Lipopolymers and synthesis thereof

Abstract

According to an example aspect of the present invention, there is provided a lipopolymer comprising at least one lipid-like moiety, connected to at least one polymer moiety via a triazole moiety, as well as a method for preparing said lipopolymer via an uncatalyzed reaction of a compound comprising an alkyne moiety with a compound comprising an azide moiety, and a use of the lipopolymers in a drug delivery system.

Description

LIPOPOLYMERS AND SYNTHESIS THEREOFFIELD

[0001] The present invention relates to lipopolymers, methods for the synthesis of lipopolymers and their use in liposomes and lipid nanoparticles.BACKGROUND

[0002] Lipopolymers are commonly referred to as hydrophilic polymers linked to a hydrophobic lipid or a lipid-like moiety. They are commonly used in liposomes for drug delivery or in lipid nanoparticles (LNPs) for gene delivery with the most prominent example being the use of LNPs in the very successful mRNA based COVID-19 vaccines. In general, drugs that have poor solubility or otherwise are sub-optimally distributed within a subject when administered as such may benefit substantially from liposomes as a delivery vehicle.

[0003] As mentioned, a common use for LNPs is the delivery of therapeutic nucleic acid strands, such as RNA, to their target cells. LNPs can protect the RNA in a biological environment from enzymatic degradation. The LNPs can be further combined with molecules allowing a specific targeting and release of the payload to the intended target. Due to their exceptionally successful adaptation for nucleic acid delivery in RNA-based COVID- 19 vaccines, great interest in the use and methods for preparing LNPs for various biomedical applications has recently emerged in the scientific community.

[0004] Currently, precise methods for modifying the chemical and physical properties of liposomes are not well characterized. For example, the mechanical stability is low, which results from their low membrane thickness and high permeability to many encapsulated compounds. These drawbacks are highlighted when liposomes are subjected to a complex and potentially challenging environment that exist, for example inside a living organism.

[0005] It is clear that there is a great need for further control of the physical and chemical properties of liposomes and LNPs to enable further uses in new biomedical applications as well as enhance the usability of the existing applications further. Onechallenge in the field of liposome and LNP development is the limited availability of new lipopolymers for fine tuning the properties of LNPs and liposomes.

[0006] One way of preparing lipopolymers is to introduce the lipid-like moiety to the polymer during polymerization. However, this method is obviously limited and is applicable for only a limited range of polymers (Simon L. et al. 2020). Furthermore, the initiation and termination efficiencies can suffer from the presence of the lipid-like moieties, leading to a less defined heterogenous mixture of polymers having highly variable lengths. In addition, synthetic procedure may be affected by the presence of the lipid-like moiety.

[0007] Another preparation method for lipopolymers is the post-polymerization method, wherein a pre-made polymer is conjugated with a lipid-like moiety after the polymerization process has been completed. This allows a more robust method for introducing lipid-like moieties to polymers, without affecting, limiting or compromising the structural characteristics of the polymer product. Furthermore, this method allows the modification of virtually any polymer having a suitable chemical handle for the conjugation of lipid-like moieties.

[0008] One commonly used post-polymerization method for obtaining lipopolymers is a coupling reaction with commercially available lipids such as DSPE. The modification generally starts from a hydroxyl end-group of the polymer which can be used for various functionalization reactions, one example being the reaction with succinic anhydride to obtain a carboxylic acid end-group. This can then be activated in order to react with the amine of the DSPE to form a lipopolymer (Wang R. et al. 2012).

[0009] These methods, however, require several method steps and harsh reaction conditions resulting in poor selectivity and susceptibility to side reactions. Furthermore, it requires the use of an additional linker, such as succinic anhydride.

[0010] Thus, there is still a need for improvement in the preparation of lipopolymers.SUMMARY OF THE INVENTION

[0011] The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims.

[0012] One aspect of the present invention provides lipopolymers that are suitable for biomedical applications. Thus, metal ions are avoided in their preparation.

[0013] In another aspect, there is provided a method for the synthesis of such lipopolymers using a post-polymerization click reaction.

[0014] In a preferred aspect, this click-reaction of the invention is designed to proceed without the commonly used copper catalysis.

[0015] Thus, the present invention relates to a lipopolymer comprising: a. at least one lipid-like moiety, b. at least one polymer moiety, wherein the at least one lipid-like moiety is connected to the at least one polymer moiety via a triazole moiety.

[0016] Further, the invention relates to a post-polymerization method for obtaining the lipopolymer disclosed above, wherein the method comprises: a. contacting a compound comprising an azide moiety with a compound comprising an alkyne moiety, one of these compounds being a polymeric compound and the other compound being a lipid-like compound, and b. allowing a reaction between the alkyne moiety and the azide moiety to proceed to obtain the lipopolymer.

[0017] Additionally, the invention relates to the use of the lipopolymers of the present invention in a drug delivery system. This use increases the stability of the drug delivery system, particularly when the drug delivery system is a liposome or a LNP.

[0018] Thus, the invention utilizes a novel modification of the well-known azidealkyne click reaction, and introduces a post-polymerization reaction method and the essential reagents for preparing lipopolymers. The reaction speed has been significantly increased by employing a highly electron deficient compound bearing at least one lipid-like moiety, for example dioctadecyl but-2-ynedioate comprising two linear lipid-like moieties and an electron-deficient alkyne moiety. This enables, for example, the efficient introduction of lipid-like moieties to polymers under mild conditions without the need for a copper catalyst.The method can be applied to a wide range of polymers since the only requirement for the polymer is the possibility to introduce an azide group or an alkyne group to one end of the polymer chain, which can be achieved for most if not all known polymers. The electrondeficient compound, typically an alkyne diester group will thus carry either the polymer, or, preferably the lipid-like moiety or moieties to form a lipopolymer in a reaction that has even more versatility as compared to known mechanisms for the synthesis of lipopolymers.

[0019] A known alternative to the conjugation method proposed herein is the known copper-catalyzed azide-alkyne click reaction. However, copper-ions used as catalysts in this method are cytotoxic and must be removed from the reaction mixture to ensure these ions are not present in the subsequent therapeutic or diagnostic composition. This purification process is not trivial and will contribute significantly to the total effort needed for obtaining said composition. The well-known dibenzocyclooctyne (DBCO) and bicyclononyne (BCN) moieties, used in some known copper-free reactions, have limited cyclic structures and lack the diester moiety and are not readily modified with lipid-like moieties as in the present invention. Thus, they are unsuitable for use in the herein described reactions. Additionally, the large size and high hydrophobicity of DBCO and BCN will have a significant steric and electronic influence on the resulting products, a problem avoided with the electron-deficient alkyne moiety disclosed here.

[0020] The present invention results in considerable advantages. Among others, the simple and modular approach of the present invention for combining hydrophilic polymers to lipid-like compounds provides a substantial increase in the number of options for the development of new lipopolymers for use in fine-tuning the properties of lipopolymers and LNPs for specific biomedical and biotechnological uses. Furthermore, due to the simple routine procedures of and fast reaction rates, a significant improvement in the time it takes to synthesise new lipopolymers is achieved. Consequently, libraries of lipopolymers and respective libraries of lipopolymers and LNPs comprising said lipopolymers become possible to construct and characterize in a high throughput manner.

[0021] In summary, the presented method and reagents provide a versatile, efficient and highly compatible pathway for the synthesis of lipopolymers for biomedical applications, which are currently in very high demand.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGURE 1 shows the reaction equation of the coupling reaction of the invention using different lipid-like compounds.

[0023] FIGURE 2 compares the lipopolymer of the invention to commercially available lipopolymers, with the compared structures shown in Fig. 2A, the relative metabolic cell viability of fibroblasts after being incubated with the lipopolymer solutions shown in Fig. 2B, and the percentage of survival of Zebrafishes in in vivo toxicity studies shown in Fig. 2C.

[0024] FIGURE 3 shows the structures of the lipopolymers obtained by carrying out coupling reactions using 6 different polymers and 2 different structures of lipid-like compounds.EMBODIMENTS

[0025] DEFINITIONSIn the present context, the term “lipopolymer” refers to an amphiphilic macromolecule comprising at least one lipid-like hydrophobic moiety and at least one hydrophilic or substantially water-soluble amphiphilic polymer moiety.The term “lipid-like moiety” refers to any hydrophobic hydrocarbon having linear, branched, cyclic or at least partially cyclic or polycyclic structure, or a combination thereof, particularly such esterified hydrocarbons. Typically, the hydrophobic character of such hydrocarbon structures is based on, among others, a relative lack of heteroatoms therein and the length of the hydrocarbon chains. In general, such natural, synthetic or semi-synthetic lipid-like moieties are for example: fatty acyls, saturated fatty acyls, unsaturated fatty acyls, prenols, isoprenols, and saccharo lipids. The molecular weight of the said lipid-like moieties is less than 3000 Da, for example less than 1500 Da, while they typically contain at least 3 carbon atoms.The term “polymer moiety” refers to a synthetic, semi-synthetic, or bioderived polymeric moiety, which is a homopolymer or a copolymer, for example an alternating copolymer, a random copolymer, a block copolymer or a graft copolymer. The polymer moiety may have a branched structure. In some embodiments, the polymer moiety is amphiphilic, i.e. at least partly hydrophilic, preferably at least 50 % of the 3D polymer structure is hydrophilic, at least at neutral or close to neutral pH within ranges of 4 - 9.A post-polymerization reaction refers to a method of preparing a lipopolymer using a modular approach, wherein the polymer having the desired properties is pre-synthesised and subsequently conjugated to a lipid-like component (moiety) to obtain the lipopolymer.

[0026] The present invention relates to lipopolymers, methods to obtain said lipopolymers and use of said lipopolymers in liposomes and LNPs.The lipopolymers of the present invention comprise at least one lipid-like moiety and at least one polymer moiety, preferably two lipid-like moieties and one polymer moiety, or two polymer moieties. The two lipid-like moieties or the two polymer moieties may have different structures or they may have the same structure. Furthermore, the at least one lipid- like moiety comprises at least 3 carbons, preferably at least 10 carbons (or 10 or more), such as 10-21 carbons, more preferably 12-21 carbons, most preferably 14 or 18 carbons.

[0027] The at least one polymer moiety is connected to the at least one lipid-like moiety via a triazole moiety.

[0028] Particularly, the triazole moiety is further connected to one or more ester moieties, or preferably a diester moiety whereby the two carbons in the triazole ring are each directly connected to the carbonyl carbons of a neighbouring ester moiety. Thus, according to this alternative, one or more lipid-like moiety or polymer moiety is connected to the triazole moiety via an ester moiety.

[0029] In one embodiment, the at least one lipid-like moiety is independently selected from the group of aliphatic alkyl chains, cyclic alkyls, branched alkyl chains, sterols, fatty acyls, saturated fatty acyls, unsaturated fatty acyls, prenols, isoprenols, saccharolipids andpolyketides, or alternatively from the group of aromatic compounds. Preferably, in a lipopolymer comprising two lipid-like moieties, the two lipid-like moieties are both selected from the group of aliphatic alkyl chains, cyclic alkyls, branched alkyl chains, sterols, fatty acyls, saturated fatty acyls, unsaturated fatty acyls, prenols, isoprenols, saccharolipids and polyketides, and most preferably, they have the same structure.

[0030] In a preferred embodiment, the lipid-like moieties are each independently selected from hydrophobic alternatives of the herein listed moieties, such as of the aliphatic alkyl chains, cyclic alkyls, branched alkyl chains, sterols, fatty acyls, saturated fatty acyls, unsaturated fatty acyls, prenols, isoprenols, saccharolipids, polyketides or aromatic compounds, while including one or more hydrophobic hydrocarbon chains or cyclic hydrocarbon structures, these hydrophobic chains or structures preferably lacking hydrophilic functional groups, such as hydroxyl, carbonyl, carboxyl, amino or sulfhydryl groups. The preferred length of these hydrocarbon chains or cyclic structures is based on the number of carbon atoms therein, as indicated above, thus particularly including 3 or more carbons, or more preferably 10 or more carbons.

[0031] Some simple hydrocarbon structures thus include linear, branched or cyclic propyl butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and undecyl moieties, while preferred structures include linear, branched or cyclic dodecyl moieties (Cl 2), tridecyl moieties (Cl 3), tetradecyl moieties (C14), pentadecyl moieties (Cl 5), hexadecyl moieties (Cl 6), heptadecyl moieties (Cl 7), octadecyl moieties (C 18), nonadecyl moieties (Cl 9), icosyl moieties (C20), and henicosyl moieties (C21).

[0032] In one embodiment, the triazole moiety is a result of a reaction between an azide and an alkyne, wherein said azide group has been introduced to the reaction as part of a polymeric compound or as part of a compound comprising the lipid-like moiety, and said alkyne group has been introduced to the reaction as part of the remaining compound, i.e. thepolymeric compound or the compound comprising the lipid-like moiety. Further, the alkyne is preferably present as part of an alkyne diester moiety.

[0033] In one embodiment, the at least one polymer moiety of the lipopolymer is a hydrophilic polymer moiety.

[0034] Thus, in some embodiments, the polymer moiety of the present invention may have a structure comprising electronegative groups, such as hydroxyl groups or amide groups. Alternatively or additionally, the polymer moiety may have a positive or a negative charge.

[0035] In some embodiments, the at least one polymer moiety is selected from the group of polyethylene glycol or poly(ethylene oxide), poly(2-methyl-2-oxazoline), poly(2- ethyl-2-oxazoline), poly(2-(3 ’-hydroxypropyl)-2-oxazoline), poly(2-methyl-2 -oxazine), poly(2-ethyl-2 -oxazine), poly(2-methyl-4-methyl-2 -oxazine), polysarcosine, poly(N- methyl-beta-alanine), polyhydroxyethylacrylamide, polyhydroxypropylmethacrylamide, polymethacrylamide, polyacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, poly(N-acryloyl morpholine, hydroxymethylcellulose, , poly(vinyl alcohol), hydroxyethylcellulose polymethylsiloxane, polyethyleneimine, poly(vinyl pyrrolidone), polyaspartamide, poly(vinylmethyl ether), dextran, polyphosphoester, polyphosphazene, poly(lipoic acid), poly(lipoamide), poly(N,N- dialkyllipoamide) and hydrophilic polypeptides and peptide sequences such as poly(L- glutamic acid). Similarly, in some embodiments, the at least one polymer moiety is selected from the group of poly(2-methoxymethyl-2-oxazoline), poly(2-methoxymethyl-2 -oxazine). Preferably, in a lipopolymer comprising two or more polymer moieties, these are all selected, independently, from the same list of polymer moieties.

[0036] In one embodiment of the invention, the polymer moiety is an amphiphilic polymer moiety that can be either a copolymer or a homopolymer, wherein the copolymer comprises a mixture of monomers, for example monomers comprising homopolymers previously mentioned. However, the polymer preferably comprises hydrophobic monomers only to such an extent that the polymer still exhibits substantial water solubility. Typically, the polymer moiety comprises at least 20%, preferably at least 40%, more preferably at least 60%, most preferably at least 80% of said hydrophilic monomers.

[0037] Examples of particularly preferred polymers include poly(sarcosine) (pSar), poly(2-methyl-2-oxazoline) (PMeOx), poly(ethylene glycol) (PEG), poly(2-ethyl-2- oxazoline) (PEtOx), poly(2-methyl-2 -oxazine) (PMeOzi), poly(N,N-dimethyl acrylamide) (PDMA), poly(2-methoxymethyl-2-oxazoline), poly(2-methoxymethyl-2 -oxazine).

[0038] In some embodiments, the hydrophilic polymer may be conjugated with a diagnostic or a therapeutic compound or with an intermediate conjugate, such as streptavidin, avidin or biotin or another affinity tag, peptide sequence with affinity to a biological entity, or a radionuclide chelator. Alternatively, the hydrophilic polymer may be conjugated with a fluorophore / fluorescent dye.

[0039] In one embodiment, the generic structure of the lipopolymer of the invention is according to Formula 1 :wherein at least one of R1, R2and R3is selected from the group category of lipid-like moieties, and at least one of R1, R2and R3is selected from the group category of polymer moieties. Typically, R3is selected from a different group category than R1and / or R2, whereas R1and R2belong to the same group category. Thus, one of R1and R2can be hydrogen, or a short hydrocarbon of <3 carbon atoms, such as a methyl or ethyl group.

[0040] In a preferred embodiment, R1, R2and R3are all selected from the group categories of lipid-like moieties and polymer moieties, with the exception that one of R1and R2can be hydrogen, whereby R1and R2belong to the same group category, whereas R3belongs to a different group category than R1and R2.

[0041] Thus, it is preferred that R1and R2belong to the same group category, except if one of R1and R2is hydrogen, whereby only the other one of R1and R2is a lipid-like moiety or a polymer.

[0042] Preferably, R1and R2are identical, thus providing symmetrical ester structures, an example including a 4-methylumbelliferon structure (C21) on both ester linkages of a fluorescent alkyne reagent shown in the following structure of Formula (2):

[0043] This fluorescent alkyne lipid reagent would yield a fluorescently labelled lipopolymer, when coupled with a polymer comprising an azide group.

[0044] However, it is also possible to obtain structures having interesting properties using asymmetrical ester options. For example, the following structure of Formula 3, including a tetradecyl chain (Cl 4) on one ester linkage and a 4-methylumbelliferon structure (C21) on the other ester linkage is still a fluorescent lipid structure, while being asymmetrical, and would yield a fluorescently labelled lipopolymer, when coupled with a polymer comprising an azide group.

[0045] In one preferred embodiment, R1represents a lipid-like moiety of the present disclosure, R2represents a second lipid-like moiety of the present disclosure, and R3represents a polymer moiety described herein.

[0046] In another preferred embodiment, R1represents a first polymer moiety of the present disclosure, R2represents a second polymer moiety of the present disclosure, and R3represents a lipid-like moiety of the present disclosure.

[0047] Most suitably, R1and R2represent lipid-like moieties, whereas R3represents a polymer moiety.

[0048] In an embodiment, the lipopolymer is obtained using a post polymerization modification method employing a molecule comprising a polymer moiety and another molecule comprising a lipid- or lipid-like moiety.

[0049] The lipopolymer herein described can be obtained for example using the postpolymerization method herein described, comprising the steps of: a. contacting a compound comprising an azide moiety with a compound comprising an alkyne moiety, one of these compounds being a polymeric compound and the other compound comprising at least one lipid-like moiety, and b. allowing a reaction between the alkyne moiety and the azide moiety to proceed to obtain the lipopolymer.

[0050] The reaction between the alkyne and azide having said moieties attached to them, facilitates the uncatalyzed formation of a triazole bond.

[0051] According to one alternative, the method could comprise the steps of: a. contacting a compound comprising an azide moiety, bonded to a polymer, with a compound comprising an alkyne diester moiety with at least one lipid- like moiety, and b. allowing a reaction between the alkyne diester moiety and the azide moiety to proceed.

[0052] According to a second alternative, the method could comprise the steps of: a. contacting a compound comprising an azide moiety, bonded to a lipid-like moiety, with a compound comprising an alkyne diester moiety with at least one hydrophilic polymer moiety, and b. allowing a reaction between the alkyne diester moiety and the azide moiety to proceed.

[0053] Most suitably, a lipid-like compound with an alkyne diester moiety is reacted with a polymeric compound having an azide moiety.

[0054] In one embodiment, the alkyne moiety and the azide moiety are introduced to the contacting step in equivalent molar amounts or with an excess of up to five-fold of eithermoiety. Preferably, the alkyne moiety to azide moiety molar ratio is 5:1 to 1 :1. More preferably, the alkyne moiety to azide moiety molar ratio is 3:1 to 1 :1, for example, the alkyne moiety to azide moiety molar ratio can be about 2:1.

[0055] In one embodiment of the post-polymerization method, the preferred solvent is trichloromethane (CHCh).

[0056] In one embodiment, the reaction between the alkyne moiety and the azide moiety takes place at an elevated temperature, such as at a temperature of > 30 °C, preferably at a temperature below the boiling point of the solvent, more preferably at a temperature of 40 - 60 °C, most preferably at a temperature of 50 - 60 °C, for example at a temperature of about 55 °C.

[0057] In a further embodiment, the reaction between the alkyne moiety and the azide moiety is allowed to proceed for at least 4 hours, preferably 6 - 120 hours, more preferably for 16 - 96 hours, for example for 72 hours.

[0058] In a specific embodiment, the post-polymerization method is performed according the reaction of Equation 1 :

[0059] Thus, the coupling reaction, or the post-polymerization method, is carried out between a compound comprising an azide moiety with a compound comprising an alkyne moiety, one of these compounds being a polymeric compound and the other compound comprising at least one lipid-like moiety. Thus, as shown in Equation 1 , one preferred option is to carry out the coupling reaction between a polymer compound comprising an azide moiety in the end of a polymer chain, and a lipid-like compound comprising an alkyne moiety and two hydrophobic hydrocarbon end-groups. The reaction can be carried out for example as shown in Equation 1 , by reacting the reactant compounds in a trichloromethane (CHCh) solvent at a temperature of 55°C for 16h. However, the reaction conditions mentioned herein and the lengths of the hydrocarbon chains are merely intended as anexample, and the reaction is suitable for use in the whole scope defined in the present disclosure.

[0060] The lipopolymers described herein, or the lipopolymers prepared by the method described herein are highly suitable for use e.g. in liposomes and LNPs or other lipid-layer based structures.

[0061] For example, the lipopolymers of the present invention may be used in liposomes as an additional component. The lipopolymers may provide beneficial properties to the liposomes of the present invention. Said properties are mostly related to stability of the liposome, preferably to stability in vivo. In a specific embodiment, the liposomes comprising lipopolymers of the present invention are used for delivering components, such as drugs or proteins, to the desired site of action for treating a subject, plants, fungi or a mammalian subject, preferably a human subject.

[0062] In one embodiment, the diameter of the liposomes is 50 to 500 nm, preferably, the diameter is 100 to 400 nm, more preferably, the diameter of the liposomes is 200 to 400 nm.

[0063] Liposome refers to a spherical self-assembled structure comprising at least one lipid bilayer, wherein the self-assembled structure is typically in an aqueous environment. The lipid bilayer of a liposome comprises mostly natural or synthetic lipids.

[0064] In some embodiments, the methods of this disclosure can be used for preparing a plurality of various lipopolymers having a plurality of structures, for example a library of lipopolymers, wherein the structures of the first lipid-like moiety, the second lipid-like moiety and the hydrophilic polymer moiety are within the scope defined by the present disclosure. In a further embodiment, these libraries are obtained and characterized in a high throughput manner due to the benefits provided by the present invention, therefore allowing fast search and characterization of structures within a lipopolymer structure space of interest.

[0065] In one embodiment, a liposome comprising a lipopolymer of the present invention may be used in a drug delivery system, cell membrane mimicking structures or in micro- or nano-reactors. In a preferred embodiment, the lipopolymer of the present invention is used in a drug delivery system, such as in a liposome comprising the lipopolymer of the present invention, for example said liposome is used for treating a subject, such as a human subject.

[0066] The terms “micro-reactor” and “nano-reactor” refers to a small confined volume enveloped by a liposome, wherein said volume comprises an isolated environment for running chemical reactions in an optimized manner.

[0067] The cell membrane mimicking structures are structures that may constitute at least a part of an artificial or a semi-artificial cell structure.

[0068] Alternatively, the lipopolymers of the present invention may be used in lipid nanoparticles. In a specific embodiment, the lipid nanoparticles comprising lipopolymers of the present invention are used for delivering genetic material, such as RNA, DNA or gene editing tools, to the desired site of action in a subject, plant, fungi or a mammalian subject, preferably a human subject.

[0069] Thus, the lipopolymers of the present disclosure may be used in drug delivery systems, such as liposomes or lipid nanoparticles, for example for the delivery of components, such as drugs or proteins, or alternatively genetic material, such as RNA, DNA or gene editing tools, to the desired site of action in a subject, plant, fungi or a mammalian subject, preferably a human subject

[0070] In one embodiment, the diameter of the lipid nanoparticles is 50 to 500 nm, preferably, the diameter is 100 to 400 nm, and more preferably, the diameter is 200 to 400 nm.

[0071] The drug delivery system refers to macro- or supramolecular structures used for encapsulating, adsorbing or otherwise capturing a therapeutic molecule inside of or onto said macro- or supramolecular structure. These supramolecular structures typically comprise the above described liposomes and / or LNPs. Such a drug delivery system may provide protection from degradation or a targeted delivery vehicle for the therapeutic molecule.

[0072] It is to be understood that the embodiments of the invention disclosed are not limited to the particular structures, process steps, or materials disclosed herein, but are extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0073] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described inconnection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.

[0074] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. In addition, various embodiments and example of the present invention may be referred to herein along with alternatives for the various components thereof. It is understood that such embodiments, examples, and alternatives are not to be construed as de facto equivalents of one another, but are to be considered as separate and autonomous representations of the present invention.

[0075] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as examples of molecular weights, monomeric unit composition, chemical structures, etc., to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

[0076] While the forgoing examples are illustrative of the principles of the present invention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the claims set forth below.

[0077] The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwiseexplicitly stated. Furthermore, it is to be understood that the use of "a" or "an", i.e. a singular form, throughout this document does not exclude a plurality.EXAMPLESExample 1 - Carrying out the coupling reaction of the invention using different lipid-like compounds, and analyzing the product lipopolymers

[0078] The coupling reaction, or the post-polymerization modification method for obtaining the lipopolymer of the invention, was performed using a PEG polymer with an azide moiety, and with 3 different symmetrical alkyne lipids (to give lipopolymers of Formula 1 with R1=R2) as shown in the equation of Fig. 1, with lipid moiety chain lengths of 12, 14 and 18 carbons. The reactions were all carried out as described in Equation 1, by reacting the reactant compounds in a trichloromethane (CHCF) solvent at a temperature of 55 °C for 16h.

[0079] The toxicity of the product lipopolymer (or “PEG lipid”), containing the triazole linker structure, was compared to commercially available lipopolymers. The structures and names of the different lipopolymers are shown in Fig. 2A.

[0080] Fig. 2B shows the relative metabolic cell viability of fibroblasts after being incubated with different concentrations of lipopolymer solutions for 72 hours. While we see pronounced cytotoxicity for the DSPE PEG at 10 mg / mE, the PEG lipid of the invention and the DSG PEG only show similar toxicity at 20 mg / mL. Importantly, we could not see a significant difference between the DSG PEG and the PEG lipid of the invention.

[0081] Fig. 2C shows the percentage of survival of Zebrafishes in a small in vivo toxicity study. DSPE PEG kills 100% of the Zebrafish embryo at 10 mg / mL. For the PEG lipid of the invention and for the DSG PEG, we could not observe a concentration dependent toxicity.

[0082] Overall, this indicates suitable cyto- and biocompatibility of the triazole linker structure used for the lipopolymers of the invention for biomedical applications.Example 2 - Carrying out the coupling reaction of the invention using different polymer compounds

[0083] The coupling reaction, or the post-polymerization modification method for obtaining the lipopolymer of the invention, was performed using 2 different symmetrical lipid- like compounds (C18 and C14), and 6 different polymers, the polymers comprising the azide moiety, and the lipid-like moieties being included in the alkyne compounds. The reactions were again all carried out as described in Equation 1 , by reacting the reactant compounds in a trichloromethane (CHCh) solvent at a temperature of 55 °C for 16h.

[0084] The used polymers were poly(sarcosine) (pSar), poly(2-methyl-2-oxazoline) (PMeOx), poly(ethylene glycol) (PEG), poly(2-ethyl-2-oxazoline) (PEtOx), poly(2-methyl- 2-oxazine) (PMeOzi), poly(N,N-dimethyl acrylamide) (PDMA). Their structures are shown in Fig. 3. All of these polymers are known to be relatively biocompatible, and all combinations of polymer compounds and lipid-like compounds led to quantitative coupling efficiencies.INDUSTRIAL APPLICABILITY

[0085] The lipopolymers described above can be used for example in drug delivery systems, such as liposomes or lipid nanoparticles, for which they increase the stability.ACRONYMS LISTLNP lipid nanoparticleDBCO dibenzocyclooctyneBCN bicyclononynePDMS poly(dimethylsiloxane)PIB poly(isobutylene)PEO poly(ethylene oxide)CITATIONSReimhult E, Virk MM. Hybrid lipopolymer vesicle drug delivery and release systems. J Biomed Res. 2021 Mar 23;35(4):301 -309. doi: 10.7555 / JBR.35.20200206. PMID: 34421006; PMCID: PMC8383167.Simon L, Marcotte N, Devoisselle J.M., Begu S. Lapinte V., Recent advances and prospects in nano drug delivery systems using lipopolyoxazolines, International Journal of Pharmaceutics, Vol. 585, 2020, 119536Wang R, Xiao R, Zeng Z, Xu L, Wang J, Application of poly(ethylene glycol)- distearoylphosphatidylethanolamine (PEG-DSPE) block copolymers and their derivatives as nanomaterials in drug delivery, International Journal of Nanomedicine 2012: 7, 4185- 4198

Claims

CLAIMS:

1. A lipopolymer comprising: a. at least one lipid-like moiety, b. at least one polymer moiety, characterized in that the at least one lipid-like moiety is connected to the at least one polymer moiety via a triazole moiety.

2. The lipopolymer according to claim 1, wherein the at least one lipid-like moiety comprises at least 3 carbons, preferably at least 10 carbons, more preferably 12 - 21 carbons, most preferably 14 or 18 carbons.

3. The lipopolymer according to any of the previous claims, wherein any one of the lipid- like moieties are independently selected from the group of aliphatic alkyl chains, cyclic alkyls, branched alkyl chains, sterols, fatty acyls, saturated fatty acyls, unsaturated fatty acyls, prenols, isoprenols, saccharolipids and polyketides.

4. The lipopolymer according to any of the previous claims, wherein the polymer moiety is a hydrophilic or substantially water-soluble amphiphilic polymer.

5. The lipopolymer according to any of the previous claims, wherein the triazole is connected to a diester moiety, which in turn is connected to either at least one lipid-like moiety or at least one polymer moiety, the diester moiety preferably being connected to at least one lipid-like moiety.

6. The lipopolymer according to any of the previous claims, having the general structure:wherein R1, R2and R3are all selected from the group categories of lipid-like moieties and polymer moieties, with the exception that one of R1and R2can be hydrogen, whereby R1and R2belong to the same group category, whereas R3belongs to a different group category than R1and R2.

7. A post-polymerization modification method for obtaining the lipopolymer of any of the previous claims, characterized by comprising: a. contacting a compound comprising an azide moiety with a compound comprising an alkyne moiety, one of these compounds being a polymeric compound and the other compound comprising at least one lipid-like moiety, thus facilitating an uncatalyzed reaction between the azide and alkyne moieties, and b. allowing a reaction forming a triazole between the alkyne moiety and the azide moiety to proceed to obtain the lipopolymer.

8. The method according to claim 7, wherein the alkyne moiety is an alkyne diester moiety.

9. The method according to claim 7 or 8, wherein the compound comprising the alkyne moiety and the compound comprising the azide moiety are introduced to the contacting step in an alkyne:azide molar ratio in the range of 1 :5 to 5:1, preferably in a molar ratio in the range of 1 : 1 to 5 : 1 , more preferably in a molar ratio in the range of 1 : 1 to 3 : 1 , most preferably in a molar ratio of 2:

110. The method according to any of claims 7 to 9, wherein the contacting step takes place in a solvent, which preferably is trichloromethane (CHCh).

11. The method according to any of claims 7 to 10, wherein the reaction between the alkyne moiety and the azide moiety takes place at an elevated temperature, preferably at a temperature of 30-60 °C, more preferably at a temperature of 40-60 °C, most preferably at a temperature of 50-60 °C.

12. The method according to any of claims 7 to 11, wherein the reaction between the alkyne moiety and the azide moiety is allowed to proceed for at least 4 hours, preferably for 6 - 120 hours, and more preferably for 16 - 96 hours.

13. Use of the lipopolymer of any of the claims 1-6, or the lipopolymer prepared in the method of any of claims 7-12, in a drug delivery system.

14. The use according to claim 13, wherein the drug delivery system is a liposome or a lipid nanoparticle.

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