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A method for reversible drug delivery to internal tissues of Drosophila embryos.

ABSTRACT: Drosophila melanogaster is a powerful model organism to elucidate basic cellular mechanisms of development. Indeed, much of our understanding of genetic pathways comes from work in Drosophila. However, mutations in many critical genes cause early embryonic lethality; thus, it is difficult to study the role of proteins that are required for early fundamental processes during later embryonic stages. We have therefore developed a method to reversibly deliver drugs to internal tissues of stage 15-16 Drosophila embryos using a 1:1 combination of D-limonene and heptane (LH). Specifically, delivery of Nocodazole was shown to be effective as evidenced by the significant decrease in microtubule density seen in muscle cells. Following complete depolymerization of the microtubule cytoskeleton, removing the Nocodazole and washing for 10 min was sufficient for the microtubule network to be re-established, indicating that drug delivery is reversible. Additionally, the morphology of LH-treated embryos resembled that of untreated controls, and embryo viability post-treatment with LH was significantly increased compared with previously reported permeabilization techniques. These advances in embryo permeabilization provide a means to disrupt protein function in vivo with high temporally specificity, bypassing the complications associated with genetic disruptions as they relate to the study of late-stage developmental mechanisms.

SUBMITTER: Schulman VK

PROVIDER: S-EPMC4049853 | biostudies-other | 2013 Jul-Sep

REPOSITORIES: biostudies-other

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A method for reversible drug delivery to internal tissues of Drosophila embryos.

Schulman Victoria K VK Folker Eric S ES Baylies Mary K MK

Fly 20130711 3

Drosophila melanogaster is a powerful model organism to elucidate basic cellular mechanisms of development. Indeed, much of our understanding of genetic pathways comes from work in Drosophila. However, mutations in many critical genes cause early embryonic lethality; thus, it is difficult to study the role of proteins that are required for early fundamental processes during later embryonic stages. We have therefore developed a method to reversibly deliver drugs to internal tissues of stage 15-16 ...[more]

PMID: 23846179

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Project description:Drug delivery to avascular, negatively charged tissues like cartilage remains a challenge. The constant turnover of synovial fluid results in short residence time of administered drugs in the joint space and the dense negatively charged matrix of cartilage hinders their diffusive transport. Drugs are, therefore, unable to reach their cell and matrix targets in sufficient doses, and fail to elicit relevant biological response, which has led to unsuccessful clinical trials. The high negative fixed charge density (FCD) of cartilage, however, can be used to convert cartilage from a barrier to drug entry into a depot by making drugs positively charged. Here we design cartilage penetrating and binding cationic peptide carriers (CPCs) with varying net charge, spatial distribution and hydrophobicity to deliver large-sized therapeutics and investigate their electro-diffusive transport in healthy and arthritic cartilage. We showed that CPC uptake increased with increasing net charge up to +14 but dropped as charge increased further due to stronger binding interactions that hindered CPC penetrability and uptake showing that weak-reversible binding is key to enable their penetration through full tissue thickness. Even after 90% GAG depletion, while CPC +14 uptake reduced by over 50% but still had a significantly high value of 148× showing that intra-tissue long-range charge-based binding is further stabilized by short-range H-bond and hydrophobic interactions. The work presents an approach for rational design of cationic carriers based on tissue FCD and properties of macromolecules to be delivered. These design rules can be extended to drug delivery for other avascular, negatively charged tissues. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) remains an untreatable disease partly due to short joint residence time of drugs and a lack of delivery methods that can effectively target the dense, avascular, highly negatively charged cartilage tissue. In this study, we designed cartilage penetrating and binding cationic peptide carriers (CPCs) that, due to their optimal charge provide adequate electrical driving force to rapidly transport OA drugs into cartilage and reach their cell and matrix targets in therapeutic doses before drugs exit the joint space. This way cartilage is converted from being a barrier to drug entry into a drug depot that can provide sustained drug release for several weeks. This study also investigates synergistic effects of short-range H-bond and hydrophobic interactions in combination with long-range electrostatic interactions on intra-cartilage solute transport. The work provides rules for rational design of cartilage penetrating charge-based carriers depending on the net charge of tissue (normal versus arthritic), macromolecule to be delivered and whether the application is in drug delivery or tissue imaging.

Dataset Information

A method for reversible drug delivery to internal tissues of Drosophila embryos.

Publications

A method for reversible drug delivery to internal tissues of Drosophila embryos.

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