Aquasomes: A novel nanoparticulate drug carrier T PDF

Save (0)



Journal of Drug Delivery Science and Technology 43 (2018) 446–452

Contents lists available at ScienceDirect

Journal of Drug Delivery Science and Technology
journal homepage:

Aquasomes: A novel nanoparticulate drug carrier T

Sritoma Banerjee∗, Kalyan Kumar Sen
Gupta College of Technological Sciences, Asansol, West Bengal, 713301, India


Keywords: Aquasomes are three layered self-assembled nanoparticulate carrier system. This three layered system contains a
Self-assembled core coated with polyhydroxy oligomer upon which biochemically active molecules are adsorbed. Ceramics are
Ceramics mainly used as core material because of high degree of order and structural regularity. Polyhydroxy oligomer
Polyhydroxy oligomer coating provides water like environment & protect biochemically active molecule from dehydration. As a whole
Stability aquasomes provide stability to biochemically active molecule. Poorly water soluble drugs, insulin, hemoglobin,

serratiopeptidase can be delivered through aquasomes. This review article includes brief introduction of
aquasome, role of core & carbohydrate, properties, methods of preparation, characterization study and appli-
cation of aquasomes.

1. Introduction der Waals forces [20]. Sugar coating produces glassy molecular layer
that adsorbs therapeutic protein or small molecule without modifica-

Potential application of nanoparticulate system as drug carrier was tion in three-dimensional conformations. Ceramic core superimposed
first proposed by Dr. Gregory Gregoriadis on 1974. He proposed lipo- with carbohydrates improved the cellular uptake in cancer cells
somes as nanoparticulate drug delivery system [1]. Liposomes, solid [21,22]. Hence, aquasomes have been extensively investigated for de-
lipids nanoparticles, dendrimers, polymers, niosomes, silicon nano- livery of both small and high molecular weight, pharmaceuticals
particles, gold nanoparticles, carbon nanotubes, and magnetic nano- [23,24]. Main advantage of aquasomes over other nanoparticulate
particles are the examples of nanocarriers for drug delivery system. carrier system is that there is no interaction between drug and carrier.
Drug is adsorbed, covalently linked on nanocarrier system or it may be Drug molecule remains stable in water like environment provided by
encapsulated inside nanocarrier system. Nanoparticles are the ideal oligosaccharide coating.
choice for drug delivery nowadays [2–6]. The advantages of nano-
particulate drug delivery system are improved drug loading and de- 1.1. Role of core & carbohydrate
livery [7,8], delivering drug to the site of action which can be called as
targeted delivery [9–13], delivering very less side effects comparing to Materials used as core are nanocrystalline tin oxide, brushite (cal-
conventional dosage form [14] & delivering drugs which are poorly cium phosphate dehydrate), carbon ceramic (diamond particles).
soluble [15–18]. Due to small amount of drug is delivered to the site of Ceramics are mainly used as core material. Since ceramics are crystal-
action thus toxicity due to large dose is eliminated. Often they are line in nature these materials provide structural regularity and high
called as nano carriers in drug delivery science. Aquasome is a self- degree of order. High degree of order provides high degree of surface
assembled nanoparticulate carrier system which was first developed by energy which leads to efficacious binding of carbohydrate onto it.
Nir Kossovsky in 1995 whose surface can be non-covalently modified Another advantage of using calcium phosphate as core material is be-
with carbohydrates [19]. It is comprised of ceramic core coated with cause of its natural presence in body. Calcium phosphate is widely used
poly hydroxyl oligomer & upon this coated core biochemically active in the form of nanorods [25–27], nanorods [28–30], biocomposites
molecule are introduced by co polymerization, diffusion or adsorption [31–33], nanoparticles [34–37], scaffolds [38–41], hydroxyapatite
method. Presence of calcium phosphate in bones makes it an ideal whiskers [42,43]. Calcium phosphate is also used in bone tissue en-
biomaterial with ideal biocompatibility, biodegradability, absence of gineering [44,45], stem cell technology [46], as coating on bone im-
toxicity and stability to use it as drug carrier. Calcium phosphate and plants [47], as adjuvants [48–52]. Hydroxyapatite was selected as a
hydroxyapatite are used as ceramic core in aquasomes. Generally core for the preparation of aquasomes. Poorly crystalline form of hy-
aquasomes assemble through non-covalent bonds, ionic bonds and Van droxyapatite found in bone is stable at physiological pH [53].

∗ Corresponding author.
E-mail address: [email protected] (S. Banerjee).
Received 23 April 2017; Received in revised form 26 October 2017; Accepted 12 November 2017
Available online 14 November 2017
1773-2247/ © 2017 Elsevier B.V. All rights reserved.


S. Banerjee, K.K. Sen Journal of Drug Delivery Science and Technology 43 (2018) 446–452

Biocompatibility, easy to manufacture and low cost makes ceramic core through ionic, non-covalent, and entropic forces. Studies have
materials a good candidate for drug delivery applications [53,54]. demonstrated that particle size of aquasomes increases as a function of
Ceramics are biodegradable in nature. Monocytes and osteoclasts are concentration of core to coat ratio. This may be credited to availability
responsible for biodegradation of calcium phosphate inside body. of free surface of the core particles with the coating material [23,62].
During biodegradation two kinds of phagocytosis takes place, one is
only calcium phosphate crystals taken up and dissolved in cytoplasm 2.4. Drug incorporation process
upon disappearing of phagosome membrane and the other is dissolution
of calcium phosphate crystals when incorporated with large volume of Biochemically active molecules are incorporated in this nanoparti-
culture medium happens after forming heterophagosome [55,56]. culate system by adsorption through ionic and non-covalent interac-

Commonly used carbohydrates are pyridoxal-5-phosphate, cello- tions. Adsorption of drug on carbohydrate coated core increases the
biose, trehalose, sucrose, lactose. Carbohydrates act as natural stabilizer drug encapsulation efficiency [62].
& dehydroprotectant by providing structural integrity, preserving mo-
lecular conformation of biochemically active molecule, delivering 2.5. Self-assemble
water like environment to the biochemically active molecule while
keeping it in dry solid state and protecting three-dimensional con- This three layered structure is based on the principle of self-as-
formations of drug molecule [57,58]. Main principle of coating is ad- sembly which is achieved by non-covalent & ionic bonds. It was ob-
sorption of carbohydrate onto core. served in the research works [23,63] that sonication process, during the

Kaushik et al., 2003 [59] analyzed the thermal stability of proteins reaction of disodium hydrogen phosphate and calcium chloride in order
in presence of trehalose. They used RNase A as a model enzyme & in- to prepare calcium phosphate influences self-assembly of crystalline
vestigate the effect of trehalose on the retention of enzymatic activity calcium phosphate. Soniction process increased the surface free energy
upon incubation at high temperatures. They observed that 2 M con- of calcium phosphate which was also reported by Vengala et al., 2013
centration of trehalose increases the transition temperature (Tm) of [64]. This surface free energy influenced self-assembly.
RNase A to 18 °C (maximum) at pH 2.5. Trehalose remained inert to
protein surfaces and stabilized the proteins at different pH which in- 3. Methods of preparation
dicated it’s applicability as universal stabilizer. Researchers have re-
ported the effect of sugar on protein, enzyme stabilization [60,61]. 3.1. Core preparation
Goyal et al., 2008 [58] found strong evidence from the data of Fourier
transform infrared (FTIR), zeta potential and differential scanning col- Preparation technique of core depends on the type of core to be
orimetry (DSC) study that there is a biochemical interaction between used. Generally nanocrystalline tin oxide, carbon ceramic (diamond),
hydroxyapatite, sugars, and BSA (protein). It increased stabilization calcium phosphate, hydroxyapatite are used as core. Among these
protein in formulation. materials nanocrystalline calcium phosphate and hydroxyapatite are

Goyal et al., 2008 [58] identified the presence of mannose-like widely used as core material for aquasomes.
binding lectins (MLBLs). Polyhydroxyl oligomers or carbohydrates
present in aquasomes are recognized by the carbohydrate recognition 3.1.1. Self-assembled nanocrystalline brushite (calcium phosphate
domains of MLBLs. These domains recognize carbohydrates present on dihydrate)
the target cells and bind to them. This is one of the important me- Self-assembled nanocrystalline brushite is prepared by different
chanism of antigen delivery with the help of aquasomes. methods which are described in Table 1.

Vengala et al., 2013 and Patil et al., 2004 [62,65] studied the effect
2. Properties of aquasomes of pH, duration & sintering on size, nature of particle & percentage

yield. Uncontrolled pH leads to formation of large, elongated particles
2.1. Nanoparticle with micrometer size range. When pH was maintained in between 8 and

10 & no sintering took place, it caused formation of elongated to
Since aquasomes are nanoparticles, they have large surface area spherical particles (≤1.0 μm) but with sintering it caused formation of

thus can be loaded with significant amount of biochemically active spherical particles in nanometer range. From the findings of Vengala
molecule through van der waals forces, entropic forces, ionic and non- et al., 2013 [62], both the uncontrolled and controlled without sin-
covalent bonds. The core material widely used is calcium phosphate tering process gave similar less percentage yield (37% in uncontrolled
(CaHPO4). The nanocrystalline, calcium phosphate ceramic core parti- pH & 36% in controlled pH) while the process with controlled pH &
cles self-assemble during the reaction process under sonication due to sintering gave percentage yield of 60%. When the slurry was stirred for
augmentation of surface free energy [23]. one day with maintaining the pH in between 8 and 10, it caused for-

mation of large, elongated particles with less percentage yield (33%) &
2.2. Calcium phosphate sintering caused formation same kind of particle with 500–1000 nm of

size with same percentage yield. When the slurry was stirred for 4–6
Calcium phosphate which is used as core material in aquasome, is days it improved particle size (250–1000 nm), type (elongated to

biodegradable in nature. Inside the body, monocytes and multicellular spherical) & percentage yield (61%) & sintering caused formation of
cells called osteoclasts are responsible for biodegradation of calcium spherical particles (100–200 nm) & percentage yield of 60%. So, while
phosphate. It is prepared from the precipitation of a monobasic sodium maintaining pH in between 8 and 10 sintering process caused formation
phosphate solution and calcium chloride solution with mechanical of spherical particle in nanometer size range with increased percentage
agitation. Researchers have shown that the process variables like the yield and if duration of stirring is increased with subsequent sintering it
level of ultrasound frequency and the effect of the sonication on the also led to formation of spherical nanoparticle with increased percen-
particle size of the inorganic cores [62]. tage yield. Patil et al., 2004 [65] found that the self-precipitation

method produced spherical particles (1–5 μm), with a very less yield
2.3. Carbohydrate coating due to formation of monolayer precipitate which occurred on container

surface. The seeding caused increase in crystallization rate with pro-
Aquasomes provides water like environment due to presence of duction of particles of irregular size and shape. It indicates that pH,

carbohydrate coating and preserves conformational stability of bio- duration of stirring & sintering has effect on particle size, shape &
chemically active molecule. Polysaccharide film stabilizes the ceramic percentage yield of ceramic core.



S. Banerjee, K.K. Sen Journal of Drug Delivery Science and Technology 43 (2018) 446–452

3.2. Coating of the core with polyhydroxy oligomer

Carbohydrate is added to a dispersion of core followed by sonication
& then lyophillization. Coating can also be done by adsorption by direct
incubation and by nonsolvent addition [66]. Cherian et al., 2000 [23]
studied the effect of core to coat ratio, sonication time, sonicator power
on particle shape & size. Cores were coated by addition of carbohydrate
to a dispersion of ceramic core followed by sonication & then lyo-
phillization. Core to coat ratio of 1:4 or 1:5 caused formation of sphe-
rical coated particles. Increase in sonicator power (up to 15 W/20 W)
caused formation of small spherical discrete particles (< 200 nm). In-
crease in sonication time (up to 60 min) caused formation of small,
spherical particles (< 200 nm) but at 90 min small aggregates began to
appear. Goyal et al., 2008 [58] applied coating of cellobiose and tre-
halose to hydroxyapatite cores. Adsorption of carbohydrates and an-
tigen were optimized by Langmuir adsorption isotherm. From the ob-
servations, it was found that binding constant of cellobiose-coated
aquasomes is greater than trehalose-coated aquasomes and trehalose-
coated aquasomes have higher amount of sugar adsorbed per milligram
than cellobiose-coated aquasomes. So trehalose has less adsorption ef-
ficiency than cellobiose, but formed stronger bond than it. When
packing is considered, trehalose packing was less than cellobiose but it
arranged in such a way that lowest adsorption energy was achieved.
Vengala et al., 2013 [62] studied the effect of carbohydrate coating on
drug loading by preparing drug loaded ceramic cores without carbo-
hydrate coating. It was observed that drug loading was less compared to
carbohydrate coated core. So, carbohydrate film on ceramic core fa-
cilitates the rate of drug adsorption.

3.3. Loading of drug

The final step is loading of drug to the coated core by adsorption
method [23,58,62,64,65] often by incubating drug in the solution
coated core. Adsorption involves non-covalent and ionic interactions
[64]. It was observed that drug concentration and incubation tem-
perature are the factors effecting drug loading [62,65]. In the research
work of Vengala et al., 2013 piroxicam [62], it was observed that drug
payload increased with increase in drug concentration. But at a certain
concentration, there was sudden unusual increase in drug loading
which was due to crystallization of drug. So, it is very much important
that drug loading must takes place by adsorption technique. Patil et al.,
2004 [65] studied loading of hemoglobin on carbohydrate coated hy-
droxyapatite. Adsorption of hemoglobin took place by closely fitting
into the cavities present in carbohydrate layer. Drug loading varied
when loaded with different carbohydrate [23,65].

4. Characterization of aquasomes

4.1. Size distribution

For morphological analysis & particle size distribution study scan-
ning 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 [67–70].

4.2. Structural analysis

Structural analysis is done by Fourier transform infrared spectro-
scopy 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 [58,64,67–70].


Table 1
Methods of preparation of self-assembled nanocrystalline brushite.

Method Description Done by

Co precipitation Diammonium hydrogen phosphate (0.19 N) solution is added drop wise to calcium nitrate solution (0.32 M) whose pH is maintained 8–10 by addition of concentrated aqueous ammonia Vengala et al., 2013 [62,64]
solution with continuous stirring at 75 °C in a three necked flask containing a reflux condenser, a thermometer fitted with a CO2 trap and a charge funnel. After 4–6 days of magnetic
stirring at 75 °C and pH 8–10, the precipitates are then filtered, washed and dried overnight at 100 °C followed by sintering to 800–900 °C.
3 ml of aliquot of 0.1 g/L methylcellulose solution (as dispersant) was mixed with 1440 ml of deionized water containing 0.152 M of calcium nitrate tetrahydrate and 0.090 M Tas et al., 2001 [76], Goyal
diammonium hydrogen phosphate. After the addition of 115 ml of 24% NH4OH into the above solution it was heated at 60–70 °C on a hot plate with vigorous mechanical stirring for 3 h. et al., 2008 [58]
Formed precipitate was recovered from the supernatant by filtration and subsequently washed five times with deionized water. After drying over night at 100 °C the precipitate was
finally calcined in an air atmosphere at 1000 °C for 6 h followed by light grinding.

Self-Precipitation pH 7.2 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 Vengala et al., 2013 [64]
phosphate (1.0 mM), magnesium chloride (1.5 mM), and disodium sulfate (0.5 mM) was adjusted to 7.26 every day with hydrochloric acid. After transferring the solution to a series of
100 ml polystyrene bottles, the bottles were tightly sealed and kept at 37± 1 °C for one week. The precipitate formed on the inner surface was filtered, thoroughly washed with double
distilled water, and dried at 100 °C.

Sonication Solution of disodium hydrogen phosphate is slowly added to solution of calcium chloride under sonication at 4 °C for 2 h. Precipitate is then separated by centrifugation & supernatant Kossovsky et al., 1996 [63],
liquid is decanted. The precipitated cores are washed, re- suspended in distilled water & filtered through membrane filter to obtain cores with desired particle size. Cherian et al., 2000 [23],

Rojas-Oviedo et al., 2007
[24], Vengala et al., 2013 [64]

PAMAM Spherical hydroxyapatite cores were formed using carboxylic acid terminated half generation poly (amidoamine) (PAMAM). Spherical shape of the cores depended on phosphate Khopade et al., 2002 [72]
saturation, rate of crystal growth and pH of the simulated boy fluid. Hydroxyapatite formed by this process was amorphous in nature with a mixture of various calcium phosphate and
showed phosphate rich apatite formation.


S. Banerjee, K.K. Sen Journal of Drug Delivery Science and Technology 43 (2018) 446–452

Table 2
Application of aquasomes.

Aquasome prepared by Active ingredient Purpose of use Significant results

Kossovsky et al., 1995 [73] Mussel adhesive protein Antigen delivery Stability of antigen due to presence of carbohydrate
Kossovsky et al., 1995 [74] Non-nuclear material from HIV-1 Antigen delivery Eliciting cellular and humoral immune response similar to live HIV
Cherian et al., 2000 [23] Insulin Insulin delivery Reduction of blood glucose level with retention of biological activity of insulin
Khopade et al., 2002 [72] Hemoglobin Oxygen carrier In vivo study results provides good potential to be used as blood substitute
Patil et al., 2004 [65] Hemoglobin Oxygen carrier Oxygen carrying capacity was same as fresh blood and hemoglobin was stable in

formulation over a period of 30 days
Goyal et al., 2006 [75] Hepatitis-B vaccine Vaccine delivery Combined Th1 and Th2 immune response
Goyal et al., 2008 [58] Bovin serum albumin Antigen delivery Preservation of structural integrity and enhanced immunological response due to

better uptake of antigen
Rawat et al., 2008 [71] Serratiopeptidase Enzyme delivery Sustained delivery of enzyme with protection of biological activity
Vengala et al., 2013 [64] Pimozide Poorly water soluble drug Sonication process yielded more amount of core and improved dissolution

delivery compared to pure drug.
Vengala et al., 2013 [62] Piroxicam Poorly water soluble drug Diffusion controlled release of drug

Nanjwade et al., 2013 [70] Etoposide Poorly water soluble drug Maximum percentage of the injected dose was in liver and spleen.


4.3. Crystallinity 4.6. Glass transition temperature

X-ray diffraction study is done to study crystalline or amorphous Differential scanning colorimetry is used to study glass transition
nature of a material. Here diffraction study of ceramic core, carbohy- temperature of carbohydrates & proteins. In the research work by Goyal
drate and drug loaded aquasomes are done & compared [71]. In the et al., 2008 [58] results of formulation loaded with BSA, model protein
study by Rojas-Oviedo et al., 2013 [24], it was observed that calcium revealed that formulations were stable at room temperature. This sta-
phosphate core, lactose individually gave identical sharp peaks for bility is provided by a synergistic effect by hydroxyapatite and carbo-
crystalline peaks but when X-ray diffraction results of carbohydrate hydrate.
coated cores were observed, it was seen that peaks represented an
amorphous structure. This may be the reason of the coating technique 4.7. Drug loading efficiency
(solubilization of carbohydrate in solvent and subsequent drying by
lyophilization) and saturation of the surface of core with carbohydrate. 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

4.4. Carbohydrate coating separated by high-speed centrifugation for 1 h at low temperature in a
refrigerated centrifuge. After centrifugation, the supernatant liquid is

Carbohydrate coating is confirmed by concanavalin-A induced ag- collected. The drug remaining in the supernatant liquid after loading is
gregation method & anthrone method [58,65,67–70]. Concanavalin-A then estimated by suitable method of analysis like by measuring ab-
induced aggregation method determines amount of sugar loaded on sorbance in UV spectrophotometer [66].
ceramic core. Concanavalin-A solution is added to suspensions of dif-
ferent carbohydrate coated core in quartz cuvettes and absorbance is

4.8. In-vitro drug release study
measured in UV-Visible spectrophotometer at a wavelength of 450 nm
as a function of time of 5 min interval. The obtained data is subtracted

In-vitro dissolution study is done in buffer media of suitable pH at
from blank experiment (without using concanavalin-A). Anthrone

37 °C with constant stirring. Sample is withdrawn time to time with
method determines unbound residual sugar. Anthrone forms green co-

replacement of similar volume of buffer. Samples withdrawn are cen-
lored substance when carbohydrates are hydrolyzed into simple sugars

trifuged at high speed. After that supernatant is collected & analyzed to
and subsequently to hydroxyl methyl furfural. Calibration curve is

determine the amount of released drug [58].
plotted and then aliquots of samples are diluted to an appropriate
concentration in boiling tubes. Anthrone reagent is added and samples
are heated in a boiling water bath and then cooled rapidly. A green 5. Applications of aquasome
colored solution is obtained and absorbance is recorded
(λmax = 625 nm) in a UV-Visible spectrophotometer using glucose as More than twenty years ago researches have been started on
standard. aquasomes. Extensive researches is still going on. An overview on ap-

plications of aquasomes and notable results observed by researchers is
depicted in Table 2. By far from the research works done by different

4.5. Zeta potential measurement researchers, different applications of aquasomes are described in detail

Zeta potential is a measure of the electrostatic attraction or repul-
sion between particles. Electrochemical equilibrium is observed and 5.1. Delivery of poorly soluble drugs
analyzed to understand the stability of a formulation. It is best known
as the stability indicator of suspension, dispersion or emulsion. Vengala et al., 2013 [62] developed and performed in vitro eva-
Adsorption of sugar can also be identified by measuring zeta potential luation of ceramic nanoparticles of piroxicam. Trehalose coated pirox-
[58]. It was observed that with the increase in saturation process by icam nano particles showed controlled release. Piroxicam loaded na-
carbohydrate on to the hydroxyapatite core, the more decrease in zeta noparticles without sugar coating showed 90% release in 1 h. Vengala
potential value. et al., 2013 [64] also developed lactose coated ceramic nanoparticle for

oral delivery of hydrophobic drug pimozide. Calcium phosphate
ceramic core was prepared by co-precipitation by reflux, co-



S. Banerjee, K.K. Sen Journal of Drug Delivery Science and Technology 43 (2018) 446–452

precipitation by sonication, self-precipitation. Co-precipitation by so- adhesive protein). Recognition of antigen by immunocompetent cells
nication method was selected based on results on percentage yield and depends specifically to chemical sequence and conformation of anti-
duration of preparation. In vitro dissolution was performed and com- genic determinant. In this study diamond nanoparticles were coated
pared with that of pure drug. Dissolution result was improved in drug with cellobiose which acts as natural stabilizer & minimizes surface
loaded formulation than pure drug. Release of pimozide loaded aqua- induced denaturation of adsorbed antigen. Kossovsky et al. [74] in
somes followed first order kinetics. Nanjwade et al., 2013 [70] for- another study developed surface-modified nanocrystalline carbon &
mulated & evaluated etoposide loaded aquasomes. Drug release from calcium phosphate ceramic particulates & evaluate the surface activity
aquasomes increased as the carbohydrate concentration increased. In of immobilized antigen (non-nuclear material from HIV-1). These im-
vivo studies were performed & it was found that maximum percentage mobilized antigens or HIV decoys could promote surface agglomeration
of the injected dose was in liver then in spleen followed by lungs and among malignant T-cells similar to live HIV. In three vaccinated animal
kidney. This result indicated drug can be targeted to organs like liver, species study it was observed that the HIV decoys elicited cellular and
spleen, lungs. humoral immune responses similar to that evoked by live HIV. Goyal

et al., 2008 [58] developed aquasomes for delivery of BSA, a model
5.2. Insulin delivery antigen. Aquasomes showed 20–30% BSA loading efficiency. Study

showed that prolonged release of antigen from aquasomes & bio-
Cherian et al., 2000 [23] developed aquasomes for the parenteral chemical nature of nano-ceramic produced better humoral response

delivery of insulin. The core was coated with disaccharides such as than pure antigen. Also it revealed that BSA-loaded aquasomes can
cellobiose, trehalose, and pyridoxal-5-phosphate. Cellobiose, trehalose elicit both Th1 & Th2 immune response. Enhanced immunological re-
& pyridoxal-5-phosphate protect the drug molecule from dehydration. sponse of BSA-loaded aquasomes is due to better presentation & uptake
Trehalose & pyridoxal-5-phosphate are more effective than cellobiose. of the antigen.
In vivo study of aquasome formulations was done using albino rats.
More effective reduction in blood glucose level was observed in pyr- 5.6. Vaccine delivery
idoxal-5-phosphate coated aquasome than trehalose or cellobiose
coated aquasomes due to the high degree of molecular preservation Goyal et al., 2006 [75] developed a nanodecoy system to design
with significant degree of retention of biological activity. The pro- hepatitis B vaccine for immunopotentiation. Preparation of nanodecoy
longed activity resulted may be due to slow release of drug from the system included formation of self-assembled hydroxyapatite core on
carrier and structural integrity (prevention from denaturation or de- which cellobiose was coated. Then hepatitis B surface antigen (HBsAg)
hydration) of the protein [34]. was adsorbed over coated core. These nanodecoy formulations also

showed to increase combined Th1 & Th2 immune response.
5.3. Enzyme delivery

6. Conclusion
Rawat M et al., 2008 [71] developed ceramic core based system for

oral administration of the acid-labile enzyme serratiopeptidase. In Various research works on aquasomes indicated that it can be used
acidic buffer, drug release followed the Higuchi model by showing a as successful nanoparticulate drug carrier. Research works suggested
low amount of drug release by diffusion up to a period of 2–6 h. In the antigen, insulin, hemoglobin, vaccine can be delivered through aqua-
alkaline medium, it showed sustained and nearly completed first-order somes. Carbohydrate coating in this unique kind of formulation pro-
release of the enzyme up to 6 h. Enzyme loaded ceramic core acted as vides natural stabilizing effect to the bioactive molecule in the for-
reservoir of the adsorbed enzyme and enzyme was protected due to the mulation by maintaining its structural integrity and molecular
encapsulation with alginate gel. conformation. Thus helps in delivering conformationally sensitive mo-

lecule to the site of action. Also aquasomes helps in delivering protein
5.4. As oxygen carrier molecule by preventing destructive denaturation. Though it has many

advantages to be used as drug carrier, extensive researches are yet re-
Khopade et al., 2002 [72] developed hydroxyapatite core by using quired to study the effect on in-vivo system, to identify if it has any

carboxylic acid– terminated half-generation poly(amidoamine) den- toxic effect in certain conditions and to prove its safety & efficacy in
drimers as templates or crystal modifiers for the delivery of he- human body.
moglobin. Aquasome formulations were in the nanometer size range.
The loading capacity was found to be approximately of 13.7 mg of Appendix A. Supplementary data
hemoglobin per gram of the core. Retention of oxygen affinity, co-
operativity & stability were observed for 30 days. In vivo studies on Supplementary data related to this article can be found at http://dx.
albino rats showed that aquasomes possess good potential for use as
blood substitute. Patil et al., 2004 [65] developed hemoglobin loaded
aquasomes. Adsorption of sugar on ceramic core and adsorption of Declaration of interest
hemoglobin on coated core both followed freundlich and langmuir
isotherm. The oxygen carrying capacity of aquasome formulation was There is no conflict of interest reported by the authors.
found to be similar to that of fresh blood. Also, the hill coefficients were
found to be good for its use as an oxygen carrier. The aquasome for- Role of the funding source
mulations neither induced hemolysis of the red blood cells nor altered
the blood coagulation time. The hemoglobin content of the formulation There is no role of any kind of funding source.
remains unchanged for over a period of 30 days. No significant increase
in arterial blood pressure and heart rate was observed in rats transfused References
with aquasome suspension on 50% exchange transfusion.

[1] G. Gregoriadis, C.P. Swain, E.J. Wills, et al., Drug-carrier potential of liposomes in
5.5. Antigen delivery cancer chemotherapy, Lancet 1 (1974) 1313–1316.

[2] T. Govender, S. Stolnik, M.C. Garnett, et al., PLGA nanoparticles prepared by na-
noprecipitation: drug loading and release studies of a water soluble drug, J. Control

Kossovsky et al., 1995 [73] first developed surface-modified dia- Release 57 (1999) 171–185.
mond nanoparticles which act as delivery vehicle for antigen (mussel [3] B. Sarmento, S. Martins, D. Ferreira, et al., Oral insulin delivery by means of solid



S. Banerjee, K.K. Sen Journal of Drug Delivery Science and Technology 43 (2018) 446–452

lipid nanoparticles, Int. J. Nanomedicine 2 (2007) 743–749. (2008) 1087–1099.
[4] V. Sanna, E. Gavini, M. Cossu, et al., Solid lipid nanoparticles (SLN) as carriers for [33] A.M. El Kady, K.R. Mohamed, G.T. El- Bassyouni, Fabrication, characterization and

the topical delivery of econazole nitrate: in-vitro characterization, ex-vivo and in- bioactivity evaluation of calcium pyrophosphate/polymeric biocomposites, Ceram.
vivo studies, J. Pharm. Pharmacol. 59 (2007) 1057–1064. Int. 35 (2009) 2933–2942.

[5] P. Jain, A. Mishra, S.K. Yadav, et al., Formulation development and characterization [34] W. Paul, C.P. Sharma, Porous hydroxyapatite nanoparticles for intestinal delivery of
of solid lipid nanoparticles containing nimesulide, Int. J. Drug Deliv. Technol. 1 insulin, Trends Biomater. Artif. Organs 14 (2001) 37–38.
(2009) 24–27. [35] I. Roy, S. Mitra, A. Maitra, et al., Calcium phosphate nanoparticles as novel non-

[6] X. Pang, F. Cui, J. Tian, et al., Preparation and characterization of magnetic solid viral vectors for targeted gene delivery, Int. J. Pharm. 250 (2003) 25–33.
lipid nanoparticles loaded with ibuprofen, Asian J. Pharm. Sci. 4 (2009) 132–137. [36] C. Lai, S.Q. Tang, Y.J. Wang, et al., Formation of calcium phosphate nanoparticles

[7] K. Ofokansi, G. Winter, G. Fricker, et al., Matrix-loaded biodegradable gelatin na- in reverse microemulsions, Mater. Lett. 59 (2005) 210–214.
noparticles as new approach to improve drug loading and delivery, Eur. J. Pharm. [37] A. Peetsch, C. Greulich, D. Braun, et al., Silver-doped calcium phosphate nano-
Biopharm. 76 (2010) 1–9. particles: synthesis, characterization & toxic effects toward mammalian & prokar-

[8] C. Choi, S.Y. Chae, J.-W. Nah, Thermosensitive poly(N-isopropylacrylamide)-b poly yotic cells, Colloids Surf. B Biointerfaces 102 (2013) 724–729.
(ɛ-caprolactone) nanoparticles for efficient drug delivery system, Polymer 47 [38] H.R. Ramay, M. Zhang, Preparation of porous hydroxyapatite scaffolds by combi-
(2006) 4571–4580. nation of the gel-casting and polymer sponge methods, Biomaterials 24 (2003)

[9] M. Yoshioka, M. Hashida, S. Muranihsi, et al., Specific delivery of mitomycin c to 3293–3302.
the liver, spleen and lung: nano- and microspherical carriers of gelatin, Int. J. [39] H.R. Ramay, M. Zhang, Biphasic calcium phosphate nanocomposite porous scaf-
Pharm. 81 (1981) 131–141. folds for load-bearing bone tissue engineering, Biomaterials 25 (2004) 5171–5180.

[10] P.D. Scholes, A.G.A. Coombes, L. Illum, et al., The preparation of sub-200 nm poly [40] K.K. Tan, G.H. Tan, B.S. Shamsul, et al., Bone graft substitute using hydroxyapatite
(lactide-co-glycolide) microspheres for site-specific drug delivery, J. Control scaffold seeded with tissue engineered autologous osteoprogenitor cells in spinal
Release 25 (1993) 145–153. fusion: early result in a sheep model, Med. J. Malays. 60 (suppl C) (2005) 53–58.

[11] A.K. Patri, J.F. Kukowska-Latallo, J.R. Baker Jr., Targeted drug delivery with [41] S.S. Kim, M.S. Park, O. Jeon, et al., Poly(lactide-co-glycolide)/hydroxyapatite
dendrimers: comparison of the release kinetics of covalently conjugated drug and composite scaffolds for bone tissue engineering, Biomaterials 27 (2006)
non-covalent drug inclusion complex, Adv. Drug Deliv. Rev. 57 (2005) 2203–2214. 1399–1409.

[12] D.A. Rothenfluh, H. Bermudez, C.P. O’Neil, et al., Biofunctional polymer nano- [42] R.K. Roeder, M.M. Sproul, C.H. Turner, Hydroxyapatite whiskers provide improved
particles for intra-articular targeting and retention in cartilage, Nat. Mater. 7 (2008) mechanical properties in reinforced polymer composites, J. Biomed. Mater Res. A
248–254. 67 (2003) 801–812.

[13] S. Dhar, N. Kolishetti, S.J. Lippard, et al., Targeted delivery of a cisplatin prodrug [43] H. Zhang, B.W. Darvell, Synthesis and characterization of hydroxyapatite whiskers
for safer and more effective prostate cancer therapy in vivo, Proc. Natl. Acad. Sci. U. by hydrothermal homogeneous precipitation using acetamide, Acta Biomater. 6
S. A. 108 (2011) 1850–1855. (2010) 3216–3222.

[14] B. Irving, Nanoparticle drug delivery systems, Inno Pharm. Biotechnol. 24 (2007) [44] H. Yoshikawa, A. Myoui, Bone tissue engineering with porous hydrxyapatite cera-
58–62. mics, J. Artif. Organs 8 (2005) 131–136.

[15] L.H. Reddy, R.S.R. Murthy, Pharmacokinetics and biodistribution studies of dox- [45] H. Zhou, J. Lee, Nanoscale hydroxyapatite particles for bone tissue engineering,
orubicin loaded poly(butyl cyanoacrylate) nanoparticles synthesized by two dif- Acta Biomater. 7 (2011) 2769–2781.
ferent techniques, Biomed. Pap. Med. Fac. Univ. Palacky. Olomouc Czech Repub. [46] H. Ohgushi, Al Caplan, Stem cell technology and bioceramics: from cell to gene
148 (2004) 161–166. engineering, J. Biomed. Mater. Res. 48 (1999) 913–927.

[16] B. Devarakonda, R.A. Hill, W. Liebenberg, et al., Comparison of the aqueous solu- [47] J.A.M. Clemens, C.P.A.T. Klein, R.C. Vriesde, et al., Healing of large (2mm) gaps
bilization of practically insoluble niclosamide by polyamidoamine (PAMAM) den- around calcium phosphate-coated bone implants: a study in goats with a follow up
drimers and cyclodextrins, Int. J. Pharm. 304 (2005) 193–209. of 6 months, J. Biomed. Mater. Res. 40 (1998) 341–349.

[17] E. Merisko-Liversidge, G.G. Liversidge, E.R. Cooper, Nanosizing: a formulation [48] M.R. Ickovic, E.H. Relyveld, E. Hénocq, Calcium-phosphate-adjuvanted allergens:
approach for poorly-water-soluble compounds, Eur. J. Pharm. Sci. 18 (2003) total and specific IgE levels before and after immunotherapy with house dust and
113–120. dematophagoides pteronyssinus extracts, Ann. Immunol. Paris. 134 (1983)

[18] Z. Luo, J. Jiang, pH-sensitive drug loading/releasing in amphiphilic copolymer 385–398.
PAE–PEG: integrating molecular dynamics and dissipative particle dynamics si- [49] E.H. Relyveld, M.R. Ickovic, E. Hénocq, et al., Calcium phosphate adjuvanted al-
mulations, J. Control Release 162 (2012) 185–193. lergens, Ann. Allergy 54 (1985) 521–529.

[19] N. Kossovsky, A. Gelman, E.E. Sponsler, H.J. Hnatyszyn, S. Rajguru, M. Torres, [50] H. Kato, M. Shibano, Relationship between hemolytic activity and adsorption ca-
et al., Surface-modified nanocrystalline ceramics for drug delivery applications, pacity of aluminum hydroxide and calcium phosphate as immunological adjuvants
Biomaterials 15 (1994) 1201–1207. for biologicals, Microbiol. Immunol. 38 (1994) 543–548.

[20] R.S. Pandey, S. Sahu, M.S. Sudheesh, J. Madan, M. Kumar, V.K. Dixit, Carbohydrate [51] N. Goto, H. Kato, J. Maeyama, et al., Local tissue irritating effects and adjuvant
modified ultrafine ceramic nanoparticles for allergen immunotherapy, Int. activities of calcium phosphate and aluminum hydroxide with different physical
Immunopharmacol. 11 (2011) 925–931. properties, Vaccine 15 (1997) 1364–1371.

[21] J.R. Kanwar, G. Mahidhara, R.K. Kanwar, Novel alginate-enclosed chitosan- cal- [52] Q. He, A.R. Mitchell, S.L. Johnson, et al., Calcium phosphate nanoparticle adjuvant,
cium phosphate-loaded iron-saturated bovine lactoferrin nano- carriers for oral Clin. Diagn Lab. Immunol. 7 (2000) 899–903.
delivery in colon cancer therapy, Nanomedicine 7 (2012) 1521–1550. [53] W. Paul, C.P. Sharma, Bioceramics, towards nano-enabled drug delivery: a mini

[22] D. Singh, S. Singh, J. Sahu, S. Srivastava, M.R. Singh, Ceramic nanoparticles: re- review, Trends Biomat. Artif. Organs 19 (2005) 7–11.
compense, cellular uptake and toxicity concerns, Artif. Cells Nanomed. Biotechnol. [54] H.J. Hnatyszyn, N. Kossovsky, A. Gelman, et al., Drug delivery systems for the fu-
17 (2014) 1–9. ture, PDA J. Pharm. Sci. Technol. 48 (1994) 247–254.

[23] A.K. Cherian, A.C. Rana, S.K. Jain, Self-assembled carbohydrate-stabilized ceramic [55] H. Baumann, J. Gauldie, The acute phase response, Immunol. Today 15 (1994)
nanoparticles for the parenteral delivery of insulin, Drug Dev. Indust Pharm. 26 74–80.
(2000) 459–469. [56] M.D. Benahmed, D. Heymann, M. Berreur, et al., Ultrastructural study of de-

[24] I. Rojas-Oviedo, R.A. Salazar-Lopez, J. Reyes-Gasga, C.T. Quirino-Barreda, gradation of calcium phosphate ceramic by human monocytes and modulation of
Elaboration and structural analysis of aquasomes loaded with indomethacin, Eur. J. this activity by HILDA/LIF cytokine, J. Histochem Cytochem 44 (1996) 1131–1140.
Pharm. Sci. 32 (2007) 223–230. [57] N. Kossovsky, A. Gelman, S. Rajguru, et al., Control of molecular polymorphisms by

[25] I. Yamaguchi, K. Tokuchi, H. Fukuzaki, et al., Preparation and mechanical prop- a structured carbohydrate/ceramic delivery vehicle – aquasomes, J. Control Release
erties of chitosan/hydroxyapatite nanocomposites, Key Eng. Mater. 192–195 (2001) 39 (1996) 383–388.
673–676. [58] A.K. Goyal, K. Khatri, N. Mishra, et al., Aquasomes-a nanoparticulate approach for

[26] M. Kikuchi, S. Itoh, S. Ichinose, et al., Self-organization mechanism in a bone like the delivery of antigen, Drug Dev. Ind. Pharm. 34 (2008) 1297–1305.
hydroxyapatite/collagen nanocomposite synthesized in vitro and its biological re- [59] J.K. Kaushik, R. Bhat, Why is trehalose an exceptional protein stabilizer? An ana-
action in vivo, Biomaterials 22 (2001) 1705–1711. lysis of the thermal stability of proteins in the presence of the compatible osmolyte

[27] H.W. Kim, J.C. Knowles, H.E. Kim, Porous scaffolds of gelatin-hydroxyapatite na- trehalose, J. Biol. Chem. 278 (2003) 26458–26465.
nocomposites obtained by biomimetic approach: characterization and antibiotic [60] T. Arakawa, S.N. Timasheff, Stabilization of protein structure by sugars,
drug release, J. Biomed. Mater Res. B Appl. Biomater. 74 (2005) 686–698. Biochemistry 21 (1982) 6536–6544.

[28] M. Chen, J. Tan, Y. Lian, et al., Preparation of Gelatin coated hydroxyapatite na- [61] M.F. Mazzobre, M. Del Pilar Buera, Combined effects of trehalose and cations on the
norods and the stability of its aqueous colloidal, Appl. Surf. Sci. 254 (2008) thermal resistance of beta-galactosidase in freeze-dried systems, Biochim. Biophys.
2730–2735. Acta 1473 (1999) 337–344.

[29] J. Zhan, Y.H. Tseng, J.C.C. Chan, et al., Biomimetic formation of hydroxyapaite [62] P. Vengala, S. Aslam, C.V.S. Subrahmanyam, Development and in-vitro evaluation
nanorods by a single-crystal-to-single-crystal transformation, Adv. Funct. Mater. 15 of ceramic nanoparticles of piroxicam, Lat. Am. J. Pharm. 32 (2013) 1124–1130.
(2005) 2005–2010. [63] N. Kossovsky, Artificial Self-assembling Systems for Gene Delivery, (1996), pp.

[30] H.N. Lim, A. Kassim, N.M. Huang, Preparation and characterization of calcium 152–168 chap. 15.
phosphate nanorods using reverse microemulsion and hydrothermal processing [64] P. Vengala, S. Dintakurthi, C.V.S. Subrahmanyam, Lactose coated ceramic nano-
routes, Sains Malays. 39 (2010) 267–273. particles for oral drug delivery, J. Pharm. Res. 7 (2013) 540–545.

[31] H.H. Beherei, G.T. El-Bassyouni, K.R. Mohamed, Modulation, characterization and [65] S. Patil, S.S. Pancholi, S. Agrawal, et al., Surface-modified mesoporous ceramics as
bioactivity of new biocomposites based on apatite, Ceram. Int. 34 (2008) delivery vehicle for haemoglobin, Drug Deliv. 11 (2004) 193–199.
2091–2097. [66] S. Kommineni, S. Ahmad, P. Vengala, et al., Sugar coated ceramic nanocarriers for

[32] K.R. Mohamed, A.A. Mostafa, Preparation and bioactivity evaluation of hydro- the oral delivery of hydrophobic drugs: formulation, optimization and evaluation,
xyapatite-titania/chitosan-gelatin polymeric biocomposites, Mater. Sci. Eng. C 28 Drug Dev. Ind. Pharm. 38 (2012) 577–586.



S. Banerjee, K.K. Sen Journal of Drug Delivery Science and Technology 43 (2018) 446–452

[67] L.P. Nori, Aquasomes: role to deliver bioactive substances, Res. J Pharma Dosage spherical hydroxyapatite cores precipitated in the presence of poly(amidoamine)
Forms Tech 2 (2010) 356–360. dendrimer, Int. J. Pharm. 241 (2002) 145–154.

[68] N. Narang, Aquasomes: self-assembled systems for the delivery of bioactive mole- [73] N. Kossovsky, A. Gelman, H.J. Hnatyszyn, et al., Surface-modified diamond nano-
cules, Asian J. Pharm. 6 (2012) 95–100. particles as antigen delivery vehicles, Bioconjug Chem. 6 (1995) 507–511.

[69] P. Vengala, D. Shwetha, A. Sana, et al., Aquasomes: a novel drug carrier system, Int. [74] N. Kossovsky, A. Gelman, E. Sponsler, et al., Preservation of surface-dependent
Res. J. Pharm. 3 (2012) 123–127. properties of viral antigens following immobilization on particulate ceramic de-

[70] B.K. Nanjwade, G.M. Hiremath, F.V. Manvi, et al., Formulation and evaluation of livery vehicles, J. Biomed. Mater. Res. 29 (1995) 561–573.
etoposide loaded aquasomes, J. Nanopharm. Drug Deliv. 1 (2013) 92–101. [75] A.K. Goyal, A. Rawat, S. Mahor, et al., Nanodecoy system: a novel approach to

[71] M. Rawat, D. Singh, S. Saraf, et al., Development and in vitro evaluation of alginate design hepatitis B vaccine for immunopotentiation, Int. J. Pharm. 309 (2006)
gel-encapsulated, chitosan-coated ceramic nanocores for oral delivery of enzyme, 227–233.
Drug Dev. Ind. Pharm. 34 (2008) 181–188. [76] A.C. Tas, X-ray diffraction data for flux grown calcium hydroxyapatite whiskers,

[72] A.J. Khopade, S. Khopade, N.K. Jain, Development of haemoglobin aquasomes from Powder Diffr. 16 (2001) 102–106.