WO2023156629A1 - Stealth-stabilized lipo-polyamino acids - Google Patents

Stealth-stabilized lipo-polyamino acids Download PDF

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Publication number
WO2023156629A1
WO2023156629A1 PCT/EP2023/054101 EP2023054101W WO2023156629A1 WO 2023156629 A1 WO2023156629 A1 WO 2023156629A1 EP 2023054101 W EP2023054101 W EP 2023054101W WO 2023156629 A1 WO2023156629 A1 WO 2023156629A1
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alkyl
cio
lipo
formula
moiety
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English (en)
French (fr)
Inventor
Carles FELIP LEON
Josep GARCIA GARCIA
Lidia HERRERA MUÑOZ
Vicent Josep Nebot Carda
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Polypeptide Therapeutic Solutions SL
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Polypeptide Therapeutic Solutions SL
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Priority to CA3251178A priority Critical patent/CA3251178A1/en
Priority to EP23705562.9A priority patent/EP4479095B1/en
Priority to ES23705562T priority patent/ES3068015T3/es
Priority to EP26155817.5A priority patent/EP4779025A2/en
Priority to CN202380028821.3A priority patent/CN119173280A/zh
Priority to JP2024548493A priority patent/JP2025508733A/ja
Publication of WO2023156629A1 publication Critical patent/WO2023156629A1/en
Anticipated expiration legal-status Critical
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    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
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    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • C—CHEMISTRY; METALLURGY
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    • C08G69/48—Polymers modified by chemical after-treatment
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present disclosure relates to lipo-polyamino acid conjugates which comprise a polyamino acid (PAA) moiety conjugated to a lipid-like moiety.
  • PAA polyamino acid
  • These lipo-polyamino acid conjugates may form self-assembled nanoparticles, such as for example lipid nanoparticles (LNPs), and can be used as non-viral vectors for delivery of active ingredients, including nucleic acids, to cells.
  • LNPs lipid nanoparticles
  • Nucleic acids have emerged in recent years to yield promising drug candidates for a wide range of diseases, including, among others, cancer, infectious diseases, and cardiovascular, inflammatory, and neurodegenerative diseases. Nucleic acids exert their action via gene inhibition, addition, replacement or editing. However, their clinical efficacy is very much limited because of their inability to cross physiological barriers. The negative charge and hydrophilicity of nucleic acids hinder their passive diffusion across plasma membranes. Moreover, the association of nucleic acids with serum proteins as well as their susceptibility to enzymatic degradation by endogenous nucleases also interferes with their efficient translation to functional response.
  • Non-viral vectors such as LNPs are known to permeate plasma membranes and thus to be able to deliver nucleic acids to cells.
  • LNPs are vesicles composed of mixtures of lipids that are physically associated with each other by intermolecular forces forming a micellar structure. LNPs typically have a small diameter of less than 200 nm. LNPs are used as drug delivery system thanks to their ability to encapsulate biologically active agents and deliver them to specific locations within the body at a desired time.
  • the lipid components of LNPs may include ionizable or cationic lipids, helper lipids such as phospholipids and structural lipids such as sterols or cholesterol. lonizable/cationic lipids, which have either a permanent or ionizable positively charged head group followed by a hydrophobic tail, can form complexes with negatively charged nucleic acids by means of electrostatic bonds.
  • LNPs One of the potential problems of LNPs is that once they are injected, they may be recognised by the immune system as foreign particles and subsequently cleared from the bloodstream by phagocytes.
  • different strategies may be used including decreasing the particle size of the vesicles or modifying their surface.
  • the LNP surface may be modified by conjugating the hydrophilic polymer polyethylene glycol (PEG) to lipids such as phospholipids or long-chain fatty acids to form PEG-lipids.
  • PEG is a hydrophilic polymer widely used in drug delivery and nanotechnology because of its stealth properties and biocompatibility. Thanks to these properties, particulate delivery systems containing PEG are able to evade the immune system, and, as a result, have a prolonged circulation time within the body.
  • hydrophilic polymers such as polyvinylpyrrolidones
  • poly (acryloyl-morpholines) or polyoxazolines have been reported as alternatives to PEG-lipids
  • no LNP containing such polymers have been marketed yet.
  • Polysarcosine is a polypeptoid based on the endogenous amino acid sarcosine (N-methylated glycine), which has previously shown potent stealth properties.
  • Lipid nanoparticles (LNPs) for therapeutic application of messenger RNA assembled using pSarcosinylated lipids have been reported as a promising tool for particle engineering (ACS Appl. Nano Mater., 2020, 3, 10634—10645) but this is still under development and no clinical data including this polymer into LNPs is presented yet.
  • the inventors have developed new lipo-polyamino acid conjugates which comprise a polyamino acid (PAA) moiety conjugated to at least one lipid-like moiety. These conjugates may form self-assembled nanoparticles, such as for example lipid nanoparticles (LNPs), and can be effectively used as non-viral vectors for delivery of active ingredients, including nucleic acids, to cells, thanks to their stealth properties and consequent improved plasma half-life time.
  • PAA polyamino acid
  • LNPs lipid nanoparticles
  • the lipo-polyamino acid conjugates show low cytotoxicity, high biodegradability and may confer to the final nanosystem enhanced efficiency, high stability due to limited or even completely suppressed aggregation issues in the bloodstream and potential different cell and tissue tropism.
  • a first aspect of the invention relates to a lipo-polyamino acid conjugate of formula (I), a salt thereof, or any stereoisomer or mixtures of stereoisomers, either of the compound of formula (I), or of any of its salts, wherein n is an integer from 5 to 250; m is 0 or 1 ;
  • X is selected from the group consisting of N, S, and 0;
  • R1 is selected from the group consisting of H, -(Ci-Ci8)alkyl, and -(C2-Ci8)alkenyl, with the condition that R1 is absent when X is 0 or S;
  • R2 is a radical selected from the group consisting of (II), (III), (IV), and (V) wherein: a, b, c, and d are independently an integer from 1 to 4;
  • R3 is H or -(Ci-Cejalkyl
  • A is selected from the group consisting of H, -(Ci-Cejalkyl, -CO(Ci-C6)alkyl, -(C5-Cio)aryl, -(C5-Cio)heteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, a protective group, a radical R4, a lipid- like moiety R5, and an active moiety; being A optionally substituted by one or more groups selected from the group consisting of -OH, halogen, -CF3, -NH2, -NH-(Ci-C4)alkyl, -NH-CO-(Ci-C6)alkyl, -(Ci-Cejalkyl, -NO2, -N 3 , -CO-(Ci-C 6 )alkyl,
  • R4 is a radical of formula (Via)
  • R4' is a radical of formula (Vlb) wherein: n' is an integer from 5 to 250; m' is O or 1 ;
  • X is selected from the group consisting of N, S, and 0;
  • Ri is a radical which has any of the meanings of Ri, with the condition that Ri” is absent when X” is 0 or S;
  • R2 is a radical which has any of the meanings of R2;
  • R3’ is a radical which has any of the meanings of R3;
  • B and B’ are independently S or CH2; e and f are independently an integer from 1 to 18;
  • A” is selected from the group consisting of H, -OH, -(Ci-C6)alkyl, -(C5-Cio)aryl, -(C5-Cio)heteroaryl, -(C6-Cio)aralkyl, -(Ci-Ce)alkyl-0-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, -(C1-C 10) alkoxy, -(C6-Cio)aryloxy, -(C6-Cio)aralkoxy, -(C5-Cio)heteroaralkoxy, -(Ci-Cio)alkyl-0-(C6-Cio)aryloxy, an amino protective group, a lipid-like moiety R5’, an amino acid-like moiety Re, and an active moiety; being A" optionally substituted by one or more groups selected from the group consisting of
  • A' is selected from the group consisting of H, -(Ci-Cejalkyl, -CO(Ci-C6)alkyl, -(Cs-Cio)aryl, -(C5-Cio)heteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(Cs-Cio)heterocycloalkyl, a protective group, a lipid-like moiety Rs, and an active moiety; being A'” optionally substituted by one or more groups selected from the group consisting of -OH, halogen, -CF3, -NH2, -NH-(Ci-C4)alkyl, -NH-CO-(Ci-C 6 )alkyl, -(Ci-C 6 )alkyl, -NO 2 , -N 3 , -CO-(Ci-C 6 )alky
  • Y and Y' are independently selected from -OH, -OCORx and -COORx;
  • Q and Q' are independently selected from -OCORy and -COORy;
  • Z and Z' are independently selected from -OCO-, -COO-, -NRz'CO-, and -CONRz'-; each Rz' is H or Rz; each Rx, Ry, and Rz is independently -(Ci-Ci8)alkyl or -(C2-Ci8)alkenyl; each g is independently an integer from 0 to 18; each h is independently an integer from 1 to 18; i is an integer from 0 to 18; each j is independently an integer from 0 to 18;
  • X' is selected from the group consisting of N, S, and 0;
  • R22, R33 and R44 are independently selected from the group consisting of hydrogen, fluorine, methyl, -CH2F, -CHF2, and -CF3; each of the dashed bonds — is independently a single bond or, alternatively, a double bond;
  • the lipo-polyamino acid conjugates of the present disclosure may be decorated with cell-targeting groups in any of A, A', A” or A'” that can actively target cells and aid in cellular entry, resulting in a conjugate which has improved cell-specific delivery.
  • the lipo-polyamino acid conjugates may also be decorated with penetration enhancing agents when an increased cell or skin penetration is desired, Further, the lipo-polyamino acid conjugates can also be used for delivering pharmaceutically, cosmetically or diagnostically active agents attached to the backbone of the conjugate in any of A, A', A” or A’”.
  • the lipo-polyamino acid conjugates of the invention may form self-assembled particles in solution such as for example liposomes or lipid nanoparticles which may be used to encapsulate and deliver molecules of different nature.
  • the lipo-polyamino acid conjugates of the invention allow carrying and/or delivering different active agents at the same time, not only bonded to the structure of the conjugate but also contained in self-assembled particles formed from the conjugated.
  • another aspect of the invention relates to a self-assembled particle comprising the lipo-polyamino acid conjugate of formula (I) as defined herein, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • the lipo-polyamino acid conjugates of the invention may be formulated in a variety of compositions, including pharmaceutical, cosmetic and diagnostic compositions, with excipients and carriers.
  • another aspect of the invention relates to a composition comprising the lipo-polyamino acid conjugate as defined herein; or alternatively, the self-assembled particle containing them, together with one or more appropriate excipients or carriers.
  • the lipo-polyamino acid conjugates of the invention may be used in medicinal, cosmetic and diagnostic applications.
  • a therapeutic product which comprises: a) a lipo-polyamino acid conjugate of formula (I) as defined herein, wherein at least one of A, A', A” or A'” is a pharmaceutically active agent; or alternatively, b) a self-assembled particle containing the lipo-polyamino acid conjugate a); or alternatively, c) a composition containing the lipo-polyamino acid conjugate a) or the self-assembled particle b); or alternatively, d) a self-assembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively, e) a composition containing the self- assembled particle d), for use in medicine.
  • the invention relates to a diagnostic product which comprises: a') a lipo- polyamino acid conjugate as defined herein, wherein at least one of A, A', A” or A'” is a diagnostically active agent; or alternatively, b') a self-assembled particle containing the lipo-polyamino acid conjugate a'); or alternatively, c') a composition containing the lipo-polyamino acid conjugate a') or the self-assembled particle b'); or alternatively, d) a self-assembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more diagnostically active agents; or alternatively, e') a composition containing the selfassembled particle d'), for use in diagnostics.
  • the invention relates to the use in cosmetics of a cosmetic product which comprises: a") a lipo-polyamino acid conjugate as defined herein, wherein at least one of A, A', A” or A'” is a cosmetically active agent; or alternatively, b”) a self-assembled particle containing the lipo-polyamino acid conjugate a'); or alternatively, c”) a composition containing a") or the self-assembled particle b”); or alternatively, d”) a self-assembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more cosmetically active agents; or alternatively, e”) a composition containing the selfassembled particle d”).
  • Another aspect of the invention relates to the use of the lipo-polyamino acid conjugate of formula (I) as defined herein, wherein A, A', A” and A'” are other than an active moiety, as a carrier.
  • FIG. 1 shows an agarose gel (2%) for the detection of free mRNA from different LNPs formulations: M) free mRNA; 1) benchmark LNPs formulation; 2) benchmark LNPs formulation + 10% triton X-100 and 2 mg/mL heparin; 3) LNP4 formulation; 4) LNP4 formulation + 10% triton X-100 and 2 mg/mL heparin; 5) LNP9 formulation; 6) LNP9 formulation + 10% triton X-100 and 2 mg/mL heparin; 7) LNP12 formulation; 8) LNP12 formulation + 10% triton X-100 and 2 mg/mL heparin; 9) LNP11 formulation; 10) LNP11 formulation + 10% triton X-100 and 2 mg/mL heparin.
  • FIG. 2 shows the in vivo mRNA transfection capacity in mice of a LNP comprising a lipo-polyamino acid conjugate according to the invention (LNP15) and comparative LNPs using DMG-PEG as lipo-shielding compound (total counts per g of tissue in liver (A), ALNs (B), spleen (C), lungs (D)).
  • LNP15 lipo-polyamino acid conjugate according to the invention
  • DMG-PEG lipo-shielding compound
  • moiety refers to a specific segment or functional group of a molecule or compound.
  • subject refers to any mammal, including both human and other mammals.
  • -(Ci-C x )alkyl refers to a saturated linear or branched hydrocarbon chain which contains from 1 to x carbon atoms and only single bonds.
  • alkyl groups may include without limitation methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, neopentyl, n-hexyl, decyl, undecyl, dodecyl, tetradecyl and hexadecyl.
  • alkenyl refers to an unsaturated branched or linear hydrocarbon chain which comprises from 2 to x carbon atoms and at least one or more double bonds.
  • alkenyl groups may include without limitation ethenyl (i.e. vinyl), allyl, propenyl, butenyl, pentenyl and hexenyl, dodecenyl, tetradecenyl and hexadecenyl.
  • alkoxy refers to an alkyl group as defined herein comprising 1 to 10 carbon atoms which is connected to the rest of the molecule via an oxygen atom.
  • alkoxy groups may include without limitation methoxy, ethoxy, 1 -propoxy, and 2-propoxy.
  • -(C5-Cio)aryl refers to an aromatic carbocyclic mono- or bicyclic ring system comprising 5 to 10 carbon ring atoms.
  • aryl groups may include without limitation phenyl, biphenyl, and naphthyl.
  • -(C6-Cio)aryloxy refers to an aryl group as defined herein comprising 6 to 10 carbon ring atoms, which is connected to the rest of the molecule via an oxygen atom.
  • aryloxy groups may include without limitation phenoxy and biphenyloxy.
  • -(C5-Cio)heteroaryl refers to an aromatic carbocyclic mono- or bicyclic ring system comprising 5 to 10 ring atoms, wherin at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, and S, the remaining ring atoms being carbon atoms.
  • heteroaryl groups may include without limitation pyridyl, azetidinyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl, isoquinolinyl, benzthiazolyl, benzimidazolyl, benzthiazolyl, quinolyl, and quinazolyl.
  • -(C6-Cio)aralkyl refers to a lower alkyl group as defined herein (e.g. -(Ci-Ce)alkyl) which is substituted by an aryl group (e.g. “-(C5-Cio)aryl) as defined herein.
  • aralkyl groups may include without limitation benzyl, phenethyl, and methylbenzyl.
  • -(C6-Cio)aralkoxy refers to a lower alkoxy group as defined herein (e.g. -(Ci-Ce)alkoxy) which is substituted by an aryl group (e.g. “-(C5-Cio)aryl) as defined herein.
  • aralkoxy groups may include without limitation benzyloxy, phenethoxy, and methylbenzyloxy.
  • heteroaralkoxy refers to a lower alkoxy group as defined herein (e.g. -(Ci-Ce) alkoxy) which is substituted by an heteroaryl group (e.g. "-(C5-Cio)heteroaryl) as defined herein.
  • heteroaralkoxy groups may include without limitation furyloxy, pyridyloxy, and azetidinyloxy.
  • heterocycloalkyl refers to a 5-10 membered mono- or bicylic (fused or bridged) saturated o unsaturated ring structure, in which one or more of the ring atoms is a heteroatom selected from N, 0, and S.
  • heterocycloalkyl groups may include without limitation include piperidyl, piperazyl, tetrahydropyranyl, tetrahydrofuryl, 1,4-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholyl.
  • protecting group refers to a grouping of atoms that when attached to a reactive group in a molecule masks, reduces or prevents that reactivity.
  • Protective groups for carboxyl and amino groups are described for example in T. W. Green and P.G. M. Wuts, Protective Groups in Organic Chemistry (Wiley, 3rd ed. 1999) in Chapter 5 (pp. 369-451) and Chapter 7 (pp. 495-653), respectively.
  • substituted means that one or more hydrogen atoms on the designated atom or group are replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded. Combinations of substituents and/or variables are permissible.
  • optionally substituted means that the number of substituents can be equal to or different from zero. Unless otherwise indicated, it is possible that optionally substituted groups are substituted with as many optional substituents as can be accommodated by replacing a hydrogen atom with a non-hydrogen substituent on any available carbon or nitrogen atom. It is possible that groups in the conjugates according to the invention are substituted with one, two, three, four or five identical or different substituents, particularly with one, two or three substituents.
  • a first aspect of the invention refers to a lipo-polyamino acid conjugate of formula (I), a salt thereof, or any stereoisomer or mixtures of stereoisomers, either of the compound of formula (I), or of any of its salts.
  • A, X, Ri, R2, R3, n, m, and A' are as defined herein.
  • I ipo-poly amino acid conjugate refers to a molecule which comprises a) a polyamino acid-based moiety and b) at least one lipid-like moiety.
  • lipid-like moiety refers to a moiety which, while it cannot be considered to be a lipid as such, structurally and/or functionally resembles a lipid.
  • lipid-like moiety is intended to include moieties that are able to form amphiphilic layers.
  • lipid refers to a group of organic compounds such as fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, sterols, and the like, which are insoluble in water.
  • such salts can be prepared by reacting the free acid or base form of a lipo-polyamino acid conjugate of the invention with a stoichiometric amount of an appropriate base or acid, respectively, in a suitable solvent such as water, an organic solvent or a mixture of them.
  • Examples of pharmaceutically, cosmetically or diagnostically acceptable salts include acid addition salts formed with inorganic acids e.g. hydrochloric, hydrobromic, sulfuric, nitric, hydroiodic, metaphosphoric, or phosphoric acid; and organic acids e.g.
  • succinic maleic, acetic, fumaric, citric, tartaric, benzoic, trifluoroacetic, malic, lactic, formic, propionic, glycolic, gluconic, camphorsulfuric, isothionic, mucic, gentisic, isonicotinic, saccharic, glucuronic, furoic, glutamic, ascorbic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), ethanesulfonic, pantothenic, stearic, sulfinilic, alginic and galacturonic acid; and arylsulfonic, for example benzenesulfonic, p-toluenesulfonic, oxalic, methanesulfonic or naphthalenesulfonic acid; and base addition salts formed with alkali metals and alkaline earth metals and organic bases such as N,N- dibenz
  • the lipo-polyamino acid conjugates of the invention and their salts may differ in some physical properties, but they are equivalent for the purposes of the present invention.
  • Some of the lipo-polyamino acid conjugates of the invention can have chiral centres that can give rise to various stereoisomers.
  • stereoisomer refers to all isomers of individual lipo- polyamino acid conjugates that differ only in the orientation of their atoms in space.
  • stereoisomer includes mirror image isomers (enantiomers), mixtures of mirror image isomers (racemates, racemic mixtures), geometric (cis/trans or syn/anti or E/Z) isomers, and isomers of lipo-polyamino acid conjugates with more than one chiral center that are not mirror images of one another (diastereoisomers).
  • the present invention relates to each of these stereoisomers and also mixtures thereof.
  • the definition of the lipo- polyamino acid conjugates of formula (I) is also intended to encompass all R- and S-isomers of a chemical structure in any ratio, e.g. with enrichment (i.e. enantiomeric excess or diastereomeric excess) of one of the possible isomers and corresponding smaller ratios of other isomers.
  • they may acquire L-configuration or D-configuration.
  • Diastereoisomers and enantiomers can be separated by conventional techniques such as chromatography or fractional crystallization. Optical isomers may be obtained using enantiospecific synthesis. Alternatively, optical isomers can be resolved by conventional techniques to give optically pure isomers. The resolution can be carried out on any chiral synthetic intermediates or on the polymers of the invention.
  • the pharmaceutically, cosmetically or diagnostically acceptable salts thereof and the stereoisomers or mixtures of stereoisomers either of any of the lipo-polyamino acid conjugates or of any of their pharmaceutically, cosmetically or diagnostically acceptable salts are always contemplated even if they are not specifically mentioned.
  • the lipo-polyamino acid conjugates of the invention may be in crystalline form either as free solvation lipo- polyamino acid conjugates or as solvates (e.g. hydrates). It is intended that all these forms are within the scope of the present invention. Methods of solvation are generally known within the art. For the purposes of the invention, the solvated forms with pharmaceutically, cosmetically or diagnostically acceptable solvents such as water, ethanol and the like are equivalent to the unsolvated form.
  • the lipo-polyamino acid conjugates of the present disclosure also include lipo- polyamino acid conjugates that differ only in the presence of one or more isotope-enriched atoms.
  • isotope-enriched atoms without limitation, are deuterium, tritium, 13 C or 14 C, or a nitrogen atom enriched in 15 N or a fluorine atom enriched in 18 F.
  • the lipo-polyamino acid conjugates of the invention are polymeric structures containing as a repeating unit a polyamino acid (PAA)-based moiety of formula (XVIII)
  • the lipo-polyamino acid conjugate of formula (I) has the formula (I'): wherein A, A', X, R1, R2, R3, n, and m are as defined herein.
  • the lipo-polyamino acid conjugates of the invention further comprise at least one lipid- like moiety.
  • the lipid-like moiety can be placed on any of the terminal ends of the PAA-based moiety. It is also possible that the lipo-polyamino acid conjugates of the invention comprise a lipid-like moiety at both ends of the molecule.
  • the lipid like moiety can be placed at the left side of the drawn molecule of formula (I) when A is R5, or at the right side when A' is R5', or at both ends when A is R5, and A is R5', but at least one of A and A' must be a lipid-like moiety (R5 in the case of A or R5' in the case of A').
  • the lipo-polyamino acid conjugates of the invention are characterized by a molecular weight, which can be average molecular weight (MW) or number average molecular weight (Mn), a degree of polymerization, and a polydispersity index.
  • the molecular weight can be measured by methods well-known in the art such as size exclusion chromatography (SEC) (also referred to as gel permeation chromatography (GPC)) matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS).
  • SEC size exclusion chromatography
  • GPC gel permeation chromatography
  • MALDI-TOF MS matrix assisted laser desorption ionization-time-of-flight mass spectrometry
  • the term "repeating unit” refers to the monomeric amino acid-based unit and is defined by square brackets ("[ ]”) depicted around it.
  • the number on the lower right of the brackets (represented by letter “n”) refers to the degree of polymerization (DP) for each monomeric unit as a statistical number.
  • the DP is calculated by dividing the corrected molecular weight (subtracting the initiator MW) of the polymer determined by SEC-MALS technique by the molecular weight of the monomer unit.
  • the DP value is reported as the central value of a gaussian distribution which comprises polymers of variable DP (depending on intrinsic polydispersity).
  • the DP value is subject to a reasonable uncertainty, due to the ring-opening polymerization mechanism, which, in the context of the present invention, may be considered within the range ⁇ 20%, preferably ⁇ 15%, more preferably ⁇ 10%, even more preferably ⁇ 5%, being particularly preferred ⁇ 2%.
  • polydispersity index (PDI) is used as a measure of broadness of molecular weight distribution. The larger the PDI, the broader the molecular weight. PDI of a polymer is calculated as the ratio of weight average (MW) by number average (Mn) molecular weight.
  • n is an integer from 5 to 250. In one embodiment, optionally in combination with any of the embodiments provided above or below, n is an integer from 5 to 150, more particularly from 5 to 100, even more particularly from 5 to 50, and even more particularly from 5 to 30 or from 5 to 25.
  • X is selected from N, S, and O. In one embodiment, optionally in combination with any of the embodiments provided above or below, X is N and Ri is H. In another embodiment, optionally in combination with any of the embodiments provided above or below, X is N and Ri is -(Ci-C jalkyl, more particularly -(Ci-Ci2)alkyl . In another embodiment, optionally in combination with any of the embodiments provided above or below, X is N and Ri is -(C2-Ci8)alkenyl, more particularly -(C2- Ci2)alkenyl.
  • X is O and Ri is absent.
  • X is S and Ri is absent.
  • m is 0 or 1 depending on the meaning of A'.
  • A' is selected from the group consisting of H, -(Ci-Cejalkyl, -(Cs-C jaryl, -(Cs-Ciojheteroaryl, -(Ce-C jaralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, an amino protective group, an amino acid-like moiety Re, and an active moiety; being A' optionally substituted by one or more groups selected from the group consisting of -OH, halogen, -CF3, -NH2, -NH-(Ci-C4)alkyl, -NH-CO-(Ci-Ce)alkyl, -(Ci
  • m is 1, and A' is selected from the group consisting of H, -OH, -(Ci-Ce)alkyl, -(Ce-Cw)aryl, -(C5-Cio)heteroaryl, -(Ce-Cwjaralkyl, -(Ci-Ce)alkyl-0-(C5-Cio)aryl, -(Ce-Cw)heterocycloalkyl, -(C1-C10) alkoxy, -(Ce-Cio)ary loxy , -(C6-Cio)aralkoxy, -(C5-Cio)heteroaralkoxy, -(Ci-Cio)alkyl-0-(C6-Cio)aryloxy, a radical R4', a lipid-like moiety Rs’, and an active moiety; being A' optionally substituted
  • R2 is a radical selected from the group consisting of (II), (III), (IV), and (V) wherein a, b, c, and d are independently an integer from 1 to 4.
  • R2 is a radical of formula (II), more particularly in the radical of formula (II), a is 1 or 2.
  • R2 is a radical of formula (III), more particularly in the radical of formula (III), b is 1 or 2.
  • R2 is a radical of formula (IV), more particularly in the radical of formula (IV), c is 1 or 2.
  • R2 is a radical of formula (V), more particularly in the radical of formula (V), d is 1 or 2.
  • R3 is H or -(Ci-Ce)alkyl. In one embodiment, optionally in combination with any of the embodiments provided above or below, in the lipo-polyamino acid conjugate of formula (I), R3 is H. According to another embodiment, optionally in combination with any of the embodiments provided above or below, in the lipo-polyamino acid conjugate of formula (I), R3 is -(Ci-Cejalkyl, more particularly R3 is -CH3.
  • A is selected from the group consisting of H, -(Ci-Ce)alkyl, -CO(Ci-C6)alkyl, -(Cs-Cio)aryl, -(Cs-Cio)heteroaryl, -(Ce-Cwjaralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, a protective group, a radical R4, a lipid-like moiety Rs, and an active moiety; and A' is selected from the group consisting of H, -OH, -(Ci-Cejalkyl, -(Cs-Cio)aryl, -(Cs-Cio)heteroaryl, -(Ce-Cwjaralkyl, -(Ci-C6)alkyl-0-(Cs-
  • one of A and A' is a lipid-like moiety R5 or R5' as defined herein, and the other one of A and A' is other than a lipid-like moiety R5 or R5', respectively.
  • A is a lipid-like moiety R5, and A' is other than a lipid-like moiety R5’; more particularly, A' is selected from the group consisting of H, -OH, -(Ci-Cejalkyl, -(Cs-Cwjaryl, -(Cs-Ciojheteroaryl, -(Ce-Cwjaralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(Cs-Ciojheterocycloalkyl, -(Ci-Cio)alkoxy, -(Ce-Cwjaryloxy, -(Ce-Cwjaralkoxy, -(Cs-Ciojheteroaralkoxy, -(Ci-Cio)alkyl-0-(C
  • A' is a lipid-like moiety R5' and A is other than a lipid-like moiety R5; more particularly, A is selected from the group consisting of H, -(Ci-Cejalkyl, -CO(Ci-C6)alkyl, -(Cs-Ciojaryl, -(Cs-Ciojheteroaryl, -(Ce-Cwjaralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(Cs-Ciojheterocycloalkyl, a protective group, a radical R 4 , and an active moiety, being A optionally substituted by one or more groups selected from the group consisting of -OH, halogen, -CF 3 , -NH 2 , -NH-(Ci-
  • the lipid-like moiety R5 in A in the lipo-polyamino acid conjugate of formula (I), and/or in A'” of the radical RT of formula (Vlb) is independently selected from the group consisting of -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, and a radical of formula (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XXI), (XXII), or (XXIII) wherein Y, Y', Q, Q', Z, g, h, I, j, t, R22, R33, R44, the dashed bonds — , and L are as defined herein.
  • the lipid- like moiety R5 in A in the lipo-polyamino acid conjugate of formula (I), and/or in A'” of the radical R4 1 of formula (Vlb) is independently selected from the group consisting of -(Ci-Ci8)alkyl, -(C2-Ci8)alkenyl, and a radical of formula (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV).
  • the lipid- like moiety R5 is -(Ci-Ci8)alkyl, more particularly -(Ce-Cisjalkyl. More particularly, the lipid-like moiety Rs has the formula (XIX): wherein k is an integer from 1 to 16, more particularly k is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16.
  • the lipid-like moiety R5 is -(C2-Ci8)alkenyl, more particularly
  • the lipid-like moiety R5 has the formula (XX): wherein k is an integer from 1 to 16, more particularly k is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16, and q is an integer from 0 to 18, more particularly q Is O, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 18.
  • the lipid-like moiety Rs has the formula (VII), wherein g is an integer from 0 to 16, more particularly, g is from 0 to 12, and even more particularly g is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • the lipid-like moiety Rs has the formula wherein each g is independently an integer from 0 to 16, more particularly, each g is independently from 0 to 12, and even more particularly each g is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • the lipid-like moiety Rs has the formula (IX), wherein Y and Y' are independently selected from -OH, -COORx, and -OCORx; and each Rx is independently -(Ci-Ci8)alkyl or -(C2-Ci8)alkenyl. More particularly, in the lipid-like moiety R5 of formula (IX), each Rx is a radical independently selected from formula (ixa) and (ixb):
  • the lipid-like moiety R5 of formula (IX) is selected from the formulas (ix1), (ix2), (ix3), (ix4), (ix5), (ix6), wherein each k is independently an integer from 1 to 16, more particularly each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; each p and each q are independently an integer from 0 to 18, more particularly each p and each q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; and each r is independently an integer from 0 to 6, more particularly each r is independently 0, 1, 2, 3, 4, 5, or 6.
  • each k has the same meaning.
  • each p has the same meaning, each r has the same meaning, and each q has the same meaning.
  • the lipid-like moiety Rs has the formula (X), wherein Q and Q' are independently selected from -OCORy and -COORy; each Ry is independently - (Ci-Ci8)alkyl or -(C2-Ci8)alkenyl; each h is independently an integer from 1 to 18, more particularly, each h is independently from 1 to 12, and even more particularly each h is independently 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12; and I is an integer from 0 to 12 more particularly, I is from 0 to 12, and even more particularly I is independently 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12. More particularly, in the lipid-like moiety R5 of formula (X), each Ry is a radical independently selected from formula (xa), (xb), and (xc):
  • k is an integer from 1 to 16, more particularly k is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16;
  • p and q are independently an integer from 0 to 18, more particularly p and q are independently 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 18;
  • r is an integer from 0 to 6, more particularly r is O, 1 , 2, 3, 4, 5, or 6.
  • the lipid-like moiety R5 of formula (X) is selected from the formulas (x1), (x2), (x3), and (x4): wherein k is an integer from 1 to 16, more particularly k is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16; each p and each q are independently an integer from 0 to 18, more particularly each p and each q are independently 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 18; each h is independently 1 , 2, 3,
  • the lipid-like moiety Rs has the formula (XI), wherein Q and Q' are independently selected from -OCORy and -COORy; each Ry is independently - (Ci-Cis)alkyl or -(C2-Cis)alkenyl; each h is independently an integer from 1 to 18, more particularly, each h is independently from 1 to 12, and even more particularly each h is independently 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12; and i is an integer from 0 to 18, more particularly, i is from 0 to 12, and even more particularly i is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12. More particularly, in the lipid-like moiety R5 of formula (XI), each Ry is a radical independently selected from formula (xia), (xib), and (xic):
  • k is an integer from 1 to 16, more particularly k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16;
  • p and q are independently an integer from 0 to 18, more particularly p and q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18;
  • r is an integer from 0 to 6, more particularly r is O, 1, 2, 3, 4, 5, or 6.
  • Q and Q' are both -OCORy.
  • Q and Q' are both -COORy.
  • the lipid-like moiety R5 of formula (XI) has the formula (xi1): wherein each p and each q are independently an integer from 0 to 18, more particularly each p and each q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; r is an integer from 0 to 6, more particularly each r is independently 0, 1, 2, 3, 4, 5, or 6; each h is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; and I is an integer from 0 to 12; more particularly, I Is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • the lipid-like moiety Rs has the formula (XII), wherein I is an integer from 0 to 18; more particularly, I is from 0 to 12, and even more particularly I is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; and each j is independently an integer from 0 to 18, more particularly, each j is independently selected from 0 to 12, and even more particularly each j is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • the lipid-like moiety Rs has the formula wherein i is an integer from 0 to 18; more particularly, I is from 0 to 12, and even more particularly I is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; and each j is independently an integer from 0 to 18, more particularly, each j is independently selected from 0 to 12, and even more particularly each j is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • the lipid-like moiety Rs has the formula (XIV), wherein Q and Q' are independently selected from -OCORy and -COORy; Z is selected from -OCO-, -COO-, - NRz'CO-, and -CONRz'-; each Rz' is H or Rz; each Ry and Rz is independently -(Ci-Cis)alkyl or -(C2- Cis)alkenyl; and I is an integer from 0 to 18, more particularly, I is from 0 to 12, and even more particularly I is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. More particularly, in the lipid-like moiety R5 of formula (XIV), each Ry is a radical independently selected from formula (xiva) and (xivb):
  • k is an integer from 1 to 16, more particularly k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16;
  • p and q are independently an integer from 0 to 18, more particularly p and q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18;
  • r is an integer from 0 to 6, more particularly r is O, 1, 2, 3, 4, 5, or 6.
  • Q and Q' are both -OCORy.
  • Q and Q' are both -COORy.
  • Z is -OCO-.
  • Z is -CONRz'.
  • the lipid-like moiety R5 of formula (XIV) is selected from the formulas (xiv1), and (xiv2):
  • each k is independently an integer from 1 to 16, more particularly each k is independently 1 , 2, 3, 4,
  • i is an integer from 0 to 12; more particularly, i is 0, 1 , 2, 3, 4, 5,
  • the lipid-like moiety Rs has the formula (XXI), wherein i is an integer from 0 to 18; more particularly, i is from 0 to 12, and even more particularly i is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12; and each j is independently an integer from 0 to 18, more particularly, each j is independently selected from 0 to 12, and even more particularly each j is independently 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12.
  • the lipid-like moiety R5 of formula (XXI) has the formula (xxi 1): wherein i is an integer from 0 to 12; more particularly, i is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12.
  • the lipid-like moiety Rs has the formula (XXII), wherein Q and Q' are independently selected from -OCORy and -COORy; each Ry is independently -(Ci-Ci8)alkyl or -(C2-Ci8)alkenyl; and I is an integer from 0 to 18, more particularly, i is from 0 to 12, and even more particularly i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. More particularly, in the lipid-like moiety Rsof formula (XXII), each Ry is a radical independently selected from formula (xxiia) and (xxiib):
  • k is an integer from 1 to 16, more particularly k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16;
  • p and q are independently an integer from 0 to 18, more particularly p and q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18;
  • r is an integer from 0 to 6, more particularly r is O, 1, 2, 3, 4, 5, or 6.
  • the lipid-like moiety R5 of formula (XXII) is selected from the formulas (xxiil), and (xxii2):
  • each k is independently an integer from 1 to 16, more particularly each k is independently 1, 2, 3, 4,
  • i is an integer from 0 to 12; more particularly, i is 0, 1, 2, 3, 4, 5,
  • the lipid-like moiety Rs has the formula (XXIII),
  • each of the dashed bonds of the alkyl chain attached to the chroman ring is a single bond
  • R22, R33 and R44 are selected from the following meanings: (I) R22, R33 and R44 are methyl, (II) R22, R44 are methyl and R33 is hydrogen, (ill) R33 and R44 are methyl and R22 is hydrogen, and (iv) R44 is methyl and R22 and R33 are hydrogen.
  • each of the dashed bonds of the alkyl chain attached to the chroman ring is a single bond and R22, R33 and R44 are methyl.
  • the formula (XXIII) has the formula (xxiii 1 ):
  • each of the dashed bonds of the alkyl chain attached to the chroman ring is a double bond
  • R22, R33 and R44 are selected from the following meanings: (I) R22, R33 and R44 are methyl, (II) R22, R44 are methyl and R33 is hydrogen, (iii) R33 and R44 are methyl and R22 is hydrogen, and (iv) R44 is methyl and R22 and R33 are hydrogen.
  • each of the dashed bonds of the alkyl chain attached to the chroman ring is a double bond and R22, R33 and R44 are methyl.
  • A is a lipid-like moiety R5 which has the formula (XXIII), X is 0 and R1 is absent, i.e., the lipo-polyamino acid conjugate of formula (I) has the formula (Ih): wherein A', R2, R3, m, n, R22, R33, R44, and the dashed bonds — are as defined herein.
  • A is a lipid-like moiety R5 which has the formula (XXIII), wherein t is 1 , i.e., the lipo-polyamino acid conjugate of formula (I) has the formula (Ij): wherein A', R2, R3, m, n, R22, R33, R44, L, and the dashed bonds — are as defined herein.
  • the linker L is a biradical selected from the group consisting of -(Ci-Ci2)alkylene- optionally substituted with one or more substituents selected from the group consisting of halogen, -OH, -NR aa Rbb, -SH, -NHNH2, -OOOR C c, -CF3, and -OCF3, wherein R aa , Rbb and R cc are radicals independently selected from the group consisting of H, -phenyl, -(Ci-Cejalkyl, -(C2-C6)alkenyl, -(Ci-Cejalkylphenyl, and -phenyl(Ci-Ce)alkyl; -(Ci-Cejalkyl-CO-; -CO-(Ci-C6)alkyl-; and a biradical
  • L is a -(Ci-Ci2)alkylene-, more particularly a -(Ci-C6)alkyl-, and is optionally substituted as defined herein.
  • L is -(Ci-Ce)alkyl-CO- or -CO-(Ci-Ce)alkyl- and is optionally substituted as defined herein.
  • L is a biradical of formula (HID), more particularly a biradical of formula (HID) wherein b1 is O, and a1 + c1 are selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and even more particularly, L is a biradical of formula (HID) selected from the group consisting of:
  • L is a biradical selected from the formulas (IVD), (VD), and (VID), more particularly, in the biradical of formula (IVD), a1 is 1 and the dashed bond is a double bond, i.e., the biradical of formula (IVD) has the formula (IVDa), and in the biradical of formula (VD), the dashed bond is more particularly a double bond, i.e., the biradical of formula (VD) has the formula (VDa).
  • L is a biradical selected from the formulas (VID), (VIID), and (VIIID), more particularly, in the biradical of formula (VIIID), a1 is 1.
  • L is a biradical of formula (IXD), more particularly a biradical of formula (IXD) wherein b1 and g1 are 0; and a1, d, d1, e1, f1 and hi are 1, i.e., a biradical of formula (IXDa).
  • L is a biradical selected from the formulas (XD) and (XID), more particularly in the biradicals of formula (XD) and (XID), b1 and g1 are 0; and a1, d, d1, e1, f1 and hi are 1, i.e. the biradicals of formula (XD), and (XID) have the formulas (XDa), and (XIDa), respectively.
  • L is a biradical selected from the formulas (XIID) and (XIIID), more particularly in the biradicals of formula (XIID) and (XIIID), b1 Is O; and a1 , d and d1 are 1.
  • L is a biradical selected from the formulas (XIVD), (XVD), (XVID), (XVIID), and (XVIIID), more particularly in the biradicals of formula (XIVD), (XVD), (XVID), (XVIID), and (XVIIID), b1 Is O; and a1 , and d are 1.
  • L is a biradical of formula (XIXD), wherein p1 is 0, and j1 and k1 are 1 .
  • L is a biradical of formula (XIXD), wherein p1 is 1 , X1' is -NH-, and j1 and k1 are 1.
  • L is a biradical selected of formula (XIXD), wherein p1 is 1 , X1' is -O-, and j1 and k1 are 1.
  • A is a lipid-like moiety R 5 selected from the group consisting of: a) -(Ci-Ci8)alkyl, more particularly -(Ce-Cisjalkyl, and even more particularly, a radical of the formula (XIX) as previously defined; b) -(C2-Ci8)alkenyl, more particularly -(Ce-Cisjalkenyl, and even more particularly, a radical of the formula (XX) as previously defined; c) a radical of formula (VII) as defined herein; d) a radical of formula (VIII) as defined herein; e) a radical of formula (IX) as defined herein, more particularly, a radical selected from the formulas (ix1), (ix2), (ix3), (ix4), (ix5), (ix6), (ix7) and (ix8) as previously
  • the lipo-polyamino acid conjugate of formula (I) has the formula (la), wherein A', R2, R3, m, n, are as defined herein, and each k is independently an integer from 1 to 16, more particularly each k is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. Even more particularly, in the lipo-polyamino acid conjugate of formula (la), each k has the same meaning.
  • A is a lipid-like moiety R5 as defined herein, m is 0, and A' is H.
  • A is a lipid-like moiety R5 as defined herein, m is 0, and A' is an amino protective group.
  • Suitable amino protective groups known in the art may be used without limitation.
  • Non-limiting examples of amino protective groups include acyl-based groups, carbamate-based groups, imide-based groups, sulfonamide-based groups, and the like.
  • the amino protective group is selected from the group consisting of acetyl, methyloxycarbonyl, benzyloxycarbonyl (Cbz), p-methoxybenzyloxycarbonyl, t-butyloxycarbonyl (Boc), 9-fluorenyl methyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl group (Troc), benzoyl (Bz), benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trimethylsilylethoxycarbonyl (Teoc), benzhydryl, triphenylmethyl (Trityl), (4-methoxyphenyl)diphenylmethyl (MMT), dimethoxytrityl (DMT), and diphenylpho
  • A is a lipid-like moiety R5 as defined herein, m is 1, and A' is a radical R4’, being R4' a radical of formula (Vlb) wherein: n' is an integer from 5 to 250;
  • X is selected from the group consisting of N, S, and 0;
  • R1 is a radical which has any of the meanings of R1, with the condition that R1” is absent when X' is 0 or S;
  • R2' is a radical which has any of the meanings of R2;
  • R3' is a radical which has any of the meanings of R3;
  • B and B’ are independently S or CH2; e and f are independently an integer from 1 to 18; and
  • A' is selected from the group consisting of H, -(Ci-Cejalkyl, -CO(Ci-C6)alkyl, -(Cs-C jaryl, -(C5-Cio)heteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, a protective group, a lipid-like moiety R5, and an active moiety; being A'” optionally substituted by one or more groups selected from the group consisting of -OH, halogen, -CF3, -NH2, -NH-(Ci-C4)alkyl, -NH-CO-(Ci-C 6 )alkyl, -(Ci-C 6 )alkyl, -NO 2 , -N 3 , -CO-(Ci-C 6 )alkyl,
  • the lipo-polyamino acid conjugate of formula (lb) is a dimer wherein Rs and A'” have the same meaning, X and X” have the same meaning, Ri and Ri” have the same meaning, R2 and R2' have the same meaning, R3 and R3’ have the same meaning, B and B' have the same meaning, e and f have the same meaning, and n and n' have the same meaning, respectively.
  • A is a lipid-like moiety Rs as defined herein, m is 0, and A' is an amino acid-like moiety Re, being Re is a radical selected from the group consisting of (XV), (XVI), and (XVII)
  • R2 is a radical of formula (II)
  • A is a lipid-like moiety R5 as defined herein
  • m is 0,
  • A’ is an amino acid-like moiety Re, being Re a radical of formula (XV), i.e., the lipo-polyamino acid conjugate of formula (I) has the formula (Ic), wherein X, R1, R3, R5, n, a, and a’ are as defined herein.
  • a and a’ have the same meaning, even more particularly, a and a’ are both 1 or 2.
  • R2 is a radical of formula (III)
  • A is a lipid-like moiety R5 as defined herein
  • m is 0, and
  • A’ is an amino acid-like moiety Re, being Re a radical of formula (XVI), i.e., the lipo-polyamino acid conjugate of formula (I) has the formula (Id),
  • X, Ri, R3, R5, n, b, and b' are as defined herein. More particularly, in the lipo-polyamino acid conjugate of formula (Id), b and b' have the same meaning, even more particularly, b and b' are both 1 or 2.
  • R2 is a radical of formula (IV)
  • A is a lipid-like moiety R5 as defined herein
  • m is 0,
  • A' is an amino acid-like moiety Re, being Re a radical of formula (XVII), i.e., the lipo-polyamino acid conjugate of formula (I) has the formula (le), wherein X, R1, R3, R5, n, c, and c' are as defined herein.
  • c and c' have the same meaning, even more particularly, c and c' are both 1 or 2.
  • R2 is a radical of formula (IV)
  • A is a lipid-like moiety R5 as defined herein
  • m is 0, i.e., the lipo-polyamino acid conjugate of formula (I) has the formula (Ig),
  • X, R1, R3, R5, n, and c are as defined herein, and A' is selected from the group consisting of H, -(Ci-C6)alkyl, -(C5-Cio)aryl, -(C5-Cio)heteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, and -(C5-Cio)heterocycloalkyl; being A' optionally substituted by one or more groups selected from the group consisting of -OH, halogen, -CF3, -NH 2 , -NH-(Ci-C4)alkyl, -NH-CO-(Ci-C6)alkyl, -(Ci-Cejalkyl, -NO 2 , -N3, -CO-(Ci-C 6 )alkyl, -CO-O-(Ci-C 6 )
  • R 2 is a radical of formula (IV)
  • A is a lipid-like moiety R5 as defined herein
  • m is 1
  • the lipo-polyamino acid conjugate of formula (I) has the formula (Ig'), wherein X, Ri, R3, R5, n, and c are as defined herein,
  • A' is selected from the group consisting of H, -OH, -(C1- Cejalkyl, -(C5-Cio)aryl, -(C5-Cio)heteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, -(Ci-Cio)alkoxy, -(C6-Cio)aryloxy,
  • m is 1, A is a lipid-like moiety R5 and A' is a lipid-like moiety Rs‘ as defined herein.
  • A is a lipid-like moiety R5 as defined herein, m is 1, and A' is an active moiety selected from the group consisting of a pharmaceutically active agent, a cell-targeting agent, a penetration enhancing agent, a cosmetically active agent, and a diagnostically active agent.
  • the active moiety in A' may be directly bound to the nitrogen atom of the lipo-polyamino acid conjugate or, alternatively, it may be bound through a linker.
  • linker There is no limitation in the nature of the linker provided that it can be attached to the rest of the molecule by chemically feasible bonds.
  • the linker may contain from 1-50 moieties, more particularly from 2 to 30 or from 2 to 20 or from 2 to 15 moieties as defined above, and may be attached to the nitrogen atom of the PAA by a chemically feasible bond which is selected from the group consisting of an amine bond, an amide bond, a carbamate bond or a urea bond.
  • the lipid-like moiety R5' in A' in the lipo-polyamino acid conjugate of formula (I), and/or in A” of the radical R4 of formula (Via) is independently selected from the group consisting of -X’(Ri’)-(Ci-Ci8)alkyl, -X'(RI')-(C2- Ci 8 )alkenyl, and a radical of formula (VII’), (VIII’), (IX’), (X’), (XI’), (XII’), (XIII’), (XIV), (XXI’), (XXII’), or (XXIII’),
  • the lipid- like moiety R5' in A' in the lipo-polyamino acid conjugate of formula (I), and/or in A” of the radical R4 of formula (Via) is independently selected from the group consisting of -X’(Ri’)-(Ci-Ci8)alkyl, -X’(Ri’)-(C2-Ci8)alkenyl, and a radical of formula (VII’), (VIII’), (IX’), (X’), (XI’), (XII’), (XIII’), or (XIV).
  • the lipid- like moiety R5’ is -X’(Ri’)-(Ci-Ci8)alkyl, more particularly -X’(Ri’)-(C6-Ci8)alkyl. More particularly, the lipid-like moiety R5’ has the formula (XIX'): wherein X' and R/ are as defined herein and k is an integer from 1 to 16, more particularly k is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16.
  • the lipid-like moiety R5’ is -X’(Ri’)-(Ci-Ci8)alkyl, wherein X' is N and R/ is -(Ci-C jalkyl, more particularly, the lipid-like moiety R5’ has the formula (XIX”): wherein X' and R? are as defined herein and each k is independently an integer from 1 to 16, more particularly each k is independently 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16, and even more particularly, each k has the same meaning.
  • the lipid-like moiety R5' is -X'(Ri')-(C2-Ci8)alkenyl, more particularly
  • the lipid-like moiety R5' has the formula (XX'): wherein X' and R? are as defined herein and k is an integer from 1 to 16, more particularly k is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16, and q is an integer from 0 to 18, more particularly q is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 18.
  • the lipid-like moiety R5' has the formula (VII'), wherein g is an integer from 0 to 18, more particularly, g is from 0 to 12, and even more particularly g is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12.
  • the lipid-like moiety R5' has the formula (VI II'), wherein each g is independently an integer from 0 to 18, more particularly, each g is independently from 0 to 12, and even more particularly each g is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • the lipid-like moiety R5' has the formula (IX'), wherein X' and R? are as defined herein, Y and Y' are independently selected from -OH, -COORx, and -OCORx; and each Rx is independently -(Ci-Ci8)alkyl or -(C2-Cis)alkenyl. More particularly, in the lipid-like moiety R5' of formula (IX'), each Rx is a radical independently selected from formula (ixa') and (ixb’):
  • Y and Y' are the same. In another particular embodiment, optionally in combination with any of the embodiments provided above or below, in the lipid-like moiety R5' of formula (IX'), Y and Y' are different.
  • the lipid-like moiety R5' of formula (IX') is selected from the formulas (1x1'), (Ix2'), (Ix3'), (Ix4'), (Ix5'), (Ix6’), (Ix7’) and (Ix8’):
  • each k has the same meaning.
  • each p has the same meaning, each r has the same meaning, and each q has the same meaning.
  • the lipid- like moiety R5' has the formula (X'), wherein X' and R? are as defined herein, Q and Q' are independently selected from
  • each Ry is independently -(Ci-Ci8)alkyl or -(C2-Ci8)alkenyl; each h is independently an integer from 1 to 18, more particularly, each h is independently from 1 to 12, and even more particularly each h is independently 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12; and I is an integer from 0 to 18; more particularly, I is from 0 to 12, and even more particularly I is independently 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12. More particularly, in the lipid-like moiety R5 of formula (X), each Ry is a radical independently selected from formula (xa'), (xb'), and (xc'):
  • Q and Q' are both -OCORy.
  • Q and Q' are both -COORy.
  • the lipid-like moiety R5' of formula (X') is selected from the formulas (xT), (x2'), (x3'), and (x4'): wherein X' and R?
  • the lipid- like moiety R5' has the formula (wherein X' and R? are as defined herein, Q and Q' are independently selected from
  • each Ry is independently -(Ci-Cis)alkyl or -(C2-Ci8)alkenyl; each h is independently an integer from 1 to 18, more particularly, each h is independently from 1 to 12, and even more particularly each h is independently 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12; and I is an integer from 0 to 18, more particularly, I is from 0 to 12, and even more particularly I is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12. More particularly, in the lipid- like moiety R5' of formula (XI'), each Ry is a radical independently selected from formula (xia'), (xib'), and (xic’):
  • xia (xia’) (xib’) (xic’) wherein X' and R? are as defined herein, the wavy bonds mean that the stereochemistry of the double bonds is undefined (i.e. it includes cis and trans isomers), k is an integer from 1 to 16, more particularly k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; p and q are independently an integer from 0 to 18, more particularly p and q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; and r is an integer from 0 to 6, more particularly r is 0, 1, 2, 3, 4, 5, or 6.
  • Q and Q' are both -OCORy.
  • Q and Q' are both -COORy.
  • the lipid-like moiety R5' of formula (XI') has the formula (xi1'): wherein X' and R? are as defined herein, each p and each q are independently an integer from 0 to 18, more particularly each p and each q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; r is an integer from 0 to 6, more particularly each r is independently 0, 1, 2, 3, 4, 5, or 6; each h is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; and I is an integer from 0 to 12; more particularly, I Is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • the lipid- like moiety R5' has the formula (XII'), wherein X' and R? are as defined herein, i is an integer from 0 to 18; more particularly, i is from 0 to 12, and even more particularly i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; and each ] is independently an integer from 0 to 18, more particularly, each j is independently selected from 0 to 12, and even more particularly each j is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • the lipid- like moiety R5' has the formula (XIII'), wherein X' and R? are as defined herein, i is an integer from 0 to 18; more particularly, i is from 0 to 12, and even more particularly i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; and each ] is independently an integer from 0 to 18, more particularly, each j is independently selected from 0 to 12, and even more particularly each j is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • the lipid-like moiety R5' has the formula (XIV), wherein X' and R? are as defined herein, Q and Q' are independently selected from -OCORy and -COORy; Z' is selected from -OCO-, -COO-, -NRz'CO-, and -CONRz'-; each Rz' is H or Rz; each Ry and Rz is independently -(Ci-Cis)alkyl or -(C2-Cis)alkenyl; and i is an integer from 0 to 18, more particularly, i is from 0 to 12, and even more particularly i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. More particularly, in the lipid-like moiety R5' of formula (XIV), each Ry is a radical independently selected from formula (xiva) and (xivb):
  • k is an integer from 1 to 16, more particularly k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16;
  • p and q are independently an integer from 0 to 18, more particularly p and q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18;
  • r is an integer from 0 to 6, more particularly r is O, 1, 2, 3, 4, 5, or 6.
  • Q and Q' are both -OCORy.
  • Q and Q' are both -COORy.
  • Z' is -OCO-.
  • Z' is -CONRz'.
  • the lipid-like moiety R5' of formula (XIV) is selected from the formulas (xivT), and (xiv2'): wherein X' and R? are as defined herein, each k is independently an integer from 1 to 16, more particularly each k is independently 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; and i is an integer from 0 to 12; more particularly, i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • the lipid-like moiety R5' has the formula (XXI'), wherein X' and R/ are as defined herein; i is an integer from 0 to 18; more particularly, i is from 0 to 12, and even more particularly i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; and each ] is independently an integer from 0 to 18, more particularly, each j is independently selected from 0 to 12, and even more particularly each j is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.
  • the lipid-like moiety R5' of formula (XXI') has the formula (xxiT): wherein X' and R? are as defined herein. In another embodiment, optionally in combination with any of the embodiments provided above or below, the lipid-like moiety R5' has the formula (XXII'),
  • each Ry is independently -(Ci-Cis)alkyl or -(C2-Cis)alkenyl; and i is an integer from 0 to 18, more particularly, i is from 0 to 12, and even more particularly i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. More particularly, in the lipid-like moiety R5' of formula (XXII'), each Ry is a radical independently selected from formula (xxiia) and (xxiib):
  • k is an integer from 1 to 16, more particularly k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16;
  • p and q are independently an integer from 0 to 18, more particularly p and q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18;
  • r is an integer from 0 to 6, more particularly r is O, 1, 2, 3, 4, 5, or 6.
  • Q and Q' are both -OCORy.
  • Q and Q' are both -COORy.
  • the lipid-like moiety R5' of formula (XXII') is selected from the formulas (xxiiT), and (xxii2'): wherein X' and R/ are as defined herein, each k is independently an integer from 1 to 16, more particularly each k is independently 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16; and i is an integer from 0 to 12; more particularly, i is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12.
  • the lipid-like moiety Rs has the formula (XXIII'), wherein t', R22, R33, R44, the dashed bonds — , and L are as defined herein.
  • each of the dashed bonds of the alkyl chain attached to the chroman ring is a single bond
  • R22, R33 and R44 are selected from the following meanings: (I) R22, R33 and R44 are methyl, (II) R22, R44 are methyl and R33 is hydrogen, (ill) R33 and R44 are methyl and R22 is hydrogen, and (iv) R44 is methyl and R22 and R33 are hydrogen.
  • each of the dashed bonds of the alkyl chain attached to the chroman ring is a single bond and R22, R33 and R44 are methyl.
  • the formula (XXIII') has the formula (xxiiiT):
  • each of the dashed bonds of the alkyl chain attached to the chroman ring is a double bond
  • R22, R33 and R44 are selected from the following meanings: (i) R22, R33 and R44 are methyl, (ii) R22, R44 are methyl and R33 is hydrogen, (iii) R33 and R44 are methyl and R22 is hydrogen, and (iv) R44 is methyl and R22 and R33 are hydrogen.
  • each of the dashed bonds of the alkyl chain attached to the chroman ring is a double bond and R22, R33 and R44 are methyl.
  • A is a lipid-like moiety R5 which has the formula (XXIII'), m is 0 and t' is 1 , i.e., the lipo-polyamino acid conjugate of formula (I) has the formula (lh'): wherein A, X, R1, R2, R3, n, R22, R33, R44, L, and the dashed bonds — are as defined herein.
  • A is a lipid-like moiety R5 which has the formula (XXIII'), wherein m is 1 , i.e., the lipo-polyamino acid conjugate of formula (I) has the formula (lj’): wherein A', R2, R3, m, n, R22, R33, R44, L, and the dashed bonds — are as defined herein.
  • the linker L is a biradical selected from the group consisting of - X1'-(Ci-Ci2)alkylene- optionally substituted with one or more substituents selected from the group consisting of halogen, -OH, -NR aa Rbb, -SH, -NHNH2, -OOOR C c, -CF3, and -OCF3, wherein R aa , Rbb and R cc are radicals independently selected from the group consisting of H, -phenyl, -(Ci-Cejalkyl, -(C2-C6)alkenyl, -(Ci-Cejalkylphenyl, and -phenyl(Ci-Ce)alkyl; -X'-(Ci-C6)alkyl-CO-; and a biradical of formula (HID'),
  • L is a -X1’-(Ci-Ci2)alkylene-, more particularly a - X1'-(Ci-C6)alkyl-, and is optionally substituted as defined herein.
  • L is -X1’-(Ci-C6)alkyl-CO- or — X1'-(Ci-C6)alkyl- and is optionally substituted as defined herein.
  • L is a biradical of formula (HID'), more particularly a biradical of formula (HID') wherein b1 is O, and a1 + c1 are selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and even more particularly, L is a biradical of formula (HID') selected from the group consisting of:
  • L is a biradical selected from the formulas (IVD'), (VD'), and (VID'), more particularly, X1' is -NH-, and even more particularly, in the biradical of formula (IVD'), a1 is 1 and the dashed bond is a double bond, i.e., the biradical of formula (IVD') has the formula (IVDa 1 ), and in the biradical of formula (VD'), the dashed bond is more particularly a double bond, and X1' is -NH-, i.e., the biradical of formula (VD') has the formula (VDa').
  • L is a biradical selected from the formulas (VID'), (VIID'), and (VIIID'), more particularly, in the biradical of formula (VIIID'), a1 is 1.
  • L is a biradical of formula (IXD'), more particularly a biradical of formula (IXD') wherein b1 and g1 are 0; a1, d, d1, e1, f1 and hi are 1, and X1' is -NH-, i.e. a biradical of formula (IXDa').
  • L is a biradical selected from the formulas (XD’) and (XID'), more particularly in the biradicals of formula (XD') and (XID'), b1 and g1 are 0; a1, d, d1, e1, f1 and hi are 1, and X1' is -NH-, i.e. the biradicals of formula (XD'), and (XID') have the formulas (XDa'), and (XI Da'), respectively.
  • L is a biradical selected from the formulas (XIID') and (XIIID'), more particularly in the biradicals of formula (XIID') and (XIIID'), b1 is O; a1, d and d1 are 1; and xr is -NH-.
  • L is a biradical selected from the formulas (XIVD'), (XVD’), (XVID’), (XVIID’), and (XVIIID’), more particularly in the biradicals of formula (XIVD’), (XVD’), (XVID’), (XVIID’), and (XVIIID’), b1 is 0; a1, and d are 1; and X1’ is -NH-.
  • L is a biradical selected from the formulas (XIXD'), wherein X1' is -NH-, and j1 and k1 are 1.
  • L is a biradical selected from the formulas (XIXD'), wherein X1' is -O-, and j1 and k1 are 1.
  • A' is a lipid-like moiety R 5 ' selected from the group consisting of: a) -X'(Ri')-(Ci-Ci8)alkyl, more particularly -X'(Ri')-(C6-Ci8)alkyl, and even more particularly, a radical of the formula (XIX') or a formula (XIX”) as previously defined; b) -X'(Ri')-(C2-Ci8)alkenyl, more particularly -X'(Ri')-(C6-Ci8)alkenyl, and even more particularly, a radical of the formula (XX') as previously defined; c) a radical of formula (VII') as defined herein; d) a radical of formula (VIII') as defined herein; e) a radical of formula (IX')
  • A' is a lipid-like moiety Rs' as defined herein, m is 1, and A is H.
  • A' is a lipid-like moiety Rs' as defined herein, m is 1, and A is a protective group.
  • A' is a lipid-like moiety Rs' as defined herein, m is 1, X is N, and A is an amino protective group; more particularly the amino protective group is selected from the group consisting of acetyl, methyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, t-butyloxycarbonyl (Boc), 9- fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloro-ethoxycarbonyl group (Troc), benzoyl (Bz), benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trimethylsilylethoxycarbonyl (Teoc),
  • A' is a lipid-like moiety R5' as defined herein, m is 1, X is 0, and A is a carboxy protective group.
  • Suitable carboxy protective groups known in the art may be used without limitation.
  • Representative carboxy protective groups include alkyl, aryl or benzyl esters, silyl esters, amides or hydrazides.
  • the carboxy protective group is selected from the group consisting of -(Ci-Cejalkyl, benzyl, p-methoxyphenyl, tri methylsilyl and [2-(trimethylsilyl)ethoxy]methyl (SEM).
  • A' is a lipid-like moiety R5' as defined herein, m is 1, X is S, and A is selected from the group consisting of -(Ci-Cejalkyl, benzyl, p-methoxyphenyl, trimethylsilyl and[2-(trimethylsilyl)ethoxy]methyl (SEM).
  • A' is a lipid-like moiety R5' as defined herein, m is 1, and A is a radical R4, being R4 a radical of formula (Via) wherein: n' is an integer from 5 to 250; m' is O or 1;
  • X is selected from the group consisting of N, S, and 0;
  • R1 is a radical which has any of the meanings of R1, with the condition that R/ is absent when X' is 0 or S;
  • R2' is a radical which has any of the meanings of R2;
  • R3' is a radical which has any of the meanings of R3;
  • B and B’ are independently S or CH2; e and f are independently an integer from 1 to 18; and
  • A” is selected from the group consisting of H, -OH, -(Ci-Cejalkyl, -(Cs-C jaryl, -(C5-Cio)heteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, -(C1-C10) alkoxy, -(Ce-Cwjaryloxy, -(Ce-Cwjaralkoxy, -(C5-Cio)heteroaralkoxy, -(Ci-Cio)alkyl-0-(C6-Cio)aryloxy, an amino protective group, a lipid-like moiety R5', an amino acid-like moiety Re, and an active moiety; being A" optionally substituted by one or more groups selected from the group consisting of -OH, halogen,
  • R5', A”, X, X', R1, R/, R1”, R 2 , R 2 ', R 3 , R 3 ', n, n', m, m', e, f, B, and B’ are as defined herein.
  • the lipo-polyamino acid conjugate of formula (lb') is a dimer wherein R5 and A” have the same meaning, m and m' have the same meaning, X and X” have the same meaning, R1 and R1” have the same meaning, R 2 and R 2 ' have the same meaning, R 3 and R 3 ' have the same meaning, B and B’ have the same meaning, e and f have the same meaning, and n and n' have the same meaning, respectively.
  • A' is a lipid-like moiety R5' as defined herein, m is 1 , and A is selected from the group consisting of H, -(Ci-Cejalkyl, -CO(Ci-C6)alkyl, -(Cs-C jaryl, -(Cs-Ciojheteroaryl, -(Ce-Cwjaralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(Cs-Ciojheterocycloalkyl, a protective group, a radical R4, a lipid-like moiety R5, an active moiety; being A optionally substituted by one or more groups selected from the group consisting of -OH, halogen, -CF3, -NH2, -NH-(Ci-C4)al
  • A' is a lipid-like moiety R5' as defined herein, m is 1 , and A is an active moiety selected from the group consisting of a pharmaceutically active agent, a cell-targeting agent, a penetration enhancing agent, a cosmetically active agent, and a diagnostically active agent.
  • the active moiety in A may be directly bound to X or, alternatively, it may be bound through a linker.
  • the linker may contain from 1-50 moieties, more particularly from 2 to 30 or from 2 to 20 or from 2 to 15 moieties as defined above and may be attached to X by a chemically feasible bond such as an ether bond, a thioether bond, or an amide bond.
  • the lipo-polyamino acid conjugates of formula (I) must contain at least one lipid-like moiety, which may be either a lipid-like moiety R5 in A, a lipid-like moiety R5' in A', or both.
  • a lipo-polyamino acid conjugate of formula (I) wherein A is a lipid-like moiety R5 may be prepared by ring opening polymerization (ROP) of a N-carboxy anhydride (NCA).
  • ROP ring opening polymerization
  • NCA N-carboxy anhydride
  • an oxazolidine-2, 5-dione derivative of an amino acid containing R 2 as side chain may react with an amine, alcohol or thiol of formula (B) which already contains the lipid-like moiety R5 and is used as initiator.
  • a polyamino acid polymer of formula (I) is obtained:
  • the above reaction may also be carried out using an NCA containing a precursor of R2 instead of a R2 moiety, and the R2 precursor is later on converted into a R2 moiety once the polymer is formed.
  • a NCA compound derived from a natural amino acid, such as glutamic acid or lysine, optionally protected with a suitable protective group may be polymerized in the presence of a compound (B) to give a lipo-polyamino acid conjugate of formula (If) wherein R2 is the side chain of the natural amino acid which has been used.
  • This precursor may be deprotected, if needed, and further reacted with other reagents to obtain the desired R2 moieties as illustrated in the examples below.
  • the obtained polyamino acids may be optionally purified by any known methods such as fractionation, precipitation, ultrafiltration, dialysis, size-exclusion chromatography, affinity chromatography or tangential flow filtration
  • the polymer conjugates of formula of formula (If) may be converted into further polymer conjugates of formula (I) by reacting the terminal amine with further groups.
  • the terminal amine may be reacted with active agents directly or linked through a linker.
  • active agents directly or linked through a linker.
  • a skilled person will easily determine the suitable methods for attaching the selected active agent to the backbone of the lipo-polyamino acid conjugates.
  • a polymer conjugate of formula (I) wherein A' is R5', and m' is 1 may be prepared from a compound of formula (If) as defined above, in which A may have any of the meanings as defined herein, by reacting it with a compound of formula R5'-(CO)Z, wherein Z may be OH or halogen.
  • the reaction may be carried out in the presence of an activating agent such as 4- (4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMMBF4), 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCI) or hydroxybenzotriazole (HOBt), preferably in the presence of a base, in a suitable solvent.
  • an activating agent such as 4- (4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMMBF4), 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCI) or hydroxybenzotriazole (HOBt), preferably in the presence of a base, in a suitable solvent.
  • Z is a halogen atom
  • the reaction may
  • a compound of formula (I) wherein A' is R5', and m' is 1 may also be prepared from a compound of formula (If) as defined above by reacting it with a compound of formula R5-H.
  • the reaction may be carried out in the presence of a carbonyl source such as 1 , 1 '-carbonyldiimidazole (GDI), N,N'- disuccinimidyl carbonate (DSC) or bis(trichloromethyl) carbonate (BTC), in a suitable solvent.
  • GDI 1 , 1 '-carbonyldiimidazole
  • DSC N,N'- disuccinimidyl carbonate
  • BTC bis(trichloromethyl) carbonate
  • the lipo-polyamino acid conjugates of the invention have amphiphilic nature since they comprise a polyamino acid-based moiety (hydrophilic part) and at least one lipid-like moiety (hydrophobic part). Thanks to their amphiphilic nature, the lipo-polyamino acid conjugates of the invention may form selfassembled particles in solution.
  • the present invention also relates to a self-assembled particle comprising the lipo-polyamino acid conjugate of formula (I) as defined herein, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, penetration enhancing agents, cell-targeting agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • self-assembled particles refers to the arrangement of the lipo-polyamino acid conjugate molecules in a solvent, in particular, small, self-assembled particle may be enclosed structures of any shape, typically spherical and/or tubular.
  • the term “self-assembled particles” intends to encompass any of a number of structures that are known in the art to be formed from amphiphilic polymers.
  • Non-limiting examples of self-assembled particles include micelles (also being referred to interchangeably herein as micellar worms or simply “worms”), inverted micelles, planar bilayers, crystal nanoparticles, liposomes, microbubbles or lipid nanoparticles.
  • the self-assembled nanoparticles and microparticles can also form gels.
  • the self-assembled particles of the invention are non-viral particles, which means that they are not able to virally infect cells.
  • the self-assembled particles may show a variety of sizes, in particular, they can be nanoparticles or microparticles.
  • a “nanoparticle”, as defined herein, is any particle of nanometric size, in particular having smallest end-to-end diameter of between 1 and 900, more particularly, between 1 and 700 nm, between 1 and 500 nm, between 1 and 300 nm, between 1 and 200 nm, and between 1 and 100 nm in size.
  • a "microparticle”, as defined herein, is typically any particle of micrometric size, having a smallest end-to-end between 1 and 100 pm in size.
  • the relevant diameter is the number average diameter.
  • the term "size" refers to a characteristic physical dimension.
  • the size of the particle corresponds to the diameter of the particle.
  • the size of the particle is determined by the diameter of the two relevant cross-section dimensions of the particle.
  • the size of the nanoparticle corresponds to the maximum edge length.
  • the set of particles can have a distribution of sizes around the specified size.
  • a size of a set of particles can refer to a mode of a distribution of sizes, such as a peak size of the distribution of sizes.
  • the term "diameter” refers to the average diameter and is also designated as Z-average or Z- ave.
  • the average diameter corresponds to the mean hydrodynamic diameter (Dh) and can be measured by dynamic light scattering (DLS) as shown in the examples below.
  • the self-assembled particles of the invention have a hydrodynamic diameter (Dh) in water from 5 to 1200 nm, more particularly, from 10 to 1100 nm, from 10 to 1000 nm, from 10 to 700, from 20 to 500 nm, from 20 to 400 nm, from 20 to 300 nm, from 30 to 200 nm, or from 50 to 150 nm.
  • the lipid-like moieties face inward and the polar head portion (the PAA) face outward.
  • the self-assembled particle is a core-shell structure comprising an inner core and an external shell. More particularly, the lipo-polyamino acid conjugate of formula (I) forms the external shell of the self-assembled particle, and an agent is optionally encapsulated or loaded in the inner core.
  • the self-assembled particle is a nanoparticle, more particularly selected from the group consisting of a micelle, an inverted micelle, a planar bilayer, a crystal nanoparticle, a liposome, microbubbles, and a lipid nanoparticle, and even more particularly, the self-assembled particle is a lipid nanoparticle or a liposome.
  • liposome refers to artificially prepared self-assembled particles composed of concentric lipidic bi-layers enclosing one or more aqueous compartments.
  • Non-limiting examples of liposomes include monolamellar liposome, multi-lamellar liposomes, multi-vesicular-liposomes and polymer-coated liposomes.
  • lipid nanoparticle refers to particles typically spherical on the order of nanometers (e.g., 1-1000 nm) that includes lipids and that is stable and dispersible in aqueous media.
  • crystal nanoparticle refers to artificially prepared crystals composed of solid lipids enclosing one or more hydrophobic compartments.
  • crystal nanoparticle refers to particles of different shapes on the order of nanometers (e.g., 1-1000 nm) that includes lipids and that is stable and dispersible in aqueous media.
  • the invention in another embodiment, optionally in combination with one or more features of the various embodiments described above or below, relates to a lipid nanoparticle (LNP) or a liposome comprising the lipo-polyamino acid conjugate of formula (I) as defined herein, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • the liposomes and lipid nanoparticles disclosed herein may comprise any suitable lipids, including ionizable lipids, cationic lipids, zwitterionic lipids, neutral lipids, or anionic lipids.
  • Suitable ionizable lipids include, without limitation, (2S)-2,5- bis(3-aminopropylamino)- N-[2-(dioctadecyl- amino)acetyl]pentanamide (DOGS), N1-[2-((1 S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]- butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 3
  • Suitable cationic lipids include, without limitation, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N- distearyl-N,N-dimethylammonium bromide (DDA13), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trirnethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)-propyl)-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3,- ditetradecyloxy)propyll-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1-(2,3,dioleyloxy)- propyl]-N, N-dimethyl-N-hydroxy ethylammonium bromide (DORIE), 3p-[N-(N', N'
  • anionic lipids include, but are not limited to, phosphatidylglycerol, diacylphosphatidylserine, diacylphosphatidic acid, N-Succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine cholesterol hemisuccinate (CHEMS), lysylphos-phatidylglycerol, N-dodecanoyl phosphatidyl ethanoloamine, cardiolipin, and combinations thereof.
  • phosphatidylglycerol diacylphosphatidylserine, diacylphosphatidic acid
  • N-Succinyl phosphatidylethanolamine N-glutaryl phosphatidylethanolamine cholesterol hemisuccinate (CHEMS)
  • CHEMS N-glutaryl phosphatidylethanolamine cholesterol hemisuccinate
  • lysylphos-phatidylglycerol N-dodecanoyl
  • Suitable neutral lipids may be uncharged or zwitterionic lipids and include, without limitation steroids, phospholipids, and combinations thereof.
  • steroids examples include, without limitation, cholesterol, progesterone, cortisone, aldosterone, estradiol, testosterone, and combinations thereof.
  • phospholipids include, but are not limited to, phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidylinositol (PI), dimyristoyl phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl serine (DMDM
  • the lipo-polyamino acid conjugate advantageously provides stealth properties.
  • stealth refers to the fact that the liposomes or lipid nanoparticles are not detected and sequestered and/or degraded by the immune system of the host to which they are administered, and consequently the length of time for which the liposomes or lipid nanoparticles can exist in vivo is increased.
  • the liposomes and lipid nanoparticles disclosed herein do not comprise (i.e., lack) a polyethyleneglycol (PEG)-lipid conjugate, that is a lipid containing polyethyleneglycol.
  • PEG polyethyleneglycol
  • the self-assembled particles of the invention in particular liposomes and lipid nanoparticles, lack polyethyleneglycol (PEG).
  • PEG polyethyleneglycol
  • the liposomes and lipid nanoparticles disclosed herein may also comprise one or more cationic polymers such as cationic amine-containing polymers.
  • these polymers may carry active ingredients, particularly nucleic acids.
  • cationic amine-containing polymers include, without limitation, poly-L- lysine, polyamidoamine, polyethyleneimine, chitosan, poly(beta-amino esters), and the like.
  • the invention in another embodiment, optionally in combination with one or more features of the various embodiments described above or below, relates to a lipid nanoparticle (LNP) or a liposome comprising the lipo-polyamino acid conjugate of formula (I) as defined herein, and one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids. More particularly, the lipid nanoparticle (LNP) or liposome comprises the lipo-polyamino acid conjugate of formula (I) as defined herein, a ionizable lipid or a cationic lipid, a phospholipid, and a sterol.
  • the lipid nanoparticle (LNP) or liposome comprises: I) the lipo-polyamino acid conjugate of formula (I) as defined herein in an amount from 0.1 to 10 mol%, more particularly from 1 to 5 mol% or from 1 to 6 mol%, even more particularly from 2 to 5% mol%; II) a ionizable lipid or a cationic lipid in an amount from 30 to 70 mol%, more particularly from 40 to 60 mol%; ill) a phospholipid in an amount from 1 to 20 mol%, more particularly from 5 to 15 mol%; and iv) a sterol in an amount from 20 to 60 mol%, more particularly from 30 to 50 mol%; wherein the percentages are expressed with respect to the sum of the mol% of the lipids and the lipo-polyamino acid conjugate of formula (I).
  • the invention in another embodiment, optionally in combination with one or more features of the various embodiments described above or below, relates to a lipid nanoparticle (LNP) or a liposome comprising the lipo-polyamino acid conjugate of formula (I) as defined herein, one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids, and one or more nucleic acids.
  • LNP lipid nanoparticle
  • a liposome comprising the lipo-polyamino acid conjugate of formula (I) as defined herein, one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids, and one or more nucleic acids.
  • the lipid nanoparticles and the liposomes containing one or more nucleic acids may be prepared by standard methods.
  • the process for the preparation of LNPs comprises: I) preparing a first alcoholic mixture comprising one or more lipids and the lipo-polyamino acid conjugate of the invention in a suitable alcohol such as for example ethanol; II) preparing a second aqueous composition comprising one or more nucleic acids and an aqueous solvent (an acidic buffer); and iii) mixing i) with ii) in a microfluidic mixing device.
  • the microfluidic mixing allows thorough and rapid mixing of the lipid phase and the nucleic acid phase in a microscale device.
  • the skilled person will be able to modulate the size of the LNPs.
  • the lipo-polyamino acid conjugate of the invention may contain one or more active moieties in its structure in any of A, A', A” or A'”.
  • active moieties are independently selected from the group consisting of a pharmaceutically active agent, a cell-targeting agent, a penetration enhancing agent, a cosmetically active agent, and a diagnostically active agent.
  • the lipo-polyamino acid conjugate of the invention may form self-assembled particles which may comprise one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • the term “pharmaceutically active agent” refers to and agent that has pharmacological activity and is used for curing, mitigating, treating or preventing a disease in a mammal, in particular a human.
  • pharmaceutical active agent refers to an agent that does not provide any therapy but is used for aesthetic purposes, for example to improve the appearance, preserve, condition, cleanse, color or protect the skin, nails or hair.
  • the term "cell-targeting agent” refers to any molecule, macromolecule, or biomacromolecule, displaying affinity for a molecule present in the human or animal body, which is able to direct the conjugates or the self-assembled particles thereof by directing them towards the target site for therapeutic treatment since e.g., it selectively binds to receptors that are expressed or over-expressed on specific cell types.
  • Celltargeting groups are well known in the art. The term therefore includes ligands for specific receptors or antigens, such as antibodies for a specific antigen, folic acid for its receptor or sugars such as galactose for its hepatic receptors.
  • the targeting agent may be attached to the lipo-polyamino acid conjugate backbone in any of A, A', A” or A'” and/or may be contained in the self-assembled particles formed from the conjugated.
  • cell-targeting groups include, but are not limited to, galactosamine, folate, a Her-2 binding peptide, TLR agonists, p-D-Glucose, Asn-Gly-Arg peptide, angiopep2, folic acid, aptamers (A-9, A10, Anti- gp120, TTA1, sgc8, Anti MUC-1, AS1411), primaquine, zidovudine, superoxide dismutase, prednisolone, platinum, cisplatin, sulphamethoxazole, amoxicillin, etoposide, mesalzine, doxorubicin, paclitaxel, 5-amino salicylic acid, denosumab, docetaxel, calcitonin, proanthocyanidin, methotrexate, camptothecin, galactose, glycyrrhetinic acid, lactose, hyaluor
  • RCPLSHSLICY laminin receptor binding peptide
  • laminin receptor binding peptide e.g. YIGSR
  • a skin homing peptide a retina homing peptide, a pancreas homing peptide, a liver homing peptide, a lymph node homing peptide, an adrenal gland homing peptide, a thyroid homing peptide, a bladder homing peptide, a breast homing peptide, a neuroblastoma homing peptide, a lymphoma homing peptide, a muscle homing peptide, a wound vasculature homing peptide, an adipose tissue homing peptide, a virus binding peptide, or a fusogenic peptide.
  • YIGSR laminin receptor binding peptide
  • penetration enhancing agent refers to moiety or compound that increases the permeability of an active agent, in particular selected from a pharmaceutically active agent, a cell-targeting agent, a cosmetically active agent, and a diagnostically active agent.
  • the penetration enhancing agent is also known as permeation enhancer.
  • penetration enhancing agents include, without limitation, cell penetrating peptides, surfactants, terpenes, sulfoxides, pyrrolidones, fatty acids, fatty alcohols, urea, azones, fatty alcohols, fatty acids, fatty esters; such as for example, lauryl sarcosine, octoxynol, phenylsulfonate, pluronic, sodium laurate, sodium oleate, sorbitan dilaurate, sorbitan dioleate, sorbitan trilaurate, sorbitan trioleate, sodium octyl sulfate, alkyl ammonium halides, decanol, dodecanol, linolenyl alcohol, oleyl alcohol, butyl acetate, cetyl lactate, lauryl lactate, myristyl lactate, diethyl sebacate, diethyl succinate, di
  • pharmaceutically active agents include low molecular weight drugs, peptides, antibodies, hormones, enzymes, nucleic acids, proteins, and combinations thereof.
  • nucleic acid refers to DNA or RNA.
  • the nucleic acid is an DNA/RNA hybrid, a short interfering RNA (siRNA), a microRNA (miRNA), a single guide RNA (sgRNA), a donorDNA, a self- amplyfing/replicating RNA, a circularRNA (oRNA), a plasmid DNA (pDNA), a closed-linear DNA (clDNA), a short hairpin RNA (shRNA), messenger RNA (mRNA), and antisense RNA (aRNA), a messenger RNA (mRNA), a CRISPR guide RNA, an antisense nucleic acid, a decoy nucleic acid, an aptamer, and a ribozyme to name a few, and encompasses both the nucleotide sequence and any structural embodiments thereof, such as double stranded, single strand
  • the nucleic acid may be a poly- or oligonucleotide, such as oligo- or poly-double stranded RNA, oligo- or polydouble stranded DNA, oligo- or poly-single stranded RNA, oligo- or poly-single stranded DNA.
  • Each of the nucleotides contained in the nucleic acid may be a naturally occurring nucleotide or a chemically-modified, non-naturally occurring nucleotide.
  • the strand length of the nucleic acid is not particularly limited and the nucleic acid may have a short strand ranging from 10 to 200 bases, preferably from 20 to 180 bases, preferably from 25 to 100 bases, preferably from 30 to 50 bases; or the nucleic acid may have a relatively long strand of from 200 to 20000 bases, more preferably of from 250 to about 15000 bases.
  • the nucleic acid is closed-linear DNA (clDNA), i.e. molecules wherein the double stranded region is flanked and protected by two single stranded loops thereby generating dumbbell-shaped molecules.
  • clDNA closed-linear DNA
  • the clDNA consists of a stem region comprising a double stranded DNA sequence of interest covalently closed at both ends by hairpin loops, the clDNA comprising at least two modified nucleotides.
  • clDNA refers to a single stranded covalently closed DNA molecule that forms a “dumbbell” or “doggy-bone” shaped structure under conditions allowing nucleotide hybridization. Therefore, although the clDNA is formed by a single stranded DNA molecule, the formation of the "dumbbell” structure by the hybridization of two complementary sequences within the same molecule generates a structure consisting on a double-stranded middle segment flanked by two single-stranded loops. The skilled in the art know how to generate clDNA from open or closed double stranded DNA using routine molecular biology techniques.
  • a clDNA can be generated by attaching hairpin DNA adaptors, for instance, by the action of a ligase, to both ends of an open double stranded DNA.
  • Hairpin DNA adaptor refers to a single stranded DNA that forms a stem-loop structure by the hybridization of two complementary sequences, wherein the stem region formed is closed at one end by a single stranded loop and is open at the other end.
  • a “modified nucleotide” is any nucleotide (e.g., adenosine, guanosine, cytidine, uracil, and thymidine) that has been chemically modified -by modification of the base, the sugar or the phosphate group- or that incorporates a non-natural moiety in its structure.
  • the modified nucleotide may be naturally or non-naturally occurring depending on the modification
  • the term “peptide” refers to molecules that comprise two or more consecutive amino acids linked to one another via peptide bonds.
  • the term peptide includes oligopeptides and polypeptides.
  • protein refers to large peptides, in particular peptides having at least about 50 amino acids. For the purposes of the invention, the terms peptide and protein are used interchangeably.
  • proteins of interest include, without limitation cytokines, interleukins, tumor necrosis factor (TNF), interferons, integrins, chimeric antigen receptors (CARs), antibodies, hormones, growth factors, enzymes), collagen, fibrinogen, elastin, tubulin, thrombin, serum albumin, erythropoietin, granulocyte colony stimulating factor (G-CSF), colony stimulating factor (CSF), and the like.
  • labeling or imaging agent refers to any substance that is used as a label, or that enhances specific structures in any imaging technique.
  • An imaging agent hence, includes optical imaging agent, magnetic resonance imaging agent, radioisotope, and contrast agent. Imaging or labelling agents are well known in the art.
  • imaging or labelling agents are gases such as sterilized air, oxygen, argon, nitrogen, fluor, perfluorocarbons, carbon dioxide, nitrogen dioxide, sulfur hexafluoride, xenon and helium; commercially available agents used in positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI).
  • gases such as sterilized air, oxygen, argon, nitrogen, fluor, perfluorocarbons, carbon dioxide, nitrogen dioxide, sulfur hexafluoride, xenon and helium
  • PET positron emission tomography
  • CAT computer assisted tomography
  • single photon emission computerized tomography single photon emission computerized tomography
  • x-ray x-ray
  • fluoroscopy and magnetic resonance imaging
  • MRI magnetic resonance imaging
  • gadolinium chelates currently available, such as diethylene triamine pentaacetic acid (DTPA) and
  • Examples of materials useful for CAT and x-rays include iodine based materials for intravenous administration, such as ionic monomers typified by diatrizoate and iothalamate, non-ionic monomers such as iopamidol, isohexol, and ioversol, nonionic dimers, such as iotrol and iodixanol, and ionic dimers, for example, ioxagalte.
  • Other useful materials include barium for oral use and non-soluble salts such as zinc acetate.
  • an imaging agent is a dye.
  • an imaging agent is a fluorescent moiety.
  • a fluorescent moiety is selected from: a fluorescent protein, a fluorescent peptide, a fluorescent dye, a fluorescent material or a combination thereof.
  • fluorescent dyes include, but are not limited to, xanthenes (e.g., rhodamines, rhodols and fluoresceins, and their derivatives); bimanes; coumarins and their derivatives (e.g., umbelliferone and aminomethyl coumarins); aromatic amines (e.g., dansyl; squarate dyes); benzofurans; fluorescent cyanines; indocarbocyanines; carbazoles; dicyanomethylene pyranes; polymethine; oxabenzanthrane; xanthene; pyrylium; carbostyl; perylene; acridone; quinacridone; rubrene; anthracene; coronene; phenanthrecene; pyrene; but
  • fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5-isothiocy anate, fluorescein-6-isothiocyanate and 6-carboxyfluorescein.
  • rhodamine dyes include, but are not limited to, tetramethylrhodamine- 6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine, diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101 sulfonyl chloride (sold under the tradename of TEXAS RED(R)).
  • cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, IRDYE680, Alexa Fluor 750, IRDye800CW, ICG.
  • fluorescent peptides include GFP (Green Fluorescent Protein) or derivatives of GFP (e.g., EBFP, EBFP2, Azurite, mKalamal , ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet). Fluorescent labels are detected by any suitable method.
  • a fluorescent label may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs), photomultipliers, etc.
  • CCDs charge coupled devices
  • photomultipliers etc.
  • the imaging agent is labeled with a positron-emitting isotope (e.g., 18F) for positron emission tomography (PET), gamma-ray isotope (e.g., 99mTc) for single photon emission computed tomography (SPECT), or a paramagnetic molecule or nanoparticle (e.g.,Gd3+ chelate or coated magnetite nanoparticle) for magnetic resonance imaging (MRI).
  • a positron-emitting isotope e.g., 18F
  • PET positron emission tomography
  • gamma-ray isotope e.g., 99mTc
  • SPECT single photon emission computed tomography
  • MRI magnetic resonance imaging
  • the imaging agent is labeled with: a gadolinium chelate, an iron oxide particle, a super paramagnetic iron oxide particle, an ultra small paramagnetic particle, a manganese chelate or gallium containing
  • gadolinium chelates include, but are not limited to diethylene triamine pentaacetic acid (DTPA), 1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA), and 1 ,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA).
  • the imaging agent is a near-infrared fluorophore for near-infra red (near-IR) imaging, a luciferase (firefly, bacterial, or coelenterate) or other luminescent molecule for bioluminescence imaging, or a perfluorocarbon-filled self-assembled particle for ultrasound.
  • the imaging agent is a nuclear probe. In some molecules, the imaging agent is a SPECT or PET radionuclide probe. In some molecules, the radionuclide probe is selected from: a technetium chelate, a copper chelate, a radioactive fluorine, a radioactive iodine, a indiuim chelate. Examples of Tc chelates include, but are not limited to HYNIC, DTPA, and DOTA.
  • the imaging agent contains a radioactive moiety, for example a radioactive isotope such as 211 At, 13 11, 125 l, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 64 Cu radioactive isotopes of Lu, and others.
  • the diagnostically active agents agent may be attached to the lipo-polyamino acid conjugate backbone in any of A, A', A” or A'” and/or may be contained in the self-assembled particles formed from the conjugated.
  • the present invention also relates to a composition
  • a composition comprising the lipo-polyamino acid conjugate, or alternatively, the self-assembled particle as defined herein, together with one or more appropriate excipients or carriers.
  • the composition is a pharmaceutical composition comprising a therapeutically effective amount of: (a) the lipo-polyamino acid conjugate as defined herein, wherein at least one of A, A', A” or A'” is a pharmaceutically active agent or alternatively, (b) a self-assembled particle containing the lipo- polyamino acid conjugate (a), or alternatively, (c) a self-assembled particle which comprises the lipo- polyamino acid conjugate as defined herein and one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof, together with one or more pharmaceutically acceptable excipients or carriers.
  • therapeutically effective amount refers to the amount of a polymer that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed.
  • the specific dose of the polymer of the invention to obtain a therapeutic benefit may vary depending on the particular circumstances of the individual patient including, among others, the size, weight, age and sex of the patient, the nature and stage of the disease, the aggressiveness of the disease, and the route of administration.
  • the term "pharmaceutically acceptable excipients or carriers” refers to components which are appropriate for use in pharmaceutical technology for the preparation of compositions for medical use. Each component should be “acceptable” in the sense of being compatible with the other ingredients of the composition. When used in contact with the tissue or organ of humans and animals should not have excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • lipo-polyamino acid conjugates described in the present disclosure, and the self-assembled particles and pharmaceutical compositions containing them may be used jointly with other, additional drugs, to provide combined therapy.
  • Said additional drugs may be a part of the same pharmaceutical composition or, alternatively, may be provided in the form of a separate composition for simultaneous or non-simultaneous administration.
  • the composition is a diagnostic composition comprising a diagnostically effective amount of: (a') the lipo-polyamino acid conjugate as defined herein, wherein at least one of A, A', A” or A'” is a diagnostically active agent or alternatively, (b') a self-assembled particle containing the lipo-polyamino acid conjugate (a'), or alternatively, (c') a self-assembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more diagnostically active agents, together with one or more diagnostically acceptable excipients or carriers.
  • the term "diagnostic composition” refers to a composition suitable for use in diagnostic, particularly in imaging diagnostic technology.
  • the term “diagnostically effective amount” as used herein, refers to the effective amount of a detection polymer that, when administered, is sufficient for the diagnosis of a disease or disorder; particularly as imaging diagnostic use as contrast imaging agent.
  • the dose of the detection polymer administered will of course be determined by the particular circumstances surrounding the case, including the polymer administered, the route of administration, the particular condition being diagnosticated, and the similar considerations.
  • the diagnostic composition of the present invention comprises one or more diagnostically acceptable excipients or carriers.
  • diagnostically acceptable refers to that excipients or carriers suitable for use in the diagnosing technology for preparing compositions with diagnostic use; particularly by imaging diagnostic use.
  • the detection of these diagnostic agents in the body of the patient can be carried out by the well-known techniques used such as in imaging diagnostic with magnetic resonance imaging (MRI) and X-ray.
  • MRI magnetic resonance imaging
  • the composition is a cosmetic composition comprising a cosmetically effective amount of: (a”) the lipo-polyamino acid conjugate as defined herein, wherein at least one of A, A', A” or A'” is a cosmetically active agent or alternatively, (b”) a self-assembled particle containing the lipo-polyamino acid conjugate (a”), or alternatively, (c”) a self-assembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more cosmetically active agents, together with one or more cosmetically acceptable excipients or carriers.
  • cosmetically effective amount refers to the effective amount of a cosmetically active agent that, when administered, is intended to improve its appearance or to beautify, preserve, condition, cleanse, color or protect the skin, nails or hair without a medical application.
  • cosmetically acceptable or “dermatologically acceptable” excipients or carriers is used interchangeably in this document and refer to components which are appropriate for use in human skin contact without toxicity, incompatibility, instability, inappropriate allergic response, among others.
  • compositions of the invention may be in solid or liquid form.
  • solid forms include frozen forms, lyophilized forms and spray-dried forms.
  • excipients or carriers include, without limitation, diluents, binders, glidants, disintegrants, lubricants colorants, mixtures thereof, and other components known in the state of the art.
  • Any administration route may be used such as e.g. oral, topical, rectal or parenteral route (including subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous route, etc.).
  • the lipo-polyamino acid conjugates of the invention, self-assembled particles, and compositions thereof may be used in therapeutic applications.
  • they may be used as non-viral vectors of general use for biomedical applications, such as vaccines or gene therapy, being effective for transfection of hosts eukaryotic cells in culture, in vivo or ex vivo, monocellular parasites and bacteria, including gene editing using the CRISP/Cas9 methodology.
  • they may be used in protein-based therapy; particularly protein-based vaccine against viral infections or as a therapeutic protein-based vaccine against cancers or infectious diseases.
  • the invention relates to a therapeutic product which is or which comprises: a) a lipo-polyamino acid conjugate of formula (I) as defined herein, wherein at least one of A, A', A” or A'” is a pharmaceutically active agent; or alternatively, b) a self-assembled particle containing the lipo-polyamino acid conjugate a); or alternatively, c) a composition containing the lipo-polyamino acid conjugate a) or the self-assembled particle b); or alternatively, d) a self-assembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof; or alternatively, e) a composition containing the selfassembled particle d), for use in medicine.
  • This aspect may also be formulated as a method for treating or preventing a disease or disorder in a subject, more particularly a mammal, and even more particularly a human, comprising administering the therapeutic product as defined herein, and one or more pharmaceutically acceptable excipients or carriers.
  • Non-limiting examples of diseases that may be treated and/or prevented by the derivatives of the present invention include neurodegenerative disorders, neurological diseases, cancer, infectious diseases, disorders related to aging, neuro-inflammation, demyelinating disorders, multiple sclerosis, ischemic disorders, ischemia-reperfusion damage, amyloidotic disease, cardiomyopathy, spinal cord injury, immune disorders, inflammatory disorders, rare diseases, wound healing, skin related diseases and lysosomal storage diseases.
  • Non-limiting examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cerebral ischaemia, post-encephalitic Parkinsonisms, dystonias, Tourette syndrome, periodic limb movement pathologies, restless legs syndrome, attention deficit hyperactivity disorders, Huntington's disease, progressive supranuclear palsy, Pick's disease, fronto-temporal dementia and neuromuscular diseases.
  • the invention relates to a therapeutic product which is or which comprises: a) a lipo-polyamino acid conjugate of formula (I) wherein any of A, A', A” or A” is an anticancer agent or an anti-infective agent as defined herein; or alternatively, b) a self-assembled particle containing the lipo-polyamino acid conjugate a); or alternatively, c) a composition containing the lipo-polyamino acid conjugate a) or the self-assembled particle b); or alternatively, d) a self-assembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more anticancer agents or anti-infective agents; or alternatively, e) a composition containing the selfassembled particle d); for use in the treatment and/or prevention of cancer or an infectious disease, respectively.
  • disorder as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disease,” “syndrome,” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms.
  • treat refers to ameliorating symptoms associated with a disease or disorder, including preventing or delaying the onset of the disease or disorder symptoms, and/or lessening the severity or frequency of symptoms of the disease or disorder.
  • the invention relates to the therapeutic product as defined herein for use (I) as transfection reagent for transfecting at least one active agent into a cell; (ii) for use in the in vivo or ex vivo production of biologies encoding a recombinant protein, a peptide or an antibody, or in the production of recombinant virus; (ill) for use as a therapeutic or prophylactic vaccine against viral infections or as a therapeutic vaccine against cancers or infectious diseases; or (iv) for use in genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
  • the present invention relates to the therapeutic product as defined herein as transfection reagent for delivering one or more nucleic acids (regardless of size and structure, circular and linear nucleic acids) to target cells, in in vivo, in vitro or ex vivo.
  • the active agent is selected from the group consisting of low molecular weight drugs, peptides, proteins, antibodies, nucleic acids, aptamers, and combinations thereof.
  • the present invention also relates to a method for in vitro, ex vivo and in vivo transferring active agents comprising the therapeutic product as defined herein.
  • the therapeutic product is a self-assembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more nucleic acids, or alternatively, a composition containing the self-assembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more nucleic acids.
  • transfection reagents of the invention are also useful for co-transfection of two or more active agents simultaneously, e.g. two or more nucleic acids, simultaneously.
  • Transfection compositions (such as kits), as well as methods of using the transfection reagents to deliver nucleic acid to target cells are also within the scope of the present invention.
  • the present invention also provides therapeutic products as defined herein for inducing a regulating effect on the expression of one or more target proteins responsible or involved in genetic hereditary diseases or complex genetic diseases, immune diseases, cancers, viral infections in various tissues/organs or tumors.
  • the present invention also relates to the in vitro or ex vivo use of therapeutic products as defined herein in the production of biologies, in particular biologies encoding a recombinant protein, a peptide or an antibody; or in the production of recombinant virus, such as adeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolytic virus, or baculovirus, or viral or virus-like particles, in particular said products comprising the lipo- polyamino acid conjugates of the invention and at least one nucleic acid molecule for transfection.
  • AAV adeno-associated virus
  • LV lentivirus
  • adenovirus adenovirus
  • oncolytic virus or baculovirus
  • viral or virus-like particles in particular said products comprising the lipo- polyamino acid conjugates of the invention and at least one nucleic acid molecule for transfection.
  • the term "biologies” refers to proteins or nucleic
  • the present invention also relates to an in vitro or ex vivo use of the therapeutic products as defined herein, in particular said products comprising the lipo-polyamino acid conjugates of the invention and at least one nucleic acid molecule for transfection, for genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
  • compositions for transfecting cells comprise the therapeutic products as defined herein, in particular said products comprising the lipo-polyamino acid conjugates of the invention and at least one nucleic acid molecule for transfection, and an acceptable excipient, buffering agent, cell culture medium, or transfection medium.
  • the present invention is also directed to the therapeutic products as defined herein for use as a therapeutic or prophylactic vaccine against viral infections, or a therapeutic vaccine against cancers.
  • the vaccine is delivered through direct administration such as systemic, intramuscular, intradermal, intraperitoneal, intratumoral, oral, topical, or sub-cutaneous administration, and, in said vaccine, the composition is in association with a pharmaceutically acceptable vehicle.
  • the vaccine can be injected directly into the body, in particular in a human individual, for inducing a cellular and/or a humoral response.
  • the cell targeting is achieved through different mechanisms and depends on the nature and properties of the transfection reagent, method or protocol composition or formulation and the route of administration.
  • the lipo-polyamino acid conjugates of the invention may be used also in cosmetic and diagnostic applications.
  • a diagnostic product which is or which comprises: a') a lipo-polyamino acid conjugate as defined herein, wherein at least one of A, A', A” or A'” is a diagnostically active agent; or alternatively, b') a self-assembled particle containing the lipo-polyamino acid conjugate I'); or alternatively, c') a composition containing the lipo-polyamino acid conjugate I') or the self-assembled particle II'); or alternatively, d') a self-assembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more diagnostically active agents; or alternatively, e') a composition containing the self-assembled particle iv'), for use in diagnostics.
  • This aspect of the invention may also be formulated as the use of a') a lipo-polyamino acid conjugate as defined herein, wherein at least one of A, A', A” or A'” is a diagnostically active agent; or alternatively, b') a self-assembled particle containing the lipo-polyamino acid conjugate a'); or alternatively, c') a composition containing the lipo-polyamino acid conjugate a') or the self-assembled particle b'); or alternatively, d') a selfassembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more diagnostically active agents; or alternatively, e') a composition containing the self-assembled particle d'), in diagnostics.
  • a' a lipo-polyamino acid conjugate as defined herein, wherein at least one of A, A', A” or A'” is a diagnostically active agent; or alternatively, b') a self-assembled particle containing the lipo- polyamino acid conjugate a'); or alternatively, c') a composition containing the lipo-polyamino acid conjugate a') or the self-assembled particle b'); or alternatively, d') a self-assembled particle which comprises the lipo- polyamino acid conjugate as defined herein and one or more diagnostically active agents; or alternatively, e') a composition containing the self-assembled particle d'), in a subject in need thereof, more particularly a mammal, and even more particularly a human.
  • the invention relates to microbubbles which comprise the lipo-polyamino acid conjugate as defined herein and one or more diagnostically active agents, in particular contrast agents, for use in diagnostics.
  • imaging diagnostic techniques suitable for the present disclosure include, but not limited to, are magnetic resonance imaging (MRI), X-ray, positron emission tomography (PET), singlephoton emission computed tomography (SPECT), fluorescence microscopy, and in vivo fluorescence.
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT singlephoton emission computed tomography
  • fluorescence microscopy and in vivo fluorescence.
  • the invention relates to the use in cosmetics of a cosmetic product which is or which comprises: a") a lipo-polyamino acid conjugate as defined herein, wherein at least one of A, A', A” or A'” is a cosmetically active agent; or alternatively, b”) a self-assembled particle containing the lipo-polyamino acid conjugate a'); or alternatively, c”) a composition containing a") or the self-assembled particle b”); or alternatively, d”) a self-assembled particle which comprises the lipo-polyamino acid conjugate as defined herein and one or more cosmetically active agents; or alternatively, e”) a composition containing the selfassembled particle d”).
  • NMR spectra were recorded at 27 °C (300 K) on a 300 UltrashieldTM from Broker (Billerica MA, USA). Data were processed with the software Topspin (Broker GmbH, Karlsrohe, Germany). Samples were prepared at a concentration of 20 - 10 mg/mL approx, in the reqoired solvent.
  • Dynamic Light Scattering (DLS) measorements were performed osing a Malvern Zetasizer NanoZS instroment, eqoipped with a 532 nm laser at a fixed scattering angle of 173 °.
  • Polymer solotions were prepared onder different conditions (MilliQ water or PBS at different concentrations and temperatores), solotions were sonicated for 10 min and allowed to age for the reqoired time, filtered throogh a 1.20 pm cellolose membrane filter and measored. Size distribotion was measored (diameter, nm) for each polymer per triplicate with n > 3 measorements, aotomatic optimization of beam focosing and attenoation was applied for each sample.
  • Ci4-PGIu(OBzl) (PGADIOL1 : 2.0 g, 0.38 mmol, PGADIOL2: 2.0 g, 1.02 mmol) as obtained in Example 1.1 was dissolved in DMF (200 mg per mL DMF) and acetic anhydride (10 eq) and DIPEA (3 eq) were added to the reaction mixture. Then, the mixture was stirred at RT for 1 hour. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate (Ci4-PGIu(OBzl)-COCH3) was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum.
  • the lipo-polymer Ci4-PGIu(OBzl)-COCH3 (Lipo-PGA1-4: 6.4 mmol) as obtained in Example 1.2 was dissolved in trifluoroacetic acid (100 mg/mL) and HBr 48% (2 eq per Glutamic acid unit) was added. The mixture was stirred at RT for 16 hours. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was washed with a mixture of Acetonitrile: H2O (8:2) until pH 4-5. Then, the product is washed again with diethyl ether, isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The polymer (C14-PGA) was isolated as a white solid.
  • Ci4-PLys(OBzl) (PLysTGI : 1.5g, 0.24 mmol, PLysTG2: 1.5g, 0.65 mmol) as obtained in Example 2.1 was capped following the procedure described above in example 1.2. Yield: 60-70%.
  • the lipo-polymer Ci4-PLys(OBzl)-COCH3 (PLys: 1.25 g, 4.7 mmol); as obtained in Example 2.2 was dissolved in chloroform (50 mg/mL) and HBr 33% (4 eq per Lysine unit) was added. The mixture was stirred at RT for 16 hours. An orange solid appears. The solid is recovered by filtration and washed with diethyl ether. The precipitate was washed with a mixture of Acetonitrile: H2O (8:2) until pH 4-5. Then, the product is washed again with diethyl ether, isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The polymer was isolated as a yellowish solid. Final purification by dialysis was performed (Mw cutt off 2000 Da) to obtain a white solid (Cn-PLys).
  • Ci4-PLys(CIAc) (PLysTG: 502 mg, 0.1 mmol) as obtained in Example 2.4 was dissolved in DMF (22 mg/mL). Then, Thioglycerol (8 eq per Lysine unit) and DIPEA (8 eq per Lysine unit) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 16h. After this time, the reaction mixture was poured into diethyl ether Precipitate was isolated by filtration, washed with diethyl ether and dried under vacuum. The product was dissolved in H 2 O, purified by sephadex column (PD-10 Desalting Columns) and lyophilized. The final product was isolated as a white solid.
  • Ci4-PLys(TFA) (Lipo-PBet x ) determined by NMR. determined by GPC, wherein the cited DP numbers are subject to a reasonable uncertainty within the range ⁇ 20%>.
  • the lipo-polymers Ci4-PLys(TFA)as obtained in Example 3.1 was dissolved in cold methanol (300 mg/mL) and cold NaOH aq (1.5 eq per Lysine unit) was added. The mixture was stirred at RT for 16 hours. The reaction mixture was quenched with HBr 48% until pH 2. The precipitate was washed with a mixture of Acetone, isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The product was dissolved in H 2 O, the pH adjusted to 4 with NaOH 1 M and lyophilized. The polymer (Cn-PLys) was isolated as a white solid.
  • the lipo-polymer Cu-PLys as obtained in Example 3.2 was dissolved in formaldehyde (1 eq per Lysine unit) and formic acid (2.3 eq per Lysine unit). The mixture was heated to 85 °C and stirred for 16 hours. The reaction mixture was concentrated by rotavapor and redissolved in methanol. The mixture was precipitated with diethyl ether, isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The product was dissolved in H2O, purified by sephadex column (PD-10 Desalting Columns) and lyophilized. The polymer (C14- PLys(Dimet)) was isolated as a white solid.
  • Ci4-PLys(Dimet) as obtained in Example 3.3 was dissolved in DMF (50 mg/mL). Then, tert-butyl bromoacetate (6 eq per Lysine unit) was added to the reaction mixture and stirred for 5 minutes at RT. After this time, TBAI (0.5 eq per Lysine unit) and DIPEA (3 eq per Lysine unit) were added. The mixture was stirred at RT for 16 hours. The reaction mixture was concentrated by rotavapor and redissolved in DCM. The mixture was precipitated with diethyl ether, isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The product was used without further purification or characterization. Yield: 75%.
  • the protected lipo-polymer Cn-PBet as obtained in Example 3.4 was dissolved in cold trifluoroacetic acid (50 mg/mL) and stirred at RT for 24 hours.
  • the reaction mixture was poured into diethyl ether to precipitate the product.
  • the precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum.
  • the solid was redissolved in H2O, dialyzed against miliQ water (Mw cutt of 3000 Da) during 24h and lyophilized.
  • the polymer was isolated as a white solid.
  • N-Carboxy-L-glutamic acid-benzyl- anhydride was added to a Schlenk tube fitted with a stirring bar and a stopper. After 3 cycles of vacuum/N 2 , monomer was dissolved in anhydrous DMF. Then, the initiator (1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPE(14:0), Avanti Polar Lipids) was diluted in chloroform (5 mL) and added to the reaction mixture, which was stirred at room temperature for 16 hours. Once NCA consumption was confirmed by IR the reaction mixture was poured into diethyl ether to precipitate the product.
  • NCA N-Carboxy-L-glutamic acid-benzyl- anhydride
  • the lipo-polymer DMPE(14:0)-PGIu(OBzl) as obtained in Example 4.1 was dissolved in trifluoroacetic acid (100 mg/mL) and HBr 48% (2 eq per Glutamic acid unit) was added. The mixture was stirred at RT for 16 hours. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was washed with a mixture of Acetonitrile: H 2 O (8:2) until pH 4-5. Then, the product is washed again with diethyl ether, isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum.
  • the polymer (DMPE(14:0)-PGA) was isolated as a white solid. Yield: 80%.
  • Boc-GABA-OH (4-(Boc-amino)butyric acid: 400 mg, 1.968 mmol) was dissolved in DCM (9 mL).
  • HOBt (1.1 eq, 2.16 mmol, 292 mg)
  • DCC dicyclohexyl carbodiimide
  • Dimyrist (ditetradecylamine: 1.1 eq, 2.16 mmol, 885 mg) was added and the reaction mixture stirred for 16 hours at room temperature. The completion of the reaction was monitored by TLC.
  • Dimyrist-GABA-PGA (PGADIOL 0.4 g, 3.3 mmol) as obtained in Example 5.3 was dissolved in DMF (50 mg/mL). Then, GDI (1.6 eq per Glutamic acid unit) was added to the reaction mixture and stirred for 30 minutes at room temperature. After this time, the 3-aminopropane-1 ,2-diol (1.3 eq per Glutamic acid unit) dissolved in 2 mL of DMF was added. The mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into acetone to precipitate the product. Precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The final product was isolated as a white solid. Yield: 50-60%.
  • oligonucleotides used were: a pDNA purchased from PlasmidFactory, with reference PF461 (pCMV-luc)), containing 6233 bp expressing luciferase and a mRNA purchased from Trilink, with reference L-1201 -1000 CleanCap Flue mRNA (5moU) expressing luciferase as reporter gene. Any other oligonucleotide with the characteristics above indicated could be used to carry out the experiments below.
  • the ionizable lipid DLin-MC3-DMA, or (6Z,9Z,28Z,31 Z)-heptatriacont-6,9,28,31 -tetraene- 19-yl 4- (dimethylamino)butanoate was purchased from Nanosoft Polymers. Shielding lipid 1 ,2-Dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) was purchased from Avanti Polar Lipids. Both polymers were used to form benchmark LNPs.
  • Structural lipids used to form LNPs were: 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC, from Avanti Polar Lipids, 850365P) and Cholesterol (Sigma-Aldrich, C8667).
  • a microfluidic HerringboneMixer from Darwing Microfluidics was used for the formulation of LNPs 1-12.
  • the reactor presents 2 inlet-channels (one for the DNA in aqueous phase and the other for the lipid mixture in ethanol) and 1 outlet-channel.
  • the specifications of the mixer are the following:
  • LNPs were diluted in 10 mM Phosphate buffer saline (PBS) pH 7.4 and concentrated with centrifugal concentrators (VivaspinTM 500, Sigma) to remove ethanol
  • Lipids were dissolved in ethanol at 46.3/9.4/42.7/1.6 molar ratio (ALC-0315/DSPC/Cholesterol/ DMG- PEG).
  • LNPs were further diluted in 10 mM Phosphate buffer saline (PBS) pH 7.4 to decrease ethanol concentration and purified with centrifugal concentrators (VivaspinTM 500, Sigma) to remove ethanol.
  • PBS Phosphate buffer saline
  • the formulation containing the polymer of the invention was prepared analogously with the difference that different LipoPAA was used instead of shielding lipid DMG-PEG and the lipid ratio was different (shown below Table 27 and 28).
  • N nitrogen
  • P negatively-charged nucleic acid phosphate
  • Size and shielding polymers were performed using a Malvern ZetasizerNanoZS instrument, equipped with a 532 nm laser at a fixed scattering angle of 173°. 50 l of the samples were measured using a quartz glass high performance cuvette (Hellma Analytics). Size distribution was measured (diameter, nm) with n > 3 measurements.
  • Table 23 shows the results obtained of Lipo-PAA2 formulated at molar ratio of 5% containing pDNA as cargo.
  • Table 25 shows the results obtained of Lipo-PAA7 formulated at molar ratio of 5% directly containing mRNA as cargo.
  • Lipo-PAA9 is similar to Lipo-PAA1, Table 26 shows the results obtained at molar ratio of 2% directly containing mRNA as cargo. Table 26. Size and PPI of Lipo-PAA9 LNPs containing mRNA.
  • LNPs were obtained with a good size (expressed as Z-average) and PDI and reproducible formulations.
  • Table 28 shows the results obtained of Lipo-PAA1, Lipo-PAA8 and Lipo-PAA10 formulated at molar ratio of 2% containing mRNA as cargo:
  • E-gel Power Snap Electrophoresis Device and E-Gel Power Snap Camera were used to perform the electrophoresis.
  • 2% agarose gels prepared that include the SYBR Gold DNA marker (E-GelTM EX Agarose Gels, 2%, Invitrogen) were used following manufacturer's instructions.
  • the LNPs (20 l) composed by the different lipo-polypeptide shieldings indicated in Figure 1 were evaluated, and also the disassembly of the LNPs in the presence of 10% triton X-100 (Scharlab.S.L) and 2 mg/mL heparin (PanReacAppliChem, Spain).
  • the equipment protocol was selected according to the type of gel used (in our case, protocol approximately 10 min, although the time can be modified according to the samples). In all cases, no free mRNA was observed when different lipo-polypeptide shieldings were tested.
  • LNPs apparent pKa values were determined by measuring the fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid (TNS) during titration with different buffer solutions: from pH 4 to 5.5 (0.5 pH unit increments) with 20 mM Acetate buffer; from pH 6 to 7.5 (0.5 pH unit increments) with 20 mM Phosphate buffer; from pH 8 to 9 (0.5 pH unit increments) with 20 mM Borate buffer.
  • TMS 2-(p-toluidino)-6-napthalene sulfonic acid
  • the pKa of the LNPs formulated with Lipo-PAA1 and Lipo-PAA2 are similar to the benchmark LNPs, which is comprised between 6.2 to 6.7. It is reported that this range of pKA is ideal to obtain the best performance of the LNPs.
  • HeLa cells were cultured in DMEM high glucose with Glutamax (Gibco- Thermo Fisher # 61965-059) supplemented with 10% of Fetal Bovine Serum (Hyclone # SV30160.03HI, provided by GE Healthcare Europe GmbH) and 1 % penicillin/streptomycin. Transfections were carried out on 96-well plates containing 10OOOcells/well in a final volume of 10Opil, and cells were incubated 24 hours at 37 °C and 5% CO2. After 24 h from cell seeding, the medium was removed and refreshed with 90pil of complete medium. 10pil of each LNPs formulation were added to the cells. In the case of positive control (LipofectamineTM 2000 Transfection Reagent, I nvitrogen TM), manufacturer guidelines were followed. After 24 hours cells were recovered and processed.
  • positive control LipofectamineTM 2000 Transfection Reagent, I nvitrogen TM
  • ATPLite reagent ATPLite PerkinElmer #6016731
  • Luminiscence was read spectrophotometrically using VictorNivo (PerkinElmer) following manufacturer's instructions and data was represented as the percentage of cell viability, taken untreated control cells as 100%.
  • the transfection efficiency and the cell viability of the LNPs formulations in HeLa cells is reported in the following table.
  • the transfection data is represented as % of the positive control LipofectamineTM 2000 being the positive control 100% after 24h of treatment and cell viability is compared to non-treated (NT) cells, being the ATP content readout of NT (non-treated) cells equal to 100%.
  • HEK293 cells were cultured in DMEM high glucose with Glutamax (Gibco- Thermo Fisher # 61965-059) supplemented with 10% of Fetal Bovine Serum (Hyclone # SV30160.03HI, provided by GE Healthcare Europe GmbH) and 1 % penicillin/streptomycin. Transfections were carried out on 96-well plates containing 30000 cells/well in a final volume of 10Opi, and cells were incubated 24 hours at 37 °C and 5% CO2. After 24 h from cell seeding, the medium was removed and refreshed with 90pl of complete medium. 1 O l of each LNPs formulation were added to the cells. After 24 hours cells were recovered and processed. 9.2 ATP Evaluation for Cell Toxicity evaluation
  • ATPLite reagent ATPLite PerkinElmer #6016731
  • Luminiscence was read spectrophotometrically using VictorNivo (PerkinElmer) following manufacturer's instructions and data was represented as the percentage of cell viability, taken untreated control cells as 100%.
  • the transfection efficiency and the cell viability of the LNPs formulations in HEK293 cells is reported in the following table.
  • the transfection data is represented as % of the positive control DMG-PEG with the formulation reported 46.3/9.4/44.3-x/x molar ratios for ALC-0315/DSPC/Cholesterol/DMG-PEG or LipoPAA being x the different molar ratios for each LipoPAA employed, and being the positive control 100% after 24h of treatment and cell viability is compared to non-treated (NT) cells, being the ATP content readout of NT (nontreated) cells equal to 100%.
  • NT non-treated
  • the transfection obtained from LNPs containing Lipo-PAA1 and Lipo-PAA8 and Lipo- PAA10 was higher than those LNPs containing DMG-PEG as well as the PEG alternative C14-PSar. Additionally, It is remarkable that, structurally different conjugates transfected the cells from 1.9 to 3.3-fold (Lipo-PAA1), from 3.1 to 3.3-fold (Lipo-PAA8), and from 3.3 to 5.1-fold (Lipo-PAA10) more efficiently than the commercial standard DMG-PEG at different concentrations, while C14PSar showed only comparable transfection to DMG-PEG.
  • lipo-PAA into LNPs, the following is obtained: i) stable nanoparticles with acceptable size and polydispersity; ii) non-cytotoxic formulation, and iii) high transfection efficiency.
  • mice were injected subcutaneously Luciferin (15 mg/kg), and after 10 minutes mice were euthanized via CO2 inhalation and organs dissected (blood, liver, spleen, lungs, kidney and ALN (Axillary Lymph Nodes).
  • Image acquisition was obtained by I VIS Spectrum (settings: Binning factor 8 F/Stop 1 Exp AUTO FOV 22,5 (D)). The weight of the different organs was obtained and then freezed with N2 and -80°C.
  • Image Analysis was performed by Living Image 4.5.4 (settings: ROI (Draw auto, threshold 2%) and data represented in total counts I tissue g.
  • mice after administration of the different formulations were weighted obtaining the following results in Table 32:
  • mice Body weight (g) of mice administered iv with the formulations at time 4 and 24h.
  • X is selected from the group consisting of N, S, and 0;
  • Ri is selected from the group consisting of H, -(Ci-Ci8)alkyl, and -(C2-Ci8)alkenyl, with the condition that Ri is absent when X is 0 or S;
  • R2 is a radical selected from the group consisting of (II), (III), (IV), and (V), wherein: a, b, c, and d are independently an integer from 1 to 4;
  • R3 is H or -(Ci-Cejalkyl
  • A is selected from the group consisting of H, -(Ci-Cejalkyl, -CO(Ci-C6)alkyl, -(Cs-C jaryl, -(Cs-Ciojheteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-C-(C5-Cio)aryl, -(Cs-Ciojheterocycloalkyl, a protective group, a radical R4, a lipid- like moiety R5, and an active moiety; being A optionally substituted by one or more groups selected from the group consisting of -OH, halogen, -CF3, -NH2, -NH-(Ci-C4)alkyl, -NH-CO-(Ci-C6)alkyl, -(Ci-Cejalkyl, -NO2, -N 3 , -CO-(Ci-C 6 )alkyl, -CO
  • A' is selected from the group consisting of H, -OH, -(Ci-Cejalkyl, -(Cs-Ciojaryl, -(Cs-Ciojheteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(Cs-Ciojheterocycloalkyl, -(C1-C10) alkoxy, -(Ce-Cwjaryloxy, -(C6-Cio)aralkoxy, -(C5-Cio)heteroaralkoxy, -(Ci-Cio)alkyl-0-(C6-Cio)aryloxy, an amino protective group, a radical R4', a lipid-like moiety R5', an amino acid-like moiety Re, and an active moiety; being A' optionally substituted by one or more groups selected from the group consist
  • R4 is a radical of formula (Via);
  • R4' is a radical of formula (Vlb); wherein: n' is an integer from 5 to 250; m' is 0 or 1 ;
  • X is selected from the group consisting of N, S, and 0;
  • R1 is a radical which has any of the meanings of R1, with the condition that R1” is absent when X” is O or S;
  • R2' is a radical which has any of the meanings of R2;
  • R3' is a radical which has any of the meanings of R3;
  • B and B’ are independently S or CH2; e and f are independently an integer from 1 to 18;
  • A” is selected from the group consisting of H, -OH, -(Ci-C6)alkyl, -(C5-Cio)aryl, -(C5-Cio)heteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, -(C1-C10) alkoxy, -(C6-Cio)aryloxy, -(C6-Cio)aralkoxy, -(C5-Cio)heteroaralkoxy, -(Ci-Cio)alkyl-0-(C6-Cio)aryloxy, an amino protective group, a lipid-like moiety R5', an amino acid-like moiety Re, and an active moiety; being A" optionally substituted by one or more groups selected from the group consisting of -OH
  • Y and Y' are independently selected from -OH, -OCORx and -COORx;
  • Q and Q' are independently selected from -OCORy and -COORy;
  • Z and Z' are independently selected from -OCO-, -COO-, -NRz’CO-, and -CONRz'-; each Rz’ is H or Rz; each Rx, Ry, and Rz is independently -(Ci-Ci8)alkyl or -(C 2 -Ci8)alkenyl; each g is independently an integer from 0 to 18; each h is independently an integer from 1 to 18; i is an integer from 0 to 18; each j is independently an integer from 0 to 18;
  • X' is selected from the group consisting of N, S, and 0;
  • R22, 33 and R44 are independently selected from the group consisting of hydrogen, fluorine, methyl, -CH2F, -CHF2, and -CF3; each of the dashed bonds — is independently a single bond or, alternatively, a double bond;
  • A' is selected from the group consisting of H, -(Ci-Cejalkyl, -(Cs-C jaryl, -(Cs-Ciojheteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, an amino protective group, an amino acid-like moiety Re, and an active moiety; when m is 1 , A' is selected from the group consisting of H, -OH, -(Ci-Ce)alkyl, -(Ce-Cwjaryl, -(Cs-Ciojheteroaryl, -(Ce-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio
  • n is an integer from 5 to 150, more particularly from 5 to 100, even more particularly from 5 to 50, and even more particularly from 5 to 30 or from 5 to 25.
  • Clause 7 The lipo-polyamino acid conjugate according to any of clauses 1 to 4, wherein X is N and R1 is -(C2-Ci8)alkenyl, more particularly -(C2-Ci2)alkenyl.
  • Clause 8 The lipo-polyamino acid conjugate according to any of clauses 1 to 4, wherein X is O and R1 is absent.
  • Clause 10 The lipo-polyamino acid conjugate according to any of clauses 1 to 9, wherein m is 0, and A' is selected from the group consisting of H, -(Ci-Cejalkyl, -(C5-Cio)aryl, -(Cs-Ciojheteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-C-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, an amino protective group, an amino acid-like moiety Re, and an active moiety; being A' optionally substituted by one or more groups selected from the group consisting of -OH, halogen, -CF3, -NH2, -NH-(Ci-C4)alkyl, -NH-CO-(Ci-Ce)alkyl, -(Ci-Ce)alkyl, -NO2, -N3, -CO
  • A' is selected from the group consisting of H, -OH, -(Ci-Cejalkyl, -(Cs-C jaryl, -(C5-Cio)heteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(Cs-Ciojheterocycloalkyl, -(C1-C10) alkoxy, -(Ce-Cio)aryloxy, -(C6-Cio)aralkoxy, -(Ce-Cio)heteroaralkoxy, -(Ci-Cio)alkyl-0-(C6-Cio)aryloxy, a radical R4', a lipid-like moiety R5', and an active moiety
  • Clause 14 The lipo-polyamino acid conjugate according to any of clauses 1 to 11, wherein R 2 is a radical of formula (IV), more particularly in the radical of formula (IV), c is 1 or 2.
  • Clause 15 The lipo-polyamino acid conjugate according to any of clauses 1 to 11, wherein R 2 is a radical of formula (V), more particularly in the radical of formula (V), d is 1 or 2.
  • Clause 16 The lipo-polyamino acid conjugate according to any of clauses 1 to 15, wherein R3 is H.
  • Clause 17 The lipo-polyamino acid conjugate according to any of clauses 1 to 15, wherein R3 is -(Ci-Cejalkyl, more particularly R3 is -CH3.
  • A is a lipid-like moiety R5, and A' is other than a lipid-like moiety R5', more particularly, A' is selected from the group consisting of H, -OH, -(Ci-Cejalkyl, -(Cs-C jaryl, -(C5-Cio)heteroaryl, -(C6-Cio)aralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, -(Ci-Cio)alkoxy, -(Ce-Cio)aryloxy, -(Ce-Cio)aralkoxy, -(C5-Cio)heteroaralkoxy, -(Ci-Cio)alkyl-0-(C6-Cio)
  • A is a lipid-like moiety R5 which is a radical of formula (IX), more particularly, a radical selected from the formulas (ix1), (ix2), (ix3), (ix4), (ix5), (ix6), (ix7) and (ix8).
  • A is a lipid-like moiety R5 which is a radical of formula (X), more particularly, a radical selected from the formulas (x1), (x2), (x3), and (x4).
  • Clause 28 The lipo-polyamino acid conjugate according to any of clauses 1 to 18, wherein A is a lipid-like moiety R5 which is a radical of formula (XIV), more particularly, a radical selected from the formulas (xiv1), and (xiv2).
  • A is a lipid-like moiety R5 which is a radical of formula (XIV), more particularly, a radical selected from the formulas (xiv1), and (xiv2).
  • Clause 30 The lipo-polyamino acid conjugate according to any of clauses 1 to 18, wherein A is a lipid-like moiety R5 which is a radical of formula (XXII), more particularly, a radical selected from the formulas (xxiil), and (xxii2).
  • Clause 31 The lipo-polyamino acid conjugate according to any of clauses 1 to 18, wherein A is a lipid-like moiety R5 which is a radical of formula (XXIII).
  • Clause 32 The lipo-polyamino acid conjugate according to any of clauses 1 to 31, wherein A is a lipid-like moiety R5, m is 0, and A' is H.
  • Clause 40 The lipo-polyamino acid conjugate according to any of clauses 1 to 9, or 16 to 17, which has the formula (Ig'), wherein A' is selected from the group consisting of H, -OH, -(Ci-Cejalkyl, -(Cs-C jaryl, -(C5-Cio)heteroaryl, -(Ce-Cwjaralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(C5-Cio)heterocycloalkyl, -(C1-C10) alkoxy, -(Ce-Cwjaryloxy, -(Ce-Cwjaralkoxy, -(C5-Cio)heteroaralkoxy, -(Ci-Cio)alkyl-0-(C6-Cio)aryloxy; being A' optionally substituted by one or more groups selected from the group consisting of -OH,
  • A is a lipid-like moiety R5, m is 1
  • A' is an active moiety selected from the group consisting of a pharmaceutically active agent, a cell-targeting agent, a penetration enhancing agent, a cosmetically active agent, and a diagnostically active agent.
  • A' is a lipid-like moiety R5' which is -X'(Ri')-(Ci-Ci8)alkyl, more particularly -X'(Ri')-(C6-Ci8)alkyl, and even more particularly, a radical of the formula (XIX') or a formula (XIX”).
  • A' is a lipid-like moiety R5' which is -X'(Ri')-(C2-Ci8)alkenyl, more particularly -X'(Ri')-(C6-Ci8)alkenyl, and even more particularly, a radical of the formula (XX').
  • A' is a lipid-like moiety R5' which is a radical of formula (IX'), more particularly, a radical selected from the formulas (ixT), (ix2’), (ix3’), (ix4’), (ix5’), (ix6’), (ix7’) and (ix8’).
  • A' is a lipid-like moiety R5' which is a radical of formula (X'), more particularly, a radical selected from the formulas (xT), (x2'), (x3’), and (x4’).
  • Clause 60 The lipo-polyamino acid conjugate according to any of clauses 1 to 9, or 12 to 17, or 46 to 58, wherein A' is a lipid-like moiety R5', m is 1, and A is H.
  • the lipo-polyamino acid conjugate according to any of clauses 1 to 9, or 12 to 17, which has the formula (lb'), more particularly the lipo-polyamino acid conjugate of formula (lb') is a dimer wherein R5' and A” have the same meaning, m and m' have the same meaning, X and X” have the same meaning, R1 and R1” have the same meaning, R2 and R2' have the same meaning, Ra and R3' have the same meaning, B and B’ have the same meaning, e and f have the same meaning, and n and n' have the same meaning, respectively.
  • A' is a lipid-like moiety R5'
  • m is 1, and A is selected from the group consisting of H, -(Ci-Cejalkyl, -CO(Ci-C6)alkyl, -(Cs-C jaryl, -(C5-Cio)heteroaryl, -(Ce-Cwjaralkyl, -(Ci-C6)alkyl-0-(C5-Cio)aryl, -(Cs-Ciojheterocycloalkyl, a protective group, a radical R 4 , a lipid-like moiety R5, an active moiety; being A optionally substituted by one or more groups selected from the group consisting of -OH, halogen, -CF3, -NH2, -NH-(Ci-C 4 )alkyl, -NH-CO-
  • Clause 64 The lipo-polyamino acid conjugate according to any of clauses 1 to 9, or 12 to 17, or 46 to 58, wherein A' is a lipid-like moiety R5', m is 1, and A is an active moiety selected from the group consisting of a pharmaceutically active agent, a cell-targeting agent, a penetration enhancing agent, a cosmetically active agent, and a diagnostically active agent.
  • a self-assembled particle comprising the lipo-polyamino acid conjugate of formula (I) as defined in any of clauses 1-64, and optionally one or more active agents selected from the group consisting of pharmaceutically active agents, cell-targeting agents, penetration enhancing agents, cosmetically active agents, diagnostically active agents, nucleic acids, peptides, proteins, and mixtures thereof.
  • the self-assembled particle according to clause 65 which is a nanoparticle, more particularly selected from the group consisting of a micelle, an inverted micelle, a planar bilayer, a crystal nanoparticle, a liposome, microbubbles, and a lipid nanoparticle, and even more particularly, the self-assembled particle is a lipid nanoparticle (LNP) or a liposome.
  • LNP lipid nanoparticle
  • the self-assembled particle according to any of the clauses 65 to 66 which is a lipid nanoparticle (LNP) and further comprises one or more lipids selected from the group consisting of ionisable lipids, cationic lipids, neutral lipids, and anionic lipids.
  • LNP lipid nanoparticle
  • Clause 68 The self-assembled particle according to any of the clauses 65 to 67, which is a lipid nanoparticle (LNP) and further comprises a ionizable lipid or a cationic lipid, a phospholipid, and a sterol.
  • LNP lipid nanoparticle
  • the self-assembled particle according to any of the clauses 65 to 68 which is a lipid nanoparticle which comprises: i) the lipo-polyamino acid conjugate of formula (I) as defined in any of the clauses 1 to 64 in an amount from 0.1 to 10 mol%, more particularly from 1 to 6 mol%, even more particularly from 2 to 5% mol%; ii) a ionizable or a cationic lipid in an amount from 30 to 70 mol%, more particularly from 40 to 60 mol%; iii) a phospholipid in an amount from 1 to 20 mol%, more particularly from 5 to 15 mol%; and iv) a sterol in an amount from 20 to 60 mol%, more particularly from 30 to 50 mol%; wherein the percentages are expressed with respect to the sum of the mol% of the lipids and the lipo- polyamino acid conjugate of formula (I).
  • Clause 70 The self-assembled particle according to clause 69, which is a lipid nanoparticle which further comprises a nucleic acid.
  • Clause 71 The self-assembled particle according to any of the clauses 65 to 70, which lacks polyethyleneglycol (PEG).
  • PEG polyethyleneglycol
  • Clause 72 A composition comprising the lipo-polyamino acid conjugate of formula (I) as defined in any of the clauses 1 to 64, or alternatively, the self-assembled particle as defined in any of the clauses 65 to 71, together with one or more appropriate excipients or carriers.
  • a therapeutic product which is: a) a lipo-polyamino acid conjugate of formula (I) as defined in any of the clauses 1 to 64, wherein at least one of A, A', A” or A'” is a pharmaceutically active agent, or alternatively, b) a self-assembled particle as defined in any of the clauses 65 to 71 containing the lipo-polyamino acid conjugate a), or alternatively, c) a composition as defined in clause 72 containing the lipo-polyamino acid conjugate a) or the selfassembled particle b), or alternatively, d) a self-assembled particle as defined in any of the clauses 65 to 71, which comprises one or more active agents selected from the group consisting of pharmaceutically active agents, nucleic acids, peptides, proteins, and mixtures thereof, or alternatively, e) a composition as defined in clause 72 containing the self-assembled particle d), for use in medicine.
  • Clause 74 The therapeutic product for use according to clause 73, for use (I) as transfection reagent for transfecting at least one active agent into a cell; (II) for use in the in vivo or ex vivo production of biologies encoding a recombinant protein, a peptide or an antibody, or in the production of recombinant virus; (ill) for use as a therapeutic or prophylactic vaccine against viral infections or as a therapeutic vaccine against cancers or infectious diseases; or (v) for use in genome engineering, for cell reprogramming, for differentiating cells or for gene-editing.
  • a diagnostic product which is: a') a lipo-polyamino acid conjugate of formula (I) as defined in any of the clauses 1 to 64, wherein at least one of A, A', A” or A'” is a diagnostically active agent, or alternatively, b') a self-assembled particle as defined in any of the clauses 65 to 71 containing the lipo-polyamino acid conjugate a'), or alternatively, c') a composition as defined in clause 72 containing the lipo-polyamino acid conjugate a') or the self- assembled particle b'), or alternatively, d') a self-assembled particle as defined in any of the clauses 65 to 71, which comprises one or more diagnostically active agents, or alternatively, e') a composition as defined in clause 72 containing the self-assembled particle d'), for use in diagnostics.
  • Clause 76 Use in cosmetics of a cosmetic product which is: a") a lipo-polyamino acid conjugate of formula (I) as defined in any of the clauses 1 to 64, wherein at least one of A, A', A” or A'” is a cosmetically active agent, or alternatively, b”) a self-assembled particle as defined in any of the clauses 65 to 71 containing the lipo-polyamino acid conjugate a"), or alternatively, c”) a composition as defined in clause 72 containing the lipo-polyamino acid conjugate a") or the selfassembled particle b”), or alternatively, d”) a self-assembled particle as defined in any of the clauses 65 to 71, which comprises one or more cosmetically active agents, or alternatively, e”) a composition as defined in clause 72 containing the self-assembled particle d”).
  • Clause 77 Use of the lipo-polyamino acid conjugate of formula (I) as defined in any of clauses 1 to 64, wherein A, A', A” and A'” are other than an active moiety, or alternatively, a self-assembled particle as defined in any of the clauses 59 to 65 containing it, as a carrier.

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WO2025021942A1 (en) 2023-07-27 2025-01-30 Bracco Suisse Sa Ultrasound responsive vesicles containing lipid-polyamino acid conjugates
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WO2025257436A3 (en) * 2024-06-14 2026-01-22 Cordenpharma International Gmbh Novel n-substituted polyglycine lipids

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