Polymer surface-coated nanoparticles for brain targeting

Double-Coated Poly (Butylcynanoacrylate) Nanoparticulate
Delivery Systems for Brain Targeting of Dalargin
Via Oral Administration
DEBANJAN DAS, SENSHANG LIN
College of Pharmacy and Allied Health Professions, St. John’s University, Jamaica, New York, 11439
Received 5 October 2004; revised 23 February 2005; accepted 23 February 2005
Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20357
ABSTRACT: The aim of this study is to evaluate oral administration of poly (butylcy-
anoacrylate) nanoparticulate delivery systems (PBCA-NDSs), double-coated with Tween
80 and poly (ethylene) glycol (PEG) 20000 for brain delivery of hexapeptide dalargin,
an anti-nociceptive peptide that does not cross blood–brain barrier (BBB) by itself.
Studies have proven the brain uptake of Tween 80 overcoated nanoparticles after
intravenous administration, but studies for brain delivery of nanoparticles after oral
administration had been limited due to reduced bioavailability of nanoparticles and
extensive degradation of the peptide and/or nanoparticles by gastrointestinal enzymes.
To address this problem, dalargin-loaded PBCA-NDS were successively double-coated
with Tween 80 and PEG 20000 in varied concentrations of up to 2% each. Measurement of
in vivo central anti-nociceptive effect of dalargin along with a dose response curve was
obtained by the tail flick test following the oral administration of PBCA-NDSs to mice.
Results from the tail flick test indicated that significant dalargin-induced analgesia was
observed from PBCA-NDSs with double-coating of Tween and PEG in comparison with
single-coating of either Tween or PEG. Hence, it could be concluded that surface coated
PBCA-NDS can be used successfully for brain targeting of dalargin or other peptides
administered orally. However, further studies are required to elucidate the exact
transport mechanism of PBCA-NDSs from gastrointestinal tract to brain.
ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1343–1353, 2005
Keywords: brain targeting; blood–brain barrier; oral absorption; nanoparticles;
peptide delivery; surfactants; dalargin; butylcyanoacrylate; Tween 80; PEG
INTRODUCTION
Number of individuals who suffer from chronic
diseases of the brain is more than the number of
people stricken with cancer and heart disease
combined. This large population suffering from
chronic brain disorders such as Alzeimer’s,
Depression/Mania, Schizophrenia, Parkinson’s,
and HIV infection to name a few, poses the need
and opportunity for the growth of brain-targeted
neuropharmaceuticals. Due to the presence of
epithelia-like tight junctions lining the brain
capillary endothelium or the so called blood–
brain barrier (BBB), more than 98% of all new
potential brain drugs do not cross the BBB.1,2
In the areas of brain delivery of drugs, there have
been a number of approaches to overcome the
BBB, such as the osmotic opening of tight
junctions,3
usage of prodrugs, and carrier systems
like targeted antibodies,4
liposomes,5–7
and nano-
particles. For almost a decade, surfactant coated
nanoparticles have been reported successfully to
transport drugs across the BBB.8–12
Nanoparticle-
mediated drug transport depends on the coating
of the particles with polysorbates, especially poly-
sorbate 80 (Tween 80). Overcoating with these
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005 1343
Correspondence to: Senshang Lin (Telephone (718)-990-
5344; Fax: (718)-990-6316; E-mail: linse@stjohns.edu)
Journal of Pharmaceutical Sciences, Vol. 94, 1343–1353 (2005)
ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association

materials leads to the adsorption of apolipopro-
tein E from blood plasma onto the nanoparticle
surface. The particles then seem to mimic low-
density lipoprotein (LDL) particles and interact
with the LDL receptor leading to their uptake by
the endothelial cells lining the BBB.13,14
Then,
the drug bound to the nanoparticles may be
released in these cells and diffuse into the interior
or the nanoparticles may be transcytosed. In ad-
dition, it has been suspected that processes such
as tight junction modulation or P-glycoprotein
active efflux system also may occur resulting in
brain uptake of nanoparticles. Up to date, many
different surfactants15
have been evaluated. Only
Tween 80 overcoat has been able to produce the
most brain targeting effect via intravenous
administration16
and the specific role of Tween
80 in brain targeting has also been conclusively
proved.17
However, studies on administration of
such nanoparticles orally have been restricted
due to the degradation of the drug and/or the
polymer nanoparticles in the gastrointestinal
media as well as due to the limited uptake of
nanoparticles across the gastrointestinal mem-
brane. So far, only one study has been reported
where nanoparticles is administered orally and
observed for brain delivery.18
The drug chosen
is Leu-enkephalin analog hexapeptide dalargin
(Tyr-D-Ala-Gly-Phe-Leu-Arg, MW 725.9) which
normally does not cross BBB by itself even after
intravenous administration.8–11
The anti-noci-
ceptive effect produced in mice brain after oral
administration of this peptide-loaded nanoparti-
cles has not been pronounced but rather pro-
longed.17
Moreover, there was no information on
the dose of dalargin used and the formulation
development especially designed for delivery of
nanoparticles through the oral route as well as
the characterization of nanoparticle formulations
by measurement of zeta potentials, release profile,
and stability in simulated gastric and intestinal
fluids.
The objective of this study was hence aimed
at brain targeting of the model peptide drug,
dalargin, via oral route. For such an objective, a
polymeric nanoparticulate drug delivery system
composed of poly (butylcyanoacrylate) (PBCA) was
fabricated. PBCA nanoparticles are expected to
be biodegraded rapidly in the body without caus-
ing any significant toxicity. Therefore, long-chain
alkylcyanoacrylates, such as n-butylcyanoacry-
late, are commercially available as Indermil1
and Liquiband1
in Europe, Canada, and USA,
while octylcyanoacrylate markets as Dermabond1
in USA.16,31
For the convention of terminology,
such nanoparticulate drug delivery systems made
with PBCA were termed as PBCA-NDSs. PBCA-
NDSs were loaded with drug and surface coated
with polyoxyethylene sorbitan monooleate (Tween
80) and poly (ethylene) glycol 20000 (PEG 20000)
in varying concentrations of up to 2% each. The
necessity of Tween 80 overcoat to affect brain
targeting of nanoparticles has been reported. In
addition to the coating of Tween 80, the second
coating of PEG 20000 was added. The rationale
of the second coat of PEG (i.e., PEGylation) was
employed for twin reasons. Firstly, PEG was
expected to protect the peptide-loaded nanoparti-
cles in the hostile gastrointestinal milieu, which
comprises of enzymes and varying levels of pH.19,20
Secondly, once nanoparticles reach the circula-
tion, PEG was expected to increase the circulation
half-life of the nanoparticles by the ‘‘dysopsonic’’
action of the long PEG chains thereby protecting
it from the rapid clearance by the reticulo-
endothelial system and mononuclear macrophage
system.21–24
This investigation was hence, aimed
to determine the feasibility of designing PBCA
nanoparticles double-coated with Tween 80 and/or
PEG 20000 for targeted delivery of peptide to brain
after oral administration.
MATERIALS AND METHODS
Materials
The monomer solution containing n-2-butylcya-
noacrylate (density 0.9580 at 208C) used for
polymerization and fabrication of PBCA-NDSs
was purchased from Glustitch Inc. (Delta, British
Columbia, Canada). Dalargin (MW 725.9) was
obtained from CSPS Pharmaceuticals Inc. (San
Diego, CA). Dextran 70 (MW 68800), naltrexone
HCl, sodium chloride, pepsin, monobasic potas-
sium phosphate, pancreatin, Mammalian Ring-
er’s solution (MRS) consisting of sodium chloride
0.96%, potassium chloride 0.04%, calcium chloride
0.03%, sodium bicarbonate 0.02%, and water
98.95%; and phosphate buffer solution (PBS)
consisting of bisodium phosphate/monobasic
potassium phosphate/sodium chloride at ratio of
7.6:1.45:4.8 w/w/w, were obtained from Sigma
Chemical Co. (St. Louis, MO). PEG 20000, What-
man glass microfiber filters (1.2 and 0.7 m) and
Whatman inorganic membrane Anotop filters
(0.02 m) were purchased from VWR International
(West Chester, PA). Mice (out-bred, albino, female
Swiss Websters, 20–25g) were obtained from
1344 DAS AND LIN
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005

Taconic Farms (Germantown, NY). Nanopure1
water (Ultrapure Water System, Barnstead,
Dubuque, IA) was used for the preparation of nan-
oparticles. All other reagents were of analytical
grade.
Fabrication, Drug Loading, and
Double-Coating of PBCA-NDSs
Typically, an anionic polymerization method was
followed8–12,14,15,18
using 0.01N HCl solution in
Nanopure1
water. Dextran 70 (1.5% w/w) was
added to it under constant magnetic stirring.
Once dextran 70 was completely solubilized in
the HCl solution, butylcyanoacrylate monomer
solution (1% v/v) was added dropwise. After 4 h of
polymerization, the milky nanoparticle solution
was neutralized with sodium hydroxide (0.1N)
and the solution was further stirred for 12 h to
ensure complete neutralization. The nanoparticle
suspension obtained was subjected to a series of
filtration steps using 5, 1.2, and 0.7 m filters by
means of a vacuum filtration assembly. The
filtered solution was ultracentrifuged for three
cycles, 1 h each at 75600g (Beckman Avanti J-25,
Fulerton, CA) with Rotor (Beckman Model Num-
ber JA 25.50). Finally, the pelleted nanoparticles
were lyophilized overnight and stored at 48C for
drug loading and subsequent surface treatments.
Drug loading on PBCA-NDSs was done by
adsorption method8–10
and was carried out in
15 mL of MRS, which is better representative of
cerebrospinal fluid. The porous nature of PBCA-
NDS25
enabled loading of dalargin by continuous
stirring of drug with PBCA-NDS in aqueous
media. Fifty micrograms of lyophilized PBCA-
NDS were re-suspended by ultrasonicating at
4.2 Khz/s for 5 min, which contained dalargin at
a concentration of 133 mg/mL. The peptide was
allowed to absorb into the nanoparticle surface for
3 h with continuous magnetic stirring at 9000 rpm.
The amount of peptide adsorbed on nanoparticles
was determined by filtering the suspension
through a 20 nm Anotop filter and the amount of
free, un-adsorbed peptide in the filtrate was
measured by UV spectroscopy. The difference of
total added drug and the amount of free or un-
adsorbed drug gave the amount of drug adsorbed/
entrapped with the PBCA-NDS. All samples were
analyzed by UV-VIS-IR spectrophotometer (model
number 14NT-UV-VIS-IR, AVIV Instruments,
Lakewood, NJ) at a preset wavelength of 220 nm
where a sharp peak, characteristic of dalargin was
obtained.8,15
The dalargin-loaded PBCA-NDSs were coated
successively with varying concentrations of up
to 2% of Tween 80 and PEG 20000 relative to the
total suspension volume of nanoparticles (Table 1).
Depending on the amount of coating of Tween and
PEG used different formulations such as T1P1
(with 1% of Tween and PEG each) or T2P2 (with
2% of Tween and PEG each) were assigned. For
each formulation, required quantities of Tween
and/or PEG were added stepwise in the above
solution under continuous magnetic stirring at
9000 rpm for 45 min. Thereafter, the solution was
centrifuged at 75600g for 20 min, the supernatant
containing un-adsorbed drug, as well as excess
Tween 80 and /or PEG 20000 was discarded. Then,
the double-coated dalargin-loaded PBCA-NDSs
were collected, lyophilized, and stored at 48C for
further use.
Characterization of PBCA-NDSs
Sample (1 mg) of dried powder obtained from the
above step was suspended in 5 mL of Nanopure
water by ultrasonication at 4.2 KHz/s for 5 min.
The homogenous suspension obtained was ana-
lyzed for particle size, size distribution, and zeta
potential by dynamic light scattering (Nicomp
380 DLS, submicron particle-sizer, Santa Bar-
bara, CA). A run time for 30 min each was allowed
for each observation, which allowed complete
stabilization of surface charge and hence, lead to
accurate measurements.
In Vitro Release Kinetics
For each formulation, 50 mg of dried powder
obtained previously was suspended in 15 mL MRS
using ultrasonication described as in previous
steps. The drug loaded and double-coated PBCA-
Table 1. Concentrations of Tween 80 and PEG 20000
Used for Double Coating of Dalargin-Loaded
PBCA-NDSs
Formulation Code Tween 80 (%)a
PEG 20000 (%)a
T0P0 0.0 0.0
T2P2 2.0 2.0
T1.5P0.5 1.5 0.5
T1P1 1.0 1.0
T0.5P1.5 0.5 1.5
T0P2 0.0 2.0
T2P2 2.0 2.0
a
Relative to the total suspension volume.
BRAIN TARGETING OF DALARGIN 1345
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NDS was placed in 15-mL screw capped tubes
and kept in a water shaker bath (Thermo Forma,
Marietta, OH), which was maintained at 378C and
at 130 cycles per min. A sample volume of 2.5 mL
was collected at predetermined time intervals
through 20 nm Anotop syringe filters and the
nanoparticle-free filtrate was analyzed for drug
content by UV spectroscopy described previously.
The sampling regimen had the following pattern:
every 15 min for the 1st h, every 30 min till the 6th
h, every 1 h till the 10th h, every 2 h till the 18th h,
every 4 h till the 34th h, and every 8 h till the end
of 50th h.
Drug Stability in Simulated Gastric and
Intestinal Fluids
The stability of peptide loaded PBCA-NDSs with
or without various coating agents were evaluated
in simulated gastric fluid (SGF) and simulated
intestinal fluid (SIF). SGF and SIF were prepared
according to USP XXVI. Briefly, SGF was pre-
pared by dissolving 2 g of NaCl and 3.2 g of pepsin
(derived from porcine stomach mucosa with an
enzyme activity of 800–2500 units per mg of
protein) in 7 mL HCl and finally made up 1000 mL
with adjustment of final pH to 1.2. SIF was
prepared by dissolving 6.8 g of monobasic potas-
sium phosphate in 250 mL water. And then, 77 mL
of 0.2N NaOH, 500 mL of water, and 10 g of
pancreatin were added. The pH was adjusted to
6.8 Æ 0.1 with 0.2N NaOH and/or 0.2N HCl.
Pancreatin was obtained as ‘‘Pancreatin Porcine
Pancreas USP’’ containing many enzymes such
as amylase, trypsin, lipase, ribonuclease, and
protease.
Fifty micrograms of each formulation of PBCA-
NDSs was suspended in 15 mL of either SGF or SIF
and placed in screw-capped tubes. The tubes were
kept in a water shaker bath, which was main-
tained at 378C and at 130 cycles per min. A specific
time period of incubation of drug-loaded PBCA-
NDS in SGF and SIF were allowed, which were 3 h
for SGF and 12 h for SIF, respectively. After these
time periods, suspensions were centrifuged at
75600g for 20 min to precipitate the PBCA-
NDS and the supernatants were discarded. The
precipitated drug-loaded PBCA-NDSs were re-
dispersed in MRS. A rigorous cycle of 20 min of
ultrasonication at 4.2 KHz/s and 5 min of vortexing
was subjected towards the nanoparticulate sus-
pension. Such cycles were carried 20 times to
ensure near complete desorption of drug from the
PBCA-NDS. Hence, the amounts of remaining or
the protected drug after the incubation of 3 h in
SGF and 12 h in SIF from each formulation were
determined. In addition, for the Formulations
T2P2 (containing 2% Tween 80 and PEG 20000
each) and T0P0 (absence of Tween and PEG), the
drug stability as a function of time was carried out
in SGF and SIF, where samples were incubated for
a specific period of time such as 5 min, 10 min,
15 min, 30 min, 1 h, 2 h, and finally 3 h in SGF and
5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and
finally 12 h in SIF, respectively. Remaining drug in
PBCA-NDS after such periods of incubation was
detected as described previously.
In Vivo Evaluation of Double-Coated
Dalargin-Loaded PBCA-NDSs—Tail Flick Test
Dalargin, which causes a central analgesic effect
in brain by binding with m opioid receptors
for pain perception, was expected to be released
from dalargin-loaded PBCA-NDS once they were
taken up in the brain. Hence, occurrence central
analgesic effect would prove the brain targeting of
PBCA-NDS after oral administration. Groups of
ten mice for each formulation were selected. All
mice were kept at ambient temperature and
humidity conditions with a 12-h light and dark
cycle and fasted overnight. Each mouse was fed
with 1 mL of drug-loaded PBCA-NDSs suspension
by oral gavaging. The dose administered corre-
sponded to 37.5 mg/kg of mouse body weight,
which was about five-fold of usual intravenous
dose for dalargin having central analgesic
actions.8
Tail was immersed in hot water main-
tained at 55–608C by a hot plate. The response
times, in seconds, taken by each mouse to with-
draw its tail by a sharp ‘‘flick’’ were recorded using
a stopwatch. The response times were then con-
verted to percentage maximum possible effect (%
MPE) by method reported elsewhere.14,15
In total
seven controls and nine formulations were eval-
uated (Table 2). Formulation T2P2þA indicates
that naltrexone HCl, an opioid antagonist (A)
with high oral bioavailability, was co-adminis-
tered at a dose of 0.1 mg/kg with Formulation
T2P2. A perception of pain would signify hence
the effect of naltrexone in brain, which displaces
dalargin from its pain receptors. This was done to
prove the presence of dalargin in brain mainly
from the drug-loaded PBCA-NDS targeted to the
brain as well as to re-establish the fact that
increase of pain threshold was caused only by
centrally acting and not by peripherally acting
mechanisms.
1346 DAS AND LIN

Dose Response Curve of Dalargin
In order to reconfirm the brain uptake and release
of dalargin from surface coated PBCA-NDSs,
a dose response study was designed with the
Formulation T2P2 that showed the maximum
anti-nociceptive effect after dosing. Groups of
ten mice each were taken and each group was
administered with varying doses of dalargin
from 7.5 to 52.5 mg/kg and observed for anti-
nociceptive effect after 60 min of dose adminis-
tration. The response times were converted to %
MPE as described above and a dose response
curve of dalargin was constructed.
Statistical Analysis
All results were expressed as mean Æ standard
deviation. A one-way ANOVA test using Statmost
3.0 (Datamost Corporation, Sandy, UT) was done to
assess any statistically significant difference among
the means of % MPE of various formulations of
PBCA-NDS in the tail flick test. A post-hoc analysis
(Duncan’s Test) was performed to determine the
groups, which show significant difference. In each
case, a p-value less than 0.05 was considered as a
representation of significant difference.
RESULTS AND DISCUSSIONS
Fabrication, Drug Loading, and
Double-Coating of PBCA-NDSs
PBCA-NDSs were obtained as a free flowing
powder and the yield was found to be 23% w/w
calculated on the initial weight of monomer
solution used. Other investigators had reported
entrapment efficiency in similar systems to be
around 25%–30% w/w.8,9
In our study, a higher
mean entrapment efficiency of 39.84 Æ 4.00% w/w
was obtained. The occurrence of higher values of
entrapment efficiency could be attributed to
smaller size ranges of nanoparticles (around
100 nm) obtained in this investigation than that
(230–260 nm) obtained by other investigators.
Smaller size ranges ensured more available sur-
face area for the adsorption of the drug on the
nanoparticle surface. Since the entrapment effi-
ciency was about 40% w/w and dalargin was
added in the concentration of 133 mg/mL to a
15 mL nanoparticle solution containing 50 mg of
nanoparticles, the amount of drug present in the
pelleted nanoparticles were 798 mg per 50 mg of
nanoparticles. This amount was used to study the
in vitro release kinetics and the stability studies
in SGF and SIF.
Characterization of PBCA-NDSs
All double-coated PBCA-NDSs formulations had
mean particle sizes of about 100 nm with a low
polydispersity index around 0.018. The uniform
size range and low polydispersity index obtained
could be attributed to the serial filtration steps
employed during the preparation and isolation of
nanoparticles from the reaction media. It can be
worthwhile to note that effect of double coats of
Tween and/or PEG did not have any significant
effects on the particles size of the nanoparticles.
Table 2. Formulations of PBCA-NDSs Used in the Tail Flick Test
Formulation Code Summary
C1 Phosphate buffer solution (PBS)
C2 PBS þ Tween (2%)
C3 PBS þ PEG (2%)
C4 PBS þ Tween (2%) þ PEG (2%)
C5 PBS þ drug
C6 PBS þ drug þ Tween (2%)
C7 PBS þ drug þ PEG (2%)
T2P2-N PBS þ drug þ Tween (2%) þ PEG (2%) þ no nanoparticles present
T0P0 PBS þ drug þ nanoparticles þ Tween (0%) þ PEG (0%)
T1.5P.5 PBS þ drug þ nanoparticles þ Tween (1.5%) þ PEG (0.5%)
T.5P1.5 PBS þ drug þ nanoparticles þ Tween (0.5%) þ PEG (1.5%)
T2P2 þ A PBS þ drug þ nanoparticles þ Tween (2%) þ PEG (2%) þ
naltrexone HCl (antagonist)

The uncoated particles (Formulation T0P0) had
the similar range of particles sizes as that of
double-coated particles with 2% of both Tween
and PEG (Formulation T2P2). Since the nano-
particle diameter did not change significantly
after coating, the exact nature of orientation of
Tween and/or PEG molecules upon naked PBCA-
NDS needs to be further investigated. However, it
can be assumed that Tween and/or PEG did not
interact with porous, polymeric PBCA nanoparti-
cles, which could have brought about a deviation
from the constant size ranges of all PBCA-NDSs
formulations.
The mean zeta potentials of different formula-
tions varied from À18.01 to À2.44 mVs (Figure 1).
The uncoated PBCA-NDS (Formulation T0P0)
had the highest negative zeta potential value of
À18.01 mV and the double coated PBCA-NDSs
with 2% Tween and 2% PEG (Formulation T2P2)
had the lowest negative value of À2.44 mVs.
Interestingly, the zeta potentials of PBCA-NDSs
showed a positive shift with increase in the coat-
ing concentrations of PEG. The shift of the shear
plane further away from the surface of a nanopar-
ticulate moiety results in a positive shift of the net
zeta potential has been reported.17,19,26
Hence,
based on the observed trend of positive shift, due to
increasing PEG concentrations, it can be sus-
pected that PEG might cause a shift of shear plane
further away from the nanoparticle surface caus-
ing the positive shift of the net zeta potentials.
It is also worthwhile to note that a high negative
zeta potential value is optimal for stabilization of
colloidal carriers, preventing their aggregation
in solution. However, since the double-coated
PBCA-NDS (Formulation T2P2) had the mean
zeta potential value of À2.44 mV and other
formulations with some concentrations of PEG
showed a positive shift, nanoparticle suspensions
prepared with such formulations were prone to
particle agglomeration in aqueous media. To re-
solve this problem, formulations except for For-
mulation T0P0 were ultrasonicated at 4.2 kHz/s
for a minute to ensure the homogenous dispersion
of nanoparticles.
In Vitro Release Kinetics
All formulations showed characteristic biphasic
release with an initial burst release followed by a
second phase with a much slower rate of drug
release (Figure 2). Release of dalargin from
PBCA-NDSs was due to gradual desorption of
adsorbed drug from the surface of the nanoparti-
cles. However, release rate of the drug was
different for each formulation suggesting that
drug had to diffuse through the polymer and
surfactant coatings employed upon the PBCA-
NDSs. After the first 3 h, except for T0P0, all
other formulations had almost similar release
rates. This enforces our findings that outward
Figure 1. Zeta potentials of different formulations of
PBCA-NDSs (n ¼ 3).
Time (hours)
0 10 20 30 40 50
CumulativeAmountReleased(%)
0
20
40
60
80
100
T0P0
T2P0
T1.5P0.5
T1P1
T0.5P1.5
T0P2
T2P2
Figure 2. Release profile of dalargin from different
formulations of PBCA-NDSs over a 50-h time span
(n ¼ 3, error bars were shown only at last two data points
to maintain clarity).
1348 DAS AND LIN

release of drug is indeed a function of polymer
and/or surfactant coatings.
The highest amount of drug release (82.03 Æ
6.33%) at 50-h of release study was obtained from
the PBCA-NDSs without any coating (Formula-
tion T0P0). With 2% coating of PEG 20000
(Formulation T0P2), the release rate was lowest
and was reduced to 50.23 Æ 4.26% over the same
period of time. A trend of decrease in release rate
with the increase in PEG coating concentration
was observed. This trend could be attributed to the
fact that the outward release of drug could be a
function of coating concentrations of PEG and not
of Tween. Drug can be imagined to slowly diffuse
out through the polymer coating, and more the
PEG coating concentration, lesser the percentage
release. Some investigators21
have reported that
folding of PEG chains occurring above a certain
molecular weight form a barrier consisting of con-
formationally random PEG chains. Furthermore,
such a folding results in unfavorable entropy
changes, which further results in compression
and stability of the coating layer.21
It can be
imagined the existence of a similar sort of a
‘‘barrier’’ caused by increasing coating concentra-
tions of PEG and resulted in the reduction of
release rate. The exact nature of such a barrier
formed by random PEG coils needs to be further
subjected to structural analysis.
Taking into account the mean zeta potentials,
Formulation T0P0 that had the highest amount of
release of 82.03% at 50-h also had the most
negative zeta potential of À18.01 mV. Formulation
T0P2 with the lowest release of 50.23% at the end
of 50 h had a near zero zeta potential of À3.61 mV.
The results indicate that higher the zeta potential,
higher the release rate at the end of 50 h time span.
Thus, it can be surmised that zeta potential had an
effect on the release profile of different formula-
tions. These findings perhaps indicate that a high
negative zeta potential ensured a better release
rate for uncoated formulation than other coated
formulations of PBCA-NDS, which might aggre-
gate over a period of time and slowed down the
drug release rate.
Drug Stability in Simulated Gastric
and Intestinal Fluids
In this investigation, drug-loaded PBCA-NDSs
were developed for oral administration, it was
important to estimate the protective effect of
double coats of PEG and Tween on the labile
nature of the peptide drug and the polymer. When
different formulations were evaluated after 3 h
of incubation in SGF (Figure 3), it was observed
that percentage of drug remaining was 86.77 Æ
1.52% for Formulation T2P2 in comparison to
65.38 Æ 2.22% for the Formulation T0P0. This
finding suggests that the percentage of drug pro-
tected increased with increasing concentrations
of PEG coating upon the PBCA-NDSs. A time
dependent stability study for 3 h in SGF (Figure 4)
for the Formulations T0P0 and T2P2 further
showed the protective effect of the PEG coating.
0
10
20
30
40
50
60
70
80
90
100
T0P0 T2P0 T1.5P0.5 T1P1 T0.5P1.5 T0P2 T2P2
PBCA-NDS Formulations
DrugRemaining(%)
Figure 3. Stability of dalargin in various formula-
tions of PBCA-NDSs after 3 h of incubation in simulated
gastric fluid (SGF) (n ¼ 3).
Time (hours)
1 2 3
DrugRemaining(%)
60
65
70
75
80
85
90
95
100
T2P2
T0P0
Figure 4. Comparative stability profiles of dalargin
in Formulations T2P2 and T0P0 in SGF (n ¼ 3).

Similarly, after the incubation of all different
formulations in SIF for 12 h (Figure 5), the percen-
tage of drug remaining increased as a function of
increasing concentrations of PEG coating. More-
over, a time dependent stability study of the
Formulations T0P0 and T2P2 (Figure 6) for 12 h
in SIF also indicated the protective action of
PEG coating. Thus, in both simulated gastric and
intestinal fluids, it was observed that with
increase in PEG 20000 coating, the percentage of
drug protected increased. In addition, it was also
interesting to note that even with the increase
of Tween alone, there had been an increase in
protective action, but not as significant as that of
PEG. For instance, after 3 h of incubation in SGF,
the percentage of drug remaining for Formula-
tions T0P0, T2P0, and T0P2 were 65.38 Æ 2.22%,
72.66 Æ 1.13%, and 85.02 Æ 1.56%, respectively.
Similarly, after 12 h of incubation in SIF, the
percentage of drug remaining for Formulations
T0P0, T2P0, and T0P2 were 42.57 Æ 1.16%, 65.02 Æ
1.45%, and 75.55 Æ 1.195%, respectively. Results
suggest that the enzyme repulsion ability was not
only contributed by the PEG but also by the Tween.
PEG was well known to form a ‘‘brush,’’ which
prevents the docking of enzymes or macrophages
on hydrophobic surface of a carrier polymer. It can
hence be hypothesized that the long chains of
Tween 80 could have also formed such a protective
brush and prevented the degradation of the drug.
The increased surface density of long chained
molecules such as Tween and PEG was able to
exert the protective effect upon the drug-loaded
PBCA-NDSs from gastrointestinal enzymes.
In Vivo Evaluation of Double-Coated
Dalargin-Loaded PBCA-NDSs—Tail Flick Test
In this test, time points for all observations
spanned for a total of 2 h and at 15 min intervals
(Figure 7). A baseline response time was recorded
using phosphate buffered saline, which served
as the suspending media for all formulations.
0
10
20
30
40
50
60
70
80
90
100
T0P0 T2P0 T1.5P0.5 T1P1 T0.5P1.5 T0P2 T2P2
PBCA-NDS Formulations
DrugRemaining(%)
Figure 5. Stability of dalargin in various formulations
of PBCA-NDSs after 12 h of incubation in simulated
intestinal fluid (SIF) (n ¼ 3).
Time (hours)
0 2 4 6 8 10 12
DrugRemaining(%)
30
40
50
60
70
80
90
100
T2P2
T0P0
Figure 6. Comparative stability profiles of dalargin
in Formulations T2P2 and T0P0 in SIF (n ¼ 3).
Time (minutes)
20 40 60 80 100 120
MPE(%)
0
20
40
60
80
10 0
T2P2
T2P2 + A
T0P2
T0.5 P1.5
T1P1
T1.5 P1.5
T2P0
T0P0
T2P2 - N
*
*
*
Figure 7. Percentage MPE of different formulations
of PBCA-NDSs vial oral administration (n ¼ 10).
*p < 0.05 when compared to T0P0 at 60 min.
1350 DAS AND LIN

After 60 min of oral administration, it was ob-
served that Formulation T2P2 showed the max-
imum anti-nociceptive effect scoring a % MPE of
93.8 Æ 6.58, followed by T2P0 (60.0 Æ 5.27) and
T1.5P0.5 (32.5 Æ 6.45). Formulation T2P2þA [i.e.,
T2P2 co-administered with central opioid antago-
nist (A) naltrexone HCl] showed a near baseline %
MPE of 5 Æ 5.45. A baseline value was observed
for the Formulation T2P2-N (i.e., physical admix-
ture of drug and excipients without nanoparticles)
showing a % MPE of just 2.5 Æ 2.27. Hence, it was
inferred that co-administration with antagonist
naltrexone did not produce a significant anti-
nociceptive effect, which was also the case with
the formulation devoid of any nanoparticles. The
maximum effect was observed after 60 min of
drug administration with return to baseline
values at the end of 2 h. Typical Straub Tail
effect10
characterized by erect tails at time points
of high % MPE were also observed. Statistically
significant differences ( p < 0.05) were observed
between the Formulations T2P2 and T0P0, T2P0
and T0P0 as well as T1.5P0.5 and T0P0. But, no
statistically significant difference was observed
between Formulations T1P1 and T0P0.
There could be a number of inferences drawn
from such observations. Firstly, the brain target-
ing of dalargin-loaded PBCA-NDSs and release of
drug in the brain interior causing dalargin-
induced analgesia was proven. This was confirmed
by the fact that with co-administration of naltrex-
one HCl (Formulation T2P2þA, % MPE of 5.0 at
60-min time point), a central opioid antagonist,
event of dalargin-induced analgesia was absent.
Antagonist had more affinity towards the opioid
receptors, which had displaced dalargin from its
binding sites, enabling mice to feel pain and
respond positively to heat stimuli in tail flick test.
Other investigators had used naloxone (0.1 mg/kg)
in similar experiments,15,18
but in this investiga-
tion, naltrexone HCl was used which has greater
oral bioavailability than naloxone. Secondly, phy-
sical admixture of drug and excipients without the
presence of PBCA-NDS (Formulation T2P2-N, %
MPE of 2.5 at 60-min time point) failed to elicit
anti-nociception, proving that brain delivery of
dalargin was only possible when the drug was
adsorbed within the nanoparticles. Thirdly, over-
coats of PEG, even at 2% concentration (Formula-
tion T0P2, % MPE of 7.5 at 60-min time point) was
unable to elicit significant anti-nociceptive effect
when compared to 2% overcoat of Tween (For-
mulation T2P0, % MPE of 60 at 60-min time point).
This pointed at the fact that brain delivery of
PBCA-NDS via oral administration with 2% PEG
coating alone is not possible even though PEG
coated PBCA-NDS shows superior protective
action in simulated gastric and intestinal fluids.
Fourthly, the increase of anti-nociceptive effect
in terms of % MPE increased as a function of
increasing concentrations of Tween 80 overcoats.
This claim is supported by the maximum % MPE
attained by different formulations at the 60-min
time point. For instance, a % MPE of 93.8 Æ 6.58
was achieved by Formulation T2P2, 60 Æ 5.27 by
Formulation T2P0, 32.5 Æ 6.45 by Formulation
T1.5P0.5, and 17.5 Æ 10.54 by Formulation T1P1,
respectively. This clearly shows that with an
increase in Tween 80 concentrations, % MPE had
increased proportionately. Thus, brain delivery of
PBCA-NDS was dependent upon the Tween 80
coating.
Dose Response Curve of Dalargin
In order to re-establish the phenomenon of brain
delivery of dalargin-loaded PBCA-NDS via the
oral route, a dose response curve was obtained
using the Formulation T2P2 (Figure 8). Formula-
tion T2P2 was chosen to construct this graph
due to the maximum effect of central analgesia
produced by this formulation in terms of max-
imum % MPE of 93.8 Æ 6.58 at the 60-min time
point. The doses increased in aliquots of 7.5 mg/kg
(IV dose) to 52.5 mg/kg (seven times of IV dose).
The smallest dose of 7.5 mg/kg failed to show any
Dose (mg/kg)
0 10 20 30 40 50
MPE(%)
0
20
40
60
80
100
Figure 8. Dose response curve of dalargin following
oral administration of various doses of Formulation
T2P2 (n ¼ 10).

effect, but with gradual increase of dose to 37.5 mg/
kg, a % MPE of 93.75 Æ 5.88 was observed at the
end of 60 min. With a further increase of dose to
45 or 52.5 mg/kg, a plateau phase was observed
with no further increase of % MPE. The graph
followed a typical sigmoidal curve proving the
relationship between pharmacodynamic response
and the amount of drug released in brain tissue.
CONCULSIONS
In the light of the success of at least three formu-
lations namely T2P2, T1.5P0.5, and T1P1 to cause
significant dalargin-induced analgesia, it can be
concluded that double-coated PBCA-NDS can
cross the gastrointestinal barrier after oral ad-
ministration and still retain its targeting pro-
perties to brain. To summarize, the novelty and
success of double-coated PBCA-NDS can be hy-
pothesized due to interplay of a number of factors
together. They could be (a) fine particle size of
around 100 nm, (b) near zero zeta potentials, and
(c) double coats of Tween 80 and PEG 20000. The
fine particle size of PBCA-NDS could have helped
in endocytic uptake, transcytosis across M-cells in
the gastrointestinal tract.27,28
Also, once is circu-
lation, particles could also escape spleenic filtra-
tion effect if their size is below 250–300 nm.29
Near zero (À2.44 mV Æ 1.18) zeta potentials of the
Formulation T2P2 could have prevented the
selective adsorption of opsonizing plasma proteins
and thereby increased the circulation half-life.30
The action of double-coats of Tween and PEG are
suspected to play the following roles. The role of
PEG 20000 coating had been the enhancement
of stability of drug-loaded in PBCA-NDS in
gastrointestinal tract and possibly, mucoadhesive
effect19,31
for better absorption and hence better
gastrointestinal uptake of nanoparticles. Apart
from that, an increase of circulation half-life by
evasion of the macrophageal clearance of PBCA-
NDS in the systemic circulation by dysopsonic
effect and a PEG mediated uptake of nanoparti-
cles across BBB can also be considered.32
The
Tween 80 coating can be believed to cause an
enhancement of oral absorption by temporary
fluidization of mucus and exposing the nanopar-
ticles to absorptive enterocytes and the M-cells.
Most importantly, as discussed earlier, Tween
80 coating had been responsible for the brain
delivery of PBCA-NDS by LDL receptor mediated
endocytic uptake across the BBB. Hence, we can
conclude that surface engineered PBCA-NDSs
with overcoats of Tween 80 and PEG 20000
represent a feasible method to deliver and target
peptides to brain via the oral route. Although
further studies using radioactive markers are
required to elucidate exact mechanisms of nano-
particular uptake through the gastrointestinal
barrier, polymeric nanoparticles continue to show
promise in delivery of macromolecules to complex
tissues by traversing biological barriers.
ACKNOWLEDGMENTS
The authors acknowledge Mr. Vishal Saxena,
St. John’s University for his assistance in animal
studies.
REFERENCES
1. Pardridge WM. 2001. Brain drug targeting: The
future of brain drug development, 1st ed. United
Kingdom: Cambridge University Press, pp 3–11.
2. Pardridge WM. 1998. CNS drug design based
on principles of blood–brain barrier transport. J
Neurochem 70:1781–1792.
3. Gummerloch MK, Neuwalt EA. 1992. Drug entry
into the brain and its pharmacologic manipulation.
In: Bradbury MWB, editor. Physiology and phar-
macology of the blood–brain barrier. Handbook
of experimental pharmacology, vol 103. Berlin:
Springer, pp 525–542.
4. Pardridge WM, Buciak JL, Friden PM. 1991.
Selective transport of an anti-transferrin receptor
antibody through the blood–brain barrier in vivo.
J Pharmacol Exp Ther 259:66–70.
5. Zhou X, Huang L. 1992. Targeted delivery of DANN
by liposomes and polymers. J Control Rel 19:269–
274.
6. Chen D, Lee KH. 1993. Biodistribution of calcitonin
encapsulated in liposomes in mice with particular
reference to the central nervous system. Biochem
Biophys Acta 1158:244–250.
7. Huwyler J, Wu D, Pardridge WM. 1996. Brain drug
delivery of small molecules using immunolipo-
somes. Proc Natl Acad Sci USA 93:14164–14169.
8. Kreuter J, Alyautdin RN, Kharkevich DA, Ivanov
AA. 1994. Passage of peptides through the blood–
brain barrier with colloidal polymer particles
(nanoparticles). Brain Res 674:171–174.
9. Schroeder U, Sabel BA. 1995. Nanoparticles, a drug
carrier system to pass the blood–brain barrier,
permit central analgesic effects of i.v. dalargin
injections. Brain Res 710:121–124.
10. Schroeder U, Sommerfiled P, Ulrich S, Sabel BA.
1998. Nanoparticle technology for delivery of drugs
across the blood–brain barrier. J Pharm Sci 87:
1305–1307.
1352 DAS AND LIN

11. Schroeder U, Sabel BA, Schroeder H. 1999. Diffusion
enhancement of drugs by loaded nanoparticles
in vitro. Prog Neuro-Psychopharmacol & Biol
Psychiat 23:941–949.
12. Schroeder U, Schroeder H, Sabel BA. 1999. Body
distribution of 3
H-labelled dalargin bound to poly
(butyl cyanoacrylate) nanoparticles after i.v. injec-
tions to mice. Life Sci 66:495–502.
13. Gessner A, Olbrich C, Schroeder W, Kayser O,
Muller RH. 2000. The role of plasma proteins in
brain targeting: Species dependent protein adsorp-
tion patterns on brain-specific lipid drug conjugate
(LDC) nanoparticles. Int J Pharm 214:87–91.
14. Kreuter J, Shamenkov D, Petrov V, Ramage P,
Cychutek K, Koch-Brandt C, Alyautdin R. 2001.
Apolipoprotein-mediated transport of nanoparti-
cles-bound drugs across the blood–brain barrier.
J Drug Target 10:317–325.
15. Kreuter J, Petrov VE, Kharkevich DA, Alyautdin
RN. 1997. Influence of the type of surfactant on the
analgesic affects induced by the peptide dalargin
after its delivery across the blood–brain barrier
using surfactant-coated nanoparticles. J Control
Rel 49:81–87.
16. Kreuter J. 2001. Nanoparticulate systems for brain
delivery of drugs. Adv Drug Del Rev 47:65–81.
17. Sun W, Xie C, Wang H, Hu Y. 2003. Specific role of
polysorbate 80 coating on the targeting of nano-
particles to the brain. Biomat 25:3065–3071.
18. Schroeder U, Sommerfeld P, Sabel BA. 1998.
Efficacy of oral dalargin-loaded nanoparticles de-
livery across blood–brain barrier. Peptides 19:777–
780.
19. Tobıó M, Sańchez A, Vial A, Soriano I, Evora C,
Vila-Jato JL, Alonso MJ. 2000. The role of PEG on
the stability in digestive fluids and in vivo fate of
PEG-PLA nanoparticle following oral administra-
tion. Coll Surf B: Biointerf 18:315–323.
20. Landry FB, Bazile DV, Spenlehauer G, Veillard M,
Kreuter J. 1996. Influence of coating agents on the
degradation of poly (lactic acid) nanoparticles in
model digestive fluids (USP XXII). STP Pharma Sci
63:195–202.
21. Gref R, Luck M, Quellec P, Marchand M, Dellach-
erie E, Harnisch S, Blunk T, Muller RH. 2000.
‘‘Stealth’’ corona-core nanoparticles surface mod-
ified by polyethylene glycol (PEG): Influences of the
corona (PEG chain length and density) and of the
core composition on phagocytic uptake and plasma
protein adsorption. Colloids and Surfaces B: Bioin-
terfaces 18:301–313.
22. Moghimi S, Muir I, Illum L, Davis S, KolhBachofen
V. 1993. Coating particles with a block copolymer
(poloxamine 908) suppresses opsonization but
permits the activity of dysopsonins in the serum.
Biochim Biophys Acta 1179:157–165.
23. Klibanov AL, Maruyama K, Torchilin VP, Huang L.
1990. Amphiphatic polyethyleneglycols effectively
prolong the circulation time of liposomes. FEBS
Lett 268:235–237.
24. Torchilin VP, Papisov MI. 1994. Hypothesis: Why
do polyethylene glycol-coated liposmes circulate so
long? J Liposome Res 4:725–739.
25. Sullivan O’, Brikinshaw C. 2002. Hydrolysis of poly
(n-butylcyanoacrylate) nanoparticles using ester-
ase. Polymer Degradation and Stability 78:7–15.
26. Fontana G, Licciardi M, Manseuto S, Schillaci D,
Giammona G. 2001. Amoxicillin-loaded polyethyl-
cyanoacrylate nanoparticles: Influence of PEG
coating on the nanoparticles size, drug release rate
and phagocytic uptake. Biomaterials 22:2857–2865.
27. Jung T, Kamm W, Breitenbach A, Kaiserling E,
Xiao JX, Kissel T. 2000. Biodegradable nanoparti-
cles for oral delivery of peptides: Is there a role for
polymers to affect mucosal uptake? Eur J Pharm
Biopharm 20:147–160.
28. Florence AT. 1997. The oral absorption of micro-
and nanoparticles: Neither exceptional nor unu-
sual. Pharm Res 14:259–266.
29. Moghimi SM, Porter CJH, Muir IS, Illum L, Davis
SS. 1991. Non phagocytic uptake of intravenously
injected microspheres in rat spleen: Influence of
particle size and hydrophilic coating. Biochem
Biophys Res Commun 177:861–866.
30. Gref R, Domb A, Quellec P, Blunk T, Muller RH,
Verbavatz JM, Langer R. 1995. The controlled
intravenous delivery of drugs using PEG-coated
sterically stabilized nanospheres. Adv Drug Del
Rev 16:215–233.
31. Vauthier C, Dubernet C, Fattal E, Pinto-Alphand-
ary H, Couvreur P. 2003. Poly (alkylcyanoacry-
lates) as biodegradable materials for biomedical
applications. Adv Drug Del Rev 55:519–548.
32. Lockman PR, Koziarza J, Roder KE, Paulson J,
Abbruscato TJ, Mumper RJ, Allen DD. 2003. In vivo
and in vitro assessment of baseline blood–brain
barrier parameters in the presence of novel nano-
particles. Pharm Res 20:705–713.

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