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Development of a transdermal drug delivery platform technology based on a designed cell-penetrating peptide

  • 권다솜 (KWONDASOM, 아주대학교 일반대학원)

초록/요약

The exploration of non-invasive drug administration routes, such as oral, nasal, ocular, and transdermal, has gained prominence as alternatives to traditional needle injections. Transdermal drug delivery stands out as an attractive method due to its ease of administration, sustained release, and minimized systemic side effects. In this study, we employ support vector machine-based models to design highly effective cell- penetrating peptides (CPPs), known for their efficient transduction and low cytotoxicity. A genomic construct, incorporating growth factors or segments of botulinum toxin, is synthesized and combined with a novel CPP named dermal penetration peptide (DPP). The induced expression of proteins is achieved through Isopropyl β-D-1-thiogalactopyranoside addition, followed by purification using Ni- NTA agarose resin and analysis through western blotting. Topical application of the newly designed peptides on human skin demonstrates their penetration ability, as confirmed through confocal imaging of frozen vertical skin sections. Skin penetration assays reveal a 3.3-fold increase in efficiency for DPP compared to the well-known CPP, TAT. The DPP-attached botulinum toxin exhibits equivalent cleavage efficacy to the non-linked counterpart in cell assays. Additionally, growth factor wound healing tests show significant wound recovery 48 hours post-treatment. Western blotting analysis confirms the expression of recombinant proteins, with specific bands detected at approximately 57.5 and 10 kDa. Cytotoxicity studies at high concentrations demonstrate the non-toxic nature of the developed materials. The results underscore the potential of DPP as a skin efficacy platform technology capable of producing substantial effects even in small quantities.

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목차

1. Introduction 1
1.1. Skin 1
1.1.1. Composition of The Dermis 1
1.1.2. Dermal Aging 2
1.2. Transdermal Drug Delivery System (TDDS) 2
1.2.1. Advantages of The TDDS 2
1.2.2. TDDS In The Skin 3
1.2.3. Cell Penetrating Peptides (CPPs) 3
1.2.3.1. Mechanism 4
1.2.3.2. Classification of CPPs 5
1.3. Cosmetic Ingredients 6
1.3.1. Botulinum Toxin (BTX) 6
1.3.1.1. Organism 7
1.3.1.2. Structure of BoNTs 7
1.3.1.3. Mode of Action of BoNTs 8
1.3.2. Epidermal Growth Factor (EGF) 11
1.3.3. Insulin-like Growth Factors (IGFs) 13
1.4. Recombinant Protein 14
1.5. Objective 15

2. MATERIALS &METHODS 16
2.1. Design of CPPs 16
2.2. Preparation of Gene construct 16
2.3. Recombinant Protein Transformation 19
2.4. Recombinant Protein Expression 20
2.5. Recombinant Protein Purification 20
2.5.1. Recombinant Protein Purification Under Native Condition (DPP-B001) 20
2.5.2. Recombinant Protein Purification Under Denaturing Condition (DPP-I001, DPP-E001) 21
2.6. Confirmation of Recombinant Protein Purification 22
2.7. Cytotoxicity Assay 23
2.8. Skin Permeation of FITC 23
2.9. Franz Diffusion Cell Assay 24
2.10. Efficacy Evaluation 25
2.10.1. SNAP-25 Cleavage Assay 25
2.10.2. In Vitro Scratch Assay 26
2.10.3. In Vitro Moisturizing Test 26

III. RESULTS&DISCUSSION 28
3.1. Design of DPPs 28
3.2. Preparation Of Gene Construct 29
3.3. Recombinant Protein Expression and Purification 31
3.4. Cytotoxicity Assessment 33
3.5. Skin Permeation of FITC 35
3.6. Franz Diffusion Cell Assay 36
3.7. Efficacy Evaluation 37
3.7.1. SNAP-25 Cleavage Assay 37
3.7.2. In Vitro Scratch Assay 38
3.7.3. In Vitro Moisturizing Test 39

IV. CONCLUSION 41

V. REFERNECE 42

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