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- Snipstad, Sofie, et al.
(författare)
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Labeling nanoparticles : Dye leakage and altered cellular uptake
- 2017
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Ingår i: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 91:8, s. 760-766
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Tidskriftsartikel (refereegranskat)abstract
- In vitro and in vivo behavior of nanoparticles (NPs) is often studied by tracing the NPs with fluorescent dyes. This requires stable incorporation of dyes within the NPs, as dye leakage may give a wrong interpretation of NP biodistribution, cellular uptake, and intracellular distribution. Furthermore, NP labeling with trace amounts of dye should not alter NP properties such as interactions with cells or tissues. To allow for versatile NP studies with a variety of fluorescence-based assays, labeling of NPs with different dyes is desirable. Hence, when new dyes are introduced, simple and fast screening methods to assess labeling stability and NP-cell interactions are needed. For this purpose, we have used a previously described generic flow cytometry assay; incubation of cells with NPs at 4 and 37C. Cell-NP interaction is confirmed by cellular fluorescence after 37C incubation, and NP-dye retention is confirmed when no cellular fluorescence is detected at 4C. Three different NP-platforms labeled with six different dyes were screened, and a great variability in dye retention was observed. Surprisingly, incorporation of trace amounts of certain dyes was found to reduce or even inhibit NP uptake. This work highlights the importance of thoroughly evaluating every dye-NP combination before pursuing NP-based applications. © 2016 International Society for Advancement of Cytometry.
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| 2. |
- Strand, Sabina P, et al.
(författare)
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Molecular design of chitosan gene delivery systems with an optimized balance between polyplex stability and polyplex unpacking
- 2010
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Ingår i: Biomaterials. - : Elsevier BV. - 0142-9612 .- 1878-5905. ; 31:5, s. 975-987
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Tidskriftsartikel (refereegranskat)abstract
- Chitosan is an attractive gene delivery vehicle, but the criteria and strategies for the design of efficient chitosan gene delivery systems remain unclear. The purpose of this work was to investigate how the strength of the charge-based interaction between chitosan and DNA determines the gene expression levels and to design chitosan vectors with an optimized balance between polyplex stability and polyplex unpacking. Using 21 formulations based on low molecular weight chitosans with constant charge density and a number-average degree of polymerization (DPn) in the range of 21-88 (M(w) 4.7-33kDa), we studied the relationship between the chain length and the formulation properties, cellular uptake of polyplexes and gene transfer efficacy. We were able to identify a narrow interval of DPn31-42 that mediated the maximum level of transgene expression. An increase in chain length and/or the amino-phosphate (A/P) ratio reduced and delayed transgene expression. Compared to DPn31, transfection with the same amount of DPn72 or DPn88 resulted in 10-fold-lower expression levels. The gene transfer pattern correlated with the ability of heparin to release DNA from the polyplexes. As a tool to facilitate the unpacking of the polyplexes, we substituted the chitosans with uncharged oligosaccharides that reduced the interaction with DNA. The substitution of chitosans that originally yielded too stable polyplexes, such as DPn72 and DPn88 resulted in a 5-10-fold enhancement of the expression levels. However, the substitution of chitosans shorter than DP28 completely abolished transfection. Tailoring of the chain length and the substitution of chitosan were shown to be feasible tools to modulate the electrostatic interactions between the chitosan and DNA and to design chitosans with an optimized balance between polyplex stability and polyplex unpacking.
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