Effect of the iontophoresis of a chitosan gel on doxorubicin skin penetration and cytotoxicity SaketAsati Dept. of Pharmaceutics NIPERA1012PE12
Flow of seminar Introduction Research Article Aim Materials & Method Result & Discusion Conclusion References
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.
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.
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.
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.
Doxorubicin hydrochloride (DOX)
Propylene glycol and ZnSO4
Natrosol 250 HHR hydroxyethylcellulose(HEC)
High molecular weight Chitosan (Hydagen® CMFP)
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.
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.
Determination of the contribution of the iontophoreticelectroosmotic 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.
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.
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, Chitosannanoparticles 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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