PHYSIOCHEMICAL STABILITY AND MASS SPECTROMETRIC ANALYSIS OF GEMINI SURFACTANT-BASED LIPOPLEXES

Cationic lipids have been comprehensively studied as non-viral vectors for gene therapy, focusing on improving the gene transfer efficiency and the safety profile. However, clinical applications of cationic lipid/DNA lipoplexes are restricted due to their low physical stability in aqueous formulations. One specific group of cationic lipids that showed efficient transfection activity is the gemini surfactants. Two main objectives were determined in this work. The first was to evaluate the feasibility of lyophilization as a formulation technique for preparing gemini surfactant-based lipoplexes with long-term stability. The second objective was to establish a universal tandem mass spectrometric “fingerprint” of novel amino acid modified gemini surfactants as a pre-requirement for the identification and quantification of gemini surfactants in different pharmaceutical matrices. In order to investigate the influence of lyophilization on the essential physiochemical properties and the in vitro transfection efficiency of gemini surfactant-lipoplexes, a diquaternary ammonium gemini surfactant (12-7NH-12) and plasmid DNA (pDNA) encoding for interferon-γ (IFNγ) were used to prepare pDNA/gemini surfactant [P/G] lipoplexes. Helper lipid DOPE [L] was incorporated in all formulations producing a [P/G/L] system. Several excipients were utilized as stabilizing agents. Lipoplexes formulated with the cryoprotectant were subjected to a lyophilization/rehydration cycle. Transfection activity was assessed by measuring the level of expressed IFNγ and cellular toxicity (MTT assay). The results showed that the physiochemical properties of gemini surfactant-based lipoplexes were dependent on the nature of the stabilizing agents used to prepare the lipoplexes. Disaccharide sugars, sucrose and trehalose, provided the most efficient cryoprotectant effect based on their ability to physically stabilize the lipoplexes during the lyophilization process. The transfection efficiency of the lyophilized lipoplexes increased 2-3 fold compared to fresh formulations upon lyophilization. This effect can be attributed to the improvement of DNA compaction and changes in the lipoplex morphology due to the lyophilization/rehydration cycles. Based on these results, we evaluated the ability of lyophilization to improve the stability of gemini surfactant-based lipoplexes. Four lyophilized formulations were stored at 25˚C for three months. The formulations were analyzed monthly for physical appearance, physiochemical properties (particle size and zeta potential, pDNA compaction, gemini surfactant:pDNA interaction) and in vitro transfection. The physiochemical properties of the lyophilized formulations were maintained throughout the three month study. All lyophilized formulations showed a loss of gene transfection activity after three months of storage. Nevertheless, no significant losses of transfection efficiency were observed for three formulations after two months storage at 25 ˚C. These findings suggest that lyophilization significantly improved the physiochemical stability of gemini surfactant-based lipoplexes compared to liquid formulations. As well, lyophilization improved the transfection efficiency of gemini surfactant-based lipoplexes. The loss of transfection activity upon storage is most probably due to the conformational changes in the supramolecular structure of the lipoplexes as a function of time and temperature, rather than to DNA degradation. To establish a foundation for employing the mass spectrometric methods in the evaluation of the chemical stability of the gemini surfactant, we evaluated the tandem mass spectrometric (MS/MS) behavior of six amino acid/di-peptide modified gemini surfactants that were synthesized based on the precursor compound 12-7NH-12. This was accomplished by using a hybrid quadrupole orthogonal time-of-flight mass spectrometer (QqToF-MS) and a triple quadrupole linear ion trap mass spectrometer (QqQ-LIT MS) equipped with electrospray ionization (ESI) source. The single stage QqToF-MS data obtained in the positive ion mode verified the molecular composition of all tested gemini surfactants. Tandem mass spectrometric (MS/MS) analysis showed common fragmentation behavior among all tested compounds, allowing for the establishment of a universal fragmentation pattern. The fragmentation pathway was confirmed by MS/MS/MS experiments utilizing a Q-TrapTM 4000 LC/MS/MS system and (MS/MS) analysis of the deuterated form of 12-7N(Glycine)-12 gemini surfactant. Unique product ions, originating from the loss of one or both head groups along with the attached tail region(s), confirmed the chemical structure of the tested compounds. In conclusion, different lyophilization strategies and analytical methods have been established to develop and examine the physiochemical stability of gemini surfactant-based lipoplex. A tandem mass spectrometric fragmentation pathway was established to enable the identification and quantification of these compounds in pharmaceutical formulations