Trends Biomater. Artif. Organs, Vol 18 (2), January 2005 http://www.sbaoi.

org

Aspirin Loaded Albumin Nanoparticles by Coacervation:

Implications in Drug Delivery
Saikat Das, Rinti Banerjee and Jayesh Bellare
School of Biosciences and Bioengineering
Indian Institute of Technology, Bombay
Powai, Mumbai – 400076, India.
E-mail: rinti@cc.iitb.ac.in
Abstract:
Application of nanotechnology in drug delivery systems has opened up new areas of research in sustained
release of various drugs. Due to their size, nanoparticles have the advantage of reaching otherwise less
accessible sites in the body by escaping phagocytosis and entering tiny capillaries. In sensitive regions
like the eye they have the advantage of causing minimal irritation. Sustained release of the drug from the
nanoparticles maintains the therapeutic concentration for long durations. We prepared aspirin loaded
albumin nanoparticles by coacervation method. Aspirin is a common anti-inflammatory and anti platelet
agent widely used for various conditions. Albumin being both bioacceptable and biodegradable has a
distinct advantage as a vehicle of drug delivery. By varying aspirin albumin ratios from 0.06 to 1.0 we
obtained stable nanoparticles of sizes 46.8 nm to 190.8 nm respectively with low polydispersity. Photon
Correlation Spectroscopy (PCS) and Transmission Electron Microscopy (TEM) of the samples were
done to characterize the nanoparticles. Drug encapsulation measured by UV spectroscopy varied from
30% to 80% for different ratios of aspirin: albumin. In vitro release study was conducted across a
Spectrapor-membrane (cut off 3500 Da) precluding albumin. In contrast to simple drug solution, whose
concentration peaks with in ½ to 1 hour, nanoparticle formulation releases aspirin at a sustained rate for
prolonged duration (50% total cumulative percentage at the end of 20 hours, 90% at 72 hrs). From the
above results we can conclude that coacervation method is well suited to produce albumin nanoparticles
and the preparative variables of the procedure can be fine tuned depending on the clinical application.
Nanoparticles thus produced, can be applied for intra-articular therapy in arthritis or as intraocular release
agents for diabetic retinopathy.
Keywords : Nanoparticles, drug delivery, TEM
Introduction: flubiprofen (Pignatello et al., 2002), rose
Bengal (Lin, Garnett et al., 2001), and
Nanoparticles are colloidal particles, which are
ganciclovir (Merodio et al., 2002) have been
less than 1 µm in diameter. They have the
delivered by entrapping in nanoparticles.
unique property to accumulate at the site of
inflammation and therefore, are very suitable Aspirin or Acetyl Salicylic Acid is a non-
for targeted drug delivery. Different kinds of steroidal anti-inflammatory drug, which acts
drugs like pilocarpine (Zimmer et al., 1995), by inhibition of prostaglandin synthesis. The
hydrocortisone (Zimmer, Maincet et al., 1994), involvement of prostaglandins in the pathology
Ibuprofen (Pignatello, Bucolo et al., 2002), of various joint and ocular disorders is well

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 Aspirin Loaded Albumin Nanoparticles by Coacervation: Implications in Drug Delivery
proven. Therefore, aspirin in widely used in
these disorders. For example, aspirin is one of
the time-tested drugs used in the treatment of
Rheumatoid and other types of arthritis. It is
used to prevent chronic deformity, which
occurs due to inflammation, synovial
proliferation and erosion of bone (Tripathi
2001). Recently, it has been shown that
prostaglandins also play a significant role in
the pathogenesis of various ocular disorders.
Ocular tissues shown to produce
prostaglandins include iris, ciliary body,
conjunctiva, cornea (Bhattacharjee et al.,
1981) and retina, while prostaglandin
production by corneal epithelial, endothelial
and trabecular meshwork cells have been
studied under tissue culture conditions.
Prostaglandins have been shown to be
important mediators of anterior chamber
inflammation and elevated levels can be
detected in aqueous humor in a variety of
inflammatory conditions (Eakins et al., 1977).
In the posterior chamber intravitreal
administration of prostaglandin E l ,
prostaglandin E2 or arachidonic acid has been
shown to have an inhibitory effect on the rabbit
electroretinogram. Furthermore,
prostaglandins have been detected in normal
human vitreous humor and implicated in
cystoid macular oedema. The regulation of
prostaglandin production in the eye would
appear to be of considerable importance in
view of their role as physiological regulators.
Various materials have been tried as vehicles
of drug delivery. Any such drug delivery
system should be able to deliver the drug in a
slow sustained manner at the site of action,
maintaining the therapeutic concentration for
longer time, thus reducing not only the
frequency of administration but also chances
of toxicity. Biodegradation is another desirable
property of such materials. After the drug has
204
been released, particles should be removable
from the site of application primarily to avoid
toxicity and secondarily for better patient
compliance to remove foreign body sensation.
Materials like poly alkyl cyanoacrylate, poly-
ε -caprolactone, polysorbate 80(Zimmer,
Maincent et al., 1994), polylactic co glycolic
acid (Ding 1998), inert resin Eudragit RS 100
 (Pignatello et al., 2002), gelatin (Leucata
1989) and albumin (Lin and Garnett 2001,
Merodio et al., 2002, Langer et al., 2003) have
been used for the preparation of the
nanoparticles. The efficacy of poly acryl
cyanoacrylate, poly-ε-caprolactone and poly
ethylene glycol for betaxolol delivery was
compared and was found that poly-å-
caprolactone nanoparticles yielded highest
pharmacological activity and the factor
thought to be responsible for this good
performance was agglomeration of the
particles. In fact poly- ε-caprolactone is a
widely studied biodegradable polymer for
nanoparticles preparation and offer good
ocular tolerance. We selected albumin as a
material for nanoparticle because it is
biocompatible and procedure of preparation
of albumin nanoparticles has been
standardized (Merodio et al., 2002). It has been
widely used for preparing nanoparticles. Both
Bovine Serum Albumin or BSA (Merodio et
al., 2002, Zimmer et al., 1994) and Human
Serum Albumin or HSA have been used. As a
major plasma protein, albumin has a distinct
edge over other materials for nanoparticle
preparation. It is both bioacceptable and
biodegradable. Moreover it is cheap and easily
available. The process of preparing
nanoparticles from albumin is not complicated
and particle size can be controlled by various
factors during preparation. Drugs entrapped
 Saikat Das, Rinti Banerjee and Jayesh Bellare
in albumin nanoparticles can be digested by
proteases and drug loading can be quantified.
The objective of the present study was to
develop aspirin loaded albumin nanoparticles
for regional drug release in inflammatory
conditions of joints and posterior chamber of
eye for diabetic retinopathy. To characterize
the particles by size and other electrochemical
properties, diameter, zeta potential,
polydispersity were determined by photon
correlation spectroscopy. Since aspirin has
high affinity for albumin the role of protein
and aspirin albumin ratio on particle diameter
and colloidal electrical properties were
determined. The entrapment of aspirin in
albumin nanoparticles was also found out and
in vitro release kinetics across spectrapor
membrane was studied. Further
pharmacological efficacy in terms of platelet
aggregation was also evaluated.
Materials and Methods:
Material
The following commercially available
materials were used as received. Bovine
Serum Albumin (Sisco Research Lab.
Bombay), 25% Glutaraldehyde (Loba
Chemie Bombay) 99% Absolute Alcohol
(Changshu Yangyuan Chemical, China),
Mannitol (S K Lab. Ahmedabad),
Phosphate Buffer (Prepared from NaH2PO4
obtained from Marc India and Na2HPO4
obtained from Qualigen fine Chemicals).
Aspirin Powder used for the experiments
was of pharmaceutical grade.
Preparation of the formulation
Aspirin loaded albumin nanoparticle were
prepared by coacervation process followed
by crosslinking with glutaraldehyde
205
(Merodio et al., 2002). Merodio et al., had
developed the ganciclovir loaded albumin
nanoparticles and we evaluated the same
process for aspirin. Aspirin was incubated
with required amount of protein solution
(2% w/v) for one hour at room temperature.
The pH pf the solution was adjusted to 5.5
by 1M HCl using digital pH meter (Orion
meter model 720). Then ethanol was added
to the solution in 2:1 ratio (v/v). The rate
of the ethanol addition was carefully
controlled at 1ml/min (Langer et al., 2003).
The coacervate so formed was hardened
with 25% glutaraldehyde (1.56 µg/mg of
protein) for 2 hours to allow cross-linking
of protein. Organic solvents were then
removed under reduced pressure by Rotary
Vacuum evaporation and the resulting
nanoparticles were purified by
centrifugation (Beckman M 21) at four
degree centigrade. Three batches of
nanoparticles were centrifuged at different
speeds corresponding to 27000g, 17200g
and 10000g. However data shown here are
from the first batch (27000g), which we
found best suited for the procedure. Pellets
of nanoparticles were then suspended in
phosphate buffer (pH 7.4; 0.1 M) and each
sample finally was lyophilized with
mannitol (2% w/v) at –48 0 C and 28 X 10
–3
M Bar pressure for 24 hours (Freeze Dry
System/Free zone, 4.5 Labconco Model
117). Albumin nanoparticle controls were
also prepared by the same procedure
without drug loading.
Determination of particle size
Lyophilized nanoparticles were suspended
in PBS to make a 1% solution. Viscosity
 Aspirin Loaded Albumin Nanoparticles by Coacervation: Implications in Drug Delivery
of each solution was measured (Contraves
LS 30 coaxial cylindrical viscometer).
Refractive index was measured using a
Paulfrich Refractometer. Then particle
sizes, polydispersity, zeta potential were
measured by Photon Correlated
Spectroscopy or PCS (BI MAS, Multiangle
sizing option on Zetaplus, Brookhaven
Instruments USA) using dynamic light
scattering principles.
Transmission Electron microscopy (TEM)
Lyophilised nanoparticles were suspended in
phosphate buffer (pH 7.4) and was observed
by transmission electron microscope (Philips,
CM 200, Accelaration voltage 200kV,
resolution 0.23 nm).
Determination of Drug Entrapment
For determination of drug entrapment, the
amount of drug present in the clear supernatant
after centrifugation was determined (w) by
UV-spectrophotometry. A standard calibration
curve of concentration versus absorbance was
plotted for this purpose. The amount of drug
in supernatant was then subtracted from the
total amount of drug added during the
coacervation process (W). Effectively, (W-w)
will give the amount of drug entrapped in the
pellet. Then percentage entrapment is given
(W − w) × 100
by . platelets was studied after adding 5 µM of
W
Study of Release Kinetics
In vitro release kinetics study across spectrapor
membrane (cut off 3500 Da) precluding
albumin, was performed in a specially
designed diffusion chamber consisting of two
compartments separated by a septum with an
orifice of 20 mm diameter for the membrane.
10mg of drug-loaded nanoparticles (batch 2)
206
suspended in 15 ml phosphate buffer (pH 7.4)
was placed in the donor compartment and the
other compartment was filled with phosphate
buffer of same volume. To determine the
amount of aspirin diffused through the
spectrapor membrane, samples (1ml) were
withdrawn from the receiver solution at
prefixed times and the drug concentration was
measured spectrophotometrically at 230nm
(Schmidt et al., 1995). After each
determination, same amount of phosphate
buffer was reintroduced in the receiver
chamber (concentration change was accounted
for).
Platelet Aggregometry
15 ml blood was collected from a healthy
volunteer and platelet rich plasma (PRP) was
obtained by centrifugation at 100g for 10
minutes. PRP was incubated with 0.06mg/ml
of aspirin loaded albumin nanoparticles at 37°
C for one hour. This was done to check
whether the amount of aspirin diffused at the
end of 48 hours is sufficient to prevent platelet
aggregation. The minimum inhibitory
concentration of aspirin for prevention of
thromboembolism can be calculated taking
into consideration the volume of distribution
of aspirin after administration which is 0.17
L/kg body weight. The recommended dose of
aspirin for prevention of thromboembolic
disorders is 60-325 mg/day. Aggregation of
ADP to 450 µ l of PRP by Platelet
Aggregometer (Chronolog Corporation) after
½ hr and 1 hr of incubation. Results were
compared with PRP control without aspirin
addition. In all cases a baseline of platelet poor
plasma was obtained as a measure of the
supernatant during 100% aggregation.
Results:
The dimensions, drug content, zetapotential
and polydispersity of various samples are
reported in Table 1. For sample 2 which was
used in subsequent release kinetics study the
entrapment was calculated to be 81.1%
 Saikat Das, Rinti Banerjee and Jayesh Bellare

Table 1: Preparative variables and Results of Aspirin Loaded Albumin Nanoparticle

Sample Protein Aspirin Total (Drug Particle Zeta Polydispersity Sample

No. (mg) (mg) +Aspirin) mg Diameter (nm) Potential (mV) Quality
1 50 50 100 190.8 30.01 0.201 10
2 70 30 100 64.6 29.13 0.222 6.3
3 90 10 100 62.9 26.05 0.062 9.2
4 125 75 100 12.4 14.97 0.005 8.7
5 150 50 200 52.4 17.29 0.281 8.6
6 170 30 200 36.2 25.55 0.04 8.9
7 190 10 200 64.8 24.50 0.005 8.2
Literatures show the evidence that the particle
size is critically affected by the amount of
alcohol added, speed of addition of alcohol,
pH and amount of glutaraldehyde. Moreover,
the amount of protein and the drug play a role
on the determination of particle size,
polydispersity and sample quality.
Polydispersity indicates the degree of non-
uniformity of the particle size. Obviously a
low polydispersity indicates more uniformity
in size distribution. Sample quality is also Fig 2: Particle Size and Aspirin Protein Ratio
related to turbidity in addition to PCS data. Black line indicates best fit line.
Polydispersity. We tried to see how protein and (pH=7.4, at 27000g speed of centrifugation)
the drug are affecting these parameters. For
our samples the range of polydispersity was
0.005 to 0.281. For most of the samples zeta
potential was also in stable range (more than
20 mV). For most of the samples the quality
was above 8 in a scale of 10.

Fig 3: Zeta Potential and proportion of protein

Zeta potential was in the stable range when the proportion
of protein was 0.04 to 0.06 and 0.08 to 1.0. However in the
range 0.06 to 0.08 zeta potential was low.

One of the samples (sample 2) was analysed

Fig 1: Particle Diameter and Proportion of Protein by TEM. The Transmission Electron
Photon Correlation Spectroscopy data on the Effect Micrographs are presented below. Diameter
of amount of protein on Particle Size (pH=7.4, at determined by TEM 21nm corroborates with
27000g speed of centrifugation) that by PCS.

207
 Aspirin Loaded Albumin Nanoparticles by Coacervation: Implications in Drug Delivery
Bar = 200nm
Fig 4: Transmission electron micrograph of
aspirin loaded albumin nanoparticles
Total cumulative release at prefixed tome
intervals in terms of percentage entrapment
was calculated and plotted in Fig 5. When we
compare the graph with that of only aspirin
solution of same amount of aspirin as
contained in the donor compartment in
nanoparticles form we found a release kinetics
which mimics pulse delivery kinetics and peak
concentration was achieved in almost ½ hr.
Discussion:
The physicochemical parameters, which
influence the particle size during the
coacervation process, have been reviewed in
the literature (Weber et al, 2000, Langer et al
2003). Studies indicate that the particle size
and the electrical properties of albumin
nanoparticles can be controlled by variation
of the preoperative parameters. The volume
of ethanol added during the desolvation
process affects both the particle size and
particle counts. We selected a 2:1 v/v ratio of
ethanol and protein solution to optimize the
process, as at this ratio the desolvation process
is 90% complete (10% undissolved protein,
Weber et al, 2000). Percentage of dissolvated
HSA in the supernatant significantly decreases
up to 25% of glutaraldehyde, beyond which
208
the undisolved nanoparticle amount is almost
constant. Pharmacokinetics of aspirin shows,
it binds with the ε-lysine amino group of
albumin. Therefore addition of excess
glutaraldehyde significantly compromise
loading of drug in the protein. However at low
concentration of glutaraldehyde significant
amount of albumin remain undisolved. (Weber
2000, Langer 2003) It may be mentioned that
pH also influence the zeta potential. Optimum
stability of zeta potential is obtained at around
pH 7 and beyond. We chose pH 7.4 because
under physiological condition pH of most of
the body fluids is around 7.4.
Figure 1 indicates that increasing the
proportion of protein significantly reduces the
particle size but the graph also shows that in
the latter half, the proportion of protein does
not have much influence on the particle
diameter. So the optimum proportion of protein
seems to be 0.5. The trend of both the results
are consistent with the earlier findings of
Langer 2003 using albumin nanoparticles.
Zeta potential is significantly affected by the
amount of protein and aspirin. In the
coacervation process, glutaraldehyde and
aspirin both compete for the amino groups of
the protein. We fixed the concentration of
glutaraldehyde at 1.56 µg / mg of protein. At
this concentration, sufficient number of amino
groups is free for aspirin binding even after
binding of all the glutaraldehyde to albumin.
Figure 3 shows the effect of proportion of
protein on the zeta potential of the particles.
Higher zeta potential (15 – 30 mV) is
associated with high stability of the colloidal
system. From the figure it may be noted that
at low proportion (0.4 to 0.6) and at high
proportion (0.8 to 1) of protein zeta potential
 Saikat Das, Rinti Banerjee, Jayesh Bellare

is in the range of stability (more than 15 mV).

However it is unclear why in the range of 0.6
to 0.8 the particles have low zeta potential.
Nevertheless we can say that the relative
amount of aspirin and albumin plays a
significant role in determining the over all
electrical properties of the nanoparticles. This
parameter of the effect of drug content on the
size and stability of nanoparticles has not been
addressed in the present literature.
The results indicate that when pH, amount of
glutaraldehyde, ethanol, rate of addition of
ethanol and salt concentration are constant the
diameter and electrical properties of
nanoparticles are determined by the percentage
of protein and aspirin protein ratio. Our study
shows that albumin has distinct advantage as
a material of nanoparticles over others because
the diameter and electrical properties can be
adjusted by variation of the preparative
variable to suit the purpose of use. By varying
aspirin albumin ratios from 0.06 to 1.0 we
obtained stable nanoparticles of sizes 46.8 nm
to 190.8 nm respectively with low
polydispersity.
Aspirin loaded albumin nanoparticles obtained
by coacervation are capable of releasing the
drug in a sustained and prolonged manner and
the drug is pharmacologically active (Figs 5
and 6). Whereas the simple drug solution
follows pulse release kinetics with peak
concentration at the end of ½ hr, the drug
released from nanoparticles follow prolonged
release kinetics with concentration building up
gradually.
Fig 5: In vitro release kinetics of aspirin loaded
albumin nanoparticles across
spectrapor membrane
The drug is released in a sustained manner slowly over
days. Total cumulative release at the end of 48 hrs 50%,
at 72 hrs is almost 90%. Also there is no “burst effect”
as found in the kinetics of free drug and the kinetics
show two plateaus.
From Fig 5 it can also be seen that the
nanoparticles formulation overcomes the burst
effect observed in the release kinetics of free
drug and also the kinetics has two phases (two
plateau kinetics). Therefore, the fraction
released during the first 10-20 hours could
correspond to the fraction of the free drug that
is released without control from the carrier.
This represents the aspirin that is physically
adsorbed on the surface of the nanoparticles.
However the drug which is covalently bound
to the nanoparticles continue to release in a
sustained manner and the total amount of drug
released in terms of percentage entrapment is
almost 90% at the end of 72hrs. It may have
significant implications in clinical settings
because it not only avoids sudden pulse release
of drugs, but also maintains the concentration
for prolonged duration. In practical situation
it would mean reduced toxicity, lesser frequent
doses of application and therapeutic coverage
for prolonged duration. This types for
formulations not only hold promise for topical
applications (like ocular use) but also for those

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 Aspirin Loaded Albumin Nanoparticles by Coacervation: Implications in Drug Delivery
patients who are dependent on life long
NSAIDS for chronic joint disorders. Similar
kinetics has been observed by Leo et al., 1999
with doxorubicin loaded gelatin nanoparticles.
Aspirin acetylates cyclooxygenase and
Thromboxane synthetase, enzymes involved
in prostaglandin synthetic pathway. In addition
to this it inhibits the release of ADP from
platelets and their sticking to each other.
Inhibition of platelets by aspirin is irreversible
and is the basis of its use in diseases like
diabetic retinopathy and thromboembolic
disorders. Platelet aggregometry study (Fig 6)
shows that aspirin released from the
nanoparticles inhibit the platelet aggregation
in a time dependent manner. Prevention of
platelet aggregation was almost 60% at the end
of one hour compared to control PRP. This
shows that drug loaded nanoparticles are
pharmacologically active and sustained release
of aspirin from the carrier prevents platelet
aggregation.
Fig 6: Platelet Aggregometric Study
Horizontal axis: Time; Vertical Axis: Degree of platelet
aggregation (in arbitary unit) on addition of ADP.
Left panel: control PRP aggregation. Middle Panel and
right panel: PRP incubated with 0.06mg/ml conc. of
aspirin; Middle panel: after ½ hr incubation. Right
panel 1hr incubation. After 1hr platelet aggregation is
3 times less than the control.
210
Aspirin is one of the oldest analgesic-anti-
inflammatory drugs and is still widely used.
It is used in various joint disorders including
osteoarthritis, rheumatoid arthritis to prevent
chronic deformity, which occurs due to
inflammation, synovial proliferation and
erosion of bone. Not only the dose of aspirin
used in these conditions is very high (3 – 5 g/
day), in most of the cases the duration of the
treatment is also long. It is also routinely
prescribed for various thromboembolic
disorders and is recommended for primary
prophylaxis like in diabetic retinopathy. There
is need for a local sustained release
formulation in joint and ocular diseases
avoiding pulse entry, side effects due to
prolonged use, large doses and frequent
dosing. Our study shows that aspirin loaded
albumin nanoparticles can be useful in these
situations because they are small in size (46
to 190.8 nm in diameter) which makes them
suitable for topical delivery and they are
capable of releasing the drug in slow sustained
manner over days.
Conclusions:
Aspirin loaded albumin nanoparticles of sizes
46.8 nm to 190.8 nm were prepared by varying
the aspirin protein ratio from 0.06 to 1.0. These
particles were stable as indicated by their zeta
potentials and uniform characterized by low
polydispersity (0.005 to 0.281). In vitro release
kinetics study shows that aspirin loaded
albumin nanoparticles are capable of releasing
the drug in a slow sustained manner (almost
90% release at the end of 72 hrs). Platelet
aggregometric study clearly shows their
pharmacological efficacy. Based on these
findings we can conclude that aspirin loaded
albumin nanoparticles are promising agents for
regional drug delivery in inflammatory
conditions of joints or thromboembolic
manifestations of eye like diabetic retinopathy.
 Saikat Das, Rinti Banerjee and Jayesh Bellare

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