INTRODUCTION:

Aquasomes are nanoparticulate carrier systems, but instead of being simple nanoparticles, they are three layered self-assembled structures consisting of a solid phase nanocrystalline core covered with oligomeric film on which biochemically active molecules are adsorbed with or without alteration¹. Specific technological strategies have been proposed in the last decade to obtain nanoparticles of a distinct type, charged with drugs that have in turn revolutionized drug administration systems, especially those of controlled release and those oriented to vectoring the active principle of release in target tissue or organs².

Aquasomes are spherical particles (5–925 nm) used for the delivery of drugs and antigens. n Different methods used in the preparation of nanoparticles use polymers and encounter difficulties such as the compatibility of solvents and other components and polymers and copolymers with the active principle and biological fluids and factors of the collection system Kossovsky proposed a system for the preparation of nanoparticles conveying the so-called aquasomes whose particle size (less than 1000 nm)³. They consist of a ceramic core, the surface of which is non-covalently modified with carbohydrates to obtain a sugar ball, which is then exposed to therapeutic agent adsorption. The center provides a nearly permanent solid with structural stability. Aquasomes provide an enticing delivery mode for therapeutic agents belonging to the protein and peptide class, as they can solve some of the inherent problems associated with these molecules. Aquasomes maintains optimum pharmacological activity and molecular confirmations. Active molecules normally possess qualities such as unique three-dimensional conformation, freedom of internal molecular rearrangement induced by molecular interactions and freedom of bulk movement but proteins undergo irreversible denaturation when desiccated, even unstable in aqueous condition. Temperature, solvents, salts in the aqueous state pH cause denaturation hence several biophysical constraints on bio-active faces. In these situations, aquasomes with natural stabilizers such as various polyhydroxy sugars serve as dehydroprotectants preserve water like state while maintaining dry solid molecules⁴. Self-assembly is appealing as an approach to macromolecular synthesis, because biomimetic processes involve more biochemically functional products. This review article focuses on the concepts of self-assembly, the complexities of preserving both immobilized surface pairs' conformational integrity and biochemical operation, and the integration of these principles into a single functional composition⁵.

Properties of aquasomes:

Aquasomes have a large size and an active surface and can therefore be loaded efficiently with substantial amounts of agents through ionic, non-co-valent bonds, van der waals and entropic forces. The physical properties of colloids show as solid particles distributed in aqueous environment⁶.

i. The mechanism of action of the aquasomes is regulated by their surface chemistry. These deliver content by combining specific targeting, molecular shielding, and slow and continuous release processes.

ii. Water-like properties of aquasomes provide a forum for maintaining bioactive conformational integrity and biochemical stability.

iii. Because of their size and structural stability, aquasomes avoid clearance through reticuloendothelial system or degradation by other environmental challenges⁷.

Material used and its importance:

Polymers and ceramics may be used to make core nanoparticles. The polymers used are albumin, gelatin or acrylates, and the diamond fragments, brushite and tin oxide core are ceramics used in aquasome preparations.

Composition of aquasomes:

i. Core material:

Ceramics and polymers are the core materials that are used the most. Polymers are used, such as albumin, gelatin, or acrylate. Ceramics are used such as diamond flakes, brushite (calcium phosphate), and tin oxide⁸.

ii. Coating material:

Commonly used coating materials are cellobiosis, pyridoxal 5 phosphate, sucrose, trehalose, chitosan, citrate and so on. As a natural stabilizer, carbohydrate plays an important role, its stabilizing efficiency has been documented. Beginning with preformed carbon ceramic nanoparticle and self assembled calcium phosphate dihydrate particles (colloidal precipitation) to which glassy carbohydrate are then allowed to adsorb as a nanometer thick surface coating a molecular carrier is formed⁹.

Principle of self assembly:

Self-assembly means that in two or three dimensional space, the constituent parts of any final product assume spontaneously defined structural orientations. The self-assembly of macromolecules in the aqueous environment, either to create smart nanostructure materials or in the course of naturally occurring biochemistry, is essentially governed by three physicochemical processes: charged group interactions, dehydration effects, and structural stability¹⁰.

i. Interaction between charged groups:

Charged group interaction, such as amino, carboxyl, sulphate, phosphate groups, facilitates the long-range approach of self-assembly sub-units. Charged group also plays a part in the stabilisation of folded tertiary. structures proteins.

ii. Hydrogen bonding and dehydration effect:

Hydrogen bond helps match and stabilize secondary protein structures like alpha helices and beta sheets in base pairs. Molecules forming hydrogen bonds are hydrophilic and this gives surrounding water molecules a considerable degree of organization. For hydrophobic molecules which can not form hydrogen bond. Their propensity to repel water, however, helps coordinate the moiety to surrounding area. The ordered water reduces the overall degree of surrounding medium's disorder/entropy. Since structured water is thermodynamically unfavorable, loose water/dehydrate from the molecule and get self-assembled¹¹.

iii. Structural stability:

There is a dipole moment in the molecules that carry less charge than formally charged groups. The dipole-associated powers are known as the van der waals. Protein structural stability in biological environment determined by the interaction of charged group and hydrogen bonds largely external to the molecule, and by van der waals largely internal molecule forces. The forces of the Vander Waals, most commonly encountered by molecular hydrophobic regions shielded from water, play a subtle but essential role in preserving molecular shape or conformation during self-assembling. The forces of the van der waals are largely responsible for molecular hardness or softness. The interaction of van der waals between hydrophobic side chain promotes the stability of compact helical structures that are thermodynamically unfavorable to expanded random coils. It is the maintenance of internal secondary structures, such as helices, which provides sufficient softness, and allows conformation to be maintained during self-assembly, small changes are required for successful antigen-antibody interactions. This can lead to altered molecular function and biological activity within biotechnological self-assembly. Therefore, to preserve maximum biological function, the van der waals must be buffered. In aquasomes, sugars help in the plasticisation of molecules¹².

Method of preparation of aquasomes:

The method of preparation of aquasomes involves three steps:

i. Formation of an inorganic core:

Calcium phosphate and diamond are the two most commonly used ceramic cores. This method involves the making of a ceramic core, and the process depends on the selected materials¹³.

ii. Synthesis of nanocrystalline tin oxide core ceramic:

It can be synthesized with direct current reactive magnetron sputtering. In a high-pressure gas mixture of orgon and oxygen, a 3-inch-diameter target of high-purity tin is sputtered. The ultrafine particles formed during the gas phase are then collected on copper tubes cooled to 770 K with flowing nitrogen.

iii. Self assembled nanocrystalline brushite (calcium phosphate dihydrate):

These can be prepared by colloidal precipitation and sonication by the reaction of disodium hydrogen phosphate and calcium chloride. Nano-crystalline carbon ceramic, diamant particles Nano-crystalline carbon ceramic, diamant particles can also be used for core synthesis after ultra-cleaning and sonication. The main feature of the different cores is that they are crystalline. When introduced into synthetic processes, they measure between 50-150 nm and exhibit an extremely clean spectrum and are therefore reactive species. Ceramic materials are structurally highly regular and are thus mostly used for core manufacturing. The high degree of order in crystalline ceramics ensures only a small effect on the nature of atoms below the surface layer when any surface modification is carried out, thus maintaining the bulk properties of ceramics. This high degree of order also provides a high surface energy level, which favors the binding of pohydroxyl oligomeric surface film. The precipitated cores are centrifuged during the reaction, and then washed with sufficient distilled water to remove formed sodium chloride. The precipitates are resuspended in distilled water to collect the particles of desired size, and passed through a fine membrane filter¹⁴.

Characterization of aquasomes:

They are characterized for the structural and morphological properties, particle size distribution and drug loading capacity.

i. Size distribution:

Scanning electron microscopy and transmission electron microscopy can characterize the morphological properties and the distribution of particle size. The particle-photon correlation spectroscopy is used for the measurement of man particle size and zeta potential¹⁴.

ii. Structural analysis:

FT-IR Spectroscopy is used for structural analysis. Using the FT-IR Potassium bromide sample disk method, the core as well as the coated core are analyzed by recording their IR spectra in the 4000-400 cm^-1 wave number range.

iii. Crystallinity:

X –ray diffraction is used to determine crystalline or amorphous behavior of ceramic core.

Role of disaccharides in aquasomes:

Disaccharides like trehalose are seen in fungi, bacteria, insects, yeast and some plants as having stress tolerance. Trehalose has an action function by preserving proteins and membranes in plant cells during the process of desiccation and thus retains cell structures, natural tastes, colors and textures. The carbohydrate hydroxyl group interacts with polar and charged protein groups similarly to water molecules alone and retains the protein's aqueous structure after dehydration. These disaccharides contain a large amount of hydroxyl group and help replace the water in proteins around polar residues, thereby maintaining their integrity in the absence of water. The studies indicated that during lyophilization the structure and function of cellular components could be protected by sugar, with Catransporting microsomes isolated from the muscles of the rabbits and lobster. The rehydrated vesicles show significantly reduced Ca-uptake and uncoupling of ATPase activity while lyophilizing Ca carrying microsomes without stabilizing sugar; Vesicles were lyophilized with as little as 0.3 g in presence. of trehalosis. Membrane is morphologically distinguishable from freshly prepared vesicles upon rehydration. Among three aquasomal layers, carbohydrate meets aquasomal targets. The hydroxyl groups on oligomer interact with polar and charged protein groups, thus preserving the aqueous protein structure upon dehydration, in the same way as with water¹⁶.

Applications:

i. Insulin Delivery:

Cherian et al. prepared aquasomes for the parenteral delivery of insulin using a ceramic calcium phosphate core. Various disaccharides such as cellobiose, trehalose, and pyridoxal-5phosphate coated the core. Subsequently the drug was loaded by adsorption method to these particles. The in vivo performance of different insulin formulations of aquasomes was assessed using albino rats. All formulations except cellobiose-coated particles have been observed for prolonged reduction of blood glucose. It has been found that pyridoxal- 5 phosphate-coated particles are more effective in reducing blood glucose levels than trehalose or cellobiose coated aquasomes. This could be due to the high degree of pyridoxal5-phosphate molecular preservation. The prolonged activity has been due to the gradual release of drugs from the carrier and the peptide's structural integrity. Accordingly, the authors proposed aquasomes as a promising carrier for the delivery of therapeutic protein and peptide drugs.14 Paul and Sharma also proved the usefulness of nanocarriers for effective insulin delivery. They prepared porous nanoparticles of hydroxyapatite trapped in matrix of alginates containing insulin for oral administration. In this study the optimum regulated insulin release was also achieved¹⁷.

ii. Oral Delivery of Enzyme:

Rawat et al proposed oral administration of the acid-labile enzyme serratiopeptidase using a nanosized ceramic core-based method. The nanocore was prepared at room temperature by colloidal precipitation under a sonication. The core then under constant stirring was coated with chitosan, after which the enzyme was adsorbed over it. The enzyme has been protected by further encapsulation of the core charged with the enzyme into alginate gel. TEM images showed that spherical shape, having an average diameter of 925 nm. Particulate enzyme-loading efficiency was found to be about 46 per cent. The data for the release of in vitro drugs followed the Higuchi model in acidic medium (pH 1.2) for up to 2 to 6 hours, whereas the alkaline medium (pH 7.4) showed a sustained and almost complete first-order release of enzyme for up to 6 hours. These aquasomes have been found to protect enzyme structural integrity so as to achieve a better therapeutic effect¹⁸.

iii. As Oxygen Carrier:

Khopade et al used carboxylic acid – terminated half-generation poly(amidoamine) dendrimers as templates or crystal alterers to prepare the hydroxyapatite center. Those cores were further coated with trehalose followed by hemoglobin adsorption. The particle size was found to be within the nanometer range, and the load capacity was found to be around 13.7 mg of hemoglobin per gram of the core. The aquasomes' oxygen-binding properties have been studied and compared with those of fresh blood and hemoglobin solution. Hill coefficient values for fresh blood, hemoglobin solution, and aquasome formulation indicated that aquasomes retained the properties of hemoglobin including its oxygen capacity. Studies in rats have shown that aquasomes possess excellent potential for use as a carrier of oxygen. The formulation has also been found to retain its oxygen-binding characteristics over a 30-day period. In another study Patil and his coworkers prepared ceramic hydroxyapatite cores by means of coprecipitation and self-precipitation. These cores were coated with different sugars including cellobiosis, trehalosis, maltose, and sucrose. Hemoglobin was subsequently adsorbed over the coated ceramic core, and the benzidine method estimated the percentage of drug loading. Aquasome formulation 's oxygen carrying capacity was found to be similar to that of fresh blood. The Hill coefficients have also been found to be perfect for its use as a carrier of oxygen. The aquasome formulations did not induce red blood cell hemolysis nor alter the time of coagulation in the blood. The loading of hemoglobin to different sugar-coated particles was found to be about 7.4%. The formulation had been able to hold the hemoglobin for 30 days. No significant increase in arterial blood pressure and heart rate was observed on 50 % exchange transfusion in rats transfused with aquasome suspension¹⁹.

iv. Antigen Delivery:

The adjuvants generally used to enhance antigen immunity tend to either alter the antigen's conformation through surface adsorption or shield the functional groups. The efficacy of a new organically modified ceramic antigen delivery vehicle was thus demonstrated by Kossovsky et al. Such particles composed of diamond substrates, which were coated in an aqueous dispersion with a glassy carbohydrate (cellobiose) film and an immunologically active surface molecule. These aquasomes (5–300 nm) provided conformational stabilisation as well as a high degree of protein-antigen exposure to the surface. Diamond, being a high surface energy material, was the first choice for cellobiose adsorption and adhesion. It provided a colloidal surface which was capable of bonding hydrogen to the protein antigen. As a dehydroprotective, the disaccharide helps to reduce the surface-induced denaturation of adsorbed antigens (muscle-adhesive protein, MAP). Conventional adjuvants had only proven marginally successful in evoking an immune response for MAP. However, a powerful and precise immune response could be elicited with the aid of these aquasomes by enhancing antigen 's availability and in vivo activity. Using coprecipitation process, Vyas et al prepared aquasomes by self-assembling hydroxyapatite. The core was coated with cellobiose and trehalose, and ultimately bovine serum albumin was adsorbed onto the coated core as a model antigen. The aquasomes were found to be spherical in shape, about 200 nm in diameter. Both concanavalin A – induced aggregation assay method and IR spectroscopy confirmed the coating of carbohydrate over the surface of the core. The efficiency of antigen loading was found to be around 20–30 %. When compared with plain bovine serum albumin, the former was found to show a better response when the immunological activity of the prepared formulation. In view of these results, it was suggested that aquasomes have superior surface immutability, in that they protect the protein structure conformation and present it to immune cells in such a way that it triggers a better immunological response²⁰. Vyas et al. suggested the use of ceramic core-based nanodecoy systems as an adjuvant and delivery mechanism for successful immunization of the hepatitis B vaccine. The self-assembling hydroxyapatite core was coated with cellobiose, and the surface antigen of hepatitis B was finally adsorbed over the coated core. The particles charged with drugs were in the range of nanometers and almost spherical in shape. The antigen-loading efficiency of the plain hydroxyapatite core (without cellobiose coating) was found to be about 50%, while the coated core was found to load about 21% antigen. In Balb / c mice, the formulation was found to be stronger than the traditional adjuvant alum accompanied by subcutaneous immunisation. It was also found that the nanodecoy systems would evoke a combined immune response to Th1 and Th2. Vyas et al demonstrated the immunoadjuvant properties of hydroxyapatite by administering protein-119 (MSP-119) with a malarial merozoite surface. Co-precipitation had prepared the hydroxyapatite nanoceramic carrier. Prepared systems were characterized for their effectiveness in crystallinity, size, shape and antigen loading. Small size and wide surface area of prepared hydroxyapatite showed good immunogenic adsorption efficiency. Prepared nanoceramic formulations have also shown a slower release of in vitro antigen and a slower biodegradability behavior, which can lead to prolonged exposure to cells and lymphocytes that present antigen. Additionally, mannose supplementation in nanoceramic formulation may lead to increased stability and immunological reactions. In nanoceramic adjuvant systems, immunization with MSP-119 induced a vigorous IgG response, with higher IgG2a than IgG1 titres. Furthermore, in spleen cells of mice immunized with nanoceramic-based vaccines, significant amounts of interferon π (IFNÿ) and interleukin2 were observed. By comparison, mice immunized either alone with MSP-119 or with alum did not show any major cytotoxic response. The antibody response to the hydroxyapatite-co-administered vaccine was a mixed Th1-Th2 compared to the alum-based Th2 response. The prepared hydroxyapatite nanoparticles exhibit physicochemical properties which point to their potential as an appropriate immunoadjuvant for immuno-potentiation use as antigen carriers. He et al. compared a new calcium phosphate nanoparticulate adjuvant with widely used aluminum (alum) adjuvants for its ability to cause immunity to herpes simplex virus type 2 and Epstein-Barr virus infections. At the site of administration, calcium phosphate was found to cause little to no inflammation, induced high titers of immunoglobulin G2a (IgG2a) antibody and neutralized antibody, and promoted a high percentage of protection against herpes simplex virus type 2 infections. Calcium phosphate has thus proved a more potent adjuvant than alum. In addition, being a natural constituent of the body, the animal studies found it to be very well tolerated and absorbed. These studies have recommended it as an adjuvant for human use because of the potency and relative absence of any side effects of calcium phosphate.

Drug delivery systems applications in allergen-specific immunotherapy tend to be a promising technique due to their ability to act as adjuvants, to carry allergens to immune-competent cells and tissues, and to reduce the amount of administration. The aim of this work was to evaluate the carbohydrate-modified ultrafine ceramic core based nanoparticles (aquasomes) as an adjuvant/delivery vehicle using ovalbumin (OVA) as an allergen model in specific immunotherapy. Prepared nanoparticles were characterized by size, shape, zetapotential, integrity of the antigen, effectiveness of surface adsorption and in vitro release. Two intradermal immunizations in BALB / c mice were studied for the humoral and cell-induced immune responses generated by OVA adsorbed aquasomes. Following proven procedure, OVA sensitized mice were treated with OVA adsorbed aquasomes, and OVA adsorbed aluminum hydroxide. Animals were challenged with OVA fifteen days after therapy, and various signs of anaphylactic shock were assessed. Developed aquasomes had a negative zeta potential (−11.3 mV) and an average size of 47 nm, with hydroxyapatite core OVA adsorption efficiency of ~60.2 μg mg−1. After two intradermal injections of OVA adsorbed aquasomes, the in vivo immune response resulted in a mixed immune response of the form Th1/Th2. OVA-sensitized mice model, treatment with OVA adsorbed aquasomes resulted in lower levels of IgE (pb0.05), serum histamine and a higher rate of survival compared to OVA adsorbed by alum. Symptoms of anaphylactic shock in aquasometrated OVA mice were weaker than that induced in the group of OVA adsorbed by the alum. Results from this study show valuable use of aquasomes in immunotherapy for allergens²¹.

V. Miscellaneous:

Mizushima and co-workers prepared spherical porous hydroxyapatite particles by spray-drying. These particles were also tested as a carrier for drug delivery such as IFNα, testosterone enanthate, and cyclosporine The in vivo release of testosterone enanthate and cyclosporine A from oil preparation was also prolonged. Thus it has been shown that the spherical porous hydroxyapatite particles are useful as a biodegradable and subcutaneously injectable drug carrier. It was proposed that the strengthening of spherical porous hydroxyapatite particles would be very useful for the controlled release of drugs²².

CONCLUSION:

Aquasomes are round particles made of calcium phosphate or ceramic diamond coated with a polyhydroxyloligomeric film and act as a nanoparticulate carrier system but, despite being simple nanoparticles, they are three layers of self-assembled structures consisting of a solid phase nanocrystalline core covered with oligomeric film on which biochemically active molecules are adsorbed with or without oligomeric film. Also in the case of conformationally sensitive drug candidates we can see stronger biological activity because of the nature of the ceramic's special carbohydrate coating. This strategy can lead to the novel delivery of other bioactive molecules in a beneficial way. The molecular plasticizer, carbohydrate, prevents the destructive interaction between drug carriers and helps preserve the spatial qualities. Crystalline nature of the core controls structural stability and overall integrity. We can say that aquasomes can be used to deliver a wide range of molecules including viral antigens, hemoglobin and insulin as a potential carrier.

REFERENCES:

1. Wani S, Yerawar A. Aquasomes: A novel nanocarrier for drug delivery. Chem Inform. 2011; 42(46): 58-64.

2. Gholap AD, Borude SS, Mahajan AM, Gholap MAD. Aquasomes: A potential drug delivery carrier. Pharmacol Online. 2011; 3: 230-237.

3. Sirikonda AK, Kumar BV, Soma Sekhar A, Gopi M, Babu HS, Rao GS. Aquasomes: A Review. Chem Inform. 2014; 45(14): 25-29.

4. Rathore P, Duggal S, Swami G. Aquasomes: a promising nanobiopharmaceutical drug delivery system for proteins and peptides. Chem Inform. 2013; 44(17): 18-25.

5. Mesariya S, Joshi K, Jain H, Upadhyay U. Aquasomes—A Self‐Assembled Nanotechnology System. Chem Inform. 2012; 43(30): 69-72.

6. Prausnitz MR. Microneedles for Transdermal Drug Delivery. Adv Drug Deliver. Rev. 2004; 56: 581-587.

7. Pillai O, Nair V, Panchagnula R, Transdermal Iontophoresis of Insulin: Influence of Chemical Enhancers. Int J Pharm. 2004; 269: 109-120.

8. Paul W, Sharma CP. Porous Hydroxyapatite Nanoparticles for Intestinal Delivery of Insulin. Trend Biomat Ar Org. 2001; 14: 37-38.

9. Rawat M, Singh D, Saraf S, Saraf S. Development and In vitro Evaluation of Alginate Gel-Encapsulated, ChitosanCoated Ceramic Nanocores for Oral Delivery of Enzyme. Drug Develop Ind Pharm. 2008; 34: 181-188.

10. Patil S, Pancholli SS, Agrawal S, Agrawal GP. Surface-modified Mesoporous Ceramics as Delivery Vehicle for Haemoglobin. Drug Del. 2004, 11, 193-199.

11. Kossovsky N, Gelman A, Hnatyszyn HJ, Rajguru S, Garrell LR, Torbati S. Surface Modified Diamond Nanoparticles as Antigen Delivery Vehicles. Bioconj Chem. 1995; 6: 507-510.

12. Crowe J. H., Crowe L.M. and Chapman D. Infrared spectroscopic studies on interactions of water and carbohydrate with a biological membrane. Arch. Biochem. Biophys. 1984; 232: 400.

13. Haberland, M.E, Fless, G.M.; Scannu, A.M. and Fogel man, A.M. Malondiaalde hyde de modification of lipoprotein produces avid uptake by human monocytes macrophages. J. boil. chem, 1992; 267: 4143-4159.

14. Dunitz, J.D. The entropic cost of bound water in crystals. biomol sci. 1994; 264-670.

15. Horbett, T.A.; Brash, J.L. proteins at interface; current issues and future prospects in; Proteins at interfaces physiochemical and biological studies ACS Symposium Series. washington: Acs, 1987; 343: 1-33.

16. Israelachvilli, J. N. Intermolecular and surface force New York. Academic press.1985. 15.

17. Cherian, A. and Jain S.K. “Self assembled carbohydrate stabilized ceramic nanoparticles for the parentral drug delivery of insulin” Drug development and industrial pharmacy 2000; 26: 459-463.

18. Jain NK, Umamaheshwari RB. Control and novel drug delivery systems. In: Jain NK, editor. Pharmaceutical product development. CBS Publishers & Distributors, New Delhi. 2006: 419-455.

19. Vyas SP, Goyal AK, Rawat A, Mahor S, Gupta PN, Khatri K, Nanodecoy system: a novel approach to design hepatitis B vaccine for immune potentiation. Int J Pharm 2006; 309: 227-233.

20. Vyas SP, Goyal AK, Khatri K, Mishra N, Mehta A, Vaidya B, et al. Aquasomes-a nanoparticulate approach for the delivery of antigen. Drug Dev Ind Pharm 2008; 34: 1297-1305.

21. Cherian, A. and Jain S.K. Self assembled carbohydrate stabilized ceramic nanoparticles for the parentral drug delivery of insulin. 2000; 15: 459-463.

22. Kossovsky, N; Gelman.A. and Sponsler, E.E. Cross linking encapsulated haemoglobin solid phase supports: lipid enveloped haemoglobin adsorbed to surfacemodified ceramic particles exhibit physiological oxygen lability artif. cells blood sub biotech. 1993; 223: 479-485.

Received on 15.06.2020 Accepted on 24.07.2020

Research J. Topical and Cosmetic Sci. 2020; 11(2):89-94.

DOI: 10.5958/2321-5844.2020.00016.3