Nanoparticles as drug carriers

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Nanoparticles as drug carriers

Abstract: The list of pharmaceutical drug carriers is now fortified with numerous nanoparticulate carriers due to persistent efforts of scientists from all over the world. Various types of nanoparticulate drug carriers have been developed which include nanoparticles – polymeric and lipidic, liposomes, dendrimers, micelles, nanoemulsions and nanosuspensions. Owing to their small size, their potential to improve therapeutic indices and safety profiles of existing drugs, and their ability to be modified by suitable ligands for specific targeting to certain body areas, drug loaded nanocarriers are being developed and clinically employed for the treatment of numerous disease conditions. Another advantage offered by these systems is that they are versatile enough to be administered by most common routes, such as oral, parenteral, dermal, pulmonary, nasal, etc. This chapter presents a brief overview of the different types of drug delivery nanoparticles, which are of particular relevance to the pharmaceutical market, because of their amenability for large-scale manufacture. These are described with the help of examples of commercially successful systems. Sections of this chapter then focus on various disease conditions where these nanoparticulate drug carriers have exhibited a distinct advantage over the conventional dosage forms. Attention has also been given to the different administration routes which have been responsible for their therapeutic and commercial success. Key words: nanoparticles, nanosuspensions, liposomes, micelles, dendrimers, cancer, infectious diseases, vaccine, nucleic acids, oral, parenteral, skin, pulmonary, nasal, brain.

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2.1 Nanoparticles as drug carriers: the scope Nanoparticles as drug delivery vehicles have undergone tremendous developments since their first conceptualization by Paul Ehrlich, back in 1954. He referred to them as ‘magic bullets’, wherein the drug was directly attached to a targeting moiety for its specific administration to the diseased tissues without affecting healthy ones. Research over the years has contributed numerous advanced drug delivery systems, mostly comprising nanoparticulate carriers, to the list of pharmaceutical products. Some of these include nanoparticles – polymeric and lipidic, liposomes, dendrimers, micelles, nanoemulsions and nanosuspensions. Their rapid development arises from their ability to overcome the drawbacks of the currently employed therapeutic drugs, which exhibit poor biopharmaceutical and pharmacokinetic properties. The majority of these are insoluble or possess poor aqueous solubility, thus presenting formulation challenges, since solubility is critical for determining the drug efficacy irrespective of the administration route. The outcome is absence of formulations containing such drugs or commercialization of less favorable formulations which complicates the prediction of bioavailability and potential side effects. In such cases nanoparticulate drug carriers provide alternative formulation strategies for these molecules thus enhancing their scope for commercialization. Nanoparticulate drug carriers can solve numerous drug delivery problems through various approaches, such as enhanced dissolution due to increased surface area of smaller particles, improved solubilization, non-invasive administration routes as alternatives to parenteral administration, formulations with higher stability and shelf-life, and enhanced absorption of insoluble moieties, thus lowering the amount of drug administered and improving the therapeutic indices and safety profiles. Additional benefits include reproducible nanoparticles, thus leading to economically beneficial and effective therapeutics, potential for prolonged drug release, potential for receptor mediated targeting through surface modification with suitable ligands and opportunities for disease specific targeting [1–5]. Drug loaded nanocarriers are being developed for most of the common routes of administration such as oral, parenteral, transdermal, pulmonary, nasal, ocular and mucosal, and are being clinically employed for the treatment of numerous disease conditions and in diagnostic imaging [2, 4]. This chapter will specifically focus on nanoparticulate systems that are of relevance to market transition, both with regards to system types and

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Nanoparticles as drug carriers

the common administration routes. Though inorganic and metal nanoparticles have applications in areas of drug delivery and diagnosis, these systems will not be discussed in this chapter because there are several other excellent reviews describing these and the limited expertise of the authors regarding these particular systems [3, 6].

2.2 Nanoparticles as drug delivery carriers 2.2.1 Nanoparticles Introduced more than 35 years ago, nanoparticles constitute a class of stable colloidal particles in the size range of 10 to 1000 nm and comprising of polymer or lipid based fabrication materials or their combination. Drug loaded nanoparticles consist of the active constituents entrapped or dissolved within the polymer/lipid matrix or adsorbed on the nanoparticle surface. The drug incorporation may be conducted either during or after the nanoparticle formulation [3, 4, 7–10]. Nanoparticles have been formulated for administration by various routes but are one of the attractive options for intravenous and pulmonary routes due to their suitable size for passage through the smaller blood vessels and the respiratory airways, respectively [3]. In addition to their size benefits, they offer the possibility of surface modification with appropriate ligands for targeting specific regions in the body, thus overcoming the problems of drug stability and toxicity [7, 11]. The rate of drug release and targeting via nanoparticulate drug delivery systems also depends on their porosity. The porosity of nanoparticles controls their water absorption capacity, which in turn plays a critical role in modulating the release rate of the encapsulated moiety for maintaining a sustained-release profile for hydrophobic drugs. One way of controlling this porosity during the manufacture is through an appropriate choice of materials used as the nanoparticle shell [3]. The majority of nanoparticles in the pharmaceutical arena have been employed for oncology-related applications. This is primarily due to their enhanced permeability and retention (EPR) in tumors and sites of inflammation and infection, these being the primary locations for widespread angiogenesis and altered tissue vasculature, leading to impaired lymphatic functions and release of permeability factors. The EPR effect may thus be exploited for selective targeting of nanoparticles to the tumor cells [9, 12]. The commonly explored Published by Woodhead Publishing Limited, 2012

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types of nanoparticulate drug delivery systems have been described as follows.

Polymeric nanoparticles Polymeric nanoparticles are one of the most widely exploited drug delivery carriers and comprise the therapeutic agent formulated using natural or synthetic polymers. The effectiveness of polymeric nanoparticles in terms of the extent of drug encapsulation or incorporation as well as the drug release depends on the physicochemical characteristics, size and morphology of the polymer. These polymer properties also play an important role in determining its biocompatibility. Additionally, some polymers may also be susceptible to changes in environmental pH, temperature and chemical composition, consequently affecting the rate and site of drug release and particle integrity in specific regions of the body [7, 13–15]. For example, our research group has developed nanoparticles from the pH-sensitive polymer Eudragit® S100 for selective colonic targeting of curcumin. The developed formulation showed significant efficacy in the murine ulcerative colitis model when compared with the un-encapsulated curcumin [16, 17]. The choice of the polymer also governs their circulation half-life and their uptake and destruction by the mononuclear phagocytic system (MPS). Particles with a higher hydrophilic shell generally adsorb lower quantities of plasma proteins and opsonins on their shells and it has been observed that nanoparticles of hydrophobic polymers coated with poly(ethylene glycol) (PEG) can easily escape phagocytosis by the macrophages of the MPS [4, 13, 18]. Additionally, PEG coating has also been reported to protect certain polymeric nanoparticles from the harsh environment of the stomach and aid in successful drug transport across the intestinal and mucosal barriers [3]. For example, Craparo et al. have explored Rivastigmine loaded nanoparticles of Pegylated, acryloylated polyaspartamide polymers where PEG coating was found to assist in reduced uptake by the macrophage cell line [19]. The most commonly employed polymers for nanoparticle formulation include poly(glycolic acid) (PGA), Poly(lactic acid) (PLA), PLGA, poly-ε-caprolactone, and poly(methyl methacrylate), because of their biocompatibility and FDA approval for human use [4, 20]. Based on the materials used in the formulation the methods of preparing polymeric nanoparticle dispersions may be categorized as those involving polymerization of monomers either by emulsion- or dispersion-polymerization, or those involving preformed polymers,

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which may be carried out by emulsification-diffusion, salting-out or nanoprecipitation methods. Detailed descriptions of these methods have already been published elsewhere [4, 7, 8].

Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) SLNs are submicron particles in the size range of 1 to 1000 nm and comprise lipophilic or hydrophilic drugs incorporated into physiological and biodegradable/biocompatible lipids, with or without emulsifiers. The most commonly employed lipids include highly purified triglyceride, complex glyceride mixtures, partial glycerides, fatty acids, pegylated lipids, steroids and waxes [4, 21, 22]. SLNs have been considered to offer the benefits of various alternative systems like polymeric nanoparticles, liposomes and emulsions while at the same time obviating their drawbacks. In general they have been found to have applications in drug targeting, controlling drug release, incorporating both hydrophilic and lipophilic drugs in significant amounts, avoiding organic solvents in formulation process and protecting drugs from harsh environments [4, 21, 23, 24]. Various approaches may be employed for formulating SLNs, which include dispersion techniques such as high shear homogenization and ultrasound [21], high pressure homogenization (HPH) [25, 26], hot homogenization of the drug containing lipid and emulsifier above the melting point of the lipid followed by cooling [27], cold homogenization for thermo-sensitive drugs which may be conducted by solvent emulsification–evaporation [25, 26], solvent emulsification–diffusion [28], solvent injection or nanoprecipitation [29], microemulsion template techniques [22] and multiple emulsion techniques [30]. Yet another distinct method for formulating SLNs is the membrane contactor method [31]. A further processing advantage of SLNs is their ability to be sterilized by filtration, heat treatment, and irradiation without alteration in their physicochemical characteristics and drug release profile, thus making them suitable for parenteral applications [21]. Excellent reviews on the details of SLN preparation and characterization, including their evaluation with respect to storage stability, toxicity potential and in vivo fate have already been published and hence will not be discussed here [21, 32–34]. From the commercial point of view, SLNs constitute an attractive delivery system due to the possibility of an easy and economically practical large-scale production, with the product fulfilling the regulatory Published by Woodhead Publishing Limited, 2012

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requirements. The high pressure homogenization technique, whose worldwide patent rights are currently with the London-based SkyePharma, fulfils the regulatory criteria and is the most commonly employed method for SLN production [35, 36]. Patents have been granted to Gasco and co-workers who developed a microemulsion template method for SLN formulation and is being further pursued by Vectorpharma, Trieste, Italy for commercial SLN production [32, 36–38]. Pharmatec, Milan, Italy has developed an SLN formulation for the oral administration of cyclosporine which exhibited in vivo advantages of absence of high plasma peak, low variability in plasma concentrations and prolonged drug release when compared with its commercial competitor Sandimmun® Optoral/ Neoral®. A commercial unit for the large-scale production of SLNs with batch sizes between 2 and 10 kg is available with this company [36, 39–41]. Similarly, DDS GmbH, Germany has developed a unit with the production capacity of up to 150 kg per hour but extendable to 500 kg per hour through the employment of high capacity homogenizers. However, despite their commercial success, SLNs are faced with several limitations including limited drug loading attributable to drug solubility issues, drug expulsion upon lipid crystallization and an upper limit of 30% on particle concentration in aqueous dispersions [36]. These problems have been proposed to be overcome by the second generation lipid nanoparticles referred to as the nanostructured lipid carriers (NLC®). Here the Lipid matrix consists of a blend of solid and liquid lipids such that the mixture retains a solid form at 40 °C and the drug exhibits a higher solubility in the liquid lipid. Many researchers have exploited lecithins, amphiphilic cyclodextrins and para-acyl-calix[4]-arenes for formulating NLCs, which have been formulated in three distinct forms namely the imperfect structured, structureless and multiple type form. At this point the authors will not discuss the various procedural and structural details of these systems due to some existing high-quality literature pertaining to these topics [21, 22, 35, 36, 42–45]. From the commercial point of view, it is possible to produce aqueous NLC dispersions with about 50–60% particle concentration using high pressure homogenization. The physical form of such high particulate dispersions varies from cream-like to solid, with further increase in particle concentration (about 80% with special multi-step processing) the final product is obtained in solid form [46, 47]. One of the commercial and economical high pressure homogenizers, approved by the regulatory agencies for manufacturing NLCs, is the APV Gaulin 5.5 with its production capability of 150 kg of dispersion per hour. With such equipment, NLCs may be developed for administration by different

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routes of administration including the oral route (as solid NLCs filled in capsules or in the form of pellets), dermal route (as cream) or by parenteral route (intravenous, intramuscular, sub-cutaneous) [36]. Our research group has exploited the afore-mentioned advantages of SLNs and NLCs for improving the delivery of various therapeutic molecules. NLCs of the anti-malarial drug β-artemether have been formulated for intravenous administration to present the drug in a solubilized form in an aqueous vehicle. This formulation speeded up the drug uptake and prolonged its release. It was found to retain its size and distribution even after sterilization by autoclaving and was found to be safe with regards to its haemolytic potential. In vivo studies indicated a better efficacy for a prolonged duration as compared to the marketed formulation of the same drug [48]. NLCs have also been formulated for Primaquine to overcome its lethal side effects. The objective was to target this drug to the liver by mimicking the natural lipoproteins and assist in reduction of therapeutic dose and drug toxicity. This formulation exhibited superior anti-malarial action as compared to the marketed formulation and a prolonged residence time in the liver (unpublished data). Our group has also formulated NLCs of benzoyl peroxide (BPO) and clindamycin phosphate to exploit the advantages of this drug combination for the treatment of acne at the same time overcoming their chemical incompatibility due to location in different compartments of the formulation. The developed NLCs exhibited superior efficacy when compared with the marketed formulation in the in vitro antimicrobial evaluation (unpublished data). Further SLNs have been formulated for tretinoin which is effective against various proliferative and inflammatory skin diseases but exhibits formulation challenges like poor solubility, skin irritation and instability when exposed to heat, light and air. The developed formulation was found to offer improved topical delivery of tretinoin and exhibited lower skin irritation when compared with the marketed cream of this drug [49]. SLNs have been developed by our group also for incorporating the antibiotic agent amphotericin B with antimycotic and anti-Leishmaniasis activity. The usefulness of this agent is limited because of its severe nephrotoxicity leading to kidney failure. The formulation was found to be safe for oral administration, increased the drug bioavailability as compared to its solubilized form and provided a controlled drug release thus indicating its potential for systemic fungal infections [50]. Finally SLNs and NLCs have also been developed for the anti-Alzheimer’s agent donepezil hydrochloride for topical administration as gels, where the formulations were found to offer good skin permeability of the drug when compared with its oil-in-water (o/w) and water-in-oil emulsion (w/o). The Published by Woodhead Publishing Limited, 2012

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formulation method is suitable for industrial application and the preliminary results indicate the formulation potential for pharmacokinetic and pharmacodynamic evaluation (unpublished data).

Protein nanoparticles Proteins form a distinct versatile form of biopolymers whose biodegradation into the constituting amino acids makes them an attractive option for nanoparticulate drug delivery systems [51]. Albumin, one of the major serum proteins, possesses suitable surface properties such as the presence of free amino and carboxylic groups available for covalent or non-covalent modification, which enhances its applicability in the design of nanoparticulate systems. Albumin nanoparticles may be formulated by dissolving the protein in water followed by its desolvation by drop-wise addition of ethanol (anti-solvent). A chemical (glutaraldehyde) or enzymatic cross-linking agent is subsequently added to assist in nanoparticle formation. This method is referred to as the coacervation or desolvation/cross-linking technique. Other common preparation methodologies include emulsion/solvent extraction and complex coacervation methods [51, 52]. In the former method an aqueous solution of the protein is emulsified in an oily phase by employing suitable stabilizers and high-speed homogenization or ultrasonic shear. The nanoparticles thus formed at the o/w interface are then obtained by the removal of oily phase employing suitable organic solvent, followed by their cross-linking with a chemical or enzymatic agent [53, 54]. The complex coacervation technique has been used for its complexation with DNA/oligonucleotides to assist their enhanced delivery by evading the MPS. In this method, charges are induced in the amphoteric protein solution by using suitable pH (below or above its isoelectric point; pI) and the charged protein then binds the nucleotide molecule by electrostatic interaction. Rhaese and co-workers used albumin in combination with the cationic polyethyleneimine (PEI) to bind DNA, where the ratio of these components was found to govern the nanoparticle features and transfection efficiency. The system was found to be less toxic than PEI alone due to the lower cationic charge. In this case 1-ethyl-3[3dimethylaminopropyl]carbodiimide (EDC) was used to cross-link the nanoparticles. Alternatively Wartlick and co-workers used glutaraldehyde as the cross-linker to formulate antisense oligonucleotide loaded albumin nanoparticles. Upon optimization of system with respect to the amounts of anti-solvent and cross-linker, they found that lower concentrations of the latter resulted in rapid intracellular degradation of the nanoparticles

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and hence higher release of the nucleotide cargo in the cytosol of the tumor cells [55, 56]. Other USFDA approved proteins which may be employed for formulating nanoparticles by the above described methodologies include the animal protein gelatin and the plant protein zein [51]. Reference [51] of this chapter has further formulation and characterization details of protein nanoparticles in drug delivery. From the commercial point of view, the protein nanoparticles were a success when the albumin-bound paclitaxel nanoparticles (ABI-007 or Abraxane™) manufactured by American Pharmaceutical Partners, Inc., USA was approved by the USFDA in January 2005 for therapy of metastatic breast cancer [4, 57–59]. This formulation is based on the ProtoSphere™ technology of Abraxis BioScience, Inc. to result in nanoparticles of the size range of 100–200 nm. The formulation is believed to act by the preferential accumulation of albumin in leaky tumor vasculature with caveolae mediated uptake as the specific internalization pathway into the cells [59, 60]. This formulation eliminated the requirement of the surfactant Cremophor EL in the earlier marketed formulation Taxol® and hence obviated the vehicle mediated hypersensitivity reactions. Higher response rates and time to tumor progression was clinically observed with Abraxane, with a reduction in overall side effects like severe neutropenia. Administration of higher doses of the drug was thus possible, which accounted for the superior anticancer efficiency of this formulation as compared to Taxol® [3, 59, 61]. Due to this initial commercial success with the infusion form of Abraxane, the formulation is being investigated for other forms of cancer as well as by alternative routes of administration, for example oral and pulmonary. Similarly albumin nanoparticles are being investigated for delivering other drug molecules [3, 62].

Nanosuspensions Dispersions of nanosize drug particles in aqueous medium provides a suitable approach for delivering drug substances insoluble in both aqueous and organic mediums and for which other formulation approaches become cumbersome. Formulation of drug nanocrystals by this approach thus results in ‘nanosuspensions’ of pure drug particles in liquid media, typically in the size range between 200 and 600 nm, and stabilized by surfactants or polymeric stabilizers [63–66]. Nanosuspensions may be produced either by ‘bottom up’ or ‘top down’ approach. The former includes precipitation process based Hydrosol technology which was developed by Sucker and co-workers and owned by Sandoz (now Published by Woodhead Publishing Limited, 2012

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Novartis). The final product is marketed by the name, Nanomorph (company: Soliqs/Abbott, formerly Knoll/BASF) [63, 65, 67–69]. This method, which is generally employed for poorly soluble drugs, involves their dissolution in suitable solvent followed by the addition of this solution to a non-solvent containing a surfactant. The rate of addition forms a critical step and has to be sufficiently rapid to enable the formation of a rapid supersaturated drug solution and formation of ultrafine drug particles, either crystalline or amorphous in nature. The advantage of this method lies in its simplicity, economic feasibility and ease of scale-up employing static blenders or micromixers; however, the disadvantages include possibility of crystal growth leading to larger particles and possibility of alteration in their physical state (e.g. amorphous to crystalline) thus affecting the drug bioavailability [63, 70]. The ‘top down’ approach may practically be enforced by two different techniques, namely high pressure homogenization and pearl/ball milling. High pressure homogenization is conducted either by microfluidization or piston-gap homogenizers. The former method was employed by Skyepharma Canada Inc. wherein the suspension of drug particles is split into two streams which collide frontally, generating sufficient shear to result in nanocrystal formation. In other types of microfluidizers, the suspension changes its flow direction leading to shear due to impact of particles against each other. The disadvantage of this method is that about 50–100 passages through the microfluidizer are generally required to result in a nanosuspension which may in turn contain a significant percentage of drug microparticles [63, 65, 67]. As an alternative to these limitations, high pressure homogenization employing piston-gap homogenizers was used for the first time by Müller et al. in 1994 to form nanosuspensions in water. This technology, known as Dissocubes® (owned by SkyePharma, formally by Drug Delivery Services GmbH), involves jet-milling of drug dispersion in a surfactant medium followed by the passage of this macrosuspension through piston-gap-type highpressure homogenizers [63–65, 71, 72]. A further generation of this technology employed homogenization of the drug particles in nonaqueous media or those with lower concentrations of water such as polyethylene glycol (PEG), glycerol or their mixtures with water as well as oils. The resulting nanosuspensions exhibit the option of being filled into capsules or being prepared as tablets or pellets if the medium consists of a mixture of water with a fast-evaporating solvent like ethanol. This technology is known as Nanopure®, the intellectual property rights for which are with PharmaSol GmbH, Germany. To combine the advantages of both, precipitation as well as high pressure homogenization, the

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company Baxter introduced the NANOEDGE™. In this technology the traditional precipitation step is followed by passage of the precipitated suspensions through high pressure homogenizers. The second step, known as the ‘annealing’ process, is believed to convert the precipitated amorphous particles into partially amorphous or crystalline particles. This avoids the risk of their natural conversion during the product shelflife and hence the changes in bioavailability or pharmacokinetic profile after oral or intravenous administration [63, 65]. Many excellent reviews are already available on the details of various formulation methods of nanosuspensions [63–67, 72]. The ‘top down’ approach, referred to as pearl/ball milling, involves production of nanosuspensions employing high-shear media mills or pearl mills in which water, drug and stabilizer are charged in a milling chamber fixed with a milling shaft. The milling media or pearls are then rotated in this mixture at a very high shear rate under controlled temperatures. Various milling media may be employed like zirconium oxide, glass or cross-linked polystyrene resin. This process, in a batch mode, yields particles smaller than 200 nm within a time span of about half to one hour. The advantages of this method include its applicability to drugs exhibiting poor solubility in both aqueous and organic media, ease of large-scale production with good inter-batch reproducibility, homogeneity of the final product and versatility with regards to the quantities of drugs which may be processed, resulting in products with high or low particle concentration. However, the drawbacks include residues of milling media in the final product. This may nevertheless be overcome by employing polystyrene resin [72, 73]. With regards to drug delivery, nanosuspensions offer numerous benefits including enhanced solubility mediated by the smaller particle size, enhanced bioavailability due to increase in adhesive nature, higher drug loading and drug stability, possibility of surface modification to achieve targeted drug delivery, and possibility of large-scale production for commercialization [64, 66, 67]. For large-scale production, the most common types of homogenizers used for this purpose are manufactured by APV Gaulin, Avestin, Stansted or Niro Soavi [63, 72]. For larger nanosuspension batches, researchers recommend use of Rannie 118 capable of handling a load of 1 ton per hour and operated at a maximum pressure of 1500 bar or Avestin 1000 capable of handling 1000 litres per hour [63]. Furthermore, the resulting products may be rendered aseptic by autoclaving or radiation sterilization or employment of aseptic manufacturing units for products of parenteral application. The investigated administration routes include oral administration (in the Published by Woodhead Publishing Limited, 2012

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form of tablets, pellets, powders or liquids filled in capsule shells as described earlier), dermal applications, pulmonary administration and administration to the central nervous system [63, 65, 66]. Our research group has recently initialized formulation of nanosuspensions of poorly soluble therapeutic agents. As a prototype, nanosuspension of the anti-malarial drug, atovaquone, was formulated using high pressure homogenization. The formulation resulted in enhanced pharmacokinetic profie and superior anti-malarial efficacy in a murine model when compared with the drug suspension and a marketed product of this drug (unpublished data). Looking at the market overview, the first nanosuspension product was introduced in 2000 by Wyeth under the brandname Rapamune® (drug: Sirolimus, an immunosuppressant); the product was developed by Elan nanosystems and was available in the form of tablets or oral solution. The tablet form resulted in a higher bioavailability as compared to the oral solution despite the very low amount of nanocrystal loading in the total tablet weight. This was followed by the product Emend, introduced in 2001 by Merck, again developed by Elan nanosystems. The product contains the anti-emetic drug aprepiptant in the form of a capsule. With the progressive availability of suitable manufacturing facilities and the ever-present challenge of limited solubility of a large number of therapeutic molecules, the nanosuspension technique has gained a rapid industrial momentum. Numerous products based on this technique are currently in various phases of clinical trials.

2.2.2 Liposomes Drug delivery research took a big leap after the introduction of liposomes in 1960s. Since then the liposomes have been one of the most widely investigated colloidal drug delivery carriers. Liposomes may be defined as colloidal vesicles comprising of one or more concentric bi-layers of phospholipids, with intermittent aqueous or buffer compartments. Their diameter may vary between 30 nm and 100 µm [3, 4, 22, 74–76]. They may be formulated to have varying physico-chemical properties depending upon their constituting lipids, which may be natural or synthetic lipids (phospho- or sphingo-lipids) or cholesterol or polymer-lipid conjugates. There are, in general, two ways of classifying liposomes, the first depending on their composition and mode of intracellular action and the second depending on their size and number of bilayers. The first classification system comprises of five sub-types namely conventional,

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pH-sensitive, cationic, long-circulating and immunoliposomes whereas the second classification system includes multilamellar vesicles (multiple lipid bi-layers), large unilamellar vesicles (group of heterogenous vesicles surrounded by single lipid layer) and small unilamellar vesicles (single lipid layer and between 25 and 50 nm in size) [3, 77]. Thus, depending on the desired application it is possible to vary the liposome type. For example small sized, long-circulating liposomes can be used to deliver the therapeutic agent to sites of tumor, inflammation or infection whereas larger liposomes can be used to target organs of MPS especially the liver and the spleen [76, 78–80]. Because of their versatile nature, liposomes can be used to incorporate a wide variety of drug substances of varying molecular weights, solubility and surface charge. Several large-scale manufacturing techniques yield liposomal products fulfilling the criteria for regulatory approval. These may be categorized as mechanical methods, those based on replacement of organic solvents by aqueous media and those based on detergent removal [81]. The mechanical methods include the film method [74] which exhibits advantages such as its versatility for various kinds of lipids, ease of manufacture and high encapsulation efficiency for both hydrophilic as well as hydrophobic drugs. However, the drawback of this method lies in the difficulty of its scale-up and the requirement of a downsizing step to result in a more homogenous formulation. The latter is commonly conducted by extrusion whereby the initial formulation is processed through filters of defined pore size. Use of high pressure homogenizers, microfluidizers and ultra sound techniques form the other mechanical methods of liposome generation. The microfluidizers used for this purpose operate in the pressure range of 0 to 200 bar and are equipped with suitable temperature control systems to generate liposomes about 50–100 nm in diameter. This technique may be adapted for aseptic manufacture of liposomal formulation for parenteral applications [81, 82]. For downsizing, extrusion polycarbonate membranes are employed which vary in diameter from 25 to 142 mm. Northern Lipids Inc. has introduced the Lipex extruder system with temperature control to process large volumes of materials. Alternatively the Maximator device may be employed for extrusion which operates at the extrusion pressure of 12 megapascal [83]. Though extrusion is advantageous in terms of generating homogenously dispersed formulations, the technique may be cumbersome for expensive materials due to losses during multiple processing steps and blockage of extrusion membranes [81]. The methods based on replacement of organic solvents by aqueous media include the ethanol injection method in which the ethanolic Published by Woodhead Publishing Limited, 2012

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solution of the lipid is rapidly injected into aqueous phase. The resulting dilution of ethanol causes precipitation of lipid bilayers [84]. Many largescale industrial production techniques were based on this method due to its applicability to a wide range of therapeutic substances like hydrophilic proteins, vaccines, antigens and amphiphilic molecules [85–90]. The Proliposome–Liposome method involves stepwise addition of aqueous phase to the ethanol solution of the lipid and offers high encapsulation efficiency for numerous drugs with varying solubility profiles. Large-scale aseptic production techniques based on this method have already been developed [91, 92]. In the reverse-phase evaporation method, a water-immiscible organic solvent containing the lipid is added to the aqueous phase to result in o/w emulsion, which on further dilution with aqueous phase results in formation of liposomes. The advantage of this method included high entrapment of drugs but the method is disadvantageous due to the possibility of residual solvent in final product. Additional drawbacks include unavailability of very large mixers and pumps to help in formulation of commercial scale emulsions and their dilution. The methods based on detergent removal employ suitable detergents like bile salts or alkylglycosides to solubilize the lipid in a micellar system. Subsequently the detergent is eliminated by dilution, dialysis, adsorption (e.g. on resins), chromatography or filtration. Liposome formation here depends on the retention of lipid-detergent micelles after removal of the free detergent [81]. Further details of all these large-scale production techniques of liposomal formulations have already been published [77, 81]. Liposomes have been a huge success with regards to their clinical applications and numerous liposomal formulations have already received market approval. Liposomal formulations of the anticancer agents, doxorubicin and daunorubicin and that of the systemic anti-fungal agent, amphotericin B are in clinical use in several countries. Liposomes possess several delivery benefits for anticancer agents like slow drug release, a good accumulation in the tumor tissues and avoidance of drug accumulation in non-cancerous tissues (suitable for cytotoxic agents with side effects like cardiotoxicity). Anthracycline antibiotics such as doxorubicin and daunorubicin are effective against a wide spectrum of cancers including those of breast, lymphatic and hematopoyetic systems, stomach, lung, bone, uterus, ovary, bladder and thyroid gland. However, their therapeutic utility is severely limited by their toxic effects like alopecia, myelosuppression and most importantly cardiotoxicity [75, 77]. Long-circulating liposomes of these drugs have proved to be beneficial in reducing their cardiotoxicity

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and promoting their accumulation in the tumor tissues. The first anthracycline loaded liposomal preparation approved by the USFDA was Doxil®, which received market permission in 1995 for the treatment of Kaposi’s sarcoma. This formulation which contains doxorubicin in Pegylated, long-circulating liposomes is marketed by the name Caelyx® in Europe. The circulation half-life of this formulation was found to be eight times superior as compared to the free drug. At the same it successfully reduced the drug cardiotoxicity, thereby permitting higher doses and increased therapeutic index [93]. Myocet™ is a non-pegylated liposomal preparation encapsulating doxorubicin citrate complex and has been approved for treating metastatic breast cancer in Europe. Though the formulation exhibited comparable efficacy and reduced cardiotoxicity when compared with the free drug (Phase III clinical trials in metastatic breast cancer), neutropenia was observed with higher doses of the formulation [94–96]. Daunoxome® is a non-pegylated liposomal injection of daunorubicin citrate used for the treatment of Kaposi’s sarcoma. The formulation was found to have a rapid clearance as compared to Doxil® which may be due to the lack of its ‘stealth’ characteristics. Furthermore dose-dependent neutropenia and mucositis was observed in patients with solid tumor and leukemia, respectively [73, 97]. Regarding the systemic antifungal agent amphotericin B, despite its success in treating serious systemic fungal infections, the administered doses are severely limited by its major side effects like nephrotoxicity. This led to the failure of its deoxycholate-based formulation, known by the name Fungizone™. However, the liposomal formulation of this drug, marketed as Ambisome®, enabled the administration of higher doses due to reduction in nephrotoxicity. This had been attributed to the ability of this formulation to selectively transfer the drug from its lipid layers to the causative fungus, thus avoiding its contact with the cell membranes [77, 98–100]. Other USFDA approved liposomal products include liposomes of verteporfin (Visudyne) for treating wet macular degeneration and those of cytosine arabinoside (DepoCyt) for the treatment of lymphomatous meningitis and neoplastic meningitis [9].

2.2.3 Micelles Micelles may be defined as nanosized, supramolecular core-shell structures which spontaneously self-assemble in aqueous solution upon the addition of amphiphiles. This self-assembly is typically driven by Published by Woodhead Publishing Limited, 2012

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hydrophobic interactions between the amphiphiles at a concentration above their critical micelle concentration (CMC) and critical micelle temperature (CMT). Amphiphilic components with low aqueous solubility may alternatively be solubilized in suitable organic solvents and then dialyzed against water or aqueous buffers [4, 101, 102]. Micelles may also be formulated using cationic polymers like poly(ethyleneimine) (PEI), poly(aspartic acid) and poly(L-lysine) (PLL). These polyioncomplex micelles are based on ionic interactions with negatively charged nucleic acids or proteins of therapeutic interest [103–105]. Unimolecular micelles constitute another type of structure which consists of covalent linkages between amphiphilic chains and single polymer molecules, having star shaped or dendritic structure. And finally there have also been reports regarding cross-linked micelles wherein a multimolecular micellar structure is stabilized by cross-linking the polymer chains. This stabilization may exist within the core region or may be throughout the micelle shell. The resulting micelles exhibit stability against dilution, shear and environmental variations in pH, salt concentration, presence of various solvents, etc. [101]. The micellar systems used for drug delivery most commonly comprise of amphiphilic di-block or tri-block co-polymers and sometimes graft polymers composed of biodegradable or low molecular weight monomeric units, which can be easily eliminated from the body to avoid toxicity. They generally vary between 50 and 200 nm in diameter and consist of hydrophobic cores which encapsulate the drugs and hydrophilic shells which prevent the micellar aggregation and protein adsorption (to prevent clearance by the MPS) [3, 101, 106, 107]. Hydrophilic shell of the micelles is more frequently composed of poly(ethylene glycol) (PEG) or poly(ethyleneoxide) (PEO) or their co-blocks while a range of polymers including poly(lactic acid) (PLA), poly(glycolic acid) (PGA) ), poly(ε-caprolactone) (PCL), phospholipids/long chain fatty acids [108] and polyethyleneoxide-polypropylene oxide block co-polymers [109] (commercially known as Pluronics/Poloxamers) generally make up their hydrophobic cores. The choice of the polymer comprising the core governs the type of drug encapsulated or kinetic stability of the micelle. These different polymers have FDA approval which makes them ideal for employment in drug delivery [101, 103]. Furthermore, the micellar surface can be decorated with suitable ligands for targeting to specific regions of the body or they may be formulated using pH-sensitive polymers to release their load in specific regions of the body [3, 103]. The self-assembly of a conventional core-shell micelle is a thermodynamic process governed by the release of organized water

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molecules from the hydrophobic components and their stabilization through interaction with hydrophilic counterparts. Thus this formation depends on the mass ratio of hydrophilic and hydrophobic blocks where a ratio of one or a slight excess of hydrophilic blocks results in conventional micelles. The effectiveness of micelles as drug delivery vehicles depends on their potential rate of disassembly (thermodynamic and kinetic stability), which governs the balance between how and when the drug is released. The thermodynamic stability, in turn, is inversely related to the CMC of the polymer whereas the kinetic stability depends on the mass ratio of hydrophobic to hydrophobic components, size of hydrophobic blocks, etc. The micelle stability is also dictated by the encapsulated hydrophobic drug [101]. Numerous approaches may be used for encapsulating drugs into the micelles. The pro-drug approach involves chemical conjugation of the drug to the core forming polymer using suitable enzyme- or pH-sensitive linker, which may then be subsequently cleaved to release the drug. This polymer pro-drug self-assembles into micelles. Its stability and nature of polymer-drug linkage can be altered to modulate the drug release and hence efficacy. This approach has been used for delivering anticancer agents, for example PEO-b-poly (aspartate doxorubicin) conjugates developed by Kataoka and coworkers [101]. The physical entrapment approach is suitable in absence of reactive functional groups in drugs and polymers to assist chemical conjugation. The drug may be physically encapsulated within the micelles by varying techniques like solvent evaporation, dialysis, direct dissolution or o/w emulsification. In this method, factors such as the molecular weight and molecular characteristics of the polymer and the drug, their compatibility, their structure, the ratio of hydrophobic to hydrophilic block length, drug to polymer ratio and solution temperature influence the amount of drug that can be loaded into the micelles. The release rate of drug is then dependant on the diffusion of drug from the micelle core, the physical state of the core, micellar stability and rate of degradation of the polymer. Finally, as mentioned earlier, charged therapeutic agents can be loaded into polymeric micelles due to ionic interactions with oppositely charged polymer core. This method of micelle loading is widely used for complexation of nucleic acids in the gene therapy approach. These ionic complexes then participate in ion exchange reactions inside the cell and in the process release their nucleic acid load. The method can also be applied for charged therapeutic proteins and charged drugs. The loading amount depends on charge density, presence of salts and length of the charged polymer blocks. Such charge interactions can also be used for Published by Woodhead Publishing Limited, 2012

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formulating environmentally tuneable delivery vehicles where drug release may be modulated by alterations in environmental pH, salt concentration, molecular mass of counterions and temperature, making them sufficiently versatile for a large number of applications [101, 110–112]. Comprehensive reviews are available regarding various theories of micelle formation, their types, mechanisms of drug loading, their cellular interaction and in vivo fate for additional knowledge of the readers [101, 103, 112–114]. With regards to drug delivery applications, polymeric micelles have been largely exploited for drug delivery to brain to treat the central nervous system (CNS) disorders, delivery of anticancer therapeutics, delivery of anti-fungal agents and for delivering nucleotide based therapeutics. Focusing the attention towards the application of micelles for the treatment of CNS disorders, our group has developed Transcutol P® (Diethylene glycol monoethyl ether) and Pluronic based micelles loaded with the anti-migraine agents, Zolmitriptan and Sumatriptan, for migraine therapy. Biodistribution and autoradiography studies conducted in rats indicated the in vivo safety of both these formulations for nasal administration and their potential for nose to brain delivery, when compared with the respective drug solutions. These investigations indicate their potential for further evaluations in higher animals and then possibly in clinical settings [115, 116]. With regards to cancer therapy, micellar systems have been used for: ■

passive targeting due to EPR effect;

■

targeted delivery to specific ligands overexpressed in tumor cells;

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pH-sensitive systems for improved delivery to the low pH regions of the tumor;

■

increasing sensitivity of drug resistant tumor tissues.

Though numerous research groups have agreed to the potential of these systems to improve therapeutic efficacy of anticancer agents via abovementioned approaches, very few delivery systems have actually been able to progress from laboratories to clinical trials [101]. Examples of these successful formulations include the formulation NK911 developed by Kataoka and co-workers which has proceeded to Phase I clinical trial at the National Cancer Center Hospital, Japan. This formulation is based on chemical conjugation approach where aspartic acid units of PEO-poly (aspartic acid) block copolymer have been partially replaced by the anthracycline agent doxorubicin. The resulting

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hydrophobic substituted copolymer is employed to form micelles which are further used for encapsulating the free drug. The formulation was well-tolerated during the trial and produced only moderate side effects at myelosuppressive doses. This study not only yielded its maximumtolerated dose but also enabled deduction of the suitable dose for Phase II clinical trials [117, 118]. The second anticancer drug loaded micelle which has reached clinical trials is SP1049C which consists of Pluronic micelles encapsulated with doxorubicin. Results of Phase I clinical trial, in patients with advanced cancer, has indicated its safety profile comparable to plain drug and efficacy in tumors resistant to doxorubicin and also in doxorubicin sensitive tumors. A phase II clinical trial, in patients with inoperable metastatic adenocarcinoma of the oesophagus, has indicated positive results in the first 10 patients and the study plans to evaluate a total of 24 patients [119–121]. Similarly, Phase I clinical trials are complete for Genexol-PM, a Cremophor EL-free, polymeric micellar system loaded with paclitaxel. This study recommended a dosage of 300 mg/m2 for Phase II trials. Multicenter phase II trials of this formulation were then conducted, along with cisplatin, in patients with advanced non-small-cell lung cancer where the formulation exhibited enhanced action. Also higher doses of the drug could be administered, without any significant toxicity, due to the absence of Cremophor EL [122, 123]. Examples as these clearly reflect that developments in micellar drug carriers have progressed tremendously in the last few years. With better availability of suitable excipients and enhanced understanding of the probable delivery mechanisms, a greater number of these carriers are anticipated to be in clinical research in the forthcoming years.

2.2.4 Dendrimers Dendrimers are well-defined, monodispersal, stable nanostructures possessing three-dimensional and highly branched architecture with possibility of high functionality [124, 125]. The various functional groups that may be attached to the globular or semi-globular organization of dendrimer branches include carbohydrates (glycodendrimers), peptides (peptide dendrimers) and silicon groups (silicon-based dendrimers), etc. [4]. The three-dimensional architecture of dendrimers may be distinguished into: (1) the core or the point of attachment of dendrons; (2) the inner shells which are defined by dendrons surrounding the core; Published by Woodhead Publishing Limited, 2012

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and (3) the outer surface comprising of polyvalent attachment sites for potentially reactive groups. The core is designated as ‘Generation zero’ or ‘G0’ and the subsequent layers between the further origin points are referred as the following ‘generations’. The size of the dendrimers, which influences their transition across different biological barriers, is governed by the number of generations present in their structure. Though various types of linkages such as polyamines (e.g. polypropylene imine; PPI) and mixtures of polyamides and amines (e.g. polyamido amine; PAMAM) are involved in various dendrimer architechtures, the PAMAM dendrimers constitute a family of one of the initial and most widely exploited systems for drug delivery [4, 124, 126]. The dendrimers may be further classified into several types depending on their structural arrangement. Apart from the PAMAM and PPI dendrimers, various other dendrimer types find application in drug delivery which include: 1. the liquid crystalline dendrimers comprising of mesogenic groups capable of displaying mesophase behavior; 2. the hybrid dendrimers comprising of block or graft polymers in which peripheral amines of polyethyleneimine cores are monofunctionalized with linear polymers; 3. the multilingual dendrimers comprising of surface with multiple copies of a specific functional group; 4. the micellar dendrimers comprising of water soluble and densely branched polyphenylenes micelles; and 5. Frechet-type dendrimers comprising of surface anchored carboxylic acid groups which imparts them solubility in water or polar solvents [125, 127]. Other types of dendrimeric systems include, the ‘dendritic boxes’ consisting of a flexible dendrimer core of PPI and a rigid surface of protected amino acids. These systems can be used to simultaneously encapsulate large and small or hydrophilic and lipophilic guest molecules or their combinations and can be tuned for their preferential release. There are also dendrimers, known as dendrophanes, with cores (cyclophane based) which specifically bind with hydrophobic molecules and those known as dendroclefts, with cores specially designed to bind hydrophilic therapeutic molecules [124]. Different synthesis approaches have been adopted for formulating dendrimeric systems for drug delivery. The divergent growth method

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synthesizes the dendrimers from the core as the site of origin. The subsequent generations are built by reacting this core with reagents consisting of at least two protected branching sites followed by the removal of protecting groups. This process is repeated until dendrimers of the desired dimensions are obtained. The PAMAM dendrimers, also referred to as the starburst dendrimers, were synthesized by this approach. However, since the method involved multiple reactions of a single molecule bearing numerous equivalent reaction sites, the yield of the final product obtained was less. The second disadvantage of this method included involvement of an excess of reactants towards the completion of synthesis to provide the necessary driving force and prevent side-reactions which poses problems in product purification. The convergent growth method, on the contrary, synthesizes the dendrimers from the periphery of the anticipated final product and gradually builds the linkages inwards. Sufficiently large dendrimer segments are then finally connected to a suitable core. The method is advantageous in terms of ease of purification of the final product, obtaining a product with fewer synthesis flaws and avoidance of formation of products with too high a number of generations due to steric hindrances between the dendrimer segments and the core. The double exponential and mixed growth approach synthesizes large dendrimers in fewer steps by reacting the monomer products of convergent and divergent growth, both of a common origin, to result in an orthogonally protected trimer which is then repeatedly reacted to yield the final product. Finally the hypercores and branched monomers growth method synthesizes large amounts of dendrimers in fewer steps, by utilizing pre-assembled oligomeric building blocks for subsequent linking [4, 125, 127]. Dendrimers can be loaded with drug molecules by various approaches such as incubation with the drug to enable its encapsulation into the empty dendrimeric spaces, formation of drug–dendrimer complexes or covalent/non-covalent attachment of therapeutic molecules on the dendrimer surface to result in pro-drugs. These approaches have been successfully used by researchers to associate PMAM, PPI and poly(etherhydroxylamine) (PEHAM) dendrimers with several drugs exhibiting anti-inflammatory (ibuprofen, indomethacin, ketoprofen, naproxen, diclofenac, etc.), anticancer (camptothecin, artemether, cisplatin, doxorubicin, etoposide, 5-fluorouracil, methotrexate, paclitaxel, etc.) and microbicidal (niclosamide, nadifloxacin, prulifloxacin, sulfamethoxazole, silver salts, etc.), actions. An excellent detailed overview of these various dendrimeric drug delivery systems has been

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presented by Sönke Svenson [129]. Dendrimers have also been widely investigated for delivering nucleic acid based therapeutics. These various therapeutic molecules have been delivered to the affected areas either due to the EPR effect of the dendrimers or their surface modification with suitable targeting ligands. The different routes of applications which have been explored for their administration include oral, pulmonary, transdermal, ophthalmic, etc. Though the biocompatibility of the dendrimers has been observed to be similar to the other nano-drug delivery vehicles, the toxicity issues arising due to their cationic nature have been tackled by their pegylation or modification with fatty acids to reduce their positive charge [4, 125, 128, 129]. Looking at the clinical application of dendrimers as drug delivery vehicles, a few of these products have already received the USFDA approval for conducting their clinical trials. An example of this includes VivaGel® (SPL7013), a vaginal water-based gel, developed by Starpharma Ltd., Australia, to prevent or decrease transmission of HIV and other sexually transmitted diseases (STDs). This formulation was postulated to act by preventing attachment of the causative organisms to the body cells and differed from barrier approaches of STD, which exhibit an inhibitory effect against the organisms. A Phase I clinical trial has demonstrated the safety of VivaGel® in human volunteers by the vaginal route. The formulation is currently in Phase II clinical trials and has a co-development agreement with Durex® condoms for coating their products [130, 131]. Starpharma is also exploring the commercial applications of their dendrimer technology in the drug delivery area with GlaxoSmithKline’s Stiefel laboratories. Furthermore, Dendritic Nanotechnologies, Inc. (DNT), a subsidiary organization of Starpharma Holdings Ltd., has designed dendrimer based products known as Priostar™ and STARBURST, for application as drug targeting carriers for cancer cells and other diseases [125, 127]. Examples such as these and the increasing dendrimer related research, over the past decade, give a clear indication of the exciting future of these systems as novel drug delivery carriers. With the above sections of this chapter giving a brief overview about the various nanoparticulate drug carriers, the following sections will now discuss the various application areas as well as the main administration routes which may be explored for drug delivery applications of these carriers.

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Figure 2.1

Types of nanocarriers

A pictorial representation of the various different types of aforementioned carriers has been depicted in Figure 2.1.

2.3 Application areas for nanoparticulate drug delivery systems Nanoparticulate drug delivery systems have been explored in numerous branches of medicine, particularly in those challenging areas where the conventional therapeutics have not yielded satisfactory results or where these novel drug delivery systems have exhibited significant advantage over their conventional counterparts. They have also been widely applied for various biological therapies including gene therapy, RNA interference (RNAi) therapy, anti-sense therapy, vaccines and cell therapy where they have resulted in improved delivery of nucleic acids and proteins employed therein [3, 5]. Some of these application areas where the nanoparticulate drug carriers have received commercial success are discussed below.

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2.3.1 Nanoparticulate drug carriers for cancer therapy The therapeutic potential of numerous anticancer agents is severely limited by their physicochemical characteristics and toxicological limitations. In many cases the hydrophilicity, charge and polarity of these agents is inapt for diseased areas, presenting problems in drug transport to the tumor and resulting in its preferential distribution to the healthy body regions. This necessitates administration of higher doses and both these factors together present a serious therapeutic barrier due to toxicity issues. Additional cellular barriers for effective cancer therapy are presented by the altered tumor vasculature, including erroneous apoptotic mechanisms and multi-drug resistance (MDR) due to P-glycoprotein (P-gp) efflux mechanisms. In this context, the nanotechnology based drug delivery systems like nanoparticles, liposomes, polymeric micelles, etc., have provided obvious benefits due to their EPR effect and ability to be modified for specific targeting. Both these properties enhance the cellular and tissue specificity of the encapsulated anticancer therapeutic, obviating unwanted side effects of the latter. Another advantage presented by these systems is that they allow suitable surface modification (PEG coating) for evasion of their uptake by the reticulo-endothelial (RES) system. This grants them sufficient circulation times inside the body to allow for their selective extravasation through the leaky tumor vasculature. Due to these advantages nanoparticulate drug carriers have been enormously researched for their applications, both as therapeutics and diagnostics. This being a major research field, it warrants an entire book for itself. In this chapter we have attempted to provide a brief overview of some of the principal therapeutic approaches. The diagnostic nanocarriers in the field of oncology have not been covered here, considering the scope of this book [132]. Various nanoparticle formulations have met with success for drug delivery to cancers. In addition to Abraxane™ mentioned previously, biodegradable nanoparticles coated with PEG and surface conjugated with folic acid have been developed for selectively targeting to the folate receptors overexpressed by several types of cancer cells. Researchers have confirmed this selective binding through appropriate experimental models. Alternatively, nanoparticles conjugated with antibodies and peptide sequences have been reported for selective targeting of tumor proteins like αvβ3 integrin and Flk-1 (Fetal Liver Kinase-1), as an antiangiogenesis strategy for treating a wide range of solid tumors [3].

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Transferrin receptors have been exploited as another mode of targeting the cancer cells using nanoparticulate drug carriers [133]. Cyclosert™ developed by Insert Therapeutics, USA (now Calando Pharmaceuticals, Inc.) is one of the earliest platform technologies, in which a therapeutic agent-cyclodextrin conjugate forms nanoparticle with a mean diameter between 30 and 60 nm. This technology limits the toxicity of the associated agents by specifically targeting them to the tumor cells followed by their release in a controlled manner. IT-101, based on this technology contains the anti-cancer compoundcamptothecin (CPT) and has been acquired by Cerulean Pharma Inc. (since known as CRLX101). The Phase I clinical trials of this formulation, in patients with relapsed or refractory solid tumor cancers like pancreatic, ovarian and non-small cell lung cancer, exhibited the longer circulation times, reduced volume of distribution and reduced clearance when compared with other CPT containing formulations. This initial success has resulted in continued enrolment of patients which was completed in January 2011. A Phase II trial in patients of non-small cell lung cancer has already been initiated [134, 135]. Lipoprotein based nanoparticles are being developed by Marillion Pharmaceuticals, USA, which consist of tumor targeting moieties attached to the protein components of lipoprotein to direct them to tumor receptors rather than the lipoprotein receptors. This technology offers the possibility of attachment of multiple copies of a single targeting moiety or different targeting moieties to the same nanoparticle, thus widening the application scope of the resulting formulation [3]. Nanoparticle formulation (nanodroplets), encapsulating anti-cancer agents, is being developed by ImaRx Therapeutics Inc., Arizona for their targeted delivery to tumor tissues aided by ultrasound application. This project has been jointly funded by National Aeronautics and Space Administration (NASA) and the National Cancer Institute in collaboration with universities of Arizona and California (Davis) [136]. Another interesting targeted system, Polymersomes, comprise of two layers of biodegradable, synthetic polymers with properties similar to the phospholipid bilayers of liposomes. This system simultaneously incorporates the hydrophobic drug, paclitaxel (within the membranes) and the hydrophilic drug, doxorubicin (interior core), in its different compartments. These spontaneously assembling nanostructures degrade in the acidic milieu of tumor tissue and facilitate a targeted release of drugs, their combination being superior to their individual actions [137]. Apart from targeting, researchers have employed nanoparticles to overcome the MDR effect and enhance the efficacy of delivered drug. An example includes poly (ethylene oxide)-poly (ε-caprolactone) Published by Woodhead Publishing Limited, 2012

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nanoparticles facilitating simultaneous administration of ceramide, a pro-apoptotic mediator, and paclitaxel. Restoration in apoptotic signaling along with the benefits of nanoparticles, with this formulation, resulted in a 100-fold increase in sensitization of MDR cancer cells to doses of paclitaxel. The results indicate the clinical potential of this strategy to overcome MDR. While discussing these potential nanoparticle systems, it will be worthwhile to mention PUREBRIGHT® technology (NOF Corporation, Japan) based on hydrogel nanoparticles. This technology utilizes nanoparticles formulated with hydrophobic polysaccharides for encapsulating proteins and anti-bodies. Cholesterol pullulan has been the polymer of choice for formulating nanoparticulate cancer vaccines encapsulating cancer-specific monoclonal anti-bodies (Her2), due to its ability to activate the immune system through dendritic cell uptake. Alnis Biosciences Inc. has developed MagNaGel™ nanoparticles comprising of magnetic iron oxide material and anti-cancer molecules entrapped within hydrogel nanoparticles with mean diameter of about 25 nm. Application of alternating magnetic fields enables heating of the magnetic substance and hence enhanced transportation of the encapsulated drug into the tumor vasculature. Additional benefit of this formulation lies in its possibility to be tracked inside the body using Magnetic Resonance Imaging (MRI). The formulation has met with success in various pre-clinical experiments [3]. Liposomes constitute another delivery system that has been successfully employed in oncology. Injectable and biodegradable liposomes incorporating antiestrogenic agents like RU 58668 have exhibited enhanced action in several oestrogen receptor expressing cancer cell lines and also in animal models. This formulation may serve as a potential therapy for treating oestrogen-dependent breast cancer and for multiple myeloma. Aphios Corporation, USA, has developed stable aqueous liposomal formulations of antiancer drugs employing the SuperFluids™ supercritical fluid technology. The products of this technology, namely Taxosomes™ encapsulating paclitaxel and Camposomes™ encapsulating CPT, have exhibited superior therapeutic action in nude mice models of breast cancer and lymphoma, respectively [138, 139]. Azaya Therapeutics Inc. has developed its Protein Stabilized Nanoparticle (PSN™) technology, based on a single-step manufacturing process, for producing liposomes loaded with anticancer agents. Owing to the preclinical success with its lead active agent, Docetaxel, the company aims to extend this technology to other hydrophobic agents like Docetaxel and Irinotecan. Apart from overcoming the formulation challenges for these agents, the technology aims at improvising their therapeutic action through targeting mechanisms [140].

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Polymeric micellar formulations are also gaining increasing importance in oncology related therapies because of their drug delivery advantages described previously. NanoCarrier Ltd., Japan has developed polymeric micelles incorporating cisplatin by its complexation with PEG-poly (glutamic acid) block copolymers. Targeting studies conducted by researchers revealed their effective passive accumulation in solid tumors and enhanced circulation, both suggesting their potential for targeted therapy of solid tumors. Clinical trials of this formulation are currently underway. Joint collaboration between NanoCarrier Ltd. and Debiopharm, Switzerland has led to the development of DACH-platinPEG-poly (glutamic acid) micelles based on Medicelle™ technology. These micelles comprise of hydrophilic–hydrophobic block copolymers and enhance the drug action due to EPR effect. Other improved micellar systems are also being developed by suitable modifications such as surface coating with hydrophilic molecules, steric stabilization or shape focused modifications [3, 141]. Other examples of nanoparticulate products either in clinical trials or in markets have already been mentioned previously while discussing the various types of nanocarriers. The nanooncology formulations involving nucleic acid delivery present a novel set of therapeutics and will be discussed at a later point in this chapter. A detailed review of nanoparticulate formulations either in clinical trials or approved by USFDA has been provided in the subsequent chapters of this book.

2.3.2 Nanoparticulate drug carriers for therapy of infectious diseases Nanoparticulate drug carriers are receiving increasing attention for the treatment of infections caused by bacteria, viruses and fungi. Previous sections of this chapter have already highlighted this fact through examples of Ambisome® and VivaGel® used for the treatment of systemic fungal infections and HIV, respectively. This is particularly due to their effectiveness in delivering existing therapeutic agents which have been rendered useless either due to emergence of drug resistant microbial strains or irritancy and toxicity of these compounds. Thus novel formulation approaches for these compounds form the need of this dayto-day combat the various deadly infections. Another problem of drug delivery, particularly with regards to bacterial infections, is their dormant existence inside the body which is reversible under suitable disease stimuli. This results in downregulation of the possible drug targets and Published by Woodhead Publishing Limited, 2012

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alteration of cellular permeability, thus hampering the efficacy of even the best therapeutic molecules. In such cases encapsulation of these drugs into nanoparticulate carriers has been visualized to enable their intracellular uptake, thus facilitating the eradication of pathogens residing therein. Researchers have adopted various strategies of formulating nanoparticulate carriers for therapy of infectious diseases and have met with considerable success with these. This has been accomplished either by targeting of encapsulated drugs or sustaining the drug release (especially for topical actives) or by enhancing the oral or intravenous bioavailability of the drug molecules [142]. NanoBio® Corporation, USA, has developed the NanoStat™ platform technology, based on nanoemulsions, for topical delivery of anti-infective products. After topical application, these nanoemulsions act by rapid penetration through the skin pores and hair shafts and kill the pathogenic organisms by physical disruption through membrane fusion. This technology has been proven to be effective against bacteria (e.g. E. coli, Salmonella), viruses (e.g. HIV, Herpes), fungi (e.g. Candida albicans), and spores (e.g. Anthrax). Phase I and Phase II clinical trials have proven the efficacy and safety of this technology, thus extending its application to numerous oral, vaginal and cutaneous infections [3]. NanoViricides Inc., USA, has developed polymeric micelle based platform technology known as NanoViricide™. This nanostructure, as against most of the current anti-virals, acts by binding to more than one site on a virus which is particularly advantageous due to the multiple binding sites involved in virus infectivity. Additionally, this product dissembles the virus particle by intercalating into the viral coat without leaving any metabolic harmful effects on the host. The company has a strong product pipeline with the technology being exploited for several drug candidates against influenza, external eye viral diseases, birdflu, AIDS, herpes and dengue. Success in the ongoing preclinical studies with these has provided a strong foundation for clinical trials [3, 143]. Scientists in France have formulated chlorhexidine-loaded nanocapsulebased gel (Nanochlorex®) for topical action against resident and transient skin bacteria. When compared with the commercial alcohol based gel, this formulation has been found to exhibit an immediate and sustained effect against Staphylococcus epidermidis, which was attributed by these researchers to the improved drug targeting to bacteria [144]. Numerous nanoparticulate formulations comprising of nanoparticles, micelles, liposomes and cyclodextrin complexes have been experimented for the treatment of infectious diseases. Amphotericin B containing targeted micelles of various biocompatible polymers have been developed

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for the treatment of Leishmaniasis; however, very few of these could make it to clinical trials due to poor pharmacokinetics and toxicity problems like haemolysis and anaphylaxis. Clinical trials of paramomycine containing topical micellar carriers exhibited improved efficacy against cutaneous Leishmaniasis [145]. Three Rivers Pharmaceuticals Inc., USA, owns the product Amphocil (USFDA approved), a disc-shaped colloidal lipid complex of amphotericin B containing cholesteryl sulphate. The complex is broken down by liver macrophages to release the active which is then carried in a bound form by the liver lipoproteins to ergosterol component of the fungal membranes. Subsequent alteration in the fungal membrane permeability leads to the leakage of the fungal contents followed by fungal death. This formulation is prescribed for opportunistic systemic fungal infections [146]. Liposomes present another success story on the commercial front with regards to the treatment of systemic fungal infections with Ambisome® being the world’s first liposomal product to receive USFDA approval for the treatment of Leishmaniasis. Since then alternative liposomal formulations have also been investigated for the treatment of parasitic infections like malaria or trypanosomiasis, but with limited success in pre-clinical or clinical investigations [145]. Scientists all over the world have been engaged in many such efforts focusing on development of various nanometric systems, for administration by different routes, to eradicate the infectious diseases accounting for significant worldwide mortality. Some of the nanotechnology based solutions, which are currently in pre-clinical development, have been extensively reviewed elsewhere [142, 145, 147–150]. Also nucleic acid loaded nanotherapeutics, used for treating infectious diseases, will be briefly discussed in the successive sections of this chapter.

2.3.3 Nanoparticulate carriers for vaccine delivery Humoral and cellular immune responses are elicited by expression of both the free antigens and antigens from plasmid DNA (pDNA). This has rendered much clinical interest in the DNA vaccines (genetic vaccines) as the new generation vaccines for diseases like tuberculosis, malaria, HIV, different cancers and hepatitis C. However, administration of ‘naked’ pDNA is met with limited clinical success due to inability of the cells to absorb it in quantities required to produce sufficient immune response, thus demanding administration of higher doses. This stimulated the scientist to resort to the exploration of nanoparticulate carriers as Published by Woodhead Publishing Limited, 2012

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new vaccine delivery systems to deliver the DNA vaccines in low doses but eliciting sufficient protective responses of the immune system [3]. Nanoparticles also serve as efficient vaccine delivery vehicles due to other properties such as size-influenced passive targeting of the antigens to the antigen-presenting cells (APCs), size-influenced targeting to the lymph nodes, ability to be administrated by multiple routes and possibility to favorably modulate the immune outcomes. Also nanoparticles have been proven to exhibit considerable transfection efficiency and traverse skin and mucosal barriers to effectively deliver the associated active to the mucosal associated lymphatic tissues (MALT) [151]. Nanoparticles of chitosan and chitosan derivatives have received significant attention for DNA vaccine delivery due to their mucoadhesive nature and ability to deliver across the mucosal surfaces. They induce stronger immune response due to their ability to be recognized by macrophages and are versatile enough to be administered by diverse administration routes. Polymeric nanoplexes, based on polymers such as PEI, PLL and poly(β-aminoester) (PBAE), involving ionic interactions between cationic polymers and the negatively charged nucleotides are also being widely explored due to their high transfection efficiency and ability to generate favorable immune response. PMAM dendrimers modified with PEG (to reduce nonspecific binding to cell membranes) and appropriate targeting ligands have also yielded promising in vivo results as effective DNA vaccine delivery agents [151]. Recently completed Phase I and II clinical trials established the clinical potential of PLGA nanoparticles loaded with DNA vaccines to elicit immune responses in patients infected with human papillomavirus virus (HPV). The studies were conducted by MGI Pharma Inc., USA, and used DNA coding for E6 and E7 genes of HPV 16 and 18 [151]. Vical Incorporated, USA, has developed vaccine delivery platform based on self-assembling nanoparticles of poloxamer CRL1005, coated with the cationic surfactant benzalkonium chloride. TransVax™, a therapeutic cytomegalovirus (CMV) vaccine based on this technology, induced cell-mediated and humoral responses in Phase I clinical trials and is currently in Phase II trials, in patients undergoing allogeneic, hematopoietic cell transplant (HCT) [152]. NanoBio® Corporation, USA, is employing its NanoStat™ platform technology for intranasal vaccination and claim a superior efficiency of this nanoemulsion to permeate the nasal mucosa and present the antigen to the APCs. The antigen is then presented by the APCs to the immune response eliciting areas of the body. Preclinical studies of this technology have been successful against respiratory syncytial virus (RSV), urinary

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tract infections, hepatitis B, anthrax and pandemic influenza, while the product for seasonal influenza is progressing towards Phase II clinical trials [153]. GlaxoSmithKline, UK, has developed Proteosomes™ which are nanoparticulate vaccine delivery carriers which form vesicles or clusters with sizes similar to small viruses. The hydrophobic porin protein component of this technology interacts with and promotes uptake of the vaccine by cells that elicit immune response, thus accounting for its efficacy. This nasal vaccine delivery technology is currently being employed to develop vaccines against influenza, RSV, plaque and allergy [3]. Aphios Corporation, USA, employs supercritical fluid technology to produce vaccine nanoparticles against influenza and HIV [154]. Here, the use of supercritical fluids has been proposed to prevent the denaturation of antigenic agents like DNA, sub-unit proteins or inactivated viruses or their combinations and improve their stability and hence effectiveness inside the body. The technology has been claimed to be economic due to lower number of manufacturing steps and is amenable to scale-up [155]. The US/Hungarian company Genetic Immunity LLC has developed a mannosylated PEI based platform vaccine technology DermaVir against HIV, as is its lead product. This transdermal vaccine product has undergone Phase I and II clinical trials where it exhibited specific action against HIV-infected cells and excellent profile for safe administration. Other pipeline products based on this technology include the ones against Chlamydia infection (ChlamyDerm), allergy (DermAll), virus-associated cancers and warts [151]. The PUREBRIGHT® technology mentioned earlier in this chapter comprises of nanoparticulate cancer vaccine, encapsulating cancerspecific monoclonal anti-bodies (Her2) and is efficacious due to selective uptake by the dendritic cells. Thus, on the whole, the future of nanoparticulate carriers as vaccine delivery vehicles looks quite promising.

2.3.4 Nanoparticulate carriers for nucleic acid delivery Nanoparticulate carriers are preferred over their viral counterparts for nucleic acid delivery due to the safety concerns of the latter. Additionally, they can be formulated to modulate the level and duration of gene expression as per the targeted disease condition. Nucleic acid delivery has been generally attempted by employing cationic nanoparticles which Published by Woodhead Publishing Limited, 2012

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effectively condense the former due to electrostatic interactions. These carriers possess the potential to rapidly escape the endo-lysosomal compartment due to charge interaction with endo-lysosomal membrane. This confers protection to the associated nucleic acid molecules from the destructive environment of these intracellular compartments and facilitates their sustained release into the cellular cytoplasm. This is particularly important for weakly expressed genes or for diseases requiring chronic protein action. Cationic agents also assist interactions with the anionic cell membranes and hence an efficient delivery of the associated load. Apart from ionic interactions, nucleic acid load may also be associated with the nanoparticles either through surface adsorption or hydrophobic interactions [3, 155]. In case of polymeric nanoparticles, especially those produced using PLGA/PLA, the nucleic acid material is generally embedded within the polymer since no charge interactions are present. However, in this case nucleic acid may remain associated with the polymer for weeks before being released, thus presenting problems of degradation. Though this extended release may be beneficial for certain applications, care needs to be taken to protect the nucleic acid during the long association period, at the same time maintaining a good association between both for having an appropriate release profile. In such cases alternative methods are adopted which involve formulation of nucleic acids with cationic materials containing suitable groups which can associate with PLGA/ PLA [156, 157]. Other than polymeric nanoparticles, researchers have developed nanoparticulate systems based on 1,2-dioleoyl-3-dimethylammonium propane (DODAP), an ionizable amino lipid, to load large quantities of nucleic acid molecules. A broader version of this technology has been applied to include other nucleic acids, like DNA, siRNA and aptamers, to form SNALPs or stable nucleic acid-lipid particles. The SNALPs were originally developed by Protiva Biotherapeutics Inc. (now known as ‘Tekmira’ after its merger with Tekmira Pharmaceuticals Corporation) and are now being used by Alnylam Pharmaceuticals. These SNALP based siRNA delivery products are currently in clinical trials. The SNALPs have a low particle size with homogenous distribution. Due to their low surface charge coupled with excellent stability and resistance to aggregation, they remain in circulation for prolonged periods. These nanoparticles, by the virtue of their EPR effect, accumulate in the tumor tissue via fenestrated vascular beds. Once at the action site, they are endocytosed by the cells through formation of a lipid bilayer (portions of cell membrane lipids) which fuses with the endosomal membrane.

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They then release the associated nucleic acid into the cellular cytoplasm for further action, that is formation of ribosome induced silencing complex (RISC) for siRNA and nuclear expression for pDNA, respectively. Other lipid based technologies include formation of SPLPs or stabilized plasmid-lipid particles in which PLL or PEI is used to pre-condense the DNA, which is then loaded into lipid bilayer through controlled mixing [155, 158]. Researchers have also attempted the use of carbonate apatite nanoparticles as bio-degradable and safer alternatives to carry nucleic acids across cellular barriers, to enable their efficient action [159]. Other studies report the use of nucleic acid as sites of liposome growth through the employment of cationic lipids or use of intercalation approach to associate nucleic acids with non-cationic carriers. To further improve the delivery of nucleic acids, scientists have also attempted receptor-mediated targeting by attaching suitable ligands like antibodies, peptides, transferrin, folic acid, etc., to the surface of polymeric or lipid carriers. Transferrin decorated cyclodextrin-based siRNA delivery technology (RONDEL™) developed by Calando Pharmaceuticals Inc., Canada, is currently undergoing clinical trials [155, 160]. Other promising approaches for siRNA delivery include the use of Pegylated PEI nanoparticles attached with suitable targeting ligands, chitosan-coated modified cyanoacrylate nanoparticles, modified phosphatidylcholine liposomes, etc. Despite its recent discovery and applications, there are numerous ongoing clinical trials of nucleic acid based products, with 80 ongoing or completed trials for anti-sense products, 70 trials of pDNA based therapeutics and 14 trials of siRNAbased therapeutics [155, 161]. The following paragraphs focus on some of these products. p53 gene delivery for tumor growth inhibition is being looked upon as a promising strategy. Following the gene delivery, sustained intracellular expression of this protein mediates its anti-cancer effects through mechanisms such as cell cycle arrest, anti-angiogenesis or apoptosis of the cancer cells. SynerGene Therapeutics (Malta) and National Institutes of Health (NIH) are jointly conducting a Phase I clinical trial of a sterically stabilized immunolipoplex formulation loaded with p53 gene in patients with advanced solid cancers. These lipoplexes have been designed for targeting the transferrin receptors of the tumor cells and also contain a PEG molecule for prolonged circulation. Clinical testing of these lipoplexes has been approved for various solid tumors such as those of breast, bladder, colon, pancreas, head and neck, prostate, brain, liver, lung, etc. Introgen, USA, has developed a liposomal nanoparticle Published by Woodhead Publishing Limited, 2012

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formulation of the tumor suppressor FUS1 gene and Phase I clinical trials of this formulation in patients with lung cancer have yielded encouraging results. Other studies are also being conducted for co-administering this gene with anti-inflammatory small molecules to reduce the nanoparticle associated inflammatory reactions or p53 gene for their possible additive effect. Researchers are currently exploring the potential of PEG-modified gelatin nanoparticles for delivering pDNA encoding VEGF-R1 or sFlt-1 for suppressing tumor growth in murine model of breast adenocarcinoma. Results indicate safety and efficacy of this system for systemic gene delivery to solid tumors and clinical trials of this method may be expected in future [3]. Compacted DNA nanoparticles comprising of positively charged peptides, known as PLASmin™ complexes, are currently being tested in Phase I/II clinical trials in cystic fibrosis patients by Copernicus Therapeutics Inc., USA, and its collaborating partners. Based on the initial results, the company foresees their application in other pulmonary diseases like asthma, genetic disorders like haemophilia, cancers and vaccines for various disorders, by administration through multiple routes [3, 162]. Discussing the nanoparticle based siRNA therapeutics in clinical trials, Alnylam Pharmaceuticals Inc., along with Tekmira, are using their SNALP technology to silence the therapeutic genes for disorders like dyslipidemia/ hypercholesterolemia (by silencing the Apo B gene to hamper cholesterol metabolism), Amyloidosis (by silencing the mutated TTR gene) and solid tumors (by silencing polo-like kinase 1 gene). These products are currently in Phase I clinical trials [158]. Calando Pharmaceutical’s leading product, CALAA-01, based on RONDEL technology, is aimed at targeting the M2 subunit of ribonucleotide reductase, using a patented siRNA molecule. Targeting of this clinically validated cancer target is being attempted in Phase I clinical trials in patients with solid tumors refractory to other standard therapies [160]. At this point the authors will like to direct the readers to comprehensive reviews by Burgess et al. and Maraganore et al. for more insights into mechanisms of pDNA and siRNA based therapy, the challenges associated therein and the developments of some of the related delivery platforms [161, 163]. These efforts have instilled interest in nucleic acid delivery technologies, which still require additional research to meet the delivery, safety and regulatory challenges. Nonetheless, with unfaltering commitment from academia, industry and investors, these technologies may be advanced to the patient population in need of such crucial innovations.

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2.4 Routes of administration of nanoparticulate drug delivery systems Having read through this book so far, it must now be evident to the reader that nanoparticulate drug delivery systems may be designed to be administered by various routes. This fact has also been exemplified through the various clinically relevant systems which have been cited in the previous sections of this chapter. To avoid their repetitive description, the following section of this chapter will be dedicated to the advantages of administering nanoparticles by certain frequently investigated administration routes. Various examples of therapeutic nanoparticles, along with their respective administration routes, will be later discussed in-depth in chapters dedicated to clinical trials and market successes of nanoparticulate drug delivery systems.

2.4.1 Oral drug delivery by nanoparticles Gastric delivery by the oral route presents numerous hurdles for conventional drug delivery vehicles due to: ■

their limited residence time in the stomach resulting in insufficient drug concentrations at the desired action sites; and

■

the presence of gastric mucous, gastric enzymes and gastric flora.

Despite these delivery barriers, the oral route is still one of the most preferred ones due to its convenience and intrinsic advantages. In such cases nanoparticles can serve as alternative delivery systems to overcome challenges of mucosal permeability, low drug solubility and absorption from the gut, gut metabolism and first-pass effect [2, 164]. Orally administered nanoparticles usually deliver the encapsulated molecules, locally or systemically, after adhesion to the intestinal surface followed by translocation across the gastrointestinal (GI) barriers. Translocation in turn may occur by different mechanisms such as internalization of nanoparticles by absorptive cells of intestine or paracellular uptake by passage between the cells of GI wall or uptake by intestinal macrophages via phagocytosis or that by M-cells of the Peyer’s patches through adsorptive clarithrin mediated endocytosis or fluid phase endocytosis and phagocytosis [164–167]. Effective drug delivery via orally administered nanoparticles is often challenged by a complex blend of pharmaceutical (particle dependent) Published by Woodhead Publishing Limited, 2012

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and biological parameters. Amongst the particle properties, the particle size and surface charge form the critical factors affecting the absorption of the nanoparticles. Particle diameter is a major parameter affecting the easy translocation of nanoparticles and it has been reported that particles with sizes between 40 and 120 nm can be taken up both by paracellular and transcellular pathways [164, 165]. The uptake of the nanoparticles also depends on the surface charge and surface characteristics of the nanoparticles. Though hydrophobic nanoparticles have been reported to have a greater uptake as compared to the hydrophilic nanoparticles, excessive hydrophobicity can compromise their translocation across the absorptive cells. This calls for attention on part of the formulator to maintain an adequate hydrophilicity–hydrophobicity balance on the nanoparticle surface by selecting appropriate formulation excipients [2, 164, 168]. Another significant determinant of effective nanoparticle internalization is its adhesion to the gastric walls by a mechanism known as bioadhesion; when the latter is restricted to gastric mucosa it may be referred to as mucoadhesion. Strategies to enhance this adhesion may lead to an increased nanoparticle absorption by increasing the transit time of the nanoparticles in the GIT, allowing them to be in contact with absorptive GI cells for longer period of time. This may be achieved either by the employment of specific mucoadhesive polymers as formulation ingredients or by decoration of nanoparticle surface with specific ligands to enhance site-specificity and uptake. As mentioned earlier, the surface charge of nanoparticles has also been explored to provide favorable interactions with gastric mucosa and enhance the bioavailability of the encapsulated therapeutic molecule. Cationic polymers have electrostatic interactions with the negatively charged gastric mucosa and in certain instances have been reported to mediate paracellular transport by opening the tight junctions of epithelial cells. The oral success of drug delivery nanoparticles also depends on their loading or the dose of active incorporated in them along with its distribution and/or its release and the type of release profile. Of these, the dose depends on the size, charge and surface properties of nanoparticles, the inherent properties of the drug and formulation excipients and method of nanoparticle formulation. Also care has to be taken with drugs to be administered in very high or low doses, since in case of former administration by nanoparticles may be unsuitable due to only a fraction of these systems being absorbed in most cases, while in case of latter internalization of too many nanoparticles may result in cellular toxicity. The site of drug release from the nanoparticles and its pattern, on the

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other hand, depends on the type of material used for formulating the systems and the type of distribution of active within the system. Alternatively, the release profile may depend on the type of dosage form into which the nanoparticles have been packaged, for example in capsules or as different tablet types [164, 165, 169]. Finally, having known the various factors responsible for effective drug delivery employing oral nanoparticles, it will be interesting to know the various application areas of these systems. These include increasing the bioavailability of encapsulated drugs, targeting vaccine antigens and nucleic acids to gut associated lymphoid tissue (GALT) while protecting them from the harsh gastric environment (lymphatic targeting), specific targeting of drugs to colon to evade their gastric irritation or to target colonic diseases, systemic targeting of drugs to specific organs or tissues as required for anticancer, anti-tubercular, anti-retroviral drugs, etc. [164]. Although much has been known about nanoparticle uptake, additional knowledge of nanoparticle interaction with specific surface receptors can increase the number of successful instances where oral nanoparticles may deliver therapeutic quantities of actives for these listed applications.

2.4.2 Parenteral drug delivery by nanoparticles Delivery by parenteral nanoparticles is especially beneficial for drugs with low bioavailability and a narrow therapeutic index, for protecting the drugs from gastric hydrolysis or enzymatic degradation, for concentrating the drugs at easily accessible sites, for directing the drugs away from sites of toxicity and for increasing circulation times of unstable or rapidly eliminated drugs. Additional advantages with some nanoparticulate drug delivery systems may include sustained or controlled drug release over prolonged time periods and formulation attributes like lack of flocculation, sedimentation as compared to parenteral systems containing larger particles. Nanoparticle delivery by this route of administration is generally restricted to anticancer, anti-infective and anti-inflammatory agents [170–172]. Specifically with reference to nanoparticles of anticancer agents, their delivery to tumor tissues is significantly enhanced by altered permeability of tumor vasculature. Additional interplay of events including presence of permeability enhancing factors, collagenases causing disintegration of matrix tissue surrounding the blood vessels and impaired lymphatic drainage, all contribute to the EPR effect of nanoparticles. This specific Published by Woodhead Publishing Limited, 2012

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drug delivery to cancerous tissues also works to the advantage of anticancer agents due to their limited therapeutic index and non-specific systemic toxicities. Infections and inflammatory situations provide similar advantages for nanoparticle delivery, except for the presence of intact lymphatic drainage, resulting in rapid clearance and lack of possibility for a sustained drug release [172]. Although the altered, leaky vasculature offers enhanced permeability to drug nanoparticles, such a diseased vasculature (as seen in tumors, infections or inflammatory conditions) is also a common attraction point for the cells of MPS like macrophages, etc. This places a limitation on the size of the nanoparticles that may be administrated, so that they enter the vascular pores but are not engulfed by macrophages. Nanoparticles having diameter less than 250 nm generally serve this purpose as the size of vascular pores normally ranges between 300 and 700 nm [172, 173]. However, engulfment of nanoparticles by macrophages may serve as an important strategy for intracellular parasitic infections caused by mycobacteria, salmonella, listeria and brucella, which primarily infect these phagocytic cells. A specific targeting of macrophages has also been achieved by their surface decoration with macrophage specific ligands [174]. While employing various types of nanoparticulate systems, like nanosuspensions, polymeric and lipidic nanoparticles, liposomes, etc., as injectables, they may be administered by routes other than intravenous delivery to avoid its inconvenience. Subcutaneous or intramuscular injections can be advantageous due to faster dissolution of nanoparticles yielding the desired systemic drug levels and prolonged drug release, leading to higher drug concentrations in limited volume of administration compartments. Furthermore, drugs with limited solubility may be directly injected into the central nervous system to achieve the desired therapeutic levels [172]. Thus although the benefits of administering drug nanoparticles by the parenteral route are many, further interdisciplinary collaborations between industry and academia are required to conduct expensive biological studies on the potential nanocarriers. With few of these products already in the market, as cited earlier, further advancement of practical applications of this technology comes across as a promising outlook.

2.4.3 Nanoparticles for drug delivery to skin Drug nanoparticles are being increasingly targeted to skin surface or furrows or hair follicles for local therapy of diseases like microbial

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infections. Additionally, their interaction with skin at cellular levels is also being explored for topical vaccine applications [175]. Successful delivery of drug nanoparticles to epidermis and dermis is severely hampered by the barrier properties of normal skin. However, studies have shown that alteration of these barrier functions, in case of diseased or aged skin, can be used to advantage for enhanced penetration of nanoparticles [175]. Though studies so far have not concluded upon an optimal particle size for skin delivery, it is now clear that this parameter is largely dependent on the properties of skin with regards to its age and form, the physicochemical properties of encapsulated drug and that of the drug carrier (shape of the carrier, excipient used for formulating nanoparticles, etc.). Additional skin penetration determinants include skin appendages like hair follicles and associated sebaceous glands (pilosebaceous units) and sweat glands which constitute significant pathways for particulate systems [176]. The pilosebaceous openings, which vary between 0.3 and 0.75 μm in diameter, serve as entry ports for rigid particulates of varying diameters. Though highest penetration has been reported by particles having diameters equivalent to the hair shafts, nanoparticles travel deep into the follicles due to surface motion of hair, where they can be used for treatment of perifollicular diseases or as reservoirs for delivering drugs to the structured cellular components [177]. Alternatively, intercorneocyte pathways serve as entry ports for flexible and soft colloidal carriers (like liposomes, micelles, hydrogel nanoparticles, etc.) which can interact with intracellular lipids. These soft shelled nanoparticles deliver their payloads either through their interaction with skin components or by intact penetration under osmotic gradient. The surface charge of nanoparticles also plays an important role in their skin penetration, with cationic vehicles exhibiting superior penetration as compared to their anionic counterparts, possibly due to their interaction with negatively charged skin lipids and proteins. Skin penetration of nanoparticles is also influenced by the nature of their dispersant, oily vehicles contributing to higher penetration in contrast to the aqueous ones [176, 178]. Finally, apart from the natural properties of skin and nanoparticles, their penetration may be enhanced by applying mechanical stress to the skin through skin flexing and massage [175]. Of the various types of lipid (vesicles like liposomes and particulates like SLNs and NLCs), polymeric (nanoparticles and dendrimers) and surfactant based (vesicles like micelles and emulsion based like nanoemulsions) nanosystems, currently being explored for drug delivery to the skin, the lipidic nanovehicles are generally better suited for delivering hydropholic drugs [178]. Controlled release SLNs are being Published by Woodhead Publishing Limited, 2012

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investigated by researchers for effectively delivering anti-inflammatory and immunosuppressive drugs. Alternative nanotechnology based delivery systems, like ‘nanopatches’, are being researched by Australian scientists as needle-free mode of delivering DNA vaccines against HIV and malaria [3]. However, while conducting such trials, attention is demanded by certain critical criterions like the formulation stability upon storage and adequate extrapolation of data from animal skin to the human skin, considering their differential composition. Another concern to be addressed is their local and systemic safety, over short-term and prolonged periods, particularly for non-biodegradable nanoparticles which may be bio-retentive in nature [175, 176]. Further investigations are also required concerning tactics to enhance delivery of drug nanoparticles through the skin, such as their combination with physical strategies like iontophoresis, application of low-frequency ultrasound and microneedles, etc. With integrated efforts in these directions the number of marketed nanosystems, delivering therapeutic molecules to the skin, is sure to increase.

2.4.4 Pulmonary drug delivery by nanoparticles The pulmonary vasculature is susceptible to a plethora of pathologies. This fact, along with its involvement in multi-functional physiological roles, makes it an attractive pharmacological target to alleviate numerous mortality and morbidity associated disorders. Though venous administration is a possible option for pulmonary drug delivery, the unfavorable pharmacokinetic properties of most of the therapeutic molecules including bio-molecules like proteins, do not allow for their safe and effective localization in the lungs. In such instances, nanoparticles can provide attractive substitutes for delivering poorly soluble therapeutic moieties, either due to mechanical or charge based retention or EPR effect. Nanoparticles administered by the pulmonary route also offer the possibility of localized drug release for prolonged durations, improve circulation times and prevent premature systemic inactivation and degradation of chemical and biological molecules. Apart from enhancing the drug properties, there is always a possibility of enhancing the carrier itself through surface modifications using suitable ligands. Thus the nanocarriers can result in overall improvement of drug delivery to the desired site via passive or active targeting [179]. Furthermore, the attributes of the pulmonary vasculature such as its extensive surface area, the insignificant activity of drug metabolizing enzymes present therein

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and the easily penetrable mucosal barrier, all contribute to the effectiveness of nanoparticle drug delivery [180]. The passive distribution and localization of nanoparticles in the lungs is largely influenced by their size, surface properties, charge and EPR effect, as is also the case with other administration routes. When considering the active targeting, the selection of a pulmonary target is mainly governed by its spatial and temporal accessibility, its reliable expression despite the diseased condition and its exclusivity to the targeted area. Additionally, targeting to a particular determinant should not adversely affect the pulmonary vasculature and should facilitate surface retention or internalization of the targeted moiety, as per the therapeutic need [179]. Another factor which may affect the pulmonary vasculature integrity is the nanoparticle itself, which necessitates attention to be rendered to certain formulation considerations for pulmonary nanoparticles. An important consideration in this matter is the biocompatibility of nanoparticle matrix. Although the material selection also depends on location, dosage and retention periods of nanoparticles as well as the upon the desired application, the general rule of thumb is to select low molecular weight, easily degradable and eliminated materials [181]. Depending on the material chosen, the pulmonary nanoparticles may then be used for imaging [182], gene delivery to the lung tissue, delivery of therapeutic enzymes like lysosomal enzymes, antithrombotic enzymes and antioxidant enzymes and delivery of anticancer agents [179]. Although targeting approach may be beneficially utilized for various types of nanoparticles to provide versatile delivery platforms for pulmonary vasculature, the immediate and delayed toxicity concerns of these targeting strategies need to be thoroughly investigated. This is particularly important for pulmonary vasculature since the circulation in this area is sensitive to inflammation mediating factors, leading to oedema and eventually fibrosis and hypertension. This becomes increasingly complex as in certain instances the degradation products of nanoparticle matrix may have pro-inflammatory properties. The general safety issues of nanoparticles as well as formulation challenges with regards to drug loading, production of reproducible systems, also encountered with nanoparticles delivered by other routes, remain of permanent concern [183, 184]. However, with careful attention to these factors along with intelligent selection of suitable targets may provide specific and sub-cellular drug nanoparticles to overcome life-threatening lung pathologies. Published by Woodhead Publishing Limited, 2012

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2.4.5 Nasal drug delivery by nanoparticles Drug delivery nanoparticles also find application via the nasal route where they have been reported to significantly improve the transport of chemical and antigenic drugs across the nasal mucosa. This route is generally preferred for potent, local and systemic drugs which require protection from gastric or hepatic inactivation, which are used for chronic disorders or which require rapid onset of action. Uptake of nanoparticles in the nasal cavity may take place either from the nasal epithelial cells or the nasal associated lymphoid tissue (NALT). The latter consists of M-cells, similar to GALT, and may hence be employed for targeting particulates encapsulating antigenic drugs [185, 186]. As with other routes of administration, numerous nanoparticle related parameters affect the transport of nanoparticles across the nasal mucosa. The ability of drug nanoparticles to penetrate into local tissues or to enter the blood stream, after delivery into the nasal cavity, is largely governed by the size of the nanoparticles. Only a limited portion of small-sized nanoparticles bear the potential to traverse the nasal mucosa through paracellular pathways due to the severe size limitation imposed by the diameters of tight junctions that interconnect the nasal epithelial cells. Generally this diameter is less than 15 nm even in presence of absorption enhancers [187]. Larger particles are generally taken up by endocytosis or receptor and/or carrier dependant transmucosal transport. Along with the size, the surface properties of the nanoparticles also govern the transport route and the amount of nanoparticles that may be transported. Certain investigations revealed that very small (10 nm) hydrophilic nanoparticles entered the rabbit nasal cells by paracellular pathways while larger (200 nm) hydrophilic ones followed the transcellular pathways. Also hydrophobic particles were internalized by transcellular pathways when compared with their hydrophilic counterparts of similar particle size. In another study it was observed that surface coating with cationic polymers like chitosan was found to enhance nanoparticle permeation attributed to the opening of tight junctions of nasal epithelial cells. Certain investigations have also suggested the enhancement of nasal mucosal permeability upon coating the particles with PEG [185, 188, 189]. To explore their benefits, nasal nanoparticles have been investigated by several researchers for administration of therapeutic molecules like insulin, but with mixed outcomes on their effectiveness. Likewise, they

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have also been investigated for delivery of vaccines where they have been found to produce or enhance an immune response, possibly due to their preferential uptake by NALT [186, 190]. Another interesting area where nasal nanoparticles are being explored by drug delivery scientists is their application for delivering drugs to the brain. The blood–brain barrier (BBB), by the virtue of the biochemical composition of its cellular components, the presence of tight junctions between brain blood vessel endothelial cells and the presence of various efflux pumps, forms a non-traversable barrier for a large number of antibiotics, anticancer drugs, macromolecular drugs like peptides and proteins and those effective in central-nervous system (CNS) disorders. Nanoparticles, on the other hand, bear the potential to use the BBB transport systems for their preferential uptake into the brain. Another means of their brain uptake is along with the carrier-mediated transport systems which deliver hydrophilic nutrients into the brain. Once administered into the nasal cavity, the drug nanoparticles exhibit numerous mechanisms of crossing BBB. These include retention inside brain capillaries giving rise to a concentration gradient across the endothelial cell layer, endothelial cell membrane fluidization by formulation surfactants, opening of tight junctions, endocytosis, transcytosis and inhibition of efflux pumps caused by appropriate formulation excipients. Such nanoparticles are being increasingly investigated for treatment of CNS disorders like Alzheimer’s disease, Parkinson’s disease, migraine, meningitis, etc., and also for treatment of obesity, though their clinical potential is still a subject of scientific deliberation [186, 191]. Here, the authors would like to draw attention to excellent reviews by Jorg Kreuter and Mistry et al. [192, 193], describing the subtle intricacies of drug delivery by nanoparticles to the brain. Although nanoparticulate carriers may serve as important and promising tools for nasal drug delivery, their toxicity to nasal cells has not been extensively researched. Studies have shown that internalization of some nanoparticulate carriers may trigger local inflammatory reaction in nasal epithelial cells or may lead to diminished movement of nasal cilia and ciliotoxicity [185]. Such instances call for more research to be conducted before nasal nanoparticles may be employed for delivering drugs and vaccines to the humans. Overviews of the various nanocarrier products or technologies which are important from commercial perspective have been listed in Table 2.1.

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Table 2.1 Type of nanocarrier

Types of nanocarrier products or technologies important from commercial perspective Commercial organization

Solid lipid Pharmatec, Milan, Italy nanoparticles (SLN) SkyePharma

Protein nanoparticles

Cyclosporine SLN High pressure homogenization technique for SLN production

Vectorpharma, Trieste, Italy

Microemulsion template method for SLN formulation

American Pharmaceutical Partners, Inc., USA

ABI-007 or Abraxane™ (Albumin nanoparticles) based on ProtoSphere™ technology

Azaya Therapeutics Inc.

Protein Stabilized Nanoparticle (PSN™) technology

Nanosuspensions Soliqs/Abbott

Nanomorph based on Hydrosol technology

Wyeth

Rapamune®

Merck

Emend

Nanoemulsions

NanoBio® Corporation, USA

NanoStat™ platform technology

Liposome

Johnson & Johnson

Doxil®

Enzon Pharmaceuticals (Europe)

Myocet™

Sopherion Therapeutics (USA)

Myocet™

NeXstar Pharmaceuticals

Daunoxome®

Astellas Pharma US, Inc.

Ambisome®

Aphios Corporation, USA

Taxosomes™ Camposomes™ based on SuperFluids™ supercritical fluid technology

NanoCarrier Ltd. Japan and Debiopharm, Switzerland

DACH-platin-PEG-poly (glutamic acid) micelles based on Medicelle™ technology

NanoViricides Inc, USA

NanoViricide™ technology

Starpharma Ltd., Australia

VivaGel®

Starpharma Holdings Ltd.

Priostar™ and STARBURST

Micelles

Dendrimer

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Hydrogel nanoparticles

NOF Corporation, Japan

PUREBRIGHT® technology

Alnis Biosciences Inc

MagNaGel™

Colloidal lipid complex

Three Rivers Pharmaceuticals Inc

Amphocil

Nanoparticulate vaccines

GlaxoSmithKline, UK

Proteosomes™ vaccine nanocarriers

Aphios Corporation, USA

Vaccine nanoparticles based on supercritical fluid technology

Genetic Immunity LLC

Mannosylated PEI based platform vaccine technology

Alnylam Pharmaceuticals

SNALP (stable nucleic acid-lipid particles) technology developed by Tekmira

Calando Pharmaceuticals Inc, Canada

RONDEL™ (cyclodextrin-based siRNA delivery) technology

Nucleic acid delivery nanoparticles

2.5 Conclusion In summary, research in drug delivery nanoparticles has progressed by leaps and bounds. Realizing their potential for overcoming insurmountable drug delivery problems of limited drug solubility and bioavailability, as discussed in the previous chapter of this book, scientists have invested considerable efforts in formulating a wide arena of such delivery systems. Various types of potential materials and hard- or soft-shelled nanoparticulate systems are being investigated for application by numerous routes. In the current scenario, the list of diseases which may be cured by these systems seems to be endless. However, the burgeoning research has also shed light on the negative facets of these fascinating drug carriers, with issues regarding their immediate and prolonged toxicity being matters increasing concern. A thorough physico-chemical characterization of these systems may serve as a first-line agenda for improvising or eliminating some potentially notorious systems. This certainly requires a follow-up by more extensive in vitro and in vivo toxicological evaluations before the systems may be progressed to animal studies and clinical trials, and further be submitted for regulatory approvals. Published by Woodhead Publishing Limited, 2012

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The following chapters of this book will be dedicated to the various methods of physico-chemical characterization and toxicological assessment of nanoparticulate drug carriers. Consequently, the readers will also be given a brief overview of the regulatory bodies responsible for approving the drug nanoparticles. Finally, some concluding chapters of this book will be focused on summarizing those nanoparticulate drug carriers which have been successful in the preliminary evaluations and are now either in clinical trials or have been approved for marketing.

2.6 References [1] Ehrlich P (1954) The partial function of cells. Int Arch Allergy Appl Immunol, 5: 67–86. [2] Bhavsar MD, Shenoy DB and Amiji MM (2006) Polymeric nanoparticles for delivery in the gastro-intestinal tract. In: Torchilin VP (Ed.), Nanoparticulates Drug Carriers, Imperial College Press, pp. 609–648. [3] Jain KK (2008) Handbook of Nanomedicine, Humana/Springer, Totowa, New Jersey. [4] Koo OM, Rubinstein I and Onyuksel H (2005) Role of nanotechnology in targeted drug delivery and imaging: a concise review. Nanomedicine: Nanotech Biol Med, 1: 193–212. [5] Jain KK (2008) Nanomedicine: application of nanobiotechnology in medical practice. Med Princ Pract, 17: 89–101. [6] Hughes GA (2005) Nanostructure-mediated drug delivery. Nanomedicine, 1: 22–30. [7] Moghimi SM, Vega E, Garcia ML, Al-Hanbali OAR and Rutt KJ (2006) Polymeric nanoparticles as drug carriers and controlled release implant devices. In: Torchilin VP (Ed.), Nanoparticulates Drug Carriers, Imperial College Press, London, pp. 29–42. [8] Schmidt C and Lamprecht A (2009) Nanocarriers. In: Lamprecht A (Ed.), Drug Delivery-Design, Manufacture and Physicochemical Properties: In Nanotherapeutics Drug Delivery Concepts in Nanoscience, Pan Stanford Publishing Pt. Ltd., Singapore, pp. 3–38. [9] Kingsley JD, Dou H, Morehead J, Rabinow B, Gendelman HE and Destache CJ (2006) Nanotechnology: a focus on nanoparticles as a drug delivery system. J Neuroimmune Pharmacol, 1: 340–350. [10] Ravi Kumar MN (2000) Nano and microparticles as controlled drug delivery devices. J Pharm Sci, 3: 234–258. [11] Moghimi SM, Hunter AC and Murray JC (2001) Long-circulating and targetspecific nanoparticles: theory to practice. Pharmacol Rev, 53: 283–318. [12] Sengupta S, Eavarone D, Capila I, Zhao G, Watson N, et al. (2005) Temporal targeting of tumour cells and neovasculature with a nanoscale delivery system. Nature, 436: 568–572. [13] D’Mello SR, Das SK and Das NG (2009) Polymeric nanoparticles for smallmolecule drugs: biodegradation of polymers and fabrication of nanoparticles.

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[14]

[15]

[16]

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