Aquasomes are novel formulation used as carriers in drug delivery of various protein and Antigen Delivery

1. PRESENTED BY: JADEJA RUTURAJSINH M.
GUIDED BY: DR. HETAL P. THAKKAR
PHARMACEUTICS (MPHARM-1) ROLL NO-26
FACULTY OF PHARMACY, THE MAHARAJA SAYAJIRAO UNIVERSITY OF BARODA 1

2. Today’s Topics
•Introduction
•Properties
•Methods of Preparation
•Evaluation
•Advantages
•Limitations
•Application
•Recent Developments
FACULTY OF PHARMACY, THE MAHARAJA SAYAJIRAO UNIVERSITY OF BARODA 2

3. Introduction
•Definition : "nanoparticulate carrier system with three layered self
assembled structures comprising of central solid nanocrystalline
core coated with polyhydroxy oligomers onto which biochemically
active molecules are adsorbed".
•First discovered by Nir Kossovsky in 1995.
•Aquasomes are spherical in shape with 60-300 nm (less than
1000nm) particles size.
•These are nanoparticulate carrier systems and are three layered
self assemble structures.
•These structures are self assembled by non covalent and ionic
bonds.
•These have been studied as carriers for bioactive molecules, mainly
proteins and peptides, as the carbohydrate coating protects them
against dehydration and stabilizes them.
•The delivery system has been successfully utilized for the delivery of
insulin, hemoglobin, and enzymes like serratiopeptidase etc…
FACULTY OF PHARMACY, THE MAHARAJA SAYAJIRAO UNIVERSITY OF BARODA 3

4. FACULTY OF PHARMACY, THE MAHARAJA SAYAJIRAO UNIVERSITY OF BARODA 4

5. Characteristics
•Water like properties.
•Aquasomes distinguish themselves from any other nanoparticulate systems,
in their conformation and the water-absorbent characteristic,
•Which allows them to transport across aqueous membranes and permits them to make covalent
bonding with different molecules and macromolecules.
• (standing them apart from liposomes because aquasomes are inorganic cores coated with polyhydroxyl
compounds which are accountable for hydrophilic nature)
•Protect and preserve fragile biological molecules.
•Maintain conformational integrity as well as high degree of surface exposure in targeting of bio-active
molecules.
•High degree of surface exposure is exploited in targeting of bioactive molecules like peptide and
protein hormones, enzymes, antigens and genes to specific sites.
•These three layered structures are self-assembled by non covalent and ionic bonds.
•The pharmacologically active molecule incorporated by co- polymerization, diffusion or adsorption to
carbohydrate surface of preformed nanoparticles.
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6. Objective
•Aquasomes protect bio-actives.
•Carriers like prodrugs and liposomes utilized but these are prone to destructive interactions
between drug and carrier.
•Worthy carrier: solid carriers whose film has been treated with a film of carbohydrate to
prevent destructive denaturing interaction between drug and solid carriers.
•Maintains molecular confirmation and optimum pharmacological activity.
•Biological molecules like proteins undergo irreversible denaturation and become non functional
when desiccated (Dried).
•natural sugar acts as dehydroprotectant.
•Sugars and polyols stabilize protein against heat denaturation and stabilization
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7. Objective
•hydroxyl group on carbohydrate interacts with polar and charged groups of biological molecules
in a manner similar to water molecules alone, Preserves the aqueous structure of biological
molecules like protein on dehydration.
•disaccharides are rich in hydroxyl groups and help to replace water around the polar residues in
proteins, thus maintaining their integrity in the absence of water.
•aquasomes with natural stabilizers like various polyhydroxy sugars act as dehydroprotectant,
maintains water like state and thereby helps to preserves the molecular conformation of
bioactive molecules in dry solid state.
•Fungal spores producing ergot alkaloids were stabilized by sucrose rich solution. Desiccation
induced molecular denaturation is reported to be prevented by certain disaccharides (Crowe et
al., 1988).
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8. Historical perspective of the
development of aquasome nanoparticles
Bhasmas and Kushtas made up of materials like calcium salts and/ or diamond – Ceramic Cores.
Quality control tests of Bhasmas include rekhapurnatvam, which indicates that all the particles
enter into the fine lines of the fingers and are not washed off easily - The particle size of the
bhasma particles has been reported to be in the range of 50 nm to a few micrometers
The characteristics of Apunarbhavtva (permanence) and Niruthatva (irreversiblity) of bhasmas
also indicate the immutability of the characters associated with the ceramic nanoparticles.
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9. Properties
•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.
•Mechanism controlled by their surface chemistry.
•Deliver their contents through a combination of specific targeting, molecular shielding and slow
sustained release.
•Preserves the conformational integrity of bioactive substances.
•Avoid clearance by reticuloendothelial system or degradation by other environmental
challenges.
•Protects the drug from harsh pH conditions & enzymatic degradation.
•calcium phosphate Biodegradation can be achieved by monocytes and osteoclasts.
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10. Properties
•Aquasome is colloidal range biodegradable nanoparticles, more concentrated in liver and
muscles. drug is absorbed on to the surface of the system without further surface modification
as in case of insulin and antigen delivery,
•They may not find any difficulty in receptor recognition on the active site so that the
pharmocological or biological activity can be achieved immediately
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11. Steps For Preparation
•Simple and straight forward approach with minimum solvent usage.
•No homogenization steps.
•3 steps of preparation by using the principle of assembly
1. Preparation of the core
i. Co precipitation
ii. Self-Precipitation
iii. Sonication
iv. PAMAM
2. Carbohydrate coating of core
3. Immobilization of drug molecule
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12. Preparation of the core
•This stage mainly depends on the selection of material for core,
- Its physical chemical properties.
•For the core material material ceramic material
•widely used as they are structurally to be known.
•Commonly used ceramic core are tin oxide, and calcium phosphate.
•Variety of techniques including- plasma condensation, reversed magnetron sputtering, colloidal
precipitation, and sonication.
1. Synthesis of Nanocrystalline Tin Oxide Core Ceramic
2. Self-assembled Nanocrystalline Brushite (Calcium Phosphate Dehydrate)
3. Nanocrystalline Carbon Ceramic, Diamond Particles
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13. Preparation of the core
1. Synthesis of Nanocrystalline Tin Oxide Core Ceramic
2. Self-assembled Nanocrystalline Brushite (Calcium Phosphate Dehydrate)
•colloidal precipitation and sonication - by reacting solution of disodium hydrogen phosphate and
calcium chloride.
•2Na2HPO4 + 3CaCl2 + H2O → Ca3(PO4)2 + 4NaCl+ 2H2 + Cl2 + (O)
3. Nanocrystalline Carbon Ceramic, Diamond Particles
3 inch diameter target of high purity tin is
sputtered in a high pressure gas mixture of
organ and oxygen.
The ultra fine particles formed in the gas
phase are then collected on copper tubes
cool to 70°k with flowing nitrogen.
Precipitated core
are centrifuged
Washed with Dis.
Water to remove
NaCl
PPt’s Resuspended
in Dis Water
Passes Through a
Tiny membrane Filter
Collection of particles
of desired size
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14. Co-precipitation
•Diammonium hydrogen phosphate (0.19 N) solution drop wise
added to calcium nitrate solution (0.32 M).
•pH maintained at 8-10 by addition of concentrated aqueous ammonia solution
with continuous stirring at 75 °C
in a three necked flask containing a
one charge funnel, a thermometer, and a reflux con-denserfitted with a CO2trap
•Stirr magnetically for 4-6 days at 75 °C & pH 8-10.
•Then, filter the precipitate, washed & dried overnight at 100 °C
followed by sintering to 800-900 °C.
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15. Self-Precipitation
•Adjust simulated body fluid containing sodium chloride (134.8 mM), potassium chloride (5.0
mM), sodium hydrogen carbonate (4.2 mM), calcium chloride (2.5 mM), disodium hydrogen
phosphate (1.0 mM), magnesium chloride (1.5 mM), and disodium sulfate (0.5 mM) to pH 7.26
every day with hydrochloric acid.
•Transfer the solution to a series of 100 ml polystyrene bottles,
seal it tightly & kept at 37±1 °C for one week.
•Filter the precipitate, thoroughly washed with double distilled water & dried at 100 °C.
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16. Sonication & PAMAM
Sonication :
•Slowly add solution of disodium hydrogen phosphate to solution of calcium chloride
under sonication at 4 °C for 2 h.
•Separate the precipitate by centrifugation & decant the supernatant.
•Wash the precipitate, re-suspend in distilled water & filter through membrane filter.
PAMAM:
•Carboxylic acid terminated half generation poly (amidoamine) (PAMAM) used.
•Forms amorphous hydroxyapatite cores with a mixture of calcium phosphate.
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17. Carbohydrate Coating Of Core
• 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.
•Coating materials : Cellobiose, citrate, pyridoxal-5- phosphate, sucrose and trehalose.
•Adsorption method -
1. Add of polyhydroxy oligomer to a dispersion of meticulously cleaned ceramics in ultra pure
water.
2. Sonicate & lyophilize it.
3. Remove excess & readily desorbing carbohydrate either by stir cell ultrafiltration or by
dialysis method.
•Lactose coated by adsorption method by direct incubation and by nonsolvent addition.
FACULTY OF PHARMACY, THE MAHARAJA SAYAJIRAO UNIVERSITY OF BARODA 17

18. Immobilization of Drug molecule
•The surface modified Nano crystalline core provide the solid phase for subsequent non
denaturing self assembly for a broad range of biological active molecule.
•The drug can be loaded by partial adsorption.
1. Prepare the solution of drug with known concentration in suitable buffer.
2. Disperse the coated particles in it.
3. Keep the dispersion overnight at low temperature or lyophilize it.
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19. Evaluation
Evaluation parameters
Evaluation of ceramic core Evaluation of sugar coating
Evaluation of drug loaded
aquasomes
• Structure analysis
• Phase analysis of core
• Particle morphology
• Colorimetric analysis
• Concanavalin-A induced aggregation
• Size & shape
• Glass transition temperature
• In vitro drug release studies
• Drug loading efficiency
• Antigen loading efficiency
• Hb loading capacity
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20. Evaluation Of Ceramic Core
•Structure analysis:
Interpretated by Fourier transformed infrared spectroscopy using hydroxyapatite powder in the
wave no. range of 4000- 400 cm-1.
•Phase analysis of core:
Cores exposed to Cu K-a radiation in a wide angle X-ray diffractometer.
•Particle morphology:
Determined by using transmission electron microscopy one drop of aqueous dispersion is placed
over a 400-mesh carbon-coated copper grid followed by negative staining with phosphotungstic
acid and placed at the accelerating voltage.
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21. Evaluation Of Sugar Coating
•Colorimetric analysis – (Anthrone method)
•Residual Sugar unbound or Remaining after coating
Preparation of aliquots of sample for calibration curve.
Transferred it to boiling tubes & diluted to an appropriate concentration.
Addition of anthrone reagent, heated in a boiling water bath & cooled rapidly.
Absorbance recorded after the formation of greenish solution using UV-visible
spectrophotometer (glucose used as standard).
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22. Continue…
•Concanavalin-A-Induced aggregation
•Determines the amount of sugar coated
Take sugar-coated HA (Hydroxyapatite) core suspensions in quartz cuvettes. (10 μg/ml)
Addition of Concanavalin-A solution to it. (100 μg/ml)
Determination of absorbance at 450 nm as a function of time of 5 min interval
using UV-visible spectrophotometer.
Obtained data subtracted from blank experiment conducted in the absence of concanavalin-A.
FACULTY OF PHARMACY, THE MAHARAJA SAYAJIRAO UNIVERSITY OF BARODA 22

23. Continue…
•Zeta Potential
•determined by a photon correlation spectroscopy.
•Zeta potential of various aquasomal formulations is obtained from the electrophoretic mobility
measurements taken with a Zetasizer.
Fifty mg of different aquasomal formulations is shaken with 1 ml of PBS (pH 7.4) buffer at 37°C for 1 h.
The HA is then washed with PBS buffer and resuspended in 10 ml of deionized water
The average of 22 measurements is used.
All measurements are made at a constant scattering angle of 90° at 25°C.
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24. Evaluation Of Drug Loaded Aquasomes
Size & shape
•Morphological examination microscope. Transmission electron
•Mean particle size & size distribution - A photon correlation spectroscopy using a Autosizer II C
apparatus and SEM.
•Chemical composition & crystalline powder diffractometry. structure X-ray
Glass transition temperature
•carbohydrates and proteins studied by using DSC analyzer.
In-process stability studies
•Stability and integrity of protein during the formulation of the aquasomes determined by using
sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).
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25. Evaluation Of Drug Loaded Aquasomes
In vitro drug release studies
•To study the release pattern of drug
Incubate a known quantity of drug loaded aquasomes in a buffer of suitable pH at 37°C with
continuous stirring.
Samples are withdrawn periodically & centrifuged at high speed for certain lengths of time.
Equal volumes of medium replaced after each withdrawal.
Supernatants are then analyzed for the amount of drug released.
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26. Evaluation Of Drug Loaded Aquasomes
Drug loading efficiency
•Ensure the amount of drug which is bound on the surface of aquasomes.
Hb loading capacity - (Drabkin's method)
•Estimated by the difference between the control sample (HbA solution) and the free hemoglobin
contained in all fractions without nanoparticles.
Antigen loading efficiency
Weigh antigen-loaded aquasome formulations, suspend it in Triton X-100 & incubate in a wrist shaker
for 1 h.
Centrifuge the sample & determine absorbance using micro- BCA methods with set a blank of
unloaded aquasomes formulation.
•Unit: g of antigen/mg of sample.
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27. Advantages
•Aquasomes conserves the structural veracity and biochemical constancy of drug particles.
•Due to their specific size and structural stability, aquasomes evade RES (reticuloendothelial
clearance) or degradation in acidic pH or so forth.
•Aquasomes displays colloidal characteristics.
•Aquasome suspension contains colloidal range biodegradable nanoparticles, chances of
accumulation in muscles and liver is high.
•Receptor recognition is not difficult as the drug is easily adsorbed on the surface of aquasomes,
hence site specific delivery of biomolecules can be achieved easily.
•Aquasomes own large size and an active surface hence, substantial amount of drug molecules
can be surface adsorbed through ionic, non-covalent bonds, van der Waals forces, and entropic
forces.
•Drug release from aquasomes can be controlled by altering their surface through combination of
specific targeting, molecular shielding, and controlled release of therapeutics.
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28. Advantages
•Increases therapeutic efficacy of pharmaceutically active agents.
•Avoid multiple-injection schedule.
•Offer favorable environment for proteins.
•Used for various imaging tests.
•Novel carrier for enzymes such as DNAses and pigment/dyes.
•Act as a vaccine delivery system
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29. Limitations
•Though aquasomes appear to be an attractive potential delivery system for a wide range of
therapeutics, certain critical aspects associated with aquasomes that have not yet been studied
include;
•their amenability to various sterilization techniques,
•their shelf life as per ICH guidelines, feasibility of scale-up and subsequent commercialization
potential, cost efficacy, reproducibility of critical parameters in terms of both in-vitro and in-vivo
characterization, and most important their safety issues.
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30. Application/Approches
•For immunotherapy
•For oral route
•For immunopotentiation
•Anti thrombic activity
•Antigen delivery
•Delivery of poorly soluble drugs
•Enzyme delivery
•Insulin delivery
•Vaccine delivery
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31. Application
•Aquasomes has got a quite versatile application potential as a carrier for delivery of vaccines,
hemoglobin, drugs, dyes, enzymes.
1. Aquasomes used as vaccines for delivery of viral antigen
2. Aquasomes as red blood cell substitutes can effectively deliver the large, complex labile molecule,
haemoglobin By incorporating in aquasome carriers, the toxicity of haemoglobin is reduced,
biological activity is preserved, haemoglobin concentration of 80% can be achieved and is
reported to deliver oxygen in a non linear manner like natural red blood cells.
3. 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.
4. Aquasomes are used for oral delivery of acid labile enzyme, serratiopeptidase. Enzyme loaded
aquasome was further protected by encapsulating in alginate gel.
•They protected structural integrity of enzymes and better therapeutic efficacy was observed.
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32. Application
Insulin Delivery For parenteral administration of insulin
•calcium phosphate ceramic core aquasomes were created.
•The core was coated with trehalose, cellobiose, and pyridoxal-5-phosphate, among other disaccharides.
•The great degree of structural stability of pyridoxal-5-phosphate may help to explain this. Due to the drug's
slow release from the carrier and the peptide's structural stability, the activity of the peptide was also
prolonged.
•Oral Delivery of Enzyme Using a nanosized ceramic core-based delivery system
•serratiopeptidase, an acid-labile enzyme, is given orally.
•At room temperature, the core was prepared by sonication and colloidal precipitation.
•The chemical was then adsorbed over this coat of chitosan, which was applied over the centre while mixing
at a consistent rate.
•Throughout the formulation process, the stability and integrity of the enzyme were evaluated using in vitro
proteolytic activity.
•The results showed that aquasomes have a great potential for preserving the structural integrity of
enzymes, leading to a more beneficial therapeutic effect.
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33. Application
As oxygen carrier
•Hemoglobin adsorption on a hydroxyapatite core that has been produced and is covered in trehalose.
• In vivo studies on rats revealed that aquasomes had a high potential for use as an oxygen transporter, with
activity lasting for 30 days.
Antigen Delivery
•They produced aquasomes using the co-precipitation method by hydroxyapatite self-assembly.
•Bovine serum albumin, a model antigen, was adsorbed onto the coated core after coating agents such
trehalose and cellobiose had been used.
•The effectiveness of antigen loading was calculated to be 20–30%.
•Aquasomes were thought to hold promise for maintaining surface immutability because they safeguard the
protein structural conformation that is delivered to immune cells, leading to a superior immunological
response.
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34. Latest developments
•In 1994, haemoglobin was adsorbed on the carbohydrate coated diamond nanoparticles and
then encapsulated in a phospholipid layer.
•Subsequently, the group successfully used these nanoparticles to deliver viral antigens and
insulin. ( potential delivery systems for protein/peptide drugs )
•In 1996, the research group christened the particles as aquasomes based on the water like
protection provided by the carbohydrate layer.
•Since then, aquasomes have been explored for various applications. The parenteral delivery of
insulin and insulin-mimetic polypeptide k50 has been reported.
•Aquasomal formulation, loaded with hemoglobin was prepared which was found to be stable
for at least thirty days.
•Their use as an adjuvant and delivery vehicle for hepatitis B vaccine for effective immunization
was reported by Vyas et al.
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35. Latest developments
•Similarly, aquasomes loaded with BSA were designed to trigger a better immunological
response.
•Aquasomes of malarial merozoite surface protein – 119 (MSP - 119) were prepared by
adsorption of antigen on self-assembling hydroxyapatite carriers.
•Aquasomes have also been used for oral delivery of enzymes like serratiopeptidase.
•Another use of aquasomes has been demonstrated by Pandey et al in a study wherein allergen
immunotherapy could be achieved in mice model using ovalbumin.
•A number of synthetic drugs have also been formulated as aquasomes. These include etoposide,
dithranol, piroxicam and pimozide etc.
•The aquasomal approach has been tried to overcome the common dissolution problems
associated with these hydrophobic drugs
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36. FACULTY OF PHARMACY, THE MAHARAJA SAYAJIRAO UNIVERSITY OF BARODA 36

37. Conclusion
•Aquasome a nanopartical shows promise as carriers for the delivery of various drugs. Aquasomes also
present interesting features in terms of drug loading and releasing properties. After association with
these nanoparticles, the biological activity measured for certain drugs was improved.
•Up to now little has been known about the invivo behavior of aquasomes as drug delivery systems. As
suggested by the number of recent papers published on this subject, aquasome may be considered as
a very active field of investigation for the development of new delivery system.
•It is specifically designed for the administration of peptides, proteins, anti-sense oligonucleotides and
even genes. However, much work is still required to evaluate their actual in-vivo potential.
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38. REFERENCES
•Chaudhari, Bharatee. (2015). aquasome.
•Gulati, Monica & Singh, Sachin & Kaur, Puneet & Yadav, Ankit & Pondman, Kirsten & Kishore,
Uday. (2014). Aquasomes: The journey so far and the road ahead.
•Jain, S.S. & Jagtap, Pramod & Dand, Neha & Jadhav, Kisan & Kadam, V.J.. (2012). Aquasomes: A
novel drug carrier. Journal of Applied Pharmaceutical Science. 2. 184-192.
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