Antitumor Composition Based on Hyaluronic Acid and Inorganic Nanoparticles, Method of Preparation Thereof and Use Thereof

Abstract

The invention relates to an antitumor composition based on hydrophobized hyaluronan and inorganic nanoparticles stabilized by oleic acid. The hydrophobized hyaluronan in the form of an acylated hyaluronan serves in the composition as a carrier of inorganic nanoparticles. Out of the group of inorganic nanoparticles, the composition may comprise superparamagnetic nanoparticles, nanoparticles of ZnO and moreover, upconversion nanoparticles. Said composition is selectively cytotoxic with respect to both suspension and adherent tumor cell lines, especially with respect to tumor cell lines of colorectum carcinoma and adenocarcinoma, lung carcinoma, hepatocellular carcinoma and breast adenocarcinoma. The highest cytotoxic effects were observed in case of the composition based on an oleyl derivative of hyaluronan with SPIONs. The composition of acylated hyaluronan with SPIONs may also be advantageously used for an in vivo detection of accumulation of the composition in the body, preferably in a tumor or in liver. Said composition is sterilizable in the final package.

Claims

1. An antitumor composition based on a C6-C18-acylated derivative of hyaluronic acid according to the general formula (I) ##STR00001## where R is H.sup.+ or Na.sup.+, and where R.sup.1 is H or C(O)C.sub.xH.sub.y or C(O)CHCH-het, where x is an integer within the range of 5-17 and y is an integer within the range of 11-35 and C.sub.xH.sub.y is a linear or branched, saturated or unsaturated C.sub.5-C.sub.17 chain and het is a heterocyclic or heteroaromatic residue, optionally containing N, S or O atoms, wherein at least in one repeating unit one or more R.sup.1 is C(O)C.sub.xH.sub.y or C(O)CHCH-het, and where n is within the range of 12 to 4000, characterized by that it further contains inorganic nanoparticles with the stabilizing oleic acid, wherein the inorganic nanoparticles are selected from the group consisting of superparamagnetic nanoparticles, upconversion nanoparticles or zinc oxide nanoparticles, especially superparamagnetic nanoparticles.

2. The antitumor composition according to claim 1, characterized by that the acylated hyaluronan is a C.sub.6-C.sub.18-acylated derivative of hyaluronic acid having saturated and unsaturated bonds, especially C.sub.18:1 acylated hyaluronic acid derivative.

3. The antitumor composition according to claim 1, characterized by that the acylated hyaluronan serves as a carrier of inorganic nanoparticles.

4. The antitumor composition according to claim 1, characterized by that the inorganic nanoparticles are superparamagnetic nanoparticles.

5. The antitumor composition according to claim 4, characterized by that it comprises superparamagnetic nanoparticles based on iron oxides, where the amount of Fe in the composition is 0.3-3% wt., preferably 1.0% wt.

6. The antitumor composition according to claim 4, characterized by that it contains superparamagnetic nanoparticles having the size of 5 to 20 nm.

7. The antitumor composition according to claim 4, characterized by that it contains superparamagnetic nanoparticles having the size of 5 to 7 nm.

8. The antitumor composition according to claim 4, characterized by that it contains superparamagnetic nanoparticles having the size of 5 nm.

9. The antitumor composition according to claim 1, characterized by that it contains zinc oxide nanoparticles in the amount of 0.3 to 3% wt.

10. The antitumor composition according to claim 1, characterized by that it contains upconversion nanoparticles in such an amount that the total content of rare-earth elements in the composition is 0.3 to 3% wt.

11. The antitumor composition according to claim 10, characterized by that it contains upconversion nanoparticles containing Er, Yb and Y.

12. The antitumor composition according to claim 1 for use in inhibition of growth of both adherent and suspension tumor cells.

13. The antitumor composition according to claim 1 for use in inhibition of growth of tumor cells derived from colorectum carcinoma and adenocarcinoma, lung carcinoma, hepatocellular carcinoma, breast adenocarcinoma, preferably colorectum carcinoma and adenocarcinoma.

14. The antitumor composition according to claim 4 for use in an in vivo detection of accumulation of the composition in the body, especially in a tumor and liver.

15. The antitumor composition according to claim 4 for use in an in vivo detection of pathological formations in the body, especially tumors.

16. The antitumor composition according to claim 1, characterized by that it is applicable in a formulation for parenteral or local administration.

17. The antitumor composition according to claim 1, characterized by that it further contains other additives used in pharmaceutical compositions, preferably sodium chloride, dextrose or buffering salts.

18. The antitumor composition according to claim 1, characterized by that it further contains a medicinal substance, preferably a cytostatic.

19. The antitumor composition according to claim 1, characterized by that it is sterilizable in the final casing by autoclaving.

20. A method of preparation of the composition defined in claim 1, characterized by that an aqueous solution of an acylated derivative of hyaluronic acid is prepared, then inorganic particles dispersed in an organic halide solvent and stabilized by oleic acid are added, said inorganic particles being selected from the group comprising superparamagnetic nanoparticles, upconversion nanoparticles or zinc oxide nanoparticles, and the resulting suspension is sonicated until a homogenous mixture is formed, and then the free inorganic nanoparticles are separated from the inorganic nanoparticles loaded in nanomicelles by centrifugation and a subsequent filtration.

21. The method according to claim 20, characterized by that the filtrate is subsequently lyophilized.

22. The method according to claim 20, characterized by that the filtrate is subsequently sterilized by autoclaving in the final casing.

23. The method according to claim 21, characterized by that the lyophilizate is subsequently dissolved in an aqueous solution and sterilized by autoclaving in the final casing.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0027] FIG. 1. TEM photo of nanoparticles (SPIONs) encapsulated in hydrophobized hyaluronan.

[0028] FIG. 2A, 2B, 2C. Inhibition of viability by the composition of acylated hyaluronan with SPIONs in tumor HT-29 line compared to the positive/neutral effect in control NHDF fibroblasts and a mouse non-tumor 3T3line.

[0029] FIG. 3. Influence of the composition of acylated hyaluronan with SPIONs on the inhibition of viability of various tumor cell lines compared to the positive effect in control NHDF fibroblasts and a mouse non-tumor 3T3 line.

[0030] FIG. 4A, 4B, 4C. Comparison of the influence of the compositions of an acylated hyaluronan with encapsulated 5 nm, 10 nm a 20 nm SPIONs on the viability of the tumor cells HT-29 and the healthy NHDF and 3T3.

[0031] FIG. 5A, 5B, 5C. Inhibition of viability by means of the composition of an acylated hyaluronan with zinc nanoparticles in the tumor HT-29 line compared to the positive/neutral effect in control NHDF fibroblasts and a mouse non-tumor 3T3 line.

[0032] FIG. 6A, 6B, 6C. Inhibition of viability by means of the composition of an acylated hyaluronan with upconversion nanoparticles in the tumor HT-29 line compared to the positive/neutral effect in control NHDF fibroblasts and a mouse non-tumor 3T3 line.

[0033] FIG. 7. Co-cultivation of healthy NHDF fibroblasts and control HT-29 cells with the composition of an acylated hyaluronan with SPIONs.

[0034] FIG. 8. Expression of the CD44 receptor on the surface of the cells NHDF, MCF-7 and MDA-MB-231 determined by means of flow cytometry.

[0035] FIG. 9. Induction of the ROS formation by means of the composition of an acylated hyaluronan with SPIONs in NHDF and HT-29.

[0036] FIG. 10. Intracellular Fe staining in tumor HT-29 and control NHDF cells after incubation with the composition of an acylated hyaluronan with SPIONs (scale: 10 m)

[0037] FIG. 11. MRI detection of time accumulation of SPIONs loaded in an acylated hyaluronan after an intravenous administration in a tumor (glioblastoma tumor, 1.1 mg Fe/kg).

[0038] FIG. 12. MRI contrast of liver after an intravenous administration of the SPION composition loaded in an acylated hyaluronan (1.1 mg Fe/kg).

[0039] FIG. 13. Fe detection in histological sections of a tumor (2 and 24 hours upon administration of the SPION composition) after staining thereof by Prussian blue.

[0040] FIG. 14. Fe detection in histological sections of liver (2 and 24 hours upon administration of the SPION composition) after staining thereof by Prussian blue.

[0041] FIG. 15. TEM photo of nanoparticles (SPIONs) encapsulated in hydrophobized hyaluronan after sterilisation.

[0042] FIG. 16. Selective cytotoxicity of the SPION composition before and after sterilisation by autoclaving.

[0043] FIG. 17A, 17B, 17C. Induction of apoptosis by means of the SPION composition in a mouse tumor lymphoma line EL4.

EXAMPLES

[0044]
SS=substitution degree=100%*molar amount of the bound substitute/molar amount of all polysaccharide dimers

[0045] The term equivalent (eq) used herein relates to a hyaluronic acid dimer, if not indicated otherwise. The percentages are weight percents, if not indicated otherwise.

[0046] Molecular weight of hyaluronic acid (source: Contipro Pharma, a.s., Doln Dobrou{hacek over (e)}, CZ) was determined by SEC-MALLS.

[0047] The term inorganic nanoparticles means inorganic nanoparticles having a diagnostic function, where the diagnostic function is an essential common property of inorganic nanoparticles used in the composition according to the invention. The diagnostic function is intended to mean the possibility to detect said particles by methods available in medicine. SPIONs may be detected by means of magnetic resonance and ZnO and upconversion nanoparticles by means of luminiscence imaging, and all these particles are just optimised for detection, and that's why they are used. Therefore, out of a set of inorganic nanoparticles, those were selected that allow for the detection (of micelles) in vivo or in vitro.

[0048] The term upconversion nanoparticles is to mean upconversion lanthanide nanoparticles, i.e. nanoparticles containing elements from the group of rare earths, since no other inorganic nanoparticles capable of an effective upconversion of energy are known.

Example 1 Preparation of Hydrophobized Hyaluronic Acid, More Specifically the Oleyl Derivative (C18:1) of Hyaluronic Acid by Means of Mixed Anhydride of Benzoic Acid and Oleic Acid

[0049] 100 g of sodium hyaluronan (250 mmol, 15 kDa) were dissolved in 2000 ml of demi water. Then 1000 ml of isopropanol were gradually added. Thereafter, TEA (70 ml, 3 eq.) and DMAP (1.52 g, 0.05 eq.) were added to the solution. At the same time, oleic acid (35.3 g 0.5 eq) was dissolved in 1000 ml of isopropanol, then TEA (70 ml, 3 eq.) and benzoyl chloride (14.4 ml, 0.5 eq.) were added to the solution. After the activation of the acid the precipitate was filtered off into the prepared HA solution. The reaction proceeded for 3 hours at room temperature. Then the reaction mixture was diluted by 1000 ml of demi water with an addition of 95 g of NaCl. The acylated derivative was isolated from the reaction mixture by precipitation by using a quadruple of absolute isopropanol. After decantation, the precipitate was repeatedly washed with an aqueous solution of isopropanol (85% vol.).

[0050] SS 13% (determined from NMR).

[0051] .sup.1H NMR (D.sub.2O): 0.88 (t, 3H, CH.sub.2CH.sub.3), 1.22-1.35 (m, 20H, (CH.sub.2).sub.10),

[0052] 1.60 (m, 2H, CH.sub.2CH.sub.2CO), 2.0 (4H, (CH.sub.2).sub.2), 2.41 (t, 2H, CH.sub.2CO), 5.41 (d, 2H, CHCH)

[0053] This example describes a general method of synthesis of a hydrophobized derivative of hyaluronan. However, the procedure is not limited to the oleyl derivative only. A detailed disclosure of the synthesis of hydrophobized derivatives is mentioned in the patent application No. CZ PV2012-842.

Example 2. Preparation of SPIONs Having an Average Size of 5 nm

[0054] 1.80 g of ferric oleate, 0.35 ml of oleic acid and 13.35 ml of 1-octadecene were added into a three-necked flask having the volume of 50 ml. The mixture was slowly heated under vacuum to 100 C., where it was maintained for 30 minutes for drawing away the volatile components. Then the mixture was heated under a mild argon flow to 280 C. and it was maintained at this temperature for 60 minutes. The mixture was bubbled through with argon during the reaction at 280 C. After cooling down to the room temperature, acetone was added to the reaction mixture and the nanoparticles were separated by centrifugation. The precipitated SPIONs were thereafter washed 4 times with a mixture of hexane/acetone (the ratio successively 1:4 to 1:1) and finally, they were dispersed in toluene and stored at 4 C. in dark.

Yield: 78%

[0055] Size of the nanoparticles: 5.2 t 0.8 nm (according to the photo from the electron microscope)

Example 3. Preparation of SPIONs Having an Average Size of 10 nm

[0056] 1.80 g of ferric oleate, 0.35 ml of oleic acid and 13.35 ml of 1-octadecene were added into a three-necked flask having the volume of 50 ml. The mixture was slowly heated under vacuum to 100 C., where it was maintained for 30 minutes for drawing away the volatile components. Then the mixture was heated under a mild argon flow to the boiling point (317 C.) and it was maintained at this temperature for 60 minutes. After cooling down to the room temperature, the SPIONs were separated in the same way as in Example 2.

Yield: 74%

[0057] Size of the nanoparticles: 9.80.5 nm (according to the photo from the electron microscope)

Example 4. Preparation of SPIONs Having an Average Size of 20 nm

[0058] 1.80 g of ferric oleate, 0.35 ml of oleic acid and 5.34 ml of 1-octadecene and 6 g of n-docosane were added into a three-necked flask having the volume of 50 ml. The mixture was slowly heated under vacuum to 100 C., where it was maintained for 30 minutes for drawing away the volatile components. Then the mixture was heated under a mild argon flow to 315 C. and it was maintained at this temperature for 60 minutes. After cooling down to the room temperature, the SPIONs were separated in the same way as in Example 2.

Yield: 56%

[0059] Size of the nanoparticles: 21.13.1 nm (according to the photo from the electron microscope)

Example 5. Preparation of ZnO Nanoparticles

[0060] Zinc acetate dihydrate (1185.30 mg; 5.4 mmol) was introduced into a three-necked flask having the volume of 250 ml and dissolved in methanol (90 ml) at room temperature. Meanwhile, a solution of tetramethyl ammonium hydroxide (1622.91 mg; 8.96 mmol) in methanol (22.39 ml) was prepared in a two-necked flask. Both above mentioned solutions were degassed in an ultrasound bath while being bubbled through with argon for 15 minutes (the temperature of the aqueous bath 50 C., output 120 W). The methanol solution of zinc acetate was heated under reflux in an oil bath (the bath temperature 60 C.). After the addition of oleic acid (310 l; 0.99 mmol) the mixture was brought to the boiling point (bath temperature 85 C.). The solution of tetramethyl ammonium hydroxide in methanol was heated under reflux (bath temperature 75 C.) and quickly added into the three-necked flask containing zinc acetate and oleic acid. The reaction mixture was refluxed while being constantly stirred (600 rpm) and bubbled through with argon for 2 minutes (bath temperature 85 C.). Then the mixture was diluted by methanol (90 ml) and cooled for 15 min on an ice bath. The cooled mixture was centrifuged for 15 min (4000g, 4 C.). The particles were washed with ethanol (325 ml), each washing step was followed by centrifugation for 10 minutes (4000g, 25 C.). The particles were dispersed in chloroform (45 ml) and stored at 4 C. in dark.

Quantum efficiency of fluorescence: 34% (determined by a relative method, standard=norharman)
Size of the nanoparticles: 3.40.3 nm (according to the photo from the electron microscope)

Example 6. Preparation of Upconversion Nanoparticles

[0061] The molar amounts corresponding to 1.60 mmol of yttrium (III) acetate, 0.36 mmol of ytterbium (III) acetate and 0.04 mmol of erbium (III) acetate were introduced into a three-necked flask having the volume of 100 ml and octadec-1-en (34 ml) and oleic acid (12.0 ml) were added. The mixture was evacuated while being stirred hard (600 rpm) and it was slowly heated on an oil bath to 80 C. At this temperature, the mixture was stirred in vacuum until total clarification and from that moment for further 90 minutes. The flask with the mixture was filled with argon and after cooling down to the room temperature in an argon atmosphere a solution of NaOH (200 mg) and NF.sub.4F (296.3 mg) in methanol (20 ml) was added, whereupon the mixture became cloudy immediately. The mixture was stirred at room temperature overnight, then methanol was slowly evaporated at 65 C. (oil bath). Then the flask with the mixture was transferred to a heating mantle controlled by a PID-controller. The mixture was gradually introduced to vacuum, in vacuum it was slowly heated to 112 C. and at this temperature it was being degassed for 30 minutes. Then the flask containing the mixture was filled with argon and under air reflux it was heated to 305 C. in a mild argon flow at speed of 2 C./min. At 305 C. the mixture was left for 110 minutes, after removing the heating it cooled down naturally to the room temperature.

[0062] The upconversion nanoparticles were precipitated from the reaction mixture by ethanol (a double volume of the reaction mixture volume) and then isolated by centrifugation (RCF 3000 g; 10 minutes). The nanoparticles (sediment) were dispersed in hexane (5 ml), precipitated by ethanol (10 ml) and separated by means of centrifugation (RCF 3000 g; 7 minutes). The nanoparticles were purified in this manner three times by the hexane/ethanol system and three times by the hexane/acetone system. Finally, the nanoparticles were dispersed in chloroform (10 ml) and stored at room temperature.

Nanoparticles composition (ICP-OES):NaYF.sub.4:Yb/Er (80 mol. % Y, 18 mol. % Yb, 2 mol. % Er
Organic component fraction (TGA): 7%
Size of the nanoparticles (electron microscope): 342 nm

Example 7. Preparation of the Composition of a Capronyl Derivative of Hyaluronic Acid (HAC6) with SPIONs

[0063] 150 mg of acylated derivative of hyaluronan (HAC6, DS=60%, Mw=38 kDa) prepared according to Example 1 was being dissolved for 2 hours in 15 ml of demi water at constant stirring on a magnetic stirrer. The SPIONs (stabilized by oleic acid, size of the nanoparticles: 5 nm), prepared according to Example 2, were transferred from the toluene medium to the chloroform medium.

[0064] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of SPIONs dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised.

[0065] The amount of the loaded Fe (ICP determination): 1.1% (wt.)

Example 8. Preparation of the Composition of a Caprylyl Derivative of Hyaluronic Acid (HAC8) with SPIONs

[0066] 150 mg of acylated derivative of hyaluronan (HAC8, DS=22%, Mw=20 kDa) prepared according to Example 1 was being dissolved for 2 hours in 15 ml of demi water at constant stirring on a magnetic stirrer. The SPIONs (stabilized by oleic acid, size of the nanoparticles: 5 nm), prepared according to Example 2, were transferred from the toluene medium to the chloroform medium.

[0067] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of SPIONs dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised. The amount of the loaded Fe (ICP determination): 1.1% (wt.)

Example 9. Preparation of the Composition of a Caprinyl Derivative of Hyaluronic Acid (HAC10) with SPIONs

[0068] 150 mg of acylated derivative of hyaluronan (HAC10, DS=15%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 2 hours in 15 ml of demi water at constant stirring on a magnetic stirrer. The SPIONs (stabilized by oleic acid, size of the nanoparticles: 5 nm), prepared according to Example 2, were transferred from the toluene medium to the chloroform medium.

[0069] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of SPIONs dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised.

[0070] The amount of the loaded Fe (ICP determination): 1.2% (wt.)

Example 10. Preparation of the Composition of a Palmitoyl Derivative of Hyaluronic Acid (HAC16) with SPIONs

[0071] 150 mg of acylated derivative of hyaluronan (HAC16, DS=9%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 2 hours in 15 ml of demi water at constant stirring on a magnetic stirrer. The SPIONs (stabilized by oleic acid, size of the nanoparticles: 5 nm), prepared according to Example 2, were transferred from the toluene medium to the chloroform medium.

[0072] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of SPIONs dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised.

[0073] The amount of the loaded Fe (ICP determination): 1.2% (wt.)

Example 11. Preparation of the Composition of a Stearyl Derivative of Hyaluronic Acid (HAC18) with SPIONs

[0074] 150 mg of acylated derivative of hyaluronan (HAC18:0, DS=9%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 2 hours in 15 ml of demi water at constant stirring on a magnetic stirrer. The SPIONs (stabilized by oleic acid, size of the nanoparticles: 5 nm), prepared according to Example 2, were transferred from the toluene medium to the chloroform medium.

[0075] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of SPIONs dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised. The amount of the loaded Fe (ICP determination): 1.0% (wt.)

Example 12. Preparation of the Composition of an Oleyl Derivative of Hyaluronic Acid (HAC18:1) with SPIONs

[0076] 150 mg of acylated derivative of hyaluronan (HAC18:1, DS=12%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 2 hours in 15 ml of demi water at constant stirring on a magnetic stirrer. The SPIONs (stabilized by oleic acid, size of the nanoparticles: 5 nm), prepared according to Example 2, were transferred from the toluene medium to the chloroform medium.

[0077] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of SPIONs dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised.

[0078] The amount of the loaded Fe (ICP determination): 1.0% (wt.)

[0079] The morphology of the clustered nanoparticles in the polymeric micelle is shown in FIG. 1.

Example 13. Preparation of the Composition of an Oleyl Derivative of Hyaluronic Acid (HAC18:1) with SPIONs

[0080] 120 mg of acylated derivative of hyaluronan (HAC18:1, DS=12%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 2 hours in 12 ml of demi water at constant stirring on a magnetic stirrer. The SPIONs (stabilized by oleic acid, size of the nanoparticles: 5 nm), prepared according to Example 2, were transferred from the toluene medium to the chloroform medium.

[0081] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 7.25 mg of SPIONs dispersed in 4 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised.

[0082] The amount of the loaded Fe (ICP determination): 1.8% (wt.)

Example 14. Preparation of the Composition of Linoleyl Derivative of Hyaluronic Acid (HAC18:2) with SPIONs

[0083] 150 mg of acylated derivative of hyaluronan (HAC18:2, DS=12%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 4 hours in 15 ml of demi water at constant stirring on a magnetic stirrer. The SPIONs (stabilized by oleic acid, size of the nanoparticles: 5 nm), prepared according to Example 2, were transferred from the toluene medium to the chloroform medium.

[0084] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of SPIONs dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised. The amount of the loaded Fe (ICP determination): 0.98% (wt.)

Example 15. Preparation of the Composition of a Linolenyl Derivative of Hyaluronic Acid (HAC18:3) with SPIONs

[0085] 150 mg of acylated derivative of hyaluronan (HAC18:3, DS=3%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 4 hours in 15 ml of demi water at constant stirring on a magnetic stirrer. The SPIONs (stabilized by oleic acid, size of the nanoparticles: 5 nm), prepared according to Example 2, were transferred from the toluene medium to the chloroform medium.

[0086] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of SPIONs dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised. The amount of the loaded Fe (ICP determination): 1.0% (wt.)

Example 16. Preparation of the Composition of an Oleyl Derivative of Hyaluronic Acid (HAC18:1) with SPIONs

[0087] 150 mg of acylated derivative of hyaluronan (HAC18:1, DS=12%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 2 hours in 15 ml of demi water at constant stirring on a magnetic stirrer. The SPIONs (stabilized by oleic acid, size of the nanoparticles: 10 nm), prepared according to Example 3, were transferred from the toluene medium to the chloroform medium.

[0088] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of SPIONs dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised.

[0089] The amount of the loaded Fe (ICP determination): 0.4% (wt.)

Example 17. Preparation of the Composition of an Oleyl Derivative of Hyaluronic Acid (HAC18:1) with SPIONs

[0090] 150 mg of acylated derivative of hyaluronan (HAC18:1, DS=12%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 2 hours in 15 ml of demi water at constant stirring on a magnetic stirrer. The SPIONs (stabilized by oleic acid, size of the nanoparticles: 20 nm), prepared according to Example 4, were transferred from the toluene medium to the chloroform medium.

[0091] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of SPIONs dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised.

[0092] The amount of the loaded Fe (ICP determination): 1.7% (wt.)

Example 18. Preparation of the Composition of an Oleyl Derivative of Hyaluronic Acid (HAC18:1) with SPIONs and Paclitaxel

[0093] 150 mg of acylated derivative of hyaluronan (HAC18:1, DS=12%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 2 hours in 15 ml of demi water at constant stirring on a magnetic stirrer. Then 5 mg of SPIONs (stabilized by oleic acid, size of the nanoparticles: 5 nm), prepared according to Example 2, were transferred from toluene to chloroform. The nanoparticles prepared in this way were mixed with 6 mg of paclitaxel dissolved in 3 ml of chloroform.

[0094] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of SPIONs with 6 mg of paclitaxel in CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles and paclitaxel were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles and paclitaxel was taken away, filtered through a 1.0 m glass filter and lyophilised.

[0095] The amount of the loaded Fe (ICP determination): 1.5% (wt.)

[0096] The amount of the loaded PTX (HPLC determination): 0.3% (wt.)

Example 19. Preparation of the Composition of an Oleyl Derivative of Hyaluronic Acid (HAC18:1) with ZnO Nanoparticles

[0097] 150 mg of acylated derivative of hyaluronan (HAC18:1, DS=12%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 2 hours in 15 ml of demi water at constant stirring on a magnetic stirrer.

[0098] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of ZnO (from Example 5) dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised.

[0099] The amount of the loaded Zn (ICP determination): 1.6% (wt.)

Example 20. Preparation of the Composition of an Oleyl Derivative of Hyaluronic Acid (HAC18:1) with Upconversion Nanoparticles

[0100] 150 mg of acylated derivative of hyaluronan (HAC18:1, DS=12%, Mw=15 kDa) prepared according to Example 1 was being dissolved for 2 hours in 15 ml of demi water at constant stirring on a magnetic stirrer.

[0101] The solution of acylated hyaluronan was transferred into a rosette sonication vessel (RZ 1, volume: 25 ml), immersed in an ice bath. First the solution was sonicated for 60 s (sonication parameters: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Further, 5 mg of upconversion nanoparticles (from Example 6) dispersed in 3 ml of CHCl.sub.3 were gradually added to said solution (sonication parameters: 200 W, amplitude 85%, cycle 0.8 s and sonotrode S2). The homogenized suspension was further sonicated for 15 min (sonication parameters: amplitude 65%, cycle 0.5 s and sonotrode S2). The free nanoparticles were separated by means of repeated centrifugation (34500 RPM, 10 min) and the resulting supernatant containing nanomicelles of hyaluronan with the loaded nanoparticles was taken away, filtered through a 1.0 m glass filter and lyophilised.

[0102] The amount of the loaded Er; Y; Yb (ICP determination): 0.02; 0.50; 0.19% (wt.)

Example 21. In Vitro Cytotoxicity of the Composition of an Acylated Hyaluronan with SPIONs

[0103] Primary cells and non-tumor and tumor lines (Table 1) were seeded in 96-well panels and cultivated for 24 hours in 37 C./5% CO.sub.2. Then the cells were treated with solutions of compositions of an acylated hyaluronan with SPION from Examples 7-12, 14 and 15 at concentrations 10, 100, 200 and 500 g/ml (concentration of polymeric micelles in the culture medium). At the same time, the viability of the acylated hyaluronan alone and of SPIONs alone was measured (in the respective concentrations). The viability of the cells was monitored in times 0, 24, 48 and 72 h by means of the MTT method and the resulting values indicate the inhibition or activation of the cell viability in the given time point. Inhibition of the cell viability of cells treated with compositions of various acylated HA derivatives with SPIONs (FIGS. 2A-C) and the influence of the composition HAC18:1+SPIONs (from Example 12) on various tumor lines (FIG. 3) was monitored. FIGS. 4A-C show the comparison of the cytotoxic action of the composition (according to Example 12, 16, 17) with 5, 10 and 20 nm SPIONs. The cell lines are described in Table 1.

TABLE-US-00001 TABLE 1 List of the tested adherent cell lines. Designation of the cell line Cell type/origin Control line NHDF Primary human dermal fibroblasts 3T3 mouse fibroblast line Tumor line HT29 human colorectal adenocarcinoma A2058 human melanoma A549 human lung carcinoma C3A human hepatocellular carcinoma MCF7 human breast adenocarcinoma HCT116 human colorectal carcinoma MDA-MB231 human breast adenocarcinoma Caco2 human colorectal adenocarcinoma

[0104] The results in FIGS. 2A-C show that unlike in control lines (NHDF and 3T3), in case of the tumor line HT29 there is a significant inhibition of the cell growth. The highest inhibitions were registered for the composition based on C18 and C18:1 acylated derivatives. The composition based on C18:1 derivatives with SPIONs even more supported the viability of NHDF and 3T3 cells. The acylated hyaluronan alone and the SPIONs did not influence the viability of any of the tested cells (data not shown).

[0105] The composition of HAC18:1 with SPIONs was further used for treating other tumor lines (FIG. 3). The experimental data showed a slow-down of growth of tumor lines A549, HCT116, C3A, MCF7 and MDA-MB231 and Caco2. An exception was only the melanoma A2058 line where inhibition showed up only when treated with the highest concentration of the composition (500 g/ml). The control fibroblasts (both NHDF and 3T3), in the contrary, were stimulated significantly by the composition and not even the highest concentration 500 g/ml has shown any cytotoxic properties.

[0106] FIGS. 4A-C confirm the selective anti-tumor activity of the composition with 5 nm SPIONs. This effect is not observed to such an extent for compositions with 10 and 20 nm SPIONs.

Example 22. In Vitro Cytotoxicity of the Composition of an Acylated Hyaluronan with Nanoparticles of Zinc Oxide

[0107] Primary human fibroblasts (NHDF), enteric tumor HT-29 cells and a mouse fibroblast 3T3 line were seeded to 96-well panels and cultivated for 24 hours in 37 C./5% CO.sub.2. Then the cells were treated with solutions of compositions of an acylated hyaluronan with zinc oxide nanoparticles from Example 19 in concentrations 10, 100, 200 and 500 g/ml (concentration of polymeric micelles). The viability of the cells was monitored in times 0, 24, 48 and 72 h by means of the MTT method and the resulting values indicate the inhibition or activation of the cell viability in the given time point (FIGS. 5A-C).

[0108] The results in FIGS. 5A-C show that unlike in control lines (NHDF and 3T3), with increasing incubation time and, further, in higher concentrations of polymeric micelles, inhibition of the tumor cell growth occurs. However, in this case, inhibition of the growth was also observed in case of concentration of 500 g/ml in 3T3 cells. In lower concentrations of the composition and also in the control NHDF cell line, no inhibition has been observed.

Example 23. In Vitro Cytotoxicity of the Composition of an Acylated Hyaluronan with Upconversion Nanoparticles

[0109] Primary human fibroblasts (NHDF), enteric tumor HT-29 cells and mouse fibroblast 3T3 line were seeded to 96-well panels and cultivated for 24 hours in 37 C./5% CO.sub.2. Then the cells were treated with solutions of polymeric micelles with upconversion nanoparticles from Example 20 in concentrations 10, 100, 200 and 500 g/ml (concentration of polymeric micelles). The viability of the cells was monitored in times 0, 24, 48 and 72 h by means of the MTT method and the resulting values indicate the inhibition or activation of the cell viability in the given time point (FIGS. 6A-C).

[0110] The results in FIGS. 6A-C show that unlike in control NHDF lines where the viability is highly increased, with increasing incubation time an inhibition of the tumor cell growth occurs. However, a slight inhibition is observed also in non-tumor 3T3 line.

Example 24. In Vitro Selective Cytotoxicity of the Composition of Acylated Hyaluronan with SPIONs

[0111] Primary human fibroblasts labelled by DiO (green) and tumor HT-29 cells labelled by DiI (red) were in the ratio of 3:1 and the total concentration of 50.000 cells/well seeded into the wells of a 24-well panel in 1 ml of RPMI 1640 (Roswell Park Memorial Institut) medium. After achieving of min 80% confluence of the cell monolayer, the cells were treated with 200 g/ml solution of the composition with SPIONs from Example 12. After 72 hours of incubation, a picture of the cells was taken by means of a fluorescence microscope Nikon Ti-Eclipse (FIG. 7).

[0112] For an explanation of the possible mechanism of the different activity towards the control cells and the tumor cells, expression of the CD44 receptor for hyaluronan was analysed by means of flow cytometry on NHDF, MCF-7 and MDA-MB-231 cells. After achieving the 80% confluence, the cells were washed with PBS, incubated for 15 min/RT with an antiCD44-FITC antibody, after the incubation they were 2 washed with PBS again and analysed on a flow cytometer MACSQuant Analyzer (Miltenyi Biotec). The results are indicated as fluorescence intensity (RFU) (FIG. 8).

[0113] Moreover, for NHDF and HT-29 cells, oxidative stress was determined after the treatment with the composition of the acylated hyaluronan with SPIONs from Example 12. The cells were cultured on 6-well panels and after achieving the 80% confluence, they were treated with a 200 g/ml solution of the composition with SPIONs for 24 hours. As far as the control cells are concerned, only the medium was exchanged for a fresh one without the content of the tested composition. After the incubation, the cells were washed and treated with DCF-DA (non-fluorescent substance which is oxidated by intracellular ROS to a fluorescent DCF, the final concentration: 1 uM) for 20 min/37 C./in dark. After the subsequent washing with PBS, the cells were analyzed on a flow cytometer MACSQuant Analyzer (Miltenyi Biotec). The results are indicated as the relative fluorescence intensity (% of the non-treated control) of DCF inside the cells (FIG. 9).

[0114] The results from FIG. 7 confirm the selective growth inhibition of the tumor HT-29 line, whereas the control fibroblasts NHDF are not influenced negatively and they reach the confluence. This effect is not caused by a different induction of ROS formation, said induction is increased in both types of cells but it is increased to the same level (FIG. 9). The explanation may be a different extent of response to said increase of the ROS production in NHDF and in HT-29 cells.

[0115] The difference of activity with respect to the control cells and to the tumor cells is not a function of expression of the main surface receptor for hyaluronan, CD44. FIG. 8 confirms a high expression of CD44 in control NHDF fibroblasts, the viability of which was increased by the composition, and a low expression in tumor MCF-7 and MDA-MB-231 lines, in which a significant viability inhibition was observed (FIG. 3).

[0116] After staining of cells (detection of the presence of Fe by means of Prussian blue) incubated with the composition of the acylated hyaluronan with SPIONs from Example 12, a different Fe ion distribution is observedwhile the dissolved Fe was detected in tumor cells, iron aggregates were detected in control cells (FIG. 10). This phenomenon could be the cause of the selective activity of the composition in tumor cells.

Example 25. Preparation of a Composition for an Intravenous Administration

[0117] 650 l of sterile 0.9% NaCl is added to 20-30 mg of the acylated hyaluronan with SPIONs from Example 12 prepared in a sterile manner, the solution is agitated from time to time until the total dissolution of the lyophilizate. The solution is injectable in vivo without problems.

[0118] The solution prepared in this way is stabile, as far as the hydrodynamic size of the particles is concerned, for at least 2 days.

Example 26. Preparation of a Composition for an Intravenous Administration

[0119] 650 l of sterile 5% dextrose is added to 20-30 mg of the acylated hyaluronan with SPIONs from Example 12 prepared in a sterile manner, the solution is agitated from time to time until the total dissolution of the lyophilizate. The solution is injectable in vivo without problems.

[0120] The solution prepared in this way is stabile, as far as the hydrodynamic size of the particles is concerned, for at least 2 days.

Example 27. In Vivo Detection of the Composition of an Acylated Hyaluronan with SPIONs

[0121] Lewis Brown Norway rats with a glioblastoma tumor were used for in vivo testing. The tumors were inoculated by injecting a suspension of 310.sup.6 glioblastoma cells into a muscle on a leg and 9 days after that the rats were administered intravenously the composition of acylated hyaluronan (HAC18:1) with SPIONs (750 l of the solution in 0.9% NaCl, with the Fe content being 1.1 mg/kg). Then the rats were analyzed by means of Bruker Biospec (4.7 T).

[0122] Accumulation of SPIONs in the tumor after the intravenous administration of the composition was confirmed in FIG. 11, where especially darkening of the edges of the tumor was detected. A visible accumulation of SPIONs was detected also in liver (FIG. 12), said composition therefore, can be used as a contrast agent for liver.

[0123] Accumulation of SPIONs was further confirmed after killing the animals on histological sections of the tumor (FIG. 13) and liver (FIG. 14), where the presence of Fe was detected by Prussian blue colouring (blue stains in Figures). The blue colouring was not detected in any of the control samples.

Example 28. Sterilization of the Composition of an Acylated Hyaluronan with SPIONs by Autoclaving

[0124] Sterilization of the composition prepared according to Example 12 (concentration: 30 mg/ml in 0.9% NaCl) was carried out in an autoclave at 121 C. for 15 minutes.

[0125] The solution was stabile after the sterilization, the SPIONs remained clustered in hyaluronan nanomicelles (FIG. 15), the selective cytotoxic effects with respect to the tumor cells were retained.

[0126] The cytotoxicity was determined on the tumor HT-29 line and control primary NHDF fibroblasts according to the procedure disclosed in Example 21. FIG. 16 shows a comparison of the composition from Example 12 before and after the sterilization by autoclaving, the selective cytotoxicity towards the tumor cells was retained even after the sterilization by autoclaving.

Example 29. Induction of Apoptosis in a Mouse Tumor Suspension Lymphoma EL4 Line

[0127] The mouse lymphoma line EL4 (used for an induction of tumors in mouse experimental models of carcinogenesis) was cultured in the RPMI 1640 (Roswell Park Memorial Institut) medium. In the exponential phase of the growth, aliquots were prepared from the cell culture in the concentration of 510.sup.5 cells/ml of the RPMI medium, which were treated with a 100, 200 and 500 g/ml solution of the composition with SPIONs from Example 9. After 72 hours of incubation, the cells were washed and coloured specifically by means of fluorescent markers of the cell death (propidium iodide, AnnexinV-FITC), which were subsequently detected on a flow cytometer MACSQuant (Miltenyi Biotec).

[0128] In FIGS. 17A-C, there is a clear induction of apoptosis (the cell population in the right lower quadrant, FIG. 17B) and a slightly increased induction of necrosis (left/right upper quadrant, FIG. 17B) after the treatment with a 100 g/ml solution of the composition with SPIONs from Example 12. The representation of the live, apoptotic and necrotic cells after the treatment by the composition in the individual concentrations is plotted in the graph (FIG. 17C).