Recent advances in galactose-engineered nanocarriers for the site-specific delivery of siRNA and anticancer drugs

Accepted Manuscript Title: Recent advances in galactose-engineered nanocarriers for the site-specific delivery of siRNA and anticancer drugs Authors: Ashay Jain, Atul Jain, Prahlad Parajuli, Vijay Mishra, Gargi Ghoshal, Bhupinder Singh, Uma Shankar Shivhare, Om Prakash Katare, Prashant Kesharwani PII: DOI: Reference:

S1359-6446(17)30340-9 https://doi.org/10.1016/j.drudis.2017.11.003 DRUDIS 2120

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Please cite this article as: Jain, Ashay, Jain, Atul, Parajuli, Prahlad, Mishra, Vijay, Ghoshal, Gargi, Singh, Bhupinder, Shivhare, Uma Shankar, Katare, Om Prakash, Kesharwani, Prashant, Recent advances in galactose-engineered nanocarriers for the site-specific delivery of siRNA and anticancer drugs.Drug Discovery Today https://doi.org/10.1016/j.drudis.2017.11.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Recent advances in galactoseengineered nanocarriers for the site-specific delivery of siRNA and anticancer drugs

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Ashay Jain1,2,3, Atul Jain2, Prahlad Parajuli4, Vijay Mishra5, Gargi Ghoshal3, Bhupinder Singh2, Uma Shankar Shivhare3, Om Prakash Katare1, and Prashant Kesharwani6 1

University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, 160 014, India UGC-Centre of Excellence in Applications of Nanomaterials, Nanoparticles and Nanocomposites, Panjab University, Chandigarh, 160 014, India 3 Dr S.S. Bhatnagar University Institute of Chemical Engineering & Technology, Panjab University, Chandigarh, 160 014, India 4 Department of Neurosurgery, Wayne State University School of Medicine and Karmanos Cancer Institute, Detroit, MI, 48201, USA 5 Department of Pharmaceutics, Lovely Institute of Technology (Pharmacy), Lovely Professional University, Phagwara (Punjab), India-144411 6 Department of Pharmaceutical Technology, School of Pharmacy, International Medical University, Malaysia Corresponding author: Kesharwani, P.K. ([email protected]; [email protected]); Katare, O.P. ([email protected]) Teaser: Galactose-functionalized nanocarriers open new avenues for the design of highly effective multifunctional, targeted drug delivery systems.

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Glycone recognition is a futuristic platform for active targeting.

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Galactose is widely explored carbohydrate ligand for targeted drug delivery.

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Recent advances of various galactosylated nano-carriers for tumor-targeting are overviewed.

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Galactosylated nano-carriers may be considered as safe and effective delivery system.

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Galactosylated nanocarriers have recently emerged as viable and versatile tools to deliver drugs at an optimal rate specifically to their target tissues or cells, thus maximizing their therapeutic benefits while circumventing off-target effects. The abundance of lectin receptors on cell surfaces makes the galactosylated carriers suitable for the targeted delivery of bioactives. Additionally, tethering of galactose (GAL) to various carriers, including micelles, liposomes, and nanoparticles (NPs), might also be appropriate for drug delivery. Here, we review recent advances in the development of galactosylated nanocarriers for active tumor targeting. We also provide a brief overview of the targeting mechanisms and cell receptor theory involved in the ligand–receptor-mediated delivery of drug carriers.

Keywords: galactose; targeted drug delivery; cancer; nanocarriers; siRNA.

Introduction

Cancer, also known as malignant neoplasm is a group of diseases [1] that, according to the WHO, affects approximately2 million new patients annually in the USA, of whom, one-third are expected to die as a result of

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their disease[1,2]. Of the total cases of cancer, 90–95% arise owing to environmental factors, whereas 5–10% are due to inherent factors [1,3].Cancer initiation depends on the trigger (either internal, such as immune factors, hormones, or inherited genetic mutations, or external, such as diet, tobacco use, X-ray and/or UV exposure, or infectious organisms) and mode of sequence of these triggers [4]. It is characterized by the continual, unregulated, abnormal proliferation and spread of cells, beyond their normal location, in a process known as ‘metastasis’ [5,6]. Anomalies in normal cells generally arise in response to acquisition of changes in the genetic material through mutations and/or epigenetic modifications that control cell division and senescence, leading to cancerous cells, in which the transformation of various genetic codes can be associated with different types and/or forms of cancer [7,8]. Cancer-related genes are broadly categorized into three types: (i) proto-oncogenes (responsible for augmenting cell division or suppressing normal cell death), whereby altered genes are called oncogenes; (ii) tumor suppressor genes (responsible for preventing cell division); and (iii) DNA repair genes (responsible for checking for the presence of mutations and repairing them) [9]. Evidence suggests that most cancers are the result of multiple rather than single factors, such as improper growth signals, antigrowth signals, apoptosis, limited replicative potential, continuous angiogenesis, tissue invasion, metastasis and cancer-related inflammation; the combination of such traits results in cells becoming cancerous [5,7]. Cancer prevention is an area where improved imaging, diagnostic and non-invasive techniques are implemented to identify neoplastic cells more precisely and to suggest treatment opportunities. For example, breast cancer can be easily detected by identifying small tumors in the dense breast tissue [10,11]. Over the past few decades, there has been significant progress in the diagnosis, prevention, treatment and management of cancer due to technological advances in imaging, gene sequencing and drug development; yet millions of people remain affected by this disease [10,12]. The systematic management of cancer includes surgery, radiation therapy, and chemotherapy, with the latter being most used. However, targeted therapies using specific ligands have become an essential component of cancer therapy to overcome the multidrug resistance that is often responsible for treatment failure [13–15]. The use of nanocarriers in cancer biology has motivated the development of site-specific targeted delivery systems that are highly efficient with minimum toxicity [16]. Nanotechnology-based drug discovery

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Nanotechnology is a multidisciplinary field of applied science for the design and application of nanometric (often 10- nm scale) functional structures [17] and has received increasing attention for use in cancer treatment. Significant research has focused on the development of biocompatible, biodegradable non-immunogenic NPs for safe and effective drug delivery [18]. Different natural or synthetic, animal-and plant-based polymers are used to encapsulate various drug moieties [19]. Nanoformulation allows slow release of a drug at therapeutic levels over an extended period of time, thus improving the efficacy of currently available drugs while reducing undesirable toxicity. Additionally, at the tumor site, NPs are able to deliver the drug closer to the intracellular site of action via endocytosis- and/or phagocytosis-mediated cellular internalization [20]. The carrier material can be specifically governed by adopting various nanotechnology approaches without altering the chemical composition of the drug [21]. In addition, nanosized carriers also enable the development of delivery vehicles for site-specific drug delivery, gene delivery, combined therapies or systems for simultaneous therapeutic, diagnostic, and monitoring applications, known collectively as ‘nanomedicine’ [22,23]. Nanocarriers used as pilot molecules to selectively deliver the drug to the intended site have been reported and are categorized as either endogenous or exogenous. A variety of site-specific and target oriented drug delivery systems have been developed and evaluated. Recently, the recognition of the potential of nanotechnology in cancer treatment was reflected by the US Food and Drug Administration (FDA) approval of first-generation nanocarriers for the therapeutic improvement of drugs (Table 1) [24]. Despite its advantages, there are also potential risks and challenges associated with this novel strategy. For instance, some cancer types have developed resistance to anticancer drugs. Nanocarrier might also alter the stability, solubility, and pharmacokinetic attributes of the agents they carry. The shelf life, aggregation, leakage, and toxicity of materials used to manufacture nanocarriers, as well as their cost and scaling-up, are other limitations to their use [25,26]. Targeted drug delivery

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Apart from the sustained and controlled delivery of drugs to the disease site, the delivery mechanism should also ensure that the cell membrane and intracellular biological events are affected by the drug in a specific and precise manner [27]. The activity of drugs often results from a concentration-dependent reversible interaction with specific active site(s) of the cell membrane (i.e., receptors) [27]. The transport of an exact amount of drug through suitable ligand-anchored drug delivery systems at the required site is essential for optimal therapeutic activity [28]. Drug targeting ensures drug accumulation specifically at the desired site (cell, tissue, or organ) at an optimal level, while circumventing neighboring organs or tissues. Thus, targeting can overcome the nonspecific toxic effects associated with conventional drug delivery systems [29]. Various types of targeting approaches are presented in Figure 1. Nanocarriers can stay in circulation for longer compared with free drug. Higher retention allows higher accumulation in disease sites, which is facilitated by abnormal and highly permeable vasculature associated with inflammation and tumor growth. This process is often referred to as the enhanced permeability and retention (EPR) effect, which improves the targeted drug delivery of ligand-modified drug nanocarriers. Drug transportation to the desired site by site-specific delivery system comprises the ability of tumor-specific genes, proteins, or drug molecules to selectively and quantitatively accumulate in the desired organ and/or area of

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the body, with minimum toxicity independent of the site and mode of intake using site-specific targeting moieties [30]. Drug molecules can biotransform and become inactive while passing through the various body compartments [30,31]. In controlled and sustained drug delivery systems, the characteristic features of targeting include: (i) the desired concentration of active molecule being available at the pharmacological site; (ii) interaction with target cells; and (iii) protection from the biochemical environment of the body, with minimum degradation and maximum availability at the site of action. The idea of active drug targeting was initiated following the observation by Paul Ehrlich, in 1902, who considered drug delivery as a ‘magic bullet’, comprising two components, with the first having the ability to recognize and bind with the target and the second being responsible for therapeutic activity over the desired site of action [32,33]. The modification of drug delivery systems at different levels can enable targeting to: (i) a discrete organ; (ii) a specific cell of the tissue and/or organ; or (iii) a specific intracellular compartment within the cell [28]. Drug allocation to nontarget organs and/or tissues is to be avoided and is a potential cause of tissue toxicity. Targeted drug delivery systems can deliver the active molecule to predetermined tissue and/or cellular compartments, with increased intrinsic activity while avoiding toxicity by minimizing drug distribution to nontarget or normal tissues and/or cells [28]. Different approaches have been adopted for targeting nanocarrier systems to specific pathological sites in the body. These include inverse targeting, dual targeting, double targeting, combo targeting, passive targeting, and active targeting (i.e., ligand-appended targeting and physical targeting) [28], as discussed below. Inverse targeting

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Based on efforts to avoid the passive uptake of colloidal drug delivery systems by obstructing the reticuloendothelial system (RES), the inverse targeting approach effectively leads to the inversion of the biodistribution pattern of the carrier, hence its name [34]. For this purpose, suppression of the activity of RES is achieved by treating with a naive nanocarrier system or other compatible macromolecules, such as dextran sulfate [35]. Physical targeting

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Locating the nanocarrier system or releasing the active component at a particular site can be programmed and examined at the external level with the help of physical means. Different drug delivery systems, such as liposomes, in situ gels, or magnetic nanocarriers, which are sensitive to temperature, pH, and/or physiological factors can be used for the sustained release of drugs at the relevant site(s) [36]. Dual targeting

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Dual targeting includes the combined activity of the carrier system with the encapsulated drug molecule. Lipids, such as ceramides and saturated fatty acids, which are derived from endogenous membranes, have their own intrinsic anticancer effect and have been found to modify the viability of tumor cells, thus synergizing with the anticancer property of the encapsulated drug [37,38]. Double targeting

Combination targeting

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Double targeting demonstrates the combined effect of spatial (specific organ) and temporal delivery (controlled rate of drug delivery) of a drug delivery system, which leads to the increased therapeutic index of the active molecules [39].

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The site-specific delivery of protein and peptides is achieved by targeting the carrier system in combination. The targeting system comprises a carrier, polymer, and homing device that are molecularly similar, and offers a straightforward approach to the target site. Nanocarriers have inherent properties to attain site-specific delivery by active targeting of the encapsulated drug, which can be used for the vectorization of modified proteins and peptides into vesicular or microparticulate carriers [40]. Passive targeting

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Passive targeting, also referred as the ‘enhanced permeation and retention (EPR) effect’, is an approach exclusively adopted by carrier systems for altering the physicochemical properties of the drug. Leaky blood capillaries result in the preferential accumulation of carriers in target tissue cells, while minimum lymphatic drainage ensures that the molecules are not removed efficiently, thus resulting in a reservoir in the tumor tissue [32,41]. The natural concentration gradient force is used in passive targeting for the biodistribution and accumulation of the drug delivery nanosystems in the target organ [42]. However, one of the key disadvantages of microparticulate carrier systems is that their rate of extravasation is dependent on the tumor microenvironment and the permeability of the vasculature. Active targeting

Kohler and Milstein first described active targeting in 1975 [43,44]. It involves redefining drug biotransformation by modification of drug carriers while maximizing the receptor-based localization of the drug by interactions of the drug nanocarrier with the receptor on target cells using specific ligands or modified analogous devices [45,46]. Much research and effort has gone into developing methods for the efficient delivery of active molecules to tumor

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cells through active targeting using ligands. Targeting ligands can be roughly categorized as proteins or peptides, antibodies and their fragments, nucleic acids, vitamins, and carbohydrates [47,48]. Cancer cells often overexpress tumor-associated as well as tumor-specific antigens. Active drug targeting involves nondestructive, chemical modification of a targeting carrier that strongly interacts with these overexpressed antigens (or receptors) in the target tissue. Targeting of, and accumulation in, the specific organ, tissue, and cells is known as first-, second- or third-order targeting, respectively. Along with decreasing the adverse effects of the toxic molecules by conveying the carrier to the specific site of action, the ligand-anchored drug nanocarrier also simplifies the process of cellular uptake by using the receptor as the mediator for endocytosis. As its name implies, active targeting is an active process that requires a significantly lower concentration gradient across the plasma membrane and some energy molecules for endocytosis across the cell membrane. Recently, researchers have shown interest in cell-penetrating peptides (CPPs) for targeting. CPPs are short translocation domain peptides (less than 40 amino acids) that translocate across cell membranes, facilitating the transport of associated nanocarriers by endocytosis [49,50]. However, some crucial factors with an important role in active targeting include: (i) the type of ligand and/or antibody attachment to the carrier; (ii) the quantitative availability of targeted receptor and/or antigen; and (iii) internalizing properties of the ligand [49]. In a nutshell, active targeting involves attaching ligands or a binding moiety that is structurally complementary to the receptor expressed on the tumor, leading to drug delivery to the specified organ or cell component.

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Receptors: the locks of cells

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Receptors are complex macromolecules comprising proteins, lipids, and carbohydrates that occur at the cellular or subcellular lipidic membrane [51]. Receptors can be extracellular (receptors expressed over the cell surface), intracellular (receptors present inside the cytoplasm on the membrane of various organelles), or in the nucleus (nuclear receptors), and are involved in interactions between cells and their extracellular milieu (cargo) to control the biochemical system at the cellular and/or tissue level [52]. Receptors have fundamental roles in various cellular functions, including mediating the trafficking of their ligands and transducing and regulating transmembrane signaling involved in growth, differentiation, metabolism, secretion, and migration. They are well known for their specificity, affinity, and reversibility of ligand binding [28]. These are same principles that apply to pharmacological drug targets, such as enzymes, transporters, and ion channels [51]. The classification of receptors is based on their molecular structure and transduction mechanism, as discussed below (Figure 2).

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Transmembrane ligand-gated and voltage-gated ion channels and ionotropic receptors

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Ligand-gated ion channels (LGICs) or ionotropic receptors are tetra- or pentameric structures that comprise a functional ion channel that is modulated by various ligand-binding cavities and has an endurance of only a few milliseconds. Such receptors are responsible for the regulation of the transport of various ions across membranes [53,54].

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G-protein-coupled receptors

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G-protein-coupled receptors (GPCRs), also known as metabotropic, seven-transmembrane or heptahelical receptors, contain a single α helical peptide represented by seven hydrophobic membrane-spanning amino acid helices. The nature of the ligand-binding site and interactions of a given receptor with G proteins across the membrane is determined by the composition and sequence of amino acids present in different loops [55]. The ligand-binding sites are located in the extracellular domain, whereas, intracellularly, these receptors are associated with a G protein, such as dopamine or metabotropic glutamate [56]. Enzyme-linked receptor tyrosine kinases

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Enzyme-linked receptor tyrosine kinases (RTKs) are protein-based receptors that extend over the membrane and can form dimers or multisubunit complexes. These receptors show inherent cytosolic enzymatic activities. They are associated with a single transmembrane α helix with an intracellular and extracellular domain; the external region contains the ligand-binding site [57]. Transcription factor receptors and nuclear hormone receptors

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Gene transcription is regulated by transcription factor receptors in the cytoplasm. They move towards the nucleus after binding with their complimentary moiety (ligand) and, thus, the latter must have sufficient lipid solubility to be able to move across the target cell membrane. Transcription factor receptors have three components: (i) C terminus; (ii) a core DNA-binding domain (responsible for recognizing the DNA sequences); and (iii) N terminus (responsible for interactions with other cellular transcription factors, so-called ‘cross-talk’). On the basis of these interactions, ligand–receptor binding can alter the activity of the receptor [58]. The responses of the receptor families described above are uneven or multidirectional because of the intensity of activation and cross-talk with other receptors. For example, cytokines can activate STATs, as can the GPCR for angiotensin II. GPCRs also modulate channel function, whereas some LGICs produce their effects in association with GPCR systems. Thus, the receptor motif remains the most critical element in assigning a receptor to a specific superfamily, whereas the related signal transduction mechanism is secondary [59]. Figure 2 depicts the various types of receptor present on the cell surface that could be explored for cell targeting. Ligands: keys to the cell

Ligands, endogenous signaling molecules or substances for receptors, act as homing devices for nanocarriers or drug molecules by producing a specific response in intracellular region (Figure 3) in the form of a conformational change following receptor binding [60]. Ligands are characterized by their affinity (strength of the attraction) and efficacy (capacity of a ligand to stimulate a biological response) [61,62]. Ligand–receptor interactions depend on charge, hydrophobicity, and molecular structure, which in turn are determined by intermolecular forces. Ligand– conformer interactions should be reversible, indicating the energetic nature of a chemical transmission process that reaches equilibrium when the ligand association rate and dissociation rate become equal [51]. Different covalent or noncovalent techniques are adopted to bind endogenous or exogenous ligands to the carrier system of the drug molecule. Drug delivery by active targeting has different patterns at different levels of targeting (i.e., first order, second order, and third order). To expedite the internalization of ligand-anchored drug nanocarriers, different cellular pathways have also been explored. Ligands provide simplicity to the recognition site over the cell membrane and specificity for the carrier and/or vector that helps in locating target selectively and delivering the drug to the desired site [28].

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Asialoglycoprotein receptors

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Similar to lipids and proteins, carbohydrates also act as building blocks of the cellular compartment and provide a novel means for potential site-specific, receptor-mediated targeted drug delivery via nanocarrier system [63][64]. Asialoglycoprotein (ASGP) receptors, which are present on various cell surfaces, can be exploited by nanocarrier systems for the site-specific delivery of therapeutic molecules, resulting in an improved therapeutic index of the active material for the treatment of diseases such as cancer [63]. In addition, site-specific drug delivery using engineered carrier systems, such as NPs, liposomes, and dendrimers, using carbohydrates as ligands could also provide several beneficial properties, including stealth characteristics, solubility, bioadhesive properties, biostability, and reduced toxicity [65,66]. Carbohydrate-based oligosaccharides and glycoconjugates can also be explored as potential drug delivery tools [63]. Thus, the use of carbohydrates as carriers as well as ligands could be useful in the development of safe, effective and stable formulations [63].

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Conjugation methods for galactosylation

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Several conjugation techniques and chemical reactions have been utilized for the derivatization of drug carrier surfaces with GAL ligands. Galactosylation of the drug delivery vehicle can be carried out by conjugating GAL directly to the carrier surface, which contains abundant surface amino groups. The conjugation method comprises opening of the D-GAL ring followed by a subsequent reaction of the aldehyde group of GAL (open chain) with the free amino groups on the surface of the nanocarrier. To tether GAL to carriers containing free amino groups on their surface (Figure 4), GAL is dissolved in buffer solution (sodium acetate buffer of pH 4.0) and stirred constantly for 4–6 h at room temperature for isomerization and ring opening. Nanocarriers comprising free amino groups are then incubated in the GAL solution for 2 days under constant stirring at ambient temperature. Reactions occur between the free aldehyde group of open-chain GAL and free amino functionalities expressed over the surface of the nanocarriers in sodium acetate buffer (pH 4.0). The above process leads to the formation of Schiff’s bases (–N=CH– ), which are reduced to secondary amines (–NH–CH2–) and reside in equilibrium with Schiff’s bases, thus achieving GAL conjugation. Garg et al. described a two-step synthesis of galactosylated liposomes, which comprised the synthesis of Opalmitoylgalactose and subsequent binding of the O-palmitoylated ester of GAL to the liposomes. Galactose was esterified with palmitoyl chloride in dimethylformamide and transformed into O-palmitoylgalactose, which was then incubated with liposomes comprising phosphatidylethanolamine to form GAL-tethered liposomes, exploiting the fact that O-palmitoylgalactose can bind with the phosphatidylethanolamine of liposomes [67]. Characteristic attributes of galactosylation in drug delivery

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The positive attributes of galactosylated carriers have made galactosylation a useful approach in modern drug delivery technologies. Galactosylated carriers confer a selective interaction of GAL with endogenous ASGP receptors. ASGP receptor-mediated endocytosis is based molecularly on GAL-binding proteins [68]. However, at the cellular level, the recognition of the glycosylated carrier is influenced mainly by the terminal carbohydrate moiety of the contributing oligosaccharide chain. Therefore, it is important to develop the carrier in such a way that the GAL units, which are required for recognition by ASGP receptors of hepatic cells, should be assimilated in the carrier and be accessible for binding with ASGP receptors [69]. The critical involvement of carbohydrate–protein affinities in various conditions, including cancer and microbial infections, is another feature that has highlighted galactosylated delivery systems as an emerging focus in biomedical research. The extent of participation of GAL in various intracellular events in different medical conditions, such as the accumulation of cancer cells, metastasis, viral agglutination, bacterial invasion, and adhesion of red blood cells, has also highlighted galactosylation as an effective paradigm in drug delivery approaches [70]. Given the involvement of GAL in numerous biological events and intercellular recognition, galactosylation is considered an intrinsic way to deliver carriers enclosing the therapeutic agent. The potential applications of galactosylated carriers for the delivery of therapeutic agents and other bioactives, and as a diagnostic tool, are discussed below. Therapeutic application of galactosylated nanocarriers

As discussed above, ASGP receptors expressed on the surface of cancer cells offer binding sites for the GAL moiety. ASGP receptors can recognize carriers with a GAL entity on their surface and internalize the carrier within the cell (Figure 5). This ligand–receptor-mediated strategy is the basis for targeted delivery of galactosylated carriers to different organs. The various biomedical applications of galactosylated carriers are summarized below. Micelles

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Micelles are colloidal, spherical, supramolecular nanostructures (10–100 nm) that generally result from the selfassembly of amphiphilic block copolymers in an aqueous milieu [71]. Over the past two decades, micelles have received significant interest from researchers owing to their multifunctional attributes, such as subtle body, potential to enclose both lipophilic and hydrophilic drugs effectively, and ability to deliver drugs to the desired site. Micelles are anticipated to improve the biopharmaceutical and pharmacokinetic characteristics of drugs and, therefore, increase their bioavailability and pharmacodynamic attributes [72]. Wang and associates successfully developed doxorubicin (DOX)-loaded GAL-engineered cross-linked micelles with ionic cores using block ionomer complexes of poly(ethylene glycol)-b-poly(2-acryloxyethyl-GAL)-b-poly(acrylic acid)/cystamine (PEG-b-PAEG-b-PAA-cl-micelles/Cys), with Cys being used as a biodegradable cross-linker [73]. Furthermore, the efficacy of PEG-b-PAEG-b-PAA-cl-micelles/Cys to deliver DOX into the cellular milieu and ligand–receptor-mediated interactions were investigated using confocal laser scanning microscope in NIH3T3 and HepG2 cells. HepG2 cells showing overexpression of ASGP receptors were the experimental group, whereas NIH3T3 cells with a minimal expression of the ASGP receptor were the control group. Following 15 min or 3 h of incubation of DOX-loaded PEG-b-PAEG-b-PAA-cl-micelles/Cys with HepG2 cells, the nucleus of these cells emitted strong fluorescence, whereas weaker fluorescence was observed in NIH3T3 cells, which were incubated with the DOX-loaded micelle formulation following 15 min or 3 h of incubation. A cytotoxicity study was also performed against these two cell lines following incubation with DOX-loaded PEG-b-PAEG-b-PAA-cl-micelles/Cys, for 24 h and 48 h. After 24 h of treatment at 0.1, 1, 10, and 100 mg/ml DOX concentrations, the percentage cell survival of HepG2 cells was 75%, 70%, 48%, and 37%, respectively. Under identical conditions, the percentage cell survival of NIH3T3 cells was greater than that observed for the treated HepG2 cells. The outcomes of this study suggest that PEG-b-PAEGb-PAA-cl-micelles/Cys had a selective affinity with HepG2 cells because of overexpressed ASGP receptor-mediated recognition and delivered DOX to the cell nuclei, thus augmenting the in vitro cytotoxicity of the encapsulated DOX [73]. Likewise, to target ASGP receptors on HepG2 cells, Wang and coworkers fabricated paclitaxel (PTX)-loaded amphiphilic diblock copolymers [poly(ɛ-caprolactone) and poly(ethyl ethylene phosphate)]-based reactive micelles functionalized with galactosamine [74]. Rhodamine 123 fluorescent dye-based flow cytometric analyses demonstrated that the cell surface attachment and cellular uptake of galactosamine-tethered micelles were remarkably enhanced as a result of the selective interaction of GAL ligands with the ASGP receptors on HepG2 cells. The outcomes of the cytotoxicity study illustrated the survival of approximately 12% of cells when incubated with galactosamine-functionalized PTX-loaded micelles (NP-Gal-PTX). By contrast, approximately 50% of cells remained viable when incubated with PTX-enclosed micelles without galactosamine conjugation (NP-PTX) at the same PTX dose. There was a distinct increase in the effectiveness of NP-Gal-PTX owing to the ASGP receptormediated cellular localization of NP-Gal-PTX by HepG2 cells. When incubated with NP-Gal-PTX, HepG2 cells were arrested in G2/M phase. At a PTX does of 1.2 mM, 67% of HepG2 cells were arrested in G2/M phase. By contrast, counts of cells arrested in G2/M phase were less in cells treated with NP-PTX. The outcomes of this study indicate that galactosamine-functionalized micelles selectively target HepG2 cells via the ASGP receptor, which could be exploited as a potential drug delivery vehicle for improved chemotherapy [74]. Li and associates developed GAL-functionalized self-assembled ribavirin-loaded micelles prepared with amphiphilic random copolymers by mingling enzymatic trans-esterification with radical polymerization [75]. The process conferred the appearance of a micelle-type structure of the amphiphilic copolymer, which was further validated by fluorescence probes using pyrene as a hydrophobic fluorescent marker. Following exposure to GALtethered micelles, MTT assays were performed using HepG2 liver carcinoma, AGS gastric cancer, and lung adenocarcinoma (SPC-A-1) cell lines. The outcomes of the study showed that GAL-anchored micelles exhibited effective toxicity selectively in HepG2 cells, possible due to the overexpression of the ASGP receptor on the surface of HepG2 cells. By contrast, AGS and SPC-A-1 cells, which did not overexpress ASGP receptors, did not exhibit a comparable growth-inhibitory effect. The improved cytotoxicity of micelles upon galactosylation indicates the involvement of ASGP receptor-mediated selective uptake of galactosylated-micelles in the cytotoxicity of tumor cells [75]. Feng et al. developed DOX-loaded nanomicelles of pH-triggered methoxyl PEG-b-poly(β-amino ester) polymers (MPEG-PBAE), which were further functionalized with GAL [N-(1-deoxylactitol-1-yl) dodecylamine (Gal-C12)] through noncovalent attachment [76]. To reach this goal, reductive amination of lactose in the presence of dodecylamine was carried out to synthesize Gal-C12, which was further purified by HPLC-MS. The binding affinity of galactosylated nanomicelles was evaluated against HepG2 cells by measuring the intracellular uptake of DOXenclosed MPEG-PBAE micelles with and without GAL modification using flow cytometry. The results illustrated that galactosylated micelles displayed improved cellular uptake compared with unconjugated micelles. It was further confirmed that the higher uptake of galactosylated micelles was due to the selective recognition of the Gal12 moiety of the ASGP receptor. Furthemore, 20 mM GAL supplied to HepG2 cells and incubated for 4 h followed

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by treatment with a galactosylated formulation for 4 h resulted in a lower uptake of Gal-12-tethered micelles; this was suggestive of a ASGP receptor-mediated endocytosis pathway that resulted in more micelles being localized to HepG2 cells. As determined by MTT assays, inhibition of cellular proliferation by GAL-modified micelles was significantly higher after 24 h compared with their unconjugated counterparts. In addition, the abundance of GAL diminished the growth inhibitory effect of ligand-coupled micelles, indicating that the endocytosis of Gal-12functionalized micelles was prevented when the ASGP receptors of HepG2 cells were saturated with additional GAL. Gal-12-coupled micelles induced cell cycle arrest in S phase at a higher level compared with DOX-loaded micelles without Gal-12 [76]. In another study, Zou and coworkers reported GAL-anchored photocrosslinked pH-sensitive degradable micelles loaded with PTX (Gal-PTX-CLMs), which were prepared by self-assembling and subsequent photocrosslinking of PEG-b-poly(mono-2,4,6-trimethoxy benzylidene-pentaerythritol carbonate-co-acryloyl carbonate) and Gal-PEGbpoly(ε-caprolactone) [77]. The results of cytotoxic experiments against HepG2 cells showed that increasing the content of GAL from 10% to 20% to 30% in Gal-PTX-CLMs reduced the cell viability from 53% to 47% to 37%, respectively. MTT assays in HepG2 cells also revealed that, with an increase in Gal from 10% to 20% to 30%, the half-maximal inhibitory concentration (IC50) of Gal-PTX-CLMs decreased from 11.7 to 2.9 to 1.1 mg/ml, which was less than that calculated for unconjugated PTX-CLMs (14.10 mg/ml), thus corroborating a receptor-mediated endocytosis mechanism. Pharmacokinetic studies reported the circulation half-lives of PTX following intravenous administration of Gal-PTX-CLMs, PTX-CLMs, and a clinically available PTX formulation, Taxol (10 mg PTX equiv/kg) in nude mice to be 3.86 h, 3.40 h ,and 0.18 h, respectively. Results of in vivo biodistribution studies in human hepatoma SMMC-7721 tumor-bearing nude mice treated with a galactosylated micelle formulation exhibited remarkably increased accumulation of the drug compared with their nontargeting PTX-CLM counterpart. Moreover, Gal-PTX-CLMs also resulted in improved antitumor activity compared with the other formulations tested. After 1 month, average tumor volumes of 35 mm3, 144 mm3, and 45 mm3 were measured in animals administered with Gal-PTX-CLMs, PTX-CLMs, and Taxol, respectively [77]. Nanoparticles

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NPs comprise either a polymeric matrix or a reservoir system in which an aqueous or oily core is enclosed by a thin polymeric shell [78]. Biodegradable polymeric nanocarriers can offer improved drug efficacy because of controlled and targeted delivery and fewer adverse effects. NPs encapsulating anticancer drugs can effectively increase the drug concentration in cancer tissues and/or cellular sites, thus improving the antitumor efficacy [79]. Cells can easily take up NPs by endocytosis and/or phagocytosis, resulting in higher accumulation of the encapsulated drug within the cells. Drug delivery to tumor cells is affected by different biological factors, organ- and/or tissue-related issues, carrier system, and the active molecule [80]. Particle size and surface characteristics, such as high curvature (resulting in a size <100 nm) and a hydrophilic surface, reduce the opsonization reactions, thus preventing their uptake by macrophages and leading to a change in the biodistribution pattern inside the body [81]. A study by Liang et al. described the formation of galactosamine-conjugated PTX-loaded self-assembled NPs (Gal-NPs) materialized with poly(ç-glutamic acid) and poly(lactide)-based block copolymers [82]. The targeting potential and growth-inhibiting efficacy of ligand-coupled polymeric NPs against HepG2 were compared with the performance of a clinically available formulation of the same chemotherapeutic agent (Phyxol). Here, rhodamine123-incorporated galactosamine-conjugated NPs resulted in increased fluorescence intensity in HepG2 cells after 30 min of incubation. Gal-NPs were first taken up by HepG2 cells via ASGP receptor- mediated recognition, followed by release of the enclosed PTX within the cytoplasm ,thus hindering cellular growth. Confocal laser scanning microscopy images of untreated HepG2 cells (control) illustrated normal nuclei, centrosomes, and microtubule networks, whereas PTX formulation-treated (Phyxol, NPs, or Gal-NPs) HepG2 cells demonstrated considerable disruption of the polar spindles and condensation of cytoplasmic microtubules, leading to cell toxicity. Additionally, HepG2 cells treated with Phyxol, NPs, or Gal-NPs showed arrested growth in G2/M phase, whereas untreated cells showed arrested growth in G0/G1 phase. However, the outcomes were less significant (P <0.05) in cells treated with NPs. This outcome might result from the active targeting ability of Gal-NPs stemming from the selective interaction of galactosamine with the hepatic tumor cells [82]. Han and coworkers proposed DOX-loaded self-assembled pH-responsive core-shell NPs (CSNPs) for the triplestage targeted delivery of DOX from the site of administration to the nuclei of affected cells [83]. The DOX-loaded cationic core comprised a TAT peptide and acid-cleavable PEG-based double modification of amino-functionalized mesoporous silica NPs. The anionic shell comprised charge-reversible GAL-functionalized poly(allylamine hydrochloride)-citraconic anhydride, which is used to target hepatocellular carcinomas (HCCs). CSNPs were assembled via electrostatic adsorption of the negatively charged shell onto the positively charged core. In vitro studies demonstrated the improved accumulation of CSNPs in tumors, while the PEG corona, involved in firststage targeting, efficiently diminished protein adsorption and phagocytic capture of CSNPs in the systemic circulation. In the mildly acidic microenvironment of the tumor, PEG was detached from the NPs because of acidic hydrolysis, leading to the exposure of GAL, which resulted in the internalization of CSNPs into HCC cells and initiated second-stage targeting. Subsequently, at the subcellular level, alteration of the charge of the anionic shell to positive occurred in response to the acidic environment of the endosomes and lysosomes (pH 5.0), leading to coreshell disassembly followed by endosomal and/or lysosomal escape and subsequent TAT-mediated delivery of DOX to the nuclei, for the third stage of targeting. The therapeutic effect of CSNPs was further tested on tumor-bearing

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mice. Intravenous administration of 2 mg/kg equivalent of DOX once every 4 days for four times showed a tumor inhibition ratio (TIR) of 43.8% for free DOX, whereas CSNPs resulted in an enriched inhibitory effect on the growth of tumors, with a TIR of 91.1% [83]. Jinxia and coworkers utilized cutting-edge polymer chemistry and co-assembly techniques for development of hierarchical polymeric NP loaded with DOX, and explored their use in cancer chemotherapy [84]. A zwitterionic nanosystem was designed based on the hierarchical self-assembly of zwitterionic poly (sulfobetaine methacrylate) blocks and a multivalent polymer fraction based on GAL. The cytotoxicity of DOX-loaded NPs was evaluated in NIH3T3 (noncancerous) and HepG2 (cancerous) cell lines using MTT=based cytotoxicity assays. DOX-loaded NPs exhibited remarkably increased inhibitory effects against HepG2 cells compared with the NIH3T3 cell line, indicating selectively targeted action. Here, obstruction of the nonspecific cellular uptake by the zwitterionic surface and acceleration of nanocarrier localization inside the HepG2 cells by GAL-triggered endocytosis were reported as two key attributes of the selectivity of the nanosystem. The selectivity of the NPs towards cancer cells was further confirmed by quantifying the accumulation of DOX-loaded NPs in HepG2 and NIH3T3 cells using confocal microscopy. Improved cellular accumulation of DOX-loaded NPs was observed in HepG2 cells, but hardly detected in NIH3T3 cells. The uptake of the NPs in HepG2 cells was reduced in the presence of free GAL, because the latter also has affinity for ASGP receptors expressed on the surface of HepG2 cells, thus competitively inhibiting the selective uptake of GAL-based NPs. Additionally, the NPs had a longer circulation profile (t1/2 = ∼14.4 h) and were expected to significantly promote drug accumulation in tumors and diminish any adverse effects of the drug [84]. Sangabathuni and coworkers studied the uptake behavior of glyco-gold NPs (G-AuNPs) with mannose and GAL modifications in different cancer cell lines [human cervical cancer cells (HeLa), HCC (HepG2) cells and human breast cancer (MDA-MB-231) cells] [85]. The results highlighted the advantages of various G-AuNP shapes in carbohydrate-mediated targeting approaches. Improved cytotoxicity and cellular uptake were seen with both mannose- and GAL-conjugated rod-shaped AuNPs because of the receptor-mediated interaction of NPs with the biological entity [85]. Zhu and associates demonstrated the targeting potential of galactose (GAL)- and PEG-co-grafted gold NPs (GALPEG-GNPs) for hepatocyte-specific ASGP receptor targeting [86]. An in vitro cell line study revealed that GALPEG-GNPs can be taken up effectively by HepG2 cells and can augment cancer cell cytotoxicity. Co-grafting with GAL and PEG increased the cellular uptake of AuNPs threefold, demonstrating that GAL can remarkably increase the cellular uptake of these NPs. Moreover, binding of GAL to internalizing epitopes, such as ASGP receptors, triggers prominent ASGP receptor-facilitated uptake. Additionally, the study demonstrated that the prepared AuNPs significantly modulated the irradiation response of HepG2 cells. GAL-PEG-GNPs demonstrated enhanced radio sensitization to HepG2cells. AuNPs also induced the generation of a huge amount of free radicals, leading to the activation of apoptotic genes involved in the sensitization mechanism of GAL-PEG-GNPs [86]. Solid lipid nanoparticles

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Solid lipid NPs (SLNs) are novel lipid-based nanocarriers that are more efficient than polymeric- and liposomebased nanocarriers [87]. SLNs are materialized with a solid-core lipid enclosed by a surfactant. They exhibit high biocompatibility with the ability to incorporate both hydrophilic and hydrophobic drugs, to protect sensitive drug molecules, and with controlled release characteristics [88]. Jain and coworkers reported the high sensitivity of GAL-decorated SLNs loaded with DOX towards human lung cancer cell lines (A549 cells) in terms of their subcellular localization, cellular toxicity, and nuclear trafficking [89]. Briefly, DOX-loaded SLNs were prepared using a solvent diffusion method involving extreme diffusion of the organic phase across the lipid phase into the adjacent aqueous phase and subsequent evaporation of the solvent resulting in the solidification of lipid particles. Furthermore, GAL conjugation was carried out by reaction of its aldehyde group with the abundant amino groups over the surface of DOX-loaded SLNs. The fabricated lipidic nanocarriers illustrated appreciable DOX entrapment. In an in vitro drug release study, the GAL-tethered SLNs demonstrated an initial burst of release followed by a sustained-release pattern. An in vitro study revealed the higher cytotoxicity of galactosylated SLNs against A549 cells, suggesting that the specific interaction between lectin receptors and GAL facilitated the cellular localization of SLNs. Results of in vivo experiments showed that the GAL-anchored DOX-loaded SLNs lengthened the circulation time and also paved the way for the selective and efficient delivery of DOX at the tumor site [89]. Liposomes

Liposomes are artificial vesicles comprising bilayers of amphiphilic lipids (i.e., phospholipids, cholesterol, and glycolipids) with a hydrophobic environment between the two membranes, and a hydrophilic inner chamber [90]. Thus, both lipophilic and hydrophilic drugs can be incorporated inside liposomes. Despite rapid degradation of liposomes in the stomach (low pH) and intestine (enzymes and bile salts) when given per-orally [91], they are regarded as an excellent delivery system for bioactives because of their biological characteristics. Owing to their reduced toxicity, biodegradability, biocompatibility, ability to entrap hydrophilic as well as hydrophobic drugs, and ease of targeted drug delivery, liposomes are widely used in academic research as well as in the pharmaceutical and cosmetic industries as a potential vehicle for targeted drug delivery [90,91]. Wang et al. described the development of PEG-galactosylated lipid-modified DOX-loaded liposomes [92]. The formulation was further evaluated for its liver-targeting potential. The prepared liposomes demonstrated exclusive

‘sustained targeting’ accompanied by slowed delivery of DOX to the liver, with reduced peak concentrations of the drug in the latter. The anticancer activity of PEG-galactosylated lipid-modified DOX was evaluated in HCC 22 (H22) tumor-bearing mice. The inhibition rate of PEG-galactosylated lipid-modified liposomes against H22 tumors was approximately 94%, which was greater than all the other tested formulations, including free DOX. Owing to the sustained uptake of PEG-galactosylated lipid-modified liposomes, the concentration of DOX in the liver escalated at a slow and steady pace, causing less damage to the liver and improving the therapeutic effectiveness of the drug against HCC [92]. Similarly, Liu and coworkers developed galactosylated liposomes loaded with norcantharidin (GAL-Lipo) to attain a hepatocyte-selective liposome system. An in vitro cell survival study illustrated that GAL-Lipo displayed enhanced cytotoxic activity (IC50 = ~25 Mmol l–1) on HepG2 cells compared with norcantharidin alone (100 Mmol l– 1) and unmodified liposomes (~40 Mmol l–1). GAL-Lipo was found to accumulate in HepG2 cells at an increased level, possibly because of clathrin-dependent ASGP receptor-mediated endocytosis and caveolin-dependent endocytosis, and inducing increased apoptosis in HepG2 cells [93].

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Dendrimers are globular, highly controlled, branched polymeric carrier systems prepared in a reiterative fashion and are monodispersive in nature, making them an effective delivery vehicle for drugs [65]. Dendrimers can also improve the characteristic attributes of drugs such as solubility and stability. Dendrimers could ultimately allow the development of a truly effective chemical moiety, unlike others that have eluded success in preclinical or clinical phases owing to suboptimal pharmacokinetic or biochemical properties [94]. Kesharwani and coworkers synthesized GAL-tethered dendrimers loaded with PTX and characterized their efficacy against HeLa and SiHa cell lines. A cell survival study demonstrated the significant cytotoxicity of GAL-anchored formulations in HeLa and SiHa cells [95]. Galactosylated carriers for the delivery of genetic material

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Galactosylated delivery systems have also been investigated for their potential significance in the transport of oligonucleotides, DNA, and genes specifically to cellular sites. They have also been studied for their ability to reduce the cellular toxicity and improve the biocompatibility of nonviral transfection agents [96]. As a result of recognition by the lectin receptor, followed by receptor-mediated endocytosis, galactosylated carriers have the ability to convey genetic material to a subcellular level [97]. Wang and coworkers fabricated GAL-based poly[6-O-methacryloyl-D-galactopyranose-co-2-(N,Ndimethylaminoethyl)methacrylate]-b-poly(pyridyl disulfide ethyl methyl acrylate) [P(MAGP-co-DMAEMA)-bPPDSMA], a redox-responsive amphiphilic diblock copolymer, to enhance the biodetection and efficacy of drug and gene delivery vehicles to target hepatomas [98]. The diblock copolymer had the ability to self-assemble into micelles and DOX was incorporated during micelle formation. Nile Red-loaded amphiphilic diblock micelles were investigated for an interaction between GAL functional groups and HepG2 cells under a fluorescence microscope. Remarkable fluorescence was recorded in HepG2 cells treated with GAL-based amphiphilic block copolymeric micelles. Ligand–receptor-mediated recognition followed by cellular internalization by HepG2 cells was the cause of this interaction [98]. Oh et al. reported a GAL-anchored liposome (Gal-DOX/siRNA-L) system for ASGP receptor-mediated selective codelivery of vimentin small interfering (si)RNA and DOX to HCC [99]. In this regard, GAL-linked cationic liposomes of DOX were developed. Furthermore, electrostatic interactions between the arginine amino groups of lipids and the phosphate group of vimentin siRNA led to the development of Gal-DOX/siRNA-L. Incubation of fluorescent dye-conjugated galactosylated liposomes with ASGP receptor-positive human HCC cells (Huh7) and ASGP receptor-negative human lung cancer cells (A549) demonstrated selective binding of galactosylated liposomes to Huh7 cells (ASGP receptor-positive), but not to A549 cells (ASGPR receptor negative). Additionally, the results of an in vitro cytotoxic study revealed that DOX-loaded galactosylated liposomes had significantly greater cytotoxic effects (IC50 = 0.2 MM) compared with free DOX (IC50 = 2.24 MM) in Huh7 cells. The results of a biodistribution study showed that Gal-DOX/siRNA-L delivered a greater amount of DOX to both normal liver and hepatic tumor tissue than DOX/siRNA-L (nontargeted liposomes) and free DOX. Overall, the results suggested that the targeting efficacy of GAL-tethered liposome was based upon the interaction of GAL with ASGP receptors [99].

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Diagnostic applications

Besides their therapeutic and biomedical applications, carbohydrate ligand-based delivery systems have potential usage as a tools for the diagnosis of various medical conditions. Galactosylated carriers have been widely utilized for in vitro organ imaging. Kikkeri and coworkers reported the potential application of the carbohydrate ligands Dgalactosamine, D-GAL, and D-mannose-coupled PEGylated quantum dots for in vitro imaging and in vivo targeting of the liver [100]. The photodynamic therapeutic efficacy of GAL-coupled carriers was also evaluated. For this purpose, a galactosyl-based amphiphilic copolymer and porphyrin- and GAL-coupled polymeric micelles enclosing monoaminoporphyrin incorporated poly(2-aminoethyl methacrylate)-polycaprolactone were fabricated and tested for possible biological activity in human laryngeal carcinoma (HEp2) and HepG2 cells. The prepared porphyrinand galactosyl-conjugated polymeric micelles demonstrated greater targeting and photodynamic therapeutic value in HepG2 cells compared with HEp2 cells [101]. The results of these studies underline the plausible use of galactosylated systems in photodynamic therapy.

Concluding remarks and prospects

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In drug delivery, the therapeutic index requires the accurate selection of a delivery system, and nanotechnology is proving to be a groundbreaking approach to the development of such systems. Over the past two decades, the use of nanotechnology-based drug delivery carriers has witnessed exponential growth, with sophisticated nanotechnologybased carriers enabling physical and chemical modifications for the conjugation of ligands over their surfaces for site-specific navigation. Carbohydrate ligands have provided opportunities to fabricate macromolecular constructs with uniquely tailored functions. Specifically, the galactosylation of macromolecular structures creates highly monodispersed systems, with effective control over the ultimate size and surface functionality. The anchoring of GAL over the façade of nanocarriers has paved the way for the efficient targeting of therapeutic agents to cancer cells both in vitro and in vivo. Collectively, passive targeting and this active targeting approach are a promising strategy for conveying anticancer agents to carcinoma cells overexpressing the ASGP receptor. This tactic could aid the delivery of diagnostic agents that are sensitive to distinct signals that are produced either outside or inside the tumor microenvironment. Although galactosylated nanocarriers have opened a new avenue in the field of drug delivery, many questions remain. Increased interest among formulation scientists is warranted to further investigate the potential of these techniques. For instance, there might be emerging opportunities for the expansion and application of galactosylated nanocarriers by using metallic nanostructures and carbon materials [102,103]. Carbon nanotubes, metallic NPs, and metallic organic frameworks are future nanocarriers that could be exploited for glycan functionalization to deliver bioactives for cancer treatment. Moreover, as described above, a variety of galactosylated carriers, such as polymeric micelle, NPs, liposomes, dendrimers, and so on, have potential applications as multifunctional delivery systems for chemotherapeutics, genes, and diagnostic agents. However, galactosylation-based delivery approaches demand more intensive investigation and validation before they can be effectively used in the clinic.

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Acknowledgments The authors are grateful for the fellowship provided by the ICAR, New Delhi, India and UGC, New Delhi, India; and the Jaswant Singh Gill Pharma Research Fellowship.

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(2013) Galactose-based amphiphilic block copolymers: synthesis, micellization, and bioapplication. Biomacromolecules 14, 1444–1451 99 Oh. H.R. et al. (2016) Galactosylated liposomes for targeted co-delivery of doxorubicin / vimentin siRNA to hepatocellular carcinoma. Nanomaterials 6, 141 100 Kikkeri, R. et al. (2009) In vitro imaging and in vivo liver targeting with carbohydrate capped quantum dots. J. Am. Chem. Soc. 131, 2110– 2112

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101 Wu, D.Q. et al. (2010) Porphyrin and galactosyl conjugated micelles for targeting photodynamic therapy. Pharm. Res. 27, 187–199 102 Kesharwani, P. et al. (2015) Validating the anticancer potential of carbon nanotube-based therapeutics through cell line testing. Drug Discov. Today 20, 1049–1060 103 Beg, S.et al. (2017) Nanoporous metal organic frameworks as hybrid polymer–metal composites for drug delivery and biomedical applications. Drug Discov. Today 22, 625–637

Biographies Ashay Jain

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Ashay Jain works in the drug delivery research group at the University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India. He is currently pursuing a PhD degree in pharmaceutical sciences under the mentorship of O.P. Katare. He has also served as lecturer of pharmaceutics in Bhagyoday Tirth Pharmacy College (India) and coordinated academic activities for Bachelor in pharmacy students. His current research interests encompass the development of nanostructured drug delivery systems for cancer with a focus on lipid and polymerbased drug delivery systems. He has co-authored more than ten publications in various international journals.

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Atul Jain

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Atul Jain earned his MSc in pharmaceutics in 2011 at Dr Hari Singh Gour Central University, Sagar University, India, and received a UGC Graduate Research Fellowship. Currently, he is working as a senior research fellow at the UGC Centre of Excellence in Applications of Nanomaterials, Nanoparticles & Nanocomposites, Panjab University. His current research interests include surface engineering, conjugation, and novel controlled and sustained drug delivery systems.

Prashant Kesharwani

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Prashant Kesharwani is currently a Ramanujan Fellow at Central Drug Research Institute (CDRI), Lucknow, India. He received his PhD in Pharmaceutical Sciences from the Dr Hari Singh Gour University in the group of N.K Jain. He is a recipient of several internationally acclaimed awards, including a Ramanujan Fellowship, DST, India 2017, Excellence Research Award 2014, Young Innovator Award (Gold medal) 2012, International Travel Award/Grant from DST (New Delhi), and INSA (CCSTDS, Chennai) 2012. He has received a ICMR Senior Research Fellowship (for his PhD) and AICTE Junior Research Fellowship (for his M. Pharm.). After his doctorate, he worked as a postdoctoral fellow in Wayne State University in Detroit (Michigan, USA). Dr Kesharwani subsequently joined the School of Pharmacy, International Medical University (Malaysia) as a lecturer in pharmaceutical technology. An overarching goal of his current research is the development of nano-engineered drug delivery systems for cancer with a focus on dendrimer-mediated drug delivery systems. Dr Kesharwani is a coauthor on more than 85 publications in international journals and two books.

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Figure 1. Different types and order of drug targeting.

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Figure 2. Examples of different types of receptor involved in cell functioning.

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Figure 3. Schematic representation of receptor-mediated cellular responses.

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Figure 4. Schematic representation of nanocarrier galactosylation.

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Figure 5. Schematic representation of asialoglycoprotein (ASGP) receptor-mediated delivery of galactosylated carriers associated with the recognition of the galactosylated carrier by the ASGP receptor-mediated endocytosis, early endosome, endosomal disruption and release of payloads into cytoplasm.

Table 1. FDA-approved nanomedicines for cancer Drug product

Active component

Formulation

Indication

Manufacturer

Oncaspar Doxil (Caelyx)

Asparaginase Doxorubicin

Pegylated asparaginase Pegylated doxorubicin

Enzon Orthobiotech, Schering-Plough

DaunoXome DepoCyt Myocet

Daunorubicin Cytarabine Doxorubicin

Liposomal daunorubicin Liposomes encapsulated cytarabine Liposome encapsulated doxorubicin

Acute lymphocytic leukemia Breast cancer, ovarian cancer HIV-related Kaposi sarcoma Lymphomatous meningitis Breast cancer

Abraxane

Paclitaxel nanospheres

Genexol-PM

Nab-paclitaxel (albumin– bound paclitaxel) + gemcitabine Paclitaxel-

Ontak

Denileukin diftitox

Marqibo

Vincristine sulfate

Albumin-bound paclitaxel nanospheres (FDA approval: Jan. 2005) Nab paclitaxel in combination with gemcitabine (FDA approval: September 2013) Paclitaxel-encapsulated polymeric micelle Recombinant DNA-derived cytotoxic protein Liposome injection

Various cancers

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Metastatic pancreatic cancer Celgene

Breast cancer, non-small cell Samyang lung cancer, ovarian cancer Cutaneous T cell lymphoma Seragen, Inc

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Philadelphia chromosomenegative lymphoblastic leukemia

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Gilead Science Skye Pharma, Enzon Elan Pharmaceuticals, Sopherion Therapeutics Abraxis Bioscience, AstraZeneca

Talon Therapeutics