Griseofulvin nanostructured lipid carriers (G-NLCs) were developed for the topical treatment of tinea pedis (athlete's foot fungus). A microemulsion method was used to prepare the G-NLCs using glyceryl monostearate, stearic acid and pluronic F68. The particles were optimized using a box-behnken design and incorporated into a carbopol gel. In vivo studies on rats showed the G-NLC gel was more effective at reducing fungal infection than the marketed antifungal cream based on histopathology and reduced oxidative stress markers. Cytotoxicity studies of the nanogel on skin cells demonstrated good biocompatibility

1. INDUSTRY DRIVEN PHARMACEUTICAL
EDUCATION AND RESEARCH : CURRENT TRENDS
AND FUTURE PROSPECTS-2023
Formulation and Pharmacological study of Griseofulvin Nanostructured
Lipid Carriers Loaded Topical gel for Tenia Pedis
Neelam Datt1*, Pankaj K. Yadav1, P. Rajasekhar2
*1. Department of Pharmaceutical Sciences, Sam Higginbottom University of
Agriculture, Technology, and Sciences.
2Department of Pharmaceutics, St. Peter’s Institute of Pharmaceutical Sciences,
Hanamkonda, Telangana.

2. 2
Introduction
Dermatophytosis
 Dermatophytosis is a superficial fungal infection caused by dermatophytes.
 They affect the skin, hair, and nails.
 The three main classes of dermatophytes are
Epidermophyton, Trichophyton, and Microsporum.
 Dermatophytes can spread by
 Direct contact from other people (anthropophilic organisms),
 Animals (zoophilic organisms),
 Soil (geophilic organisms)and
 Indirectly from fomites.

3. 3
Antifungals for treatment
 Two to three weeks treatment is required for skin lesions
whereas four-six weeks for feet inflammation.
 For the most part, similar antifungal treatments are used
worldwide.
Classification of antifungal therapy based on their structure
Antifungal class Examples Mechanism of Action
Antibiotics
Polyenes
Heterocyclic
benzofuran
Amphotericin B,
Nystatin,
Natamycin
Griseofulvin
Binds to ergosterol moiety in the membranes
of fungus . This stimulates granulocytes, T
cells and B cells, ultimately lead to death of
fungi.
Interaction with ergosterol, inhibits growth
by , disrupting mitotic spindle formation
Classification Continue….

4. 2A. Microemulsion Method for Griseofulvin Nanostructured Lipid Carriers(G-NLCs) (Waghule, T. et al .2020)
Surfactant and emulsifier was dissolved in aqueous
phase and melted at 70◦C
Drug + Lipids was dissolved in alcoholic phase
and melted at 70◦C
Mixed Isothermally at same
temperature
Micro emulsion was sonicated for 45 min
Centrifuged at 6000 RPM ,4ºC for 1 hrs.
Microemulsion was then poured in a cold aqueous solution (2-10°C) under mechanical stirring at 3000 RPM for 3 hrs.
32
Method Optimization and Formulation Development
Nanostructured Lipid Carriers separated and Lyophilized

5. Optimization of Process Parameters For G-NLCs using Box Behnken
Design
Variables Levels
Independent variables Low Medium High
X1 = GMS: Stearic acid 1: 0.25 1:0.50 1:1
X2 = Pluronic F68: Tween 80 1:1 1:1.5 1:2
X3 = Sonication time (min) 45 60 75
Dependent variables Constraints
Y1 = Particle size (nm) Minimize
Y2= Entrapment efficiency (%) Maximize
33
The topical gel containing an Optimized batch of G- NLCs, was formulated
by incorporating the lyophilized G-NLCs in optimized concentration of 1.5%
Carbopol 940P gelling agent and evaluated for various parameters .
Optimized batch of G-NLCs gel was performed for pharmacological activity .

6. Pharmacological Study (In-Vivo)
1. Animals: Healthy male Wistar rats (180-250 g).
2. Experimental Groups: Animals were divided into four groups (n = 5).
3. Disease Induction
45
S. No Experimental Groups Treatment
1. Group I (Control) Blank Gel
2. Group II (Standard) Marketed antifungal Cream
3. Group III (Test 1)
Test = Tenia corporis
Test formulation (G-NLCs
Gel)

7. 46
Animal model (Shimamura T et al. 2012; Koga et al.2012)
An area of 3 cm2 on the rat's dorsal site was trimmed and formed hair free
A cell suspension containing T. ruburum(Tenia pedis), M. canis(Tenia corporis) was
applied on every day for signs and symptoms of infections.
The topical treatments were started after 7 days of growth of fungal infection observed
by visual, appearances, & continued up to 21 days till the confirmations of the
complete skin healing.
 Histopathological examination
 The skin tissue specimen was preserved in 10% w/v buffered Formalin solution and
fixed in blocks and stained with Hematoxylin-eosin for histopathological examination.
Specimens were examined under a light microscope
 The histopathological examinations were done under different magnifications.

8. 47
Biochemical Estimations
Rats were dissected under ether anaesthesia killed and then . The dorsal surface skin of experimental
animal (all groups) was incised & 10 % w/v homogenate solvents were prepared to employ 1.15 %
weight/volume potassium chloride in an ice bath to stop free radical generation. After that homogenates
of animals, was centrifuged at 4000 RPM for up to 10 min. & supernatants was isolated for various kinds
of estimations.
 Catalase (CAT) activity
In this assay, supernatant of homogenate tissues (10% w/v) was combined with 3mL of hydrogen
peroxide (30 Mm/L) in 50 millimolar phosphate buffer solution(pH 7.4). The absorbance was examined
spectrophotometrically at 240 nm. The obtained values were represented in nanomoles/mg of protein.
Hydrogen peroxides calibration plot was prepared using colorimetric apparatus.

9. 48
Glutathione (reduced) estimation : Tissue homogenate was isolated with equivalent quantity
of 20 percent TCA (trichloroacetic acid) containing 1mM EDTA and after 5 minutes, centrifugation was
done at 2000 rpm for 10 minutes. In supernatant (0.1 nM) 5, 5′-Dithiobis (2-nitrobenzoic acid) and 0.3 M
PBS made with 1% of Na3C6H5O7 i.e., sodium salt of citric acid solution was added, and absorbance was
observed at 412 nm against a blank.
Lipid (Malondialdehyde) estimation : Tissue homogenate with (8.1 % of SLS), (of 20 percent
acetic acid) & (0.8% TBA) was mixed with distilled water and kept for 95 minutes at 95 degrees
Celsius, volume up to 5 mL was made up using distilled water. Later, 5 mL of pyridine: n-butanol (1:15)
was added to solution and were mixed using vortex mixture for 2 minutes. Centrifuged for10 minutes
at 3000 rpm. The isolated organic layer was observed at 532 nm. Concentration of LPO was
represented as nmoles MDA/mg protein using following equation. (Ohkawa et al.1979)

10. 98
Histology Of G-NLCs gel For Tenia pedis Infection
Histopathology image H &E Stain at
(A) 100X and (B) at 400X
magnifications (Control Group 2 –
For tenia pedis
Histopathology image of
H&E Stain 100X
Magnifications.
Test(T1b) – G-NLCs Gel
for tenia pedis
(A) H&E-stained images at
100X and (B) at 400X
magnification. (Standard – For
tenia pedis

11. 100
Biochemical Analysis of G-NLCs Gel Against Tenia pedis Infections
LPO (nmoles/mg) GSH (nmoles/mg) Catalase (nmoles/mg)
0
10
20
30
Concentration
Normal Control
Positive Control
Test(G-NLCs Gel)
Standard
+
✱
✱
++
✱✱
✱✱
+++
✱✱✱
✱✱✱
Lipid peroxidation (LPO), Glutathione (GSH) and Catalase concentration in different Treatment
Groups. Normal control, Positive Control = No treatment; Test = G-NLCs gel; Standard = Cream
(Marketed). ANOVA was used accompanied by a t-test for unpaired comparison (n=5). Values
are expressed as Mean ± SEM, ‫٭‬P < 0.05, ‫٭٭‬P < 0.01, ‫٭٭٭‬P P< 0.001 when Groups compared;
III-II, IV-II, and +P < 0.05, ++P < 0.01, +++P < 0.001 when group compared; II-I

12. HET-CAM Study
12
Before Exposure After Exposure
(-) ve Control
Group
(0.9% NaCl)
(+) ve Control
Group
(0.1 M NaOH)
Test Group
(Nanogel)

13. Cytotoxicity Study of Griseofulvin-
Loaded Nanogel on HaCaT cells
Sample
Concentration
(μg/mL)
Cell viability (%)
Replicate Values
Mean ± SD
1 2 3
Control 109.7561 90.2439 100 100 ± 9.75
1 97.560976 95.12195 107.3171 100 ± 6.45
10 90.243902 100 95.12195 95.12 ± 4.87
50 100 78.04878 100 92.68 ± 12.67
100 85.365854 104.878 87.80488 92.68 ± 10.63
250 97.560976 102.439 109.7561 103.25 ± 6.13
500 68.292683 87.80488 117.0732 91.05 ± 24.55
1000 109.7561 80.4878 112.1951 100.81 ± 17.64
13

14. Cell Viability (%) of Griseofulvin-Loaded Nanogel on HaCaT Cells using
MTT Assay. Data mentioned here as mean ± SD (n = 3) using GraphPad
Prism 9.0.0
Control 1 10 50 100 250 500 1000
0
20
40
60
80
100
120
140
Cell
Viability
(%)
HaCaT
(MTT)
Concentration (μg/mL)
Griseofulvin-Loaded Nanogel
Cytotoxicity Evaluation of Griseofulvin-Loaded Nanogel on HaCaT cells

15. 15
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