Ms. Aishwarya Chagan Patil*, Ms. Shital Sanjay Shewale, Ms. Yogita Tukaram Rayate, Dr. Manojkumar M. Nitalikar, Dr. ShreenivasK.Mohite
Department of Pharmaceutics, Rajarambapu College of Pharmacy Kasegaon, Tal –Walwa, Tist – Sangali
Aquasomes are one of the most recently developed delivery system for bioactive molecules like peptide, protein, hormones, antigens and genes to specific sites. Aquasomes are spherical in shape with 60–300 nm particles size. These are nanoparticulate carrier systems but instead of being simple nanoparticles these are three layered self assembled structures, comprised of a solid phase nanocrystalline core coated with oligomeric film to which biochemically active molecules are adsorbed with or without modification. These structures are self assembled by non covalent and ionic bonds. The solid core provides the structural stability, while the carbohydrate coating protects against dehydration and stabilizes the biochemically active molecules. The delivery system has been successfully utilized for the delivery of insulin, hemoglobin, and enzymes like serratiopeptidase etc. This reviews the principles of self assembly, the challenges of maintaining the conformational integrity and biochemical activity of immobilized surface pairs, the convergence of these principles into a single functional composition and its application in various fields of pharmacy.
Nanobiopharmaceutics involves delivery of biopharmaceutical product through different biomaterials like multifunctional nanoparticles, quantum dots, aquasomes, superparamagnetic iron oxide crystals, liposomes, niosomes and dendrimers . There are different types of ‘somes’ like Aquasomes (Carbohydrates-ceramic nanoparticles) are the nano-biopharmaceutical carrier system contains the particle core composed of nanocrystalline calcium phosphate or ceramic diamond, and is covered by a polyhydroxyl oligomeric film [1, 2].
Kossovsky proposed a system to prepare nanoparticles transporting the so-called aquasomes, whose particle size (lower than 1000 nm), is appropriate to parenteral administration because it prevents the obstruction into the bloodstream capillaries. Alternatively aquasomes are called as “bodies of water” [3, 4].
These three layered structure are self assembled by non-covalent bonds. Principal of “self assembly of macromolecule” is governed by three physiochemical process:
1. 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. The intrinsic chemical groups or adsorbed ions from the biological milieu lend to most biological and synthetic surfaces a charge polarity. Most biochemically relevant molecules, in fact are amphoteric. The interactions of charged groups such as amino-, carboxyl-, sulfate-, and phosphate-groups, facilitate the long range approach of self assembling subunits. The long range interaction of constituent subunits beginning at an intermolecular distance of around 15 nm, is the necessary first phase of self assembly. With hydrophobic structures, long range forces may extend up to 25 nm. Charged groups also play a role in stabilizing tertiary structures of folded proteins [1, 5].
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 [1, 5].
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 [1, 5].Conformational integrity of aquasomes exploited as a red blood cell substitutes, vaccines for delivery of viral antigen (Epstein-Barr and Immune deficiency virus) to evoke correct antibody and as targeted system for intracellular gene therapy. Enzyme activity and sensitivity towards molecular conformation made aquasome as a novel carrier for enzymes like DNAses and pigment/dyes .
Properties 13, 14, 15:
1) Aquasomes water like properties provides a platform for preserving the conformational Integrity and bio chemical stability of bio-actives.
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 due to their size and structure stability, avoid clearance by reticuloendothelial system or degradation by other environmental challenges
4) Aquasomes possess large size and active surface hence can be efficiently loaded with substantial amounts of agents through ionic, non covalent bonds, van der waals forces and entropic forces. As solid particles dispersed in aqueous environment, exhibit physical properties of colloids.
5) The drug delivery vehicle aquasome is colloidal range biodegradable nanoparticles, so that they will be more concentrated in liver and muscles. Since the drug is adsorbed on to the surface of the system without further surface modification they may not find any difficulty in receptor recognition on the active site so that the pharmacological or biological activity can be achieved immediately. In normal system, the calcium phosphate is a biodegradable ceramic. Biodegradation of ceramic in vivo is achieved essentially by monocytes and multicellular cells called osteoclasts because they intervene first at the biomaterial implantation site during inflammatory reaction. Two types of phagocytosis were reported when cells come in contact with biomaterial; either calcium phosphate crystals were taken up alone and then dissolved in the cytoplasm after disappearance of the phagosome membrane or dissolution after formation of heterophago-somes. Phagocytosis of calcium phosphate coincided with autophagy and the accumulation of residual bodies in the cell 12.
Characterization of Aquasomes:
Aquasomes are mainly characterized for structural analyses, particle size, and morphology. These are evaluated by X-ray powder diffractometery, transmission electron microscopy, and scanning electron microscopy. The morphology and the size distribution were obtained through images of scanning electron microscopy. The chemical composition and the crystalline structure of all samples were obtained through X-ray powder diffractometery. In this technique, the x-ray diffraction pattern of the sample is compared with the standard diffractogram, based on which the interpretations are made 16.
METHOD OF PREPARATION OF AQUASOMES(4, 6, 7, 8, 9):
The general procedure consists of an inorganic core formation, which will be coated with Lactose forming the polyhydroxylated core that finally will be loaded by model drug.By using the principle of self-assembly, the aquasomes are prepared in three steps i.e.,
1) Preparation of core
2) Coating of core
3) Immobilization of drug molecule.
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 magnetron sputtering, 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 polyhydroxy oligomeric surface film. The precipitated cores are centrifuged and then washed with enough distilled water to remove sodium chloride formed during the action. The precipitates are resuspended in distilled water and passed through a fine membrane, filter to collect the particles of desired size. Two ceramic cores that are most often used are diamond and calcium phosphate. The equation for the reaction is as follows;
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 (polyhydroxy oligomers) coating to adsorb epitaxially on to the surface of the nano-crystalline ceramic cores. The processes generally entail the addition of polyhydroxy oligomer 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 electron microscopy. The morphology and the size distribution were obtained through images
of scanning electron microscopy
Formulation of Aquasomes:
1. Principles of Self Assembly 10, 11:
Self assembly implies that the constituent parts of some final product assume spontaneously prescribed structural orientations in two or three dimensional space. The self assembly of macro molecules in the aqueous environment, either for the purpose of creating smart nano- structured materials or in the course of naturally occurring biochemistry, is governed basically by three physicochemical processes: the interactions of charged groups, dehydration effects and structural stability.
2. Interactions between Charged Groups:
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. The intrinsic chemical groups or adsorbed ions from the biological milieu lend to most biological and synthetic surfaces a charge polarity. Most biochemically relevant molecules, in fact are amphoteric. The interactions of charged groups such as amino-, carboxyl-, sulfate-, and phosphate-groups, facilitate the long range approach of self assembling subunits. The long range interaction of constituent subunits beginning at an intermolecular distance of around 15 nm, is the necessary first phase of self assembly. With hydrophobic structures, long range forces may extend up to 25 nm. Charged groups also play a role in stabilizing tertiary structures of folded proteins.
3. Hydrogen Bonding and Dehydration Effects:
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.
4. Structural Stability:
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. Van der Waals forces, most often experienced by the relatively hydrophobic molecular regions that are shielded from water, play a subtle but critical role in maintaining molecular conformation during self assembly. Van der Waals forces largely internal to the molecule also play a small but measurable role in the interaction of polypeptides with carbohydrates and related polyhydroxyloligomers. When molecules change their shape substantially following an interaction, the energy minima assumed upon conformational denaturation tend to preclude reversal.
1. Oxygen carrier Patil et al., made an attempt to deliver hemoglobin using colloidal ceramic carbohydrate composites termed aquasomes. This study demonstrates that the hemoglobinadsorbedaquasomes can carry the oxygen satisfactorily, and it also establishes the superiority of hemoglobin aquasomal formulation over the other methods acting as artificial blood substitute. The self-assembling surface modified nanocrystalline ceramic core capable of nondenaturing attachment can be used for various applications like delivery of bioactive molecules as well as viruses.
2. For immunotherapy Pandey et al., prepared aquasomes for delivery of model allergen without altering the antigenic and immunogenic properties of the protein/allergen. His report demonstrates that OVA adsorbed aquasomes are able to induce a strong T cell specific proliferative response with a cytokine profile suggestive of a Th1 response, prevention of anaphylactic reactions and maintenance of low titers of IgE, without abrogation of Th2-mediated responses. This suggests that aquasomes could have possible implications in the future of peptide-based vaccines against allergic disorders.
3. For oral route Kommineni et al. carried out a technological innovation for the delivery aquasomes via the peroral route. Piroxicam loaded aquasomes with their nanometric dimensions, low drug dose, and water like properties were prepared by using two techniques; namely, coprecipitation by refluxing and coprecipitation by sonication.
4. For immunopotentiation Goyal et al., 2008 prepared aquasomes that develops immune responses to recombinant or synthetic epitopes which is of considerable importance in vaccine research for immunopotentiation. Bovine serum albumin-immobilized aquasomes were around 200 nm in diameter and spherical in shape and had approximately 20–30% BSA-loading efficiency. The formulated aquasomes was compared with plain bovine serum albumin (BSA) and transport of immunogen to APCs was found to be a promising target for gene therapy. Thus, the enhanced transport of conformationally stable antigen leads to better presentation to APCs. APCs contain both MHC-I and MHC-II molecules leading to processing and presentation of antigen via both endocytic and cytosolic pathways leading to elicit both humoral and cellular responses. Aquasome formulations could elicit combined T-helper 1 (Th1) and Th2 immune response and proved to be slightly better carrier system compared with other ceramic-based antigen nanocarriers.
5. Anti thrombic activity Leclerc et al., 2003 formulated nanoparticles based on heparin-poly (isobutylcyanoacrylate) copolymers to carry hemoglobin. His work constitutes the demonstration of hemoglobin loaded on nanoparticle surface, rather than being encapsulated. Antithrombic activity of native nanoparticles was evaluated by anti-Xa factor activity assay using a coagulometer ST1. Binding of nanoparticles to von 100 Asian Journal of Pharmaceutics - April-June 2012 Narang: Aquasomes Willebrand factor (vWF) was measured. Results have shown that the heparin on the nanoparticles surface preserved most its antithrombic activity and its capacity to recognize the vWF. The bound hemoglobin also maintained its capacity to bind ligands. One ml of nanoparticles (with a size of 100 nm) suspension can be loaded with up to 2.1 mg of hemoglobin, which preserves its ligand binding capacity as well as make suitable tools in the treatment of thrombosis oxygen deprived pathologies. These nanoparticles maintain the heparin antithrombic properties and inhibit complement activation.
6. Antigen delivery The immunity can be increased by adjuvants which have a tendency either to shield the functional groups or to alter the conformation of the antigen through surface adsorption or to. So Kossovsky et al. demonstrated the efficacy of a new organically modified ceramic antigen delivery vehicle. 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 adsorption and adhesion of cellobiose. It provided a colloidal surface capable of hydrogen bonding to the proteinaceous antigen. The disaccharide, being a dehydro-protectant, helps to minimize the surfaceinduced 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.
1. Size and shape:
The morphological examination of prepared systems is performed using a transmission electron microscope following the negative staining of phosphotungstic acid solution. The mean particle size and size distribution are determined by a photon correlation spectroscopy using a Autosizer II C apparatus and SEM. Aquasomes are mainly characterized for structural analyses, particle size, and morphology. The chemical composition and the crystalline structure of all samples were obtained through X-ray powder diffractometry.
2.Glass transition temperature:
DSC studies have been extensively used to study glass transition temperature of carbohydrates and proteins. Asian Journal of Pharmaceutics - April-June 2012 99 Narang: Aquasomes glass to rubber state can be measured using a DSC analyzer as a change in temperature upon melting of glass. In-process stability studies using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) can be performed to determine the stability and integrity of protein during the formulation of the aquasomes. (heparin coated nanoparticles coupled with hemoglobin.)
OTHER EVALUATION PARAMETERS(23):
1. In vitro drug release studies:
The in vitro release kinetics of the loaded drug is determined to study the release pattern of drug from the aquasomes by incubating a known quantity of drug loaded aquasomes in a buffer of suitable pH at 37°C with continuous stirring. Samples are withdrawn periodically and centrifuged at high speed for certain lengths of time. Equal volumes of medium must be replaced after each withdrawal. The supernatants are then analyzed for the amount of drug released by any suitable method.
2. Drug loading efficiency:
This test is done to ensure the amount of drug which is bound on the surface of aquasomes. Spectrophotometric analysis of hydrophobic drugs like indomethacin and piroxicam are done by using 0.1 N methanolic hydrochloric acid solutions.
3. The Hb loading capacity:
It is estimated by the difference between the control sample (HbA solution) and the free hemoglobin contained in all fractions without nanoparticles. The spectrophotometric measurements of hemoglobin are done according to Drabkin’s method.
4. The antigen-loading efficiency for the aquasomes:
The formulation’s loading efficiency can be determined as reported in literature. Accurately weight antigen-loaded aquasome formulations were suspended in Triton X-100 and incubated in a wrist shaker for 1 h. Then, samples are centrifuged at and absorbance is determined using microBCA methods with set a blank of unloaded aquasomes formulation. Antigen loading is expressed as per unit weight of aquasomes particles (g of antigen/mg of sample).
5. Effect of cellobiose and trehalose on antigen:
DSC analysis of aquasome formulations is done by DSC analyzer having a sample cell (containing formulation) and a reference cell (filled with buffer only).
Aquasomes, the self-assembling surface-modified nanocrystalline ceramic cores, seem to have potential and promising carriers capable of preserving the structural integrity of protein pharmaceuticals and carrier for delivery of broad range of molecules including viral antigens, heamoglobin and insulin, thus promoting a better therapeutic effect. Also, these formulations have been found to evoke a better immunological response and could be used as immunoadjuvants for proteinaceous antigens. This approach thus provides pharmaceutical scientists with new hope for the delivery of bioactive molecules.
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Received on 06.06.2018 Accepted on 29.06.2018
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