Chitosan-based nanoscale systems for doxorubicin delivery: Exploring biomedical application in cancer therapy

REVIEW ARTICLE Chitosan-based nanoscale systems for doxorubicin delivery: Exploring biomedical application in cancer therapy Milad Ashrafizadeh 1 | Kiavash Hushmandi 2 | Sepideh Mirzaei 3 | Saied Bokaie 2 | Ashkan Bigham 4 | Pooyan Makvandi 5 | Navid Rabiee 6 | Vijay Kumar Thakur 7,8 | Alan Prem Kumar 9,10 | Esmaeel Sharifi 11 | Rajender S. Varma 12 | Amir Reza Aref 13,14 | Marcin Wojnilowicz 15,16 | Ali Zarrabi 17 | Hassan Karimi-Maleh 18,19,20 | Nicolas H. Voelcker 15,16,21 | Ebrahim Mostafavi 22,23 | Gorka Orive 24,25,26,27 1 Faculty of Engineering and Natural Sciences, Sabanci University, Üniversite Caddesi, Tuzla, Istanbul, Turkey 2 Department of Food Hygiene and Quality Control, Division of Epidemiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 3 Department of Biology, Faculty of Science, Islamic Azad University, Science and Research Branch, Tehran, Iran 4 Institute of Polymers, Composites and Biomaterials - National Research Council (IPCB-CNR), Naples, Italy 5 Istituto Italiano di Tecnologia, Center for Materials Interfaces, Pontedera, Pisa, Italy 6 School of Engineering, Macquarie University, Sydney, New South Wales, Australia 7 School of Engineering, University of Petroleum & Energy Studies (UPES), Dehradun, Uttarakhand, India 8 Biorefining and Advanced Materials Research Center, Scotland's Rural College (SRUC), Edinburgh, UK 9 NUS Centre for Cancer Research (N2CR), Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore 10 Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Kent Ridge, Singapore 11 Department of Tissue Engineering and Biomaterials, School of Advanced Medical Sciences and Technologies, Hamadan University of Medical Sciences, Hamadan, Iran 12 Regional Center of Advanced Technologies and Materials, Czech Advanced Technology and Research Institute, Palacky University, Olomouc, Czech Republic 13 Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA 14 Xsphera Biosciences Inc., Boston, Massachusetts, USA 15 Commonwealth Scientific and Industrial Research Organisation (CSIRO) Manufacturing, Clayton, Victoria, Australia 16 Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia 17 Department of Biomedical Engineering, Faculty of Engineering and Natural Sciences, Istinye University, Istanbul, Turkey 18 School of Resources and Environment, University of Electronic Science and Technology of China, Chengdu, PR China 19 Department of Chemical Engineering, Quchan University of Technology, Quchan, Iran 20 Department of Chemical Sciences, University of Johannesburg, Doornfontein Campus, Johannesburg, South Africa 21 Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, Clayton, Victoria, Australia 22 Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California, USA 23 Department of Medicine, Stanford University School of Medicine, Stanford, California, USA 24 NanoBioCel Research Group, School of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz, Spain Abbreviations: AA, acrylic acid; ABC, ATP-binding cassette; AGO, amine-functionalized GO; BSA, bovine serum albumin; CHOL, cholesterol; CMC, carboxymethyl CS; CSO, CS oligosaccharide; CXB, celecoxib; DCA, deoxycholic acid; DOX, doxorubicin; FA, folic acid; GA, glycyrrhetinic acid; GO, graphene oxide; GSH, glutathione; HA, hyaluronic acid; HSPC, hydrogenated soy phosphatidyl choline; IA, itaconic acid; miRNA, microRNA; M-MSNs, magnetic mesoporous silica nanoparticles; MOFs, metal organic frameworks; OA, oleanolic acid; P-gp, P-glycoprotein; PHA, pheophorbide A; PTX, paclitaxel; RAPA, rapamycin; ROS, reactive oxygen species; SA, stearic acid; shRNA, short hairpin RNA; siRNA, small interfering RNA; SOC, N-succinyl-N0-octyl chitosan. Received: 15 February 2022 Revised: 12 March 2022 Accepted: 17 March 2022 DOI: 10.1002/btm2.10325 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers. Bioeng Transl Med. 2023;8:e10325. wileyonlinelibrary.com/journal/btm2 1of29 https://doi.org/10.1002/btm2.10325 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 25 University Institute for Regenerative Medicine and Oral Implantology–UIRMI(UPV/EHU-Fundaci on Eduardo Anitua), Vitoria-Gasteiz, Spain 26 Bioaraba, NanoBioCel Research Group, Vitoria-Gasteiz, Spain 27 Singapore Eye Research Institute, Singapore Correspondence Pooyan Makvandi, Istituto Italiano di Tecnologia, Center for Materials Interfaces, viale Rinaldo Piaggio 34, 56025 Pontedera, Pisa, Italy. Email: [email protected] Nicolas H. Voelcker, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria 3052, Australia. Email: [email protected] Gorka Orive, Bioaraba, NanoBioCel Research Group, Vitoria-Gasteiz, Spain. Email: [email protected] Funding information National Institute of Biomedical Imaging and Bioengineering, Grant/Award Number: 5T32EB009035; University of the Basque Country; Ministry of Economy, Grant/Award Number: PID2019-106094RB-I00/AEI Abstract Green chemistry has been a growing multidisciplinary field in recent years showing great promise in biomedical applications, especially for cancer therapy. Chitosan (CS) is an abundant biopolymer derived from chitin and is present in insects and fungi. This polysaccharide has favorable characteristics, including biocompatibility, biodegradability, and ease of modification by enzymes and chemicals. CS-based nanoparticles (CS-NPs) have shown potential in the treatment of cancer and other diseases, affording targeted delivery and overcoming drug resistance. The current review emphasizes on the application of CS-NPs for the delivery of a chemotherapeutic agent, doxorubicin (DOX), in cancer therapy as they promote internalization of DOX in cancer cells and prevent the activity of P-glycoprotein (P-gp) to reverse drug resistance. These nanoarchitectures can provide co-delivery of DOX with antitumor agents such as curcumin and cisplatin to induce synergistic cancer therapy. Furthermore, co-loading of DOX with siRNA, shRNA, and miRNA can suppress tumor progression and provide chemosensitivity. Various nanostructures, including lipid-, carbon-, polymericand metal-based nanoparticles, are modifiable with CS for DOX delivery, while functionalization of CS-NPs with ligands such as hyaluronic acid promotes selectivity toward tumor cells and prevents DOX resistance. The CS-NPs demonstrate high encapsulation efficiency and due to protonation of amine groups of CS, pH-sensitive release of DOX can occur. Furthermore, redoxand light-responsive CS-NPs have been prepared for DOX delivery in cancer treatment. Leveraging these characteristics and in view of the biocompatibility of CS-NPs, we expect to soon see significant progress towards clinical translation. KEYWORDS chitosan, drug resistance, gene therapy, stimuli-responsive nanocarriers, synergistic therapy 1|INTRODUCTION Cancer treatment requires development of therapeutic strategies for minimizing growth and migration of tumor cells to improve overall survival of patients. For exerting such activities, antitumor compounds should be effectively internalized by cancer cells and induce a therapeutic effect at the cellular level by affecting the molecular pathways and mechanisms responsible for organelle organization and function, such as mitochondria and endoplasmic reticulum. 1–8 Due to advancement in the fields of medicinal chemistry, various antitumor compounds, namely cisplatin, paclitaxel, docetaxel, and doxorubicin (DOX) among others, have been developed in cancer therapy. 9,10 Chemotherapy is currently a first-line option for the treatment of cancer patients to eradicate tumor progression and improve prognosis. Furthermore, chemotherapy is preferred to surgery, as it is a noninvasive strategy in cancer treatment. However, some of the cancers are inherently resistant to chemotherapy, or attained drug resistance during the treatment affecting antitumor agent or its target. As chemoresistance threatens the life of many people around the world, there have been incessant efforts in understanding underlying factors in this process. The drug resistance is a multifactorial condition and each factor can independently participate in decreasing cytotoxicity of antitumor agent. The enhanced drug efflux, diminution in drug uptake, mutation, drug inactivation, apoptosis machinery impairment, signaling networks (upregulation of tumor-promoting factors and downregulation of tumor-suppressor), and phenotype switching are the mechanisms that can lead to cancer drug resistance. 11–14 Given the importance of drug resistance in chemotherapy failure, scientists have followed some strategies for overcoming this condition by applying nanostructures that improve drug delivery potential, enhance 2of29 ASHRAFIZADEH ET AL. 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License intracellular accumulation, and provide targeted delivery and codelivery with other antitumor agents or nucleic acid therapeutics. 15,16 The aim of present review is to discuss the role of chitosan (CS) for the delivery of DOX as one of the most well-known chemotherapeutic agents in cancer therapy and introducing CS chemistry, structure, and potential applications in medicine. The function of DOX in cancer suppression, factors responsible for its resistance and role of nanoparticles in reversing DOX resistance are discussed with emphasis on CS-based nanostructures for its delivery; pHand redox-sensitive assorted CS nanoparticles are highlighted includingtheiruseforco-deliveryofDOX with antitumor agents and nucleic acid therapeutics. Finally, the modification of various nanoparticles and appropriate solutions for their clinical applications are described to shed a light on the deployment of these nanostructures for cancer chemotherapy. 2|CHITOSAN: CHEMISTRY AND BIOMEDICAL APPLICATION The green technology is one of the newest and most recent approaches for the development of nanopharmaceuticals in treatment of diseases. The green chemistry approach utilizes compounds and agents derived from nature for synthesis and modification of nanocarriers to improve their characteristics and make them better options for disease treatment. In this strategy, the hazardous material application is avoided and in turn, safe, biorenewable and biocompatible agents isolated from nature are utilized to develop nanoparticles. Since green-based nanocarriers demonstrate good safety profile and biocompatibility, the way for their clinical application is paved. The delivery of drugs and nucleic acid therapeutics is essential in cancer therapy due to their low accumulation at tumor site and emergence of drug resistance; hence, green synthesis or green modification of nanoparticles can be beneficial in this case for improving efficacy in cancer therapy. 17–19 In the present review, our aim is to highlight greener modifications of nanoparticles with CS as a natural compound to show its potential for cancer chemotherapy and possible clinical applications in the near future. After cellulose, chitin is the most abundant natural polymer 20 and CS is derived from chitin (Figure 1), 22 an essential component comprising shells of insects, crustaceans, and cell walls of fungi. 23–25 The annual production of chitin is estimated to be 10–100 Gt and the commercialized chitin/CS can result from seafood waste in which αand β-chitins are derived from shells of crab and shrimp, while CS is prepared by deacetylation of chitin. 23,24,26–28 The amount of deacetylation seems to be more than 60% in commercialized CS where Japan is considered as the major producer of CS. Based on the estimates, the value of CS market has been 6.8 B$ in 2019. 20 The unique chemical structure of CS has made it a suitable option for biomedical and engineering applications. The most important feature of CS is its great solubility in aqueous solution due to the presence of amino groups at C2 position. In aqueous acidic solvents, CS undergoes protonation to generate NH 3+ that is beneficial in the design of nanoarchitectures and their synthesis via bottom-up approach. Importantly, amino and acetylamino groups in CS are main sources of nitrogen for generating fertilizers and N-doped carbon materials for deployment as catalyst. 20,29 The application of CS in industry has demonstrated potential in reducing environmental pollution as a biodegradable and renewable abundant material that should not be discarded in to scarce landfills. The aim of green chemistry is to limit industrial production of hazardous compounds and prevent destructive impacts, both short-term and long-term, on ecosystem. 30–32 Besides, green chemistry is beneficial in decreasing energy consumption and substituting conventional solvents with newer options that are renewable and demonstrate low destructive impact on environment. 33 The precursor of CS, chitin is made of up to 3000 repeating units comprising N-acetyl-D-glucosamine 34 that are interconnected via β(1 !4) glycosidic bonds. The chitin exhibits high similarity to cellulose in terms of chemical structure with the difference that hydroxyl group at position C2 is substituted by acetamido group. 35 There are various kinds of chitin including α,β, and γ, which show variation in hydration, size, and number of chains 36 and are present in various structures and sources. For instance, α-chitin is found in shells and cell walls, β-chitin is present in endocycleton of squid pens 36,37 and γ-chitin is observed in stomach lining of squid and cuttlefish. 36 There are two saccharides, namely, N-acetyl-D-glucosamine and β 1–4D-glucosamine in the CS structure and during the deacetylation of chitin, N-acetyl-D-glucosamine monomers are transformed into Dglucosamine to generate CS. The LD 50 of CS is 16 g/kg body weight and it shows a great safety profile. The various kinds of CS are categorized based on molecular weight and deacetylation degree 38 ;CSisa polycation and its charge density is determined by pH and deacetylation degree including the solubility aspects. The CS oligomers display solubility in acidic and basic media but with increase in its molecular weight, it is only soluble in acidic media even with a higher deacetylation degree. Consequently, significant efforts have been made in synthesizing CS derivatives that are soluble under neutral and basic pH conditions by altering acetylation, polymerization, and quaternization 39 ; pKa value is suggested to be 6.5 and protonation of NH 2 groups provides CS solubility in acidic media 40 as has been affirmed that protonation of 50% of amine groups leads to CS solubility. 41 The molecular weight and deacetylation degree determine the viscosity of CS and reduction in molecular weight significantly FIGURE 1 Chemical structures of chitin and CS via deacetylation.Source: Reprinted with permission from Ref 21 ASHRAFIZADEH ET AL.3of29 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License diminishes CS's viscosity. 41–43 The biocompatibility and biodegradability are other beneficial characteristics of CS. 44–46 The biomedical application of CS nanostructures 47 has garnered much attention in recent years, especially in cancer therapy, which has been investigated in detail; CS nanoparticles (CS NPs) can mediate drug and nucleic acid therapeutic delivery, 48 chemotherapy, 49 phototherapy, 50 and imaging in cancer treatment. 51 The redox-sensitive micelles with carboxymethyl CS decoration can be utilized for NIR imaging of liver cancer cells and simultaneously, photoand chemo-therapy. 49,52 Another study evaluated the potential of gold (Au)-embedded CS nanostructures for delivery of drugs in a pH-sensitive manner and providing fluorescence imaging. 53 Notably, the surface modification of CS nanostructures is of importance for nucleic acid therapeutic delivery and synthesizing vectors that can form stable complexes with genetic tools. In a recent study, CSAu nanostructures have been applied in photoacoustic imaging-guided nucleic acid therapeutic delivery and photothermal therapy to exert a synergistic impact for cancer suppression. 54 The CS can be utilized for the development of pulmonary drug delivery systems. Notably, CS-based nano-scale delivery systems for pulmonary delivery are of importance in treatment of related cancers such as lung tumor. Furthermore, it can be used in treatment of infectious diseases such as COVID-19 that is inflicting nowadays. 55–60 The CS-based nanostructures have demonstrated great efficacy in prolonged delivery of drugs 61,62 that is of utmost importance in cancer therapy. Due to CS's positive charge, it can easily form complexes with negatively charged nucleic acids. A recent study has exploited CS-hyaluronate-SPION nanoparticles forthedeliveryofsiRNA and EP4 antagonist in cancer therapy; these nanoparticles effectively suppress HIF-1α/EP4 axis in impairing growth and invasion of tumor cells. 63 Furthermore, CS NPs can mediate pulmonary delivery of CRISPR/Cas9 system, as a new emerging genetic tool. 56 Besides cancer therapy, CS has a therapeutic impact in alleviating osteoarthritis 64 and, recently, a hydrogel system based on lactate-modified CS has been prepared for osteoarthritis treatment due to its antioxidant activity, where it exhibits high biocompatibility. 65 Furthermore, CS-based complexes can ameliorate osteoarthritis by enhancing proliferation rate of chondrocytes and preventing apoptosis. 66,67 In another study, CS NPs were used for the delivery of rosuvastatin to decrease cholesterol levels upon atherosclerosis treatment and to prevent the development of cardiovascular diseases. 68 Further, CS NPs can be utilized in anti-inflammatory formulations by delivery of drugs such as diclofenac sodium 69 and dexamethasone, 70 among others. Overall, studies highlight the fact that CS-based NPs demonstrate good biomedical applications. 71–76 The following sections explore the role of CS NPs in the delivery of anticancer drug— doxorubicin (DOX). 3|DOXORUBICIN: MECHANISM OF ACTION AND RESISTANCE The DOX is an anthracycline antibiotic derived from Streptomyces peucetius caesius with high antitumor activity 77–79 as it displays efficacy even at low doses in suppressing different neoplasms. 80 The animal experiments evaluating anticancer activity of DOX have affirmed its potential in minimizing tumor progression and improving survival of animal models. 81 Antisuppressive activity of DOX has been successfully demonstrated in preclinical models and clinical trials on various cancers, including leukemia, lymphoma, sarcoma, and urogenital cancers, among others. 80,82 DOX mainly targets genetic components in nucleus and mitochondria inhibiting the cell growth and division. However, DOX alone is not cellselective and affects also the function of healthy cells. This antitumor agent is capable of intercalating with DNA to prevent DNA replication and protein synthesis. Furthermore, DOX stimulates DNA damage in tumor cells by preventing the activity of topoisomerase II enzymes. Additional investigations revealed that DOX enhances the production of reactive oxygen species (ROS) to induce DNA damage and destroy cell membrane via direct interaction. 83–86 However, DOX resistance evolution in tumor cells is considered a major challenge as various underlying molecular pathways and mechanisms are responsible for the development of DOX resistance. 87–89 The breast tumor is a heterogeneous cancer with different subtypes and its incidence rate is various based on geographical differences. Although chemotherapy is used for breast cancer treatment, its therapy is still a challenge. 90 The lncRNA H19 is involved in triggering DOX resistance in breast tumor (invitroandinvivo)viaPARP1downregulation. 91 The lncRNA TUG1 decreases miRNA-9 expression via sponging to induce DOX resistance in breast cancer. 92 The circRNA-0002060 mediates the DOX resistance in osteosarcoma via miRNA-198 downregulation and subsequent increase in the expression level of ABCB1. 93 The TCF4 and EIF5A2 are other molecular pathways that are affected in cancer cells to regulate DOX chemotherapy response. 93,94 However, antitumor agents, such as trabectedin and resveratrol among others, have shown potential in reversing DOX resistance. 95,96 Different studies provide novel insights and pathways for development of DOX resistance. The DOX resistance in osteosarcoma can be mediated by TCF4 overexpression. In this case, circ0001721 promotes TCF4 expression via miRNA-758 downregulation to induce DOX resistance. 94 Another experiment reveals that circATXN7 increases HOXA11 expression via miRNA-149-5p downregulation to increase breast cancer progression and to inhibit DOX resistance. 97 Furthermore, EMT is responsible for cancer metastasis 98 and its induction can lead to DOX resistance in tumor cells. Therefore, targeting these pathways can effectively suppress DOX resistance in cancer. For instance, silencing RNF8, 99 CIP2A, 100 and miRNA-21 101 can inhibit DOX resistance in various tumors and impairs their proliferation. A more advanced strategy in reversing DOX resistance is the application of nanoparticles for targeted delivery of DOX at the tumor site and co-delivery of DOX with other antitumor agents or nucleic acid therapeutics. 88,102–104 4of29 ASHRAFIZADEH ET AL. 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4|NANOSTRUCTURES OF CHITOSANDOXORUBICIN 4.1 |Stimuli-responsive nanocarriers 4.1.1 | pH-responsive The tumor microenvironment has a mild acidic pH (approximately 6.5) that is lower than physiological condition and such difference in pH level can be exploited for drug release at tumor site; functionalization of nanomaterials and their drug conjugates can lead to release at this mild acidic pH. Based on this system, the bond between the material and the drug degrades under acidic pH of tumor site to mediate drug release. 105 Various kinds of bonds including imine, hydrazone, oxime, amide, acetals, and orthoester can be deployed for synthesizing pHsensitive nanocarriers and drug release at tumor site 106 as has been discussed in this section. Figure 2provides a schematic representation of stimuli-responsive CS-based nano-scale delivery systems in cancer therapy. Recently, a CS-based polymeric prodrug, that is pH sensitive and can provide a platform for co-delivery of DOX and siRNA, has been synthesized. The DOX and Bcl-2-siRNA can be conjugated to CSmodified polymeric nanoparticles and then, synergistic cancer therapy is provided. These nanostructures are internalized by hepatoma cells via glycyrrhetinic acid receptor-mediated endocytosis. After 10 h, nanostructures can release siRNA and DOX as much as 90.2% and 81.3%, respectively. This CS-based polymeric prodrug efficiently suppresses tumor progression (HepG2 cells) by 88% via mediating both chemoand nucleic acid-therapy. 107 The CS is a pH-sensitive agent due to the presence of amine groups ( NH 2 ) that undergoes protonation in acidic pH 108,109 ; higher pH significantly decreases the solubility of CS. 109 On the other hand, polyvinylpyrrolidone (PVP) is often utilized for the synthesis of nanoparticles, but it significantly decreases the initial burst release. 110 For overcoming such issues, the combination of PVP and CS has been suggested to improve the solubility of CS at high pH levels and mechanical characteristic of PVP, simultaneously. 109–111 A recent study demonstrated that CS/PVP/α-Fe 2 O 3 nanocomposites for the delivery of DOX, where nanoparticles had a spherical structure, and they could load Fe 2 O 3 in CS/PVP. The nanostructures showed a particle size of 247 nm and due to conjugation of α-Fe 2 O 3 , they demonstrated prolonged release and increased retention of DOX at tumor FIGURE 2 Active targeting and stimuli-responsive drug release of CS-doxorubicin nanocarrier. The surface modification of CS-based nanostructures with ligands can increase their internalization in tumor cells. Besides, External and internal stimuli can be utilized for developing smart nanocarriers in cancer therapy. CSC, cancer stem cell ASHRAFIZADEH ET AL.5of29 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License site. The CS/PVP-based nanocomposites release DOX in a pHsensitive manner, mimicking pH level of tumor microenvironment and they induced apoptosis to significantly decrease the viability of breast cancer (MCF-7 cells). 112 There are several reasons for using pH-sensitive nanocarriers for cancer therapy and also delivery of chemotherapeutic agents. The nonspecificity of systemic chemotherapy can negatively affect normal and healthy cells after intravenous injection. 113,114 Besides, repeating injections can lead to pain, infection, and hospitalization. Therefore, the application of nanoparticles for sustained delivery of chemotherapeutic agents eliminates the need for repeated injections and can improve the chance in the fight against cancer. 114–116 Due to glycolysis phenomenon in the tumor microenvironment and conversion of glucose to lactate, pH level is significantly diminished, which is beneficial for cancer progression. 117,118 A recent experiment has advanced graphene-CS nanocomposites for DOX delivery, which were stabilized with bovine serum albumin (BSA); the presence of BSA is beneficial in preventing burst release of drug from CS nanocomposites. They show uniform release over 24 h and can release drug for up to 28 days (84% of drug). Such prolonged release of DOX from CS-decorated nanocomposites that is pH-sensitive can improve cytotoxicity against cancer cells. 119 In another study, a composite structure consists of halloysite nanotubes as a natural aluminosilicate ceramic and CS was designed for pH-responsive release of DOX for breast cancer therapy. The drug-loaded carrier showed a sustained release in cell lysate and the mechanism of action was to penetrate into mitochondria followed by inducing damage. Moreover, the IC 50 of nanocarrier against MCF-7 cells was found 1.17 μgml 1 lower than that of free DOX (2.43 μgml 1 ). The in vivo studies revealed that the tumor inhibition ratio of the nanocarrier was 83.5%, whereas the free DOX showed 46.1%. Notably, the treated mice with the DOX-loaded carrier survived over 60 days without a significant systemic cytotoxicity (Figure 3). 120 The UV-triggered injectable CS hydrogels are extensively applied in biomedicine. 121–124 Different UV-crosslinkable CS derivatives have been designed via covalent attachment of UV-responsive components 125–128 to improve their solubility. The “thiol-ene”click chemistry can be utilized for the development of pH-responsive UV crosslinkable CS hydrogel. The UV crosslinking ability and pHsensitive capacity of CS ensue from allyl groups on C 6 site and amine FIGURE 3 A pH-responsive CS carrier in combination with halloysite nanotubes for breast cancer therapy. (a,b) The synthesis procedure followed by loading of DOX molecules on the chitosan (COS)- halloysite nanotubes (HNTs) and the uptake process by which the drug-loaded carrier induce cytotoxicity toward cancerous cells. (c) Tumor volume changes after being treated with different samples including control (CON), free DOX, unloaded carrier, and DOX-loaded carrier; significant difference with the control group ***P< 0.001, unloaded carrier at ###P< 0.001, and DOX at &P< 0.01. (d) The drug release from the carrier at different pH. (e) The excised tumors removed at the end of treatment after being treated with different samples. (f) The histology that was accomplished through H&E staining on the 14th day of treatment. Source: Reprinted from Ref 120 with permission from ACS 6of29 ASHRAFIZADEH ET AL. 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License groups on C 2 site. At various pH levels, CS-based hydrogels show different behaviors, and their swelling and shrinkage can lead to the release of DOX in a pH-sensitive manner. 129 The purpose of using CS in the modification of nanoparticles is its capacity in functionalizing or loading various antitumor drugs (DOX), targeting ligands (aptamer), coating polymers and imaging probes. 130 For this purpose, Au nanoparticles were modified with CS and DNA aptamer to mediate selective targeting to glioblastoma cells. Obtained nanostructures were utilized for delivery of 5-flourouracil (5-FU) and DOX in glioblastoma suppression. The prepared CS-Au NPs had a particle size of 196.2 nm with zeta ζ-potential of 16.26 mV and they significantly enhanced the cytotoxicity of DOX and 5-FU against glioblastoma cells and induced cell death and G0/G1 cell cycle arrest. 131 Based on these results, it can be concluded that CS is a promising agent for the synthesis of pH-responsive nanocarriers for DOX delivery and cancer suppression 132 and can be easily functionalized by other ligands to improve the selectivity of NPs toward cancer cells, while simultaneously providing co-delivery of DOX and other antitumor agents such as 5-FU. By providing pH-sensitive feature, these nanocarriers release DOX at the tumor site and also mediate sustained delivery, which are both beneficial in cancer suppression. 4.1.2 | Redox responsive The presence of redox imbalance is another unique feature of tumor microenvironment that is responsible for enhancing tumor proliferation rate. The production of ROS, initiated by inflammatory cells, endothelial cells, and cancer-associated fibroblasts, induces an aerobic glycolysis and significantly increases tumor progression. On the other hand, glutathione (GSH) is an enzyme that regulates oxidative stress and diminishes ROS levels. In the pharmaceutical industry, there have been efforts in synthesizing redox-sensitive nanomaterials. Preparation of such nanostructures usually involves incorporation of disulfide as it undergoes degradation by GSH. 133,134 This section focuses on redox-sensitive CS-NPs and disulfide bond decomposition by GSH at the tumor site for DOX release. As has been discussed in the previous sections, a combination of CS with other agents is applied to synthesize NPs and improve features that are important for cargo delivery. The CS oligosaccharide (CSO) and stearic acid (SA) can be utilized for the preparation of glycolipid-like copolymer 135 and ensued CSO-SA copolymer has high stability that can be further exploited for drug delivery applications 136–139 as it exhibits high internalization and cellular uptake. 136 However, micelles prepared from CSO-SA have a major challenge to efficiently release the drug in vitro due to slow degradation kinetics of amide bond. To improve drug release profile from CSO-SA-based NPs, DOX can be conjugated with CSO-SA via disulfide bond. This approach is advantageous in synthesizing CSO-SAbased nanoparticles that are redox sensitive and due to higher GSH levels in cells, they release DOX to suppress breast cancer progression (MCF-7 cells). 140 In another study, carboxymethyl CS (CMC)-based micelles for delivery of DOX in cancer therapy were prepared. The poly-ε-caprolactone (PCL)-SS-CMC self-assembled into micelles for improvement of their selectivity against cancer cells (liver and cervical cancers) and were modified with glycyrrhetinic acid (GA). Then, DOX and another antitumor agent known as pheophorbide A (PHA) were loaded on these NPs, which showed a release profile up to 86.3% and 92.1% of DOX and PHA, respectively, after 48 h. This approach is beneficial in enhancing intracellular accumulation of DOX and PHA by providing GA receptor-mediated endocytosis and redox-sensitive system. 49 Although a few experiments have evaluated the potential of redox-sensitive CS-based NPs for delivery of DOX, 140 they highlight the fact that disulfide bond between CS and DOX can be easily degraded in the presence of GSH. This system is biocompatible and its modification with ligands can be performed to promote its potential in DOX delivery. 4.1.3 | Light responsive A few experiments have exploited the role of CS-based nanostructures for light-mediated release of DOX, which deployed nanobubbles. 141 The nanobubbles have spherical core–shell structure and their surface can be conjugated with functional groups. The nanobubbles possess enhanced permeability and retention (EPR) effect that is of importance for crossing over endothelial barrier. The nanobubbles can be administered via intravenous route and in order to improve their biocompatibility and biodegradability, surface modification of nanoparticles with PLGA and PCL can be performed. 142 In an experiment, modification of nanobubbles with CS has been conducted. The CS nanobubbles can release DOX in vitro upon irradiation and significantly enhance uptake of DOX in mammalian cells. These biocompatible nanobubbles can be used for inhibiting breast tumor suppression (MCF-7) via light-mediated DOX delivery. 141 A lightresponsive theranostic platform composed of DOX-loaded gold nanoparticles and CS was reported for breast cancer therapy. Applying NIR irradiation not only facilitated the payload release in the cancer cells but also caused the gold nanoparticles to elevate the inner temperature up to level, which leads apoptosis. Besides photothermal therapy, it was observed that the liberated DOX caused an oxidative stress through generation of ROS. 143 Through Figure 4, the preparation and the mechanism of action of the light-responsive platform are indicated. 4.1.4 | Thermosensitive The thermosensitive hydrogels have garnered much attention in recent years as an implantable delivery system. Thermosensitive hydrogels are injected into tumor site in a liquid state and undergo conversion into a solid gel at body temperature. Besides, hydrogels can provide the prolonged release of antitumor drugs at the tumor site. 144–146 A study describes the synthesis of thermosensitive CS hydrogel to encapsulate liposomal DOX with a particle size of 94.2 nm and encapsulation efficiency as much as 98%. This ASHRAFIZADEH ET AL.7of29 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License thermosensitive hydrogel changes to a solid gel at body temperature and provides sustained release of DOX. These hydrogels demonstrated a high safety profile and biocompatibility, and simultaneously, they showed capacity in suppressing tumor progression (H22 hepatoma cells and tumor-bearing mice). 147 The application of CS prevents the burst release of DOX from hydrogels. Notably, CS-DOX conjugate in hydrogels does not affect the antitumor activity of DOX and it is comparable to free DOX. Both in vitro and in vivo experiments have shown the potential of hydrogels containing CS-DOX conjugate for cancer suppression (A549 lung cancer cells and nude mice). 148 Furthermore, loading liposomal DOX in hydrogel does not negatively affect the entrapment efficiency of liposomes. For instance, an experiment synthesized CS-based thermosensitive hydrogel containing liposomal DOX for topical cancer treatment (hepatoma). The entrapment efficiency was 90% and after loading liposomal DOX in the hydrogel, its entrapment efficiency did not change. 149 Notably, carbon nanotube (CNT)-CS can be loaded in thermosensitive hydrogels for controlling DOX release. Their exposure to irradiation provides a photothermal effect of CNTs that is beneficial in destroying hydrogel structure and mediating DOX delivery. 150 Therefore, a thermosensitive hydrogel can be synthesized first for conversion to solid gel at body temperature and in the next step, CS-carbon nanotubes are loaded into the hydrogel for regulating DOX release upon irradiation. 150 The succeeding section focuses on multisensitive CS-based nanocarriers for DOX delivery. 4.1.5 | Multiresponsive There have been many efforts in developing multifunctional CS-based NPs for DOX delivery in cancer treatment including nanocomposites that are triple sensitive. For preparing such systems, CS is utilized as a pH-sensitive agent, g-poly(N-vinylcaprolactam) (PNVCL) as a thermosensitive agent and H6R6 as a cell-penetrating peptide. Then, DOX and oleanolic acid (OA) are loaded on nanocomposites with a particle size of 190 nm, the loading efficiency being 13.2% and 7.3% for DOX and OA, respectively. These nanocomposites are accumulated in the tumor microenvironment and the drug is released at the tumor site. The in vitro and in vivo experiments demonstrated the potential of DOX and OA-loaded CS/PNVCL/H6R6 nanocomposites in suppressing cancer progression and apoptosis induction (SKOV3 ovarian cancer cells and nude mice). 151 Another strategy for improving the physicochemical properties of CS exploited its conjugation with PEG, which significantly enhances solubility and biocompatibility. The hollow mesoporous silica NPs have been modified with CS and PEG with loading efficiency of 32.8%. The DOX is loaded in PEF-CSsilica nanoparticles and there is no release of DOX at low levels of GSH and pH 7.4. However, a mild acidic pH or higher levels of GSH can induce DOX release in breast cancer suppression. 152 Another study prepared alginate/CS-based NPs for delivery of DOX that are pHand light-responsive where alginate improved stability of CSbased NPs and could release DOX in pHand light-sensitive manner after irradiation and in mildly acidic pH (Figure 5). 153 Based on these FIGURE 4 The light-responsive DOX-loaded gold-chitosan nanocomposite. (a) The preparation of CS-gold nanorattles (AuNRT), DOX loading and the effect of NIR irradiation on the payload release. (b) The thermal images related to the nanocomposite after being irradiated with 785 nm laser for 15 min with various concentrations including (i) water as the control, (ii) 75, (iii) 150, (iv) 200, (v) 500, and (vi) 800 μgml 1 . (c) Measurements of temperature increase after applying on/off cycles at a power density of 5 W/cm 2 .Source: Reprinted from Ref 143 with permission from ACS 8of29 ASHRAFIZADEH ET AL. 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License studies, CS and its combination with other agents can be beneficial in the development of multifunctional nanostructures for DOX delivery (Table 1). 159–161 Besides internal-responsive CS-NPs for DOX delivery, there is another category termed hyperthermia-based external-stimuli drug delivery systems. These systems are responsive to an external stimulus, like magnetic field and light capable of increasing the temperature of the tumor microenvironment followed by killing the cancerous cells. 162 Moreover, after being triggered by the mentioned stimuli agents, the anticancer drug release rate undergoes a significant increase thus improving the efficiency of the delivery system. 163 This strategy has been exemplified by a multifunctional chemophototherapeutic delivery system where black phosphorus nanosheets have been adopted as an inorganic light-responsive material; the DOX has been first loaded onto the nanosheets followed by being surface modified with CS-PEG and folic acid to form the final platform (BP-DcF). Next, small interfering RNA and programmed death-ligand 1 (PL) are encapsulated into the delivery system. Upon irradiation of near-infrared, a hyperthermia effect and burst release of DOX could be observed and the combination of both chemo and phototherapies culminated in an effective tumor cells apoptosis (Figure 6). 164 Overall, some technical conclusions about CS-based nanocarriers for DOX delivery can be provided. Due to protonation of amine groups of CS at acidic pH that is similar to tumor microenvironment pH, the pH-sensitive release of DOX occurs upon CS modification of nanocarriers. Notably, by loading some kinds of nanoparticles such as Fe 3 O 4 in CS-based nanocarriers, multifunctional nanoarchitectures are designed that can provide stimulus-responsive release of DOX and simultaneous imaging. 4.2 |Reversing drug resistance There are several reasons responsible for role of NPs in reversing chemoresistance. Frequent application and high doses of chemotherapeutic agents can result in drug resistance development and in targeted delivery, a low amount of anticancer agent is loaded that reduces chance of drug resistance, while it maintains tumorsuppressor activity. 165–167 Additionally, increasing accumulation of FIGURE 5 Multiresponsive CS-NPs for DOX delivery. (a) (i) Step-by-step synthesis of multiresponsive (pH, thermo, and redox responsive) DOX/(oleanolic acid [OA[)@functionalized cell-penetrating peptide (H6R6)-chitosan (CS)-g-poly(N-vinylcaprolactam) (PNVCL) NPs. (ii) The applicability of nanoparticles for anticancer therapy; the improved permeation and retention leads the NPs to remain in the tumor environment followed by triggering triple sensitivity to release doxorubicin (DOX) and OA. Source: Reprinted from Ref 151 with permission from Elsevier. (b) (i) Synthesis of DOX-loaded alginate (ALG)/chitosan (CS) stabilized perfluorohexane (PFH) NPs through nano-emulsion technique. (ii) Double sensitivity (ultrasound and pH) of the DOX-loaded nanodroplets against cancer cells. Source: Reprinted from Ref 153 with permission from Elsevier ASHRAFIZADEH ET AL.9of29 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License micelles and carbon nanomaterials should be evaluated in terms of co-delivery of DOX and other drugs. 4.5 |Lipid nanoparticle modification Liposomes are synthetic lipid NPs that have been first discovered in the 1960s and comprise a lipid bilayer with an aqueous core. 230 The liposomal nanocarriers can be exploited for the delivery of both hydrophilic and hydrophobic drugs. The hydrophilic drugs can be loaded in the core, while hydrophobic drugs can be loaded in lipid bilayer. The drugs loaded in liposomes are protected against inactivation in blood circulation, dilution, and degradation. The clinical application of liposomes has some impediments such as their rapid clearance, immune system activation, and accumulation in other organs. 231 Recent experiments have exploited liposomes for delivery of DOX or its co-delivery with other agents such as hispolon, curcumin, and linalool in combination cancer therapy. 232–235 Therefore, liposomal nanocarriers can provide delivery of DOX at the tumor site and this section focuses on CS-modified liposomes in DOX delivery. An experiment has conjugated DOX to amphiphilic stearolylspermine anchor to produce a prodrug. Then, this prodrug has been loaded into liposomal nanocarriers for colorectal cancer therapy. For improving the stability of DOX-loaded liposomes, their modification with CS and PEG has been made. Furthermore, stearoyl chains promote local microfluidity of liposomes and spermine via amine groups interacts with phosphate groups of lipids in improving liposome stability. In addition to stability, CS-PEG modification of liposomes prevents aggregation and this coating mediates charge neutralization. These neutral liposomal nanocarriers demonstrated cytotoxicity against A549 (lung cancer) and Caco-2 cells (colorectal tumor) and they showed high stability. 236 Another study described the preparation of liposomes using hydrogenated soy phosphatidylcholine (HSPC) and cholesterol (CHOL) and glycol CS conjugation has been performed during film preparation. Then, DOX was embedded in preformed liposomes via transmembrane pH gradient loading strategy. These CS-based liposomes enhance internalization of DOX in tumor cells (HT1080 cells) and were successful in enhancing the therapeutic efficacy of DOX in vitro and in vivo. This system was pH-sensitive and could be exploited for the treatment of other cancer types. 237 Another kind of lipid-based nanoparticles that can be utilized for cancer therapy are micelles as they are considered as promising vectors in drug delivery and cancer treatment due to their ease of synthesis and chemical modification. Furthermore, the size of micellar NPs is tunable and they can enhance drug solubility in water and significantly promote blood circulation of drugs. The increased bioavailability of the drug, lowering adverse impacts and high accumulation at tumor site are other benefits of using micelles for drug delivery. The micelles can provide pH-responsive release of DOX at the tumor site and provide co-delivery of DOX with other antitumor agents such as cisplatin for synergistic cancer therapy. Furthermore, surface modification of micelles, for instance, with phenyboronic acid promotes selectivity toward tumor cells and enhances the tumor-suppressor activity of DOX. 238–242 The DOX-loaded micelles can be prepared using alginate and CS in a water-in-oil emulsion method with a spherical particle size of 80 nm. This is an interesting method for loading DOX in nanocarriers and uses an aqueous phase dispersed in a cyclohexane/dodecylamine organic phase. These nanocarriers showed high cellular uptake by breast cancer cells and can suppress proliferation of 4 T1 cells. 243 The CS-modified micelles can also provide co-delivery of DOX and curcumin in liver cancer therapy. The CS-cystamine-poly(ε-caprolactone) copolymer micelles have been prepared for curcumin and DOX codelivery and then, modification with GA has been performed in enhancing their cellular uptake. They showed drug loading efficiency of 19.8% and 8.9% for DOX and curcumin, respectively. They had a spherical shape with a particle size of 110 nm. The GA modification enhanced its internalization in cells via endocytosis and exposure to the tumor microenvironment induced changes in charge of nanocarriers from negative to positive. These CS-modified micelles are pHand redox sensitive and 10 mM of GSH induces the release of DOX (80.6%) and curcumin (67.2%). This combination therapy exerts a synergistic impact and is beneficial in suppressing the progression of hepatoma cells. 244 Overall, CS derivatives that can self-assemble into micelles, are able to provide nanocarriers that are biocompatible, have low immunogenicity, and provide nanoplatforms for drug delivery. 245–247 An experiment has prepared N-succinyl-N0-octyl chitosan (SOC)-based micelles for DOX delivery and increasing the ocetyl chain amount, promotes capacity of these micelles in DOX loading. Drug loading and ocetyl chain number determine the size of micelles and they have a particle size of 100–200 nm. They showed high antitumor activity against various cancer types including HepG2, A549, BGC, and K562 cells. 248 Hence, similar to liposomes, micellar nanoparticles are potential vectors for DOX delivery and cancer suppression as well as preventing drug resistance development. 249,250 4.6 |Metal nanoparticle modification Metal–organic frameworks (MOFs) can be considered as ideal options for drug delivery due to their nanoscale size, high surface area, and porosity as well as adjustable size. 251,252 To improve the property of MOFs in drug release, their modification with polymers has been performed; CS modification of MOFs renders them pH-sensitive feature and provides a condition for sustained release of DOX in cancer chemotherapy. Furthermore, CS can be functionalized by folic acid (FA) for selective targeting of tumor cells overexpressing folate receptor. Then, DOX can be loaded in CS-modified MOFs with a high drug loading capacity (1.63 g). Notably, MOFs can encapsulate carbon dots for providing imaging. These CS-based metal NPs provide simultaneous chemotherapy and bioimaging that are beneficial in cervical cancer treatment. 253 When exposed to mild acidic pH of the tumor microenvironment, CS layers located on the surface of MOFs would collapse and swell, leading to the release of DOX at the tumor site and subsequent breast cancer suppression. 254 16 of 29 ASHRAFIZADEH ET AL. 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License TABLE 3 A summary of CS-based nanostructures for DOX delivery in cancer suppression Nanostructure Particle size (nm); zeta potential (mV); encapsulation efficiency or drug loading (%) Cancer type In vitro/ in vivo Cell line/animal model Remarks Referencs Estrogen-functionalized CS nanoparticles 198.2 and 206.4 nm; 28.3 and 30.6 mV; up to 66.33% Breast cancer In vitro MCF-7 cells High biocompatibility and antineoplastic activity 278 CS-raloxifene nanoparticles 26.85 and 34.75 nm; 0.17 and 0.49 mV; up to 98% Breast cancer In vitro MCF-7 cells Decreasing proliferation rate by 60% Nanoparticles inhibit cancer progression via suppressing estrogen receptor 279 DOX-loaded LGCC NPs 200 nm; 20–35 mV; up to 86.4% Breast and liver cancers In vitro; in vivo QGY-7703 and 4 T1 cells H22 hepatocarcinoma model Penetrating directly via cell membrane and circumventing endocytic vesicles Cargo release under high GSH levels Endosomal and lysosomal escape High nuclear distribution 262 Catechol-modified CS-hyaluronic acid nanoparticles 160 nm; 19.8 mV Oral cancer In vitro HN22 cells Negative charge and spherical shape High mucoadhesive ability Prolonged release of DOX Reducing cancer proliferation 280 Ethyl cellulose/CS/g-C 3 N 4 /MoS 2 core– shell nanofibers 285–370 nm Breast and cervical cancers In vitro MCF-7 and HeLa cells Sustained delivery of DOX Inducing cell death up to 89% and 85% in MCF-7 and HeLa cells, respectively in 7 days 281 Aptamer-functionalized CS-bases silica nanostructures 87 nm; 35.9 to 32.3 mV Breast cancer In vitro; in vivo MCF-7 and 4 T1 cells C26 tumor-bearing mice Enhanced cellular uptake Targeted delivery of DOX and antimiRNA-21 in cancer suppression 282 PEGylated CS nanoparticles 169–192 nm; up to 43 mV Breast cancer In vitro MCF-7 cells Functionalization of CS nanoparticles with anti-hMAM and anti-HER2 promotes selectivity toward cancer cells Exerting dose-dependent toxicity against cancer cells 283 CMC/PCL nanofibers 300 nm; higher than 30 mV; 90% Breast cancer In vitro MCF-7 cells Lack of initial burst release Sustained release for 7 and 25 days Cytotoxicity against tumor cells up to 85% 284 HMSN grafted with CS-copper sulfide composites 150 nm; 19.6 mV; 46.1% Breast cancer In vitro; in vivo MDA-MB-231 cells Mouse model of breast cancer High biocompatibility Increased cellular uptake by cancer cells Apoptosis induction Increasing survival of mice 285 (Continues) ASHRAFIZADEH ET AL.17 of 29 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License The interesting note is the modification of Fe 3 O 4 nanoparticles with CS that provides a drug delivery system that can mediate bioimaging. The surface modification of Fe 3 O 4 with CS is of importance for loading DOX. The CS can form a stable complex with DOX and produces NH 2 -Zn(II)-DOX structure. Exposing to certain pH destroys the bond between DOX and CS and leads to the pH-sensitive release of DOX and Fe 3 O 4 nanoparticles can provide magnetic resonance imaging. 255 As has been mentioned in the Introduction section, CS can undergo changes by chemicals and enzymes. There have been efforts in the chemical modification of CS and improving its properties. It has been reported that CS modification with guanidine moieties significantly elevates its intracellular accumulation; guadinylated CS can form chelates with copper via copper-nitrogen coordination. The CS-copper complexes enhance the production of ROS in impairing lung cancer progression and exert synergistic cancer therapy with DOX. 256 Based on these experiments, modification of metalbased nanostructures with CS improves their biocompatibility and loading of DOX. Notably, further progress can be made by the modification of CS-metal NPs. A recent experiment has synthesized CS-Au nanostructures and for enhancing their selectivity toward lung (A549) and breast (4 T1) cancer cells, their modification with nucleolin aptamer (AS1411) was conducted. These nanocomplexes targeted the tumor cells and effectively penetrated into them, leading to DOX accumulation and subsequent cancer progression suppression. 257 Therefore, CS is used as a reducing and stabilizing agent for loading DOX on Au NPs and further modification of CS-Au NPs with polyethylene glycol promoters their blood circulation that is of importance for elevating cytotoxicity of DOX against lung cancer cells. 258 Taking everything together, efficacy of metal-based NPs for DOX delivery and cancer suppression can significantly enhance by CS modification. 259–261 4.7 |Carbon-based nanoparticle modification A recent experiment has provided novel insights about the surface modification of graphene composites by CS and its impact on DOX conjugation and release. The concentration or pH of the solution (level of CS protonation) affects the aggregation and dispersion of CS on the surface of graphene composites. At low concentration levels of CS and DOX, the bare surface of graphene composites enhances. Increasing the numbers of NH 2 CS and DOX molecules promotes the adsorption of DOX on bare surface areas of graphene and mediates encapsulation of DOX by CS clusters on the surface or results in conjugation with CS chains. On the other hand, when levels of NH 3 CS and DOX increase, there will be higher positive charges and lower bare surface area of graphene that can provide conditions for the release of DOX. Therefore, at mild acidic pH of the tumor microenvironment, protonation of CS occurs and leads to the release of DOX from nanocarriers. 262 Graphene oxide (GO) is a derivative of graphite or graphene and has a two-dimensional plate-like structure. The GO sheets have both sides and due to their large surface area, they are considered as promising structures for the delivery of antitumor TABLE 3 (Continued) Nanostructure Particle size (nm); zeta potential (mV); encapsulation efficiency or drug loading (%) Cancer type In vitro/ in vivo Cell line/animal model Remarks Referencs CS-, PEGand PVA-modified MgFe 2 O 4 ferrite magnetic nanoparticles 78–140 nm; below 21 mV Breast and colorectal cancers In vitro Caco-2 and SKBR-3 cells Decreasing cancer cell viability in a concentration-dependent manner pH-sensitive release of DOX 85.86% release of DOX after 72 h 286 CS hydrogel beads 13.5 mV Breast cancer In vitro MCF-7 cells High swelling rate (426%) and drug release (81.33% in 144 h) at pH of 5.8 High biocompatibility Decreasing proliferation rate of MCF-7 cells 287 CMCS/MAGG hydrogel - Breast cancer In vitro MCF-7 cells pH-responsive swelling of hydrogels 67.06% release of DOX after 5 days in pH of 5.5 32.13% release of DOX at pH of 7.4 High biocompatibility Cytotoxicity against MCF-7 cells 287 Abbreviations: CMC, N-carboxymethyl chitosan; CS, chitosan; DOX, doxorubicin; GSH, glutathione; HMSN, hollow mesoporous silica nanoparticle; NPs, nanoparticles; PCL, poly(ε-caprolactone); PVA, polyvinyl alcohol, PEG, polyethylene glycol. 18 of 29 ASHRAFIZADEH ET AL. 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License drugs. Furthermore, aromatic molecules and nucleobases can be loaded on GO composites via π–πstacking and hydrogen bonding interactions. 263,264 For improving the biodegradability of CS and its solubility, conjugation of CS with other agents such as acrylic acid (AA) and itaconic acid (IA) monomers has been performed to improve its functionality via COOH group. Furthermore, modification by folic acid (FA) selectively targets tumor cells overexpressing folate receptor. A recent experiment has first converted GO sheets to aminefunctionalized GO (AGO) to provide a function of a cationic polyelectrolyte. Next, CS and FA conjugation via N,N0-dicyclohexylcarbodiimide was performed. Then, CS was chemically modified by AA and IA monomers by binding to COOH group via ethyleneglycol dimethacrylate as cross-linker and potassium peroxydisulfate as an initiator. Subsequently, DOX was embedded into FA-chemically modified chitosan (CMCS)/AGO nanocomposites via π–πstacking interactions. The prepared nanocomposites demonstrated drug loading capacity as much as 95% and they released DOX at a pH-sensitive manner (release of DOX at pH 5.3 compared to pH 7.4). These DOX-loaded nanoparticles were able to significantly reduce the viability of tumor cells including HeLa and MCF-7 cells. 265 Another experiment has prepared injectable hydrogels for the controlled release of DOX. This hydrogel is based on cross-linking of graphene, CS, and cellulose nanowhiskers where Schiff base reaction by a synthetic dialdehyde has been used. This hydrogel is responsive to pH and other stimuli by adding benzaldehyde and amino acid cysteine and can be administered subcutaneously to deliver both DOX and curcumin in synergistic cancer therapy. 266 In addition, CNTs can be modified with CS to provide pH-sensitive release of DOX in cancer therapy; CNTs can be noncovalently wrapped with CS and then loaded with DOX. Due to the deprotonation form of CS, DOX release does not occur at physiological pH, whereas protonation of CS at pH 5–6.5 leads to DOX release due to charge–charge repulsion between CS and DOX, resulting in controlled drug release 267 that is beneficial for cancer therapy. Another experiment prepared graphene FIGURE 8 Modification of various kinds of carbon-based nanostructures with CS for DOX delivery and cancer suppression. (a) Surface modification of single-walled carbon nanotubes (SWCNs) with CS followed by loading doxorubicin (DOX) and paclitaxel (PTX); (i and ii) DOX and PTX loading on the bare SWCNs from front and side views; a close view of DOX (iii) and PTX (iv) orientation on the side of SWCNs; CS modification on the surface of SWCNs (v and vi); the final drug-loaded CS-coated SWCNs from front and side views (vii and viii). Source: Reprinted from Ref 277 with permission from RSC. (b) A schematic on the synthesis and applicability of ternary DOX-loaded graphene oxide nanoparticles (GON)-CS-dimethylmaleic anhydride (DMMA) for cancer therapy. Source: Reprinted from Ref 276 with permission from ACS publication. (c) A schematic on the synthesis of folic acid (FA)-anchored O-carboxymethyl CS (OCMC)-Fe 3 O 4 modified with carbon dots (CDs) for DOX delivery to the cancer cells. Source: Reprinted from Ref 253 with permission from ACS publication. NH 2 -H 2 BDC, 2-amino terepthalic acid; IRMOF-3, metal organic framework ASHRAFIZADEH ET AL.19 of 29 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License and CNTs for co-delivery of DOX and paclitaxel (PTX). For improving loading efficiency and release features, further functionalization by CS has been performed. Furthermore, CS provides pH-sensitive manner release of DOX and PTX and slow release of these antitumor drugs promotes tumor-suppressor activity. 268 Based on these experiments, carbon-based NPs can provide delivery of DOX in cancer therapy and CS modification improves their beneficial features. 99,269–275 Figure 7 demonstrates modification of carbon-based nanomaterials with CS and then, conjugation of folic acid as ligand on nanocarriers to mediate their internalization in cervical cancer cells via endocytosis, resulting in a significant increase in accumulation of DOX in tumor cells. 253,276,277 Table 3provides a summary of various nanoparticles modified by CS for purpose of DOX delivery in cancer suppression. Taking everything together, these studies highlight the fact that various kinds of lipid-, carbonand metal-based nanostructures can be modified with CS in improving their characteristics. The nanoparticles demonstrate low particle size, sustained drug release, and high encapsulation efficiency. Furthermore, CS modification may significantly enhance biocompatibility and stability of nanocarriers. Modification of CS-based nano-scale delivery systems with ligands such as HA and folic acid increases their selectivity toward tumor cells for specific accumulation of DOX. The CS modification appears to be vital for metaland carbonbased nanocarriers, as they may show toxicity toward normal cells and such modification improves their biocompatibility(Figure 8). 278–287 4.8 |Directions for clinical application The current review article demonstrated that rational integration of engineering and biology appears to be promising in treatment of cancer. The gold standard for improving prognosis and survival of cancer patients is chemotherapy. However, chemotherapy failure is a common outcome in cancer patients due to drug resistance. Therefore, there is an urgent need toward development of targeted delivery systems for chemotherapeutic agents. Since DOX is frequently used in clinic for treatment of cancer patients, resistance to its anticancer activities is common. Although problem (drug resistance) is obvious and one of solutions is application of nanocarriers, there are still a number of challenges for use of DOX-loaded nanostructures in clinical course. The first problem is related to biocompatibility of nanoparticles for DOX delivery in cancer patients. The second problem is affordability and final difficulty is related to large-scale production of nanocarriers. All of these problems can be solved using greener modification of nanoparticles. Throughout this review article, it was shown that surface modification of various nanocarriers by CS promotes their biocompatibility, safety profile and increases their stability. Furthermore, CS is a natural product that is affordable and can be used for synthesis on larger scale. A number of clinical studies (clinicaltrials.gov) have been conducted on CS application in patients; however, there is no experiment pertaining to the use of CS-based nanocarriers for DOX delivery in treatment of cancer patients that exploits the afore mentioned benefits of CS. Hopefully, it will occur in the near future. 5|CONCLUSION AND REMARKS The CS is among the most abundant polysaccharides that have received much attention in recent years and a large number of experiments have been performed in revealing therapeutic potentials of CS and its efficacy in NPs synthesis and their modification. Furthermore, as CS is a nature-derived agent, it is affordable and can be utilized in both preclinical and clinical studies. Regardless of the protective impacts of CS in diabetes, anti-inflammatory diseases and cardiovascular diseases among others, CS appliance in cancer has undergone a surge due to its cytotoxicity against tumor cells and ability in nanostructure preparation with high biocompatibility. On the other hand, cancer treatment depends on overcoming drug resistance to improve chemotherapy efficacy in tumor cell suppression. Among various chemotherapeutic agents, DOX is a well-known compound and that is why DOX it has been discussed in this review article. Frequent application of DOX leads to drug resistance and as DOX is an FDAapproved agent and is utilized in the clinic for the treatment of cancer patients, efforts should be made in reversing this resistance. The present review focused on CS-NPs for DOX delivery in cancer treatment. Different kinds of stimuli-responsive CS-based nanostructures have been developed for DOX delivery including pH-, redox-, thermoand multi-sensitive nanocarriers. The pH of the tumor microenvironment tends to be acidic and is lower than physiological pH. Therefore, pHsensitive CS-based NPs can deliver DOX at tumor site. Due to glycolysis and high proliferation rate of tumor cells, redox-sensitive NPs are also of importance and GSH levels can induce DOX release from CS nanostructures. The thermosensitive CS-based hydrogels have been also developed that can be transformed to solid gel at body temperature and provide sustained release of DOX that is of importance for enhancing cytotoxicity against cancer cells and preventing the drug resistance development. Based on the documented experiments, CS has antitumor activity and it can exert a synergistic impact with DOX. Furthermore, CSbased NPs promote DOX internalization in cancer cells. The enhanced cellular uptake is vital for promoting the sensitivity of cancer cells to DOX chemotherapy. Furthermore, using other agents such as TPGS with CS can prevent P-gp activity in inhibiting DOX efflux from tumor cells. Hence, CS-based NPs can prevent the development of DOX resistance. Notably, CS-based NPs can provide co-delivery of DOX with antitumor drugs and nucleic acid therapeutics. This strategy can induce apoptosis in tumor cells in providing DOX sensitivity. Furthermore, the proliferation rate and the invasion of tumor cells undergo a decrease, and the pathway is paved for DOX to induce its tumorsuppressor activity. Besides, various kinds of NPs including carbon-, lipid-, polymerand metal-based nanostructures can be modified with CS to improve their stability and biocompatibility and provide conditions for DOX complex formation. A search at clinicaltrials.gov shows that CS is currently applied in the clinical course for the treatment of various cancer such as prostate and breast cancers (NCT03202446; NCT03712371). Therefore, future experiments can be directed toward using DOX-loaded CS-based nanoarchitectures in the clinic. The application of some of the CS-modified nanoparticles in clinical 20 of 29 ASHRAFIZADEH ET AL. 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License course still needs extensive investigation. For instance, CS-modified metal nanostructures still require examination in terms of biocompatibility, as metal nanoparticles are toxic toward normal cells. The same explanation can be provided for carbon-based nanomaterials. However, lipid-based nanoparticles have demonstrated long-term biocompatibility, especially liposomes and their modification with CS not only increases their biocompatibility but also improves drug release profile and their stability, paving the way for clinical application. ACKNOWLEDGMENT Gorka Orive wishes to thank the Spanish Ministry of Economy, Industry, and Competitiveness (PID2019-106094RB-I00/ AEI/10.13039/501100011033) and technical assistance from the ICTS NANBIOSIS (Drug Formulation Unit, U10) at the University of the Basque Country. The authors also appreciate the support from the Basque Country Government (Grupos Consolidados, No ref: IT907-16). Ebrahim Mostafavi would like to acknowledge the support from the National Institute of Biomedical Imaging and Bioengineering (5T32EB009035). Alan Prem Kumar was supported by a grant from the Singapore Ministry of Education (MOET2EP30120-0016). CONFLICT OF INTERESTS The authors declare no conflict of interest. PEER REVIEW The peer review history for this article is available at https://publons. com/publon/10.1002/btm2.10325. DATA AVAILABILITY STATEMENT Data sharing is not applicable to this article as no new data were created or analyzed. ORCID Pooyan Makvandi https://orcid.org/0000-0003-2456-0961 Esmaeel Sharifi https://orcid.org/0000-0003-3400-3106 Marcin Wojnilowicz https://orcid.org/0000-0002-6480-6615 Ali Zarrabi https://orcid.org/0000-0003-0391-1769 Ebrahim Mostafavi https://orcid.org/0000-0003-3958-5002 Gorka Orive https://orcid.org/0000-0002-0773-300X REFERENCES 1. Kumar KSS, Girish YR, Ashrafizadeh M, et al. AIE-featured tetraphenylethylene nanoarchitectures in biomedical application: bioimaging, drug delivery and disease treatment. Coord Chem Rev. 2021;447:214135. 2. Makvandi P, Chen M, Sartorius R, et al. Endocytosis of abiotic nanomaterials and nanobiovectors: inhibition of membrane trafficking. Nano Today. 2021;40:101279. doi:10.1016/j.nantod.2021.101279 3. Mirzaei S, Mohammadi AT, Gholami MH, et al. Nrf2 signaling pathway in cisplatin chemotherapy: potential involvement in organ protection and chemoresistance. Pharmacol Res. 2021;167:105575. 4. Ashrafizaveh S, Ashrafizadeh M, Zarrabi A, et al. Long non-coding RNAs in the doxorubicin resistance of cancer cells. Cancer Lett. 2021;508:104-114. 5. Mirzaei S, Gholami MH, Hushmandi K, et al. The long and short noncoding RNAs modulating EZH2 signaling in cancer. J Heamto Oncol. 2022;15(1):1-34. 6. Ashrafizadeh M, Mirzaei S, Hashemi F, et al. New insight towards development of paclitaxel and docetaxel resistance in cancer cells: EMT as a novel molecular mechanism and therapeutic possibilities. Biomed Pharmacother. 2021;141:111824. 7. Mirzaei S, Gholami MH, Zabolian A, et al. Caffeic acid and its derivatives as potential modulators of oncogenic molecular pathways: new hope in the fight against cancer. Pharmacol Res. 2021;171:105759. doi:10.1016/j.phrs.2021.105759 8. Ashrafizadeh M, Zarrabi A, HashemipourM,etal.Sensingthescentof death: modulation of microRNAs by curcumin in gastrointestinal cancers. Pharmacol Res. 2020;160:105199. doi:10.1016/j.phrs.2020.105199 9. Akbari E, Mousazadeh H, Hanifehpour Y, et al. Co-loading of cisplatin and methotrexate in nanoparticle-based PCL-PEG system enhances lung cancer chemotherapy effects. J Cluster Sci. 2021;1-12. doi:10. 1007/s10876-021-02101-9 10. Rabiee N, Bagherzadeh M, Ghadiri AM, et al. Calcium-based nanomaterials and their interrelation with chitosan: optimization for pCRISPR delivery. J Nanostructure Chem. 2021;1-14. doi:10.1007/ s40097-021-00446-1 11. Ward RA, Fawell S, Floc'h N, et al. Challenges and opportunities in cancer drug resistance. Chem Rev. 2020;121(6):3297-3351. 12. Kirtonia A, Ashrafizadeh M, Zarrabi A, et al. Long noncoding RNAs: a novel insight in the leukemogenesis and drug resistance in acute myeloid leukemia. J Cell Physiol. 2021;237(1):450-65. 13. Mirzaei S, Zarrabi A, Hashemi F, et al. Regulation of nuclear factorKappaB (NF-κB) signaling pathway by non-coding RNAs in cancer: inhibiting or promoting carcinogenesis? Cancer Lett. 2021;509:63-80. 14. Ashrafizadeh M, Zarrabi A, Hushmandi K, et al. Association of the epithelial–mesenchymal transition (EMT) with cisplatin resistance. Int J Mol Sci. 2020;21(11):4002. 15. Mostafavi E, Soltantabar P, Webster TJ. Nanotechnology and picotechnology: a new arena for translational medicine. Biomaterials in Translational Medicine. Elsevier; 2019:191-212. 16. Dehshahri A, Ashrafizadeh M, Ghasemipour Afshar E, et al. Topoisomerase inhibitors: pharmacology and emerging nanoscale delivery systems. Pharmacol Res. 2020;151:104551. doi:10.1016/j.phrs. 2019.104551 17. Jahangirian H, Ghasemian lemraski E, Webster TJ, RafieeMoghaddam R, Abdollahi Y. A review of drug delivery systems based on nanotechnology and green chemistry: green nanomedicine. Int J Nanomed. 2017;12:2957-2978. 18. Makvandi P, Ghomi M, Ashrafizadeh M, et al. A review on advances in graphene-derivative/polysaccharide bionanocomposites: therapeutics, pharmacogenomics and toxicity. Carbohydr Polym. 2020; 250:116952. doi:10.1016/j.carbpol.2020.116952 19. Ashrafizadeh M, Ahmadi Z, Mohamadi N, et al. Chitosan-based advanced materials for docetaxel and paclitaxel delivery: recent advances and future directions in cancer theranostics. Int J Biol Macromol. 2020;145:282-300. doi:10.1016/j.ijbiomac.2019.12.145 20. Takeshita S, Zhao S, Malfait WJ, Koebel MM. Chemistry of chitosan aerogels: three-dimensional pore control for tailored applications. Angew Chem. 2021;60(18):9828-9851. 21. Jin T, Liu T, Lam E, Moores A. Chitin and chitosan on the nanoscale. Nanoscale Horizon. 2021;6(7):505-542. 22. Sharifi E, Chehelgerdi M, Fatahian-Kelishadrokhi A, YazdaniNafchi F, Ashrafi-Dehkordi K. Comparison of therapeutic effects of encapsulated mesenchymal stem cells in Aloe vera gel and chitosanbased gel in healing of grade-II burn injuries. Regen Ther. 2021;18: 30-37. doi:10.1016/j.reth.2021.02.007 23. Kumar MR, Muzzarelli RAA, Muzzarelli C, Sashiwa H, Domb AJ. Chitosan chemistry and pharmaceutical perspectives. ACS Publ. 2004;104(12):6017-6084. ASHRAFIZADEH ET AL.21 of 29 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 24. Ifuku SJM. Chitin and chitosan nanofibers: preparation and chemical modifications. Molecules. 2014;19(11):18367-18380. 25. Zafari M, Boroujeni MM, Omidghaemi S, et al. Physical and biological properties of blend-electrospun polycaprolactone/chitosanbased wound dressings loaded with N-decyl-N, N-dimethyl1-decanaminium chloride: an in vitro and in vivo study. J Biomed Mater Res. 2020;108(8):3084-3098. 26. Yan N, Chen XJNN. Sustainability: don't waste seafood waste. Nature. 2015;524(7564):155. 27. Prashanth KVH, Tharanathan RN. Chitin/chitosan: modifications and their unlimited application potential - an overview. Trends Food Sci Technol. 2007;18(3):117-131. doi:10.1016/j.tifs.2006. 10.022 28. Bellich B, D'Agostino I, Semeraro S, Gamini A, Cesàro A. “The good, the bad and the ugly”of chitosans. Mar Drugs. 2016;14(5):99. 29. Verma S, Nadagouda MN, Varma RS. Porous nitrogen-enriched carbonaceous material from marine waste: chitosan-derived carbon nitride catalyst for aerial oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid. Sci Rep. 2017;7(1):13596. 30. Varma RS. Biomass-derived renewable carbonaceous materials for sustainable chemical and environmental applications. ACS Sustain Chem Eng. 2019;7(7):6458-6470. doi:10.1021/acssuschemeng. 8b06550 31. Nik AB, Zare H, Razavi S, et al. Smart drug delivery: capping strategies for mesoporous silica nanoparticles. Microporous Mesoporous Mater. 2020;299:110115. 32. Maghsoudi S, Shahraki BT, Rabiee N, et al. Burgeoning polymer nano blends for improved controlled drug release: a review. Int J Nanomed. 2020;15:4363. 33. Özel N, Elibol MJCP. A review on the potential uses of deep eutectic solvents in chitin and chitosan related processes. 2021;262:117942. doi:10.1016/j.carbpol.2021.117942 34. Kadokawa J-I. Dissolution, derivatization, and functionalization of chitin in ionic liquid. Int J Biol Macromol. 2019;123:732-737. 35. Shahidi F, Abuzaytoun R. Chitin, chitosan, and co-products: chemistry, production, applications, and health effects. Advances in Food and Nutrition Research. Academic Press; 2005:93-135. 36. Kim S-K. Chitin, Chitosan, Oligosaccharides and their Derivatives: Biological Activities and Applications. CRC Press; 2010. 37. Reys L, Silva SS, Oliveira JM, et al. Revealing the potential of squid chitosan-based structures for biomedical applications. Biomed Mater. 2013;8(4):045002. 38. Ferreira LM, Dos Santos AM, Boni FI, et al. Design of chitosan-based particle systems: a review of the physicochemical foundations for tailored properties. Carbohydr Polym. 2020;250:116968. doi:10. 1016/j.carbpol.2020.116968 39. Aranaz I, Harris R, Heras A. Chitosan amphiphilic derivatives. Chemistry and applications. Curr Organ Chem. 2010;14(3):308-330. 40. Domard A. pH and c.d. measurements on a fully deacetylated chitosan: application to CuII—polymer interactions. Int J Biol Macromol. 1987;9(2):98-104. 41. Rinaudc M, Pavlov G, Desbrières J. Solubilization of chitosan in strong acid medium. Int J Polymer Anal Charact. 1999;5(3):267-276. 42. Chattopadhyay D, Inamdar MS. Aqueous behaviour of chitosan. Int J Polym Sci. 2010. doi:10.1155/2010/939536 43. Wang W, Xu D. Viscosity and flow properties of concentrated solutions of chitosan with different degrees of deacetylation. Int J Biol Macromol. 1994;16(3):149-152. 44. Bagheri M, Validi M, Gholipour A, Makvandi P, Sharifi E. Chitosan nanofiber biocomposites for potential wound healing applications: antioxidant activity with synergic antibacterial effect. Bioeng Transl Med. 2022;7(1):e10254. 45. Abadehie FS, Dehkordib AH, Zafaric M, et al. Lawsone-encapsulated chitosan/polyethylene oxide nanofibrous mat as a potential antibacterial biobased wound dressing. Eng Reg. 2021;2:219-226. 46. Nikbakht M, Karbasi S, Rezayat SM, Tavakol S, Sharifi E. Evaluation of the effects of hyaluronic acid on poly (3-hydroxybutyrate)/chitosan/carbon nanotubes electrospun scaffold: structure and mechanical properties. Polym-Plast. Technol Mater. 2019;58(18):2031-2040. 47. Jhaveri J, Raichura Z, Khan T, Momin M, Omri A. Chitosan nanoparticles-insight into properties, functionalization and applications in drug delivery and theranostics. Molecules. 2021;26(2):272. 48. Hajebi S, Hajebi S, Bagherzadeh M, et al. Stimulus-responsive polymeric nanogels as smart drug delivery systems. Acta Biomater. 2019; 92:1-18. 49. Rabiee N, Bagherzadeh M, Ghadiri AM, et al. Turning toxic nanomaterials into a safe and bioactive nanocarrier for co-delivery of DOX/pCRISPR. ACS Appl Bio Mater. 2021;4:5336-5351. doi:10. 1021/acsabm.1c00447 50. Nasseri B, Kosemehmetoglu K, Kaya M, Piskin E, Rabiee N, Webster TJ. The pimpled gold nanosphere: a superior candidate for plasmonic photothermal therapy. Int J Nanomed. 2020;15:2903. 51. Rabiee N, Yaraki MT, Garakani SM, et al. Recent advances in porphyrin-based nanocomposites for effective targeted imaging and therapy. Biomaterials. 2020;232:119707. 52. Lee S, Jo G, Jung JS, Yang DH, Hyun H. Near-infra-red fluorescent chitosan oligosaccharide lactate for targeted cancer imaging and photothermal therapy. Artif Cells Nanomed Biotechnol. 2020;48(1): 1144-1152. doi:10.1080/21691401.2020.1817054 53. Ma K, Cheng Y, Wei X, Chen D, Zhao X, Jia P. Gold embedded chitosan nanoparticles with cell membrane mimetic polymer coating for pH-sensitive controlled drug release and cellular fluorescence imaging. J Biomater Appl. 2021;35(7):857-868. doi:10.1177/ 0885328220952594 54. Dai X, Zhao X, Liu Y, et al. Controlled synthesis and surface engineering of Janus chitosan-gold nanoparticles for photoacoustic imaging-guided synergistic gene/photothermal therapy. Small. 2021; 17(11):e2006004. doi:10.1002/smll.202006004 55. Rasul RM, Tamilarasi Muniandy M, Zakaria Z, et al. A review on chitosan and its development as pulmonary particulate anti-infective and anti-cancer drug carriers. Carbohydr Polym. 2020;250:116800. doi:10.1016/j.carbpol.2020.116800 56. Miao YQ, Chen MS, Zhou X, et al. Chitosan oligosaccharide modified liposomes enhance lung cancer delivery of paclitaxel. Acta Pharmacol Sin. 2021;42(10):1714-1722. doi:10.1038/s41401-020-00594-0 57. Vikas, Viswanadh MK, Mehata AK, et al. Bioadhesive chitosan nanoparticles: dual targeting and pharmacokinetic aspects for advanced lung cancer treatment. Carbohydr Polym. 2021;274: 118617. 58. Fernández- Alvarez F, García-García G, Arias JL. A tri-stimuli responsive (maghemite/PLGA)/chitosan nanostructure with promising applications in lung cancer. Pharmaceutics. 2021;13(8):1232. doi:10. 3390/pharmaceutics13081232 59. Safarzadeh M, Sadeghi S, Azizi M, Rastegari-Pouyani M, Pouriran R, Haji Molla Hoseini M. Chitin and chitosan as tools to combat COVID-19: a triple approach. Int J Biol Macromol. 2021;183:235244. doi:10.1016/j.ijbiomac.2021.04.157 60. Jaber N, al-Remawi M, al-Akayleh F, al-Muhtaseb N, al-Adham ISI, Collier PJ. A review of the antiviral activity of chitosan, including patented applications and its potential use against COVID-19. J Appl Microbiol. 2022;132(1):41-58. doi:10.1111/jam.15202 61. Lotfy VF, Basta AH. Optimizing the chitosan-cellulose based drug delivery system for controlling the ciprofloxacin release versus organic/inorganic crosslinker, characterization and kinetic study. Int J Biol Macromol. 2020;165:1496-1506. 62. Zhang X, Pan Y, Li S, et al. Doubly crosslinked biodegradable hydrogels based on gellan gum and chitosan for drug delivery and wound dressing. Int J Biol Macromol. 2020;164:2204-2214. doi:10.1016/j. ijbiomac.2020.08.093 22 of 29 ASHRAFIZADEH ET AL. 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 63. Karpisheh V, Fakkari Afjadi J, Nabi Afjadi M, et al. Inhibition of HIF1α/EP4 axis by hyaluronate-trimethyl chitosan-SPION nanoparticles markedly suppresses the growth and development of cancer cells. Int J Biol Macromol. 2021;167:1006-1019. doi:10.1016/j.ijbiomac. 2020.11.056 64. Hamza RZ, Al-Salmi FA, El-Shenawy NS. Chitosan and lecithin ameliorate osteoarthritis symptoms induced by monoiodoacetate in a rat model. Molecules. 2020;25(23):5738. doi:10.3390/ molecules25235738 65. Scognamiglio F, Travan A, Donati I, Borgogna M, Marsich E. A hydrogel system based on a lactose-modified chitosan for viscosupplementation in osteoarthritis. Carbohydr Polym. 2020;248: 116787. doi:10.1016/j.carbpol.2020.116787 66. Liu Z, Mo X, Ma F, et al. Synthesis of carboxymethyl chitosanstrontium complex and its therapeutic effects on relieving osteoarthritis. Carbohydr Polym. 2021;261:117869. doi:10.1016/j.carbpol. 2021.117869 67. Medina-Cruz D, Mostafavi E, Vernet-Crua A, et al. Green nanotechnology-based drug delivery systems for osteogenic disorders. Expert Opin Drug Deliv. 2020;17(3):341-356. 68. Chen L, Wang C, Wu Y. Cholesterol (blood lipid) lowering potential of Rosuvastatin chitosan nanoparticles for atherosclerosis: preclinical study in rabbit model. Acta Biochim pol. 2020;67(4):495-499. 69. Gull N, Khan SM, Butt OM, et al. Inflammation targeted chitosanbased hydrogel for controlled release of diclofenac sodium. Int J Biol Macromol. 2020;162:175-187. doi:10.1016/j.ijbiomac.2020.06.133 70. Shen K, Tang Q, Fang X, et al. The sustained release of dexamethasone from TiO(2) nanotubes reinforced by chitosan to enhance osteoblast function and anti-inflammation activity. Mater Sci Eng C Mater Biol Appl. 2020;116:111241. doi:10.1016/j.msec.2020.111241 71. Wang X, Almoallim HS, Cui Q, Alharbi SA, Yang H. In situ decorated au NPs on chitosan-encapsulated Fe(3)O(4)-NH(2) NPs as magnetic nanocomposite: investigation of its anti-colon carcinoma, antigastric cancer and anti-pancreatic cancer. Int J Biol Macromol. 2021; 171:198-207. doi:10.1016/j.ijbiomac.2020.12.037 72. Sarkar S, Das D, Dutta P, Kalita J, Wann SB, Manna P. Chitosan: a promising therapeutic agent and effective drug delivery system in managing diabetes mellitus. Carbohydr Polym. 2020;247:116594. doi:10.1016/j.carbpol.2020.116594 73. Othman SI, Alturki AM, Abu-Taweel GM, Altoom NG, Allam AA, Abdelmonem R. Chitosan for biomedical applications, promising antidiabetic drug delivery system, and new diabetes mellitus treatment based on stem cell. Int J Biol Macromol. 2021;190:417-432. doi:10.1016/j.ijbiomac.2021.08.154 74. Arnaldi P, Pastorino L, Monticelli O. On an effective approach to improve the properties and the drug release of chitosan-based microparticles. Int J Biol Macromol. 2020;163:393-401. doi:10.1016/ j.ijbiomac.2020.07.016 75. Shi W, Ching YC, Chuah CH. Preparation of aerogel beads and microspheres based on chitosan and cellulose for drug delivery: a review. Int J Biol Macromol. 2021;170:751-767. doi:10.1016/j. ijbiomac.2020.12.214 76. Silva TND, Reynaud F, Picciani PHS, de Holanda e Silva KG, Barradas TN. Chitosan-based films containing nanoemulsions of methyl salicylate: formulation development, physical-chemical and in vitro drug release characterization. Int J Biol Macromol. 2020;164: 2558-2568. doi:10.1016/j.ijbiomac.2020.08.117 77. Carvalho FS, Burgeiro A, Garcia R, Moreno AJ, Carvalho RA, Oliveira PJ. Doxorubicin-induced cardiotoxicity: from bioenergetic failure and cell death to cardiomyopathy. Med Res Rev. 2014;34(1): 106-135. 78. Soltantabar P, Calubaquib EL, Mostafavi E, Biewer MC, Stefan MC. Enhancement of loading efficiency by coloading of doxorubicin and quercetin in thermoresponsive polymeric micelles. Biomacromolecules. 2020;21(4):1427-1436. 79. Rabiee N, Haris MH, Ghadiri AM, Moghaddam FM, Fatahi Y. Polymer-coated NH2-UiO-66 for the codelivery of DOX/pCRISPR. ACS Appl Mater Interfaces. 2021;13(9):10796-10811. 80. Bonadonna G, Monfardini S, De Lena M, Fossati-Bellani F, Beretta G. Phase I and preliminary phase II evaluation of adriamycin (NSC 123127). Cancer Res. 1970;30(10):2572-2582. 81. Bonadonna G, Monfardini S, de Lena M, Fossati-Bellani F. Clinical evaluation of adriamycin, a new antitumour antibiotic. Br Med J. 1969;3(5669):503-506. 82. Carvalho C, Santos RX, Cardoso S, et al. Doxorubicin: the good, the bad and the ugly effect. Curr Med Chem. 2009;16(25):3267-3285. 83. Hortobagyi GJ. Anthracyclines in the treatment of cancer. Drugs. 1997;54(4):1-7. 84. Aubel-Sadron G, Londos-Gagliardi DJB. Daunorubicin and doxorubicin, anthracycline antibiotics, a physicochemical and biological review. Biochimie. 1984;66(5):333-352. 85. Ashrafizaveh S, Ashrafizadeh M, Zarrabi A, et al. Long non-coding RNA in the doxorubicin resistance of cancer cells. Cancer Lett. 2021; 508:104-114. doi:10.1016/j.canlet.2021.03.018 86. Maghsoudi S, Shahraki BT, Rabiee N, et al. Recent advancements in aptamer-bioconjugates: sharpening stones for breast and prostate cancers targeting. JDrugDelivSciTechnol. 2019; 53:101146. 87. Mirzaei S et al. The involvement of epithelial-to-mesenchymal transition in doxorubicin resistance: possible molecular targets. Eur J Pharmacol. 2021;908:174344. 88. Mirzaei S, Zarrabi A, Hashemi F, et al. Nrf2 signaling pathway in chemoprotection and doxorubicin resistance: potential application. Drug Discov. 2021;10(3):349. 89. Ashrafizadeh M, Zarrabi A, Hashemi F, et al. Polychemotherapy with curcumin and doxorubicin via biological nanoplatforms: enhancing antitumor activity. Pharmaceutics. 2020;12(11):1084. 90. Wang C, Kar S, Lai X, et al. Triple negative breast cancer in Asia: an insider's view. Cancer Treat Rev. 2018;62:29-38. doi:10.1016/j.ctrv. 2017.10.014 91. Wang Y, Zhou P, Li P, Yang F, Gao XQ. Long non-coding RNA H19 regulates proliferation and doxorubicin resistance in MCF-7 cells by targeting PARP1. Bioengineered. 2020;11(1):536-546. doi:10.1080/ 21655979.2020.1761512 92. Wang S, Cheng M, Zheng X, et al. Interactions between lncRNA TUG1 and miR-9-5p modulate the resistance of breast cancer cells to doxorubicin by regulating eIF5A2. Onco Targets Ther. 2020;13: 13159-13170. 93. Ji Y, Liu J, Zhu W, Ji J. circ_0002060 enhances doxorubicin resistance in osteosarcoma by regulating the miR-198/ABCB1 Axis. Cancer Biother Radiopharm. 2020. doi:10.1089/cbr.2020.4240 94. Guan H, Xu H, Chen J, et al. Circ_0001721 enhances doxorubicin resistance and promotes tumorigenesis in osteosarcoma through miR-758/TCF4 axis. Cancer Cell Int. 2021;21(1):336. 95. Xiong L, Lin XM, Nie JH, Ye HS, Liu J. Resveratrol and its nanoparticle suppress doxorubicin/docetaxel-resistant anaplastic thyroid cancer cells in vitro and in vivo. Nanotheranostics. 2021;5(2):143-154. doi:10.7150/ntno.53844 96. Casagrande N, Borghese C, Favero A, Vicenzetto C, Aldinucci D. Trabectedin overcomes doxorubicin-resistance, counteracts tumorimmunosuppressive reprogramming of monocytes and decreases xenograft growth in Hodgkin lymphoma. Cancer Lett. 2021;500:182193. doi:10.1016/j.canlet.2020.12.015 97. Wang H, Shan S, Wang H, Wang X. CircATXN7 contributes to the progression and doxorubicin resistance of breast cancer via modulating miR-149-5p/HOXA11 pathway. Anticancer Drugs. 2022;33(1): e700-e710. doi:10.1097/CAD.0000000000001243 98. Cheng J-T, Wang L, Wang H, et al. Insights into biological role of LncRNAs in epithelial-mesenchymal transition. Cell. 2019;8(10): 1178. doi:10.3390/cells8101178 ASHRAFIZADEH ET AL.23 of 29 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 99. Xie M, Li J, Deng T, Yang N, Yang M. Modification of magnetic molybdenum disulfide by chitosan/carboxymethylcellulose with enhanced dispersibility for targeted photothermal/chemotherapy of cancer. J Mater Chem B. 2021;9(7):1833-1845. doi:10.1039/ D0TB01664K 100. Zhu Z, Wei Z. CIP2A silencing alleviates doxorubicin resistance in MCF7/ADR cells through activating PP2A and autophagy. Clin Transl Oncol. 2021;23(8):1542-1548. doi:10.1007/s12094-02102616-7 101. Zhao W, Ning L, Wang L, et al. miR-21 inhibition reverses doxorubicinresistance and inhibits PC3 human prostate cancer cells proliferation. Andrologia. 2021;53(5):e14016. doi:10.1111/and.14016 102. Ashrafizadeh M, Mirzaei S, Gholami MH, et al. Hyaluronic acidbased nanoplatforms for doxorubicin: a review of stimuli-responsive carriers, co-delivery and resistance suppression. Carbohydr Polym. 2021;272:118491. doi:10.1016/j.carbpol.2021.118491 103. Paskeh MDA, Saebfar H, Mahabady MK, et al. Overcoming doxorubicin resistance in cancer: siRNA-loaded nanoarchitectures for cancer gene therapy. Life Sci. 2022;298:120463. doi:10.1016/j.lfs.2022.120463 104. Ashrafizadeh M, Saebfar H, Gholami MH, et al. Doxorubicin-loaded graphene oxide nanocomposites in cancer medicine: stimuliresponsive carriers, co-delivery and suppressing resistance. Expert Opin Drug Deliv. 2022;1-28. doi:10.1080/17425247.2022.2041598 105. Makvandi P, Jamaledin R, Chen G, et al. Stimuli-responsive transdermal microneedle patches. Mater Today. 2021;47:206-222. doi:10. 1016/j.mattod.2021.03.012 106. Zhuo S, Zhang F, Yu J, Zhang X, Yang G, Liu X. pH-sensitive biomaterials for drug delivery. Molecules (Basel, Switzerland). 2020;25(23): 5649. doi:10.3390/molecules25235649 107. Yan T, Zhu S, Hui W, He J, Liu Z, Cheng J. Chitosan based pHresponsive polymeric prodrug vector for enhanced tumor targeted co-delivery of doxorubicin and siRNA. Carbohydr Polym. 2020;250: 116781. doi:10.1016/j.carbpol.2020.116781 108. Wang R, Shou D, Lv O, Kong Y, Deng L, Shen J. pH-controlled drug delivery with hybrid aerogel of chitosan, carboxymethyl cellulose and graphene oxide as the carrier. Int J Biol Macromol. 2017;103:248-253. 109. Marsano E, Bianchi E, Vicini S, et al. Stimuli responsive gels based on interpenetrating network of chitosan and poly (vinylpyrrolidone). Polymer. 2005;46(5):1595-1600. 110. Hasan A, Waibhaw G, Tiwari S, Dharmalingam K, Shukla I, Pandey LM. Fabrication and characterization of chitosan, polyvinylpyrrolidone, and cellulose nanowhiskers nanocomposite films for wound healing drug delivery application. J Biomed Mater Res A. 2017;105(9):2391-2404. 111. Risbud MV, Hardikar AA, Bhata SV, Bhonde RR. pH-sensitive freeze-dried chitosan–polyvinyl pyrrolidone hydrogels as controlled release system for antibiotic delivery. J Controlled Release. 2000; 68(1):23-30. 112. Gerami SE, Pourmadadi M, Fatoorehchi H, Yazdian F, Rashedi H, Nigjeh MN. Preparation of pH-sensitive chitosan/polyvinylpyrrolidone/α-Fe(2)O(3) nanocomposite for drug delivery application: emphasis on ameliorating restrictions. Int J Biol Macromol. 2021;173:409-420. doi:10.1016/j.ijbiomac.2021.01.067 113. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68(1):7-30. doi:10.3322/caac.21442 114. Bae YH, Park K. Targeted drug delivery to tumors: myths, reality and possibility. J Control Release. 2011;153(3):198-205. doi:10.1016/j. jconrel.2011.06.001 115. Shi Y, Li LC. Current advances in sustained-release systems for parenteral drug delivery. Expert Opin Drug Deliv. 2005;2(6):1039-1058. doi:10.1517/17425247.2.6.1039 116. Sartipzadeh O, Naghib SM, Shokati F, et al. Microfluidic-assisted synthesis and modelling of monodispersed magnetic nanocomposites for biomedical applications. J Nanotechnol Rev. 2020;9(1):1397-1407. 117. Yin Q, Shen J, Zhang Z, Yu H, Li Y. Reversal of multidrug resistance by stimuli-responsive drug delivery systems for therapy of tumor. Adv Drug Deliv Rev. 2013;65(13–14):1699-1715. doi:10.1016/j.addr. 2013.04.011 118. Pouysségur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature. 2006; 441(7092):437-443. doi:10.1038/nature04871 119. Gooneh-Farahani S, Naghib SM, Naimi-Jamal MR, Seyfoori A. A pHsensitive nanocarrier based on BSA-stabilized graphene-chitosan nanocomposite for sustained and prolonged release of anticancer agents. Sci Rep. 2021;11(1):17404. 120. Yang J, Naghib SM, Shokati F, et al. Enhanced therapeutic efficacy of doxorubicin for breast cancer using chitosan oligosaccharidemodified halloysite nanotubes. Nanotechnol Rev. 2016;8(40):2657826590. 121. Gao X, Zhou Y, Ma G, et al. A water-soluble photocrosslinkable chitosan derivative prepared by Michael-addition reaction as a precursor for injectable hydrogel. Carbohydr Polym. 2010;79(3): 507-512. 122. Kufelt O, El-Tamer A, Sehring C, Meißner M, Schlie-Wolter S, Chichkov BN. Water-soluble photopolymerizable chitosan hydrogels for biofabrication via two-photon polymerization. Acta Biomaterialia. 2015;18:186-195. 123. Li B, Wang L, Xu F, et al. Hydrosoluble, UV-crosslinkable and injectable chitosan for patterned cell-laden microgel and rapid transdermal curing hydrogel in vivo. Acta Biomater. 2015;22:59-69. 124. Zhou Y, Ma G, Shi S, Yang D, Nie J. Photopolymerized water-soluble chitosan-based hydrogel as potential use in tissue engineering. Int J Biol Macromol. 2011;48(3):408-413. 125. Carvalho IC, Mansur HS. Engineered 3D-scaffolds of photocrosslinked chitosan-gelatin hydrogel hybrids for chronic wound dressings and regeneration. Mater Sci Eng C. 2017;78:690-705. 126. Cho IS, Cho MO, Li Z, et al. Synthesis and characterization of a new photo-crosslinkable glycol chitosan thermogel for biomedical applications. Carbohydr Polym. 2016;144:59-67. 127. He M, Jiang Z, Yang Y, Peng Y, Liu W. Synthesis of a chitosan-based photo-sensitive hydrogel and its biocompatibility and biodegradability. Carbohydr Polym. 2017;166:228-235. 128. Nada AA, Ali EA, Solimanc AAF. Biocompatible chitosan-based hydrogel with tunable mechanical and physical properties formed at body temperature. Int J Biol Macromol. 2019;131:624-632. 129. Ding H, Li B, Jiang Y, et al. pH-responsive UV crosslinkable chitosan hydrogel via "thiol-ene"click chemistry for active modulating opposite drug release behaviors. Carbohydr Polym. 2021;251:117101. doi:10.1016/j.carbpol.2020.117101 130. Fathi M, Majidi S, Zangabad PS, Barar J, Erfan-Niya H, Omidi Y. Chitosan-based multifunctional nanomedicines and theranostics for targeted therapy of cancer. Med Res Rev. 2018;38(6): 2110-2136. 131. Sathiyaseelan A, Saravanakumar K, Mariadoss AVA, Wang MH. pHcontrolled nucleolin targeted release of dual drug from chitosan-gold based aptamer functionalized nano drug delivery system for improved glioblastoma treatment. Carbohydr Polym. 2021;262: 117907. doi:10.1016/j.carbpol.2021.117907 132. Raja MA, Arif M, Feng C, Zeenat S, Liu CG. Synthesis and evaluation of pH-sensitive, self-assembled chitosan-based nanoparticles as efficient doxorubicin carriers. J Biomater Appl. 2017;31(8):1182-1195. doi:10.1177/0885328216681184 133. Mirhadi E, Mashreghi M, Faal Maleki M, et al. Redox-sensitive nanoscale drug delivery systems for cancer treatment. Int J Pharm. 2020; 589:119882. doi:10.1016/j.ijpharm.2020.119882 134. Ahmadi S, Rabieec N, Bagherzadeh M, et al. Stimulus-responsive sequential release systems for drug and gene delivery. Nano Today. 2020;34:100914. 24 of 29 ASHRAFIZADEH ET AL. 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 135. Hu F-Q, Zhao M-d, Yuan H, You J, Du Y, Zeng S. A novel chitosan oligosaccharide–stearic acid micelles for gene delivery: properties and in vitro transfection studies. Int J Pharm. 2006;315(1–2):158-166. 136. Li J, Huo M, Wang J, et al. Redox-sensitive micelles self-assembled from amphiphilic hyaluronic acid-deoxycholic acid conjugates for targeted intracellular delivery of paclitaxel. Biomaterials. 2012;33(7): 2310-2320. 137. Yan J, Du Y-Z, Chen F-Y, You J, Yuan H, Hu F-Q. Effect of proteins with different isoelectric points on the gene transfection efficiency mediated by stearic acid grafted chitosan oligosaccharide micelles. Mol Pharm. 2013;10(7):2568-2577. 138. Zhao M-D, Hu F-Q, Du Y-Z, et al. Coadministration of glycolipid-like micelles loading cytotoxic drug with different action site for efficient cancer chemotherapy. Nanotechnology. 2009;20(5):055102. 139. You J, Wang Z, Du Y, et al. Specific tumor delivery of paclitaxel using glycolipid-like polymer micelles containing gold nanospheres. Biomaterials. 2013;34(18):4510-4519. 140. Su Y, Hu Y, du Y, et al. Redox-responsive polymer-drug conjugates based on doxorubicin and chitosan oligosaccharide-g-stearic acid for cancer therapy. Mol Pharm. 2015;12(4):1193-1202. doi:10.1021/ mp500710x 141. Zhou X, Guo L, Shi D, Duan S, Li J. Biocompatible chitosan Nanobubbles for ultrasound-mediated targeted delivery of doxorubicin. Nanoscale Res Lett. 2019;14(1):24. 142. Xu RX. Multifunctional microbubbles and nanobubbles for photoacoustic imaging. Contrast Media Mol Imaging. 2011;6(5):401-411. 143. Sarkar A, Roy S, Sanpui P, Jaiswal A. Plasmonic gold nanorattle impregnated chitosan nanocarrier for stimulus responsive theranostics. ACS Appl Bio Mater. 2019;2(11):4812-4825. doi:10. 1021/acsabm.9b00568 144. Ruel-Gariepy E, Leroux J-C. In situ-forming hydrogels—review of temperature-sensitive systems. Eur J Pharm Biopharm. 2004;58(2): 409-426. 145. Ruel-Gariepy E, Chenite A, Chaput C, Guirguis S, Leroux J. Characterization of thermosensitive chitosan gels for the sustained delivery of drugs. Int J Pharm. 2000;203(1–2):89-98. 146. Lalloo A, Chao P, Hu P, Stein S, Sinko PJ. Pharmacokinetic and pharmacodynamic evaluation of a novel in situ forming poly (ethylene glycol)-based hydrogel for the controlled delivery of the camptothecins. J Control Release. 2006;112(3):333-342. 147. Ren S, Dai Y, Li C, et al. Pharmacokinetics and pharmacodynamics evaluation of a thermosensitive chitosan based hydrogel containing liposomal doxorubicin. Eur J Pharm Sci. 2016;92:137-145. doi:10. 1016/j.ejps.2016.07.002 148. Cho YI, Park S, Jeong SY, Yoo HS. In vivo and in vitro anti-cancer activity of thermo-sensitive and photo-crosslinkable doxorubicin hydrogels composed of chitosan-doxorubicin conjugates. Eur J Pharm Biopharm. 2009;73(1):59-65. doi:10.1016/j.ejpb.2009.04.010 149. Wang W, Zhang P, Shan W, Gao J, Liang W. A novel chitosan-based thermosensitive hydrogel containing doxorubicin liposomes for topical cancer therapy. J Biomater Sci Polym Ed. 2013;24(14):1649-1659. doi:10.1080/09205063.2013.789357 150. Dong X, Wei C, Liang J, Liu T, Kong D, Lv F. Thermosensitive hydrogel loaded with chitosan-carbon nanotubes for near infrared light triggered drug delivery. Colloids Surf B Biointerfaces. 2017;154:253262. doi:10.1016/j.colsurfb.2017.03.036 151. Chen X, Niu S, Bremner DH, et al. Co-delivery of doxorubicin and oleanolic acid by triple-sensitive nanocomposite based on chitosan for effective promoting tumor apoptosis. Carbohydr Polym. 2020; 247:116672. doi:10.1016/j.carbpol.2020.116672 152. Jiao J, Li X, Zhang S, et al. Redox and pH dual-responsive PEG and chitosan-conjugated hollow mesoporous silica for controlled drug release. Mater Sci Eng C Mater Biol Appl. 2016;67:26-33. doi:10. 1016/j.msec.2016.04.091 153. Gao Y, Ma Q, Cao J, et al. Bifunctional alginate/chitosan stabilized perfluorohexane nanodroplets as smart vehicles for ultrasound and pH responsive delivery of anticancer agents. Int J Biol Macromol. 2021;191:1068-1078. doi:10.1016/j.ijbiomac.2021.09.166 154. Karthika V, AlSalhi MS, Devanesan S, Gopinath K, Arumugam A, Govindarajan M. Chitosan overlaid Fe(3)O(4)/rGO nanocomposite for targeted drug delivery, imaging, and biomedical applications. Sci Rep. 2020;10(1):18912. 155. Liao T, Liu C, Ren J, et al. A chitosan/mesoporous silica nanoparticle-based anticancer drug delivery system with a "tumortriggered targeting"property. Int J Biol Macromol. 2021;183:20172029. doi:10.1016/j.ijbiomac.2021.06.004 156. Mu Y, Wu G, Su C, et al. pH-sensitive amphiphilic chitosanquercetin conjugate for intracellular delivery of doxorubicin enhancement. Carbohydr Polym. 2019;223:115072. doi:10.1016/j. carbpol.2019.115072 157. Nogueira-Librelotto DR, Scheeren LE, Macedo LB, Vinardell MP, Rolim CMB. pH-sensitive chitosan-tripolyphosphate nanoparticles increase doxorubicin-induced growth inhibition of cervical HeLa tumor cells by apoptosis and cell cycle modulation. Colloids Surf B Biointerfaces. 2020;190:110897. doi:10.1016/j.colsurfb.2020.110897 158. Huang N, Wang J, Cheng X, Xu Y, Li W. Fabrication of PNIPAMchitosan/decatungstoeuropate/silica nanocomposite for thermo/pH dual-stimuli-responsive and luminescent drug delivery system. J Inorg Biochem. 2020;211:111216. doi:10.1016/j.jinorgbio.2020. 111216 159. Verma NK, Purohit MP, Equbal D, et al. Targeted smart pH and thermoresponsive N,O-carboxymethyl chitosan conjugated Nnanogels for enhanced therapeutic efficacy of doxorubicin in MCF-7 breast cancer cells. Bioconjug Chem. 2016;27(11):2605-2619. doi:10.1021/ acs.bioconjchem.6b00366 160. Xia B, Zhang W, Tong H, Li J, Chen Z, Shi J. Multifunctional chitosan/porous silicon@au nanocomposite hydrogels for long-term and repeatedly localized combinatorial therapy of cancer via a single injection. ACS Biomater Sci Eng. 2019;5(4):1857-1867. doi:10.1021/ acsbiomaterials.8b01533 161. Kong M, Zuo Y, Wang M, Bai X, Feng C, Chen X. Simply constructed chitosan nanocarriers with precise spatiotemporal control for efficient intracellular drug delivery. Carbohydr Polym. 2017;169:341350. doi:10.1016/j.carbpol.2017.03.090 162. Bigham A, Foroughi F, Rezvani Ghomi E, Rafienia M, Neisiany RE, Ramakrishna S. The journey of multifunctional bone scaffolds fabricated from traditional toward modern techniques. Bio-Design Manufact. 2020;3(4):281-306. doi:10.1007/s42242-020-00094-4 163. Bigham A, Aghajanian AH, Saudi A, Rafienia M. Hierarchical porous Mg2SiO4-CoFe2O4 nanomagnetic scaffold for bone cancer therapy and regeneration: surface modification and in vitro studies. Mater Sci Eng C. 2020;109:110579. 164. Ou W, Byeon JH, Thapa RK, Ku SK, Yong CS, Kim JO. Plug-and-play Nanorization of coarse black phosphorus for targeted chemophotoimmunotherapy of colorectal cancer. ACS Nano. 2018;12(10): 10061-10074. doi:10.1021/acsnano.8b04658 165. Mirzaei S, Gholami MH, Hashemi F, et al. Employing siRNA tool and its delivery platforms in suppressing cisplatin resistance: approaching to a new era of cancer chemotherapy. Life Sci. 2021; 277:119430. 166. Delfi M, Sartorius R, Ashrafizadeh M, et al. Self-assembled peptide and protein nanostructures for anti-cancer therapy: targeted delivery, stimuli-responsive devices and immunotherapy. Nano Today. 2021;38:101119. 167. Mirzaei S, Hushmandi K, Zabolian A, et al. Elucidating role of reactive oxygen species (ROS) in cisplatin chemotherapy: a focus on molecular pathways and possible therapeutic strategies. Molecules. 2021;26(8):2382. ASHRAFIZADEH ET AL.25 of 29 23806761, 2023, 1, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10325 by Universidad Del Pais Vasco, Wiley Online Library on [06/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License