INTRODUCTION:
Conventional formulations
intended for topical and dermatological administration of drugs, such as
creams, foams, pessaries and gels, are considered to
reside for a relatively short period of time at the targeted site. . To overcome many of these above-mentioned drawbacks,
attempts have been made to introduce lipid nanoparticles
into the cosmetic and pharmaceutical fields.
During the recent decades
several studies have suggested that novel drug delivery systems based on lipid nanoparticles that have the potential of increasing cutaneous drug delivery of both hydrophilic and lipophilic drugs compared to the above mentioned
conventional formulations1,2.
These lipid-based systems,
well known as solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC)2, are
composed of a solid matrix of physiological nature which might thereby fulfills
the many promising aspects of the topical route and should in addition provide
a controlled and prolonged release of drugs. Their lipid based composition and
small particle size contribute to the enhancement of penetration of compounds
incorporated into these particles. With aging the stratum corneum
barrier becomes fragile and the recovery is delayed3, hence lipids
play a crucial role for the water impermeability barrier function of the skin.
In order to maintain locally a certain drug level and enable lower dosing
frequency and lower amount of administered drug, Nystatin-loaded
SLN and NLC formulations have been proposed in the present work as newly
controlled and prolonged delivery systems. These
properties bring many other advantages such as occlusion and skin hydration,
absorption-increasing effects, active penetration enhancement, and controlled-release
properties4, 5.SLN and NLC systems differ because SLNs were
developed by exchanging the liquid lipid (oil) of oil-in-water (o/w) emulsions
by a solid lipid6, which can bring many advantages in comparison to
a liquid core6. SLN is a less ordered lipid matrix with many
imperfections, which can accommodate a higher amount of drug11, 13.
The mixture obtained with solid and liquid lipids needs to be solid at least at
400C to make sure that it does not melt at room and body temperature
if used for drug delivery. Advantages of using lipids as carrier systems
for skin administration are related to their
physiological and well-tolerated nature, which reduces the risk of toxicological problems and local irritancy 6,
7. A range of very different lipids with
generally recognized as safe status has been used to produce SLNs and NLCs, ranging from highly purified lipids, for example, tristearin in SLNs, to mixtures of
mono-, di-, and triacylglycerols,
including monoacid and polyacid acylglycerols.
OBJECTIVES:
So the aim is,
1) To prepare solid lipid nanoparticles of
Nystatin.
2) To reach at optimum formulation using Box Behnken
Design.
3) To characterize the prepared optimum formulation.
4) To prepare and characterize gel containing solid lipid nanoparticles.
5) Ex-vivo study of plain and nanoparticulate
gels to investigate localization of Nystatin in skin.
6) Primary Skin Irritation test on rabbits.
MATERIALS
AND METHODS:
List of materials and equipment:
|
Sr. No.
|
Materials
|
|
1
|
Nystatin
|
|
2
|
Propylene Glycol
|
|
3
|
Polyethylene Glycol 400
|
|
4
|
Glyceryl Monostearate
|
|
5
|
Emulcire 61
|
|
6
|
Compritol 888 ATO
|
|
7
|
Precirol ATO 5
|
|
8
|
Gelucire
|
|
9
|
Stearic acid
|
|
10
|
Span 60
|
|
11
|
Tween 80
|
|
12
|
Soy lecithin
|
|
13
|
Poloxamer188
|
|
14
|
Carbopol 940
|
|
Sr. No.
|
Equipment
|
|
1
|
Electronic
Balance
|
|
2
|
Mechanical stirrer
|
|
3
|
Heating Mantle
|
|
4
|
Probe sonicastor
|
|
5
|
Brookfield Viscometer
|
|
6
|
Ultra-Centrifugation
|
|
7
|
Bath sonicator
|
|
8
|
Particle size analyzer (Laser diffraction)
|
|
9
|
UV-Visible Spectrophotometer
|
|
10
|
Differential Scanning Calorimetry
|
|
11
|
Fourier
Transform Infra-red Spectroscopy
|
|
12
|
Transmission
Electron Microscopy
|
|
13
|
X-Ray
Diffraction
|
|
14
|
Stability
chamber
|
|
15
|
Design Expert
|
Selection
of lipid:
Selection
of lipid was done on the basis of maximum solubility of Nystatin
in different lipids and also on melting point of lipid as the type of
drug-lipid matrix and drug release pattern will depends on it. Various co
solvents were also used to aid the solubility of Nystatin
in lipid. Out of different lipids used, Nystatin
showed maximum solubility in mixture of glyceryl monostearate (GMS) and propylene glycol (PG) 10.
Nystatin
was only soluble in gelucire and GMS near about 65-70oC,
but after cooling the gelucire did not solidify up to
desired extent hence the GMS was used. The solubility was increased by addition
of PG as Nystatin is soluble in PG at hot condition.
Due to low melting point of GMS (60oC) and high melting point of Nystatin (160oC)11, possible
structure of SLN will be homogeneous matrix with prolong release of drug12.
A. Characterization of drug:
Instrumental analytical techniques were performed to
characterize the physicochemical properties of drug and to determine the
changes that might occur in drug after preparation of solid lipid nanoparticle14.
1. Differential
Scanning Calorimetry study:
DSC
is a highly useful means of characterizing material with respect to crystalline
behavior and physical changes in the formulation. DSC gives the specific thermogram for Nystatin (Fig. 1)
which is used to identify the Nystatin in Ny-SLN.
Figure 1: Differential scanning thermograms
of pure drug Nystatin.
2. Fourier
Transforms Infrared (FTIR) Study:
FTIR
shows the characteristic peak for NH, OH stretch – 3350-3331cm-1, Lactone carbonyl stretch- 1701cm-1, Carboxylate ion – 1566-1546cm-1, C-OH stretch-1068cm-1
(Fig.2).
Fig. 2: Fourier Transform Infrared spectrums of Nystatin.
3. Melting
Point Determination:
Melting
point of pure drug Nystatin was found to be between
158-1600C
4. Analytical
method development (UV spectrophotometric method).
Preparation of calibration curve of Nystatin:
Calibration
curve was prepared in methanol and saline phosphate buffer pH 7.4, at λmax 304 nm where Nystatin
shows the maximum linearity. For the Nystatin in
methanol, the concentration range 2 -12 µg/ml has regression coefficient (R2)
0.999 and regression equation y = 0.0899x – 0.01 ; while the Nystatin in saline phosphate buffer pH 7.4 in the
concentration range 2- 16 µg/ml has regression coefficient (R2)
0.9991 and regression equation y =
0.0239x + 0.0823 .
A. Optimization
of Pre-emulsion:
In
the preparation of SLN different surfactants were used to determine their
effect on particle size, Span 60 and Soy Lecithin were used in lipid phase and Tween 80 and Poloxamer 188 were
used in aqueous phase. Lesser particle size was found in lecithin-Tween 80 combinations and Span 60-Tween 80 combinations,
but while preparing pre-emulsion due to high temperature the sticky mass was
found at the bottom of container and also the dispersion was not stable for
longer period of time. The Span 60-Tween 80 combination formulation exhibited
lesser particle size as well as rigid particles and is stable over longer
period of time. The poly dispersity index was
slightly greater than 0.3. Effect of various formulation variables on average
particle size and entrapment efficiency was determined by varying the
concentration of GMS, Span 60 and Sonication Time. As the concentration of GMS increases,
particle size and entrapment efficiency of SLN increases. While increase in
concentration of surfactant i.e. Span 60, particle size decreases and
entrapment efficiency increases due to more solubilization
of drug in lipid.
B. Experimental
design:
Solid
lipid nanoparticles of Nystatin
(Ny-SLN) were prepared using probe sonication
technique. The experiments were designed to study the effect of three
independent variables at three levels on particle size and percent drug
entrapment efficiency
C. Analysis of Experimental results:
Analysis of experimental results was done by using the Stat-Ease
Design Expert. After filling
The data in the design, linear and quadratic model were suggested
to run the design,
Table 4: Values of particle size and %EE of optimized dispersion of Ny-SLN
|
Code
|
Dispersion composition
|
Particle size (nm)
|
Entrapment efficiency (%)
|
|
Drug: Lipid
|
Span 60 Soni.
time
|
|
S1
|
1: 7.2
|
3.45 11.125
|
210.33
|
89.4779
|
|
S2
|
1: 7.2875
|
3.45 11.125
|
210.206
|
89.4881
|
|
S3
|
1: 7.375
|
3.45 11.125
|
210.17
|
89.5361
|
|
S4
|
1: 7.2875
|
3.5 11.25
|
214.504
|
89.5369
|
|
S5
|
1: 7.375
|
3.5 11.25
|
214.49
|
89.5417
|
Table 5: Comparison of experimental results
with predicted responses
|
Formulation code
|
Response
|
Predicted value
|
Experimental value
|
Percent error
|
|
S1
|
Particle size (nm)
Entrapment (%)
|
210.33
89.4779
|
219
91.87
|
0.0369
0.011
|
|
S2
|
Particle size (nm)
Entrapment (%)
|
210.206
89.4881
|
218
92.06
|
0.0385
0.0107
|
|
S3
|
Particle size (nm)
Entrapment (%)
|
210.17
89.5361
|
217.5
92.72
|
0.0297
0.0098
|
|
S4
|
Particle size (nm)
Entrapment (%)
|
214.504
89.5329
|
225
92.86
|
0.0404
0.0117
|
|
S5
|
Particle size (nm)
Entrapment (%)
|
214.49
89.5417
|
224.36
92.88
|
0.0422
0.00119
|
|
Mean (±
S.E.M.) of Percentage Error
|
0.0338±0.002
0.00211
|
F. Evaluation
of solid lipid nanoparticles:
The
prepared Ny-SLN dispersion was characterized with
respect to the particle size, shape, entrapment efficiency, crystallinity
and stability study.
Particle size analysis:
the
particle size analysis of the optimized sample S3 was 217 nm. Particle size of
other samples is shown in above Table 5. The effect of Span 60 concentration,
sonication time and Drug: Lipid ratio on the particle size can be seen from
values for sample S1, S2, S4 & S5 (219 nm, 218 nm, 225 nm and 224.36 nm
respectively). Increased span 60 concentration decreases the particle size
which can be explained by reduction in interfacial tension between aqueous and
lipid phase which lead to the formation of emulsion droplet of smaller size
thereby effectively stabilize the particles
by forming a steric barrier on particle
surface and by protecting the particle
from coagulation. A particle size distribution curve of sample S3 is shown in
Fig. 4 having average particle size 217 nm. The poly dispersity
index was slightly greater than 0.3 as the SLN was prepared by the probe
Sonication method and homogeneous distribution of power density is necessary to
obtain narrow size distribution. In the particle size distribution curve, the
small peak observed near the 7000 nm which is due to poly dispersity
of nanoparticles because the particles located in
different volume of sample will experience different dispersing forces and
therefore degree of particle disruption will vary within the sample volume.
Figure
4: Particle size distribution curve of Sample S3.
G. Entrapment efficiency:
Entrapment efficiency of Ny-SLN
sample S3 was found to be 92.72%. A high amount of drug could be incorporated
in nanoparticle dispersion. Such high incorporation
was possible because of lipid solubility of Nystatin
and use of PG as cosolvent and also span 60 as a
lipid surfactant helps to solubilize the Nystatin in to lipid which further increases entrapment of
drug. It can be seen that, high lipid and span 60 concentrations show positive
influence on entrapment efficiency while sonication time has less impact.
Sample S3 was selected as optimized SLN dispersion since it showed less
particle size & high entrapment efficiency (92.72 %) as compared to other dispersions.
H.
Differential scanning calorimetry (DSC):
DSC
is a highly useful means of characterizing material with respect to crystalline
behavior and physical changes in the formulation. Nystatin
alone and in formulation was studied using DSC. The DSC thermogram
of GMS show the melting process taking place at 62°C and Nystatin
show peak at 160°C (Fig. 5). Thermogram of Ny-SLN showed an endotherm at
60.55°C, which can be attributed to melting of Nystatin
in GMS.
Figure
5: Differential scanning thermograms of bulk material
of (a) Nystatin, (b) Ny-SLN
and (c) GMS
I. X-ray diffraction:
X-ray investigations have been most valuable in the
elucidation of the manner of arrangement of lipid molecules, their
multiple-melting phenomena, phase behavior and the characterization and
identification of the structure of lipid and drug molecules. X-ray Diffraction
data (Fig. 6) was good in agreement with results established by DSC asurements.
Figure
6: X-ray Diffractograms of (a) GMS, (b) Ny-SLN.
J. Fourier Transform Infrared study
From
FTIR study, the characteristic peak of drug such as of NH,OH stretch (3350-3331
cm-1), lactone carbonyl stretch (1701 cm-1),
carboxylate ion stretch (1566-1546 cm–1)
disappeared and were replaced by the peak of GMS of H-OH (3300-3311 cm-1), COOH OH stretch (2914-2848 cm-1)
and C=O stretch (1730 cm-1) while remaining peaks also either
shifted or replaced in the IR shown in (Fig. 7) spectra of formulation. This
established drug entrapment in lipid matrix.
Figure
7: Fourier transforms infrared spectrums of (a) GMS, (b) Ny-SLN.
Stability
study:
After
one month storage the SLN dispersion at various temperature parameters showed
little difference in particle size and entrapment efficiency. There is no change
in clarity and phase separation was observed. The average particle size and
entrapment efficiency of optimized sample S3 stored for 1 month was 229 nm ±
2.88 and 89.37 % ± 0.5263 content. Centrifugation at 3000 rpm for 30 min showed
there is no precipitation and the Ny-SLN had a good
physical stability. Changes in particle size and entrapment efficiency were due
to polymorphic transition of the lipid which leads to expulsion of drug from
SLN (transformation of higher energy α and β’ modification to the
lower energy β modification).This may imply that the transition of
dispersed GMS in SLN from ß´ form to stable ß form might occur extremely
slowly.
K. Preparation and evaluation of Ny-SLN gels:
Gel of Ny-SLN was prepared by
using different concentrations of carbopol 940 (0.3%-
1%) out of that 0.4% concentration was selected. The criteria for the selection
of 0.4 % Carbopol gel are the consistency of gel
rheological pattern, drug release from the gel and hydrating and film forming
properties.
Drug content in Ny-SLN
gel was found to be 98.73 % ± 0.39 (n=3).
L.
Rheological study of gel:
The
rheological behavior of 0.4 % Carbopol 940 gel
containing Ny-SLN kept at different temperature.
Rheological study was performed in Brookfield Viscometer CAP 2000+2, the results were recorded after one week of storage at
5ºC, 25ºC and at 40ºC, show pseudo plastic flow behavior at all temperature
conditions.
CONCLUSION:
The
main purpose of this thesis has been the investigation of the latest
developments of innovative solid lipid carriers, particularly solid lipid nanoparticles (SLN) for topical delivery of antifungal
drug. Fungal infections to the skin due to various fungi can be treated by
antifungal drugs. Nystatin is an antifungal antibiotic
drug which have barrel like nonpolar structure capped
by polar group, penetrate the fungal cell membrane, acting as “false membrane
component” and bind closely with ergosterol, causing
membrane disruption, cessation of membrane enzymatic activity and loss of
cellular constituents specially potassium ions. Although Nystatin
is one of the active molecules in case of various fungal infections it is given
topically. To get symptomatic relief, fast action and also patient compliance, topical
drug delivery of Nystatin is desirable. Hence, in the
present work an attempt has been made to prepare topical formulation of Nystatin loaded SLN to improve the drug targeting to skin
and accumulating the drug in skin for prolonged release.
Following conclusion can be drawn from the study:
1 For the selected SLN delivery system GMS,
Span 60 and tween 80 were used as formulation
ingredients determined by pre-optimization study.
2 Box Behnken
design was used for optimization study and searched optimum formulation with
low particle size and high entrapment efficiency.
3 The optimized formulation was evaluated
for particle size, percent entrapment efficiency, XRD, FTIR and DSC to confirm
the formation of SLN and entrapment of Nystatin in
it.
4 Nystatin SLN was
formulated as gel by using different concentration Carbopol
940 from with 0.4 % gel was finalized and pseudo plastic behavior was observed
by rheology study.
5 When Ny-SLN gel compared with the marketed gel, drug release was
found to be sustained in case of SLN gel and more drug penetrated in to the
skin layer which was desired.
6 To study the occlusive behavior of SLN, Ex vivo skin hydration and In vitro occlusion study were
performed. From Ex vivo skin hydration study, it was found that due to
formation of compact film trans epidermal water loss
from skin reduces due to which moisture content of skin increases lead to more
hydration with increase thickness of stratum corneum.
Similar type of data obtained by occlusion study showing the
change in weight of beakers which indicate loss of water.
7 After various process conditions, nystatin in the Ny-SLN was found
to be effective against Candida albicans NCIM 3471.
8 Skin irritation test of Ny-SLN gel was performed on rabbit. The test article, Ny-SLN gel and plain gel was evaluated for primary skin
irritation in accordance with the guidelines of the Consumer Product Safety
commission and primary irritation index was calculated to be 0.00.
9 The result of stability study indicates no
significant difference between the parameters tested before and after the
stability studies.
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