Lipid nanoparticles containing conjugated oligoelectrolytes

Abstract

The present disclosure provides a lipid nanoparticle comprising a helper lipid at about 5 mol% to about 80 mol% relative to a total lipid content; an ionisable lipid at about 20 mol% to about 95 mol% relative to the total lipid content; a conjugated oligoelectrolyte at about 0.005 mol% to about 5 mol% relative to the total lipid content, wherein the conjugated oligoelectrolyte is configured to interact with at least the helper lipid in order 10 to stabilise the lipid nanoparticle. The present disclosure also provides methods of fabricating the lipid nanoparticle and methods of use thereof.

Description

Lipid Nanoparticles Containing Conjugated OligoelectrolytesTechnical FieldThe present invention relates, in general terms, to lipid nanoparticles containing conjugated oligoelectrolytes.BackgroundMessenger RNA (mRNA) is a transient intermediator between genes and proteins. Investigations of mRNA structure and function resulted in the development of in vitro- transcribed (IVT) mRNA. In recent times, mRNA is used as a form of therapeutics providing the basis for mRNA as a new class of drug. mRNA has shown therapeutic potential in a range of applications, including viral vaccines, protein replacement therapies, cancer immunotherapies, cellular reprogramming and genome editing. Fundamentally, mRNA therapy involves the use of synthetic mRNA molecules to instruct cells in the body to produce specific proteins that play a clinically relevant role. Unlike conventional gene therapies that often rely on introducing transient or permanent DNA modifications, mRNA therapeutics leverage the body's natural protein synthesis machinery to achieve the intended treatment results. One of the most notable applications of mRNA technology has been in the rapid development of vaccines during the COVID-19 pandemic, with remarkable clinical efficacy and safety profiles.Despite the clear advantages of the mRNA technology, it shares common limitations with other nucleic-acid based therapeutics. To achieve therapeutic effects, mRNA molecules have to reach specific target cells and produce sufficient proteins of interest. In other words, they require effective in vivo delivery systems to defend them from enzymatic degradation and quick elimination by the reticuloendothelial system (RES), enhance delivery to target cells, and limit exposure to off-target cells. However, targeted delivery and endosomal escape remain challenging for mRNA delivery systems, highlighting the need for safe and effective mRNA delivery materials.A variety of materials have been developed for mRNA delivery, including lipids, lipid-like materials, polymers and protein derivatives. In particular, lipid nanoparticles have been thoroughly investigated and successfully entered the clinic for the delivery of small molecules, siRNA drugs and mRNA. Notably, two authorized coronavirus disease 2019 (COVID-19) vaccines, mRNA-1273 and BNT162b, use lipid nanoparticles to deliver antigen mRNA. Many other lipid nanoparticle-mRNA formulations have been developed and are under clinical evaluation for the prevention and treatment of virus infections, cancer and genetic diseases.LNPs are nano-sized, biocompatible vesicles of a non-viral origin that encapsulate anionic mRNA molecules, aiding them in surmounting physiological barriers and facilitating their efficient delivery into cells. Once inside the cells, the sequence encoded in the mRNA is translated into proteins that elicit specific functions or immune responses. LNPs are thus crucial for a mRNA therapeutic effectiveness. They are engineered to enhance the uptake of mRNA by cells, promote intracellular release of mRNAs, and mitigate potential side effects. This is achieved through optimization and formulation of the four main lipid components: ionizable lipids, zwitterionic helper lipids, cholesterol, and PEG-lipids. Although mRNA vaccines have shown the potential of this platform, there is renewed and ongoing interest in developing novel LNP formulations that are highly efficient and adaptable to various applications, including achieving organspecific and cell-specific delivery.However, there are still issues with the use of lipid nanoparticles as a drug delivery agent. The transient nature of the mRNA molecule is a benefit, preventing it from continually generating the antigen after the immune response has been triggered. The same property, however, is a downside for stability and storage. To facilitate global delivery, it is vital to ensure the mRNA is stabilised for prolonged periods prior to dosing of a patient. Since hydrolysis can cause the mRNA to break down or degrade when stored at elevated temperatures, the Pfizer-BioNTech and Moderna SARS-CoV-2 vaccines are stored at -70°C and -20°C respectively, to slow this process.Frustratingly, while low temperature storage in the frozen state can slow the degradation of the mRNA, it can have a detrimental effect on the physical stability of the lipid nanoparticles. During freezing, water forms ice crystals, which concentrates the nanoparticles and can lead to aggregation. To reduce the aggregation upon freezing and storage, sugars (eg, trehalose and sucrose) are added, which entrap thenanoparticles in an amorphous matrix which isolates them and reduces their mobility, which prevents damaging aggregation.Another way to make mRNA vaccines more stable is to add stabilisers (such as sugars or solvents) and remove water from the product through a process called lyophilisation or freeze-drying. This has been shown to allow some mRNA vaccines to be stored in a refrigerator instead of a freezer. However, during the freezing step of the lyophilisation, the same aggregation process must be prevented.Because of the use of various stabilisers, side effects are also associated with the use of lipid nanoparticles in treatment. Side effects include difficulty breathing, swelling of face and throat, fast heartbeat, rash, dizziness, weakness, chest pain, headache, muscle pain, chills, joint pain, fever, and nausea. Myocarditis (inflammation of the heart muscle) and pericarditis (inflammation of the lining outside the heart) have occurred in some people who have received mRNA COVID-19 vaccines, including COMIRNATY and Pfizer-BioNTech COVID-19 vaccines. Myocarditis and pericarditis following COMIRNATY have occurred most commonly in adolescent males 12 through 17 years of age.It would be desirable to overcome or ameliorate at least one of the above-described problems.SummaryThe present disclosure concerns a lipid nanoparticle, comprising : a) a helper lipid at about 5 mol% to about 80 mol% relative to a total lipid content; b) an ionisable lipid at about 20 mol% to about 95 mol% relative to the total lipid content; c) a conjugated oligoelectrolyte at about 0.005 mol% to about 5 mol% relative to the total lipid content, wherein the conjugated oligoelectrolyte is configured to interact with at least the helper lipid in order to stabilise the lipid nanoparticle.In some embodiments, the conjugated oligoelectrolyte is configured to interact with a payload within the lipid nanoparticle in order to stabilise the payload.In some embodiments, the conjugated oligoelectrolyte is intercalated with at least the helper lipid.In some embodiments, the helper lipid is about 10 mol% relative to the total lipid content.In some embodiments, the helper lipid is a phospholipid.In some embodiments, the helper lipid is a phosphocholine or phosphatidylethanolamine.In some embodiments, the helper lipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dimyristoyl-sn- glycero-3-phosphocholine (DMPC), l-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-Dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or a combination thereof.In some embodiments, the ionisable lipid is about 50 mol% relative to the total lipid content.In some embodiments, the ionisable lipid is ionisable into a cationic lipid.In some embodiments, the ionisable lipid is selected from (6Z,9Z,28Z,31Z)- Heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), [(4-Hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), 9Z,12Z-octadecadienoic acid, 3-[4,4-bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester (LP01), and a combination thereof.In some embodiments, the conjugated oligoelectrolyte is a compound of Formula (I) or a salt or solvate thereof:whereinRi is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy or optionally substituted thio or optionally substituted phosphoryl;R2 is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; m is an integer selected from 1 to 7; n is an integer independently selected from 0 to 4; and q is an integer independently selected from 1 to 5; or a compound of Formula (II) or a salt or solvate thereof:wherein each Ri is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy or optionally substituted thio or optionally substituted phosphoryl; q is an integer selected from 1 to 5; q' is an integer selected from 1 to 5; wherein each L2 is independently selected from optionally substituted ethylene, or optionally substituted phenylethylene;Li is a n-conjugated core comprising monomeric unit A and monomeric unit D:wherein each A is independently selected from optionally substituted alkenylene, optionally substituted arylene or optionally substituted heteroarylene; each D is independently selected from optionally substituted alkenylene, optionally substituted arylene or optionally substituted heteroarylene; t is an integer selected from 1 to 5; u is an integer selected from 1 to 5; wherein * represents a bond to another monomeric unit or to L2; wherein monomeric units A and monomeric units D are alternatively bonded to each other; wherein the compound of Formula (I) has a substantially linear topology.In some embodiments, the conjugated oligoelectrolyte is selected from List 1 or List 2.In some embodiments, the lipid nanoparticle further comprises steroid at about 20 mol° / o to about 60 mol% relative to the lipid nanoparticle.In some embodiments, the steroid is selected from cholesterol, fecosterol, sitosterol, ergosterol, campersterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alphatocopherol or a derivative thereof.In some embodiments, the lipid nanoparticle further comprises a polymer conjugated lipid about 0.5 mol% to about 2 mol% relative to the lipid nanoparticle.In some embodiments, the polymer is polyethylene glycol (PEG) or polysarcosine.In some embodiments, the lipid nanoparticle is characterised by an average particle size of about 50 nm to about 250 nm.In some embodiments, the lipid nanoparticle is characterised by a photoluminescence lifetime of at least 0.05 ns.In some embodiments, the lipid nanoparticle is characterised by a photoluminescence fold increase of at least 2 relative to free conjugated oligoelectrolyte.In some embodiments, the lipid nanoparticle further comprises a payload in a cavity of the lipid nanoparticle.In some embodiments, the payload is characterised by an anionic charge.In some embodiments, the payload is selected from a nucleic acid, a drug, a protein or a combination thereof.In some embodiments, the payload is selected from mRNA, nucleoside-modified mRNA, plasmid DNA, DNA, or a combination thereof.In some embodiments, the lipid nanoparticle is characterised by a COE to polynucleotide nitrogen-to-phosphate (N / P) ratio of less than 2, and a total N / P ratio of more than 0.5.In some embodiments, the lipid nanoparticle is characterised by a payload encapsulation efficiency of more than about 40%.The present disclosure also concerns a method of detecting a lipid nanoparticle as disclosed herein in a cell, comprising imaging the cell using a fluorescence detector.The present disclosure also concerns a method of fabricating a lipid nanoparticle, comprising : a) mixing a helper lipid and an ionisable lipid in a solvent to form a mixture; b) introducing the mixture of step a) into a non-solvent in order to fabricate a precursor lipid nanoparticle; and c) adding a conjugated oligoelectrolyte to the precursor lipid nanoparticle of step b) to form the lipid nanoparticle; wherein the lipid is about 5 mol% to about 80 mol% relative to the mixture; wherein the ionisable lipid is about 20 mol% to about 95 mol% relative to the mixture; and wherein the conjugated oligoelectrolyte is about 0.005 mol% to about 5 mol% relative to the mixture.wherein the conjugated oligoelectrolyte is configured to interact with the helper lipid in order to stabilise the lipid nanoparticle.The present disclosure also concerns a method of fabricating a lipid nanoparticle, comprising : a) mixing a helper lipid, an ionisable lipid and a conjugated oligoelectrolytes in a solvent to form a mixture; b) introducing the mixture of step a) into a non-solvent in order to fabricate a lipid nanoparticle; and wherein the lipid is about 5 mol% to about 80 mol% relative to the mixture; wherein the ionisable lipid is about 20 mol% to about 95 mol% relative to the mixture; and wherein the conjugated oligoelectrolyte is about 0.005 mol% to about 5 mol% relative to the mixture, wherein the conjugated oligoelectrolyte is configured to interact with the helper lipid in order to stabilise the lipid nanoparticle.The present disclosure also concerns a lipid nanoparticle composition, comprising a lipid nanoparticle as disclosed herein and at least one excipient.The present disclosure also concerns a kit for forming a lipid nanoparticle, comprising : a) a helper lipid at about 5 mol% to about 80 mol% relative to a total lipid content; b) an ionisable lipid at about 20 mol% to about 95 mol% relative to the total lipid content; c) a conjugated oligoelectrolyte at about 0.005 mol% to about 5 mol% relative to the total lipid content, the conjugated oligoelectrolyte for interacting with the helper lipid in order to stabilise the lipid nanoparticle.The present disclosure also concerns a method of stabilising a lipid nanoparticle formulation, comprising mixing a conjugated oligoelectrolyte with the lipid nanoparticle formulation, wherein the conjugated oligoelectrolyte is about 0.005 mol% to about 5 mol% relative to the lipid nanoparticle formulation, wherein the conjugated oligoelectrolyte is configured to interact with a helper lipid of the lipid nanoparticle formulation in order to stabilise the lipid nanoparticle.In some embodiments, the lipid nanoparticle formulation is selected from Comirnaty and Spikevax.The present disclosure also concerns a method of delivering a payload to a cell, comprising incubating the cell with a lipid nanoparticle as disclosed herein.In some embodiments, the method is characterised by a cell viability more than about 50%.In some embodiments, the lipid nanoparticle is characterised by an in vitro delivery efficiency of more than about 5%.In some embodiments, when the payload is a nucleic acid, the method is characterised by a transfection efficiency of more than 25% relative to a lipid nanoparticle without the conjugated oligoelectrolyte.The present disclosure also concerns a method of delivering a therapeutic payload to a subject in need thereof, comprising administering a therapeutic amount of lipid nanoparticle as disclosed herein to the subject, wherein the lipid nanoparticle comprises the therapeutic payload.The present disclosure also concerns a lipid nanoparticle as disclosed herein for use in delivering a therapeutic payload to prevent and / or treat a disease or condition.The present disclosure also concerns a use of a lipid nanoparticle as disclosed herein in the manufacture of a medicament for delivering a therapeutic payload to prevent and / or treat a disease or condition.In some embodiments, the disease or condition is selected from a viral disease or condition, and cancer.Brief description of the drawingsEmbodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:Figure 1. Cartoon illustration of previous work with modifying bilayer membrane systems using COE-S6 such as lighting up extracellular vesicles and stabilizing bacterial membranes. Conceptual illustration of the lipid nanoparticle (LNP) containing conjugated oligoelectrolytes (COEs) in the present work. COE-S6 was incorporated ex- situ into mRNA LNPs via membrane intercalation. Upon intercalation, the unique bola- like structure of COE-S6 permits interaction with the lipid bilayer membrane, the intercalated COE-S6, and the encapsulated mRNA which modulates particle size, elevates cellular uptake, facilitates endosomal escape, and higher protein expression. Figure 2. The molecular structure of COE-S6.Figure 3. Photophysical and binding properties of the COE-S6 incorporated LNP system, (a) Ex-situ incorporation of COE-S6 onto mRNA LNPs proceeds with the addition of an aqueous COE solution to the purified LNPs before vortexing for uniform mixing, (b) Normalized photoluminescence emission spectra of COE-S6 in different environments (PBS and LNP). (c) Photoluminescence lifetime spectra of COE-S6 in PBS and LNP. (d) Normalized fluorescence anisotropy of COE-S6 titrated against increasing concentrations of LNP. The dissociation constant (Kd) was derived after fitting to a nonlinear regression curve.Figure 4. Particle characteristics of the COE-S6 incorporated LNP system, (a-c) DLS characterizations of the mRNA LNPs. (d) The mRNA encapsulation efficiencies of the LNPs as determined using Quant-it™ RiboGreen assay, (e) Cryo-TEM micrographs of the control and LNP with 0.2% COE-S6 incorporated. The particle size is given by the diameter as measured using Image! (n = 50). Scale bar = 100 nm. Each value represents the mean ± SD (n = 50). (****p<0.0001).Figure 5. The serum stability study of the COE-S6 incorporated LNPs. The LNPs were incubated in 10% fetal bovine serum (FBS) at 37 °C. The particle sizes were measured at 0 h and 24 h. The values at each point represent the mean ± SD (n = 3).Figure 6. Transfection efficiencies and cell viabilities of LNPs incorporated with different amounts of COE-S6 tested at 24 h against (a) HEK293T, (b) HeLa, and (c) A549, respectively. The LNPs were formulated using an mRNA encoding firefly luciferase. Untreated cells and naked mRNA were used as the negative controls. Commercial Lipofectamine MessengerMAX was used as a positive control. Each value represents the mean ± SD (n = 6).Figure 7. In vitro transfection of HEK293T cells using LNPs formulated with mCherry mRNA. (a-c) Quantification of in vitro mCherry mRNA LNP transfection efficiencies, (d)Confocal micrographs of mCherry-expressing HEK293T cells at 24 h post-transfection. The nuclei were stained with SYTO Deep Red. Scale bar = 50 pm.Figure 8. The effect of COE-S6 pre-treatment on the transfection of HEK293T cells using LNPs formulated with mCherry mRNA. HEK293T cells were pre-treated with 1 pM COE-S6 for 1 h, washed, transfected with the pristine mRNA-LNP, and imaged after 24 h of incubation. The nuclei were stained with SYTO Deep Red. Scale bar = 50 pm.Figure 9. Cellular uptake and endosomal escape studies of the Cy5 mRNA LNPs. (a-c) Cellular uptake of Cy5 mRNA LNPs by HEK293T as assessed by flow cytometry, (d) Confocal micrographs of HEK293T cells after 24 h incubation with the Cy5 mRNA LNPs and the corresponding intensity profile plots. FLuc mRNA was labeled with Cy5. Endosomes / lysosomes were stained with lysotracker red. Scale bar = 10 pm. Each value represents the mean ± SD (n =3). (ns: not significant, *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001).Figure 10. The colocalization analyses of COE-S6 with the acidic endosomes / lysosomes and mRNA. The acidic endosomes / lysosomes were labeled with Lysotracker red and the mRNA was labeled with Cy5. The pseudocolors red, green, and blue were assigned to lysotracker, mRNA, and COE-S6, respectively. The Pearson's correlation coefficient value, R, was given by Image! using the plugin Coloc2. Scale bar = 10 pm.Figure 11. The two different methods to incorporate COEs into the LNP system, (a) In- situ incorporation involves the addition of the COE into the lipid mixture (ethanol phase) before mixing with the aqueous phase via various manufacturing methods and purification to form the LNPs. (b) Ex-situ incorporation proceeds with the addition of an aqueous COE solution to the purified LNPs before vortexing for uniform mixing.Figure 12. Particle characterizations of COE-S6-incorporated LNP systems. The LNPs were formulated using an in-house synthesized mRNA encoding mScarlet (1732 bases, 558.3 kDa) as a representative example, (a) Comparison of LNP particle sizes with 0.2% COE-S6 incorporated prepared via the two different methods, (b) Particle size distribution profiles, and (c) PDI of the particles. Neat LNP without any COE incorporated was used as the control (0%). Each value represents the mean ± SD (n = 3).Figure 13. Transfection efficiencies and cell viabilities of LNPs incorporated with 0.2% of COE-S6 prepared using the two different methods (Figure 11) and tested at 24 h against (a) HEK293T, (b) HeLa, and (c) A549, respectively. The LNPs were formulated using an mRNA encoding firefly luciferase. Untreated cells and naked mRNA were usedas the negative controls. Commercial Lipofectamine MessengerMAX was used as a positive control. Each value represents the mean ± SD (n = 6).Figure 14. The molecular structure of BO.Figure 15. Transfection efficiencies and cell viabilities of LNPs incorporated with different amounts of BO tested at 24 h against HEK293T cells. The LNPs were formulated using an mRNA encoding firefly luciferase. Untreated cells and naked mRNA were used as the negative controls. Commercial Lipofectamine MessengerMAX was used as a positive control. Each value represents the mean ± SD (n = 6).Figure 16. Photophysical and binding properties of the COE-S6 incorporated LNP system, (a) Normalized photoluminescence emission spectra of COE-S6 (1 pM) in different environments (IX PBS, 1 mM SUVs, and 1 mM mRNA-LNPs). (b) Photoluminescence decay profiles of COE-S6 (1 pM) in 1 mM SUVs and 1 mM mRNA- LNPs. (c) Normalized fluorescence anisotropy values of COE-S6 (1 pM) titrated against increasing concentrations of SUVs or mRNA-LNPs. The dissociation constant (Kd) was derived after fitting to a nonlinear regression curve, (d) The CD spectra of mRNA complexed with COE-S6 at different N / P ratios (0 to 2.5).Figure 17. In vivo delivery and translation efficiency of the mRNA-LNPs. (a) in vivo translation efficiency visualized using IVIS at different time points, (b) Quantification of bioluminescence values at different time points, (c) Organ biodistribution of the mRNA- LNPs at 6 hours postinjection.Figure 18. Body weight monitoring of mice across 15 days after treatment with mRNA- LNPs at an mRNA dosage of 0.1 mg / kg via intravenous administration. PBS was used as a negative control. Each value represents the mean ± SD (n = 3).Figure 19. Hematological parameters evaluated at 15 days post-treatment of mRNA- LNPs at an mRNA dosage of 0.1 mg / kg via intravenous administration. PBS was used as a negative control. Each value represents the mean ± SD (n = 3).Figure 20. Representative images of hematoxylin and eosin (H&E) stained tissue samples from different organs at 15 days post-treatment of mRNA-LNPs at an mRNA dosage of 0.1 mg / kg via intravenous administration. PBS was used as a negative control.Figure 21. Representative confocal micrographs of HEK293T cells at 24 hours posttransfection with Pfizer mRNA-LNPs encoding the full-length spike protein. The spike protein expression was probed with a secondary antibody conjugated with Alexa Fluor 647. The nuclei were stained with Nucspot® 555 / 570. Scale bar = 50 pm.Figure 22. Representative confocal micrographs of HEK293T cells at 24 hours posttransfection with Moderna mRNA-LNPs encoding the full-length spike protein. The spike protein expression was probed with a secondary antibody conjugated with Alexa Fluor 647. The nuclei were stained with Nucspot® 555 / 570. Scale bar = 50 pm.Figure 23. Representative confocal micrographs of HEK293T cells showing the expression of full-length spike protein between commercial lipofectamine, Pfizer mRNA- LNPs, and Moderna mRNA-LNPs. Scale bar = 50 pm.Figure 24. Representative confocal micrographs of primary cortisol neuronal cells at 24 hours post-transfection with Moderna mRNA-LNPs encoding mCherry protein. The nuclei were stained with SYTO Deep Red. Scale bar = 50 pm.Figure 25. Structures of additional COEs screened.Figure 26. Heatmap representation of the fold-changes in mean fluorescence intensity of emiRFP703 relative to pristine mRNA-LNP control. The LNP formulation from Moderna was used and the details of the reporter protein emiRFP703 are shown. Each point on the heatmap represent the mean value (n = 3).Figure 27. Western blot analysis of the expression levels of full-length spike protein for HEK293T cells post-transfection with respective mRNA-LNPs for 24 h. Untreated cells and lipofectamine were used as controls. GAPDH was used to normalize the protein concentration.Figure 28. Transfection efficiencies and mean fluorescence intensities at 24 h of the different cell lines (a, e) PC3, (b, f) LNCaP, (c, g) 22rvl, and (d, h) DU145 posttransfection with Moderna mRNA-LNPs encoding emiRFP703 as assessed by flow cytometry. Each value represents the mean + SEM (n = 3).Figure 29. Cell viability assays, (a) Cell viabilities of different cell lines at 24 h posttreatment with different concentrations of COE-S6. (b) Cell viabilities of different cell lines at 24 h post-treatment with different doses of emiRFP703 mRNA-LNPs. (c) Cell viabilities of different cell lines at 24 h post-treatment with different doses of PTEN mRNA-LNPs. (d) Real time cell viabilities of different cell lines at 24 h and 48 h posttreatment with 2.5 pg / mL of PTEN mRNA-LNPs. Each value represents the mean + SEM (n = 3).Figure 30. Western blot analysis of the expression levels of PTEN for the different cell lines post-transfection with respective mRNA-LNPs for 24 h. Untreated cells and lipofectamine were used as controls. GAPDH was used to normalize the protein concentration.Figure 31. Normalised band intensities from the Western Blot analysis for the different cell lines (a) 22rvl, (b) LNCaP, and (c) PC3.Figure 32. Apoptotic activity of the different cell lines (DU 145, 22rvl, and LNCaP) as assessed by flow cytometry at 24 h post-treatment with PTEN mRNA-LNPs. Each value represents the mean + SEM (n = 3).Figure 33. Representative confocal micrographs of PC3 cells at 24 h post-treatment with PTEN mRNA-LNPs. The nuclei were stained with SYTO Deep Red. Scale bar = 200 pm.Figure 34. Representative confocal micrographs and quantification of cell migration at 24 h post-treatment with PTEN mRNA-LNPs. The length of the wound was measured using ImageJ. The migration rate is given by subtracting the final length from the initial length, divided by the initial length and multiplying by 100%. Each value represents the mean + SEM (n = 3).Figure 35. Representative confocal micrographs of LNCaP spheroids at different day points post-treatment with PTEN mRNA-LNPs. Each value represents the mean + SEM (n = 3).Figure 36. Representative confocal micrographs of 22rvl spheroids at different day points post-treatment with PTEN mRNA-LNPs. Each value represents the mean + SEM (n = 3).Figure 37. Representative confocal micrographs of DU145 spheroids at different day points post-treatment with PTEN mRNA-LNPs. Each value represents the mean + SEM (n = 3).Detailed description"Alkyl" refers to monovalent alkyl groups which may be straight chained or branched and preferably have from 1 to 10 carbon atoms or more preferably 1 to 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, / so-propyl, n-butyl, isobutyl, n-hexyl, and the like."Alkenyl" refers to a monovalent alkenyl group which may be straight chained or branched and preferably have from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and have at least 1 and preferably from 1-2, carbon to carbon, double bonds. Examples include ethenyl (-CH=CH2), n-propenyl (-CH2CH=CH2), / so-propenyl (-C(CH3)=CH2), but-2-enyl (-CH2CH=CHCH3), and the like."Alkynyl" refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1, and preferably from 1-2, carbon to carbon, triple bonds. Examples of alkynyl groups include ethynyl (-C= CH), propargyl(-CH2CH CH), pent-2-ynyl (-CH2OCCH2-CH3), and the like."Alkoxy" refers to the group alkyl-O- where the alkyl group is as described above. Examples include, methoxy, ethoxy, n-propoxy, / so-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like."Alkenyloxy" refers to the group alkenyl-O- wherein the alkenyl group is as described above."Alkynyloxy" refers to the group alkynyl-O- wherein the alkynyl groups is as described above."Halo" or "halogen" refers to fluoro, chloro, bromo and iodo."Acyl" refers to groups H-C(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl- C(O)- and heterocyclyl-C(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein."Oxyacyl" refers to groups HOC(O)-, alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl-OC(O)-, and heterocyclyl-OC(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein."Amino" refers to the group -NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein."Aminoacyl" refers to the group -C(O)NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein."Acylamino" refers to the group -NR"C(O)R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein."Acyloxy" refers to the groups -OC(O)-alkyl, -OC(O)-aryl, -C(O)O-heteroaryl, and -C(O)O-heterocyclyl where alkyl, aryl, heteroaryl and heterocyclyl are as described herein."Aminoacyloxy" refers to the groups -OC(O)NR"-alkyl, -OC(O)NR"-aryl, -OC(O)NR"-heteroaryl, and -OC(O)NR"-heterocyclyl where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.'Cyano' refers to the group -CN."Oxyacylamino" refers to the groups -NR"C(O)O-alkyl, -NR"C(O)O-aryl, -NR"C(O)O-heteroaryl, and NR"C(O)O-heterocyclyl where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein."Oxyacyloxy" refers to the groups -OC(O)O-alkyl, -O-C(O)O-aryl, -OC(O)O- heteroaryl, and -OC(O)O-heterocyclyl where alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein."Thio" refers to groups H-S-, alkyl-S-, cycloalkyl-S-, aryl-S-, heteroaryl-S-, and heterocyclyl-S-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein."Phosphoryl" refers to the groups -P(O)(R'")(OR"") where R'" represents OR"" or is hydroxyl, alkyl or amino and R"" is alkyl, cycloalkyl, aryl or arylalkyl, where alkyl, amino, alkenyl, aryl, cycloalkyl, and arylalkyl are as described herein."Optionally substituted" is taken to mean that a group may or may not be further substituted or fused (so as to form a condensed polycyclic group) with one or more groups selected from hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl.alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, acylamino, cyano, halogen, nitro, phosphono, sulfo, phosphorylamino, phosphinyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, oxyacyl, oxime, oxime ether, hydrazone, oxyacylamino, oxysulfonylamino, aminoacyloxy, trihalomethyl, trialkylsilyl, pentafluoroethyl, trifluoromethoxy, difluoromethoxy, trifluoromethanethio, trifluoroethenyl, mono- and di-alkylamino, mono-and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclyl amino, and unsymmetric di-substituted amines having different substituents selected from alkyl, aryl, heteroaryl and heterocyclyl, and the like, and may also include a bond to a solid support material, (for example, substituted onto a polymer resin). For instance, an "optionally substituted amino" group may include amino acid and peptide residues.The present disclosure is predicated on the understanding that membrane-intercalating conjugated oligoelectrolytes (COEs) with a central hydrophobic conjugated backbone flanked by pendant cationic groups may be applied to lipid nanoparticles (LNPs). The LNPs may be loaded with therapeutic agents such as nucleic acids, proteins, or anionic drugs. By furnishing the particle surface with a positive zeta potential and fortifying the particle assembly via hydrophobic associations, the intercalated COEs contribute to both the colloidal and structural stability of the LNPs. Upon intercalation in the lipid layer, the unique bola-like structure of the COEs permits their interaction with the loaded payload within the core of the lipid nanoparticle, modulating particle size, facilitating payload release, and enhancing therapeutic efficacy (Figure 1). The present disclosure is expected to contribute to the field of nanomedicine concerning storage stability, shelf life and lowering dosage requirements.In particular, a representative COE molecule, COE-S6, was used to alter the intricate structures of mRNA-LNPs which has a range of distinct lipid phases, further defined by an interior aqueous core containing anionic mRNA. It is anticipated that upon membrane intercalation, the bola-like architecture of COE-S6 would interact with both the lipid membrane and the loaded mRNA molecules, thereby modulating the overall physical properties of the mRNA-LNPs.Further, in some embodiments, an ex situ method to "dope" COE-S6 into pre-formed mRNA-LNPs is disclosed. This method fully exploits its spontaneous membraneintercalation property to improve pre-formed mRNA-LNP characteristics through a straightforward add-and-mix procedure (Figure 3A). An optimized incorporation of 0.2% COE-S6 into mRNA-LNPs relative to total lipid contents resulted in remarkable enhancements across all relevant key metrics, including increased cellular uptake, improved endosomal escape, and ultimately leading to significantly higher in cellular transfection and in vivo translation efficiencies. This approach, combined with the vast molecular structural diversity of transmembrane-spanning COEs, could provide new options for developing more effective mRNA-LNPs.Conjugated oligoelectrolytes (COEs) are a class of synthetic water-soluble conjugated molecules containing a n-conjugated core and pendant ionic groups. COEs have a lipid bilayer-mimicking property. When the backbone of a polyphenylene vinylene structure having 6 phenyl units is about 3.4 nm, this is comparable to the thickness of a lipid bilayer (-4 nm). The terminals of the backbone structure are functionalised with hydrophilic groups, allowing interactions between the hydrophilic and hydrophobic moieties of the compounds with that of the phospholipids can be further driven. This electrostatic affinity and hydrophobic matching allows COEs to favourably intercalate within the lipid bilayers of cell membrane. Depending on their structural design, COEs exhibit a range of functionalities, such as stabilizing or disrupting bacterial membranes, boosting the capabilities of biohybrid electrodes, lighting up biological membranes for various downstream characterizations, enabling organelle-specific assay development, and permitting extended in vivo tumor monitoring.It was found that the mechanism of gene delivery enhancement by using the lipid nanoparticles of the present disclosure is unlike the cationic polythiophenes. In the case of cationic polythiophenes, an excitation light source is required to generate reactive oxygen species from the polythiophenes that facilitate both endolysosomal membrane disruption and transgene release. However, even in the absence of an excitation light source, the lipid nanoparticles comprising the conjugated oligoelectrolytes are able to achieve a significant enhancement in transfection efficiency as disclosed herein. Without wanting to be bound by theory, it is believed that the symmetrical bola-like molecular structure of the COEs is able to interact with the loaded nucleic acid upon intercalation. This interaction brings about two outcomes; reducing particle size, and facilitating payload release by bringing it closer to the particle surface.Further, the stabilizing effect of COEs on lipid nanoparticles was found to be more robust than the conventional cationic surfactants. The commonly used cationic surfactants have a single cationic group flanked by hydrophobic lipid tails. For example, the didodecyldimethylammonium bromide (DMAB) comprises a quarternary ammonium group flanked by two lipid tails. The cationic surfactants stabilize nanoparticles by imparting a positive surface zeta potential on the nanoparticles (causes charge repulsion between nanoparticles so minimizes aggregation). COEs intercalate into the lipid nanoparticles provide a positive surface zeta potential with the pendant ionic groups and additionally structural reinforcement via the hydrophobic conjugated backbone. The intercalated COEs, therefore, contribute to both the colloidal and structural stability of the nanoparticles.Accordingly, the present disclosure concerns a lipid nanoparticle, comprising : a) a helper lipid at about 5 mol% to about 80 mol% relative to a total lipid content; b) an ionisable lipid at about 20 mol% to about 95 mol% relative to the total lipid content; c) a conjugated oligoelectrolyte at about 0.005 mol% to about 5 mol% relative to the total lipid content, wherein the conjugated oligoelectrolyte is configured to interact with at least the helper lipid in order to stabilise the lipid nanoparticle.In some embodiments, the conjugated oligoelectrolyte is configured to interact with a payload within the lipid nanoparticle in order to stabilise the payload.The interaction or intercalation of COEs into at least the phospholipid layer of the LNPs improves their structural integrity via hydrophobic associations between the conjugated backbone of the COE and the lipid layer. This contributes to the colloidal stability of the nanoparticles such that the solution properties that directly affect therapeutic efficacy (e.g., particle size and zeta potential) can be retained in different environments. This may extend a shelf life of the LNPs and minimizes wastage of expired solutions. The small particle size of the LNPs regardless of the manufacturing method may increase cellular uptake and prolongs in vivo circulation time, both of which increase the bioavailability of payloads to target sites and thus efficacy. The LNPs may be store at higher temperatures (+8 to 20 °C) and may be lyophilized and stored as a dry formulation.Further, the unique bola-like structure of the COEs permits their interaction with the core of the lipid nanoparticle which may encapsulate a payload. The payload maybe anionic. This interaction is expected to bring about two outcomes: reducing particle size and facilitating payload release by bringing it closer to the particle surface. Both outcomes are expected to enhance the therapeutic efficacy of the LNPs thus allowing a lower dosage requirement and thus reduce costs. The shelf-life may also be extended.The COEs are fluorescent and their incorporation into the LNPs provides an opportunity to optically label the nanocarriers. The COEs can be structurally varied to achieve distinct emission wavelengths, enabling a wide range of options for theranostics. The ease of incorporation into LNPS (in- and ex-situ) eliminate the need for intricate training. As COEs are small molecules, batch-to- batch variation is avoid, which is commonly affiliated with polymers based LNPs.As used herein, "lipid nanoparticle" (LNP) is a nanoparticle composed of lipids. A lipid nanoparticle is typically spherical with an average diameter between 10 nm and 1000 nm. A monolayer of lipid encapsulates an interior or core. Lipid nanoparticle may have a liquid core or a solid core. Solid lipid nanoparticles possess a solid lipid core matrix that can solubilize lipophilic molecules. The lipid core is stabilized by surfactants (emulsifiers) which forms an external layer. The emulsifier used depends on administration routes and is more limited for parenteral administrations. The term lipid is used here in a broader sense and includes triglycerides (e.g. tristearin), diglycerides (e.g. glycerol bahenate), monoglycerides (e.g. glycerol monostearate), fatty acids (e.g. stearic acid), steroids (e.g. cholesterol), and waxes (e.g. cetyl palmitate). All classes of emulsifiers (with respect to charge and molecular weight) have been used to stabilize the lipid dispersion. A combination of emulsifiers may prevent particle agglomeration more efficiently. Biological membrane lipids such as phospholipids, sphingomyelins, bile salts (sodium taurocholate), and sterols (cholesterol) may also be utilized as stabilizers. Biological lipids having minimum carrier cytotoxicity and the solid state of the lipid permit better controlled drug release due to increased mass transfer resistance. LNPs used in mRNA vaccines for SARS-CoV-2 (the virus that causes COVID-19) are made of four types of lipids: an ionizable cationic lipid (whose positive charge binds to negatively charged mRNA), a PEGylated lipid (for stability), a helper lipid such as phospholipid (forstructure), and cholesterol (for structure). Because of rapid clearance by the immune system of the positively charged lipid, neutral ionizable amino lipids may be used.This is contrasted from liposomes, which are closed lipid bilayer vesicles that form spontaneously in water. Liposomes consist of one or several lipid bilayers, ranging in size between 20 and -1000 nm. Hydrophilic drugs can be enclosed in the aqueous interior of liposomes, while hydrophobic drugs can be entrapped in the hydrocarbon chain region of the lipid bilayer. The structures of liposomes depend strongly on how they are prepared. Liposomes may be either unilamellar (small unilamellar vesicles (SUV) with diameters of 20-100 nm, large unilamellar vesicles (LUV) with diameters of 100-1000 nm, or giant unilamellar vesicles (GUV) with diameters >1000 nm) or multilamellar vesicles (MLV), with diameters of >500 nm, in which concentric bilayers form an onion-like multilayer structure.In some embodiments, the conjugated oligoelectrolyte is intercalated with at least the helper lipid. In this regard, the conjugated oligoelectrolytes and the helper lipid forms an exterior lipid layer. COE is inserted within the helper lipid layer which forms the surface of the lipid nanoparticle. In some embodiments, the conjugated oligoelectrolyte is intercalated with the phospholipid and the ionisable lipid to form an exterior lipid layer.In some embodiments, the conjugated oligoelectrolytes interacts with a payload in the core of the lipid nanoparticle (if present). The conjugated oligoelectrolytes may interact with the payload via electrostatic interaction due to their pendant ionic groups.In some embodiments, the helper lipid is about 5 mol% to about 80 mol% relative to a total lipid content of the lipid nanoparticle. The total lipid content refers to a sum of all the lipids used (and COE if present) when forming the lipid nanoparticle. In some embodiments, the helper lipid is about 5 mol% to about 75 mol%, about 5 mol% to about 70 mol%, about 5 mol% to about 65 mol%, about 5 mol% to about 60 mol%, about 5 mol% to about 55 mol%, about 5 mol% to about 50 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 40 mol%, about 5 mol% to about 35 mol%, about 5 mol% to about 30 mol%, about 5 mol% to about 25 mol%, about 5 mol% to about 20 mol%, about 5 mol% to about 15 mol%, or about 5 mol% to about 10 mol%.In some embodiments, the helper lipid is about 10 mol% relative to the total lipid content.Helper lipids contribute to the stability and delivery efficiency of lipid nanoparticles. Helper lipids with cone-shape geometry favoring the formation hexagonal II phase, such as dioleoylphosphatidylethanolamine (DOPE), can promote endosomal release of ONs. Meanwhile, cylindrical-shaped lipid phosphatidylcholine can provide greater layer stability, which is important for in vivo application of lipid nanoparticles.The helper lipid may be a phospholipid. Phospholipids are a class of lipids whose molecule has a hydrophilic "head" containing a phosphate group and two hydrophobic "tails" derived from fatty acids, joined by an alcohol residue (usually a glycerol molecule). The phosphate group may be modified with simple organic molecules such as choline, ethanolamine or serine. In some embodiments, the lipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol.In some embodiments, the helper lipid is a phosphatidylcholine. In some embodiments, the helper lipid is selected from l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1.2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l,2-Dimyristoyl-sn-glycero-3- phosphocholine (DMPC), l-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or a combination thereof.In some embodiments, the helper lipid is a phophatidylethanolamine. For example, the helper lipid may be l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).In some embodiments, the ionisable lipid is about 20 mol% to about 95 mol% relative to the total lipid content. In other embodiments, the ionisable lipid is about 25 mol% to about 95 mol%, about 30 mol% to about 95 mol%, about 35 mol% to about 95 mol%, about 40 mol% to about 95 mol%, about 40 mol% to about 90 mol%, about 40 mol% to about 85 mol%, about 40 mol% to about 80 mol%, about 40 mol% to about 75 mol%, about 40 mol% to about 70 mol%, about 40 mol% to about 65 mol%, about 40 mol° / o to about 60 mol%, about 40 mol% to about 55 mol%, or about 40 mol% to about 50 mol%. In other embodiments, the ionisable lipid is about 50 mol% relative to the total lipid content.Ionizable lipids are capable of modulating their charge depending on the environmental pH. For example, the lipid may remain neutral at physiological pH, but are protonated at low pH, making it positively charged. In some embodiments, the ionisable lipid is ionisable into a cationic lipid. In some embodiments, the ionisable lipid is selected from (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), and [(4-Hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), 9Z,12Z-octadecadienoic acid, 3-[4,4-bis(octyloxy)- l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester (LP01), and a combination thereof. The structure of SM-102 isOther ionisable lipids that may be used, but not limited to, are:Other examples of cationic and / or ionisable lipids may be found in ACS Nano. 2021, 15(11), 16982-17015, the reference of which is incorporated herein.In some embodiments, the conjugated oligoelectrolyte is about 0.01 mol% to about 5 mol%, or about 0.02 mol% to about 5 mol% relative to the total lipid content. In some embodiments, the conjugated oligoelectrolyte is about 0.02 mol% to about 4.8 mol%, about 0.02 mol% to about 4.6 mol%, about 0.02 mol% to about 4.4 mol%, about 0.02 mol% to about 4.2 mol%, about 0.02 mol% to about 4 mol%, about 0.02 mol% to about 3.8 mol%, about 0.02 mol% to about 3.6 mol%, about 0.02 mol% to about 3.4 mol%, about 0.02 mol% to about 3.2 mol%, about 0.02 mol% to about 3 mol%, about 0.02 mol% to about 2.8 mol%, about 0.02 mol% to about 2.6 mol%, about 0.02 mol%to about 2.4 mol%, about 0.02 mol% to about 2.2 mol%, or about 0.02 mol% to about 2 mol%.In some embodiments, the COE is a compound of Formula (I) or a salt or solvate thereof:whereinRi is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy or optionally substituted thio or optionally substituted phosphoryl;R2 is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; m is an integer selected from 1 to 7; n is an integer independently selected from 0 to 4; and q is an integer independently selected from 1 to 5.The backbone oligophenylene vinylene should be preferably in a linear configuration; i.e. E (trans) configuration.In some embodiments, m is an integer selected from 3 to 7. In other embodiments, m is an integer selected from 4 to 7, or 4 to 6. In other embodiments, m is 3, 4, 5, 6 or 7. In some embodiments, m is 4, 5 or 6. In other embodiments, m is 4.In some embodiments, n is an integer independently selected from 0 to 3, 0 to 2, or 0 to 1.In some embodiments, Ri is independently selected from optionally substituted alkyl, optionally substituted alkoxy, optionally substituted acyl, optionally substituted oxyacyl,optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted oxyacyloxy or optionally substituted thio or optionally substituted phosphoryl. In other embodiments, Ri is optionally substituted alkoxy, optionally substituted oxyacyl or optionally substituted amino. In another embodiment, Ri is optionally substituted polyethoxy, wherein the monomeric unit is from 3 to 10. In other embodiments, the chain length of Ri is from about 3 to about 10. In other embodiments, Ri is optionally substituted C3- C10 alkoxy, optionally substituted C3-C10 alkylamino, optionally substituted C3-C10 dialkylamino, optionally substituted C3-C10 alkyloxyacyl or optionally substituted polyethoxy.In some embodiments, the optional substituent at Ri is independently selected from oxy, oxyacyl, acyl, amino, phosphoryl, thiol, alkyl, alkenyl, alkynyl, oxyalkyl, alkylacyloxy, sulfonyl, chlorate or its charged species thereof. In some embodiments, the optional substituent at Ri is independently selected from hydroxyl, carboxyl, phosphate, amino, alkylamino, dialkylamino, chlorate, sulphate, acetate or its charged species thereof. In some embodiments, the optional substituent at Ri is a tertiary amino. The tertiary amino may be neutralised by a counterion, which can be a halide.In other embodiments, the optional substituent at Ri is a hydrophilic moiety. In other embodiments, the optional substituent at Ri is a charged moiety. Examples of hydrophilic and / or charged moieties are trialkylammonium halide. For example, the charged moiety can be trimethylammonium iodide. In this embodiment, Ri terminates with trimethylammonium, and thereby imparts a positive charge when substituted to Ri (for example, alkyl). Other cationic charged groups include but are not limited to pyridinium, pyrrolidinium, imidazolium, guanidinium, sulfonium, thiouronium, and phosphonium. Other anionic charged groups include but not limited to chlorate, sulphate, phosphate, acetate, carboxyl, hydroxide. The hydrophilic and / or charged moieties can also in zwitterionic form that contains both cationic and anionic charged groups through covalent bonds. The excess charges can be neutralized by acceptable cations or anions.Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.Examples of the optional substituents on Ri can be selected from:In some embodiments, R2 is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl. In other embodiments, R2 is independently selected from halogen, cyano or optionally substituted alkyl. In other embodiments, R2 is independently selected from halogen, cyano, methyl, ethyl or propyl.In some embodiments, q is an integer from 1 to 4. In other embodiments, q is an integer from 1 to 3. In this regard, there are on compound of Formula (I), a total of at least 2 Ri groups, at least 3 Ri groups, at least 4 Ri groups, at least 5 Ri groups or at least 6 Ri groups.For compound of Formula (I) to maintain its linear configuration, Ri are preferentially positioned at the meta and / or para positions of the terminus phenyl groups. In some embodiments, Ri is present at the meta and para positions of the terminus phenyl groups. In other embodiments, Ri is present at the meta or para positions of the terminus phenyl groups.Examples of compound of Formula (I) or a salt or solvate are as follows (List 1) :In some embodiments, the COE is selected from COE-S6.In some embodiments, the COE is compound of Formula (II) or a salt or solvate thereof:wherein each Ri is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy or optionally substituted thio or optionally substituted phosphoryl; q is an integer selected from 1 to 5; q' is an integer selected from 1 to 5; wherein each l_2 is independently selected from optionally substituted ethylene, or optionally substituted phenylethylene;Li is a n-conjugated core comprising monomeric unit A and monomeric unit D:wherein each A is independently selected from optionally substituted alkenylene, optionally substituted arylene or optionally substituted heteroarylene; each D is independently selected from optionally substituted alkenylene, optionally substituted arylene or optionally substituted heteroarylene; t is an integer selected from 1 to 5; u is an integer selected from 1 to 5; wherein * represents a bond to another monomeric unit or to L2; wherein monomeric units A and monomeric units D are alternatively bonded to each other; wherein the compound of Formula (I) has a substantially linear topology.In some embodiments, A and D are not both alkenylene. In other embodiments, A and D are not both phenylene. In other embodiments, A and D are not alkenylene andphenylene. In some embodiments, Li is not butadienylene, polyalkenylene, phenylalkenylene and polyphenylalkenylene. In some embodiments, each A is independently selected from alkenylene substituted with cyano, optionally substituted monocyclic heteroarylene or optionally substituted fused heteroarylene. In some embodiments, each D is independently selected from alkenylene, phenylene, optionally substituted fused arylene, optionally substituted monocyclic heteroarylene or optionally substituted fused heteroarylene.In some embodiments, the bonds connecting monomeric units A and monomeric units D in Li are substantially aligned along a longitudinal axis of the compound. In other embodiments, the monomeric units A and monomeric units D in Li are substantially aligned along a longitudinal axis of the compound. In this regard, bonds connecting monomeric units A and monomeric units D when offset from the longitudinal axis of the compound are within the scope of the invention.The compounds of Formula (I) are linear in order to accommodate its position within the lipid bilayer. In some embodiments, the compounds are not branched; i.e. the monomeric units only extend along a single chain. In some embodiments, the compounds of Formula (I) are symmetrical in nature. In this regard, in some embodiments, the compounds of Formula (I) have a C2v point group.In some embodiments, L2 is independently selected from optionally substituted ethylene, or optionally substituted phenylethylene. In other embodiments, L2 is independently selected from:wherein * represents a bond to a monomeric unit and to a terminal phenyl moiety in compound of Formula (I).Li is a n-conjugated core. A conjugated system is a system of connected p orbitals with delocalized electrons in a molecule, which in general lowers the overall energy of the molecule and increases stability. Lone pairs, radicals or carbenium ions may be part of the system, which may be cyclic, acyclic, linear or mixed.In some embodiments, when Li comprises 6 membered aryl or heteroaryl, or when Licomprises fused aryl or heteroaryl having a 6 membered ring, the monomeric units are 1,4 conjugated on the 6 membered ring. In other embodiments, when Li comprises 5 membered aryl or heteroaryl, or when Li comprises fused aryl or heteroaryl having a 5 membered ring, the monomeric units are 1,4 conjugated or 2,5 conjugated on the 5 membered ring.In some embodiments, Li is selected from:As mentioned, each A and D can be the same moiety such that Li is an alternating n- conjugated core. Alternatively, each A and D can be different. As Li comprises an alternating donor / acceptor composition of structural units (relative to each other), structures in which the alternating donor / acceptor composition is not adhered to are excluded from the scope of this invention. For example, butadienylene, polyalkenylene, phenylalkenylene and polyphenylalkenylene are excluded.In some embodiments, the optional substituent on Li is selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, and optionally substituted alkynyloxy. In other embodiments, the optional substituent on D is selected from halogen, cyano, alkyl, alkenyl, alkoxy, and alkenyloxy. In some embodiments, the optional substituent on Li is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl.As used herein, monomeric unit D is an electron donating (rich) moiety relative to monomeric unit A. In this regard, monomeric unit A is an electron withdrawing / accepting (poor) moiety. When in sequence the D-A combination gives rise to intramolecular charge transfer excited states with optical absorption and emission further into the red, as compared to a sequence of similar moieties (-Dn- or -An-) in conjugation.In some embodiments, A is an electron accepting moiety. An electron acceptor is achemical entity that accepts electrons transferred to it from another moiety or compound. In some embodiments, A has electron accepting substituents. In other embodiments, A has electron withdrawing substitutents.In some embodiments, A is independently selected from optionally substituted alkenylene or optionally substituted heteroarylene. In some embodiments, A is independently selected from cyano substituted alkenylene or optionally substituted heteroarylene. In some embodiments, the cyano substituted alkenylene is monosubstituted alkenylene or di-substituted alkenylene. In other embodiments, the optionally substituted heteroarylene is optionally substituted monocyclic heteroarylene or optionally substituted fused heteroarylene. The heteroarylene can be 5 membered heteroarylene or a 6 membered heteroarylene. The heteroarylene can be a fused heteroarylene. In some embodiments, the heteroarylene is a fused 5,5 membered heteroarylene, fused 5,6 membered heteroarylene, fused 6,6 membered heteroarylene, fused 5,5,6 membered heteroarylene, fused 5,6,6 membered heteroarylene, fused 6,6,6 membered heteroarylene, fused 5, 5, 6, 6 membered heteroarylene, fused 5, 6, 6, 6 membered heteroarylene, or fused 6, 6, 6, 6 membered heteroarylene.In some embodiments, the optional substituent on A is selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, and optionally substituted alkynyloxy. In other embodiments, the optional substituent on A is selected from halogen, cyano, alkyl, alkenyl, alkoxy, and alkenyloxy.In some embodiments, A is selected fromwherein represents a bond to D or to L2; each Xi is independently selected from C, O, N, S and Se; each X2 if present is independently selected from C, O, N, S and Se; when X2 is present, at least one of Xi and X2 is 0, N or S;R is independenly selected from H, halo, cyano, and optionally substituted alkyl.In some embodiments, A is a moiety of Formula (III) :wherein represents a bond to D or to L2;R2, R3, R4 and Rs are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R2 and Rs are linked to form optionally substituted heterocyclyl, optionally substituted heteroaryl; orR4 and Rs are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl.In some embodiments, R2 and Rs are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl. In other embodiments, R2 and Ra are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl. In other embodiments, R2 and R3 are independently selected from H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted Ca-Cs alkenyl, optionally substituted Ci-Cs alkoxy, and optionally substituted C2-C6 alkenyloxy. In other embodiments, R2 and Rs are independently selected from H, halogen, and Ci-Cs alkyl.In some embodiments, R2 and Rs are linked to form optionally substituted heteroaryl such that it forms a conjugated n system with the phenyl moiety.In some embodiments, R4 and Rs are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl. In other embodiments, R4 and Rs are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl. In other embodiments, R4 and Rs are independently selected from H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted Ci-Ce alkoxy, and optionally substituted C2-C6 alkenyloxy. In other embodiments, R4 and Rs are independently selected from H, halogen, and Ci-Cs alkyl.In some embodiments, R4 and Rs are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl. In other embodiments, R4 and Rs are linked to form optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl. In some embodiments, R4 and Rsare linked to form optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, R4 and Rs are linked to form optionally substituted heteroaryl such that it forms a conjugated n system with the phenyl moiety.In some embodiments,, wherein R4 and Rs are as disclosed herein.In some embodiments,, wherein R4 and Rs are as disclosed herein.,In some embodiments, t is an integer selected from 1 to 4, 1 to 3, 1 to 2, 2 to 4, 3 to 4 or 3 to 5.In some embodiments, D is an electron donating moiety. An electron donor is a chemical entity that donates electrons transferred from it to another moiety or compound. In some embodiments, D has electron donating substituents.In some embodiments, D is independently selected from optionally substituted alkenylene, optionally substituted arylene or optionally substituted heteroarylene. In some embodiments, D is independently selected from alkenylene, arylene or optionally substituted heteroarylene. In other embodiments, the arylene is phenylene. In other embodiments, the optionally substituted heteroarylene is optionally substituted monocyclic heteroarylene or optionally substituted fused heteroarylene. The heteroarylene can be 5 membered heteroarylene or a 6 membered heteroarylene. The heteroarylene can be a fused heteroarylene. In some embodiments, the heteroarylene is a fused 5,5 membered heteroarylene, fused 5,6 membered heteroarylene, fused 6,6 membered heteroarylene, fused 5,5,6 membered heteroarylene, fused 5,6,6 membered heteroarylene, fused 6,6,6 membered heteroarylene, fused 5, 5, 6, 6 membered heteroarylene, fused 5, 6, 6, 6 membered heteroarylene, or fused 6, 6, 6, 6 membered heteroarylene.In some embodiments, the optional substituent on D is selected from halogen, cyano,optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, and optionally substituted alkynyloxy. In other embodiments, the optional substituent on D is selected from halogen, cyano, alkyl, alkenyl, alkoxy, and alkenyloxy.In some embodiments, D is a moiety selected fromwherein ''' represents a bond to A or to L2; each Xi is independently selected from C, O, N, S, and Se; each X2 if present is independently selected from C, O, N, S and Se; when X2 is present, at least one of Xi and X2 is 0, N or S;R is independently selected from H, halo, cyano, and optionally substituted alkyl.In some embodiments, D is an optionally substituted 5 membered heteroarylene.In some embodiments, D is a moiety of Formula (IV):wherein Y is NR, O, S, or Se;Re and R? are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; orRe and R? are linked to form optionally substituted cycloalkyl, optionally substitutedcycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl; andR is selected from H, halo, cyano, and optionally substituted alkyl.In some embodiments, Rs and R? are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkoxy, or optionally substituted alkenyloxy. In other embodiments, Rs and R? are independently selected from H, halogen, optionally substituted alkyl, or optionally substituted alkoxy. In other embodiments, Rs and R? are independently selected from H, halogen, optionally substituted Ci-Cs alkyl, optionally substituted C2-C6 alkenyl, optionally substituted Ci- Cs alkoxy, or optionally substituted C2-C6 alkenyloxy. In other embodiments, Rs and R7 are independently selected from H, halogen, optionally substituted Ci-Cs alkyl, or optionally substituted Ci-Cs alkoxy. In other embodiments, Rs and R? are independently selected from H, halogen, or optionally substituted Ci-Cs alkyl.In some embodiments, Rs and R7 are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl. In other embodiments, Rs and R? are linked to form optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.In some embodiments, D is, wherein Rs and R7 are as disclosed herein.In some embodiments,In some embodiments, u is an integer selected from 1 to 4, 1 to 3, 1 to 2, 2 to 4, 3 to 4, or 3 to 5.In some embodiments, t and u together is at least 3. In other embodiments, t and u together is at least 4 or 5.In some embodiments, the COE or salt thereof is selected from List 2:The lipid layer of the lipid nanoparticle substantially comprises at least the helper lipid, and preferably also the ionisable lipid. Other components may also be present. In some embodiments, the lipid layer further comprises a steroid. The steroid may be cholesterol, fecosterol, sitosterol, ergosterol, campersterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, or a derivative thereof. In some embodiments, the steroid is cholesterol or a derivative thereof.In some embodiments, the steroid is about 20 mol% to about 60 mol% relative to the total lipid content. In other embodiments, the steroid is about 20 mol% to about 55 mol%, about 20 mol% to about 50 mol%, about 25 mol% to about 50 mol%, about 30 mol% to about 50 mol%, about 30 mol% to about 48 mol%, about 30 mol% to about 46 mol%, about 30 mol% to about 44 mol%, about 30 mol% to about 42 mol%, or about 30 mol% to about 40 mol%. In other embodiments, the steroid is about 38 mol% to about 39 mol%.In some embodiments, the lipid layer further comprises a polymer conjugated lipid. The polymer may be polyethylene glycol (PEG). The polymer conjugated lipid may be a PEGylated 1,2-dimyristoyl-sn-glycerol (PEG2000-C-DMG), wherein the PEG has a molecular weight of 2000 g / mol. Alternatively, the polymer conjugated lipid may be PEGylated l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG2000).The polymer may be polysarcosine. Polysarcosine is composed of repeated units of the natural amino acid sarcosine (N-methylglycine) and is biodegradable. In some embodiments, the polysarcosine conjugated lipid comprises a polysarcosine having a formulawherein R H, a hydrophilic group or a functional group optionally comprising a targeting moiety; and x is a integer between 2 and 200.In some embodiments, the polysarcosine conjugated lipid is selected from the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate, and a mixture thereof.In some embodiments, the polysarcosine conjugated lipid isIn some embodiments, the polymer conjugated lipid is about 0.5 mol% to about 2 mol% relative to the lipid nanoparticle. In some embodiments, the polymer conjugated lipid isabout 0.6 mol% to about 2 mol%, about 0.7 mol% to about 2 mol%, about 0.8 mol% to about 2 mol%, about 0.9 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 1.1 mol% to about 2 mol%, about 1.2 mol% to about 2 mol%, about 1.3 mol% to about 2 mol%, about 1.4 mol% to about 2 mol%, or about 1.5 mol% to about 2 mol%. In some embodiments, the polymer conjugated lipid is about 1.5 mol%.In some embodiments, the lipid nanoparticle is characterised by an average particle size of about 50 nm to about 250 nm. In some embodiments, the average particle size is about 50 nm to about 200 nm, about 50 nm to about 180 nm, about 50 nm to about 160 nm, about 50 nm to about 140 nm, about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 50 nm to about 80 nm, about 50 nm to about 75 nm, about 50 nm to about 70 nm, or about 50 nm to about 65 nm.In some embodiments, the lipid nanoparticle is characterised by a photoluminescence lifetime of at least 0.05 ns. This corresponds to roughly 5 % quantum yield. In some embodiments, the photoluminescence lifetime is at least 0.1 ns, 0.2 ns, 0.3 ns, 0.4 ns, 0.5 ns, 0.6 ns, 0.7 ns, 0.6 ns, 0.8 ns, 0.9 ns, 1 ns, 1.1 ns, or 1.2 ns.In some embodiments, the lipid nanoparticle is characterised by a photoluminescence fold increase of at least 2 relative to free conjugated oligoelectrolyte. In some embodiments, the lipid nanoparticle is characterised by a photoluminescence fold increase of at least 2.5 relative to free conjugated oligoelectrolyte. In some embodiments, the lipid nanoparticle is characterised by a photoluminescence fold increase of at least 2.8 relative to free conjugated oligoelectrolyte.The lipid nanoparticle may further comprise a payload in the cavity of the lipid nanoparticle. In this regard, the lipid nanoparticle serves to carry and protect a payload. The payload may be characterised by an anionic charge. In some embodiments, the payload is selected from a nucleic acid, a drug, a protein or a combination thereof. In some embodiments, the payload is selected from mRNA, nucleoside-modified mRNA, plasmid DNA, DNA, or a combination thereof.In some embodiments, the payload is selected from:In some embodiments, the payload is patisiran. Patisiran is sold under the brand name Onpattro, is a medication used for the treatment of polyneuropathy in people with hereditary transthyretin-mediated amyloidosis, a fatal rare disease that is estimated to affect 50,000 people worldwide. It is the first small interfering RNA-based drug approved by the U.S. Food and Drug Administration (FDA) and the first drug approved by the FDA to treat this condition. It is a gene silencing drug that interferes with the production of an abnormal form of transthyretin.In some embodiments, the lipid nanoparticle is characterised by a payload encapsulation efficiency of more than about 40%. In some embodiments, the lipid nanoparticle is characterised by a payload encapsulation efficiency of more than about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, or about 94%.In some embodiments, the lipid nanoparticle is characterised by a nucleic acid encapsulation efficiency of more than about 40%. In some embodiments, the lipid nanoparticle is characterised by a nucleic acid encapsulation efficiency of more than about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, or about 94%.Characteristically, the fluorescence emission of COEs enhance significantly upon their intercalation into lipid layers from the aqueous solution. This "light up" mechanism confers a high signal-to-noise ratio for COEs when they are localized within the more hydrophobic environment of lipid layers. Additionally, COEs have a distinct chemical structure from many commercially available lipophilic dyes, which usually contain a surfactant-like structure, i.e., one side of the molecule is hydrophobic, and the other side is hydrophilic. These surfactant-like structures will induce micelle-like aggregation in the aqueous solutions. For example, the commonly used membrane dye, PKH-26, has been shown to form aggregates, which have a similar size and fluorescence intensity compared to small particles such as the exosomes, thereby leading to false-positive signals. These phenomena can be avoided in the case of COEs given that their emission has been shown to greatly intensify after intercalation into the lipid layer.The present disclosure also concerns a method of detecting a lipid nanoparticle as disclosed herein in a cell, comprising imaging the cell using a fluorescence detector.As the lipid nanoparticle comprising COE emits fluorescence, fluorescence based techniques can be used to detect cells incorporated with the lipid nanoparticles. Examples of fluorescence based techniques include, but is not limited to, fluorescence microscopy, confocal microscopy, plate readers, fluorometer, fluorescence spectroscopy, and flow cytometry (such as fluorescence activated cell sorting).For example, the cell may be excited by an electromagnetic radiation (source) in a wavelength range of about 300 nm to about 1000 nm. By using different types of COE, the excitation may be tunable within this wavelength range. The fluorescence emission may be in the wavelength of about 300 nm to about 2000 nm, and may also be tunable.In some embodiments, the method is a method of tracking the lipid nanoparticle. Accordingly, the method further comprises imaging the cell at multiple time points in order to track the movement of the lipid nanoparticle.In some embodiments, the lipid nanoparticle further comprises a dye. The dye may be for staining lysosome or other components in the cell. The dye can be used to stain cell membranes, nucleus, DNA, RNA, or other organelles in the cell. The dye can be a fluorescence probe, such as FM4-64, FM 2-10, FM 1-43, Propidium Iodide, SYTO 82, SYTO 83, SYTO 84, SYTO 85, YOYO®-3 iodide, YO-PRO™-3 Iodide, BOBO™-3 Iodide, Ethidium Homodimer-1, Ethidium Homodimer-2, Ethidium monoazide, Acridine Orange, CellMask™ Plasma Membrane Stains or Di-4-ANEPPS.In some embodiments, the lipid nanoparticle is characterised by a COE to polynucleotide nitrogen-to-phosphate (N / P) ratio of less than 2. In some embodiments, the N / P ratio is less than 1.8, 1.6, 1.4, 1.2, 1, 0.9, 0.8, 0.7, 0.6 or 0.5. In some embodiments, the N / P ratio is about 0.1 to about 1, about 0.1 to about 0.9, about 0.1 to about 0.8, about 0.1 to about 0.7, about 0.1 to about 0.6, or about 0.1 to about 0.5.In some embodiments, the lipid nanoparticle is characterised by a total N / P ratio of more than 0.5. This ratio also includes the nitrogen group from the ionizable lipid. In some embodiments, the N / P ratio is more than 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, or 2.The present disclosure also concerns a method of detecting a lipid nanoparticle as disclosed herein in a subject in need thereof, imaging the subject using a fluorescence detector.The present disclosure also concerns a method of fabricating a lipid nanoparticle, comprising :a) adding a conjugated oligoelectrolyte to a precursor lipid nanoparticle to form the lipid nanoparticle; wherein the precursor lipid nanoparticle comprises a helper lipid and an ionisable lipid; wherein the helper lipid is about 5 mol% to about 80 mol% relative to the lipid nanoparticle; wherein the ionisable lipid is about 20 mol% to about 95 mol% relative to the lipid nanoparticle; and wherein the conjugated oligoelectrolyte is about 0.005 mol% to about 5 mol% relative to the lipid nanoparticle, wherein the conjugated oligoelectrolyte is configured to interact with the helper lipid in order to stabilise the lipid nanoparticle.In some embodiments, the method further comprises a step before step a) of mixing the helper lipid and ionisable lipid in a solvent to form a mixture and introducing the mixture into a non-solvent in order to fabricate the precursor lipid nanoparticle.The present disclosure also concerns a method of fabricating a lipid nanoparticle, comprising : a) mixing a helper lipid and an ionisable lipid in a solvent to form a mixture; b) introducing the mixture of step a) into a non-solvent in order to fabricate a precursor lipid nanoparticle; and c) adding a conjugated oligoelectrolyte to the precursor lipid nanoparticle of step b) to form the lipid nanoparticle; wherein the lipid is about 5 mol% to about 80 mol% relative to the mixture; wherein the ionisable lipid is about 20 mol% to about 95 mol% relative to the mixture; and wherein the conjugated oligoelectrolyte is about 0.005 mol% to about 5 mol% relative to the mixture, wherein the conjugated oligoelectrolyte is configured to interact with the helper lipid in order to stabilise the lipid nanoparticle and to interact with a payload in order to stabilise the payload.The present disclosure also concerns a method of fabricating a lipid nanoparticle, comprising :a) mixing a helper lipid, an ionisable lipid and a conjugated oligoelectrolytes in a solvent to form a mixture; b) introducing the mixture of step a) into a non-solvent in order to fabricate a lipid nanoparticle; and wherein the lipid is about 5 mol% to about 80 mol% relative to the lipid nanoparticle; wherein the ionisable lipid is about 20 mol% to about 95 mol% relative to the lipid nanoparticle; and wherein the conjugated oligoelectrolyte is about 0.005 mol% to about 5 mol% relative to the lipid nanoparticle, wherein the conjugated oligoelectrolyte is configured to interact with the helper lipid in order to stabilise the lipid nanoparticle.In some embodiments, the conjugated oligoelectrolyte is configured to interact with a payload in order to stabilise the payload.The solvent may be an organic solvent. Such solvents can be either polar or non-polar, and / or either protic or aprotic. Solvent systems refer to combinations of solvents which resulting in a final single phase. Both 'solvents' and 'solvent systems' can include, and is not limited to, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, dioxane, chloroform, diethylether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, nitromethane, propylene carbonate, formic acid, butanol, isopropanol, propanol, ethanol, methanol, acetic acid, ethylene glycol, diethylene glycol or water.The non-solvent induces the lipids to phase separate, and thus agglomerate to form lipid nanoparticles.The present disclosure also concerns a lipid nanoparticle composition, comprising a lipid nanoparticle and at least one excipient. Excipients include any and all solvents, dispersion media, inert diluents, or other liquid vehicles, dispersion or suspension aids, granulating agents, surface active agents, disintegrating agents, isotonic agents, thickening or emulsifying agents, preservatives, binding agents, lubricants, buffering agents, oils, and the like, as suited to the particular dosage form desired or as approved by any regulatory body. Various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof is disclosed in G. A. R.Remington : The Science and Practice of Pharmacy, 21st ed. (2006), Lippincott Williams & Wilkins. Except insofar as any conventional excipient is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.For example, cryoprotectants, such as sucrose, may be added as an excipient. This allows the lipid nanoparticle composition to be freeze-dried without damaging the lipid nanoparticle' structure. The cryoprotectant may be added at about 0.1 wt% to about 30 wt%.The present disclosure also concerns a kit for forming a lipid nanoparticle, comprising : a) a helper lipid at about 5 mol% to about 80 mol% relative to a total lipid content; b) an ionisable lipid at about 20 mol% to about 95 mol% relative to the total lipid content; c) a conjugated oligoelectrolyte at about 0.005 mol% to about 5 mol% relative to the total lipid content, the conjugated oligoelectrolyte for interacting with the helper lipid in order to stabilise the lipid nanoparticle and interacting with a payload in order to stabilise the payload.The kit may comprise an exact amount of lipid, ionisable lipid and conjugated oligoelectrolyte, such that a user may mix these components together to fabricate the lipid nanoparticle. The kit may comprise lyophilised lipid and / or lyophilised ionisable lipid. The kit may further comprise a solvent and optionally a non-solvent for fabricating the lipid nanoparticles. The kit may further comprise excipients for stabilising the lipid nanoparticle during synthesis. For example, the kit may comprise a cryoprotectant such as sucrose so that the as formed lipid nanoparticle in solution may be freeze dried. The kit may further comprise instructions for use to fabricate the lipid nanoparticles. The instructions can comprise the method as disclosed herein.The present disclosure also concerns a method of stabilising a lipid nanoparticle formulation, comprising mixing a conjugated oligoelectrolyte with the lipid nanoparticle formulation, wherein the conjugated oligoelectrolyte is about 0.005 mol% to about 5 mol% relative to the lipid nanoparticle formulation, wherein the conjugated oligoelectrolyte is configured to interact with a helper lipid of the lipid nanoparticleformulation in order to stabilise the lipid nanoparticle. In some embodiments, the conjugated oligoelectrolytes is configured to interact with a payload in order to stabilise the payload.Accordingly, the conjugated oligoelectrolyte may be used to stabilise commercially available products, such as Comirnaty and Spikevax. For example, Comirnaty comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate), 2-(polyethylene glycol 2000)-N,N-ditetradecylacetamide, l,2-distearoyl-sn-glycero-3- phosphocholine, and cholesterol. The lipid nanoparticle of the present disclosure thus comprises: a) ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate); b) 2-(polyethylene glycol 2000)-N,N-ditetradecylacetamide; c) l,2-distearoyl-sn-glycero-3-phosphocholine; d) cholesterol; and e) conjugated oligoelectrolyte.The present disclosure also concerns a method of delivering a payload to a cell, comprising incubating the cell with a lipid nanoparticle as disclosed herein.In some embodiments, the method is characterised by a cell viability more than about 50%. In some embodiments, the method is characterised by a cell viability more than about 60%, about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, or about 90%.In some embodiments, the lipid nanoparticle is characterised by an in vitro delivery efficiency of more than about 5%. In some embodiments, the lipid nanoparticle is characterised by a delivery efficiency of more than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90%.In some embodiments, when the payload is a nucleic acid, the method is characterised by a transfection efficiency more than 25% relative to a lipid nanoparticle without the conjugated oligoelectrolyte. In some embodiments, the lipid nanoparticle is characterised by a transfection efficiency of more than about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90%.The present disclosure also concerns a method of delivering a therapeutic payload to a subject in need thereof, comprising administering a therapeutic amount of lipid nanoparticle as disclosed herein to the subject, wherein the lipid nanoparticle comprises the therapeutic payload.The present disclosure concerns a method of preventing and / or treating a viral disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a lipid nanoparticle to the subject, wherein the lipid nanoparticle comprises the therapeutic payload.The present disclosure concerns a method of modulating an immune response in a subject in need thereof, comprising administering a therapeutically effective amount of a lipid nanoparticle to the subject, wherein the lipid nanoparticle comprises the therapeutic payload.The present disclosure also concerns a lipid nanoparticle as disclosed herein for use in delivering a therapeutic payload to treat a disease or condition.The present disclosure concerns a lipid nanoparticle for use in preventing and / or treating a viral disease or condition, wherein the lipid nanoparticle comprises the therapeutic payload.The present disclosure concerns a lipid nanoparticle for use in modulating an immune response, wherein the lipid nanoparticle comprises the therapeutic payload.The present disclosure also concerns use of a lipid nanoparticle as disclosed herein in the manufacture of a medicament for delivering a therapeutic payload to treat a disease or condition.The present disclosure concerns a use of a lipid nanoparticle in the manufacture of a medicament for preventing and / or treating a viral disease or condition, wherein the lipid nanoparticle comprises the therapeutic payload.The present disclosure concerns a use of a lipid nanoparticle in the manufacture of a medicament for modulating an immune response, wherein the lipid nanoparticle comprises the therapeutic payload.The disease or condition may be COVID-19 or polyneuropathy or influenza.Viral diseases are infections caused by viruses. A viral disease (or viral infection) occurs when an organism's body is invaded by pathogenic viruses, and infectious virus particles (virions) attach to and enter susceptible cells. Some of the most common types of viral diseases include: common cold, respiratory infections, digestive system infections, viral hemorrhagic fevers, sexually transmitted infections (STIs), exanthematous (rashcausing) infections, neurological infections, and congenital infections.In some embodiments, the viral disease or condition is caused by a virus selected from Human papillomavirus (HPV), H1N1, SARS-CoV-2, H5N1, H7N9, Rabies virus (RV), Feline Infectious Peritonitis virus (FIPV), and Herpes simplex virus (HSV).An immune response is a physiological reaction which occurs within an organism in the context of inflammation for the purpose of defending against exogenous factors. These include a wide variety of different toxins, viruses, intra- and extracellular bacteria, protozoa, helminths, and fungi which could cause serious problems to the health of the host organism if not cleared from the body. In addition, there are other forms of immune response. For example, harmless exogenous factors (such as pollen and food components) can trigger allergy; latex and metals are also known allergens. A transplanted tissue (for example, blood) or organ can cause graft-versus-host disease. A type of immune reactivity known as Rh disease can be observed in pregnant women. These special forms of immune response are classified as hypersensitivity. Another special form of immune response is antitumor immunity.The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body ofinvading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.The lipid nanoparticles may also be used to deliver a therapeutic payload for treating cancer. For example, the cancer may be prostate cancer.The present disclosure concerns a pharmaceutical composition comprising a polynucleotide encapsulated in a lipid nanoparticle as disclosed herein, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof.The lipid nanoparticle can be administered to a subject as a pharmaceutically acceptable salt thereof. Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. In particular, the present disclosure includes within its scope cationic salts eg sodium or potassium salts, or alkyl esters (eg methyl, ethyl) of the phosphate group.Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.The lipid nanoparticle may be in crystalline form either as the free nanoparticle or as a solvate (e.g. hydrate) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.The lipid nanoparticle, or a pharmaceutically acceptable salt, or solvate thereof is administered to the patient in a therapeutically effective amount. As used herein, a therapeutically effective amount is intended to include at least partially attaining the desired effect, or delaying the onset of, or inhibiting the progression of, or halting or reversing altogether the onset or progression of macular degeneration.As used herein, the term "effective amount" relates to an amount of lipid nanoparticle which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated.The lipid nanoparticle may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be preparedby any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.The lipid nanoparticle may be injected directly to the eye, and in particular the vitreous of the eye. The lipid nanoparticle can be administered to the vitreous of the eye using any intravitreal or transscleral administration technique. For example, the lipid nanoparticle can be administered to the vitreous of the eye by intravitreal injection. Intravitreal injection typically involves administering a lipid nanoparticle or a pharmaceutically acceptable salt, solvate or prodrug in a total amount between 0.1 ng to 10 mg per dose.Injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion. Carriers can include, for example, water, saline (e.g., normal saline (NS), phosphate-buffered saline (PBS), balanced saline solution (BSS)), sodium lactate Ringer's solution, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances, such as wetting or emulsifying agents, buffers, and the like can be added. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants.Other modes of administration including topical or intravenous administration may also be possible. For example, solutions or suspensions of the lipid nanoparticle may be formulated as eye drops, or as a membranous ocular patch, which is applied directly to the surface of the eye.The lipid nanoparticle may also be suitable for intravenous administration. For example, a lipid nanoparticle or a pharmaceutically acceptable salt, or solvate thereof may be administered intravenously at a dose of up to 16 mg / m2.The lipid nanoparticle may also be suitable for oral administration and may be presented as discrete units such as capsules, sachets or tablets each containing a predeterminedamount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water- in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. In another embodiment, the lipid nanoparticle or a pharmaceutically acceptable salt, solvate or prodrug is orally administerable.A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.The lipid nanoparticle may be suitable for topical administration in the mouth including lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.The lipid nanoparticle may be suitable for topical administration to the skin may comprise the lipid nanoparticle dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Transdermal patches may also be used to administer the lipid nanoparticle.The lipid nanoparticle may be suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants.buffers, bactericides and solutes which render the lipid nanoparticle isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, lipid nanoparticle may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.Preferred unit dosage composition or combinations are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.The lipid nanoparticle may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and / or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of Wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.ExamplesPhotophysical and binding properties of COE-S6 incorporated LNPs.The molecular structure of the COE used (S6 or COE-S6) is shown in Figure 2. The lipid nanoparticle formulation was prepared using the microfluidic method following a protocol published in Nat. Protoc. 2023, 18(1), 265-291. Alternatively, the mRNA-LNPs were prepared using the microfluidic method following a modified Onpattro formulationpublished elsewhere and comprised DLin-MC3-DMA / cholesterol / DSPC / PEG2000-DMG fixed at a molar ratio of 50 / 38.5 / 10 / 1.5.The membrane intercalation of COEs into the lipid bilayer occurs spontaneously. Hence, we explored a facile method to incorporate COE-S6 by adding an aqueous solution of COE-S6 into purified LNP solutions to access the COE-S6-incorporated LNPs (Figure 3a). To demonstrate the binding of COE-S6 to the LNPs, we first studied the photophysical properties of COE-S6 in buffer, COE-S6-modified LNPs and COE-S6 in small unilamellar vesicles (SUVs) using UV-visible (UV-vis) absorption and photoluminescence (PL) spectroscopies (Figure 3 and 16). The SUVs were prepared from l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and l-palmitoyl-2- oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) sodium (POPG) at a molar ratio of 85 / 15 and were used as the model bilayer membrane. Upon intercalation of COE-S6 into LNPs, the lipid bilayer regions of LNPs with a lower polarity experience increased structural rigidity due to the interaction with the hydrophobic conjugated backbone of COE-S6. When the conjugated backbone of COE-S6 is in an environment of lower polarity relative to the buffer, its PL emission intensity will increase several fold. This is shown in Figure 3b, whereby there is an increase in PL emission relative to COE-S6 in PBS. It is worth noting that the increase appears to be less pronounced than that of COE-S6 in SUVs, most reasonably due to variations in lipid bilayer homogeneity or differences in association constants.The PL decay profiles of fluorophores such as COE-S6 provide insights into their surrounding environment. Thus, we measured the PL decay profiles of COE-S6 in PBS, LNPs and SUVs (Figure 3c and 16b) and determined their PL lifetimes by fitting them to a single exponential curve (Table 1). The PL lifetime values (1.9 and 1.3 ns for COE- 56 in PBS and LNPs, respectively) are consistent with a well-solvated COE-S6, indicating successful membrane intercalation. COE-S6 in LNPs exhibited a longer PL lifetime value of 1.3 ns as compared to 0.9 ns for COE-S6 in SUVs.Table 1. Photophysical properties of COE-S6 in PBS and LNPs.Samp “le A a .bs A emm PL fold PL lifetime Stokes shift(nm) (nm) increase (ns) (nm)' COE-S6 in PBS ' 409 ' 518 ' - ' 1.9 ' 109COE-S6 in LNPs 415 511 2.8 1.3 96We then determined the binding dissociation constant of COE-S6 to pre-formed LNPs using a fluorescence anisotropy (FA) assay. The FA is the phenomenon where a fluorophore emits light of uneven intensities along distinct polarization axes in response to a binding event. When a fluorophore binds to a larger molecule such as proteins or LNPs, the rate at which it tumbles will decrease appreciably. In this case, the binding of the fluorogenic COEs to the LNPs should cause a reduction in its tumbling rate and an accompanying increase in the FA of the system. We titrated different amounts of LNPs (as determined by the total lipid concentration) to a fixed amount of COE-S6 at 1 pM until saturation (Figure 3d). The anisotropy values of COE-S6 when titrated with SUVs reached a significantly higher value than when COE-S6 is titrated with mRNA-LNPs which is expected due to the uniform lipid bilayer. Interestingly, the titration of mRNA- LNPs to COE-S6 gave a lower dissociation constant (7.6 pM) as compared to SUVs (14.4 pM). We point out, however, that the mRNA-LNP organization is considerably more complex than the SUVs, enabling a wider variety of interactions and localizations (See Figure 1 for one possible interaction and localization).The secondary structure of mRNAs is correlated to their translatability into functional proteins in cellulo. Interaction with strongly binding oppositely charged species can induce denaturation, which may give rise to diminished, or inhibition, of expression. Thus, it is crucial to determine whether the binding of COE-S6 to mRNA will introduce any adverse changes to the functional secondary structure. We thus employed circular dichroism (CD) spectroscopy to examine the secondary structure of the mRNA and the effects of binding COE-S6. Firefly luciferase (FLuc) mRNA was used as a representative mRNA. It is worth noting that a study has shown that by introducing some folding to the mRNA secondary conformation using cationic lipids, the translation efficiency can be elevated while overbinding leads to denaturation and poor translation. The CD spectra of mRNA complexes with COE-S6 at different nitrogen-to-phosphate (N / P) ratios are shown in Figure 16d. We observed a gradual denaturation of the mRNA from N / P ratios 0.5 to 2.5. Notably, some folding was observed at N / P = 0.5 and the mRNA was completely denatured from N / P = 1. As such, we chose to incorporate COE-S6 into the mRNA-LNPs up to the ratio N / P = 0.5 for subsequent experiments and this corresponds to 0.2% COE-S6 relative to total lipid concentration.Suspension properties of the COE-S6 incorporated LNPs.To study how the incorporation of COE-S6 into the LNP influences its physical properties such as suspension properties, dynamic light scattering studies were conducted (Figure 4 and Table 2). 0-0.2% (molar % relative to the total lipids) of COE-S6 was incorporated into the LNP as described above; i.e. ex situ at concentrations ranging from 0.02 to 0.2% (molar % relative to the total lipid content). As seen in Figure 4a, the particle sizes showed a decreasing trend from 84.5 ± 1 to 67.9 ± 0.8. The zeta potentials of the LNPs showed negligible changes, maintaining neutral with values between -2.0 to -4.0 following incorporation of up to 0.2% COE-S6(Figure 4b). Successful incorporation of the COE into the LNPs can also be observed with the monodispersed particle size distribution profiles (Figure 4c) with little change to the polydispersity index (PDI). The encapsulation efficiencies were also unaffected by the incorporation of COE-S6 and remained above 90% as determined using the RiboGreen assay (Figure 4d). The results of the suspension properties are summarized in Table 2.The morphology of LNPs reveals critical insights into their payload encapsulation and release properties. For instance, replacing the commonly used cholesterol in mRNA- LNPs with various phytosterols resulted in morphological changes that led to variations in mRNA transfection efficiencies. To study whether incorporating COE-S6 into the LNPs will induce any changes to their morphology, we utilized cryo-TEM microscopy to visualize the LNPs. A noteworthy point of using the technique cryo-TEM is that the LNP solution will be rapidly plunged into liquid ethane, forming vitreous ice that preserves the original LNP morphology in the solution state. This contrasts with room temperature TEM whereby the original morphology is often lost due to dehydration. As shown in Figure 4e, the LNPs adopt a spherical morphology, consistent with published data on the Onpattro formulation. Incorporating 0.2% COE-S6 into the LNPs did not alter the morphology. However, there is a reduction in particle size from 84.5 ± 1 to 67.9 ± 0.8 nm as analyzed using Image!, which agrees with the DLS results.Table 2. Particle characteristics of the pristine and COE-S6 incorporated LNPs.COE-S6 Z-average PDI Zeta potential mRNA incorporated (d.nm)a(mV) encapsulation(%) efficiency (%)b0 84.5 ± 1.0 0.07 ± 0.02 -3.0 ± 0.4 85.0 ± 2.00.02 80.7 ± 1.4 0.08 ± 0.03 -4.0 ± 0.6 87.0 ± 3.00.04 75.4 ± 1.2 0.10 ± 0.01 -3.5 ± 1.0 88.0 ± 2.50.06 73.4 ± 1.0 0.10 ± 0.02 -3.0 ± 1.1 87.0 ± 3.00.08 71.0 ± 1.6 0.14 ± 0.02 -2.0 ± 0.9 89.0 ± 2.80.1 70.3 ± 1.1 0.12 ± 0.02 -3.5 ± 1.0 88.0 ± 3.60.2 67.9 ± 0.8 0.14 ± 0.03 -3.7 ± 1.0 87.0 ± 4.0aMean Z-average diameters by dynamic light scattering from three individual measurements in tris-HCI buffer (pH = 7.4).^Determined using Quant-it™ RiboGreen assay.Serum stability of COE-S6 incorporated LNPs.We also assessed how the incorporation of COE-S6 into the LNPs can impact serum stability. The serum stability of nanocarriers can provide critical insights into their colloidal stability. For example, a serum-stable nanocarrier can have prolonged in vivo circulation time, an important parameter for increased bioavailability to target sites. Additionally, good colloidal stability may permit lower storage requirements, reducing costs associated with cold chain transport. We show in Figure 5 that among the concentrations tested, at 0.06% of COE-S6 incorporation, we saw a profound enhancement in serum stability (in terms of particle size) relative to the pristine LNP after 24 h of incubation.Transfection efficiencies of COE-S6 incorporated LNPs.We then evaluated the transfection efficiencies of the COE-S6 incorporated LNPs across three different cell lines, HEK293T, A549, and HeLa. The LNPs were incorporated with varying amounts of COE-S6 (0.02-0.2%) relative to total lipids. The cell viabilities of the corresponding transfection tests are also presented to ensure that any low transfection values are not a direct consequence of carrier cytotoxicity. From Figure 6, the cells retained >80% viabilities across all COE concentrations and cell lines, showing insignificant cytotoxic effects of incorporating COE-S6 into the LNPs. Interestingly, there is a general rising trend of transfection efficiencies (represented by the luminescence values, with an increasing amount of COE-S6 incorporated into the LNPs up to 0.2%. At0.2% incorporation of COE-S6, luminescence values increased by 23.3-fold, 14.8-fold, and 14.3-fold for HEK293T, HeLa, and A549 cells, respectively compared to the unmodified mRNA-LNP control. Beyond 0.2%, however, we saw decreasing efficiencies (data not shown), likely attributed to the increasing denaturation of mRNA (Figure 3e). LNP with 0.2% COE-S6 incorporated was therefore selected to be the optimal formulation for subsequent experiments.Encouraged by the increase in transfection efficiencies by incorporating COE-S6 using the ex situ method, we sought to determine if we could achieve the same result using COE-S6-mRNA-LNPs prepared by the more traditional in situ formulation method. For these tests, we selected 0.2% COE-56 loading due to the consistently high transfection enhancement across the three cell lines. Thus, mRNA-LNPs were incorporated with 0.2% of COE-S6 using the in situ method, where COE-S6 was pre-mixed with other lipids in the ethanol phase before production (Figure 11A). Again, we observed heightened transfection efficiencies for these in situ generated COE-S6-mRNA-LNPs with a 23.7- fold, 14.7-fold, and 13-fold increase in luminescence values for HEK293T, HeLa, and A549 cells, respectively compared to the unmodified mRNA-LNP controls (Figure 13). Indeed, the increase in transfection efficiencies is similar for both ex situ and in situ modification protocols. It is also worth pointing out that the enhancement by the COE- 56 incorporation enabled the mRNA-LNPs to outperform the commercial control, LipofectamineTM MessengerMaxTM, a formulation optimized for mRNA transfection, at the same mRNA treatment dosage.We formulated the LNPs with a different mRNA (mCherry) for quantification with flow cytometry and direct visualization using confocal laser scanning microscopy (CLSM) (Figure 7). HEK293T cells seeded on 24-well plates were treated with the LNPs at 0.5 pg mRNA / well and assessed for mCherry-positive cells after 24 h. As observed from Figures 7a-c, we saw a dramatic increase in transfection efficiencies between pristine LNP and LNP + 0.2% COE-56, from 24.2 ± 1.6 to 98.7 ± 0.6%, respectively. This optimized formulation also outperformed the commercial Lipofectamine which only showed 39.1 ± 1.6% transfection efficiency. However, we noticed that the mean fluorescence intensities (MFI) of mCherry in Lipofectamine-treated cells were greater than in the COE-S6 incorporated LNP-treated cells. This may indicate that the Lipofectamine transfects the cells unevenly such that only a portion of the cell population exhibits high protein expression. In contrast, our optimized formulation transfects thecells evenly, resulting in relatively equal protein expression throughout the cell population.To visualize the transfection enhancement, we employed CLSM (Figure 7d). HEK293T cells seeded on 8-well chamber slides were treated with the LNPs at 0.5 pg mRNA / well and observed for mCherry expression after 24 h. Comparing the neat / unmodified LNP to the COE-S6 incorporated LNPs, there was an obvious elevation in expressions, reflected by the increased intensities of the mCherry channel. The uneven mCherry expression in the lipofectamine-treated cells was also consistent with our flow cytometry observations.We then asked whether a pre-treatment with COE-S6 would achieve the same enhancement in transfection efficiency. Hence, we pre-treated HEK293T cells with 1 pM COE-S6 for 1 h, washed with PBS, transfected with pristine mCherry mRNA LNPs, and imaged using confocal microscopy after 24 h (Figure 8). No qualitative enhancement in transfection efficiency was seen, and the mCherry expression profile appears identical to the LNP control presented in Figure 7d. This proves that the transfection enhancement is unlikely due to the effects of free or associated COE-S6 on the cells (if any), and is ascribed to the LNP properties.Cellular uptake and endosomal escape studies of COE-S6 incorporated LNPs.We have demonstrated that there is a significant boost in the transfection efficiencies of LNPs incorporated with 0.2% COE-S6. However, little is known about the exact factors contributing to this observation. Two main factors that govern the in cellula efficacy of nanocarriers are cellular uptake and endosomal escape ability.To study the cellular uptake profiles of the LNPs, we prepared LNPs with Cy5-labeled mRNA and employed flow cytometry. HEK293T cells seeded on 24-well plates were treated with the Cy5-labeled mRNA LNPs (pristine and LNP + 0.2% COE-S6) at 0.5 pg mRNA / well, incubated for different time points (0.5-24 h) and assessed for Cy5-positive cell populations using flow cytometry. In terms of Cy5-positive cells, we observe a significant increase in uptake for cells treated with COE-S6 incorporated LNPs up to 2 h. At this time point, HEK293T cells exhibited 48.5 ± 1.2% and 77.3 ± 3.2% Cy5 positivity for pristine mRNA-LNP and mRNA-LNP + 0.2% COE-S6, respectively. No differences from 4 h onwards (Figure 9a). However, we do note that there is asignificant increase in the MFI of Cy5 across all time points for the LNP + 0.2% COE- S6-treated cells (Figures 9b, c). For instance, at 24 h, the Cy5 MFI for the mRNA-LNP control was 8570 ± 39, whereas the COE-S6 formulation showed a higher MFI of 11100 ± 218. The higher MFI indicates that a greater number of particles were taken up by the cells. Therefore, while the uptake in terms of cells positive for Cy5 may be similar, more particles were taken up by the HEK293T cells for the LNP + 0.2% COE-S6 formulation on a per-cell basis.To assess the endosomal escape ability of the LNPs, we treated HEK293T cells seeded on 8-well chamber slides with the same Cy5 mRNA LNPs at 0.5 pg mRNA / well and imaged them after 24 h. The endolysosomal compartments were stained with lysotracker red. We then evaluated the endosomal escape ability by analyzing the fluorescence correlation between the Cy5 mRNA and lysotracker red (i.e., a poor correlation denotes good endosomal escape ability and vice versa). The results are displayed in Figure 9d. After 24 h, the Cy5 mRNAs in the pristine LNP-treated cells appear largely colocalized with the endolysosomal compartments with a high correlation coefficient value of 0.6. However, with the LNP + 0.2% COE-S6-treated cells, there is a poor colocalization correlation (R = 0.2), indicating good endosomal escape capability.Due to the inherent fluorogenic properties of COE-S6, we were also able to visualize its intracellular spatial distribution after transfection. We then performed colocalization analyses of COE-S6 with lysotracker and mRNA. We noted a poor correlation with lysotracker, suggesting that upon the endosomal escape of the LNPs, COE-S6 relocated to other parts of the cytosol. A slight correlation with mRNA was observed, implying that some mRNA may remain bound to COE-S6 even after its release to the cytosol.Formulation preparation methodsTwo different incorporation methods are disclosed. First, the COEs are incorporated in- situ (Figure Ila) by adding into the lipid mixture (ethanol phase) before mixing with the aqueous phase via various manufacturing methods (pipetting, vortexing, or microfluidic) to form the LNPs. The remaining ethanol is then removed by dialysis or ultrafiltration. In the second method, a known amount of COE dissolved in an aqueous solution is added ex-situ to the purified LNP solution (Figure lib) before vortexing for uniform mixing. For the experiments up till this point, we have used method B due to its ease of incorporation. To expand the utility of developing LNPs containing COEs, wealso explored method A (with the optimized 0.2% concentration) and assessed its effects on solution properties and transfection efficiencies.To compare the two different methods, 0.2% (molar % relative to the total lipids) of COE-S6 was incorporated into the LNP via the two different methods described above (A and B). The LNPs were formulated using an in-house synthesized mRNA encoding mScarlet (1732 bases, 558.3 kDa). As seen in Figure 12a, the particle sizes showed a reduction from 165 ± 1 to 56 ± 0.3 and 62 ± 0.4 for incorporation methods A and B, respectively. Successful incorporation of the COE into the LNPs can also be observed with the monodispersed particle size distribution profiles (Figure 12b) with little change to the polydispersity index (PDI) (Figure 12c and Table 3). The zeta potentials of the LNPs showed negligible changes after incorporation since only 0.2% was incorporated. The results of the solution properties are summarized in Table 3.Table 3. Particle characteristics of COE-S6-incorporated LNP systems in suspension.The LNPs were formulated using mRNA encoding mScarlet.Sample Particle Size (BIB) PDI Zeta Potential (mV)0% 165 1 0.04 -5.60.2% (A) 56 * 0.3 0.09 -7.1 0.10 -9.3Encouraged by the enhanced transfection efficiencies by incorporating COE-S6 using method B as shown above, we then sought to find out if we could achieve the same result using method A. 0.2% was selected due to the consistently high transfection enhancement across the three cell lines. The LNPs were then incorporated with 0.2% of COE-S6 using method A. Again, we observed heightened transfection efficiencies relative to the neat LNP (Figure 13). We saw no significant differences in the transfection efficiencies with the 0.2% COE-S6 incorporated LNP prepared via the two different methods.In Vivo Translation Efficiency and Organ Biodistribution of COE-S6-mRNA-LNPs We administered mRNA-LNPs encoding FLuc to healthy BALB / c mice intravenously to determine the in vivo translation efficiency and organ biodistribution using an in vivo imaging system (IVIS) (Figure 17). At 6 h post-injection, mice injected with mRNA-LNP +0.2% COE-S6 showed a 1.75-fold increase in bioluminescence compared to thoseinjected with mRNA-LNP controls (Figure 17a and 17b). Bioluminescence gradually decreased, with almost no expression observed at 48 h in both groups. The incorporation of COE-S6 into the mRNA-LNPs did not change the organ biodistribution profile, with accumulation primarily in the liver at 6 h post-injection (Figure 17c). The in vivo toxicity was also assessed. Healthy BALB / c mice were injected intravenously with the mRNA-LNPs at a mRNA dosage of 0.1 mg / kg. At 15 days post-treatment, blood samples were collected for hematological parameter evaluation, and major organs (heart, liver, lungs, kidneys, and spleen) were harvested for hematoxylin and eosin (H&E) staining. No significant differences were observed in the hematological parameters or H&E-stained organ sections (Figure 18, 19, and 20), indicating that incorporating 0.2% COE-S6 into the mRNA-LNPs did not appear to pose any toxicity issues. Overall, these specific results highlight the potential of COE-S6 incorporation to enhance mRNA-LNP performance in vivo without an obvious compromise in biocompatibility.Intracellular tracking with COE-LNP formulationDue to the inherent fluorogenic properties of the COE, visualization of the LNP's intracellular spatial distribution after transfection is possible. In our preliminary experiments using the representative COE-S6, we were able to perform colocalization analyses of COE-S6 with lysotracker (stain for acidic endolysosomal compartments) and mRNA (Figure 10). We noted a poor correlation with the lysotracker, suggesting that upon the endosomal escape of the LNPs, COE-S6 relocated to other parts of the cytosol. A slight correlation with mRNA was observed, implying that some mRNA may remain bound to COE-S6 even after its release to the cytosol.This contrasts with commercially available products whereby an additional fluorophore must be included in their formulation for tracking experiments. In those cases, the fluorophore may affect the properties and efficacy of the pristine LNP, potentially rendering inaccurate experimental conclusions. In our case, the LNP is already fluorescent due to the incorporated COE thus eliminating the need for additional formulation and ensuring reliable conclusions. Finally, the broad emission spectrum of our COE library enables us to select a suitable wavelength for distinct studies, such as the near-infrared range for in vivo studies.LNP with BOUsing a different representative COE compound, BO (Figure 14), we conducted a similar transfection screening test using incorporation method B. BO has been published previously (Sci. Adv. 2023, 9(2), eade2996), the reference of which is herein incorporated. We evaluated the transfection efficiencies of the BO incorporated LNPs against HEK293T cells. The LNPs were incorporated with varying amounts of BO (0.02- 0.2%). The cell viabilities of the corresponding transfection tests are also presented to ensure that any low transfection values are not a direct consequence of carrier cytotoxicity. From Figure 15, the cells retained >80% viabilities across all COE concentrations, showing insignificant cytotoxic effects of incorporating BO into the LNPs. We observed a general rising trend of transfection efficiencies (represented by the luminescence values), with an increasing amount of BO incorporated into the LNPs up to 0.2%. This observation is similar to the results obtained using COE-S6.Enhancing transfection efficiencies of spike-protein mRNA-LNPsBuilding upon our prior findings on the use of COE-S6, we extended our investigation by formulating spike-protein mRNA-loaded lipid nanoparticles (mRNA-LNPs) using both Pfizer's and Moderna's LNP formulations. COE-S6 was incorporated into these systems to evaluate its potential in enhancing transfection efficiency. Optimization studies identified the ideal COE-S6 incorporation percentages as 0.2% for Pfizer's formulation and 0.5% for Moderna's formulation, based on transfection performance metrics. Representative confocal micrographs, as depicted in Figures 21, 22 and 23, illustrate the resulting spike protein expression across experimental conditions. Notably, LNPs incorporating COE-S6 demonstrated a marked enhancement in spike protein expression levels when compared to both lipofectamine and LNP controls.Transfection of hard-to-transfect primary cortisol neuronal cellsTo further evaluate the versatility and effectiveness of COE-S6-enhanced LNP formulations, we conducted transfection experiments on hard-to-transfect primary cortical neuronal cells using mCherry-mRNA-loaded LNPs. These LNPs were formulated with 0.2% COE-S6, utilizing the Onpattro-inspired formulation described in the manuscript. The results, visualized in the representative confocal micrographs (Figure 24), revealed a significant enhancement in mCherry protein expression compared to the control groups, which included both unmodified LNPs and lipofectamine. The observed increase in expression exhibits the potential of COE-S6 incorporation toovercome transfection barriers in challenging cellular models, such as primary neuronal cells, which are typically resistant to mRNA delivery.Incorporation of other COE structuresWe screened other COE compounds for optimizing mRNA delivery and transfection efficiency. Figure 25 shows the structures of the COEs screened. For this study, we prepared mRNA-loaded LNPs encoding emiRFP703 and transfected HEK293T cells to evaluate their performance. The transfection efficacy was quantified by assessing the mean fluorescence intensities (MFI) of emiRFP703-positive cells. A heatmap was generated to represent the fold-change in MFI relative to the pristine LNP control, providing a visual overview of the performance across different COE formulations (Figure 26).From the heatmap analysis, COE-FL emerged as the top-performing candidate, demonstrating an ~80-fold increase in MFI compared to the control. To further contextualize this result, we compared the spike protein expression levels achieved with 0.2% COE-S6 and 0.5% COE-FL, corresponding to different positions on the heatmap. The comparison using western blot analysis (Figure 7) revealed that while 0.2% COE- 56 produced a significant increase in protein expression, 0.5% COE-FL demonstrated a substantially higher enhancement in the form of darker and thicker band, aligning with its superior performance in the emiRFP703 screening. The comparative analysis of COEs indicates that there is the potential of similarly related chemical structures that can provide further enhancements in desirable metrical parameters.Enhancing PTEN-null prostate cancer therapy via conjugated oligoelectrolytes mRNA-lipid nanoparticles (COE-mRNA-LNPs)The tumor suppressor gene PTEN (phosphatase and tensin homolog) plays a crucial role in regulating cell growth, survival, and proliferation by antagonizing the PI3K-AKT signaling pathway. PTEN loss or mutation is a common genetic alteration in prostate cancer, contributing to tumor progression, resistance to therapy, and poor clinical outcomes. Restoring PTEN function has emerged as a promising therapeutic strategy for prostate cancer management. In this context, lipid nanoparticles (LNPs) have gained significant attention as an innovative platform for protein replacement therapy. LNPs offer a highly efficient delivery system for encapsulating and protecting therapeutic proteins, enabling targeted intracellular delivery and functional restoration of PTEN incancer cells. This approach holds substantial potential for addressing PTEN-associated prostate cancers and overcoming current treatment limitations. In this work, we hypothesis that incorporation of COEs into PTEN mRNA-LNPs is able to enhance the transfection, leading to greater anti-cancer effects.We first screened the optimal incorporation content of COE-S6 into Moderna's mRNA- LNPs encoding emiRFP703 by preparing formulations with varying COE-S6 concentrations and assessing their transfection efficiencies in different prostate cancer cell lines (PC3, LNCaP, 22Rvl, and DU145) using flow cytometry (Figure 28). This allowed us to determine the concentration of COE-S6 that maximized transfection efficiency and emiRFP703 expression, as reflected by both the percentage of transfected cells and the mean fluorescence intensity (MFI) within each cell line. The results provided a comprehensive understanding of how COE-S6 incorporation impacts the performance of mRNA-LNPs across diverse cellular environments.We subsequently evaluated the cell viabilities of prostate cancer cell lines under various treatment conditions to assess the cytotoxicity and therapeutic potential of our formulations (Figure 29). Initially, we tested the cytotoxicity of COE-S6 across different prostate cancer cell lines (PC3, LNCaP, 22Rvl, and DU145) at a range of concentrations. Results indicated that COE-S6 was generally non-toxic at concentrations up to 31.3 pM, demonstrating its suitability for further incorporation into lipid nanoparticle systems. Next, we investigated the potential cytotoxic effects of emiRFP703 mRNA-LNPs across our testing concentration range. These LNPs exhibited no significant cytotoxicity, confirming their biocompatibility and safety for transfection applications.Finally, we evaluated the therapeutic performance of PTEN-mRNA-LNPs, both with and without the optimal incorporation of 0.5% COE-S6. Remarkably, the inclusion of COE- 56 significantly enhanced the anticancer effects, as evidenced by increased cell death in the prostate cancer cell lines. This was further corroborated by real-time viability assays, which demonstrated pronounced reductions in cell viability at both 24- and 48- hours post-treatment for COE-56-incorporated PTEN-mRNA-LNPs compared to controls. To further validate that the enhanced cytotoxic effects observed in PTEN-null prostate cancer cell lines were attributable to the restored expression of PTEN, we conducted a western blot analysis (Figures 30 and 31). This assay allowed us to directly measure PTEN protein levels in cells treated with COE-S6-incorporated PTEN-mRNA-LNPs. Theresults confirmed a significant elevation in PTEN expression in treated cells compared to untreated controls or cells treated with LNPs lacking COE-S6. This increase in PTEN expression strongly correlates with the observed therapeutic effects, highlighting the role of COE-S6 in improving mRNA delivery and subsequent protein translation. These findings provide direct biochemical evidence linking COE-S6-enhanced mRNA delivery to the functional restoration of PTEN, thereby reinforcing the potential of this approach for targeting PTEN-deficient cancers.To further investigate the mechanism underlying the enhanced anticancer effects of COE-S6-incorporated mRNA-LNPs, we performed an apoptosis assay to evaluate whether these formulations promote increased apoptotic events in PTEN-null prostate cancer cell lines (Figure 32). The restoration of PTEN expression is expected to reestablish its tumor suppressor function, which includes inducing apoptosis. This hypothesis was validated in LNCaP cells, a PTEN-null cell line, where treatment with COE-S6-mRNA-LNPs resulted in a marked increase in apoptosis. Specifically, approximately 40% of the cells were in the early apoptotic phase following treatment with COE-S6-incorporated PTEN-mRNA-LNPs, compared to only 18% observed with the pristine LNP control. In PTEN-competent cell lines, there were insignificant apoptotic events. This substantial increase in early apoptosis highlights the functional impact of PTEN restoration on cellular pathways leading to programmed cell death. These results provide compelling evidence that COE-S6 enhances the therapeutic efficacy of mRNA- LNPs by facilitating PTEN expression and subsequently triggering apoptosis in PTEN- deficient cancer cells, supporting its potential utility in PTEN-null prostate cancer therapy.We further conducted morphological studies on PC3 cells following treatment with the COE-S6-incorporated PTEN-mRNA-LNPs (Figure 33). Morphological changes in cell structure can provide additional visual evidence of apoptotic processes. Observations at 24 hours post-treatment revealed a distinct rounding of the cells, a hallmark feature of apoptosis. This cellular rounding was consistent with the early apoptotic events identified in the biochemical assays and further confirmed that the treatment effectively induced programmed cell death in PTEN-null cells. These morphological changes, along with the apoptosis assay results, strengthen the evidence that COE-S6 enhances the delivery and expression of PTEN, effectively reinstating its tumor-suppressive functions.Together, these findings offer a multi-faceted validation of the therapeutic potential of COE-S6-mRNA-LNPs in targeting PTEN -deficient cancer cells.Wound healing assays were performed to investigate the impact of PTEN restoration on the migratory behavior of invasive cancer cells (Figure 34). This assay is commonly used to evaluate cell migration, a critical aspect of cancer metastasis. Since PTEN is a well-known tumor suppressor involved in inhibiting cell migration and invasion, restoring its expression in PTEN-null cell lines is expected to suppress migratory activity, leading to decreased migration rates. The results demonstrated a striking reduction in migration rates in the PTEN-null cell line PCS following treatment with COE-S6- incorporated PTEN-mRNA-LNPs. Representative images illustrate significantly slower wound closure in treated PC3 cells compared to untreated controls, highlighting the effectiveness of PTEN restoration in halting migratory processes. In contrast, a PTEN- competent cell line, which naturally maintains basal PTEN levels, showed no significant differences in migration rates upon treatment, indicating that the effect is specific to PTEN-deficient cells. These findings further confirm the functional role of PTEN in regulating cell migration and suggest that COE-S6-enhanced mRNA-LNP formulations can effectively target invasive cancer cell behavior, potentially mitigating metastasis in PTEN-deficient cancers.To further evaluate the therapeutic potential of PTEN restoration in a more physiologically relevant environment, we transitioned to 3D culture models using tumor spheroids. These models mimic the three-dimensional architecture and cell-cell interactions of tumors in vivo, providing a robust platform for studying the effects of PTEN restoration on tumor growth. Since PTEN is known to regulate cell proliferation and survival, restoring its levels in PTEN-null cells is expected to arrest spheroid growth. Upon treatment with COE-S6-incorporated PTEN-mRNA-LNPs, we observed a significant reduction in spheroid growth in the PTEN-null cell line LNCaP (Figure 35). Representative micrographs showed visibly smaller spheroids compared to untreated controls, indicating that PTEN restoration effectively suppressed tumor cell proliferation within the 3D microenvironment. In contrast, spheroids derived from PTEN-competent cell lines did not exhibit any significant changes in size following treatment, continuing to grow as expected within the monitored timeframe (Figures 36 and 37).These results highlight the specificity of PTEN-mRNA-LNPs in targeting PTEN-deficient tumor models and displayed the therapeutic potential of COE-S6-enhanced LNP systems in halting tumor progression. The use of 3D spheroid models further strengthens the translational relevance of this approach, warranting future investigations in more complex in vivo systems.Table. Particle size of mRNA-LNPs used.SummaryWe have demonstrated remarkably simple ex situ and in situ methods to incorporate COEs into mRNA-LNPs. Using the representative example of COE-S6 one finds that a relatively low amount (0.2%) relative to total lipid content leads to several positive outcomes. These include increased cellular uptake, improved endosomal escape, and a remarkable rise in in cellula transfection efficiency from 24.2 ± 1.6% to 98.7 ± 0.6%. Moreover, the incorporation of COE-S6 into the mRNA-LNPs resulted in a 1.75-fold enhancement in in vivo protein expression upon intravenous administration into BALB / c mice. This improvement in in vivo performance did not come at the cost of safety; the administration of COE-S6-mRNA-LNPs did not present toxicity issues over a 15-day period. This straightforward strategy for integrating COEs into pre-formed mRNA-LNPs presents a fundamentally new strategy to boost the performance of existing mRNA- based therapeutics across a range of applications, such as vaccines for infectious diseases and protein replacement therapies. We also note that although mRNA served as the payload in this study, it is reasonable to expect that this strategy to modify LNP properties may be extended to other payloads, including DNA, small interfering RNA (siRNA), and proteins. Moreover, considering the diverse chemical structural space available for transmembrane-spanning COEs, significant advancements may be attained post the design of molecular structures specific to improving the properties of existing or new formulations. Such enhancements may include greater increases in transfection efficiencies, more precise tailoring of mRNA-LNP physical properties, and the integration of multifunctionalities such as optical reporting or targeting capabilities. In summary, the ex situ and in situ strategies for COE integration into pre-formed LNPs describedherein is a promising platform for the future development and improvements of effective therapies and diagnostics.Materials and MethodsMaterialsDLin-MC3-DMA ((6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was purchased from SINOPEG. 1,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC) and cholesterol were purchased from Sigma-Aldrich. PEG2000-C-DMG was purchased from MedChemExpress. FLuc and mCherry mRNA were purchased from TriLink BioTechnologies. RealTime-Glo™ MT Cell Viability Assay kit and ONE-Glo™ Luciferase Assay System were purchased from Promega Corporation. Quant- it™ RiboGreen assay kit was purchased from Invitrogen.InstrumentsUV-Vis-NIR absorption spectra were recorded on a Shimadzu UV-3600i plus spectrophotometer. Photoluminescence (PL) spectra were measured on a Horiba Fluorolog-3 fluorescence spectrometer equipped with an R13456 PMT detector. The absorbance or fluorescence readings were recorded using the TECAN plate reader (Spark®) for microplate-based assays. The confocal micrographs were obtained using the Olympus FV3000 confocal laser scanning microscope. Dynamic light scattering (DLS) measurements were performed using the Zetasizer Ultra (Malvern Panalytical). Flow cytometry data was collected using CytoFLEX LX (Beckman Coulter). Circular dichroism measurements were performed using the Chirascan™ spectrometer. The mRNA-lipid nanoparticles were prepared using the NanoAssemblr® Ignite instrument from Precision Nanosystems.Cells and MediaHEK293T, HeLa, and A549 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells were maintained in serum-containing DMEM media at 37 °C, 5% COz, and 95% relative humidity. Serum-containing DMEM media was supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate, 100 units / mg penicillin, and 100 pg / mL streptomycin.Preparation of Small Unilamellar VesiclesTo prepare the small unilamellar vesicles (SUVs), POPC and POPG were dissolved in chloroform at a molar ratio of 85: 15 in a glass via I .

[0001] The mixture was dried by rotary evaporation and further dried overnight under a vacuum to form a thin lipid film. PBS buffer was added to rehydrate the film at a 5 mg / ml concentration. The solution was then heated at 45 °C for 2 hours under constant stirring at 300 rpm. The resulting solution was then extruded 20 times using a 100 nm membrane at 45 °C to produce a stock of 5 mg / ml SUVs, which was stored at 4 °C until further use.Preparation of mRNA-Lipid NanoparticlesThe mRNA-lipid nanoparticles (mRNA-LNPs) were prepared using the microfluidic mixing method. The lipids were dissolved and mixed in ethanol while mRNA was dissolved in 10 mM citrate buffer (pH = 4.0). The molar ratio of the lipids (DLin-MC3- DMA / cholesterol / DSPC / PEG2000-C-DMG) was fixed at 50 / 38.5 / 10 / 1.5. The FLuc and mCherry mRNA concentrations in citrate buffer were fixed at 22.2 and 18.9 pg / mL, respectively. The lipids solution (ethanol phase) loaded onto a 1 mL syringe (TERUMO, Tokyo, Japan) and mRNA solution (aqueous phase) loaded onto a 3 mL syringe (TERUMO, Tokyo, Japan) were rapidly mixed using the NanoAssemblr® Ignite. The settings are as follows: Total injection volume = 3.3 mL, total flow rate = 12 mL / min, flow rate ratio (aqueous / ethanol) = 3 / 1, and total waste volume = 0.3 mL. The resulting mixture was then diluted in IX PBS and purified (to remove ethanol and acidic buffer) by ultrafiltration using Amicon® Ultra-15 Centrifugal filter lOkDa MWCO (2000g, 4 °C, 45 min). This cycle was repeated at least 3 times to ensure adequate buffer exchange. The purified mRNA-LNP solutions were used immediately or stored at 4 °C until further use (within 1 week of storage). To prepare COE-S6-incorporated mRNA-LNPs, different amounts of COE-S6 (at a molar percent relative to total lipid concentration) were either added to the ethanol phase (in-situ method) or added to the purified mRNA-LNPs, vortexed and equilibrated at room temperature for 10 min (ex-situ method). For determining the encapsulation efficiencies using Quant-it™ RiboGreen and particle characterizations using DLS, the LNP formulations were diluted to an mRNA concentration of 0.5 pg / mL before testing.Photoluminescence MeasurementsSamples of 1 pM COE-S6 in different environments (IX PBS, 1 mM SUVs, and 1 mM mRNALNPs were excited at 405 nm, and the PL emission spectra were recorded from 425-700 nm. For PL lifetime measurements, the samples were excited using a 402 nmlaser diode source (DeltaDiode™ DD-405L), and the emission was recorded at 490 nm. The decay profiles were fitted to a single exponential curve and processed using OriginPro.Fluorescence Anisotropy AssayThe fluorescence anisotropy (FA) saturation binding assay was set up as described elsewhere. The solutions were prepared in IX PBS to a final volume of 100 pL in a 96- well flat black plate (Greiner). The total lipid concentration of LNPs was varied from 1- 200 pM while the concentration of COE-S6 was kept constant at 1 pM. The fluorescence polarization measurements were performed at 25 °C at excitation / emission wavelengths of 410 / 520 nm. The G factor was fixed at 1. The resulting anisotropy values were normalized and fitted to a nonlinear regression curve using GraphPad Prism to give the dissociation constant (Kd) values.Circular Dichroism (CD) MeasurementsFLuc mRNA sample solutions (0.3 mg / mL) mixed with COE-S6 at different N / P ratios (0.05 to 2.5) in IX PBS were added to a quartz cuvette (1 mm pathlength, Hellma®). The CD spectra were monitored from 190 to 350 nm at 20 °C. The sampling time per point was fixed at 1 s. A solution of naked Flue mRNA was used as a control.Cryo-TEM Measurements3 pL of the Flue mRNA LNP solution (1 mg / mL total lipids) was pipetted onto a Lacey carbon grid layered with ultrathin carbon film (Tedpella ultrathin carbon film on Lacey carbon support film, 400 mesh, copper) and was glow-discharged in the air for 20 s. The grid was then blotted for 1.5 s (blot force 1) at 4 °C and 100% humidity, before plunging into liquid ethane using a vitrification device (FEI Vitrobot Mark IV). The micrographs were recorded using a 200 kV Tecnai Arctica cryo-transmission electron microscope equipped with a Falcon 3EC direct electron detector. Images were collected at a magnification of 53,000 x, yielding a pixel size of 2.01 A / px.In Cellula mRNA Transfection StudiesThe screening of formulations for optimal transfection efficiency was first evaluated using firefly luciferase (FLuc) mRNA in different cell lines (HEK293T, HeLa, and A549). The cells were seeded in white 96-well plates (Nunc™) at densities of 1 x 104cells / well in 100 pL of cell culture media. After 24 h of incubation, the culture media was replacedwith the FLuc mRNA LNP solutions (containing 0.25 |j mRNA) diluted in Opti-MEM (Gibco™) and further incubated for 24 h. At the end of the transfection, 50 pL of a 3X working solution of RealTime-Glo™ MT Cell Viability Assay reagent was added to quantify the cell viabilities and further multiplexed by adding 150 pL of ONE-Glo™ Luciferase Assay reagent to quantify the firefly luciferase expression (final volume in each well = 300 pL).The formulation with the optimal transfection efficiency was further studied by switching to mCherry mRNA. For the flow cytometry measurements, HEK293T cells were seeded in a 24-well plate (Corning Inc.) at densities of 5 x 104cells / well in 500 pL of cell culture media. After 24 h of incubation, the cell media was replaced with the mCherry mRNA LNP solutions (containing 0.5 pg mRNA) diluted in Opti-MEM (Gibco™) and further incubated for 24 h. At the end of the transfection, the treatment solution was aspirated, and the cells were washed with PBS, detached using trypsin, harvested via centrifugation, and resuspended in PBS containing 2% FBS. The samples were assessed for transfection efficiency using a flow cytometer (Cytoflex LX, Beckman Coulter), Y610- mCherry channel. Untreated cells were used as a negative control to calibrate background fluorescence. For the confocal imaging experiments, HEK293T cells were seeded in 8-well chamber slides (ibidi, p-Slide) at densities of 5 x 104cells / well in 500 pL of cell culture media. After 24 h of incubation, the cells were transfected similarly to the flow experiments. A working solution of SYTO Deep Red was added 0.5 h before the designated time point for nuclear staining. The treatment media was then aspirated, and the cells were washed 3 times with PBS. Phenol red-free DMEM was then added to the wells before live imaging using confocal microscopy.Cellular Uptake StudyFLuc-mRNA was covalently labeled with Cy5 LabellT nucleic acid labeling kit (Mirus, USA) according to the manufacturer's protocol. HEK293T cells were seeded in a 24-well plate (Corning Inc.) at densities of 5 x 104cells / well in 500 pL of cell culture media. After 24 h of incubation, the cell media was replaced with Cy5-mRNA LNP solutions diluted in Opti-MEM (Gibco™) and further incubated for the different time points (0.5- 24 h). At each time point, the Cy5-mRNA LNP solution was aspirated, and the cells were washed with PBS, detached using trypsin, harvested via centrifugation, and resuspended in PBS containing 2% FBS. The samples were assessed using a flowcytometer (Cytoflex LX, Beckman Coulter), R660-APC channel. Untreated cells were used as a negative control to calibrate background fluorescence.Endosomal Escape StudyFLuc-mRNA was covalently labeled with Cy5 LabellT nucleic acid labeling kit (Mirus, USA) according to the manufacturer's protocol. HEK293T cells were seeded in 8-well chamber slides (ibidi, p-Slide) at densities of 5 x 104cells / well in 500 pL of cell culture media. After 24h of incubation, the cell media was replaced with Cy5-mRNA LNP solutions diluted in Opti-MEM (Gibco™) and further incubated for 24h. A working solution of lysotracker red was added 0.5 h before the designated time point. The treatment media was then aspirated, and the cells were washed 3 times with PBS. Phenol red-free DMEM was then added to the wells before live imaging using confocal microscopy. Pearson's correlation coefficient was computed using Image! analysis (Coloc2 plugin).In Vivo Luc mRNA DeliveryThe animal experiments were conducted according to the guidelines established and approved by the A*STAR-Institutional Animal Care and Use Committee (A*STAR- IACUC), protocol number 201561. Female BALB / c mice aged 6-8 weeks old (n = 3 per treatment group and n = 2 for PBS control group) were intravenously injected with IX PBS or FLuc mRNA-LNPs (mRNA-LNP control or mRNA-LNP + 0.2% COE-S6) at an mRNA dosage of 0.1 mg / kg. At different time points post injection (6, 24, and 48 h), the mice were anesthetized using 2.5% isoflurane, injected intraperitoneally with D-Luciferin (150 mg / kg), and imaged with IVIS (PerkinElmer, Waltham, USA). After live imaging, the mice were euthanized using CO2, and the major organs (heart, liver, lungs, spleen, and kidneys) were harvested for ex vivo imaging and quantification of bioluminescence using the Living image 2.5 software as photons / sec / ROI. The data was expressed as total radiance (photons / sec / ROI).In Vivo Toxicological EvaluationThe animal experiments were conducted according to the guidelines established and approved by the A*STAR-Institutional Animal Care and Use Committee (A*STAR- IACUC), protocol number 211629. Healthy BALB / c mice (n = 3) were intravenously injected with IX PBS, mRNA-LNP control, and mRNA-LNP + 0.2% COE-S6. At 15 days post-treatment, blood samples were collected to evaluate hematological parameters.The major organs of the mice (heart, liver, lungs, kidneys, and spleen) were harvested for H&E evaluation.Cells and MediaDU145 (HTB-81), LNCaP clone FGC (CRL-1740), PC3 (CRL-1435), and 22Rvl (CRL- 2505) were purchased from ATCC (Manassas, Virginia). DU145 was grown in DMEM medium supplemented with 10% FBS (HyClone, Cytiva, Marlborough, Massachusetts) 1% penicillin / streptomycin (P / S) (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts), and Sodium pyruvate (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts). LNCaP and 22Rvl were grown in RPMI medium supplemented with 10% FBS and 1% P / S. PC3 was grown in Ham's F12k (Kaighn's) media supplemented with 10% FBS and 1% P / S. All cell lines were maintained in the incubator at 37 °C with 5% CO2.Cell ViabilityA 5xl03seeding density was utilized for DU145, PC3, and 22Rvl in a 96-well plate (Corning®, Corning, New York). 2xl04LNCaP cells were seeded into a 96-well plate. DU145, PC3, and 22Rvl cell lines were grown for 24 hours while LNCaP cell line was grown for 48 hours before treatment with LNPs (PTEN and emiRFP mRNA) in Opti-MEM (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts) at a dose of 0 to 2.5ug / mL. The toxicity of COE-S6 was also assessed using a treatment concentration ranging from 0 to 250 iM. Cell viabilities were assessed 24- or 48-hours post-treatment with Cel ITiter- Glo® 2.0 or RealTime-Glo™ MT Cell Viability Assay (Promega, Madison, Wisconsin). Cell viabilities of samples (n=9) were then analyzed and quantified by a microplate reader (Spark®, TECAN, Mannedorf, Switzerland).In Cellula emiRFP-mRNA Transfection StudiesAll cell lines were seeded in a 24-well plate (Corning®, Corning, New York) where DU145, PC3, and 22Rvl were grown for 24 hours whereas LNCaP was grown for 48 hours before treatment. emiRFP mRNA-LNP was mixed with 0.1 to 1.0 mol% of COE-S6, vortexed, and allowed to equilibrate for 15 minutes. Treatment of the sample wells (n=3) was conducted and the cells were allowed to grow for 24 hours at 37°C with 5% CO2. Samples were harvested and live cell mean fluorescence intensities and transfection efficiencies were analyzed immediately using a flow cytometer (CytoFLEX, Beckman Coulter, Brea, California).Spheroids Culture and Anticancer StudiesCell lines were seeded in 96-Well plates (Nunclon™ Sphera™, Thermo Fisher Scientific, Waltham, Massachusetts) at a seeding density of IxlO3and were centrifuged at 200- 500 x g for 10 minutes at 4°C. All cell lines were allowed to grow for 72 hours in the incubator at 37°C with 5% CO2. Cell media was changed at the 48-hour time point and treatment with LNPs in opti-MEM began at the 72-hour time point. Upon treatment, spheroids were imaged (CQ1, Yokogawa, Tokyo, Japan) in 24-hour intervals until the 168thhour time point. The diameter of spheroids (n=4) was assessed with ImageJ software (National Institute of Health, United States of America).Apoptosis AssayAll cell lines were seeded in a 6-well plate (Nunc™, Thermo Fisher Scientific, Waltham, Massachusetts) with seeding densities of 4xl05for DU145, 22Rvl cell lines, and 6xl05for LNCaP cell lines. DU145 and 22Rvl cell lines were grown for 24 hours while LNCaP cell line was grown for 48 hours before treatment with LNPs in Opti-MEM at a dose of 1.25 to 2.5ug / mL. LNCaP (n=3), DU145, and 22Rvl samples (n = l) were stained with annexin-Alexa Fluor 488nm and Propidium Iodide (Dead Cell Apoptosis Kit, Invitrogen™, Waltham, Massachusetts) as per manufacturer's protocol and analyzed with flow cytometry (CytoFLEX, Beckman Coulter, Brea, California).Wound Healing AssayAll cell lines were seeded in a 24 well plate (p-Plate 24 Well, Ibidi, Grafelfing, Germany) with seeding densities of IxlO5for DU145, PC3, 22Rvl cell lines and 1.5xl05for LNCaP cell line. DU145, PC3, and 22Rvl cell lines were grown for 24 hours while LNCaP cell line was grown for 48 hours before treatment. Sample wells (n=2) were scratched using a lOOuL pipette tip (Axygen®, Union City, California), washed with Dulbecco's Phosphate Buffered Saline (DPBS) (HyClone, Cytiva, Marlborough, Massachusetts) before treatment at a dose of 1.25 to 2.5ug / mL and immediately imaged (CQ1, Yokogawa, Tokyo, Japan) at time point 0 and 24 hours with 2x magnification. Distance of the wound was assessed with ImageJ software (National Institute of Health, United States of America) and migration rate was calculated : (0-hour distance - 24-hour distance) / 0- hour distance x 100%.Western Blot AnalysisAll cell lines were seeded in a 6-well plate (Nunc™, Thermo Fisher Scientific, Waltham, Massachusetts) with seeding densities of 4xl05. PC3 and 22Rvl cell lines were grown for 24 hours while LNCaP cell line was grown for 48 hours before treatment with LNPs in Opti-MEM at a dose of 1.25 to 2.5ug / mL. Cells were harvested post-treatment and lysed with lysis buffer (RIPA lysis buffer, Thermo Fisher Scientific, Waltham, Massachusetts). Lysates were collected and the protein concentration was quantified using BCA assay (Pierce™ BCA Protein Assay Kits, Thermo Fisher Scientific, Waltham, Massachusetts) following the manufacturer's protocol. Western blot loading samples (n= l) were prepared to a protein concentration of 40ug / 40uL using loading buffer (4X Bolt™ LDS Sample Buffer, Invitrogen™, Waltham, Massachusetts) and deionized water. Samples were then heated for 10 minutes at 70°C. Samples were loaded into a gel (Bolt™ Bis-Tris Plus Mini Protein Gels 4-12%, Thermo Fisher Scientific, Waltham, Massachusetts) and allowed to equilibrate for 10 minutes before running the gel at 150V for 25 minutes on a power supply (PowerEase™ Touch Power Supply, Invitrogen™, Waltham, Massachusetts). The sample gel was then transferred to a blot using a transfer device (IBIot™ 3 Western Blot Transfer Device Invitrogen™, Waltham, Massachusetts) and incubated for 3 hours with the PTEN primary antibody (PTEN Monoclonal Antibody (2F4C9), Invitrogen™, Waltham, Massachusetts) with a 1 :250 dilution and secondary antibody (Goat anti-Mouse IgG Fc Secondary Antibody, Invitrogen™, Waltham, Massachusetts) with a 1: 1000 dilution using the iBind™ device (iBind™ Flex Western Device, Invitrogen™, Waltham, Massachusetts). The blot was then washed twice for 3 minutes with deionized water and brought to incubate in the chemiluminescent substrate (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific, Waltham, Massachusetts) as per manufacturer's protocol. The blot was then imaged, and the density of bands was analyzed with an imaging system (iBright™ CL1500 Imaging System Invitrogen™, Waltham, Massachusetts). GAPDH was used to normalize protein expression of PTEN.SynthesisUnless otherwise stated, all reagents and chemicals were purchased from Sigma-Aldrich or TCI chemicals and were used as received. The 1H NMR spectra were measured on Bruker AV 400 spectrometer in deuterated chloroform or dimethyl sulfoxide (DMSO). Chemical shifts were reported as 6 value (ppm) relative to the solvent peak.l,2-bis(4-bromophenyl)ethene (compound 6-3) To a solution of 4-bromobenzaldehyde (5 g, 27.02 mmol) and diethyl (4- bromobenzyl)phosphonate (9.96 g, 32.43 mmol) in THF (80 mL) was added t-BuOK (1 M, 54 mL) dropwise at 0 °C under Nz and the mixture was stirred at 25 °C for 12 h. TLC (Petroleum ether: Ethyl acetate=5: l) showed the starting material was consumed completely and a new spot was formed. The reaction mixture was quenched by the addition of saturated aqueous NH4CI (50 mL) at 0 °C and then diluted with HzO (500 mL), filtered and the filtrate was washed by EtOAc (100 mL). The filter cake was concentrated under reduced pressure to afford compound 6-3 (8 g, 23.67 mmol) as a white solid.XH NMR (400 MHz, CHLOROFORM-d) 6 = 7.48 (bd, J = 8.0 Hz, 2H), 7.36 (bd, J = 8.4 Hz, 2H), 7.02 (s, 1H).4,4'-(ethene-l,2-diyl)dibenzaldehyde (compound 6)To a solution of l,2-bis(4-bromophenyl)ethene (10.5 g, 31.06 mmol) in THF (200 mL) was added n-BuLi (2.5 M, 43 mL) dropwise at -70 °C under Nz and the mixture was stirred at -70 °C for 3 h. A solution of morpholine-4-carbaldehyde (10.73 g, 93.19 mmol) in THF (20 mL) was added dropwise at -70 °C, and the mixture was warmed to 25 °C and stirred at 25 °C for 2 h. LCMS showed the starting material was consumed completely and the desired mass was detected. The reaction mixture was quenched by the addition of saturated aqueous NH4CI (50 mL) at 0 °C, and then diluted with HzO (100 mL) and extracted with DCM (80 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over NazSC , filtered and concentrated under reduced pressure. The crude product was triturated by EtOAc (50 mL) at 25 °C to afford compound 6 (4 g, 54% yield) as a yellow solid.XH NMR (400 MHz, CHLOROFORM-d) 6 = 10.02 (s, 2H), 7.90 (d, J = 8.4 Hz, 4H), 7.69 (d, J = 8.0 Hz, 4H), 7.29 (s, 2H). tetraethyl (l,4-phenylenebis(methylene))bis(phosphonate) (compound 3)To a solution of 3,4,5-trihydroxybenzaldehyde (50 g, 324.42 mmol) in acetone (1500 mL) was added K2CO3 (269.02 g, 1.95 mol) and 1,6-diiodohexane (500 g, 1.48 mol) at 25 °C and the mixture was stirred at 60 °C for 48 h. LCMS showed the starting material was consumed completely and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove acetone. The residue was diluted with HzO (2 L) and extracted with EtOAc (1 mL x 3). The combined organic layers were washed with brine (300 mL x 2), dried over NazSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (330 g Silica Flash Column, Eluent of 0~l l% Ethyl acetate / Petroleum ether @ 80 mL / min) to afford compound 3 (57 g, 22% yield) as a yellow oil. LCMS ESI [M+H]+ = 785.0;TH NMR (400 MHz, CHLOROFORM-d) 6 = 9.94 - 9.70 (m, 1H), 7.08 (s, 2H), 4.10 - 4.00 (m, 6H), 3.20 (t, J = 7.2 Hz, 6H), 1.90 - 1.72 (m, 12H), 1.58 - 1.44 (m, 12H). diethyl (4-(3,4,5-tris((6-iodohexyl)oxy)styryl)benzyl)phosphonate (compound 5)To a solution of tetraethyl (l,4-phenylenebis(methylene))bis(phosphonate) (38.59 g, 102.00 mmol) and 3,4,5-tris((6-iodohexyl)oxy)benzaldehyde (40 g, 51.00 mmol) in THF (400 mL) was added t-BuOK (1 M, 77 mL) dropwise at 0 °C under N2 and the mixture was stirred at 20 °C for 3 h. LCMS showed the starting material was consumed completely and the desired mass was detected. The reaction mixture was quenched by adding saturated aqueous NH4CI (300 mL) at 0° C, and then diluted with H2O (300 mL) and extracted with DCM (500 mL x 3). The combined organic layers were washed withbrine (100 mL x 2), dried over Na?S04, filtered, and concentrated under reduced pressure. The crude product was triturated with MeCN (200 mL) at 25 °C for 30 min and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (330 g Silica Flash Column, Eluent of 0~55% Ethyl acetate / Petroleum ether @ 80 mL / min) to afford compound 5 (7.5 g,15% yield) as a yellow oil. LCMS ESI [M + H]+ = 1009.1;XH NMR (400 MHz, CHLOROFORM-d) 5 = 7.44 (d, J = 8.0 Hz, 2H), 7.31 - 7.26 (m, 2H), 7.03 - 6.90 (m, 2H), 6.70 (s, 2H), 4.09 - 3.92 (m, 10H), 3.33 - 3.04 (m, 8H), 1.94 - 1.69 (m, 12H), 1.59 - 1.42 (m, 12H), 1.25 (t, J = 7.2 Hz, 6H) l,2-bis(4-((E)4-((E)-3,4,5-tris((6-iodohexyl)oxy)styryl)styryl)phenyl)ethene (compound 7)To a solution of (E)-4,4'-(ethene-l,2-diyl)dibenzaldehyde (500 mg, 2.12 mmol) and diethyl (4-(3,4,5-tris((6-iodohexyl)oxy)styryl)benzyl)phosphonate (5.55 g, 5.50 mmol) in THF (50 mL) was added t-BuOK (1 M, 5 mL) at 0 °C and the mixture was stirred at 20 °C for 12 h. TLC (Dichloromethane : Petroleum ether=3: l) showed the starting material was consumed completely and a new main spot was detected. The reaction mixture was quenched by adding saturated aqueous NH4CI (50 mL) at 0 °C, and then diluted with H2O (30 mL) and extracted with DCM (50 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over NazSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (120 g Silica Flash Column, Eluent of 0~80% DCM / Petroleum ether @ 80 mL / min) to afford compound 7 (2.8 g, 68% yield) as a yellow solid.XH NMR (400 MHz, CHLOROFORM-d) 3 = 7.63 - 7.40 (m, 16H), 7.13 (s, 6H), 7.07 - 6.95 (m, 4H), 6.73 (s, 4H), 4.09 - 3.93 (m, 12H), 3.22 (t, J = 7.2 Hz, 12H), 1.94 - 1.72 (m, 24H), 1.55 - 1.45 (m, 24H).COE-S6To a solution of (E)-l,2-bis(4-((E)-4-((E)-3,4,5-tris((6- iodohexyl)oxy)styryl)styryl)phenyl)ethene (2.8 g, 1.44 mmol) in CHCI3 (50 mL) was added trimethylamine (2 M, 30 mL in MeOH) and the mixture was stirred at 25 °C for 48 h. The reaction mixture was concentrated under reduced pressure. The mixture was dissolved in THF (100 mL) and H2O (20 mL). Trimethylamine (2 M, 36 mL in MeOH) was added and the mixture was stirred at 25 °C for 48 h. LCMS showed the starting material was consumed completely and the desired mass was detected. The reactionmixture was concentrated under reduced pressure. The residue was purified by prep- HPLC (column: Phenomenex Luna C18 200 x 40mm x 10 pm; mobile phase: [water(HCI)-MeCN]; gradient: 10%-40% B over 12 min) to afford the desired product, which contained I and Cl salt). The aqueous solution of the product was transferred to Ion exchange resin (Amberlite® IRA402 (Cl)) to afford the yellow eluent, which was lyophilized to afford the final compound (1.51 g, 73% yield, 96.56% purity, as a single Cl salt) as a yellow solid.(400 MHz, DMSO-d6) 6 = 8.03 - 7.39 (m, 16H), 7.31 (bs, 6H), 7.22 (s, 4H), 6.93 (s, 4H), 4.04 (bt, J = 6.0 Hz, 8H), 3.89 (bt, J = 6.0 Hz, 4H), 3.33 (bd, J = 12 Hz, 12H), 3.08 (s, 54H), 1.82 - 1.64 (m, 24H), 1.56 - 1.46 (m, 12H), 1.41 - 1.29 (m, 12H).Statistical AnalysisThe statistical analyses of data were conducted using unpaired student's t-test or oneway analysis of variance (ANOVA) with Tukey's post-hoc test using Origin. All experiments were repeated at least three times and data are presented as mean ± standard deviation.It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

Claims

Claims1. A lipid nanoparticle, comprising: a) a helper lipid at about 5 mol% to about 80 mol% relative to a total lipid content; b) an ionisable lipid at about 20 mol% to about 95 mol% relative to the total lipid content; c) a conjugated oligoelectrolyte at about 0.005 mol% to about 5 mol% relative to the total lipid content, wherein the conjugated oligoelectrolyte is configured to interact with at least the helper lipid in order to stabilise the lipid nanoparticle.

2. The lipid nanoparticle according to claim 1, wherein the conjugated oligoelectrolyte is configured to interact with a payload within the lipid nanoparticle in order to stabilise the payload.

3. The lipid nanoparticle according to claim 1 or 2, wherein the conjugated oligoelectrolyte is intercalated with at least the helper lipid.

4. The lipid nanoparticle according to any one of claims 1 to 3, wherein the helper lipid is about 10 mol% relative to the total lipid content.

5. The lipid nanoparticle according to any one of claims 1 to 4, wherein the helper lipid is a phospholipid.

6. The lipid nanoparticle according to any one of claims 1 to 5, wherein the helper lipid is a phosphocholine or phophatidylethanolamine.

7. The lipid nanoparticle according to any one of claims 1 to 6, wherein the helper lipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dipalmitoyl-sn-glycero- 3-phosphocholine (DPPC), l,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1- Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or a combination thereof.

8. The lipid nanoparticle according to any one of claims 1 to 7, wherein the ionisable lipid is about 50 mol% relative to the total lipid content.

9. The lipid nanoparticle according to any one of claims 1 to 8, wherein the ionisable lipid is ionisable into a cationic lipid.

10. The lipid nanoparticle according to any one of claims 1 to 9, wherein the ionisable lipid is selected from (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), 8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), and [(4- Hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), 9Z,12Z-octadecadienoic acid, 3-[4,4-bis(octyloxy)-l-oxobutoxy]-2-[[[[3- (diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester (LP01), and a combination thereof.

11. The lipid nanoparticle according to any one of claims 1 to 10, wherein the conjugated oligoelectrolyte is a compound of Formula (I) or a salt or solvate thereof:whereinRi is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy or optionally substituted thio or optionally substituted phosphoryl;R2 is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; m is an integer selected from 1 to 7; n is an integer independently selected from 0 to 4; and q is an integer independently selected from 1 to 5; or a compound of Formula (II) or a salt or solvate thereof:wherein each Ri is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy or optionally substituted thio or optionally substituted phosphoryl; q is an integer selected from 1 to 5; q' is an integer selected from 1 to 5; wherein each l_2 is independently selected from optionally substituted ethylene, or optionally substituted phenylethylene;Li is a n-conjugated core comprising monomeric unit A and monomeric unit D:wherein each A is independently selected from optionally substituted alkenylene, optionally substituted arylene or optionally substituted heteroarylene; each D is independently selected from optionally substituted alkenylene, optionally substituted arylene or optionally substituted heteroarylene; t is an integer selected from 1 to 5; u is an integer selected from 1 to 5; wherein * represents a bond to another monomeric unit or to L2; wherein monomeric units A and monomeric units D are alternatively bonded to each other; wherein the compound of Formula (I) has a substantially linear topology.

12. The lipid nanoparticle according to any one of claims 1 to 11, wherein the conjugated oligoelectrolyte is selected from13. The lipid nanoparticle according to any one of claims 1 to 12, wherein the lipid nanoparticle further comprises steroid at about 20 mol% to about 60 mol% relative to the lipid nanoparticle.

14. The lipid nanoparticle according to any one of claims 1 to 13, wherein the steroid is selected from cholesterol, fecosterol, sitosterol, ergosterol, campersterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol or a derivative thereof.

15. The lipid nanoparticle according to any one of claims 1 to 14, wherein the lipid nanoparticle further comprises a polymer conjugated lipid about 0.5 mol% to about 2 mol% relative to the lipid nanoparticle.

16. The lipid nanoparticle according to any one of claims 1 to 15, wherein the polymer is polyethylene glycol (PEG) or polysarcosine.

17. The lipid nanoparticle according to any one of claims 1 to 16, wherein the lipid nanoparticle is characterised by an average particle size of about 50 nm to about 250 nm.

18. The lipid nanoparticle according to any one of claims 1 to 17, wherein the lipid nanoparticle is characterised by a photoluminescence lifetime of at least 0.5 ns.

19. The lipid nanoparticle according to any one of claims 1 to 18, wherein the lipid nanoparticle is characterised by a photoluminescence fold increase of at least 2 relative to free conjugated oligoelectrolyte.

20. The lipid nanoparticle according to any one of claims 1 to 19, further comprising a payload in a cavity of the lipid nanoparticle.

21. The lipid nanoparticle according to claim 20, wherein the payload is characterised by an anionic charge.

22. The lipid nanoparticle according to claim 20 or 21, wherein the payload is selected from a nucleic acid, a drug, a protein or a combination thereof.

23. The lipid nanoparticle according to any one of claims 20 to 22, wherein the payload is selected from mRNA, nucleoside-modified mRNA, plasmid DNA, DNA, or a combination thereof.

24. The lipid nanoparticle according to any one of claims 1 to 23, wherein the lipid nanoparticle is characterised by a COE to polynucleotide nitrogen-to-phosphate (N / P) ratio of less than 2, and a total N / P ratio of more than 0.5.

25. The lipid nanoparticle according to any one of claims 1 to 24, wherein the lipid nanoparticle is characterised by a payload encapsulation efficiency of more than about 40%.

26. A method of detecting a lipid nanoparticle according to any one of claims 1 to 25 in a cell, comprising imaging the cell using a fluorescence detector.

27. A method of fabricating a lipid nanoparticle, comprising: a) mixing a helper lipid and an ionisable lipid in a solvent to form a mixture; b) introducing the mixture of step a) into a non-solvent in order to fabricate a precursor lipid nanoparticle; and c) incubating a conjugated oligoelectrolyte with the precursor lipid nanoparticle of step b) to form the lipid nanoparticle; wherein the lipid is about 5 mol% to about 80 mol% relative to the mixture; wherein the ionisable lipid is about 20 mol% to about 95 mol% relative to the mixture; and wherein the conjugated oligoelectrolyte is about 0.005 mol% to about 5 mol% relative to the mixture, wherein the conjugated oligoelectrolyte is configured to interact with the helper lipid in order to stabilise the lipid nanoparticle.

28. A method of fabricating a lipid nanoparticle, comprising: a) mixing a helper lipid, an ionisable lipid and a conjugated oligoelectrolytes in a solvent to form a mixture; b) introducing the mixture of step a) into a non-solvent in order to fabricate a lipid nanoparticle; and wherein the lipid is about 5 mol% to about 80 mol% relative to the mixture; wherein the ionisable lipid is about 20 mol% to about 95 mol% relative to the mixture; and wherein the conjugated oligoelectrolyte is about 0.005 mol% to about 5 mol% relative to the mixture, wherein the conjugated oligoelectrolyte is configured to interact with the helper lipid in order to stabilise the lipid nanoparticle.

29. A lipid nanoparticle composition, comprising a lipid nanoparticle according to any one of claims 1 to 25 and at least one excipient.

30. A kit for forming a lipid nanoparticle, comprising: a) a helper lipid at about 5 mol% to about 80 mol% relative to a total lipid content; b) an ionisable lipid at about 20 mol% to about 95 mol% relative to the total lipid content; c) a conjugated oligoelectrolyte at about 0.005 mol% to about 5 mol% relative to the total lipid content, the conjugated oligoelectrolyte for interacting with the helper lipid in order to stabilise the lipid nanoparticle.

31. A method of stabilising a lipid nanoparticle formulation, comprising mixing a conjugated oligoelectrolyte with the lipid nanoparticle formulation, wherein the conjugated oligoelectrolyte is about 0.005 mol% to about 5 mol% relative to the lipid nanoparticle formulation, wherein the conjugated oligoelectrolyte is configured to interact with a helper lipid of the lipid nanoparticle formulation in order to stabilise the lipid nanoparticle.

32. The method according to claim 31, wherein the lipid nanoparticle formulation is selected from Comirnaty and Spikevax.

33. A method of delivering a payload to a cell, comprising incubating the cell with a lipid nanoparticle according to any one of claims 1 to 25.

34. The method according to 33, wherein the method is characterised by a cell viability more than 50%.

35. The method according to 33 or 34, wherein the lipid nanoparticle is characterised by an in vitro delivery efficiency of more than about 5%.

36. The method according to any one of claims 33 to 35, wherein when the payload is a nucleic acid, the method is characterised by a transfection efficiency more than 25% relative to a lipid nanoparticle without the conjugated oligoelectrolyte.

37. A method of delivering a therapeutic payload to a subject in need thereof, comprising administering a therapeutic amount of lipid nanoparticle according to any one of claims 1 to 25 to the subject, wherein the lipid nanoparticle comprises the therapeutic payload.

38. A lipid nanoparticle according to any one of claims 1 to 25for use in delivering a therapeutic payload to prevent and / or treat a disease or condition.

39. Use of a lipid nanoparticle according to any one of claims 1 to 25in the manufacture of a medicament for delivering a therapeutic payload to prevent and / or treat a disease or condition.

40. The method, lipid nanoparticle for use or use according to any one of claims 37 to 39, wherein the disease or condition is selected from a viral disease or condition, and cancer.

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