2. CONTENT
Introduction
Properties
Rational Behind Development Of aquasomes
Material used and its importance
Formulation of aquasomes
Principle of self assembly
Methods of preparation
Characterization
Applications
3. Aquasomes are nanoparticulate carrier system but instead of being simple nanoparticle these are
three layered self assembled structures.
It comprised of a solid phase nanocrystalline core coated with oligomeric film on which
biochemically active molecules are adsorbed with or without modification.
Aquasomes are like “bodies of water" and 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 is exploited in targeting of bioactive molecules.
Aquasomes are the nanobiopharmaceutical carrier system contains the particle core composed
of nanocrystalline calcium phosphate or ceramic diamond, and is covered by a polyhydroxyl
ligomeric film.
Aquasomes are spherical 60-300nm particles used for drug and antigen delivery.
Introduction
4.
5. • Aquasomes water like properties provides a platform for preserving the conformational
Integrity and bio chemical stability of bio-actives.
• Aquasomes due to their size and structure stability, avoid clearance by reticuloendothelial
system or degradation by other environmental challenges.
• 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, they exhibit physical
properties of colloids
Properties
6. Rational Behind Development Of Aquasomes
There are several reasons behind the development of this novel drug delivery system comprising
natural material: some of them are described here as;
1. The careers like prodrug, macromolecules and liposomes have served to attain the intended
purpose, but they all are prone to have biological constraints. The destructive interaction between
the drug career and the drug are often present several limitations for the development of the
newer carrier system.
2. The intrinsic biophysical constraints, dehydration and conformational changes caused by the
drug delivery system can lead to adverse or allergic reactions with some adverse pharmacological
activities. By incorporation such molecules in aquasomes with natural stabilizers , one can
preserve the molecular conformation since these natural sugars act as dehydroprotactants.
3. There are several systemic biophysical and intrinsic biophysical constraints, which tend to
destabilize the drug. These can be overcome by using an natural stabilizer like sugar.
7. Material used and its importance:
• Metal nano-biomaterials are used for the preparation of aquasomes.
• For preparation of nanoparticles core both polymers and ceramic can be used.
Polymers used are albumin, gelatin or acrylates and ceramics used are diamond
particles (nano-crystalline carbon ceramic), brushite, and tin oxide core.
• Ceramic materials were widely used because ceramics are structurally the most
regular materials known, being crystalline high degree of order ensures.
8. FORMULATION OFAQUASOMES:
A. PRINCIPLE OF SELFASSEMBLY
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 macromolecules in the aqueous environment for the purpose of
creating smart nanostructure materials is governed basically by three physicochemical
processes:
1. The interactions of charged groups
2. Hydrogen bonding
3. structural stability
24
9. 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 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.
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.
10. • The method of preparation of aquasomes involves three steps.
• The general procedure consists of Formation of an inorganic core, followed by Coating of the
core with polyhydroxy oligomer, and finally loading of the drug of choice to this assembly.
B. METHOD OF FORMATION OF AQUASOMES
11. I- FORMATION OF AN INORGANIC CORE
It involves the fabrication of a ceramic core, and the procedure depends upon the
materials selected. The two most commonly used ceramic cores are calcium phosphate
and diamond.
a)Synthesis of nanocrystalline tin oxide core ceramic - It can be synthesized by direct
current reactive magnetron sputtering.
b)Self assembled nanocrystalline brushite (calcium phosphate dihydrate) - These can
be prepared by colloidal precipitation and sonication by reacting solution of disodium
hydrogen phosphate and calcium chloride.
c)Nanocrystalline carbon ceramic, diamond particles - These can also be used for the
core synthesis after ultra cleansing and sonication.
26
12. II- COATING OF THE CORE WITH POLYHYDROXY OLIGOMER
• In the second step, ceramic cores are coated with carbohydrate (polyhydroxyl
oligomer).
• The coating is carried out by addition of carbohydrate into an aqueous dispersion of
the cores under sonication.
• These are then subjected to lyophilization to promote an irreversible adsorption of
carbohydrate onto the ceramic surface.
• The unadsorbed carbohydrate is removed by centrifugation.
• The commonly used coating materials are cellobiose, citrate,pyridoxal-5- phosphate,
trehalose and sucrose.
27
13. III- LOADING OF THE DRUG OF CHOICE TO THIS ASSEMBLY
• The final stage involves the loading of drug to the coated particles by adsorption.
• For that, a solution of known concentration of drug is prepared in suitable pH
buffer, and coated particles are dispersed into it.
• The dispersion is then either kept overnight at low temperature for drug loading or
lyophilized after some time so as to obtain the drug-loaded formulation (i.e.,
aquasomes).
28
14. CHARACTERIZATION
• Size distribution
• Structural analysis
• Crystallinity
• Glass transition temperature
• In vitro drug release studies
• Entrapment efficiency
• Zeta potential
• Polydispersibility index
• Optical microscopy
29
15. 1. Size distribution
• For morphological analysis & particle size distribution study scanning electron microscopy
(SEM) & transmission electron microscopy (TEM) techniques are used. In SEM, for
determination of particle size, samples were placed on the surface of a specimen stub coated
with gold using a double-sided adhesive tape. In case of TEM, particle size is determined
after negative staining with phosphotungstic acid.
2. Structural analysis
• Structural analysis is done by Fourier transform infrared spectroscopy in the wave number
range of 400–4000 cm−1. KBr (Potassium bromide) pellet method is used. Ceramic core,
carbohydrate coated core, drug loaded formulation, and drug are analyzed by this method.
Determination of stability of drug in formulation can be determined by FTIR analysis.
16. 3. Crystallinity
• X-ray diffraction study is done to study crystalline or amorphous nature of a
material. Here diffraction study of ceramic core, carbohydrate and drug loaded
aquasomes are done & compared . In the study by Rojas-Oviedo et al., 2013 [24],
it was observed that calcium phosphate core, lactose individually gave identical
sharp peaks for crystalline peaks but when X-ray diffraction results of
carbohydrate coated cores were observed, it was seen that peaks represented an
amorphous structure. This may be the reason of the coating technique
(solubilization of carbohydrate in solvent and subsequent drying by
lyophilization) and saturation of the surface of core with carbohydrate.
4. Glass transition temperature
• Differential scanning colorimetry is used to study glass transition temperature of
carbohydrates & proteins. In the research work by Goyal et al., 2008 [58] results
of formulation loaded with BSA, model protein revealed that formulations were
stable at room temperature. This stability is provided by a synergistic effect by
hydroxyapatite and carbohydrate.
17. 5. In-vitro drug release study
• In-vitro dissolution study is done in buffer media of suitable pH at 37 °C with constant stirring.
Sample is withdrawn time to time with replacement of similar volume of buffer. Samples
withdrawn are centrifuged at high speed. After that supernatant is collected & analyzed to
determine the amount of released
6. Drug loading efficiency
• The drug loading efficiency can be determined by incubating the aquasome formulations
without drug in a known concentration of the drug solution for 24 h at 4 °C. After 24 h, the
supernatant liquid is separated by high-speed centrifugation for 1 h at low temperature in a
refrigerated centrifuge. After centrifugation, the supernatant liquid is collected. The drug
remaining in the supernatant liquid after loading is then estimated by suitable method of
analysis like by measuring absorbance in UV spectrophotometer.
7. Zeta potential measurement
• Zeta potential is a measure of the electrostatic attraction or repulsion between particles.
Electrochemical equilibrium is observed and analyzed to understand the stability of a
formulation. It is best known as the stability indicator of suspension, dispersion or emulsion.
Adsorption of sugar can also be identified by measuring zeta potential. It was observed that
with the increase in saturation process by carbohydrate on to the hydroxyapatite core, the more
decrease in zeta potential value.
18. 1) Aquasomes used as vaccines for delivery of viral antigen i.e., Epstein-Barr and
Immune deficiency virus 31 to evoke correct antibody, objective of vaccine
therapy must be triggered by conformationally specific target molecules.
2) 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 4.
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.
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 32.
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.
APPLICATIONS
30
