Inclusion Complexes of Melphalan with Gemini-Conjugated ß-Cyclodextrin: Physicochemical Properties and Chemotherapeutic Efficacy in In-Vitro Tumor Models.

ß-cyclodextrin (ßCD) has been widely explored as an excipient for pharmaceuticals and nutraceuticals as it forms stable host-guest inclusion complexes and enhances the solubility of poorly soluble active agents. To enhance intracellular drug delivery, ßCD was chemically conjugated to an 18-carbon chain cationic gemini surfactant which undergoes self-assembly to form nanoscale complexes. The novel gemini surfactant-modified ßCD carrier host (hereafter referred to as 18:1ßCDg) was designed to combine the solubilization and encapsulation capacity of the ßCD macrocycle and the cell-penetrating ability of the gemini surfactant conjugate. Melphalan (Mel), a chemotherapeutic agent for melanoma, was selected as a model for a poorly soluble drug. Characterization of the 18:1ßCDg-Mel host-guest complex was carried out using 1D/2D 1H NMR spectroscopy and dynamic light scattering (DLS). The 1D/2D NMR spectral results indicated the formation of stable and well-defined 18:1ßCDg-Mel inclusion complexes at the 2:1 host-guest mole ratio; whereas, host-drug interaction was attenuated at greater 18:1ßCDg mole ratio due to hydrophobic aggregation that accounts for the reduced Mel solubility. The in vitro evaluations were performed using monolayer, 3D spheroid, and Mel-resistant melanoma cell lines. The 18:1ßCDg-Mel complex showed significant enhancement in the chemotherapeutic efficacy of Mel with 2-3-fold decrease in Mel half maximal inhibitory concentration (IC50) values. The findings demonstrate the potential applicability of the 18:1ßCDg delivery system as a safe and efficient carrier for a poorly soluble chemotherapeutic in melanoma therapy.

Molecular Engineering as an Approach To Modulate Gene Delivery Efficiency of Peptide-Modified Gemini Surfactants.

The unique molecular structure confers the diquaternary ammonium gemini surfactants with enhanced nucleic acid complexation ability, bottom-up design flexibility, and relatively low cytotoxicity. To capitalize on their potential as gene delivery vectors, novel structural modifications should be explored. In this work, 22 novel peptide-modified gemini surfactants with various alkyl tails and peptide spacer modifications were evaluated. This work represents the first report of dendrimer-like gemini surfactants and first evaluation of the impact of incorporating a hydrocarbon linker into the peptide chain. Our aim was to establish a structure activity relationship of the peptide-modified gemini surfactants and to identify the fundamental architectural requirements needed for the ultimate gene delivery systems. In vitro assessment revealed that the highest transfection efficiency and lowest cytotoxicity were associated with the glycyl-lysine modified gemini surfactants having the hexadecyl tail, 16-7N(G-K)-16. In fact, it showed an 8-fold increase in secreted protein with 20% increase in cell viability relative to the first-generation unsubstituted gemini surfactants. Further increase in the size of the attached peptides resulted in a decrease in the transfection efficiency and cell viability. Whereas the incorporation of a hydrocarbon linker into the peptide chain decreased the transfection efficiency of compounds with dipeptides, it increased the transfection efficiency of compounds with larger peptide chains. Such an increase was more prominent with the incorporation of a longer hydrocarbon linker. We conclude that a balance between the hydrophilic and hydrophobic characteristics of the compound is necessary since it results in physicochemical parameters conducive to the gene delivery process.

Design and Evaluation of Gemini Surfactant-Based Lipoplexes Modified with Cell-Binding Peptide for Targeted Gene Therapy in Melanoma Model.

Purpose Achieving successful gene therapy requires delivery of a gene vector specifically to the targeted tissue with efficient expression and a good safety profile. The objective of this work was to develop, characterize and determine if a novel gemini surfactant-based lipoplex systems, modified with a cancer-targeting peptide p18-4, could serve this role. Methods The targeting peptide p18-4 was either chemically coupled to a gemini surfactant backbone or physically co-formulated with the lipoplexes. The influence of targeting ligand and formulation strategies on essential physicochemical properties of the lipoplexes was evaluated by dynamic light scattering and small angle X-ray scattering techniques. In vitro transfection activity and cellular toxicity of lipoplexes were assessed in a model human melanoma cell line. Results All lipoplexes zeta potential and particle size were optimal for cellular uptake and physical stability of the system. The lipoplexes adopted an inverted-hexagonal lipid arrangement. The lipoplexes modified with the peptide showed no significant changes in physicochemical properties or lipoplex assembly. The modification of the lipoplexes with the targeting peptide significantly enhanced protein expression 2-6 fold compared to non-modified lipoplexes. In addition, p18-4 modified lipoplexes significantly improved the safety of the lipoplexes. The ability of the p18-4 modified lipoplexes to selectively express the model protein was confirmed by using healthy human epidermal keratinocytes (HEKa). Conclusion The gemini surfactant-based lipoplexes modified with p18-4 peptide showed significantly higher efficiency and safety compared to the system that did not contain a cancer targeting peptide and provided evidence for their potential application to achieve targeted melanoma gene therapy.

Design and Evaluation of RGD-Modified Gemini Surfactant-Based Lipoplexes for Targeted Gene Therapy in Melanoma Model.

PURPOSE: We have developed and evaluated novel peptide-targeted gemini surfactant-based lipoplexes designed for melanoma gene therapy. METHODS: Integrin receptor targeting peptide, cyclic-arginylglycylaspartic acid (cRGD), was either chemically coupled to a gemini surfactant backbone or physically co-formulated with lipoplexes. Several formulations and transfection techniques were developed. Transfection efficiency and cellular toxicity of the lipoplexes were evaluated in an in vitro human melanoma model. Physicochemical properties were examined using dynamic light scattering, zeta-potential, and small-angle X-ray scattering measurements. RESULTS: RGD-modified gemini surfactant based lipoplexes showed significant enhancement in gene transfection activity in A375 cell lines compared to the standard non-targeted formulation, especially when RGD was chemically conjugated to the gemini surfactant (RGD-G). The RGD had no effect on the cell toxicity profile of the lipoplex systems. Targeting specificity was confirmed by using an excess of free RGD and negative control peptide (RAD) and was demonstrated by using normal human epidermal keratinocytes. Physicochemical characterization showed that all nanoparticles were in the optimal size range for cellular uptake and there were no significant differences between RGD-modified and standard lipoplexes. CONCLUSIONS: These findings indicate the potential of RGD-modified gemini surfactant-based lipoplexes for use in melanoma gene therapy as an alternative to conventional chemotherapy.

Evaluation of ß-cyclodextrin-modified gemini surfactant-based delivery systems in melanoma models.

Novel drug delivery systems are developed to improve the biological behavior of poorly soluble drugs and to improve therapeutic outcomes. In melanoma therapy, the goal is efficient drug delivery and mitigation of drug resistance. Melphalan (Mel), a currently used therapeutic agent for melanoma, requires solvent system for solubilization, leading to poor chemical stability. Moreover, drug resistance often renders the drug inefficient in clinical setting. A novel ß-cyclodextrin-modified gemini surfactant (CDgemini) delivery system was developed to incorporate Mel in order to improve its physicochemical and biological behavior. Melphalan nanoparticles (Mel-NP) showed optimal particle size in the 200-250 nm range for endocytosis and induced significantly higher cell death compared with Mel (50% of inhibitory concentration [IC50] of 36 µM for the complexes vs 82 µM for Mel). The CDgemini delivery system did not alter the pathway of the cellular death triggered by Mel and caused no intrinsic toxicity to the cells. The Mel-NP complexes induced significant cell death in melanoma cells that were rendered resistant to Mel. These findings demonstrate in principle the applicability of the CDgemini delivery system as safe and efficient alternative to the current melanoma therapy, especially in chemoresistant cases.

Di-Peptide-Modified Gemini Surfactants as Gene Delivery Vectors: Exploring the Role of the Alkyl Tail in Their Physicochemical Behavior and Biological Activity.

The aim of this work was to elucidate the structure-activity relationship of new peptide-modified gemini surfactant-based carriers. Glycyl-lysine modified gemini surfactants that differ in the length and degree of unsaturation of their alkyl tail were used to engineer DNA nano-assemblies. To probe the optimal nitrogen to phosphate (N/P) ratio in the presence of helper lipid, in vitro gene expression and cell toxicity measurements were carried out. Characterization of the nano-assemblies was accomplished by measuring the particle size and surface charge. Morphological characteristics and lipid organization were studied by small angle X-ray scattering technique. Lipid monolayers were studied using a Langmuir-Blodgett trough. The highest activity of glycyl-lysine modified gemini surfactants was observed with the 16-carbon tail compound at 2.5 N/P ratio, showing a 5- to 10-fold increase in the level of reporter protein compared to the 12 and 18:1 carbon tail compounds. This ratio is significantly lower compared to the previously studied gemini surfactants with alkyl or amino- spacers. In addition, the 16-carbon tail compound exhibited the highest cell viability (85%). This high efficiency is attributed to the lowest critical micelle concentration of the 16-tail gemini surfactant and a balanced packing of the nanoparticles by mixing a saturated and unsaturated lipid together. At the optimal N/P ratio, all nanoparticles exhibited an inverted hexagonal lipid assembly. The results show that the length and nature of the tail of the gemini surfactants play an important role in determining the transgene efficiency of the delivery system. We demonstrated here that the interplay between the headgroup and the nature of tail is specific to each series, thus in the process of rational design, the contribution of the latter should be assessed in the appropriate context.

Lysine-functionalized nanodiamonds as gene carriers: development of stable colloidal dispersion for in vitro cellular uptake studies and siRNA delivery application.

PURPOSE: Nanodiamonds (NDs) are emerging as an attractive tool for gene therapeutics. To reach their full potential for biological application, NDs should maintain their colloidal stability in biological milieu. This study describes the behavior of lysine-functionalized ND (lys-ND) in various dispersion media, with an aim to limit aggregation and improve the colloidal stability of ND-gene complexes called diamoplexes. Furthermore, cellular and macromolecular interactions of lys-NDs are also analyzed in vitro to establish the understanding of ND-mediated gene transfer in cells. METHODS: lys-NDs were synthesized earlier through covalent conjugation of lysine amino acid to carboxylated NDs surface generated through re-oxidation in strong oxidizing acids. In this study, dispersions of lys-NDs were prepared in various media, and the degree of sedimentation was monitored for 72 hours. Particle size distributions and zeta potential measurements were performed for a period of 25 days to characterize the physicochemical stability of lys-NDs in the medium. The interaction profile of lys-NDs with fetal bovine serum showed formation of a protein corona, which was evaluated by size and charge distribution measurements. Uptake of lys-NDs in cervical cancer cells was analyzed by scanning transmission X-ray microscopy, flow cytometry, and confocal microscopy. Cellular uptake of diamoplexes (complex of lys-NDs with small interfering RNA) was also analyzed using flow cytometry. RESULTS: Aqueous dispersion of lys-NDs showed minimum sedimentation and remained stable over a period of 25 days. Size distributions showed good stability, remaining under 100 nm throughout the testing period. A positive zeta potential of >+20 mV indicated a preservation of surface charges. Size distribution and zeta potential changed for lys-NDs after incubation with blood serum, suggesting an interaction with biomolecules, mainly proteins, and a possible formation of a protein corona. Cellular internalization of lys-NDs was confirmed by various techniques such as confocal microscopy, soft X-ray spectroscopy, and flow cytometry. CONCLUSION: This study establishes that dispersion of lys-NDs in aqueous medium maintains long-term stability and also provides evidence that lysine functionalization enables NDs to interact effectively with the biological system to be used for RNAi therapeutics.

Impact of phospholipids on plasmid packaging and toxicity of gemini nanoparticles.

Understanding the relationship of structural modifications on the assembly and disassembly of synthetic or non-viral gene delivery is crucial with regard to their rational development. This study describes the use of fluorescence correlation spectroscopy (FCS), as a new tool, to investigate the effect of systematic chemical modifications to dicationic N,N-bis(dimethylalkyl)-α,ω-alkanediammonium surfactants (gemini surfactants) on the self-assembly and physical properties of a series of gemini nanoparticles (gemini NPs). A systematic screening of 27 gemini-plasmid (GP) complexes and gemini NPs showed that their final morphology is governed by the pre-compaction of plasmid by the gemini surfactants. The assembly process of gemini-plasmid intermediate complex (GP) and the final gemini NP (or gemini-plasmid-lipid complex, GPL) was monitored by the tracking of the Cy5-labeled plasmid. Based on diffusion properties, GP complexes were larger than gemini NPs (300-500 nm for GP and 200-300 nm for GPLs). Stoichiometric analysis of the raw intensity histograms showed that both GPs and GPLs particles were composed of multiple plasmids. The final GPLs contain fewer plasmids (2-20 per particle) compared to the intermediate GP (5-35 per particle). The addition of phospholipids dispersed and stabilized GPs to form GPL, but the type of phospholipid (DOPE or DD 1:3) had little effect on the final size of the particles. The FCS data were both validated and complemented by the results of studies of dynamic light scattering (DLS), atomic force microscopy (AFM), X-ray scattering and dye-exclusion assays. A model for gemini NP assembly involving supramolecular aggregate intermediates is proposed.

A 1H NMR Study of Host/Guest Supramolecular Complexes of a Curcumin Analogue with ß-Cyclodextrin and a ß-Cyclodextrin-Conjugated Gemini Surfactant.

Host systems based on ß-cyclodextrin (ßCD) were employed as pharmaceutical carriers to encapsulate a poorly soluble drug, curcumin analogue (NC 2067), in order to increase its water solubility. ßCD was chemically conjugated with an amphiphilic gemini surfactant with the ability to self-assemble and to form nanoscale supramolecular structures. The conjugated molecule, ßCDgemini surfactant (ßCDg), was shown to be a promising drug delivery agent. In this report, its physicochemical properties were assessed in aqueous solution using 1D and 2D 1H NMR spectroscopy. The results showed that the apolar hydrocarbon domain of the gemini surfactant was self-included within the ßCD internal cavity. The host/guest complexes composed of native ßCD or ßCDg with NC 2067 were examined using 1D/2D ROESY NMR methods. The stoichiometry of ßCD/NC 2067 complex was estimated using Job's method via 1H NMR spectroscopy. The binding geometry of NC 2067 within ßCD was proposed using molecular docking and further supported by 1D and 2D ROESY NMR results. Addition of NC 2067 to ßCDg revealed minimal changes to the overall structure of the ßCDg system, in agreement with the formation of a ßCDg/NC 2067 ternary complex.

Development of amino acid substituted gemini surfactant-based mucoadhesive gene delivery systems for potential use as noninvasive vaginal genetic vaccination.

AIM: Recently, we synthesized amino acid- and peptide-substituted gemini surfactants, 'biolipids' that exhibited high transfection efficiency in vitro. In this study, we developed these plasmid DNA and gemini surfactant lipid particles for noninvasive administration in vaginal cavity. MATERIAL & METHODS: Novel formulations of these gene delivery systems were prepared with poloxamer 407 to induce in situ gelling of the formulation and diethylene glycol monoethyl ether to improve their penetration across mucosal tissue. RESULTS: Poloxamer at 16% w/v concentration in diethylene glycol monoethyl ether aqueous solution produced dispersions that gelled near body temperature and had a high yield value, preventing leakage of the formulation from the vaginal cavity. Intravaginal administration in rabbits showed that the glycyl-lysine-substituted gemini surfactant led to a higher gene expression compared with the parent unsubstituted gemini surfactant. CONCLUSION: This provides a proof-of-concept that amino acid substituted gemini surfactants can be used as noninvasive mucosal (vaginal) gene delivery systems to treat diseases associated with mucosal epithelia.

Characterization of the host-guest complex of a curcumin analog with ß-cyclodextrin and ß-cyclodextrin-gemini surfactant and evaluation of its anticancer activity.

BACKGROUND: Curcumin analogs, including the novel compound NC 2067, are potent cytotoxic agents that suffer from poor solubility, and hence, low bioavailability. Cyclodextrin-based carriers can be used to encapsulate such agents. In order to understand the interaction between the two molecules, the physicochemical properties of the host-guest complexes of NC 2067 with ß-cyclodextrin (CD) or ß-cyclodextrin-gemini surfactant (CDgemini surfactant) were investigated for the first time. Moreover, possible supramolecular structures were examined in order to aid the development of new drug delivery systems. Furthermore, the in vitro anticancer activity of the complex of NC 2067 with CDgemini surfactant nanoparticles was demonstrated in the A375 melanoma cell line. METHODS: Physicochemical properties of the complexes formed of NC 2067 with CD or CDgemini surfactant were investigated by synchrotron-based powder X-ray diffraction, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. Synchrotron-based small- and wide-angle X-ray scattering and size measurements were employed to assess the supramolecular morphology of the complex formed by NC 2067 with CDgemini surfactant. Lastly, the in vitro cell toxicity of the formulations toward A375 melanoma cells at various drug-to-carrier mole ratios were measured by cell viability assay. RESULTS: Physical mixtures of NC 2067 and CD or CDgemini surfactant showed characteristics of the individual components, whereas the complex of NC 2067 and CD or CDgemini surfactant presented new structural features, supporting the formation of the host-guest complexes. Complexes of NC 2067 with CDgemini surfactants formed nanoparticles having sizes of 100-200 nm. NC 2067 retained its anticancer activity in the complex with CDgemini surfactant for different drug-to-carrier mole ratios, with an IC50 (half-maximal inhibitory concentration) value comparable to that for NC 2067 without the carrier. CONCLUSION: The formation of host-guest complexes of NC 2067 with CD or CDgemini surfactant has been confirmed and hence the CDgemini surfactant shows good potential to be used as a delivery system for anticancer agents.

The development and assessment of high-throughput mass spectrometry-based methods for the quantification of a nanoparticle drug delivery agent in cellular lysate.

The safe use of lipid-based drug delivery agents requires fast and sensitive qualitative and quantitative assessment of their cellular interactions. Many mass spectrometry (MS) based analytical platforms can achieve such task with varying capabilities. Therefore, four novel high-throughput MS-based quantitative methods were evaluated for the analysis of a small organic gene delivery agent: N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3). Analysis utilized MS instruments that detect analytes using low-resolution tandem MS (MS/MS) analysis (i.e. QTRAP or linear ion trap in this work) or high-resolution MS analysis (i.e. time of flight (ToF) or Orbitrap). Our results indicate that the validated fast chromatography (FC)-QTRAP-MS/MS, FC- LTQ-Orbitrap-MS, desorption electrospray ionization-collision-induced dissociation (CID)-MS/MS and matrix assisted laser desorption ionization-ToF/ToF-MS MS methods were superior in the area of method development and sample analysis time to a previously developed liquid chromatography (LC)-CID-MS/MS. To our knowledge, this is the first evaluation of the abilities of five MS-based quantitative methods that target a single pharmaceutical analyte. Our findings indicate that, in comparison to conventional LC-CID-MS/MS, the new MS-based methods resulted in a (1) substantial reduction in the analysis time, (2) reduction in the time required for method development and (3) production of either superior or comparable quantitative data. The four new high-throughput MS methods, therefore, were faster, more efficient and less expensive than a conventional LC-CID-MS/MS for the quantification of the G16-3 analyte within tissue culture. When applied to cellular lysate, no significant change in the concentration of G16-3 gemini surfactant within PAM212 cells was observed between 5 and 53 h, suggesting the absence of any metabolism/excretion from PAM212 cells.

Multi-stage tandem mass spectrometric analysis of novel ß-cyclodextrin-substituted and novel bis-pyridinium gemini surfactants designed as nanomedical drug delivery agents.

RATIONALE: This study aimed at evaluating the collision-induced dissociation tandem mass spectrometric (CID-MS/MS) fragmentation patterns of novel ß-cyclodextrin-substituted- and bis-pyridinium gemini surfactants currently being explored as nanomaterial drug delivery agents. In the ß-cyclodextrin-substituted gemini surfactants, a ß-cyclodextrin ring is grafted onto an N,N-bis(dimethylalkyl)-α,ω-aminoalkane-diammonium moiety using variable succinyl linkers. In contrast, the bis-pyridinium gemini surfactants are based on a 1,1'-(1,1'-(ethane-1,2-diylbis(sulfanediyl))bis(alkane-2,1-diyl))dipyridinium template, defined by two symmetrical N-alkylpyridinium parts connected through a fixed ethane dithiol spacer. METHODS: Detection of the precursor ion [M](2+) species of the synthesized compounds and the determination of mass accuracies were conducted using a QqTOF-MS instrument. A multi-stage tandem MS analysis of the detected [M](2+) species was conducted using the QqQ-LIT-MS instrument. Both instruments were equipped with an electrospray ionization (ESI) source. RESULTS: Abundant precursor ion [M](2+) species were detected for all compounds at sub-1 ppm mass accuracies. The ß-cyclodextrin-substituted compounds, fragmented via two main pathways: Pathway 1: the loss of one head-tail region produces a [M-(N(Me)2-R)](2+) ion, from which sugar moieties (Glc) are sequentially cleaved; Pathway 2: both head-tail regions are lost to give [M-2(N(Me)2-R)](+), followed by consecutive loss of Glc units. Alternatively, the cleavage of the Glc units could also have occurred simultaneously. Nevertheless, the fragmentation evolved around the quaternary ammonium cations, with characteristic cleavage of Glc moieties. For the bis-pyridinium gemini compounds, they either lost neutral pyridine(s) to give doubly charged ions (Pathway A) or formed complementary pyridinium alongside other singly charged ions (Pathway B). Similar to ß-cyclodextrin-substituted compounds, the fragmentation was centered on the pyridinium functional groups. CONCLUSIONS: The MS(n) analyses of these novel gemini surfactants, reported here for the first time, revealed diagnostic ions for each compound, with a universal fragmentation pattern for each compound series. The diagnostic ions will be employed within liquid chromatography (LC)/MS/MS methods for screening, identification, and quantification of these compounds within biological samples.

A general liquid chromatography tandem mass spectrometry method for the quantitative determination of diquaternary ammonium gemini surfactant drug delivery agents in mouse keratinocytes' cellular lysate.

Development of a simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of diquaternary ammonium gemini surfactants, utilized as gene deliver agents, is required for the evaluation of their post transfection fate. The continued development of efficient and safe gene delivery agents will benefit directly from an understanding of their rate of uptake, metabolism and excretion. A method is reported that is specific for the quantification of twenty-nine individual diquaternary ammonium gemini surfactant molecules and was validated for N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3) within PAM212 cell lysate according to USFDA bioanalytical method validation guidelines. The 10min chromatographic separation procedure utilized an Agilent Zorbax CN column (100mm×2.1mm with 3µ particles) with LC-MS grade water and acetonitrile, both containing 0.3% (v:v) formic acid and 1mM triethylamine. Extraction of the gemini surfactant from PAM212 keratinocyte cell lysate was performed using octanol and 10µL aliquots were injected onto the column. The standard curve was linear from 0.30µg/mL to 220µg/mL (r(2)≥0.999) for G16-3 and precision and accuracy were within USFDA specified limits. G16-3 analyte was assessed as stable during storage in the auto-injector, bench-top, freeze-thaw cycling and long-term (60 days) storage at -20°C. Evaluation of the cellular uptake and fate of G16-3, during both the incubation and post incubation periods, has established the potential of the application of the LC-MS/MS quantification method for monitoring diquaternary ammonium gemini surfactants in transfection studies.

Development of lyophilized gemini surfactant-based gene delivery systems: influence of lyophilization on the structure, activity and stability of the lipoplexes.

PURPOSE: Cationic gemini surfactants have been studied as non-viral vectors for gene therapy. Clinical applications of cationic lipid/DNA lipoplexes are restricted by their instability in aqueous formulations. In this work, we investigated the influence of lyophilization on the essential physiochemical properties and in vitro transfection of gemini surfactant-lipoplexes. Additionally, we evaluated the feasibility of lyophilization as a technique for preparing lipoplexes with long term stability. METHODS: A gemini surfactant [12-7NH-12] and plasmid DNA encoding for interferon-γ were used to prepare gemini surfactant/pDNA [P/G] lipoplexes. Helper lipid DOPE [L] was incorporated in all formulation producing a [P/G/L] system. Sucrose and trehalose were utilized as stabilizing agents. To evaluate 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 at different time-points for physiochemical properties and in vitro transfection. RESULTS: The results showed that both sucrose and trehalose provided anticipated stabilizing effect. The transfection efficiency of the 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. The physiochemical properties of the lyophilized formulations were maintained throughout the stability study. All lyophilized formulations showed a significant 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. CONCLUSION: Lyophilization significantly improved the physical stability of gemini surfactant-based lipoplexes compared to liquid formulations. As well, lyophilization improved the transfection efficiency of the 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.

Lysine-functionalized nanodiamonds: synthesis, physiochemical characterization, and nucleic acid binding studies.

PURPOSE: Detonation nanodiamonds (NDs) are carbon-based nanomaterials that, because of their size (4-5 nm), stable inert core, alterable surface chemistry, fluorescence, and biocompatibility, are emerging as bioimaging agents and promising tools for the delivery of biochemical molecules into cellular systems. However, diamond particles possess a strong propensity to aggregate in liquid formulation media, restricting their applicability in biomedical sciences. Here, the authors describe the covalent functionalization of NDs with lysine in an attempt to develop nanoparticles able to act as suitable nonviral vectors for transferring genetic materials across cellular membranes. METHODS: NDs were oxidized and functionalized by binding lysine moieties attached to a three-carbon-length linker (1,3-diaminopropane) to their surfaces through amide bonds. Raman and Fourier transform infrared spectroscopy, zeta potential measurement, dynamic light scattering, atomic force microscopic imaging, and thermogravimetric analysis were used to characterize the lysine-functionalized NDs. Finally, the ability of the functionalized diamonds to bind plasmid DNA and small interfering RNA was investigated by gel electrophoresis assay and through size and zeta potential measurements. RESULTS: NDs were successfully functionalized with the lysine linker, producing surface loading of 1.7 mmol g(-1) of ND. These modified NDs formed highly stable aqueous dispersions with a zeta potential of 49 mV and particle size of approximately 20 nm. The functionalized NDs were found to be able to bind plasmid DNA and small interfering RNA by forming nanosized "diamoplexes". CONCLUSION: The lysine-substituted ND particles generated in this study exhibit stable aqueous formulations and show potential for use as carriers for genetic materials.

Design and evaluation of cyclodextrin-based delivery systems to incorporate poorly soluble curcumin analogs for the treatment of melanoma.

Various analogs of curcumin show high in vitro cytotoxic activity and are potential candidates for treating a deadly skin disease, melanoma. Due to the low solubility of the drugs, a new delivery agent, namely a cationic gemini surfactant-conjugated ß-cyclodextrin, was designed to incorporate novel drug candidates of the 1,5-diaryl-3-oxo-1,4-pentadienyl family. Based on physicochemical parameters, such as particle size and zeta potential, a schematic model for the potential interaction of the drug with the delivery agent was developed. The drug formulations were highly efficient in inhibiting the growth of melanoma cells, with IC(50) values significantly lower than melphalan, the drug currently used for the treatment of in-transit melanoma. CDgemini formulations showed excellent cellular selectivity, triggering apoptosis in the A375 cell line while showing no cytotoxicity to healthy human epidermal keratinocytes. The goal is to develop this novel nanoparticle approach into a non-invasive therapy for in-transit melanoma metastasis that lacks adequate treatment to date.

Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles.

BACKGROUND: Gene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency. RESULTS: Nanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-γ) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression. CONCLUSION: Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the unsubstituted parent gemini surfactant. Glycyl-lysine substitution in the gemini spacer improved buffering capacity and imparted a pH-dependent increase of particle size. This property conferred to the P/12-7NGK-12/L nanoparticles the ability to escape efficiently from clathrin-mediated endosomes. Balanced binding properties (protection and release) of the 12-7NGK-12 in the presence of polyanions could contribute to the facile release of the nanoparticles internalized via caveolae-mediated uptake. A more efficient endosomal escape of the P/12-7NGK-12/L nanoparticles lead to higher gene expression compared to the parent gemini surfactant.

Tandem mass spectrometric analysis of novel diquaternary ammonium gemini surfactants and their bromide adducts in electrospray-positive ion mode ionization.

Gemini surfactants are cationic lipids which are utilized for both in vitro and in vivo gene delivery. Structurally, they are comprised of two hydrophobic tail regions with polar head termini that are attached to one another through a spacer region. Structural elucidation and characterization of 29 novel diquaternary ammonium gemini surfactant molecules were achieved using a quadrupole time-of-flight mass spectrometer (QqToF-MS) and a quadrupole-hexapole-quadrupole mass spectrometer (QhQ-MS). The tested compounds were categorized into four distinct structural families based upon the composition of the spacer region. Single stage (MS), tandem stage (MS/MS) and quasimulti-stage (quasi MS(3)) mass spectrometric analysis allowed for confirmation of each gemini surfactant's molecular composition and structure through the identification of common and unique product ions. Identification of similarities in the gemini surfactants' fragmentation behaviour resulted in the production of a universal fragmentation pathway that can assist in the future MS/MS analysis of novel quaternary ammonium gemini surfactants, with unique product ions being indicative of specific structural elements. Furthermore, evidence for the association of agemini surfactant with bromine counter ion was confirmed during MS analysis of tested gemini surfactants regardless of their chemical composition; previously, evidence for bromine and gemini surfactant association was only observed with compounds bearing short alkyl spacer regions. MS/MS analysis of the bromine adducts was also confirmatory to the molecular structure.Understanding the ionization and fragmentation behaviour of gemini surfactants, including bromine adducts, will allow for future qualitative and quantitative identification of these novel drug delivery agents within biological samples.

Amino acid-substituted gemini surfactant-based nanoparticles as safe and versatile gene delivery agents.

Gene based therapy represents an important advance in the treatment of diseases that heretofore have had either no treatment or cure. To capitalize on the true potential of gene therapy, there is a need to develop better delivery systems that can protect these therapeutic biomolecules and deliver them safely to the target sites. Recently, we have designed and developed a series of novel amino acid-substituted gemini surfactants with the general chemical formula C(12)H(25) (CH(3))(2)N(+)-(CH(2))(3)-N(AA)-(CH(2))(3)-N(+) (CH(3))(2)-C(12)H(25) (AA= glycine, lysine, glycyl-lysine and, lysyl-lysine). These compounds were synthesized and tested in rabbit epithelial cells using a model plasmid and a helper lipid. Plasmid/gemini/lipid (P/G/L) nanoparticles formulated using these novel compounds achieved higher gene expression than the nanoparticles containing the parent unsubstituted compound. In this study, we evaluated the cytotoxicity of P/G/L nanoparticles and explored the relationship between transfection efficiency/toxicity and their physicochemical characteristics (such as size, binding properties, etc.). An overall low toxicity is observed for all complexes with no significant difference among substituted and unsubstituted compounds. An interesting result revealed by the dye exclusion assay suggests a more balanced protection of the DNA by the glycine and glycyl-lysine substituted compounds. Thus, the higher transfection efficiency is attributed to the greater biocompatibility and flexibility of the amino acid/peptide-substituted gemini surfactants and demonstrates the feasibility of using amino acid-substituted gemini surfactants as gene carriers for the treatment of diseases affecting epithelial tissue.