Gemini Amphiphile-Based Lipoplexes for Efficient Gene Delivery: Synthesis, Formulation Development, Characterization, Gene Transfection, and Biodistribution Studies

Abstract

Some quaternary gemini amphiphiles (GAs) were synthesized as nonviral gene delivery carriers. The critical miceller concentration values of these amphiphiles are indicative of their superior surface-active properties. All of the synthesized GAs, alone or along with lipids like cholesterol and/or dioleoylphosphatidyl ethanolamine (DOPE), were formulated as liposomes. Formulations of GAs with DOPE showed average particle diameters of 326–400 nm with positive ζ-potential (30.1–46.4 mV). The lipoplexes of theses formulations showed complete pDNA retention at the base at a N/P ratio higher than 1.0 in gel retardation study. The GAs were effective in condensing pDNA into a ψ-phase, as indicated by circular dichroism study, and provided complete protection of the pDNA against the enzyme DNase at a N/P ratio more than 1. In vitro cell line studies showed that GA liposomal formulations caused β-gal expression and offered a higher transfection efficiency than that of liposomes prepared with the help of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP)/DOPE and dicyclocarbodiimide (DCC)/DOPE but comparable to those of Lipofectamine 2000 in A549 and HeLa cell lines. Modulation of head group polarity significantly affected the transfection efficacy of GAs. The cell viabilities of almost all of the formulations were comparable to those of the standards (DCC/DOPE and DOTAP/DOPE liposomes). Incorporation of cholesterol [GA/DOPE/cholesterol in the ratio of 1:1:1] further improved the serum compatibility of the formulations and improved the transfection efficacy when evaluated in A549 and HeLa cell lines. Fluorescence-assisted cell sorting studies showed comparable number of transfected cells to Lipofectamine 2000 in the HeLa cell line. Intracellular trafficking studies using confocal microscopy indicated transfection of the HeLa cells with the reporter gene within 30 min of lipoplex treatment. γ-Scintigraphy using 99mTc-labeled lipoplexes showed higher concentrations of the lipoplexes in vital tissues like liver, spleen, lungs, and kidneys.

Further Studies on Cationic Gemini Amphiphiles as Carriers for Gene Delivery─The Effect of Linkers in the Structure and Other Factors Affecting the Transfection Efficacy of These Amphiphiles

“…GAs with monohydroxyethyl groups offered slightly better results than the GAs having di­(hydroxyethyl) head groups. These observations were in tune with our earlier observations . Tetradecyl ( 9CIIIa , 10BIIIa , and 10CIIIb ) and hexadecyl ( 9AIa , 9BIb , 9CIa , and 10AIa ) lipophilic chains offered better transfection than the remaining two lipophilic chains (i.e., dodecyl and octadecyl).…”

“…That means, the chain length equivalent to C 5 with a higher polarity than the hydrocarbons is responsible for offering a higher transfection efficacy than the rest of the linkers. The best transfection efficacy was shown by the GA formulation ( 10BIIIa ), which was even better than that shown by the standard formulations ( Lipofectamine , DCC–DOPE , or DOTAP–DOPE ), and also by the earlier reported best GA ( 5d ) having the same structural features as 10BIII except for the spacer. Among the head groups, mixed-type results were obtained for monohydroxyethyl and di­(hydroxyethyl) head groups, both of which offered a higher transfection efficacy than the dimethyl head group.…”

“…In our earlier study, it was observed that the tetramethylene hydrocarbon chain as the spacer offered a higher transfection efficacy than the hexamethylene chain. In the current study, it was found out that the polar linker diethyleneoxy ( 10BIIIa and 10CIIIb ) offered better results than the more rigid p -xylenyl linker ( 9BIb , 9CIa , or 9CIIIa ) in both the series in both of the cancer cell lines.…”

“… 44 Tetradecyl ( 9CIIIa , 10BIIIa , and 10CIIIb ) and hexadecyl ( 9AIa , 9BIb , 9CIa , and 10AIa ) lipophilic chains offered better transfection than the remaining two lipophilic chains (i.e., dodecyl and octadecyl). The impact of tetradecyl and hexadecyl lipophilic chains on transfection efficacy was observed to be higher than that of dodecyl and octadecyl chains in our report 44 also. Overall, much higher transfection was observed in HeLa cell lines than in A549 cell lines for all the reported GAs.…”

Further Studies on Cationic Gemini Amphiphiles as Carriers for Gene Delivery─The Effect of Linkers in the Structure and Other Factors Affecting the Transfection Efficacy of These Amphiphiles

“…GAs with monohydroxyethyl groups offered slightly better results than the GAs having di­(hydroxyethyl) head groups. These observations were in tune with our earlier observations . Tetradecyl ( 9CIIIa , 10BIIIa , and 10CIIIb ) and hexadecyl ( 9AIa , 9BIb , 9CIa , and 10AIa ) lipophilic chains offered better transfection than the remaining two lipophilic chains (i.e., dodecyl and octadecyl).…”

“…That means, the chain length equivalent to C 5 with a higher polarity than the hydrocarbons is responsible for offering a higher transfection efficacy than the rest of the linkers. The best transfection efficacy was shown by the GA formulation ( 10BIIIa ), which was even better than that shown by the standard formulations ( Lipofectamine , DCC–DOPE , or DOTAP–DOPE ), and also by the earlier reported best GA ( 5d ) having the same structural features as 10BIII except for the spacer. Among the head groups, mixed-type results were obtained for monohydroxyethyl and di­(hydroxyethyl) head groups, both of which offered a higher transfection efficacy than the dimethyl head group.…”

“…In our earlier study, it was observed that the tetramethylene hydrocarbon chain as the spacer offered a higher transfection efficacy than the hexamethylene chain. In the current study, it was found out that the polar linker diethyleneoxy ( 10BIIIa and 10CIIIb ) offered better results than the more rigid p -xylenyl linker ( 9BIb , 9CIa , or 9CIIIa ) in both the series in both of the cancer cell lines.…”

“… 44 Tetradecyl ( 9CIIIa , 10BIIIa , and 10CIIIb ) and hexadecyl ( 9AIa , 9BIb , 9CIa , and 10AIa ) lipophilic chains offered better transfection than the remaining two lipophilic chains (i.e., dodecyl and octadecyl). The impact of tetradecyl and hexadecyl lipophilic chains on transfection efficacy was observed to be higher than that of dodecyl and octadecyl chains in our report 44 also. Overall, much higher transfection was observed in HeLa cell lines than in A549 cell lines for all the reported GAs.…”

Physical–Chemical Characterization of Bilayer Membranes Derived from (±) α-Tocopherol-Based Gemini Lipids and Their Interaction with Phosphatidylcholine Bilayers and Lipoplex Formation with Plasmid DNA

“…Except for the lipoplexes of THM in which the intensity of the 275 nm band increased a little, for all other lipoplexes, the same intensity was held (TH5S and TH12S) or flattened (TH4S, TH6S, and TH8S). These observations indicate the formation of perhaps more ordered and condensed DNA structures (ψ-DNA), but the extent of interaction and the changeover of the conformation of pDNA were individual properties of each lipid, and these were also dependent on the spacer length and the nature of headgroups. − …”

Further Studies on Cationic Gemini Amphiphiles as Carriers for Gene Delivery─The Effect of Linkers in the Structure and Other Factors Affecting the Transfection Efficacy of These Amphiphiles

“…GAs with monohydroxyethyl groups offered slightly better results than the GAs having di­(hydroxyethyl) head groups. These observations were in tune with our earlier observations . Tetradecyl ( 9CIIIa , 10BIIIa , and 10CIIIb ) and hexadecyl ( 9AIa , 9BIb , 9CIa , and 10AIa ) lipophilic chains offered better transfection than the remaining two lipophilic chains (i.e., dodecyl and octadecyl).…”

“…That means, the chain length equivalent to C 5 with a higher polarity than the hydrocarbons is responsible for offering a higher transfection efficacy than the rest of the linkers. The best transfection efficacy was shown by the GA formulation ( 10BIIIa ), which was even better than that shown by the standard formulations ( Lipofectamine , DCC–DOPE , or DOTAP–DOPE ), and also by the earlier reported best GA ( 5d ) having the same structural features as 10BIII except for the spacer. Among the head groups, mixed-type results were obtained for monohydroxyethyl and di­(hydroxyethyl) head groups, both of which offered a higher transfection efficacy than the dimethyl head group.…”

“…In our earlier study, it was observed that the tetramethylene hydrocarbon chain as the spacer offered a higher transfection efficacy than the hexamethylene chain. In the current study, it was found out that the polar linker diethyleneoxy ( 10BIIIa and 10CIIIb ) offered better results than the more rigid p -xylenyl linker ( 9BIb , 9CIa , or 9CIIIa ) in both the series in both of the cancer cell lines.…”

“… 44 Tetradecyl ( 9CIIIa , 10BIIIa , and 10CIIIb ) and hexadecyl ( 9AIa , 9BIb , 9CIa , and 10AIa ) lipophilic chains offered better transfection than the remaining two lipophilic chains (i.e., dodecyl and octadecyl). The impact of tetradecyl and hexadecyl lipophilic chains on transfection efficacy was observed to be higher than that of dodecyl and octadecyl chains in our report 44 also. Overall, much higher transfection was observed in HeLa cell lines than in A549 cell lines for all the reported GAs.…”

Physical–Chemical Characterization of Bilayer Membranes Derived from (±) α-Tocopherol-Based Gemini Lipids and Their Interaction with Phosphatidylcholine Bilayers and Lipoplex Formation with Plasmid DNA

“…Except for the lipoplexes of THM in which the intensity of the 275 nm band increased a little, for all other lipoplexes, the same intensity was held (TH5S and TH12S) or flattened (TH4S, TH6S, and TH8S). These observations indicate the formation of perhaps more ordered and condensed DNA structures (ψ-DNA), but the extent of interaction and the changeover of the conformation of pDNA were individual properties of each lipid, and these were also dependent on the spacer length and the nature of headgroups. − …”

Further Studies on Cationic Gemini Amphiphiles as Carriers for Gene Delivery─The Effect of Linkers in the Structure and Other Factors Affecting the Transfection Efficacy of These Amphiphiles

“…GAs with monohydroxyethyl groups offered slightly better results than the GAs having di­(hydroxyethyl) head groups. These observations were in tune with our earlier observations . Tetradecyl ( 9CIIIa , 10BIIIa , and 10CIIIb ) and hexadecyl ( 9AIa , 9BIb , 9CIa , and 10AIa ) lipophilic chains offered better transfection than the remaining two lipophilic chains (i.e., dodecyl and octadecyl).…”

“…That means, the chain length equivalent to C 5 with a higher polarity than the hydrocarbons is responsible for offering a higher transfection efficacy than the rest of the linkers. The best transfection efficacy was shown by the GA formulation ( 10BIIIa ), which was even better than that shown by the standard formulations ( Lipofectamine , DCC–DOPE , or DOTAP–DOPE ), and also by the earlier reported best GA ( 5d ) having the same structural features as 10BIII except for the spacer. Among the head groups, mixed-type results were obtained for monohydroxyethyl and di­(hydroxyethyl) head groups, both of which offered a higher transfection efficacy than the dimethyl head group.…”

“…In our earlier study, it was observed that the tetramethylene hydrocarbon chain as the spacer offered a higher transfection efficacy than the hexamethylene chain. In the current study, it was found out that the polar linker diethyleneoxy ( 10BIIIa and 10CIIIb ) offered better results than the more rigid p -xylenyl linker ( 9BIb , 9CIa , or 9CIIIa ) in both the series in both of the cancer cell lines.…”

“… 44 Tetradecyl ( 9CIIIa , 10BIIIa , and 10CIIIb ) and hexadecyl ( 9AIa , 9BIb , 9CIa , and 10AIa ) lipophilic chains offered better transfection than the remaining two lipophilic chains (i.e., dodecyl and octadecyl). The impact of tetradecyl and hexadecyl lipophilic chains on transfection efficacy was observed to be higher than that of dodecyl and octadecyl chains in our report 44 also. Overall, much higher transfection was observed in HeLa cell lines than in A549 cell lines for all the reported GAs.…”

Physical–Chemical Characterization of Bilayer Membranes Derived from (±) α-Tocopherol-Based Gemini Lipids and Their Interaction with Phosphatidylcholine Bilayers and Lipoplex Formation with Plasmid DNA

“…Except for the lipoplexes of THM in which the intensity of the 275 nm band increased a little, for all other lipoplexes, the same intensity was held (TH5S and TH12S) or flattened (TH4S, TH6S, and TH8S). These observations indicate the formation of perhaps more ordered and condensed DNA structures (ψ-DNA), but the extent of interaction and the changeover of the conformation of pDNA were individual properties of each lipid, and these were also dependent on the spacer length and the nature of headgroups. − …”