Chondroitin sulfate derived theranostic and therapeutic nanocarriers for tumor-targeted drug delivery

Journal Pre-proof Chondroitin sulfate derived theranostic and therapeutic nanocarriers for tumor-targeted drug delivery Abdur Rauf Khan, Xiaoye Yang, Xiyou Du, Haotong Yang, Yuanxiu Liu, Abdul Qayyum Khan, Guangxi Zhai

PII:

S0144-8617(20)30011-4

DOI:

https://doi.org/10.1016/j.carbpol.2020.115837

Reference:

CARP 115837

To appear in:

Carbohydrate Polymers

Received Date:

19 November 2019

Revised Date:

22 December 2019

Accepted Date:

6 January 2020

Please cite this article as: Khan AR, Yang X, Du X, Yang H, Liu Y, Khan AQ, Zhai G, Chondroitin sulfate derived theranostic and therapeutic nanocarriers for tumor-targeted drug delivery, Carbohydrate Polymers (2020), doi: https://doi.org/10.1016/j.carbpol.2020.115837

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Chondroitin sulfate derived theranostic nanocarriers for tumor-targeted drug delivery.

and

therapeutic

Abdur Rauf Khan1, Xiaoye Yang1, Xiyou Du1, Haotong Yang1, Yuanxiu Liu1, Abdul Qayyum Khan2, Guangxi Zhai1*

1. Department of Pharmaceutics, Key Laboratory of Chemical Biology (Ministry of

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Education), School of Pharmaceutical Sciences, Shandong University, Jinan, 250012, PR China.

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2. Pakistan Council of Scientific and Industrial Research, Lahore, Pakistan.

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* Corresponding author:

Professor

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Department of Pharmaceutics

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Guangxi Zhai, Ph.D

School of Pharmaceutical Sciences, Shandong University 44 WenhuaXilu, Jinan 250012, China Tel.: (86) 531-88382015.

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E-mail: [email protected]

Highlights

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 Chondroitin sulfate (CS) and its proteoglycans have therapeutic and theranostic potential.  CS nanoformulations are promising carrier for tumor targeting  Rationally designed CS nanocarriers could overcome biological barriers for tumor CS nanoparticles could be exploited for immunotherapy and gene therapy of tumor

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Abstract

The standard chemotherapy is facing the challenges of lack of cancer selectivity and development of drug resistance. Currently, with the application of nanotechnology, the rationally

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designed nanocarriers of chondroitin sulfate (CS) have been fabricated and their unique features of low toxicity, biocompatibility, and active and passive targeting made them drug delivery

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vehicles of the choice for cancer therapy. The hydrophilic and anionic CS could be incorporated as a building block into- or decorated on the surface of nanoformulations. Micellar nanoparticles (NPs) self-assembled from amphiphilic CS-drug conjugates and CS-polymer conjugates,

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polyelectrolyte complexes (PECs) and nanogels of CS have been widely implicated in cancer directed therapy. The surface modulation of organic, inorganic, lipid and metallic NPs with CS promotes the receptor-mediated internalization of NPs to the tumor cells. The potential contribution of CS and CS-proteoglycans (CSPGs) in the pathogenesis of various cancer types, and CS nanocarriers in immunotherapy, radiotherapy, sonodynamic therapy (SDT) and

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photodynamic therapy (PDT) of cancer are summarized in this review paper. Keywords: Cancer; Chondroitin sulfate; Nanoparticles; Immunotherapy; Chemotherapy; Receptor-mediated endocytosis.

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1. Introduction

Despite of rapid advancement in the field of nanotechnology, cancer remains one of the major challenges to human health. According to the World Health Organization fact sheets, cancer is

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the major cause of death worldwide after cardiovascular diseases with high mortality rate (WHO, 2019). It is estimated that a total of 1,762,450 new patients will be diagnosed and

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606,880 patients will die of cancer in the United States in 2019 (Siegel, Miller, & Jemel, 2019). A cancer is an abnormal and uncontrolled growth of cells in the body, capable of invading or spreading to the surrounding or distant tissues. In recent decades, a wide research on pathology

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and multifactorial and highly complex pathogenesis of cancer has brought about a revolutionary breakthrough in the treatment and diagnosis of cancer. The currently available options for the treatment of cancer based on chemotherapy and radiotherapy have been failed to get desired outcomes owing to off-target side effects. To tackle the shortcomings of conventional therapies, a smartly engineered nanomedicine with the applications of pharmaceutical nanotechnology is

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highly demanded that can realize site-specific delivery and tumor microenvironment responsive, "off-on" switchable release of a drug with stability and safety in biological system. Various biopolymers have been investigated and had shown success in designing biomaterials suitable for tumor therapy. The polysaccharide biopolymers such as chitosan (CHS), chondroitin sulfate (CS), dextran, heparin and hyaluronic acid with non-immunogenic and non-toxic profiles, and plenty of pharmaceutical merits have urged scientists to construct chemotherapeutic and 3|Page

theranostic nanocarriers of these biopolymers (Yang, Du, Liu, & Zhai, 2015). CS is a sulfated glycosaminoglycan (GAG) made up of repeated disaccharides with variable amount of sulfate groups attached at different positions of CS. The various functional groups on CS chain such as carboxyl, hydroxyl and amino groups endow CS modifications with other hydrophobic moieties/drug molecules and probes. Amidation and esterification are commonly used chemical reactions for CS modification and synthesis of derivatives suitable for fabrication of drug delivery systems (Zhao, Liu, Wang, & Zhai, 2015). These modifications render scientists to

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conjugate chemotherapy drugs or macromolecules such as doxorubicin (DOX), paclitaxel (PTX) and ES2 (IVRRADRAAVP) peptide with CS and fabricate tumor-targeting multifunctional selfassembled NPs (Li et al., 2019; Onishi et al., 2017; Xing et al., 2019). The amphiphilic polymers

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are synthesized by covalent conjugation of CS polymer backbone with other hydrophobic polymers such as poly (d,l-lactideco-glycolide) (PLGA), poly(lactic acid) (PLA), deoxycholic

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acid (DOCA) and polycaprolactone (PCL), and exploited for engineering of tumor-site directed nanocarriers (Chang et al., 2017; Liu et al., 2017; Soe et al., 2019; Zhang et al., 2017). The

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polyanionic nature of CS confers its electrostatic association with cationic polymers to make PECs. PEC of CS with cationic CHS, ellagic acid, DOX and poly [(2-dimethylamino) ethyl

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methacrylate] (PDMAEM) have been designed for chemotherapy (Abd Elwakil et al., 2018; Bonkovoski et al., 2015; Jardim, Siqueira, Bao, Sousa, & Parize, 2020; Varshosaz, Sadeghi, & Asheghali, 2017). Nanogels constructed of CS are other promising carriers for the tumor-targeted

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delivery of macromolecules and drug moieties. In addition to recent progress in the development of nanovehicles based on CS as a polymer backbone, surface modified NPs with CS have also been widely studied in cancer nanotechnology. Surface decoration serves multipurpose of providing colloidal stability to NPs, receptor-mediated internalization to tumor cells and on-site delivery of cargo molecules. Various NPs fabricated of polymeric, lipid or metallic construction

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materials have been functionalized with CS and have shown promising results of active targeted delivery of chemotherapeutics, excellent gene transfection efficiency of nucleic acid and immunotherapy of tumor (Gurav et al., 2016; Hattori et al., 2016; Okubo, Miyazaki, Yuba, & Harada, 2019; Toth et al., 2019). The worthwhile literature published in recent years reported profound significance of stromal and cell surface CSPGs in the growth, vascularization and metastasis of tumor. The pathogenesis of melanoma, triple negative breast cancer, glioblastoma, ovarian carcinoma, neuroblastoma, 4|Page

osteosarcoma and chondrosarcoma is linked with over-expressed CSPGs (Ilieva et al., 2018). Different classes of CSPGs have variable effects on tumor, some with pro-tumoral and others with anti-tumor potential (Wegrowski, & Maquart, 2006; Wu, Chang, Lin, Chen, & Chen, 2014). The advanced multifunctional nanovehicles of CS have promising attributes to be exploited in PDT, SDT, immunotherapy, radiotherapy and magnetic therapy of tumor. Immunotherapy of cancer is a hot research topic worldwide and cancer immunotherapy clinical trials based on monoclonal antibodies (mAbs) directed against CSPGs have shown positive outcomes. Chimeric

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antigen receptor (CAR) T-cells therapy is a promptly emerging immunotherapy approach and has been approved by Food and Drug Administration (FDA) for the treatment of acute lymphoblastic leukemia in children and advanced lymphomas in adults (Hopfinger, Jager, &

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Worel, 2019). CAR T-cells targeting CSPG4 had shown therapeutic efficacy and safety in phase1 clinical trials of tumor immunotherapy (Chuntova, Downey, Hegde, Almeida, & Okada, 2019).

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Further clinical trials are of need to fully explore the clinical benefits of CAR T-cells in immunotherapy and engineering of multifunctional CAR T-cells targeting multiple antigens are

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mandatory for successful tumor elimination. Thus, CSPGs are excellent target of targeting modalities such as mAbs or CAR T-cell favoring theranosis and immunotherapy of tumor. This

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review summarizes the expression and pathological functions of CS and CSPGs in proliferation and metastasis of different tumors. The therapeutic as well as theranostic applications of various nanocarriers constructed of CS building block or functionalized with CS in immunotherapy,

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PDT, SDT, active-targeting therapy and immunotherapy of tumor are reviewed in detail.

2. Structure and oncogenic/anti-oncogenic aspects of CS and CSPGs CS is a naturally occurring polysaccharide belongs to GAG that is a predominant constituent of extracellular matrix (ECM) and widely expressed on the cell surface. Each chain of CS is

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consisted of alternating disaccharide units of N-acetylgalactosamine and glucuronic (Mikami, & Kitagawa, 2013). There are about over 100 individual sulfated sugar units of CS chain with variable quantities of sulfate group attached at various positions, and marked with identification letter (for example, CS A-E) as shown in Fig.1 (Vessella et al., 2019). The covalent attachment of sulfate group to the chondroitin confers anionic charge to CS (Mizumoto et al., 2012), and commercially available salt of CS contains sodium cation to counter negative charge of sulfate group. CS is abundantly present in bone, skin, cartilage, nerve, ECM, vein, adipose tissue, 5|Page

spleen, bone marrow, lung, heart and muscles (Volpi, 2019). Chondrocytes, macrophages, melanocytes, glial cells, and smooth muscles have low to moderate level of CS. The GAG chain of CS is covalently attached to serine moiety of protein molecules to form CSPGs (Ilieva et al., 2018). CSPGs are classified on the basis of their localization and functions into ECM, cell surface, neural and non-classified PGs. Cell surface PGs fall into syndecans, lecticans and glypicans families. The neural CSPGs are appican, amyloid-precursor-like protein 2, neuroglycan, neurocan, phosphacan/RPTPbeta/PTPzeta, BEHAB/brevican. Thrombomodulin,

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betaglycan, endocan, and neuron-glial antigen 2 (NG2) are non-classified PGs. ECM PGs are aggrecan, versican, small leucine-rich PG (SLRP) and testicans. Decorin, lumican and fibromodulin are members of SLRP family found in collagen fibrils, cornea and tendon,

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respectively. Perlecan and agrin are infrequently CS-conjugated basement membrane PGs (Wegrowski et al., 2006). CSPGs have a strong hand in the growth, development and

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homeostasis of tissues. Inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1α, interferon gamma (IFN-γ), and transforming growth factor beta (TGF-β),

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and hypoxia-induced mechanisms involving hypoxia-inducible factors regulate the expression of CSPGs (Ilieva et al., 2018). CSPGs have a prominent role in neural development, spinal injury

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and neural disorders. The stromal and cell surface CSPGs have been found to play a vital role in the proliferation and metastasis of tumor (Ilieva et al., 2018). ECM regulates the cell activity and coordinates with the cells through CSPGs. Tumor cells proliferation and migration are not

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possible without CSPGs-assistance. The augmented level of stromal CSPGs has been found in many tumors. The enhanced expression of decorin, a member of SLRP family, was found in malignancy of colorectal carcinoma, adenocarcinoma, melanoma and osteosarcoma, whereas carcinoma of lung, ovary, breast and liver had decreased level of decorin. Biglycan and lumican prevented proliferation of pancreatic cancer cells and growth of melanoma, respectively, via up-

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regulation of p27 and cyclin A. Agrecan expression was amplified in benign and malignant cartilage tumor but diminished in squamous cell carcinoma (Wegrowski et al., 2006).

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Fig.1: Structure of different types of CS (Zhao, Liu, Wang, & Zhai, 2015).

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The PGS present on the cell surface are necessary for survival of cells. The surface PGs have prominent position in growth and angiogenesis of tumor. The surface PGs are classified into type-1 trans-membrane PGs and glycosylphosphatidylinositol (GPI)-attached proteins.

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Syndecans and glypicans, cell-surface heparin sulfate-PGs, have tremendous involvement in tumor proliferation. The trans-membrane CSPGs class contains type III TGF‐beta receptor

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betaglycan, CD44 and NG2 (Wegrowski et al., 2006). CD44-CSPGs are involved in the migration and invasion of melanoma cells, while NG2 have effect on vascularization and angiogenesis of tumor. Betaglycan is another trans-membrane CSPG with core protein consisted of 853 amino acids. It has receptor for TGF-beta and binding of small amount of TGF-beta triggers signaling pathway related to tumor growth. CSPG4 genes encode NG2 protein. NG2 is

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expressed by mesenchymal stem cells, chondroblasts, osteoblasts, immature keratinocytes, muscle progenitors and melanocytes during developmental stage, and during differentiation expression of NG2 is reduced. The sulfation of CS and its glycosylation with core protein is processed in Golgi complex (Wegrowski et al., 2006). The glycosylated NG2 has molecular weight of 300 kDa, and NG2 protein consists of three domains; extracellular region of 2,225 amino acids, trans-membrane portion of 25 amino acids and tail consist of 76 amino acids (Yadavilli, Hwang, Packer, & Nazarian, 2016). The trans-membrane serine residue is present in CSPG4 similar to other CSPGs. The ecto-domain is comprised of three sub-regions, D1-D3, with 7|Page

binding sites for various ligands. D1-subpart is consisted of N-terminus globular region of 1-640 amino acid residues of CSPG4 protein which is further encompassed of two-laminin G-type portions, eight cysteine residues and four pairs of glycine/serine. The binding sites at laminin Gtype region are responsible for association with integrin, carbohydrates and cell matrix. The D2 domain represents 641 to 1590 amino acids of core protein. CS is covalently attached to PG at this region, and proteins (collagens V and VI) and growth factors (fibroblast growth factor (FBGF) and platelet-derived growth factor (PDGF)) have binding sites at D2-region.

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Juxtamembrane sub-region (D3) which is C-terminus globular part starts from 1591 to 2221 amino acids of core protein. It has acceptor sites for lectin and integrin and D3-region mediates NG2 shedding (Rolih et al., 2017). The intracellular domain of NG2 has binding sites for multi-

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PZD domain protein-1, Glutamate receptor-interacting (GRIP)-1 protein and syntenin-1 (Campoli, Ferrone, & Wang, 2010). Tumor cells are migrated through interaction of GRIP-1 and

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syntenin-1 with cytoplasmic NG2 domain. Binding of β1 integrin to the cytoplasmic NG2 domain and collagens II, V, and VI, galectin, laminin, and tenascin to NG2 facilitate adhesion of

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NG2 to cell surface. The proliferation, migration, invasion of tumor and healthy tissues are regulated by NG2-mediated focal adhesion kinase and mitogen-activated protein (MAP) kinase

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signaling pathways (Yadavilli et al., 2016). The structure and functions of CSPG-4 are

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elaborated in Fig. 2 and Table-1.

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Fig. 2: Pictorial depiction of structure and functions of CSPG-4 in tumor (Mayayo et al., 2011). Table-1: Classification of CSPG families and their oncogenic/anti-oncogenic effects. Type

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ECM CSPGs

Family

Hyalectan (aggrecan, versican)

SLRP

Testicans

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Pathological Functions in tumor Protumoral effects by increasing cell motility and migration Regulatory effects on tumor progression such as biglycan and decorin have antioncogenic effects. Mediates growth and metastasis of prostate cancer.

Ref. Wegrowski et al., 2006 Wegrowski et al., 2006

Cheng, Montmasson, Terradot,

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Basement membrane CSPGs

Perlecan

Agrin Cell surface transmembrane CSPGs

Syndecans

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Lecticans

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Glypicans

Endocan

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Betaglycan

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Thrombomodulin

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Cell surface nonclassified transmembrane CSPGs

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NG2/human melanoma

Neural CSPGs

Amyloid precursor like protein‐ 2

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Neurcan

Neuroglycan C

phosphacan/RPTPbeta/PTPzeta

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Rousselle, 2016 The fragment of perlecan Wegrowski et al., (endorepelin) regulates tumor 2006 angiogenesis Facilitates progression and Wegrowski et al., angiogenesis of oral cancer. 2006 Modulate cell proliferation, Howell, migration, adhesion, and & Gottschall, tumorigenesis processes. It 2012 can act as prognostic marker of tumor progression. Mediate glioma cell invasion Gary, Kelly &, Hockfield, 1998 Over-expression in glioma Saito et al., 2017 correlates with dissemination and poor prognosis of tumor Anti-tumor potential. Prognostic marker of vascular Iqbal, 2000; Wu and lymphatic tumor and lung et al., 2014 cancer. It influences progression, Nishida, proliferation, migration, Miyazono, & invasion and angiogenesis of Ehata, 2018 tumor. Pro-tumoral effects. Helpful Ates, Gedik, for prognosis of breast tumor. Sunar, & Altundag, 2018 NG2 contributes to cell Maritzen, growth, migration, detachment Schachtner, & from the substratum and Legler, 2015 metastasis. Involves in the abnormal growth, migration, and invasion of tumor cells Stimulation of tumor growth, and neuroblastoma cells to enhance malignant phenotypes

Pandey 2016

et

al.,

Rauch, Feng, & Zhou, 2001; Su et al., 2017

The binding of pleiotrophin to Nakanishi et al., neuroglycan C mediates cell 2010 migration, angiogenesis, and tumor cell proliferation Variable functions in cell Xia et al., 2019 proliferation, adhesion and migration. Helpful in detection

of tumor. Effective role in glioma cells Lu et al., 2019 motility, adhesion, growth and differentiation.

BEHAB/brevican

3. CS and CSPGs as theranostic and therapeutic targets across different types of cancer

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3.1 Melanoma Melanoma is characterized by the rapid and abnormal growth of mutated melanocytes that are

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responsible for production of melanin pigment. Melanoma, the most serious form of skin cancer, originates from melanocytes. Melanoma becomes metastatic when it spreads to other organs

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such as lungs, liver and brain. Late-stage melanoma become chemo-resistant and has a high mortality rate (Liu et al., 2018). CSPG-4 also known as melanoma linked antigen is highly found in the melanoma cells and could be exploited as a marker for diagnosis and immunotherapy of

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melanoma. CSPG-4 has been reported as immunohistochemical marker and could be targeted for immunotherapy of canine malignant melanoma (Mayayo et al., 2011). Similarly, scientists

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considered the prominent role of NG2, rat analogue of human CSPG, in the angiogenesis of tumor (Staub, Hinzmann, & Rosenthal, 2002). The anticancer T cells are functionally impaired by the programmed cell death in melanoma and Abs blocking the binding of programmed cell

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death receptor ligand-1 (PD-L1) to the programmed cell death receptors have an excellent scope in therapy of melanoma. Therefore, bispecific Ab, PD-L1xCSPG4, simultaneously binding to PD-L1 and CSPG-4 was designed and exhibited strong PD-L1 blocking activity as a result of enhanced avidity of binding to PD-L1/ CSPG4 cancer cells (Koopmans et al., 2019). Various immunotherapy approaches based on targeting CSPGs for tumor therapy are illustrated in Fig.3

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and summarized in Table-2.

3.2 Triple-negative breast cancer Triple-negative breast cancer (TNBC) is a widely growing cancer expressing CSPGs on the cell surface. Scientists are trying very hard to find suitable ways of overcoming pathological entanglement 11 | P a g e

of CSPGs and targeting CSPGs in TNBC is a highly attractive approach these days. Although high expression of CSPGs does not correlate well with TNBC but it is associated with prognosis and relapse of breast tumor (Hsu, Yokoyama, Chu, & Hou, 2013). NEDD9 has a vital role in promoting migration, invasion and ultimately breast cancer malignancy. To understand mechanism of NEDD9promoted invasion and growth of breast tumor cells, microarray study was conducted on NEDD9 over-expressing cells, HCC38. CD44 and serglycin, core proteins, were upregulated in NEDD9 cells than HCC38 vector cells while down-regulation of syndecan-1, syndecan-2, and versican

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was observed in NEDD9 cells (Iida et al., 2015). The CS component of ECM has been found to accelerate the invasion of breast cancer cell line BT-549. N-cadherin promotes the cell motility and invasion by FGF receptor signaling mechanism. The CS binds to cadherin and cleaves the

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cadherin to produce catenin (Nadanaka, Kinouchi, & Kitagawa, 2018). Neutrophils recirculate in breast cancer ECM and secrete a proliferation-inducing ligand (APRIL), responsible for

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like receptor 4 (TLR4) (Bat-Erdene et al., 2018).

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migration and invasion of tumor cells. CSPGs activated the production of APRIL through toll-

3.3 Colorectal cancer

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The CS and its PGs provide protection against colorectal cancer (CRC). Various research articles published in recent decades focused on effective participation of CS in prevention and decreasing risk of CRC. The Nurses’ Health study and Health Professionals Follow-up study

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demonstrated that CS supplements used for the treatment of joint diseases reduced the risk of CRC (Daniells, 2016). VITamins And Lifestyle (VITAL) and another cohort study mentioned that osteoarthritis drugs reduced the risk of CRC (Kantor et al., 2016; Satia, Littman, Slatore, Galanko, & White, 2009). CS decreases the translocation of nuclear factor κβ (NF-kβ) which plays a prominent role in immune response and tumor angiogenesis. Synthesis of CS demands an

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enzyme chondroitin synthase-1 (CHSY1) which has a connection with tumorigenesis of several tumor types. CHSY1 has a considerably high expression in the colorectal tissue. Knockdown of CHSY1 resulted in significantly reduced proliferation of CRC (Zeng et al., 2018). Serglycin (SRGN) has a critical role in the metastasis of CRC. The hypoxia-inducible transcription factor-1 alpha (HIF-1α) regulates the expression of SRGN gene. In in vitro experiments, cell migration and invasion were potentiated with the over-expression of SRGN. HIF-1α synchronized SRGN signaling and transcription led to proliferation and metastasis of CRC (Xu et al., 2018). 12 | P a g e

Table-2: Immunotherapeutic approaches targeting CSPGs for cancer therapy. Target

Targeting agent

CSPG-4

mouse monoclonal Canine anti-human Melan-A malignant Ab. melanoma

CSPGs were identified as Abs based Mayayo et al., Immunohistochemical immunotherapy 2011 marker of melanoma

CS

IO3D9, IO3H10, Metastatic IO3H12, and IO4C2 melanoma Abs

Abs displayed strong immunoreactivity in the ECM of metastatic melanoma

CSPG-4

bispecific Abs L1xCSPG4

CSPG-4

mAb 225.28 labeled TNBC with 212Pb and loaded with α-particle

CSPG-4

TPCS2a (fimaporfin) TNBC and Immunotoxin 225.28-saporin

CS

shRNA fragment CRC targeting human CHSY1 gene loaded in lentivirus

CS-E

GD3G7 Ab, Ab GD3A11

Outcomes

Future prospects

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Purification and analysis of CS epitopes involved in the pathogenesis of melanoma Abs potentiated the IFN-γ Abs based production and activation immunotherapy of anticancer-T cells in of melanoma melanoma cells mAb 225.28 exhibited Radiohigher uptake and dose- immunotherapy dependent growth of TNBC. inhibition of SUM159 or 2LMP cells than control. TPCS and saporin had Photochemical shown more combined effect internalization than alone in killing tumor (PCI) based cell lines therapy of TNBC Knockdown of CHSY1 Immunotherapy altered the expression of based on NF-kβ. knockdown of CHSY1

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PD- Melanoma

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CSPG-4

Tumor type

anti-CSPG4 electrovaccine

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OC

DNA Osteosarcoma

Strong expression of the GD3G7 and GD3A11epitopes in the malignant tumor than benign and low malignant tumor Post-electrovaccination sera effectively inhibited the proliferation of sarcoma cells and decreased viability of osteospheres.

Ref.

Smetsers al., 2004

et

Koopmans et al., 2019

Kasten et al, 2018

Eng et 2018

al.,

Zeng et al., 2018

Prognosis and Vallen et al., immunotherapy 2012; Van der of malignant OC Steen et al., 2016

Translation of Riccardo CSPGs-targeted al., 2019 active immunotherapy from animal to the clinical settings

et

Melanoma

CSPG-4

CAR T-cells

Brain tumor

CSPG-4

CAR T-cells

Glioma

CSPG-4

αCSPG4(scFv)‐MAP

TNBC

Glioma CAR T Lohmueller, therapy & Finn, 2017 Translation of CAR T-cells based therapy to the clinic. Targeted immunotherapy of TNBC

Beard et al., 2014

Amoury et al., 2016

CS and CSPGs are intermediate players in the angiogenesis of ovarian carcinoma (OC). CSPGs are the core component of ECM and regulate the various pathophysiological processes of cell

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migration, differentiation and metastasis of ovarian cancer (Vallen, van der Steen, van Tilborg, Massuger, & van Kuppevelt, 2014). The carbohydrate sulfotransferases (CHSTs) have a central position in the amount and degree of sulfation of CS. CS-E subgroup potentiates the expression of vascular endothelial growth factor (VEGF) and resultantly proliferation of tumor cells (Oliveira-Ferrer, Heßling, Trillsch, Mahner, & Milde-Langosch, 2015). Various biomarkers in

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the microenvironment and stroma of tumor such as MMPs, CD44 and CS-E are under the direct consideration for tumor targeted therapy of OC (Vos et al., 2019). The search of biomarkers for prognosis of OC is undergoing and a new prognostic biomarker, 4,6-disulfated (GD3G7 epitope), was evaluated (Vallen, Massuger, Dam, Bulten, & van Kuppevelt, 2012). Similarly, the significance of CS in the prognosis of OC was determined using the single chain Ab, GD3A11. The healthy ovaries showed minimal expression of GD3A11, and GD3A11 was significantly expressed in the ECM of malignant OC than benign tumor. There was no 14 | P a g e

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Chen et al., 2019

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3.4 Ovarian carcinoma

Photothermal therapy of solid tumor

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CSPG4-CAR T-cells

Photothermal therapy mediated infiltration and excellent cytotoxicity of CAR T -cells in tumor. Deteriorated the growth of tumor in neurospheres and xenograft mice model CAR T-cells actively targeted CSPG-4 and inhibited tumor growth and progression in tumor model Fusion protein targeted and accumulated at tumor tissue retarded tumor growth in in vivo model

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CSPG-4

CAR T-cells efficiently CAR T-cells Pellegatta inhibited the growth of based al., 2018 tumor neurospheres immunotherapy of glioma

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CART-cells targeting Glioblas-toma to EGFRvIII

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NG2

significant correlation between the results of debulking surgery or clinical remission following the chemotherapy with the expression of epitope (Van der Steen et al., 2016). In near future,

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single chain Abs could be useful for the diagnosis and immunotherapy of OC.

Fig.3: Immunotherapy strategies targeting CSPGs expressed in tumor tissues: 1. Macrophagemediated phagocytosis and Abs-dependent cytotoxicity; 2. Cytotoxic fusion protein (CFP); 3. CAR T- cells directing CSPG; 4. Redirection of cytotoxic T cells by bispecific T cell engager

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antibodies (BiTEs) (Ilieva et al., 2018).

3.5 Glioblastoma Glioblastoma is a highly proliferating type of brain tumor with high malignancy and mortality. CSPGs have an evident contribution in the growth and development of brain. CSPGs regulate the 15 | P a g e

migration of neurons, gliogenesis, and synaptic plasticity, and participate in the axonal guidance (Schwartz & Domowicz, 2018). Elimination of CSPGs deposited in the glioma using chondroitinase ABC or CSPG inhibitor could be fruitful in the treatment of glioma (Dmitrieva et al., 2011). Long-term chemotherapy of glioma with temozolomide (TMZ) led to P-glycoprotein (P-gp) MDR. Furanodienone (FUR), a diene‐type sesquiterpene, inhibited the expression of CSPGs and CSPG4‐Akt‐ERK signals and subsequently, exhibited antitumor effect in TMZresistant glioma cells (Chen et al., 2019). The current studies discovered that various CSPGs

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endowed cancer stem cells (CSC) with the potential of resistance to chemotherapy (Vitale et al., 2019). CAR T-cells targeting NG2 could be efficient in tumor directed delivery. For the first time, clinical trial of CAR T-cells targeting EGFRvIII on 10 patients bearing recurrent glioma

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was conducted. CAR T-cells had shown safety and no off-tumor toxicity. Although initial results forecasted the therapeutic efficacy and safety of CAR T-cells but further work has to be done to

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overcome heterogeneous expression of antigens (O'Rourke et al., 2017).

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3.6 Sarcoma

Sarcoma is the malignant tumor of mesenchymal tissues with high metastatic capacity. CS and

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CSPGs have the same position in adhesion, migration, differentiation and angiogenesis of sarcoma as in other tumors. Immunotherapy of chondrosarcoma based on Abs targeting CSPGs could enhance the survival of patients and abolish metastasis. In this context, anti-CSPG4 DNA

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based electrovaccine was introduced into the dog with canine osteosarcoma and sera was collected and, vaccine-induced Abs were evaluated for inhibition of proliferation in sarcoma cells and osteospheres. Abs effectively inhibited the proliferation of sarcoma cells and decreased viability of osteospheres (Riccardo et al., 2019). Soft tissue sarcoma and its precursor mesenchymal cells are over-expressed with CSPG4. The effect of NG2-Abs on the growth and

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initiation of sarcoma was investigated in vivo. The decreased expression of NG2 in mice and human sarcoma was correlated with reduction in tumor volume and growth of cells. The sarcoma cells were infected with the virus expressing short hairpin RNA (shRNA) and subsequently down-regulation of NG2 was observed (Hsu et al., 2018). Pericytes are precursor cells from which mesenchymal tumors originate. Depletion of tumor suppressor gene TRP53 in the precursor cells expressing NG2/CSPG4 mediated osteoarcoma and soft tissue sarcoma in mice (Sato et al., 2016). CHSY1 involved in the biosynthesis of CSPGs and knock-down of CHSY1 16 | P a g e

gene decreased the proliferation of CRC. Similarly, the expression of CHSY1 was connected with the tumorigenesis of sarcoma with myxoid substance (Momose et al., 2016).

3.7 Non-small-cell lung cancer Non-small-cell lung cancer (NSCLC) is a worldwide threatening cancer with high death rate in the Western and Asian countries (Barta, Powell, & Wisnivesky, 2019). Altered glycosylation or expression of PGs attributed towards proliferation of lung cancer. Abnormal glycosylation may

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result from altered expression and glycosylation of mucins, dysregulation of O-glycans level and modified branching of N-glycans (Chugh et al., 2015). Samples from normal healthy tissues and

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lung cancer tissues were collected and structure and expression of different PGs in tissue samples were compared. The content of CS and DS were twice in cancerous tissue than normal tissue (Li et al., 2017). Further research was done to match-up the content and expression of PGs in

-p

cancerous and normal tissues. The concentration of CS in tumor tissues was remarkably high compared to healthy tissues. These findings explained the pathophysiological significance of

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PGs in the prognosis of lung cancer (Rangel et al., 2018). The crosstalk of ECM and cancer cells is regulated by CD44 receptors. The SRGN-mediated NSCLC via CD44 axis was explored

lP

with the help of gain-of-function and loss-of-function strategies. The SRGN was over-expressed in carcinoma and stroma of NSCLC (Guo et al., 2017). Versican is another member of CSPGs

ur na

family contributing towards proliferation of NSCLC (Pirinen et al., 2005).

4. CS-based nanocarriers for tumor-targeted drug delivery In recent decades, a lot of work has been done in designing of self-assembled NPs of amphiphilic polymers with the advantage of lack of organic solvents or surfactants which are required for the fabrication of formulations. Amphiphilic molecules are constructed of hydrophilic and

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hydrophobic parts, and CS as a hydrophilic in nature is an ideal candidate for construction of amphiphiles (Yang et al., 2015). Conjugation of hydrophobic moiety with CS constructs amphiphilic copolymer which self-assembles upon contact with water to form micelles or micellar nanocarriers (Carvalho et al., 2018). There are two main strategies of introducing hydrophobic therapeutic moiety to the nanocarriers: (1) conjugation of hydrophobic drug with hydrophilic CS via a linker or chemical bond to form CS-drug conjugate; (2) CS is conjugated with hydrophobic polymer to form amphiphilic copolymer which upon self-assembly 17 | P a g e

encapsulates poorly soluble drug in the hydrophobic core (Fajardo et al., 2014). The anionic nature CS favors the formation of electrostatic-association with cationic polymers to form PECs. Similarly, CS is suitable for construction of hydrophilic nanogels with three-dimensional physical structure (Onishi, Ikeuchi-Takahashi, Kawano, & Hattori, 2019). In addition, CS also serves as a coating material for decoration of various nanoformulations such as lipid, polymeric, gold and magnetic NPs to achieve active delivery of drugs to the tumor site (Fig. 6 and Table-3).

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4.1 Self-assembled NPs of CS-drug conjugates Grafting of poorly-soluble therapeutically active molecules to the hydrophilic CS via spacer or

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linker results in the formation of prodrugs or drug conjugates. CS has many functional groups such as carboxyl and hydroxyl groups for covalent conjugation of drug, and sulfate and carboxylate for ionic bonding of drug. CS is covalently linked to a drug via ester or amide bond

-p

directly or by the use of linkers. Spacers are used when CS and drug have no mutual reacting functional groups and have the same functional groups. Linker is a chemical moiety with two

re

reactive functional groups, one reacts with CS and other makes a bond with the drug such as cystamine (CYS) (Yu et al., 2013). The CS-drug conjugate self-assembles to form micellar

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nanocarriers in aqueous solution. The drug forms the core of micelles, protecting hydrophobic drug from aqueous environment and in this way the CS-drug conjugate has eminent significance of improving solubility of drug. The prodrug strategy has additional benefits of potentiating

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bioavailability, plasma half-life and environmental stability of drug. The pH- and enzymetriggered drug release is also realized with the help of prodrug design. The enhanced permeability and retention (EPR) effect of the tumor endows tumor-targeting potential to nanocarriers or macromolecules (Najjar & Karaman, 2019). The nanosystem with pH-dependent release behavior was formulated based on CS-DOX prodrug. The drug conjugate was

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synthesized using acid-labile linker, adipic acid dihydrazide (ADH). CS was first grafted with ADH and then conjugated with DOX to form CS-DOX conjugate. An in vitro release study demonstrated pH-dependent release of DOX with more than 60% DOX was released at acidic pH, while only 20% was released at pH 7, after incubation for 7 days. The DOX conjugate had less cytotoxicity towards lungs carcinoma cells than free DOX but more tumor growth inhibition in mice bearing lung carcinoma cells. The enhanced tumor growth inhibition was linked to the EPR effect of conjugate and pH-dependent release of DOX at tumor site (Onishi et al., 2017). A 18 | P a g e

short peptide, ES2, with sequence of IVRRADRAAVP has an anti-tumorigenic potential. It had established anti-proliferative properties and also inhibitory effect on migration of endothelial cells. AF is another short peptide selectively binds to VEGFR1 and blocks the VEGF binding to its receptor and neovascularization. A synthetic peptide ES2-AF was synthesized by solid phase method, and to prevent the enzymatic degradation of peptide in the body, CS- peptide conjugate was fabricated. The proton nuclear magnetic resonance (1H NMR) spectroscopic analysis confirmed the structure of conjugate and zeta potential measurement ensured the stability of

of

micellar system in aqueous solution. CS served dual purpose to prevent enzymatic degradation of peptide, and its binding with CD44 receptors enabled the targeted delivery of conjugate to the tumor cells. The glycosylation modified peptide was more efficient in inhibiting the proliferation

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and metastasis of endothelial cells than peptide alone. The enzyme linked immunosorbent assay (ELISA) showed that C-conjugated peptide blocked binding of VEGF to its receptor in an

-p

effective way than ES2-AF. In vivo experiments revealed more plasma half-life, targeted in vivo distribution and metabolic stability of CS-ES2-AF compared to CS2-AF. In a summarized

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manner, CS modification of peptide improved stability, anti-neovascularization activity and antitumor efficacy (Xing et al., 2019). Golgi apparatus has significant influence on the metastasis

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of tumor. CS could accumulate in the Golgi apparatus and Golgi apparatus targeting NPs of CS were developed and characterized in vitro and in vivo. Retinoic acid (RA) has antitumor efficacy of influencing differentiation, proliferation and metastasis of tumor, and Golgi apparatus

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morphology. RA is enabled to reach the target cell and Golgi apparatus effectively, thus CSretinoic acid (RA) prodrug was synthesized and micelles were formulated encapsulating PTX. The prodrug NPs were internalized by the tumor cells via endocytosis and acid-labile bond between RA and CS was broken down at the acidic tumor environment to release RA. The prodrug disturbed the morphology of Golgi apparatus and subsequently inhibited protein

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expression responsible for metastasis of tumor. In mice bearing tumor, PTX- loaded micelles inhibited steps involved in the tumor growth and metastasis. The combined therapy had shown synergistic effect on tumor metastasis and prodrug NPs were highly efficient in realizing targeting of Golgi apparatus (Li et al., 2019). A composite nanosystem with dual properties of prodrug micelle and gel is a novel strategically designed nanoplatform of CS. B-cell lymphomaextra-large (Bcl-XL) belongs to Bcl-2 family with inhibitory effect on tumor apoptosis, leading to chemo-resistance. COX-2 enzyme controls the level of this protein by regulating the 19 | P a g e

phosphorylation of Akt and CS have shown anti-inflammatory effect through decreased synthesis of COX-2 enzyme which could be helpful to deal with bottleneck of chemo-resistance. A nanocomposite system of hydrogel shell and prodrug core loading two drugs, simultaneously, was fabricated with the purpose of enhancing sensitivity of cancer to chemotherapeutics. A prodrug of PTX conjugated with CS was self-assembled to load PTX and sunitinib, simultaneously. A CS hydrogel shell was encapsulated with two free drugs and drugs loaded micelles, and glutathione S-transferase-mediated disulfide bond was introduced in shell enabling

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the shell degradation at acidic tumor environment. The two drugs loaded in shell were instantly released after breakdown of shell to provide synergistic antitumor effect followed by slow release of drugs loaded in the core to maintain therapeutic level of drugs in resistant tumor. The

ro

CS released from degraded shell down-regulated Bcl-XL and effectively sensitized tumor to the chemotherapy. A programmable nanocomposite system with bimodal release profile and CS

-p

enhanced sensitization of tumor provides a rational way of drug transportation to the resistant

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tumor (Zhang et al., 2019).

4.2 Micellar nanovehicles of CS-polymer conjugates

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CS is a multifunctional hydrophilic polymer conjugating with a variety of hydrophobic polymers to design amphiphilic copolymer which self-assembles to form micelles or micellar nanocarrier on contact with aqueous solution based on inter or intramolecular linkage of hydrophobic entity

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(Liu et al., 2018). The various hydrophobic moieties that form inner core of the micelles are PCL, CHS, histamine, bile acid (BA), PLA, PLGA, protoporphyrin (PpIX), alkanethiol, DOCA (Fig. 5) (Chang et al., 201; Fajardo et al., 2014; Liu et al., 2017; Zhang et al., 20177). In recent decades, CS based core-shell NPs have been exploited for active-and passive-targeted delivery of therapeutics to the tumor site. The various critical attributes of hydrophobic moieties are kept

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under consideration while their selection in the design of self-assembled NPs (Tesauro et al., 2019). The hydrophobic moiety should encapsulate hydrophobic drug depending upon the compatibility of core and drug which works on the principle of "like dissolves like". It should be non-immunogenic and non-toxic (Najjar et al., 2019). The redox-sensitive conjugate of CS with PLGA (CS-PLGA) was constructed via CYS as a linker. NPs of CS-PLGA were formulated and loaded with DOX. The nanoconstruct had a high drug loading of 15% and redox-sensitive in vitro release of DOX. The NPs released drug in A549 cells which confirmed NPs realized redox20 | P a g e

sensitive intracellular release of DOX. In vitro cell viability of free DOX and NPs were comparable. The redox-sensitive NPs of PLGA-CS are promising nanocarriers for tumortargeting (Lv et al., 2013). Similarly, CS-PLGA NPs have drawn attention of scientists for chemotherapeutic drug delivery. Different chain lengths of hydrophobic PLGA were selected to compare the effect of chain length on in vitro and in vivo characteristics of NPs. The NPs were homogenous in size and had low critical micelle concentration. Effective encapsulation of DOX and pH-dependent release of DOX was realized with the nanovehicles. The NPs exhibited

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accelerated cell uptake, safety profile and inhibited cells growth in a concentration-dependent manner. The in vivo experiments in Kunming mice revealed improved plasma half-life and 2times more tolerance of DOX-loaded NPs than free DOX. Overall, CS-PLGA micellar NPs had

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excellent features to be used as a carrier for cancer therapy. Micelles with tunable morphology and physiochemical properties are obtained by varying the proportion of hydrophobic and

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hydrophilic segments. The morphology of NPs was found to affect in vivo pharmacokinetics, bio-distribution and cell uptake. PCL-grafted CS copolymer (CP) was researched and effect of

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PCL contents on the morphology of NPs was studied using coarse-grained molecular dynamics (CGMD) simulation. 63 monomers of low, medium and high contents containing PCL were

lP

grafted to 120 monomers of each CS backbone. The morphology of low and medium CP conjugates was spheroid whereas, conjugate containing high contents of PCL was rod-shape in appearance. Radius of gyration demonstrated that three-dimensional morphology was

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dependent on number of CP chains. When CP chains were three in number, the shape of high CP was nanorod, while low CP changed from nanospheriod at 3 CP chain to nanodisk at beyond 3 numbers of chains. The self-diffusion coefficients of CS and PCL sections were inversely related with the number of PCL molecules in the conjugate (Chang et al., 2017). CSDOCA NPs with switchable ‘on-off’ release profile have also been investigated to inhibit the

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growth and metastasis of melanoma. These NPS had dual release behavior with response to redox environment of tumor and enzymatically breakdown of CS at the tumor site. CYS was used as redox sensitive linker to design CS-DOCA conjugate which could self-assemble to make NPs. The NPs had desired loading and enhanced release of DTX was observed with breakdown of S-S bond at acidic tumor microenvironment and CS backbone with action of hyaluronidase-1. The in vivo distribution of DTX realized with NPs was significantly higher than that of Taxotere®. The NPs inhibited metastasis by inhibiting the expression of protein associated with 21 | P a g e

metastasis and efficiently killed tumor cells (Zhao et al., 2015). Thus, polysaccharide based nanocarriers with dual triggers initiated release of drug and efficient way of killing tumor cells and inhibiting tumor metastasis could be fruitful drug delivery systems for melanoma therapy. Similarly, CS-DOCA nanovehicels have also been designed and characterized for tumor delivery of DOX. The micelles had improved physiochemical characteristics with reduction-induced release pattern. In human gastric cancer HGC-27 cells, confocal laser scanning microscope confirmed reduction-triggered release of DOX, and redox NPs were more cytotoxic to HGC-27

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cells than non-redox responsive NPs (Liu et al., 2017). Chemo-SDT works on the principle of low intensity ultrasound-based activation and enhanced

ro

permeability of sonosensitizers to the local tumor tissues without damaging to normal peripheral tissues. The combination of SDT with nanotechnology is a revolutionary approach for

-p

chemotherapy of malignancy difficult to respond traditional treatments. The CS has shown promising attributes for SDT of malignant tumor. In the light of above, redox-/enzyme/ultrasound-responsive polymer backbone of CS-chlorin e6 (Ce6)-lipoic acid was synthesized

re

and self-assembled NPs were fabricated for management of proliferation and metastasis of melanoma (Fig.5). The micellar NPs were cross-linked and had homogenous size distribution,

lP

high loading and enzyme-/redox-triggered release. The cross-linked NPs were able to generate reactive oxygen species (ROS) which in turn damaged mitochondria and induced apoptosis through caspase pathway. Chemo-SDT deteriorated the proliferation and metastasis of melanoma

ur na

through reduced expression of metastasis-associated protein. In addition to anti-proliferation and anti-metastasis of melanoma, NPs were also capable to induce immune response via tumorspecific antigen secretion (Fig. 4) (Liu et al., 2018). The combined SDT with intelligent versatile nanocarriers paves a way for improved management and therapy of tumor. Similar to SDT, PDT works on the concept of light-triggered activation of photosensitizers (PS) to produce ROS in the

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tumor tissues, leading to apoptotic or necrotic death of tumor cells. Ce6 is highly efficacious second generation PS requiring low drug and light doses. CS-Ce6 graft was constructed via esterification and investigated as nanoplatform for PDT of tumor. The nanodrugs were small in size and critical self-quenching concentration had inverse relation with contents of Ce6. The smart self-quenched nanodrug was photo-inactive in aqueous solution and upon irradiation with light, the permeability of conjugate to the cells was enhanced, and enzyme in the cells released PS form conjugated nanodrug. Exposure of nanodrug to laser light rapidly generated O2. 22 | P a g e

Enhanced cytotoxicity in Hela cells treated with combined red light and nanodrug at variable doses of Ps was noted compared with only nanodrug or free PS without exposure to light. The nanodrug was effectively taken up by the Hela cells (Li, & Na, 2011). In recent years, MDR is challenging to the successful chemotherapy of tumor and PDT of tumor is a reliable approach to overcome MDR-related failure of chemotherapy. P-gp and its transporters are one of the culprits for MDR and competitive inhibition of transporters could help to overcome MDR. A multifunctional nanosystem was designed based on CS-PpIX complex, loading apatinib and

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DOX, simultaneously. The anionic CS binding with CD44 provided MDR cells targeted delivery. ROS generated from exposure of nanomicelles to red-light dissociated micelles to release encapsulated drugs. The apatinib competitively inhibited enzymatic activity of P-gp

ro

transporter and retained DOX within MDR cells. The ROS through reduced mitochondria membrane potential and with DOX via DNA damage induced apoptosis of cells. Thus, PDT in

-p

combination with chemotherapy is an effective way to combat tumor, especially, life threatening MDR tumor (Wei et al., 2018). Folic acid (FA) receptors are over-expressed on the surface of

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colorectal cells and binding of ligands with FA receptors results in cell internalization. Tumor resistance could be overcome with active-and passive-targeting of tumor via FA decorated

lP

nanovehicles. CHS as a hydrophobic segment was conjugated to CS to form amphiphile which was decorated with FA. The self-assembled micelles were formulated from CS/CHS/FA conjugate and were encapsulated with bortezomib. The micelles had homogenous size

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distribution and pH-dependent release in tumor simulated acidic environment. The result of in vitro cell uptake and apoptosis studies indicated that colorectal cell line expressing FA receptors had enhanced uptake and apoptosis of FA-decorated NPs than A5A9 cancer cells of lung with absence of FA receptors. The in vivo experiments based on intravenously administered NPs to the xenograft mouse model showed safety profile and efficacy of FA-NPs. Hence, FA-

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functionalized micelles could be smart nanocarriers for CRC targeting (Soe et al., 2019).

23 | P a g e

of ro -p

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Fig. 4: Schematic representation of self-assembly, cell uptake and apoptosis mechanism of

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ur na

lP

crosslinked NPs based on chemo-SDT. Reprinted with permission from (Liu et al., 2018).

24 | P a g e

of ro -p re lP ur na

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Fig. 5: Hydrophobic modification of CS with: (A) PLGA via ADH linker; (B) Histamine; (C) PpIX; (D) DOCA via CYS linker.

4.3 Polyelectrolyte nanocomplexes

PECs are another well-known class of NPs fabricated by electrostatic interaction between oppositely charged polyelectrolytes with the advantage of no requirement of organic solvent or energy. The polyanionic nature of CS confers its electrostatic association with cationic polymer 25 | P a g e

to make PEC. CHS is the widely searched polycationic counterpart of CS for PEC NPs generation. CS/CHS PECs were formulated using ionic gelation method and effect of lecithin on in vitro characteristics of NPs was studied such as drug loading, drug release and cell cytotoxicity. The NPs were of small size with desired morphology and stability. The lecithin had benefited of enhancing encapsulation efficiency of curcumin loaded NPs and provided fast release in buffer solution than NPs formed without lecithin addition. The lecithin/CS/CHS NPs had comparable cell viability with free curcumin in breast cancer cells. These results promoted

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the positive impact of lecithin addition on the in vitro attributes of PECs efficiently designed for tumor therapy (Jardim et al., 2020). In addition to CHS, PDMAEMA was utilized, for the first time, to prepare PECs. The effect of external conditions such as pH and temperature on the

ro

design, structure, stability and release profile of NPs was evaluated. PDMAEMA showed lower critical solution temperature (LCST) at pH 8.0 while PECs exhibited LCST at all pH conditions.

-p

In addition, LCST value of PECs was less than PDMAEMA at pH 8.0. The thermo-responsive PECs showed pH-and temperature-dependent release with more release of CS from PECs at pH

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8 and 60 °C than pH 6.0 and 36.5 °C, respectively. CS nanocomplexs of PDMAEMA were effective in improving cell viability of PDMAEMA and opened a gateway for exploration of new

lP

biomaterials in nanotechnology based therapy of tumor (Bonkovoski et al., 2015). PECs of CS with cationic therapeutic molecules have also been studied for cancer treatment. Asialoglycoprotein receptors (ASGPRs) are over-expressed in hepatic cells and responsible for In this context, CS-PECs with DOX were compared with DOX

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endocytosis of CS.

encapsulating CS-coated gelatin NPs to target HepG2 cells of hepatocellular carcinoma. The PECs and CS-coated NPs had considerable in vitro characteristics in term of size, charge and morphology. Although, the cytotoxicity study in HepG2 cells revealed more cell-killing ability of CS-coated NPs than free drug but were less potent than PECs. The inadequate coating of CS

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on the surface of NPs was declared responsible for less cytotoxicity of CS-coated NPs. The nanocomplexes of CS-DOX have a bright future in chemotherapy of hepatocellular carcinoma (Varshosaz et al., 2017). The knowledge of phytochemistry is vital for screening of new chemical constituents of plants valuable for tumor therapy. Ellagic acid, a plant-derived polyphenol, demonstrated anti-tumor efficacy in lung carcinoma. Systemic delivery of nanovehicles to the tumor site has many drawbacks and inhalable drug delivery is reliable alternate of it. There are many challenges associated with the inhaled therapy of tumor such as small sized NPs are 26 | P a g e

exhaled out before reaching the desired site and microparticles (MPs) undergo phagocytosis by macrophages. To avoid this problem, combined properties of NPs and MPs were exploited by formulating nanocomposite. A nanocomplex of lactoferrin (Lf) and CS was formulated on the basis of electrostatic association. The nanocrystal of ellagic acid was prepared and loaded along with DOX into nanocomplex. Spray dried technology was used to formulate inhalable nanocomposite of PECs with inert carrier. The in vitro and in vivo chemotherapeutic efficacy of composite system was investigated. The inhalable nanosystem provided fast release of ellagic

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acid and DOX with deep deposition to the lung. The composite was internalized via transferrin (Tf) and CD44 receptors-mediated endocytosis into A549 lung cancer cells and showed excellent

ro

antitumor efficacy (Abd Elwakil et al., 2018).

Prodrug-based micelles

CS

CS

nanocomposite system of hydrogel shell and prodrug core

CS

Jo

CS-Polymer conjugate as nanocarriers

Ligand

DOX

N/A

ES2peptide

ES2 peptide

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CS

Therapeutic molecule

PLGA/CS

PLGA/CS

27 | P a g e

In vitro Results cells Lewis lung The enhanced tumor growth carcinoma inhibition was linked to the EPR cells effect of conjugate. The glycosylation-modified A549 peptide had improved stability, carcinoma anti-neovascularization activity cells and antitumor efficacy. The combined therapy had shown synergistic effect on tumor 4T1 cells metastasis, and prodrug NPs were highly efficient in targeting of Golgi apparatus. The release of CS from degraded A549 shell down-regulated Bcl-XL carcinoma expression and effectively cells sensitized tumor to chemotherapy. The NPs released the drug in A549 cells which confirmed NPs A549 cells realized redo-sensitive intracellular release of DOX.

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Polymer/ Material

lP

Nanovehicle

-p

Table-3: Summary of CS-based nanocarriers for tumor-targeted drug delivery.

RA/PTX

PTX and sunitinib

DOX

DOX

N/A

N/A

N/A

N/A

MCF-7 cells

Ref. Onishi et al., 2017 Xing et al., 2019

Li et al., 2019

Zhang et al., 2019

Lv et al., 2013

Zhang et The NPs exhibited accelerated al., 2017 cell uptake, safety profile, and inhibited cells growth.

CS/e6/ lipoic acid

N/A

N/A

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CS/PpIX

CS/CHS

PECs

CS/CHS/ lecithin

28 | P a g e

N/A

HGC-27 cells

N/A

Apatinib/ DOX

Bortezomib

Curcumin

N/A

Hela cells

N/A

B16F10 cells

FA

A5A9 cells

N/A

of

DOX

B16F10 cells

The in vivo distribution of DTX realized with NPs was significantly higher than that of Taxotere®. The NPs inhibited metastasis and efficiently killed tumor cells. The confocal microscopy confirmed reduction-triggered release of DOX, and redox NPs were more cytotoxic to HGC-27 cells than non-redox- responsive NPs.

Liu et al., 2018

Liu et al., 2017

ro

N/A

ur na

CS/Ce6

DTX

N/A

-p

DOCA/CS

N/A

re

DOCA/CS

N/A

lP

PCL/CS

Chang et The morphology of NPs was al., 2017 found to effect in vivo pharmacokinetics, biodistribution and cell uptake.

MCF-7 cells

Liu et al., Chemo-SDT deteriorated the 2018 proliferation and metastasis of melanoma through reduced expression of metastasisassociated protein. Li et al., The enhanced cytotoxicity in 2011 Hela cells treated with combined red light and nanodrug at variable doses of Ps was noted. The ROS through reduced mitochondria membrane potential and with DOX via DNA damage induced apoptosis of cell. The xenograft mouse model showed safety profile and efficacy of FA-NPs. The lecithin had benefited of enhancing encapsulation efficiency of curcumin loaded NPs and provided fast release in buffer solution than NPs formed without lecithin addition.

Wei et al., 2018

Soe et al., 2019 Jardim et al., 2020

N/A

HepG2 cells

CS

Ellagic acid /DOX

Lf

A549 cells

CS-decorated polymeric NPs

The endocytic uptake of DOX- Park et al., loaded nanogels to the cytoplasm 2010 of Hela cells was dependent on CD44 receptors-mediated endocytosis. Wang et The monodispersed nanogels had al., 2017 200nm size. Nanocarriers were superior in killing cells in vitro than free MTX.

-p

CDDP

Amino malonic acid

CS

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A549T and Hela cells

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N/A

CHS/ PLGA

PAMAM

Hela cells

lP

MTX

CS

PLGA

29 | P a g e

N/A

ur na

CS/PEGPAEU

DOX

The PECs and CS coated NPs had Varshosaz al., considerable in vitro et characteristics. CS-coated NPs 2017 had more cell-killing than free drug. Abd The inhalable nanosystem Elwakil et provided fast release of ellagic al., 2018 acid and DOX with deep deposition to the lung.

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Vero cells

DOX

CS

CS-dendrimers

N/A

CS/DOX

Acetylated CS

Nanogel

N/A

ro

PDMAEM A/CS

Bonkovos CS nanocomplexs of PDMAEMA ki et al., were effective in improving cell 2015 viability of PDMAEMA.

CPT

HCPT

miR-34a

CS

A5A9 cells

CT-26 cells

C26 cells

PC-3, A549 and 3T3 cells

Gil et al., Target specific delivery of CS2017 nanogel. Injectable hydrogel showed in vivo gelation and biodegradability in rat. After treatment with CS-NPs, less viable cells and higher percentage of apoptotic cells were found compared to cellulose coated NPs. The CS-modified NPs were taken-up by the colon cancer cells and promoted apoptosis through inhibition of topoisomerase.

Zu et al., 2019

Liu et al., 2019

Chen et al., 2017 The dendrimers delivered miR34a exaggerated apoptosis and mediated cell cycle arrest.

CS-liposomes

Cationic lipoplex

Phosphatid ylcholine

Antigen

MGluCS/ CHexCS

NIH3T3 cells

DOX

HAuCl4

30 | P a g e

MCF-7luciferase cells

CS

MCF7 cells

DNA

CS

293 T, CRL5802, and U87MG cells

N/A

CS

A431 cells.

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Jo SPIO

N/A

DOX

CS

The physiochemical properties of Au – NPs were dependent on Zhang et proportional contributions of al., 2018 PLGA, DSPE-PEG-COOH and lecithin in construction of NPs. Okubo et CS-modified liposomes were al., 2019 superior to dextran-conjugated liposomes as dextran induced less immune response.

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Choriocarcinoma JGE3 cells

SPIO

SPIO/PEI

A549 cells

plCSABP

SiRNA

Magnetic fluid

CS-Gold NP

CS/ HA

DOX

DOTAP/ cholesterol

CS-capped SPIONs

Magnetoplex

Plasmid DNA

Fernandez A hybrid nanosystem with -Piñeiro et excellent transfection capability al., 2018 could be translated from industry to the clinics.

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PLGA/ DSPEPEGCOOH

CS

Kim et al., The SLN were highly effective in 2019 inhibition of tumor growth and prevention of tumor emergence.

-p

Sorbitan ester

DTX

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DOPC/ DOTAP

lP

CS-lipid-based NPs

SK-BR-3, B16F10, CT26, 4 T1 cells

HCT116 cells

The sequentially injected CS / lipoplex transfected RNA to tumor being mice and inhibited Hattori et tumor growth. al., 2016 Compared to free DOX solution, CS-NPs had shown more anti- Mallick et al., 2016 tumor activity in MCF7 cells. The gene transfection efficiency (Lo et al., of magneto-nanoplex in cell lines 2015). was significantly higher than PEI/DNA and commercial Magnetofection™ reagent. Toth et al., The fluidic system showed 2019 colloidal and chemical stability related to redox-condition and salt concentration. Gurav et CS-NPs provided pH-responsive al., 2016 release and more cytotoxicity of DOX to the cancer and resistant tumor cells than DOX.

4.4 Nanogel Nanogels are hydrophilic polymers with three-dimensional structure cross-linked physically or chemically to form aqueous dispersion. Nanogels are NPs with desirable features of small size, high stability, biocompatibility, biodegradability, low immunogenicity, high drug loading, variable morphology, environmental protection of drug molecules, pervious reservoir for drugs and stimuli-triggered drug release endow them as an excellent choice for chemotherapeutic drug delivery. The physical methods of nanogel formation are freeze thawing, hydrogen bonding,

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heating/cooling, inotropic gelation, while chemical methods are chemical cross-linking, chemical-mediated grafting, radiation-mediated grafting, enzymatic reactions and radical

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polymerization (Dave, & Gor, 2018). Among other polysaccharide materials-based nanogels, nanogels constructed of CS are promising carriers of macromolecules and drug molecules for the

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tumor-targeted delivery. CS with different degrees of acetylation was worked out by chemical modification with acetic anhydride and CS with highest degree of acetylation was chosen to

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make nanogel formulations. To determine whether acetyl group conferred hydrophobic properties to gels or not, the partition equilibrium constant of nanogels was determined which

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was nearly equal to the micelles (7.88 × 105). The carboxyl and sulfate groups of swellable polysaccharide endowed stability to nanosystem with surface charge of more than -40. The nanocarriers were highly efficient to load DOX with encapsulation efficiency of 90%. The

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endocytic uptake of DOX-loaded nanogels to the cytoplasm of Hela cells was dependent on CD44 receptors-mediated endocytosis (Park, Park, & Na, 2010). Thus, acetylated CS nanogels have salient features towards anti-tumor drug transportation. Nanogels formed by self-assembly of hydrophilic CS and hydrophobic counterpart, methotrexate (MTX), tested to improve solubility profile of MTX with additional benefit of achieving tumor-directed therapy.

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Amphiphilic bioconjugate of CS and MTX was designed and NPs were generated. The Fouriertransform infrared (FTIR) and 1H NMR spectra confirmed the structure of conjugate with 13.65 degree of MTX substitution. The monodispersed nanogels had 200nm size. Nanocarriers were superior in killing cells in vitro, while less cytotoxic in in vivo animal experiments than free MTX (Wang, Zhao, Chen, Qin, & Zhu, 2017). The encouraging results highlight positive future prospects of self-assembled nanogels of CS in tumor treatment. Cisplatin (CDDP) is extensively used anticancer drug for treatment of solid tumors, NSCLC, cervical, ovarian, TNBC, bladder, head 31 | P a g e

and neck cancer but severe side effects limited its clinical efficacy. Nano-size drug delivery vehicles could overcome its harmful effects but hampered by non-specific tissue- targeting and stability problems. CS nanogels incorporated in hydrogel to improve tumor tissue- targeting of CDDP were designed. CS-aminomalonic acid conjugate was constructed via EDC- mediated coupling reaction to crosslink with CDDP via metal-ligand interaction. The poly (ethylene glycol)-poly (βamino ester urethane) (PEG-PAEU) hydrogels responsive to temperature and pH conditions were formulated and loaded with CS-CDDP nanogels. In tumor microenvironment tertiary amine

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groups of hydrogel network were ionized and made ionic interaction with CS-nanogel. The hydrogel-nanogel showed sol-gel transition at tumor and physiological conditions, respectively. The exposure of nanogel-hydrogel system to A549 tumor cells slowly released CS-nanogel from

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hydrogel to bind CS with CD44 receptor and mediate endocytosis. The stimuli-responsive release of CDDP was observed. Slow endocytosis-mediated internalization of CS-nanogel and

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slow release of CDDP from nanogel were responsible for less cytotoxic activity of nanogel compared to free CDDP. The specific binding of CS-nanogel to A549 cells expressing CD44

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receptors and no binding to NIH 3T3 cells indicated target specific delivery of CS-nanogel. Injectable hydrogel showed in vivo gelation and biodegradability in rat (Gil, Thambi, Phan, Kim,

lP

& Lee, 2017). Injectable nanogel-hydrogel hybrid system is a useful application of nanotechnology for tumor-specific delivery of anticancer drugs.

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4.5 CS-functionalized nanoformulations The design of cargo carriers with the help of recently introduced nanotechnology based approaches helped scientists to treat and manage life-threatening cancer. In addition to serve as a skeleton or building block for construction of nanocarriers, CS has also been investigated for functionalization of multitask-oriented NPs (Guelfi et al., 2017). The CS has multifunction as a

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coating material of tumor-targeting NPs such as realizes tumor-directed delivery of cargo molecule, provides colloidal stability to NPs and protection from macrophages phagocytic system, enhances drug loading and cell uptake, and inhibits tumerigenesis, proliferation, and inflammation-related response of tumor (Gurav et al., 2016; Liu et al., 2019; Toth et al., 2019). Various NPs constructed of organic-, inorganic-and lipid-based materials have been decorated with CS and investigated for tumor-targeting therapy (Fig. 5).

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4.5.1 CS-decorated polymeric NPs In recent decades, many biocompatible, biodegradable and non-toxic polymers have been utilized for preparation of nanoformulations such as CHS, poly (glycolic acid) (PGA), PLA, PLGA and PEG (Chen et al., 2019; Dauda et al., 2017; Zhang, Yang, Ji, Liu, & Zhai, 2016). Various ligands have been conjugated with polymeric NPs for guidance and internalization of NPs to the targeted cells (Zuo, Chen, Cooper, & Xu, 2017). CS is extensively decorated ligand on the surface of PLGA NPs for site-specific targeting and delivery of anticancer agents. PLGA

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NPs had shown high encapsulation efficiency for hydrophobic molecules and were chosen to deliver camptothecin (CPT) to colon cancer cells. The nanoprepartions were coated with CS and

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had neutral surface charge, small polydispersity index (PDI) and small size. Compared to cellulose coated NPs, CS-NPs had more anti-cancer activity which was indication of CD44

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mediated endocytosis of CS-NPs. Moreover, less viable cells and higher percentage of apoptotic cells were found after treatment with CS-NPs compared to cellulose coated NPs. The in vivo

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anti-tumor experiment revealed more cytotoxic efficacy of CS-coated NPs in colon tumor bearing mice than cellulose coated NPs. Taken together, CS-coating on the surface of NPs is an

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encouraging technique for tumor-specific delivery of therapeutics (Zu et al., 2019). Another group of researcher loaded hydroxyl CPT (HCPT) into CHS/PLGA hybrid NPs. CHS conferred positive charge to bind with negatively charged CS. Uniform sized particles with little increase

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in size and zeta potential and reduced PDI were obtained after CS modification. Both CS-coated and uncoated NPs had good encapsulation efficiency. The drug release study demonstrated stable release profile in acidic environment due to positive charge on CHS conferred stability to NPs and at neutral pH, the enhanced solubility of CHS resulted in destruction of hydrophobic structure of NPs and fast drug release. The CS-modified NPs were taken-up by the colon cancer

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cells and promoted apoptosis through inhibition of topoisomerase. The in vivo colon cancer bearing mice experiments declared superior cytotoxic effect of CS-modified NPs in term of maintaining body weight of mice and reducing nodules of tumor (Liu et al., 2019). PLGA NPs of CS-DOX conjugate were studied for their impact on potentiating anti-tumor efficacy and tumortargeted transportation of DOX in in vivo and in vitro experiments. The structure of conjugate was confirmed by FTIR and NMR spectroscopies. The synthesized NPs were well-characterized of mono-dispersed size distribution, spherical shape and desirable surface charge. The NPs

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exploited CD44-mediated endocytosis of CS for improved cell uptake and cytotoxicity towards U251 cells. Intravenous administration of NPs to the mice bearing U251 cells inhibited tumor growth, and enhanced anti-tumor capacity and plasma concentration of DOX with less toxic effect on cardiac tissues. As a whole, CS-DOX-PLGA NPs are excellent alternate of DOX with less cardiac toxicity, and improved anti-cancer activity and pharmacokinetic parameters (Liu et al., 2019). In parallel with polymeric NPs, dendrimers which are three dimensional network of polymer material have attained considerable recognition for tumor therapy. Dendrimers have

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characteristic features of enhancing the solubility of cargo molecules. The positive charge enables them to increase cellular uptake and interact with negative charge nucleic acid (Khan, Liu, Khan, & Zhai, 2017). The gene therapy of tumor is hampered by immunogenicity and

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toxicity related shortcomings of viral vector. Cationic dendrimers are an excellent replacement of viral vector for gene therapy of tumor. The Michael addition of CS to the surface of PAMAM

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dendrimers and then loading of miR-34a to the CS-modified dendrimers for inhibition of proliferation of adenocarcinoma cells could achieve an efficient gene therapy of tumor. The

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modified dendrimers facilitated endocytic uptake to the carcinoma cells and endosomal escape of miR-34a. The dendrimers delivered miR-34a exaggerated apoptosis and mediated cell cycle

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arrest, leading to inhibition of cell proliferation. In a xenograft mice model of adenocarcinoma, dendrimers impeded growth of tumor and mediated apoptosis which were the indication of potentiated delivery of miR-34a to the tumor tissue. Hence, dendrimers are promising drug

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delivery vehicles for gene therapy of tumor (Chen, Liu, Liang, Huang, & Li, 2017).

4.5.2 CS-modified lipid based NPs Lipid materials have gained substantial interest of scientists for construction of NPs with desire of ameliorating drug loading, providing release of a cargo in a controlled manner and stability to

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drug, surface modification and ease of fabrication at industrial level (Spencer, Puranik, & Peppas, 2015; Yingchoncharoen, Kalinowski, & Richardson, 2016). All these unique attributes made lipid NPs as nanovehicles of choice for tumor-targeted cargo delivery. Oral administration of oncology drugs has not gained success owing to considerable objections. Frequent administration of available chemotherapeutic formulations have drawback of high plasma concentration-related side effects and damage to immune cells (La-Beck, & Gabizon, 2017). The design of oral oncology drug delivery systems with less harmful effects is in dire need of this 34 | P a g e

advanced era of nanotechnology. Orally delivered cationic solid lipid NPs (SLN) were decorated with anionic CS-conjugated to glycocholic acid (GA). A wide-spectrum anticancer drug, DTX, was encapsulated in CS-SLN with the intentions of killing cancer cells and also uplifting inherent immune system capable of fighting with tumor. The orally administered SLNs were absorbed from distal ilium through GA- mediated BA absorption route and transported via lymphatic system avoiding first pass metabolism. The SLN maintained plasma level of DTX for 24 h without effecting immune system. The level of cytotoxic T cells was noted to be high

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compared to decrease level of tumor-associated macrophages and regulatory T cells. The SLN were highly effective in inhibition of tumor growth and prevention of tumor emergence. SLN were not only able to inhibit tumor growth but also capable of boosting inherent immune system.

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Thus, the intestinal lymphatically administered SLN with immune-responsive effect could be an encouraging treatment of recurrence tumor after complete remission (Kim, Youn, & Bae, 2019).

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We have discussed role of different genes in the developmental process of different tumors in detail and down-regulation or up-regulation of various genes is helpful for tumor therapy. These

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days, extensive research work is being done on gene therapy of tumor. Lipid NPs constructed of sorbitan ester were decorated with CS/hyaluronan and loaded with plasmid DNA. Lipid-polymer

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hybrid nanosystem was characterized for physicochemical properties, stability and transfection efficiency. The electrostatic interaction of positively charged surface of lipid NPs with anionic CS/hyaluronan contributed to decrease the size of hybrid NPs. Effective incorporation of CS/HA

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kept liquid dispersion and freeze-dried forms of NPs stable at different temperature, overcoming compromised transfection efficiency due to instability of NPs in aqueous suspension. The hybrid nanocarriers efficiently transfected A549 cell line and had shown safety profile in connection to cell viability. A hybrid nanosystem with stability in lyophilized form and excellent transfection capability could be translated from industry to the clinics for gene therapy of tumor (Fernandez-

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Piñeiro, Pensado, Badiola, & Sanchez, 2018). CS-A is highly presented in various cancers and placental trophoblasts. A malarial protein VAR2CSA have been found to interact with CS-A. A peptide (plCSA-BP) constructed from malarial protein VAR2CSA could bind with CS-A expressed on placental trophoblasts. Lipid-polymer NPs were fabricated via sonication method to reduce fabrication time and were loaded with DOX. PLGA and 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[carboxy(PEG)]-COOH (DSPE-PEG-COOH) formed the core and shell of NPs, respectively and lecithin was located between core and shell of NPs. NH2 groups of of 35 | P a g e

pICSA-BP was coupled with –COOH group of PEG to form amide bond via EDC-mediated bioconjugation. The size, distribution and loading efficiency of NPs were dependent on proportional contributions of PLGA, DSPE-PEG-COOH and lecithin in construction of NPs. The uptake of NPs by placental choriocarcinoma JGE3 cells was dependent on culture time and temperature and the concentration of fetal bovine serum (FBS) in the medium. Thus, pICSA-BP conjugated NPs comprise an effective delivery system for tumor targeted therapy of

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chemotherapeutics (Zhang, Zheng, Cai, & Fan, 2018).

Fig. 6: Diagrammatic presentation of CS-functionalized NPs for tumor-targeted drug delivery.

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4.5.3 CS-coupled liposomes Liposomes are lipid vesicles with self-aggregation behavior and capable of carrying wide variety of cargo molecules such as hydrophilic therapeutics, lipophilic drugs and macromolecules. The inherent lipophilic nature of liposomes made them excellent nanocarriers for transportation across the lipid membrane of cancerous cells. Liposomes are associated with severe limitations in chemotherapy of tumor such as in vivo instability, short circulation time and non-specific drug delivery (Bagari et al., 2011). Many novel chemotherapeutic approaches have been implicated to

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provide tumor site-targeted liposomal delivery of anti-cancer agents with improved plasma stability and less off-target effects. Strategy based on surface modification of liposomes with

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targeting moiety could lead to ligand-receptor interaction-related endocytosis of nanocarriers (Riaz at al., 2018). Among recently explored various ligands, CS with capability of mediating

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CD44-coupled endocytosis is considered ligand of the choice for tumor-targeted delivery. Cancer immunotherapy is hot topic among research community these days. The inherent immune system

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of cancer patients is compromised and activation of immune system through induced T lymphocytes could be effective way of tumor management (Sanghera, & Sanghera, 2019).

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Specially designed liposomal system could deliver cancer antigen to dendritic cells, leading to induction and migration of T lymphocytes to tumor tissue for killing tumor cells (Fucikova, Palova-Jelinkova, Bartunkova, & Spisek, 2019). Liposomes conjugated with CS derivative

dendritic

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loaded with cancer antigen could fuse with dendritic endosome to deliver cancer antigen into cytosol.

3-Methylglutarylated

CS

(MGlu-CS)

and

2-carboxycyclohexane-1-

carboxylated CS (CHex-CS) were synthesized as derivatives of CS and decorated with liposomes through 1-aminodecane linker. Subcutaneous injection of CS-liposomes to the tumor mice model transported antigen to the cytosol of dendritic cells via low pH-based fusion with the endosomal

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membrane and initiated cytokine production and activation of immune response. CS-modified liposome were superior to dextran-conjugated liposomes as dextran induced less immune response due to hydrophobicity of linker and CS also prevented non-targeted binding to skinresident cells (Okubo, Miyazaki, Yuba, & Harada, 2019). Cationic lipoplex consisting of cationic liposomes and siRNA could transfect RNA into liver metastases from breast tumor. Intravenous administration of cationic lipoplex to breast tumor-metastasized liver induced mice model followed by 1 minute CS injection is a novel way of systemically delivering siRNA to liver. The

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systemically transfected luciferase RNA deteriorated the enzymatic action of luciferase in metastasized liver. The repeated injections of CS / lipoplex were safe to liver tissue and not induced inflammatory response compared to PGA/ lipoplex injection induced hepatic damage and inflammatory cytokines production. The sequential injection of CS and lipoplex inhibited tumor growth in mice bearing liver metastasis. Cationic lipolex administration resulted in lung accumulation of RNA but sequential injection of lipolpex 1 min after CS injection avoided lung accumulation. The sequential injection technique could avoid modification of lipoplex with CS

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and effectively deliver siRNA to liver (Hattori et al., 2016).

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4.5.4 CS-coated metallic NPs

Many metals such as gold, silver, iron, zinc and platinum, etc., have received prominent position in the field of cancer nanotherapy owing to nano-scale dimensions suitable for engineering of

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multifunctional NPs (Ruenraroengsak et al., 2019; Zhao et al., 2019). CS-functionalized gold NPS and superparamagnetic iron oxide NPs (SPIONs) are pharmaceutically versatile

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nanovehicles for tumor-specific therapy. Surface functionalization of SPIONs with biopolymer provides hydrophilicity to the surface, confers colloidal stability, prevents protein adsorption and

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encourages receptor-mediated active transport to the tumor-site (Hirsch et al., 2013). CS capped SPIONs have dual advantages of magnetically-facilitated and CD44-induced active-transport to the tumor tissues (Mallick et al., 2016). The bidentate chelation of CS with SPIONs formed CS-

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capped SPIONs and CS-capped SPIONs were investigated for loading of DOX and their efficacious contribution in targeting tumor cells. The DOX was loaded onto NPs via physical interaction with drug loading of 2% (w/w). X-ray powder diffractogram and FTIR spectra confirmed crystalline state of iron oxide and bidentate chelation of CS with iron oxide, respectively. Fast release of DOX with first order kinetics was realized by NPs. Compared to

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free DOX solution, NPs had shown more anti-tumor activity in MCF7 cells (DOX-NPs had low value of half maximal inhibitory concentration (IC50) than free DOX) (Mallick et al., 2016). CScapped SPIONs could be splendid nanocarriers for dual targeting of tumor tissues. SPIONs owing high specificity and innovative architecture could be used for transfection of gene. Strategically maneuvered SPIONs could be smart transgene vehicles for gene therapy of tumor. PEI-CS copolymer was synthesized and decorated on SPIONs via physical interaction to fabricate novel magnetoplex (CPIO) with DNA as a gene nanocarrier. Flow cytometry analysis 38 | P a g e

determined the internalization of magneto-nanoplex, and magnetic field enhanced the internalization of magneto-nanoplex to U87 cells than nanoplex alone. The gene transfection efficiency of magneto-nanoplex in 293 T, CRL5802, and U87-MG cell lines was significantly higher than gold standard, PEI/DNA and commercial Magnetofection™ reagent. The magnetoplex was safer than PEI/DNA. In addition, magnetic field- triggered CPIO/miRNA delivery exhibited more expression of miRNA-128 than without. Magnetic field placed near tumor site helped to lodge more CPIO/Cy5-DNA. Overall, magnetofection technique based on magnetic

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field-triggered delivery of gene could improve lodgment of DNA/RNA at tumor-targeted site and is an attractive strategy for gene therapy of glioma (Lo et al., 2015). The aqueous formulations of SPIONs have extensive theranostic and therapeutic prospects in tumor. A magneto-fluid

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nanoparticulate system demands non-toxicity, homogenous size and in vivo colloidal stability. The architecture of fluidized system requires coating of SPIONs with organic acids, neutral

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biopolymer or polyelectrolyte. CS is a worthwhile biopolymer for providing electrolyte stabilization to fluidic nanosystem. CS also has additional benefit of its interaction with CD44

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receptor activates signaling pathway of tumor. Magnetic NPs coated with CS have been used as magnetic resonance imaging (MRI) contrast agent and for other theranostic purposes. The post-

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synthesis coating approach was applied for fabrication of core-shell fluidic NPs of SPIONs. The as-synthesized magnetic fluid had colloidal stability for in vivo applications and was further characterized for its physicochemical properties. The spectroscopic studies confirmed inner-

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sphere metal–carboxylate complexes of CS-A with SPIONs. Under simulated biological conditions, the fluidic system showed colloidal and chemical stability related to the redox condition and salt concentration. The bioapplications of nanosystem were confirmed by coagulation kinetics which showed low value of equilibrium concentration of non-absorbed CS and suitability of nanosystem for biomedical applications. The magneto-fluidic NPs had shown

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safety in cytotoxicity study with no considerable effect on proliferation of tumor cells (Toth et al., 2019). A magneto-fluidic nanosystem could be contributive carrier of chemotherapeutics and employed for cancer theranosis. The site-specificity, tunable physicochemical properties, low toxicity, biocompatibility and ease of fabrication are unique attributes made gold NPs as promising nanovehicles for anti-cancer drugs delivery. Gold NPs opened a new avenue to design a promising nanoplatform for MDR

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tumor. Chemotherapeutics have immunosuppressant and thrombo-inflammatory effects which are leading cause of MDR along with efflux pump. To achieve synergistic effect of overcoming MDR and thrombo-inflammation, gold NPs were strategically designed and coated with CS. CSgold NPs were scrutinized for DOX delivery to tumor cells and tumor cells expressing MDR1 gene. CS was conjugated with hydrazide moiety and disulfide group to cap with gold NPs and act as reducing agent, respectively. NPs with small size and polydispersity were achieved with 3:1 molar ratio of CS and gold tetrachloride (HAuCl4) DOX was linked to hydrazide group on CS via pH-

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sensitive hydrazine connection to realize drug release at acidic -microenvironment of cancer. CS-NPs provided pH-responsive release and more cytotoxicity of DOX to human colon carcinoma cell lines, ovarian carcinoma cell line, and DOX resistant cell line expressing P-gp than free

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DOX. The NPs were internalized and transported into nucleus of cancer cells. These findings ensured CS-NPs overcome drug efflux pump and MDR of tumor, effectively.

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Further, immunosuppressant effect of DOX and NPs was evaluated using ELISA test. DOX exhibited platelet aggregation, a leading cause of thrombocytopenia, which was overcome

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by CS-NPS. DOX unregulated the thrombin–antithrombin (TAT) complex which was an indicator of coagulation. Whereas, CS-NPs decreased level of TAT. Free DOX had platelet

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aggregation and coagulation activation effects. Thus CS-gold NPs could overcome thrombotic complication of chemotherapy (Gurav et al., 2016).

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5. Limitations and future prospects

The biological properties of CS such as anti-angiogenesis and anti-metastasis make it attractive for treatment of tumor. CS can be utilized as a targeting moiety considering receptor-mediated endocytosis to tumor cells (Abd Elwakil et al., 2018). Based on theranostic and therapeutic

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applications of CS and CSPGs, CS nanovehicles could be exploited for cancer therapy and imaging (Lee, Chung, Cho, & Kim, 2015). Compared with other nanocarriers, CS-based nanoformulations had improved physiochemical properties, and lack of immune system recognition and accumulation to non-targeted tissues. These nanocarriers are facing problems of low drug loading and encapsulation efficiency, serum instability, off-target accumulation, external stimuli insensitivity, cost effectiveness and large scale manufacturing. There are still many hurdles to realize the applications of CS mediated nanovehicles in clinics and no formulation of CS for treatment of tumor has gained access to clinical trials. The pharmacology and toxicology of CS nanopreparations loading drug should be evaluated 40 | P a g e

guided by suitable indexes and methods. On account of immune modulation effect of CS, specially designed CS nanoparticles could serve dual purpose of enhancing immunity of cancer patient as well as treating the tumor (Liu et al., 2018). Multiple pathways-targeting nanocarriers could be beneficial to enhance antitumor response. It is expected that innovative nanomaterials of CS will be fabricated to overcome biological obstacles such as mononuclear phagocyte system, complex tumor microenvironment, intratumoral pressure, endosomal escape or multidrug resistance that have limited the therapeutic potential of chemotherapeutics (Sau et al., 2019).

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6. Conclusion

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Cancer is a life threatening disease and complete eradication of cancer is a major challenge to the available chemotherapy, radiotherapy and surgical interventions. A great attention has been

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given for inclusion of CS natural polysaccharides into highly efficient and effective nanoparticulate systems for cancer management. The hydrophilic and anionic CS with versatile

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functional groups on backbone can be applied for generation of amphiphilic polymers with drug molecules or hydrophobic polymer and ultimately, self-assembly to NPs. PECs could be formulated by electrostatic interaction of CS with cationic polymers, and physical or chemical

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crosslinking of CS also generates hydrophilic nanogels. CS-functionalized NPs had shown great potential in cancer management and treatment. The robust chemotherapy approaches working on

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the principle of external magnetic field, ultrasonic wave, radiations and light-triggered activetargeting of CS nanocarriers to the over-expressed CSPGs in tumor tissue had achieved considerable interest of the researchers for tumor therapy. Our group of researchers implied crosslinked and self-assembled NPs of CS for SDT of tumor and reported additional benefit of NPs favoring immune response. The cancer research community is focusing on immunotherapy;

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especially, mAbs and CAR T-cells targeting CSPGs antigens in tumor tissues are being widely investigated in clinical trials and had shown positive results. Further work is required for translation of CAR T-cells based immunotherapy from laboratory to the clinical settings. In near future, highly efficacious drug delivery vehicles constructed of CS with high payload of drugs, carrying wide range of bioactive molecules, and multiple antigens-directed CAR T-cells, and providing designated-site delivery without toxicity to normal tissues are expected to take place in the industry for successful chemotherapy of tumor.

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Acknowledgements This work is supported by the Major Research Project of Shandong Province, P.R.China (No.2018GSF118004) and Shandong Provincial Major Science &Technology Innovation Project, P.R.China (2018CXGC1411) and Major Basic Research Projects of Shandong Natural Science Foundation, P.R.China (No. ZR2018ZC0232).

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