Visualization of plasmid delivery to keratinocytes in mouse and human epidermis

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Abstract

The accessibility of skin makes it an ideal target organ for nucleic acid-based therapeutics; however, effective patient-friendly delivery remains a major obstacle to clinical utility. A variety of limited and inefficient methods of delivering nucleic acids to keratinocytes have been demonstrated; further advances will require well-characterized reagents, rapid noninvasive assays of delivery, and well-developed skin model systems. Using intravital fluorescence and bioluminescence imaging and a standard set of reporter plasmids we demonstrate transfection of cells in mouse and human xenograft skin using intradermal injection and two microneedle array delivery systems. Reporter gene expression could be detected in individual keratinocytes, in real-time, in both mouse skin as well as human skin xenografts. These studies revealed that non-invasive intravital imaging can be used as a guide for developing gene delivery tools, establishing a benchmark for comparative testing of nucleic acid skin delivery technologies.

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Microneedles for transdermal drug delivery.

Mark Prausnitz (2004)

The success of transdermal drug delivery has been severely limited by the inability of most drugs to enter the skin at therapeutically useful rates. Recently, the use of micron-scale needles in increasing skin permeability has been proposed and shown to dramatically increase transdermal delivery, especially for macromolecules. Using the tools of the microelectronics industry, microneedles have been fabricated with a range of sizes, shapes and materials. Most drug delivery studies have emphasized solid microneedles, which have been shown to increase skin permeability to a broad range of molecules and nanoparticles in vitro. In vivo studies have demonstrated delivery of oligonucleotides, reduction of blood glucose level by insulin, and induction of immune responses from protein and DNA vaccines. For these studies, needle arrays have been used to pierce holes into skin to increase transport by diffusion or iontophoresis or as drug carriers that release drug into the skin from a microneedle surface coating. Hollow microneedles have also been developed and shown to microinject insulin to diabetic rats. To address practical applications of microneedles, the ratio of microneedle fracture force to skin insertion force (i.e. margin of safety) was found to be optimal for needles with small tip radius and large wall thickness. Microneedles inserted into the skin of human subjects were reported as painless. Together, these results suggest that microneedles represent a promising technology to deliver therapeutic compounds into the skin for a range of possible applications.

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Transport into and out of the nucleus.

I Macara (2001)

A defining characteristic of eukaryotic cells is the possession of a nuclear envelope. Transport of macromolecules between the nuclear and cytoplasmic compartments occurs through nuclear pore complexes that span the double membrane of this envelope. The molecular basis for transport has been revealed only within the last few years. The transport mechanism lacks motors and pumps and instead operates by a process of facilitated diffusion of soluble carrier proteins, in which vectoriality is provided by compartment-specific assembly and disassembly of cargo-carrier complexes. The carriers recognize localization signals on the cargo and can bind to pore proteins. They also bind a small GTPase, Ran, whose GTP-bound form is predominantly nuclear. Ran-GTP dissociates import carriers from their cargo and promotes the assembly of export carriers with cargo. The ongoing discovery of numerous carriers, Ran-independent transport mechanisms, and cofactors highlights the complexity of the nuclear transport process. Multiple regulatory mechanisms are also being identified that control cargo-carrier interactions. Circadian rhythms, cell cycle, transcription, RNA processing, and signal transduction are all regulated at the level of nucleocytoplasmic transport. This review focuses on recent discoveries in the field, with an emphasis on the carriers and cofactors involved in transport and on possible mechanisms for movement through the nuclear pores.

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Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.

Adil Daud, Ronald C DeConti, Stephanie Andrews … (2008)

Gene-based immunotherapy for cancer is limited by the lack of safe, efficient, reproducible, and titratable delivery methods. Direct injection of DNA into tissue, although safer than viral vectors, suffers from low gene transfer efficiency. In vivo electroporation, in preclinical models, significantly enhances gene transfer efficiency while retaining the safety advantages of plasmid DNA. A phase I dose escalation trial of plasmid interleukin (IL)-12 electroporation was carried out in patients with metastatic melanoma. Patients received electroporation on days 1, 5, and 8 during a single 39-day cycle, into metastatic melanoma lesions with six 100-mus pulses at a 1,300-V/cm electric field through a penetrating six-electrode array immediately after DNA injection. Pre- and post-treatment biopsies were obtained at defined time points for detailed histologic evaluation and determination of IL-12 protein levels. Twenty-four patients were treated at seven dose levels, with minimal systemic toxicity. Transient pain after electroporation was the major adverse effect. Post-treatment biopsies showed plasmid dose proportional increases in IL-12 protein levels as well as marked tumor necrosis and lymphocytic infiltrate. Two (10%) of 19 patients with nonelectroporated distant lesions and no other systemic therapy showed complete regression of all metastases, whereas eight additional patients (42%) showed disease stabilization or partial response. This report describes the first human trial, to our knowledge, of gene transfer utilizing in vivo DNA electroporation. The results indicated this modality to be safe, effective, reproducible, and titratable.

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Author and article information

Journal

Journal ID (nlm-ta): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group

ISSN (Electronic): 2045-2322

Publication date (Electronic): 15 November 2011

Publication date Collection: 2011

Volume: 1

Electronic Location Identifier: 158

Affiliations

[1 ]simpleMolecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine , CA, USA

[2 ]simpleDepartment of Pediatrics, Stanford University School of Medicine , CA, USA

[3 ]simpleDepartment of Radiology, Stanford University School of Medicine , CA, USA

[4 ]simpleDepartment of Microbiology & Immunology, Stanford University School of Medicine , CA, USA

[5 ]simpleSchool of Chemical and Biomolecular Engineering, Georgia Institute of Technology , Atlanta, GA, USA

[6 ]simpleDepartment of Microbiology and Immunology, Emory University School of Medicine , Atlanta, GA 30322

[7 ]simpleTransDerm Inc. , Santa Cruz, CA, USA

[8 ]simpleWelsh School of Pharmacy, Cardiff University , Cardiff CF10 3NB, UK

[9 ]simpleDepartment of Dermatology Yale University School of Medicine , New Haven, CT, USA

[10 ]simpleDepartment of Immunology, Yale University School of Medicine , New Haven, CT, USA

[11 ]Current address: Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

[12 ]Current address: Department of Developmental and Cell Biology, University of California at Irvine, Irvine, CA

Author notes

[a ] Roger.Kaspar@ 123456TransDermInc.com

Article

Publisher Item ID: srep00158

DOI: 10.1038/srep00158

PMC ID: 3240989

PubMed ID: 22355673

SO-VID: 8415ce1e-27e1-4aa0-9641-75ea0c578eb0

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This work is licensed under a Creative Commons Attribution-NonCommercial-ShareALike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/

Visualization of plasmid delivery to keratinocytes in mouse and human epidermis

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Abstract

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RNA drug delivery

Most cited references 55

Microneedles for transdermal drug delivery.

Transport into and out of the nucleus.

Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.

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