Hybrid Nanocapsules as Vehicles for Drug Delivery

Many natural and artificial objects are subjected to degradation by the surrounding environment. It is particularly important to protect compounds that can be damaged by irradiation, heating, oxidizing (reducing) agents, polymers, moisture, etc. at the molecular level during their storage and targeted delivery. Such substances are of practical importance in medicine (drugs), gene engineering (DNA, RNA), biotechnology (proteins, individual cells, enzymes), and in the food industry. The development of protective microenvironments combining the defense property with other properties, such as, for instance, the possibility of controlled release of the secured compound at the desired site in aggressive media, is of particular interest. Hybrid nanocapsules are probably the most suitable materials for this purpose. The use of hybrid nanocapsules is not casual because these latter aim to mimic the most sophisticated entities found in nature, working as carrier and vehicle to deliver and release substances or cells. Thus, liposomes, taken as models of biological membranes, are of fundamental importance because they serve as selective barriers for transport, as fluid matrixes for biosynthetic transformations, and as boundaries for the transfer of energy and information. Lipid bilayer vesicles are also well known nanomaterials, which have been extensively employed as supramolecular assemblies to construct molecular devices [151-159].

The performance of any of these biomimetic applications strongly depends on the membrane fluidity. For instance, the interest in using phospholipids vesicles as nanocapsules and vehicles to entrap, deliver and control-release functional substances (e.g. proteins, enzymes and drugs) resides in two main features [160] . Firstly, the bilayer can behave as a barrier and provide protection to the molecules encapsulated in the interior aqueous phase from the aggression of external denaturizing agents. Secondly, entrapped molecules can be control-released in response to a variety of physical and chemical stimuli (including temperature, pressure, light, pH, and ions) that, as mentioned above, determine the bilayer fluidity and thus, its permeability [161] . In general, liposomes change their aggregate morphology by collapse and fusion when they interact with polyelectrolytes having the opposite charge. Obviously, a drastic increase in the morphological stability of liposomes seems to be quite desirable if one intends to apply these structures for practical purposes.

An example where the fragility of the liposome structure limits its applicability is that reported recently by Matsui et al. [ 162] . As the number of functions of nucleic acids continues to expand, there is a growing need for efficient delivery of functional nucleic acids. Since the discovery of lipofection [163]. cationic lipids have been widely used as transfection agents in gene delivery [164] . They form liposomal bilayers in water. Polyanionic DNAs are readily bound to the resulting cationic liposomes to give complexes (lipoplexes) which are inserted in the cells via endocytosis to ultimately result in expression of the encoded gene. The bilayer-keeping forces are not very strong, and liposomes are not so rigid or robust. They are potentially fusible with cell membranes and hence toxic. They also easily undergo DNA-mediated/induced cross-linking/fusion to give larger particles (>> 100 nm) that have lower endocytosis susceptibility [164-167] and poorer vascular mobility. This problem has been challenged, occasionally, in the context of artificial viruses, with different strategies of mostly multicomponent surface coverage such as stabilized plasmid-lipid particles [168], saccharide-manipulated particles [169], dimerizable cationic detergents [170, 171], liposome-m-DNA systems [172, 173], glycoviruses [174], and PEG-stabilized plasmid nanoparticles [319].

The Nara group recently reported a ceramic-coated liposome and called it "cera-some" [ 175] , A cerasome is a novel bioinspired organic-inorganic hybrid composed of a liposomal membrane with a ceramic surface. The cerasomes were prepared from organoalkoxysilane proamphiphiles (1 and 2 in Fig. 15.15) under sol-gel reaction conditions [162] , Formation of the bilayer vesicular structures of the cerasomes was confirmed by negative-staining transmission electron microscopy (TEM). The obtained diameter from TEM observation was 70-300 nm and 20-100 nm for the cerasomes prepared from 1 and 2 (Fig. 15.15), respectively. These cerasomes had characteristic properties as lipid bilayer vesicles, e.g. phase transition behavior from gel to liquid-crystalline state. The authors have focused on the consequence of surface rigidification of the cerasome as a gene carrier. They report that the cerasome retains its integrity in the complexation with plasmid DNA (pGL3) and the resulting DNA complex of infusible or monomeric cerasome in a viral size (~70 nm) exhibits a remarkable transfection performance (high activity, minimized toxicity and serum-compatibility). They experimentally examined the cerasome 1-mediated transfection of HeLa (uterine) and HepG2 (hepatic) cells using a fixed amount (200 ng, 0.6 nM or 6.25 mM P) of pGL3 and a variable amount

Fig. 15.15 Structures of lipids 1 and 2 and freeze-fracture TEM images of the liposomes formed with Lipid 1 (a) or 2 (b) in water at 12.5 mM. Approximately ten independent images were taken for each lipid; three of them, including the smallest one and the largest one, are shown. (Reprinted with permission from [162]; copyright ACS publications)

Fig. 15.15 Structures of lipids 1 and 2 and freeze-fracture TEM images of the liposomes formed with Lipid 1 (a) or 2 (b) in water at 12.5 mM. Approximately ten independent images were taken for each lipid; three of them, including the smallest one and the largest one, are shown. (Reprinted with permission from [162]; copyright ACS publications)

of lipoid 1 [ 162] . The results indicate that neither the transfection-responsible cerasome-plasmid complex (~70 nm) nor the DNA-free cerasome (60-70 nm) is toxic under the present transfection conditions. Reference lipid 2 . on the other hand, turned out to be far less active and far more toxic. Interestingly, the big difference in activity of 1/2 = 102-3 (serum-free) is in agreement with the size-activity correlation revealed for glyco viruses [174] . This large activity ratio can thus be explained primarily in terms of the difference between the sizes of monomeric 1-plasmid complex (~70 nm) and fused 2-plasmid complex (>200 nm), although a possible difference in aggregation behavior should also be taken into account. The presented monocomponent silicon strategy provides a simple and widely applicable new tool to overcome these general problems inherent to the current technology of artificial gene delivery. Size control is particularly crucial for in vivo applications. Particles of viral size (mostly 30-100 nm) have not only good diffusion in vascular periphery, but also good permeation into malignant tissues by the so-called EPR effect [176].

The recently developed layer-by-layer (LbL) technology [177, 178] also offers the opportunity to fabricate multifunctional nanoengineered microcontainers. This technology is based on sequential deposition of oppositely charged polyelectrolytes on a surface of variable shape, which allows the formation of multilayer shells from a wide range of components with nanometer precision. Templating of LbL polyelectrolyte films on the surface of the micron- and submicron-sized isolated particles followed by their dissolution leads to the formation of hollow polyelectrolyte capsules [179-181] . The initial colloidal core determines the size of the capsules, which can be varied from 50 mm to 50 nm. The polyelectrolyte shell is permeable for small molecules and ions [182, 183]. while large molecules and nanoparticles can be entrapped inside or banned from the capsule interior. Sustained release of the encapsulated furosemide, dextran and fluorescein molecules was demonstrated [ 184-186] . Different additional functionalities (magnetic, luminescent, sensing) can be imparted to the capsule shell by introducing nano-Fe3O4, q-CdS, etc. as shell constituent [187, 188] . The protective character of polyelectrolyte microcapsules has been demonstrated in a recent work [189]. The protecting ability of the developed polyelectrolyte capsules was monitored by oxidation of encapsulated bovine serum albumin by H2O2 dissolved in aqueous solution. Two approaches for designing protective capsule microcontainers are demonstrated (Fig. 15.16): The "passive armor" approach is composed of a sacrificial reducing agent as a shell constituent, while the "active armor" approach includes the catalyst for H.O2 decomposition deposited onto the shell as the outer layer. In the latter case, the protective material is not consumed during the H2O2 treatment, thus prolonging the protection activity of the microcapsule. Advances in the field of capsules are in the direction of the combined use of LbL and organoalkoxysilane-type lipids (Si-lipid) with a polymerizable moiety in the headgroup, which yields robust lipid coatings on core-shell colloids [ 190] . The particle-supported Si-lipid membrane was found to be highly stable upon exposure to Triton X-100 and ethanol solutions, even at high concentrations. The enhanced stability of the Si-lipid film is attributed to the polymerized moiety in the headgroup of the synthetic lipids and H-bonding interactions between adjacent

Fig. 15.16 Schematic overview of the "passive" and "active" protection approaches for nanoen-gineered polyelectrolyte capsules. (Reprinted with permission from [189]; copyright ACS publications)

Si-lipid molecules. The robustness of the inorganic lipid-based shell allows the removal of the core without significant delamination of the Si-lipid, providing a viable approach to the preparation of lipid-functionalized capsules.

There has also been increased interest in mesoporous silica materials for their use as carriers in controlled drug release. Amorphous mesoporous silica materials have been investigated as drug supports because of their nontoxic nature, adjustable pore diameter and very high specific surface area with abundant Si-OH bonds on the pore surface [191-200]. Several research groups have investigated the conventional mesoporous silica materials (such as MCM-41 and SBA-15) used as drug-delivery systems. These systems exhibit sustained-release properties, but their drug storage capacity is relatively low and, also, the irregular bulk morphology is not perfect for drug delivery. To overcome these disadvantages, one strategy is to synthesize hollow mesoporous silica (HMS) spheres on the nanoscale with pore channels penetrating from the outside to the inner hollow core. Recently, Li et al. have successfully synthesized HMS spheres with a 3D pore-network shell [201, 202]. Later on, they proposed a strategy to combine the advantages of HMS spheres with a 3D pore network and polyelectrolyte multilayers with a stimuli-responsive property [203] (Fig. 15.17).

Nonetheless, inorganic hollow silica or polyorganosiloxane nanoparticles can also be prepared by direct template approaches. Schacht et al. have prepared the hollow silica spheres in oil/water emulsions, which resulted from an interfacial reaction [204]. Pinnavaia and coworkers introduced the silica into the interlayer regions of the multilamellar vesicles to form nearly spherical particles with stable lamellar mesostructures [205]. The ultrastable mesostructured silica vesicles were also reported by the same group using the neutral gemini surfactants as the

Fig. 15.17 TEM micrographs of HMS (a and b), the IBU-HMS system (c), and the [email protected] PEM system (d). (Reprinted with permission from [203]; copyright Wiley-VCH publications)

structure-directing agents [206]. An organic/inorganic hybrid vesicle was prepared by gelation in the vesicles given by a lipid bearing an alkoxysilyl head, and a hierarchical vesicle assembly was made through an LbL approach [ 175, 207] . The colloids have been used as the templates for forming the inorganic shells. The linear polysiloxane colloids, as the templates formed in situ in an emulsion polymerization, were further coated with a cross-linked polysilsesquioxane shell by gelation of trimethoxymethylsilane, and the hollow particles were formed through extraction of the soluble core by a solvent [208, 209]. Functional molecules such as dyes were encapsulated in the siloxane network and in the cavity of the hollow particles [210].

In a very recent report, the hybrid core shell particles were prepared by coating the surfaces of monodisperse polystyrene beads with uniform silica shells [211]. Hollow silica spheres could be obtained by removing the polymer cores via calcination or by wet etching with toluene. The surfaces of block copolymer micelles have also been coated with a silica layer by reacting with an active silicate, making use of a few trimethoxysilane groups born on the corona-forming blocks of the micelles [212]. Recently, the same group has reported preliminary results on the preparation of such hollow nanoparticles using a novel reactive diblock copolymer, poly(ethyleneoxide)-fcfoc£-poly[3-(trimethoxysilyl) propyl-methacrylate], which was synthesized by atom-transfer radical polymerization [213]. Pictures obtained by typical TEM and scanning electron microscopy -clearly showing the three-dimensional vesicular morphology- of the hybrid vesicles are presented in Fig. 15.18. The formation of these unique vesicles by this block copolymer was rather remarkable. In a recent work, the authors have studied how the preparation conditions and the copolymer compositions influenced the formation of these novel hybrid vesicles [214]. They conclude that the inorganic components impart stability.

Fig. 15.18 (a) TEM images of the vesicles after addition of 1.3 wt% TEA in methanol/water 45:55 (w/w) at a polymer concentration of 0.45 g/L and (b) SEM image of the particles. (Reprinted with permission from [213] ; copyright ACS publications)

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