Introduction

Colorectal cancer (CRC) is the third most prevailing cancer worldwide. In 2020, CRC caused 506,449 deaths in Asia, 244,824 in Europe, 63,987 in North America and 69,435 in Latin America and the Caribbean.1 The morbidity is expected to increase up to 1.1 million worldwide by 2030.2 CRC is usually seen in patients under the age of 50 and it is associated with dietary characteristics and eating habits that contribute to bacterial dysbiosis in the gastrointestinal tract (GIT) and colon cancer.3

CRC is a type of malignancy that affects the large intestine and rectum regions.4 It is caused by the deactivation of the p53 pathway or adenomatous polyposis coli gene, the accumulation of mutations such as in K-ras, the transforming growth factor-beta pathway and the genesis of small polyps. The American Joint Committee on Cancer divided CRC into five phases with different treatment strategies for each stage.5 Stage 0, the presence of abnormal colon cells or polyps on the mucosa, is completely curable with a surgical resection when found early enough. Similarly, surgical excision is the conventional treatment for stages I–II in most cases but with a 5-year survival rate of 37–74%. However, the survival rate drops to 6% in advanced stages of CRC6 and adjuvant pharmaceutical therapies such as chemotherapy are usually recommended following surgical resection.7 In stages III–IV, adjuvant chemotherapy like oxaliplatin, 5-fluorouracil, cisplatin, doxorubicin and other emerging medicines are used. However, the administration of these medicines, has been linked to adverse effects, such as vomiting, hair loss and nausea and not demonstrated the expected efficacy.8 Variety of drawbacks also associated with anticancer drugs, such as hydrophobicity, low water solubility, inadequate biodistribution, and susceptibility to multiple drug resistance.9 This has urged the need for innovative approaches to enhance the physicochemical and pharmacodynamic properties of standard chemotherapeutics, or achieve target-specific delivery to optimise treatment effectiveness while reducing off-target side effects.10

Nanoparticles are among the most promising treatment modalities for cancers including CRC. Nanoparticles taken intravenously can circulate for extended period of time with reduced clearance rates from the renal system due to their ultra-small size and superior drug loading and encapsulation efficacy.11 Furthermore, since nanoparticles allow for continuous or controlled release, the physical encapsulation of anticancer drugs improves their pharmacokinetic properties while reducing the number of dosages administered. This controlled release can circumvent multiple drug resistance since the cells actively ingest bigger amounts of the drug intracellularly.12–14 Nanoparticles surface can be further modified with targeting ligands to achieve drug targeting by increasing the selectivity towards the desired cells/tissues while reducing the availability of the anticancer drug at the normal tissues.15 In addition, amphiphilic lipid-based nanoparticles, such as micelles, possess a hydrophilic surface and a hydrophobic core and able to increase the solubility of hydrophobic drugs.16

Oral drug delivery is the most preferred and convenient route for drug administration and can be utilised for both systemic or local drug delivery.17 The high surface area (>300 m2) of gastrointestinal track coated with a viscous mucosal layer facilitates drug adhesion and absorption.18 Nevertheless, oral drug delivery can be challenging due to the complexity of GIT and obstacles that impact drug delivery like poor drug solubility, stability, and permeability.17 Moreover, oral drugs may also have an unpleasant taste, induce gastric irritation, and be subjected to intestinal and hepatic barriers metabolism.19,20 Oral route can be utilised to target a specific location in the GIT following proper design of the drug or the delivery carrier.21 Several studies utilised oral route to target CRC throughout the rational design of cancer target discovery and development (CTDD).22

Numerous studies demonstrated the suitability of colon as a site for drugs absorption as it has less degradation and thus contributing to a greater drug bioavailability.23 Special considerations are involved while designing CTDD as the drug need to be protected from gastric pH and enzymes. The colon’s physiology differs greatly from that of the rest of the GIT. CTDD is affected by the inconsistency of colonic pH, transit time, and fluid volume, which are highly variable depending on the colon part, food, and metabolic enzymes.24 Other important factors impacting CTDD efficacy include colonic fluid viscosity and the presence of microbial enzymes.25

Nanoparticles for drug delivery are prepared using numerous polymers, which range between natural, synthetic and semisynthetic.26–28 Amongst these polymers, chitosan and its derivatives attracted much attention for oral route of administration owing to their suitable properties. Owing to the presence of amino groups, chitosan carries a cationic charge that is responsible for its permeation enhancing and mucoadhesion effect. It is also biodegradable, biocompatible and non-toxic.29 Chitosan is a linear polysaccharide, comprises of randomly repeated units of N-acetyl-D-glucosamine and D-glucosamine linked by β(1→4) glycosidic bonds.30 Chitosan is a natural polymer obtained from chitin, the second most abundant naturally occurring polymer following cellulose. Both chitin and chitosan are extensively used in several applications such as food technology31 and medical and pharmaceutical applications including wound healing,32 tissue engineering33 and gene delivery.34 Chitosan has been formulated into chitosan nanoparticles (CSNPs) with attracted features especially in ocular and oral delivery. CSNPs are considered a very promising strategy to overcome the low stability and bioavailability of many active ingredients. Chitosan can efficiently bind to the negatively charged mucus membrane, increasing the retention time and enhancing the probability of their cellular uptake. Moreover, chitosan was also used as a coating agent due to its unique properties that enhances the permeation of anti-cancer drugs by transiently opening the tight junction between epithelial cells. Owing to its biodegradability via glycosidic linkage lysis by specified enzymes from the colonic microflora,35 chitosan is very valuable for CRC targeting. Recently, Patil and Killedar developed water-insoluble gallic acid and quercetin into chitosan nanoparticles to assemble a stable and targeted colorectal cancer formulation.36 Kankala et al successfully prepared a versatile drug delivery platform based on nanocarriers by coating the ultrasmall Pt nanoparticles by a composite layer of chitosan over the Zn-doped MSNs to combat MDR in cancer efficaciously.37 In 2017, 5-fluorouracil loaded PLGA-nanoparticles modified with chitosan were developed for target treatment of colorectal cancer. Coated NPs showed significant inhibition of colon cancer cells (HT-29) compared to uncoated NPs or free drug solution.38

Despite the availability of different types and grades of chitosan that differ mainly in the molecular weight and degree of deacetylation, original (un-modified) chitosan may lack the optimal properties for specific applications. Therefore, chemical modifications have been performed on chitosan structure mainly on the functional groups (-NH2 and -OH). Modified chitosan derivatives have shown enhanced properties like bioactivity, biocompatibility and non-toxicity.39,40 These chitosan modifications also facilitated CRC targeting where the drug need to be protected from acidic and enzyme degradation with minimal loss until it reaches the colon.41,42 In 2021, Wang and his team combined of oxaliplatin and resveratrol successfully into N,O-carboxymethyl chitosan nanoparticles as a promise strategy for colorectal cancer therapy. The inhibition efficiency and anti-colon cancer activity of the combined treatment were more stronger than either type of nanoparticle alone or the free drugs.43 In the same year, colon site-specific mucoadhesive system was developed by Samprasita et al composed of a-Mangostin-loaded thiolated chitosan NPs following by genipin (GP) crosslinking and the surface modification by EudragitVR L100.44

This review emphasized on chitosan nanoparticle applications in CRC. We first reviewed CRC pathology and therapy and explained the oral route of drug administration. Then, we explored nanotechnology in drug delivery and targeting before moving to detailed explanation of chitosan and CSNPs and their applications especially in CRC.

Colorectal Cancer

Colorectal cancer is a type of malignancy that affects the large intestine and rectum regions.4 In 2020, the number of death caused by colorectal was 506,449 in Asia, 244,824 in Europe, 63,987 in North America and 69,435 in Latin America and the Caribbean.1 Colorectal cancer statistics are expected to grow with a morbidity estimation of 1.1 million worldwide by 2030.2 Despite continual diagnosis and therapy advancements for both primary and metastatic type of CRC, long-term survival and cure rates are remained low. Males have a greater death rate from CRC than females, and this rate may rise as a result of several stimuli factors including age, unhealthy eating habits, obesity, smoking, alcohol use, high body mass index, sedentary lifestyle, and poor physical activities.45 Adopting a healthy lifestyle appears to be the most important factor in preventing colorectal cancer.

Pathophysiology

Colorectal cancer is the third most prevailing cancer worldwide, usually seen in patients under the age of 50 and it is associated with dietary characteristics and eating habits that contribute to bacterial dysbiosis in the GIT and colon cancer.3 Colorectal cancer is now common in China, India, Singapore, Japan, and South Korea, despite the fact that it was long thought to be a disease only seen in Western nations. It is a multistage disease that can take 10–40 years to evolve from adenoma to carcinoma. Colorectal cancer is caused by the deactivation of the p53 pathway or adenomatous polyposis coli gene, the accumulation of mutations such as in K-ras, the transforming growth factor-beta pathway and the genesis of small polyps. These small polyps are prone to be transformed to large polyps, leading to tumour initiation. A preneoplastic indication of colon tissue is aberrant crypt foci where chemoprevention can be achieved by blocking or delaying the carcinogenesis process.46

T-lymphokine-activated killer cell-originated protein kinase signalling abnormalities promote cancer growth and is abundantly expressed and active in colorectal cancer, where it also stimulates cancer cell survival, inflammation and proliferation. Moreover, T-LAK-derived protein kinase, which interacts with the binding domain of DNA of the tumour suppressor p53 protein and controls the expression of transcription for target p21, has been suggested as a potential therapeutic target to fight colorectal cancer. Also, 3-Deoxysappanchalcone, a compound originated from Caesalpiniasappan L. is a natural component that capable of apoptosis induction via a p53-dependent pathway and hence, inhibiting colon cancer cell proliferation through direct targeting of signaling pathway of T-lymphokine-activated killer cell-originated protein kinase.47 The Tumor, Node, Metastasis (TNM) classification of cancer and the International Union for Cancer Control as well as the Classification of Malignant Tumors provide the foundation for therapeutic decisions. Colorectal cancer is categorised with reference to lymph node involvement (N stage), local invasion depth (T stage), and distant metastases (M stage).48 The laterality of initial tumours is critical for a patient with synchronous metastatic colon cancer. Individuals with right-sided colon cancer had a considerably poorer overall survival rate than patients with left-sided colon cancer.49

Biomarkers of Colorectal Cancer

Despite the accelerated progress of medicine, the prognosis of CRC patients is largely determined by the disease’s stage at the time of diagnosis.50 Considering the fact that early discovery of CRC reduces related morbidity and mortality and that detection of its precursor lesion can even reduce the occurrence, current CRC screening systems still have a number of flaws.51 As a result, it is critical to develop novel non-invasive biomarkers that can detect tumour in the asymptomatic CRC early stages, when the disease is still treatable.

CRC patients, on the other hand, have been treated for decades with relatively reliable diagnostic methods like as colonoscopy and histological study of tumour tissue, as well as imaging modalities such as x-rays, computed tomography, and magnetic resonance imaging. Nevertheless, based on these investigations, clinical illness assessment and tumour stage classification are unable to suggest clinically predictive information and relevant prognostic at the patient level.52 As a result, both clinical and basic scientists are continually searching for more precise individual biomarkers to reduce the severe side effects of chemotherapy, disease relapse, and treatment resistance, which would ultimately improve patients’ longevity and quality of life.

The presence of tumour traces in the blood and other bodily fluids has been recognised as a possible individual biomarker with a diagnostic, prognostic, and predictive value. The ability of detection of so-called circulating tumour cells (CTCs) in the blood samples of patients has been proven in multiple investigations to have a deleterious impact on their survival.53–55 The detection of CTCs from blood is comparable to that of conventional tumour tissue biopsy, hence, the term “liquid biopsy” has been introduced. Liquid biopsy, in contrast to tissue biopsy, has various advantages, including easy and quick procedure, reduced cost, and minimum risk and pain for patients due to its limited invasiveness.

It has recently been discovered that the existence of circulating tumour DNA (ctDNA) is just as important as the presence of CTCs.56 Furthermore, the biological function of microRNAs (miRNAs) in CRC pathogenesis is becoming clearer, and their existence in peripheral blood has been proposed as a major predictive/prognostic biomarker.57 As a result, the concept “liquid biopsy” has been widely known and is now utilised to detect all circulating tumour features, primarily ctDNA, CTCs, circulating miRNAs, as well as others such mRNAs, exosomes, proteins, and others) in peripheral blood or other biological fluids.58

Figure 1 Examples of nanoparticles used in the therapeutic management of colorectal cancer.

In the context of multiple clinical investigations, the liquid biopsy/circulating biomarker concept is steadily nearing clinical practice Colorectal cancer is typically sporadic, although having a family history of the disease increases your chances of getting it. Prevalent mutations in colorectal cancer includes KRAS, TP53, BRAF, transforming-growth factor-beta receptor 2 (TGFBR2), catenin-beta 1 (CTNNB1), APC, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit-alpha (PIK3CA), ERBB2, SMAD4, AT-rich interactive domain 1 A (ARID1A), SRY (sex-determining region Y) box 9 (SOX9) and family with sequence resemblance 123B (FAM123B).59 By interfering with essential signalling pathways, mutations in these genes cause tumorigenesis. The mutations of WNT signalling pathway are seen in the early stages of colorectal cancer, with TP53 mutations appearing later.60 Current colorectal cancer biomarker research aims to target these molecules, as well as a few others, in order to improve cancer management. The gold standard procedure for colon inspection is colonoscopy, which is used for a successful screening programme. However, the discomfort during the examination, high cost and the examination recommended interval of 10 years are the main limitations of this technique. Non-invasive screening fecal tests including fecal immunochemical test (FIT), guaiac fecal occult blood test (gFOBT) and fecal DNA tests are able to detect blood in stool in high sensitivity manner.61 The FIT method uses antibodies, whereas the gFOBT method uses chemicals; however, the faecal DNA test examines colorectal cancer-related DNA biomarkers in faeces from cells released from the colon and rectal lining.

Need for Novel Oral Drug Delivery Systems

Colorectal polyps, an abnormal noncancerous growth in the inner lining of the intestine, are the most common cause of CRC. However, only about 10% of polyps have been seen to progress to aggressive malignancy. This change is very slow, taking more than 10 to 20 years to complete, and it is more likely as polyps become larger.62 Later, malignant cells infiltrate muscle and lymph nodes prior migrating to other tissues, such as the lungs and liver. CRC is divided into five phases by the American Joint Committee on Cancer, where the treatment strategy is highly related to the corresponding stage.5 Stage 0 is characterised by the presence of abnormal colon cells or polyps on the mucosa when found early enough, and it is completely curable following a surgical resection. In most cases, surgical excision is the conventional treatment for stages I–II, with a 5-year survival rate of 37–74%. Unfortunately, the survival rate in advanced stages of CRC drops to 6% due to the considerable risk of recurrence and metastasis to distant organs.6 As a result, adjuvant pharmaceutical therapies such as chemotherapy are usually recommended in the advanced stages of CRC to lower the risk of recurrence and metastasis following surgical resection.7

In stages III–IV, adjuvant chemotherapy like oxaliplatin, 5-fluorouracil, cisplatin, doxorubicin and other medicines are used to suppress tumour growth and extend survival. Mere administration of these medicines, however, has not demonstrated the expected efficacy at these stages and had been linked to a number of dose-dependent adverse effects, such as vomiting, hair loss and nausea.8 Chemotherapeutics are given in multi-cycles to reduce their adverse effects, while varying the doses, frequency, and duration of each cycle based on the individual’s condition. Anticancer drugs also have a variety of drawbacks, including the hydrophobicity, low water solubility, inadequate biodistribution, and susceptibility to multiple drug resistance.9 This has urged researchers to conduct innovative approaches such as using nanotechnology-based drug carrier system to enhance the physicochemical and pharmacodynamic properties of standard chemotherapeutics, or achieving target-specific delivery of cytotoxic agents to various types of tumour tissues to optimise treatment effectiveness while reducing off-target effects.10

Nanoparticles are among the most promising treatment modalities for CRC and other cancers, as their properties are considered as a benefit in addressing the systemic drug delivery issues that diagnostics and anticancer drugs encounter. The surface area-to-volume ratio of nanoparticles varies between 1 and 100 nm, allowing for greater drug loading through encapsulation inside the core and adsorption on the surfaces of the nanocarrier. Nanoparticles can circulate for extended periods of time and have reduced clearance rates from the renal system due to their ultra-small size and superior drug loading and encapsulation efficacy.11 Furthermore, since nanoparticles allow for continuous or controlled release, the physical confinement of diagnostics agents and anticancer drugs improves their pharmacokinetic properties while reducing the number of dosages administered. This controlled release can circumvent multiple drug resistance since the cells actively ingest bigger amounts of the drug intracellularly. Furthermore, the nanoparticles facilitates the anticancer mobility in the systemic circulation, increases cellular uptake and offers benefits over the bulk equivalent.63

Another advantage of using nanoparticles is the ability to customise their surface with targeting ligands (peptides or small-molecule ligands) to increase their selectivity towards the desired cells/tissues while reducing the availability of the chemotherapeutics at the normal tissues. Furthermore, amphiphilic lipid-based nanoparticles, such as micelles, possess a hydrophilic surface and a hydrophobic core and able to increase the solubility of hydrophobic drugs. In addition, quantum dots, nanoshells, nanosomes, and paramagnetic nanoparticles have structural properties that can be exploited for imaging and diagnostic purposes. Figure 1 illustrates different types of nanoparticles employed for colorectal cancer targeting.

Figure 2 Deacetylation of chitin into chitosan.

Emerging Oral Therapeutic Agents for Treating Colorectal Cancer (Synthetic & Natural)

The main treatment for stage 0 (the early stage) and stages I to III of non-metastatic CRC is radiotherapy with/without chemotherapy whereas chemotherapy is the option for stage IV (metastatic CRC (mCRC)). Several therapeutic agents were emerged as potential therapy for CRC with some gained regulatory approvals. They include synthetic and natural materials as small molecules or macromolecules. In this regard, we discuss the orally administered emerging therapeutic agents for CRC and divided them into synthetic and natural.

Emerging Synthetic Therapeutic Agents

KRAS (Kirsten rat sarcoma virus) gene is a member in tyrosine kinase signaling pathways. It is one of the important identified oncogenes whereby downregulating these pathways becomes a target for tyrosine kinase inhibitors (TKIs).64 Targeting growth factor receptors is one of the important mechanisms for the emerging therapeutic agents against CRC. These growth factors include platelet-derived growth factor receptors (PDGFRs), fibroblast growth factor receptors (FGFRs), vascular endothelial growth factor receptors (VEGFRs) and others.

Tyrosine Kinase Inhibitors Targeting VEGF or KIT

Nintedanib is a TKI that inhibits some of Src tyrosine kinases and targets several growth factor receptors including PDGFRs α and β, FGFRs 1–3 and VEGFRs 1–3.65 Nintedanib is approved in Europe and the USA, with 150 mg twice daily as a standard adult dose.64

Regorafenib is a multi-TKI with activities against Ret, Kit, PDGFR, and Raf kinases. It also inhibits VEGFR 2 and VEGFR 3, which is responsible for its antiangiogenic activity.66 Regorafenib gained FDA and EMA approval for the treatment of mCRC.64

Axitinib is an antiangiogenic agent inhibiting VEGF and PDGF. Axitinib is an effective agent against mCRC.66

Apatinib (rivoceranib) is a TKI and VEGFR 2 inhibitor showing antiangiogenic and antineoplastic effects. Apatinib decreases tumor microvessel density and inhibits VEGF-stimulated endothelial cell migration and proliferation.66 In a study involved patients undergone at least two standard treatments, Apatinib 500 mg once daily was effective against refractory mCRC. However, the dose was then reduced to avoid adverse events.66 In VEGFA-overexpressed tumors, Apatinib decreases the hyperangiogenesis and hypoxia inside the tumor microenvironment, converting it from an immunosuppressive into an immunostimulatory one.66

Famitinib is a TKI that inhibits VEGFRs 2 and 3. In a clinical trial, Famitinib increased the progression-free survival in patients with refractory mCRC compared to the placebo. It showed an acceptable safety profile but the overall survival did not increase.64

Surufatinib (Sulfatinib) is a TKI exhibiting activities on anti-angiogenesis and regulating cancer immunity. Surufatinib inhibits VEGFRs 1–3, FGFR1 and colony stimulating factor 1 receptor (CSF1R). Surufatinib safety and efficacy is being investigated in a clinical trial (NCT04653480) in second-line RAS/BRAF mutant and microsatellite stable CRC.64

Masitinib is a TKI with a specific KIT targeting, which is mutated or overexpressed in many cancers. Activated c-KIT receptor and tryptase have important role in the cancer tissue angiogenesis, which help the cancer cell in the invasion and metastasis.66

BRAF Inhibitors

BRAF is an enzyme in the RAF/MEK/ERK signaling pathway. BRAFV600E mutation is upregulated in many human cancers. In CRC, around 15% of the patients have mutated BRAF. BRAF inhibition showed in success in BRAF-mutant metastatic melanoma, therefore it can be also employed in mCRC treatment.64 Vemurafenib and Encorafenib are among the inhibitors of BRAFV600E-mutated kinase. Encorafenib showed modest antitumor activity in patients with refractory mCRC bearing BRAFV600E mutation.66

MEK Inhibitors

MEK1 and MEK2 are dual-specificity Thr/Tyr kinases. Binimetinib is a mitogen-activated inhibitor of both enzymes while Cobimetinib is a specific inhibitor of MEK1. In vitro, Cobimetinib induces the apoptosis of HCT-116 CRC cells and inhibits cell proliferation.64

STAT3 Inhibitors

In colon cancer and other cancers, a signal transducer and activator of transcription factor 3 (STAT3) is active and have a significant role in cancer progression. In a clinical trial Phase III, Napabucasin as an inhibitor of STAT3 increased the survival of patients with mCRC compared with placebo.66

PD1/PDL-1 Inhibitors

Programmed cell death 1 (PD-1) receptor is found on the immune cell surface including T cells. PD-1 ligand (PDL-1) was detected in several cancers and when binds to PD-1, it inhibits the activation of T cells. Therefore, interfering with the PD-1/PDL1 pathway restores T cell function and this leads to an improvement of the immune response against cancer.66 Combination therapy of capecitabine and oxaliplatin together with immune checkpoint blockers anti-PD-1 and anti-PDL1 was explored in murine syngeneic preclinical models that are similar to the tumor type in patients.66

PDZ Domains Inhibitors

PDZ domains represents the most abundant protein–protein interaction domains of the human proteome. PDZ is composed of 266 different domains located in 150 proteins. PDZ aberrant expression was found in CRC and other cancers.64

By themselves, these domains do not have any enzymatic activity but they orchestrate several pathways. Therefore, inhibition of PDZ domains results in multiple enzyme inhibition simultaneously.

NHERF1 (Na+/H+ exchanger 3 regulating factor 1) is a PDZ containing protein overexpressed in the cytoplasm and nuclei of CRC cells. Therefore, a new class of anticancer drugs can be utilized based on the inhibition of NHERF1. Several small molecules were designed employing molecular and chemical approaches then evaluated as NHERF1 inhibitors.66

Cancer Stem Cells

Cancer stem cells (CSCs) are resistant to the radiation and chemotherapeutic agents. They involve in the self-renewal of many cancers. Colorectal CSCs and other solid tumor stem cells have an insistent activation of several signal transduction pathways, whereby Wnt/β-catenin pathway plays an important role in stemness maintenance and drug resistance of colorectal CSCs.66 CD44 is a well-documented surface marker involved in the proliferation of cancer cells. It has an important role in regulating CSC stemness properties. CD44 was found to regulate the in vitro and in vivo growth of colon cancer xenografts in animals.66 Therefore, CD44 can be utilized in CRC treatment options.

The anti-diabetes drug Metformin downregulates CD44 in primary oral cancer cells.66 Combined with doxorubicin, Metformin inhibits both cancer stem cells and non-stem cancer cells in culture. Therefore, a new use of Metformin may emerge in the anticancer treatment plans.64

The antibiotic Salinomycin inhibits CSCs in different human cancers. The mechanism is suggested to be by interfering with ABC drug transporters and CSC pathways including the Wnt/β-catenin signaling pathway. Salinomycin derivative with cyclopropylamine at position C20 inhibits HMLER CD24-low/CD44-high cells. With high selectivity against CSCs, Salinomycin and its derivative could have the potential to prevent cancer resistance.66

The COX2 inhibitor celecoxib is used as nonsteroidal anti-inflammatory drug. Synergistically, celecoxib and the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin, may eradicate CSCs with CD44 overexpression.66

γ-secretase inhibitors like MK-0752 are being investigated in various CSCs as modulators of the Notch signaling pathway. MK-0752 and ridaforolimus (MK-8669) as a combination are being clinically investigated (phase 1 clinical trial) in patients suffering from advanced and refractory pancreatic and colorectal cancer.66

Emerging Natural Therapeutic Agents

Natural products serve as a main source of new anticancer drugs. It is estimated that about half of the anticancer drugs currently in use are derived from natural products whether directly or not directly. Anticancer natural products fall into several categories based on their structure including alkaloids, polysaccharides, polyphenols, diterpenoid and unsaturated fatty acids.67 The use of chemotherapy for the treatment of CRC is commonly associated with side effects and drug resistance. Therefore, there is a need to find new and more effective treatments for CRC. Several natural products exhibit anti-CRC effects.68

Alkaloids

Irinotecan (CPT-11) is a water-soluble camptothecin derivative. Its mechanism involves the inhibition of DNA topoisomerase I, blocking DNA replication and inhibiting RNA synthesis.67 Irinotecan has a proven anti-cancer effect against mCRC. Irinotecan has been used for standard CRC treatment. In combination with 5-fluorouracil/formyltetrahydrofolate (CF), it has better anti-cancer efficacy against the advanced mCRC.68

SN38 is the active analogue of irinotecan. It inhibits the growth of several cancer cells.67 Its mechanism involves anti-angiogenic activity due to the inhibition of VEGF and hypoxia-inducible factor 1 alpha expression. SN38 inhibits both the endothelial cell proliferation or lumen formation and the angiogenic activity of glioma cells.

Despite of the variable anti-tumor activities of CPT-11 and SN38, they both have significant toxicity including cholinergic syndrome, neutropenia and delayed diarrhea. More research is being performed to improve their efficacy and safety through chemical structure modification and combination chemotherapy.68

Polysaccharides

Fucoidan is a sulfated polysaccharide isolated from the seaweed Sargassum hemiphyllum. It has been shown to induce apoptosis in HT-29 and HCT-116 cells.69 Double-blind, randomized clinical trials was conducted in Taiwan and demonstrated that low-molecular-weight fucoidan (0.8 KDa) has significant activity against CRC in humans in combination with chemotherapy, with relatively few side effects.67

Polyphenols

Resveratrol is a polyphenolic compound with significant inhibitory effects against wide range of cancers. Resveratrol induces apoptosis in CRC by inducing Fas redistribution and inhibiting Wnt signaling.70 The grape seed extract, a rich source of Resveratrol, was shown to inhibit PI3K pathway and induce apoptosis in CaCo-2 cells.71

Curcumin is a polyphenol isolated from Curcuma longa with multiple effects against malignancy. Curcumin exhibits anti-angiogenic properties, and increases the expression of the p53 and cip1/p21. It is able to induce the apoptosis of CRC.72

Terpenoids

Andrographolide is the major bioactive component of Andrographis paniculata. This plant has been used as a traditional herbal medicine in Asia for thousands of years. Andrographolide exhibits anti-cancer effects due to its DNA-binding activity and inhibition of NF-κB activation.68 The combination of andrographolide and 5-fluorouracil induces the apoptosis of HCT-116 cells as a result of andrographolide binding and upregulation of BAX protein expression. The safety and efficacy of the combination treatment of andrographolides and capecitabine was shown in elderly patients with locally advanced CRC.73

Unsaturated Fatty Acids

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are polyunsaturated fatty acids (PUFAs) with significant inhibitory effect against angiogenic factors like platelet growth factor, VEGF, and endothelial cell growth factor. Therefore, they are effective against CRC, and other cancers.74 DHA and EPA as free acids or in the form of phospholipids inhibits the growth of CRC cells, and the studies have shown the strong growth-inhibitory effects of PUFAs in the HT-29 cell line more than in the Caco-2 and DLD-1 cell lines.68,75

Oral Route of Drug Delivery

Oral drug delivery has been recognised as the most attractive approach of drug administration among various others and can be utilised for systemic or local drug delivery as well as enhance the drug solubility and dissolution of low solubility drugs.17 It is the most preferred way by patients, ease of administration, convenience for self-administration, noninvasive, and feasibility for the formulations.19,20 Furthermore, a high surface area (>300 m2) of gastrointestinal track coated with a viscous mucosal layer facilitates drug adhesion and absorption.18 Nevertheless, oral drug delivery can be challenging due to the complexity of GIT, even among healthy people, which poses some obstacles that impact delivery such as poor drug solubility, stability, and permeability.17 Drugs that are administered through the oral route have a slower absorption as they must be solubilised in the gastric fluid to be absorbed.20 The main factors affecting GIT absorption of the drug is drug physicochemical characteristics (polymorphism, solubility, partition coefficient, and pH-pKa), biological factors (blood flow at absorption site, mucus, absorption surface area, ionic concentration, enzymes, and microbiome) as well as maintenance of sync condition (drug concentration at the absorption site).76 Moreover, oral drugs may also have an unpleasant taste, induce gastric irritation, and/or be subjected to intestinal and hepatic barriers.19,20

The rectal route of administration is an alternative route of drug delivery that has some disadvantages in comparison to the oral route. The low surface area and rectal fluids can lead to irregular or incomplete absorption of the drug. Table 1 shows a comparison between characteristics of different parts of GIT.18 Different segments of GIT vary anatomically and physiologically and impact the extent of dissolution and absorption of the drugs. Hence, the basic characteristics of the GIT should be considered during the formulation design of oral drug delivery system.

Table 1 Characteristics of Different Parts of the GIT

Factors Influencing Gastrointestinal Tract

Regardless of absorption mode, each digested substance will encounter three major biological environments along with the gastrointestinal tract including the lumen (internal space), mucus, and tissue.

Lumen pH

The pH is the first biological barrier to any orally delivered drug, which denature/depurination most of the administered drugs and substantially reduces their effectiveness. The pH of GIT varies based on GIT part and fed state to unfed state. The pH inside the gastric environment is extremely acidic (pH 1–2) in unfed state and it could rise to pH 5 in fed state. The pH increases rapidly in duodenum (pH 6) and rises along the small intestine to reach pH 7.4 at the terminal ileum.18 Cecum pH decreases just below pH 6 and again increases in the colon and reaches pH 6.7 at the rectum.77 Nevertheless, the pH ranges can vary with factors like dietary intake, microbial metabolism, and diurnal cycle.78 gastrointestinal pH changes affect the extent of drug molecules ionisation, subsequently impact drug absorption.79,80

Enzyme

Enzymatic degradation of dosage forms and drugs can occur throughout the GIT and hence can affect their stability. The major site of the enzymes that are involved in food digestion is mouth (salivary amylase), stomach (pepsin).81 Besides that, pancreatic enzymes secreted into the intestinal lumen including trypsin, amylase, lipase, and peptidases. Those pancreatic enzymes are not considered as the main challenge, although they are considered as a biological barrier. These enzymes are especially abundant in duodenum, and their concentrations are reduced significantly in the later intestinal parts. duodenum transit time is relatively short (10% of the whole small intestine residence time) and is not enough for the enzymatic degradation of the drugs. Duodenal pH is lower compared to the later small intestine parts, and thence the unwanted drug release inside the duodenum can be prevented via increasing delivery carriers pKa.

Microbiome

The intestinal microbiome has over 500 bacterial species,82 and they are important for digestion and metabolism of the ingested food as well as for intestinal health.83 The colon contains the bulk of the intestinal microbiome and they ferment carbohydrates as the main source of nutrition.83 The composition of the microbiome varies between individuals based on genetic and environmental factors.84 Nevertheless, the dominant species appear to be consistent and represent the majority of the colonic flora.82 These bacteria secrete many enzymes such as azoreductase, α-arabinosidase, β-galactosidase, β-glucuronidase, β-xylosidase, deaminase, nitroreductase, urea hydroxylase.85 These enzymes are exploited for CTDD.86

The density of the microbiome inside the small intestinal is lower compared to that of the large intestine. This is due to the intestinal fluid volume, rapid luminal flow, and bactericidal compounds secretion in this part of the GIT.87 Small intestine microbiome composition can highly fluctuate over a short period as it is affected by dietary intake variations.88 The colonic microbiome is claimed to play a crucial role in the regulation of metabolic function.87 Small intestinal microbiota influences oral drug absorption have not yet been elucidated.

Mechanical Degradation

Mechanical degradation inside the lumen including the peristalsis of the GIT muscles, the shear stresses by gastric juice flow rate within the lumen as well as the osmolarity along the GIT are factors that reduce the drug efficiency. Flowing gastric juice could reduce the contact time between the epithelial layer and drugs, hence impeding their absorption. Biologics and microencapsulation are the major elements sensitive to mechanical destruction through mechanical stresses.

Transit Time

Gastrointestinal transit time is the key factor for drugs that have region-specific targeting or absorption characteristics. Gastric transit time varies from several minutes to hours, depending on several factors, such as age, gender, body posture, osmotic pressure, dietary intake, physical activity, etc.89 The transit time in gastric can vary from 0 to 6 h based on fast/fed state,81 while small intestine transit time is considered constant at approximately 4 h.90 On the other hand, colon transit time is extremely variable from 6 to 70 h.91 Gender is highly affecting the colonic transit time and females have significantly longer colonic transit times compared to males.92

Mucus

Mucus is the second barrier for effective oral drug delivery. Mucus facilitates the digested moiety passage and protects the underlying epithelium from mechanical stress and pathogens by capturing any foreign moieties (especially hydrophobic molecules). Although mouth and oesophagus lack a distinct mucus layer, they are washed by salivary gland mucus.93 The small intestine has a loose and unattached mucosal layer, while the stomach and colon have a two-layered mucus system composed of an outer loosely adherent layer and an inner firmly adherent layer.93 It is claimed that mucus is the primary barrier against oral drug bioavailability due to the difficulty to diffuse into the mucus layer. While the inner mucus layer facilitates the absorption and improves the drug uptake efficiency, justifying the dual role of mucus in the absorption/desorption of oral administrated drugs. Goblet cells are continuously secreted mucus; consequently, it is cleared in each part of the GIT (50–270 min).20 This defines the final mucus layer thickness (Table 1).

Fluid Volume

Controlling fluidity is crucial for gastrointestinal function.81 The fluid environment allows digestive enzymes to contact with food, and this aids in transit the intestinal contents along the GIT without damaging the epithelial lining and it facilitates the nutrients and drugs solubility and absorption.81 The daily water balance in a healthy adult human GIT is determined by the secretion from saliva (1.5 L), gastric juice (2.5 L), pancreatic, and other intestinal components (~2.5 L), and absorption from the small and large intestine (7 L and 1.9 L, respectively).81 The pH is influenced by fluid-to-matter ratios, which can potentially alter drug absorption and delivery, especially in lower GIT. For instance, diet can have a big impact on the GIT free fluid volume, bile salts, and digestive enzyme levels.81 Moreover, intestinal fluid secretion affects the viscosity of the mucous-gel layer,81,93 which can alter the drug’s cellular uptake at the site of action. Increased fluid secretion and reduced fluid reabsorption dilute the digestive enzymes and affect the intestinal microbiota which in turn alter the digestion of carbohydrates94 and contribute to changes in intestinal transit time.95 Variations in intestinal fluid volume can impact the way drugs are processed in the GIT.

Tissue

The drug molecule properties determine where it will be absorbed and how it will cross the intestinal epithelial cells. Drugs may be absorbed in four ways: transcellular (transcytosis of the drug by crossing epithelial cells membrane), paracellular (permits only small hydrophilic molecules diffusion through epithelial cell spaces), facilitated transport, and carrier-mediated transcellular.96 Tight junctions, that affect the paracellular absorption pathway for orally delivered drugs, are the primary extracellular biological barrier against oral administration.97

The size and chemistry of the drug affect their transport through tissue. Generally, hydrophilic small drugs prefer paracellular routes, while hydrophobic drug molecules prefer to be absorbed via transcellular routes.98,99 Passive drug diffusion via intercellular pathways is only conceivable for substances as small as 0.5–3 nm, which is too small for most drug molecules to be delivered.100,101

Approaches for Oral Colon Delivery in Colorectal Cancer

Numerous studies demonstrated that the colon is a suitable site for drugs absorption as it has less reactive enzymes against drugs, CTDD protects the drug from hydrolysis by enzymes and decreases their degradation in the duodenum and jejunum, subsequently greater drug systemic bioavailability23 and prolonged colonic residence time of the drug is accompanied by the synergistic effect of absorption enhancers on the drug absorption.102 The goal of developing oral CTDD systems is to avoid or limit the release of drugs in the upper GIT while allowing explicit drug release in the colorectal area for local and/or systemic delivery. To accomplish this, the physiological parameters that affect colonic drug delivery must be taken into consideration. CTDD can be characterised as either prodrug, pH-dependent systems, time-dependent systems, or ligand/receptor-mediated systems. Each of these formulation methods has its own set of benefits and drawbacks. A combination of two or more formulation systems has been examined to overcome the limits of a single system and to maximise CTDD.

Nanotechnology has significantly advanced current drug formulations for improved absorption and site-specificity. The use of nanosystems facilitates the delivery of active compounds to cancer tissues in the colon. The usage of nanoparticles in drug formulations has a lower dose requirement compared to the amount needed for conventional dosing. Nanoparticles can be tailored with various charges to treat colonic diseases. Some examples of known polycation nanoparticles can interact with inflamed tissues of the intestine, while polyanion nanoparticles interact with cationic components (cytokines) in colorectal cancer. Nanoparticles are also prone to be uptaken by macrophages at the inflamed areas of the colon, thus allowing the drug to remain in the colon for a longer time.

Moreover, nanometre-scale sizes of nanoparticles contribute to their efficacy as drug delivery systems. Nanosized particles possess increased epithelial permeability and retention, that promotes selective delivery into inflamed tissues,103 prevents removal through diarrhoea,104 and enhances uptake by immune cells accumulated in the inflamed colon portions. Bioadhesion is the attachment of anionic nanoparticles to inflamed tissues by electrostatic interactions with positively charged proteins like eosinophil and transferrin.105 Cationic nanocarriers are used in the nanoparticles designed as a mucoadhesive system, which is attached to the anionic intestinal mucosa.106 This cationic delivery system can be used in GIT targeting as it enhances the drug cellular uptake into the mucosal surface. pH-dependent nanocarriers have been also used for CTDD. The ideal polymer should withstand gastric acidic pH and proximal small intestine while being dissolved in pH of the terminal ileum and colon. Thus, pH-dependent drug delivery systems with dissolution threshold of pH 6.0–7.0 are expected to delay drug dissolution and prevent unwanted drug release in the upper GIT before reaching the colon.107 This makes the delayed-release profile of the drug formulation.

Nanoparticles with different polymers that are capable of entrapping drugs into their matrix until further release in the colon, have been designed. Polymeric nanoparticles selectively release drugs through the enzymatic activity of colonic bacteria or by manipulating colon transit time for time-dependent release nanoparticles.

Oral drug administration in the form of a prodrug can enhance its bioavailability and prevent early degradation in the upper GIT. Colon-targeted prodrug is the inactive derivative of a drug molecule that becomes an active ingredient upon enzymatic hydrolysis in the colon. One strategy is disulfide bond conjugates formation between the prodrug and the carrier. The disulfide bond is stable in the upper GIT and can be disintegrated via extracellular bacterial reduction process or intracellular enzymes. This results in the carrier breakage and prodrug release into the colon.

For a more successful local therapy of colorectal cancer with less toxic side effects, ligand/receptor-mediated drug delivery systems can improve target specificity through the interaction of specific receptors expressed at disease locations and the targeted ligands on the carrier surface have been investigated. It can be also combined with other delivery strategies to improve its GIT stability and site-specificity. Folic acid is a receptor-selective targeting ligand that targets folic acid receptors overexpressed in many types of tumours including colorectal cancer.108 Folate receptor functionalised nanoparticles can enhance selective drug uptake into cancer cells. Folic acid functionalised liposomes enhanced the cytotoxicity of daunorubicin via facilitating folate receptor-mediated drug uptake.109 5-fluorouracil loaded Folic acid conjugated liposomes enhance the selective drug delivery to cancer cells as it showed higher anti-cancer activity and significantly decrease the volume of the tumour when compared to free 5-fluorouracil.110 Hyaluronic acid is a polysaccharide that has a high affinity for CD44 receptors, which is overexpressed in many tumours including colorectal cancer.111 Previous studies have examined the effectiveness of hyaluronic acid nanoparticles as CTDD for colorectal cancer.112,113 Hyaluronic acid-functionalised camptothecin/curcumin-loaded polymeric nanoparticles (HA-CPT/CUR-NPs) showed significant targeting capability towards Colon-26 cancer cells.113 Peptide is gaining popularity as a possible ligand for targeted drug delivery. Peptides have several pros, such as simplicity, low cost, chemical variety, biocompatibility, and stimulus responsiveness.114,115 Moreover, peptide ligands show higher binding affinity and specificity with receptors compared to small molecule ligands.108,116 In addition, the metabolic instability caused by proteases can be addressed by peptide sequence alteration, encouraging the use of peptide ligands in targeted drug delivery systems. Peptide functionalised nanoparticles are mainly being investigated as a viable strategy for tumour-targeted drug delivery. For instance, TWYKIAFQRNRK, TK peptide, has been investigated for CTDD of anticancer drugs. TK peptide has a high affinity to α6β1 integrin, that is upregulated in colorectal cancer. Hence, TK peptide-functionalised PEG-PLA micelles loaded with doxorubicin showed significantly stronger toxicity and more effectively penetrated the tumour spheroids, indicating TK peptide as a potential targeting ligand for CTDD.117

Polysaccharide as CTDD

Natural polysaccharides like pectin, chitosan, xylan, and guar gum have been used in CTDD systems, as they can remain intact in the upper GIT, but are degraded by microflora to release the drug.118 Moreover, polysaccharide structural modifications or derivatives can enhance drug release behaviour, site-specificity, and stability.119 Polysaccharide mucoadhesiveness may be beneficial for drug absorption through extended contact between drug delivery carriers and the mucosal surface. Polysaccharide-based drug delivery systems provide additional advantages such as low toxicity, low cost, immunogenicity, biodegradability, and biocompatibility.120 As a result, the microbiota-triggered polysaccharide-based system offers a viable approach for CTDD. Nevertheless, polysaccharide-based delivery systems have certain potential limitations, such as variable polysaccharide chemistry and a wide range of molecular weights.120 Moreover, the rate of drug release is affected by the type and concentration of polysaccharides.

Chitosan and Its Physicochemical Properties

Chitosan is a linear polysaccharide, comprises of randomly repeated units of N-acetyl-D-glucosamine and D-glucosamine linked by β(1→4) glycosidic bonds.121 Chitosan is the most valuable derivative of chitin, the second most abundant naturally occurring polymer following cellulose.122 Chitin can be found in the exoskeleton of insects and crustaceans, in addition to the cell walls of certain fungi.123 Chitosan, the partially deacetylated form of chitin, can be obtained by chitin deacetylase enzyme or chemical alkaline treatment using concentrating NaOH as shown in (Figure 2).124,125 Chitin polymer can be called chitosan when the degree of deacetylation (DD) exceeds 50%.126 Both chitin and chitosan have attracted tremendous attention in various fields, such as food technology,31 textile industry,127 and diverse medical and pharmaceutical applications including wound healing,32 tissue engineering33 and gene delivery.34

Figure 3 Schematic illustration of the interaction between chitosan loaded nanoparticles with the mucus layer.

Unlike chitin, chitosan is readily soluble at pH below its pKa (pKa≈6.5) ascribing to the protonation of its amino group (-NH2) at C-2 in each repeating D-glucosamine unit turning the chitosan into a cationic polymer.30 Besides this cationic nature that is responsible for chitosan permeation enhancing and mucoadhesion effects, being also biodegradable, biocompatible and non-toxic enable chitosan to be a polymer of choice for various drug delivery applications.29

Chitosan has become of great interest to be formulated into CSNPs. CSNPs are considered a very promising strategy to overcome the low stability and bioavailability of many active ingredients by combining the favourable natural characteristics of both the polymer itself and nanotechnology. Chitosan can efficiently bind to the negatively charged mucus membrane, increase the retention time and enhance the probability of their cellular uptake. Moreover, chitosan is known for transiently opening the tight junction between epithelial cells, which allows the permeation of the hydrophilic loaded drug.128

Interaction Between Chitosan and Colon Mucosa

Mucus sticks to most particles, it is as a tenacious barrier prevents their penetration to the epithelial surface. Mucus