Drug Delivery Into The Skin By Degradable Particles

Anish Shrestha6th Semester, B Pharm.

School of Health and Allied SciencesPokhara University, PO Box 427, Lekhnath-12, Kaski,

NEPAL

OVERVIEW

• Introduction• Objective• Material and Method• Results• Discussion• References

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.

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.

ObjectiveIn the study,

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

Materials and Methods

• Particle preparation• Skin model• Application protocols• Skin samples

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

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.

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.

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.

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.

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.

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.

RESULTS

Typical images of the particles obtained by scanning electronicmicroscopy

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

• 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.

Fig. 3. Follicular penetration. Fluorescence LSM analysis of histological sectionsdemonstrating the penetration of the particles into the hair follicles: (a) –

application of particles alone (application protocol no. 1). (b) – application ofparticles previous to protease administration (protocol no. 2).

• 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

• 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

• 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.

DISCUSSION

• 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.

• 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

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.

• 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.

Conclusion• Obtained results convincingly shows that the

degradable particles can be a promising tool in the drug delivery.

References

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

Thank you