Functional liposomes in the cancer-targeted drug delivery.

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Nanotechnology in Biomaterials

Functional liposomes in the cancer-targeted drug delivery

Journal of Biomaterials Applications 0(0) 1–14 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328215578111 jba.sagepub.com

Dena Tila1, Saeed Ghasemi2, Seyedeh Narjes Yazdani-Arazi3 and Saeed Ghanbarzadeh3,4

Abstract Cancer is considered as one of the most severe health problems and is currently the third most common cause of death in the world after heart and infectious diseases. Novel therapies are constantly being discovered, developed and trialed. Many of the current anticancer agents exhibit non-ideal pharmaceutical and pharmacological properties and are distributed non-specifically throughout the body. This results in death of the both normal healthy and malignant cells and substantially leads to accruing a variety of serious toxic side effects. Therefore, the efficient systemic therapy of cancer is almost impossible due to harmful side effects of anticancer agents to the healthy organs and tissues. Furthermore, several problems such as low bioavailability of the drugs, low drug concentrations at the site of action, lack of drug specificity and drug-resistance also cause many restrictions on clinical applications of these drugs in the tumor therapy. Different types of the liposomal formulations have been used in medicine due to their distinctive advantages associated with their structural flexibility in the encapsulation of various agents with different physicochemical properties. They can also mediate delivery of the cargo to the appropriate cell type and subcellular compartment, reducing the effective dosage and possible side effects which are related to high systemic concentrations. Therefore, these novel systems were found very promising and encouraging dosage forms for the treatment of different types of cancer by increasing efficiency and reducing the systemic toxicity due to the specific drug delivery and targeting. Keywords Functional liposomes, cancer, drug targeting, tumor therapy, drug specificity

Introduction Cancer is essentially a pathology with various mechanisms and a wide variety of therapies has been developed for the treatment of these diseases over the past few decades. Despite the presence of many effective chemotherapeutic agents, existing anticancer drugs have non-ideal pharmaceutical and pharmacological properties including low aqueous solubility, irritant properties, lack of stability, rapid metabolism, unfavorable pharmacokinetics and non-selective drug distribution, which can lead to a number of adverse consequences. Due to the lack of optimal therapeutic activity, dose-limiting side effects and poor patient’s life quality, effective cancer therapy remains an important challenge. Therefore, the efficacy of cancer treatments is determined by the ability to balance their benefits against their toxicity.1,2 The use of targeted drug delivery systems can improve the pharmacological properties of loaded drugs by modifying their pharmacokinetics and biodistribution. Tumor-specific

drug delivery systems have become increasingly interesting in cancer therapy, as the use of chemotherapeutics is often limited due to the severe side effects. Conventional drug delivery systems have shown low efficiency, and as a result, continuous search for more advanced drug delivery systems have attracted great importance. Lipid-based vesicular systems such as liposomal drug delivery systems have been investigated to help 1

Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 2 Department of Medicinal Chemistry, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran 3 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 4 Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran Corresponding author: Saeed Ghanbarzadeh, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. Email: [email protected]

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therapeutic agents accumulate at specific disease sites in the body. Liposomes, initially called smectic mesophases, are unilamellar or multilamellar spherical vesicles and primarily included phospholipids, either from plant or animal source. They were first discovered by A.D. Bangham at the Agricultural Research Council Institute of Animal Physiology at Babraham, Cambridge, when he and his coworkers observed that phospholipids formed spherical, self-closed vesicles with concentric lipid bilayers and hydrophilic inner core over dispersion in water spontaneously. For the first time, in 1960s, Bangham et al. used a liposomal structure as a model to study the effect of narcotics on lipid bilayer membranes and a few years later, Allison and Gregoriadis described the use of a similar liposomal system as an immunological adjuvant.3–7 Because of the biocompatibility, biodegradability, low toxicity and immunogenicity, as well as the capability for incorporation of the various molecules at high concentrations, and protection of the biologically active molecules by the lipid bilayer (from chemicals and enzymes), liposomal formulations are promising and encouraging drug carriers for drug delivery. Furthermore, due to the high formulation versatility, where their surfaces can be easily modified with a variety of functional devices, liposomes have attracted the greatest interest as encouraging pharmaceutical carriers for several practical applications.4,8–10 However, no drug delivery system shows ideal characteristic and this is the case with liposomes as well. As liposomes are used to enhance the efficacy of a drug, the cost of production and all of the other implications must be taken into account. The high cost of production is because of the high costs associated with the raw materials used in lipid excipients and expensive equipment needed to manufacturing.11,12 In some cases, liposomal formulations such as the cationic formulations tend to be cytotoxic especially when liposomal doses are very high. Lack of targeted therapy and appearance of adverse effects such as the hand-foot syndrome after intravenous administration of anti-cancer liposomes are another complication in the administration of liposomal formulations. Other problems in manufacturing of liposomal formulations are sterilization, batch to batch reproducibility, low drug entrapment, particle size controlling, production of large batch sizes, short shelf life and stability, as well as removal from circulation by the reticuloendothelial system.11,13–15 Liposomebased drug formulations have not come into the market in great numbers up to now. For pharmaceutical products to be available for the market, it is necessary to be stable for at least a 1.5 to 2 years. To achieve for this with liposomal formulation, it is very difficult if the liposomes remain in suspension form. Some methods may be used to enhance the shelf life of liposomes, such as

freeze-drying after production.13 Chemical and physical degradation plays a major role in the stability of liposomes. The chemical degradation of liposomes is attributed to oxidation and hydrolysis. To decrease oxidation and hydrolysis, use of fresh and new reagents with the highest quality, avoiding from methods that have high temperatures, use of inert atmosphere to store liposomes, deoxygenation of aqueous solutions and doing all of manufacturing in the absence of oxygen should be undertaken. Physical degradation can be prevented by incubating the liposomes at a temperature close to the phase transition temperature, until the arrangement of the lipids equalizes.16–18 Fusion between liposomes is quite common and avoided by addition of cholesterol into the lipid mixture to raise the TC of the lipids. Both of these processes influence the in vivo performance of the drug formulation, and therefore may affect the therapeutic index of the drug. On the other hand, large liposomes may be rapidly cleared by RES leading to low therapeutic plasma concentrations of the drug and reduced area under the curve (AUC).14,19 Besides, identification of a suitable method for sterilization of liposome formulations is another major challenge because phospholipids are thermolabile and sensitive to sterilization procedures involving the use of heat, radiation or chemical sterilizing agents. The routine method available for sterilization of liposomal formulations after manufacturing is filtration through sterile 0.22 mm membranes. However, filtration is not suitable for large vesicles (>0.22 mm) and also is not able to remove viruses. Another option is filtering the initial solutions through 0.45 lm regenerated cellulose filters and glass fiber filters before starting production, thereafter the entire production process must be done under aseptic conditions. Sterilization by other approaches such as g-irradiation and exposure to chemical sterilizing agents are not recommended due to the degradation of liposome components and leaving toxic contaminants,15,20–22 Finally, the amount of drug that liposomes can encapsulated is often very low. Liposome formulation of a drug could only be effective if the encapsulation efficiency is fairly high and could be delivered in a reasonable amount of lipid, since lipids in high doses could be toxic and also cause nonlinear pharmacokinetics of liposomal formulations. When entrapment of the drug is especially low, methods like active loading can be used to improve the entrapment by using an uncharged drug that can easily cross the lipid bilayer in uncharged form, but changes to the charged species once inside the liposome. Another new approaches that provide high encapsulation efficiencies for hydrophilic drugs is active loading of the amphipathic weak acidic or basic agents in blank liposomes which can increase the encapsulation efficiency.11,17,20,23 These drawbacks limit the use of liposomal formulations for use as an ideal carrier for anticancer drugs.



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Various types of liposomes can be prepared by different preparation methods, depending on the desirable application, sizes and characteristics. The most simple and widely used method for preparation of munilamellar vesicles (MLV) is the thin-film hydration procedure in which a thin film of lipids is hydrated with an aqueous buffer at a temperature above the transition temperature of lipids. Furthermore, several methods have been developed for the preparation of large unilamellar vesicles (LUV), including solvent (ether or ethanol) injection, detergent dialysis, calcium-induced fusion, remote loading and reverse phase evaporation (REV) techniques.20,23–27 In the last decades, liposomes have gained considerable attention as systemic drug delivery vehicles following recent approvals of several vesicle-formulated drugs. The products on the market which contain liposomes are very limited. Generally, there are two separate classes of drugs (infections and cancer) (which loaded in liposomes) were routinely investigated for the treatment in humans and the growing number of liposomal formulations are under clinical evaluation or in the market [e.g. DoxilÕ (Doxorubicin), Dauno-XomeÕ (Daunorubicin citrate), AmbiosomeÕ (Amphotericin B), VentusÕ (Prostaglandin E1) and AtragenÕ (Tretinoin)].11,28–32 For years, liposomes have been explored as carriers of antitumor agents in an attempt to alter favorably the pharmacokinetics and organ distribution of these drugs and possibly to increase their therapeutic index.33–35 Liposomes are used to administer drugs by several routes such as topical, oral and parenteral and have many applications in the fields of immunology, tumor therapy, vaccine adjuvant, antimicrobial therapy, gene therapy as well as delivery of radiopharmaceuticals for diagnostic imaging. As drug carriers, liposomes have various favorable characteristics for cancer therapy such as low toxicity, long-term blood circulation and accumulation in tumors by enhanced permeability and retention effect.36–38 Liposomal formulations of hydrophobic drugs have been shown to overcome the solubility problem and the solvent-induced side effect. However, despite of their unquestionable advantages, conventional liposomes are not site-specific drug delivery systems and the cellular uptake of conventional or non-specific liposomes is limited. To improve these limitations and further enhancement of their selective drug delivery, clinically active functional liposomal drug delivery systems containing macromolecules (such as antisense, oligonucleotide aptamers and genes) were developed. Addition of surface ligands, functional polymer antibodies, carbohydrates or peptides are necessary to deliver liposomes content to the targeted sites by in vivo transport and delivery, cellular uptake, endocytosis and other processes.39–42 The combination of a stable and sealed

formulation with a specific targeting and release mechanism enables sufficient drug concentration at the desired site maintaining a low systemic concentration. Liposomes can also be surface functionalized via chemical conjugation like incorporation of pro-drug molecules to the liposome surface as lipid conjugates to facilitate controlled release by developing environmental changes such as low pH, enhanced temperature, high levels of reducing agents and enzymatic cleavage.43–49 For drugs with intracellular targets or receptors, it is essential to cross the plasma membrane for pharmacological activity. Tumors are one of the primary sites for accumulation of liposomes, where they concentrate due to the higher permeability of the vascular endothelium surrounding tumors. For establishment of safe and effective tumor therapy, accurate drug delivery is a promising approach which enables specifically targeted attack of malignant cells with cytotoxic drugs.50–52 Primarily, cytotoxic drug incorporation into liposomes has been reported since 1970. Furthermore, several liposome-based drug delivery systems have been approved by the FDA and many others are in the late-stage clinical trials. Liposomes can be used to increase cytosolic delivery of many active agents. To achieve maximum efficacy, controlled release of therapeutic agents from liposomes at the tumor site is necessary.53–55 In this manuscript, we reviewed different types of liposomes and their structural formulations which were used in the cancer-targeted drug delivery.

Stimuli–responsive liposomes Strategies used to enhance liposome-mediated drug delivery in vivo include the enhancement of stability and circulation time in the bloodstream, targeting the specific tissues or cells, and facilitation of intra-cytoplasmic delivery. First-generation liposomes have emerged as one of the first nano-medicines used clinically for localized delivery of chemotherapy. Second-generation liposomes, such as stimuli-responsive liposomes, have the potential not only to provide site-specific chemotherapy, but also to provide triggered drug release and thus greater spatial and temporal control of therapy.56–59 Engineer-made liposomes which provide higher concentration of therapeutic or diagnostic materials in diseased site have attracted great interest in medicine and especially in cancer chemotherapy. The diseased tissues such as solid tumors microenvironment are characterized by oxygen depletion or hypoxia and certain abnormalities in temperature and pH. These characteristics were widely investigated to design stimuli–responsive drug liposomal formulations for site-specific delivery of active agents.29,60,61 Stimuli–responsive liposomes are active delivery vesicles which will be changed via an external signal and



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release their loaded agents in the site of action. Many stimuli-sensitive liposomes have been and are being developed that avoid degradation of loaded drugs and release their containing substance in one single burst as a result of destabilization of the liposome membrane caused by certain internal or external stimuli (such as changes of physiological pH, tissue specific enzymes, physiological temperature or electrolyte concentration, etc.).57,62–65 It was shown that specific cellular and extracellular stimulus, with chemical, biochemical or physical origin, can modify the structural composition or conformation of the liposomes and thus induce release of the active agents to specific biological environment and target tissues. This allows liposomes to release the encapsulated payload in response to particular pathological condition present in the diseased tissues which leads to reduction in the side effects. These functional liposomes simulate numerous controlled biological feedbacks which are usual in nature where the presence of any physical, chemical or physicochemical factors can control a series of biochemical processes.36,43,56,66

pH-sensitive liposomes Controlled drug release using liposomal targeting results in the accumulation of the drug at the site of action. The concept of pH-sensitive liposomes arose from the fact that certain enveloped viruses develop strategies to take advantages of the acidification of the endosomal lumen to infect cells. Accordingly, it was found that some pathological tissues such as tumors, inflamed and infected areas exhibit an acidic environment as compared to normal tissues.31,44,45 Therefore, specific conditions found in the target tissues can be employed as a trigger for controlled release by the incorporation of pH-sensitive components into the liposomal bilayer. Furthermore, pH-dependent release via membrane fusion, can escape endolysosomal degradation after endocytosis and deliver active therapeutic agent to the cytoplasm of target cells. Therefore, different applications of pH-sensitive liposomes were investigated for the transport and specific delivery of potent drugs (for the treatment of cancer as well as pulmonary and infectious diseases), vaccination (as immunological adjuvants), imaging (carrying contrasting agents) and delivery of nucleic acids (which are aiming at gene therapy applications).43,44,67–71 pH-sensitive liposomes are commonly stable at physiological pH; however, they undergo destabilization under acidic conditions leading to the release of their aqueous-loaded drugs and effective delivery of drug or gene fragments into the cytoplasm via the endocytotic pathway. Different classes of pH-sensitive liposomes have been previously studied based on the different

pH-sensitivity triggering mechanisms. Therefore, a high local drug level at the target site is obtained due to their controlled release. The most proposed concept involves the combination of phosphatidylethanolamine or its derivatives with compounds containing an acidic group which acts as a stabilizer at neutral pH. The drawback of this system is that the same groups (such as carboxylate groups that are negatively charged at a neutral pH) can interact with plasma proteins resulting in the elimination of liposomes from the circulation. Another mechanism is pH-dependent hydrolysis of the non-charged cleavable components which are integrated in the membrane that occurs by the thiolysis of membrane lipids containing disulfide bonds.44,72–74 Furthermore, pH-sensitive liposomes can be prepared simply by addition of pH-sensitive units to the liposomal dispersion or by mixing pH-sensitive lipids and polymers during the vesicles preparation. Such liposomes stay intact at physiological pH but destabilize and acquire fusogenic properties under acidic conditions. After being endocytosed, pH-sensitive liposomes fuse with the endovacuolar membrane on the condition of lower pH value inside the endosome, and by destabilization of that, can release their content into the cytoplasm. Since most liposomes are internalized by endocytosis, pH-sensitive liposomes undergo destabilization at this step and thus prevent degradation at the lysosomal level, which can promote cytosolic delivery of the intact contents. As a result, pH-sensitive liposomes can be designed efficiently to release their encapsulated contents, particularly biological macromolecules, such as proteins and peptides, drugs, enzymes, antibodies and antisense oligonucleotide as well as plasmids into cytoplasm before reaching the lysosome, ensuring the activity of loaded drugs.70,75,76 Different types of macromolecules which formed pH-sensitivity in liposomes were used in pH-sensitive liposome as mentioned further in the text. Polymorphic lipids. The typical polymorphic lipid used to form pH-sensitivity in pH-sensitive liposomes is the unsaturated phosphatidylethanolamine, including dioleoyl phosphatidyl ethanolamine (DOPE), palmitoyloleoyl phosphatidyl ethanolamine and diacetylenic-phosphatidyl-ethanolamine. To formulate pH-sensitive liposomes, DOPE is commonly used with mild acidic amphiphiles that act as stabilizers at neutral pH, such as oleic acid, cholesteryl hemisuccinate and palmitoyl homocysteine. Their carboxyl groups were protonated in the acidic environment and three dimensional volume of the hydrophilic side got small and lost their remedy to the phospholipid, which will lead to membrane destabilization of pH-sensitive liposomes and release of encapsulated molecules into the cytoplasm.77–80



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Cage-lipid derivatives. This kind of liposome that contains the derivatives of phosphatidylethanolamine or annular lipid compositions with alkyl ether, such as N-citraconyl-dioleoyl-phosphatidyl-ethanolamine, Ncitraconyl-dioleoyl-phosphatidylserine and poly(ethylene-glycol)-N-distearolyphosphatidyl-ethanolamine can reversibly display the ability to form non-bilayer phase simply with the drug-permeable membranes or with the fusion competent. This process is performed by reversible covalent modifying of a nucleophilic functionality on the head group of the lipid or cleaving the alkyl group of the liposome in the blood circulation to expose the long-chain of fatty acids. This can destabilize the stability of the biofilm and thus increase the permeability of entrapped drugs.44,75,76 Synthetic fusogenic peptides and proteins. Another novel type of pH-sensitive liposomes is developed by introducing the pH-sensitive peptide or proteins, such as GALA (a peptide composed of repeating sequences of Glu-Ala-Leu-Ala), the N-terminus of hemaglutinin (INF peptides from influenza) or the listeriolysin O into the phospholipid double-fusion peptide or protein. In this type of liposomes, the peptide or protein is inactive when such liposomes are in the neutral pH environment. However, in the acidic environment, the conformation of the fusion peptide or protein is changed and the fusion between liposomal and cell membrane is stimulated and consequently, the pHsensitive liposomes release the encapsulated drugs eventually.44,76 pH-sensitive polymers. These synthetic polymers have attracted growing scientific attention for the design of pH-sensitive liposomes and are based on poly (alkyl acrylic acid)s, succinylated PEG and N-isopropylacrylamide (NIPAM) copolymers. These polymers showed interesting features in the release of drugs upon an external stimulation and can interact with the lipid bilayer, which promotes the fusion between liposomes and endsomal membrane. At high to near to neutral pH, the head groups of mildly acidic amphiphiles such as oleic acid, N-palmitoylhomocystein, palmitoylhomocysteine, NIPAM, methacrylic acid and cholesteryl hemisuccinate are predominantly charged and their presence in the lipid mixture prevents the formation of inverted hexagonal (HII) phase and other inverted phases.70,71,81–83 Different types of these polymer offer unique advantages and disadvantages that may vary in potential depending on the desired application purpose and preparation methods. pH-sensitive liposomes can significantly increase cytoplasmic delivery of various fluorescent markers with various molecular sizes, ribozymes, enzymes, cytotoxic agents, proteins, RNA and

DNA to cells with considerable efficiency. However, till now none of this kind of preparations was used in clinical applications due to their complications, since a clinically viable pH-sensitive liposomal formulation requires several essential properties including efficient pH-triggered release, plasma stability and enough long circulation time in vivo. Moreover, after being injected into the body, pH-sensitive liposomes can still be phagocytized by reticuloendothelial system to some extent, which is an important limitation to the in vivo use and the main barrier for the delivery of drugs and gene to the pathological organs. Furthermore, physicochemical and biological stability, acid sensitivity and bioavailability, control of particle size, batch to batch reproducibility and sterilization problem are other drawbacks. Therefore, many researches focused on the clinical and therapeutic side of pH-sensitive liposomes and would enable their commercial utility in cancer treatment.

Thermo-sensitive liposomes A potential form of targeted drug delivery, which currently attracts enhanced interest, involves the use of thermo-sensitive liposomes in some diseases like cancer chemotherapy. The idea of using temperaturesensitive nanocarriers naturally came from the fact that many pathological areas demonstrate distinct hyperthermia. Furthermore, there are various means to heat the required area in the body. The therapeutic effects of liposomal formulations can be also enhanced by receptor targeting and by triggering drug release within the tumor, by decomposition of the formulation at increased temperature. Hence, targeted temperaturesensitive liposomes have attracted much attention and are considered to be a promising tool to achieve sitespecific delivery of drugs. Thermo-sensitive liposomes were first formulated by Yatvin et al.84 These liposomes have been prepared using lipids whose membranes undergo a gel-to-liquid crystalline phase transition a few degrees above physiological temperature.84–86 Attractive strategies for the development of traditional thermo-sensitive vesicles includes utilization of phospholipids having phase transition temperature (Tm) between 41 and 42 C and undergoing a gelto-liquid phase transition at several degrees above physiological temperature. Thermo-sensitive liposomes with long circulating properties have also been studied using PEG or oligoglycerol-moieties. Furthermore, an interesting methodology involving target temperaturesensitive magnetic liposomes for thermo-chemotherapy has been reported recently which focuses on magnetic hyperthermia-triggered drug release.87–89 Temperaturesensitive liposomes frequently include dipalmitoylphosphatidylcholine (DPPC) as the key component, since



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liposomes usually become leaky at a gel-to-liquid crystalline phase transition and this transition for DPPC takes place at 41 C. Temperature sensitization of liposomes has been frequently attempted using thermosensitive polymers. To this point, functional liposomes whose contents release behavior, surface properties and affinity to cell surface can be controlled in a temperature-dependent manner, have been developed according to this strategy. Liposomes can also be made with temperature-sensitive property via the incorporation of grafting of certain polymers, which display a lower critical solution temperature (CST) slightly above the physiological temperature. Because these polymers are soluble below CST and precipitate by the temperature increase above the CST, they can damage the liposomal membrane during precipitation and allow the drug release. The most common demonstrative example of this class of polymers are poly-NIPAM and poly [(2-(2ethoxy)ethoxyethyl vinyl ether)] (EOEOVE).88,90–94 Hayashi et al. developed novel temperature-sensitive liposomes having surface properties that can be controlled by temperature and were designed as liposomes coated with poly NIPAM, which exhibits a hydrated coil to dehydrated globule transition at 32 C. Nevertheless, thermo-sensitive polymers are not biodegradable and toxicity remains a problem in vivo that limits biological application. Another encouraging anticancer therapy is temperature-sensitive liposome in combination with mild hyperthermia (39–42 C). A combination of thermo-sensitive liposome with mild hyperthermia has demonstrated better therapeutic efficacy than simple liposomal chemotherapy, by increasing the intra-tumoral drug concentration.85 A temperature-sensitive folatetargeted doxorubicin-containing magnetic liposome also been developed by Pradhan et al. for thermochemotherapy of cancer.86

PEGylated stealth liposomes Nanoparticles and other macromolecules introduced into the bloodstream are distributed throughout the body via the vascular system. Clearance of these species is mediated by the renal system or the mononuclear phagocytic system (MPS), also known as RES. Nanoparticles up to 8 nm are cleared rapidly by the kidneys with minimal catabolism. Particles larger than 8 nm evade the renal system typically and are cleared by the MPS, which represents the primary means of nanoparticle clearance from blood circulation. Shielding nanoparticles from opsonization, known as stealth, is a critical feature in nanoparticle design.95,96 Stealth liposomes can be formed by introducing coating nanoparticles with hydrophilic polymers to escape from MPS detection. In intravenous administration, for

reaching to the target tissue or cells and organize cytoplasmic delivery, it is necessary for liposomes to be stable in biological fluids and display long circulation times. However, after intravenous administration, the conventional liposomes are recognized rapidly and uptaken by the cells of the reticuloendothelial system mainly in liver or spleen and removed from the circulation. This leads to short plasma half-lives and therefore, their limited clinical potential. In addition, liposomes can enter cells mainly via the endocytotic pathway, where liposomes and their encapsulated drugs or gene which cannot escape the endosome are exposed to the risk of being degraded by lysosomal enzymes, resulting in significant decrease of the drug efficacy.97–99 Early research into stealth liposomes involved mimicking the natural cellular polysaccharide coating (glycocalyx) using monosialote trahexosylganglioside gangliosides and hydrated phosphatidylinositol as chemically inert coatings for liposomes. In order to achieve prolonged and sustained drug delivery, conventional liposomes are being surface modified with inert, biocompatible and hydrophilic polymers such as polyethylene glycol (PEG). PEGylation inhibits liposomes aggregation and non-specific interactions by altering the physicochemical properties of liposomes surface (particularly surface charge and hydration). In addition, the PEG polymers also create a physical barrier to opsonins and other serum proteins, which prevent their adsorption to the liposomes surface and the subsequent liposomes clearance. Some of the opsonizing proteins that are responsible for recognizing liposomes have been identified as immunoglobulins, fibronectin, beta2-glycoprotein, C-reactive protein (CRP) and beta 2-macroglobulin.96,99–101 For liposomes, the optimal loading of PEG has been shown to be between 5 and 9 mol% of PEG 2000, resulting in complete nanoparticle surface coverage to prevent opsonization. Below this range, polymer surface coverage is incomplete, which may allow opsonins to associate with the particle’s surface and target it for phagocytosis. Above 9 mol%, although surface coverage is complete, the high density of polymer on the surface causes the PEG to adopt a more brush-like morphology, which destabilizes the lipid bilayer due to the strain induced by the lateral repulsion of the PEG molecules.101,102 Polymers like poly(vinyl pyrrolidone) (PVP), poly(acryl amide) (PAA), poly[N-(2-hydroxypropyl) methacrylamide], amphiphilic poly-N-vinylpyrrolidones, L-aminoacid-based biodegradable polymer–lipid conjugate, polyvinyl alcohol, amphipatic polymers poly(2methyl-2-oxazoline) and (PMOZ) poly(2-ethyl-2-oxazoline) (PEOZ) have also been used previously for preparation of long circulating liposomes. The use of lipids with high transition temperatures and the integration of cholesterol or lipid conjugates such as



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phosphatidylethanolamine–polyethylene glycol (PE– PEG), also resulted in a significant reduction in leakage of the loaded cargo during circulation or in the extracellular environment. Furthermore, addition of these lipids can also decrease non-specific interactions between the liposomes with serum proteins, and hence inhibition of liposome clearance by the reticuloendothelial system.102–104 PEGylation of liposomes is now a clinically accepted practice, offering the highest degree of control over physicochemical properties of liposomes surface. In addition, PEGylation is combined easily with ligand targeting, which has benefits for tumor targeting and enhanced intracellular uptake. Active targeting of PEGylated liposomes involves the conjugation of targeting ligands to the surface of liposomal formulations. These ligands can include antibodies, engineered antibody fragments, proteins, peptides, small molecules and aptamers. The active targeting mechanism takes advantages of highly specific interactions between the targeting ligand and certain tissues or cells which promotes the accumulation of liposomal formulations. In the case of weak binding ligands, low affinity can be balanced by increased avidity through the surface functionalization of multiple molecules.104–108 Currently, several liposomal anticancer drugs are available in the clinic or are in advanced stages of clinical development including: PEGylated liposomal Doxorubicin (Doxil/CaelyxÕ ), non-PEGylated liposomal Doxorubicin (MyocetÕ ), liposomal Daunorubicin (DaunoXomeÕ ), liposomal Cytarabine (DepoCyteÕ ) and liposomal Cisplatin (LipoplatinÕ ). Lipoplatin is used for the treatment of epithelial malignancies, and liposomal formulations of Anthracyclines are used for the treatment of ovarian and breast cancer or HIV-associated Kaposi’s sarcoma. DepoCyteÕ was approved for the treatment of lymphomas with meningeal spread and is the only liposomal formulation administered by intrathecal infusion.11,29,30,109,110 The clinical use of liposomal formulations of conventional cytostatic drugs was focused initially on Anthracyclines since these cationic amphiphiles allow for an efficient and stable liposomal entrapment. More importantly, Anthracyclines bear a high risk for acute and cumulative cardiotoxicity which limits their use in clinical applications. This problem may be addressed using appropriate liposomal formulations since an altered pharmacokinetics of liposomal Anthracyclines offers the possibility to avoid high plasma peaks owing to the drug retention within the liposomal formulation. In addition, a reduced distribution of the liposomal Anthracyclines to the heart muscle was observed using PEGylated liposomal formulations.111–113 Therefore, PEGylation remains the significant choice for the development of many nanoparticle-based pharmaceutical

dosage forms such as liposomes for use as anticancer drug carriers.

Immunoliposomes The use of antibodies attached to the surface of liposomes to form immunoliposomes, where they are selectively adsorbed to a chosen antigenic site, is now a well-established approach to liposome targeting. The first report on antibody-targeted liposomes came from Torchilin et al. three decades ago. These antibodytargeted liposomes were shown to be able to specifically bind to the antigen that is expressed on the target cells. In contrast to many liposome studies which have concentrated on targeting than actual drug delivery, immunoliposomes bearing antibodies have been used to investigate for the delivery of several anticancer agents such as Adriamycin and Duanomycin.114–116 Recently, this novel generation of active targeted liposomal formulation, based on the use of targeting ligands (such as antibodies and peptides which specially bind to receptor structures over-expressed at the tumor sites), were investigated. By using antibodies attached to the liposomes surface to form immunoliposomes, they can adsorbed selectively to a chosen antigenic site, and they are now considered as a well-established approach to liposome targeting. They generally display improved target cell recognition and uptake which consequently facilitate antitumor agent’s therapeutic efficacy.116,117 There are many challenges to translating active targeted liposomes into routine clinical use. The targeting and retention of drug-carrying immunoliposomes to specific antigenic sites must be followed by the release of the drug. This can occur by passive diffusion through the liposomal bilayer, but this may be too slow to maintain an effectively high drug concentration in the vicinity of the cell membrane. Cell populations are heterogeneous and the expression of receptors or antigens on tumor cell surfaces is also different. In addition, both the presence of antigens and the expression of receptors on the surface of the cancerous cells are transient and dynamic. Furthermore, despite modification with ligands, the initial tumor accumulation is still based on passive extravasation which results in a lack of deep penetration into the tumor.42,118,119 Active targeting of liposomes to tumor cells is generally attempted by conjugating ligands to the liposomal surface which allow a specific interaction with the tumor cells. Several types of ligands have been used for this purpose, including antibodies or antibody fragments, vitamins, glycoproteins, peptides and oligonucleotide aptamers.116,120–122 The main benefit of the actively targeted nano-medicines over passively targeted formulations is their ability to be retained within tumors for a



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longer period of time because of their binding to /or uptake by cancer cells, which inhibits their rapidly reentering to systemic circulation. Active targeting using immunoliposomes has several advantages than antibody–drug conjugates. Immunoliposomes can carry a significant larger number of drug molecules compared with simple conjugates and also can encapsulate drugs with different physicochemical properties. Drugs encapsulated in immunoliposomes can also reach their intracellular target by diffusion after release from immunoliposomes associated with target tissue. Therefore, unlike antibodies–drug conjugates, in some cases immunoliposomes do not have to undergo receptor–mediated endocytosis to deliver their contents intra-cellularly.116,121,123,124 Selectivity provided by antibodies can be used, not only for direct treatment but also for targeting other anticancer drugs. In this sense, the conjugation of complete or fragmented antibodies to liposomes has resulted in the next generation of drug delivery systems. In this strategy, the liposome acts as a drug carrier, and the antibody allows bringing the drug system to its target specifically.123,125,126 The immune-liposome preparation is based on the some chemical strategies including use of free functional groups (like amino groups, carboxyl groups and carbohydrate chains present in the antibody molecule) or modification of existing functional groups (disulfide, amine, carboxyl and carbohydrate groups) in the antibodies with appropriate crosslinking reagents bearing functional groups. Other strategies are use of free functional groups presented in phospholipids like hydroxyl and amine groups, modification of the existing functional groups of the phospholipids by means of suitable crosslinking reagents containing reactive functionalities, and finally utilization of various functionalized PEG derivatives which act as a linker between antibodies and liposomes.127–129

Cationic liposomes (Lipoplexes) Cationic liposomes are usually employed as a gene delivery system due to their low toxicity and immunogenicity, potential for oncogenicity, size independent delivery of nucleic acids as well as ease of preparation and quality control. Lipid base membranes are mainly composed of a combination of natural or synthetic phospholipids as well as cholesterol, but additional lipids carrying neutral, cationic or anionic groups are often involved.21,130 Amphiphilic lipids also make up the predominant part of biological membranes and are widely used for the liposome production. The length and saturation of the lipid chains as well as the conformation of the polar headgroups are important determinants for the membrane fluidity, stability and permeability. In addition, the head groups have an

impact on the charge of the outer and inner membrane surfaces. Any lipophilic or amphiphilic components, integrated into the membranes, influence the functional complexity and biological characteristics of the membrane and the outer surface of the membrane.131–133 Cationic lipids are composed of a charged headgroup, comprising of a hydrophobic moiety and a linker (spacer, backbone) where this linker acts as a connector between hydrophobic domain and cationic head. The type of the spacer chain with regard to chain length, saturation and symmetry has an impact on membrane fluidity and thereby the transfection efficiency of the cationic liposomal systems. Some of the used cationic lipids used in cationic liposomes are N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)1-propanaminium bromide (DMRIE) and 3b-[N(dimethylaminoethane) carbamoyl]cholesterol (DC-Chol).132,134,135 Nucleic acid-based bioactive materials have recently emerged as a new class of next generation therapeutics. However, their development has been restricted due to the relatively weak delivery into target cells. They have become a promising drug target for the treatment of multiple diseases including different types of cancer. There have also some successful applications of siRNA-loaded liposomal dormulations in the treatment of age-related macular degeneration and respiratory syncytial virus infection. However, significant barriers are still present in the clinical applications of siRNA drugs, including poor cellular uptake, instability under physiological conditions, off-target effects and possible immunogenicity.136–140 The successful application of siRNA for cancer therapy requires the development of clinically suitable, safe and effective drug delivery systems. Cationic lipids interact and form particulate complexes with negatively charged molecules in the liposomal membrane and are often used for the formulation of nucleic acids and introducing plasmid DNA (pDNA), small interfering RNA (siRNA) or micro-RNA into the cells. Cationic liposomes have been investigated as a means to increase the stability of nucleic acid therapeutics in the bloodstream and improve their cellular delivery.19,40,136,141 Therefore, nucleic acid therapeutics, siRNA and plasmid DNA have been extensively studied for drug delivery by using cationic liposomes. In comparison with other gene delivery modes (such as viral vectors), cationic liposomes are (a) technically simple and quick to formulate, (b) not as biologically hazardous as viral vectors, (c) freely available commercially and (d) could be tailored for specific applications. The variability in morphology and size of lipoplexes may be attributed to a variety of factors, including the lipidic composition of the vesicles, the manner in which the complexes are made, the lipid to nucleic acid ratio, the size of the nucleic acid construct, and the technique



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used to treat and visualize these complexes.133,142–144 Depending on the positive (cationic lipid) to negative (phosphate group on nucleic acid) charge ratio, lipoplexes may enter the cells through electrostatic interaction with such charged residues at the cell surface as sialic acid moieties, or by hydrophobic interaction with the hydrophobic areas of the plasma membrane.133,145,146

Fusogenic liposomes The therapeutic application of many anticancer drugs is limited, or not effective, because of their poor cellular uptake, lack of specifity toward tumor tissues and the ability of cancer cells to develop resistance to chemotherapeutical agents. To this point, multiple attempts have been made to develop a tumor-targeted pharmaceutical carrier with the ability to provide an effective cellular internalization of an anticancer drug directly into the cytoplasm by passing the endocytic pathways (with protecting drugs from lysosomal degradation), thus enhancing the drug efficacy. The interactions between the liposomal vesicles and cells can occur via one or more processes including stable physical adsorption, endocytosis, lipid exchange and fusion. The fusion event is attractive because it opens the possibility of easy release of the encapsulated drugs into the cytoplasm or cell organelles which is potentially dangerous for the stability of drugs. A number of different approaches have been used to create phospholipid liposomes that have the capacity to fuse with cellular membranes. In some fusogenic liposomes, fusogenicity comes from membrane-associated proteins or peptides. In other types, it depends on specific interactions between the liposomes and target membrane receptors. Furthermore, liposomes containing negatively charged phospholipids become fusogenic in the presence of calcium.71,147,148 The development of strategies to increase the ability of liposomes to mediate intracellular delivery of biologically active molecules has attracted great interest for intensive research activity. The application of such strategies resulted in the formation of liposomes which could constitute crucial tools to improve the therapeutic efficiency of many drugs that exert their effect at the intracellular level. The phospholipids by which liposomes are conventionally made lead to the formation of lipid bilayers that closely resemble the cell and biological membranes. This allows an easy and deep interaction of these drug carriers with the cells and an improved release of the encapsulated drugs.149,150 In general, fusogenic liposomes bilayer shows an enhanced ability of interacting in their liquid crystalline phase with cell membranes leading to lipid mix and thus the release of the vesicle content inside the cytoplasm.71,148,151

Fusogenic liposomes can be produced by incorporating special lipids such as DOPE, which can promote destabilization of the bilayer and increase the vesicles fluidity and able them to promote the destabilization of biological membranes. Using these liposomes, researchers have delivered intact macromolecules such as proteins and DNA into the tissue cells as well as cultured cells for targeting anticancer agents, cell labeling, improving antibacterial effect and targeting specific intracellular organelles.41,152,153 Liposomes made of DOPE mixed with other lipid components, such as cholesterol, are able to release their load into the cytoplasm upon a brief contact with eukaryotic cells. Finally, other phospholipids such as DPPC are necessarily required to produce stable vesicles, since DOPE alone would form an inverted hexagonal phase instead of a lamellar phase.154,155

Conclusion Conventional drug delivery systems have shown a low efficiency, and a continuous search for more advanced drug delivery principles is therefore of great importance. The mechanism of many anticancer agents currently used for tumor treatment is based on cytotoxic effects and high concentrations and selective drug delivery to the tumor site are necessary. However, the systemic toxicity of chemotherapy has various and major undesirable side effects, limiting dose and therapeutic window. Therefore, tumor-specific drug delivery has attracted increasing interest in cancer chemotherapy. Since the discovery of liposomes in 1964, the field and applications of liposomes have broadened considerably. Liposomes have been used as drug delivery systems for years with a few formulations commercially available. Liposomes are composed of the different synthetic or naturally occurring lipids, where each have their own uses, advantages and disadvantages. After the primary use of first-generation liposomes, second-generation liposomes, like stimuli-responsive liposomes, PEGylated stealth liposomes, immunoliposomes, cationic liposomes and fusogenic liposomes have the potential to not only provide site-specific chemotherapy, but also triggered drug release and thus greater spatial and temporal control of therapy. However, there are still some drawbacks limiting the application of different types of liposomal formulation as optimum anticancer drug delivery carriers which are necessary to be more studied.

Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.



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Declaration of conflicting interests The authors report no conflicts of interest.

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Application of Various Types of Liposomes in Drug Delivery Systems.

Liposomes in Drug Delivery: How It All Happened.

Application of liposomes in medicine and drug delivery.

Liposomes in topical ophthalmic drug delivery: an update.

Micromixer Based Preparation of Functionalized Liposomes and Targeting Drug Delivery.

Multifunctional liposomes for MRI and image-guided drug delivery.

Liposomes as a drug delivery system for antisense oligonucleotides.

Phosphatidylethanolamine liposomes: drug delivery, gene transfer and immunodiagnostic applications.

Tablets of pre-liposomes govern in situ formation of liposomes: concept and potential of the novel drug delivery system.

The application of EDTA in drug delivery systems: doxorubicin liposomes loaded via NH4EDTA gradient.

Two cholesterol derivative-based PEGylated liposomes as drug delivery system, study on pharmacokinetics and drug delivery to retina.

Dyphylline liposomes for delivery to the skin.

Multifunctional liposomes for nasal delivery of the anti-Alzheimer drug tacrine hydrochloride.

Bleomycin A6-loaded anionic liposomes with in situ gel as a new antitumoral drug delivery system.

A review of mechanistic insight and application of pH-sensitive liposomes in drug delivery.

Enhanced Specificity and Drug Delivery in Tumors by cRGD-Anchoring Thermosensitive Liposomes.

Controlled-release drug delivery system based on fluocinolone acetonide-cyclodextrin inclusion complex incorporated in multivesicular liposomes.

Gold Nanoantenna-Mediated Photothermal Drug Delivery from Thermosensitive Liposomes in Breast Cancer.

Hyperthermia-induced drug delivery from thermosensitive liposomes encapsulated in an injectable hydrogel for local chemotherapy.

Synthesis and evaluation of glyco-coated liposomes as drug carriers for active targeting in drug delivery systems.

A pH-Responsive Drug-Delivery Platform Based on Glycol Chitosan-Coated Liposomes.

Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery.

Nanotechnology approaches for antibacterial drug delivery: Preparation and microbiological evaluation of fusogenic liposomes carrying fusidic acid.

Motion Compensated Ultrasound Imaging Allows Thermometry and Image Guided Drug Delivery Monitoring from Echogenic Liposomes.