Liposome-targeted delivery for highly potent drugs

Abstract

Considerable adverse side effects and cytotoxicity of highly potent drugs for healthy tissue require the development of novel drug delivery systems to improve pharmacokinetics and result in selective distribution of the loaded agent. The introduction of targeted liposomal formulations has provided potential solutions for improved drug delivery to cancer cells, penetrating delivery through blood–brain barrier and gene therapy. A large number of investigations have been developed over the past few decades to overcome pharmacokinetics and unfavorable side effects limitations. These improvements have enabled targeted liposome to meet criteria for successful and improved potent drug targeting. Promising in vitro and in vivo results for liposomal-directed delivery systems appear to be effective for vast variety of highly potent therapeutics. This review will focus on the past decade’s potential use and study of highly potent drugs using targeted liposomes.

Keywords:

• Liposome
• highly potent drugs
• liposome targeted delivery

Introduction

Liposomes abundance utilizations for therapeutic benefit have been continuously expanded due to their flexible structures and practical function since their introduction in 1960s (Balazs and Godbey 2011). Liposomes advantages over other drug delivery systems are their capability for transporting and protecting diverse therapeutic biomolecules including both hydrophilic and hydrophobic drugs, their desirable biocompatibility and biodegradability, different range of morphologies and sizes based on easy manipulation of their composition, low toxicity, nonimmunogenicity and low costs (Tiwari et al. 2012, Voinea and Simionescu 2002). Hence, there are numerous lipid formulations for liposomal drug and gene delivery application. Moreover, constant improvement and modification to control their biological properties and promote their pharmacokinetics characteristics have been investigated via addition of different lipids and targeting moieties (Kong et al. 2012, Sercombe et al. 2015).

Among variety of liposome modifications, active targeting emerges as a useful and common strategy in order to enhance liposomal delivery system’s desired properties such as targeting ability, increased selectivity, increased cellular internalization, prolonged duration of exposure, minimized adverse effects, and improved therapeutic index (Puri et al. 2009, Zamboni 2005). As a result, expanding number of receptors has been significantly progressed to target therapeutic cargo’s delivery in a preferred manner (Deshpande et al. 2013). The different types of liposomal drug delivery systems including conventional, PEGylated, ligand targeted and theranostic liposomes have shown in Figure 1. All types of liposomes enclose an aqueous core and based on their versatile structure, they are capable of carrying both hydrophobic drugs (in their phospholipid bilayer) and hydrophilic drugs (in their lumen) (Daraee et al. 2016, Sercombe et al. 2015). However, low encapsulation efficiency of hydrophilic drugs resulted in undertaking many strategies in order to increase encapsulation such as the preparation method of the liposome, particularly and expansively the cycles of freeze and thaw and the reverse phase evaporation, the proper selection of the composition, the formulation of liposomes with larger particle size, the adjustment of the zeta potential to maximize electrostatic interaction between the charged drug (Eloy et al. 2014).

Figure 1. Schematic representation of the different types of liposomal drug delivery systems including (A) conventional (B) PEGylated (C) ligand-targeted and (D) theranostic liposomes. In addition, the site of hydrophobic and hydrophilic drug placing has shown. (Taken from Susan Hua work, with permission from author and journal) (Sercombe et al., 2015).

In addition to these statements, rapid advances in medicine and biotechnology result in growing number of highly potent compounds to overcome incurable and fatal diseases such as cancers, diabetes, etc. Despite the fast discovery and development race in this field, many highly potent drug candidates have encountered serious barriers because of their limited bioavailability, low solubility, poor efficacy, rapid clearance, low stability, high nonspecific toxicity to healthy organs and increased occurrence of side effects (Basavaraj and Betageri 2014, Naseri et al. 2015). Therefore, the essence for designing and creating of novel drug delivery systems to improve the pharmacokinetics and pharmacodynamics of each therapeutic and to overcome stated delivery obstacles was widely felt. Among available drug delivery platforms, the inherent advantages of liposomes that are mentioned earlier make them the most commonly studied candidate to deliver drugs, proteins, polypeptides, genes, and nucleic acids (Chime and Onyishi 2013). The focus of this review is to provide detailed descriptions of different lately investigated surfaced modified liposomes as a promising targeted delivery system for potent drugs because of recent increased interests in the use of targeted liposomes.

Anticancer drugs

Growing use of potent drugs to overcome fatal disease has become a serious concern. According to their unwanted side effects and damages on healthy and normal tissues, their common and unavoidable usages have resulted in fundamental restriction. Hence, with the advent of extensive adverse effects of highly potent chemical drugs and the significant need to improve their therapeutics, closed phospholipid bi-layered structures called liposome have received a substantial attention as a pharmaceutical carrier during the past 30 years (Deshpande et al. 2013). Using liposome-mediated delivery reduces toxicity and increases efficacy by modifying therapeutic index. Furthermore, merging the concept of passive and active targeting for liposomes has shown promising results such as reduced systemic toxicity, increased cellular uptake, selective drug targeting and controlled release in vitro, in vivo and clinical data. Passive targeting (enhanced permeability and retention (EPR) effect mediated) requires the longevity of the pharmaceutical carrier in the blood by incorporating into a macromolecule or nanoparticle and its accumulation in pathological sites and active targeting requires the conjugation of specific ligand to the surface of pharmaceutical carriers to recognize and bind pathological cells (Rahman et al. 2016, Singh and Lillard 2009). There are several encouraging reports from early clinical applications and clinical trials based on using liposomes for different cancers treatment, Alzheimer's and Parkinson's disease, inflammatory diseases, parasitic diseases, fungal infections, and bacterial infections (Lopez-Berestein and Fidler 1989, Spuch and Navarro 2011).

Cisplatin is a highly potent alkylating antineoplastic agent and commonly used for the treatment of many cancers including melanoma, lung, lymphoma, ovarian, testicular, cervical, bladder, testicular, head and neck cancers. Due to its high toxicity (nephrotoxicity, ototoxicity, and neurotoxicity), designing proper delivery systems during treatments remains still as significant challenges (Apps et al. 2015). Wang et al. have developed conjugated sodium alginate (SA) to cisplatin, incorporated into PEGylated liposomes and were modified with epidermal growth factor (EGF) to specifically target EGFR-expressing tumors (Wang et al. 2014). The in vitro experiments on SKOV3 cells and in vivo xenograft experiments have revealed enhanced delivery of cisplatin and its antitumor efficacy to EGFR-positive ovarian tumor cells that is caused tumor-specific accumulation and induced cell-cycle arrest and apoptosis in a time-dependent manner, as well as reducing nephrotoxicity and lower body weight loss in mice. In addition, Hirai et al. have established a novel in vivo antitumor therapy with Sialyl Lewis^X conjugated cisplatin-loaded liposome to selectively target tumors and to substantially reduce the toxic side effects of high dose of cisplatin (Hirai et al. 2010). In another study, Lv et al. have investigated cisplatin liposome modified with transferrin (TF) in order to target glioma aggressive type of primary intracranial neoplasm and subsequently result into passing of drug through the blood–brain barrier (Lv et al. 2013). Their novel drug delivery system has exhibited potential ability to cross the BBB in a clathrin-mediated endocytosis way and subsequently was targeted to glioma cells and also was resulted in a higher cytotoxicity and much more potent sequential-target to glioma cells in vitro.

Oxaliplatin (trans-l-diaminocyclohexane oxalatoplatinum, l-OHP) is a cisplatin derivative that was designed to improve side effects such as toxicity to the kidney and peripheral nerve system (Apps et al. 2015). Suzuki et al. have described novel target-sensitive TF–PEG-liposomes, called pendant-type PEG immunoliposomes against solid tumors (Suzuki et al. 2008). Their investigation has showed high concentration of l-OHP being maintained in mouse tumor tissue and that tumor growth was more suppressed in various types of tumors that overexpress TF receptors.

Paclitaxel, a potent chemotherapeutic agent and a microtubule-stabilizing drug, is used for the treatment of breast, ovarian, colon, head and neck and nonsmall cell lung cancers (Peltier et al. 2006). A novel float receptor (FR)-targeted liposomal paclitaxel formulation by Wu et al. was composed of DPPC/DMPG/mPEG-DSPE/folate-PEG-DSPE has exhibited excellent colloidal stability and good drug loading properties in addition to the prolonged circulation time and efficiently taken up by FR + KB, human oral carcinoma cells (Wu et al. 2006). In another study, the in vitro and in vivo drug delivery characteristics of a paclitaxel-loaded anti-HER2 PEGylated immunoliposomal formulation have been prepared by Yang et al. which showed efficient and specific delivery of paclitaxel, slower disease progression, and appropriate response rate in patients with human epidermal growth factor receptor-2 (HER2)-overexpressing tumors (Yang et al. 2007).

In the earlier stages of targeting of liposomes with HER2 receptor, one of the thoroughly studied models for immunoliposomal formulations was the application of the whole part of mAb as a target moiety, but advances in antibody engineering have allowed the use of antibody fragments such as Fab′ and single-chain fragment variable (scFv) as targeting agents (Kontermann 2006, Park et al. 2002). scFvs are recombinant molecules in which the variable regions of heavy and light immunoglobulin chains encoding antigen-binding domains are engineered into a single polypeptide (Nelson 2010). In recent years, scFv has gained popularity over traditional targeting agents because of several potential advantages including slower clearance, reduce immunogenic risk (engineer fully human fragments), lower production cost, using phage display and select desired affinity and specificity, better identification and purification with engineering tags into scFv constructs (Cheng and Allen 2010).

Tamoxifen (TAM) is an antagonist of the estrogen receptor in breast tissue. It has been used in hormone (antiestrogen) therapy for early breast cancer and other types of cancer to block the actions of estrogen and also is able to reverse multidrug resistance (MDR) protein efflux of drugs (Sugimoto et al. 2003). Epirubicin is a new potent anthracycline derivative of doxorubicin (DOX) used for chemotherapy and a broad-spectrum antineoplastic agent. Epirubicin has been clinically used in treating parenchymal organ cancers, breast cancer, ovarian cancer, non-Hodgkin’s lymphomas, pancreatic cancer, soft-tissue sarcomas, acute leukemia, gastric cancer, small-cell lung cancer plus inhibiting brain tumor cells in vitro (Safra 2003). Tian et al. have developed a new kind of functionalized epirubicin liposomes, modified with two targeting ligands, TAM and TF which significantly transport drug-loaded liposomes across the blood–brain barrier (BBB) and then target the brain glioma cells in vitro and in animals (Tian et al. 2010). Another newly liposomal nanostructure of functional targeting epirubicin, dually modified with aminophenyl glucose and cyclic pentapeptide, was made by Zhang et al. to treat brain glioblastoma (Zhang et al. 2015). The designated nanocarrier have exhibited the ability to be transported across the blood–brain barriers via the carrier-mediated transcytosis, efficient in killing glioblastoma cells and in destroying glioblastoma neovasculature in vitro and in vivo, targeting effects via the integrin β3 receptor and prolonged circulation in the blood system which presented functional targeting epirubicin liposomes as an effective therapy for disabling neovascularization in the brain glioblastoma region and treating brain glioblastoma.

Lonidamine a derivative of indazole-3-carboxylic acid is a potent apoptotic inducer of cancer cells. It had been used to treat nonsmall-cell lung carcinoma (NSCLC), benign prostate hypertrophy (BPH) metastatic, brain tumors, and breast cancer. It has revealed a serious side effect on liver. Li et al. have developed mitochondria-specific targeting lonidamine liposomes modified with a dequalinium polyethylene glycol distearoylphosphatidylethanolamine (DQA-PEG2000-DSPE) conjugate as a cotherapy with targeting epirubicin liposomes to circumvent drug-resistant lung cancer and NSCLC (Li et al. 2013). The results have suggested enhanced accumulation in tumor tissue via the long-circulatory effect and reduced immediate aggregation in live tissue.

Doxorubicin, an anthracycline antitumor antibiotic used in chemotherapy for the treatment of wide range of cancers, including breast, ovary, gastric, liver, kidney, endometrial, blood cancers like leukemia and lymphoma and many types of carcinoma (solid tumors) and soft-tissue sarcomas. It has showed serious adverse effects including threatening heart damage, nausea or vomiting, low blood counts, hair loss which were consider as its limiting factor for the usage of this common anticancer drug (Safra 2003). Gabizon et al. and Li et al. have prepared doxorubicin-loaded stealth liposomes conjugated transferrin that have revealed prolonged residence time in the circulation and low reticulo-endothelial system (RES) uptake in tumor-bearing mice. All these were resulted in enhanced extravasation of the liposomes into the solid tumor tissue and enhanced intracellular uptake of the entrapped DOX and improved therapeutic efficacy of liver cancer (Gabizon et al. 2004, Li et al 2009). Based on Tuscano et al. report, xenograft mouse model of delivering CD22-targeted PEGylated liposomal doxorubicinin a B-cell non-Hodgkin's lymphoma have exhibited improved cytotoxicity, higher drug accumulation in tumor and greater tumor volume reduction, so that survival was occurred more effectively than nontargeted liposomal DOX (Tuscano et al. 2010). Moreover, in recent years, aptamers with the highest binding affinity and specificity as novel strategies and promising molecules in drug delivery are the most frequently used ligands for targeting (Nimjee et al. 2005). In this regard, Song et al. have developed a novel DOX-loaded DOTAP:DOPE liposomes delivery system by conjugating a target tumor cell-specific aptamer that was selected among 10 potential generated aptamers. In vitro and in vivo studies on the selected aptamer (SRZ1) have revealed promoted uptake efficiency, enhanced DOX accumulation, highest and specific binding affinity to 4T1 cells and significant suppression of tumor growth in tumor-bearing mice (Song et al. 2015).

Additionally phase-1 clinical study of anti-EGFR immunoliposomes loaded with DOX (anti-EGFR ILs-DOX) in patients with solid tumors was assessed by Mamot et al. where produced nanobody-targeted liposomes in the dose of 50 mg doxorubicin per m² have showed promising antitumor activity and the drug was well tolerated (Mamot et al. 2012). Since nanobodies discovery in 1993, a number of variable domain of heavy-chain antibodies with (1) high specificity and affinity (2) more stability and selectivity (3) low toxicity and immunogenicity (4) ease of production, are being utilized as therapeutic targets in many fields of medicine (Oliveira et al. 2013, Steeland et al. 2016).

In addition, Van der Meel et al. have utilized simultaneous liposomal targeting, using EGFR and insulin-like growth factor 1 receptor (IGF-1R) loaded with kinase inhibitor (AG538). This multivalent nanobody liposomal system loaded with kinase inhibitor for cancer treatment was able to block both EGFR and IGF-1R activation and induce a strong inhibition of tumor cell proliferation (van der Meel et al. 2012).

According to serious side effects of DOX and its extensive use, the necessity of designing effective delivery system and improving selective localization of this commonly used chemotherapy drug to reduce its cytotoxicity, plenty of researches have been reported for innovative targeted liposomal doxorubicin formulation (Chang et al. 2013, Mamot et al. 2003, Medina et al. 2011, Paliwal et al. 2011, Pastorino et al. 2003, Wang et al. 2012).

Daunorubicinis chemotherapeutic of the anthracycline antitumor antibiotic family and widely used in treating a variety of malignancies and specific types of leukemia (acute myeloid leukemia and acute lymphocytic leukemia). However, its clinical utility is hampered by the myelosuppression, cardiotoxicity and resistance. Ying et al. have prepared novel dual-targeting daunorubicin PEGylated liposomes by conjugating with p-aminophenyl-α-D-mannopyranoside (MAN) and TF which transported drug across the BBB to treat brain glioma (Ying et al. 2010). This liposomal systems have exhibited significant increase in transport of the drug-loaded liposomes across the BBB model and antineoplastic effect to C6 glioma cells in vitro and enhanced median survival time on C6 glioma-bearing rats in vivo. In another study, Zhang et al. have developed mitochondrial-targeting liposomes that incorporate daunorubicin plus quinacrine for relapsed breast cancer treatment which is arised from the cancer stem cells (Zhang et al. 2012). The designated system has showed well-distributed particle size with high encapsulation efficiency and delayed drug release besides long circulatory effect and selective accumulation in mitochondria that eventually cause apoptosis of MCF-7 cancer stem cells. Daunorubicin-loaded folate polyethylene glycol–cholesterol hemisuccinate (folate–PEG–CHEMS) anchored liposomes is another study that have developed by Xiong et al. (2011). The novel targeted liposomal daunorubicin has showed improved in vitro cytotoxicity effect on L1210JF cells and in vivo significant prolonged life span on an L1210JF murine leukemia tumor model and enhanced in vitro and in vivo endocytosis of liposomes into L1210JF cells via FR cell-specific delivery.

Deferoxamine (DFO), a potent iron chelator used to remove excess iron from the body. With its increased usage due to its short half-life and poor absorption, considerable toxicity for cardiovascular and pulmonary has been seen. Thus, an efficient delivery system that intensifies its cell-specific bioavailability is essential. Schuster et al. have developed DFO-loaded PEGylated immunoliposomes that specifically targets fibroblast activation protein (FAP) by single-chain fragment variable (scFv) (Schuster et al. 2015). It has exhibited specific binding and uptake into FAP-expressing cells and significant reduction in the collagen deposition. There are also further examples of various highly potent drugs that were applied in the form of immunoliposomes to (1) increase specificity of cells, (2) decrease cytotoxicity (3) provide high rates of uptake (4) enhance drug delivery, such as indinavir, vincristine, ceramides, mitoxantrone, mitoxantrone, irinotecan that were explained briefly in Table 1.

Table 1. Highly potent chemical drugs delivered using targeted liposomes.

Drug	Targeting ligand	Receptor	Explanation	References
Cisplatin	– Tf. – 26-mer DNA aptamer – cRGDyk (RGD peptide)	– TfR – Nucleolin (NCL) a bcl-2 mRNA-binding protein – Integrin receptors	Binding to DNA and results in DNA strands cross-linking and apoptosis	[Iinuma et al. 2002, Krieger et al. 2010] [Cao et al. 2009] [Wang et al. 2014]
Oxaliplatin	– Cetuximab (CTX), a mAb – Tf	– epidermal growth factor receptor (EGFR) – TF receptor	A platinum-based antineoplastic agent that inhibits DNA synthesis in cells	[Zalba et al. 2015] [Maruyama 2011]
Paclitaxel	– Herceptin – Hyaluronic acid (HA) – Folate – RGD peptide	– HER2 – Cluster of differentiation 44 (CD44) – Folate receptor (FR) – Integrin receptors	A cytoskeletal drugs that target tubulin and generally cause apoptosis	[Yang et al. 2007] [Ravar et al. 2016] [Wu et al. 2006] [Zhao et al. 2009]
Epirubicin	– C225-Fab′ – PTD (HIV-1) peptide– Tf	– EGFR or EGFR variant III (EGFRvIII) – Heparan sulfate receptors and the positively charged amino acids – TfR	Intercalating DNA strands result in inhibiting DNA and RNA syntheses which lead to cell death.	[Mamot et al. 2005] [Ju et al. 2014] [Gandhi et al. 2015]
Lonidamine	– Dequalinium (DQA)	– Mitochondrial molecule	Inhibitboth respiration and glycolysis under conditions of hypoxia leading to a decrease in cellular ATP and causes apoptosis.	[Li et al. 2013]
Doxorubicin	– hCTMO1 mAb – Tf – Folic acid – Hyaluronan (HA) – RNA aptamer	– MUC-1 antigen – TfR – Float receptor – CD44 – Prostate-specific membrane antigen (PSMA)	Intercalating DNA and inhibit macromolecular biosynthesis and stop the process of DNA replication.	[Al-Ahmady et al. 2014] [Wu et al. 2007] [Goren et al. 2000] [Eliaz and Szoka 2001] [Baek et al. 2014]
Daunorubicin	– P-aminophenyl-α-D-manno-pyranoside(MAN) and Tf	– Stereo-specific glucose carriers and TfR	Act similar to doxorubicin and slows or stops the growth of cancer cells in the body.	[Ying et al. 2010]
Deferoxamine	– Float	– Float receptor	Binding iron and inactivates P4H and interferes with the collagen synthesis machinery and reduce excessive collagen deposition.	[Mathias et al. 1996]
Indinavir	– Anti-HLA-DR Fab□ fragments	– HLA-DR-expressing cells	A protease inhibitor used as a highly active antiretroviral component.	[Gagné et al. 2002]
Vincristine	– mBAFF – Anti-CD19 IgG2a	– BAFF receptor – CD19 epitope at the cell surface	During the metaphase binds to the tubulin protein and stops the cell from separating its chromosomes leading to apoptosis.	[Zhang et al. 2008] [Sapra et al. 2004]
Ceramides	– Tf	– TfR	Enhances membrane rigidity and stabilizes smaller lipid platforms and participates in a cellular signaling, includes cell death	[Koshkaryev et al. 2012]
Mitoxantrone	– LHRH analogs	– LHRH receptors	A type-II topoisomerase inhibitor; disrupts DNA synthesis and DNA repair	[He et al. 2010]
Irinotecan	– Folic acid – Octreotide	– Float receptor – Somatostatin receptor	Prevents DNA from unwinding by inhibition of topoisomerase	[Zhang and Yao 2012] [Iwase and Maitani 2010]

Peptide/protein drugs

Until now, US Food and Drug Administration (FDA) have approved more that 200 proteins and peptides as therapeutic modalities to treat variety of human diseases based on their high potency and specificity besides less toxicity to normal tissues (Cryan 2005). Hence, designing an appropriate delivery system for them become a challenge due to their (1) immunogenicity, (2) inadequate stability and rapid clearance, (3) short plasma half-life and shelf-life, (4) low penetration across biological membranes (Lu et al. 2006). Therefore, among different novel drug delivery systems, liposomes are one of the most promising systems that have the potential to overcome current limitations and consider as next generation protein therapeutics (Pisal et al. 2010). Protein and peptide drugs can be either encapsulated or chemically conjugated to the surface group of liposomes (Martins et al. 2007). Long circulation and target sensitive liposomes are two approaches that were followed extensively to obtain liposomal protein and peptide delivery; however, considerable researches have been created for active and antibody-targeted structures (Huang and Zhou 1992, Lu et al. 2006).

Hua et al. developed tissue plasminogen activator (tPA) loaded microbubbles and targeted by arginine–glycine–aspartic acid–serine peptide for the ultrasound-assisted drug thrombolysis. Results have represented that in vivo tPA release and thrombolytic efficacy were promising which was lead to significant hemorrhagic risk decreases (Hua et al. 2014). Mastrobattista et al. have also entrapped pH-dependent fusogenic peptide (diINF-7) together with diphtheria toxin A chain (DTA) inside immunoliposomes, targeted to the epidermal growth factor receptor (EGFR) on the surface of ovarian carcinoma cells. Their results have showed great enhance in cytosolic delivery of immunoliposome-entrapped proteins by pH-dependent destabilization of endosomal membrane after cellular uptake of liposomes (Mastrobattista et al. 2002).

As already mentioned elsewhere passive targeting besides pegylation of liposomal protein and peptide are prevalent and well-worked structures to overcome their instability and short half-life (Deshpande et al. 2013, Milla et al. 2012). The preclinical efficacy of the cytokine-loaded liposomes to deliver interleukin-12 (IL-12) into several human tumors and to reactive tumor-associated T cells in situ by Simpson-Abelson et al. have exhibited intratumoral liposome IL-12 delivery and local and sustained release of IL-12 from liposomes, which causes reactivates resident lymphocytes to secrete IFN-γ in different epithelial tumor types (Simpson-Abelson et al. 2009). Based on Kanaoka et al. report, liposomal IL-2 resulted in better distribution, enhanced the duration time in the systemic circulation and improved therapeutic effect against tumor after subcutaneous administration to mice (Kanaoka et al. 2003).

In another study, Kisel et al. have prepared liposomal carrier for oral delivery of insulin. In vitro oral administration of liposomes resulted in hyperinsulinemia that was attended by a decrease in blood glucose concentration as a consequence of interlocking of receptors with highly resistant lipid–insulin complexes (Kisel et al. 2001).

Also another recent crucial development in the field of liposomal protein delivery systems is simultaneous administration of multiple therapeutic agents such as chemotherapeutics and proteins for achieving enhanced therapeutic effects and elevated tumor inhibition efficiency in treatment of different types of cancers (Eldar-Boock et al. 2013, He et al. 2016). Cabanes et al. have assessed in vivo therapeutic effect of Doxil and liposomal IL-2 which was resulted in long-term survivors against M109 pulmonary metastases in mice. Their research has showed that synergistic liposome-based chemoimmunotherapy is highly efficacious approach to eradicate regionally spread tumors and metastasis (Cabanes et al. 1999). Accordingly, delivery of the dual or multiple cargos, such as protein and peptides beside chemotherapeutic drugs, will enhance sensitivity of tumors to therapeutic agents and reduced dose of toxic drugs and adverse effects (Mo et al. 2014). In Table 2, there are given some extra examples of targeted and non-targeted liposomal protein delivery systems.

Table 2. Highly potent protein and peptide drugs delivered using targeted liposomes.

Active targeted liposomal protein and peptide
Protein/Peptide	Target moiety	Therapy	References
RGD peptide plus streptokinase	RGD	Arterial thrombi	[Vaidya et al. 2011]
Monoclonal IgG2a antibody 34A	Monoclonal IgG2a antibody 34A	Lung cancer	[Maruyama et al. 1997]
Monoclonal IgG1, antibody 21B2	Monoclonal IgG1, antibody 21B2	Solid tumors	[Maruyama et al. 1997]
EYFP and CNA35	Cysteine		[Reulen et al. 2007]
Growth factors such as NTs, GDNF, VEGF, FGF2, EGF	TAT, RVG, and transferrin receptor antibody	Ischemic stroke	[Rhim and Lee 2016]
Eptifibatide	RGD	Atherosclerosis	[Bardania et al. 2016a,b]
Codelivery/passive targeted liposomal protein and peptide
Protein/peptide	Drugs	Therapy	References
IL-2 plus transforming growth factor-β (TGF-β)^a		Tumor immunotherapy	[Park et al. 2012]
γ-Interferon (INF-γ)	Cisplatin	Cancer of the sigmoid colon andmetastatic nodules in the liver	[Kohara et al. 1995]
Tumor necrosis factor-a (TNF)	Liposomal DOX	Soft-tissue sarcoma	[Ten Hagen et al. 2002]
IL-2	–	Hepatic metastases	[Kanaoka et al. 2002]
Calcitonin	–	Assessment of mucoadhesive function for oral administration	[Takeuchi et al. 2003]
Vasoactive intestinal peptide (VIP)	–	Chronic ocular pathologies such as autoimmune uveitis	[Lajavardi et al. 2009]
Asialoerythropoietin (AEPO)	–	Cerebral ischemia–reperfusion (I/R)	[Ishii et al. 2012]
Insulin	–	Diabetes mellitus-oral route-intestinal route-pulmonary route	[Jain et al. 2007] [Bi et al. 2008] [Ramadas et al. 2000]
Recombinant IL-2	–	Immunoregulatory response in cancer	[Kanaoka et al. 2001]
Myelin basic protein (MBP)	–	Allergic encephalitis	[Belogurov et al. 2013]
Superoxide dismutase (SOD)	–	Rheumatoid arthritis, cancer, and respiratory distress syndrome	[Xu et al. 2012]
CD39	–	Thromboregulatory	[Haller et al. 2006]

^a TGF-β receptor-I inhibitor represents a role of a small hydrophobic protein drug molecule via blockade signaling and its combination with IL-2 in liposomes, induce antitumor responses.

miRNA, siRNA, and DNA therapeutics

The potential use of gene therapy via viral and nonviral vectors holds great promise and has been equipped modern medicine with new achievements that were unthinkable two decades ago (Mali 2013). High safety, low immunogenicity, large capacity and also possibility of repeat administration of nonviral vectors in comparison to virus-derived vectors, has made them as a popular choice in targeted transgenic delivery system (Ramamoorth and Narvekar 2015).

The first common nonviral gene transfer vectors were naked-DNA or plasmid DNA (pDNA) that was placed in liposomes in order to enhance cellular DNA uptake (Al-Dosari and Gao 2009). Recently, siRNAs, microRNAs, single-stranded antisense oligonucleotides (ODNs) and ribozymes as four major types of anti-mRNA strategies have been broadly used in the treatment of cancers, infections, hematological diseases, antiviral diseases, neurodegenerative diseases, dominantly inherited genetic disorders, cardiovascular disorders, and pain research and many other illnesses (Majzoub et al. 2016, Movahedi et al. 2015). Since application of small interfering RNA (siRNA) as a potent sequence-selective inhibitors of transcription, encountered with serious limitation due to (1) strong electrostatic repulsion between anionic cell surface charge and anionic phosphate backbone charge of naked siRNA (2), activating immunogenic responses (3), poor tissue and cell membrane penetration (4), low transfection efficiency (5), rapid clearance and poor pharmacokinetic property (6), arising unfavorable biological responses such as off-target effects and interferon response (Kanasty et al. 2013). Therefore, the essence of designing efficient delivery system in order to deliver and internalize the siRNA to the cells of target tissue still remains a challenge (Shim and Kwon 2010). Hence, various successful examples of liposomal delivery systems have been developed contain using cationic or ionizable lipids, shielding lipids, cholesterol, and targeting ligands in order to achieve cell-specific delivery system (Huang and Liu 2011, Whitehead et al. 2009). Among them, the ability of targeted anti-mRNA liposomal delivery systems in covering protection of entrapped anti-mRNA formulation from renal clearance and nuclease degradation besides providing efficient cellular uptake and endosomal escape is of utmost importance (Gandhi et al. 2014, Pereira et al. 2013).

Asai et al. have developed tetraethylenepentamine-based polycation liposomes, active targeted with Ala-Pro-Arg-Pro-Gly (APRPG-TEPA-PCL) for the systemic delivery of siRNA used for targeting angiogenic endothelium. Intravenous injection of APRPG–TEPA–PCL into Colon26 NL-17 carcinoma-bearing mice have showed liposome accumulation in the tumor, which have confirmed APRPG–PEG conjugation to liposome and it was a useful strategy for both drug- and gene-targeted delivery to angiogenic vessels in tumors (Asai et al. 2011). In another study, Sato et al. have demonstrated potential therapeutic efficacy of encapsulated siRNA, vitamin A–coupled liposomes for reversing human liver cirrhosis. Their study has exhibited prolonged survival time and liver fibrosis resolve in rat (Sato et al. 2008). In addition, Feng et al. have concluded that folate receptor-targeted liposomes, delivering MYCN siRNA into LA-N-5 cells, have efficient suppressive effect on MYCN mRNA expression both in vitro and in vivo and capable of treating various tumors (Feng et al. 2010). In another study, Yu-Wai-Man et al. have optimized receptor-targeted liposome peptide nanoparticles incorporating MRTF-B siRNA to develop a safe and efficient nonviral siRNA delivery system to prevent fibrosis after glaucoma filtration surgery and other contractile scarring conditions in the eye. Their results suggested that after a single transfection treatment the MRTF-B gene in human Tenon’s fibroblasts was efficiently silenced and collagen matrix contraction was completely blocked (Yu-Wai-Man et al. 2016). Furthermore, Li et al. have developed aptamer-targeted PEGylated liposome (AS1411ePEG-liposome) to deliver anti-BRAF siRNA (siBraf) for melanomas treatment. AS1411, an aptamer as a targeting probe was resulted in the inhibition of the melanoma growth in vitro and in vivo because of higher selective accumulation of the siRNA in tumor cells comparing with normal cells and significant siRNA silencing activity in A375 tumor xenograft mice (Li et al. 2014).

Over the past decade, among small RNAs, microRNAs (miRNAs) approximately 21-nucleotide-long, have become a major focus of researches in target gene therapy by gene silencing via the recruitment of corresponded mRNA into the RNA-induced silencing complex and cleavage of the target mRNA resulting in selective inhibition of expressed unwanted protein (Mourelatos 2008, Zhang et al. 2012). The most common nonviral strategy for disease treatment thorough miRNAs transfection is liposomal delivery system that frequently has been used both in vitro and in vivo however understanding the molecular mechanisms of these therapeutic vehicles needs substantial investigation in order to be employed in clinical trials (Zhang et al. 2012). Cyclic RGD peptide (cRGD)-functionalized a PEGylated LPH (liposome–polycation–hyaluronic acid) nanoparticle for the targeted delivery of anti-miRNA antisense oligonucleotides for antiangiogenesis therapy has been reported by Liu et al. In vitro and in vivo assessments of cRGD-modified LPH nanoparticles have exhibited effective antiangiogenesis activities of suppressing endothelial cell migration and blood tube formulation (Liu et al. 2010). Also Liu et al. have investigated in vitro and in vivo tumor-targeting ability of IL-4Rα aptamer–liposome–CpG oligodeoxynucleotide (ODN) delivery system. The IL-4Rα–aptamer–liposome–CpG has demonstrated better tumor distribution and enhanced antitumor activity compared to the control group in vitro and in vivo. The developed immunotherapy system has inhibited distinct myeloid-derived suppressor cell populations in tumors and bone marrow and improved immunotherapy efficacy (Liu et al. 2016). In another study, Pore et al. have described new strategy for gene therapy by using glucocorticoid receptor (GR)-targeted liposome, delivering a specially designed artificial miRNA-plasmid against Hsp90 (amiR-Hsp90). Their straightforward strategy for simultaneous targeting of multiple pro-cancerous factors, have shown significant anticancer effect without eliciting nonspecific side effects on two different tumor models in mice (Pore et al. 2013).

Similar to siRNA, naked-DNA or pDNA allows the treatment of many diseases at their root cause. However, they also suffer from the low efficiency of penetration into the nucleus (Moghimi et al. 2016). Therefore, much progress has been made in recent decades after investigation of lipid-mediated gene delivery systems (Felgner et al. 1987). As a result, highly efficient formulations for in vitro applications of liposomal-based gene delivery system have been evaluated; however; designing appropriate synthetic vectors for in vivo applications remains a challenge (Ewert et al. 2016). Reddy et al. have demonstrated folate-targeted cationic liposomal vectors containing protamine-condensed plasmid DNA, can significantly enhance folate-mediated endocytosis and target transgenes to FR expressing cancer cells both in vitro and in vivo. They have also concluded from gene therapy of peritoneal cancers with folic acid (FA)-targeted cationic lipid that this novel liposomal vector can be a more efficient alternative to conventional chemotherapy (Reddy et al. 2002). In another study, Gira˜o da Cruz et al. have designed transferrin-associated lipoplexes to transfer DNA into C6 glioma cells and primary hippocampal and cortical neurons. Their results were promising for enhanced internalization and transfection of Tf-associated lipoplexes by neuronal cells and approved usefulness of these lipid-based carriers to transfer gene to cells (da Cruz et al. 2004). In addition, Simo˜es et al. have applied transferring with cationic liposome/DNA complex to gene therapy. Their study have revealed high levels of transfection besides having the advantage of being active in the presence of serum and being nontoxic due to application of the ternary complexes of transferrin/cationic liposomes/DNA (Simoes et al. 1998). And in the last example, Charoensit et al. have demonstrated incorporation of All-trans–retinoic acid with IL-12 plasmid DNA into cationic liposome (ATRA-cationic liposome/IL-12 pDNA complexes) which was significantly improved therapeutic efficacy in metastatic lung tumor after intravenous injection in a mouse model (Charoensit et al. 2010).

In addition to targeted liposomal gene delivery systems, targeted codelivery of potent drugs (especially chemotherapeutic drugs for cancer treatment) and therapeutic gene in order to suppress cancer growth besides reducing adverse effects of chemotherapeutics and improve the chemo-sensitivity, have been shown to be successful (Glasgow and Chougule 2015). Chen Wang et al. have investigated nanoscale liposomal codelivery of siRNA and DOX targeted with NGR (aspargine–glycine–arginine) peptide as a new strategy for cancer therapy and to overcome the drug resistance of cancer cells. They have accomplished novel therapeutic agent against drug-resistant tumors, which was successfully enhance drug potency (Chen et al. 2010). Based on Wang et al. report, codelivery of DNA and DOX incorporated into float-coated PEGylated liposomes, have showed effective DNA-binding ability, high gene transfection efficiency and sustained drug release profile (Wang et al. 2010). In another study, Skandrani et al. have prepared lipid nanoliposome functionalized with polyethyleneimine (PEI) moieties for codelivery of pDNA and paclitaxel. They have demonstrated the codelivery of paclitaxel with PEI/pDNA, efficiently internalized both cargo into cells and loading nanovectors with fluorescent molecules create multiactive vectors for theranostics in gene therapy (Skandrani et al. 2014). In Table 3, there are given several other important examples of liposomal potent and specific gene delivery systems which also include miRNA and oligonucleotides.

Table 3. Highly potent nucleic acid therapeutics delivered using targeted liposomes.

Therapeutic gene	Targeted moiety	Application	References
siRNA	– Vitamin A – RGD peptide Aptamer	– Fibrosis associated with chronicpancreatitis – Cancer RNAi therapy melanomas treatment	[Ishiwatari et al. 2013] [Yonenaga et al. 2012]
Plasmid DNA	– Transferrin – HIV-1 Tat protein – Folate – RI7/8D3 MAbs – TAT peptide – Anti-TfR scFv – RGD peptide	– Amplify transfection efficacy in gene delivery via enhancement in endocytosis and fusion – Cell-specific gene transfer – Human oral and prostate cancer treatment – Targeted gene therapy including vector-specific transient transfection – Localized in vivo gene therapy and transfection with various protocols of cell – Systemic gene therapy of various human cancers – Gene therapy	[Simoes et al. 1999] [Hyndman et al. 2004] [Hattori and Maitani 2004] [Rivest et al. 2007] [Torchilin et al. 2003] [Xu et al. 2002] [Majzoub et al. 2014]
miRNA/siRNA	– GC4 scFv	– Targeted cancer therapy	[Chen et al. 2010]
Oligonucleotides	– CD20 Ab (rituximab) – Transferrin Aptamer	– Treatment of chronic lymphocytic leukemia – Treatment of Acute myelogenous leukemia (AML) and chronic myeloid leukemia treatment selectively targeting and introducing CpG into CT26 carcinoma cells,	[Yu et al. 2013] [Mendonça et al. 2009, Zhang et al. 2009] [Liu et al. 2016]
Codelivery
Gene + Drug	Target moiety	Application	References
siRNA + DOX	– Anisamide (AA) ligand	– Overcome drug resistance in cancer treatment	[Chen et al. 2010]
GFP plasmid + calcein	– Folate ligand	– Drug delivery and gene transfection	[Javadi et al. 2013]
siRNA + DOX	– Folate receptor	– Tumor targeting and cancer treatment	[Yang et al. 2014]

Targeted liposomes under clinical trials

Immunoliposomes are one of the pioneer examples used in clinical trials and they have had the greatest impact especially in cancer treatment to date. Although there are some drawbacks for immunoliposome-based drug delivery systems in clinical development, prospects of further clinical acceptance of them are gradually evaluated by newer approaches (Basavaraj and Betageri 2014, Eloy et al. 2014). Examples of ligand-targeted nanoliposome formulations undergoing clinical evaluation were presented briefly in Table 4.

Table 4. Examples of ligand-targeted nanoliposome formulations undergoing clinical evaluation (Goodall et al. 2015, van der Meel et al. 2013).

Ligand	Product name	Bioactive compound	Type of drug	Clinical indication	Phase
Antitransferrin receptor scFv	SGT-53	p53 gene	Nucleic acid	Solid tumors	Phase II
Antibody fragment (Fab′)	Anti-EGFR ILs-DOX	Doxorubicin	Chemical	Solid tumors	Phase I
Single domain antibody (dAb) fragment (VH)	Lipovaxin-MM	Melanoma antigens + IFNγ	Proteins	Melanoma vaccine	Phase I
Transferrin	MBP-426	Oxaliplatin	Chemical	Gastroesophageal adenocarcinoma	Phase II
Antibody fragment (scFv)	MM-302	Doxorubicin	Chemical	Breast cancer	Phase I
Antibody fragment (scFv)	SGT-94	RB94 plasmid DNA	Nucleic acid	Solid tumor	Phase I

Conclusions

Highly standard approaches in pharmacology have been achieved via current progressions in the field of liposomal drug delivery systems. Targeted liposomal formulations for highly potent drugs present an ideal strategy to overcome potent therapeutics barriers such as off-target distribution and lack of efficacy. Feasibility of novel therapeutic concepts with desired attributes have been provided via substantial improvements in liposomal-based delivery system’s preclinical studies. Continual advances in application of targeted liposomal delivery systems for novel potent therapeutics point to the need for consideration of penetration and localization of sufficient drug content to modify therapeutic impacts. Therefore, with new targets and drugs being evaluated, necessity for improving drug stability and exposure besides drug accumulation enhancement in the target tissue is expanding. In this context, advance in the design of novel-directed liposomal delivery platform facilitates fast and effective therapeutic response and led to further improvement in liposome’s future clinical applications concept for potent drug, protein, and gene carriers. In the near future with new targets and drugs being evaluated, the applications of directed liposomes will undergo a growth; however, practical and affordable application of them are still remained a challenge in late-stage clinical trials.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.