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Pharmaceutical Research, Vol
...
5, May 2004 (© 2004)

Research Paper

Chitosan Nanoparticles as New
Ocular Drug Delivery Systems: in
Vitro Stability, in Vivo Fate, and
Cellular Toxicity
Angela M
...
S
...
To assess the potential of chitosan (CS) nanoparticles for
ocular drug delivery by investigating their interaction with the ocular
mucosa in vivo and also their toxicity in conjunctival cell cultures
...
Fluorescent (CS-fl) nanoparticles were prepared by ionotropic gelation
...
The
in vivo interaction of CS-fl nanoparticles with the rabbit cornea and
conjunctiva was analyzed by spectrofluorimetry and confocal microscopy
...

Results
...
In vivo
studies showed that the amounts of CS-fl in cornea and conjunctiva
were significantly higher for CS-fl nanoparticles than for a control
CS-fl solution, these amounts being fairly constant for up to 24 h
...
Cell survival at 24 h after incubation with
CS nanoparticles was high and the viability of the recovered cells was
near 100%
...
CS nanoparticles are promising vehicles for ocular drug
delivery
...


INTRODUCTION
Topical application of drugs into the eye is severely limited by the protective physiological mechanisms that exist in
the precorneal area resulting in considerable drug loss
...
Being
conscious of these limitations, an important effort in ocular
drug delivery has been to improve the bioavailability and to
prolong the residence time of drugs instilled topically onto the
eye (1)
...

2
IOBA-University of Valladolid, Edificio de Ciencias de la Salud,
Ramón y Cajal 7, Valladolid 47005, Spain
...
es)

of success (2)
...
This improved ocular
penetration was partially attributed to the interaction and
further transport of these colloidal carriers across the corneal
epithelium (8)
...

Nevertheless, despite these interesting data, a limitation of
these particulate colloidal carriers is their inability to persist
at the eye’s surface for extended periods of time and, hence to
provide a prolonged drug delivery to the eye
...
Among them, the cationic polysaccharide chitosan (CS) exhibits several favorable biological properties, such as biodegradability (12), nontoxicity (13), biocompatibility (14), and mucoadhesiveness
...
This
unique combination of properties makes it a novel versatile
biopolymer, which fulfils the requirements for its application
in the ophthalmic field
...
Calvo et al
...
The authors concluded that it was not only the positive charge but the specific
nature of CS that was responsible for the enhanced bioavailability of the drug incorporated into CS-coated nanocapsules
...
Nevertheless, it should be noted that these authors followed the retention of 125I-labeled bovine serum albumin used as a marker rather than the fate of the CS-coated
liposomes
...
More
recently, Felt et al
...
In
addition, all types of CS investigated exhibited an excellent
tolerance
...
Important advantages of
these nanoparticles include their rapid preparation under extremely mild conditions and also their ability to incorporate
bioactive compounds (20)
...
Thus, the main objective of this work
was to quantify and to investigate the mechanism of interaction between CS nanoparticles and the corneal and conjunc-

803

0724-8741/04/0500-0803/0 © 2004 Plenum Publishing Corporation

de Campos et al
...
With this idea in mind, the polymer
was previously labeled with sodium fluorescein
...
S
...
Sigma
Chemical Co
...
Louis, MO, USA) supplied the sodium
tripolyphosphate (TPP) and sodium fluorescein
...
Culture plastic material
was from Nunc (Roskilde, Denmark), and reagents for cell
culture were from Gibco (Life Technologies, Inchinnan, UK)
...
0 and
2
...
Animals were allowed
free access to food and water during the experiments
...

The mean particle size and size distribution of the nanoparticles were determined by photon correlation spectroscopy
(PCS)
...
Each analysis was
performed at 25°C with an angle detection of 90°
...
Nanoparticles suspension was diluted with
10−3 M KCl and placed in the eletrophoretic cell, where a
potential of ±150 mV was established
...
)3 ס‬
Stability of Chitosan Nanoparticles in the Presence of
Mucus Components
Incubation with Lysozyme

Synthesis of the Chitosan-Fluorescein (CS-fl) Conjugate
The covalent attachment of fluorescein to CS was
achieved by the formation of amide bonds between primary
amino groups of the polymer and the carboxylic acid groups
of fluorescein
...
5-g sample of CS was dissolved in 200 ml
of (1%, v/v) acetic acid aqueous solution, and the pH value
was adjusted to 6
...
Demineralized water was
added to this solution to make the final volume 250 ml
...
Thereafter, both solutions were mixed together, and,
to catalyze the formation of amide bonds, EDAC [1-ethyl-3(3-dimethylaminopropyl) carbodimide hydrochloride] was
added in a final concentration of 0
...
The reaction mixture
was incubated under permanent stirring for 12 h in the dark at
room temperature
...
05 N NaOH solution, and finally against demineralized water and then lyophilized
...

Preparation of Chitosan Nanoparticles
Nanoparticles were spontaneously obtained upon addition of a TPP aqueous solution to the CS solution, according
to the procedure previously developed by our group (20)
...
05% (w/v) acetic acid solution at a
concentration of 0
...
5 with a
0
...
TPP was dissolved in purified
water at a concentration of 0
...
Following this, 0
...
5 ml of the CS-fl solution,
thereby leading to the formation of fluorescence-labeled CS
nanoparticles
...
4
...
The samples were stained

The stability of CS nanoparticles was analyzed following
their incubation at 37°C in a solution of lysozyme in purified
water (1 mg/ml) under moderate stirring
...
The physicochemical properties of the
nanoparticles (mean particle size and zeta potential) were
monitored during the incubation process
...
The first
method was based on the measurement of the viscosity of a
mucin dispersion in water (0
...
Dispersion viscosity measurements were carried
out by Cannon-Fenske viscometer 5354/2 (Dien, France)
...
1°C
...
The second method evaluated the influence of the
mucin on the zeta potential of the nanoparticles
...
At given time intervals (30, 60, 120,
240 min) during incubation, the zeta potential of the nanoparticles was determined as previously described
...
The nanoparticles
were not isolated because of the absence of toxic ingredients
in the final preparation; consequently, the suspension contained CS in the form of nanoparticles and also CS in solution
...
9 mg/ml
...
Two control formulations were also administered: one was a solution of fluorescein-labeled CS (CS-fl
solution) and the other a fluorescein aqueous solution
...
The rabbits were sacrificed 1,
2, 4, 8, and 24 h after the last instillation, the eyes enucleated,
and the cornea and conjunctiva excised
...
5 M NaOH solution
...
Afterwards, the fluorescein was extracted using buthanol as an extraction solvent
...
Buthanol solution fluorescence was measured by
spectrofluorimetry (Luminescence Spectrometer LS50 B,
Perkin Elmer, Norwalk, CT, USA), and the fluorescein quantities were calculated (n ‫
...

Confocal Laser Scanning Microscopy
Three instillations of 25 ␮l of the CS-fl nanoparticles
suspension containing as well CS in solution were administered to the cul-de-sac of conscious rabbits at 10-min intervals
...
Two hours later,
rabbits were killed with an intravenous injection of an overdose of sodium pentobarbital given via a marginal ear vein
...

The system (Confocal Bio-Rad MRC 1024E5, Barcelona,
Spain) consists of a computer-controlled laser scanner assembly with a Nikon fluorescence microscope (Cambridge, MA,
USA)
...
Images were assembled in an integral image processor and displayed on a
digital video monitor
...
2, clone 1-5c-4) was used for the toxicity experiments (68-72 passages)
...
1 ␮g/ml cholera
toxin, 10% fetal bovine serum, 50 U/ml penicillin, 50 ␮g/ml
streptomycin, and 2
...
The culture conditions were standard (incubation at 37°C in a 5% CO2/95%
O2 atmosphere), the medium was changed every 2–3 days,
and cell growing assessed daily by phase-contrast microscopy
...
The suspensions were diluted in acetate buffer pH
6
...
25, 0
...
0, 2
...
The dilution buffer was chosen
because of the instability of the particles in a phosphate buffer
pH 7
...
After 30 min incubation, nanoparticle suspension was
washed out and supplement-free culture medium added
...
5 h at 37°C
...
005% benzalkonium chloride (BAC) (positive control), acetate buffer pH 6
...
After 24 h and
several washes in Ca2+ and Mg2+-free PBS, cell survival (percentage of cell recovery) and the viability of those surviving
cells were assessed
...

Cell Culture Studies: Scanning Electron Microscopy
Scanning electron microscopy (SEM) was used to monitor potential cell alterations or membrane damage in NHC
cells after exposure to chitosan nanoparticles
...
NHC cells were incubated with
nanoparticles and controls as above and washed
...
0% glutaraldehyde in 0
...
4) for 10 min at 37°C, washed in 0
...
A JEOL T300 microscope
with a Maiya Rolf Holder CS-I photographic system (Tokyo,
Japan) was used to examine the cells
...
0)
...
05
...
The association of fluorescein to the polymer was first identified by
spectrofluorimetry
...
On the other hand, using IR spectroscopy we
could see that the amine peak in CS-fl spectrum (at 1562
cm-1) decreased whereas the peak due to amide group (at
1659 cm-1) increased, when compared with the unmodified CS
spectrum
...
Therefore, the result of this reaction is a fluorescent yellow polymer
that has a lower number of free CS amino groups
...
Therefore, once identified the formation of the

de Campos et al
...
The results
showed that the zeta potential of unmodified CS nanoparticles was very similar (+37
...
9 mV) to that of CS-fl nanoparticles (+35
...
17 mV)
...
6 ± 8
...
34, and transmission electron microscopy evidenced spherical CS-fl nanoparticles with a solid and consistent structure,
characteristics that were very similar to those previously reported for unmodified CS nanoparticles (20)
...

Stability of CS-fl Nanoparticles in the Presence of
Mucus Components
The stability of colloidal particles in biological fluids containing important amounts of proteins and enzymes is a crucial issue
...
Surprisingly, despite the importance of the size,
there are very few articles on the stability of colloidal particles
in biological fluids
...
Similarly, we have observed that poly-␧-caprolactone (PECL)
nanocapsules suffer an immediate aggregation process upon
their incubation with lysome (23)
...

This previous information led us to consider the importance
of performing a preliminary study of the stability of the particles in the presence of two major components of the precorneal fluid: lysozyme and mucin
...
The results presented in Table I indicate
that the size was slightly reduced upon incubation with the
enzyme
...
We should, however, be cautious in the
interpretation of these data as it has been reported that lysozyme does interact with the acetamide groups but it does not
with the free amino groups (24)
...
This minor
degradation might however be enough to generate some polymer fragments, which could eventually detach from the nanoparticles
...
(25) observed that the
molecular weight of 77% deacetylated CS went down to half
of the original value in 12 h
...
This could be understood by the fact that lysozyme is
a cationic protein (pI: 10
...
5) and, consequently, its interaction with the nanoparticles might not affect significantly
their zeta potential
...
Therefore,
the major conclusion from this stability study is that the integrity of CS nanoparticles is not significantly compromised
by the presence of lysozyme in the tears fluid
...
This viscosity was also compared with that of a CS
solution incubated in the same conditions
...
Therefore, a significant increase in this parameter should, preferably, be avoided
...
This has been attributed to the mucin carboxylic acid groups, which are ionized at a pH value of 5
...
6) and therefore freely accessible for interaction with the
positively charged amine groups (24)
...
757 ± 0
...
This could be explained by the
low flexibility of the CS chains forming the solid particles
...
Thus, the lack of
viscosimetric changes in the mucin-nanoparticle dispersion
suggested that, under the current experimental conditions, no
significant interactions of mucin with the nanoparticle
ocurred
...
Nevertheless, despite these viscosity
data, results of the zeta potential of the nanoparticles upon
incubation in a mucin dispersion suggest that a certain interaction may occur between mucin and CS nanoparticles
...
Physicochemical Properties of CS-fl Nanoparticles Before and After Incubation with Lysozyme
(n ‫)3 ס‬
␨ Potential (mV)

Particle size (nm)
Time

Control

After incubation
with LZM

Control

After incubation
with LZM

Initial
30 min
1h
2h
4h

394 ± 6
382 ± 17
377 ± 13
377 ± 13
375 ± 15

394 ± 6
367 ± 14
363 ± 15
355 ± 8
348 ± 8

37
...
9
38
...
2
38
...
3
38
...
4
37
...
7

+37
...
9
+37
...
5
+37
...
4
+37
...
3
+36
...
3

LZM, lysozyme
...
Cinematic Viscosity Values of a Mucin Dispersion (0
...
009 ± 0
...
954 ± 0
...
942 ± 0
...
921 ± 0
...
887 ± 0
...
767 ± 0
...
764 ± 0
...
761 ± 0
...
758 ± 0
...
759 ± 0
...
757 ± 0
...
CS, chitosan
...
1) support this observation
...
This
reduction could be attributed to the ionic interaction between
the negatively charged particles and chitosan nanoparticles
...

In vivo Study: Quantitative and Qualitative Evaluation of
the Interaction of CS Nanoparticles with the
Ocular Mucosa
As indicated in the “Materials and Methods” section,
isolation of CS-fl nanoparticles is not required for in vivo
administration because the only major component in the suspending medium is free CS-fl in solution
...
This parameter (nanoparticles yield) could be determined following centrifugation of the nanoparticles and further quantification of
CS-fl by spectrofluorimetry
...
8% of
CS-fl was in solution and hence 73
...

Figs
...
1
...


Fig
...
Evaluation of fluorescein concentration in (A) rabbit cornea
and (B) conjunctiva after the instillation of CS-fl nanoparticles ( ),
CS-fl solution (ᮀ), and fluorescein solution ( ) (n ‫
...
These results show that the behavior of the CS-fl
nanoparticles is remarkably different from that of CS-fl solution
...
In fact, the differences in fluorescein concentrations for
the nanoparticles and the CS solution were statistically significant at all times assayed with the exception of time l h
...
(conjunctiva) (p < 0
...
These results indicate that the interaction of CS with the ocular surface (either cornea or conjunctiva) is more persistent when it
is in a nanoparticulated form
...
The second important observation
from Figs
...
05)
...
In other
words, CS-fl in solution is cleared from the eye faster than
CS-fl nanoparticles
...
05)
...
the cornea
...
In order to elucidate whether the particles are
simply able to stick to the ocular mucosa or further able to
enter the epithelia, we examined cross sections of the corneal
and conjunctival epithelia by confocal laser scanning microscopy
...

3
...
These particle-like signals could be associated to the CS-fl nanoparticles
as only a light and diffuse fluorescence is visible following
application of a simple fluorescein solution
...
This
behavior is slighly different from that of other types of nanoparticles, such as poly(alkylcyanoacrylate) (28) and PECL
nanoparticles (8), the transport of which was found to occur
by a transcellular pathway
...
Previous studies carried out in Caco-2 cell cultures have suggested that CS mol-

Fig
...
Confocal fluorescence images at different levels from the rabbit corneal epithelium (5 ␮m sequential cross sections from the corneal surface) at 1 h postinstillation of (C) CS-fl nanoparticles, (B)
CS-fl solution
...


de Campos et al
...
More precisely, this behavior has been attributed to the interaction of the positively charged amino
groups of CS with negatively charged sites of the cell surfaces
and tight junctions (30)
...
An alteration in the relative concentration of specific ion species in
the pore volume would result in changes in tight junction
resistance, which might lead to loosening or opening of the
pore
...
In fact, the suspension of CS nanoparticles contains approximately 50% of CS in solution
...
This
intercellular penetration rather than simple mucoadhesion
could explain the high interaction and long residence time of
the particles in the cornea
...
This could also explain the
greater persistence of the particles as compared to the CS-fl
solution
...
4) were drastically different
from those of the cornea
...

Additionally, this image suggests that the particles are located
inside the cells rather than in the intercellular spaces
...
In fact, this epithelium is more heterogeneous than that of the cornea as it contains not only regular
epithelial cells, but also goblet cells and antigen presenting
cells (APC)
...
The internalization of CS
nanoparticles by conjunctival epithelial cells could also be

Fig
...
Confocal fluorescence image at 10 ␮m from the surface of a
conjunctiva excised after 1 h postinstillation of CS-fl nanoparticles in
rabbit
...
Percentage of Recovered Cells and Their Percentage of Viability (Assessed by the Trypan Blue Dye Exclusion Test) After 24-h
Exposure to the Different Nanoparticles Concentrations in Acetate Buffer (pH 6
...
25 mg/ml)

CS np
(0
...
9

100
...
95 ± 1
...
0 ± 2
...
0 ± 50
...
75 ± 10
...
2 ± 1
...
3 ± 17
...
4 ± 1
...
65 ± 23
...
25 ± 3
...
55 ± 1
...
15 ± 1
...
42 ± 4
...
7 ± 1
...
005% benzalkonium chloride in culture medium; CS np, chitosan nanoparticles suspended
in acetate buffer
...
Therefore, more detailed
studies using cell cultures could be useful to further elucidate
the mechanism of interaction and internalization of the CS
nanoparticles within these cells
...
3A and 3B
...
Consequently, CS-fl nanoparticles may have
a greater change to adhere to the conjuntiva, and to subsequently enter some epithelial cells, rather than to the cornea
...
The similar survival
values observed for the buffer and for the various concentrations of nanoparticles indicates that no inherent toxicity can

be attributed to the nanoparticles at concentrations as high as
2 mg/ml
...

In order to investigate further the tolerance of the particles by the conjunctival cells, we observed the appearance of
the cells after exposure to the nanoparticles and corresponding controls, by SEM
...
5A–5C
show the general healthy state of the cells, characterized by a
well-preserved cell surface
...
This suggests that the suspensions of nanoparticles cause some deleterious effect that could be simply
attributed to the buffer that was used for the resuspension of
the nanoparticles
...
25–1
...
5B and 5C)
...
5A)
...
Cells exposed to
BAC showed enormous alterations and flattering (Fig
...

Some membrane alterations and cell loss were observed for
those cells exposed to acetate buffer, mostly small holes in the
membrane and microvilli loss resulting in a plain surface (not
shown)
...

CONCLUSIONS

Fig
...
Scanning electron microphotographs of Chang cells exposed
to culture medium, chitosan nanoparticles, and BAC for 24 h
...
(B) (CS nanoparticles, 0
...
(C) (CS nanoparticles, 1 mg/ml)
...
Magnification
×750 (bar ‫␮ 51 ס‬m)
...
Current studies are
aimed at investigating in further detail how the interaction
and internalization of these particles occurs and their toxicity
following repeated administration
...
The first author wishes to thank the Brazilian Ministry of Education (CAPES) for her fellowship
...
D
...


810
electron microscope and from Prof
...
Caruncho for the interpretation of the confocal images is gratefully acknowledged
...


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