**6. Clinical applications of liposomes**

New drug delivery systems such as liposomes are developed when the existing formulations are not satisfactory. Among all the nanomedicine platforms, liposomes have demonstrated one of the most established nanoplatforms with several FDA-approved formulations for cancer treatment, and had the greatest impact on oncology to date, because of their size, biocompat‐ ibility, biodegradability, hydrophobic and hydrophilic character, low toxicity and immuno‐ genicity [118]. A vast of literature describes the feasibility of encapsulation of a wide range of drugs, including anti-cancer and antimicrobial agents, peptide hormones, enzymes, other proteins, vaccines and genetic materials, in the aqueous or lipid phases of liposomes which showed enhanced therapeutic activity and/or reduced toxicity in preclinical models and in humans when compared to their non-liposomal formulations.

Liposome applications in drug delivery depend, and are based on, physicochemical and colloidal characteristics such as composition, size, loading efficiency and the stability of the carrier, as well as their biological interactions between liposomes and cells. Based on these liposome properties, several modes of drug delivery can be listed: the major ones are enhanced drug solubilization (e.g. amphotericin B, minoxidil), protection of sensitive drug molecules (e.g. cytosine arabinose, DNA, RNA, antisense olgionucleotides, ribozymes), enhanced intracellular uptake (all agents, including antineoplastic agents, antibiotics and antivirals) and altered pharmacokinetics and biodistribution of the encapsulated drug.

prior to bilayer disruption. The techniques used for this quantification depend on the nature of the encapsulant and include spectrophotometry [106, 107], fluorescence spectroscopy [108], enzyme-based methods [109] and electrochemical techniques. If a separation technique such as HPLC of field-flow fractionation (FFF) is applied, the percent encapsulation can be ex‐ pressed as the ratio of the unencapsulated peak area to that of a reference standard of the same initial concentration [110, 111]. This method can be applied if the liposomes do not undergo any purification following preparation. Either technique serves to separate liposome encap‐ sulated materials from those that remain in the extravesicular solution and hence can also be used to monitor the storage stability in terms of leakage or the effect of various disruptive conditions o the retention of encapsulants. Some authors have combined the size distribution and encapsulation efficiency determination in one assay by using FFF-MALS (multi angled light scattering) coupled to a concentration detector suitable for the encapsulant [112].

Since techniques used to separate free materials from liposome-encapsulated contents can potentially cause leakage of contents and, in some cases, ambiguity in the extent of separation, research using methods that do not rely on separation are of interest. Reported methods have included 1H NMR where free markers exhibited pH sensitive resonance shifts in the external medium versus encapsulated markers [113]; diffusion ordered 2D NMR which relied on differences in diffusion coefficients of entrapped and free marker molecules [114]; fluorescence methods where the signal from unencapsulated fluorophores was quenched by substances present in the external solution [115]; electron pin resonance (ESR) methods which rely on the signal broadening of unencapsulated markers by the addition of a membrane-impermeable

The drug release from liposomes can be followed by the usage of a well calibrated in vitro diffusion cell in order to predict pharmacokinetics and bioavailability of drug before expensive and time-consuming in vivo studies. For the determination of pharmacokinetic performance of liposomal formulations, dilution-induced drug release in buffer and plasma was employed and for the determination of drug bioavailability, another procedure is followed which involves the liposome degradation in the presence of mouse-liver lysosome lysate [93].

New drug delivery systems such as liposomes are developed when the existing formulations are not satisfactory. Among all the nanomedicine platforms, liposomes have demonstrated one of the most established nanoplatforms with several FDA-approved formulations for cancer treatment, and had the greatest impact on oncology to date, because of their size, biocompat‐ ibility, biodegradability, hydrophobic and hydrophilic character, low toxicity and immuno‐ genicity [118]. A vast of literature describes the feasibility of encapsulation of a wide range of drugs, including anti-cancer and antimicrobial agents, peptide hormones, enzymes, other proteins, vaccines and genetic materials, in the aqueous or lipid phases of liposomes which showed enhanced therapeutic activity and/or reduced toxicity in preclinical models and in

Liposome applications in drug delivery depend, and are based on, physicochemical and colloidal characteristics such as composition, size, loading efficiency and the stability of the

agent [116, 117].

16 Application of Nanotechnology in Drug Delivery

**6. Clinical applications of liposomes**

humans when compared to their non-liposomal formulations.

Although lipid based formulations have advantages as drug carriers, drug-delivery systems based on unmodified liposomes are limited by their short blood circulation time, instability in vivo and lack of target selectivity [119, 120]. To increase accumulation of liposomal formula‐ tions in the desired cells and tissues, the use of targeted liposomes including surface-attached ligands such as; antibodies, folates, peptides and transferrin that are capable of recognizing and binding to the desired cells. Despite of some improvements in targeting efficiency by these immunoliposomes, the majority of these modified liposomes were still eliminated rapidly by the reticulo endothelial system, primarily in the liver [120]. Better target accumulations are expected if liposomes can be made to remain in the circulation long enough.

Schematic drawing of cytosolic delivery and organelle-specific targeting of drug loaded nanoparticles (i.e. most frequently liposomes) via receptor-mediated endocytosis is shown in Figure 3.

**Figure 3.** Schematic drawing of the cytosolic delivery and organelle-specific targeting of drug loaded nanoparticles via receptor-mediated endocytosis. After receptor mediated cell association with nanoparticles, the nanoparticles are engulfed in a vesicle known as an early endosome. Nanoparticles formulated with an endosome disrupting property disrupt the endosomes followed by cytoplasmic delivery. On the other hand, if nanoparticles are captured in early en‐ dosomes, theymaymake theirway to lysosomes as late endosomes where their degradation takes place. Only fraction of non-degraded drug released in the cytoplasm interacts with cellular organelles in a random fashion. However, cyto‐ solic delivery of a fraction of organelle-targeted nanoparticles via endosomal escape or from lysosomes travel to the targeting organelles to deliver their therapeutic cargo [121].

Different methods have been suggested to achieve liposomes with high stability and long circulation times in vivo, including the surface coating of the liposomes with inert, biocom‐ patible polymers such as PEG (stealth liposomes), which forms a protective layer over the liposome surface and slow down liposome recognition by opsonins and therefore subsequent clearance of liposomes [80, 84]. Long circulating liposomes are now being investigated in detail and are widely used in vitro and in vivo studies due their flexibility and also they found their place in the clinical applications. The flexibility allows a relatively small number of surfacegrafted polymer molecules to create an impermeable layer over the liposome surface [122, 123]. Long-circulating liposomes demonstrate dose-dependent, non-saturable, log-linear kinetics and increased bioavailability [124].

conditions, liposomes even in the presence of the mucoadhesive had migrated toward the

Liposomes as Potential Drug Carrier Systems for Drug Delivery

http://dx.doi.org/10.5772/58459

19

Lung is a natural target for the delivery of therapeutic and prophylactic agents such as peptides and proteins. The past 15 years have been marked by intensive research efforts on pulmonary drug delivery not only for local therapy but also for systemic therapy as well as diagnostic purposes, primarily due to the several advantages the pulmonary route offers over other routes of drug administration. Drugs that undergo gastrointestinal degradation (such as proteins and

Targeted drug delivery to the lungs has evolved to be one of the most widely investigated systemic or local drug delivery approaches. The use of drug delivery systems for the treatment of pulmonary diseases is increasing because of their potential for localized topical therapy in the lungs. This route also makes it possible to deposit drugs more site-specific at high concen‐ trations within the diseased lung thereby reducing the overall amount of drug activity while reducing systemic side effects. To further exploit the other advantages presented by the lungs, as well as to overcome some challenges, scientists developed interests in particulate drug delivery systems for pulmonary administration, such as liposomes, micelles, nano-and micro-

The use of liposomes as drug carriers for pulmonary delivery has been reported for different kinds of therapeutics such as anti-microbial agents, cytotoxic drugs, antioxidants, anti-asthma compounds and recombinant genes for gene therapy in the treatment of cystic fibrosis.

Liposomes as carrier systems for pulmonary delivery offer several advantages over aerosol delivery of the corresponding non-encapsulated drug. Liposomes might be used to solubilize poorly soluble drugs, provide a pulmonary sustained release reservoir prolonging local and systemic therapeutic drug levels, facilitate intracellular delivery of drugs especially to alveolar macrophages, tumor cells or epithelial cells, prevent local irritation of lung tissue and reduce the drug's toxicity, target specific cell populations using surface bound ligands or antibodies

Local delivery of medication to the lungs is highly desirable, especially in patients with specific pulmonary diseases such as cystic fibrosis, asthma, chronic pulmonary infections or lung cancer. The principal advantages include reduction of systemic side effects and application of higher doses of the medication at the site of drug action. Although simple inhalation devices and aerosols containing various drugs have been used since the early 19th century for the treatment of respiratory disorders, the past 15 years have been marked by intensive research efforts on pulmonary drug delivery not only for local therapy but also for systemic therapy as well as diagnostic purposes due to the several advantages the pulmonary route offers over other routes of drug administration. Lung is a natural target for the delivery of therapeutic and prophylactic agents such as peptides and proteins due to the large surface area available for absorption, the very thin absorption membrane and the elevated blood flow which rapidly distributes molecules throughout the body. Moreover, the lungs exhibit relatively low local

and be absorbed across the epithelium to reach the systemic circulation intact [132].

conjuctival sac with very little activity remaining at the corneal surface.

peptides) are ideal candidates for pulmonary delivery.

**6.2. Pulmonary applications**

particles based on polymers.

The studies that attempt to combine the properties of long-circulating liposomes and immu‐ noliposomes in one preparation place themselves in the literature as the further development in the liposomal formulations as drug carriers [125, 126]. In the early experiments, simple coimmobilization of an antibody and PEG on the surface of the same liposome has been per‐ formed despite the possibility of PEG creating steric hindrance for target recognition with the targeting moiety [125]. To achieve better selectivity of PEG-coated liposomes, it is advanta‐ geous to attach the targeting ligand via a PEG spacer arm, so that the ligand is extended outside the dense PEG brush which reduces steric hindrance of binding to the target [127]. The use of PEG-conjugated immunoliposomes for increasing drug carrying capacity of monoclonal antibody has been demonstrated [128]. In addition to costly monoclonal antibodies, common molecules such as folic acid, trensferrin and RGD peptides have also been studied for tumor targeting with enhanced selective uptakes [120].

Encouraging results of liposomal drugs in the treatment or prevention of a wide spectrum of diseases in experimental animals and in human, indicate that more liposome-based products for clinical and veterinary applications may be forthcoming. These could include treatment of eye and skin diseases in therapeutic applications, antimicrobial and anticancer therapy in clinical applications, metal chelation, enzyme and hormone replacement therapy, vaccine and diagnostic imaging, etc. Some of the liposome applications in terms of drug delivery are discussed below.

#### **6.1. Ocular applications**

The eye is protected by three highly efficient mechanisms (a) an epithelial layer which is the barrier to penetration (b) tear flow (c) the blinking reflex. All these mechanisms are responsible for the poor drug penetration into the deeper layers of the cornea and the aqueous humor and for the rapid wash out of drugs from the corneal surface. Initially, in 1981 the enhanced efficiency of liposomes encapsulated idoxuridine in herpes simplex infected corneal lesions in rabbits was reported [129]. In 1985, it was concluded that ocular delivery of drugs can be either promoted or impeded by the use of liposome carriers, depending on the physicochemical properties of the drugs and the lipid mixture employed [130]. The use of mucoadhesive polymers, carbopol 934P and carbopol 1342 to retain liposomes at the cornea was proposed [131]. While precorneal retention times were indeed significantly enhanced under appropriate conditions, liposomes even in the presence of the mucoadhesive had migrated toward the conjuctival sac with very little activity remaining at the corneal surface.

#### **6.2. Pulmonary applications**

Different methods have been suggested to achieve liposomes with high stability and long circulation times in vivo, including the surface coating of the liposomes with inert, biocom‐ patible polymers such as PEG (stealth liposomes), which forms a protective layer over the liposome surface and slow down liposome recognition by opsonins and therefore subsequent clearance of liposomes [80, 84]. Long circulating liposomes are now being investigated in detail and are widely used in vitro and in vivo studies due their flexibility and also they found their place in the clinical applications. The flexibility allows a relatively small number of surfacegrafted polymer molecules to create an impermeable layer over the liposome surface [122, 123]. Long-circulating liposomes demonstrate dose-dependent, non-saturable, log-linear

The studies that attempt to combine the properties of long-circulating liposomes and immu‐ noliposomes in one preparation place themselves in the literature as the further development in the liposomal formulations as drug carriers [125, 126]. In the early experiments, simple coimmobilization of an antibody and PEG on the surface of the same liposome has been per‐ formed despite the possibility of PEG creating steric hindrance for target recognition with the targeting moiety [125]. To achieve better selectivity of PEG-coated liposomes, it is advanta‐ geous to attach the targeting ligand via a PEG spacer arm, so that the ligand is extended outside the dense PEG brush which reduces steric hindrance of binding to the target [127]. The use of PEG-conjugated immunoliposomes for increasing drug carrying capacity of monoclonal antibody has been demonstrated [128]. In addition to costly monoclonal antibodies, common molecules such as folic acid, trensferrin and RGD peptides have also been studied for tumor

Encouraging results of liposomal drugs in the treatment or prevention of a wide spectrum of diseases in experimental animals and in human, indicate that more liposome-based products for clinical and veterinary applications may be forthcoming. These could include treatment of eye and skin diseases in therapeutic applications, antimicrobial and anticancer therapy in clinical applications, metal chelation, enzyme and hormone replacement therapy, vaccine and diagnostic imaging, etc. Some of the liposome applications in terms of drug delivery are

The eye is protected by three highly efficient mechanisms (a) an epithelial layer which is the barrier to penetration (b) tear flow (c) the blinking reflex. All these mechanisms are responsible for the poor drug penetration into the deeper layers of the cornea and the aqueous humor and for the rapid wash out of drugs from the corneal surface. Initially, in 1981 the enhanced efficiency of liposomes encapsulated idoxuridine in herpes simplex infected corneal lesions in rabbits was reported [129]. In 1985, it was concluded that ocular delivery of drugs can be either promoted or impeded by the use of liposome carriers, depending on the physicochemical properties of the drugs and the lipid mixture employed [130]. The use of mucoadhesive polymers, carbopol 934P and carbopol 1342 to retain liposomes at the cornea was proposed [131]. While precorneal retention times were indeed significantly enhanced under appropriate

kinetics and increased bioavailability [124].

18 Application of Nanotechnology in Drug Delivery

targeting with enhanced selective uptakes [120].

discussed below.

**6.1. Ocular applications**

Lung is a natural target for the delivery of therapeutic and prophylactic agents such as peptides and proteins. The past 15 years have been marked by intensive research efforts on pulmonary drug delivery not only for local therapy but also for systemic therapy as well as diagnostic purposes, primarily due to the several advantages the pulmonary route offers over other routes of drug administration. Drugs that undergo gastrointestinal degradation (such as proteins and peptides) are ideal candidates for pulmonary delivery.

Targeted drug delivery to the lungs has evolved to be one of the most widely investigated systemic or local drug delivery approaches. The use of drug delivery systems for the treatment of pulmonary diseases is increasing because of their potential for localized topical therapy in the lungs. This route also makes it possible to deposit drugs more site-specific at high concen‐ trations within the diseased lung thereby reducing the overall amount of drug activity while reducing systemic side effects. To further exploit the other advantages presented by the lungs, as well as to overcome some challenges, scientists developed interests in particulate drug delivery systems for pulmonary administration, such as liposomes, micelles, nano-and microparticles based on polymers.

The use of liposomes as drug carriers for pulmonary delivery has been reported for different kinds of therapeutics such as anti-microbial agents, cytotoxic drugs, antioxidants, anti-asthma compounds and recombinant genes for gene therapy in the treatment of cystic fibrosis.

Liposomes as carrier systems for pulmonary delivery offer several advantages over aerosol delivery of the corresponding non-encapsulated drug. Liposomes might be used to solubilize poorly soluble drugs, provide a pulmonary sustained release reservoir prolonging local and systemic therapeutic drug levels, facilitate intracellular delivery of drugs especially to alveolar macrophages, tumor cells or epithelial cells, prevent local irritation of lung tissue and reduce the drug's toxicity, target specific cell populations using surface bound ligands or antibodies and be absorbed across the epithelium to reach the systemic circulation intact [132].

Local delivery of medication to the lungs is highly desirable, especially in patients with specific pulmonary diseases such as cystic fibrosis, asthma, chronic pulmonary infections or lung cancer. The principal advantages include reduction of systemic side effects and application of higher doses of the medication at the site of drug action. Although simple inhalation devices and aerosols containing various drugs have been used since the early 19th century for the treatment of respiratory disorders, the past 15 years have been marked by intensive research efforts on pulmonary drug delivery not only for local therapy but also for systemic therapy as well as diagnostic purposes due to the several advantages the pulmonary route offers over other routes of drug administration. Lung is a natural target for the delivery of therapeutic and prophylactic agents such as peptides and proteins due to the large surface area available for absorption, the very thin absorption membrane and the elevated blood flow which rapidly distributes molecules throughout the body. Moreover, the lungs exhibit relatively low local metabolic activity, and unlike the oral route of drug administration, pulmonary inhalation is not subject to first pass metabolism [133].

studies have demonstrated the accumulation of liposomes in certain tumors in a higher concentration than found in normal tissues [138, 139]. Anthracyclines are drugs which stop the growth of dividing cells by intercalating into the DNA and therefore kill predominantly quickly dividing cells. These cells are not only in tumors but are also in hair, gastrointestinal mucosa, and blood cells; therefore, this class of drugs is very toxic. Many research efforts have been directed towards improving the safety profile of the anthracyclines cytotoxics, doxoru‐ bicin and daunorubicin, along with vincristine. Encapsulation of these drugs into the lipo‐ somes showed reduced cardiotoxicity, dermal toxicity and better survival of the experimental animals compared to the controls receiving free drugs [138]. Such beneficial effects of liposomal anthracyclines have been observed with a variety of liposome formulations regardless of their lipid composition and provided that lipids used high cholesterol concentration of phospholi‐ pids with high phase transition temperature are conducive to drug retention by the vesicles

Liposomes as Potential Drug Carrier Systems for Drug Delivery

http://dx.doi.org/10.5772/58459

21

Active targeting of cancer drugs to the tumors is shown schematically in Figure 4.

Currently several liposomal formulations are in the clinical practice containing different chemotherapeutics such as doxorubicin (Doxil1/Caelyx1), doxorubicin (Myocet1), daunoru‐ bicin (DaunoXome1) and cytarabine (DepoCyte1) for treating the ovarian cancer, AIDS related Kaposi's sarcoma, multiple myeloma, lymphomas, leukemia with meningeal spread. Several other liposomal chemotherapeutic drugs containing doxorubicin, annamycin, mitoxantrone, cisplatin, oxaliplatin, camptothecine, 9-nitro-20 (S)-camptothecin, irinotecan, lurtotecan, topotecan, paclitaxel, vincristine, vinorelbine and floxuridine are at the various stages of

Two liposomal formulations have been approved by the US Food and Drug Administration (FDA) and are commercially available for the treatment of AIDS-related Kaposi's sarcoma. Doxil, first liposomal drug approved by FDA and has been on the market since 1995, is a

in the systemic circulation [45].

**Figure 4.** Representation of active targeting via receptors [121].

clinical trials [140].

Inhaled drug delivery devices can be divided into three principal categories: nebulizers, pressurized metered-dose inhalers and dry powder inhalers; each class presents unique strengths and weaknesses. A good delivery device has to generate an aerosol of suitable size and provide reproducible drug dosing. It must also protect the physical and chemical stability of the drug formulation.

For controlled delivery of drug to the lung, liposomes are one of the most extensively inves‐ tigated systems in recent studies given that they can be prepared with phospholipids such as egg phosphatidylcholine (PC), distearoyl phosphatidylcholine (DSPC) and dipalmitoylphos‐ phatidylcholine (DPPC) endogenous to the lung.

A significant disadvantage of many existing inhaled drugs is the relatively short duration of resultant clinical effects, which requires most medications to be inhaled at least twice daily. This often leads to poor patient compliance. A reduction in the frequency of dosing would be convenient, particularly for chronic diseases such as asthma. The advantages of such an approach include reduced dosing, increased effectiveness of rapidly cleared medicine and enhanced residence time at the target site for the treatment of infection. Many challenges exist in developing controlled release inhalation medicine, which is reflected in the fact that no commercial product exists. Cytotoxic agents, bronchodilators, anti-asthma drugs, antimicro‐ bial and antiviral agents and drugs for systemic action, such as insulin and proteins are being investigated.

#### **6.3. Cancer therapy**

The numerous anti-cancer agents that have a high cytotoxic effect on the tumor cells in vitro exhibit a remarkable decrease of the selective ant-tumor effect for in vivo procedures applicable in the clinical treatment. One of the significant limitations of the anti-cancer drugs is their low therapeutic index meaning that the dose required to produce an anti-tumor effect is toxic to normal tissues. The low therapeutic index of these drugs results from the inability to achieve therapeutic concentrations at the specific target sites, tumors. Further, it results from the nonspecific toxicity to normal tissues such as bone marrow, renal, gastrointestinal tract, and cardiac tissue and also from the problems associated with a preparation of a suitable formu‐ lation of the drugs [134].

Many different liposome formulations of various anticancer agents were shown to be less toxic than the free drug so that most of the medical applications of liposomes that have reached the preclinical stage are in cancer treatment [135-137]. Entrapment of these drugs into liposomes resulted in increased circulation lifetime, enhanced deposition in the infected tissues, and protection from the drug metabolic degradation, altered tissue distribution of the drug, with its enhanced uptake in organs rich in mononuclear phagocytic cells (liver, spleen and bone marrow) and decreased uptake in the kidney, myocardium and brain. To target tumors, liposomes must be capable of leaving the blood and accessing the tumor. However, because of their size liposomes cannot normally undergo transcapillary passage. In spite of this, various studies have demonstrated the accumulation of liposomes in certain tumors in a higher concentration than found in normal tissues [138, 139]. Anthracyclines are drugs which stop the growth of dividing cells by intercalating into the DNA and therefore kill predominantly quickly dividing cells. These cells are not only in tumors but are also in hair, gastrointestinal mucosa, and blood cells; therefore, this class of drugs is very toxic. Many research efforts have been directed towards improving the safety profile of the anthracyclines cytotoxics, doxoru‐ bicin and daunorubicin, along with vincristine. Encapsulation of these drugs into the lipo‐ somes showed reduced cardiotoxicity, dermal toxicity and better survival of the experimental animals compared to the controls receiving free drugs [138]. Such beneficial effects of liposomal anthracyclines have been observed with a variety of liposome formulations regardless of their lipid composition and provided that lipids used high cholesterol concentration of phospholi‐ pids with high phase transition temperature are conducive to drug retention by the vesicles in the systemic circulation [45].

Active targeting of cancer drugs to the tumors is shown schematically in Figure 4.

**Figure 4.** Representation of active targeting via receptors [121].

metabolic activity, and unlike the oral route of drug administration, pulmonary inhalation is

Inhaled drug delivery devices can be divided into three principal categories: nebulizers, pressurized metered-dose inhalers and dry powder inhalers; each class presents unique strengths and weaknesses. A good delivery device has to generate an aerosol of suitable size and provide reproducible drug dosing. It must also protect the physical and chemical stability

For controlled delivery of drug to the lung, liposomes are one of the most extensively inves‐ tigated systems in recent studies given that they can be prepared with phospholipids such as egg phosphatidylcholine (PC), distearoyl phosphatidylcholine (DSPC) and dipalmitoylphos‐

A significant disadvantage of many existing inhaled drugs is the relatively short duration of resultant clinical effects, which requires most medications to be inhaled at least twice daily. This often leads to poor patient compliance. A reduction in the frequency of dosing would be convenient, particularly for chronic diseases such as asthma. The advantages of such an approach include reduced dosing, increased effectiveness of rapidly cleared medicine and enhanced residence time at the target site for the treatment of infection. Many challenges exist in developing controlled release inhalation medicine, which is reflected in the fact that no commercial product exists. Cytotoxic agents, bronchodilators, anti-asthma drugs, antimicro‐ bial and antiviral agents and drugs for systemic action, such as insulin and proteins are being

The numerous anti-cancer agents that have a high cytotoxic effect on the tumor cells in vitro exhibit a remarkable decrease of the selective ant-tumor effect for in vivo procedures applicable in the clinical treatment. One of the significant limitations of the anti-cancer drugs is their low therapeutic index meaning that the dose required to produce an anti-tumor effect is toxic to normal tissues. The low therapeutic index of these drugs results from the inability to achieve therapeutic concentrations at the specific target sites, tumors. Further, it results from the nonspecific toxicity to normal tissues such as bone marrow, renal, gastrointestinal tract, and cardiac tissue and also from the problems associated with a preparation of a suitable formu‐

Many different liposome formulations of various anticancer agents were shown to be less toxic than the free drug so that most of the medical applications of liposomes that have reached the preclinical stage are in cancer treatment [135-137]. Entrapment of these drugs into liposomes resulted in increased circulation lifetime, enhanced deposition in the infected tissues, and protection from the drug metabolic degradation, altered tissue distribution of the drug, with its enhanced uptake in organs rich in mononuclear phagocytic cells (liver, spleen and bone marrow) and decreased uptake in the kidney, myocardium and brain. To target tumors, liposomes must be capable of leaving the blood and accessing the tumor. However, because of their size liposomes cannot normally undergo transcapillary passage. In spite of this, various

not subject to first pass metabolism [133].

20 Application of Nanotechnology in Drug Delivery

phatidylcholine (DPPC) endogenous to the lung.

of the drug formulation.

investigated.

**6.3. Cancer therapy**

lation of the drugs [134].

Currently several liposomal formulations are in the clinical practice containing different chemotherapeutics such as doxorubicin (Doxil1/Caelyx1), doxorubicin (Myocet1), daunoru‐ bicin (DaunoXome1) and cytarabine (DepoCyte1) for treating the ovarian cancer, AIDS related Kaposi's sarcoma, multiple myeloma, lymphomas, leukemia with meningeal spread. Several other liposomal chemotherapeutic drugs containing doxorubicin, annamycin, mitoxantrone, cisplatin, oxaliplatin, camptothecine, 9-nitro-20 (S)-camptothecin, irinotecan, lurtotecan, topotecan, paclitaxel, vincristine, vinorelbine and floxuridine are at the various stages of clinical trials [140].

Two liposomal formulations have been approved by the US Food and Drug Administration (FDA) and are commercially available for the treatment of AIDS-related Kaposi's sarcoma. Doxil, first liposomal drug approved by FDA and has been on the market since 1995, is a formulation of doxorubicin precipitated in sterically stabilized liposomes and has been on the market since 1995 [141], while DaunoXome, approved six months later than Doxil, is daunor‐ ubicin encapsulated in small liposomes with very strong and cohesive bilayers, which can be referred as mechanical stabilization [142].

The commercial PTX preparation (Taxol®) is formulated in the vehicle composed of Cremo‐ phor EL® (polyethoxylated castor oil used as a solubilizing surfactant) and dehydrated ethanol, which provides a homogenous preparation. However, some drawbacks have been reported for its clinical applications of this formulation such as severe hypersensitivity reactions, neurotoxicity and neutropenia [151, 152]. It was reported that these adverse effects associated with this formlation would be due to Cremophor EL rather than PTX itself [153]. PTX solubilized in Cremophor EL shows also an incompatibility with the polyvinyl chloride of the administration sets [152]. Furthermore, the short-term stability of PTX upon dilution

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23

Special requirements regarding a proper filter device as well as appropriate containers and infusion bags for the storage and administration of the drug have to be fulfilled in order to overcome the problems of incompatibility and instability during the clinical application of Taxol®. Hence, the development of an improved delivery system for PTX is of high impor‐ tance. Current approaches are focused mainly on the development of formulations that are devoid of Cremophor EL, investigation of the possibility of a large-scale preparation and a request for a longer-term stability. There are some promising possibilities to replace Taxol® by a less irritable preparation such as micelle formulations, water-soluble prodrug prepara‐ tions, enzyme-activatable prodrug preparations conjugated with antibodies or albumin,

The preparation of an optimal PTX formulation requires important considerations such as the optimization of the liposomal composition, the balance of the PTX amount encapsulated in the liposomes and the stability of the prepared PTX liposomes during storage in aqueous media [163]. The main characteristics of PTX molecule are asymmetry, bulkiness, hydrophobicity, low solubility and tendency to crystallization in aqueous media. All these factors affect the

Liposomes provide suitable environment enhancing the solubility of the hydrophobic nature PTX by associating the molecule within the membrane bilayers. Commonly prepared formu‐ lations of PTX with liposomes were able to encapsulate the highest achievable content of PTX, 3-4 mol% with stability for weeks to months whereas 4-5 mol% paclitaxel was stable in the time range of just several hours to a day, and 8% paclitaxel loading only resulted in 15 minutes of liposome stability. Generally, increasing the encapsulated amount of PTX causes a reduction in the stability of the liposomal-PTX formulation due to the crystallization of the drug molecule. Thereby, to achieve a high drug/lipid ratio while retaining the long-term physicalchemical stability, a freeze-drying method is employed to obtain a dry drug-lipid powder, which is rehydrated in an aqueous solution immediately before use [58]. The encapsulation of PTX into liposomes enhances the drug therapeutic efficacy, thus, the same therapeutic effect could be reached by a decreased PTX-dose. On the other hand, the maximum tolerated dose (MTD) of liposome-encapsulated PTX increased compared with the Taxol® [Straubinger, R.M. and S.V. Balasubramanian, Preparation and Characterization of Taxane-containing Lipo‐

Taxane liposomes have shown slower elimination, higher antitumor activity against various murine and human tumors and lower systemic toxic effect compared to Taxol® [58]. They

with aqueous media can result in possible drug precipitation [154].

parenteral emulsions, microspheres, cyclodextrins, and nanocrytals [155-162].

final design and preparation of a suitable drug formulation.

somes, Methods Enzymol. 391 (2005) 97-117.]. [163].

DaunoXome is composed of small unilamellar vesicles containing distearoylphosphatidyl‐ choline-cholesterol (2:1) with daunorubicin loaded by a pH gradient [137]. These liposomes are selectively stable in the circulation because they are small and their membrane is electrically neutral and mechanically very strong [142]. This reduces the charge-induced and hydrophobic binding of plasma components but does not protect against van der Waals adsorption. Also, uncharged liposomes are colloidally less stable than charged ones.

Doxil is a liquid suspension of 80-100 nm liposomes (2000PEG-distearoylphosphatidyletha‐ nolamine-hydrogenated-soya-bean phosphatidylcholine-cholesterol, 20 mM) loaded with doxorubicin HCl by ammonium sulfate gradient technique and additionally precipitation with encapsulated sulfate anions. These liposomes circulate in patients for several days, which increase their chances of extravasating at sites with a leaky vascular system. Their stability is due to their surface PEG coating as well as to their mechanically very stable bilayers [141, 142].

Cytarabine (Ara-C) is an effective hydrophilic chemotherapeutic agent used widely for the treatment of acute myelogenous leukaemia and lymphocytic leukeamia [143]. It has often been utilized in the combination chemotherapy, against solid tumors and leukaemias. Cytarabine is a cell cycle-dependent drug; hence, prolonged exposure of cells to cytotoxic concentrations is critical to achieve maximum cytotoxic activity. The toxicity of cytarabine is reduced if it is able to maintain an effective therapeutic level for a long period of time and, thus, it is a suitable candidate for administration in a controlled-release dosage form. Liposome encapsulated liposomes (DepoCytTM) are now commercially available.

Etoposide (VP-16-213) is another successful chemotherapeutic agents used for the treatment of human cancers. The drug is currently in its third decade of clinical use and is a front line therapy for a variety of malignancies, including leukaemias, lymphomas and several solid tumors [144]. It has a short biological half-life (3.6 h) with a terminal half-life of 1.5 h intrave‐ nously and a variable oral bioavailability ranging from 24% to 74%. Although intraperitoneal injection would result in initial high local tumor concentrations, prolonged exposure of tumor cells may not be possible [145].

The harmful and even destructive effect of cytotoxic drugs on healthy body cells makes it necessary to search for new delivery methods for drugs like cytarabine and etoposide. There are many articles describing the results of investigations of incorporation of cytarabine [146] and etoposide [147] into liposome. However, there is no information about their simultaneous incorporation, in spite of the fact that these two drugs have been used for more than 30 years.

Taxanes are complexes of diterpenoid natural products and semisynthetic analogs. Presently, these drugs belong to prominent anticancer agents used for combined chemotherapy [148]. Paclitaxel (PTX), the prototype of this class, emerges from a natural source [149]. This drug have been used for various cancers including ovarian, breast, head and neck, and non-small cell lung cancers [150].

The commercial PTX preparation (Taxol®) is formulated in the vehicle composed of Cremo‐ phor EL® (polyethoxylated castor oil used as a solubilizing surfactant) and dehydrated ethanol, which provides a homogenous preparation. However, some drawbacks have been reported for its clinical applications of this formulation such as severe hypersensitivity reactions, neurotoxicity and neutropenia [151, 152]. It was reported that these adverse effects associated with this formlation would be due to Cremophor EL rather than PTX itself [153]. PTX solubilized in Cremophor EL shows also an incompatibility with the polyvinyl chloride of the administration sets [152]. Furthermore, the short-term stability of PTX upon dilution with aqueous media can result in possible drug precipitation [154].

formulation of doxorubicin precipitated in sterically stabilized liposomes and has been on the market since 1995 [141], while DaunoXome, approved six months later than Doxil, is daunor‐ ubicin encapsulated in small liposomes with very strong and cohesive bilayers, which can be

DaunoXome is composed of small unilamellar vesicles containing distearoylphosphatidyl‐ choline-cholesterol (2:1) with daunorubicin loaded by a pH gradient [137]. These liposomes are selectively stable in the circulation because they are small and their membrane is electrically neutral and mechanically very strong [142]. This reduces the charge-induced and hydrophobic binding of plasma components but does not protect against van der Waals adsorption. Also,

Doxil is a liquid suspension of 80-100 nm liposomes (2000PEG-distearoylphosphatidyletha‐ nolamine-hydrogenated-soya-bean phosphatidylcholine-cholesterol, 20 mM) loaded with doxorubicin HCl by ammonium sulfate gradient technique and additionally precipitation with encapsulated sulfate anions. These liposomes circulate in patients for several days, which increase their chances of extravasating at sites with a leaky vascular system. Their stability is due to their surface PEG coating as well as to their mechanically very stable bilayers [141, 142]. Cytarabine (Ara-C) is an effective hydrophilic chemotherapeutic agent used widely for the treatment of acute myelogenous leukaemia and lymphocytic leukeamia [143]. It has often been utilized in the combination chemotherapy, against solid tumors and leukaemias. Cytarabine is a cell cycle-dependent drug; hence, prolonged exposure of cells to cytotoxic concentrations is critical to achieve maximum cytotoxic activity. The toxicity of cytarabine is reduced if it is able to maintain an effective therapeutic level for a long period of time and, thus, it is a suitable candidate for administration in a controlled-release dosage form. Liposome encapsulated

Etoposide (VP-16-213) is another successful chemotherapeutic agents used for the treatment of human cancers. The drug is currently in its third decade of clinical use and is a front line therapy for a variety of malignancies, including leukaemias, lymphomas and several solid tumors [144]. It has a short biological half-life (3.6 h) with a terminal half-life of 1.5 h intrave‐ nously and a variable oral bioavailability ranging from 24% to 74%. Although intraperitoneal injection would result in initial high local tumor concentrations, prolonged exposure of tumor

The harmful and even destructive effect of cytotoxic drugs on healthy body cells makes it necessary to search for new delivery methods for drugs like cytarabine and etoposide. There are many articles describing the results of investigations of incorporation of cytarabine [146] and etoposide [147] into liposome. However, there is no information about their simultaneous incorporation, in spite of the fact that these two drugs have been used for more than 30 years. Taxanes are complexes of diterpenoid natural products and semisynthetic analogs. Presently, these drugs belong to prominent anticancer agents used for combined chemotherapy [148]. Paclitaxel (PTX), the prototype of this class, emerges from a natural source [149]. This drug have been used for various cancers including ovarian, breast, head and neck, and non-small

uncharged liposomes are colloidally less stable than charged ones.

liposomes (DepoCytTM) are now commercially available.

cells may not be possible [145].

cell lung cancers [150].

referred as mechanical stabilization [142].

22 Application of Nanotechnology in Drug Delivery

Special requirements regarding a proper filter device as well as appropriate containers and infusion bags for the storage and administration of the drug have to be fulfilled in order to overcome the problems of incompatibility and instability during the clinical application of Taxol®. Hence, the development of an improved delivery system for PTX is of high impor‐ tance. Current approaches are focused mainly on the development of formulations that are devoid of Cremophor EL, investigation of the possibility of a large-scale preparation and a request for a longer-term stability. There are some promising possibilities to replace Taxol® by a less irritable preparation such as micelle formulations, water-soluble prodrug prepara‐ tions, enzyme-activatable prodrug preparations conjugated with antibodies or albumin, parenteral emulsions, microspheres, cyclodextrins, and nanocrytals [155-162].

The preparation of an optimal PTX formulation requires important considerations such as the optimization of the liposomal composition, the balance of the PTX amount encapsulated in the liposomes and the stability of the prepared PTX liposomes during storage in aqueous media [163]. The main characteristics of PTX molecule are asymmetry, bulkiness, hydrophobicity, low solubility and tendency to crystallization in aqueous media. All these factors affect the final design and preparation of a suitable drug formulation.

Liposomes provide suitable environment enhancing the solubility of the hydrophobic nature PTX by associating the molecule within the membrane bilayers. Commonly prepared formu‐ lations of PTX with liposomes were able to encapsulate the highest achievable content of PTX, 3-4 mol% with stability for weeks to months whereas 4-5 mol% paclitaxel was stable in the time range of just several hours to a day, and 8% paclitaxel loading only resulted in 15 minutes of liposome stability. Generally, increasing the encapsulated amount of PTX causes a reduction in the stability of the liposomal-PTX formulation due to the crystallization of the drug molecule. Thereby, to achieve a high drug/lipid ratio while retaining the long-term physicalchemical stability, a freeze-drying method is employed to obtain a dry drug-lipid powder, which is rehydrated in an aqueous solution immediately before use [58]. The encapsulation of PTX into liposomes enhances the drug therapeutic efficacy, thus, the same therapeutic effect could be reached by a decreased PTX-dose. On the other hand, the maximum tolerated dose (MTD) of liposome-encapsulated PTX increased compared with the Taxol® [Straubinger, R.M. and S.V. Balasubramanian, Preparation and Characterization of Taxane-containing Lipo‐ somes, Methods Enzymol. 391 (2005) 97-117.]. [163].

Taxane liposomes have shown slower elimination, higher antitumor activity against various murine and human tumors and lower systemic toxic effect compared to Taxol® [58]. They have also shown antitumor effect in Taxol-resistant tumor models [164]. Abraxane®, the only nonliposomal preparation of PTX, (albumin nanoparticle-based PTX preparation) and Lipusu® (liposomal PTX approved by State FDA of China) have entered the field of clinical applications. LEP-ETU (NeoPharm) and EndoTAG®-1 (Medigene) have reached the phase II of the clinical trials. Generally, liposomes and protein nanoparticles represent a promising approach to the optimization of PTX delivery. Their commercialization is at the doorstep of modern drug delivery market.

ical range or lower, thus making it possible to encapsulate thermally labile compounds. Additionally, they can be prepared and stored in the presence oxygen without any degradation. According to the clinical experiments, in vivo and in vitro, these new drug delivery vehicles are not toxic. Thus, the biocompatibility and better stability of archaeo‐ somes in numerous conditions offer advantages over conventional liposomes for their usage in biotechnology including vaccine and drug/gene delivery [167]. Consequently, they can be considered as better carriers than conventional liposomes, especially for protein and peptide delivery due to their high stability. Li et al. showed the superiority of archaeo‐ somes over conventional liposomes in their study in which they used insulin as a model

Liposomes as Potential Drug Carrier Systems for Drug Delivery

http://dx.doi.org/10.5772/58459

25

Another development aiming to enhance tissue targeting is virosomes in which the liposome surface is modified with fusogenic viral envelope proteins [170]. Virosomes have been used for the intracellular delivery of drugs and DNA [171, 172] as well as the basis of the newly developed vaccines which are very effective in the delivery of protein antigens to the immune system [173]. As a result, a whole set of virosomes-based vaccines have been developed for human and animal use. Special attention has been paid to the delivery of influenza vaccine using virosomes containing the spike proteins of influenza virus. Virosome-based vaccines were found to be highly immunogenic and well tolerated in children. A similar approach was used to prepare virosomal hepatitis A vaccine that elicited high antibody titres after primary and booster vaccination of infants and young children which was also confirmed for the healthy adults and elderly patients [174-176]. In general, virosomes can provide an excellent opportunity for the efficient delivery of both various antigens and many drugs, including nucleic acids, cytotoxic drugs and toxoids [177, 178], although they might present certain

Niosomes, exhibiting a similar behavior to liposomes, are the vesicles that are made up of nonionic surfactants (e.g. alkyl ethers and alkyl esters) and cholesterol. These structures are stable on their own and they increase the stability of the encapsulated drugs. No special conditions are needed for handling and storage of these surfactants. Niosomes improve the oral bioavailability of poorly absorbed drugs, and enhance skin penetration of drug. When compared with liposomes, their oral absorption is better due to the replacement of phospho‐ lipids with nonionic surfactants which are less susceptible to the action of bile salts, parenteral, as well as topical routes. These delivery systems are biodegradable, biocompatible and nonimmunogenic. Niosomes improve the therapeutic performance of drug molecules by delaying the clearance from the circulation and protecting the drug from biological environment [179].

The transdermal delivery is one of the most important routes of drug administration. The main factor which limits the application of transdermal route for drug delivery is the permeation of drugs through the skin. Human skin has selective permeability for drugs. Lipophilic drugs can pass through the skin but the drugs which are hydrophilic in nature can not pass through. Water soluble drugs either show less or no permeation. To improve the permeation of drugs through the skin various mechanisms have been investigated, including use of chemical or physical enhancers, such as iontophoresis, sonophoresis, etc. Liposomes and niosomes are not

problems associated with their stability, leakiness and immunogenicity.

peptide for its oral delivery [169].
