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Oligonucleotide uptake in cultured keratinocytes: influence of confluence, cationic liposomes, and keratinocyte cell
P J White1, R D Fogarty, S C McKean
1Center for Hormone Research, Royal Children's Hosptial, Parkville, Victoria, Australia.
Abstract:
The success of anti-sense strategies has been limited, at least in part, by the poor uptake of these agents into the target cells. In keratinocytes, there is conflicting evidence as to the amount and location of oligonucleotide uptake into these cells, with variable proportions of cells reported to take up oligodeoxynucleotide, and also cytoplasmic and nuclear localization reported. In this study, the uptake of oligodeoxynucleotides in cultured normal human keratinocytes and the HaCaT cell line was quantitated in the presence of various lipids designed to enhance uptake and in varying culture conditions. About 12% of cells in a confluent normal human keratinocyte culture showed nuclear uptake, with a small and variable proportion showing cytoplasmic localization after 24 h incubation with 1 microM oligodeoxynucleotide. Uptake of oligodeoxynucleotide was found to be increased by liposome encapsulation (to a maximum of 28.1% +/- 2.1% of cells), low confluence (39.5% +/- 2.5%), and further increased by a combination of the two conditions (55.4% +/- 4.3%). HaCaT cell populations showed sparse but consistent uptake of oligodeoxynucleotide, with about 1% of cells showing nuclear localization in the presence of 1 microM oligodeoxynucleotide, increasing to 13.5% +/- 4.9% in the presence of cationic lipid (Tfx-50) in low confluence HaCaT monolayers. We conclude that normal keratinocytes exhibit reliable, substantial uptake of oligonucleotides in conditions controlled for confluence and aided by liposome encapsulation.
Insights
Oligonucleotide delivery to keratinocytes is challenging. Liposome encapsulation and low cell density significantly enhance uptake in normal human keratinocytes and HaCaT cells, improving anti-sense strategy potential.
Area of Science:
- Cell Biology
- Molecular Biology
- Drug Delivery
Background:
- Anti-sense strategies are hindered by poor cellular uptake of therapeutic agents.
- Evidence regarding oligonucleotide uptake in keratinocytes is conflicting, with variable cell proportions and localization reported.
- Understanding and optimizing oligonucleotide delivery is crucial for therapeutic applications in skin cells.
Purpose of the Study:
- To quantify oligodeoxynucleotide uptake in normal human keratinocytes and HaCaT cells.
- To evaluate the efficacy of various lipids and culture conditions in enhancing cellular uptake.
- To resolve conflicting data on oligonucleotide uptake and localization in keratinocytes.
Main Methods:
- Quantification of oligodeoxynucleotide uptake in cultured normal human keratinocytes and HaCaT cells.
- Assessment of uptake in the presence of liposome encapsulation and varying confluence levels.
- Utilizing cationic lipid (Tfx-50) to evaluate its effect on uptake in HaCaT cells.
Main Results:
- In confluent normal human keratinocytes, ~12% showed nuclear uptake and a small proportion showed cytoplasmic localization.
- Liposome encapsulation increased uptake to 28.1% +/- 2.1%, low confluence to 39.5% +/- 2.5%, and combined conditions to 55.4% +/- 4.3%.
- HaCaT cells showed sparse uptake (~1%), enhanced to 13.5% +/- 4.9% with cationic lipid at low confluence.
Conclusions:
- Normal keratinocytes demonstrate reliable and substantial oligonucleotide uptake under controlled conditions.
- Liposome encapsulation and low cell confluence are effective strategies to enhance oligonucleotide delivery.
- Optimized delivery conditions improve the potential of anti-sense strategies in keratinocyte research and therapy.