A dual catalytic functionalized hollow mesoporous silica-based nanocarrier coated with bacteria-derived exopolysaccharides for targeted delivery of irinotecan to colorectal cancer cells

Abstract

In this study, we introduced a multifunctional hollow mesoporous silica-based nanocarrier (HMSN) for the targeted delivery of irinotecan (IRT) to colorectal cancer cells. Due to their large reservoirs, hollow mesoporous silica nanoparticles are suitable platforms for loading significant amounts of drugs for sustained drug release. To respond to pH and redox, HMSNs were functionalized with cerium and iron oxides. Additionally, they were coated with bacterial-derived exopolysaccharide (EPS) as a biocompatible polymer. In vitro analyses revealed that cytotoxicity induced in cancer cells through oxidative stress, mediated by mature nanocarriers (EPS.IRT.Ce/Fe.HMSN), was surprisingly greater than that caused by free drugs. Cerium and iron ions, in synergy with the drug, were found to generate reactive oxygen species when targeting the acidic pH within lysosomes and the tumor microenvironment. This, in turn, triggered cascade reactions, leading to cell death. In vivo experiments revealed that the proposed nanocarriers had no noticeable effect on healthy tissues. These findings indicate the selective delivery of the drug to cancerous tissue and the induction of antioxidant effects due to the dual catalytic properties of cerium in normal cells. Accordingly, this hybrid drug delivery system provides a more effective treatment for colorectal cancer with the potential for cost-effective scaling up.

Introduction

Colorectal cancer (CRC) ranks as the third most prevalent and second most lethal cancer globally. The current treatment modalities for colon cancer involve surgical resection, radiotherapy, and chemotherapy. Despite their extensive use, these therapies often yield poor clinical outcomes, leading to frequent recurrence and metastasis.

Irinotecan (IRT) is an approved first-line chemotherapy drug for metastatic colorectal cancer (mCRC) [1,2]. IRT is an alkaloid derived from camptothecin (CPT) that undergoes conversion to its bioactive metabolite, 7-ethyl-10-hydroxy-camptothecin (SN-38), within cells through the action of carboxylase and butyrylcholinesterase. Notably, SN-38 exhibits a potency 100 to 1000 times greater than its parent compound. Inside malignant cells, both compounds stabilize the separable complex between topoisomerase I and DNA. This leads to DNA breaks, prevents replication, and triggers apoptotic cell death [3,4]. Despite its significant antitumor activity and clinically improved survival rates, IRT also has notable disadvantages, including dose-dependent side effects such as diarrhea and myelosuppression, a relatively short half-life, a pH-dependent chemical structure, and the need for frequent infusions, along with limited doses and strict administration regimens [[5], [6], [7]].

Due to these drawbacks, and considering the threat posed by cancer, which emerges from intelligent eukaryotic life, it becomes crucial to develop smart and comprehensive strategies for the effective treatment of this disease. It is worth noting that the effectiveness of a medicinal compound is not solely determined by its inherent strength. Rather, several factors come into play in the drug’s effectiveness under physiological conditions, including the formulation method and the delivery mechanism within the body [8,9]. One of the most successful strategies in this field involves the use of drug delivery systems (DDSs) that aim to enhance targeted delivery methods and ensure the controlled release of therapeutic agents at the right time and place [10,11].

One of the most common inorganic materials used in biomedical applications is silica-based nanoparticles (SNPs). These nanoparticles have been generally recognized by the Food and Drug Administration (FDA) as safe materials (Generally Recognized as Safe – GRAS) for human consumption. Moreover, the FDA has also approved SNPs, known as Cornell dots (C dots), for targeted molecular imaging in phase I human clinical trials [12,13]. In addition to its privileged biocompatibility and tunable physicochemical properties, SNP is suitable for use as a nanocarrier for the targeted delivery of therapeutic agents due to its extended circulation life, proper bioavailability, and favorable biodistribution [12]. Among the various structural shapes and arrangements of SNPs, mesoporous silica nanoparticles (MSNs) and hollow mesoporous silica nanoparticles (HMSNs) have been evaluated and researched. Due to their unique physicochemical properties, researchers investigate the applications of HMSNs in various fields such as catalysis, absorption, substance separation, and targeted drug delivery. As drug nanocarriers, both MSNs and HMSNs have several significant advantages, including well-established morphology and structure, as well as nano-scale dimensions [13,14]. Moreover, these nanostructures provide a high specific surface area, large pores with adjustable sizes, and, in the case of HMSNs, large cavities and reservoirs. They also exhibit a diversity of frameworks and structures, are easily accessible and producible, and provide the possibility of scaling up [15,16].

To date, a variety of functional silica-based drug delivery systems for treating several types of tumors have been developed and documented (Table SI 1). However, the majority of reported studies have utilized model and generic anticancer drugs. Further research is needed on the targeted delivery of newer and more specific antineoplastic drugs. Meanwhile, challenges such as limitations in the amount of drug loaded, sustained release, biocompatibility, and biostability affect the success rate of the proposed nanocarriers. Ongoing efforts are aimed at improving these favorable characteristics.

In relation to biomedical applications, especially in combination therapies, cerium (IV) oxide, or ceria, is gaining increasing significance and value [17]. Its attractiveness in drug delivery is primarily due to its ability to convert between two oxidative states, namely Ce3+ and Ce4+. Therefore, nanoceria crystals can exhibit antioxidant or pro-oxidant properties depending on environmental conditions. In other words, it has been demonstrated that the intracellular redox state of treated cells with nanoceria affects its antioxidant activity, promoting cell viability, or its pro-oxidase activity, leading to apoptosis following reactive oxygen species (ROS) production [18]. Another notable point about nanoceria is its dual catalytic property, which can be stimulated by specific wavelengths of UV or IR radiation, known as photocatalytic activity [19,20].

In biomedical applications, particularly in drug delivery systems, iron oxide nanoparticles (IONPs) are extensively utilized due to their biocompatibility, abundance, affordability, chemical stability, and unique magnetic properties [21,22]. Moreover, IONP-based strategies have recently been actively and intelligently considered in cancer-targeting approaches, such as Fenton therapy, which relies on inducing cell damage through the intracellular production of ROS [23,24]. Although IONPs have numerous applications in drug delivery, their most significant drawback in biomedical applications is their propensity to aggregate. This propensity increases particle size and leads to the loss of features, resulting in defects in their expected performance. The main causes of this phenomenon are the high specific surface area, van der Waals interactions, and dipole-dipole interactions between these nanoparticles. For this reason, in the synthesis and utilization of IONPs, surface modification or neutralization of these factors plays a crucial role in controlling dimensions and preventing nucleation. So far, various organic and inorganic materials, such as cetyltrimethylammonium bromide (CTAB), chitosan, polyvinylpyrrolidone (PVP), gold, etc., have been used to modify the surface of IONPs to prevent their accumulation due to electrostatic forces [25,26].

Polysaccharides are carbohydrate macromolecules, constituting the largest and most influential category of biological compounds produced by bacteria, fungi, and algae. Microbial polysaccharides are divided into three categories depending on their cellular location: i) polysaccharides forming the cell wall (structural polysaccharides) such as teichoic acids, lipopolysaccharides (LPS), and peptidoglycans; ii) cytosolic polysaccharides (intracellular polysaccharides, IPS) that provide energy and serve as a carbon source for the cell; and iii) exopolysaccharides (EPS) that form biofilms or capsules, secreted into the extracellular environment [27]. Polysaccharides obtained from different microorganisms (bacteria, molds, and yeast) have recently gained interest in the pharmaceutical and food industries due to their solubility in water, stable physicochemical properties, biodegradability, antitumor properties, and immune system stimulation. Their rheological properties can also be improved by using them as an alternative polymer [[28], [29], [30]]. Moreover, studies have shown that their use in both in vitro and in vivo conditions is safe and harmless, without causing any significant immunological response [[31], [32], [33]].

A wide variety of EPS derivatives are available that can be functionalized through bioconjugates [29,34,35]. In recent years, various EPSs from different strains of Lactobacillus plantarum have been structurally characterized [31,[36], [37], [38], [39], [40]]. The primary building blocks of these heteropolysaccharides are glucose, galactose, and mannose. In some strains, these building blocks are detected and isolated in the form of polysaccharides, such as dextran, mutan, levan, etc. [41,42]. The average molecular weight of these compounds ranges between 10 and 104 kDa [41]. Numerous studies have also investigated their antitumor, antioxidant, and biological properties [[43], [44], [45]]. EPS glycans are recognized for their use as purified and isolated glycan compounds, such as dextran, levan, pullulan, etc., and have been applied in targeted drug delivery research. To maximize their synergy, considering intact EPS as a mixture of carbohydrate biopolymers may be a novel approach. Different systems could be created using these functionalized composite biopolymers to achieve controlled drug release and the targeted accumulation of therapeutic agents. In line with the use of combined treatment methods and smart approaches to fighting CRC, our study aimed to enhance drug delivery using a large-capacity system that responds to changes in the acidity and redox status of the environment. To achieve this goal, a multifunctional nanocarrier based on HMSNs was designed, synthesized, and characterized. The design of this combined treatment system has taken into account not only the synergistic effects of different components, including chemotherapy drugs and surface modification but also the potential for scaling up the approach using available and low-cost compounds. This HMSN-based nanocarrier is functionalized with ceria and iron oxide. These two metal oxides, in addition to their unique characteristics, interact during oxidation-reduction reactions. The acidity of the environment influences these interactions, indicating that the tumor microenvironment is the target of pro-oxidant effects caused by ceria and the Fenton catalytic cascade initiated by iron oxide. Subsequently, the functionalized nanocarriers were loaded with IRT and coated with extracted bacterial EPS. The presence of a hydrophilic polysaccharide coating not only enhances the biocompatibility of our desired drug delivery system but also, through the development of in vivo protective effects, leads to extended blood circulation time and consequently a longer and more sustained anti-cancer effect. Finally, after characterizing the synthesized nanocarrier, in vitro and in vivo experiments were conducted to assess their antitumor properties.