This document summarizes a study investigating the penetration of degradable particles into hair follicles for drug delivery purposes. Bovine serum albumin (BSA) particles loaded with a fluorescent dye (FITC) were fabricated and applied to pig ear skin using different protocols. Laser scanning microscopy found the particles penetrated deeply into hair follicles, with an average depth of 886 μm for one protocol. Releasing protease after particle application increased fluorescence intensity in upper follicles. The results support degradable particles around 500 nm as a promising approach for targeted drug delivery via hair follicles.

1. Drug Delivery Into The Skin By
Degradable Particles
Anish Shrestha
6th Semester, B Pharm.
School of Health and Allied Sciences
Pokhara University, PO Box 427, Lekhnath-
12, Kaski, NEPAL

2. OVERVIEW
• Introduction
• Objective
• Material and Method
• Results
• Discussion
• References

3. Introduction
• Until recently, the only relevant penetration
pathway for topically applied substances through
the skin barrier was the intercellular route i.e.
through the lipid layers gathered around
corneocytes.
• But, the hair follicles may also represent an
efficient penetration pathway.

4. Hair follicles – interesting target for
drug delivery
• Surrounded by a close network of capillaries and
dendritic cells.
• Host of the stem cells important for regenerative
medicine and immunomodulation.
• A long term reservoir for drugs.
• No drug loss.
• Particle size of approx. 500nm penetrate better.

5. Objective
In the study,
The penetration of degradable particles based on a
two component system into the hair follicles was
investigated under in vivo conditions.

6. Materials and Methods
• Particle preparation
• Skin model
• Application protocols
• Skin samples

7. Particle preparation
 Bovine serum albumin ( BSA ) ,fabricated based on co-
precipitation of BSA molecules with Calcium carbonate
followed by cross linkage of BSA molecules and
dissolution of the calcium carbonate template.
 Model drug-fluorescein isothiocyanate ( FITC) was
loaded into BSA particles.
 Drug loaded particles were dispersed in Phosphate
Buffer saline (PBS) at pH 7.4

8. Contd….
• BSA-FITC particles had a uniform spherical
morphology (Average diameter of 500 ± 30 nm.)
• Initiation of decomposition of BSA particles with a
protease solution.
• Protease solution (0.1 U µ1ˉ¹, PBS pH 7.4) hydrolyzes
the peptide bonds within BSA molecules and breaks
down the particles into amino acid residues.
• Investigated by Transmission Microscopy.

9. Skin Model
• Pig ear skin was chosen.
• Six pig ears from freshly slaughtered pigs.
• Porcine ears, washed with cold water, dried.
• Formulation applied to the ears on marked areas of
6 cm².

10. Application Protocols
• Application protocol no.1
– Particle containing formulation applied onto the skin
surface at concentration of 10 µl/cm².
– Massage applied immediately for 2 min.
– After penetration time of 30 min, Biopsies were
removed.

11. Application protocol no. 2
• Particle containing formulation applied onto the
skin surface.
• After penetration time of 30 min, 5 µl/cm² of the
protease solution was applied onto the same skin
area pre-treated with the particles.
• After 30 min, Biopsies were removed.

12. Application Protocol No. 3
• The particle-containing formulation (60 µl) was
mixed with the Protease solution (30 µl) and was
stored for 30 min in a glass vessel before
application onto the skin.
• 30 min after application of the mixture onto skin,
biopsies were taken.

13. Skin Samples
• After application and penetration, punch biopsies
with a diameter of 4 mm were taken.
• Histological sections with a thickness of 5µm were
prepared.
• Fluorescence mode of a LSM used to visualize the
distribution of dye in the hair follicles.
• An argon laser at 488 nm was used for dye
excitation.

14. RESULTS

15. Typical images of the particles obtained by scanning electronic
microscopy

16. Fig; The kinetics of the degradation
process of the particles after contact
with the formulation containing 0.1
U ll1 protease
T= 0 min
T= 5 min
T= 25 min
T= 60 min

17. • Laser scanning microscopic analysis of the
degradation process of the particles on the
microscopic glass slides showed an increase in the
fluorescence intensity during the degradation of
the particles.

18. Fig. 3. Follicular penetration. Fluorescence LSM analysis of histological sections
demonstrating the penetration of the particles into the hair follicles: (a) –
application of particles alone (application protocol no. 1). (b) – application of
particles previous to protease administration (protocol no. 2).

19. • Analyzing 20 hair follicles, an average penetration
depth of the dye-loaded particles of 886 ± 101 µm
was found. ( for application protocol no. 1),
• If the particles were applied 30 min previous to the
protease application (application protocol no. 2),
again the fluorescence of the particles could be
detected deep inside the hair follicles up to
approximately 841 ± 159 µm

20. • In the deeper parts of hair follicle, the fluorescence
intensity was comparable.
• In the upper parts, the hair follicles showed higher
FI upto a depth of approx 280 ± 80 µm

21. • In the case of application protocol no. 3 (particles
and protease were stored 30 min in a glass vessel
before application), an intensive fluorescence
signal could be detected up to a depth of 486 ± 80
µm.
• In deeper parts of the hair follicles, no
fluorescence signal was found.

22. DISCUSSION

23. • The particles used had an average size of 500 ± 30
nm.
• In the study, the particles dissolved when they
contacted the protease, due to a hydrolytic reaction
cleaving the peptide bonds within protein
molecules into small peptide fragments and
producing amino acid residues.
•
• Finally, the BSA particles were disassembled and
the model drug, the fluorescent dye, was released.

24. • Using the application protocol no. 1, a penetration
of the particles deep into the hair follicles up to
approx. 900 µm was detected.
• When utilizing application protocol no. 2, the
particles were able to penetrate deeply into the hair
follicles within 30 min
• As a result, the fluorescence intensity of the dye
increased strongly in the upper part of the hair
follicles

25. Contd…..
• The utilization of application protocol no. 3
revealed an intensive fluorescence signal in the
upper 500 µm of the hair follicles.
• Though penetration was less, Fluoresence intesity
was relatively higher.

27. • The results obtained in the present study confirm
that particles at a size of approx. 500 nm penetrate
the deepest into the hair follicles.
• The model drug (fluorescent dye) was released
close to the target structures in the hair follicles.
• The transport of the dye into the hair follicles was
more efficient when the particles were used as a
carrier system.

28. Conclusion
• Obtained results convincingly shows that the
degradable particles can be a promising tool in the
drug delivery.

29. References
• W.C. Mak et al. Drug delivery into the skin by
degradable particles, European Journal of
Pharmaceutics and Biopharmaceutics 79 (2011)
23–27.

30. Thank you