1. Effect of the iontophoresis of a chitosan gel on doxorubicin skin penetration and cytotoxicity<br />SaketAsati<br />Dept. of Pharmaceutics<br />NIPERA1012PE12<br />
2. Flow of seminar<br />Introduction<br />Research Article<br />Aim<br />Materials & Method<br />Result & Discusion<br />Conclusion<br />References<br />
3. Human skin<br /><ul><li> Most extensive and readily accessible organs of the human body.
4. It receives about 1/3 of the blood circulation through the body.
5. Adult body covers a surface area approximately 2m2
6. Average thickness of human skin: 0.5 mm.
7. The stratum corneum is effectively a 10-15μm thick.
8. The epidermis, is approximately 100 to150 µm thick.
9. Skin is a net negatively charged membrane under normal physiological condition.</li></li></ul><li>Skin structure<br />
10. Iontophoresis<br />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.<br />Four components needed for effective Iontophoresis delivery:<br />• Power source in order to generate controlled direct current.<br />• Electrodes that contain the drug and disperse the drug.<br />• Negatively or positively charged aqueous medication of small molecule size (nearly <8000 Daltons).<br />• Treatment site (localized).<br />
11. Mechanism involved<br /> In Iontophoresis, electrodes are present both anode and cathode.<br /> Anode represents a positively charged chamber whereas cathode represents a negatively charged chamber.<br /> Now the cationic drugs are kept under the anode or the anionic drugs are placed under the cathode.<br />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.<br />
12. Cont….<br />The amount of drug delivered is directly proportional to the quantity of electrical charge passed. <br />Electromigration and electroosmosis are two different mechanisms contributing to the iontophoretic flux.<br />In electromigration, the applied electrical potential gradient causes electrorepulsion between the positive electrode and a cationic drug.<br />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.<br />
13. Cont….<br />
14. Advantages over other drug delivery systems:<br />When compared with injections:<br /><ul><li> Free from pain and invasion.
15. Minimizes the needle-pricking accidents.
16. Allows the drug delivery by skin contact itself can be used outside hospitals.</li></ul>When compared to pills:<br /><ul><li>Minimizes the on-set time
17. Adverse effects alleviation
18. Through this process, it is possible to delivery the drugs which dissolve and lose their potency and efficacy in the digestive organs.</li></li></ul><li>Cont….<br />When compared to patches (adhesives):<br /><ul><li>Shortens the on-set time
19. Drugs can de delivered quantitatively
20. Reduces the residual drug amount.</li></ul> <br />
21. Chitosan gel<br />Chitosan is a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine. <br />The amino group in chitosan makes chitosan water soluble and cationic which readily binds to negatively charged surfaces such as mucosal membranes.<br />Chitosan enhances the transport of polar drugs across epithelial surfaces, and is biocompatible and biodegradable.<br />
22. Research article<br />
23. Aim<br />To determine the effect of iontophoresis on skin permeation & retension of doxorubicin (DOX).<br />To determine the effect of chitosan gel on the electroosmotic flow.<br />To determine the effect of low electric current on the melanoma cells.<br />
29. High molecular weight Chitosan (Hydagen® CMFP)
30. Deionized water
31. Pig ear skin
32. B16F10 Melanoma cells</li></li></ul><li>Preparation of DOX formulation<br /><ul><li>DOX 0.5% was dispersed in water, hydroxyethylcellulose (HEC) gel and chitosan gel.
33. All formulations contained 119 mmol/L of NaCl and were adjusted to pH 5.5.
34. The HEC gel consisted of 1.5% of the polymer, 5% (w/w) of propylene glycol, NaCl, and water.
35. The chitosan gel consisted of 2% of the polysaccharide dispersed in an acetic acid solution with 0.5% propylene glycol , NaCl and water.</li></li></ul><li>Iontophoretic experiments<br />Experiments were performed in vitro using vertical, flow-through diffusion cells.<br /> 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.<br />Iontophoretic delivery of DOX was measured at a fixed pH (5.5) from different formulations.<br />The anode compartment was filled with 1.0mL of the various formulations containing 0.5% (w/w) of DOX.<br />
36. Cont….<br />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.<br />The receiving solution was stirred at 300 rpm and kept at 37 °C by a circulating water system.<br />“Passive” experiments were also performed with donor formulations containing 0.5% DOX.<br />In these experiments, all conditions were identical to those described above except that no current was applied.<br />
37. Result:1<br /><ul><li>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.</li></li></ul><li>Skin Uptake<br />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.<br />This part of the skin, which had been exposed to the anode, was then tape-stripped 15 times.<br />The tape strips were subsequently immersed in 5 mL methanol/water (1:1) in a vial for 24 h to extract the permeated drug.<br />
38. Cont….<br />Subsequently, an aliquot of the resulting solution was subjected to protein precipitation and HPLC analysis to evaluate the compounds in the SC.<br />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).<br />Then analyzed by a HPLC-fluorometric assay to determine the quantity of drug in the epidermis and dermis (‘‘viable skin’’).<br />
39. Result:2<br />Iontophoresis of the HEC gel delivered large amounts of DOX to the SC when compared to the chitosan gel. <br />But in the viable epidermis, the HEC gel delivered almost the same amount of DOX as the chitosan gel.<br /> Cationic charged chitosan compete with DOX for the membrane binding sites and allow the drug to penetrate into the deeper layers of the skin.<br />
40. Cont….<br />
41. Determination of the contribution of the iontophoreticelectroosmotic flow<br />Acetaminophen (electroosmotic marker) was delivered in the different vehicles (HEC and chitosan gels) .<br />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%).<br />These samples were placed in the anode compartment with 0.5 mA/cm2 of electrical current for 6 h.<br />
42. Result:3<br />It was observed that acetaminophen electrotransport (electroosmosis) was dramatically reduced when DOX was added to the HEC gel.<br />Chitosan seems to interact with negative charges of the skin, thereby reducing electro-osmotic flow.<br />Electroosmotic marker transport from chitosan gel almost disappeared when DOX was added to the formulation.<br />
43. Cont….<br />
44. In vitro cytotoxicity measurement of DOX in the presence of low electrical current<br />Tumour cells were placed in 24-well plates with 2 mL of media per well at a density of 3×105 cells per well.<br /> 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. <br />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.<br />
45. Cont….<br />Electrochemosensitivity was evaluated by the MTT assay.<br />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.<br />
46. Result:4<br />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).<br />Thus, iontophoresis increased DOX cytotoxicity independent of the formulation utilized.<br />
47. Cont….<br />
48. Conclusion<br />Iontophoresis significantly increased not only the permeation, but also the skin retention of DOX.<br />Iontophoresis of chitosan gels significantly decreased the electrosmotic flow, they improved DOX diffusion throughout the deeper layers of the skin.<br />The application of the low electrical current to melanoma cells did not kill the cells directly, but did increase DOX cytotoxicity.<br />
49. References<br />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.<br />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.<br />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.<br />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.<br />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.<br />
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