Aquasomes: Role to Deliver Bioactive Substances

N.L Prasanthi^*, S.S. Manikiran, C. Sowmya Krishna and N. Rama Rao

Chalapathi Institute of Pharmaceutical Sciences, Lam, Guntur- 522034 Andhra Pradesh

ABSTRACT:

Aquasomes are one of the most recently developed delivery systems; these are nanoparticulate carrier systems with three-layered self-assembled structures. They comprise a central solid nanocrystalline core coated with polyhydroxyoligomers onto which biochemically active molecules are adsorbed. Three types of core materials are mainly used for producing aquasomes: tin oxide, nanocrystalline carbon ceramics (diamonds) and brushite (calcium phosphate dihydrate). Calcium phosphate is the core of interest, owing to its natural presence in the body. The brushite is unstable and converts to hydroxyapatite upon prolong storage. Hydroxyapatite seems, therefore, a better core for the preparation of aquasomes. It is widely used for the preparation of implants for drug delivery. The solid core provides the structural stability, while the carbohydrate coating protects against dehydration and stabilizes the biochemically active molecules. This property of maintaining the conformational integrity of bioactive molecules has led to the proposal that aquasomes have potential as a carrier system for delivery of peptide, protein, hormones, antigens and genes to specific sites.

KEYWORDS: Aquasomes, nanoparticels, bioactives, nanocrystalline core.

INTRODUCTION:

The global market for advanced drug delivery systems was more than €37.9 billion in 2000 and is estimated to grow and reach €75B by 2005 (i.e., controlled release €19.8B, needle-less injection €0.8B, injectable/impantable polymer systems €5.4B, transdermal €9.6B, transnasal €12.0B, pulmonary €17.0B, transmucosal €4.9B, rectal €0.9B, liposomal drug delivery €2.5B, cell/gene therapy €3.8B,miscellaneous €1.9B). Developments within this market are continuing at a rapid pace, especially in the area of alternatives to injected macromolecules, as drug formulations seek to cash in on the €6.2B worldwide market for genetically engineered protein and peptide drugs and other biological therapeutics.

Advances have since been made in the area of vesicular drug delivery, leading to the development of systems that allow drug targeting, entrapping large size drug moieties and sustained or controlled release of conventional medicines. In recent years, much revolutionary explorations were come across the formulation and development of dosage forms of small size to improve the performance of the drug. Different types of pharmaceutical carriers are present. They are particulate, polymeric, macromolecular and cellular carrier. Particulate type carrier also known as a colloidal carrier system, includes lipid particles (low and high density lipoprotein-LDL and HDL, respectively), microspheres, micellar solutions, vesicle and liquid crystal dispersions, as well as nanoparticle dispersions consisting of small particles of 10–400 nm diameter show great promise as drug delivery systems^1-4. When developing these formulations, the goal is to obtain systems with optimized drug loading and release properties, long shelf-life and low toxicity. The incorporated drug participates in the microstructure of

the system, and may even influence it due to molecular interactions, especially if the drug possesses amphiphilic and/or mesogenic properties⁵. Some of the carrier systems are shown in Table 1.

A new class of solid drug carriers, aquasomes has emerged during the last decade. Aquasomes are three-layered structures (i.e., core, coating, and drug) that are self-assembled through non-covalent bonds, ionic bonds, and Vander Wals forces⁶. They consist of a ceramic core whose surface is non-covalently modified with carbohydrates to obtain a sugar ball, which is then exposed to adsorption of a therapeutic agent. The core provides structural stability to a largely immutable solid. Alternatively aquasomes are called as “bodies of water”, their water like properties protect and preserve fragile biological molecules, and this property of maintaining conformational integrity as well as high degree of surface exposure are exploited in targeting of bio-active molecules like peptide and protein hormones, antigens and genes to specific sites. These carbohydrate stabilize nanoparticles of ceramic are known as “aquasomes” which was first developed by Nir Kossovsky. The pharmacologically active molecule incorporated by co polymerization, diffusion or adsorption to carbohydrate surface of pre formed nanoparticles. These three layered structure are self assembled by non-covalent bonds. Principle of “self assembly of macromolecule” is governed by three physiochemical process^7,8 i.e.

1. Interaction between charged group - Interaction between charged group, the interaction of charged group facilitates long range approach of self assembly sub units charge group also plays a role in stabilizing tertiary structures of folded proteins.

2. Hydrogen bonding and dehydration effect - Hydrogen bond helps in base pair matching and stabilization secondary  protein structure such as alpha helices and beta sheets. Molecules forming hydrogen bonds are hydrophilic and this confers a significant degree of organization to surrounding water molecules. In case of hydrophobic molecules, which are incapable of forming hydrogen bond, their tendency to repel water helps to organize the moiety to surrounding environment, organized water decreases  level of entropy and is thermodynamically  unfavorable, the molecule dehydrate and get self assembled.

3. Structural stability of protein in biological environment determined by interaction between charged group and Hydrogen bonds largely external to molecule and by van der waals forces largely internal to molecule, experienced by hydrophobic molecules, responsible for hardness and softness of molecule and maintenance of internal secondary structures, provides sufficient softness, allows maintenance of conformation during self assembly. Self assembly leads to altered biological activity, van der waals need to be buffered. In aquasomes, sugars help in molecular plasticization.

Strategies used in chemical synthesis of aquasomes ( three layered nanostructures) are, i) arrays of co-valently linked atoms generated with well defined composition, connectivity and shape ii) covalent polymerization [9] used for preparing molecules with high molecular weight, low weight substance allowed to react with itself to produce molecule comprising many covalently linked monomers iii) self–organizing synthesis, relies on weaker and less directional bonds as ionic, hydrogen and van der waals.Molecules adjust their own position to reach thermodynamic minimum, true nanostructures prepared iv) molecular self assembly, it combines features of preceding strategies, involves formation of intermediate structural complexity through co valent synthesis; formation of stable structure through ionic, hydrogen and van der waals links and use of multiple copies. For final assembly, non covalent connection between molecules must be stable.

Rational behind development of aquasomes:

Generally colloidal drug carriers like produgs, liposomes and macromolecules have the biophysical constains due to interaction between drug and the carrier. In such sictuvations aquasomes are worth promising carriers. Aquasomes maintains molecular confirmation and optimum pharmacological activity. Normally, active molecules possess following qualities i.e. a unique three-dimensional conformation, a freedom of internal molecular rearrangement induced by molecular interactions and a freedom of bulk movement but proteins undergo irreversible denaturation when desiccated, even unstable in aqueous state. In the aqueous state pH, temperature, solvents, salts cause denaturation hence bio-active faces many biophysical constrain^10,11. In such case, aquasomes with natural stabilizers like various polyhydroxy sugars act as dehydroprotectant maintains water like state thereby preserves molecules in dry solid state.

Preparation of aquasomes:

Aquasomes are generally prepared by the principles of self-assembly. The principle having three steps^12,15.

1. Preparation of the core:

The first step of aquasome preparation is the fabrication of the ceramic core. The process of ceramic core preparation depends on the selection of the materials for core. These ceramic cores can be fabricated by colloidal precipitation and sonication, inverted, plasma condensation and other processes. For the core, ceramic materials were widely used because ceramics are structurally the most regular materials known. Being crystalline, the high degree of order in ceramics ensures that any surface modification will have only a limited effect on the nature of the atoms below the surface layer and thus the bulk properties of the ceramic will be preserved. The high degree of order also ensures that the surfaces will exhibit high level of surface energy that will favor the binding of polyhydroxyoligomeric surface film.

Table 1: Some novel carriers for drug delivery

Carrier	Description	Application
Aquasomes	Three-layered self-assembly compositions with ceramic nanocrystalline particulate core loaded with glassy layer of polyhydroxy compounds	Molecular shielding, specific targeting
Archaeosomes	Vesicles composed of glycerolipids of Archaea with potent adjuvant activity	Potent adjuvant activity
Cryptosomes	Lipid vesicles with a surface coat composed of PC and of suitable polyoxyethylene derivative of phosphatidylethanolamine	Ligand-mediated drug targeting
Discomes	Niosomes solubilized with nonionic surfactant solution(polyoxyethylene cetyl ether glass)	Ligand-mediated drug targeting
Emulsomes	Nanosized lipid particles (bioadhesive nanoemulsions) consisting of microscopic lipid assembly with apolar core	Parenteral delivery of poorly water-soluble drugs
Enzymosomes	Liposomes designed to provide a mini bioenvironment in which enzymes are covalently immobilized or coupled to the surface of liposomes	Targeted delivery to tumor cells
Erythrosomes (Proteoliposomes)	Human erythrocyte cytoskeletons used as a support to which lipid bilayer is coated	Effective targeting of macromolecular drugs
Ethosomes	Lipid-based soft, malleable vesicles containing a permeation enhancer and composed of phospholipids, ethanol, and water	Targeted delivery to deep skin layers
Genosomes	Artificial macromolecular complexes for functional gene transfer. Cationic lipids are most suitable because they possess high biodegradability and stability in the bloodstream.	Cell-specific gene transfer
Novasomes	Consist of glyceryl dilaurate, cholesterol, and polyoxyethylene 10-stearyl ether at a weight-percent ratio of 57:15:28, respectively	Drug delivery to pilosebaceous compartment
Photosomes	Photolyase encapsulated in liposomes that release the contents by phototriggered changes in membrane permeability characteristics	Photodynamic therapy
Proteosomes	High-molecular-weight multi-subunit enzyme complexes with catalytic activity that is specifically due to assembly pattern of enzymes	Better catalytic activity turnover than non-associated enzymes, may serve as adjuvant as well as protein carrier
Transferosomes (elastic liposomes)	Modified lipid-based soft, malleable carriers tailored forenhanced systemic delivery of drugs	Noninvasive delivery of drugs into or across the deeper skin layers and/or the systemic circulation
Vesosomes	Nested-bilayer compartments with “interdigitated” bilayer phase formed by adding ethanol to a variety of saturated phospholipids	Multiple compartments of the vesosomes give better protection to the interior contents in serum
Virosomes	Liposomes spiked with virus glycoprotein, incorporated into the liposome bilayers based on retrovirus-derived lipids	Immunological adjuvants

Two ceramic cores that are most often used are diamond and calcium phosphate. Generally cores are crystalline and they measure between 50-150 nm. Other are nanocrystalline tin oxide core ceramic, nanocrystalline brushite, nanocrystalline carbon ceramic and diamond particles.

2. Carbohydrate coatings:

The second step involves coating by carbohydrate on the surface of ceramic cores. There are number of processes to enable the carbohydrate (polyhydroxyoligomers) coating to adsorb epitaxially on to the surface of the nano-crystalline ceramic cores. The processes generally entail the addition of polyhydroxyoligomer to a dispersion of meticulously cleaned ceramics in ultra pure water, sonication and then lyophilization to promote the largely irreversible adsorption of carbohydrate on to the ceramic surfaces. Excess and readily desorbing carbohydrate is removed by stir cell ultra-filtration. The commonly used coating materials are cellobiose, citrate, pyridoxal-5-phosphate, sucrose and trehalose.

3. Immobilization of drugs:

The surface modified nano-crystalline cores provide the solid phase for the subsequent non denaturing self assembly for broad range of biochemically active molecules. The drug can be loaded by partial adsorption.

The preparation procedure of aquasomes is depicted in Figure 1 and 2.

Figure 1: Synthesis of aquasomes consists of fabricating a nanocrystalline core of a calcium phosphate (brushite) colloidal precipitate or ceramic diamond. The core is coated with a polyhydroxyl oligomeric film, and the coated particles are then allowed to adsorb a drug or antigen. The final product consists of three layers: drug (or antigen), polybydroxyl oligomeric film, and the nanocrystalline ceramic core.

The above figure can be depicted in the following way,

Figure 2: Synthesis of aquasomes

Properties:^16,17

1) Aquasomes possess large size and active surface hence can be efficiently loaded with substantial amounts of agents through ionic, non co-valent bonds, van der waals forces and entropic forces. As solid particles dispersed in aqueous environment, exhibit physical properties of colloids.

2) Aquasomes mechanism of action is controlled by their surface chemistry.Aquasomes deliver contents through combination of specific targeting, molecular shielding, and slow and sustained release process.

3) Aquasomes water like properties provides a platform for preserving the conformational integrity and bio chemical stability of bio-actives.

4) Aquasomes due to their size and structure stability, avoid clearance by reticuloendothelial system or degradation by other environmental challenges.

5) In normal system, calcium phosphate is biodegradable. Biodegradation in vivo achieved by monocytes and multicellular cells called osteoclast.Two types of phagocytosis reported, either crystals taken up alone and then dissolved in cytoplasm after disappearance of phagosome membrane or dissolution after formation of heterophagosome.^.

6) Aquasomes are mainly characterized for structural analyses, particle size, and morphology these are evaluated by X-ray powder diffractometry, transmission electron microscopy, and scanning electron microscopy. The X-ray analysis of the samples and drug loading efficiency and in vivo performance.

Applications:

1) Aquasomes as red blood cell substitutes, haemoglobin immobilized on oligomer surface because release of oxygen by haemoglobin is conformationally sensitive. By this toxicity is reduced, haemoglobin concentration of 80% achieved and reported to deliver blood in non linear manner like natural blood cells. Khopade et al prepared hydroxyapatite core by using carboxylic acid–terminated half-generation poly(amidoamine) dendrimers as templates or crystal modifiers. These cores were further coated with trehalose followed by adsorption of hemoglobin. The size of the particles was found to be in the nanometer range, and the loading capacity was found to be approximately 13.7 mg of hemoglobin per gram of the core^18,19.

2) Aquasomes used as vaccines for delivery of viral antigen i.e. Epstein-Barr and Immune deficiency virus to evoke correct antibody, objective of vaccine therapy must be triggered by conformationally specific target molecules. Kossovsky et al demonstrated the efficacy of a new organically modified ceramic antigen delivery vehicle. These particles consisted of diamond substrate coated with a glassy carbohydrate (cellobiose) film and an immunologically active surface molecule in an aqueous dispersion. These aquasomes (5–300 nm) provided conformational stabilization as well as a high degree of surface exposure to protein antigen. Diamond, being a material with high surface energy, was the first choice for surface capable of hydrogen bonding to the pertinacious antigen. The disaccharide, being a dehydro-protectant, helps to minimize the surface-induced denaturation of adsorbed antigens (muscle adhesive protein, MAP). For MAP, conventional adjuvants had proven only marginally successful in evoking an immune response. However, with the help of these aquasomes a strong and specific immune response could be elicited by enhancing the availability and in vivo activity of antigen^20,21.

3) Aquasomes have been used for successful targeted intracellular gene therapy, a five layered composition comprised of ceramic core, polyoxyoligomeric film, therapeutic gene segment, additional carbohydrate film and a targeting layer of conformationally conserved viral membrane protein.

4) Aquasomes for pharmaceuticals delivery i.e. insulin, developed because drug activity is conformationally specific.Bio activity preserved and activity increased to 60% as compared to i.v. administration and toxicity not reported. Cherian et al prepared aquasomes using a calcium phosphate ceramic core for the parenteral delivery of insulin. The core was coated with various disaccharides such as cellobiose, trehalose, and pyridoxal-5-phosphate. Subsequently the drug was loaded to these particles by adsorption method²².

5) Aquasomes also used for delivery of enzymes like DNAase and pigments/dyes because enzymes activity fluctuates with molecular conformation and cosmetic properties of pigments are sensitive to molecular conformation. Saraf et al proposed the use of a nanosized ceramic core–based system for oral administration of the acid-labile enzyme serratiopeptidase. The nanocore was prepared by colloidal precipitation under sonication at room temperature. The core was then coated with chitosan under constant stirring, after which the enzyme was adsorbed over it. The enzyme was protected by further encapsulating the enzyme-loaded core into alginate gel^23,24.

6) Miscellaneous

a) Mizushima and co-workers prepared spherical porous hydroxyapatite particles by spray-drying. These particles were tried as a carrier for the delivery of drugs such as IFNα, testosterone enanthate, and cyclosporin A. Spherical porous hydroxyapatite was found to have an average diameter of 5 μm with approximately 58% porosity. These particles could be injected subcutaneously through a 27-gauge needle. IFNα was adsorbed well to spherical hydroxyapatite particles. Addition of HAS and zinc (for reinforcement) to IFNα-adsorbed hydroxy- apatite particles caused marked prolongation of release in vivo²⁵.

b) Oviedo and co-workers prepared aquasomes loaded with indomethacin through the formation of an inorganic core of calcium phosphate covered with a lactose film and further adsorption of indomethacin as a low-solubility drug^..

CONCLUSION:

Aquasomes, the self-assembling surface-modified nanocrystalline ceramic cores, appear to be promising carriers for the delivery of a broad range of conformational sensitive molecules with better biological activity due to presence of unique carbohydrate coating over the ceramic core. This approach thus provides pharmaceutical scientists with new hope for the delivery of a broad range of molecules including viral antigens, bioactive molecules. Further study of aquasomes is necessary to confirm their efficiency as well as safety, to establish their clinical usefulness and to launch them commercially.

ACKNOWLEDGEMENTS

The authors are thankful to Chalapathi Educational Society, Guntur for providing the necessary facilities.

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Received on 26.08.2010

Accepted on 11.09.2010

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Research Journal of Pharmaceutical Dosage Forms and Technology. 2(6): Nov.-Dec. 2010, 356-360