Advances in liposomal drug delivery to cancer: An overview

Journal of Drug Delivery Science and Technology 56 (2020) 101549

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Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst

Advances in liposomal drug delivery to cancer: An overview a

Shivani Saraf , Ankit Jain Sanjay K. Jaina,∗ a b

a,b

a

a

T a

, Ankita Tiwari , Amit Verma , Pritish Kumar Panda ,

Pharmaceutics Research Projects Laboratory, Department of Pharmaceutical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar, M.P., 470 003, India Department of Materials Engineering, Indian Institute of Science, Bangalore, 560012, Karnataka, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Liposomes Targeted drug delivery Stimuli Ligand Theranostic application

Liposomes are biodegradable and biocompatible lipid bilayer vesicles which are widely exploited as preferred carriers for smart delivery of both hydrophobic as well as hydrophilic bioactives. Structural fabrication of liposomes for ligand anchoring, long-circulation and stimuli-responsiveness are advancing features to meet the needs of clinical and industrial demands. Recent studies report newer developments in multipronged liposomes for synchronized theranostic manifestations in cancer treatment. This review gives an insight to advances in ligand targeted liposomes (like folate, mannose, transferrin, hyaluronic acid, antibody, aptamer, and peptide, etc.), stimuli-triggered liposomes (stimuli such as pH, temperature, and hypoxia, etc.) and liposomes mediated autophagy modulation, and theranostic liposomes for the diagnosis and treatment of cancer. It also includes patents, clinical studies and marketed liposomal products. This assemblage of advances would be of great interest to budding scientists and pharmaceutical companies engaged in the development of liposomes.

1. Introduction Liposomes are bilayer vesicles made by phospholipids enclosing hydrophilic core [1,2]. These have been extensively investigated as a carrier of choice for the delivery of therapeutic agents from last few decades as they are suitable to entrap a category of drugs which may be hydrophilic as well as lipophilic in nature [3–5]. Liposomal formulations have shown promising results in drug delivery. Number of approved liposomes for the treatment of cancer include Onivyde™, Marqibo®, Doxil®, Visudyne® and Depocyt®. There are numerous advantages of liposomes like cell-like membrane structure, biocompatibility, low immunogenicity, protection of the active groups, enhancement of half-life, safety and efficacy [6]. However, these conventional liposomes demonstrate some limitations such as low entrapment and higher escaping tendency of hydrophilic and amphiphilic drugs from liposomal vesicles, and accelerated blood clearance (ABC) by the reticulo-endothelial system (RES). Opsonins recognize liposomes as a foreign material leading to uptake by macrophages of mononuclear phagocyte system (MPS) and RES. The physicochemical properties of liposomes such as net surface charge, size, hydrophobicity and hydrophilicity play an important role in their clearance from the systemic circulation/body. Surface hydophobicity and size (greater than 200 nm) promote opsonization and RES uptake (Bar-David et al., 2019).

Sterically stabilized liposomes (PEGylated or coated with other hydrophilic polymers) have been developed to overcome the problem of RES uptake. PEGylation is the most common approach used for the development of long circulatory liposomes. Moreover, some other PEG alternatives like sialic acid, polyvinyl alcohol, and poly-N-vinylpyrrolidones have also been utilized for the same purpose. These alternatives are found to improve the blood circulation time but lack the selectivity for target site [7]. Therefore, targeted liposomes were then developed for imparting the selective delivery of drug to the desired site. Various ligands such as transferrin, mannose, folate, peptide, asialoglycoprotein, and antibody are being utilized in targeting of liposomes [8,9]. Recently, features of tumor microenvironment (like acidic pH, slightly elevated temperature, and hypoxia) have also been exploited using stimuli triggered drug delivery systems to deliver payloads in tumor tissues [10–14]. Stimuli responsive materials are employed to alter their features in response to fluctuations in the environment. They can specifically release the drug in the presence of stimuli (internal or external) [15,16]. Advances in nanotechnology have offered an opportunity to develop more specific, individualized therapies for cancer [17]. The theranostic system holding both diagnostic moiety and therapeutic agent in a single liposomal system can monitor the drug localization at a particular site, visualize its bio-distribution and evaluate the therapeutic efficacy [18].

∗ Corresponding author. Pharmaceutics Research Projects Laboratory, Head, Department of Pharmaceutical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar, M.P., 470 003, India. E-mail address: [email protected] (S.K. Jain).

https://doi.org/10.1016/j.jddst.2020.101549 Received 3 September 2019; Received in revised form 15 November 2019; Accepted 24 January 2020 Available online 25 January 2020 1773-2247/ © 2020 Published by Elsevier B.V.

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Abbreviations RES ABC SA PSA GPCRs LTLS FR Tf TfR BBB MDR

TET VC TPGS

Reticulo-endothelial system Accelerated blood clearance Sialic acid Poly sialic acid G-protein coupled receptors Ligand targeted liposomes Folate receptors Transferrin Transferrin receptors Blood brain barrier Multi-drug resistance

QDs DOX DHA HA HCQ Cur PEG apt RGD

Tetrandrine Vincristine D-alpha-tocopheryl polyethylene glycol-1000 succinate mono-ester Quantum dots Doxorubicin Dihydroartemisinin Hyaluronic acid Hydroxychloroquine -Curcumin Polyethylene glycol Aptamer (apt) Arginylglycylaspartic acid

prevents the liposomal blood clearance by avoiding the recognition by opsonins [20,21]. However, the integrity of pegylation was reduced upon systemic injection which may affect the long circulatory behavior. Novel PEG-dendron-phospholipid constructs were developed for the production of super stealth liposomes. PEG chains were attached with distearoyl phosphoethanolamine lipids using a β-glutamic acid dendron. These super stealth liposomes showed good stability, increased the blood circulation time, improved the bio-distribution, and enhanced the antitumor efficacy [22,23]. The long-circulating liposomes containing PEG-derivatized phospholipids are found to preferentially localize at the tumor site due to enhanced permeability and retention (EPR) effect [24]. Wolfram et al. (2014) investigated the stability of PEGylated and non PEGylated liposomes in serum (in terms of homogeneity, size, and zeta potential). The size and homogeneity of liposomal formulations (PEGylated and non-PEGylated) were reduced in the presence of serum. The zeta potential of pegylated liposomes was constant, whereas non pegylated liposomes showed decrease in zeta potential. All formulations displayed the stability in serum up to 12 days [25]. However, there are some pitfalls of PEGylation:

In this review, the application potential of various types of ligands (such as transferrin, mannose, folate, peptide, asialoglycoprotein, antibody, and aptamer) appended liposomes and stimuli-responsive liposomes (stimuli such as pH, temperature and hypoxia responsive) have been exhaustively discussed. Research on these liposomes for cancer treatment has been expanding every year as observed in the chronological increasing publications shown in Fig. 1. 2. Types of liposomes 2.1. Long circulatory liposomes The major problem in employing liposomes as potential drug delivery system is their ABC and RES uptake. The opsonins present in blood are responsible for rapid opsonization of liposomes. Many attempts have been made to escape the RES-uptake of liposomes and to enhance the circulation time by changing the size or modifying the surface of liposomes. The second generation liposomes are a type of modified liposomes in which their surface is modified using glycoprotein, oligosaccharides, polysaccharides and synthetic polymers for enhancing its circulation time [3,19].

• Cellular uptake blocked phenomenon: The “cloud” of hydrophilic •

2.1.1. PEGylation Opsonins proteins recognize liposomes as foreign particles leading to uptake by macrophages in the MPS and RES. Various techniques have been employed to attain prolonged blood circulation of liposomes such as coating the liposomal surface with PEG which is an inert and biocompatible polymer. PEG forms a coat over the liposomes and

steric barrier increases the blood circulation time of liposomes but it hampers their affinity/interaction towards targeted cells [26]. ABC phenomenon: Repeated dosing of PEGylated liposomes through parenteral route negates the prolonged residence property referred to as the “ABC phenomenon”. Since PEGylated carriers generate anti-PEG IgM antibody and consequently increase the systemic elimination of these carrier systems owing to complement

Fig. 1. Chronological increment in scientific interest on liposomes for cancer treatment (Publications and Patents) (https://www.ncbi.nlm.nih.gov/pubmed/?term= liposomes+cancer; https://patents.google.com/?q=liposomes&q=cancer&oq=liposomes+cancer; https://clinicaltrials.gov/). 2

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because of the enhanced intracellular uptake by receptor-mediated endocytosis [45]. In another study, pH-sensitive PEGylated liposomes (pPSL) were formulated and evaluated for cancer targeted intracellular administration of SN25860, a dinitrobenzamide mustard prodrug which is activated by the E. Coli nitro-reductase nfB. pPSL displayed 21–24 fold higher anti-proliferative potency in comparison to non pHsensitive PSL and free drug, respectively. Cells which were treated with pPSL showed 1.6–2.5 times more intracellular accumulation in comparison to non pH-sensitive PSL [46]. Zhang et al. (2016) developed dual-functional paclitaxel-encapsulated liposomes which were surface modified with arginylglycylaspartic acid (RGD) and a pH-sensitive antimicrobial peptide [D]-H6L9 for efficient tumor targeting. These liposomes enhanced the cellular toxicity against C26 cells in comparison to the liposomes functionalized with RGD or [D]-H6L9, and a noteworthy tumor suppression was also observed in C26 tumor models [47].

activation [27].

• Metabolic byproduct: PEG shows a complex degradation inside the •

body and accumulates within the lysosomes, which is responsible for various untoward effects [28]. The molar ratio of DSPE-PEG2000: The ratio of DSPE-PEG2000 and lipids also influences the properties of stealth liposomes. The molar ratio 0.1:1 (DSPE-PEG2000: phospholipids) forms the stable stealth liposomes due to self-assemblage of DSPE-PEG2000 in the phospholipid bilayers. Higher molar ratio has been found to hinder the inclusion of DSPE-PEG2000 in pre-formed liposomes. Moreover, micelles are formed at higher molar ratio eliciting the de-PEGylation of the stealth liposomes [29].

2.1.2. Alternative long circulatory polymers Similar to polyethylene glycol, PSA (poly sialic acid) modified system also forms a “water cloudy” barrier, due to its high hydrophilicity and chain flexibility. These modifications are reported to avoid the RES uptake of carrier by eliminating the binding of plasma proteins and extend the blood circulation time. Sialic acid (SA)/PSA has been exploited to overcome the problems of PEGylation [30]. SA serves the dual purpose as it acts an endogenous substance can preferentially interact with selectin receptors that are over-expressed on tumor and endothelial cell surface, and it also works as a PEG alternative [31–33]. SA modified carrier can deliver chemotherapeutic agent to these cells selectively via receptor-mediated endocytosis, and thus avoids the issue of cellular uptake blocked phenomenon [34]. The SA and PSA are metabolized into CO2 and water that are not harmful and can be easily eliminated from the body. Therefore, SA/PSA can be a better alternative to PEGylation for improving the circulation half-life and overcoming the problem of ABC [35]. Recent studies have reported the use of other alternatives such as poly [N-(2-hydroxypropyl) methacrylamide)] [36], polyvinyl alcohol [37], L-amino-acid-based biodegradable polymer–lipid conjugates [38], and poly-N-vinylpyrrolidones polymers in the preparation of long-circulating liposomes [39].

2.2.2. Temperature sensitive liposomes Temperature is used as a trigger to release drug from temperaturesensitive liposomes (TSL). Local hyperthermia is found to enhance the permeability of tumor cells and hence the drug/drug delivery system gets accumulated more inside the tumor. TSL can be divided into two categories: traditional TSL containing the temperature sensitive materials and liposomes modified with temperature-sensitive polymers. These liposomal systems exhibit a dramatic increase in permeation of the lipid membrane at its gel-to-sol crystalline phase transition temperature. These systems remain intact and stable at normal physiological condition (37 °C) but display a temperature triggered drug delivery above 37 °C under an influence of internal or external stimulus [48]. TSL are the nanocarriers which rapidly release the entrapped drug at hyperthermic temperature, typically above ~40 °C. Zhang et al. (2014) developed DOX loaded thermosensitive liposomes for the targeted delivery of drug to the tumor. In vitro drug release profile depicted that the rate of DOX release from the thermosensitive liposomes was high at 42 °C as compared to 37 °C, which reflected a temperature triggered drug release from the liposomes. Tumor growth suppression study was performed in xeno-transplanted tumor model of human breast cancer in nude mice. Docetaxel-encapsulated liposomes-treated group (10.0 mg/kg) displayed a comparatively lesser tumor volume (61.99 ± 26.88 mm3) and tumor weight (0.07 ± 0.03 g), and a higher tumor suppression rate (94.3%) compared to the free drugtreated group. Suppression rate of docetaxel-encapsulated liposomestreated group was around 1.3 folds higher than that of free drug-treated group [35]. Recently, gold nanorod mediated mild hyperthermia (MHT) was used for improving the delivery of gemcitabine. Gemcitabine loaded liposomes were prepared for the treatment of pancreatic cancer. Pancreatic cancer CAPAN-1 tumor model was used for the determination of antitumor efficacy. The accumulation of liposomes in pancreatic cancer after i.v. infusion was enhanced by three folds after MHT therapy in comparison to infusion without MHT. MHT mediated liposomes improved the transport and antitumor efficiency of gemcitabine and tumor size was significantly reduced [49].

2.2. Stimuli-sensitive liposomes The complex microenvironment of tumor plays a crucial role in occurrence of drug resistance [40]. Therefore, tumor microenvironment responsive strategies can be exploited to target solid tumor. Stimulisensitive system can trigger the release of drug as a result of destabilization mechanism caused by an external or internal stimuli such as pH, temperature, redox potential, enzymes, and electrolyte concentration [11,41,42]. 2.2.1. pH-sensitive liposomes The concept of pH-sensitive carriers was introduced in 1980 [43]. Later on, it was found that the pH of tumor microenvironment is different from the pH of normal cells. Therefore, the pH-sensitive liposomal formulation can be a promising approach for improving the drug accumulation at the tumor site (intracellular or extracellular). The ligand coupled pH-sensitive system can further improve the targeting ability and enhance the therapeutic effects [43]. The pH-sensitive liposomes can remain stable at the physiological pH but after reaching the tumor they display a pH-triggered drug release by destabilization of lipid bilayer [44]. pH-sensitive liposomes are employed as a substitute to conventional liposomes for effective delivery of bioactive/antigen/ DNA to the target cell. These liposomes undergo destabilization at the acidic pH of the endocytotic vesicle as they are comprised of pH-sensitive lipid components. Thus, the encapsulated moiety is internalized into the cell by destabilization or it fuses with the endosomal membrane [43]. Hyaluronic acid targeted pH-responsive stealth liposomes (SL-pHHA) were developed and these liposomes demonstrated a greater efficacy as compared to free DOX towards CD44 receptor over-expressing cells and relatively lesser toxicity to non-cancerous cells. SL-pH-HA were observed to be more efficacious than non-targeted formulation

2.2.3. Dual pH and temperature sensitive liposomes Methoxy diethylene glycol methacrylate, methacrylic acid, and lauroxy tetraethylene glycol methacrylate were copolymerized to synthesize dual-signal-sensitive copolymers, which rendered temperature sensitivity and pH sensitivity. This copolymer was anchored on the surface of liposomes. These water soluble copolymers with varying solubility were hydrophilic in nature at neutral pH and low temperature; merely they turned into hydrophobic aggregates in acidic pH and high-temperature. Modification of liposomes with these copolymers showed an increased drug release at weakly acidic pH with rising temperature. However, no temperature-dependent release was observed under neutral conditions. Interactions between copolymermodified liposomes and HeLa cells showed that the copolymer functionalized liposomes depicted a rapid and effective adsorption onto the 3

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3.1.2. Selection of the targeting ligands Numerous targeting ligands such as monoclonal antibody (mAb) or fragments of antibody, natural or synthetic ligands like carbohydrates, glycoproteins, and peptides or receptor ligands can be used in LTLs. Targeted liposomal delivery of anticancer drugs offers several advantages i.e. higher drug: carrier ratio as compared to antibody:drug or antibody-toxin conjugates. The scFv (single-chain fragment variable) fragments are one of the targeting ligands used in LTLs. These systems are found to increase the binding avidity and display a high affinity towards the receptors [55,56]. Many other ligands like hyaluronic acid, folic acid, and transferrin are also reported with LTLs. The selection of appropriate ligand depends on the expression of receptors in a particular type of cancer.

cell surface and a gradual internalization potential via endocytosis as compared to the unmodified liposomes [50]. 2.2.4. Hypoxia responsive liposomes Hypoxic cells in tumor are deprived of blood circulation and deeply embedded inside the tumor cells leading to hampered drug delivery. The conventional therapies such as radiotherapy, chemotherapy, and photodynamic therapy face the problem of resistance due to hypoxic condition of tumor. Thus, recent studies report that the hypoxia responsive liposomes may improve the delivery of current therapeutics agents [51]. Hypoxia-responsive liposomes were prepared by incorporating nitroimidazole derivative into the membrane of liposomes. The nitroimidazole derivative showed a reductive metabolism in hypoxic condition of tumor and depicted a triggered drug release due to liposomal degradation. These drug delivery carriers release the drug (DOX) in an oxygen-dependent manner which was also confirmed by confocal laser scanning microscope and near-infrared imaging. The hypoxia responsive liposomes demonstrated a selective cytotoxicity in hypoxic tumor cells. These outcomes suggested that hypoxia responsive liposomes can be a promising strategy for the treatment of cancer [52].

3.1.3. Selection of the therapeutic agents The selection of the drug/active pharmaceutical ingredient is important in designing of LTLs. Selection of a drug is based on physiochemical and pharmacological properties, and intended purpose of the delivery. Potent therapeutic agents can be efficiently delivered through LTLs. The lipid: drug ratio may be maintained at an optimum level so that, the patient receives a small amount of lipid and the harmful reactions can be avoided. Ideally, drug release in sustained manner is better for optimum bioavailability in general ailments however if drug is released before reaching the target then the availability of drug is decreased at the target site [57].

3. Targeted liposomes The targeted liposomes are the third generation liposomes which are prepared by modifying their surface using suitable ligands (Fig. 2). The liposomal systems can be exploited for active and passive targeting. Liposomal formulations in case of passive targeting show a higher localization of drug inside the tumor cells due to EPR effect. Apart from the passive targeting, the third generation liposomes also have the active and physicochemical targeting potential. Functionalized liposomes can deliver the drug to a specific site like cell organelles due to selective affinity for a particular receptor using different types of targeting ligands such as peptides, and antibody [3,53].

3.1.4. Selection of target tissues or cells LTLs may efficiently deliver the drug to the target when the target sites are located in the systemic circulation or are easily reachable from the vasculature. Solid tumors having the angiogenic vasculature are readily accessible target for LTLs. Numerous category of molecules, including proteins e.g., integrins, are expressed in vascular endothelial cells, and could possibly be selected as targets to provide a new insight for the development of LTLs for solid tumor [57].

3.1. General principles of ligand targeted liposomes (LTLS) 3.2. Ligand targeted liposomes (LTLS) 3.1.1. Selection of target receptors Cancer cells over-express various kinds of receptors. Selection of appropriate targeting receptor is a crucial step in designing of LTLs. The most appropriate receptors are those which are expressed stably and homogeneously in cancer and have non-significant level of expression in normal cells. Moreover, some antigens are also present in cancer and these may be helpful in targeting of tumor cells [54].

The conventional liposomal systems have some limitations such as RES uptake, opsonization, and immunogenicity. To overcome these limitations ligand targeted liposomes were developed. The LTLs selectively delivered the drug to the target site and improved the anticancer activity. LTLs also offer many other advantages like increased cellular uptake, improved biodistribution and cell internalization.

Fig. 2. Plausible modification potential of liposomes for targeting, diagnosis or theranostics. 4

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dual-functional liposomes, along with a pH-sensitive anti-microbial peptide [D]-H6L9 and RGD were developed for tumor targeting. Integrin αvβ3 are receptors are commonly expressed in many tumors, and could be targeted by ligands like cyclic RGD. RGD-conjugated delivery could be an excellent approach to target tumors with great efficiency. TAT is a cell penetrating peptide and found to improve photodynamic therapy in Hela tumor-bearing mice model. Before entering tumor cells, histidine-rich peptide [D]-H6L9 was deactivated in systemic circulation as well as in healthy tissues at pH 7.4. On arriving tumor cells, RGD recognized tumor cells (C26 cells) which expressed or overexpressed integrin αvβ3 receptors. After the cell penetration, [D]-H6L9 triggered the drug release selectively in acidic endo-lysosomal compartment (pH 6.3) [47]. Suga et al. (2017) synthesized the ligand peptide-lipid derivatives with serine-glycine units (SG)n to improve the targeting efficiency of ligand peptide-grafted PEGylated liposomes. A ligand peptide KCCYSL (KCC) was chosen to bind with human epidermal growth factor receptor-2 (HER2/EGFR2). KCC-(SG)n-lipid derivatives were developed (where, n = 3, 5, 7) and their cellular response was assessed in breast cancer (BC) cells. KCC-(SG)n/PEGylated liposomes showed an improved cellular response in HER2-positive breast cancer cells [71].

3.2.1. Folate-mediated targeting Folate receptors (FR) are commonly found to over-express in tumor cells and hence, folate mediated targeting is one of the potential strategies for tumor targeting. Previously, it has been investigated that FRmediated endocytosis can deliver macromolecules and liposomes inside the cell, and therefore, folate-targeted liposomal drug delivery has grabbed the attention of the researchers [58–61]. Daunorubicin and doxorubicin (DOX) loaded liposomes have been developed for targeting tumor cells through FR for better cytotoxicity. Recently, folate-conjugated DOX-encapsulated liposomes were employed for the therapy of acute myelogenous leukemia. Folate-targeted liposomes have also been employed as a tool for boron neutron capture therapy and for immunotherapy with haptens [20]. Supramolecular vesicular aggregates (SVAs) were prepared by self-assembling of liposomes and polyasparthydrazide co-polymers conjugated to folic acid for the active targeting of solid tumor. The antitumor efficacy was evaluated in breast cancer model (NOD-SCID mice bearing MCF-7 human xenograft). The folate modified system reduced the tumor volume significantly as compared to PEGylated liposomes and free gemcitabine [62,63]. 3.2.2. Transferrin mediated targeting Transferrin receptors (TfR) are over expressed on the surface of cancer cells and these are used for targeting liposomes to tumor cells [64]. Glioma is one of the commonest tumors, and the treatment outcomes are not satisfactory since the complications arise due to the complex blood brain barrier (BBB), the multidrug resistance (MDR) and invasiveness of the cancer cells and also due to the vasculogenic mimicry (VM). To meet the above challenges, vincristine (VC) and tetrandrine (TET) liposomes were prepared and modified with transferrin (Tf) to target TfR which was over-expressed on endothelial layers of the BBB and on the surface of the tumor cells [65]. These liposomes crossed the BBB and eventually reached the cancer cells [66,67]. In vitro results revealed that Tf modified VC and TET liposomes might potentiate the passage across the BBB, improved the cellular uptake, hindered the MDR, and inhibited the cancer cell invasion, and VM channels. Similarly, it is also reported that Tf conjugated VC and TET liposomes can delay the circulation time, localize more in the brain tumor tissues and thus enhance the treatment efficacy in vivo [68]. Brain cancer targeting is difficult due to complex nature of BBB and limited permeation. Liposomes bearing D-alpha-tocopheryl polyethylene glycol 1000 succinate mono-ester (TPGS) having theranostic properties have been used for brain-targeted imaging as well as therapy. Moreover, Tf modified TPGS coated liposomes co-delivered docetaxel and quantum dots (QDs) were developed for theranosis of the brain. These liposomes were below 200 nm size with about 71% of drug encapsulation efficiency. The invivo results revealed that TfR-targeted liposomes could be a promising delivery system for brain targeting due to its good BBB permeability, nano-size and a potential to provide theranostic application [69].

3.2.4. Antibody-mediated targeting Antibody fragments are significantly used as targeting agents due to their excellent selectivity and affinity towards the tumor cells [57]. Anti-CD19-targeted and anti-CD20-targeted liposomes were developed for targeting doxorubicin against internalizing epitopes (e.g., CD19) and non-internalizing epitopes (e.g., CD20), respectively. Anti-CD19targeted liposomal formulation improved the therapeutic activity due to rapid internalization into human B-lymphoma (Namalwa) cells, whereas anti-CD20-targeted liposomal formulation was not internalized. Anti-CD19-targeted liposomal formulation enhanced the survival rate in comparison to anti-CD20-targeted liposomal formulation or non-targeted liposomes [72,73]. The antibody CC52 conjugated PEGylated liposomes have been used to target colon adenocarcinoma CC531 lines. In another study, liposomes comprising fenretinide (an antitumor drug) were developed to target the ganglioside GD2 and they induced apoptosis in neuroblastoma and melanoma cell lines resulting in enhanced anti-neuroblastoma activity both in vitro and in vivo in a mice model [74]. 3.2.5. Aptamer-mediated targeting Aptamers are single stranded nucleotides, introduced in 1990 and are recently used as new class of ligands. Aptamers have a great demand over antibodies due to their low manufacturing cost, greater specificity, good affinity for their targets; and no effects on binding properties by their chemical modifications. Besides, they also effectively penetrate in the target cells due to smaller size. These are also found to be more stable in varying pH and temperature, and are tolerable to degradation on physical and chemical changes. The apt TSA14 (an anti-breast cancer RNA apt) was conjugated on the surface of PEGylated liposomes bearing DOX (PL-DOX). Apt-targeted PL-DOX showed a superior cytotoxic effect as compared to non-targeted PL-DOX in HER2-overexpressing TUBO breast cancer tumor model [75]. In another study, aptamer sgc8 conjugated liposomes were prepared for targeting leukemia CCRF-CEM cells. The sgc8 apt showed good binding affinity to its target protein tyrosine kinase 7 as it was observed that apt-conjugated liposomes efficiently bind to the tumor cells and effectively released fluorescein isothiocyanato-dextran into cells [51].

3.2.3. Peptides mediated targeting Peptides are employed as an advanced tool for the delivery of drug and gene. Moreover, they have the potential to cross the endothelial and epithelial barriers, and can reach the cytoplasm of target cells. Peptides are comparatively small, economic and stable in different biological conditions. Various peptides based delivery system like peptide-drug conjugates (PDC), peptide-modified drug delivery systems (PMDS), and cell penetration peptides based delivery systems are developed for targeted delivery of drugs [70]. Zhang et al. (2016) conjugated a histidine-rich peptide ([D]-H6L9), which has antimicrobial properties, to the surface of DOTAP-soybean phospatidylcholine-DSPEPEG2000 liposomes. The Mir-10b antagomir (antagomir-10b) and paclitaxel were loaded in these liposomes. The results of the in vivo studies showed that tumor growth was suppressed and lung metastases were also reduced in an animal model. In acidic pH, the histidines of [D]H6L9 noticeably escaped the endosomes/lysosomes by membrane rupture mechanism upon protonation phenomenon. In another study, the

3.2.6. Asialoglycoprotein receptors (ASGPR) mediated targeting Hepatocellular carcinoma is a leading cancer and causes high rate of mortality. Doxil (a DOX loaded liposomes) is found to have inadequate efficacy against hepatocellular carcinoma but has shown promising results in platinum-resistant ovarian carcinoma. Liposomes are eliminated by RES after i.v. injection and are primarily taken up by phagocytic Kupffer cells in the liver. Asialoglycoprotein receptors (ASGPR) 5

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[51].

are over expressed in hepatocellular carcinoma cells and could be selected as a ligand for targeted drug delivery [76]. Number of studies reported that alteration with D-galactose or N-acetylgalactosamine residues can efficiently deliver the drugs to target site via ASGPR dependent pathway. Zhou et al. (2012) synthesized the lactosylated liposomes comprising calcein (Lac-L-calcein) or DOX for targeted delivery of DOX to hepatocellular carcinoma (HCC) [77]. These liposomes were developed by polycarbonate membrane extrusion method. Bio-distribution and antitumor efficacy were studied in nude mice with HepG2 xenografts. The cellular uptake and cyto-toxicity were enhanced in lactosylated liposomes as compared to non-targeted liposomes. The blood circulatory time was prolonged and the tumor inhibition was increased in case of targeted liposomes as compared to non-targeted liposomes. They found relatively higher accumulation of targeted system in HCC cells as compared to non-targeted system.

3.2.8. Hyaluronic acid mediated targeting Hyaluronic acid (HA) belongs to the category of glycosaminoglycan [78]. HA is a biodegradable and biocompatible polysaccharide. HA is the basic component of human extracellular matrix and plays a significant role in various cellular processes like cell migration, cell adhesion, wound healing, and innate immunity. Many types of tumor over express the CD44 receptor and thereby offering it a suitable target for the treatment of cancer. Thus, targeting of nanocarriers to the CD44 over-expressing tumors can be promoted by altering the surface of nanocarriers with HA moieties [78]. Photothermal and chemotherapy combined delivery system was prepared for improving the tumor targeting. In this system, a HA derivative i.e. hyaluronic acid hexadecylamine (HA-C16) was anchored on the liposomal surface, and biocompatible magnetic nanoparticles and the anticancer drug docetaxel (DTX) were entrapped therein. Moreover, the chemo-therapeutic activity was assessed in a human breast cancer cell line (MCF-7) [79]. The liposomes remarkably improved the therapeutic efficacy (IC50, 0.69 ± 0.1036 μg/mL), which was quite less than those obtained for radiation monotherapy or DTX monotherapy [80]. In another research, stealth HA-targeted pH-sensitive (SLpH-HA) liposomes containing DOX were prepared for the treatment of breast cancer by targeting CD44 receptor. In vitro release data revealed the pH-dependent release of DOX from SL-pH-HA, i.e. rapid at mild acidic pH (~5) than physiological pH (~7.4). SLpH-HA was investigated for their cytotoxic activity on CD44 receptors expressing MCF-7 cells. The IC50 of SL-pH-HA and SL-HA after 48 h were determined to be 1.9 and 2.5 mM, respectively [45]. In

3.2.7. Mannose receptors mediated targeting DOX and dihydroartemisinin (DHA) encapsulated liposomes were functionalized with mannose for targeting tumor. The mannose receptor (MR, CD206) shows its overexpression in the drug resistant human colon cancer cell line and therefore depicts a better cellular uptake of therapeutic moieties by receptor mediated endocytosis. Delivery of the Mannose targeted liposomes subcutaneously in HCT8/ ADR tumor xenograft model revealed higher tumor suppression rate in comparison to free DOX or free DOX + DHA. Various mechanisms involved in anti-MDR effect of the Mannose targeted liposomes include selective accumulation of the bioactives in the nucleus, increased apoptosis, down regulation of Bcl-xl, and the stimulation of autophagy

Fig. 3. Schematic overview of different targeting approaches of liposomes based on tumor microenvironment and ligands. Quantum dot (QD); Hypoxia-inducible factor 1 (HIF-1); Vascular endothelial growth factor (VEGF); Protein kinase C (PKC); Phosphoinositide 3-kinases (PI3Ks); sphingosine kinase (SPK); protein kinase B/Akt (PKB/Akt), RAF-Rapidly Accelerated Fibrosarcoma, MEK- Mitogen-activated protein kinase kinase, ERK-Extracellular Receptor Kinase. 6

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3.3.2. Autophagy inhibition mediating targeting Autophagy is defined as a scavenging pathway by which cells eat parts of them and recycle the breakdown components, to sustain survival in starvation (Fig. 5). Tumor cells could have high levels of autophagy and could rely on autophagy for survival [83,84]. Autophagy is also triggered in hypoxic cancer areas and imparts a survival advantage as a salvage mechanism [85]. The researches revealed that autophagy suppression can compromise tumor survival and might be a good strategy for targeting cancer [86,87]. Autophagy, in which intracellular constituents undergo degradation in the lysosomes, permits tumor cells to endure under metabolic and therapeutic stress. Hydroxychloroquine (HCQ) which is a lysosomotropic agent can curb autophagy and has been employed in various preclinical and clinical trials for rendering sensitivity to tumors during chemotherapy. HCQ loaded liposomes functionalized with pH-sensitive TH-RGD targeting peptide showed effective delivery of HCQ in B16F10 tumor cells and lysosomes. The tumor growth suppression capability of DOX was enhanced by the codelivery of these modified liposomes with either free DOX or liposomal DOX [88].

another study, hyaluronan (HA) was conjugated to dipalmitoyl phosphatidylethanolamine, DPPE by an amidification method to form novel HA-DPPE macromolecules. HA-DPPE was utilized for coating of liposomes using post insertion technique. Liposomes were prepared using dipalmitoyl phosphatidylcholine and cholesterol (DPPC/Chol: 95/5 M ratio) and incubated with HA-DPPE at 55 °C in order to form hyaluronan-modified liposomes. HA coating avoided the binding of serum proteins and accelerated the blood clearance phenomenon. Thus, HA modified liposomes enhanced the plasma half-life and site-specific targeting [81]. Schematic representation of various targeted liposomes for tumor targeting and explored ligands has been depicted in Fig. 3.

3.3. Miscellaneous approaches 3.3.1. Poly (carboxybetaine) (PCB) modified liposomes PEGylated liposome-based drug carriers have shown a promising potential for cancer targeted therapy because of their longer circulation time. PEGylation substantially decreases their cellular uptake, which noticeably impairs the in vivo tumor retention and anticancer efficacy of drug-encapsulated systems. Above all, it has been evidenced that PEGylated topotecan loaded liposomes when injected repeatedly exhibit “ABC phenomenon”, which reduces the tumor accumulation of drug encapsulated in liposomes and confronts enormous obstacles to the clinical utility of liposome-based systems. Li et al. (2015) prepared a zwitterionic poly (carboxybetaine) (PCB) functionalized liposomal carrier. They explored the application of alternative polymers for extending the circulation time of topotecan loaded liposomes. PCB has higher capability in prolonging the blood retention without hindering the uptake and endosomal evasion of the liposomes, which is rather disimilar from PEGylation. Moreover, PCBylation can alter the cellular uptake behavior and evade ABC process of liposomes, which alleviates the tumor accumulation and thus increases the anticancer efficacy of liposomes. The presence of PCB can evade protein adsorption and increase the liposomal stability similar to PEG. The endocytosis was observed in uptake of pH-sensitive PCBylated liposomes into the cells opposed to PEGylated systems. Moreover, these formulations would obviate ABC phenomenon and improve the retention of drug-loaded liposomes in vivo. PCBylated drug-entrapped liposomes showed greater drug retention and cellular uptake thereby remarkably suppressed the tumor growth and furnished a promising potential for cancer treatment (Fig. 4) [82].

3.3.3. Curcumin loaded liposomes The encapsulation of partially water soluble drugs in liposomes enhances their bioavailability resulting in enhanced stability and antitumor activity, but reduced unwanted effects. Curcumin (Cur) is a herbal polyphenol compound which possesses remarkable anticancer potential in pancreatic adenocarcinoma (PA). Various liposomal preparations loaded with Cur were developed and investigated for different physicochemical parameters and stability at 4 °C and 37 °C in human plasma in vitro. The most efficient formulation amongst all with respect to these parameters was found to be PEGylated one. It was observed that the cholesterol-free preparation made from hydrogenated soy PC with 0.05/10 drug-to-lipid molar ratio was stable and depicted 96% entrapment efficiency. Cur encapsulated liposomes depicted a better anti-cancerous effect in the PA cancer cell lines AsPC-1 and BxPC-3, along with low toxicity to a normal cell line (NHDF). Moreover, apoptosis activated by Cur in PA cells was linked with morphological alterations like cell shrinkage, and cytoplasmic blebbing followed by an increased production of intracellular reactive oxygen species (ROS) and caspase 3/7 activation [89]. 3.3.4. Theranostic TPGS–doxorubicin liposomes Muthu et al. (2017) described TPGS-DOX complex as a prodrug to increase the therapeutic activity and decrease the toxicity. The

Fig. 4. Schematic representation of ABC process of PCBylated and PEGylated drug-loaded liposomes. 7

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Fig. 5. Molecular signaling of autophagy and its inhibitorsmammalian target of rapamycin complex 1 or mechanistic target of rapamycin complex 1(mTORC1); unc-51-like kinase 1(ULK1); AMP-activated protein kinase (AMPK); Ambra1 (activating molecule in Beclin-1-regulated autophagy); Vacuolar protein sorting 34 (Vps34); Phosphatidylinositol-3-phosphate (PI3P); Hydroxychloroquine (HCQ); Chloroquine (CQ); Bafilomycin A1 (Baf A1).

theranostic liposomes for tumor treatment in conjunction with diagnostic applications. Singh et al. (2016) developed TPGS modified theranostic liposomes conjugated with transferrin, which comprised of both docetaxel and quantum dots (QDs) for the purpose of imaging and therapy of brain cancer. The liposomes conjugated with and without transferrin were prepared and characterized. The transferrin conjugated liposomes depicted a sustained drug release for about 72 h. The in-vivo outcomes depicted that transferrin receptor-targeted liposomes can be a promising delivery system for brain theranostics because of their nanosize and permeability across BBB which improved the brain targeting of docetaxel and QDs as compared to the non-targeted formulations [69]. Table 1 summarizes different targeted liposomal systems for the treatment of various cancers.

pharmacokinetic outcomes of TPGS-coated liposomes in rats depicted 24 h prolonged circulation time as compared to the PEGylated liposomes. In addition, the researchers developed conventional, PEG and TPGS-coated liposomes and investigated them for the cellular uptake and cytotoxicity on brain cancer cells. The outcomes displayed an efficient in vitro cellular uptake and cytotoxicity in case of TPGS modified liposomes as compared to PEG-coated liposomes [90]. The theranostic liposomes bearing quantum dots and apomorphine were developed for brain targeting. Bioimaging studies were performed to monitor fluorescence from quantum dots. Theranostic liposomes offered a remarkable distribution in tissues in comparison to the free quantum dots [91]. Quantum dots loaded liposomes showed enhanced brain uptake. The fluorescence for the liposomal formulation was noticed up to 1 h. Nevertheless, free quantum dots were quickly removed from the brain and their retention in liver was observed up to 35 min. This study proposed a viability of bio-imaging in animal models employing

3.3.5. Gemcitabine loaded liposomes PEGylated liposomes of gemcitabine were prepared for the 8

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Table 1 Functionalized targeted liposomal chemotherapeutic drug delivery systems for the treatment of cancer. Liposomes type

Ligand

Active pharmaceutical ingredients

Cancer type

References

Plain liposomes

Aspartic acid Anti-CA IX antibody and CPP33 cellpenetrating Peptide Transferrin Transferrin Transferrin Hyaluronic acid Hyaluronic acid Asparagine Glycine Arginine (NGR) peptide RGD peptide RGD peptide RIPL peptide

Paclitaxel Triptolide

Bone metastasis Lung cancer

[106] [107]

Resveratrol 5-Fluorouracil Cordycepin Daunorubicin and honokiol Sorafenib Doxorubicin

Glioblastoma Colon cancer Liver Cancer Breast cancer Cancer Breast adenocarcinoma

[108] [109] [110] [111] [112] [113]

Paclitaxel and Curcumin Shikonin Docetaxel

[114] [115] [116]

GE11 peptide

Magnetic targeting

Docetaxel (DTX) and siRNA against the ABCG2 gene Cordycepin Doxorubicin Paclitaxel and Imatinib Losartan Docetaxel Rapamycin and polypyrrole (photosensitizer) Doxorubicin Docetaxel Doxorubicin

Lung cancer Breast cancer Hepsin-expressing cancer cells Laryngeal cancers Liver Cancer Breast cancer Breast cancer Tumor Cancer Breast cancer Cancer Breast cancer Tumor

[110] [118] [119] [120] [121] [79] [122] [123] [124]

Magnetic targeting Nitroimidazoles (radiosensitizer)

Curcumin Doxorubicin

Cancer(MCF-7 cells) Malignant glioma

[125] [126]

Hydroxycamptothecin

Tumor (Huh-7 cells)

[127]

Cationic liposomes

pH sensitive liposomes pH sensitive liposomes Photothermal therapy Thermosensitive liposomes Thermosensitive liposomes Thermo-responsive magnetic liposomes Magnetic liposomes Hypoxic radiosensitizer-prodrug liposomes Thermosensitive magnetic liposomes

Transferrin HER3 aptamer Folic acid TH peptides Eph A10 Trastuzumab (monoclonal antibody) iRGD (CCRGDKGPDC)

[117]

molecules. The superior biocompatibility and biodegradability of liposomal systems offer their clinical potential in the delivery of various anti-cancer agents bringing [95] various clinical products, like Doxil™, Onivyde™, and Marqibo® [96]. Slingerland et al. (2013) performed a bioequivalence study of the novel formulation of liposome-entrapped paclitaxel i.e. LEP Easy-to-Use (LEP-ETU) developed by NeoPharm and paclitaxel formulated with castor oil and assessed the tolerability of LEP-ETU. The preclinical studies of LEP-ETU have revealed its higher efficacy with minimum toxicity. The results from phase I clinical trial displayed that LEP-ETU is well tolerated than paclitaxel at the higher maximum-tolerated dose [97]. Growth factor receptor-bound protein-2 (Grb-2) liposomes were prepared which inhibited the production of the growth factor receptor-bound protein-2 and thereby reduced the proliferation of tumor cell. Grb-2 is an antisense oligodeoxy nucleotide. The results of phase 1 clinical trial demonstrated that it could help in the treatment of relapsed or refractory acute myeloid leukemia. Further, many other clinical outcomes like anticancer activity, optimal therapeutic dose, safety, maximum tolerated dose have been observed in clinical studies [98,99]. MM-302 is HER2-targeted PEGylated liposomes that were prepared for the encapsulation of doxorubicin and delivered in HER2-overexpressing tumor cells. In this study, the feasibility as well as the preclinical activity of transtuzumab in combination with MM-302 was evaluated. MM-302 and trastuzumab target the HER2 receptor. It binds with the HER2-overexpressed tumor cells both in vitro and in vivo, respectively. Furthermore, it has been noticed that the antibody trastuzumab did not interrupt the mechanism of action of MM-302 for the delivery of doxorubicin to the target site of the nucleus of the tumor cell and triggered the DNA damage. Furthermore, MM-302 did not disturb the function of trastuzumab and helped in blocking the p-Akt signalling. The MM-302 formulation enhanced the accumulation of both doxorubicin and trastuzumab in human xenograft tumors. It also enhanced the expression of the DNA damage marker p-p53. Thus, the combination therapy of MM302/trastuzumab for HER2-positive metastatic breast cancer gave positive results in a randomized phase II

treatment of multiple myeloma cancer. The growth inhibition rate was determined in vitro using U266 (autocrine, interleukin-6-independent) and INA-6 (IL-6-dependent) multiple myeloma cell lines. The formulation improved the growth inhibition rate as compared to free drug. Liposomal delivery enhanced the cellular uptake and apoptosis. Interaction of liposomes with multiple myeloma cells was confirmed by confocal laser scanning microscopy [92]. The liposomal delivery improved the pharmacokinetics and bio-distribution of gemcitabine. In another study, gemcitabine loaded pegylated liposomes were developed for pancreatic cancer (PC). The in vitro and in vivo antitumor activities were determined using human PC cell lines, BXPC-3 and PSN-1. The results demonstrated that the liposomes decreased the cell viability in time and dose-dependent manner as compared to free drug. The in vivo study was carried out in a cohort of SCID mice bearing BxPC-3 or PSN-1 xenografts. The liposomes showed higher growth inhibition as compared to free drug. The pharmacokinetic analysis demonstrated that liposomal formulation showed higher systemic bioavailability as compared to the free drug [93]. The in vivo efficiency of the liposomal gemcitabine (5 mg/kg) was investigated with respect to marketed product GEMZAR® (50 mg/kg) in anaplastic thyroid carcinoma xenograft model. The amount of drug in the liposomal formulation was 10 fold less than that in the marketed product. The results of bio-distribution and pharmacokinetic profile demonstrated that the liposomes increased the accumulation of drug inside the tumor enhanced the plasma half-life and also reduced the side effects [94].

4. Clinical studies The translation of preclinical studies of the nanotechnology to clinical practices requires some advanced models and methodologies. These models help in the prediction of the biofate of nanocarriers inside the body to improve the clinical applicability of nanocarriers. Clinically, liposomes based delivery systems are being used for the delivery of bio-actives such as gene, drug and other biological 9

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antigen and drugs can also be encapsulated into liposomes to kill the tumor cells.The present invention revealed the method of liposomal targeted delivery of antigen for modulating immune cells or drugs to target cells [104]. PEGylated liposomes were comprised of an immunostimulatory agent, a diagnostic marker and a maleimide. These components were attached via the covalent bond(s) to a PEG molecule on the exterior surface of the liposomes. These PEGylated liposomes are anionic or neutral in nature. The targeting moiety has shown more affinity to the folate receptors (alpha and beta) and also for the epitopes present on the surface of antigens of the tumor cells. These epitopes are over-expressed in cancer cells and used as biomarkers for targeting of cancer. All the ingredients used in liposomal formulation are compatible with gemcitabine. Cytidine deaminase is an enzyme produced in tumor microenvironment by tumor associated macrophages and/or associated with hypoxic tumor microenvironment and it converts gemcitabine into inactive form in the blood plasma of the cancer patients. It may be used as a novel method for the treatment of cancer/ human immunodeficiency virus [105]. Some of the patents granted for liposomal drug delivery are reported in Table 3 and marketed liposomal products approved in the last two decades are summarized in Table 4 [96].

clinical trial [100]. Several liposomal formulations are under investigation in different stages of clinical trial and some of them are reported in Table 2. 5. Patented and marketed liposomal products A patent is an intellectual property right granted for a novel invention, either product or a process which has some industrial utility. The patent provides the solution for the technical problems and gives an exclusive right to the inventor to exclude others to sell or use the patent for the limited period of time. The peptide-lipid conjugates are used for the destabilization of liposomes in vicinity of target peptidasesecreting cells. Peptide-lipid conjugates were incorporated into liposomal formulation. The peptide was conjugated to phosphatidylethanolamines. Various conditions like tumor, microbial infection and inflammation showed the presence of peptidase-secreting cells. The liposomes remained intact until the peptide was conjugated to lipid. In the presence of peptidase in target cell the peptide portion was removed from lipid and the contents were released due to destabilization of liposomes. Therefore, the active pharmaceutical ingredients were selectively delivered to peptidase-secreting cells [101]. This invention comprised of a serum stable liposomal system comprising the pH sensitive substituent (titratable acidic polymer) and thermo-sensitive polymers [(poly(N-acryloyl pyyrolidine), poly(N-acryloyl piperidine, a poly(acryl-L-amino acid amide)]. Thermo-sensitive polymer based liposomal system displayed the lower critical solution temperature behavior. PEG or ganglioside-derivatized lipids were utilized for the preparation of long circulatory liposomes. The liposomal formulation showed aggregation and dehydration of lipid membrane which results in the leakage of their contents. The formulation displayed significant drug release at pH 4.5. They demonstrate that N-isopropylacrylamide (“NIPA”) and methacrylic acid (“MAA’) displayed temperature and pH responsive behavior, respectively [102]. Antibody binding fragments like Fab, F(ab’), Fab'or a single antibody chain polypeptide were used for brain targeting. These fragments bind with receptors present on vascular endothelial cells of brain such as transferrin, insulin receptor, IGF-I or IGF-2 receptor. The invention claim the targeting of brain using antibody binding fragment, liposomal formulation administered intravenously or intraarterially and antibody binding fragments which specifically bind to transferrin, insulin receptor, IGF-I or IGF-2 receptor. Liposomal encapsulation of drugs or diagnostic agents prevents the premature degradation, and ameliorates side effects [103]. This patent demonstrated liposomal targeting agents for targeting the tumor cells over-expressing sialic acid binding Ig like lectin (Siglec). The targeting liposomal agent contains a glycan ligand (binding moiety) of the Siglec on the target cell. Biological agents like

6. Challenges in liposomal drug delivery systems Liposomes confront multitude of defense systems which are intended at identification by the macrophages followed by their degradation, and removal of liposomal systems which enter the body. The defense mechanisms are opsonization, uptake by reticuloendothelial system and induction of immunity. Although the above mentioned hindrances need to be evaded for optimum functioning of the liposomes, few factors like enhanced permeability and retention (EPR) effect could be explored for effective drug delivery. In spite of substantial research in the past five decades, there has been no progress in the clinical transformation of liposome based delivery systems. Liposomes have manifested substantial therapeutic advantages in biomedical applications; nevertheless the main grounds for their limited applicability is due to the process involved in manufacturing, regulations laid by the government and intellectual property (IP). Restrictions in the development are focused on cost and quality assurance. Quality assurance comprises consequences encompassing the process of manufacturing and stability of the liposomes, which are influenced by, (i) reliability and reproducibility, (ii) unavailability of equipments, (iii) chemical instability of the entrapped agent during the manufacturing, and (iv) stability concerns of the developed system on long term storage. Desirable methods which involve neither complex manufacturing-steps nor the organic solvent have been effectively produced in order to

Table 2 Clinical studies of liposomes and ligand mediated liposomes. Types of liposomal systems or products

Therapeutic agents

Targeting ligand(s)

Type of cancer

Clinical Status

Reference

Anti-EGFR Immunoliposomes Anti-CD19/CD20 Liposomes OSI-211 liposomes T7/TAT-Liposomes-PTX

Doxorubicin

Cetuximab Fab Fragment Anti-CD19 and anti-CD20 monoclonal antibodies

Breast cancer

Phase II

[128]

B cell lymphoma

[129]

lurtotecan (LRT) Paclitaxel

Preclinical Stage Phase II Preclinical stage

LEM-ETU liposomes

Mitoxantrone

Phase 1

[95]

P-selectin/avβ3 Integrin liposomes Platar Integrin avβ3 peptide/[D]-H6L9 liposomes RGD/TF-LP

Fluorescent marker

Preclinical Stage

[133]

Solid tumors Colon cancer

Preclinical Stage

[20] [47]

Paclitaxel

Brain glioma

Preclinical Stage

[134]

Onco TCS

Vincristine

Doxorubicin

Platinum compounds Paclitaxel

Ligand peptide (HAIYPRH), cationic cell penetrating peptide (TAT)

Peptides targeting P-selectin and avβ3 integrin Integrin avβ3 peptide, [D]-H6L9 peptide Cyclic arginine glycine aspartic acid (RGD) and transferrin (TF)

Ovarian cancer Lung cancer Leukemia, breast, stomach, liver and ovarian cancers Metastatic breast cancer

Non-Hodgkin's lymphoma

10

[130,131] [132]

[20]

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Table 3 Patents of liposome based drug delivery systems. Patent No.

Description

References

US6339069B1

Liposomes were loaded with peptide-lipid conjugates with an aim of destabilizing the formulation in target peptidase-secreting cells, and thereby, to deliver the carrier to the target cells. This invention relates to the pH sensitive liposomes which are stable in plasma. Further the technique of rendering pH sensitivity to liposomes of various compositions. This invention reported the ligand mediated liposomes as systemic delivery system to the target site. The compositions and techniques for celltargeted gene transfer and gene therapy for cancers have also been discussed. The invention reported the targeting of various agents like drugs and proteins to the brain. Antibody binding fragments modified liposomes bind specifically to receptors on the vascular endothelium of the blood-brain barrier. The receptors for targeting the BBB include transferrin receptor, insulin receptor, IGF-I and IGF-II receptors, among others. The invention relates the delivery vehicle with a ligand i.e. hyaluronan having the affinity towards CD44 receptors. Favorably, the carrier is liposomes while other nano-carriers were also investigated. The invention also encompasses techniques of targeting drug to CD44 overexpressing cells. The present invention furnishes the liposomal delivery system for targeting the cells expressing sialic acid binding Ig-like lectin (Siglec). Further, it rendered the techniques of liposomal system for targeted delivery of antigen and for altering immune activity. This revealed PEGylated liposomes entrapped gemcitabine and a targeting agent; pharmaceutical composition and techniques constituting liposomes. Liposomes consisting at least one poly-unsaturated fatty acid (omega-3 fatty acid, omega-6 fatty acid, and omega-9 fatty acid), a β-glucan, a cholesterol, and a doxorubicin for the treatment of cancer. The beta-glucan and doxorubicin were loaded in the liposomal vesicles. The invention introduced a targeted system for the diagnosis and treatment of cancer comprising peptide (IL4RPep) conjugated liposomes which depicted affinity towards interleukin-4 receptors, and a method of preparation thereof.

[101]

US 6,426,086 B1 US6749863B1 749 US20020025313A1

US6593308B2

US20130164364A1 US20170319482A1 US9655847B1 US9833464B2

Drug

Year of Approval

Manufacturer

Myocet® DaunoXome® Marqibo®

Doxorubicin Daunorubicin Vincristine

2000 1996 2012

Onivyde™ Visudyne® Depocyt® Doxil®

Irinotecan Verteporphin Cytarabine/Ara-C Doxorubicin

2015 2000 1999 1995

Elan Pharmaceuticals NeXstar Pharmaceuticals Spectrum Pharmaceuticals Launches Merrimack Pharmaceuticals Novartis SkyPharma Inc. Sequus Pharmaceuticals

[135] [103]

[136]

[104] [105] [137] [138]

efficacy. In future, it is very essential to acquire a comprehensive perception about the physiological and pathophysiological variations existing in patients to examine the effects of the administered liposomal delivery system. This would help in the development of efficient liposomes for personalized cancer treatments. Recently, software based screening and key optimization of critical quality and process parameters are being employed for the development of stable and economic liposomes. This holistic approach uses design of experiments based optimization to bring precise development of liposomal products at industrial outset. To prepare liposomes for in vivo purpose, it is essential to prepare them with optimum in vivo kinetics, and good throughput conditions and particular liposome compositions. Formulation by design could be a potential strategy to identify desired liposome compositions through high-throughput screening methods, the design and optimization of critical components (factors) for the development of liposomes. Several optimization techniques (Factorial designs, Box-Behnken Designs, Fractional factorial designs, Plackett - Burman designs) have been used for the preparation of efficient liposomal drug delivery systems [4,140]. It has also been expected that the future researches will emphasize on the development of safe and stable liposomal delivery systems with enhanced efficacy for tumor targeting and capability to carry a broad range of anticancer agents with favorable outcomes.

Table 4 Marketed liposomal products approved in last two decades. Product Name

[102]

produce the liposomes industrially [64]. The modification of the liposomal surface with ligands or their coating poses challenges leading to the complexity in the functionality of the liposomes. IP of liposomal could be a confounding concern and can probably lead to higher development costs. Eventually, clinical trials of the liposomes based formulations are usually more complicated as compared to the conventional liposomes, since many control groups are necessitated to account for various facets of the drug carrier system [139]. 7. Conclusion and prospects

Declaration of competing interest Since cancer is a multi-factorial disease it demands multi-modality based therapeutic strategy for attaining desirable outcomes. The liposomes offer many beneficial potential aspects for the treatment of cancer like surface modifiability using ligands, PEGylation and stimuli sensitive component to impart selective biological manifestation activities that remarkably increase the applicability of liposomes in the field of cancer. Although PEGylation has been explored as golden standard for the evasion of RES uptake but it is still a dilemma with respect to cellular internalization of the liposomes. Hence, more studies are needed to investigate the effects of PEG on cellular uptake. In addition, the ligand functionalized liposomes selectively target the drugs to the cancer site and increase the effectiveness of the therapeutic agents. Further, better understanding of molecular biology can also help in the exploration and selection of existing and newer ligands for the efficient treatment of cancer. Tumor microenvironment based drug delivery still demands advances for the effective treatment of cancer. Various stimuli responsive liposomes can lead to preferential release of the drug in response to particular stimuli such as temperature, pH, redox, and hypoxia resulting in improvement in the therapeutic

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