Stabilization of lipid/DNA complexes during the freezing step of the lyophilization process: the particle isolation hypothesis

Abstract

The instability of nonviral vectors in aqueous suspensions has stimulated an interest in developing lyophilized formulations for use in gene therapy. Previous work has demonstrated a strong correlation between the maintenance of particle size and retention of transfection rates. Our earlier work has shown that aggregation of nonviral vectors typically occurs during the freezing step of the lyophilization process, and that high concentrations of sugars are capable of maintaining particle size. This study extends these observations, and demonstrates that glass formation is not the mechanism by which sugars protect lipid/DNA complexes during freezing. We also show that polymers (e.g., hydroxyethyl starch) are not capable of preventing aggregation despite their ability to form glasses at relatively high subzero temperatures. Instead, our data suggest that it is the separation of individual particles within the unfrozen fraction that prevents aggregation during freezing, i.e., the particle isolation hypothesis. Furthermore, we suggest that the relatively low surface tension of mono- and disaccharides, as compared to starch, allows phase-separated particles to remain dispersed within the unfrozen excipient solution, which preserves particle size and transfection rates during freezing.

Solid lipid based nanocarriers: An overview / Nanonosači na bazi čvrstih lipida: Pregled

“…Sugars isolate individual particles in unfrozen fraction and thereby prevent particle aggregation during freezing above Tg. Sugar vitrification is not required for this effect (54).…”

Effect of Lyophilization and Freeze-thawing on the Stability of siRNA-liposome Complexes

“…Lyophilization is another method for addressing this problem and has been attested to be effective for long-term storage of nucleic acid formulations (7,13,14) and thus enables storage at room temperature. Besides cost reduction in transportation and storage, lyophilization is preferred over freezing due to better stability of biomolecules resulting from the removal of non freezable water associated with most biomolecules (15).…”

Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

“…21,22 In the present study, we choose lyophilization for fabricating the miRNA reverse transfection formulations and demonstrated that this is able to load nucleic acid complexes onto a surface effectively and to make long-term storage of nucleic acid formulations possible. [23][24][25][26][27] Encouragingly, we found that the miRNA lipoplexes could be loaded onto the tissue culture plate surface evenly by lyophilization and that they could retain their intact structure. Lyoprotectants are generally used for lipoplex lyophilization, but it has been reported that ordinary lyoprotectants, such as glucose, can interfere with cell differentiation.…”

“…21,22 Lyophilization enabling long-term storage of nucleic acid formulations has recently been demonstrated to be able to load nucleic acid complexes onto surfaces. [23][24][25][26][27] Hence, reverse transfection formulations on a surface formed by lyophilization may provide an easy and efficient transfection approach for miRNA delivery. To our knowledge, although DNA and siRNAs have been plated on tissue culture plates for reverse transfection, there has been no such attempt using miRNAs.…”