INTRODUCTION
Nanoencapsulation is one of the most important subcategories of controlled-release bionanotechnology. Normally, active substances are encapsulated in submicro-meter-sized devices made of barrier materials. These materials are designed to control the rate of release. This concept has been largely inspired by spontaneous assembling of the phospholipid liposomes as a model of biological membranes.[1] As in Nature, one has to develop preparations of nanovehicles that allow precise control over their structure and morphology. In this context, the self-assembled superstructures of surfactants (micelles, liposomes)1-2-1 and/or polymers (nanoparticles) have proven to be valuable tools.[3]
Nanoparticles may be defined as being submicrometer (from 10 to 1000 nm) colloidal systems generally, but not necessarily, made of polymers (biodegradable or not).[4-6] Depending on the process used for their preparation, two different types of nanoparticles can be obtained, namely, nanospheres and nanocapsules. Unlike nanospheres (matrix systems where the bioactive substance is dispersed throughout the particles), nanocapsules exhibit a membrane-wall structure with an aqueous or oily core containing the bioactive substance. Thus, nanocapsules may be considered as a ”reservoir” or ”envelop” system. Because nanoparticles have very high surface areas, the active substance may also be adsorbed or conjugated onto the surface.[6] At present, micellar/liposomal systems have also been included under the term ”nanoparticles.”
Another type of nanometer-sized carriers is an inclusion complex or clathrate, which can be assembled by inclusion of bioactive substances into molecular cavities of the so-called cavitands,[7] or dendrimers.[8,9] Natural examples of such internal-cavity-containing molecules are cyclodextrins, which are used widely for preparation of various drug formulations.1-10-1 The outer diameter of molecular nanocapsules is in the range of 3-50 nm. The nanometer size ranges of liposomes, nanoparticles, and clathrates offer certain distinct advantages for drug delivery. Because of their subcellular size, they can penetrate deep into tissues through fine capillaries, cross the fenestration present in the epithelial lining (e.g., liver), and generally are taken up efficiently by the cells to perform the so-called intracellular trafficking.1-6,11,12-1 This allows direct delivery of therapeutic agents to target sites in the body followed by the controlled release.[13]
CONTROLLED RELEASE
Controlled-release nanotechnology can solve a variety of problems regarding the effective delivery of a bioactive compound to a target to achieve good local and systemic tolerance during and after application.1-14-1 In controlled-release systems, a drug or some other active agent is incorporated into a self-assembled carrier. The main condition of success is to design tissue-friendly and biodegradable accompanying materials.1-4-6-1 The delivery carriers are usually required for the following reasons: 1) many effective drugs are characterized by poor aqueous solubility and need to be solubilized; 2) many drugs, such as proteins, are very fragile and need a microen-vironment providing protection from hydrolysis or enzymatic degradation; 3) many drugs are highly toxic and require a carrier to shield tissues until the drug release at the targeted tissue occurs; 4) the release itself has to be designed to allow controlling; and 5) targeted delivery of drug can be attained by conjugating a specific vector to the carrier.1-15-1
The rate of releasing the agent depends on the nature of the carrier as well as various environmental factors (such as nature of solvent, osmotic pressure, temperature, pH of media, and so on). The controlled release, depending on rate of releasing, can be classified as triggered, pulsing, or sustained. The rate of delivery of any bioactive substance to a target tissue or reaction site is highly critical. Key advantages to the use of this technology are prolonged activity, fewer doses, fewer undesirable side effects, and reduced toxicity.
Three mechanisms of controlled release can be considered: diffusion controlled (through membranes and from matrices), chemically controlled (erosion and cleavage of polymer chain or spacer connecting bioactive agent with carrier, if any), and solvent activated (osmotic pressure and swelling). Each mechanism has certain advantages and must be selected for design of carrier depending on the agent to be released as well as the conditions required to yield favorable release. Recently, significant effort has been devoted to develop nanotech-nology for controlled drug delivery devices because it offers a suitable means of delivering low molecular weight drugs, as well as macromolecules such as peptides, proteins, DNA, or genes by either localized or targeted delivery to the tissue of interest.[6,11,16,17]
LIPOSOMES
Liposomes (or vesicles) are topologically closed nano-sized lamellar aggregates of highly ordered lipid molecules that are normally dispersed in a hydrophilic solvent, typically water. The aggregates may be formed by the combination of both polar and nonpolar residues in the same lipid molecule, which is described as amphiphilic. In aqueous medium the amphiphilic molecules arrange the ordered micellar or lamellar structures depending on the concentration, in which the hydrophobic regions are brought into proximity with each other while the polar groups are exposed to water. The amphiphiles are capable of forming a variety of phases due to steric factors such as head group size, variation in the number of acyl chains, or to electrostatic effects arising from attractive or repulsive forces between adjacent polar head groups.[18] The different phases can interconvert, either by inward or outward migration of particular amphiphiles changing their shape. Heating, for example, increases the dynamic motion of hydrocarbon chains, effectively broadening the nonpolar region. Changes in pH can affect an ionization of polar head groups and so alter their diameter, as can changes in their hydration level.[19] Thus, bilayer to nonbilayer transition can take place, resulting in loss of membrane barrier function. These transitions can be exploited to design liposomal carrier systems with preset release properties that are triggered by changes in their microenvironment.
Normal micelles, which have only short-range order, form spontaneously when the amphiphiles are added to water. The liquid crystalline phases, which exhibit long-range order, like lamellar (cubic) and normal hexagonal phases, do not disperse spontaneously. When the lamellar phase is diluted with excess aqueous phase, it converts into spherical liposomes (Fig. 1),[2] which are widespread carriers for drugs, cosmetics, and many other types of actives.[19] Because of the presence of the aqueous core and the hydrophobic lipid bilayers, liposomes can accommodate both hydrophilic and hydrophobic actives. The liposomes can be constructed with widely different physical structures, lipid composition, and surface properties, thus enabling a great deal of control over entrapment and release of their contents.
Fig. 1 Schematic structure of liposomes: (a) multilamellar vesicles, MLV, and (b) small and large unilamellar vesicles, SUV and LUV.
Three kinds of liposomes are in common use. Large spherical liposomes, each consisting of numerous concentric bilayers, alternating with layers of water, are known as multilamellar vesicles (MLVs) (Fig. 1a).[2] The MLVs are simple to produce, but the volume available for solute entrapment is limited. By gentle swirling, individual lamellae are able to detach to form large unilamellar vesicles (LUVs) (Fig. 1b).[20] Because the interior of LUVs is not occupied by internal lamellae, there is ample space for incorporation of actives. This also means that cells taking up LUVs are less subject to lipid overload. However, LUVs are more fragile than MLVs. In general, MLVs and LUVs vary in their outer diameter, from 100 nm up to 10 mm. Their large size range is considered to be a drawback for many medical applications requiring par-enteral administration because it leads to rapid clearance from the bloodstream by the cells of the reticuloendo-thelial system (RES).
Small unilamellar liposomes (SUVs), with outer diameter in the range of 20-100 nm,[21] can be obtained by ultrasonication of MLVs.[18] Because of their small size, clearance from the systemic circulation is significantly reduced, but SUVs have a much lower capacity for drug entrapment, typically less than 1% of the material available.
Preparation of Liposomes
Multilamellar vesicles
Multilamellar vesicles (Fig. 2a) may be prepared from natural or synthetic lipids by suspending them in an aqueous solution maintained at a temperature above the melting point Tc of the lipid. For unsaturated phospho-lipids such as egg and soy phosphatidylcholine, which have Tc values below 0°C, this can be done by stirring in an inert atmosphere of nitrogen or argon to avoid lipid oxidation.[2- A small amount of the active solution is entrapped within the interlamellar spaces when lipid is hydrated, followed by the liposome formation. The loaded liposomes can be separated from nonencapsulated solute using centrifugation or dialysis.
Fig. 2 Freeze-fracture transmission electron micrographs of liposomes: (a) MLV constituted from 0.1 M mixture of ammonium bromide/hexanol = 9/1/20, and (b) SUV prepared from 3wt.% ammonium bromide in water.
The encapsulation efficiency can be increased by inclusion of a charged amphiphile, such as phosphati-dylglycerol or phosphatidic acid, at a molar ratio of 1020%, or by preliminary freeze-drying of the lipid from an organic solution followed by formation of an expanded foam with an increased surface area, which increases the amount of aqueous phase that can be in-corporated.[22- The most advanced method, which was designed to achieve high levels of entrapment, particularly of sensitive biomacromolecules such as proteins and nucleic acids, is dehydration/rehydration method.[23-It allows a prolongation of exposure of solute to the lipid before its final lamellar structure has been fixed. This may be achieved by preparing MLVs in water followed by converting these to SUVs. Thus, when SUVs are mixed with a solution of the active to be entrapped, most of the amphiphile is directly exposed to the solute. At this stage, water is removed by freeze-drying to produce the vesicles in a metastable state enriched with active. Following the hydration stage, the liposomes are diluted with an isotonic buffer[23- to provide an osmotic gradient between the internal and external phases and avoid redistribution of active, and finally washed to remove the nonencapsulated material.
Lipids dissolved in organic solvents can be hydrated in solution without prior solvent removal. If the solvent is water miscible, such as ethanol or propylene glycol, it may be removed at the end of MLV preparation by dialysis or filtration. Water-immiscible solvents such as ether, chloroform, or methylene chloride may also be used, then later removed through evaporation. High-capacity multi-vesicular liposomes were created in w/o/w double emulsion, containing active in the inner entrapped aqueous phase.1-24-1 Composition and conditions are chosen such that each droplet of the organic chloroform-ether disperse phase contains multiple droplets of the initial aqueous solution of active. The organic solvents are removed under a nitrogen flow, wherein the lipid is deposited around the internal aqueous, drug-containing nanodroplets, which form separate compartments within a single liposome. The method allows encapsulation efficiencies of up to 90%, but requires specialized lipids and rather complex preparation conditions.
Different technology has been applied so far to produce a new solid lipid carrier system, the so-called solid lipid nanoparticles (SLNs).[25-28- It is rather effective for encapsulating drugs that are poorly soluble in both aqueous and organic media.[25- Solid lipid nanoparticles combine the advantages of both liposomes and polymer nano-particles. In SLNs the liquid lipid in emulsion is replaced by a solid lipid, e.g., high-melting glycerides (trilaurin, tribehenin) or waxes (cetyl palmitate); the particles are stabilized against aggregation by natural emulsifiers (lecithin), synthetic surfactants (Miranol, Plantaren, Tween 80), or sterically stabilizing polymers (Pluronic F68).[26,27- To prepare SLN dispersions, melted lipid mixed with an active was added to a surfactant solution of distilled water at elevated temperature. After stirring, the crude preemulsion was homogenized under high pressure between 500 and 1500 bar, applying several homogeni-zation cycles.1-26,27- Mean particle size of prepared SLNs ranged from 100 to 300 nm.
Within SLNs, actives with a melting point below the melting point of the lipid matrix preferentially distribute to the surface of the particles.1-27-1 Wide-angle X-ray scattering investigations suggested that good entrapment efficacy in SLNs can be achieved with lipids of low crystalline order and metastable polymorphs of the p' form.[28:l This allows optimization of lipid composition in favor of formation of this metastable polymorph.
Small unilamellar vesicles
Small unilamellar vesicles (Fig. 2b) can be formed easily by high-power probe or bath sonication of MLVs in an inert atmosphere by cooling to dissipate local over-heating[18- and avoid disruption of lipid molecules. Alternatively, MLVs can be converted into SUVs by use of high-pressure homogenization, such as in the case of SLN, or extrusion. High-pressure extrusion involves forcing MLVs through porous membranes[29- at temperatures above Tc. As a result, lamellar fragments break away and close to form small vesicles of similar diameter to that of the pore. A particular advantage of the method is that the disruptive effects of sonication are avoided. Rapid injection of an ethanol solution of lipid into an aqueous solution also leads to production of vesicles 30 to 110 nm in diameter.[30] This method has two disadvantages as compared to sonication: high polydispersity and dilute suspension of liposomes obtained. Acidic phospholipids such as phosphatidic acid and phosphatidylglycerol are able to form unilamellar liposomes simply by transiently increasing the pH.[18] However, this process is critical to ionic strength, presence of other lipid components, and rate of titration.
Large unilamellar vesicles
Methods for preparing LUVs fall into two categories. The first involves removal of a lipid solubilizing agent, whereas the second applies physical modification of preformed bilayer. In the first case, the lipid is initially dissolved by an aqueous solution of the surfactant (either ionic—cholate, deoxycholate, or nonionic—Triton 100, octylglucoside) to form mixed lipid-surfactant micelles followed by removal of the surfactant by dialysis, diafil-tration, or gel chromatography.1-31-1 With another method, a volatile solvent such as diethyl ether, petroleum ether, or dichlorofluoromethane containing dissolved lipid is infused slowly into the aqueous phase, which is maintained at a temperature above the boiling point of the solvent so that bubbles are formed.[32] The lipid molecules form a multilayer around the vapor-water interphase, and as the solvents evaporate, uni- and oligolamellar liposomes with the size range of 100-400 nm remain in dispersion. Encapsulation efficiencies up to 46% were reported.
In the more advanced reverse-phase evaporation method, the w/o emulsion containing excess lipid in the organic phase (diethyl ether) is subject to rotary evaporation. At this stage the emulsion inversion takes place. This involves collapse of inverted micelles so that their aqueous contents form the new continuous phase, while their lipid components convert into a vesicular form.[33] In the absence of cholesterol, these vesicles have an outer diameter in the range of 50-500 nm, whereas with 50 mol% cholesterol, mean diameter is about 500 nm. High encapsulation efficiencies up to 65% can be achieved using hydrophilic solutes.
Physical modification of existing bilayers involves the above-mentioned extrusion method, which is effective for obtaining both SUVs and LUVs, and exposure of SUVs to alternate cycles of freezing and thawing. These procedures lead to fusion of the SUVs followed by formation of LUVs.[29] An elegant method using fusion is based on electrostatic effect. SUVs composed of negatively charged phospholipids are mixed with calcium ions, which cause the vesicles to aggregate and then fuse.[34] This results in formation of ”cochleate cylinders,” which are rolled-up portions of lipid bilayer.
Chelation of Ca2+ by adding EDTA results in conversion of the cochleates to LUVs.
Applications of Liposomes
Liposomes have been widely investigated as delivery systems for treatment of cancer, as well as bacterial, fungal, viral, and parasitic diseases.[35] Liposome-based gene transfection systems have been promoted as means to achieve the transfection efficacy of viral constructs without any associated risks. Liposomes themselves can serve as immunological adjuvants. They are applied as vehicles to deliver various radioisotopes and contrast agents for use in diagnostic imaging. In general, several beneficial properties of the liposomal form of drugs and the ways of providing their targeted delivery are essential for polymer nanoparticles as well. However, because of the noncovalent interactions responsible for their formation liposomes have only limited stability and are subject to structural changes.[18]
Efficiency of both the liposomal forms and nano-particles depends on the rate of releasing of an active substance and their clearance from the blood. Compounds with low molecular weight release rapidly. Depending on molecular weight and the ability to withstand enzymatic attack, drugs could then act either locally (e.g., hydrolysis of stored sucrose by liposomal fructofuranosidase) or, after diffusion through the lysosomal membrane, in other cell compartments (e.g., inhibition of DNA-directed RNA synthesis by liposomal actinomycin D).[35] It was shown that the rate of clearance of injected liposomes from the blood is rather rapid, dose dependent, and biphasic. Neutral MLVs and SUVs exhibit a slower rate of clearance than charged MLVs.[35] Understanding of liposomal fate and behavior led to several proposed applications. The most important fact is that liposomes with entrapped material were shown to end up in the fixed macrophages of the RES, mainly in the liver and spleen.[36] Fast blood clearance is the principal obstacle to use of liposomes for drug delivery via the parenteral route. Liposome clearance can be substantially reduced by inclusion of mono-sialoganglioside GMj, hydrogenated phosphatidylinositol, or PEG-substituted phosphatidylethanolamine into the bilayer.[37] The same effect may be achieved by PEG coating of liposomes—PEGylation.[38] This modification hampers detection by the RES, and led to their being called StealthTM liposomes. The long-circulating PEG-ylated liposomes are considered suitable for targeted drug delivery to tumors and inflammatory foci, as well as for diagnostic imaging applications, e.g., magnetic resonance and scintigraphic imaging.
Two types of targeting are under way: passive and active. Passive targeting is limited in its scope, leads to the rather wide distribution of the drug-filled liposomes in tissues, and depends dramatically on time of circulation. Thus, tremendous efforts are being directed at present toward the development of control together with modification of the liposome surface with molecules having recognition properties. Typical examples of such modification are antigenic determinants, including various types of membrane receptors, or antibodies (either intact or as active fragments), other proteins, lipoproteins, glycopro-teins, and so on, which may associate with bilayers as they do in the living cells. One such way is ligand-coupling strategy, which was originally realized with tumor-specific antibodies, known as immunoliposomes.[39- Most of the subsequent studies have used chemical coupling to link a targeting moiety to an amphiphilic molecule inserted into the liposome bilayer. The most widely used approach involves the use of heterobifunctional cross-linking reagents for introducing thiol-ether-based linkages between the lipid anchor and the ligand (e.g., immunoglobulin).
The way in which liposomes induce immune responses to antigens associated with them is still not clear, but has been attributed to a depot mechanism and the ability of liposomes and antigen content to migrate to regional lymph nodes. A novel concept, namely, production of the required vaccine antigen by the host cells in vivo, promises to alter vaccination, especially where vaccines are either ineffective or unavailable. The concept entails the direct injection of antigen-encoding plasmid DNA, which, after its uptake by cells, finds its path to the nucleus where it transfects the cells.[41- Antigen so produced is recognized by the host as foreign and then subjected to pathways leading to protective immunity. Simple mixing of antigen-encoding plasmid DNA or DNA itself and cat-ionic SUV leads to neutralization of polyanionic nucleic acid chain to form a compact nanostructure, known as a lipoplex.[42- Each of these nanoparticles carries a small net positive charge, which enables them to interact with the negatively charged cell membranes and gain entry by receptor-mediated endocytosis to perform transfection.
POLYMER NANOSPHERES
Manufacture of Nanospheres
The first approaches used to produce polymer nano-particles were derived from the field of latex engineering. These methods were based on in situ polymerization of monomers.[43,44- Despite the actual technological advances, polymerization-based methods have some drawbacks and limitations. Thus, alternative methods based on the dispersion of well-characterized preformed polymers or natural biopolymers[4,5- have been proposed.[45- Together with polymerization-based methods, these new techniques allow the production of both nanospheres and nanocapsules (Fig. 3).
Fig. 3 Schematic structure of polymer nanoparticles: (a) nanospheres and (b) nanocapsules.
Polymerization methods
Two different approaches have been considered for the preparation of nanospheres by polymerization methods, depending on whether a monomer to be polymerized is emulsified in a nonsolvent phase (emulsion polymerization) or dissolved in a solvent that is a nonsolvent for the resulting polymer (dispersion polymerization).
In two types of emulsion polymerization, either conventional (o/w emulsion) or inverse (w/o emulsion), the monomer is emulsified in the nonsolvent phase with surfactant, leading to the formation of monomer-swollen micelles and stabilized monomer droplets. The polymerization reaction takes place in the presence of an initiator that creates free reactive monomer radicals. These collide with the unreactive monomers and initiate polymer chain growth, or nucleation.[44- The reaction stops when full consumption of monomer or initiator is achieved. The drug to be incorporated to the nanospheres may be present during the polymerization process. Two different mechanisms of nucleation were considered. First, the so-called micellar polymerization mechanism treats the monomer-swollen micelles as the site of nucleation and propagation.[46,47- In this case, monomer droplets serve as monomer reservoirs. The monomer molecules reach the micelles by diffusion through the continuous phase. The second mechanism is valid for monomers that are sufficiently soluble in the continuous phase. In this case, homogeneous nucleation followed by propagation can occur directly in this phase, leading to formation of oligo-mers.[47- When the oligomers have reached a certain length, they form primary particles stabilized by surfactant molecules present in the system. In the case of inverse emulsion polymerization, when we deal with w/o system, the water-soluble monomers cannot diffuse from the micelles through the organic phase because of their low partition coefficient. The resulting nano-spheres contain fewer polymer chains and a narrower size distribution as compared to the conventional emulsion polymerization.[48]
Both the conventional and inverse emulsion polymerization reactions were applied for the production of biodegradable poly(alkyl cyanoacrylate) (PACA) nano-spheres.[49,50] In the case of conventional emulsion polymerization, the alkyl cyanoacrylate monomer is added to an aqueous acidic solution of surfactant under vigorous stirring to polymerize it following the anionic mechanism. Drug is dissolved in the polymerization medium either before the addition of monomer or at the end of the polymerization reaction. Nanospheres (Fig. 4a) are sedi-mented by ultracentrifugation of the obtained suspension. In the case of inverse emulsion polymerization, drug is dissolved in a small amount of water or hydrophilic solvent and emulsified in an organic phase (e.g., isooctane, cyclohexane, and hexane) in the presence of surfactants. Alkyl cyanoacrylate monomers are then added directly or dissolved in an organic solvent to the preformed w/o emulsion under stirring.[49] The system becomes milky, and nanospheres with a diameter of 200-300 nm with a narrow polydispersity are formed.
Dispersion polymerization deals with polymerization of monomers dissolved in an aqueous medium that acts as a precipitant for the polymer to be formed. Nucleation is induced in the aqueous monomer solution,[51] and the presence of stabilizers or surfactants is not absolutely necessary for the formation of nanospheres.
As mentioned earlier, polymerization methods have significant limitations, especially for formation of na-nospheres.[45] First, it is very difficult to predict the molecular weight of the resulting material. This is a major drawback because the molecular weight influences the biodistribution and release of the polymer carrier. Second, the presence of free radicals or numerous H+ ions generated by anionic polymerization process can inhibit drug activity. Third, the presence of toxic unreacted monomer, initiator, and surfactant molecules requires time-consuming and, sometimes, inefficient procedures for their elimination.
Fig. 4 (a) Electron scanning micrograph of insulin-loaded poly(ethylcyanoacrylate) nanospheres obtained by a polymerization technique. (From Radwan, M.A.; Aboul-Enein, H.Y. J. Microencapsul. 2002, 19 (2), 225-235. Copyright 2002, Taylor & Francis, Ltd.) (b) Transmission electron micrograph of procaine hydrochloride loaded poly(lactide-co-glycolide) nano-spheres obtained by precipitation method.
