Abstract
Colon cancer ranks third amongst type of cancer and second in terms of mortality. Various natural and synthetic anticancer agents have been used for the treatment of colon cancer. However, most anticancer agents have poor aqueous solubility and thus low absorptions and bioavailability. Moreover, the chemotherapeutic agents are unable to differentiate between normal and healthy cells and thus kill both types of cells. Nanocarriers have surfaced as a budding anticancer agent delivery system that could enhance the solubility and bioavailability of the anticancer agents and at the same time specifically deliver them to the cancerous cells. In this regard, gold, polymeric, and solid lipid nanoparticles, dendrimers, liposomes, niosomes, and carbon nanotubes have shown promising results as targeted anticancer drug delivery agents with enhanced bioavailability. The objective of this review article is to highlight these nanocarriers and present a detailed overview of how they have enhanced the efficacy of both synthetic and natural anticancer agents.
European Journal of Medicinal Chemistry Reports 10 (2024) 100137 Available online 23 February 2024 2772-4174/© 2024 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Enhancement of the efficacy of synthetic and natural anticancer agents through nanocarrier for colon cancer treatment Kaushik Mukherjee , Pallobi Dutta , Sourav Dey , Tapan Kumar Giri * Department of Pharmaceutical Technology, Jadavpur University, Kolkata, 700032, West Bengal, India A R T I C L E I N F O Keywords: Colon cancer Nanocarriers Monoclonal antibodies Anticancer agents A B S T R A C T Colon cancer ranks third amongst type of cancer and second in terms of mortality. Various natural and synthetic anticancer agents have been used for the treatment of colon cancer. However, most anticancer agents have poor aqueous solubility and thus low absorptions and bioavailability. Moreover, the chemotherapeutic agents are unable to differentiate between normal and healthy cells and thus kill both types of cells. Nanocarriers have surfaced as a budding anticancer agent delivery system that could enhance the solubility and bioavailability of the anticancer agents and at the same time specifically deliver them to the cancerous cells. In this regard, gold, polymeric, and solid lipid nanoparticles, dendrimers, liposomes, niosomes, and carbon nanotubes have shown promising results as targeted anticancer drug delivery agents with enhanced bioavailability. The objective of this review article is to highlight these nanocarriers and present a detailed overview of how they have enhanced the efficacy of both synthetic and natural anticancer agents.
1. Introduction
Colon cancer is one of the most common type of cancer with a high mortality rate [1]. It is a type of adenocarcinoma and if left untreated, may develop into malignant tumors [2]. The most common causative factors of colon cancer are related to food habits, lifestyle, and genetic mutations. A recent study reveals that environmental factors also promote colon carcinogenesis. The pathophysiology of colon cancer is well established, whereby, it first appears as colorectal polyps (abnormal growths in the inner lining of the colon) and subsequently invades the colonic muscular layers and colon wall [3]. The most common indications of colon cancer are tiredness, constipation, rectal bleeding, bloody stool, and weight loss. Diverse treatment modules for colon cancer are available, which include surgery, radiation, and chemotherapy. Surgical intervention (resection) is a widely adopted treatment option and is critical in controlling the progression of colon cancer, especially in mature stages [4]. Apart from the pain, diarrhoea, and constipation, the prime limitation of surgical intervention is that while removing the cancerous tissues, some part of the healthy colon tissue is also removed [2]. Radiation therapy, which lasts for a few weeks to several months, is also associated with adverse skin reactions, fatigue, gastrointestinal upset, bloody stools and infertility in both males and females. Cancer chemotherapy is by far the most commonly used and successful treatment module for colon cancer. Various natural and synthetic anticancer agents have been developed which can effectively kill the cancer cells either by halting cell division or inhibiting the proliferation of the cancer cells [2]. Capecitabine, fluorouracil, oxaliplatin, paclitaxel, and irinotican are the most prescribed synthetic colon anticancer agents. Likewise, resveratrol, curcumin, quercetin, and lycophene are widely explored natural colon cancer therapeutics. Table 1 lists the various drugs used for the treatment of colon cancer. The major concern of the chemotherapeutic agents is their poor aqueous solubility, which limits their absorption and lowers the bioavailability [1,5]. Additionally, the non-specific distribution of the therapeutic agents is a key issue of systemic chemotherapy. Therapeutic concentrations of the anticancer agents in the tumor cells are achieved at the massive cost of contaminating the healthy cells. This poor specificity leads to serious toxicological side effects and drug resistance [6]. With the advent of advancement in technology, nanotechnology has emerged as the “solution to all problems” in delivering anticancer therapeutics. The size range of nanoparticles (10–400 nm) facilitate them an easy penetration into the cancer cell membrane. Nanoparticles of the size range above 400 nm are also able to get inside the cancer cells by the process of endocytosis [2]. Various nanocarriers like liposomes, niosomes, dendrimers are known to encapsulate hydrophobic drugs * Corresponding author. E-mail address: tapan_ju01@rediffmail.com (T.K. Giri). Contents lists available at ScienceDirect European Journal of Medicinal Chemistry Reports journal homepage: www.editorialmanager.com/ejmcr/default.aspx https://doi.org/10.1016/j.ejmcr.2024.100137 Received 29 September 2023; Received in revised form 16 February 2024; Accepted 22 February 2024 European Journal of Medicinal Chemistry Reports 10 (2024) 100137 inside their structures and successfully deliver them to the target site, thereby enhancing bioavailability [1,19,20]. Additionally, nanoparticles are amenable to various surface modifications, through which the cancer cells can be specifically targeted. Detection of the nanoparticles by the reticuloendothelial system (RES) leads to their expeditious removal from circulation. PEGylation of the nanoparticle surface imparts them with hydrophilic surface properties, making them invisible to the RES. Since cancer cells have an overexpression of the folate receptors, folic acid conjugation to the nanoparticle surface aids in the selective uptake of the nanoparticles by the cancerous cells, thereby promoting targeted delivery of the nanocarrier [21]. The objective of this review article is to discuss the efficacy of synthetic as well as natural chemotherapeutic agents when delivered through nanocarriers for targeting colon cancer. We also aim to discuss the pathophysiology of colon cancer, recent treatment modules and drugs under clinical trial for the treatment of colon cancer.
2. Pathophysiology of colon cancer
Colon cancer is the most complicated (multistep) disease with well- understood molecular genetics [22] as depicted in Scheme 1. The first stage of malignancy is marked by the development of specific types of neoplastic polyps throughout the intestinal mucosa. For assessing the possibility of malignancy, information on polyp histology is important. The two predominant histologic categories are adenomatous and hyperplastic. According to histology, hyperplastic polyps have less cytoplasmic mucus and more glandular cells, but they typically have no nuclear stratification, hyperchromatism, or abnormality. Adenomatous nuclei are often palisade-like, bigger, cigar-shaped, and hyperchromatic. Adenomas can be tubular or villous in nature. Villous adenomas possess digitiform villi grouped in a pattern-like frond, whereas tubular adenomas histologically consist of branching tubules. Epidemiologic, medical, pathologic, and molecular genetic results show that adenomas (the adenoma-to-carcinoma sequence) are the primary cause of the majority of colon malignancies. Colon cancer has been linked to several hyperplastic polyps, although most of them appear to have little to no connection [23]. Risk factors for malignancy in hyperplastic polyps include the development of an adenoma inside the polyp (a combination of hyperplastic-adenomatous polyps), an enormous polyp size (more than 1 cm diameter), the appearance of more than twenty hyperplastic K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 growths throughout the colon, and genetic history of hyperplastic polyposis [24]. Hyperplastic polyps appear to be associated with colon cancer via serrated adenoma [25]. An abnormal cryptepithelial development and nuclear atypia separate a serrated adenoma from an usual hyperplastic polyp that also occurs within the hyperplastic polyp [26]. Serrated adenomas differ from ordinary adenomas in that they usually contain BRAF (proto-oncogene) genetic alterations and show significant DNA methylation but deficit mutations in the adenomatous polyposis coli (APC) gene [27]. A serrated adenoma represents an indicator lesion for high microsatellite-instability colorectal carcinoma (MSI-H) and is responsible for 15% of all sporadic malignancies in the colon. Similar to serrated adenomas, MSI-H colon lesions lack alterations in the cancer-causing APC gene or the K-ras oncogene but possess vast methylation of DNA and BRAF gene alterations. Gene expression can be stopped and silenced by DNA methylation at the promoter area without causing DNA mutation [28]. Microsatellite instability (MSI) is a condition when DNA methylation, for instance, prevents DNA mismatch repair (dMMR) genes like the MutL homolog 1 or 2 (MLH1 or MLH2) gene from functioning. Somatic mutations of spastic oncogenes (rat Sarcoma (RAS), myelocytomatosis oncogene (MYC), and SRC) have connections with CRC having the most clinical relevance [29,30]. RAS mutation variations (HRAS, NRAS, KRAS) are present in half of CRC sporadic cases and are now being explored on CRC screening by stool-DNA testing, the lack of epidermal growth factor receptors (EGFR) targeted therapeutic response and possible direct targeted drugs. The possibility of specific MMR gene mutations in human mutS homolog 2 and 6 (hMSH2, hMSH6), human Mismatch Repair System Component homolog 1 and 2 (hPMS1, hPMS2), and hMLH1, and hMLH3—all of which interact with MLH1 and are each thought to be present in almost 15% of all sporadic CRC and therefore calls for universal testing. These mutations can result in a Lynch-like condition including MSI-H. Cyclooxygenase (COX-2) and peroxisome proliferator-activating receptor (PPAR) genes have been found to be linked to CRC carcinogenesis and are currently being investigated for chemo-protection.
3. Conventional treatment of colon cancer
Treatment choices for individuals vary and are evaluated depending on the size of the tumour, the stage of the screening, the area of the colon where the tumour is located, the likelihood that cancer will return, and the patient’s state of health. In addition to surgical therapy, developing various powerful cytotoxic and targeted medicines has increased survival [31]. There are several different types of treatment available for colon cancer, such as surgery, radiation therapy, and administration of chemotherapeutic agents.
3.1. Surgery
Surgical intervention, often known as surgical resection, is the most commonly utilized therapy in many patients. Controlling the spread of cancer, especially, when it is at an advanced or mature stage, requires surgical excision [4,32]. Tumour stage and localization have an impact on the surgical strategy for CRC [33]. The primary objective of colon cancer surgery is to do a significant resection, which involves removing the main tumour and local lymphatics with precise surgical margins. Laparoscopic or open surgery can be used to accomplish surgical treatments [34]. One of the primary drawbacks of surgical resection is that, while removing the malignant tumour, a portion of the healthy colon may also be removed. In addition, surgery can be painful and might lead to constipation and diarrhoea.
3.2. Radiotherapy
Radiation therapy is a different method of treating colon cancer, in Scheme 1. Schematic representation of the pathophysiology of colon cancer. K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 which malignant tumors are exposed to several radiation treatments over the course of several weeks or months [35]. Radiation therapy is used before surgery much more commonly than after surgery to reduce the size and stage of advanced cancers and hence avoid local recurrence. It is used to treat advanced malignancies as a palliative and to decrease tumors before surgery, as well as to eliminate any cancer cells that could survive the procedure. Radiation therapy can have numerous adverse effects, including exhaustion, skin responses, and digestive disturbance, but it can also occasionally result in bloody stools and issues with fertility in both male and female sufferers.
3.3. Chemotherapeutic drugs
Anticancer drugs can also be used to treat cancer. Anticancer drugs that are produced synthetically are used to eradicate cancer cells by preventing cancer cells from dividing or reducing the growth of cancer cells. These anticancer drugs successfully treat cancerous cells. The structures of various chemotherapeutic agents used in the treatment of colon cancer are depicted in Fig. 1. The conventional drugs used as chemotherapeutic agents in the management of colon cancer are of synthetic origin and natural origin. 1 Synthetic drugs The various synthetic anticancer agents that are used in the treatment of colon cancer are given in Table 2. 2 Natural products The various natural anticancer agents that are used in the treatment of colon cancer are given in Table 3.
4. Why need nanocarrier for colon cancer treatment
Conventional treatments for cancer include surgery, radiation, and chemotherapy. It is challenging to totally cure the disease due to the limits of these treatments. Patients eventually develop undesirable side effects like anaemia, diarrhoea, neutropenia, gastrointestinal (GI) poisoning, nausea, pustules, vomiting, hematologic disorders, tiredness, and liver damage as an outcome of conventional chemotherapy for colon cancer which involves delivering drugs to non-target areas [47,48]. As a result of the increased chances of adverse effects, which eventually culminate in drug resistance, the results of treatment so far have not been satisfactory [49]. Surgery is therefore the main form of therapy, and it cures colon cancer in around 50% of suffering people. However, the recurrence of colon cancer after surgery is a serious downside that frequently results in fatalities. Despite long-standing attempts to enhance patient survival by early diagnosis and efficient treatment methods, the success rate is very low, and the use of radiation therapy, chemotherapeutic agents, and/or other treatments requires further development. The discovery of molecular biomarkers with a diagnostic and/or therapeutic utility has been intensively studied in order to combat the fundamental negative repercussions. The genetic and epigenetic K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 pathways appear to be interrelated in promoting the growth of colon cancer. Most chemotherapy drugs lack the ability to distinguish between malignant and healthy cells, which can have harmful side effects and systemic toxicity. The maximum allowed systemic dose is severely constrained by the severe adverse effects in other tissues, which prevents proper drug concentrations from reaching the tumour. In recent years, novel techniques have been discovered to address these limitations while simultaneously increasing the efficacy of drugs [50]. The development of nanomaterial science has made it feasible to create nanomaterials that can be employed for the early diagnosis and management of cancer in an efficient manner. Nanoparticles (NPs) have lately gained greater interest due to their biocompatibility and other physicochemical properties which include the ability to conjugate, degrade in ambient biological environments, high surface-to-volume ratios, and intriguing functional groups on the surface [51,52]. Nanoscale compounds such as metal, oxide of metal, polymeric material, and biodegradable biopolymer have resulted in important breakthroughs in nano-drug preparations for targeted therapy. Nano-bioconjugation is important in targeted cancer treatment due to the formation of covalent bonds of cancer receptors, cytotoxic chemotherapeutic drugs, ligands, antibodies, and immunotoxins [53]. NP systems can administer chemotherapeutic agents to tumour locations using either active or passive targeting, increasing treatment efficacy and decreasing negative side effects. They are widely recommended across the world for selectively detecting cancer cells and treating them with minimum side effects due to their non-toxicity, and they can easily be enclosed with other drugs in combination with biomarkers that are fluorescent-tagged cancer-specific.
5. Nanoparticulate drug delivery for colon cancer
Paul Ehrlich was the first to propose targeted treatment as an approach to achieve therapeutically successful dosages to the target site while protecting healthy tissue. A successful nano-drug carrier system must include a site-specific drug nanocarrier be coupled with particular biomolecules, and be capable of overcoming multiple biological barriers, with or without the assistance of the body’s immune system [54, 55]. When given intravenously, nanoparticles might be taken up by the reticuloendothelial system (RES) or macrophage system by means of a process called opsonization [56,57]. As a result, for NPs to be effective as drug carriers, they must be capable of targeting polyps that are situated outside the organs with a high concentration of mononucleus phagocytes. The size of nanocarriers is thought to be an essential consideration in determining drug delivery effectiveness with fewer adverse effects. In general, nanoparticles with sizes ranging from 10 to 800 nm should be permeable through cancerous tissue but not healthy tissue. The primary drawback of drug delivery nanocarriers is the premature release of drugs and the exclusion of nanocarriers from circulation when detected by the body’s self-defence systems, which finally deprives the particles [58]. Recently, certain methods utilizing polymer microspheres and NPs have been created expressly for the treatment of colon cancer. The initial strategy relies on oral prodrug delivery; the prodrug is converted into an active ingredient through digestive enzymes and microorganisms as it travels across the gastrointestinal tract and successfully fights against cancerous cells when it enters the colonic region. The second method is to create a delivery system dependent on the time it takes to travel from the upper part of the GI tract to the colon area. The third strategy involves employing pH-sensitive polymers to provide a pH-dependent drug delivery. Many biological enzymes tend to be less reactive in the colonic area due to their neutral pH against the acidic environment of the small intestine. The pH-sensitive polymers used to encapsulate drugs give them the ability to withstand an acidic environment while still releasing them in a pH-neutral environment. The fourth strategy makes use of the colonic microflora. The enzymes secreted by the microbes residing in the colon can break down the polymer substrate and discharge the drug molecules in the site of polyps in the colon, enhancing colon-specific treatment of colon cancer. Nanocarrier surface chemistry plays a vital role in enhancing the duration of circulation of nanocarriers in blood for drug targeting. Surface modification has been explored extensively over the past decade in an effort to create "stealth" particles or PEGylated NPs, which can evade macrophages [59]. A wide variety of nanotechnologies involving biodegradable polymers, like polycaprolactone (PCL) or poly-lactic-co-glycolic acid (PLGA), chitosan (CS), lipids (solid-lipid NPs, liposomes, nano-liposomes), as well as other nanosystems such as quantum dots (QDs), micelles, iron oxide magnetic nanoparticles (MNPs), mesoporous silica NPs (MSNs), and carbon nanotubes (CNs) and have been researched (Fig. 2) [60]. The most significant features of NP-based drug delivery systems include sustained release of drugs, lower adverse reactions, and extended circulation throughout the body [61].
5.1. Liposomal nanocarriers for colon cancer treatment
Liposome is firstly approved nanocarriers which is mainly used for drug delivery as described by FDA [62]. Cholesterol and other phospholipids—i.e., nonimmunogenic biopolymers—are used to create K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 anionic, cationic, and neutral liposome nanoparticles (LNPs). Furthermore, lipid bilayers were easily loaded onto receptors and ligands that are particular to cancer cells to improve the site-specific delivery of therapeutic agents.
5.1.1. Synthetic anticancer agents loaded liposomal nanocarriers for colon cancer treatment
HCT8/ADR cancer cells that are abundant in mannose receptors make up human colon tumors. The anti MDR effects of dihydroartemisinin (DHA), when combined with doxorubicin (DOX) in drug tolerant human colon tumor HCT8/ADR cells were evaluated [24]. They created a technique for co-encapsulating two medications into mannosylated liposomes (Man-liposomes), which targets tumors. The Man-liposomes with a zeta potential of − 15.8 mV and a mean diameter of 158.8 nm changed how Dox was distributed within cells, which led to a significant entry of Dox into the cells and the highest cytotoxicity level. The nuclear accumulation of drug is particularly required to enhance cell death, autophagy and to reduce the regulation of Bcl-xl. The treatment of CRC frequently targets the EGFR. Delivery of EGFR-targeted nanoparticles in a mouse model significantly improved the inhibition of CRC growth [63,64]. CPT-11 (irinotecan) is an efficacious chemotherapy drug for CRC. In vitro and in vivo evaluations of the CPT-11-loaded 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[a mino(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG 2000) revealed its therapeutic potential and targeting specificity on colon cancer cells [65]. Using a self-assembly process, the DSPE-PEG2000 liposome (Lipo-CPT-11) loaded with CPT-11 was created [66]. The DSPE-PEG2000 loaded with CPT-11 targeting EGFR liposome complex (EGFR-Lipo-CPT-11) was created by combining the monoclonal antibody that is specifically directed against the EGFR with the DSPE-PEG2000 liposome. The EGFR-targeting DSPE-PEG2000 liposome treatment shown significantly greater anticancer activity in vitro and in vivo as compared to the non-target DSPE-PEG2000 liposome loaded with CPT-11. Various features of copper (cu) complexes such as bioavailability, structural flexibility, redox characteristics can offer themselves to be used in newly developed medications for cancer treatment [67,68]. In case of colon cancer, a cu complex with phenanthroline ligands (Cuphen) was created, which has strong antiproliferative effects in vitro in pH-sensitive long circulating liposomes [69]. The pH-sensitive liposomal formulations containing Cuphen were created, characterized, and validated, and the antiproliferative activities in cancer cells were verified. The effects of cuphen on cell migration and cell cycle were also assessed. In both human and mouse colon cancer cells, the aquaporin-3 (AQP3)-mediated inhibition of glycerol permeability has been studied. AQP3 is known to be involved in the pathophysiology of CRC. The tumor microenvironment, which consists of both tumor and non tumor cells, has received significant attention in recent treatment approaches to cancer cells. Macrophages, also known as tumor-associated macrophages (TAMs), are significant components of tumor locations. TAMs are a crucial group of inflammatory cells in solid tumors and have a significant impact on the release of inflammatory cytokines. Additionally, due to their amphiphilic nature, LNPs ensure the delivery of anticancer medications which are hydrophobic in nature. The macrophages present in tumor microenvironment are classified into two polarization states such as M2 (pro tumor) and M1 (anti tumor). These two phenotypes in particular exhibit a divergent impact on the cancer’s immune system. Due to the release of vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and matrix metalloproteinase-9 (MMP-9), the M2 phenotype is also called as a pro angiogenic, pro-tumorigenic, and pro-metastatic phenotype. Through the direct elimination of cancer cells through the release of reflexive nitric oxide (No) and the induction of immunological responses in the tumor, the M1 phenotype strengthens the antitumor responses. It has been demonstrated that the cytokine profile in the tumor microenvironment correlates with the polarization of macrophages. M1 phenotype containing high Major Histocompatibility Complex supply kills pathogens with the help of Type 1 T helper (Th1) cytokines including interleukin 12 (IL-12), TNF-α, and interferon gamma (IFN-γ). TGF- β and interleukin-2 induce macrophage polarization into the phenotype of M2, which plays a role in promoting tumor development and proliferation of cell. The anticancer activity of macrophages is now dependent on a number of signaling pathways, namely nuclear factor-κB(NF-KB) and signal transducer and activator of transcription-1 (STAT1), which suppress the release of pro-inflammatory cytokines. IFN-γ activates the NF-KB pathway, results in increasing the M1 phenotype in TAMs [70]. The efficacy of PEGylated and non-PEGylated liposome containing IFN-γ to induce M1 phenotype macrophage in vitro was tested [71]. The in vivo biodistribution and anticancer effects of LNPs were tested on mice containing C26 tumors. It can be revealed that the liposomal formulation containing IFN-γ is more potent than free IFN-γ because the liposomal formulations directly target the tumor site and provide the anticancer effects in tumor cells.
5.1.2. Natural anticancer agents loaded liposomal nanocarriers for colon cancer treatment
Curcumin which is derived from turmeric (Curcuma longa L.), is not only used as spice but also it has therapeutic effects. Various pharmacological effects such as antibacterial, antioxidant, anti-inflammatory, anticoagulant, antitumor are there in curcumin. Low solubility, high first pass metabolism, poor absorption limit its applicability. In order to improve delivery kinetics and solubility β-cyclodextrin-curcumin (βCDC) inclusion complexes were developed and this complexes were hydrophilic in nature [72]. Both the hydrophilic and hydrophobic curcumin were entrapped into the liposomes separately and checked for in vitro cytotoxicity. The βCD-C complexes showed enhanced solubility as compared to free curcumin and successful loading into the liposome was achieved by thin film hydration approach. MTT assay confirmed that all curcumin loaded formulations were efficacious in arresting cell proliferation. Artemisinin (ART), a sesquiterpene, is obtained from Artemisia annua. In spite of having antimalarial properties it is currently reported as a novel anticancer medication. A transferrin-conjugated liposome loaded with ART (ART-L-Tf) was created and compared with ART loaded stealth liposomes(ART-L) [73]. Drug entrapment efficiency, transferrin coupling moieties, size and stability were the important parameters and the liposomes were characterised based upon them. The results confirmed that the enhanced delivery of ART-L-Tf as compared to ART-L in targeting cancer cell and improved cytotoxicity because of iron ions resulted in concomitant synergism due to transferrin receptor overexpression on tumor surface.
5.2. Gold nanocarriers for colon cancer treatment
Chemotherapy is one of the most popular cancer treatments, but it has a number of drawbacks, including a number of adverse effects from chemical interactions with tumor and normal cells that are non-specific, low solubility, and poor biodistribution [8]. One of the most potential solutions to the drawbacks is the use of drug delivery systems, which might offer a more effective targeted transport of molecules, boost solubility, improve biodistribution, and prevent rapid removal of the therapeutic from the organism. One of the materials that is now being explored is AuNPs, which have special adjustable features that make them useful for CRC bioimaging, diagnostics, and treatment [74]. The high surface-to-volume ratio of AuNPs facilitates both their functionalization for precise targeting and use in drug delivery. AuNPs can also be altered to meet the intended application depending on their physical characteristics (size, form). AuNPs are simple to make and have tunable features that enable them to be made in a variety of forms and sizes. Depending on their size, shape, and physical properties, AuNPs can take the form of nanorods, nanospheres, nanoshells, nanotriangles, nanohexagons, nanostars, nanobranched, and hollow spheres. AuNPs show surface functionalization, high surface-volume ratio and distinctive optical features due to Surface Plasmon Resonance (SPR) phenomenon. K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 SPR happens when photons or other radiations excite the gold surface layer’s electrons, causing them to absorb and scatter light. Surface enhanced fluorescence (SEF), photothermal conversion, and colorimetric reactions can all be brought about by SPR of AuNPs. AuNPs can be conjugated with biomolecules to offer particular identification thanks to their surface functionalization. By covalent or non-covalent chemical bonding, physical encapsulation, or both, AuNPs can be functionalised to carry different medicines.
5.2.1. Synthetic anticancer agents loaded gold nanocarriers for colon cancer treatment
Anti-EGFR antibodies coated AuNPs were created and loaded with 5- fluorouracil (5-FU) and were intended to be targeted at CRC cells that overexpress EGFR [75]. The prime objective of the experiment was to enhance the effectiveness of 5-FU. The drug antibody combination not only enhanced the apoptosis rate but also decreased cell proliferation. Treatment of metastatic CRC has benefited greatly from the use of anti-EGFR specified monoclonal antibodies (ex: panitumumab and cetuximab). However, a variety of resistance mechanisms restrict the pharmacological effect of anti-EGFR targeted monoclonal antibodies. Development of cetuximab-conjugated AuNPs (cetuximab-AuNPs) to augment the cytotoxicity against cancer cells as a result of the discovery of novel uses for AuNPs, as well as to cause an alteration in the expression of the associated biomarkers on cancer cell surface is reported in literature [19]. The expression of cell surface biomarkers as well as size dependent cytotoxicity study against CRC cell line were performed in response to the treatment with Cetuximab-AuNPs and Cetuximab. AuNPs with a size of 60 nm had the greatest efficacy on cytotoxicity and the cell counting kit-8 (CCK-8) assay confirmed that. When cetuximab was coupled to AuNPs, it was discovered that three cell surface biomarkers with increased heterogeneity were expressed, including melanoma cell adhesion molecule (MCAM), epithelial cell adhesion molecule (EpCAM), and human epidermal growth factor receptor-3 (HER-3). DOX loaded AuNP-based drug delivery system in CRC using G-C rich oligonucleotide (ONT) coated AuNPswith DOX intercalated into them [76] (Fig. 3). Numerous DOX binding sites are present on ONT-modified AuNPs, which makes it easier to deliver large amounts of medication into cancer cells. Using the MTT assay, the anticancer activity of DOX-loaded AuNPs coated with ONTs (Doxorubicin-Oligomer-AuNP, DOA) was performed in the human CRC cell line SW480. They proved that CRC cell can successfully uptake DOA. By using low concentration significant therapeutic effect was achieved. The efficiency and organization of vascular perfusion are crucial for the delivery of oxygen and drugs to tumors. However, solid stress within the tumor microenvironment causes improperly compressed tumor vasculature, which leads to the creation of hypoxic regions and impairs intratumoral medication delivery. The potential of gold nanoparticles (AuNPs) to constrict the arteries of CRC, improve vascular perfusion, and improve medication delivery in CRC were examined [77]. Through the Akt-signaling pathway, it was found that AuNPs could decrease the level of tumor stromal collagen I, lowered the density of CRC-associated fibroblasts (CAFs), and reduced the expression of profibrotic signals like VEGF, transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), in both vivo and in vitro. Therefore, AuNPs could decrease the tumor stress, which would then result in improved vesicle perfusion. Therefore, AuNPs reduced hypoxia and improved both medication and oxygen delivery to tumor cells. Methotrexate-loaded gold nanoparticles (AuNPs) were formulated [78]. Both the efficacy and anticancer activity were tested in colon cancer cell. The drug conjugated AuNPs not only increased the therapeutic efficacy by inducing apoptosis but also decreased the effective dose of MTX in colon cancer cell line. The anticancer activity proceeded via internalization of MTX, attaching to dihydrofolate reductase (DHFR) and interrupting the folic acid synthesis. This has led to the widespread usage of AuNPs in biomedical applications such as drug administration, photothermal treatment, radiation, and imaging.
5.2.2. Natural anticancer agents loaded gold nanocarriers for colon cancer treatment
Trans-resveratrol has cancer-targeting capabilities that could be used in conjunction with radionuclide imaging to identify malignant spots throughout the body. In order to increase the solubility of resveratrol and to provide biological and chemical protection, radio labelled resveratrol loaded gold nanoparticles that were biocompatible water soluble carriers were developed [79]. As a result, they were suitable for a variety of in vivo applications.
5.3. Dendrimer nanocarriers for colon cancer treatment
Dendrimers are defined as a monomeric, hyperbranched nanomaterials with numerous branches that look like trees starting from central core [80]. The branches of this dendrimer contain various cationic, anionic and neutral groups which can be further modified with a variety of functional groups to target specific site. Dendrimers are produced through a series of synthetic stages in which the incorporation of the repeating of monomer units results in dendrimers with multiple generations. The addition of monomeric branches to the core during synthesis is referred to as a dendrimer generation. In spite of being opposite to linear polymers dendrimers have special polymeric features such as molecular weight, monodispersity, controlled size, and form. Dendrimers are also excellent nanocarriers for encasing or conjugating molecules due to their interior molecular spaces and periphery functional groups. The active medicinal components and targeting ligands can be loaded onto the surface of dendrimers through chemical linkages, hydrogen bonds, or hydrophobic interactions. Dendrimers are also a prime possibility for site specific delivery of biological molecules such as medications, genetic material, or bioimaging agents due to their solubility, biodegradable backbones, stability and penetrating capacity. There are specific dendrimers available to target tumor site such as poly (propyleneimine) (PPI), polyamidoamine (PAMAM), and poly(L-lysine) (PLL). In cancer therapy dendrimers are used specifically to target solid tumors and a major idea in this process is the "Enhanced Permeability K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 and Retention" (EPR) effect, also known as primary targeting or passive accumulation.
5.3.1. Synthetic anticancer agents loaded dendrimer nanocarriers for colon cancer treatment
Enhancement of the anticancer activity of Capecitabine (CPB) using G4 PAMAM dendrimers and eliminating the non-specific side effects on other organs like bone marrow and liver has been reported [23]. Without conjugating any targeted ligand, the researchers looked at the effects of dendrimer delivery and unconjugated CPB in reducing tumors in mice models. The displayed a noticeable shrinkage in size of tumor compared to the free form, as well as less negative effects on the liver and blood. Only static tumors with high permeability are relevant to the passive targeting technique or the EPR effect. However, the permeability of colon cancer cells in an advanced stage is generally weak or unequal across the entire heterogeneous tumor population. These issues can only be solved via site specific targeting technique with the help of targeting ligands. It is important to attach certain types of ligands on nanocarrier’s surfaces and in case of dendrimers, terminal functional groups can be modified with this ligand to target specific overexpressed receptor on cancer cells [81]. In order to carry camptothecin (CPA) a pegylated polyamidoamine dendrimers G4 (PAMAM) has been developed [82]. AS1411 anti-nucleolinaptamers can be functionalised with dendrimers for site-specific binding to colon carcinoma cells overexpressing nucleolin receptor. Both HT29 and C26 CRC cell line were more sensitive to the specific CPA-loaded pegylated dendrimers than the CHO cell line, according to an MTT assay. On mice with C26 tumors, the exact same approach was successfully tried in vivo. Irinotecan is clinically used to treat CRC, but due to its limited therapeutic index, it is not as effective as it may be. Development of a 5 generation L-lysine dendrimer which could be further modified with polyoxazoline in order to improve the therapeutic index of SN-38, an active metabolite of irinotecan is also reported [83]. The drug’s release rate was altered by attaching SN-38 to dendrimer with the help of different linker. By controlling release rate of the drug from the linker and extending circulation time of the dendrimer, an effective dose of SN-38 could be administered, leading to a considerable regression of the SW620 tumors. Narmani and co-workers developed oxaliplatin loaded polyamidoamine dendrimers G4 (PAMAM) imprinted with polyethylene glycol (PEG) and folic acid (FA) (PEG-PAMAM G4-FA-OX) as novel nanocomplex for targeted delivery of oxaliplatin (OX) to tumor cells [84]. Firstly, polyethylene glycol (PEG) was used to imprint the surface of polyamidoamine dendrimers G4 (PAMAM G4) nanoparticles and then folic acid (FA) was used to functionalize them. Oxaliplatin (OX)’s anti-cancer efficacy was modified utilizing a nanocarrier based method with enhanced targeting specificity towards CRC cells expressing folic acid receptors (FAR). OX was found to have a better cellular absorption in the SW480 cell line. Cell viability tests clearly proved the effects of PEG-PAMAM-FA-OX on the inhibition of cancer cell proliferation.
5.4. Niosomal nanocarriers for colon cancer treatment
5.4.1. Synthetic anticancer agents loaded Niosomal nanocarriers for colon cancer treatment
Synthetic chemotherapeutic agents are associated with poor solubility, low absorptions and low oral bioavailability. Niosomes (also known as non-ionic surfactant vesicles) are nanoparticulate drug delivery systems that are bilayered vesicles having both polar and non-polar regions and are capable of encapsulating hydrophobic and hydrophilic drugs inside their vesicles [85]. They are cheap and highly stable nanocarriers that can be used to alleviate the problems associated with the delivery of hydrophobic chemotherapeutic agents and are now widely researched for the same. Successful niosomal delivery of oxaliplatin and paclitaxel have been reported in literature with enhanced bioavailability and reduced toxicity for colon cancer treatment [86]. The effect of cholesterol and surfactant molar ratio, surfactant type (tween 80, span 60 and D-α-tocopheryl polyethylene glycol 1000 succinate (TPS)) on the zeta potential and particle size of the niosomes were evaluated. The particle size range of the niosomes were between 189.2 ± 13.4 nm to 293.3 ± 17.2 nm. A polydispersity index less than 0.3 indicated formulation homogeneity. Niosomes prepared with tween 80 and TPS displayed a decrease in particle size with increase in cholesterol content, while the reverse phenomenon was observed with niosomes prepared with span 60. A higher surfactant decreased in the noisome particle size. The surfactant and cholesterol ratio had a positive effect on the zeta potential (32.7 ± 1.01 and 31.69 ± 0.98 mV) of the niosomes. Paclitaxel and oxaliplatin entrapment onto the niosomes were 93.51 ± 2.97 % and 90.57 ± 2.05 % respectively. In-vitro oxaliplatin and paclitaxel release was done using the dialysis bag method. Oxaliplatin release from the niosomes (87.5 ± 1.99%) was significantly higher as compared to the release of free oxaliplatin (19.4 ± 1.76%) after 24h. Similarly, paclitaxel release from niosomes was 80.81 ± 2.98% and free paclitaxel release was 14.77 ± 0.98% after 24h.The smaller vesicular size coupled with the presence of the surfactants resulted in improvement in drug release from the niosomes. Cytotoxicity of paclitaxel and oxaliplatin loaded niosomes was studied against HT-29 cells using the MTT assay method and the results were compared with free drugs and plain niosomes. It was observed that the niosomal drugs had a two-fold increase in the cytotoxicity as compared to free drugs. The cytotoxicity effect was also dose dependent. A remarkable decrease in the IC50 value (two-fold for oxaliplatin and three-fold for paclitaxel) from the niosomal drugs compared to the free drugs indicated the significant ability of the noisome formulation to accentuate the cellular uptake of the drugs. The apoptotic effect was significantly higher from the niosomal drugs when compared with blank niosomes and free drugs. These results indicated an enhancement of the bioavailability, reduction in toxicity to normal cells and improvement in the therapeutic effect of the drugs from the noisome nanocarrier formulations.
5.4.2. Natural anticancer agents loaded Niosomal nanocarriers for colon cancer treatment
Chemotherapy of various synthetic anticancer agents is associated with the limitations of drug resistance upon long term treatment, low immune response, hormonal imbalance, and fatigue [87]. This results in high prevalence and recurrences of CRC. Research shows that alternative treatment regimens consisting of probiotics (mainly from bacteria and yeasts) and natural bioactive (plant secondary metabolite like silibinin, curcuminoids, resveratrol, chrysin, and helenalin) are equally effective as chemotherapy, and additionally not prone to the development of drug resistance. Literature also reveals that dead probiotics or their secretions show same or even greater biological activity. Few researchers developed curcumin loaded niosomal nanoparticles for evaluating their anticancer activity on human colon cancer cell lines [1]. The niosomes were also loaded with the yeast Saccharomyces cerevisiae. Attachment of polyethylene glycol (PEGylation) was done to the niosomal external surface to enhance the pharmaceutical attributes of the noisome vesicles. The thin film hydration method was used to prepare the PEGylated niosomes. The preparation method involved dissolving the required quantities of PEG, cholesterol, and span 60 in methanol and chloroform. Then removal of solvents was done by a rotary evaporator. The resulting dispersion was brought to room temperature and then phosphate buffer solutions were added to it and again connected to the rotary evaporator. The final dispersion was ultrasonicated and stored at 4 ◦C for further use. The mean diameter of curcumin free and curcumin loaded niosomes was 139 ± 5.67 and 201 ± 9.94 nm respectively, as measured by the dynamic light scattering method. SEM pictures indicated smooth, spherical niosomes particles which were devoid of any pores or perforations (Fig. 4). Curcumin encapsulation efficiency was around 88% onto the niosomes. In vitro curcumin release after 96h at physiological pH 7.4 and cancer cell pH 5 was 59% and 74% respectively. The curcumin release was biphasic, where 49% and 29% of the K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 curcumin was released with 10h of dissolution study at the respective pH. This was due to the dissolution of curcumin at the niosomal surfaces. The cytotoxic effect of free curcumin and niosomes loaded with curcumin were investigated on SW480 CRC cell lines. The IC50 values of free curcumin (16.72 μM) and curcumin loaded niosomes (11.86 μM) indicated that curcumin loaded niosomes had more lethal cytotoxic effect than free niosomes at the same dose. This is due to enhanced solubility, absorption, and overall optimum bioavailability of curcumin from the niosomes. Colorectal metastatic genes (COL10A1, MMP2, and MMP9) expression study was performed on SW480 cell lines using RT-PCR. The study revealed that the expression of metastatic genes was significantly downregulated on curcumin loaded niosomal groups as compared to the free curcumin group, due to enhanced bioavailability of curcumin from the niosomes. Flow cytometry and annexin assay were performed to study the proapoptotic effect of curcumin on SW480 cell lines. Free curcumin induced apoptosis on the cell lines. Niosomal curcumin induced both late and early apoptosis, and the apoptotic potential was significantly more as compared to free curcumin. Cell cycle analysis (of SW480 cell lines) was performed to analyze the cell growth inhibition due to treatment with curcumin. The study indicated normal cell growth for the control group. However, the niosomal curcumin treated group revealed an increase in the cell number in the G2/M phase, indicating cell cycle was arrested in the G2/M phase. The author concluded that niosomal curcumin could be used for the treatment of CRC.
5.5. Polymeric nanocarriers for colon cancer treatment
5.5.1. Synthetic anticancer agents loaded polymeric nanocarriers for colon cancer treatment
Doxycycline is an extended spectrum antibiotic mainly used for the treatment of acne [88]. It was repurposed as an efficacious anticancer agent because of its ability to inhibit mitrochondrial biogenesis in cancer cells [89,90]. However, doxycycline is extremely harmful to normal cells due to enzymatic changes and inhibitions in protein synthesis [91]. It is reported that polymeric nanoparticles (PLN) can effectively release the drug at the target site, resulting in enhanced drug efficacy and therapeutic effect and significant attenuation in side effects [9]. Doxycycline loaded pH sensitive PLNs were synthesised by nanoprecipitation technique using hydroxypropylmethyl cellulose phthalate HP55 (HPMCP 55) or eudragit S100 (ES100) [92]. The therapeutic potential of the doxycycline PLNs was tested against experimentally induced colon cancer in mice. The type of polymer had an impactive effect on the drug release from the PLNs. Doxycycline release from eudragit PLNs were much higher than from HPMCP 55 PLNs. Moreover, HPMCP 55 PLNs dissolved at greater pH than eudragit PLNs and both polymers showed a more delayed release of doxycycline than the pure drug. Since HPMCP 55 PLNs showed better drug release, it was selected for in-vivo studies. The authors used 1,2-Dimethylhydrazine to induce colon cancer on mice. Histopathological study indicated that the control group (treated with normal saline) colon tissues showed normal mucosa, submucosa and musculosa. The negative control group (treated with only 1,2, -Dimethylhydrazine) showed hyperplasia, irregular mucosa, distorted crypts and disintegrated goblet cells. Group treated with free doxycycline showed moderate dysplasia, lesser inflammatory cells and higher count of goblet cells. The group treated with HPMCP 55 PLNs showed almost normal mucosa, submucosa, crypts and very few inflammatory cells. Tumour scores of the colonic tissues were also generated. Cryptic distortions score for negative control group were higher than the control group, the same was reduced significantly in HPMCP 55 PLNs group. The same pattern of scores in the groups were also observed for dysplasia, depletion in goblet cells, and hyperplasia. Western blot studies indicated that the HPMCP 55 PLNs group had reduced angiogenic factors CD31 and VEGDas compared to free doxycline treated group. ELISA study pointed out the normal levels of proangiogenic factors (VEGF and IL-6) in the control group. There was 3–4fold increase in the levels of proangiogenic factors in the negative control group. The values were somewhat reduced in the free doxycline treated group and significantly reduced in HPMCP 55 PLNs group. The combined results of histopathological images, tumour scores, Western blot and ELISA study indicated the efficacy of PLNs towards improvement in therapeutic anticancer effect of doxycycline with reduced toxicity towards the normal cells.
5.5.2. Natural anticancer agents loaded polymeric nanocarriers for colon cancer treatment
Natural products obtained from foods have shown promising anticancer activity. Plants produce a low molecular weight polyphenolic compound called flavonoids, which is known to have immense health benefits and are found in foods [93]. Quercetin is one of such flavonoid compounds that possess anticancer activity, can monitor the cell cycle, induce apoptosis, and inhibit tyrosine kinase activity [94,95]. Another polyphenolic compound caffeic-acid phenethyl ester (CPE) also have apoptotic inducing effects in cancer cell lines, additionally, it shows selective cytotoxicity to cancer cells without posing any harm to the healthy cells [96,97]. Both quercetin and CPE have hydrophobicity, low stability, and diminished bioavailability [98]. Nanoparticulate delivery systems could alleviate the above problems. Biodegradable and biocompatible polymeric nanoparticulate delivery systems are much K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 preferred because of their enhanced encapsulation efficiency, lower side effects and controlled release attributes. In this regard, poly (lactic-co-glycolic acid) (PLGA) nanoparticles are widely used because of their easy elimination by normal metabolic pathways. Combined use of polyphenolic compounds has shown greater anticancer activities, than when used alone [99]. Colpan and his coworkers reported the development of quercetin-CPE PLGA nanoparticles and investigated its anticancer potential on human colorectal HT-29 cells [100]. Depending upon the formulation variables, the average particle size of the nanoparticles was 237.8–512.6 nm. The polydispersity index revealed uniform particle size of the nanoparticles. Zeta potential values of the nanoparticles were in between − 22.8 and − 6.87 mV, indicating stable nanoparticles. Reaction yield of the nanoparticles was calculated at 64% (w/w) for CPE and 70.09% (w/w) for quercetin. In vitro quercetin and CPE release was performed for 30days at pH 7.4 and pH 5.5. At pH 7.4, the release study indicated a burst release in the first 7 days of the dissolution study releasing 55.56% and 36.59% (w/w) of CPE and quercetin respectively. Thereafter, quercetin and CPE release were sustained, and after 30 days 47.19% and 63% (w/w) of quercetin and CPE were released from the nanoparticles respectively. At cancerous cell pH 5.5, the release profile was similar to that in physiological pH 7.4, showing a burst release in the early days of the study, after which the release was sustained. After 28 days, the cumulative release of CPE and quercetin was 86.34% and 82.44% (w/w) respectively. The fast release of the bioactive at pH 5.5 was due to the pH responsive degradation of PLGA at low pH. In vitro cytotoxicity of free quercetin-CPE and nanoparticles were evaluated for 24h and 48h against HT-29 cells. The nanoparticles indicated a time and dose dependent effect on cell proliferation. At 24h, the IC50values of free quercetin-CPE and quercetin-CPE nanoparticles were 53.4 mg/mL and 11.2 mg/mL respectively. It decreased to 15.5 mg/mL and 8.2 mg/mL respectively after 48h. Higher doses of free quercetin-CPE and quercetin-CPE nanoparticles decreased cell viability up to 32.1% and 19.9% respectively. More importantly, lower doses of quercetin-CPE nanoparticles displayed greater cytotoxic activity than free quercetin-CPE on HT-29 cells after 48 h treatment. The increase in the cytotoxicity profile of the nanoparticles is due to their increased stability and controlled release attributes. Metastasis of HT-29 cells was also studied. It showed that migration of the free HT-29 cells was increased after 72h in the control group. However, it was 27% and 4% for free quercetin-CPE and quercetin-CPE nanoparticles after the same time period. Cell cycle proliferation was studied using Proliferating cell nuclear antigen (PCNA). Overexpression of PCNA would indicate proliferation in the cell cycle. PCNA positive cell count was significantly less in the quercetin-CAPE nanoparticles treated group than in the control group. The cells treated with free quercetin-CAPE didn’t show any reduction in the PCNA positive cell count. The results demonstrated improved anticancer activity of the quercetin-CAPE nanoparticles.
5.6. Solid lipid nanocarriers for colon cancer treatment
5.6.1. Synthetic anticancer agent loaded solid lipid nanocarriers for colon cancer treatment
Chemotherapy is one of the first line treatment regimens in cancer patients. Chemotherapy is indispensable in late stage (stage 3 and 4) cancer where metastasis is confirmed. Though chemotherapy is a widely accepted treatment protocol, it is associated with several drawbacks. Chemotherapy drugs, being primarily given as intravenous infusion, travel throughout the body through the systemic circulation and so affects both healthy and cancer cells. The efficacy of cancer chemotherapy is thus significantly compromised owing to its toxic effects on the healthy, normal cells [101]. Targeting of the anticancer drug to the disease location paves the way for specific attack on the cancer cells, without affecting the healthy cells. Targeted delivery of anticancer drugs can be achieved by a wide variety of ways. Ligand decorated nanoparticulate delivery systems seems to be a promising approach where the ligand attached to the nanocarrier surface will specifically bind to the receptors having affinity for the ligand. Additionally, the EPR effect (enhanced permeability and retention) synergistically improves the active targeting method via receptor facilitated accumulation of drug at tumour site. Another approach of the targeted delivery is coating the delivery agent with a pH responsive polymer [102–104]. Various cellulose derivatives and acrylic polymers are used for pH responsive coating of the delivery agent [105,106]. Rajpoot et al. reported the development of folic acid fabricated solid lipid nanoparticles (SLN) using 1, 2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE) and tristearin [107]. The SLN were loaded with irinothecan. Further, the SLN were encapsulated on Eudragit S100 coated alginate microbeads. The microbeads were thus dual targeted i.e. ligand targeted by attachment of folic acid and pH sensitive due to eudragit coating. At first, the SLN were synthesised and then folic acid was conjugated onto it. Following this, the SLN were dispersed in sodium alginate solution and then extruded into calcium chloride solution to effect the formation of alginate beads. Later on, the SLN loaded alginate beads were dropped on eudragit solution for surface coating of the microbeads. The microbeads could thus be administered orally and can be targeted to the colon tumors due to their dual-targeting feature. Fig. 5 shows the possible mechanism of the colon targeting of the microbeads. The particle size of the SLN were between 157.5 ± 3.64 nm to 168.6 ± 3.18 nm and the poly dispersity index was between 0.210 ± 0.02 to 0.249 ± 0.02. The drug loading and entrapment efficiency of the SLN was 36% and 76% respectively. The zeta potential (− 30.2 ± 1.5 mV) indicated the formation of stable nanoparticles. The high negative value of the nanoparticles was due to the presence of free carboxylic acid of folic acid attached onto SLN surfaces. This provided sufficient repellent forces among the nanoparticles which hindered its agglomeration and provided optimum stability. The uncoated microbeads had a particle size of 0.95 ± 0.042 mm and entrapment efficiency of 86%. Weight gain of microbeads after eudragit coating was around 9.99%. In vitro drug dissolution study pointed out that there was no drug dissolution from the microbeads up to 4h. This is due to the presence of eudragit at the microbead surface which ionizes only at alkaline pH. As ionization of the eudragit takes place, the coating layer is disrupted which exposes the drug loaded nanoparticles. Irinotican release after 12 h from the nanoparticles was around 30%. Irinotican release was found to decrease with the increase in the thickness of the eudragit coating. Cytotoxicity study was conducted on COLO-205 cells. Blank SLN beads didn’t show any inhibitory effect on cells. Folic acid-irinotican SLN, irinotican SLN and free irinotican solution demonstrated decrease in IC50 values. The IC50 values of Folic acid-irinotican SLN, irinotican SLN and free irinotican solution were found to be 7.0 μg/mL, 8.4 μg/mL, and 15.0 μg/mL, respectively Least growth of cell was observed in case of Folic acid-irinotican SLN. This is due to the specific uptake of the Folic acid-irinotican SLN by the COLO-205 cells, which demonstrated the selectivity towards colon cancer cells. Eudragit coated irinotican-SLN beads and eudragit coated irinotican-folic acid-SLN beads were radiolabelled with 99mTc and in vivo pharmacokinetic study was conducted on mice. The results suggested eudragit coated irinotican-folic acid-SLN beads dispensed greater (19.62 ± 0.78%) amount of drug as compared to eudragit coated irinotican-SLN beads (7.63 ± 0.49%) in the colon tumor after 48 h, confirming its targeting ability to colon tumors. Further, eudragit coated irinotican-folic acid-SLN beads displayed significantly enhanced antitumor effect against HT-29 cells bearing Balb/c mice over eudragit coated irinotican-SLN. The authors advocated the enhanced and selective anticancer activity of the eudragit coated irinotican-folic acid-SLN beads.
5.6.2. Natural anticancer agent loaded solid lipid nanocarriers for colon cancer treatment
Multiple drug loading onto a single nanocarrier offers significant benefits in the management of cancer. It provides a synergistic anticancer effect because of different mechanism of action of the individual K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 anticancer agents leading to decrease in drug resistance, lower chances of disease recurrences and above all, enhanced anticancer therapeutic effect. Trans resveratrol (TRV) is reported to provide cardio protection, vasodilatation and cancer prevention [108]. Ferulic acid (FUA) also possesses a broad spectrum of pharmacological effects, of which inhibition of carcinogenesis of colon cancer is noteworthy [109]. TRV and FUA loaded folic acid conjugated SLN have been prepared for targeted delivery of the natural anticancer agents directly to the cancerous cells without compromising the drug efficacy [110]. Surface fabrication of the SLN were done by chitosan coating to get sustained release effect and improve the delivery efficacy and absorption level of the SLN. In vitro release of TRV and FUA indicated a burst release followed by sustained release up to 48h. TRV and FUA release after 48h was 42.87 ± 3.97 % and 45.24 ± 4.17 % respectively. The existence of free TRA and FUA at the SLN surface was responsible for the burst release. As both the bioactives was embedded at the intermolecular spaces of the SLN, the sustained release profile was achieved. The IC50 value of the chitosan coated and folic acid conjugated SLN was 10 μg/ml, while that for uncoated and unconjugated SLN was 25 μg/ml on the HT-29 cells. These results indicated that chitosan coated and folic acid conjugated SLN displayed better cytotoxicity as compared to uncoated and unconjugated SLN. This was due to the folic acid functionalization of the SLN, which could selectively and effectively deliver the bioactive load onto the cancer cells via the folate receptor mediated endocytosis. The targeted cellular delivery of the conjugated SLN was determined on HT-29 cancerous cells (possessing folate receptors) and NIH 3T3 cells normal cells (lacking folate receptors). It was observed that the NIH 3T3 cells didn’t show any signs of apoptosis, while the number of apoptotic cells significantly rose in case of the HT-29 cancerous cells. This confirmed the cancer cell specific delivery of the bioactives via the chitosan coated and folic acid conjugated SLN. Inhibition of the cell growth was studied by analysing the advancement of the cell cycle. It was observed that there was a significant increase in the cell toll in G0/G1 phase in case of folic acid conjugated SLN, indicating the cell cycle was restrained in G0/G1 phase. Overall, the study indicated that TRV-FUA loaded, chitosan coated and folic acid conjugated SLN could be effectively used for targeted delivery and colon cancer treatment.
5.7. Carbon nanotubes for colon cancer treatment
5.7.1. Synthetic anticancer agent loaded carbon nanotubes for colon cancer treatment
The benefits of targeted therapy in cancer treatment havecenters already been discussed in this article. Recently, CNs have surfaced as a budding nanocarrier delivery system for delivering the drugs to the cancer cells. CNs are nanoneedles which offer significant benefits as nanocarriers for internalization (drug targeting). Their huge surface area (both internal and external) and amenable surface chemistry for attachment with numerous active centres make them a dynamic option for targeted cancer therapy [111,112]. Additionally, toxicity profiles due to their metal impurities and aspect ratio can be easily overcome by suitable purification and functionalization procedures [113]. Moreover, their hydrophobic character (which makes them incompatible in the biological system) can be fabricated by colloidal stabilization and chemical functionalization [114]. In this regard, biopolymer based nanocarriers (like nanocellulose) have come up as a budding option for efficient functionalization/stabilization of CNs in aqueous systems in an environment friendly and biocompatible framework [115]. Nanocellulose are crystalline fibrous nanostructures obtained from plants (hence biocompatible) and can also be developed by green processes. Nanocelluloses can interact with CNs by physical interactions to form hybrid nanostructures which have sufficient stability in water system [116]. It has been seen that the most commonly used colon cancer drugs like fluorouracil, irinotican, capecitabine can be attached to the external surface of the CNs by physical or chemical processes [117,118]. Formulation of water dispersible single walled CNs (SWCNs) and nanocrystalline cellulose (NCC) and covalently surface loaded with capecitabine is cited in literature [119]. Folic acid (targeting molecule) and fluorescein (a fluorophore) were covalently linked with the SWCNs to aid in the revelation and targeting of the cancer cell. The functionalization of the SWCNs are depicted in Fig. 6. Functional group tracking of the SWCNs were performed by Kaiser test and thermogravimetric analysis. Cell culture analysis was carried out on Caco-2/TC7 human enterocyte cell line. The cells undergo growth-dependent automatic discernment in culture medium leading to the evolution of monolayer of cells signifying the functional and morphological attributes of the full-grown enterocytes. The cell differentiation begins at 7 days and is K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 over after 15 days. The experiments on undifferentiated (cancerous) and differentiated (normal) cells were carried out between 5 and 15 days. The SWCNs were then administered to human colon cells. Survival of the cells was measured by the MTT assay. The in-vitro studies showed that the SWCNs-NCC were selectively cytotoxic to cancer cells at a concentration of 5 μg/ml. The notable findings of the study indicated that the SWCNs-NCC showed no effects on the normal cells but had a significant decline in the cancer cells population. The functionalised SWCNs-NCC hybrids displayed an enhanced cytotoxic activity than capecitabine against the Caco-2 cancer cell line. Additionally, confocal microscopy fluorescence imaging using cell cultures pointed out the significant potential of SWCNs-NCC nanohybrid platform for colon cancer theranostics.
5.7.2. Natural anticancer agent loaded carbon nanotubes for colon cancer treatment
Studies show that Lycopene (LYC), found in tomatoes, has potential as a natural cancer preventive agent and antioxidant properties [120]. However, its low solubility and bioavailability pose challenges in delivering the bioactive [121]. Nanomaterials, particularly SWCNs hold K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 promise in enhancing drug delivery for cancer treatment. Functionalised SWCNs can act as carriers for anticancer drugs. A nanotube-based dosage form to deliver isolated lycopene specifically to treat CRC has been formulated. Stability of the f-SWCNs was ensured using Phosphatidylcholine (PC) and Polyvinylpyrrolidone K30 (PVP) [122]. Cytotoxicity tests on HT29 and COLO320DM cells were carried out using various assays. Zeta potential revealed anionic LYC adhering to modified SWCNTs due to electrostatic and π–π stacking interactions. f-SWCNTs bears 90.00% loading efficiency and 93.81% In vitro LYC was released after 1 h. In vitro cytotoxicity tests were conducted using the MTT, SRB, and trypan blue assay method. f-SWCNs exhibited higher cell inhibition (83.29–85.27%) compared to plain LYC (38.58–68.44 as indicated by MTT assay method. Similar results were also obtained from SRB and trypan blue assay method. The results of the three different assay methods indicated that the cell penetration capability of the f-SWCNs were significantly better as compared to pure LYC, which resulted in better cell inhibition. The f-SWCNs were encapsulated in gelatin capsules and the capsules were coated with eudragit L100. Eudragit L100 disintegrates only in colonic pH and thus colon specific release of the f-SWCNs can be achieved. In vitro LYC release studies were performed in simulated gastric fluid pH 1.2, followed by pH 6.5 to mimic the small intestinal pH and finally in pH 7.2 to mimic the colonic pH. Capsule shells were almost intact in pH 1.2 for 3h. LYC release in pH 6.5 was around 40.56 ± 0.35% after 4h of release study, and at the end of 12h in pH 7.2, LYC release was 81 ± 0.55%. In vivo X-ray imaging study indicated that the capsules were intact in pH 1.2 and pH 6.5 and disintegrated only after 8h, confirming the effectiveness of the enteric-coated capsule in preventing LYC release in gastric and small intestinal region. The authors concluded that the in vitro and in vivo studies confirmed the colon specific delivery of LYC through f-SWCNs loaded gelatin capsules.
6. Recent developments in mAb loaded nano carriers for colon cancer treatment
Immunotherapy based on monoclonal antibody (mAb) is now the primary component of targeted cancer treatment, avoiding the potential limitations of surgery, chemotherapy and radiation therapy. Antibodies are able to directly target cancer cells and simultaneously provide longterm anticancer immune responses. mAbs have a variety of mechanisms by which they induce apoptosis, the primary of which is by blocking the growth factor receptor signalling. Growth and survival signalling of protumors are disturbed when mAbs adhere to the target growth factor receptor and fabricate their activation or hinder ligand binding [123]. Bevacizumab is the first mAb based angiogenesis inhibitor approved by USFDA for advanced colon cancer treatment in 2004, which directly interacts with extracellular vascular endothelial growth factor (VEGF) [124]. Bevacizumab is humanized IgG1 that binds to human VEGF-A (primary regulator of angiogenesis) and interacts with VEGF receptor tyrosine kinases, thereby blocking angiogenesis [125,126]. However, mAb based treatment is quite challenging, attributable to very low percentage of mAb distribution to the tumour site, primarily due to the large size of the mAb and barrier effect of the binding site. To circumvent the limitation, large doses are needed to be frequently administered, which results in therapy cost escalation and patient non-compliance. The incorporation of bevacizumab onto nanocarriers will circumvent the limitations of mAb therapy, improve efficacy, reduce toxic effects, modulate bevacizumab release profile and potentiate tumour cell targeting [127,128]. Nanocarriers could also enhance the process of passive and active targeting. Researchers developed bevacizumab loaded PEGylated PLGA nanoparticles surface characterized with human antibody fragment specific to human CD44v6 [121]. CD44v6 is well known for its involvement in development of primary tumors and its metastasis due to its co-receptor function for VEGFR-2 [129]. It is overexpressed in almost half of colon cancer cells and assumes a pivotal role in its metastasis and thus has become a potential target for colon cancer diagnosis and therapy. The nanoparticles were first prepared and then conjugated with antibody fragment specific for CD44v6 receptor. The nanoparticles had a particle size of 120–250 nm, zeta potential of − 5 to − 10mV and PDI between 0.1 and 0.25. CD44v6 functionalised nanoparticles had greater particle size and lesser charge as compared to non-functionalised nanoparticles, indicating successful conjugation of the v6 with the nanoparticles. The increase in particle size of the functionalised nanoparticles is due to the large size of the mAb. ELISA indicated conjugation efficiency at 86 ± 5%. The drug loading and association efficiency of bevacizumab is 7.9 ± 0.2% and 86.5 ± 1.8% respectively. The cytotoxicity (after 24h) of free bevacizumab, blank nanoparticles and bevacizumab nanoparticles were assessed by metabolic activity using MKN74-CD44v6 cell lines. Blank nanoparticles were non-cytotoxic in the cell lines, as PLGA nanoparticles are nontoxic in nature. Cytotoxicity was observed both in case of free bevacizumab and loaded nanoparticles, however, free bevacizumab was more cytotoxic than bevacizumab loaded nanoparticles. The low toxicity of bevacizumab nanoparticles is due to the sustained release of bevacizumab from the nanoparticles. The specific binding of the v6 nanoparticles onto the tumour cells with or without overexpression of CD44v6 was done by fluorescence activated cell sorting technique. Blank nanoparticles, negative control antibody fragment nanoparticles, and v6 functionalised nanoparticles were exhibited to MKN74-CD44v6+ and MKN74-CD44std cells and their closeness to v6 receptors were determined. The study revealed a positive significant difference in attaching to the surface of MKN74-CD44std cells for v6 functionalised nanoparticles compared to non-functionalised nanoparticles. However, the surface attachment was more prominent in the MKN74-CD44v6+ cell line, confirming the presence of v6 antibody fragment of the functionalised nanoparticles surface. Functionalised nanoparticles had a two-fold accentuation in cell attachment compared to non-functionalised nanoparticles. This confirms the specificity of the functionalised nanoparticles to the CD44v6 surface receptor. Cellular uptake studies were done on the same cells and at the same concentrations of the nanoparticles to confirm that the nanoparticles remain attached to the CD44v6 cells. Functionalised nanoparticles had a greater prominent internalization in MKN74-CD44v6+ cells than the blank and negative control nanoparticles, indicating the potential of functionalised nanoparticles to be internalised easily than the non-functionalised nanoparticles. Quantification of the intracellular VEGF levels were done to determine the intracellular bevacizumab concentrations. Intracellular bevacizumab levels were significantly less after free bevacizumab administration than those with bevacizumab functionalised nanoparticles. Again, intracellular bevacizumab levels were notably higher in functionalised nanoparticles incubated cells than those in non-functionalised nanoparticles. This demonstrated functionalised nanoparticles were able to internalize and intracellularly deliver bevacizumab successfully. v6 functionalised nanoparticles exhibited a notable decrease in the intracellular VEGF levels in comparison to non-functionalised nanoparticles and free bevacizumab. This was due to higher internalization of bevacizumab achieved from functionalised nanoparticles as compared to non-functionalised nanoparticles.
7. Drugs under clinical trial for colon cancer treatment
Pharmaceutical companies have recently been working to create new treatments for people with advanced colon cancer. A few promising medicines for metastatic CRC are in Phase 3 development. Encorafenib, a small-molecule BRAF inhibitor, and binimetinib, an oral MEK1/2 inhibitor, are being developed by Array Biopharma for the second-line therapy for people who have BRAF-mutant metastatic CRC along with cetuximab [130]. Masitinib developed by AB Science is an oral tyrosine kinase inhibitor (TKI) of the phenylaminothiazole class that targets both the wildtype and mutant versions of c-Kit, Lyn tyrosine kinase, plateletderived growth factor receptor (PDGFR) alpha/beta, and fibroblast K. Mukherjee et al. European Journal of Medicinal Chemistry Reports 10 (2024) 100137 growth factor receptor 3 (FGFR3) [131]. Mast cell invasion and histamine levels are decreased by inhibiting the c-Kit pathway. In patients with CRC, a high mast cell count is typically indicative of a poor prognosis. Second-line therapy for mCRC patients who are still suffering after receiving conventional chemotherapy. Sumitomo Dainippon Pharma and Boston Biomedical developed an oral drug called napabucasin (BBI-608) to block the signal transducer and activator of transcription 3 (STAT3) pathway that leads to the development of cancer [132]. Cancer stem cells have the "stemness" quality, which allows them to regenerate themselves and differentiate into different kinds of cancer cells. This enables the stem cells to behave like seeds, assisting the cancer in the patient to recur or spread. The STAT3 pathway was discovered to be a crucial mechanism for preserving cancer stemness in preclinical investigations. In CRC patients, high STAT3 expression has been linked to a poor prognosis. In mCRC patients, it is given as a second-line treatment with the FOLFIRI regimen (5-fluorouracil, leucovorin, and irinotecan), either with or without bevacizumab, a drug that suppresses the development of specific types of blood vessels into cancer cell.
8. Conclusions and future perspectives
The complexity of cancer cells coupled with complicated pathways of tumorigenesis and recent chemotherapeutic modules for colon cancer treatment, which includes large doses of cytotoxic drugs, have culminated in serious adverse effects to the cancer patient. Additionally, these chemotherapeutic agents are often associated with poor aqueous solubility which results in poor absorption and lower bioavailability characteristics. The conventional delivery agents are unable to deliver the therapeutic agent at the tumor cell, which results in cytotoxicity to the normal healthy cells. Hence, the “need of the hour” is to develop targeted antitumor drug delivery with enhanced efficacy and reduced drug toxicity. Nanocarriers have surfaced as a budding anticancer agent delivery system that could enhance the solubility and bioavailability of the anticancer agents and at the same time specifically deliver them to the cancerous cells. Nanocarriers have successfully encapsulated hydrophobic drugs inside their structures and delivered them to the target site, thereby enhancing bioavailability. Additionally, the nanocarriers are amenable to various surface modifications, through which the cancer cells can be specifically targeted. In this review article we have emphasized the various nanocarriers which have successfully delivered the chemotherapeutic agent to the cancer cell. We have highlighted the effectiveness of the nanocarriers in enhancing the efficacy of synthetic and natural colon cancer chemotherapeutic agents. We have discussed the diverse targeting mechanisms of the nanocarriers and have also given some insights on the latest treatment modules of colon cancer. The future perspective is to develop nanocarriers which can alter the biodistribution, cellular uptake and pharmacokinetics of medications. Controlled release of the medication inside the tumor can greatly enhance the efficacy of the medication and reduce the adverse effects. This would result in better patient compliance. Though easier said than done, recent results show that nanocarriers will take the centre stage in treatment of cancer. CRediT authorship contribution statement Kaushik Mukherjee: Writing – original draft, Data curation, Conceptualization. Pallobi Dutta: Writing – original draft, Data curation, Conceptualization. Sourav Dey: Writing – original draft, Data curation. Tapan Kumar Giri: Writing – review & editing, Supervision, Conceptualization. Declaration of competing interest No conflict of interest, financial or otherwise. Data availability No data was used for the research described in the article.