Amphiphilic conjugate of hyaluronic acid oligomer, micellar composition containing it, method for their preparation and use

Abstract

The invention relates to an amphiphilic conjugate of a hyaluronic acid oligomer, which is prepared by a condensation reaction of an oxyamine group attached to a non-polar chain with the anomeric end of a polar hyaluronic acid oligosaccharide, and to micellar compositions prepared from the amphiphilic conjugates of hyaluronic acid oligosaccharides. The invention further relates to a method for preparing the amphiphilic conjugate and a method for preparing the micellar composition containing it, optionally with one or more active substances. The micellar composition based on amphiphilic conjugates can be used, for example, in pharmaceutical and cosmetic applications where the transport of less polar active substances in a polar environment is required. Formula (I), where M is hydrogen, sodium or potassium, n is the number of disaccharide units of the hyaluronic acid oligomer in the range of 1 to 4, X is a non-polar linear chain of 16 to 20 carbons with the possible content of multiple bonds, for example hexadecyl, octadecyl or cA-octadec-9-enyl.

Description

[0001] Amphiphilic conjugate of hyaluronic acid oligomer, micellar composition containing it, method for their preparation and use

[0002] Technical field

[0003] The invention relates to an amphiphilic conjugate of a hyaluronic acid oligomer, which is prepared by means of a condensation reaction of an oxyamine group bound to a non-polar chain, with the anomeric end of a polar hyaluronic acid oligosaccharide, and micellar compositions prepared from amphiphilic conjugates of hyaluronic acid oligosaccharides. The thus prepared water-soluble amphiphilic conjugates spontaneously form micellar formations in an aqueous environment, mainly with dimensions of 7 to 100 nm, in which less polar molecules can be bound by physical interactions. The core of the micellar composition is formed by a nonpolar linear chain, and the surface of the micelle is formed by hyaluronic acid oligosaccharide containing polar functional groups, such as carboxyl. Encapsulation of less polar active substances into micelles allows effective transport of active substances in a physiological environment. Micellar compositions based on amphiphilic conjugates can be used in pharmaceutical and cosmetic applications where the transport of less polar active substances in a polar environment is required.

[0004] State of art

[0005] Hyaluronic acid

[0006] Hyaluronic acid or its sodium salt is a non sulfated glycosaminoglycan composed of two repeating units of D-glucuronic acid and A-acetyl-D-glucosamine. Hyaluronic acid oligosaccharides (oliHA) contain 1 to 15 repeating disaccharide units and are prepared from polymeric hyaluronic acid by enzymatic degradation and subsequent chromatographic separation (Sindelaf M. et al., Colloids and Surface B: Biointerfaces, 208, 112095, 2021). OliHAs exhibit a wide range of biological functions related mainly to their binding to proteins (Garantziotis S. et al., Matrix Biology, 7, 79, 1-10, 2019).

[0007] Oximes at the anomeric end of saccharides

[0008] Oxyamine groups of the general formula NH2-O-R react readily with aldehydes of the general formula R'-CHO to form oximes of the general formula R1-CH=N-O-R, and this condensation reaction is a robust tool for the modification, or rather the attachment, of macromolecules. In the case of saccharides that contain an aldehyde group masked in the form of cyclic acetals at their anomeric end, the reactivity with oxyamines is significantly lower. To accelerate the reaction with the saccharide anomeric end of glucose (Glc), N-acetylglucosamine (GlcNAc) or glucuronic acid (GlcA), for example, aniline or its compounds, such as 2-amino-5- methoxybenzoic acid (PAN-C, pKa 5.6), 3,5-diaminobenzoic acid (MDA-C, pKa 5.1), 4- methoxyaniline (PAN, pKa 5.3) or 1,4-diaminobenzene (PDA, pKa 6.1) are therefore used (Ostergaard M. et al., Bioconjugate Chemistry, 29, 1219-30, 2018). The study showed that the reaction of O-benzyl-N-methyl hydroxylamine is 20 times slower than the reaction of O- benzylhydroxylamine. The reaction of glucose saccharides was found to proceed most rapidly at pH 4. For Glc and GlcNAc, PAN-C is the most effective catalyst, while for GlcA it is aniline. PDA provides oximes most rapidly at higher pH (pH 6-7), increasing the reaction rate 34-fold for Glc, 50-fold for GlcA, and 75-fold for GlcNAc compared to the uncatalyzed reaction.

[0009] While hydrophilic oxyamines bind to the reducing end of saccharides or oligosaccharides in acidic aqueous media, usually with acetate buffer (Baudendistel O. R. et al., Chemistry-A Europrean Journal, 22, 17359-65, 2016), or with aniline catalysis (McReynolds K. D. et al., Tetrahedron Lett. 55, 2270-73, 2014), for the binding of hydrophobic oxyamines it was necessary to use other solvents, such as methanol (Silva J. et al., J. Mater. Chem. B, 10, 5016-27, 2022) or a mixture of acetate buffer with isopropanol in a ratio of 1 / 1 (Renaudet O. et al., PLoS ONE, 5, el 1216, 2010), or a mixture of DMF-AcOH (Cheng S. et al., European Journal of Medicinal Chemistry, 134, 43-51, 2017), or a mixture of CHC13-MeOH-H2O-AcOH (Ishida J. et al., Bioorganic & Medicinal Chemistry Letters, 24, 1197-200, 2014). In general, it can be stated that for the successful condensation of oxyamine with aldehyde polar solvents are preferred, which, on the other hand, are not preferred for the conjugation of molecules with low polarity described in the following paragraph due to the low solubility of non-polar substances.

[0010] Amphiphilic conjugates of oliHA with amino compounds with reductive amination

[0011] Liposomes based on oliHA (4, 6, 8 saccharides) prepared by coupling phosphatidylethanolamine lipids to the anomeric end of oliHA by reductive amination were used as carriers of doxorubicin, with the particle size of these liposomes 127-134 nm (Eliaz R. E. et al., Cancer Res. 61, 2592-601, 2001).

[0012] Similarly, the active substance gemcitabine was introduced into a liposome formed from an amphiphilic conjugate of l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and oliHA (12 and 32 HA disaccharides) (Apricco S., 2013). The synthesis of the amphiphilic conjugate itself proceeds in the DMSO / methanol / chloroform system for 96 hours at 60 °C. The average size of the liposome was in the range of 150-190 nm.

[0013] The amphiphilic glycoside oliHA (5.6-8.9 kDa) with bound poly(lactic-co-glycolic acid) (lactic and glycolic acid copolymer) (Mw 13.6 kDa) was prepared by binding ethylenediamines to the anomeric end by reductive amination. This amino conjugate of oliHA was subsequently bound to the carboxyl end of poly(lactic- -gly colic acid) activated with N- hydroxy succinimide. The resulting copolymer formed nanoparticles up to 200 nm with a CMC of 7.9-19.9 mg / L and a typical "core-shell” structure. With increasing hyaluronan length, the particle size and CMC increased. Docetaxel was successfully bound to the carrier and showed increased cytotoxicity against MDA-MB-231 cancer cells overexpressing CD44 (Huang J. et al., Biomaterials, 35, 550-66, 2014).

[0014] Fluorescent 2-aminoacridone was bound to oliHA (HA4-HA22) by reductive amination in DMSO / AcOH at 90 °C / 30 min. Interactions with HA-binding proteins, fibrinogen and immunoglobulin G were monitored by capillary affinity electrophoresis. The interaction between oliHA and HA-binding proteins was manifested by the disappearance of the oligosaccharide signal. The interaction increased with the length of the oligomers. The interaction of HA6 and HA8 with HA-binding proteins was not evident even at a protein concentration of 125 nM, but only at a concentration of 1000 nM (Kinoshita M. et al., Journal of Chromatography B, 816, 289-95, 2005).

[0015] HA conjugates with amino compounds without reductive amination

[0016] This section describes conjugates of polymeric and oligomeric hyaluronic acid with oxyamines.

[0017] Hyaluronan fragments (4 kDa) were linked by the reaction of star-like glyco-oxyamines with the anomeric end of oliHA. To increase the reactivity of the reducing end, toxic aniline was used, which stabilizes the iminium intermediate and thus accelerates the reaction. The number of oligosaccharides linked to the branched polyglycerol is limited to 4-6 chains regardless of the higher amount of oxyamine groups of the branched molecule in the "core". The reaction proceeded in a mixture of acetate buffer (pH 4.5) / DMSO (1 / 1) (Novoa-Carballal R. et al., Chemistry - a European Journal, doi: 10.1002 / chem.201802243, 2018).

[0018] 2-(Octadecyloxy)-l,3-dioxan-5-amine, with an orthoester group sensitive to acidic environments, for example in tumors, was attached to the carboxyl of hyaluronic acid by an amide bond (11 kDa, DS 23%, particle size 136 nm) and the derivative was used as a carrier for the drug doxorubicin. The particle size increased with pH decrease to 4 (600 nm), while it decreased with the presence of the drug (Qiu L. et al., Material Science & Engineering C, 78, 912-22, 2017)

[0019] Nonpolar octadecyl amine was grafted in a formamide / DMF solvent mixture onto the carboxyl of HA (25kDa) as an amide with a degree of modification of 12-20%. The conjugate formed micelles of size 170-460 nm with a CMC of 10-40 mg / L, where 10-30% (w / w) paclitaxel was subsequently bound (Liu Y, 2011).

[0020] Oxyamine with PEG linker and biotin can be attached to the anomeric end of oliHA4 up to HA 360 kDa. The reaction proceeds in acetate buffer at pH 4.5 and 37 °C, under aniline catalysis (Thakar D. et al., Chem. Commun., 50, 15148-151, 2014).

[0021] In a review publication (Kim S. et al., Expert Opinion on Drug Delivery, 7, 49-62, 2010), the biodistribution of active substances in various micelles is discussed, where effective particle sizes in the range of 10-200 nm were observed, and the adverse effect of irregularity of the micelle structure on its stability in the bloodstream, precisely because of hydrophobic interactions on the micelle surface, was also discussed.

[0022] In the publication (Ondreas F. et al., Applied Surface Science, 593, 153440, 2022) polymeric micelles formed by HA (Mw 15 kDa) with OH groups esterified with oleic acid and their interaction with BSA were described. It was found that BSA interacts significantly with micelles and functions as a physical crosslinker, where a hydrophobic interaction of BSA with the oleyl chain is assumed.

[0023] Polymeric micelles from an amphiphilic HA derivative (28 kDa), where the fatty chain (Cl 8) is bound by an amide bond to the HA carboxyl in the presence of a redox-cleavable cysteamine (-S-S- bond), were studied in detail in the publication (Jeong G-W. et al., Carbohydrate Polymers, 209, 161-71, 2019). The CMC values were in the range of 0.03 to 0.08 mg / mL and the particle sizes ranged from 230 to 305 nm without bound doxorubicin and 280 to 390 nm with bound doxorubicin. The high binding capacity was interesting, reaching up to 3% (w / w) for doxorubicin.

[0024] A pH-sensitive amphiphilic conjugate of HA oligosaccharide modified as an amide at the carboxyl by linking histidine as a linker and a hydrophobic moiety formed by menthone 1,2-glycerol ketal (Chen D. et al., Drug Delivery, 23, 798-803, 2016) was used to bind the model substance curcumin. The particle size ranged from 120 to 170 nm at pH 7.4 in PBS. As expected, faster curcumin release was observed at pH 5 than at pH 7.4.

[0025] The influence of the presence of a sterically accessible non-polar chain was observed in the development of an antimicrobial and hemocompatible biomaterial binding albumin from the bloodstream (Felgueiras H. P. et al., ACS Appl. Mater. Interfaces, DOI: 10.1021 / acsami.6bl6415, 2017). A HA derivative (330 kDa) with octadecyl acrylate bound to the carboxyl group of HA via an amide was used as a matrix. It was shown that significantly less human serum albumin (HSA) was absorbed (270 ng / cm2) on the HA precursor without the presence of non-polar octadecyl than on the final amphiphilic derivative with octadecyl with a polymer modification degree of 20% (1100 ng / cm2). No differences in the absorption rate between HSA and bovine serum albumin (BSA) were observed.

[0026] Peptides with a palmitoylated N-terminus were linked to the anomeric end of saccharides (including HA) by an O-glycosidic bond, where click chemistry (alkyne-azide) was used for the final connection of the polar saccharide part and the non-polar peptide part (Stupp S. I. et al., WO2016 / 168302). oliHA can also be linked to polymeric HA using a bisoxyamine linker, which reacts to form oximes with both the anomeric end of oliHA and the aldehyde group generated on polymeric HA at position 6 of glucosamine (Buffa R. et al., WO2015 / 135511). This involves the connection of two polar substrates, where an aqueous environment with acetic acid catalysis can be used.

[0027] In general, it can be stated that to ensure the firm binding of non-polar molecules to the anomeric end of oliHA, reductive amination is most often used, which converts aminoglycosides into hydrolytically very stable secondary amines. The disadvantage of these procedures is that they allow the formation of conjugates without the possibility of re-release of oligosaccharides in the native form, because they are associated with an irreversible reductive change of the terminal saccharide cycle. In the case of the connection of the amine and the terminal anomeric center of oliHA, which allows the re-release of oliHA in the native form, procedures for the preparation of conjugates connecting molecules with similar polarity are described. However, these procedures are not suitable for the preparation of the material covered by this invention, which connects a very polar oliHA with a very non-polar linear chain by a hydrolytically cleavable imine bond without the presence of linkers. The procedure described in this invention uses the solvent DMSO with the presence of acetic acid at a higher temperature. This combination enables the preparation of amphiphilic conjugates, which in an aqueous environment create better defined, degradable micellar compositions with the possibility of binding the active substance, while these compositions exhibit acceptable properties (CMC, particle size, binding capacity, stability). In less defined micellar compositions, it can be assumed that the non-polar parts of the conjugate are better accessible for unwanted interactions, which significantly complicate the transport of the entire micelle, and therefore the active substance to the target. In better defined compositions, parts containing polar oliHA are accessible, and their interactions with cell receptors such as CD44, TLR, RHAMM are, on the contrary, desirable, as they can significantly enhance, or target, the distribution of the active substance into cells (Nesporova K. et al., International Journal of Pharmaceutics, 511, 638-47, 2016).

[0028] Summary of the invention

[0029] The subject of the invention is a chemically modified hyaluronic acid oligosaccharide in the form of an amphiphilic conjugate, and micellar compositions containing it, optionally with a possible non-covalently bound active substance. The hyaluronic acid oligosaccharide or its salt is chemically modified at its anomeric end with a hydrophobic chain to form an amphiphilic conjugate according to Scheme 1, where there is an equilibrium between four mutually isomeric chemical structures (I) to (IV).

[0030] Scheme 1 General formula of amphiphilic conjugates of hyaluronic acid oligomers where M can be hydrogen, sodium or potassium, n is the number of disaccharide units of the hyaluronic acid oligomer in the range of 1 to 4, X is a non-polar linear chain of 16 to 20 carbons with the possible content of multiple bonds, for example hexadecyl, octadecyl or cz -octadec-9-enyl.

[0031] The micellar composition contains the following components listed in weight percentages: water 96 to 99.8% one or more active substances in total amount 0 to 1% one or more biocompatible salts, such as inorganic salts, for example NaCl, or a mixture of NaCl, KC1, NaEbPC and Na2HPO4 in a total amount

[0032] 0.01 to 1% a conjugate of hyaluronic acid oligomer according to at least one of the isomeric formulae shown in the Scheme 1 0.01 to 2%

[0033] Active substances include, in particular, less polar vitamins, drugs, cytostatics, steroids and cosmetically active substances, such as curcumin, amphotericin B, cannabidiol (CBD), dexamethasone or quercetin, while the composition may contain multiple active substances simultaneously.

[0034] Acceptable physical properties of final compositions'.

[0035] Particle size (DLS method with particle size distribution by volume) - range 7-100 nm, preferably 7-15 nm.

[0036] The size of 1-4 nm represents approximately the length of one molecule of the amphiphilic conjugate, particles with a diameter of 200 to 500 nm represent large, poorly ordered structures, where the accessibility of both polar and non-polar parts of the composition on the surface of the structure can be assumed.

[0037] For each Example, a number is given that expresses the median particle size and represents at least 90% of the material present. If multiple numbers are given, this means that the particle distribution was clearly multimodal and multiple particle sizes are present, representing more than 10% of the material present, the median values of which are given.

[0038] Critical micellar concentration (CMC) - less than 0.1 mg / mL. At concentrations higher than 0.5 mg / mL, the fraction of amphiphilic molecules that do not assemble into the desired micelles is already unsatisfactory, especially considering that the final composition will be applied in a highly diluted form.

[0039] Binding capacity - at a carrier concentration of 0.1 mg / mL, the amount of the bound active substance or the sum of the active substances higher than 1% (by weight), calculated with respect to the weight of the carrier. At lower binding capacity, conjugates are less effective as earners. Oxime bond stability pH 6 to 7.5 - half-life of hydrolysis of the bond between the anomeric carbon end of oliHA and the amino group of alkyloxyamine longer than 30 days.

[0040] Interaction with human serum albumin (HSA) - it is acceptable if the interactions are minimal or similar to the interactions of native hyaluronic acid with HSA. Stronger interactions are undesirable from the point of view of problematic transport of the final composition, for example in blood, where these interactions significantly complicate transport.

[0041] OliHA can have a number of disaccharide units in the range of 2 to 5. With a higher number of disaccharide units, the availability and reactivity of the anomeric end of oliHA with sterically bulky non-polar molecules are problematic, and an increase in CMC can also be expected due to a more significant representation of the polar component of the amphiphilic conjugate. With one disaccharide unit, the solubility in water is significantly reduced, which complicates the application of the final formulation. The length of the non-polar linear chain is critical for maintaining the final properties of the compositions. In the case of chains shorter than C16 (C12, Comparative Examples 1, 5, 6 and 7), extremely low binding capacity was observed. The particle size and critical micellar concentration were also problematic. In case of longer linear chains containing, for example, more than 20 carbon atoms, whose solubility in a medium suitable for the synthesis of amphiphilic conjugates is very low, attempts to prepare these materials were unsuccessful. The anomeric end of the oligosaccharide chain is also an important structural parameter. If glucuronic acid is present at the anomeric end and not N- acetylglucosamine, the resulting composition is not sufficiently stable (Comparative Examples 8 - 13) and the particle sizes and CMC are also problematic. Also important is the fact that the non-polar linear chain is bound directly to the polar oliHA without the presence of a linker. Analogous and linear amphiphilic conjugates with a 1,3-bisaminooxypropyl bridge between oliHA and the non-polar chain were prepared and studied (Comparative Examples 14 - 16), where the particle size and stability surprisingly turned out to be problematic. For general comparison, an amphiphilic derivative of polymeric hyaluronic acid modified with oleic acid was also included, where an unacceptable particle size and also interaction with the HSA protein were observed (Comparative Example 26). Amphiphilic conjugates of HA oligomers with oleic acid bound to the OH groups of the oligosaccharide by an ester bond showed unacceptable particle size and binding capacity.

[0042] Furthermore, the invention relates to a method for preparing the above conjugate, where the polar oliHA reacts with the oxyamine group of a non-polar linear chain in DMSO in the presence of acetic acid for 5 to 20 days. Specifically, a 5 to 20 wt% solution of hyaluronic acid oligosaccharide in dimethyl sulfoxide reacts at its reducing end with 1.2 to 2.2 equivalents of oxyamine of the general formula H2N-O-X, where X is a linear chain of carbons in the number of 16 to 20 with a possible content of multiple bonds, in the presence of 1.5 to 4 equivalents of acetic acid at a temperature of 40 to 60 °C for 5 to 20 days to form a conjugate. Acetic acid refers to concentrated acetic acid. The covalent bond is formed at the reducing end without the need for protection of hydroxyl and / or carboxyl groups and their subsequent deprotection. When using other solvents or solvent mixtures such as water, water / acetic acid (Buffa R. et al., WO2015 / 135511), water / acetic acid / aniline (ThakarD. et al., Chem. Commun., 50, 15148-151, 2014), water / DMSO / acetic acid (Novoa-Carballal R. et al., Chemistry - A European Journal, doi: 10.1002 / chem.201802243, 2018), water / CHCh / MeOH / AcOH (Ishida J. et al., Bioorganic & Medicinal Chemistry Letters, 24, 1197-200, 2014), water / THF, water / propan-2-ol, DMF / acetic acid (Cheng S. et al., Journal of Medicinal Chemistry, 134, 43-51, 2017), formamide, the conversions of the reactions were very low, with an increase in temperature leading to the formation of undesirable by-products mainly related to the degradation of the oligosaccharide. The resulting conjugate is subsequently isolated by precipitation with mixtures containing chloroform or dichloromethane in combination with hexane or cyclohexane or heptane, which are preferably in a volume ratio of 4:3 to 1 : 1, for example 4:3 or 5:4 or 1 : 1, preferably a mixture containing chloroform and hexane, which are preferably in a volume ratio of 4:3. The isolated conjugate may then be subjected to washing with a mixture containing chloroform or dichloromethane in combination with hexane or cyclohexane or heptane, which are in the mixture preferably in a volume ratio of 3 :2 to 1 : 1, more preferably with a mixture of chloroform and hexane in a volume ratio of 3 :2, to remove dimethyl sulfoxide. This is followed by a washing step with a mixture containing chloroform or dichloromethane in combination with hexane or cyclohexane or heptane, which are in the mixture preferably in a volume ratio of 7: 1 to 15: 1, for example 8: 1 or 10: 1 or 12: 1, more preferably with a mixture of chloroform and hexane in a volume ratio of 10: 1, to remove oxyamine residues.

[0043] The invention further relates to a method for preparing a micellar composition. The micellar composition without active substance is prepared by mixing the amphiphilic conjugate with phosphate buffer (PBS) or physiological saline at a concentration of 0.1 to 2 mg / mL, the resulting solution being mechanically stirred at a temperature of 15 to 25 °C for 0.1 to 24 h, and then filtered through a 1 pm filter or centrifuged. The non-covalent binding of the active substance is carried out by dissolving the active substance in an organic solvent, for example methanol, acetone, ethanol or isopropanol, or a mixture thereof, followed by evaporation of the solvent. Subsequently, the amphiphilic conjugate of oliHA in PBS or physiological saline at a concentration of 0.1 to 2 mg / mL is added to a vessel containing the active substance. The solution is mechanically stirred for 24 to 72 h at a temperature of 15 to 25 °C and the unbound active substance residue is removed either by a 1 pm filter or by centrifugation. The composition can be subsequently sterilized by filtration through a 0.22 pm filter.

[0044] It follows from the above that for the successful preparation of an amphiphilic oliHA conjugate with acceptable properties, a surprisingly narrow range of both chemical structure parameters and reaction condition parameters is required for the preparation of these chemical structures. These ranges are not apparent from the prior art.

[0045] Furthermore, the invention relates to a method of using compositions based on amphiphilic conjugates of oliHA and an active substance, where the amphiphilic conjugate has the advantage of connecting parts with significantly different polarity using a hydrolytically cleavable imine bond allowing reversible release of oliHA in its native form. This is important mainly from the point of view of the safety of the resulting composition, since native oliHA occurs naturally in the body. In addition, the described combination allows the preparation of better defined micellar compositions with acceptable physical properties with a significant content of the active substance, where the non-polar parts of this composition are less accessible to undesirable interactions that can cause significant problems, for example, during intravenous applications (interaction with serum components). Mainly polar parts of the composition containing oliHA are accessible, which can effectively bind to cell receptors of the CD44, TLR, RHAMM type, and thus significantly improve the distribution of the active substance. Therefore, the solution described in this invention is suitable as a carrier of less polar active substances applied, for example, intravenously, for targeted transport of these substances to cells that have receptors on their surface sensitive to the presence of hyaluronic acid fragments, such as some cancer cell lines. The described application options are supported by Examples 46, 47 and 48, which describe the advantageous properties of the conjugates during penetration through the skin or into cells.

[0046] The micellar composition according to the invention can be used in the field of cosmetics or pharmacy, preferably for topical or intravenous applications of active substances.

[0047] Description of the drawings

[0048] Fig. 1 shows the enthalpy changes per mole of HSA depending on the molar ratio HSA:HA (Example 46).

[0049] Fig. 2 shows fluorescence images of curcumin penetration into the skin (Example 47). Fig. 3 shows images demonstrating the passage of curcumin through the cell membrane (Example 48).

[0050] Examples

[0051] The term equivalent (eq) as used herein refers to the molar amount of hyaluronic acid oligosaccharide, unless otherwise stated. Percentages are given as weight percent unless otherwise stated.

[0052] The term critical micelle concentration (CMC) as used herein refers to the concentration at which amphiphilic molecules self-assemble into larger structures under given conditions. oliHA4, 6, 8, 10 is a hyaluronic acid oligosaccharide with the number of saccharide cycles 4, 6, 8 and 10, where the reducing end is N-acetylglucosamine. oliHANA4, 6, 8, 10 is a hyaluronic acid oligosaccharide with the number of saccharide cycles 4, 6, 8 and 10, where the reducing end is glucuronic acid.

[0053] OliHA is a hyaluronic acid oligomer with an unspecified number of disaccharide units in the range of 1 to 10, and an unspecified chain termination (glucuronic acid or A-acetyl-D- glucosamine).

[0054] The term "amphiphilic" means that the substrate contains two parts that differ significantly in their polarity, or water affinity.

[0055] The term "conjugate" means a compound formed by the connection of two or more chemical compounds by means of a covalent bond. The conjugate according to the present invention is formed by the connection of a HA oligomer with a non-polar chain to form an amphiphilic conjugate HA oligomer - non-polar chain.

[0056] The term "hyaluronic acid oligomer" means a hyaluronic acid oligomer containing alternating repeating saccharide cycles of P-(l,4)-A-acetyl-D-glucosamine and P-(l,3)-D- glucuronic acid, wherein P-(l,4)-A-acetyl-D-glucosamine is preferably at the reducing end of the oligosaccharide, which can be chemically modified with oxyamines to form oximes. Preferably, the number of saccharide cycles is 4 to 10.

[0057] The term phosphate buffer, or PBS, is known to the person skilled in the art. It may, for example, be an aqueous solution of 8 mg / mLNaCl, 0.2 mg / mLKCl, 1.44 mg / mLlSfeHPCU and 0.24 mg / mL KH2PO4, which has a pH of 7.4.

[0058] The term physiological saline means a 0.9 wt% aqueous solution of NaCl.

[0059] FBS means fetal bovine serum.

[0060] POAis 1,3-bisaminooxypropane Example 1. Preparation of dodecyl oxyamine (Comparative Example)

[0061] The synthesis is based on the nucleophilic substitution of the bromo derivative with N- hydroxyphthalimide. The mentioned equivalents (eq) are related to the molar amount of the bromo derivative.

[0062] 10 g of 1 -bromododecane (1 eq), A-hydroxyphthalimide (1.1 eq) and 40 mL of anhydrous THF were placed to a round-bottomed flask and the mixture was suspended for 1 h at 20 °C. DIPEA (1.2 eq) and DMSO (20 mL) were added to the suspension. The mixture was stirred at 75 °C for 18 h. The product was isolated by precipitation in chilled distilled water (300 mL) and filtration. The precipitate was washed with 4x200 mL of distilled water, then with 20 mL of isopropanol and dried at 20 °C. The resulting dodecyloxyamine phthalimide was further suspended in isopropanol (10% w / v) at 20 °C and then hydrazine hydrate (8 eq) was added. The suspension was stirred at 75 °C for 20 h. The suspension was filtered through a frit, the precipitate was washed with 2x100 mL of isopropanol. The filtrate was evaporated under reduced pressure to give dodecyl oxyamine in 75% yield.

[0063] Example 2. Preparation of hexadecyl oxyamine

[0064] The synthesis is based on the nucleophilic substitution of the bromo derivative with N- hydroxyphthalimide. The mentioned equivalents (eq) are related to the molar amount of the bromo derivative.

[0065] 10 g of 1 -bromohexadecane (1 eq), A-hydroxyphthalimide (1.2 eq) and 40 mL of anhydrous THF were placed to a round-bottomed flask and the suspension was stirred for 1 h at 20 °C. DIPEA (1.3 eq) and DMSO (20 mL) were added to the suspension. The mixture was stirred at 75 °C for 24 h. The product was isolated by precipitation in chilled distilled water (300 mL) and filtration. The precipitate was washed with 4x200 mL of distilled water, then with 20 mL of isopropanol and dried at 20 °C. The resulting hexadecyloxyamine phthalimide was further suspended in isopropanol (10% w / v) at 20 °C and then hydrazine hydrate was added (8 eq). The suspension was stirred at 75 °C for 20 h. The suspension was filtered through a frit, the precipitate was washed with 2x100 mL of isopropanol. The filtrate was evaporated under reduced pressure to give hexadecyloxyamine in 69% yield.

[0066] Example 3. Preparation of octadecyl oxyamine The synthesis is based on the nucleophilic substitution of the bromo derivative with N- hydroxyphthalimide. The mentioned equivalents (eq) are related to the molar amount of the bromo derivative.

[0067] 10 g of 1 -bromooctadecane (1 eq), A-hydroxyphthalimide (1.2 eq) and 40 mL of anhydrous THF were placed to a round-bottomed flask and suspension was stirred for 1 h at 20 °C. DIPEA (1.3 eq) and DMSO (20 mL) were added to the suspension. The mixture was stirred at 75 °C for 24 h. The product was isolated by precipitation in chilled distilled water (300 mL) and filtration. The precipitate was washed with 4x200 mL of distilled water, then with 20 mL of isopropanol and dried at 20 °C. The resulting hexadecyloxyamine phthalimide was further suspended in isopropanol (10% w / v) at 20 °C and then hydrazine hydrate was added (8 eq). The suspension was stirred at 75 °C for 20 h. The suspension was filtered through a frit, the precipitate was washed with 2x100 mL of isopropanol. The filtrate was evaporated under reduced pressure to give hexadecyloxyamine in 67% yield.

[0068] Example 4. Preparation of cv.s-octadec-9-enyloxyamine

[0069] Oleyl alcohol (55 mmol) was dissolved in 45 mL of chloroform. The solution was cooled with ice to 0 to 5 °C, pyridine (111 mmol) was added, followed by 1.5 eq of tosyl chloride. The reaction mixture was stirred under cooling for 5 h and then gradually warmed to room temperature. The pyridinium salt (Py.HCl) gradually precipitated from the reaction mixture, which was subsequently removed by filtration. The filtrate was extracted successively with 2x30 mL of 0. IM HC1, 30 mL of distilled water and finally 30 mL of IM NaHCCL. The organic phase was dried by adding ISfeSCU and evaporated under reduced pressure.

[0070] The resulting oleyl tosylate (50 mmol) (1 eq) was mixed with A-hydroxyphthalimide (2.2 eq) and 40 mL of anhydrous THF and the suspension was stirred for 1 h at 20 °C. DIPEA (2.5 eq) and DMSO (20 mL) were added to the suspension. The mixture was stirred at 75 °C for 18 h. The product was isolated by extraction into a water / hexane mixture. The organic phase was extracted twice with water with the addition of NaCl to improve phase separation. The organic phase was evaporated under reduced pressure and a pale orange suspension was obtained with a yield of 75% of oleyloxyamine phthalimide. This was further suspended in ethanol (10% w / v) at 20 °C, bubbled with nitrogen and then hydrazine hydrate (3 eq) was added. The suspension was stirred at 75 °C for 20 h. The suspension was filtered through a frit, the precipitate was washed with 2x100 mL of ethanol. The filtrate was evaporated under reduced pressure to give cA-octadec-9-enyloxyamine in a yield of 67%. Example 5. Preparation of dodecyl oxyamine conjugate with oliHA4 (Comparative

[0071] Example)

[0072] 100 mg of oliHA4 were dissolved in 1 mL of DMSO and 2.2 eq of dodecyl oxyamine and 2 eq of AcOH were added to the reaction mixture. The reaction mixture was stirred for 7 days at 40 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of CHCL / hexane mixture (10 / 1). NMR, D2O 7.58 ppm (cis, -CH=N-), 6.91 ppm (trans, -CH=N-), 4.03 ppm (-O-CH2-).

[0073] Particle size - the most abundant populations with size 4 nm and 14 nm in PBS, 1 mg / mL, (Examples 30 and 45)

[0074] CMC = 0.8 mg / mL (Example 44)

[0075] Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL - 0% (Examples 33 and 43)

[0076] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 0.17% (Examples 36 and 43)

[0077] Example 6. Preparation of dodecyloxyamine conjugate with oliHA6 (Comparative Example)

[0078] 100 mg of oliHA6 were dissolved in 1 mL of DMSO and 2.2 eq of dodecyl oxyamine and 2 eq of AcOH were added to the mixture. The reaction mixture was stirred for 10 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and 3 mL of hexane. The gel -like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (10 / 1). NMR, D2O 7.58 ppm (cis, -CH=N-), 6.92 ppm (trans, -CH=N-), 4.03 ppm (-O-CH2-).

[0079] Particle size - the most abundant populations with size 47 nm and 328 nm in PBS, 1 mg / mL, (Examples 30 and 45)

[0080] CMC = 0.3 mg / mL (Example 44)

[0081] Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL - 0% (Examples 33 and 43))

[0082] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 0.08% (Examples 36 and 43) Example 7. Preparation of dodecyl oxyamine conjugate with oliHAlO (Comparative

[0083] Example)

[0084] 100 mg of oliHAlO were dissolved in 1 mL of DMSO and 2.2 eq of dodecyloxyamine and 2 eq of AcOH were added to the mixture. The reaction mixture was stirred for 15 days at 60 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and 3 mL of hexane. The gel -like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (10 / 1). NMR, D2O 7.58 ppm (cis, -CH=N-), 6.91 ppm (trans, -CH=N-), 4.03 ppm (-O-CH2-).

[0085] Particle size - the most abundant populations with size 2 nm and 39 nm in PBS, 1 mg / mL, (Examples 30 and 45)

[0086] CMC = 0.8 mg / mL (Example 44)

[0087] Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL - 0% (Examples 33 and 43)

[0088] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 0.13% (Examples 36 and 43)

[0089] Example 8. Preparation of hexadecyl oxyamine conjugate with oliHANA4 (Comparative Example)

[0090] 100 mg of oliHANA4 were dissolved in 1 mL of DMSO and 1.2 eq of hexadecyl oxyamine and

[0091] 2 eq of AcOH were added to the mixture. The reaction mixture was stirred for 5 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and

[0092] 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (10 / 1). NMR, D2O 7.63 ppm (cis, -CH=N-), 6.95 ppm (trans, -CH=N-), 3.96 ppm (-O-CH2-).

[0093] Particle size - the most abundant populations with size 9 nm and 200 nm in PBS, 1 mg / mL (Examples 30 and 45) stability - in the PBS (pH 6.1) at the conjugate concentration 10 mg / mL, after 2 days at 20 °C, formation of a solid hexadecyloxyamine was observed.

[0094] 50% decrease of oximes after 12 days in PBS (pH 6.2), 10 mg / mL

[0095] 20% decrease of oximes after 20 days in PBS (pH 7.4), 10 mg / mL Example 9. Preparation of hexadecyl oxyamine conjugate with oliHANA6 (Comparative Example)

[0096] 100 mg of oliHANA6 were dissolved in 1 mL of DMSO and 2.2 eq of hexadecyl oxyamine and

[0097] 2 eq of AcOH were added to the mixture. The reaction mixture was stirred for 3 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and

[0098] 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (10 / 1). NMR, D2O 7.63 ppm (cis, -CH=N-), 6.94 ppm (trans, -CH=N-), 3.96 ppm (-O-CH2-).

[0099] Particle size - the most abundant populations with size 9 nm and 100 nm in PBS, 1 mg / mL (Examples 30 and 45) stability - in the PBS (pH 6.1) at the conjugate concentration 10 mg / mL, after 2 days at 20 °C, formation of a solid hexadecyloxyamine was observed.

[0100] 50% decrease of oximes after 13 days in PBS (pH 6.2), 10 mg / mL

[0101] 20% decrease of oximes after 20 days in PBS (pH 7.4), 10 mg / mL

[0102] Example 10. Preparation of hexadecyloxyamine conjugate with oliHANA10 (Comparative Example)

[0103] 100 mg of oliHANA10 were dissolved in 1 mL of DMSO and 2.2 eq of hexadecyl oxyamine and

[0104] 2 eq of AcOH were added to the mixture. The reaction mixture was stirred for 3 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and

[0105] 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (10 / 1). NMR, D2O 7.62 ppm (cis, -CH=N-), 6.94 ppm (trans, -CH=N-), 3.96 ppm (-O-CH2-).

[0106] Particle size - the most abundant populations with size 200 nm and 30 nm in PBS, 1 mg / mL (Examples 30 and 45) stability - in the PBS (pH 6.1) at the conjugate concentration 10 mg / mL, after 2 days at 20 °C, the formation of a solid hexadecyloxyamine was observed.

[0107] 50% decrease of oximes after 14 days in PBS (pH 6.2), 10 mg / mL

[0108] 15% decrease of oximes after 20 days in PBS (pH 7.4), 10 mg / mL

[0109] Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL - 1.99% (Examples 33 and 43) Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 0.99% (Examples 36 and 43)

[0110] Example 11. Preparation of octadecyl oxyamine conjugate with oliHANA4 (Comparative Example)

[0111] 100 mg of oliHANA4 were dissolved in 1 mL of DMSO and 2.2 eq of octadecyl oxyamine and

[0112] 2 eq of AcOH were added to the mixture. The reaction mixture was stirred for 5 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and

[0113] 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (10 / 1). NMR, D2O 7.63 ppm (cis, -CH=N-), 6.95 ppm (trans, -CH=N-), 3.96 ppm (-O-CH2-).

[0114] Particle size - the most abundant population with size 120 nm in PBS, 1 mg / mL (Examples 30 and 45)

[0115] CMC = 0.1 mg / mL, (Example 44)

[0116] Example 12. Preparation of octadecyl oxyamine conjugate with oliHANA6 (Comparative Example)

[0117] 100 mg of oliHANA6 were dissolved in 1 mL of DMSO and 2.2 eq of octadecyl oxyamine and

[0118] 2 eq of AcOH were added to the mixture. The reaction mixture was stirred for 3 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and

[0119] 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (10 / 1). NMR, D2O 7.63 ppm (cis, -CH=N-), 6.94 ppm (trans, -CH=N-), 3.96 ppm (-O-CH2-).

[0120] Particle size - the most abundant populations with size 10 nm, 70 nm and 300 nm in PBS, 1 mg / mL (Examples 30 and 45)

[0121] CMC = 0.13 mg / mL (Example 44)

[0122] Example 13. Preparation of octadecyl oxyamine conjugate with oliHANA10 (Comparative Example)

[0123] 100 mg of oliHANA10 were dissolved in 1 mL of DMSO and 2.2 eq of octadecyl oxyamine and 2 eq of AcOH were added to the mixture. The reaction mixture was stirred for 3 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCh / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCh / hexane mixture (10 / 1). NMR, D2O 7.62 ppm (cis, -CH=N-), 6.94 ppm (trans, -CH=N-), 3.96 ppm (-O-CH2-).

[0124] Particle size - the most abundant populations with size 10 nm and 250 nm in PBS, 1 mg / mL (Examples 30 and 45)

[0125] CMC = 0.23 mg / mL (Example 44)

[0126] Example 14. Preparation of hexadecanoic acid conjugate with oliHA4-POA (Comparative Example)

[0127] 4 eq of POA.HC1 and 4 eq of acetic acid were added to an 8.5% aqueous solution of oliHA4 (100 mg), the reaction mixture was stirred for 48 h at room temperature. The acidic solution (approx. pH 2.5) was then adjusted using 10% aqueous ISfeCCh solution to pH 5-6. The solution was subsequently precipitated with 6-fold of isopropanol, the solid portion was washed with 3- fold of isopropanol and 3-fold of isopropanol / water mixture 20 / 1. Subsequently, the product oliHA4-POA was dried in a drying oven at 40 °C.

[0128] 100 mg of oliHA4-POA were dissolved in water to a concentration 40 mg / mL, then 2 mL of THF and 3.2 eq of pyridine were added gradually. After mixing, 1.5 eq of hexadecanoic acid anhydride were added and the reaction mixture was stirred for 48 h at 20 °C. The reaction mixture was precipitated with 6-fold of isopropanol, the white precipitate was washed with 3- fold of isopropanol and dried at 40 °C for 15 h. Subsequently, the residues of non-polar substances were removed by washing with chloroform, mixture chloroform / hexane 10 / 1 (2 mL). NMR, D2O 7.59 ppm (cis, -CH=N-), 6.92 ppm (trans, -CH=N-), 2.35 ppm (-CO-CH2-). Particle size - the most abundant populations with size 8 nm and 250 nm in PBS, 1 mg / mL (Examples 30 and 45) unsatisfactory stability - in the PBS (pH 6.1) at the conjugate concentration 10 mg / mL, after 2 days at 20 °C formation of a solid was observed

[0129] Example 14a. Preparation of cv.s-octadec-9-enyl acid conjugate with oliHA4-POA (Comparative Example) (HA4-POA-C18: 1)

[0130] 4 eq of POA.HC1 and 4 eq of acetic acid were added to an 8% aqueous solution of oliHA4 (100 mg), the reaction mixture was stirred for 48 h at room temperature. The acidic solution (approx. pH 2.5) was then adjusted using 10% aqueous Na2CCh solution to pH 5-6. The solution was subsequently precipitated with 6-fold of isopropanol, the solid portion was washed with 3-fold of isopropanol and 3-fold of isopropanol / water mixture (20 / 1). Subsequently, the product oliHA4-POA was dried in a drying oven at 40 °C.

[0131] 100 mg of oliHA4-POA were dissolved in water to a concentration 40 mg / mL, then 2 mL of THF and 3.2 eq of pyridine were added gradually. After mixing, 1.5 eq of c / .s-octadec-9-enyl acid anhydride were added and the reaction mixture was stirred for 48 h at 20 °C. The reaction mixture was precipitated with 6-fold of isopropanol, the white precipitate was washed with 3- fold of isopropanol and dried at 40 °C for 15 h. Subsequently, the residues of non-polar substances were removed by washing with chloroform, mixture chloroform / hexane 10 / 1 (2 mL). NMR, D2O 7.58 ppm (cis, -CH=N-), 6.91 ppm (trans, -CH=N-), 2.34 ppm (-CO-CH2-). Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 1.6% (Examples 36 and 43)

[0132] Skin Penetration - Mean Curcumin Fluorescence Value in Stratum Corneum and Epidermis = 1.443 photon / pixel (Example 47)

[0133] Example 15. Preparation of cv.s-octadec-9-enyl acid conjugate with oliHA6-POA (Comparative Example) - (HA6-POA-C18:1)

[0134] 4 eq of POA.HC1 and 4 eq of acetic acid were added to an 8% aqueous solution of oliHA6 (100 mg) and the reaction mixture was stirred for 48 h at room temperature. The acidic solution (approx. pH 2.5) was then adjusted using 10% aqueous Na2CO2solution to pH 5-6. The solution was subsequently precipitated with 6-fold of isopropanol, the solid portion was washed with 3- fold of isopropanol and 3-fold of isopropanol / water mixture (20 / 1). Subsequently, the product oliHA6-POA was dried in a drying oven at 40 °C.

[0135] 100 mg of oliHA4-POA were dissolved in water to a concentration 40 mg / mL, then 2 mL of THF and 3.2 eq of pyridine were added gradually. After mixing, 1.5 eq of oleic acid anhydride were added and the reaction mixture was stirred for 48 h at 20 °C. The reaction mixture was precipitated with 6-fold of isopropanol, the white precipitate was washed with 3-fold of isopropanol and dried at 40 °C for 15 h. Subsequently, the residues of non-polar substances were removed by washing with chloroform, chloroform / hexane mixture 10 / 1 (2 mL). NMR, D2O 7.59 ppm (cis, -CH=N-), 6.92 ppm (trans, -CH=N-), 2.35 ppm (-CO-CH2-).

[0136] Particle size - the most abundant populations with size 8 nm and 200 nm in PBS, 1 mg / mL (Examples 30 and 45) stability - in the PBS (pH 6.1) at the conjugate concentration 10 mg / mL, after 2 days at 20 °C formation of a solid was observed Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 0.9% (Examples 36 and 43)

[0137] Skin Penetration - Mean Curcumin Fluorescence Value in Stratum Corneum and Epidermis = 1.527 photon / pixel (Example 47)

[0138] Example 16. Preparation of cv.s-octadec-9-enyl acid conjugate with oliHAlO-POA (Comparative Example) (HA10-POA-C18: 1)

[0139] 4 eq of POA.HC1 and 4 eq of acetic acid were added to a 7.5% aqueous solution of oliHAlO (100 mg), the reaction mixture was stirred for 48 h at room temperature. The acidic solution (approx. pH 2.5) was then adjusted using 10% aqueous ISfeCCE solution to pH 5-6. The solution was subsequently precipitated with 6-fold of isopropanol, the solid portion was washed with 3- fold of isopropanol and 3-fold of isopropanol / water mixture 20 / 1. Subsequently, the product oliHAlO-POA was dried in a drying oven at 40 °C.

[0140] 100 mg of oliHA4-POA were dissolved in water to a concentration 40 mg / mL, then 2 mL of THF and 3.2 eq of pyridine were added gradually. After mixing, 1.5 eq of oleic acid anhydride were added and the reaction mixture was stirred for 48 h at 20 °C. The reaction mixture was precipitated with 6-fold of isopropanol, the white precipitate was washed with 3-fold of isopropanol and dried at 40 °C for 15 h. Subsequently, the residues of non-polar substances were removed by washing with chloroform, chloroform / hexane mixture 10 / 1 (2 mL). NMR, D2O 7.59 ppm (cis, -CH=N-), 6.92 ppm (trans, -CH=N-), 2.35 ppm (-CO-CH2-).

[0141] Particle size - the most abundant populations with size 10 nm and 300 nm in PBS, 1 mg / mL (Examples 30 and 45)

[0142] CMC = 0.15 mg / mL (Example 44)

[0143] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 0.6% (Examples 36 and 43)

[0144] Skin Penetration - Mean Curcumin Fluorescence Value in Stratum Corneum and Epidermis = 1.569 photon / pixel (Example 47)

[0145] Example 17. Preparation of hexadecyl oxyamine conjugate with oliHA4

[0146] 100 mg of oliHA4 were dissolved in 1 mL of DMSO and 1.5 eq of hexadecyl oxyamine and 2 eq of AcOH were added to the reaction mixture. The reaction mixture was stirred for 5 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / cyclohexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCh / cyclohexane mixture (10 / 1). NMR, D2O 7.57 ppm (cis, -CH=N-), 6.90 ppm (trans, - CH=N-), 4.03 ppm (-O-CH2-).

[0147] Particle size - the most abundant populations with size 7 nm in PBS, 1 mg / mL (Examples 30 and 45) stability -10% decrease of oximes after 20 days in PBS (pH 6.2), 10 mg / mL

[0148] 4% decrease of oximes after 20 days in PBS (pH 7.4), 10 mg / mL

[0149] CMC = 0.02 mg / mL (Example 44)

[0150] Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL - 5.9% (Examples 33 and 43)

[0151] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 4.0% (Examples 36 and 43)

[0152] Example 18. Preparation of hexadecyl oxyamine conjugate with oliHA6

[0153] 100 mg of oliHA6 were dissolved in 1 mL of DMSO and 1.5 eq of hexadecyl oxyamine and 2 eq of AcOH were added to the reaction mixture. The reaction mixture was stirred for 5 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of dichloromethane and 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the dichloromethane / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (10 / 1). NMR, D2O 7.57 ppm (cis, -CH=N-), 6.90 ppm (trans, -CH=N-), 4.03 ppm (-O- CH2-).

[0154] Particle size - the most abundant populations with size 8 nm in PBS, 1 mg / mL, (Example 30 a 45) stability - 10% decrease of oximes after 20 days in PBS (pH 6.2), 10 mg / mL

[0155] 0% decrease of oximes after 20 days in PBS (pH 7.4), 10 mg / mL

[0156] CMC = 0.04 mg / mL (Example 44)

[0157] Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL - 5.3% (Examples 33 and 43)

[0158] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 2.5% (Examples 36 and 43) Example 19. Preparation of hexadecyl oxyamine conjugate with oliHAlO

[0159] 100 mg of oliHAlO were dissolved in 1 mL of DMSO and 1.5 eq of hexadecyl oxyamine and 2 eq of AcOH were added to the reaction mixture. The reaction mixture was stirred for 5 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and 3 mL of heptane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / heptane mixture (10 / 1). NMR, D2O 7.57 ppm (cis, -CH=N-), 6.90 ppm (trans, -CH=N-), 4.03 ppm (-O-CH2-). Particle size - the most abundant populations with size 10 nm and 100 nm in PBS, 1 mg / mL (Examples 30 and 45) stability - 15% decrease of oximes after 20 days in PBS (pH 6.2), 10 mg / mL

[0160] 0% decrease of oximes after 20 days in PBS (pH 7.4), 10 mg / mL

[0161] CMC = 0.098 mg / mL (Example 44)

[0162] Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL - 7.2% (Examples 33 and 43)

[0163] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 1.8% (Examples 36 and 43)

[0164] Example 20. Preparation of octadecyl oxyamine conjugate with oliHA4 - (HA4-C18)

[0165] 100 mg of oliHA4 were dissolved in 2 mL of DMSO and 2 eq of octadecyl oxyamine and 4 eq of AcOH were added to the reaction mixture. The reaction mixture was stirred for 20 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and 4 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (8 / 1). NMR, D2O 7.57 ppm (cis, -CH=N-), 6.90 ppm (trans, -CH=N-), 4.03 ppm (-O-CH2-). Particle size - the most abundant populations with size 8 nm in PBS, 1 mg / mL (Examples 30 and 45) particle size with bound curcumin - the most abundant populations with size 8 nm and 100 nm, in PBS, 1 mg / mL (Examples 36 and 45) particle size with bound amphotericin B - 94 nm, in PBS, 0.1 mg / mL (Examples 40 and 45) particle size with bound CBD - 7 nm, in PBS, 0.1 mg / mL (Examples 42 and 45)

[0166] CMC = 0.01 mg / mL (Example 44) Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL - 5.2% (Examples 33 and 43)

[0167] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 3.3% (Examples 36 and 43)

[0168] Binding capacity of amphotericin B at the conjugate concentration 0.1 mg / mL - 13.6% (Examples 40 and 43)

[0169] Binding capacity of CBD at the conjugate concentration 0.1 mg / mL - 1.5% (Examples 42 and 43)

[0170] Example 21. Preparation of octadecyl oxyamine conjugate with oliHA6 - (HA6-C18)

[0171] 100 mg of oliHA6 were dissolved in 2 mL of DMSO and 2 eq of octadecyl oxyamine and 4 eq of AcOH were added to the reaction mixture. The reaction mixture was stirred for 18 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (4 / 3). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (10 / 1). NMR, D2O 7.57 ppm (cis, -CH=N-), 6.90 ppm (trans, -CH=N-), 4.03 ppm (-O-CH2-). Particle size - the most abundant populations with size 9 nm in PBS, 1 mg / mL (Examples 30 and 45) particle size with bound curcumin - the most abundant populations with size 9 nm and 120 nm, in PBS, 1 mg / mL (Examples 36 and 45) particle size with bound amphotericin B - 27 nm, in PBS, 0.1 mg / mL (Examples 40 and 45) CMC = 0.01 mg / mL (Example 44)

[0172] Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL - 1.1% (Examples 33 and 43)

[0173] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 3.0% (Examples 36 and 43)

[0174] Binding capacity of amphotericin B at the conjugate concentration 0.1 mg / mL - 29.4% (Examples 40 and 43) interaction with HSA using ITC (Example 46): molar ratio HSA / conjugate 0.4, AH = -5 kJ / mol, molar ratio HSA / HA (Mw 15000) 0.4, AH = -3.5 kJ / mol molar ratio HSA / conjugate 0.6, AH = -2 kJ / mol, molar ratio HSA / HA (Mw 15000) 0.6, AH = -3 kJ / mol - AH values similar for conjugate and native HA

[0175] Example 22. Preparation of octadecyl oxyamine conjugate with oliHAlO

[0176] 100 mg of oliHAlO were dissolved in 2 mL of DMSO and 2 eq of octadecyl oxyamine and 4 eq of AcOH were added to the reaction mixture. The reaction mixture was stirred for 22 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (8 / 1). NMR, D2O 7.57 ppm (cis, -CH=N-), 6.90 ppm (trans, -CH=N-), 4.03 ppm (-O-CH2-).

[0177] Particle size - the most abundant populations with size 10 nm in PBS, 1 mg / mL (Examples 30 and 45) particle size with bound curcumin - the most abundant populations with size 10 nm and 130 nm, in PBS, 1 mg / mL (Examples 36 and 45) particle size with bound amphotericin B - 23 nm, in PBS, 0.1 mg / mL (Examples 40 and 45) CMC = 0.02 mg / mL (Example 44)

[0178] Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL - 1.9% (Examples 33 and 43)

[0179] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 2.1% (Examples 36 and 43)

[0180] Binding capacity of amphotericin B at the conjugate concentration 0.1 mg / mL - 35.3% (Examples 40 and 43)

[0181] Example 23. Preparation of cv.s-octadec-9-enyl oxyamine conjugate with oliHA4 - (HA4- C18:l)

[0182] 100 mg of oliHA4 were dissolved in 1 mL of DMSO and 1.7 eq of cz -octadec-9-enyloxyamine and 4 eq of AcOH were added to the reaction mixture. The reaction mixture was stirred for 20 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (12 / 1). NMR, D2O 7.57 ppm (cis, -CH=N-), 6.90 ppm (trans, -CH=N-), 4.03 ppm (-O-CH2-). Particle size - the most abundant populations with size 8 nm in PBS, 1 mg / mL (Examples 30 and 45) CMC = 0.07 mg / mL (Examples 44)

[0183] Skin Penetration - Mean Curcumin Fluorescence Value in Stratum Corneum and Epidermis = 2.002 photon / pixel (Example 47)

[0184] Example 24. Preparation of cv.s-octadec-9-enyloxyamine conjugate with oliHA6 - (HA6- C18:l)

[0185] 100 mg of oliHA6 were dissolved in 1 mL of DMSO and 1.5 eq of cz -octadec-9-enyloxyamine and 4 eq of AcOH were added to the reaction mixture. The reaction mixture was stirred for 20 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (10 / 1). NMR, D2O 7.57 ppm (cis, -CH=N-), 6.90 ppm (trans, -CH=N-), 4.03 ppm (-O-CH2-). Particle size - the most abundant populations with size 9 nm in PBS, 1 mg / mL (Examples 30 and 45) particle size with bound amphotericin B - 27 nm, in PBS, 0.1 mg / mL (Examples 40 and 45) particle size with bound dexamethasone - 9 nm, in PBS, 0.1 mg / mL (Examples 41 and 45) CMC = 0.07 mg / mL (Examples 44)

[0186] Binding capacity of amphotericin B at the conjugate concentration 0.1 mg / mL - 32.6% (Examples 40 and 43)

[0187] Binding capacity of dexamethasone at the conjugate concentration 0.1 mg / mL - 2.3% (Examples 41 and 43)

[0188] Skin Penetration - Mean Curcumin Fluorescence Value in Stratum Corneum and Epidermis = 2.002 photon / pixel (Example 47)

[0189] Cell penetration: HaCaT 4.91 photon / pixel, HT29 4.01 photon / pixel (Example 48)

[0190] Interaction with HSA using ITC (Example 46): molar ratio HSA / conjugate 0.4, AH = -6.5 kJ / mol, molar ratio HSA / HA (Mw 15000) 0.4, AH = -3.5 kJ / mol molar ratio HSA / conjugate 0.6, AH = -4.5 kJ / mol, molar ratio HSA / HA (Mw 15000) 0.6, AH = -3 kJ / mol molar ratio HSA / (conjugate + curcumin) 0.4, AH = -12 kJ / mol, molar ratio HSA / (HA (Mw 15000) + curcumin) 0.4, AH = -10.5 kJ / mol molar ratio HSA / (conjugate + curcumin) 0.6, AH = -8.5 kJ / mol, molar ratio HSA / (HA (Mw 15000) + curcumin) 0.6, AH = -8 kJ / mol - AH values similar for conjugate and native HA

[0191] Example 25. Preparation of cv.s-octadec-9-enyloxyamine conjugate with oliHAlO - (HA10- C18: l)

[0192] 100 mg of oliHA6 were dissolved in 1 mL of DMSO and 2.2 eq of cv.s-octadec-9-enyl oxyamine and 4 eq of AcOH were added to the reaction mixture. The reaction mixture was stirred for 20 days at 50 °C. The product was isolated by precipitation of the reaction mixture using 4 mL of chloroform and 3 mL of hexane. The gel-like precipitate was isolated by centrifugation and then washed 3 times with 3 mL of the CHCL / hexane mixture (3 / 2). Subsequently, the residues of non-polar substances were removed by washing with 3 mL of the CHCL / hexane mixture (10 / 1). NMR, D2O 7.57 ppm (cis, -CH=N-), 6.90 ppm (trans, -CH=N-), 4.03 ppm (-O-CH2-). Particle size - the most abundant populations with size 10 nm in PBS, 1 mg / mL (Examples 30 and 45) particle size with bound curcumin - 10 nm, in PBS, 0.1 mg / mL (Examples 33 and 45) particle size with bound curcumin - 9 nm, in 0.9% NaCl, 0.1 mg / mL (Examples 34 and 45) particle size with bound curcumin - 8 nm, in 0.9% NaCl, 1 mg / mL (Examples 35 and 45) CMC = 0.1 mg / mL (Example 44)

[0193] Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL in PBS - 1.95% (Examples 33 and 43)

[0194] Binding capacity of curcumin at the conjugate concentration 0.1 mg / mL in 0.9% NaCl - 2.18% (Examples 34 and 43)

[0195] Binding capacity of curcumin at the conjugate concentration 1 mg / mL in 0.9% NaCl - 2.99% (Examples 35 and 43)

[0196] Skin penetration - Mean fluorescence value in stratum comeum and epidermis = 1.741 photon / pixel (Example 47)

[0197] Cell penetration: HaCaT 4.06 photon / pixel, HT29 2.82 photon / pixel (Example 48)

[0198] Example 26. Preparation of oleic acid ester with hyaluronic acid - oleyl-HA (Comparative Example)

[0199] 200 g of hyaluronic acid (Mw 17 kDa) were dissolved in 4000 mL of distilled water. After 5 minutes, 1.2 g of 4-dimethyl aminopyridine were added and the mixture was stirred for 1.5 h. Then 1200 mL of isopropanol were added and the mixture was homogenized for 1 h. To this solution a solution of oleic acid 75 mL in 2400 mL of isopropanol was added, separately activated using 90 mL of tri ethylamine and 25 mL of benzoyl chloride, and the whole mixture T1 was stirred for 2.5 h at 20 °C. The resulting mixture is subsequently precipitated with 18 L of isopropanol and washed with a mixture of isopropanol / water 5 / 1 and isopropanol / water 20 / 1. The solid is then dried at 40 °C for three days. The degree of modification 7%, which means that on average 7 disaccharide units out of a hundred are modified, was determined from NMR, (D2O) by comparing the signals at 0.88 ppm (-CH3 oleyl) and 2.0 ppm (-CH3 HA). particle size 100-300 nm (Examples 28 and 45)

[0200] Binding capacity of curcumin at concentration oleyl-HA 1 mg / mL - 19% (Examples 36 and 43)

[0201] Skin penetration - Mean fluorescence value in stratum comeum and epidermis = 1.017 photon / pixel (Example 47) interaction with HSA using ITC (Example 46): molar ratio HSA / oleyl-HA 0.4, AH = -34 kJ / mol, molar ratio HSA / HA(Mw 15000) 0.4, AH = -3.5 kJ / mol molar ratio HSA / oleyl-HA 0.6, AH = -20 kJ / mol, molar ratio HSA / HA(Mw 15000) 0.6, AH = -3 kJ / mol molar ratio HSA / (oleyl-HA + curcumin) 0.4, AH = -40 kJ / mol, molar ratio HSA / (HA(Mw 15000) + curcumin) 0.4, AH = -10.5 kJ / mol molar ratio HSA / (oleyl-HA + curcumin) 0.6, AH = -27 kJ / mol, molar ratio HSA / (HA(Mw 15000) + curcumin) 0.6, AH = -8 kJ / mol - AH values for oleyl-HA and native HA are significantly different

[0202] Example 27. Preparation of oleic acid ester with oliHA6 (Comparative Example)

[0203] A solution of 60 mg of oleic anhydride in 3 mL of THF was added to a solution of 100 mg of oliHA, 20 mg of tri ethylamine and 3 mg of DMAP in 3 mL of distilled water. The mixture was stirred for 2 h at 20 °C, then precipitated with 20 mL of isopropanol. The precipitate was washed 3 times with 10 mL of isopropanol, then dried under reduced pressure. The resulting solid was subsequently separated by chromatography, where the fraction with one oleyl per molecule of oliHA6 was separated.

[0204] Particle size - 145 nm, in PBS, 1 mg / mL (Examples 30 and 45) particle size with bound curcumin - 131 nm, in PBS, 1 mg / mL (Examples 36 and 45)

[0205] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 0.9% (Examples 36 and 43) Example 28. Preparation of micellar composition without active substance

[0206] Amphiphilic conjugate of oliHA prepared according to one of the Examples 5 - 25 was dissolved in phosphate buffer (PBS with pH 7.4 consisting of 8 mg / mL NaCl, 0.2 mg / mL KC1,

[0207] 1.44 mg / mL ISfeHPCU and 0.24 mg / mL KH2PO4) with concentration 0.02 mg / mL with mechanical stirring for 0.1 h at 15 °C.

[0208] Example 29. Preparation of micellar composition without active substance

[0209] Amphiphilic conjugate of oliHA prepared according to one of the Examples 5 - 25 was dissolved in phosphate buffer (PBS with pH 7.4 consisting of 8 mg / mL NaCl, 0.2 mg / mL KC1,

[0210] 1.44 mg / mL Na2HPO4 and 0.24 mg / mL KH2PO4) with concentration 2 mg / mL with mechanical stirring for 1 h at 25 °C.

[0211] Example 30. Preparation of micellar composition without active substance

[0212] Amphiphilic conjugate of oliHA prepared according to one of the Examples 5 - 25 was dissolved in phosphate buffer (PBS with pH 7.4 consisting of 8 mg / mL NaCl, 0.2 mg / mL KC1,

[0213] 1.44 mg / mL Na2HPO4 and 0.24 mg / mL KH2PO4) with concentration 1 mg / mL with mechanical stirring for 24 h at 20 °C.

[0214] Example 31. Preparation of micellar composition without active substance

[0215] Amphiphilic conjugate of oliHA prepared according to one of the Examples 5 - 25 was dissolved in physiological saline (0.9 wt% NaCl) with concentration 0.02 mg / mL with mechanical stirring for 1 h at 20 °C.

[0216] Example 32. Preparation of micellar composition without active substance

[0217] Amphiphilic conjugate of oliHA prepared according to one of the Examples 5 - 25 was dissolved in physiological saline (0.9 wt% NaCl) with concentration 1.2 mg / mL with mechanical stirring for 5 h at 20 °C.

[0218] Example 33. Preparation of micellar composition with bound curcumin

[0219] 0.1 mL of curcumin solution in acetone with concentration 21 mg / mL was transferred to a suitable vessel and the solvent was evaporated. 3 mL solution of the amphiphilic oliHA conjugate prepared according to one of the Examples 5 - 25 with concentration 0.1 mg / mL in PBS were added to the vessel containing curcumin. The solution was then mechanically stirred for 48 h at 20 °C and filtered through a 1 pm filter. Absorbance at 425 nm was determined using UV-VIS spectrophotometry and from the calibration curve, the amount of curcumin bound to the carrier was evaluated as the weight percentage of curcumin, with the solubility in the respective solvent (0.86 pg / mL) subtracted to the weight of the carrier.

[0220] Example 34. Preparation of micellar composition with bound curcumin

[0221] 0.1 mL of curcumin solution in acetone with concentration 21 mg / mL was transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL solution of the amphiphilic oliHA conjugate prepared according to one of the Examples 17 - 25 with concentration 0.1 mg / mL in physiological saline (0.9 wt% NaCl) were added to the vessel containing curcumin. The solution was then mechanically stirred for 24 h at 25 °C and filtered through a 1 pm filter. Absorbance at 425 nm was determined using UV-VIS spectrophotometry and from the calibration curve, the amount of curcumin bound to the carrier was evaluated as the weight percentage of curcumin, with the solubility in the respective solvent (0.86 pg / mL) subtracted to the weight of the carrier.

[0222] Example 35. Preparation of micellar composition with bound curcumin

[0223] 0.1 mL of curcumin solution in acetone with concentration 21 mg / mL was transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL solution of the amphiphilic oliHA conjugate prepared according to one of the Examples 17 - 25 with concentration 1 mg / mL in physiological saline (0.9 wt% NaCl) were added to the vessel containing curcumin. The solution was then mechanically stirred for 72 h at 25 °C and filtered through a 1 pm filter. Absorbance at 425 nm was determined using UV-VIS spectrophotometry and from the calibration curve, the amount of curcumin bound to the carrier was evaluated as the weight percentage of curcumin, with the solubility in the respective solvent (0.86 pg / mL) subtracted to the weight of the carrier.

[0224] Example 36. Preparation of micellar composition with bound curcumin

[0225] 0.1 mL of curcumin solution in acetone with concentration 21 mg / mL was transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL solution of the amphiphilic oliHA conjugate prepared according to one of the Examples 5 - 25 with concentration 1 mg / mL in PBS) were added to the vessel containing curcumin. The solution was then mechanically stirred for 48 h at 20 °C and filtered through a 1 pm filter. Absorbance at 425 nm was determined using UV-VIS spectrophotometry and from the calibration curve, the amount of curcumin bound to the carrier was evaluated as the weight percentage of curcumin, with the solubility in the respective solvent (0.86 pg / mL) subtracted to the weight of the carrier.

[0226] Example 37. Preparation of micellar composition with bound curcumin

[0227] 0.1 mL of curcumin solution in ethanol with concentration 3 mg / mL was transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL solution of the amphiphilic oliHA conjugate prepared according to one of the Examples 17 - 25 with concentration 0.5 mg / mL in PBS were added to the vessel containing curcumin. The solution was then mechanically stirred for 48 h at 20 °C and filtered through a 1 pm filter. Absorbance at 425 nm was determined using UV-VIS spectrophotometry and from the calibration curve, the amount of curcumin bound to the carrier was evaluated as the weight percentage of curcumin, with the solubility in the respective solvent (0.86 pg / mL) subtracted to the weight of the carrier.

[0228] Example 38. Preparation of micellar composition with bound curcumin

[0229] 0.1 mL of curcumin solution in isopropanol with concentration 1.5 mg / mL was transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL solution of the amphiphilic oliHA conjugate prepared according to one of the Examples 17 - 25 with concentration 2 mg / mL in PBS) were added to the vessel containing curcumin. The solution was then mechanically stirred for 48 h at 20 °C and filtered through a 1 pm filter. Absorbance at 425 nm was determined using UV-VIS spectrophotometry and from the calibration curve, the amount of curcumin bound to the carrier was evaluated as the weight percentage of curcumin with the solubility in the respective solvent (0.86 pg / mL) subtracted to the weight of the carrier.

[0230] Example 39. Preparation of micellar composition with bound curcumin

[0231] 0.1 mL of curcumin solution in acetone with concentration 21 mg / mL was transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL solution of the amphiphilic oliHA conjugate prepared according to one of the Examples 17 - 25 with concentration 1 mg / mL in 0.01% solution of NaCl were added to the vessel containing curcumin. The solution was then mechanically stirred for 72 h at 25 °C and filtered through a 1 pm filter. Absorbance at 425 nm was determined using UV-VIS spectrophotometry and from the calibration curve, the amount of curcumin bound to the carrier was evaluated as the weight percentage of curcumin, with the solubility in the respective solvent (0.86 pg / mL) subtracted to the weight of the carrier.

[0232] Example 40. Preparation of micellar composition with bound amphotericin

[0233] 1.2 mL of amphotericin B solution in methanol with concentration 0.5 mg / mL was transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen or in a vacuum. 3 mL of solution of the amphiphilic oliHA conjugate prepared according to one of the Examples 17 - 25 with concentration 0.1 mg / mL in PBS was added to the vessel containing amphotericin B. The solution was then mechanically stirred for 24 h at 20 °C and filtered through a 1 pm filter. The amount of amphotericin B bound to the carrier was expressed as a weight percentage of amphotericin B, with the solubility in the respective solvent (0.05 pg / mL) subtracted to the weight of the carrier.

[0234] Example 41. Preparation of micellar composition with bound dexamethasone

[0235] 0.2 mL of dexamethasone solution in acetone with concentration 15 mg / mL was transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL solution of the amphiphilic oliHA conjugate prepared according to one of the Examples 17 - 25 with concentration 0.1 mg / mL in PBS were added to the vessel containing dexamethasone. The solution was then mechanically stirred for 48 h at 25 °C and filtered through a 1 pm filter. The amount of dexamethasone bound to the carrier was expressed as the weight percentage of dexamethasone, with the solubility in the respective solvent (77.26 pg / mL) subtracted to the weight of the carrier.

[0236] Example 42. Preparation of micellar composition with bound cannabidiol

[0237] 0.2 mL of a solution of cannabidiol in acetone with a concentration of 15 mg / mL was transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL of solution of the amphiphilic oliHA conjugate prepared according to one of the Examples 17 - 25 with concentration 0.1 mg / mL in PBS, were added to the vessel containing cannabidiol. The solution was then mechanically stirred for 48 h at 20 °C and filtered through a 1 pm filter. The amount of cannabidiol bound to the carrier was expressed as a weight percentage of cannabidiol, with the solubility in the respective solvent (this was below the detection limit, 0.15 pg / mL, which was used instead in the calculation) subtracted to the weight of the carrier. Example 42a. Preparation of micellar composition with bound curcumin and quercetin

[0238] 0.2 mL of curcumin solution in acetone (15 mg / mL) and 0.2 mL of quercetin solution (15 mg / mL) in acetone were transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL of the HA4-C18 amphiphilic conjugate solution prepared according to Example 20 with concentration 1 mg / mL in PBS were added to the vessel containing curcumin and quercetin. The solution was then mechanically stirred for 24 h at 20 °C and filtered through a 1 pm filter.

[0239] Particle size with bound curcumin and quercetin - the most abundant population with size 7 nm, in PBS, 1 mg / mL

[0240] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 3.55% Binding capacity of quercetin at the conjugate concentration 1 mg / mL - 0.21%

[0241] Example 42b. Preparation of micellar composition with bound curcumin and quercetin

[0242] 0.2 mL of curcumin solution in acetone (15 mg / mL) and 0.2 mL of quercetin solution (15 mg / mL) in acetone were transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL of the amphiphilic conjugate HA10-C18: l solution prepared according to the Example 25 with concentration 1 mg / mL in PBS were added to the vessel containing curcumin and quercetin. The solution was then mechanically stirred for 24 h at 20 °C and filtered through a 1 pm filter.

[0243] Particle size with bound curcumin and quercetin - the most abundant population with size 10 nm, in PBS, 1 mg / mL

[0244] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 2.49% Binding capacity of quercetin at the conjugate concentration 1 mg / mL - 0.12%

[0245] Example 42c. Preparation of micellar composition with bound curcumin and cannabidiol 0.2 mL of curcumin solution in acetone (15 mg / mL) and 0.2 mL of cannabidiol solution in acetone (15 mg / mL) were transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL of the HA4-C18 amphiphilic conjugate solution prepared according to Example 20 with concentration 1 mg / mL in PBS were added to the vessel containing curcumin and cannabidiol. The solution was then mechanically stirred for 24 h at 20 °C and filtered through a 1 pm filter.

[0246] Particle size with bound curcumin and cannabidiol - the most abundant population with size 8 nm, in PBS, 1 mg / mL

[0247] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 4.54% Binding capacity of cannabidiol at the conjugate concentration 1 mg / mL - 2.24%

[0248] Example 42d. Preparation of micellar composition with bound curcumin and cannabidiol 0.2 mL of curcumin solution in acetone (15 mg / mL) and 0.2 mL of cannabidiol solution in acetone (15 mg / mL) were transferred to a suitable vessel and the solvent was evaporated under a gentle stream of nitrogen. 3 mL of the amphiphilic conjugate HA10-C18: l solution prepared according to Example 25 with concentration 1 mg / mL in PBS were added to the vessel containing curcumin and cannabidiol. The solution was then mechanically stirred for 24 h at 20 °C and filtered through a 1 pm filter.

[0249] Particle size with bound curcumin and cannabidiol - the most abundant population with size 11 nm, in PBS, 1 mg / mL

[0250] Binding capacity of curcumin at the conjugate concentration 1 mg / mL - 2.11%

[0251] Binding capacity of cannabidiol at the conjugate concentration 1 mg / mL - 2.40%

[0252] Example 43. Determination of the active substance binding capacity.

[0253] The film was prepared in glass vials by dissolving the active substance in acetone or methanol or in ethanol or in isopropanol (1 to 21 mg / mL) and evaporating the solvent under gentle nitrogen purge. Subsequently, 3 mL of the amphiphilic oliHA conjugate solution in PBS with concentration 0.1 to 1 mg / mL were added to each vial. The solution was mechanically stirred at room temperature for 48 h and filtered. The amount of dissolved active substance was determined from a calibration curve measured using a spectrometer or HPLC and is expressed as a percentage by weight of active substance relative to the weight of the carrier.

[0254] Example 44. Determination of critical micelle concentration (CMC), general procedure

[0255] The method is based on the fluorescence of pyrene in the environment of an amphiphilic derivative. A series of pyrene films was prepared in glass vials: a solution of pyrene in acetone (0.1 mg / mL) was evaporated with a gentle stream of nitrogen. A solution of the amphiphilic derivative in PBS at a concentration of: 0.001-10.0 mg / mL according to its solubility was added to the vial. The concentration of pyrene in the derivative solution was 0.8 pg / mL. The obtained solution was mechanically stirred for 20 h at 25 °C. Subsequently, the fluorescence intensity of pyrene was measured at an excitation wavelength of 334 nm and emission between 345-550 nm. To determine the CMC, the ratio of the first peak (L) at about 370 nm (dependent on the polarity of the environment) and the third peak (independent on the polarity of the environment around pyrene) at about 380 nm (L), the so-called "polarity index", plotted against the amphiphilic derivative concentration logarithm is used (Ondreas F. et al., Applied Surface Science, 546, 149161, 2021).

[0256] Example 45. Particle size determination, general procedure

[0257] Hydrodynamic particle size was determined by dynamic light scattering (DLS) at 25 °C on a Zetasizer Nano-ZS (Malvern Instrument) equipped with a 633 nm He-Ne laser and a dynamic light scattering detector after 24 h of stabilization. The particle size in solution was calculated using Zetasizer Software 7.11 and the particle size distribution by volume was evaluated. The result is a number that expresses the mean particle size by volume and represents at least 90% of the material present. If multiple numbers are given, this means that the particle distribution was clearly multimodal and multiple particle sizes are present, representing more than 10% of the material present, the mean values of which are given.

[0258] Example 46. Determination of interaction with human serum albumin (HS A) using isothermal titration calorimetry (ITC)

[0259] ITC analysis was performed using a MicroCai PEAQ-ITC (Malvern Panalytical). Titration parameters were set: T = 37 °C, reference power = 5 pcal / s, feedback = high, stirring speed = 750 rpm, initial delay = 60 s, time between injections = 150 s, injection time = 4 s. Solutions of amphiphilic conjugates in PBS 280 pL with a concentration of 0.1 mg / mL were titrated, adding a total of 36.4 pL of HSA solution in PBS with a concentration of 40 mg / mL. The titration consisted of 19 injections, with the first injection volume being 0.4 pL. The temperature and reference power were stabilized before each measurement. From the measured data, titration of 40 mg / mL HSA / PBS into PBS was subtracted as a control. The enthalpy change per mole of HSA is plotted as a function of the molar ratio of HSA:HA. The enthalpy change values at molar ratios of HSA:HA = 0.4 and 0.6 are compared.

[0260] Example 47. Determination of the penetration of curcumin as a model active substance into the skin

[0261] Skin penetration tests were performed in Franz diffusion cells according to EFSA guidelines (European Food Safety et al., https: / / doi.Org / 10.2903 / j.efsa.2017.4873, 2017). Porcine ear skin from slaughterhouses (Bocus, Letohrad, Czech Republic) was processed into 600 pm thick slices and clamped in the cell with the stratum corneum facing upwards. After 30 minutes of equilibration, a 1 cm2diffusion area was filled with 130 pL of sample solution and covered. BSA (40 mg / mL) was used as an acceptor at 32 °C. Penetration experiments were conducted for 24 hours. Washed and frozen tissues were then cut into 10 pm thick slices using a Leica CM1950 cryostat (Leica Biosystems, Germany). Fluorescence images were acquired using a Leica TCS SP8 X confocal microscope (Leica Microsystems) equipped with a Leica HC PL APO CS2 objective (63x, 1.40 NA, oil). The excitation wavelength was 405 nm. Emission was monitored at 425-550 nm using a hybrid single-molecule detector in photon counting mode. Each section was recorded in a z-stack with a step of 2.5 pm, covering the entire thickness of each section. A projection of the maximum intensity was performed on the z-stack and used for further analysis. The acquired images were analyzed using Fiji software (Schindelin J. et al., Nature Method 9, 676-82, 2012). To measure the mean fluorescence intensity, the images were manually segmented, excluding the background from the analysis and separating the dermis and stratum corneum+epi dermis.

[0262] In the oils (jojoba, almond), which were used as controls, the concentration of curcumin was 10 pg / mL. The administered amount was 150 pL. The total administered amount of curcumin was therefore 1.5 pg. The mean fluorescence value in the stratum corneum + epidermis was determined for individual carriers with bound curcumin and compared with the standard jojoba oil (0.84 photon / pixel), almond oil (0.66 photon / pixel) and the negative control, where only background fluorescence of the skin was observed (0.14 photon / pixel).

[0263] The figures in Example 47 demonstrate that the compositions covered by this invention transport the administered substance through the stratum corneum and epidermis and therefore have a potential as carriers of active substances in topical applications.

[0264] Example 48. Determination of cell membrane permeability

[0265] HaCaT cells (human keratinocytes) were cultured in DMEM medium supplemented with 10% FBS, glutamine (0.3 mg / mL), glucose (1.8 mg / mL), penicillin (100 units / mL) and streptomycin (0.1 mg / mL) in an atmosphere of 5% CO2 at 37 °C. HT29 cells (colorectal adenocarcinoma) were cultured in McCoy's medium supplemented with 10% FBS, glutamine (0.3 mg / mL), penicillin (100 units / mL) and streptomycin (0.1 mg / mL) in an atmosphere of 5% CO2 at 37 °C. After reaching 80% confluence, the cells were passaged and plated on 8-well slides (Cellvis) at 10,000 cells / well. The medium was then removed and the cells were challenged with 300 pL of RPMI medium with 0% albumin containing 0.2 mg / mL of the test substance. The challenged cells were imaged on a Leica TCS SP8 X confocal microscope (Leica Microsystems) equipped with a Leica HC PL APO CS2 objective (63x, 1.20 NA, water) 30 minutes after challenge. The excitation wavelength was 405 nm. The emission was monitored at 425-550 nm using a hybrid single-molecule detector in photon counting mode. The scanned area was recorded in a z-stack with a step of 0.36 pm, covering the entire thickness of the scanned cells. Projection of the maximum intensity was performed on the z-stack and used for further analysis.

[0266] The images in Example 48 demonstrate that the compositions covered by this invention transport the administered substance (curcumin) into the cell membrane and therefore have potential as carriers of active substances.

Claims

C LAIMS1. An amphiphilic conjugate of hyaluronic acid oligomer according to isomeric formulaeI to IVwherein M is hydrogen, sodium or potassium, n is the number of disaccharide units of the hyaluronic acid oligomer in the range of 1 to 4,X is a linear chain of 16 to 20 carbons with possible multiple bonds.

2. The amphiphilic conjugate according to claim 1, wherein X is selected from the group comprising hexadecyl, octadecyl, cA-octadec-9-enyl.

3. A micellar composition characterized in that it contains: water in the amount of 96 to 99.8 wt%, one or more biologically compatible salts in the total amount 0.01 to 1 wt%, the conjugate of hyaluronic acid oligomer as claimed in claim 1 or 2 in the amount of 0.01 to 2 wt%.

4. The micellar composition according to claim 3, characterized in that it further contains one active substance in an amount of up to 1 wt%, wherein the active substance is selected from the group comprising vitamins, drugs, cytostatics, steroids andcosmetically active substances, or a mixture thereof, such as curcumin, amphotericin B, cannabidiol (CBD), dexamethasone or quercetin.

5. The micellar composition according to claim 3, characterized in that it further contains more than one active substance in the total amount of up to 1 wt%, wherein the active substance is selected from the group comprising vitamins, drugs, cytostatics, steroids and cosmetically active substances, or a mixture thereof, such as curcumin, amphotericin B, cannabidiol (CBD), dexamethasone or quercetin.

6. The micellar composition according to any one of claims 3 to 5, characterized in that the biocompatible salt is selected from the group comprising NaCl, KC1, NaJfcPC and Na2HPO4, or any combination thereof.

7. A method for preparing the conjugate of isomeric formulas I to IV defined in claim 1, characterized in that 5 to 20 wt% solution of hyaluronic acid oligosaccharide in dimethyl sulfoxide reacts at its reducing end with 1.2 to 2.2 equivalents of an oxyamine of the general formula H2N-O-X, where X is a linear chain of 16 to 20 carbons with a possible content of multiple bonds, in the presence of 1.5 to 4 equivalents of acetic acid at a temperature of 40 to 60 °C for 5 to 20 days to form the conjugate as defined in claim 1, which is isolated from the reaction mixture by precipitation with a mixture containing chloroform or dichloromethane in combination with hexane or cyclohexane or heptane.

8. The method for preparing the conjugate according to claim 7, characterized in that for precipitation, a mixture of chloroform and hexane is used, which has a volume ratio in the range of 4:3 to 1 : 1, preferably 4:3.

9. The method for preparing the conjugate according to claim 7 or 8, characterized in that the isolated conjugate is subsequently washed with a mixture containing chloroform or dichloromethane in combination with hexane or cyclohexane or heptane, preferably a mixture containing chloroform and hexane in a ratio of 3 :2 to 1 : 1, preferably 3 :2, in order to remove dimethyl sulfoxide, and then subjected to further washing with a mixture containing chloroform or di chloromethane in combination with hexane or cyclohexane or heptane, preferably a mixture containing chloroform and hexane in a ratio of 7: 1 to 15: 1, preferably 10: 1, in order to remove oxyamine residues.

10. The method for preparing the conjugate according to any one of claims 7 to 9, characterized in that the oxyamine of general formula H2N-O-X is selected from the group comprising hexadecyloxyamine, octadecyloxyamine, cis-octadec-9- enyloxyamine.

11. A method for preparing the micellar composition as defined in claim 3, characterized in that the amphiphilic conjugate as defined in any one of claims 1 and 2 is mixed with phosphate buffer or physiological saline at a concentration of 0.1 to 2 mg / mL, the resulting solution is mechanically stirred for 0.1 to 24 h at the temperature of 15 to 25 °C, then filtered through a 1 pm filter or centrifuged, the resulting solution forming a micellar composition.

12. A method for preparing the micellar composition as claimed in claims 4 to 6, characterized in that the active substance is dissolved in an organic solvent, which is subsequently evaporated, then a solution of the amphiphilic conjugate as claimed in claim 1 or 2 in phosphate buffer or in physiological saline at a concentration of 0.1 to 2 mg / mL is added to the active substance and the resulting solution is mechanically stirred for 24 to 72 h at a temperature of 15 to 25 °C, then filtered through a 1 pm filter or centrifuged, the resulting solution forming a micellar composition.

13. The method for preparing the micellar composition according to claim 12, characterized in that after the filtration step through a 1 pm filter or after the centrifugation step, the micellar composition is subjected to sterilization by filtration through a 0.22 pm filter.

14. The method for preparing the micellar composition according to claim 12 or 13, characterized in that the organic solvent is selected from the group comprising methanol, acetone, ethanol, isopropanol, or a mixture thereof.

15. A micellar composition as defined in any one of claims 3 to 6 for use as a cosmetic product.

16. A micellar composition as defined in any one of claims 3 to 6 for use as a pharmaceutical product.

17. A micellar composition as defined in any one of claims 3 to 6 for use as a cosmetic or pharmaceutical product for topical applications of active substances.

18. A micellar composition as defined in any one of claims 3 to 6 for use as a cosmetic or pharmaceutical product for intravenous administration of active substances.

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