49 research outputs found
Endosomal DNA release studies using giant unilamellar vesicles as model endosomal membranes
Endosomal DNA release is one of the main barriers to successful non-viral gene delivery, since the inability of DNA to escape from the endosome at an early stage leads to its degradation through trafficking to the lysosomal compartment. It is therefore essential to understand the interactions between commonly used gene delivery vectors and endosomal membranes. While membrane interactions are often studied using small unilamellar vesicles (SUVs) as model bilayers, it is proposed that giant unilamellar vesicles (GUVs) present more realistic models due to their larger size, their superior lipid packing due to reduced surface curvature and the ability to visualise them using light or confocal microscopy. GUVs composed of a mixture of neutral or neutral and negatively charged lipids, representing early or late stage endosomal membranes respectively were prepared by electroformation in calcein, followed by the addition of cobalt chloride to quench background fluorescence. GUVs were then observed by confocal fluorescence microscopy before and after the addition of lipid:DNA complexes composed of equimolar mixture of dimethyldioctadecylammonium bromide (DDAB) with the helper lipid dioleoylphosphatidyl-ethanolamine (DOPE) incorporating a 10 mol% rhodamine-labelled DOPE at a 4:1 lipid:DNA charge ratio. Furthermore, in order to visualise the DNA in relation to the encapsulated calcein (green) and the lipid (red), 4 ,6-diamidino-2-phenylindole (DAPI) was added to highlight the DNA blue. Both endosomal models formed spherical GUVs approximately 10–90 m in diameter and were visible as green calcein-encapsulating vesicles. Upon the addition of lipid:DNA complexes to the early endosomal model, a large number of GUVs were shown to lose fluorescence due to calcein leakage, which was concentration dependent first order kinetics. This was also associated with visible alignment of the lipid (red) and the DNA (blue) around the GUV with possible pore formation and DNA translocation across the endosomal membrane. When lipid:DNA complexes were added to the late endosomal membrane model (which incorporated a small percentage of anionic lipid), calcein leakage and pore formation on the surface of the GUV membranes were clearly visible. Additionally, and exclusively to this model, however, a high number of GUVs were shown to deform after the addition of the complexes with or without calcein leakage. This was thought to be due to electrostatic interactions between the cationic DDAB and the anionic lipid domains of the endosomal membrane. In conclusion, it is thought that DDAB-DOPE:DNA complexes interact with both early and late endosomal membranes, causing pore formation and DNA translocation across the membrane, however the nature of the interaction changes as the endosomes traffic from early to late stages
Effect of the helper lipid DOPE on endosomal membrane integrity
The zwitterionic helper lipid Dioleoylphospatidylethanolamine (DOPE) is usually incorporated with cationic lipid gene delivery vectors to improve their transfection efficiency by facilitating escape of the DNA from the endosomal compartment. In this study the effect of DOPE on the integrity of model early and late endosomal monolayers was investigated through Langmuir trough and neutron reflectivity experiments. Mixtures of either neutral or neutral and anionic lipids representing early or late endosomal membranes respectively were dissolved in chloroform and spread on the surface of a Langmuir trough in MES buffer at pH 5 to mimic the endosomal environment then compressed to 30mN/m. Lipoplexes composed of the cationic lipid Dimethyldioctadecylammonium bromide (DDAB) or equimolar mixtures of DDAB:DOPE at 2:1 NP charge ratio were injected in the subphase and changes in surface pressure and neutron reflectivity were measured with time while holding constant area. Interestingly, injections of DDAB:DOPE lipoplexes resulted in a marked increase in surface pressure by up to 15mN/m to 45mN/m in both early and late endosomal models whereas injections of the same concentration of DDAB lipoplexes in the absence of DOPE did not result in a change in surface pressure. Simultaneous neutron reflectivity measurements, however, showed that DDAB in both formulations has been incorporated in both early and late endosomal membrane models within several minutes of injection. This difference in the two experimental techniques is thought to be due to DOPE’s ability to form non-lamellar hexagonal phases when the cationic lipid undergoes charge neutralisation upon interaction with the endosomal anionic lipids
Understanding and optimising the transfection of lipopolyplexes formulated in saline: the effects of peptide and serum
Lipopolyplexes (LPDs) are of considerable interest for use as gene delivery vehicles. Here LPDs have been prepared from cationic vesicles (composed of a 1 : 1 molar ratio of DOTMA with the neutral helper lipid, DOPE), singly branched cationic peptides and plasmid DNA. All peptides contained a linker sequence (cleaved by endosomal furin) attached to a targeting sequence selected to bind human airway epithelial cells and mediate gene delivery. The current study investigates the effects of novel Arg-containing cationic peptide sequences on the biophysical and transfection properties of LPDs. Mixed His/Arg cationic peptides were of particular interest, as these sequences have not been previously used in LPD formulations. Lengthening the number of cationic residues in a homopolymer from 6 to 12 in each branch reduced transfection using LPDs, most likely due to increased DNA compaction hindering the release of pDNA within the target cell. Furthermore, LPDs containing mixed Arg-containing peptides, particularly an alternating Arg/His sequence exhibited an increase in transfection, probably because of their optimal ability to complex and subsequently release pDNA. To confer stability in serum, LPDs were prepared in 0.12 M sodium chloride solution (as opposed to the more commonly used water) yielding multilamellar LPDs with very high levels of size reproducibility and DNA protection, especially when compared to the (unilamellar) LPDs formed in water. Significantly for the clinical applications of the LPDs, those prepared in the presence of sodium chloride retained high levels of transfection in the presence of media supplemented with fetal bovine serum. This work therefore represents a significant advance for the optimisation of LPD formulation for gene delivery, under physiologically relevant conditions, in vivo
The discovery and enhanced properties of trichain lipids in lipopolyplex gene delivery systems
The formation of a novel trichain (TC) lipid was discovered when a cationic lipid possessing a terminal hydroxyl group and the helper lipid dioleoyl l-α-phosphatidylethanolamine (DOPE) were formulated as vesicles and stored. Importantly, the transfection efficacies of lipopolyplexes comprised of the TC lipid, a targeting peptide and DNA (LPDs) were found to be higher than when the corresponding dichain (DC) lipid was used. To explore this interesting discovery and determine if this concept can be more generally applied to improve gene delivery efficiencies, the design and synthesis of a series of novel TC cationic lipids and the corresponding DC lipids was undertaken. Transfection efficacies of the LPDs were found to be higher when using the TC lipids compared to the DC analogues, so experiments were carried out to investigate the reasons for this enhancement. Sizing experiments and transmission electron microscopy indicated that there were no major differences in the size and shape of the LPDs prepared using the TC and DC lipids, while circular dichroism spectroscopy showed that the presence of the third acyl chain did not influence the conformation of the DNA within the LPD. In contrast, small angle neutron scattering studies showed a considerable re-arrangement of lipid conformation upon formulation as LPDs, particularly of the TC lipids, while gel electrophoresis studies revealed that the use of a TC lipid in the LPD formulation resulted in enhanced DNA protection properties. Thus, the major enhancement in transfection performance of these novel TC lipids can be attributed to their ability to protect and subsequently release DNA. Importantly, the TC lipids described here highlight a valuable structural template for the generation of gene delivery vectors, based on the use of lipids with three hydrophobic chains
Novel cationic lipopolyplexes as gene therapy vectors
A major obstacle in the development of gene therapy is delivery of therapeutic genes to the desired cell/tissue. The objective of our study is to use a non-viral ternary system (lipopolyplexes) to encapsulate and deliver therapeutic DNA. Our lipopolyplexes comprise a glycerol-based cytofectin, a targeting peptide and plasmid DNA. Novel derivatives of the cationic lipids DOTMA and DOTAP have been synthesized and tested in a breast cancer cell line. A range of branched cationic peptides varying in number of residues, composition and linker to a targeting head group were also designed and prepared. The bio-physical studies demonstrated that all LPD complexes were positively charged, small (60-80 nm) and were shown to effectively condense DNA. Gel assays showed which peptides were able to protect DNA more effectively and gave high transfection efficiency. Further studies are underway investigating these systems in siRNA delivery
Tuning a gene delivery vector: the role of peptide sequence and peptide branching in delivering of DNA and siRNA to the cytoplasm
Tuning a gene delivery vector: the role of peptide sequence and peptide branching in delivering of DNA and siRNA to the cytoplas
Stability testing of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine: a translational study in UK vaccination centres
Objective - The roll-out of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine has brought many logistical challenges, such as the absence of comprehensive stability data leading to strict handling instructions during dilution and administration. Accidental mishandling therefore presents challenging clinical dilemmas, which often led vaccine providers to err on the side of caution and discard mishandled vials rather than risk administering ineffective vaccine. This study aims to answer key questions about the vaccine’s stability to allow for a more informed decision-making process should a non-conformity occur.
Methods - Residual vaccine in freshly used, but appropriately stored vials collected from vaccination centres in Brighton, UK, were tested after exposure to various handling conditions and analysed by dynamic light scattering to determine the size of the lipid-mRNA nanoparticles, and gel electrophoresis to visualise the mRNA integrity and separation from the lipid formulation.
Results - Knocking or dropping vaccine samples from small heights resulted in lowest levels of instability, indicating low risk of compromising clinical efficacy. However, repeated drawing and injecting through 23 G needles at high speed and, more significantly, shaking and vortexing led to progressive increase in the size and polydispersity index of the lipid-mRNA nanoparticles, coupled with or caused by up to ~50% release of mRNA from the lipid formulation. This is thought to impact the vaccine’s efficacy due to lack of free mRNA protection and cellular internalisation.
Conclusions - These results reiterate the importance of adhering to the manufacturer’s instructions on handling, especially with regard to shaking and exposing the vaccine to excessive vibration
The discovery and enhanced properties of trichain lipids in lipopolyplex gene delivery systems
The formation of a novel trichain (TC) lipid was discovered when a cationic lipid possessing a terminal hydroxyl group and the helper lipid dioleoyl l-α-phosphatidylethanolamine (DOPE) were formulated as vesicles and stored. Importantly, the transfection efficacies of lipopolyplexes comprised of the TC lipid, a targeting peptide and DNA (LPDs) were found to be higher than when the corresponding dichain (DC) lipid was used. To explore this interesting discovery and determine if this concept can be more generally applied to improve gene delivery efficiencies, the design and synthesis of a series of novel TC cationic lipids and the corresponding DC lipids was undertaken. Transfection efficacies of the LPDs were found to be higher when using the TC lipids compared to the DC analogues, so experiments were carried out to investigate the reasons for this enhancement. Sizing experiments and transmission electron microscopy indicated that there were no major differences in the size and shape of the LPDs prepared using the TC and DC lipids, while circular dichroism spectroscopy showed that the presence of the third acyl chain did not influence the conformation of the DNA within the LPD. In contrast, small angle neutron scattering studies showed a considerable re-arrangement of lipid conformation upon formulation as LPDs, particularly of the TC lipids, while gel electrophoresis studies revealed that the use of a TC lipid in the LPD formulation resulted in enhanced DNA protection properties. Thus, the major enhancement in transfection performance of these novel TC lipids can be attributed to their ability to protect and subsequently release DNA. Importantly, the TC lipids described here highlight a valuable structural template for the generation of gene delivery vectors, based on the use of lipids with three hydrophobic chains
Chitosan-coated mesoporous MIL-100(Fe) nanoparticles as improved bio-compatible oral nanocarriers
Nanometric biocompatible Metal-Organic Frameworks (nanoMOFs) are promising candidates for drug delivery. Up to now, most studies have targeted the intravenous route, related to pain and severe complications; whereas nanoMOFs for oral administration, a commonly used non-invasive and simpler route, remains however unexplored. We propose here the biofriendly preparation of a suitable oral nanocarrier based on the benchmarked biocompatible mesoporous iron(III) trimesate nanoparticles coated with the bioadhesive polysaccharide chitosan (CS). This method does not hamper the textural/ structural properties and the sorption/release abilities of the nanoMOFs upon surface engineering. The interaction between the CS and the nanoparticles has been characterized through a combination of high resolution soft X-ray absorption and computing simulation, while the positive impact of the coating on the colloidal and chemical stability under oral simulated conditions is here demonstrated. Finally, the intestinal barrier bypass capability and biocompatibility of CS-coated nanoMOF have been assessed in vitro, leading to an increased intestinal permeability with respect to the noncoated material, maintaining an optimal biocompatibility. In conclusion, the preservation of the interesting physicochemical features of the CS-coated nanoMOF and their adapted colloidal stability and progressive biodegradation, together with their improved intestinal barrier bypass, make these nanoparticles a promising oral nanocarrier