Estudio del Transporte de Péptidos a Través de Membranas con Permeabilidad Selectiva

En este trabajo se estudió el transporte de tres péptidos a través de una membrana sintética tipo piel (Strat-M® SKBM02560). Los péptidos utilizados en este estudio (DM 1.14, DM1.16 y DM 1.18) son modificaciones de dos péptidos derivados de la Dermaseptina (DM 1.4 y DM 1.6) y se sintetizaron con la...

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Jaramillo Ramírez, Sandra Nataly
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2019
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Universidad Nacional de Colombia
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Química y ciencias afines
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network_name_str Universidad Nacional de Colombia
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dc.title.spa.fl_str_mv Estudio del Transporte de Péptidos a Través de Membranas con Permeabilidad Selectiva
title Estudio del Transporte de Péptidos a Través de Membranas con Permeabilidad Selectiva
spellingShingle Estudio del Transporte de Péptidos a Través de Membranas con Permeabilidad Selectiva
Química y ciencias afines
title_short Estudio del Transporte de Péptidos a Través de Membranas con Permeabilidad Selectiva
title_full Estudio del Transporte de Péptidos a Través de Membranas con Permeabilidad Selectiva
title_fullStr Estudio del Transporte de Péptidos a Través de Membranas con Permeabilidad Selectiva
title_full_unstemmed Estudio del Transporte de Péptidos a Través de Membranas con Permeabilidad Selectiva
title_sort Estudio del Transporte de Péptidos a Través de Membranas con Permeabilidad Selectiva
dc.creator.fl_str_mv Jaramillo Ramírez, Sandra Nataly
dc.contributor.advisor.spa.fl_str_mv Barragán Ramírez, Daniel Alberto
dc.contributor.author.spa.fl_str_mv Jaramillo Ramírez, Sandra Nataly
dc.subject.ddc.spa.fl_str_mv Química y ciencias afines
topic Química y ciencias afines
description En este trabajo se estudió el transporte de tres péptidos a través de una membrana sintética tipo piel (Strat-M® SKBM02560). Los péptidos utilizados en este estudio (DM 1.14, DM1.16 y DM 1.18) son modificaciones de dos péptidos derivados de la Dermaseptina (DM 1.4 y DM 1.6) y se sintetizaron con la metodología en fase sólida (SPPS). El análisis de la estructura secundaria y el de algunas propiedades fisicoquímicas de los péptidos se hizo con herramientas de la bioinformática. Para el estudio del transporte a través de membrana se diseñaron celdas verticales de difusión tipo Franz. El transporte se llevó a cabo en presencia de solución amortiguadora de fosfato, a concentración isotónica con la sangre humana y a 37ºC. La caracterización del montaje experimental de las celdas de Franz y la implementación de la técnica analítica (RP-HPLC) para la cuantificación se hizo utilizando cafeína como sustancia testigo del transporte a través de membrana. Los datos experimentales del transporte se registraron como cantidad acumulada de sustancia transportada por unidad de área en función del tiempo. Después de someter a un análisis estadístico los datos experimentales, para tener confianza de que hay diferencias significativas en el transporte de las sustancias estudiadas, se estimaron los parámetros difusivos de la cafeína y de los péptidos mediante ajuste a la solución de un modelo tipo Fick, bajo la aproximación de dosis infinita. Los resultados obtenidos muestran que los parámetros difusivos hallados para cada sustancia tienen un valor característico, y que el péptido DM 1.14 propuesto en este trabajo es el que se transporta en mayor cantidad a través de la membrana sintética tipo piel. Estos resultados son promisorios hacia el desarrollo de nuevos productos farmacéuticos basados en biomoléculas sintéticas y de administración tópica para ser usados en el tratamiento, o como coadyuvante del tratamiento de enfermedades de origen parasitario.
publishDate 2019
dc.date.issued.spa.fl_str_mv 2019
dc.date.accessioned.spa.fl_str_mv 2020-02-10T17:04:03Z
dc.date.available.spa.fl_str_mv 2020-02-10T17:04:03Z
dc.type.spa.fl_str_mv Documento de trabajo
dc.type.driver.spa.fl_str_mv info:eu-repo/semantics/workingPaper
dc.type.version.spa.fl_str_mv info:eu-repo/semantics/publishedVersion
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dc.type.content.spa.fl_str_mv Text
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url https://repositorio.unal.edu.co/handle/unal/75572
dc.language.iso.spa.fl_str_mv spa
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Y. Gofman, T. Haliloglu, and N. Ben-Tal, “Monte Carlo simulations of peptide-membrane interactions with the MCPep web server,” Nucleic Acids Res., vol. 40, pp. 358–363, 2012.
B. Agoram, W. S. Woltosz, and M. B. Bolger, “Predicting the impact of physiological and biochemical processes on oral drug bioavailability,” Adv. Drug Deliv. Rev., vol. 50, pp. S41–S67, Oct. 2001.
K. Rajarathnam and J. Rösgen, “Isothermal titration calorimetry of membrane proteins — Progress and challenges,” Biochim. Biophys. Acta - Biomembr., vol. 1838, no. 1, pp. 69–77, Jan. 2014.
B. Sinkó et al., “Skin-PAMPA: A new method for fast prediction of skin penetration,” Eur. J. Pharm. Sci., vol. 45, no. 5, pp. 698–707, 2012.
T. Uchida, W. R. Kadhum, S. Kanai, H. Todo, T. Oshizaka, and K. Sugibayashi, “Prediction of skin permeation by chemical compounds using the artificial membrane, Strat-M,” Eur. J. Pharm. Sci., vol. 67, pp. 113–118, 2015.
G. Ottaviani, S. Martel, and P. A. Carrupt, “Parallel artificial membrane permeability assay: A new membrane for the fast prediction of passive human skin permeability,” J. Med. Chem., vol. 49, no. 13, pp. 3948–3954, 2006.
M. R. Prausnitz and R. Langer, “Transdermal drug delivery,” Nat. Biotechnol., vol. 26, no. 11, pp. 1261–1268, 2009
A. Nair et al., “Basic considerations in the dermatokinetics of topical formulations,” Brazilian J. Pharm. Sci., vol. 49, no. 3, pp. 423–434, 2013.
M. . Lane, P. Santos, A. . Watkinson, and J. Hadgraft, Passive Skin Permeation Enhancement. 2012.
M. Prausnitz, S. Mitragotri, and R. Langer, “Current status and future potential of transdermal drug delivery,” Nat. Rev. Discov., vol. 3, no. 2, pp. 115–124, 2004
R. J. Scheuplein, “Percutaneous absorption after twenty-five years: or ‘old wine in new wineskins,” Journal of Investigative Dermatology, vol. 67. pp. 31–38, 1976.
J. E. Riviere,J.D. Brooks, W. T: Collard, J. Deng, G. De Rose, S. P. Mahabir, D. A. Merritt, A. A. Marchiondo, “Prediction of formulation effects on dermal absorption of topically applied ectoparasiticides dosed in vitro on canine and porcine skin using a mixture-adjusted quantitative structure permeability relationship,” J Vet Pharmacol Ther,vol. 5. pp. 435–444, 2014.
J. A. Bouwstra, P. L. Honeywell-Nguyen, G. S. Gooris, and M. Ponec, Structure of the skin barrier and its modulation by vesicular formulations, Prog Lipid Res vol. 42, no. 1. 2003.
S. K. Li and K. D. Peck, “Passive and iontophoretic transport through the skin polar pathway,” Skin Pharmacol. Physiol., vol. 26, no. 4–6, pp. 243–253, 2013.
P. Desai, P. Shah, P. Hayden, and M. Singh, “Investigation of follicular and non-follicular pathways for polyarginine and oleic acid-modified nanoparticles.,” Pharm. Res., vol. 30, no. 4, pp. 1037–49, 2013.
N. Kanikkannan and J. Babu, “Structure Activity Relationship of Chemical Penetration Enhancers,” in Percutaneous Penetration Enhancers, vol. 19, 2015, pp. 39–54
P. Desai, R. R. Patlolla, and M. Singh, “Interaction of nanoparticles and cell-penetrating peptides with skin for transdermal drug delivery.,” Mol. Membr. Biol., vol. 27, no. 7, pp. 247–259, 2010.
M. Chen, M. Zakrewsky, V. Gupta, AC: Anselmo, DH. Slee, JA. Muraski, S. Mitragotri, “Topical delivery of siRNA into skin using SPACE-peptide carriers.,” J. Control. Release, vol. 179, pp. 33–41, 2014
S. Kumar, M. Zakrewsky, M. Chen, S. Menegatti, J. a Muraski, and S. Mitragotri, “Peptides as skin penetration enhancers: Mechanisms of action,” J. Control. Release, vol. 199, pp. 168–178, 2014.
Y. Chen, P. Quan, X. Liu, M. Wang, and L. Fang, “Novel chemical permeation enhancers for transdermal drug delivery,” Asian J. Pharm. Sci., vol. 9, no. 2, pp. 51–64, 2014.
S. Kumar, M. Chen, A. C. Anselmo, J. a. Muraski, and S. S. Mitragotri, “Enhanced epidermal localization of topically applied steroids using SPACETM peptide.,” Drug Deliv. Transl. Res., pp. 523–530, 2015.
R. R. Patlolla, P. R. Desai, K. Belay, and M. S. Singh, “Translocation of cell penetrating peptide engrafted nanoparticles across skin layers,” Biomaterials, vol. 31, no. 21, pp. 5598–5607, 2010.
L. Lopes et al., “Enhanced skin penetration of P20 phosphopeptide using protein transduction domains.,” Eur. J. Pharm. Biopharm., vol. 68, no. 2, pp. 441–5, 2008.
E. L. Snyder and S. F. Dowdy, “Cell penetrating peptides in drug delivery,” Pharm. Res., vol. 21, no. 3, pp. 389–393, 2004.
S. Kumar, P. Sahdev, O. Perumal, and H. Tummala, “Identification of a novel skin penetration enhancement peptide by phage display peptide library screening,” Mol. Pharm., vol. 9, no. 5, pp. 1320–1330, 2012.
W. Guanshun and G. Wang, Antimicrobial peptides: discovery, design and novel therapeutic strategies. 2010
M. Chen, V. Gupta, A. C. Anselmo, J. A. Muraski, and S. Mitragotri, “Topical delivery of hyaluronic acid into skin using SPACE-peptide carriers,” J. Control. Release, vol. 173, pp. 67–74, 2014.
M.-P. M. Schutze-Redelmeier, S. Kong, M. B. Bally, and J. P. Dutz, “Antennapedia transduction sequence promotes anti tumour immunity to epicutaneously administered CTL epitopes.,” Vaccine, vol. 22, no. 15–16, pp. 1985–91, 2004.
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M. Cohen-Avrahami, D. Libster, A. Aserin, and N. Garti, “Penetratin-induced transdermal delivery from HII mesophases of sodium diclofenac,” J. Control. Release, vol. 159, no. 3, pp. 419–428, 2012.
T. Ogiso and A. Yono, “In Vitro Skin Penetration and Degradation of Peptides and Their Analysis Using a Kinetic Model,” Pharm. Soc. Japan, vol. 23, no. 11, pp. 1346–1351, 2000.
J. J. Rouse, S.-F. Ng, F. D. Sanderson, V. Meidan, and G. M. Eccleston, “Validation of a Static Franz Diffusion Cell System for In Vitro Permeation Studies,” AAPS PharmSciTech, vol. 11, no. 3, pp. 1432–1441, 2010
M. Debandi and N. François, “Evaluación de distintas membranas para la libreración de principios activos anticelulíticos,” Av. en Ciencias e Ing., vol. 2, no. 2, pp. 97–105, 2011.
S. F. Ng, J. Rouse, D. Sanderson, and G. Eccleston, “A Comparative study of transmembrane diffusion and permeation of ibuprofen across synthetic membranes using franz diffusion cells,” Pharmaceutics, vol. 2, no. 2, pp. 209–223, 2010.
V. Shah, J. Elkins, S. Y. Lam, and J. Skelly, “Determination of in vitro drug release from hydrocortisone creams,” Int. J. Pharm., vol. 53, no. 1, pp. 53–59, 1989
S. T. Wu, G. K. Shiu, J. E. Simmons, R. L. Bronaugh, and J. P. Skelly, “In vitro release of nitroglycerin from topical products by use of artificial membranes,” J. Pharm. Sci., vol. 81, no. 12, pp. 1153–1156, 1992
D. Karadzovska and J. E. Riviere, “Assessing vehicle effects on skin absorption using artificial membrane assays,” Eur. J. Pharm. Sci., vol. 50, no. 5, pp. 569–576, 2013.
Y. G. Anissimov, O. G. Jepps, Y. Dancik, and M. S. Roberts, “Mathematical and pharmacokinetic modelling of epidermal and dermal transport processes,” Adv. Drug Deliv. Rev., vol. 65, no. 2, pp. 169–190, 2013
F. Yamashita and M. Hashida, “Mechanistic and empirical modeling of skin permeation of drugs,” Adv. Drug Deliv. Rev., vol. 55, no. 9, pp. 1185–1199, 2003.
S. Mitragotri et al., “Mathematical models of skin permeability: An overview,” Int. J. Pharm., vol. 418, no. 1, pp. 115–129, 2011
B. Godin and E. Touitou, “Transdermal skin delivery: predictions for humans from in vivo, ex vivo and animal models.,” Adv. Drug Deliv. Rev., vol. 59, no. 11, pp. 1152–61, 2007.
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N. J. Greenfield, “Using circular dichroism spectra to estimate protein secondary structure,” Department of Neurosciencie and Cell Biology, New Jersey, 2007.
dc.rights.spa.fl_str_mv Derechos reservados - Universidad Nacional de Colombia
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rights_invalid_str_mv Atribución-SinDerivadas 4.0 Internacional
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dc.publisher.department.spa.fl_str_mv Escuela de química
dc.publisher.branch.spa.fl_str_mv Universidad Nacional de Colombia - Sede Medellín
institution Universidad Nacional de Colombia
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spelling Atribución-SinDerivadas 4.0 InternacionalDerechos reservados - Universidad Nacional de ColombiaAcceso abiertohttp://creativecommons.org/licenses/by-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Barragán Ramírez, Daniel Alberto81552183-b7d1-4ee0-ab0f-0f190d01f8be-1Jaramillo Ramírez, Sandra Natalyf7cb4dbd-9abb-4402-af69-e07a459e132c2020-02-10T17:04:03Z2020-02-10T17:04:03Z2019https://repositorio.unal.edu.co/handle/unal/75572En este trabajo se estudió el transporte de tres péptidos a través de una membrana sintética tipo piel (Strat-M® SKBM02560). Los péptidos utilizados en este estudio (DM 1.14, DM1.16 y DM 1.18) son modificaciones de dos péptidos derivados de la Dermaseptina (DM 1.4 y DM 1.6) y se sintetizaron con la metodología en fase sólida (SPPS). El análisis de la estructura secundaria y el de algunas propiedades fisicoquímicas de los péptidos se hizo con herramientas de la bioinformática. Para el estudio del transporte a través de membrana se diseñaron celdas verticales de difusión tipo Franz. El transporte se llevó a cabo en presencia de solución amortiguadora de fosfato, a concentración isotónica con la sangre humana y a 37ºC. La caracterización del montaje experimental de las celdas de Franz y la implementación de la técnica analítica (RP-HPLC) para la cuantificación se hizo utilizando cafeína como sustancia testigo del transporte a través de membrana. Los datos experimentales del transporte se registraron como cantidad acumulada de sustancia transportada por unidad de área en función del tiempo. Después de someter a un análisis estadístico los datos experimentales, para tener confianza de que hay diferencias significativas en el transporte de las sustancias estudiadas, se estimaron los parámetros difusivos de la cafeína y de los péptidos mediante ajuste a la solución de un modelo tipo Fick, bajo la aproximación de dosis infinita. Los resultados obtenidos muestran que los parámetros difusivos hallados para cada sustancia tienen un valor característico, y que el péptido DM 1.14 propuesto en este trabajo es el que se transporta en mayor cantidad a través de la membrana sintética tipo piel. Estos resultados son promisorios hacia el desarrollo de nuevos productos farmacéuticos basados en biomoléculas sintéticas y de administración tópica para ser usados en el tratamiento, o como coadyuvante del tratamiento de enfermedades de origen parasitario.In this work, the transport of three different peptides through a synthetic membrane skin-type (Strat-M® SKBM02560) was studied. The peptides used in this work (DM 1.14, DM1.16 y DM 1.18) were derived from the Dermaseptin (DM 1.4 y DM 1.6) and were synthetized with the solid phase peptide synthesis methodology (SPPS). The secondary structure and some of the physicochemical properties of the peptides were analyzed with tools of bioinformatics. To study the transport through the membrane some Franz type diffusion vertical cells were designed. The transport occurred in presence of a phosphate buffer solution, an isotonic concentration with the human blood at 37°C. The characterization of the experimental assembly of the Franz’ cells and the execution of the analytical technique (RP-HPLC) to quantify, was made using the caffeine as the reference substance of the transport through the membrane. The experimental data was recorded as an accumulate quantity of the substance transport by unit of area as a function of time. After performing a statistical analysis on experimental data, to stablish an confident result between the significant differences in the transport among the studied substances, some parameters of diffusion were estimated for caffeine and peptides by adjusting the solution of a type-Fick model under the approximation to the infinite doses. The results obtained show that the substances of this study have characteristically diffusive parameter values and the peptide DM 1.14, proposed in this work, has the highest quantity of transport through the synthetic skin type membrane. The results obtained in this work are promising to the development of new pharmaceutical products based on synthetic biomolecules and with a topic administration can be used as treatment or coadjutant of the treatment in conditions caused by parasitesMaestríaspaQuímica y ciencias afinesEstudio del Transporte de Péptidos a Través de Membranas con Permeabilidad SelectivaDocumento de trabajoinfo:eu-repo/semantics/workingPaperinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_8042http://purl.org/coar/version/c_970fb48d4fbd8a85Texthttp://purl.org/redcol/resource_type/WPEscuela de químicaUniversidad Nacional de Colombia - Sede MedellínE. Heinz, “Transport through biological membranes.,” Annu. Rev. Physiol., vol. 29, no. May, pp. 21–58, Mar. 1967Y. Gofman, T. Haliloglu, and N. Ben-Tal, “Monte Carlo simulations of peptide-membrane interactions with the MCPep web server,” Nucleic Acids Res., vol. 40, pp. 358–363, 2012.B. Agoram, W. S. Woltosz, and M. B. Bolger, “Predicting the impact of physiological and biochemical processes on oral drug bioavailability,” Adv. Drug Deliv. Rev., vol. 50, pp. S41–S67, Oct. 2001.K. Rajarathnam and J. Rösgen, “Isothermal titration calorimetry of membrane proteins — Progress and challenges,” Biochim. Biophys. Acta - Biomembr., vol. 1838, no. 1, pp. 69–77, Jan. 2014.B. Sinkó et al., “Skin-PAMPA: A new method for fast prediction of skin penetration,” Eur. J. Pharm. Sci., vol. 45, no. 5, pp. 698–707, 2012.T. Uchida, W. R. Kadhum, S. Kanai, H. Todo, T. Oshizaka, and K. Sugibayashi, “Prediction of skin permeation by chemical compounds using the artificial membrane, Strat-M,” Eur. J. Pharm. Sci., vol. 67, pp. 113–118, 2015.G. Ottaviani, S. Martel, and P. A. Carrupt, “Parallel artificial membrane permeability assay: A new membrane for the fast prediction of passive human skin permeability,” J. Med. Chem., vol. 49, no. 13, pp. 3948–3954, 2006.M. R. Prausnitz and R. Langer, “Transdermal drug delivery,” Nat. Biotechnol., vol. 26, no. 11, pp. 1261–1268, 2009A. Nair et al., “Basic considerations in the dermatokinetics of topical formulations,” Brazilian J. Pharm. Sci., vol. 49, no. 3, pp. 423–434, 2013.M. . Lane, P. Santos, A. . Watkinson, and J. Hadgraft, Passive Skin Permeation Enhancement. 2012.M. Prausnitz, S. Mitragotri, and R. Langer, “Current status and future potential of transdermal drug delivery,” Nat. Rev. Discov., vol. 3, no. 2, pp. 115–124, 2004R. J. Scheuplein, “Percutaneous absorption after twenty-five years: or ‘old wine in new wineskins,” Journal of Investigative Dermatology, vol. 67. pp. 31–38, 1976.J. E. Riviere,J.D. Brooks, W. T: Collard, J. Deng, G. De Rose, S. P. Mahabir, D. A. Merritt, A. A. Marchiondo, “Prediction of formulation effects on dermal absorption of topically applied ectoparasiticides dosed in vitro on canine and porcine skin using a mixture-adjusted quantitative structure permeability relationship,” J Vet Pharmacol Ther,vol. 5. pp. 435–444, 2014.J. A. Bouwstra, P. L. Honeywell-Nguyen, G. S. Gooris, and M. Ponec, Structure of the skin barrier and its modulation by vesicular formulations, Prog Lipid Res vol. 42, no. 1. 2003.S. K. Li and K. D. Peck, “Passive and iontophoretic transport through the skin polar pathway,” Skin Pharmacol. Physiol., vol. 26, no. 4–6, pp. 243–253, 2013.P. Desai, P. Shah, P. Hayden, and M. Singh, “Investigation of follicular and non-follicular pathways for polyarginine and oleic acid-modified nanoparticles.,” Pharm. Res., vol. 30, no. 4, pp. 1037–49, 2013.N. Kanikkannan and J. Babu, “Structure Activity Relationship of Chemical Penetration Enhancers,” in Percutaneous Penetration Enhancers, vol. 19, 2015, pp. 39–54P. Desai, R. R. Patlolla, and M. Singh, “Interaction of nanoparticles and cell-penetrating peptides with skin for transdermal drug delivery.,” Mol. Membr. Biol., vol. 27, no. 7, pp. 247–259, 2010.M. Chen, M. Zakrewsky, V. Gupta, AC: Anselmo, DH. Slee, JA. Muraski, S. Mitragotri, “Topical delivery of siRNA into skin using SPACE-peptide carriers.,” J. Control. Release, vol. 179, pp. 33–41, 2014S. Kumar, M. Zakrewsky, M. Chen, S. Menegatti, J. a Muraski, and S. Mitragotri, “Peptides as skin penetration enhancers: Mechanisms of action,” J. Control. Release, vol. 199, pp. 168–178, 2014.Y. Chen, P. Quan, X. Liu, M. Wang, and L. Fang, “Novel chemical permeation enhancers for transdermal drug delivery,” Asian J. Pharm. Sci., vol. 9, no. 2, pp. 51–64, 2014.S. Kumar, M. Chen, A. C. Anselmo, J. a. Muraski, and S. S. Mitragotri, “Enhanced epidermal localization of topically applied steroids using SPACETM peptide.,” Drug Deliv. Transl. Res., pp. 523–530, 2015.R. R. Patlolla, P. R. Desai, K. Belay, and M. S. Singh, “Translocation of cell penetrating peptide engrafted nanoparticles across skin layers,” Biomaterials, vol. 31, no. 21, pp. 5598–5607, 2010.L. Lopes et al., “Enhanced skin penetration of P20 phosphopeptide using protein transduction domains.,” Eur. J. Pharm. Biopharm., vol. 68, no. 2, pp. 441–5, 2008.E. L. Snyder and S. F. Dowdy, “Cell penetrating peptides in drug delivery,” Pharm. Res., vol. 21, no. 3, pp. 389–393, 2004.S. Kumar, P. Sahdev, O. Perumal, and H. Tummala, “Identification of a novel skin penetration enhancement peptide by phage display peptide library screening,” Mol. Pharm., vol. 9, no. 5, pp. 1320–1330, 2012.W. Guanshun and G. Wang, Antimicrobial peptides: discovery, design and novel therapeutic strategies. 2010M. Chen, V. Gupta, A. C. Anselmo, J. A. Muraski, and S. Mitragotri, “Topical delivery of hyaluronic acid into skin using SPACE-peptide carriers,” J. Control. Release, vol. 173, pp. 67–74, 2014.M.-P. M. Schutze-Redelmeier, S. Kong, M. B. Bally, and J. P. 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W5pbyBww7pibGljby4gU2kgbm8gZnVlc2UgZWwgY2FzbywgYWNlcHRvIHRvZGEgbGEgcmVzcG9uc2FiaWxpZGFkIHBvciBjdWFscXVpZXIgaW5mcmFjY2nDs24gZGUgZGVyZWNob3MgZGUgYXV0b3IgcXVlIGNvbmxsZXZlIGxhIGRpc3RyaWJ1Y2nDs24gZGUgZXN0b3MgYXJjaGl2b3MgeSBtZXRhZGF0b3MuCkFsIGhhY2VyIGNsaWMgZW4gZWwgc2lndWllbnRlIGJvdMOzbiwgdXN0ZWQgaW5kaWNhIHF1ZSBlc3TDoSBkZSBhY3VlcmRvIGNvbiBlc3RvcyB0w6lybWlub3MuCg==

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