Recent strategies towards the surface modification of liposomes: an innovative approach for different clinical applications

Abstract

Liposomes are very useful biocompatible tools used in diverse scientific disciplines, employed for the vehiculation and delivery of lipophilic, ampiphilic or hydrophilic compounds. Liposomes have gained the importance as drug carriers, as the drugs alone have limited targets, higher toxicity and develop resistance when used in higher doses. Conventional liposomes suffer from several drawbacks like encapsulation inefficiencies and partially controlled particle size. The surface chemistry of liposome technology started from simple conventional vesicles to second generation liposomes by modulating their lipid composition and surface with different ligands. Introduction of polyethylene glycol to lipid anchor was the first innovative strategy which increased circulation time, delayed clearance and opsonin resistance. PEGylated liposomes have been found to possess higher drug loading capacity up to 90% or more and some drugs like CPX-1 encapsuled in such liposomes have increased the disease control up to 73% patients suffering from colorectal cancer. The surface of liposomes have been further liganded with small molecules, vitamins, carbohydrates, peptides, proteins, antibodies, aptamers and enzymes. These advanced liposomes exhibit greater solubility, higher stability, long-circulating time and specific drug targeting properties. The immense utility and demand of surface modified liposomes in different areas have led their way to the modern market. In addition to this, the multi-drug carrier approach of targeted liposomes is an innovative method to overcome drug resistance while treating ceratin tumors. Presently, several second-generation liposomal formulations of different anticancer drugs are at various stages of clinical trials. This review article summarizes briefly the preparation of liposomes, strategies of disease targeting and exclusively the surface modifications with different entities and their clinical applications especially as drug delivery system.

Introduction

Liposomes are made up of one or more double-layered phospholipid vesicles that enclose a discrete aqueous space, used in diverse scientific disciplines from mathematics, biophysics, biochemistry to biology, etc. The drug-loaded liposomes have improved their therapeutic index by modifying absorption, decreasing metabolism, enhancing half-life or lessening toxicity (Sakamoto et al. 2010). The liposome characteristics vary greatly with size, charge, lipid composition and preparation methods. Liposomes are biocompatible, non-toxic and biodegradable entities which help to enhance the efficacy, improve the stability and minimize the toxicity of encapsulated drugs (Samad et al. 2007). Liposomes are widely used for enhanced solubility, specific drug targeting, proper delivery and controlled release of various formulations (Yun et al. 2015). Recently, mathematical models by partial differential equations and graphical representations have been proposed to study the liposome-encapsulated drug kinetics, drug release, interation with proteins and tumor cell receptors, internalization through endocytosis and plasma clearance (Chakravarty and Dalal 2018).

Conventional liposomes (basic forms) are considered as first-generation liposomes, composed of phospholipid bilayer with anionic, cationic or neutral phospholipids and cholesterol enclosing an aqueous space (Riaz et al. 2018). One of the major shortcomings of the these traditional liposomes is short life span during intravenous circulation due to uptake by reticuloendothelial system (RES). Phagocytes with the help of opsonins identify them as foreign bodies and engulf them quickly. Keeping these limitations of liposomes in view, different researchers have engineered the surface of these particles with various substances, thus enhancing the repulsion with serum components (Steichen et al. 2013) (Fig. 1).

Fig. 1.

Modification of liposome surface with different ligands (targeted liposomes). The different modifications can be achieved using small molecules, peptides, carbohydrates, aptamers, proteins/enzymes. These entities can be bonded to liposome phospholipid bilayer directly or through polyethylene glycol

The first innovative modification of liposomes leads to the conjugation of polyethylene glycol (PEG) to lipid anchor, which resulted in significant circulation time, stress stabilization and protection against opsonins. These additional features resulted in delayed clearance from hepatic RES, so greatly enhancing circulation time (Kamaly et al. 2012). The PEGylated liposomes act efficiently as drug carriers and show enhanced accumulation near tumors (Torchilin and Lukyanov 2003). In addition to PEG, the surface modification of liposomes has been performed with suitable ligands like small molecules, vitamins (Sperling and Parak 2010), carbohydrates (Faraasen et al. 2003), polysaccharides (Sihorkar and Vyas 2001), peptides (Torchilin 2005), aptamers (Farokhzad et al. 2006), antibodies (Anabousi et al. 2005) and enzymes based on their application (Nobs et al. 2004).

These additional components have drastically enhanced the applications of liposomes in different fields like drug delivery, cancer detection and therapy. The utilization of supplementary biomolecules on the surface of liposomes works as ligands for the receptors expressed as docking sites on the interested tissues. The targeted delivery of different formulations, common drugs and anticancer agents at the required location, i.e., tumor has revolutionized the field of liposome applications. Targeted liposomes are more specific, tissue oriented, have minimum off-target effects, and are required in lesser quantity as compared to conventional liposomes. Immunoliposomes (antibody coupled liposomes) have been used as the most efficient targeted liposome to bind with their ideal antigens (Salim et al. 2014).

Targeting of ligands on liposomes is usually performed by utilizing three types of reactions as by the formation of amide bond, thioester bond and disulfide bond (Nobs et al. 2004). Liposome functionalization with different specific ligands like small molecules, vitamins, peptides, antibody fragments or whole antibody, aptamers or enzymes enhances the efficiency of targeted delivery of anticancer agents at the requisite site (Riaz et al. 2018).

During 1970s, the liposomes were first used as drug delivery system for humans, which has now gradually improved as a special treatment system, and currently several commercial liposomal formulations are available in the market against several diseases including cancer (Lasic and Papahadjopoulos 1995; Singh et al. 2012). Liposomal formulation of amphotericin B and doxorubicin (Lopez-Berestein et al. 1985; Gabizon et al. 1989) was the first clinical trial vehicle used for human beings. Nowadays, there are different liposomal products available in the market which include antibacterial, antifungal and mostly anticancer formulations. In addition to this, liposomal formulations are available as therapeutic agents to treat eye, skin and respiratory disorders (Adjei 1997).

There are several special liposomal formulations available for the clinical practice which include chemotherapeutics as cytarabine (DepoCyte1) daunorubicin (DaunoXome1), doxorubicin (Doxil1/Caelys1), doxorubicin (Myocet1) use for the treatment of AIDS related Kaposi’s sarcoma, ovarian cancer, lymphomas, multiple myeloma, leukemia, etc. In addition to this, several other liposomal formulations of mitoxantrone, camptothecine, annamycin, cisplatin, 9-nitro-20(S)-camptothecine, lurtotecan, paclitaxel, iritotecan, vincristine, and floxuridine are at various stages of clinical trials (Slingerland et al. 2012).

Here, in this review, we are neither focusing on the structure of the phospholipid bilayer molecules nor on the structure of the liganded molecules. We have evaluated the latest updates on liposome preparation, strategies of its targeting, multidrug delivery and the surface modifications of liposomes with various ligands by consulting competitive literature available.

Liposome preparation

The preparation and size reduction of liposomes by conventional techniques is popular due to its simplicity and least requirements of sophisticated equipment. However, the growing demand and point-of-care applications of liposomes have motivated the improvement of conventional methods to second generation liposomal formulations (Nguyen et al. 2016). The preparation of liposomes basically involves three main steps as: vesicle formation, vesicle volume reduction and its purification. The ordinary liposomes are simply prepared by adding phospholipids to an aqueous medium and these phospholipids organize and form vesicles with one or more bilayers (Lopes et al. 2013; Singh 2018).

Different strategies are employed to prepare liposomes, depending upon the desired formulation. These methods differ on their approach to overcome the hydrophobic nature of lipids when mixed with water. Accordingly, these methods are classified as detergent solubilization, solvent evaporation, mechanical agitation, solvent injection. All these methods involve passive drug loading (Çagdaş et al. 2014).

Liposomes are most commonly prepared by lipid hydration and the organic solvent replacement by an aqueous medium. These methods can be performed either by reverse-phase evaporation or by organic-solvent injection. The lipid hydration is followed by manual stirring or by vortex (Bangham’s method), followed by solvent removal, under reduced pressure or by the method of rotary evaporation, until a thin film is formed. Aqueous medium is used to hydrate the thin film above the phase-transition temperature, which results in multilamellar vesicle (MLV) liposome formation (Bangham et al. 1965; Wagner and Vorauer-Uhl 2011). Furthermore, different strategies like sonication, extrusion, homogenization or microfluidization convert MLV to large unilamellar vesicles (LUV) or small unilamellar vesicles (SUV). The final purified liposome preparation is performed by either centrifugation, dialysis, ultrafiltration or by column chromatography.

In addition to this, several stratagies are further applied to prepared targeted liposomes based on scale of production, liposome lipid structure and charge, drug encapsulation efficiency, physicochemical characteristics of encapsulating drugs and the administrative route (Lasch et al. 2003). Targeted liposome preparation is also performed by checking the nature of ligand attached (small molecule, peptide, protein, aptamer, antibody fragments, whole antibody, etc.), and drug release as long release or triggered release (Storm and Crommelin 1998).

The most common methods of liposome characterization include Bartlet method, thin layer chromatography (TLC), size-exclusion chromatography, high-performance liquid chromatogragphy (HPLC), gas chromatography (GC), nuclear magnetic resonance (P³¹-NMR), electron microscopy and X-ray diffraction and scattering (Edwards and Baeumner 2006).

Strategies for liposome targeting to disease sites

Different approaches have been adopted for targeting liposomes to the disease sites, especially the tumor (Deshpande et al. 2013). These approaches include passive/spontaneous liposomal targeting and active liposomal targeting.

Passive targeting

The basis for the passive targeting of liposomes at the disease sites and especially at tumor is mainly due to the leaky structure of tumor-associated blood vessels. Depending upon the cancer type, the size of the gaps between the endothelial cells ranges between 100 and 780 nm in tumor capillaries; while in normal endothelium, it is between 5 and 10 nm (Haley and Frenkel 2008). In addition to this, due to enhanced permeability and retention (EPR) effect at the site of tumor, this strategy is effective for targeting liposomes at such sites. Therefore, if liposomes are prepared with permissible size to extravasate in cancer tissue, an ideal passive targeting strategy is achieved (Lehtinen et al. 2012; Kim and Huang 2012).

A good example of passive targeting include the use of Sclareol solid lipid nanoparticles (Sclareol-SLNs), with an average particle size of 88 ± 5 nm, against human lung epithelial cancer cells, which showed sustained drug release even after a period of 48 h as compared to the free drug alone (Paszko and Senge 2012).

Active targeting

Keeping in view the various limitations of passively targeted liposomes, the new strategies led to the discovery of actively targeted liposomes. These liposomes are designed to lessen the off-target effects and are prepared by conjugating their surface with targeting moieties. The targeting moieties include small molecules, peptides, aptamers, antibody fragments or whole antibodies (Bryne et al. 2008; Egusquiaguirre et al. 2012). To efficiently aim liposomes at cancer cells, it is a prime requisite to attach the specific targeting moiety/moieties in enough amount to achieve maximum affinity for the tumor cell surface receptors (Egusquiaguirre et al. 2012). Recently, the strategy of tumor-specific active targeting has significantly increased by focusing on different macromolecules overexpressed in different cancer cells as:

1. Targeting cell surface receptors: The approach to target cell surface receptors expressed on cancer cells is the most common strategy to deliver the drugs by liposomes liganded with antibodies or other receptor specific ligands. The different targeting receptors in focus are as:

   Targeting folate receptors: Folate receptors are frequently overexpressed in certain lung, ovarian, colon, breast, brain and kidney tumors. Due to the overexpression of such receptors on cancer cells as compared to the normal cells, this receptor has been efficiently used as a targeting ligand for specific drug delivery liposomes against different tumor cells (Low et al. 2007).

   Targeting transferrin receptors: Increased expression of transferrin receptors on cancer cells is associated with higher iron demand due to quickly proliferating cancer cells (Ying et al. 2010). This overexpression of transferrin has led to design of transferrin targeted anticancer therapy (Li et al. 2009). Transferin-targeted stealth liposomes containing doxorubicin against liver cancer cells have been found to possess improved intracellular uptake, biodistribution and pharmacokinetic profile and has led to enhanced therapeutic efficacy (Li et al. 2009).

   Targeting EGFRs: The epidermal growth factor receptors (EGFRs) mediates repair, growth and differentiation of non-cancerous cells (Lehtinen et al. 2012). Many solid tumors like non-small-cell lung cancer, colorectal cancer, squamous cell carcinoma of kidney, ovary, pancreas and prostate and breast cancer show its overexpression, thus making it a smart target for therapeutic drug delivery (Danhier et al. 2010). In tumor cells, this growth factor mediates several processes like angiogenesis, proliferation and metastasis.

2. Targeting the tumor microenvironment: There are several reasons which indicate that targeting the tumor microenvironment is advantageous as compared to targeting the cell surface receptors. Some of the reasons are: vascular destruction reduces the tumor growth and metastasis, the blockade for the liposome diffusion through the tumor can be overcome directly by tumor vasculature targeting, neovascular endothelial cell phenotypic variation can be curbed, which can prevent the resistance development, and in addition to this, for any cancer type, the tumor vasculature is non-specific (Byrne et al. 2008).

   Targeting VEGF: Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) are well known as proangiogenic proteins and are well-defined targets for antiangiogenic therapy (Li et al. 2008; Wicki et al. 2012). Bevacizumab is a monoclonal antibody used as an antihuman VEGF, which has been approved by United States Food and Drug Administration (US-FDA) and works as an anticancer drug. In addition to this drug, different inhibitors of tyrosine kinase receptors like VEGFRs or basic FGFRs have been used as anticancer agents (Katanasaka et al. 2008).

   Targeting VCAM: As Vascular cell adhesion molecules (VCAM) are involved in inflammatory complications like cancer, they are good targets for anticancer therapy. Tumor vessels show overexpression of VCAM-1, and thus represents an interesting target for anticancer drug delivery (Kang et al. 2011).

   Targeting B-Raf: Recently, an antitumor drug, vemurafenib, has been used for some cancer patients. It acts as a targeting agen for ^V600EB-Raf protein expressed in melanoma and such patients showed 80% regression during the first 2 months of treatment cycle. However, majority of the patients developed drug resistance, even though initially they were responsive to this drug (Aryal et al. 2011; Khdair et al. 2009).

   Targeting matrix metalloproteases: These endopeptidases are involved in remodeling of tissues, tumor invasiveness, apoptosis resistance and metastasis. Inhibitors of MMP family proteins have been used to suppress angiogenesis in tumor-bearing mice (Hatakeyama et al. 2007).

Liposomal formulation with multidrug delivery approach

Drug resistance is a major complication towards a good outcome during a disease treatment. A single liposomal formulation with both hydrophilic and hydrophobic regions, can be designed for loading different types of anti-cancer drugs with synergizing drug ratios. The liposomal formulation delivers simultaneously the drugs with optimizing ratios at the tumor site, and thus used to decrease the resistance development. Two drugs, Plumbagin and Celecoxib, were identified to kill synergistically the melanoma cells as compared to the normal cells. The traditional use of these drugs in combined form was not certified due to its poor bioavailability and the toxicological issues. A nanoliposomal formulation of plumbagin and celecoxib, called Celeplum-777, was prepared against melanoma cells. This formulation is stable and releases the drugs at an ideal ratio to kill the melanoma cells only, inhibiting tumor growth by 72% without any apparent toxicity (Gowda et al. 2017).

Two antitumor drugs (Gemcitabine and Paclitaxel) were investigated to study the multidrug carrier approach of liposomes. The PEGylated liposomes encapsulated with these two drugs were found to possess high drug loading capacity (~ 90% for Gemcitabine and ~ 80% for Paclitaxel) and no destabilizing effect. Treatment of human breast cancer cells (Michigan Cancer Foundation-7 cells) with this formulation showed stronger cytotoxic activity as compared to freed and individual drug liposomal preparation. In addition to this, the formulation possessed a controlled release to these two compounds (Cosco et al. 2011).

Co-encapsulation of two hydrophilic drugs (irinotecan and floxuridine) as a liposomal formulation, also named as CPX-1 was found to be well tolerated during in vivo conditions, improved the pharmacokinetic profiles of these drugs, and increased the disease control in about 73% patients suffering from colorectal cancer (Harasym et al. 2007; Batist et al. 2009). In addition to this, the encapsulated drugs did not show any destabilizing behavior to colloidal structure and showed synergistic action on different cancer cell lines (Tardi et al. 2009).

Liposomes as carriers of prodrugs and conversion to active metabolites

A prodrug is a compound with little or no pharmacological properties, which are converted to active form (active drugs) by specific chemical reactions or enzymes in vivo or by a combination of the two mechanisms (Rautio et al. 2018). Prodrugs are designed to achieve bioavailability, when its active form is poorly absorbed by gastrointestinal (GI) tract (Nofsinger et al. 2014).

Liposomes are very efficient vehicles to carry prodrugs to achieve a proper pharmacokinetic goal of its active form. Lipophilic forms of methotrexate and melphalan as prodrugs have been loaded as unilamellar liposomes to overcome the resistance exhibited by human leukemia cells for initial drugs across the membrane (Kuznetsova et al. 2009). Methotrexate is widely used as an autoimmune and anticancer drug (McGuire 2003). The therapeutic efficacy of this native drug is impeded due to its toxicity and resistance by tumor cells (Pui 1995). The phospholipid derivative of methotrexate, esterified by lysophosphatidylcholine, acts as an active agent against leukemia (CEM/MTX) cells (Kozak et al. 2001).

Irinotecan hydrochloride (CPT-11) is a prodrug which is converted to its active form SN-38 at the tumor site. The active drug possesses efficient antitumor activity when used at proper concentration. PEGylated liposome preparation of CPT-11 enhances its antitumor activity due to increased blood circulation time. The formation of SN-38 within liposomal membranes occurs by incubation with carboxylesterase, present within the liposomes (Sadzuka 2000).

Surface modification of liposomes

Liposomes have been potentially used as drug carriers against different diseases including cancer therapy. To facilitate their use specifically and effectively, it is important to identify the chemistry of targeted cell surface receptors, identification of ligands to be used on liposome surface against these receptors, and delaying of fast clearance of liposomes by RES (Zhao et al. 2008). Here, we are describing the different liposomal surface modifications that address their efficient targeting against different diseases, especially cancer.

1. Surface modifications of liposomes with vitamins

The employment of vitamins as liposome ligands for efficient and targeted drug delivery has opened a vast field of their applications. Different types of cancer cells overexpress vitamin receptors more as compared to the normal cells; so, the knowledge of receptors for the docking of vitamin-liganded liposomes is crucial. The expression of different vitamins receptors are often raised in malignant phenotypes (Kuldo et al. 2005). Different vitamins used as liposome ligands for receptors on malignant cells are mostly folate, although tocopherol, pyridoxal phosphate or pyridoxine have also been used (Drummond et al. 2000) (Table 1).

Table 1.

Surface-engineered liposomes functionalized with different vitamins for varied clinical and research applications

S. no	Vitamin	Liposome applications	References
1	Biotin	As DNA sensors	Patolsky et al. (2000)
		Oral delivery of insulin	Zhang et al. (2014)
		Delivery of quantum dots conjugated to epidermal growth factor ligand to target EGFR	Sigot et al. (2010)
		Used for analysis of solutes during capillary electrophoresis	Yang et al. (1998)
2	Nicotinamide	Radiolabelling liposome	Laverman et al. (2000)
		To study the thermal stability of formaldehyde dehydrogenase	Yoshimoto et al. (2011)
3	Vitamin A	siRNA carrier to ameliorate skin fibrosis	Yamakawa et al. (2018)
4	Pyridoxal phosphate	To study polarization anisotropy and fluorescence lifetime of liposomes	Greenaway and Ledbetter (1987)
5	Riboflavin	Riboflavin photolysis stability	Ahmad et al. (2015)
6	Tocopherol	Lipopolysaccharide induced lung injury	Suntres and Shek (1998)
		Delivery of docetaxel	Raju et al. (2013)
		Added to liposomes to enhance their antioxidant activity	Fex and Johannesson (1987)
7	Folic acid	Macrophage targeting with ovarian carcinoma	Turk et al. (2004)
		Oligodeoxynucleotide targeting to cancer cells	Leamon et al. (2003)

Vitamin E has been used as a liposomal ligand as d-alpha tocopheryl polyethylene glycol succinate (TPGS). It is a water-soluble amphiphilic structure with PEG as hydrophilic portion and tocopherol succinate as lipophilic group. Due to its hydrophilic–lipophilic balance value, TPGS works as an excellent solubilizer, emulsifier and bioavailability enhancer for hydrophobic drugs (Duhem et al. 2014). In addition to this, TPGS amplifies the drug absorption and its cytotoxicity, and slows down the multi-drug resistance (Pérez-Herrero and Fernández-Medarde 2015). Furthermore, to prevent the free radical induced damage of liposomes, vitamin E is used to prevent oxidative stress during its storage (Halliwell et al. 1995).

The tumor cells overexpress folate receptors on their plasma membranes which has a specific affinity with folic acid. The principle used for the conjugation of folate to liposomes is to prepare folate-linked peptides, cholesterol or phospholipids first, before the preparation of tumor specific liposomes. The normal tissues usually lacks the expression of folate receptors on their cells (O’Shannessy et al. 2015). So, this advantage of expression of folic acid receptor only by cancer cells has been utilized as a selective marker for liposomes for specific location and shipment of therapeutics to defective cells (Lu and Low 2002). Different types of chemotherapeutics, imaging agents, genes and antisense oligonucleotides have been delivered to tumor cells utilizing folate–folate receptor binding (Steichen et al. 2013).

During the earlier days of folate-conjugated liposome preparation, phospholipids were directly conjugated with folic acid. However, later it was observed that folate receptor expressed on tumor cells did not bind with folate-conjugated liposomes when folic acid was directly bonded to the phospholipids (Drummond et al. 2000). This limitation led to the use of many linkers like polyethylene glycol (PEG) and hydrazine in addition to direct binding of folic acid to cholesterol, protein or other spacers (D’souza and Shegokar 2016). The use of PEG in folate-conjugated liposomes enhanced their efficiency by increasing the solubility and half-life of the drug as well as improved the drug reserve at the tumor spot (Pasut et al. 2008). Folic acid binding with PEG enhanced the liposome retention near the tumor and enhanced its uptake by folate receptor-mediated endocytosis (Kim et al. 2008).

The overall data available which illustrate the use of vitamins other than folate as liposome ligands are few. However, the fame of folate targeting laid a good foundation for other vitamins as ligands for drug delivery by liposome (Kratz et al. 2013). Besides specific targeting, vitamins play some distinct roles in liposome technology. The advantage of non-covalent interaction between biotin and streptavidin has been utilized to prepare biotinylated liposomes for different purposes (Jain and Cheng 2017).

Different vitamins like biotin, nicotinamide, pyridoxal phosphate, pyridoxine, riboflavin have been liganded with liposomes for various diagnostic and therapeutic agents (Table 1).

• 2.Surface modification of liposomes with carbohydrates

Several carbohydrates have been decorated on liposome surfaces to enhance drug delivery mission and have disclosed great opportunities in clinical field (Chua et al. 1984). It has been a great challenge to point out a ligand that contributes sufficient selectivity for the targeted cells. The binding of glycan ligands expressed on liposomes, by glycan binding protein receptors on different cells, has helped in targeted drug delivery tasks. The mannose has contributed sufficiently as mannosylated liposomes which work as productive drug carriers with no toxicity and very low intrinsic immunogenicity (Kawakami et al. 2000; Tsumoto et al. 2009) (Table 2).

Table 2.

Surface-modified liposomes liganded with different carbohydrates for varied clinical and research purposes

S. no	Carbohydrate	Application of liposome	References
1	Glucose	Tumor study	Luciani et al. (2004)
		Drug delivery for brain capillary endothelia cells	Xie et al. (2012)
2	Mannose	Efficient formation of giant liposomes	Tsumoto et al. (2009)
3	Galactose	To study galactose receptors on macrophages and to study the targeted delivery of galactose to hepatocytes	Managit et al. (2005)
4	Sucrose	Doxirubicin-loaded liposomes for cancer treatment	Song et al. (2009)
5	Maltose	Doxirubicin transport for cancer treatment	Song et al. (2009)
6	Lactose	To study liposome size and stability	Zhu et al. (2005)
7	Oligosaccharides	Therapeutic inhibitor designs	Galustian et al. (2004)
8	Lectins	Pulmonary drug delivery	Abu-Dahab et al. (2001)
9	Tomato lectin, wheat germ agglutinin	Oral administration of insulin	Zhang et al. (2005)

Immunocompetent cells like dendritic cells and macrophages usually express mannose receptors, which are regarded as good recognition sites for liposomes with mannose, fructose and N-acetyl glucosamine. So, mannose-mediated drug transportation systems have been efficiently used for immune cells and this facility has been used to modify immune reactions (Wijagkanalan et al. 2008).

A study was conducted based on liposomes modified with disaccharides (sucrose or maltose) to study the intracellular uptake of anticancer drugs by certain cancer cells. The study showed that disaccharides-liganded liposomes work as excellent tools for drug targeting as malignant cells usually express lectins, the sugar binding proteins. In vitro uptake and cytotoxicity pattern against cancer cells by liposomes liganded with disaccharides were higher than sterically stabilized liposomes (Song et al. 2009).

Doxirubicin-containing liposomes, reshaped with a carbohydrate ligand, have been used for Human Burkitt lymphoma Daudi B-cell lines to find out the targeted delivery of specific chemotherapeutic medicines (Sapra and Allen 2003). These liposomes efficiently dock with B-cell-specific surface protein CD22 (Goldenberg 2001). Sialic acid is a group of diverse acidic monosaccharides, usually found as glycans of glycosphingolipids and glycoproteins, widely found on animal cell membranes. Different modifications of sialic acid diversify its applications for different purposes (Spevak et al. 1993). B-cell surface protein (CD 22) specifically binds with sialic acid and exhibits highly productive endocytosis, leading to rapid internalization of doxirubicin-loaded liposomes (Sun et al. 2016).

Glycoproteins play a major role in different cellular activities like recognition, immune response, inflammation, differentiation (Gordon 2002). Keeping this in view, lectins like concanavalin A have been used as highly specific binding tools for carbohydrates, which are conjugated to liposomal membranes (Abu-Dahab et al. 2001; Vyas et al. 2001).

Lectins have a unique ability to interact with exposed carbohydrate residues on other glycoproteins found on the epithelial cell membranes, so working as efficient bioadhesives. The binding specificity of wheat germ agglutinins liganded on liposomes has been used for transport of some cytotoxic drugs like doxorubicin to tumor cells (Murata et al. 2013). The lectin-mediated drug transport is an alternate to receptor-mediated transport of some ligands like vit. B12 or transferrin. The lectin-mediated liposomes have desired drug release characteristics including antibiotics, antitumor drugs, or plasmid and gene transport (Li et al. 2014).

Insulin has been transported through lectin-liganded liposomes by oral administration and this has led to the idea that peptide and protein drugs can be transported by same idea. Different lectins like Ulex europaeus agglutinin, tomato lectin and wheat germ agglutinin have been conjugated on liposome lipid bilayer (Kuzma 2009). Efficient hypoglycemic activity was found by these lectin-liganded insulin liposomes on diabetic mice as compared to abdominal cavity insulin injection treatment (Zhang et al. 2005; Du et al. 2009).

• 3.Surface modification of liposomes with peptides, proteins, antibodies and antibody fragments

One of the greatest applications of liposomes is considered to be tumor treatments. The alteration of liposomes with small peptides, antibody fragments, or whole antibodies has been engineered with encouraging strategy against tumor (Sapra and Allen 2003). Different types of human cancer cells like bladder, lung, ovarian and breast cancers express EGFR, which is known as a significant target for therapy. This receptor is also known to be associated with different advanced diseases with poor prognosis (Herbst and Shin 2002) (Table 3).

Table 3.

Liposome surface engineered with antibody fragments and monoclonal antibodies used as targeted delivery of various anti-cancer drugs

S. no	Antibody/antibody fragment	Liposome loaded drugs for different cancers	References
1	Mab CC52	Liposome carrying 5-fluorodeoxyuridine against Colon cancer	Koning et al. (2002)
2	Anti B cell lymphoma Mab LL2	Liposome carrying doxorubicin against Lymphoma	Lundberg et al. (2000)
3	Anti CD 32	Liposome carrying oligonucleotides against leukemia	Ma and Wei (1996)
4	Mab AF 20	Liposome carrying fluorescent dye used against hepatocarcinoma	Moradpour et al. (1995)
5	Mab 2C5	Liposome carrying doxorubicin used against mammary adenocarcinoma and lung carcinoma	Lukyanov et al. (2004)
6	Anti CD 133 Mab	Liposomes containing bevacizumab against glioblastoma	Shin et al. (2015)
7	Anti CD 19 and anti CD 37 Mab	Liposome carrying fluorescent dye against leukemia	Yu et al. (2013)
8	Anti transferrin ScFv antibody fragment	Liposome carrying plasmid DNA against prostate cancer sever cancer cell lines	Xu et al. (2002)
9	Anti T24	Liposome carrying pheophorbide a and used against bladder carcinoma	Bergstrom et al. (1994)
10	Anti FAP Sc Fv antibody	Liposome carrying fluorescent dye against metastatic fibrosarcoma	Tansi et al. (2016)
11	Anti-ovarian carcinoma Fab’	Liposome used against ovarian carcinoma	Moradpour et al. (1995)
12	Fab’	Liposome with doxorubicin against advanced solid tumors against EGFR receptor	Mamot et al. (2012)
13	scFv	Liposome carrying P53DNA plasmid against transferin of refractory cancers	Senzer et al. (2013)
14	scFv	Liposome with transtuzumab against advanced breast cancer	Miller et al. (2016)

Different approaches have been applied to prepare immunoliposomes, either as whole antibodies directly bound to phospholipids by surface adsorption or through hapten binding. The antibodies are also attached by direct covalent bonding as a whole protein. The antibody can also be bound to liposome surface with the help of linkers like PEG or using the technique of avidin–biotin interaction (Torchilin 2005) (Fig. 2).

Fig. 2.

Immunoliposomes: The different antibodies/antibody fragments can be bound to liposome phospholipid surface by employing different procedures. These techniques include: (a) direct adsorption of antibody; (b) hapten binding; (c) binding of whole antibody; (d) covalent binding of Fab’; (e) binding of whole antibody by utilizing PEG spacers; (f) and (g) binding of whole antibody through avidin–biotin with either the biotin or avidin bound to the liposome surface

A novel approach has been applied to engineer and minimize conventional antibodies to form different antibody fragments like Fab, scFv, bispecific antibody, nanobody (Van Driel et al. 2016), bifunctional antibody (Holliger and Hudson 2005), minibody and diabody. These strategies have been applied to form different nanoparticle-antibody fragments and smaller-sized liposomes, which function as important biomedical tools in diagnostic and therapeutic areas (Richards et al. 2017).

The use of immunoliposomes for targeted drug delivery has emerged as an encouraging strategy for drug transportation and delivery for the cells presenting the antigen for the matching antibodies (Zhang et al. 2016). Both non-covalent and covalent strategies have been applied to use monoclonal antibodies or their fragments to be used as ligands on liposomal surface. The important targets with such liganded liposomes have been suggested for the treatment of different diseases like EGFR- and HER2-positive malignancies, neurodegenerative diseases, autoimmune and infectious diseases, cardiovascular and inflammatory disorders. However, immunoliposomes are still struggling for the clinical approval to be used widely (Wang and Thanou 2010).

Immunoliposomes targeted against EGFR have been efficient in delivery of doxorubicin against tumor cells; so, better anti-tumor results have been obtained when working on animal xenograft models (Mamot et al. 2003). Besides antibody fragments or whole antibodies, the peptide ligands have also been observed to possess specific interaction with receptors present on tumor neovasculature. This interaction was utilized for direct intervention of chemotherapeutics, gene constructs or other proteins (Das et al. 2009; Torchilin and Lukyanov 2003).

On the basis of comparative sequence and structural analysis of proteins and phage display screening, different attempts have been tried to search for effective ligands for many targets on cancer cells (Wu et al. 2016). Based on these trials, the screening of a novel peptide (GE11) for EGFR was reported. EGFR over-expressing cancer cells showed promising results for gene transfection (Song et al. 2008).

The protein data bank of EGFR is well known, and thus provided a straightforward template for computer-assisted design (CAD) for a novel ligand suitable for cancer therapy (Feiner 2019). Recently, a growth factor antagonist named as antagonist G (H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH2) was used on liposomes (Moreira et al. 2001). This antagonist G is a broad-range molecule which blocks competitively the action of multiple neuropeptides like vasopressin, bradykinin, gastrin-releasing peptide) by binding with different receptors through the residues D-Trp-NmePhe-D-Trp-Leu, which is found on small cell lung cancer cell surface (Arai et al. 2015).

• 4Surface modification of liposomes with aptamers

Aptamers are short single-stranded DNA or RNA oligonucleotides (usually 25–90 nucleotide bases) which can bind through unique three-dimensional pattern with corresponding targets like proteins and cells (Płotka-Wasylka et al. 2016). Aptamers have been revealed as superior and fastest emerging new tools to target cancer biomarkers quite efficiently and are employed as effective ligands for drug delivery and anti-cancer therapy (Table 4). These ligands usually bind with affinities in nanomolar–picomolar range to target molecules (Dreaden et al. 2012; Yüce et al. 2015).

Table 4.

Liposome surface liganded with aptamers used as targeted delivery for several cancers

S. no	Aptamer	Application of liposome	References
1	NCL-Aptamer	Cisplatin encapsulated liposome as chemotherapeutic agent against broad range of cancer	Cao et al. (2009)
2	sgc8 aptamer	Liposomes for leukemia CEM-CCRF cells	áO’Donoghue (2010)
3	NX 1838	Aptamer having specific binds with VEGF on cancer cells	Brody and Gold (2000)
4	Anti-CD44	Selective targeting of cancer cells	Alshaer et al. (2014)
5	DAG-NX213	Specificity for VEGF, the inducer of angiogenesis	Yang et al. (2011)
6	AS1411	Cytotoxicity to MCF-7 breast cancer cells	Xing et al. (2013)
7	Macugen	Treatment of macular age-related macular degeneration	Sun et al. (2014)
8	BOCK	Used to recognize distinct binding sites on thrombin	Jung et al. (2010)
9	TASSET	Used to recognize distinct binding sites on target protein	Jung et al. (2010)
10	xPSM-A9	Used against prostate-specific membrane antigen expressed on prostate cancer cells	Baek et al. (2014)
11	IL-4R⍺	Suppression of tumor growth by targeting tumor microenvironment	Liu et al. (2017)

Aptamers are created by systematic evolution of ligands by exponential enrichment (SELEX) technique, which specifically bind with broad range of targets like cells, viruses, proteins, and small molecules (Daniels et al. 2003). For the targeted delivery of anticancer drugs, the nucleic acid aptamers have won the race as compared to antibodies. There are several reasons for aptamers to be efficient ligands as they are resistant to organic solvent, resistant to variation in temperature and pH and can have mass production through chemical synthesis. Besides this, aptamers are resistant to denaturation–renaturation cycles and are less immunogenic than antibodies (Niemeyer 2001; Shah et al. 2014; Yu et al. 2012).

Aptamer-liganded liposomes are most efficient drug-delivery systems. The US-FDA has approved a number of liposome-based therapies in different clinics for disease treatments (Cao et al. 2009). Aptamers have also been chemically modified with varied functional groups on either ends to enhance site-specific conjugation. Nucleic acid aptamers exhibit swift penetration to target cells and good serum retention due to better stability (Lee et al. 2016). Tumor growth and metastasis are contributed by some immunosuppressive cells like tumor-associated macrophages, myeloid-derived suppressor cells, and tumor-resident regulatory T cells (Fujimura et al. 2010). Targeting these immunosuppressive cells can improve the efficacy of cancer therapy.

The use of an IL-4Ra RNA aptamer blocks human IL-4 receptor and also suppresses myeloid-derived suppressor cells (Liu et al. 2017). CpG oligodeoxynucleotide-10 displays exceptional anti-tumor activity as aptamer (IL-4Ra)-liganded liposome selectively targets IL-4Ra receptor expressed on CT26 carcinoma cells, which expresses good level of IL-4Ra receptor. The efficient uptake of CpG by tumor cells noticeably inhibit in vivo CT26 tumor growth and this cancer-targeting aptamer delivery by liposomes may support to be an efficient approach for successful victory against immunosuppression and facilitating immunotherapy (Andon et al. 2017).

• 5.Surface modification of liposomes with enzymes

Liposomes liganded with enzymes (enzymosomes) are emerging as a novel drug transportation and delivery system for site-specific action. The enzymosomes avail the same peculiar nature of an enzyme, binding to its specific substrate and catalyzing the normal product formation (Petrak 2005). The enzyme is covalently bound by physical adsorption, acetylation, direct conjugation, etc. to the surface of liposome to form the enzymosome. Enzymosomes prove to be efficient drug transportation and release vehicles and minimize unwanted side effects of some traditional treatment ways (Shefrin et al. 2017). They show efficient long duration treatment for some diseases and are promising substitutes of conventional management of anti-platelet activities, gout etc.

The covalent bonding of the enzymes with the liposome surface attributes the biological activity and conformational structural stability of the enzyme as reported with d-amino acid oxidase and catalase (Shefrin et al. 2017). Most commonly used enzymes used for the preparation of enzymosomes to enhance the potential of anti-tumor drugs include alkaline phosphatase, β-glucosidase, β-lactamase, and carboxypeptidase (Table 5).

Table 5.

Liposome surface engineered with different enzymes to study different biochemical activities

S. no	Enzymosome	Liposome application	References
1	Brain glutamate-decarboxylase	To study the comparison of pyridoxal phosphate (PLP) activation and inhibition kinetics by PLP oxime-o-acetic acid between free and liposome bound form	Covarrubias and Tapia (1980)
2	SOD	To study retention, efficiency, enzymatic activity, incorporation, protein–lipid ratio and zeta potential between conventional and long circulating liposome (LCL)	Gaspar et al. (2003)
3	SOD	SOD-liposome and SOD-enzymosome were used to compare their therapeutic activities on different animal models for oxidative stress and liver ischemia,	Corvo et al. (2015)
4	Catalase	To study controlled oxidation of glucose by encapsulated glucose oxidase with decomposition of H2O2	Yoshimoto et al. (2010)
5	Catalase–sSOD	Stability studies including half-life and activity	Skólmowska and Kmieć (2011)
6	C. utilis Uricase	Comparison between in vitro uricolytic activity of free and PEGylated uricase and in vivo uricase studies of its enzymosome in animal models	Tan et al. (2012)
7	NTP Dase-1	To study the inhibition of platelet activation induced by ADP, collagen or thrombin	Chaikof et al. (2006)
8	β-glucuronidase	Immunoliposome directed against human ovarian cancer cells to study anti-tumor activity	Fonseca et al. (1999)
9	Carboxypeptidase	To study membrane targeting and biological functions	Mima et al. (2006)
10	Carbonic anhydrase	to study protein membrane interaction and refolding of denatured enzyme	Yoshimoto et al. (1998)
11	Glucose oxidase	To study the sensitivity of liposomes for glucose concentration	Jo et al. (2008)

Carbonic anhydrase molecules have been encapsulated inside the liposomes for checking the permeability of some substrates including CO₂ through lipid bilayer (Renggli et al. 2011). This approach has also been applied to study the liposomal reaction kinetics (Ramundo‐Orlando et al. 2007). A novel enzymosome containing uricase and catalase has been prepared recently against anti-hyperuricemia complication, and the use of these enzymosomes has proved to be much efficient than conventional uricase application and traditional chemotherapy (Zhou et al. 2016). In addition to this, these enzymosomes showed higher acid–base, thermal, hypothermal stability and much lowering effects of uric acid (Zhou et al. 2016).

To enhance the utility of superoxide dismutase (SOD), many attempts were made including covalent attachment with albumin, PEG, dextran and encapsulation within liposomes, but it was not quite efficient in its activity in different organs. In comparison with all these different methods of SOD vehiculation, SOD surface liganded liposomes have been found to possess enhanced incorporation efficiency and half-life (Bansal et al. 2011; Corvo et al. 1999). The covalent-liganded form of SOD on the liposome surface is assumed to be more advantageous rather than its encapsulated form, as its release at the inflammation site may not be required to achieve its therapeutic activity (Bansal et al. 2011).

Even though enzymosomes are emerging as novel drug delivery systems, there are some drawbacks with this type of liposome as well. The production cost of enzymosomes is usually higher, and the constituent phospholipids may undergo hydrolysis or oxidation. In some conditions, low solubility and shorter half-life curtail the bioavailability (Bansal et al. 2012).

Conclusion

Development of nanoscale drug transport and delivery setup with liganded liposomes provides an efficient solution to improve disease therapy. Liposomes have played a great role in drug targeting against cancer tissue. Drug distribution by liganded liposome has improved the therapeutics as compared to the free drugs. Free drugs are inefficient in targeting tumors as reticuloendothelial system remarkably reduces its peak levels. Long circulating liposomal formulations, including PEGylated liposomes, are engineered to survive identification and removal by reticuloendothelial system. Liposomal formulation of Doxorubicin, encapsulated in PEGylated liposomes (Doxil), was the first anti-cancer drug, approved by US-FDA. The efficiency of liposome targeting has been drastically improved by surface modification with vitamins, carbohydrates, aptamers and monoclonal antibodies or their fragments directed against cancer-associated antigens. These modified conventional liposomes are efficient in carrying anti-angiogenesis drugs and antisense oligonucleotides (aptamers) for delivery with improved accumulation and prolonged exposure near tumors. Nowadays, there are different liposomal products available in the market which include antibacterial, antifungal and mostly anticancer formulations. In addition to this, liposomal formulations are available as therapeutic agents to treat eye, skin and respiratory disorders. A novel multidrug carrier approach of liposomes like (Gemcitabine and Paclitaxel) has been discovered and treatment of some human diseases with such formulations has shown stronger cytotoxic activity as compared to individual free or liposomal preparations. The future engineered liposomal therapeutics based on different surface modifications are emerging as new pharmacological tools. The technology of surface modification of liposomes will include much better and specific ligands with improved target delivery of specific drugs against different diseases.

Author contributions

AAK and KSA have contributed in concept, design, diagrams and information collection of the work. SAA and AAA has reviewed it thoroughly and AHR has contribution in reviewing and final approval of the work.

Compliance with ethical standards

Conflict of interest

All the authors state that there is no conflict of interest.