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.

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.

Trichain cationic lipids: the potential of their lipoplexes for gene delivery.

Lipoplexes (LDs) have been prepared from DNA and positively charged vesicles composed of the helper lipid, dioleoyl l-α-phosphatidylethanolamine (DOPE) and either a dichain (DC) oxyethylated cationic lipid or their corresponding novel trichain (TC) counterpart. This is the first study using the TC lipids for the preparation of LDs and their application. Here the results of biophysical experiments characterising the LDs have been correlated with the in vitro transfection activity of the complexes. Photon correlation spectroscopy, zeta potential measurements and transmission electron microscopy studies indicated that, regardless of the presence of a third chain, there were little differences between the size and charge of the TC and DC containing LDs. Small angle neutron scattering studies established however that there was a significant conformational re-arrangement of the lipid bilayer when in the form of a LD complex as opposed to the parent vesicles. This re-arrangement was particularly noticeable in LDs containing TC lipids possessing a third chain of C12 or a longer chain. These results suggested that the presence of a third hydrophobic chain had a significant effect on lipid packing in the presence of DNA. Picogreen fluorescence and gel electrophoresis studies showed that the TC lipids containing a third acyl chain of at least C12 were most effective at complexing DNA while the TC lipids containing an octanoyl chain and the DC lipids were least effective. The transfection efficacies of the TC lipids in the form of LDs were found to be higher than for the DC analogues, particularly when the third acyl chain was an octanoyl or oleoyl moeity. Little or no increase in transfection efficiency was observed when the third chain was a methyl, acetyl or dodecanoyl group. The large enhancement in transfection performance of the TC lipids can be attributed to their ability to complex their DNA payload. These studies indicate that presence of a medium or long third acyl chain was especially beneficial for transfection.

Delivery of siRNA using ternary complexes containing branched cationic peptides: the role of peptide sequence, branching and targeting.

Ternary nanocomplexes, composed of bifunctional cationic peptides, lipids and siRNA, as delivery vehicles for siRNA have been investigated. The study is the first to determine the optimal sequence and architecture of the bifunctional cationic peptide used for siRNA packaging and delivery using lipopolyplexes. Specifically three series of cationic peptides of differing sequence, degrees of branching and cell-targeting sequences were co-formulated with siRNA and vesicles prepared from a 1 : 1 molar ratio of the cationic lipid DOTMA and the helper lipid, DOPE. The level of siRNA knockdown achieved in the human alveolar cell line, A549-luc cells, in both reduced serum and in serum supplemented media was evaluated, and the results correlated to the nanocomplex structure (established using a range of physico-chemical tools, namely small angle neutron scattering, transmission electron microscopy, dynamic light scattering and zeta potential measurement); the conformational properties of each component (circular dichroism); the degree of protection of the siRNA in the lipopolyplex (using gel shift assays) and to the cellular uptake, localisation and toxicity of the nanocomplexes (confocal microscopy). Although the size, charge, structure and stability of the various lipopolyplexes were broadly similar, it was clear that lipopolyplexes formulated from branched peptides containing His-Lys sequences perform best as siRNA delivery agents in serum, with protection of the siRNA in serum balanced against efficient release of the siRNA into the cytoplasm of the cell.

Manipulating the pH response of 2,3-diaminopropionic acid rich peptides to mediate highly effective gene silencing with low-toxicity.

Cationic amphipathic pH responsive peptides possess high in vitro and in vivo nucleic acid delivery capabilities and function by forming a non-covalent complex with cargo, protecting it from nucleases, facilitating uptake via endocytosis and responding to endosomal acidification by being released from the complex and inserting into and disordering endosomal membranes. We have designed and synthesised peptides to show how Coulombic interactions between ionizable 2,3-diaminopropionic acid (Dap) side chains can be manipulated to tune the functional pH response of the peptides to afford optimal nucleic acid transfer and have modified the hydrogen bonding capabilities of the Dap side chains in order to reduce cytotoxicity. When compared with benchmark delivery compounds, the peptides are shown to have low toxicity and are highly effective at mediating gene silencing in adherent MCF-7 and A549 cell lines, primary human umbilical vein endothelial cells and both differentiated macrophage-like and suspension monocyte-like THP-1 cells.

Gene delivery using ternary lipopolyplexes incorporating branched cationic peptides: the role of Peptide sequence and branching.

Cationic peptide sequences, whether linear, branched, or dendritic, are widely used to condense and protect DNA in both polyplex and lipopolyplex gene delivery vectors. How these peptides behave within these particles and the consequences this has on transfection efficiency remain poorly understood. We have compared, in parallel, a complete series of cationic peptides, both branched and linear, coformulated with plasmid DNA to give polyplexes, or with plasmid DNA and the cationic lipid, DOTMA, mixed with 50% of the neutral helper lipid, DOPE, to give lipopolyplexes, and correlated the transfection efficiencies of these complexes to their biophysical properties. Lipopolyplexes formulated from branched Arg-rich peptides, or linear Lys-rich peptides, show the best transfection efficiencies in an alveolar epithelial cell line, with His-rich peptides being relatively ineffective. The majority of the biophysical studies (circular dichroism, dynamic light scattering, zeta potential, small angle neutron scattering, and gel band shift assay) indicated that all of the formulations were similar in size, surface charge, and lipid bilayer structure, and longer cationic sequences, in general, gave better transfection efficiencies. Whereas lipopolyplexes formulated from branched Arg-containing peptides were more effective than those formulated from linear Arg-containing sequences, the reverse was true for Lys-containing sequences, which may be related to differences in DNA condensation between Arg-rich and Lys-rich peptides observed in the CD studies.

Effective endogenous gene silencing mediated by pH responsive peptides proceeds via multiple pathways.

Cationic amphipathic histidine rich peptides possess high plasmid DNA and siRNA delivery capabilities. To further understand the pH responsive siRNA delivery process and evaluate the capabilities of such peptides we have investigated their ability to mediate specific silencing of endogenous GAPDH gene activity in MCF-7 and A549 cells and compared this with plasmid DNA delivery. A substantial and selective reduction of both GAPDH activity and expression was achieved using pH responsive peptide vectors, which compared favourably with that mediated by commercially available non-viral vectors in terms of efficacy and toxicity. Furthermore, by comparing the efficacy of both gene delivery and silencing mediated by a series of such peptides, their sensitivities to known inhibitors of endocytotic processes, and their route of uptake via confocal live cell imaging, we show that both plasmid DNA and siRNA are internalised via endocytosis. However siRNA entry facilitated by LAH4-L1, proceeds via a cholesterol dependent mechanism, in contrast to DNA transfer which is associated with clathrin dependent endocytosis. Furthermore, using peptides that respond at increasingly acidic pH, we demonstrate that the route of entry for the siRNA that ultimately mediates silencing is peptide specific and whilst some pH responsive peptides promote the escape of labelled siRNA from endosomes, others may promote entry via alternative mechanisms.

Lipopolyplex ternary delivery systems incorporating C14 glycerol-based lipids.

The structure, biophysical properties and biological behavior of lipopolyplex ternary gene delivery vectors incorporating novel C14 glycerol based lipids of varying alkyl chain geometry (containing cis, trans or alkyne double bonds) have been studied in the presence and absence of a bifunctional targeting peptide designed to both condense DNA and confer integrin-specific targeting. In vitro transfection studies in breast cancer MDA-MB-231 cells revealed that ternary formulations of lipid:peptide:DNA (LPD) complexes prepared using the aforementioned lipids possessed highly synergistic transfection activity up to 2500-fold higher than their respective lipid:DNA (LD) or peptide:DNA (PD) counterparts. Furthermore, the small structural differences in the lipid alkyl chain geometries also resulted in pronounced differences in transfection within each type of formulation, whereby the trans lipids showed best activity when formulated as LD complexes, whereas the cis lipids were superior in LPD formulations. Confocal fluorescence internalization studies using labeled components of the formulations showed both the lipid and the DNA of LD complexes to be trapped in endocytic compartments, whereas in the case of LPD complexes, the DNA was clearly released from the endosomal compartments and, together with the peptide, internalized within the cell nucleus. Physicochemical characterization of the formulations carried out by light and neutron scattering, zeta potential measurement, and negative staining electron microscopy detected major structural differences between LD and LPD complexes. Gel electrophoresis assays additionally showed differences between the individual lipids tested in each type of formulation. In conclusion, the superior transfection of the trans lipids in the LD complexes was thought to be attributed to superior DNA binding caused by a more closely matched charge distribution of the more rigid, trans lipids with the DNA. In the case of the LPD complexes, the DNA was thought to be predominantly condensed by the cationic portion of the peptide forming a central core surrounded by a lipid bilayer from which the targeting sequence partially protrudes. The more fluid, cis lipids were thought to confer better activity in this formulation due to allowing more of the targeting peptide sequence to protrude.

Lipid chain geometry of C14 glycerol-based lipids: effect on lipoplex structure and transfection.

The effects have been determined of a systematic alteration of the alkyl chain geometry of a C14 analogue of DOTMA on the detailed molecular architecture of the resulting cationic vesicles formed both in the absence and presence of 50 mol% DOPE, and of the lipoplexes prepared from these vesicles using either calf thymus or plasmid DNA. The C14 DOTMA analogues studied involved cis- or trans-double bonds at positions Δ9 or Δ11, and a compound (ALK) featuring an alkyne at position C9. For all of these analogues, examination by light scattering and neutron scattering, zeta potential measurement, and negative staining electron microscopy showed that there were no significant differences in the structures or charges of the vesicles or of the resulting lipoplexes, regardless of the nature of the DNA incorporated. Differences were observed, however, between the complexes formed by the various lipids when examining the extent of complexation and release by gel electrophoresis, where the E-lipids appeared to complex the DNA more efficiently than all other lipids tested. Moreover, the lipoplexes prepared from the E-lipids were the most effective in transfection of MDA-MB-231 breast cancer cells. As indicated through confocal microscopy studies, the E-lipids also showed a higher internalisation capacity and a more diffuse cellular distribution, possibly indicating a greater degree of endosomal escape and/or nuclear import. These observations suggest that the extent of complexation is the most important factor in determining the transfection efficiency of the complexes tested. At present it is unclear why the E-lipids were more effective at complexing DNA, although it is thought that the effective area per molecule occupied by the cationic lipid and DOPE head groups, and therefore the density of positive charges on the surface of the bilayer most closely matches the negative charge density of the DNA molecule. From a consideration of the geometry of the cationic lipids it is anticipated that the head groups of the E-lipids would occupy a smaller area per molecule than the ALK or Z-lipids.

Stabilized integrin-targeting ternary LPD (lipopolyplex) vectors for gene delivery designed to disassemble within the target cell.

Recent research in the field of nonviral gene delivery vectors has focused on preparing nanoparticles that are stabilized by the incorporation of a PEG coating and where one of the vector components is also cleavable. Here,we describe the synthesis, formulation, transfection properties, and biophysical studies of a PEG-stabilized ternary lipopolyplex vector in which, for the first time, both the lipid and peptide components are designed to be cleaved once the vector has been internalized. A series of cationic lipids, bearing short tri- or hexaethylene glycol groups, attached to the headgroup via an ester linkage, has been prepared. Trifunctional peptides have also been prepared, consisting of a Lys(16) sequence at the N-terminus (to bind and condense plasmid DNA); a spacer group (containing a sequence recognized and cleaved by endosomal enzymes) and an optional PEG4 amino acid; and an integrin-targeting cyclic peptide sequence (allowing the resulting nanoparticle to be internalized via receptor-mediated endocytosis). Differing combinations of these lipids and peptides have been formulated with DOPE and with plasmid DNA, and complex stability, transfection, and cleavage studies carried out. It was shown that optimal transfection activities in a range of cell types and complex stabilities were achieved with lipids bearing short cleavable triethylene glycol moieties, whereas the incorporation of PEG4 amino acids into the cleavable peptides had little effect. We have synthesized appropriate fluorescently labeled components and have studied the uptake of the vector, endosomal escape, peptide cleavage, and plasmid transport to the nucleus in breast cancer cells using confocal microscopy. We have also studied the morphology of these compact, stabilized vectors using cryo-EM.

The effect of electrolyte on the encapsulation efficiency of vesicles formed by the nonionic surfactant, 2C18E12.

Encapsulation efficiencies of vesicles formed by the nonionic surfactant 1,2-dioctadecyl-rac-glycerol-3-omega-methoxydodecylethylene glycol (abbreviated as 2C18E12) and its phospholipid counterpart, distearoylphosphatidylcholine (DSPC) at 298 K, were determined by the entrapment of the water-soluble dye, carboxyfluorescein (CF) to be 0.045+/-0.001 and 0.03+/-0.04 L mol(-1) for 2C18E12 vesicles prepared using low osmolarity (270 m Osm) Krebs-Henseleit (K-H) buffer and a modified 'high salt' (1600 m Osm) variant of K-H buffer, respectively, and 0.64+/-0.01 and 0.31+/-0.04 Lmol(-1) for DSPC vesicles prepared under the same conditions and in the same buffers. Freeze fracture electron microscopy studies confirmed the presence of vesicles when 2C18E12 and DSPC were dispersed in water and both buffer solutions. Small angle neutron scattering (SANS) studies, using D2O in place of H2O, showed that when 2C18E12 vesicles were prepared in the 'high salt' variant of K-H buffer as opposed to K-H buffer or water, a higher proportion of multilamellar vesicles (MLV) were formed. Furthermore when prepared in the 'high salt' variant of K-H buffer, the 2C18E12 bilayers were thinner, and when present in the form of MLV exhibited a smaller layer of water separating the bilayers. However, even in the absence of electrolyte, 2C18E12 formed surprisingly thin bilayers due to the penetration of the polyoxyethylene chains into the hydrophobic chain region of the bilayer. Due to the dehydrating effect of the high concentration of electrolyte present in the 'high salt' variant of K-H, the polyoxyethylene head groups penetrated further into the hydrophobic region of the bilayer making the bilayer even thinner. In the case of the DSPC vesicles, although the SANS study showed an increase in the relative proportion of multilamellar to unilamellar vesicles when samples were prepared in the 'high salt' variant of K-H buffer, no differences were observed in the thickness and the d-spacing of the vesicle bilayers. Variable temperature turbidity measurements of 2C18E12, and DSPC vesicles prepared in water indicated phase changes at 320+/-0.5 and 327+/-0.5 K, respectively, and were unchanged when the 'high salt' variant of K-H buffer was used as hydrating medium. Taken together, these results suggest that a low phase transition temperature was not the reason for the poor entrapment efficiency of 2C18E12 vesicles but rather the very 'thin' hydrophobic barrier formed by the penetration of the polyoxyethylene chains into the hydrophobic region of the bilayer.