68,99 €
Mehr erfahren.
- Herausgeber: Bentham Science Publishers
- Kategorie: Fachliteratur
- Sprache: Englisch
Promising Cancer Therapeutic Drug Targets: Recent Advancements offers a comprehensive overview of novel and emerging strategies in cancer therapy. The book explores cutting-edge approaches such as exosomal delivery systems, CRISPR/Cas9 gene editing, and immunotoxins, alongside targeting mechanisms like cancer stem cells, apoptosis pathways, and key signaling processes. A strong emphasis is placed on the therapeutic potential of natural compounds in disrupting cancer progression and enhancing treatment response. Chapters are organized around molecular targets, therapeutic pathways, and bioactive agents to provide a clear, thematic structure. Key Features - In-depth exploration of CRISPR/Cas9, exosomes, and stem cell-targeted therapy - Focus on natural compounds in cancer treatment - Insights into key signaling pathways (Hippo, Hedgehog, STAT3) - Discussion on apoptosis and drug resistance mechanisms.
Das E-Book können Sie in Legimi-Apps oder einer beliebigen App lesen, die das folgende Format unterstützen:
Veröffentlichungsjahr: 2025
Ähnliche
BENTHAM SCIENCE PUBLISHERS LTD.
End User License Agreement (for non-institutional, personal use)
This is an agreement between you and Bentham Science Publishers Ltd. Please read this License Agreement carefully before using the book/echapter/ejournal (“Work”). Your use of the Work constitutes your agreement to the terms and conditions set forth in this License Agreement. If you do not agree to these terms and conditions then you should not use the Work.
Bentham Science Publishers agrees to grant you a non-exclusive, non-transferable limited license to use the Work subject to and in accordance with the following terms and conditions. This License Agreement is for non-library, personal use only. For a library / institutional / multi user license in respect of the Work, please contact: [email protected].
Usage Rules:
All rights reserved: The Work is the subject of copyright and Bentham Science Publishers either owns the Work (and the copyright in it) or is licensed to distribute the Work. You shall not copy, reproduce, modify, remove, delete, augment, add to, publish, transmit, sell, resell, create derivative works from, or in any way exploit the Work or make the Work available for others to do any of the same, in any form or by any means, in whole or in part, in each case without the prior written permission of Bentham Science Publishers, unless stated otherwise in this License Agreement.You may download a copy of the Work on one occasion to one personal computer (including tablet, laptop, desktop, or other such devices). You may make one back-up copy of the Work to avoid losing it.The unauthorised use or distribution of copyrighted or other proprietary content is illegal and could subject you to liability for substantial money damages. You will be liable for any damage resulting from your misuse of the Work or any violation of this License Agreement, including any infringement by you of copyrights or proprietary rights.Disclaimer:
Bentham Science Publishers does not guarantee that the information in the Work is error-free, or warrant that it will meet your requirements or that access to the Work will be uninterrupted or error-free. The Work is provided "as is" without warranty of any kind, either express or implied or statutory, including, without limitation, implied warranties of merchantability and fitness for a particular purpose. The entire risk as to the results and performance of the Work is assumed by you. No responsibility is assumed by Bentham Science Publishers, its staff, editors and/or authors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products instruction, advertisements or ideas contained in the Work.
Limitation of Liability:
In no event will Bentham Science Publishers, its staff, editors and/or authors, be liable for any damages, including, without limitation, special, incidental and/or consequential damages and/or damages for lost data and/or profits arising out of (whether directly or indirectly) the use or inability to use the Work. The entire liability of Bentham Science Publishers shall be limited to the amount actually paid by you for the Work.
General:
Any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims) will be governed by and construed in accordance with the laws of Singapore. Each party agrees that the courts of the state of Singapore shall have exclusive jurisdiction to settle any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims).Your rights under this License Agreement will automatically terminate without notice and without the need for a court order if at any point you breach any terms of this License Agreement. In no event will any delay or failure by Bentham Science Publishers in enforcing your compliance with this License Agreement constitute a waiver of any of its rights.You acknowledge that you have read this License Agreement, and agree to be bound by its terms and conditions. To the extent that any other terms and conditions presented on any website of Bentham Science Publishers conflict with, or are inconsistent with, the terms and conditions set out in this License Agreement, you acknowledge that the terms and conditions set out in this License Agreement shall prevail.Bentham Science Publishers Pte. Ltd. 80 Robinson Road #02-00 Singapore 068898 Singapore Email: [email protected]
PREFACE
The development of a population of cells that can invade tissues and spread to distant sites, resulting in significant morbidity, is what is known as cancer. Cancer is an abnormal growth of cells brought on by multiple changes in the gene expression, which result in a dysregulated balance of cell proliferation and cell death. A group of illnesses affecting higher multicellular organisms include cancer. The capacities to invade locally, disseminate to nearby lymph nodes, and metastasize to distant organs in the body distinguish malignant cancer from benign tumors. The acquisition of multidrug resistance and relapse pose the biggest challenge in the development of anticancer drugs. Traditional cancer treatments directly affect the DNA of the cell, but modern anticancer medications use molecularly focused therapy, such as focusing on proteins that have an aberrant expression in cancer cells. Conventional methods for completely eliminating cancer cells were found to be ineffective. Although targeted chemotherapy has been beneficial in treating some cancers, its efficacy has frequently been constrained by drug resistance and adverse effects on healthy tissues and cells. The aberrant tumor signaling, however, involves pathways for phosphoinositide 3-kinase (PI3K)/Akt, mammalian target of rapamycin (mTOR), Wnt/-catenin, mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3 (STAT3), and notch signaling. Targeted chemotherapy has been beneficial in some cases of cancer, but its efficacy has frequently been constrained by drug resistance and adverse effects on healthy tissues and cells. On the other hand, the majority of researchers are interested in the promising field of immunotherapy. Targeting cancer stem cells and microRNAs generally play a vital role in cancer medication development together with aberrant tumor signaling pathways. The main cause of medication resistance and tumor recurrence is recognized to be cancer stem cells. MicroRNAs are brief non-coding molecules that are 20-22 nucleotides long. It has the propensity to control a number of important signaling pathways that encourage cancer. Therefore, the current book discusses several of these important anticancer targets, such as cancer stem cells, microRNAs, (PI3K)/Akt, mTOR, Wnt/-Catenin, MAPK, STAT3, and notch signaling pathways. Additionally, numerous clinical trial phases for promising natural and synthetic medication candidates are outlined.
List of Contributors
Exosomal Delivery of CRISPR/CAS9 Assembly: Approach towards Cancer Therapeutics
Kaumudi Pande1,2,#,PP Mubthasima1,2,#,Rajalakshmi Prakash1,2,#,Anbarasu Kannan1,2,*Abstract
Exorbitant cancer malignancy is at the helm of multiple organ malfunction in humans and is considered a cause of increased cancer mortality worldwide. Clustered regularly interspaced short palindromic repeats (CRISPR) are powerful machinery for the therapeutic approach to tumors because of their substantial peculiarity, focusing on modulatory molecules, both oncogenes and tumor suppressors, to preclude tumor metastasis and enable apoptosis. Exosomes are considered an ideal delivery system because of their specificity and ability to prevent premature release of cargo. Exosomes are accessed as an effective conveyance of CRISPR/Cas9 elements and other attractive biomolecules to recipient cancer cells. The CRISPR/Cas9 loaded exosomes are endocytosed for further alteration of cellular metabolic pathways, either by knock-in or knock-out of the designed destined gene using sgRNA and Cas9 protein. The current study provides a platform to address the alliance between the CRISPR/Cas9 model and exosomes, depicting a remarkable therapeutic approach against cancer and other fatal diseases.
INTRODUCTION
Cancer can be defined as the ungoverned multiplication of cells that propagate far and wide through discrete organs. The unbeatable accumulation of aggressive
cells creates an aching mass called “Tumor” that is benign in the preliminary phase, however, it undergoes extension in later phases. The characteristics of cancer cells include evasion of cell cycle regulatory checkpoints, chromosomal aberrations leading to a gain of function of specific oncogenes influencing conventional splitting of cells, subjugation of tumor suppressors by mutation or its related molecules, blocking of the body’s immune system and its regular functions, prevention of caspase-mediated cell death, acquisition of mesenchymal cellular attributes, and blood vessel organization.
As reported by GLOBOCAN 2020, there are 19.3 million unprecedented cases of cancer with a survival rate of 9.3 million for the early detection of the disease [1]. The understanding of the occurrence of cancer can be extrinsic components, for instance, consumption of excessive nicotine present in cigarettes [2], consumption promoting foodstuffs such as processed meat, alcohol, junk food, and drinks, exposure to deadly radiation [3], and viral infections [4, 5]. Intrinsic abiogenetic records increase vulnerability to tumorigenesis [6]. By employing diagnostic tools corresponding to national databases, doctors can reveal different stages of cancer and advance further. The tumor of stage 0 is benign and exists at the place of its origin, and its early recognition can be remediable by eliminating the tumor through surgery. Stage I tumor is a minor protuberance that is settled at one site and neither augmented intensively in adjacent tissues nor stretched in another place towards lymph nodes. Stages II and III are regarded as the initiation of the migratory ability of cancer cells within easy-to-reach tissues along with lymph nodes. Treatment options for stages I-III include surgery, chemotherapy, and radiation therapy. Stage IV exhibits a highly precarious patient condition with a weakly favorable medicament since the tumor circulated rapidly to distant organs and recommended Targeted Immunotherapy in addition to the mentioned ones.
Cancer examinations can be performed through imaging trials, Endoscopy, Bioscopy, and discrete body fluid tests. Cancer metastasis is related to the expansion of unregulated cells that disrupt their prevailing morphology and attain locomotion efficiency in various parts of the human body (Fig. 1). These metastatic cells breached the epithelial tissue lining and entered the biological fluids, such as blood, milk, saliva, semen, vaginal fluid, and urine, and further interfaced with healthy cells for their transition into tumor cells. This evolution of genes favors the synthesis of growth-promoting proteins, whereas it obstructs programmed cell death molecules [7]. Portments of metastatic carcinoma include pain, vomiting, body weight reduction, exhaustion, feverishness, infrequent excretion, and hemorrhage [8]. Therapeutic options for cancer that are currently used worldwide include chemotherapy, radiation therapy, and surgical removal of solid tumors coupled with either one of the aforementioned therapies. Targeted therapeutic interventions are of prime focus these days in order to avoid any cytotoxicity exerted by chemotherapy on adjacent normal cells.
Fig. (1)) Cancer metastasis.Exosomes are nanovesicles of approximately 30-150 nm liberated by all types of living cells under natural as well as pathological conditions. The breakthrough in the discipline of extracellular vesicles emerged in the year 1983 searched out in Reticulocytes for endocytosis of iron [9] and it is composed of transferrin receptors, sphingomyelin, amino acid and glucose transporters liberated apart from the cell, therefore, designated as “Exosomes” [10]. Exosomes are produced by cellular machinery and further move out and mobilize in the extracellular space, interacting with other cells for the purpose of conveying specific molecules, consequently influencing the cellular metabolism of recipient cells. They are an exemplary means of proteins (CD63, CD81/82, Alix, TSG101, Rab) [11], nucleic acid family [12], and fatty acids (ATP-binding cassette transporter A1, low-density lipoprotein receptor, low-density lipoprotein receptor) [13], which are involved in immune response, modulate the tumor microenvironment, cancer infiltration, and evade cell death. Further studies have demonstrated that exosomes serve as clusters of biomarkers for the preliminary identification of malignancy.
Exosomes perform their function in cancer therapeutics by impeding the activity of constitutively expressed growth-promoting genes at former neoplastic spots, controlling their cellular habitat, and preventing affinity to different organs and tissues. Exosomes obtained from natural sources, such as milk- or plant-derived exosomes, have many qualities that can assist in directing man-made therapeutic medicines [14]. Macrophage-derived exosomes are effective in reducing the number of tumor cells. Exosomes carry the Major Histocompatibility Complex and Heat shock proteins, which generate innate and adaptive immunity in recipient cells. Thus, exosomes can be employed in novel cancer therapies to destroy tumor cells [15].
CRISPR/Cas system is an adaptive immune system in bacteria. Single-guide RNA (gRNA) and CRISPR-associated endonucleases (Cas) are the main components of the CRISPR/Cas system. Cas9 is an endonuclease that functions as a pair of molecular scissors to cleave the target DNA sequence. Single-guide RNA is the combination of tracrRNA and crRNA. crRNA contains two main parts: a region that binds to tracrRNA and a spacer sequence that directs the complex to the target DNA. When tracrRNA binds to a crRNA, a functional guide RNA is formed for Cas9 recognition. Multiple crRNAs can be incorporated into a crRNA array and then packaged with tracrRNA to form sgRNA [16].
There were no sources of data in the current study. Systems use different RNP complexes and further distinguish themselves by the presence of a specific “signature protein” responsible for DNA degradation, namely, Cas3, Cas9, and Cas10 for types I, II, and III, respectively [17]. CRISPR triggers DNA repair by creating a double-strand break in the DNA. This break results in two types of genome modifications: knock-ins (KI) through homologous recombination and constitutive knockout (KO) through non-homologous end-joining [18].
CRISPR/Cas9 system is used in various applications, including genome editing, screening, chromatin immunoprecipitation, transcriptional activation and repression, epigenetic editing with live imaging of DNA/mRNA, and therapeutic applications [19]. Simplicity and high efficiency are the main advantages of the CRISPR/Cas9 system over other gene-editing systems. The main disadvantage of the CRISPR system is its off-target effects [20]. In this chapter, we discuss the role of the CRISPR/Cas9 system in cancer therapeutics and exosomes as a potential delivery system for CRISPR.
CRISPR as a potential therapeutic agent for metastasis
Molecules Involved in Metastasis
The expanded knowledge associated with the anomalous evolution of growth-promoting molecules has enabled the identification of novel indicators for cancer metastasis (Table 1). The hallmarks of tumor malignancy include disruption of DNA integrity by depletion of the tumor suppressor phosphoprotein p53 due to mutations in the coding region (2–11) in high-grade pelvic serous carcinoma [21]. The overexpression of mouse double minute 2 homolog (MDM2) in ductal carcinoma causes nuclear interaction with p53, leading to its aggregation, and has been reported as an additional channel for cancer development [22]. The establishment of cancer metastasis implicated the genesis of fresh blood vessels at the metastatic niche to obtain nourishment from the primitive veins. Angiogenesis encouraging C–C chemokine ligand 2 is a marker for advanced-stage breast cancer by causing the admission of C-C chemokine receptor type 2, resulting in inflammation and high vesicular compactness. The role of interleukin 6 in the initiation of angiogenesis is boosted by C–C chemokine ligand 2, which has a lower number of cancer victims [23]. The δ-catenin enciphered by the CTNND2 gene on chromosome 5 is involved in the attachment of cells to adjacent cells, and its expression increases in prostate cancer for the renewal of cell division by influencing cyclin D1 and phosphorylation of Histone3, therefore, maintaining cell viability. Its participation in lowering epithelial molecule E-cadherin results in the loss of surface attachment and cell migration in vitro and a two-fold increase in tumor measurement in mice depicting gene sequence modification at the 5’ untranslated site in δ-catenin, enabling rapid growth of tumor cells [24].
PTK2 protein tyrosine kinase 2 (FAK) is a cytoplasmic protein that is confined to focal adhesions and undergoes phosphorylation at the tyrosine 397 region for its activation in response to growth factors and unclasps from the NH2-terminal Protein4.1-ezrin-radixin-moesin (FERM) domain for interaction with Phosphoinositide-3-Kinase Regulatory Subunit 2 to instigate AKT inclined towards multiple intracellular oncogenic pathways. Further studies have shown that nearly 92% of small-cell lung cancer tissue sections express FAK at varied intensities, and FAK mRNA is expressed in 23% of lung carcinomas [25]. The intervention of FAK in metastatic cascade was found to be examined in 38.6% of Nonsmall cell lung cancers with greater immune-reactivity in lymph nodal tissue specimens and 18% of 5-year life expectancy, subsequently accelerating cancer metastasis [26].
Wiskott-Aldrich syndrome verprolin-homologous 3 (WAVE3) is an actin cytoskeleton restructuring protein formed by gene sequence present on chromosome 13 of q arm region 12, comprised of 502-amino acids polypeptide sequence [27]. WAVE3 combines with the Arp2/3 complex for actin polymerization for cell mobility and has three times greater expression at the final stages of breast cancer. Conquering the farther body organs, for instance, the lungs by MDA-MB-231 adenocarcinoma cells, WAVE3 encourages tumor initiation in the lungs; however, the infusion of MDA-MB-231 cells with inactivated WAVE3 leads to the severe immunodeficient mice detected with a lower rate of cancer metastatic cells, thereby, inert WAVE3 declines the migratory potential of cancerous cells [28]. Surprisingly, we found that WAVE3 was immensely present in the metastatic grade of prostate cancer cells, mainly in the cytoplasmic compartment, and its silencing prevented the disruption of basement membrane curbing invasion [29].
Prostaglandin-endoperoxide synthase-2 (PTGS2) is an inflammatory agent of 70 kDa molecular weight that catalyzes the processing of arachidonic acid into prostaglandin H2. Recent studies have suggested that PTGS2 escalates Prostaglandin E2, fostering malignancies in a variety of cancer types, particularly colorectal, breast, head, and neck cancer [30], prostate cancer [31], and melanoma [32]. The crucial activity of PGE2 enables Src in the presence of cytosolic β-arrestin to stimulate the epidermal growth factor receptor to phosphorylate AKT on Serine -473 residue to trigger the PI 3-kinase/AKT mechanism in colorectal cancer [33].
Cyclooxygenase (COX-2) emerged as a predicted marker for breast cancer, ascertained mostly in post-menopause advanced stage in 83 patients and exhibiting lymph node metastasis in 92.6% of COX-2 positive patients, influenced by the RAS-MAPK schematic approach [34]. Prominin-1 (CD133) located in chromosome 4 is a glycoprotein often overexpressed in brain cancer confirmed by the histological glioma sections in the second and third grades [35]. CD133 is sufficient for the automatic regeneration of cancer stem cells, facilitating tumor initiation in ovarian and colon cancers. Its multipurpose regulation of cell proliferation molecules boosts cancer metastasis, notably observed in Wnt signaling engaged in the complex formation of C133 with β-catenin and NF-κB signalling influenced by CD133 in epithelial to mesenchymal transition (EMT), further advancing cancer metastasis [36].
Matrix metallopeptidase 9 (MMP-9) is a proteolytic enzyme that provides cell mobility by deteriorating the extracellular matrix, attaining mesenchymal characteristics in tumor cells. The prevalence of metastasis in the brain (p=0.0062) was reported to be evoked by breast carcinoma cells in the presence of MMP-9 in the serum, which describes colonization in other organs [37]. The fraction of MMP-9/MMP-2 in serum samples of hepatocellular carcinoma cases also increased significantly with respect to further staging of cancer [38]. Numerous tumor accretion molecules contribute to genetic modification, and cellular transformation, surpass physical barriers, diffuse in body fluids, and invade different body parts; therefore, there is a requirement for an effective technique that specifically targets oncogenes to stop cancer metastasis.
Metastatic Molecules Targeted by CRISPR/Cas9 System
CRISPR/Cas9 is a promising gene editing tool. CRISPR can precisely edit genes in both model organisms and humans and can act as a potential agent in cancer therapeutics. Metastasis is an important issue in cancer treatment. Targeting metastatic molecules using the CRISPR/Cas9 system can be used as a novel therapeutic agent in cancer treatment (Table 1).
Several oncogenes that are modulated using CRISPR are discussed here; δ-catenin is overexpressed in many cancers, including prostate, breast, lung, and ovarian cancers. It acts as an oncogene that promotes the malignancy of lung adenocarcinoma. In lung adenocarcinoma, β-catenin enhances invasion and colonization via maintenance of cancer stem cells. δ-Catenin is encoded by the CTNND2 gene, the knockdown of which in an animal model by the CRISPR/Cas9 systemleads to the loss of tumorigenicity and metastatic ability [39]. Another study focused on CD133 as a stem cell marker and revealed that CD133 plays an essential role in the invasion and proliferation of colon cancer cells. Knockout of CD133 encoding gene using the CRISPR/Cas9 system in colon cancer has remarkable inhibitory effects on cell migration and invasion. In addition, CD133 knockout cells show decreased expression of the EMT marker vimentin [40].
Yet another molecule, Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that integrates mitogenic signaling, cell survival, and cytoskeleton remodeling. FAK1 localizes to nuclease during cell migration. Ablation of FAK by CRISPR/ Cas9 editing results in susceptibility to ionizing radiation, impaired oxidative phosphorylation, and basal DNA damage [41].
In addition to oncogenes, tumor suppressor genes such as p53 are mutated in many cancers. They respond to many cellular stressors, including DNA damage, hypoxia, oncogene activation, and reactive oxygen species (ROS). Upon activation, this leads to cell cycle arrest to restore genetic integrity, apoptosis, senescence, or ferroptosis to eliminate unrecoverable cells. Mutations in p53 facilitate cancer progression by accelerating cell proliferation, promoting cancer metastasis, inducing chemoresistance and radio resistance, and facilitating a pro-oncogenic tumor microenvironment. The use of CRISPR/Cas9 gene editing machinery will replace the mutant p53 gene with a functional gene that restores the normal functioning of p53. CRISPR/Cas9 can also be employed in a p53 genetic sensor system, which precisely and efficiently kills p53-deficient cancer cells [42].
Prostaglandin-end peroxide synthase 2 (PTGS2) plays an essential role in melanoma development and progression. PTGS2 is frequently expressed in malignant melanoma. Knockdown of PTGS2 using the CRISPR/Cas9 system in melanoma cells leads to inhibition of cancer cell migration, proliferation, and invasiveness. It also reduces tumor development and metastasis in vivo [43]. In addition to WAVE3, a member of the WASP/WAVE family of actin-cytoskeleton remodeling proteins plays a vital role in cancer cell invasion and migration in triple-negative breast cancer (TNBC). In addition, WAVE3 plays an essential role in cancer stem cell (CSC) maintenance and further leads to chemo resistance in cancer. Knockout of WAVE3 via CRISPR/Cas9 significantly attenuates CSC subpopulation and inhibits the transcription of CSC transcription factors [44]. Thus, CRISPR/Cas9 gene editing machinery can act as a potential cancer therapeutic agent to prevent cancer metastasis. The list of oncogenic molecules whose expression levels are modulated using CRISPR/Cas9 is depicted in Table 1.
Exosomes as a potential carrier (Therapeutic carrier): Exosomes-based delivery of small molecules, Bioactive molecules, siRNA
Exosomes are extracellular vesicles produced by all cell lines and play a vital role in intercellular communication. Exosomes are present in various body fluids, including blood, saliva, and urine. These are also present within the tissue matrix, known as matrix-bound nanovesicles (MBV). Exosomes originate in multivesicular bodies and are released into the extracellular space by the fusion of multivesicular bodies to the lipid bilayer [46]. Exosomes accommodate complex cargo, such as proteins, RNA, DNA, and lipids, and are delivered to specific target cells that reprogram the recipient cell. Exosome cargo can vary based on its origin. Exosomes represent the metabolic state of the cell line from which they get originated [47]. Exosomes deliver their cargo to recipient cells through receptor-ligand interactions, direct fusion of membranes, or internalization via endocytosis. Once internalized into the recipient cell, it fuses with the limiting membrane of the endosome, leading to horizontal genetic transfer by releasing cargo to the cytoplasmic space of the target cell [48]. Exosomes play an essential role in cancer progression. They carry pro-tumorigenic signals from cancerous cells and deliver them to non-cancerous cells, causing reprogramming of recipient cells [49].
Differential Centrifugation/Ultracentrifugation, chromatography, density gradient centrifugation, kit-based methods, and magnetic beads can be used to isolate exosomes from various sources [50]. The isolated exosomes were characterized by transmission electron microscopy (TEM), nano-sight, and western blotting [51]. Using TEM and Nano sight, the size of the exosome is estimated, and by using western blotting for exosome-specific markers such as CD9, CD81, and CD64, expression levels are estimated [52].
Numerous nano-based drug formulations have been developed to improve the therapeutic efficacy of molecular and chemical drugs (Table 2). However, the cytotoxicity of materials and rapid clearance by the reticuloendothelial system (RES) or mononuclear phagocyte system (MPS) are major problems encountered in the clinical translation of these nano-based systems [53]. Exosomes have an advantage over other nano-based drug formulations owing to their natural biocompatibility. In addition, they have high stability, low immunogenicity, and a long circulation time. Some exosomes can even have a high capacity to escape from degradation or clearance by the immune system [54]. Exosomes of different origins have been used in cancer therapeutic applications. Cancer cell line-derived exosomes, normal cell line-derived exosomes, bovine milk-derived exosomes, and macrophage-derived exosomes are examples of the exosomes used in cancer therapeutics [14, 55-60]. Various plants and vegetables release exosome-like nanoparticles. They also contain lipids, proteins, and miRNAs, and because of their absence of toxicity and easy internalization by mammalian cells, they act as a good delivery system for cancer therapeutics. Plant-derived nanovesicles (PDNVs) can be used as effective delivery systems for small molecule agents and nucleic acids with therapeutic effects (siRNAs, miRNAs, and DNAs). Plant-derived nanovesicles alone have immunomodulatory, anti-inflammatory, and regenerative properties to treat liver diseases, inflammatory bowel disease (IBD), and cancer. PDNVs are stable in the gastrointestinal tract; therefore, drugs incorporated into PDNVs can be administered orally [61].
Exosomes are modified to improve drug delivery by incorporating penetrating peptides such as iRGD, RGD, and RVG. These peptides are incorporated into exosomes in various ways. The plasmid contains an exosomal membrane protein Lamp2b gene fused with the targeting peptide RVG (rabies viral glycoprotein peptide) that has to be expressed in cells. The exosomes produced by the cell line have Lamp2b linked with RGV; RVG directs exosomes to organs that express the acetylcholine receptor [62]. Similarly, both iRGD- and RGD-tagged exosomes can be created [63, 64]. This penetrating peptide can be incorporated into the surface of exosomes by a chemical method, directly conjugating the peptide to the exosome surface using chemicals.
Therapeutic agents are incorporated into exosomes using both passive and active encapsulation methods. Passive encapsulation includes the incubation of the drug with exosomes or donor cells. Exosomes are incubated with the drug, and the drug moves passively based on its concentration gradient, hydrophobicity, and the efficiency of encapsulation changes [65]. The drug is incubated with donor cells to allow the incorporation of the drug into its derived exosomes, which is the principle behind the passive encapsulation method [66]. Active cargo loading includes sonication, extrusion, freeze-thaw cycles, electroporation, incubation with membrane permeabilizers, and the click chemistry method for direct conjugation. When the sonication mechanical shear force is compromised, the integrity of the membrane allows the diffusion of drugs through the membrane. Exosomes from donor cells were mixed with a drug using the extrusion method, and the mixture was loaded into a syringe-based lipid extruder with 100-400 nm porous membranes under controlled temperature. During extrusion, the exosome membrane is disrupted and vigorously mixed with the drug [67]. In the electroporation method, an electric field creates tiny pores in the exosome membrane, allowing drug diffusion through the membrane [68]. In the freeze-thaw method, the drug was incubated at room temperature and then kept at -80 °C, and the process was repeated for 3-4 cycles. This cycle increases the membrane permeability [69]. The use of permeabilizers, such as saponin, increases the permeability of the exosome membrane, allowing the diffusion of drugs through the membrane [70]. The drug can be directly conjugated to the surface of exosomes using the click chemistry method [71].
Several previously published studies have highlighted the incorporation of various bioactive molecules into exosomes of different origins. Paclitaxel is a potent chemotherapeutic agent incorporated into macrophage cell-derived exosomes via sonication that increases drug delivery efficiency by 50 times in drug-resistant MDCK-MDR1 (P-gp+) cells [12]. Curcumin is an anti-inflammatory molecule that can target inflammatory cell lines. Curcumin was incorporated into EL4 (mouse lymphoma cell line) exosomes by simple incubation and delivered to the cell line. Exosomes can increase the solubility and stability of curcumin in vitro and its bioavailability in vivo. It also increases the anti-inflammatory activity of curcumin by accumulating high levels of curcumin in cellular targets [55]. Bovine milk can act as a carrier for chemotherapeutic/chemopreventive agents and can serve as a scalable source of exosomes. Milk exosomes exhibited cross-species tolerance with no adverse immune or inflammatory responses. Doxorubicin and Paclitaxel are two different types of chemotherapeutic agents incorporated into bovine milk exosomes via simple incubation, which showed significantly higher efficacy than free drugs and increased tumor targetability [14]. Doxorubicin encapsulates in MCS (mesenchymal cell) derived exosomes via electroporation and delivers them to colorectal cancer, exhibiting higher tumor accumulation and faster liver clearance than free DOX. In addition, it showed increased inhibition of tumor growth [56]. Erastin is a ferroptosis (lipid peroxide-driven cell death caused by inhibition of the cystine/glutamate transporter) inducer; its low water solubility and renal toxicity have limited its application in therapeutics. Incorporating erastin into HLF1 cell-derived exosomes via sonication increases the uptake efficiency of erastin into MDA-MB-231 (breast cancer) cells compared with free erastin. EXO-erastin has a stronger inhibitory effect on proliferation and migration than free erastin [57]. Exosomes derived from mouse immature dendritic cells (imDCs) were engineered by incorporating iRGD peptides to increase the efficiency of drug delivery. iRGD peptides are specific to integrins; cells expressing integrin take up more exosomes and increase delivery efficiency. Doxorubicin is incorporated into iRGD-tagged exosomes, showing an increased inhibitory effect compared to imDC-derived exosomes [58].
