Gemini surfactant-based nanoparticles T-box1 gene delivery as a novel approach to promote epithelial stem cells differentiation and dental enamel formation.

Enamel is the highest mineralized tissue in the body protecting teeth from external stimuli, infections, and injuries. Enamel lacks the ability to self-repair due to the absence of enamel-producing cells in the erupted teeth. Here, we reported a novel approach to promote enamel-like tissue formation via the delivery of a key ameloblast inducer, T-box1 gene, into a rat dental epithelial stem cell line, HAT-7, using non-viral gene delivery systems based on cationic lipids. We comparatively assessed the lipoplexes prepared from glycyl-lysine-modified gemini surfactants and commercially available 1,2-dioleoyl-3-trimethylammonium-propane lipids at three nitrogen-to phosphate (N/P) ratios of 2.5, 5 and 10. Our findings revealed that physico-chemical characteristics and biological activities of the gemini surfactant-based lipoplexes with a N/P ratio of 5 provide the most optimal outcomes among those examined. HAT-7 cells were transfected with T-box1 gene using the optimal formulation then cultured in conventional 2D cell culture systems. Ameloblast differentiation, mineralization, bio-enamel interface and structure were assessed at different time points over 28 days. Our results showed that our gemini transfection system provides superior gene expression compared to the benchmark agent, while keeping low cytotoxicity levels. T-box1-transfected HAT-7 cells strongly expressed markers of secretory and maturation stages of the ameloblasts, deposited minerals, and produced enamel-like crystals when compared to control cells. Taken together, our gemini surfactant-based T-box1 gene delivery system is effective to accelerate and guide ameloblastic differentiation of dental epithelial stem cells and promote enamel-like tissue formation. This study would represent a significant advance towards the tissue engineering and regeneration of dental enamel.

Cellular Uptake and Distribution of Gemini Surfactant Nanoparticles Used as Gene Delivery Agents.

Gemini surfactants are promising molecules utilized as non-viral gene delivery vectors. However, little is known about their cellular uptake and distribution after they release their therapeutic cargo. Therefore, we quantitatively evaluated the cellular uptake and distribution of three gemini surfactants: unsubstituted (16-3-16), with pyridinium head groups (16(Py)-S-2-S-16(Py)) and substituted with a glycyl-lysine di-peptide (16-7N(GK)-16). We also assessed the relationship between cellular uptake and distribution of each gemini surfactant and its overall efficiency and toxicity. Epidermal keratinocytes PAM 212 were treated with gemini surfactant nanoparticles formulated with plasmid DNA and harvested at various time points to collect the enriched nuclear, mitochondrial, plasma membrane, and cytosolic fractions. Gemini surfactants were then extracted from each subcellular fraction and quantified using a validated flow injection analysis-tandem mass spectrometry (FIA-MS/MS) method. Mass spectrometry is superior to the use of fluorescent tags that alter the physicochemical properties and pharmacokinetics of the nanoparticles and can be cleaved from the gemini surfactant molecules within biological systems. Overall, a significantly higher cellular uptake was observed for 16-7N(GK)-16 (17.0%) compared with 16-3-6 (3.6%) and 16(Py)-S-2-S-16(Py) (1.4%), which explained the relatively higher transfection efficiency of 16-7N(GK)-16. Gemini surfactants 16-3-16 and 16(Py)-S-2-S-16(Py) displayed similar subcellular distribution patterns, with major accumulation in the nucleus, followed by the mitochondrion, cytosol, and plasma membrane. In contrast, 16-7N(GK)-16 was relatively evenly distributed across all four subcellular fractions. However, accumulation within the nucleus after 5 h of treatment was the highest for 16(Py)-S-2-S-16(Py) (50.3%), followed by 16-3-16 (41.8%) and then 16-7N(GK)-16 (33.4%), possibly leading to its relatively higher toxicity. Graphical Abstract.

Peptide-Modified Gemini Surfactants: Preparation and Characterization for Gene Delivery.

Diquaternary ammonium-based gemini surfactants have been investigated widely as nonviral gene delivery systems. These unique cationic lipids have versatility in their chemical structure, show relatively low toxicity, are able to compact genetic material (pDNA, RNA) into nano-sized lipoplexes, and can be easily produced. In addition, the gemini surfactants show significant improvement in the transfection activity and biocompatibility compared to other cationic lipids used as nonviral gene delivery agents. The successful applications of gemini surfactant-based lipoplexes as topical gene delivery systems in animal models indicate their potential as noninvasive carriers for genetic immunization, theranostic agents, and in other gene therapy treatments. Detailed physicochemical characterization of gemini surfactant lipoplexes is a key factor in terms of formulation optimization and elucidation of the cellular uptake and stability of the lipoplexes system. In this chapter, we describe in detail different formulation methods to prepare gemini surfactant lipoplexes and comprehensive physicochemical characterization. In addition, we illustrate general protocols for in vitro evaluations.

A 89Zr-labeled lipoplex nanosystem for image-guided gene delivery: design, evaluation of stability and in vivo behavior.

BACKGROUND: With the advances in radiopharmaceutical research, the development of image-guided therapy has become a major interest. While the development of theranostic nanotherapeutics is frequently associated with cancer chemotherapy, phototherapy and radiotherapy, there is little information available on the in vivo monitoring of gene delivery systems and the application of image-guided approach in gene therapy. The goal of this work was to determine the in vivo behavior of DNA delivery nanosystems - based on cationic gemini surfactants - designed for image-guided gene therapy. We tested the feasibility of monitoring tumor accumulation of gene delivery nanoparticles by positron emission tomography. METHODS: To be able to conjugate radiotracers to the nanoparticles, a deferoxamine-modified gemini surfactant was synthesized, DNA-containing lipoplex nanoparticles were formulated, and radiolabeled with Zirconium-89 (89Zr). The pharmacokinetics and biodistribution of 89Zr labeled surfactant and 89Zr labeled nanoparticles were monitored in mice by microPET/CT imaging and ex vivo gamma counting. RESULTS: Modification of the nanoparticles with deferoxamine did not alter their physicochemical properties. The radiolabeled nanoparticles (labeling efficiency of 95±3%) were stable in PBS and serum. The biological half-life of the 89Zr labeled nanoparticles was significantly higher compared to 89Zr labeled surfactant. As expected, the nanoparticles had significantly higher liver accumulation than the radiolabeled surfactant alone and lower kidney accumulation. Tumor uptake was detected at 2 hours post injection and decreased throughout the 3-day monitoring. CONCLUSION: We propose that radiolabeling DNA delivery lipoplex nanosystems is a promising approach for the design and optimization of image-guided nanomedicines, especially in the context of cancer gene 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.

The development of simple flow injection analysis tandem mass spectrometric methods for the cutaneous determination of peptide-modified cationic gemini surfactants used as gene delivery vectors.

Diquaternary ammonium gemini surfactants are a class of non-viral gene delivery vectors, primarily studied for their dermal applications. However, their biological fate has rarely been investigated. In this work, we developed simple flow injection analysis tandem mass spectrometric methods, (FIA)-MS/MS, to understand the fate and biodistribution of topically applied gemini surfactant-based therapeutics in an ex-vivo skin model. Three peptide-modified gemini surfactants with varied structures and transfection efficiencies were evaluated. For each compound, two methods were developed to quantify their presence in skin tissue and in phosphate buffered saline (PBS). The methods were developed using single-point calibration mode. Skin penetration was assessed on CD1 mice dorsal skin tissue mounted in a Franz diffusion cell after extraction. Amongst the five evaluated liquid-liquid extraction protocols, the Folch method provides the highest extraction efficiency for all compounds. Weak cationic exchange solid phase extraction was also used to further isolate gemini surfactants from endogenous skin lipids. FIA-MS/MS analysis of the skin revealed that all compounds were detected in the skin with minimal partition into the PBS compartment, which represents circulation. Interestingly, the detected amounts of gemini lipids in the skin were correlated with their transfection efficiencies.

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.

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.