The structure of the lipoplex formed from DNA and the sugar-based cationic gemini surfactant 1, which exhibits excellent transfection efficiency, has been investigated in the pH range 8.8-3.0 utilizing small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-TEM). Uniquely, three well-defined morphologies of the lipoplex were observed upon gradual acidification: a lamellar phase, a condensed lamellar phase, and an inverted hexagonal (H(II)) columnar phase. Using molecular modeling, we link the observed lipoplex morphologies and physical behavior to specific structural features in the individual surfactant, illuminating key factors in future surfactant design, viz., a spacer of six methylene groups, the presence of two nitrogens that can be protonated in the physiological pH range, two unsaturated alkyl tails, and hydrophilic sugar headgroups. Assuming that the mechanism of transfection by synthetic cationic surfactants involves endocytosis, we contend that the efficacy of gemini surfactant 1 as a gene delivery vehicle can be explained by the unprecedented observation of a pH-induced formation of the inverted hexagonal phase of the lipoplex in the endosomal pH range. This change in morphology leads to destabilization of the endosome through fusion of the lipoplex with the endosomal wall, resulting in release of DNA into the cytoplasm.

The superior surfactant properties of cationic gemini surfactants are applied to the complex problem of introducing genes into cells. Of almost 250 new compounds tested, of some 20 different structural types, a majority showed very good transfection activity in vitro. The surfactant is shown to bind and compact DNA efficiently, and structural studies and calculations provide a working picture of the "lipoplex" formed. The lipoplex can penetrate the outer membranes of many cell types, to appear in the cytoplasm encapsulated within endosomes. Escape from the endosome--a key step for transfection--may be controlled by changes in the aggregation behavior of the lipoplex as the pH falls. The evidence suggests that DNA may be released from the lipoplex before entry into the nucleus, where the new gene can be expressed with high efficiency.

Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12–18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO‐K1). Transfection efficiencies peaked at 2.7 times that of the commercial standard Lipofectamine Plus/2000 for pure solutions of the compound bearing unsaturated (oleyl) alkyl tails. For those compounds bearing saturated alkyl tails, transfection efficiency peaked at a tail length of 16, at a level similar to Lipofectamine Plus/2000. All of the amphiphiles formed bilayer vesicles at physiological pH. Some of the amino groups at the surface were protonated, and vesicles therefore bore a positive charge. Increased protonation with reduced pH resulted in greatly increased monomer solubility and a morphology change from vesicle to micelle at characteristic pH values, dependent on the tail length. For the compounds promoting high transfection efficiency, this characteristic pH was within the range found in the endosomal compartment (7.4–4.0). Formation of mixed micelles between gemini surfactant and membrane phospholipids at reduced pH may therefore provide a method of endosome rupture and subsequent escape of entrapped DNA, thus discarding the need for extra fusogenic or endosomolytic agents. The positive charge on the vesicles at physiological pH drives the colloidal association with DNA. Small angle X‐ray scattering measurements indicate that lamellar aggregates are formed, which have a d spacing of 48–54 Å. Preliminary differential scanning calorimetric measurements suggest that reduction of pH causes a disordering of the hydrocarbon region of the DNA–surfactant complex.

The pH-dependent aggregation behavior of one representative of a recently described series of sugar-amine gemini surfactants has been investigated. The surface tension (γ ) and turbidity of hexane-1,6-bis(hexadecyl-1 -deoxyglucitylamine) drop steeply between pH 5.5 and 4.0, consistent with a vesicle-to-micelle transition. The critical micelle concentration (cmc) at low pH (3.0) was determined by surface tension measurements to be 1 × 10 −3 M. This value is high, as γ is at the cmc (57 mN m −1 ). The area per headgroup (A h ) extracted from the slope of the curve of γ vs concentration below the cmc is 109Å2 . In an attempt to obtain a reasonable estimate of the headgroup area at higher pH, surface pressure vs area measurements were performed on a monolayer supported on pure water (pH 6), providing an A h of ca. 69Å 2 . The dependence of A h on pH is consistent with the proposed vesicle-to-micelle transition. Measurements of the gel-to-liquid crystalline phase transition using differential scanning calorimetry at a range of pHs revealed a drop in both the phase transition temperature and the transition enthalpy with decreasing pH. The pH dependence of the aggregation behavior can thus be summarized as follows: (1) pH 7.5-5.5, bilayer vesicles; (2) pH 5.5-4.0, a "drop region" where aggregate morphology is sensitive to small changes in pH; and (3) pH < 4.0, micelles. C 2001 Academic Press

Die oberflächenaktiven Eigenschaften kationischer Gemini‐Tenside werden zur Lösung des komplexen Problems der Gentransfektion in Zellen verwendet. Von nahezu 250 getesteten Substanzen, denen etwa 20 verschiedene Struktur‐Typen zugrunde liegen, zeigten viele eine gute In‐vitro‐Transfektionsaktivität. Das Tensid bindet und komprimiert DNS wirkungsvoll. Strukturelle und rechnerische Untersuchungen liefern ein brauchbares Modell des geformten Lipoplexes. Der Lipoplex kann durch die äußeren Membranen verschiedener Zelltypen dringen und so ins Cytoplasma gelangen. Die Freisetzung aus dem Endosom – ein Schlüsselschritt der Transfektion – könnte durch Veränderungen im Aggregationsverhalten des Lipoplexes bei fallendem pH‐Wert kontrolliert werden. Dies deutet auf ein Loslösen der DNS aus dem Lipoplex vor dem Eintritt in den Zellkern hin, wo das neue Gen mit hoher Effizienz exprimiert werden kann.