Effect of the iontophoresis of a chitosan gel on doxorubicin skin penetration and cytotoxicity

Saket AsatiDept. of PharmaceuticsNIPERA1012PE12

Flow of seminar

Introduction

Research Article

Aim

Materials & Method

Result & Discusion

Conclusion

References

Human skin

Most extensive and readily accessible organs of the human

body.

It receives about 1/3 of the blood circulation through the body.

Adult body covers a surface area approximately 2m2

Average thickness of human skin: 0.5 mm.

The stratum corneum is effectively a 10-15μm thick.

The epidermis, is approximately 100 to150 µm thick.

Skin is a net negatively charged membrane under normal

physiological condition.

Skin structure

Iontophoresis

Iontophoresis involves the application of a weak (low) electric

current to the skin and allowing the drugs into the body, through

the skin, by a potential gradient.

Four components needed for effective Iontophoresis delivery:

• Power source in order to generate controlled direct current.

• Electrodes that contain the drug and disperse the drug.

• Negatively or positively charged aqueous medication of small

molecule size (nearly <8000 Daltons).

• Treatment site (localized).

Mechanism involved

 In Iontophoresis, electrodes are present both anode and cathode.

Anode represents a positively charged chamber whereas cathode

represents a negatively charged chamber.

Now the cationic drugs are kept under the anode or the anionic

drugs are placed under the cathode.

When a low voltage density current is applied, due to ‘Electro

repulsion’, the ions will be repelled into the skin from the active

electrode that is having the drug ions.

Cont….

The amount of drug delivered is directly proportional to the quantity of

electrical charge passed.

Electromigration and electroosmosis are two different mechanisms

contributing to the iontophoretic flux.

In electromigration, the applied electrical potential gradient causes

electrorepulsion between the positive electrode and a cationic drug.

Electroosmosis causes a convective solvent flow in the anode-to cathode

direction, which enhances the transport of cations and of neutral polar

compounds, while diminishing the overall electrotransport of anions.

Cont….

Advantages over other drug delivery systems:

When compared with injections:

Free from pain and invasion.

Minimizes the needle-pricking accidents.

Allows the drug delivery by skin contact itself can be used outside

hospitals.

When compared to pills:

Minimizes the on-set time

Adverse effects alleviation

Through this process, it is possible to delivery the drugs which

dissolve and lose their potency and efficacy in the digestive organs.

Cont….

When compared to patches (adhesives):

Shortens the on-set time

Drugs can de delivered quantitatively

Reduces the residual drug amount.

 

Chitosan gel

Chitosan is a linear polysaccharide composed of randomly

distributed β-(1-4)-linked D-glucosamine and N-acetyl-D-

glucosamine.

The amino group in chitosan makes chitosan water soluble and

cationic which readily binds to negatively charged surfaces such

as mucosal membranes.

Chitosan enhances the transport of polar drugs across epithelial

surfaces, and is biocompatible and biodegradable.

Research article

Aim

To determine the effect of iontophoresis on skin permeation &

retension of doxorubicin (DOX).

To determine the effect of chitosan gel on the electroosmotic flow.

To determine the effect of low electric current on the melanoma

cells.

Materials

Doxorubicin hydrochloride (DOX)

Methanol

Tetrahydrofuran

Propylene glycol and ZnSO4

Natrosol 250 HHR hydroxyethylcellulose(HEC)

High molecular weight Chitosan (Hydagen® CMFP)

Deionized water

Pig ear skin

B16F10 Melanoma cells

Preparation of DOX formulation

DOX 0.5% was dispersed in water, hydroxyethylcellulose (HEC)

gel and chitosan gel.

All formulations contained 119 mmol/L of NaCl and were

adjusted to pH 5.5.

The HEC gel consisted of 1.5% of the polymer, 5% (w/w) of

propylene glycol, NaCl, and water.

The chitosan gel consisted of 2% of the polysaccharide dispersed

in an acetic acid solution with 0.5% propylene glycol , NaCl and

water.

Iontophoretic experiments

Experiments were performed in vitro using vertical, flow-

through diffusion cells.

Skin was mounted in the diffusion cell with the dermal side

facing downward into the receiving medium of 6.0 mL of

isotonic buffer (HEPES 25 mmol/L, NaCl 133 mmol/L), pH

7.4.

Iontophoretic delivery of DOX was measured at a fixed pH

(5.5) from different formulations.

The anode compartment was filled with 1.0mL of the various

formulations containing 0.5% (w/w) of DOX.

Cont….

DOX transport from the anode compartment was followed over a

period of 6 h at a constant current of 0.5 mA/cm2 generated by a

Kepco APH 500DM apparatus.

The receiving solution was stirred at 300 rpm and kept at 37 °C by

a circulating water system.

“Passive” experiments were also performed with donor

formulations containing 0.5% DOX.

In these experiments, all conditions were identical to those

described above except that no current was applied.

Result:1

DOX passive permeation

from these formulations

showed that the drug does

not cross the skin in

quantifiable amounts after

6h. Therefore,iontophoresis

facilitates DOX skin

permeation.

Skin Uptake

After a 6 h experimental period, the skin was removed from

thediffusion cell and pinned to a piece of Parafilm™ with the

SC face up.

This part of the skin, which had been exposed to the anode,

was then tape-stripped 15 times.

The tape strips were subsequently immersed in 5 mL

methanol/water (1:1) in a vial for 24 h to extract the

permeated drug.

Cont….

Subsequently, an aliquot of the resulting solution was subjected

to protein precipitation and HPLC analysis to evaluate the

compounds in the SC.

The remaining skin was cut into small pieces and homogenized

by a tissue homogenizer for 2 min with 5 mL of methanol/water

(1:1).

Then analyzed by a HPLC-fluorometric assay to determine the

quantity of drug in the epidermis and dermis (‘‘viable skin’’).

Result:2

Iontophoresis of the HEC gel delivered large amounts of

DOX to the SC when compared to the chitosan gel.

But in the viable epidermis, the HEC gel delivered almost the

same amount of DOX as the chitosan gel.

Cationic charged chitosan compete with DOX for the

membrane binding sites and allow the drug to penetrate into

the deeper layers of the skin.

Cont….

Determination of the contribution of the iontophoretic electroosmotic flow

Acetaminophen (electroosmotic marker) was delivered in the

different vehicles (HEC and chitosan gels) .

Gel formulations were prepared as described before and

8.5 mmol/L of acetominophen was incorporated into the gels

in the presence and absence of DOX (0.5%).

These samples were placed in the anode compartment with

0.5 mA/cm2 of electrical current for 6 h.

Result:3

It was observed that acetaminophen electrotransport

(electroosmosis) was dramatically reduced when DOX was added

to the HEC gel.

Chitosan seems to interact with negative charges of the skin,

thereby reducing electro-osmotic flow.

Electroosmotic marker transport from chitosan gel almost

disappeared when DOX was added to the formulation.

Cont….

In vitro cytotoxicity measurement of DOX in the presence of low electrical current

Tumour cells were placed in 24-well plates with 2 mL of media

per well at a density of 3×105 cells per well.

After 24 h of culture, the media was removed and fresh media

with different concentrations of DOX formulations (10–50

ng/mL) was added to the wells.

In addition, a constant current of 0.5 mA/cm2 was applied in the

wells for 1 h via Ag/AgCl electrodes connected to a power

supply.

Cont….

Electrochemosensitivity was evaluated by the MTT assay.

The medium was replaced and 100 μL of MTT (5 mg/mL)

was added, and then the cells were incubated for a further 3 h

at 37 °C.

Result:4

It was observed that DOX formulations (HEC and chitosan,

both at 0.5%) showed no significant difference in cell toxicity

compared to control (drug solution).

Thus, iontophoresis increased DOX cytotoxicity independent

of the formulation utilized.

Cont….

Conclusion

Iontophoresis significantly increased not only the

permeation, but also the skin retention of DOX.

Iontophoresis of chitosan gels significantly decreased the

electrosmotic flow, they improved DOX diffusion throughout

the deeper layers of the skin.

The application of the low electrical current to melanoma

cells did not kill the cells directly, but did increase DOX

cytotoxicity.

References

K.A. Janes, M.P. Fresneau, A. Marazuela, A. Fabra, M.J. Alonso, Chitosan nanoparticles as delivery systems for doxorubicin, J. Control Release 73 (2–3)(2001) 255–267.

R.H. Guy, Y.N. Kalia, M.B. Delgado-Charro, V. Merino, A. Lopez, D. Marro, Iontophoresis: electrorepulsion and electroosmosis, J. Control Release 64 (1–3) (2000) 129–132.

G.L. Bidwell III, I. Fokt, W. Priebe, D. Raucher, Development of elastin-like polypeptide for thermally targeted delivery of doxorubicin, Biochem. Pharmacol. 73 (5) (2007) 620–631.

P. Glikfeld, C. Cullander, R.S. Hinz, R.H. Guy, A new system for in vitro studies of iontophoresis, Pharm. Res. 5 (7) (1988) 443–446.

E.R. Scott, A.I. Laplaza, H.S. White, J.B. Phipps, Transport of ionic species in skin: contribution of pores to the overall skin conductance, Pharm. Res. 10 (12) (1993) 1699–1709.

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