Dioleoyltrimethylammoniumpropane Research Articles

Evaluation of the passive permeability of antidepressants through pore-suspended lipid bilayer

HypothesisThe antidepressant drug imipramine, and its metabolite desipramine show different extents of interaction with, and passive permeation through, cellular membrane models, with the effects depending on the membrane composition. Through multimodal interrogation, we can observe that the drugs have a direct impact on the physicochemical properties of the membrane, that may play a role in their pharmacokinetics. ExperimentsMicrocavity pore-suspended lipid bilayers (MSLBs) of four different compositions, each with a different headgroup charge namely; zwitterionic dioleoylphosphatidylcholine (DOPC), mixed DOPC and negatively charged dioleoylphosphatidylglycerol (DOPG) (3:1), mixed DOPC and positively charged dioleoyltrimethylammoniumpropane (DOTAP) (3:1), and with increasing complex composition mimicking blood-brain-barrier (BBB) were prepared on gold and polydimethylsiloxane (PDMS) substrates using a Langmuir-Blodgett-vesicle fusion method. The molecular interaction and permeation of antidepressants, imipramine, and its metabolite desipramine with the lipid bilayers were evaluated using highly sensitive label-free electrochemical impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS). Drug-induced membrane packing/fluidity alterations were assessed using fluorescence lifetime imaging (FLIM) and fluorescence lifetime correlation spectroscopy (FLCS) of MSLB over microfluidic PDMS array. FindingsUsing EIS to evaluate in real-time membrane admittance changes, we found that imipramine greatly increases the ion permeability of negatively charged DOPC:DOPG (3:1) membranes. The effect was observed also at neutral (DOPC) and to a lesser extent at positively charged DOPC:DOTAP(3:1) membranes. In contrast, desipramine had a much weaker impact on ion permeability across all bilayer compositions. Temporal capacitance data show that desipramine intercalates at negatively charged membrane thereby increasing the thickness of the membrane. The overall kinetics of the imipramine permeation is higher than that of desipramine. This was confirmed using SERS, which also provides an evaluation of drug passive permeation based on arrival time across the membrane. Using FLCS, we found that imipramine increases the lipid membrane fluidity, whereas desipramine lowers it, with the exception of the negatively charged membrane. A translocation rate pharmacokinetics model was established for the first time at the MSLB platform by real-time monitoring of the variation in membrane resistance of pristine DOPC and blood-brain-barrier (BBB) membrane.

Design, Physical Characterizations, and Biocompatibility of Cationic Solid Lipid Nanoparticles in HCT-116 and 16-HBE Cells: A Preliminary Study.

In this study, pEGFP-LUC was used as a model plasmid and three distinct cationic lipids (dioleyloxy-propyl-trimethylammonium chloride [DOTMA], dioleoyl trimethylammonium propane [DOTAP], and cetylpyridinium chloride [CPC]) were tested along with PEG 5000, as a nonionic surfactant, to prepare glyceryl monostearate (GMS)-based cationic solid lipid nanoparticles (cSLNs). Both the type and quantity of surfactant had an impact on the physicochemical characteristics of the cSLNs. Thermal analysis of the greater part of the endothermic peaks of the cSLNs revealed they were noticeably different from the individual pure compounds based on their zeta potential (ZP ranging from +17 to +56 mV) and particle size (PS ranging from 185 to 244 nm). The addition of cationic surfactants was required to produce nanoparticles (NPs) with a positive surface charge. This suggested that the surfactants and extensive entanglement of the lipid matrix GMS provided support for the behavioral diversity of the cSLNs and their capacity to interface with the plasmid DNA. Additionally, hemolytic assays were used to show that the cSLNs were biocompatible with the human colon cancer HCT-116 and human bronchial epithelial 16-HBE cell lines. The DOTMA 6-based cSLN was selected as the lead cSLN for further ex vivo and in vivo investigations. Taken together, these new findings might provide some guidance in selecting surfactants to prepare extremely efficient and non-toxic cSLN-based therapeutic delivery systems (e.g., gene therapy).

The electrostatic basis of diacylglycerol pyrophosphate-protein interaction.

Phosphatidic acid (PA) is an anionic lipid with a simple phosphate headgroup that recruits multiple proteins for its diverse functional roles in all eukaryotes. Its charge, shape and Electrostatic Hydrogen bond switch mechanism forms the basis of its interaction with membrane components. PA is phosphorylated to Diacylglycerol pyrophosphate (DGPP) in many signaling events in plants, yeast, and some parasites. DGPP has a pyrophosphate headgroup with similar ionization properties to PA, except that its charge at any given physiological pH is higher. The synthesis of DGPP in the signaling cascade potentially alters membrane dynamics and properties due to the differences in the physicochemical parameters between the two lipids. Thus, DGPP is expected to differ from PA in its interaction with membrane components. However, the overall effect on the biophysical properties of DGPP's interactions with proteins and ions are unexplored. In this study, we investigated the effect of the interactions of DGPP with model peptides KALP23 and WALP23 by 31P MAS NMR spectroscopy. In addition, we also analyzed the effect of DGPP with two positively charged lipids, dioleoyl trimethylammonium propane (DOTAP) and ethyl -phosphatidylcholine (EtPC) as well as with Magnesium and Calcium cations. Results from this study highlight a unique model for DGPP-protein interactions and has important implications in how DGPP interacts with membrane components in the signaling pathway.

Efficient liposome fusion to phase-separated giant vesicles

Lateral phase heterogeneity in biomembranes can govern cellular functions and may serve as a platform for enrichment or depletion of membrane-anchored molecules. In this work, we address the question of how the process of membrane fusion is affected by the membrane phase state (fluid or gel) and by phase coexistence, as well as the effects of fusion-mediated incorporation of exogeneous lipids on phase separation. Our system is based on the fusion of cationic fluid large unilamellar vesicles (LUVs) composed of dioleoyl trimethylammonium propane (DOTAP) and dioleoyl phosphoethanolamine (DOPE) with neutral and anionic giant unilamellar vesicles (GUVs) composed of phosphatidylcholine and phosphatidylglycerol. By changing the lipid composition of the GUVs, we modulated the phase state and charge of the different phases (charged or neutral, fluid or gel) and identified systems in which we can target fusion to specific domains on phase-separated membranes. Fusion efficiency was quantified using fluorescence microscopy-based lipid and content mixing assays, and flow chamber devices were used to assess the real-time sequence of events of the fusion process. To investigate the bilayer thermal behavior, differential scanning calorimetry (DSC) experiments were performed on LUVs. The results show that fusion is extensive in single-component GUVs only for fluid and negatively charged acceptor membranes. On the other hand, in phase-separated GUVs, high fusion efficiency was observed even when the gel phase was anionic and phase separation somewhat increased the fusion efficiency. Extensive fusion led to dissolution of the gel domains as a result of extensive incorporation of lipids in the fluid state from the fusogenic liposomes. Altogether, these findings have the potential to unravel the important role of membrane phase state, phase separation, charge, and the effects of extensive fusion on membrane organization and may give insights in the regulation of the interactions between cells and liposomes that are used in drug delivery systems.

Preparation of stable polymer-liposome complexes by a novel approach employing a one-pot method

The purpose of this study was to prepare stable polymer-liposome complexes using a new, improved one-pot method. Complexes prepared by the conventional method were used as reference. The one-pot method combines the vesicle formation and complexation with the polymer in a single step allowing for fewer steps compared to the conventional thin-film method followed by coating. This has the possibility of complexing the polymer on the surface and within the aqueous core of the liposomes. Soya phosphatidylcholine (SoyPC) in combination with different amounts of either dioleoyl trimethylammoniumpropane (DOTAP, positively charged) or egg phosphatidylglycerol (Egg PG, negatively charged) were complexed with alginate or chitosan, respectively. All the alginate-liposome complexes were larger compared to the naked liposomes and the zeta potential changed from positive to negative after complexation with the polymer. Polydispersity index (PDI) analysis showed narrow size distributions implying homogenous populations. The best stability was obtained by employing 20 mol% DOTAP in the membrane. This formulation was stable during the whole four months period when the stability test was performed. The chitosan-liposome complexes were generally difficult to extrude and were larger than the alginate-liposome complexes due to the need for a larger pore size in the membrane during extrusion. Based on these results, the one-pot method can be used for preparing stable alginate-liposome complexes. For the chitosan-liposome complexes alternative methods for size reduction should be investigated.

Cationic lipid-based formulations for encapsulation and delivery of anti-EFG1 2' OMethylRNA oligomer.

The effective protection and delivery of antisense oligomers to its site of action is a challenge without an optimal strategy. Some of the most promising approaches encompass the complexation of nucleic acids, which are anionic, with liposomes of fixed or ionizable cationic charge. Thus, the main purpose of this work was to study the complexation of cationic liposomes with anti-EFG1 2'OMe oligomers and evaluate the complex efficacy to control Candida albicans filamentation in vitro and in vivo using a Galleria mellonella model. To accomplish this, cationic dioleoyl-trimethylammoniumpropane (DOTAP) was mixed with three different neutral lipids dioleoyl-phosphocholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE) and monoolein (MO) and used as delivery vectors. Fluorescence Cross Correlation Spectroscopy measurements revealed a high association between antisense oligomers (ASO) and cationic liposomes confirming the formation of lipoplexes. In vitro, all cationic liposome-ASO complexes were able to release the anti-EFG1 2'OMe oligomers and consequently inhibit C. albicans filamentation up to 60% after 72h. In vivo, from all formulations the DOTAP/DOPC 80/20 ρchg=3 formulation proved to be the most effective, enhancing the G. mellonella survival by 40% within 48h and by 25% after 72h of infection. In this sense, our findings show that DOTAP-based lipoplexes are very good candidates for nano-carriers of anti-EFG1 2'OMe oligomers.

The Electrostatic Basis of Diacylglycerol Pyrophosphate-Protein Interaction.

Diacylglycerol pyrophosphate (DGPP) is an anionic phospholipid formed in plants, yeast, and parasites under multiple stress stimuli. It is synthesized by the phosphorylation action of phosphatidic acid (PA) kinase on phosphatidic acid, a signaling lipid with multifunctional properties. PA functions in the membrane through the interaction of its negatively charged phosphomonoester headgroup with positively charged proteins and ions. DGPP, like PA, can interact electrostatically via the electrostatic-hydrogen bond switch mechanism but differs from PA in its overall charge and shape. The formation of DGPP from PA alters the physicochemical properties as well as the structural dynamics of the membrane. This potentially impacts the molecular and ionic binding of cationic proteins and ions with the DGPP enriched membrane. However, the results of these important interactions in the stress response and in DGPP’s overall intracellular function is unknown. Here, using 31P MAS NMR, we analyze the effect of the interaction of low DGPP concentrations in model membranes with the peptides KALP23 and WALP23, which are flanked by positively charged Lysine and neutral Tryptophan residues, respectively. Our results show a significant effect of KALP23 on the charge of DGPP as compared to WALP23. There was, however, no significant effect on the charge of the phosphomonoester of DGPP due to the interaction with positively charged lipids, dioleoyl trimethylammonium propane (DOTAP) and dioleoyl ethyl-phosphatidylcholine (EtPC). Divalent calcium and magnesium cations induce deprotonation of the DGPP headgroup but showed no noticeable differences on DGPP’s charge. Our results lead to a novel model for DGPP—protein interaction.

Encapsulation of a CpG oligonucleotide in cationic liposomes enhances its local antitumor activity following pulmonary delivery in a murine model of metastatic lung cancer

Immunotherapy brings new hope to the fight against lung cancer. General immunostimulatory agents represent an immunotherapy strategy that has demonstrated efficacy with limited toxicity when delivered intratumorally. The goal of this study was to enhance the antitumor efficacy of unmethylated oligodeoxynucleotides containing CpG motifs (CpG) and polyinosinic-polycytidylic acid (poly I:C) double-stranded RNA following their local delivery in lung cancer by encapsulating them in liposomes. Liposomes encapsulation of nucleic acids could increase their uptake by lung phagocytes and thereby the activation of toll-like receptors within endosomes. Liposomes were prepared using a cationic lipid, dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the main phospholipid in lung surfactant. The liposomes permanently entrapped CpG but could not efficiently withhold poly I:C. Both poly I:C and CpG delayed tumor growth in the murine B16F10 model of metastatic lung cancer. However, only CpG increased IFN-γ levels in the lungs. Pulmonary administration of CpG was superior to its intraperitoneal injection to slow the growth of lung metastases and to induce the production of granzyme B, a pro-apoptotic protein, and IFNγ, MIG and RANTES, T helper type 1 cytokines and chemokines, in the lungs. These antitumor activities of CpG were strongly enhanced by CpG encapsulation in DOTAP/DPPC liposomes. Delivery of low CpG doses to the lungs induced increased inflammation markers in the airspaces but the inflammation did not reach the systemic compartment in a significant manner. These data support the use of a delivery carrier to strengthen CpG antitumor activity following its pulmonary delivery in lung cancer.

Binding of warfarin differently affects the thermal behavior and chain packing of anionic, zwitterionic and cationic lipid membranes

Warfarin is a coumarin derivative drug widely used for its anticoagulant properties. The interaction of warfarin with fully hydrated lipid bilayers has been studied by combining differential scanning calorimetry, spectrophotometry, electron spin resonance of chain-labelled lipids and molecular docking. Bilayers formed by lipids with different chemico-physical properties were considered, namely dimyristoyl-phosphatidylcholine (DMPC), dimyristoyl-phosphatidylglycerol (DMPG), and dioleoyltrimethyl-ammoniumpropane (DOTAP). We observed in all cases the binding of warfarin in proximity of the surface of the bilayers, leading to a variety of distinct effects on key molecular properties of the membranes. The drug associates with the lipid bilayers in the deprotonated open chain form, with an association constant similar for DMPC and DMPG (1.27·104 and 2.82·104 M−1, respectively) and lower for DOTAP (0.46·104 M−1). In DMPC bilayers, which are zwitterionic and with saturated symmetrical chains, warfarin at 10 mol% suppresses the pre-transition, slightly stabilizes the fluid state and reduces the cooperativity of the main transition. Moreover, it alters the lateral packing density of the chain segments close to the polar/apolar interface at any temperature through the gel phase. In anionic DMPG bilayers, the drug slightly perturbs the thermotropic phase behavior, and at 10 mol% markedly loosens the compact gel phase packing of the first chain segments. In cationic DOTAP bilayers, possessing unsaturated acyl chains, the drug induces a slightly higher degree of order and motional restriction in the outer hydrocarbon region in the frozen state. In all cases, as a surface adsorbed molecule, warfarin does not affect the segmental chain order and dynamics for temperatures in the fluid phase. The overall results provide an outline of the action of warfarin on membranes formed by lipids of different types.

Mechanistic profiling of the release kinetics of siRNA from lipidoid-polymer hybrid nanoparticles in vitro and in vivo after pulmonary administration

Understanding the release kinetics of siRNA from nanocarriers, their cellular uptake, their in vivo biodistribution and pharmacokinetics is a fundamental prerequisite for efficient optimisation of the design of nanocarriers for siRNA-based therapeutics. Thus, we investigated the influence of composition on the siRNA release from lipid-polymer hybrid nanoparticles (LPNs) consisting of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) (PLGA) intended for pulmonary administration. An array of siRNA-loaded LPNs was prepared by systematic variation of: (i) the L5 content (10–20%, w/w), and (ii) the L5:siRNA ratio (10,1–30:1, w/w). For comparative purposes, L5-based lipoplexes, L5-based stable nucleic acid lipid nanoparticles (SNALPs). and dioleoyltrimethylammoniumpropane (DOTAP)-modified LPNs loaded with siRNA were also prepared. Release studies in buffer and lung surfactant-containing medium showed that siRNA release is dependent on the presence of both surfactant and heparin (a displacing agent) in the release medium, since these interact with the lipid shell structure thereby facilitating decomplexation of L5 and siRNA, as evident from the retarded siRNA release when the L5 content and the L5:siRNA ratio were increased. This confirms the hypothesis that siRNA loaded in LPNs is predominantly present as complexes with the cationic lipid and primarily is located near the particle surface. Cellular uptake and tolerability studies in the human macrophage cell line THP-1 and the type I-like human alveolar epithelial cell line hAELVi, which together represents a monolayer-based barrier model of lung epithelium, indicated that uptake of LPNs was much higher in THP-1 cells in agreement with their primary clearance role. In vivo biodistributions of formulations loaded with Alexa Fluor® 750-labelled siRNA after pulmonary administration in mice were compared by using quantitative fluorescence imaging tomography. The L5-modified LPNs, SNALPs and DOTAP-modified LPNs displayed significantly increased lung retention of siRNA as compared to L5-based lipoplexes, which had a biodistribution profile comparable to that of non-loaded siRNA, for which >50% of the siRNA dose permeated the air-blood barrier within 6 h and subsequently was excreted via the kidneys. Hence, the enhanced lung retention upon pulmonary administration of siRNA-loaded LPNs represents a promising characteristic that can be used to control the delivery of the siRNA cargo to lung tissue for local management of disease.

Cationic Nanoliposomes Are Efficiently Taken up by Alveolar Macrophages but Have Little Access to Dendritic Cells and Interstitial Macrophages in the Normal and CpG-Stimulated Lungs.

The purpose of this study was to assess whether cationic nanoliposomes could address tumor vaccines to dendritic cells in the lungs in vivo. Nanoliposomes were prepared using a cationic lipid, dimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol) or dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the most abundant phospholipid in lung surfactant. The liposomes presented a size below 175 nm and they effectively entrapped tumor antigens, an oligodeoxynucletotide containing CpG motifs (CpG) and the fluorescent dye calcein used as a tracer. Although the liposomes could permanently entrap a large fraction of the actives, they could not sustain their release in vitro. Liposomes made of DOTAP were safe to respiratory cells in vitro, while liposomes composed of DC-cholesterol were cytotoxic. DOTAP nanoliposomes were mainly taken up by alveolar macrophages following delivery to the lungs in mice. Few dendritic cells took up the liposomes, and interstitial macrophages did not take up liposomal calcein more than they took up soluble calcein. Stimulation of the innate immune system using liposomal CpG strongly enhanced uptake of calcein liposomes by all phagocytes in the lungs. Although a small percentage of dendritic cells took up the nanoliposomes, alveolar macrophages represented a major barrier to dendritic cell access in the lungs.

Application of a Quality-By-Design Approach to Optimise Lipid-Polymer Hybrid Nanoparticles Loaded with a Splice-Correction Antisense Oligonucleotide: Maximising Loading and Intracellular Delivery.

Antisense oligonucleotides (ASOs) are promising therapeutics for specific modulation of cellular RNA function. However, ASO efficacy is compromised by inefficient intracellular delivery. Lipid-polymer hybrid nanoparticles (LPNs) are attractive mediators of intracellular ASO delivery due to favorable colloidal stability and sustained release properties. LPNs composed of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) were studied for delivery of an ASO mediating splice correction of a luciferase gene transcript (Luc-ASO). Specific purposes were: (i) to increase the mechanistic understanding of factors determining the loading of ASO in LPNs, and (ii) to optimise the LPNs and customise them for Luc-ASO delivery in HeLa pLuc/705 cells containing an aberrant luciferase gene by using a quality-by-design approach. Critical formulation variables were linked to critical quality attributes (CQAs) using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS). A series of CQAs were identified based on the quality target product profile. The L5 content and L5:Luc-ASO ratio (w/w) were determined as critical formulation variables, which were optimised systematically. The optimised Luc-ASO-loaded LPNs, defined from the OOS, displayed high loading and mediated splice correction at well-tolerated, lower doses as compared to those required for reference L5-based lipoplexes, L5-modified stable nucleic acid lipid nanoparticles or LPNs modified with dioleoyltrimethylammonium propane (conventional cationic lipid). The optimal Luc-ASO-loaded LPNs represent a robust formulation that mediates efficient intracellular delivery of Luc-ASO. This opens new avenues for further development of LPNs as a broadly applicable technology platform for delivering nucleic acid cargos intracellularly.

Polymeric nanoparticles for the delivery of miRNA to treat Chronic Obstructive Pulmonary Disease (COPD)

RNA interference (RNAi) based therapeutics are considered an endogenous mechanism for modulating gene expression. In addition, microRNAs (miRNAs) may be tractable targets for the treatment of Chronic Obstructive Pulmonary Disease (COPD). In this study miR146a was adsorbed onto poly (glycerol adipate-co-ω-pentadecalactone), PGA-co-PDL, nanoparticles (NPs) to reduce target gene IRAK1 expression. NPs were prepared using an oil-in-water single emulsion solvent evaporation method incorporating cationic lipid dioleoyltrimethylammoniumpropane (DOTAP). This resulted in NPs of 244.80 ± 4.40 nm at 15% DOTAP concentration, zeta potential (ZP) of +14.8 ± 0.26 mV and miR-146a (40 µg/ml) maximum adsorption onto 15% DOTAP NPs was 36.25 ± 0.35 µg per 10 mg NP following 24 h incubation. Using the MTT assay, it was observed that over 75% at 0.312 mg/ml of A549 cells remained viable after 18 h exposure to cationic NPs at a concentration of 1.25 mg/ml. Furthermore, the in vitro release profile of miR-146a from loaded NPs showed a continuous release up to 77% after 24 h. Internalization of miR-146a loaded cationic NPs was observed in A549 cell lines using fluorescence and confocal microscopy. The miR146a delivered as miR-146a-NPs had a dose dependent effect of highest NPs concentrations 0.321 and 0.625 mg/ml and reduced target gene IRAK1 expression to 40%. In addition, IL-8 promoter reporter output (GFP) was dampened by miR-146a-NPs. In conclusion, miR-146a was successfully adsorbed onto PGA-co-PDL-DOTAP NPs and the miR-146a retained biological activity. Therefore, these results demonstrate the potential of PGA-co-PDL NPs as a delivery system for miR-146a to treat COPD.

Targeting of high mobility group A2 by small interfering RNA-loaded nanoliposome-induced apoptosis and migration inhibition in gastrointestinal cancer cells.

Considering the complex nature of gastrointestinal cancer, different methods including surgery, radiotherapy, and chemotherapy are considered for the treatment. Novel strategies including silencing of oncogenes using safe delivery systems could be considered as a novel approach in colorectal cancer treatment. The aim of this study was to investigate the silencing effect of high mobility group A2 (HMGA2) small interfering RNA (siRNA)-loaded nanoliposomes on gastrointestinal cancers. The siRNA-lipoplexes were prepared using dioleoyl trimethylammonium propane (DOTAP)/cholesterol (Chol)/1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) through the freeze-drying of a monophase solution method. The size, polydispersity index (PDI), and zeta-potential of nanoliposomes were determined using Zetasizer analyzer. The morphology of the nanoliposomes was determined by transmission electron microscopy (TEM). The agarose gel-retardation assay was carried out to confirm the loading of siRNAs into liposome. The silencing of the HMGA2 in cancer cells was evaluated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of liposomes on cell cytotoxicity was studied by MTT assay. The inhibitory effect of siRNA-loaded liposomes was evaluated by a wound-healing assay. The apoptosis induction was investigated via the annexin V/propidium iodide assay. The size, PDI, and zeta-potential of the prepared liposomes were found to be 350 nm, 0.67, and 86.3 mV, respectively. They were spherical in shape and could efficiently associate with siRNA. The results of gene silencing showed that the optimum condition of HMGA2 silencing was 80 pmol HMGA2 and 24 hours after treatment in each cancer cell lines. MTT assays indicated that silencing of HMGA2 in optimal condition could reduce the viability of the cancer cells more than 60% in the three cell lines. The result of the apoptosis assay showed more than 50% of the cell deaths related to the apoptosis in all three cell lines. The gene expression evaluation confirmed that apoptosis was induced via the intrinsic pathway inducing both caspase-3 and -9 expressions. Also, the reduction in Bcl2 expression confirmed the activation apoptosis pathway in the treated cancer cells. The wound-healing assay showed the suppression of cancer cell migration after treatment with the prepared nanoliposomes. The results of this study showed the HMGA2 siRNA-loaded nanoliposomes could be effective in the treatment of gastrointestinal cancers.

Immunogenicity Testing of Lipidoids In Vitro and In Silico: Modulating Lipidoid-Mediated TLR4 Activation by Nanoparticle Design

Therapeutics based on small interfering RNA (siRNA) have promising potential as antiviral and anti-inflammatory agents. To deliver siRNA across cell membranes to reach the RNAi pathway in the cytosol of target cells, non-viral nanoparticulate delivery approaches are explored. Recently, we showed that encapsulation of siRNA in lipid-polymer hybrid nanoparticles (LPNs), based on poly(DL-lactic-co-glycolic acid) (PLGA) and cationic lipid-like materials (lipidoids), remarkably enhances intracellular delivery of siRNA as compared to siRNA delivery with LPNs modified with dioleoyltrimethylammoniumpropane (DOTAP) as the lipid component. However, the potential immune modulation by these cationic lipids remains unexplored. By testing lipidoids and DOTAP for innate immune-receptor-activating properties in vitro, we found that neither lipidoids nor DOTAP activate human Toll-like receptor (TLR) 2, 3, 7, and 9. However, in contrast to DOTAP, lipidoids are strong agonists for TLR4 and activate murine antigen-presenting cells in vitro. This agonistic effect was further confirmed in silico using a prediction model based on crystal structures. Also, lipidoids formulated as lipoplexes or as stable nucleic acid lipid particles, which was the reference formulation for siRNA delivery, proved to activate TLR4. However, by combining lipidoids with PLGA into LPNs, TLR4 activation was abrogated. Thus, lipidoid-mediated TLR4 activation during siRNA delivery may be modulated via optimization of the formulation design.

Competition of PEG coverage density and con-A recognition in mannose/PEG bearing nanoparticles

Cell specific ligand molecules are attached to polyethylene glycol (PEG) modified nanoparticles to enhance the drug delivery efficiency. The tethered PEG would interfere in ligand recognition as well as providing biocompatibility to the nanoparticles. The denser PEG can give the greater biocompatibility, while should more hamper the ligand recognition. Therefor it is important to tune PEG density at an appropriate amount to compensate these two factors. In this study, we prepared a series of nanoparticles composed of α-mannose-bearing lipid, dioleoyltrimethyl ammoniumpropane (DOTAP) and 1,2-distearoyl-sn-glycero -3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). The nanoparticles were characterized with dynamic light scattering (DLS), field flow fractionation (FFF), small angle X-ray scattering (SAXS), and quartz-crystal microbalance (QCM). Based on the structural parameter obtained from DLS, SAXS, and FFF, we determined the PEG crowding parameter (Ͽ) quantitatively; when Ͽ=1.0, the PEG chain occupies the same value on the surface as when the chain is present in the unperturbed state, and at Ͽ=1.0, the PEG chains start to contact each other. We found that the recognition ability had the maximum around Ͽ⿼0.75 and there was the critical composition at Ͽ=1.0 for which the recognition was drastically reduced. The present results demonstrated that quantitative characterization and controlling the PEG density are key to designing effective targeting delivery system.

Effect of Polyelectrolyte Stiffness and Solution pH on the Nanostructure of Complexes Formed by Cationic Amphiphiles and Negatively Charged Polyelectrolytes.

The interaction between amphiphiles and polyelectrolytes has been widely investigated in recent years due to their potential application in industry and medicine, with special focus on gene therapy. The cationic lipid dioleoyl trimethylammonium propane, DOTAP, and the oppositely charged polyelectrolytes, sodium poly(acrylic acid) and sodium poly(styrenesulfonate), form multilamellar complexes in water. Because of the different molecular stiffness of the two polyelectrolytes, they form different nanostructured complexes. Also, because of the different ionization behavior of the two polyelectrolytes, pH differently affects the complexation of the polyelectrolytes with didodecyldimethylammonium bromide (DDAB), another cationic surfactant. We used cryogenic temperature transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS) to compare the nanostructures formed. Our results show that although the basic nanostructures of the complexes are always lamellar (multilamellar or unilamellar) the morphology of the complexes is affected by the polyelectrolyte rigidity and the solution pH.

Macrophage-Targeting Gene Delivery Using a Micelle Composed of Mannose-Modified Lipid with Triazole Ring and Dioleoyl Trimethylammonium Propane.

Gene carriers with cell specific ligand molecules are needed for the treatment of several diseases. Mannose is known to be recognized and incorporated into the cells through mannose recognition lectins that are exclusively expressed on macrophages. In this study, we synthesized two types of mannose-modified lipids with different stereoisomer (α-mannose and β-mannose). To make a complex with plasmid DNA (pDNA), termed “lipoplex,” we prepared a two-component micelle made from cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP); and mannose-modified lipid (D/α-Man or D/β-Man). The prepared D/α-Man lipoplexes were able to bind to one of the α-mannose lectins concanavalin A (ConA) immobilized on gold substrate in the quartz-crystal microbalance sensor cell. D/β-Man lipoplexes did not show any frequency changes. These results indicate that the mannose residues were exposed on the lipoplexes, leading to not only the binding to ConA but also the prevention of nonspecific interactions with proteins. Both lipoplexes showed high transfection efficiencies to RAW264.7 cells which have several kinds of mannose lectins. This delivery system to macrophages may overcome the problems for gene therapy and may be used for the treatment of immune diseases involved in macrophages.

Mechanistic profiling of the siRNA delivery dynamics of lipid–polymer hybrid nanoparticles

Understanding the delivery dynamics of nucleic acid nanocarriers is fundamental to improve their design for therapeutic applications. We investigated the carrier structure–function relationship of lipid–polymer hybrid nanoparticles (LPNs) consisting of poly(dl-lactic-co-glycolic acid) (PLGA) nanocarriers modified with the cationic lipid dioleoyltrimethyl-ammoniumpropane (DOTAP). A library of siRNA-loaded LPNs was prepared by systematically varying the nitrogen-to-phosphate (N/P) ratio. Atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM) combined with small angle X-ray scattering (SAXS) and confocal laser scanning microscopy (CLSM) studies suggested that the siRNA-loaded LPNs are characterized by a core–shell structure consisting of a PLGA matrix core coated with lamellar DOTAP structures with siRNA localized both in the core and in the shell. Release studies in buffer and serum-containing medium combined with in vitro gene silencing and quantification of intracellular siRNA suggested that this self-assembling core–shell structure influences the siRNA release kinetics and the delivery dynamics. A main delivery mechanism appears to be mediated via the release of transfection-competent siRNA–DOTAP lipoplexes from the LPNs. Based on these results, we suggest a model for the nanostructural characteristics of the LPNs, in which the siRNA is organized in lamellar superficial assemblies and/or as complexes entrapped in the polymeric matrix.

Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry

Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed ‘lipoplex’, we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis.