thereby weakening the tumor microenvironment. Synergistic effects with conventional chemotherapy further underscore apigenin's potential as a chemo-sensitizer and in combination therapy, offering a promising avenue for reducing drug resistance in CRC. The findings suggest that apigenin nanoarchitectures could be a powerful strategy for enhancing CRC treatment outcomes, providing a foundation for further clinical development.

Graphical abstract

Introduction

Colorectal cancer (CRC) is the third most prevalent diagnosed cancer globally in males and females and the second most prevalent cause of cancer-related death [1]. More than one million cases are newly diagnosed per year, with around 600,000 people losing their lives to the disease annually [2]. The epidemiology of CRC shows considerable variation across different regions of the world, as well as among various age, gender and racial groups [3]. By 2035, deaths due to rectal and colon cancer are expected to rise by 60 % and 71.5 %, respectively. However, estimates vary by country, contingent on their economic status, highlighting CRC as a potential marker of a nation’s socioeconomic development [4,5]. This type of cancer arises from mutations in the epithelial tissues of the colon and rectum, impacting tumor suppressor genes, oncogenes and genes responsible for DNA repair processes [6]. Additionally, it is the principal cause of death of the gastrointestinal cancers. Risk factors for developing this cancer include poor dietary habits, smoking, inflammatory bowel disease, polyps, genetic predispositions and aging [4]. Main symptoms of CRC are rectal bleeding, abdominal pain, modification in bowel habits, unexplained weight loss and anemia [7,8]. CRC diagnosis mainly depends on colonoscopy, which can be limited by resource constraints. Predictive models based on symptoms have limited accuracy. The diagnostic process is complex and involves interactions between the patient, physician and healthcare system [9].

Surgery is a common treatment approach for CRC, especially for stages 0 through II. For more advanced stages, additional treatments such as adjuvant therapy, chemotherapy and targeted therapy are typically required alongside surgery [10]. Although the 5-year survival rate of patients with early-stage CRC (stages I and II) is above 60 %, more than half are diagnosed at stage III or later, when distant metastasis has occurred, causing the 5-year survival rate to drop to 10 % [11]. The primary strategy for treating CRC involves a combination of chemotherapy and surgery, which is the only way to improve survival outcomes [12]. Since the 1950s, the foundation of CRC treatment has been chemotherapy using 5-fluorouracil (5-FU) [13]. In recent years, new chemotherapy drugs such as oxaliplatin, irinotecan and capecitabine have been developed and utilized. Standard treatment for advanced CRC typically involves combining 5-FU and leucovorin with either oxaliplatin or irinotecan [14,15]. However, the limitations of chemotherapy are significant, including low selectivity, inadequate concentrations in tumor tissues and systemic toxicity [16]. To address this challenge, the U.S. Food and Drug Administration (FDA) recently approved six targeted therapies aimed at metastatic CRC (mCRC). These include bevacizumab, aflibercept, regorafenib, cetuximab and panitumumab [[16], [17], [18], [19], [20]]. Notwithstanding the development of these novel approaches, drug resistance still remains a significant challenge during CRC treatment, with the primary reasons including aberrant anti-tumor drug metabolism, transportation or target [21].

Chemotherapy resistance hampers the effectiveness of current cancer treatments, including those for colorectal cancer [22]. There are different mechanisms of drug resistance in cancer treatment, some of which are tumor heterogeneity, tumor microenvironment (TME), cancer stem cells (CSCs), and multi-drug resistance (MDR) [[23], [24], [25], [26]]. Apigenin, a flavone commonly present in various plants, is known for its bioactive properties, including anti-inflammatory, antioxidant, anticancer effects and an ability to lower blood pressure [27]. This flavone (a subclass of bioflavonoids) is predominantly found in its glycosylated form in substantial amounts in various vegetables, fruits, herbs, tea, beer and wine [28]. Apigenin has been shown to play a role in inhibiting other key characteristics of cancer, such as cell invasion and metastasis, by modulating various signaling pathways associated with the development of different types of cancer [29]. Nanostructures and nanoarchitectures are engineered to enhance the delivery, stability and effectiveness of a therapeutic agent [30,31]. As mentioned earlier before, apigenin has remarkable anticancer attributes and nanoarchitectures that can guide a substance with precision to the tumor site and prolong its half-life [32]. The aim of this study is to explore the potential of apigenin-loaded nanoarchitectures as a targeted therapeutic strategy to overcome drug resistance in colorectal cancer.

Mechanisms of drug resistance

The treatment of cancer typically involves procedures such as surgery, radiotherapy and chemotherapy. However, the development of resistance to chemotherapy remains a recurring challenge in managing both localized and widespread disease [33]. Additionally, cancer drug resistance can be acquired through various mechanisms [34]. Currently, most of chemotherapy failures are associated with drug resistance during cancer invasion and metastasis [35,36]. Tumor heterogeneity is a major factor contributing to drug resistance [37]. This situation can result in the development of distinct cell groups with varying molecular characteristics. Tumor heterogeneity refers to the variation in tumor cell populations and genetic differences, which can occur both within and between tumor locations (spatial heterogeneity) and over time as the molecular profile of cancer cells evolves [38,39]. Another factor contributing to drug resistance is the TME. The TME is a complex and dynamic "rogue organ" that includes the blood supply, stroma, lymphatic and nervous systems, immune cells, and extracellular matrix (ECM) [40]. By fostering an immunosuppressive and nutrient-poor environment, the TME enhances tumor growth, proliferation and phenotypic diversity. Interactions between cells and between cells and the ECM in the TME result in the release of soluble factors that aid in immune evasion and ECM remodeling, which in turn contributes to resistance to therapy [41,42]. Additionally, CSCs are thought to be a key player in drug resistance and cancer recurrence, largely because of their capacity for self-renewal as well as their capability to differentiate into various types of cancer cells [43].

CSCs use various tactics to avoid treatment, including the increased production of ATP-binding cassette (ABC) transporters. For example, ABCB1, which encodes P-glycoprotein (P-gp) and ABCG2 and was first discovered in cells resistant to mitoxantrone, are known to help CSCs evade chemotherapeutic drugs [[44], [45], [46], [47]]. Another mechanism that resists cancer therapy is multi drug resistance (MDR). MDR during chemotherapy relates to the capacity of cancer cells to withstand and survive exposure to multiple anti-cancer drugs [48]. The mechanism of MDR can arise from an enhanced expulsion of drugs from the cells, resulting in decreased drug uptake within those cells [49]. Basically, the diminished therapeutic effectiveness of many drugs can be generally attributed to the inherent overexpression of ABC transporter proteins on the cell membrane. This leads to reduced drug uptake, enhanced drug detoxification and increased DNA repair [50,51]. Cell death is facilitated by three key processes: necrosis, apoptosis and autophagy [52]. Apoptosis can be initiated via both internal and external pathways. In the intrinsic pathway, which occurs within the mitochondria, anti-apoptotic proteins such as Bcl-2 and AKT counteract cell death, whereas pro-apoptotic proteins such as Bax, Bak, and caspase-9 promote it [53,54]. In the extrinsic pathway, cell death is facilitated by ligands and receptors such as FAS and TNF-R, along with adaptor proteins and caspases-3, -6, -7, and -8. This process leads to the breakdown of actin and nuclear lamin proteins, ultimately causing apoptosis [55,56]. Increased chemotherapy resistance in tumor cells is often due to elevated expression of anti-apoptotic genes, like as Bcl-2 and AKT, combined with a reduction in levels of pro-apoptotic genes like Bax and Bcl-xl [57,58]. Additionally, drug resistance can arise from p53 gene mutations [59], which normally induces apoptosis as a result of cellular stress and DNA damage [60]. Moreover, there are other mechanisms of drug resistance which have their own unique approach to combat therapy. These strategies are inactivation of the anticancer drugs, reducing the absorption of the drugs, changing the drug metabolism and changing the chemotherapeutic agent’s targets [[61], [62], [63]] (Fig. 1).

Fig. 1.

Key mechanisms of drug resistance in CRC. The figure highlights: (1) Tumor Heterogeneity - variation in tumor cell characteristics; (2) TME - supports immune evasion and ECM remodeling; (3) CSCs - contribute to resistance via drug transporter overexpression; (4) Apoptosis Evasion - upregulation of anti-apoptotic genes and p53 mutations; and (5) Epigenetic Regulation - frequent changes in gene expression during cancer progression. These factors collectively drive chemoresistance in CRC. Created with BioRender.com.

Nanotechnology in cancer treatment

As defined by the FDA, a drug is a substance that is "intended to diagnose, cure, mitigate, treat or prevent disease" as well as a substance that affects any aspect of the human or animal body other than food. The drugs should be able to target the disease-causing cell with an exact therapeutic concentration. Conversely, the rate and stability of release, as well as the ability to target cells and tissues, are not controlled [64]. Drug delivery systems are designed to overcome these challenges. An effective drug delivery system regulates the rate of drug release and improves its effectiveness [65]. As early as the 1970s, nanoparticles (NPs) were being developed as potential drug delivery systems [66]. The term "nanomaterials" refers to materials containing very small components and/or structural features with dimensions ranging from 1 nm to 100 nm. Due to a nanoscale materials' reduced size, their surface area and number of surface molecules increase exponentially, resulting in complex biophysical chemical interactions at the bio-nano interfaces [67]. A nanotechnology approach involves concerted research in the areas of chemistry, engineering and biology, with its application in medicine aimed at detecting cancer accurately and early and with personalized treatment [68]. Interest in using nanoparticles in cancer therapy and diagnosis is on the increase, with many prospects at the clinical trial stage and many gaining U.S. patents [69,70]. There have been numerous studies evaluating the effectiveness of nanotechnology as a cancer treatment method including drug delivery [[71], [72], [73], [74], [75]], vaccine [76,77], theranostic [78,79], thermodynamics approach [80,81], gene therapy [[82], [83], [84]] and immunotherapy [85,86]. Progress on NP-based drugs for cancer treatment is gradually moving forward as a result of this translational progress. There are now a few FDA-approved drugs including Doxil®, Abraxane®, Marqibo®, Onivyde®, and Eligard® [70] (Fig. 2).

Fig. 2.

Specification of FDA Approved Nanopharmaceuticals. PEG: Ethylene Glycol; PLGA: poly (lactic-co-glycolic acid; PLA: Poly-lactic acid. Created with BioRender.com.

Using nanocarriers for drug delivery to colorectal cancer

In traditional intravenous chemotherapy for CRC, drug was distributed both to the desired (cancer site) and undesired (normal cells) sites. Site-specific release of chemotherapeutics in the cancerous region of the colon can subdue these adverse effects [87]. Efforts are being made by drug delivery scientists to enhance drug permeability through colonic epithelium, so that chemotherapeutics can be delivered locally and systemically. This hypothesis has been used to target chemotherapeutic agents to colon via oral administration in a number of formulation and preclinical studies. There are four basic approaches to colon specific drug delivery via oral routes: (1) Depending on passage time, drug delivery can be controlled; (2) Delivery of drugs based on pH; (3) Drug delivery using enzymes; and (4) Systems based on pressure. Additionally, prodrugs based systems, microbial triggered approaches, commensal bacteria, and hydrogels are used for colon specific delivery [87,88]. Each formulation approach has its own merits and limitations. In order to overcome the limitations associated with each formulation approach and to maximize colon-specific drug delivery, a combination of two or more formulation approaches has been used. Drug delivery researchers have gained interest in carbohydrate-drug design, especially in regards to colon-specific drug delivery [89]. Natural polymers play a vital role in colon-targeted delivery since anaerobic bacteria in the colon are able to recognize and degrade various substrates [88]. As a first-line and second-line treatment for metastatic CRC, oxaliplatin with leucovorin (FOLFOX) plus 5-FU is an FDA-approved chemotherapeutic agent [90]. Nevertheless, since these treatments have a short half-life and are prone to resistance, they must be given in large doses along with combinational therapy, which is highly toxic to healthy colon cells and causes gastrointestinal and cardiac complications [91,92]. Generally, through enhanced permeability and retention (EPR), nanocarriers can passively reach tumor sites [93]. The screening and identification of particular overexpressed cancer cell receptors in the TME provides an effective method for selective drug delivery to cancer cells in the TME. It is called targeted drug delivery, and specific moieties such as various aptamers are conjugated on the surface of DDSs to improve their identification of cancer cell receptors. Clinical outcomes can be improved by maximizing anticancer drug availability at tumor sites through targeted drug delivery [94,95]. For example, in rectal cancer tissue, epithelial cell adhesion molecules such as EpCAM and CD326 are abundantly overexpressed in comparison with adjacent normal tissues [96], therefore, it is an ideal biomarker for active targeting of CRC. It has been reported that Xie et al. increased DOX's therapeutic efficacy against SW620 colon cancer cells by modifying mesoporous silica NPs (MSNs) with a DNA EpCAM aptamer and show significantly increased toxicity of DOX when compared to non-targeted MSNs [97]. The use of MSNs covalently bonded with poly (oligo (ethylene glycol) monomethyl ether methacrylate) and targeting peptides (RDG) is another example of a hybrid nanocarrier. Using this system, 5-FU can be delivered in a targeted and effective manner. Compared to free drug, hybrid nanocarriers have better efficacy against CRC, thus showing the potential of hybrid nanosystems as cancer therapies [98]. By improving the circulation time and pharmacokinetic properties of the drug, nanocarrier-based delivery could overcome these side effects [99]. More studies of nanocarriers in drug delivery to colorectal cancer are presented in Table 1, which shows the efficiency of these systems.

Table 1.

Summary of preclinical studies of nanoparticles for drug delivery to colorectal cancer.

Nanoparticle	Drug	Models	Outcome	Ref.
Liposomes	IRI hydrochloride and curcumin	SW620 cells / rat	•Both drugs were co-delivered to tumor cells in highly synergistic ratios, and they were steadily released. •Enhancing SW620 cell proliferation inhibition. •A tumor inhibition rate of 94.43 % indicates strong effect.	[100]
Liposomes	IRI and DOX	HCT 116, SK-HEP-1, A549 / mice	•Delayed and extended release after Liposomes-IRI administration. •Inhibition rates: 98.35 %, 95.77 %, 87.40 %. •No colon inflammation at 5 mg/kg dose.	[101]
Liposomes	Oxaliplatin and IRI hydrochloride	CT-26, HCT-116 / BALB/c mice	•Higher sensitivity to co-loaded liposomes. •Superior in vivo anti-tumor activity. •Less toxicity vs solution formulations.	[102]
Polymeric micelles	IRI	BALB/c mice	•Tumor inhibition: 89 % (vs 68.7 % in free drug). •No weight loss or morbidity. •No alteration in kidney function.	[103]
FA–grafted SLN	IRI	COLO-205 cells / BALB/c mice	•Higher cytotoxicity than solution and uncoupled SLNs. •Drug released only in intestinal region (pH > 7.0).	[104]
Chitosan-coated SLN	5-FU, cinnamon, oregano	HCT116 cell	•Cinnamon and oregano inhibited HCT 116. •5-FU + extracts induced highest inhibition, apoptosis, caspase-3 activation.	[105]
HA-SiNP	5-FU	COLO-205 cells	•Improved efficacy and reduced side effects. •HA contributed to release modulation.	[106]
PEGylated MWCNT	Oxaliplatin	HT-29 cell	•43.6 % loading efficiency. •Sustained release for 6 hours (34 % released).	[107]

DOX: doxorubicin; IRI: Irinotecan; 5-FU: 5-Flourouracil; HA: hyaluronic acid; SiNP: silica nanoparticles; FA: Folic acid; SLN: Solid lipid nanoparticles; MWCNT: multi-walled carbon nanotubes; PEG: polyethylene glycol

Table 1is provided as a detached high-resolution file. Citation: [[100], [101], [102], [103], [104], [105], [106], [107]].

Nanocarriers in overcoming colorectal cancer drug resistance

Chemotherapy resistance has increased over the past century, resulting in cancer recurrences and thus decreasing the success rate and completion of cancer treatment. Ninety percent of chemotherapy failures are the result of drug resistance [108]. It is common knowledge that MDR relates is a result of cancer cells capacity to develop simultaneous resistance towards several structurally unrelated chemotherapeutic agents that have unrelated mechanisms of action and endure the treatment course [109]. There are multiple factors that contribute to this phenomenon, including tumor burden, the microenvironment, heterogeneity, growth dynamics and physical impediments [110]. In stage IV CRC, current therapies only marginally improve survival, largely due to drug resistance [111]. During treatment, colon cancerous cells develop inherent resistance to the majority of anticancer medications P-glycoprotein (P-gp) is the primary cause of drug resistance in colorectal cancer cells [110]. Colorectal cancer cells often express it more than normal colorectal cells. multi-drug resistant gene 1 (MDR1) expresses p-gp protein [112]. The P-gp must be disabled and blocked for efficient medication delivery to colorectal cancerous cells [113]. An MDR1 inhibitor and tumor uptake enhancer were fabricated with camptothecin (CPT)-loaded poly (lactic-co-glycolic acid) NPs with Pluronic F127 and chitosan surfaces. CPT and other NPs had much higher IC₅₀ values than NPs-P/C1 in in vitro cytotoxicity tests at 24 and 48 hours. The NPs-P/C1 formulation showed the highest level of cellular uptake (85.5 %). NP surfaces coated with Pluronic F127 and chitosan significantly enhanced CPT's therapeutic efficacy, induced tumor cell apoptosis, and reduced systemic toxicity [114]. The poly (lactic-glycolic acid) nanoparticles with α-tocopherol polyethylene glycol 2000 succinate (TPGS2k) were modified to delivery DOX and simvastatin (SV). SV-based nano-delivery system with cholesterol depletion in lipid rafts reduces P-gp expression directly, potentially reversing drug resistance [115]. Researchers evaluated poly (butyl cyanoacrylate) nanoparticles (PBCA-NPs), double-coated with Tween 80 and polyethylene glycol 20,000, to deliver DOX and overcome MDR caused by P-gp and breast cancer resistance protein (BCRP) in cancer cells. In the cell uptake study, double-coated PBCA-NPs significantly enhanced DOX accumulation. Compared with free doxorubicin, single-, and un-coated PBCA-NPs, double-coated PBCA-NPs significantly enhanced DOX sensitivity in P-gp overexpressing cells [116].

Furthermore, increased expression of ABCG2, a drug efflux pump, is a common cause of resistance to irinotecan and SN38 [117]. The in vitro study of ABCG2 has shown that high expression of it results in decreased potency of drugs [118]. A study was conducted to characterize SN38-loaded polyethylene glycol (PEG)-PLGA [poly (lactic-co-glycolic acid)]-verapamil nanoparticles, to identify this NPs' ability to inhibit drug resistance through inhibition of ABCG2 expression. BAX expression increased while ABCG2 expression decreased [119].

Apigenin: A therapeutic agent for cancer treatment

Apigenin, a naturally occurring flavonoid found in plants such as chamomile, parsley, and celery, has garnered considerable attention in oncology due to its multifaceted anti-cancer properties. Its potential as a therapeutic agent stems from its ability to target a wide range of molecular pathways involved in cancer progression, metastasis, and resistance to treatment [120]. One of the hallmark mechanisms through which apigenin exerts its anti-cancer effects is the induction of apoptosis in malignant cells. This is achieved by modulating key signaling cascades, including the PI3K/AKT and MAPK pathways, which are often dysregulated in cancer. By restoring the balance in these pathways, apigenin promotes programmed cell death while sparing normal cells, a property that sets it apart from many conventional chemotherapeutic agents. Furthermore, its capacity to arrest the cell cycle at various stages adds another layer of efficacy, halting the proliferation of cancer cells across multiple types, including breast, prostate, and colon cancers [121,122]. In addition to its pro-apoptotic properties, apigenin demonstrates significant anti-metastatic potential. Metastasis, a leading cause of cancer-related mortality, involves processes such as epithelial-mesenchymal transition (EMT), angiogenesis, and ECM remodeling [123]. Apigenin inhibits these processes by downregulating key mediators, including matrix metalloproteinases (MMPs), and modulating critical signaling pathways like those involving VEGF and integrins [124]. Another compelling aspect of apigenin's therapeutic profile is its immunomodulatory effects. It has been shown to enhance the body's immune response to tumors by regulating the production of inflammatory cytokines and supporting the activity of immune cells like natural killer cells and cytotoxic T lymphocytes. This property makes apigenin a promising candidate for integration into immunotherapeutic regimens, potentially boosting the effectiveness of immune checkpoint inhibitors and other immunomodulatory treatments [125]. Moreover, apigenin's compatibility with conventional chemotherapeutics and its ability to counteract drug resistance add to its appeal. Many cancers develop resistance to standard therapies, a challenge that apigenin addresses by sensitizing cancer cells to drugs and mitigating resistance mechanisms. For instance, studies have shown that apigenin enhances the cytotoxic effects of certain chemotherapeutic agents while simultaneously reducing their toxic side effects on normal tissues. This dual benefit underscores its potential role as an adjuvant therapy in oncology [126]. Despite these promising findings, challenges remain in the clinical translation of apigenin. Its limited bioavailability due to poor solubility and rapid metabolism has prompted research into novel delivery systems, including nanoparticles and liposomes, which aim to enhance its stability and therapeutic efficacy [127,128].

Unique structural and physical properties of apigenin in colorectal cancer therapy

Apigenin, a flavonoid classified as 4′,5,7-trihydroxyflavone, exhibits a planar structure with hydroxyl groups at positions critical for its bioactivity. These hydroxyl groups facilitate interactions with proteins and cell membranes, enhancing its therapeutic properties. Apigenin's lipophilic nature allows for integration into lipid membranes, increasing its cellular uptake. However, its poor water solubility and rapid metabolism pose challenges for systemic administration. To address these limitations, recent advancements have focused on utilizing nanocarriers to improve its bioavailability and delivery to CRC tissues [121,129]. Compared to conventional chemotherapeutics like 5-FU or irinotecan, apigenin distinguishes itself by modulating multiple oncogenic pathways simultaneously, such as PI3K/Akt/mTOR and Wnt/β-catenin. This multi-target approach not only enhances efficacy but also reduces the likelihood of resistance development. For example, apigenin inhibits VEGF expression, curtailing angiogenesis and depriving tumors of essential nutrients—a mechanism less pronounced in traditional chemotherapies [121,122]. Furthermore, apigenin’s antioxidant properties and its role in downregulating inflammatory mediators like NF-κB contribute to a reduction in the tumor-promoting microenvironment, a feature underexplored in standard therapies [129].

Nanocarriers for delivery of apigenin in colorectal cancer

A PLGA nanoparticle loaded with apigenin was developed by Dutta et al. The method of multiple emulsion solvent evaporation was used to formulate PLGA nanoparticles. Nanoparticles were conjugated with aptamers to target the epithelial cell adhesion molecule (EpCAM) on the surface of CRC cells. These systems have a size of 226 nm. ANPs, aptamer-apigenin nanoformulation, showed the highest cytotoxicity in HCT-116 cells at a concentration of 15.4 µM after incubation for 48 hours. In comparison to free-drug, the IC₅₀ value of apt-ANP on HCT-116 cells indicates its potential cytotoxicity. Apt-ANP enhanced therapeutic efficacy on CRC cells, while greatly reducing off-target cytotoxicity on normal cells. There is greater colonic accumulation and less hepatic clearance of apt-ANP compared with other ANPs [130]. Additionally, Hyaluronic acid (HA)-coated PLGA nanoparticles loaded with apigenin (API) were prepared to target CD44-expressing colon cancer cells. In the system, chitosan acts as an intermediary, binding HA and PLGA at the same time via electrostatic interactions. A specific binding of HA to CD44 is observed on colon cancer cells. In terms of sustained release ability, HA-PLGA-API-NPs were superior. The mean particle size of HA-PLGA-API-NPs was 254 ± 6.23 nm. At 48 hours, 68 % of the APIs from HA-PLGA-API-NPs had been released. When HT-29 cells with high CD44 expression were treated with HA-PLGA-API-NPs, cellular uptake was increased. When HA-PLGA-NPs concentrations were less than 0.1 mg/L, HT-29 cell viability remained above 80 %, but decreased to around 65 % at 5 mg/L when HA-PLGA-NP concentrations were increased. A comparison between PLGA-API-NPs and HA-PLGA-API-NPs showed that HA-PLGA-API-NPs target HT-29 mice with enhanced specificity [131]. Both studies demonstrate that polymer nanocarriers can be highly effective in active tumor targeting while reducing side effects.

Alfaleh et al. developed lipid polymer hybrid nanoparticles (LPHyNPs) to deliver apigenin. Nanoprecipitation methods with slight modifications were used to prepare LPHyNPs. This nanocarrier had a size of 234.80 ± 12.28 nm. It is possible to entrap drugs inside NPs' cores. Studies have shown that LPHyNPs released AGN more sustainably than AGN suspensions. AGN was released from LPHyNPs over 72 hours. It was found that AGN suspension exhibited an IC₅₀ of50.93 ± 2.86 µg/mL, whereas LPHyNPs exhibited an IC₅₀ of 124.77 ± 7.01 µg/mL. Thus, as discussed above, LPNHyNPs were five times more potent than AGN suspension. Flow cytometry was used to evaluate its effectiveness against CRC in terms of apoptosis and cell cycle arrest, which demonstrated impressive results. Moreover, compared to blank LPHyNPs and AGN suspension, JNK and MDR-1 gene expression levels were significantly reduced [132]. In a study by Sen et al, apigenin and 5-FU drug-loaded liposomal formulations were synthesized and produced through a modified thin film hydration process. Apigenin encapsulation efficiency is about 89.98 % and 5-FU encapsulation efficiency is about 38.6 %. DSPC LUVs loaded with apigenin and 5-FU were 95.97 ± 0.14 nm and 105.2 ± 0.42 nm respectively. Dual-drug liposomes were significantly more toxic against two human colon cancer cell lines than individual and combinatorial free drugs. It was found that apigenin was moderately toxic to HT-29 cells (IC₅₀ = 45.96 μM), whereas HCT-15 showed increased sensitivity (IC₅₀ = 43.28 μM). Colorectal cells treated with apigenin-loaded vesicles exhibited increased cytotoxicity (IC₅₀ = 26.71 μM and 28.22 μM) and thus, potentiated apigenin's effects. As a result of the dual-drug liposomes, angiogenesis was inhibited more effectively, cell proliferation was reduced, and apoptosis levels were increased. According to the study, the liposomal drug-loaded nano-carrier and apigenin's synergistic effect on 5-FU treatment resulted in an enhanced passive targeting in vivo [133]. Hong et al. encapsulated apigenin with whey protein isolate (WPI) using a pH-cycle method. Nanocapsules exhibited good storage stability and hydrodynamic diameters of 180–240 nm. It was observed that nanodispersions remained stable during storage, however, after encapsulation, apigenin became amorphous. Compared to untreated cells, 20 μM/L of free and nanoencapsulated apigenin increased apoptosis by 2.25 and 3.29 folds, respectively. A comparison of apigenin nanoencapsulated versus unencapsulated in vivo, demonstrated that the nanoencapsulated apigenin was more effective at inducing apoptosis, as well as having a greater bioavailability in colon mucosa and blood. As a result, the present study may contribute to the development of functional beverages with lipophilic phytochemicals that can prevent disease [134].

Mechanism of action of apigenin-loaded nanoparticles on drug resistant colorectal cancer

Chemoresistance presents a major barrier to effective CRC therapy and is a primary contributor to treatment failure and disease recurrence, particularly in metastatic cases, where over 90 % of patients ultimately experience resistance to chemotherapy [135]. Resistance to 5-fluorouracil (5-FU), a cornerstone chemotherapeutic agent in CRC. For patients receiving 5-FU-based treatments, 3- and 5-year survival rates are around 72.2 % and 60 %, respectively. However, since most cases are diagnosed at advanced stages, the overall 5-year survival drops sharply to 18.3 % [136]. Similarly, acquired resistance to epidermal growth factor receptor (EGFR)-targeted agents such as cetuximab and panitumumab often emerges within 3 to 12 months of treatment initiation. Moreover, re-challenging patients with the same or similar chemotherapy regimens typically results in diminished therapeutic responses due to established chemoresistance. This phenomenon is driven by various factors, including tumor heterogeneity and the influence of the tumor microenvironment, both of which play critical roles in the persistence and evolution of resistant cancer cell populations [137].

Apigenin has shown potential in combating drug-resistant CRC [138]. As previously mentioned, nanostructures and nanoarchitectures have great benefits in the efficacy of therapy (Fig. 3). These benefits are improved bioavailability, targeted delivery and sustained release [139,140]. There are several mechanisms by which apigenin-loaded nanoparticles can overcome drug resistance in CRC treatment. One of these mechanisms is enhanced cellular uptake. In this mechanism, encapsulating apigenin in nanoparticles enhances solubility and stability, leading to improved absorption and retention within cancer cells [141]. Nanoparticles can also be designed for specific targeting of receptors that are overexpressed on cancer cells, facilitating a higher concentration of apigenin within the TME. This feature improves targeted delivery in CRC treatment [142]. Moreover in drug-resistant CRC cells, efflux pumps such as P-gp frequently expel therapeutic drugs, lowering their concentration within the cells [143]. Nanoparticles can circumvent these pumps, allowing apigenin to stay inside the cells long enough to have its intended effect [144]. Apigenin also has the ability to induct apoptosis by two mechanisms. One of which is mitochondrial pathway activation where apigenin can induce apoptosis through disruption of the mitochondrial membrane potential, resulting in cytochrome c release and the subsequent activation of the caspase cascade [145]. The other mechanism is the modulation of Bcl-2 family proteins. This process observes apigenin reducing the levels of anti-apoptotic proteins such as Bcl-2 while increasing pro-apoptotic proteins like Bax. Thus, shifting the balance toward cell death in drug-resistant CRC cells [146,147]. Apigenin also can inhibit proliferation of CRC cells by two unique approaches. The first is cell cycle arrest, where, apigenin-loaded nanoparticles can cause cell cycle arrest during the G2/M phase, inhibiting CRC cell growth. This effect occurs by blocking cyclin-dependent kinases (CDKs) and decreasing the levels of cyclins that are essential for cell cycle progression [148,149]. The second approach is suppression of oncogenic pathways and in here apigenin disrupts critical signaling pathways that drive cancer cell proliferation, including the PI3K/Akt/mTOR pathway. This results in the suppression of downstream targets that are essential for cell growth and survival [150]. Apigenin can also inhibit angiogenesis, which has a great impact on CRC treatment. Apigenin has the ability to suppress VEGF expression, a vital component in the angiogenesis process that provides blood to tumors. By curbing angiogenesis, apigenin effectively deprives the tumor of necessary nutrients and oxygen, thereby restricting its growth and potential to metastasize [151,152]. Furthermore apigenin-loaded nanoparticles have significant influence on reduction of inflammation. Nanoparticles loaded with apigenin can suppress the NF-κB signaling pathway, which is frequently overactive in drug-resistant CRC and plays a role in inflammation and cancer progression. By curbing inflammation, apigenin may also weaken the tumor-promoting microenvironment [153,154]. Additionally, apigenin has synergistic effects with chemotherapy which are beneficial in the treatment of drug resistant CRC. There are two ways in which these nanoparticles can perform. The first being chemo-sensitization, where apigenin can make drug-resistant CRC cells more responsive to conventional chemotherapy drugs. For example apigenin can re-sensitize CRC cells to 5-FU by inhibiting the nucleotide synthetic enzyme thymidylate synthase (TS), while the combined apoptotic effects of 5-FU and apigenin are regulated through the activity of functional P53 [121,155]. The second mechanism is via combination therapy. When combined with chemotherapeutic agents, apigenin-loaded nanoparticles can boost the cytotoxic impact of these drugs, which may enable the use of lower doses and lead to fewer side effects. For example apigenin can synergistically increase the anti-cancer activity of cisplatin when treating lung cancer cell lines [156].

Fig. 3.

Molecular mechanisms of apigenin-loaded nanoparticles in the treatment of chemoresistant CRC. The diagram showcases various delivery platforms, including PLGA nanoparticles, Hyaluronic Acid-Coated PLGA nanoparticles, Lipid Polymer Hybrid nanoparticles and Dual-Drug Liposomes, highlighting the mechanisms by which apigenin nanoparticles enhance cellular uptake, bypass efflux pumps, induce apoptosis, inhibit angiogenesis and suppress oncogenic pathways. Created with BioRender.com.

Conclusion

The use of apigenin nanoarchitectures as a targeted therapy offers a promising new direction in combating drug-resistant CRC. The ongoing challenge of drug resistance in CRC, driven by factors like tumor heterogeneity, an immunosuppressive TME and CSCs, highlights the urgent need for innovative treatment strategies. Although traditional chemotherapy has some success, it is often hampered by issues such as systemic toxicity, low selectivity and the development of MDR. Consequently, there is a pressing need for novel approaches that can improve drug delivery, counteract resistance mechanisms and enhance patient outcomes in CRC treatment. Nanotechnology, particularly through the application of apigenin-loaded nanoparticles, provides a versatile solution to these challenges. The studies discussed in this review illustrate the potential of polymeric, lipid-based and protein-coated nanocarriers to significantly improve the bioavailability, stability and targeted delivery of apigenin to CRC cells. These advanced delivery systems allow for a more effective concentration of apigenin within the TME, bypassing efflux pumps and boosting therapeutic impact on drug-resistant cancer cells. The ability of these nanocarriers to selectively target CRC cells while minimizing unintended side effects is particularly noteworthy, as it represents a key advancement in reducing the systemic toxicity associated with conventional chemotherapy. Furthermore, the range of anti-cancer effects exerted by apigenin-loaded nanoparticles including the induction of apoptosis through mitochondrial disruption and modulation of Bcl-2 family proteins, as well as the inhibition of crucial oncogenic pathways and angiogenesis, demonstrates their broad therapeutic potential. The observed synergistic effects when apigenin is combined with standard chemotherapy agents further emphasize its role as an effective chemosensitizer, potentially enhancing the efficacy of existing treatments and allowing for lower dosages with fewer side effects.

Challenges and future directions

Apigenin-loaded nanoparticles offer a promising strategy for combating drug-resistant colorectal cancer, overcoming limitations of conventional therapies. Their advantages—enhanced bioavailability, precise tumor targeting, and multi-modal anti-cancer effects—address critical challenges in CRC treatment. By bypassing drug resistance mechanisms, inducing apoptosis, and suppressing oncogenic pathways, apigenin nanoparticles enhance therapeutic outcomes while minimizing systemic toxicity. Moving forward, the focus must shift to clinical translation, optimizing formulations, and evaluating their efficacy in combination therapies. This innovative approach heralds a significant advance in the fight against drug-resistant cancers, with the potential to improve patient survival and quality of life.

The promising preclinical outcomes of apigenin-loaded nanoarchitectures in targeting drug-resistant CRC underscore the need for further exploration, particularly in clinical settings. As we move forward, several key areas require attention to translate these findings into viable treatment options for patients. The next logical step is to initiate clinical trials to evaluate the safety, tolerability and efficacy of apigenin-loaded nanoparticles in humans. Initial studies should focus on determining the maximum tolerated dose (MTD), pharmacokinetics and pharmacodynamics of these nanoformulations. These early-phase trials will help establish a safety profile and identify any potential adverse effects, which is crucial before moving on to larger, more definitive studies. Subsequent clinical trials should compare the effectiveness of apigenin-loaded nanoparticles with standard therapies, including conventional chemotherapy and targeted treatments. These studies should assess not only the therapeutic outcomes but also the impact on drug resistance, progression-free survival and overall survival rates in patients with drug-resistant CRC. Comparative studies could also explore the potential of these nanoformulations to reduce the required doses of chemotherapeutic agents, thereby minimizing toxicity while maintaining or enhancing efficacy.

Data availability

No datasets were generated or analysed during the current study.

CRediT authorship contribution statement

Pouya Goleij: Writing – review & editing, Writing – original draft, Investigation, Conceptualization. Saeid Ferdousmakan: Writing – review & editing, Visualization. Mohammad Amin Khazeei Tabari: Writing – review & editing, Writing – original draft, Data curation. Alireza Amini: Writing – review & editing, Writing – original draft. Danaé S Larsen: Writing – review & editing, Visualization. Maria Daglia: Writing – review & editing, Supervision, Methodology. Alireza Javan: Visualization. Tian Li: Writing – review & editing, Supervision. Haroon Khan: Writing – review & editing, Supervision. Yifei Xu: Writing – review & editing, Supervision, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.