PROCESS OF PREPARING POLYMERIC NANOPARTICLES THAT CHELATE RADIOACTIVE ISOTOPES AND HAVE A SURFACE MODIFIED WITH SPECIFIC MOLECULES TARGETING THE PSMA RECEPTOR AND THEIR USE

Abstract

Process for preparation of polymeric nanoparticles that chelate radioactive isotopes and have their surface modified with specific molecules targeting PSMA receptor on the surface of cancer cells, with a targeting agent modified by a linker molecule attaching to free aldehyde groups present in the dextran chain. Polymeric nanoparticles that chelate radioactive isotopes synthesized according to the claimed process for use in therapy and diagnostics of prostate cancer and metastatic cancer cells as well as other affected cells for which the nanoparticles show the affinity.

Claims

1. A process for preparing polymeric nanoparticles that chelate radioactive isotopes and have their surface modified with specific molecules targeting the PSMA receptor on the surface of cancer cells, characterized in that it comprises the stages in which: a) a dextran chain is oxidized to polyaldehyde by means of periodate, b) a targeting agent that is α,α-urea of glutamic acid and lysine, the targeting agent modified by a linker molecule is attached to free aldehyde groups present in the dextran chain, c) a folding agent in the form of hydrophobic diamine or polyamine is attached, with one or two amino groups of the folding agent attaching to aldehyde groups, d) the resulting imine bonds are reduced to amine bonds, e) to the free amino group of the attached folding agent, a chelator molecule is attached via an amide bond, f) the resulting mixture is purified, g) the nanoparticle fraction is subjected to lyophilization.

2. The process according to claim 1, wherein the mixture from stage f) is purified by dialysis.

3. The process according to claim 1, wherein the cells on which the PSMA receptor is present are prostate cancer cells and metastatic prostate cancer cells.

4. The process according to claim 1, wherein the cells on which the PSMA receptor is present are breast, lung, colon and pancreatic cancer cells.

5. The process according to claim 1, wherein the substitution of the aldehyde groups with the targeting agent is from 1 to 50%.

6. The process according to claim 5, wherein the substitution of the aldehyde groups with the targeting agent is from 2.5 to 5%.

7. The process according to claim 1, wherein the chelators are derivatives of DOTA, DTPA and/or NOTA.

8. (canceled)

9. The process according to claim 1, wherein the linker is 2,5-dioxopyrrolidin-1-yl 2,2-dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azatricos-23-ate (PEG.sub.5).

10. The process according to claim 1, wherein the folding agent are lipophilic diamines selected from the group consisting of dodecylamines, diaminooctanes, diaminodecanes (DAD), polyether diamines, polypropylene diamines and block copolymer diamines.

11. The process according to claim 1, wherein the obtained nanoparticles are radiochemically labelled with such isotopes in which the breakdown pathway involves beta plus decay, beta minus decay, gamma emitter decay.

12. Polymeric nanoparticles chelating radioactive isotopes, with a surface modified by specific molecules targeting the PSMA receptor as obtained according to the process of claim 1, for use in diagnostics and therapy.

13. Polymeric nanoparticles chelating radioactive isotopes according to claim 12 for use in Positron Emission Tomography PET and PET/MRI diagnostics.

14. Polymeric nanoparticles chelating radioactive isotopes according to claim 12 for use in focal brachytherapy.

15. Polymeric nanoparticles chelating radioactive isotopes prepared according to the process of claim 1 for use in the therapy and diagnostics of prostate cancer and metastatic cancer as well as other cancers with affected cells to which the nanoparticles show the affinity.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0056] The figures enclosed to the description which illustrate the invention present what follows:

[0057] FIG. 1—fluorescence assay of the PSMA receptor enzyme activity inhibition for nanoparticles with aldehyde groups substituted with the GuL targeting agent in 10% (BCS 0277), 30% (BCS 0290) and 2.5% BCS 0319) and without the substitution (Control without nanoparticles) for various concentrations of nanoparticle solutions used in the analysis, i.e. 16 μg, 4 μg, 1.6 μg, 0.4 μg, 0.16 μg.

[0058] FIG. 2—fluorescence assay of the PSMA inhibition by nanoparticles with GuL without the linker (408) and with the linker (277) for various quantities of the targeting agent, i.e. 8000 ng, 800 ng, 80 ng and 8 ng.

[0059] The object of the invention is illustrated in the preferred embodiments described below.

EXAMPLE 1

[0060] Preparation of Nanoparticles with 10% Substitution of Aldehyde Groups with the GuL Targeting Agent at 90% Substitution with the DAD Folding Agent (BCS277)

[0061] 1.1. Oxidation of Dextran to Polyaldehyde Dextran (PAD)

[0062] Dextran Oxidation Reaction:

[0063] 5.00 g of dextran was dissolved in 100 ml ultrapure water. 0.66 g of sodium periodate was added. The oxidation reaction was continued overnight in the dark at room temperature. The product was purified through dialysis for 72 hours in one hundred-fold volume of the ultrapure water, with the water changed at least twice. The water was removed by evaporation at 40° C.

[0064] Determination of Aldehyde Groups in PAD:

[0065] 100 μl of 0.8 mM hydroxylamine hydrochloride solution, 300 μl of 0.6 M acetate buffer with pH 5.8 and 20-100 μl of PAD was added to a 2 ml tube, and then ultrapure water (0-80 μl) was added up to a total volume 500 μl. The assay was conducted for three different PAD volumes (20, 60 and 100 μl). A control sample was prepared: 100 μl of 0.8 mM hydroxylamine hydrochloride solution, 300 μl of 0.6 M acetate buffer with pH 5.8 and 100 μl of ultrapure water was added to a tube. The samples were mixed, incubated at 95° C. for 15 minutes, and then incubated at room temperature for 5 minutes. 500 μl of 0.05% TNBS solution was added to every sample. The samples were mixed, incubated in the dark at room temperature for 60 minutes. Once the incubation was completed, the sample absorbance was measured at the wavelength of 500 nm. 300 μl of 0.6 M of acetate buffer with pH 5.8 mixed with 200 μl of ultrapure water was used as a blank sample. On the basis of these determinations, the content of aldehyde groups of 480.3 μmol/1 g PAD was determined.

[0066] 1.2. Reaction of Glu-CO-Lys(OBu.sup.t).sub.3NH.sub.2 with the Linker PEGs

##STR00007##

[0067] 10.40 mg (0.0205 mmol) of the linker (compound 1) was dissolved in 0.5 ml of anhydrous methylene chloride. Afterwards, 10.00 mg (0.0205 mmol) of α,α-urea of glutamic acid and lysine in the form of tert-butyl triesters (compound 2) and 4 μl of DIPEA were added. The reaction was carried out for 24 h at room temperature. After that time, 150 μl of TFA was added, and stirring was continued over the next 24 h at room temperature. The solvent was evaporated, the oily residue was dissolved in 0.5 ml of ultrapure water, and then alkalised with a 5M sodium hydroxide solution to pH>11 against a universal indicator paper. Thus prepared aqueous solution of linker-modified GuL (compound 5) was used for the next stage of the synthesis without purification.

[0068] 1.3. Formation of Dextran Nanoparticles with Attached Targeting Agent Glu-CO-Lys.

##STR00008##

[0069] 427 mg of PAD (containing 205.1 μmol CHO) was dissolved in 4.3 ml of ultrapure water to give a 10% (w/v) solution. The aqueous solution of linker-modified Glu-CO-Lys (compound 5) was added to this mixture. In thus prepared reaction mixture, a 0.5M NaOH solution was used to bring the pH to 11.00, and the mixture was stirred at 30° C. for 60 minutes, resulting in modified polyaldehyde dextran (compound 6). After this time, 2.27 ml of a 2% (w/v) ultrapure water solution of 1,10-diaminodecane dihydrochloride was added, and the reaction mixture thus obtained was stirred at 30° C. for 10 minutes, with pH controlled and adjusted to 10 every 20 minutes. After the end of the reaction, a 0.5M HCl solution was used to bring the pH to 7.4. Afterwards, 1.60 ml of a 1% (w/v) ethanol solution of sodium borohydride was added. The reduction reaction was carried out at 37° C. for 60 minutes. After the end of the reaction, the pH was brought to 7.4 with a 0.5M HCl solution. The final product 8 was purified by dialysis in one hundred-fold volume of the ultrapure water for 48 h, with water changed six times. Water was removed from thus purified nanoparticles by lyophilisation.

[0070] 1.4. DOTA Chelator Attachment to Nanoparticles Containing the GuL Targeting Agent

##STR00009##

[0071] 100 mg of nanoparticles lyophilisate (compound 8) was dissolved in 2.0 ml of 0.1M phosphate buffer of pH 8.0. Afterwards, 0.5 ml of DOTA-NHS suspension in ultrapure water, containing 18.5 mg of the chelator, was added. Thus prepared reaction mixture was stirred at room temperature for 90 minutes. The product was purified by dialysis against one hundred-fold volume of 10 mM acetate buffer solution with pH of 5.0 for 48 hours, with the buffer solution changed six times. Water was removed from thus purified nanoparticles (compound 9) by lyophilisation.

EXAMPLE 2

[0072] Preparation of Nanoparticles with 30% Substitution of Aldehyde Groups with the GuL Targeting Agent at 70% Substitution with the DAD Folding Agent (BCS290)

[0073] 2.1. Oxidation of Dextran to Polyaldehyde Dextran (PAD)

[0074] Dextran Oxidation Reaction:

[0075] 5.00 g of dextran was dissolved in 100 ml ultrapure water. 0.66 g sodium periodate was added. The oxidation reaction was continued overnight in the dark at room temperature. The product was purified through dialysis for 72 hours in one hundred-fold volume of ultrapure water, with the water changed at least twice. The water was removed by evaporation at 40° C.

[0076] Determination of Aldehyde Groups in PAD:

[0077] 100 μl of 0.8 mM hydroxylamine hydrochloride solution, 300 μl of 0.6 M acetate buffer with pH of 5.8 and 20-100 μl of PAD were added to a 2 ml tube, and then ultrapure water (0-80 μl) was added up to a total volume of 500 μl. The assay was conducted for three different PAD volumes (20, 60 and 100 μl). A control sample was prepared: 100 μl of 0.8 mM hydroxylamine hydrochloride solution, 300 μl of 0.6 M acetate buffer with pH of 5.8 and 100 μl of ultrapure water were added to a tube. The samples were mixed, incubated at 95° C. for 15 minutes, and then incubated at room temperature for 5 minutes. 500 μl of 0.05% TNBS solution was added to every sample. The samples were mixed, incubated in the dark at room temperature for 60 minutes. Once the incubation was completed, the sample absorbance was measured at wavelength of 50) nm. 300 μl of 0.6 M acetate buffer of pH 5.8 mixed with 200 μl of ultrapure water was used as a blank sample. Such assays determined a content of aldehyde groups of 508.1 μmol/1 g PAD.

[0078] 2.2. Reaction of Glu-CO-Lys(OBu.sup.t).sub.3NH.sub.2 with the Linker PEGs.

##STR00010##

[0079] 15.50 mg (0.0307 mmol) of the linker (compound 1) was dissolved in 0.75 ml of anhydrous methylene chloride. Afterwards, 15.00 mg (0.0307 mmol) α,α-urea of glutamic acid and lysine in the form of tert-butyl triesters (compound 2) and 6 μl of DIPEA were added. The reaction was carried out for 24 h at room temperature. After that time, 234 μl of TFA was added, and stirring was continued over the next 24 h at room temperature. The solvent was evaporated, the oily residue was dissolved in 0.75 ml of ultrapure water, and then alkalised using 5M sodium hydroxide solution to pH>11 against a universal indicator paper. Thus prepared aqueous solution of linker-modified GuL (compound 5) was used for the next stage of the synthesis without purification.

[0080] 2.3. Formation of Dextran Nanoparticles with Attached Targeting Agent GuL.

##STR00011##

[0081] 200 mg of PAD (containing 101.6 μmol CHO) was dissolved in 2.0 ml of ultrapure water to give a 10% (w/v) solution. The aqueous solution of linker-modified GuL (compound 5) was added to that mixture. In thus prepared reaction mixture, a 0.5M NaOH solution was used to bring the pH to 11.00, and the mixture was stirred at 30° C. for 60 minutes, resulting in modified polyaldehyde dextran (compound 6). After this time, 0.87 ml of 2% (w/v) ultrapure water solution of 1,10-diaminodecane dihydrochloride was added, and thus obtained reaction mixture was stirred at 30° C. for 10 minutes, with pH controlled and adjusted to 10 every 20 minutes. After the end of the reaction, 0.5M HCl solution was used to bring the pH to 7.4. Afterwards, 0.88 ml of 1% (w/v) ethanol solution of sodium borohydride was added. The reduction reaction was carried out at 37° C. for 60 minutes. After the end of the reaction, the pH was brought to 7.4 using 0.5M HCl solution. The final product 8 was purified by dialysis in one hundred-fold volume of the ultrapure water for 48 h, with water changed six times. Water was removed from thus purified nanoparticles by lyophilisation.

[0082] 2.4. DOTA Chelator Attachment to Nanoparticles Containing the GuL Targeting Agent

##STR00012##

[0083] 100 mg of nanoparticles lyophilisate (compound 8) was dissolved in 2.0 ml of 0.1M phosphate buffer of pH 8.0. Afterwards, 0.5 ml of DOTA-NHS suspension in ultrapure water, containing 18.5 mg of chelator was added. Thus prepared reaction mixture was stirred at room temperature for 90 minutes. The product was purified through dialysis against one hundred-fold volume of 10 mM acetate buffer with pH of 5.0 for 48 hours, with the buffer solution changed six times. Water was removed from thus purified nanoparticles (compound 9) by lyophilisation.

EXAMPLE 3

[0084] Obtaining Nanoparticles with 5% Aldehyde Group Substitution with the GuL Targeting Agent at 95% Substitution with the DAD Folding Agent (BCS 318)

[0085] 3.1. Oxidation of Dextran to Polyaldehyde Dextran (PAD)

[0086] Dextran Oxidation Reaction:

[0087] 5.00 g of dextran was dissolved in 100 ml ultrapure water. 0.66 g sodium periodate was added. The oxidation reaction was continued overnight in the dark at room temperature. The product was purified through dialysis for 72 hours in one hundred-fold volume of ultrapure water, with the water changed at least twice. The water was removed by evaporation at 40° C.

[0088] Determination of Aldehyde Groups in PAD:

[0089] 100 μl of 0.8 mM hydroxylamine hydrochloride solution, 300 μl of 0.6 M acetate buffer with pH of 5.8 and 20-100 μl of PAD were added to a 2 ml tube, and then ultrapure water (0-80 μl) was added up to total volume 500 μl. The assay was conducted for three different PAD volumes (20, 60 and 100 μl). A control sample was prepared: 100 μl of 0.8 mM hydroxylamine hydrochloride solution, 300 μl of 0.6 M acetate buffer with pH of 5.8 and 100 μl of ultrapure water were added to a tube. The samples were mixed, incubated at 95° C. for 15 minutes, and then incubated at room temperature for 5 minutes. 500 μl of a 0.05% TNBS solution was added to every sample. The samples were mixed, incubated in the dark at room temperature for 60 minutes. Once the incubation was completed, the sample absorbance was measured at the wavelength of 500 nm. 300 μl of 0.6 M of acetate buffer with pH 5.8 mixed with 200 μl of ultrapure water was used as a blank sample. Such assays determined a content of aldehyde groups of 480.3 μmol/1 g PAD.

[0090] 3.2. Reaction of Glu-CO-Lys(OBu.sup.t).sub.3NH.sub.2 with the Linker PEG.sub.5.

##STR00013##

[0091] 10.40 mg (0.0205 mmol) of the linker (compound 1) was dissolved in 0.5 ml of anhydrous methylene chloride. Afterwards, 10.00 mg (0.0205 mmol) of α,α-urea of glutamic acid and lysine in the form of tert-butyl triesters (compound 2) and 4 μl of DIPEA were added. The reaction was carried out for 24 h at room temperature. After that time, 150 μl of TFA was added, and the mixing was continued over the next 24 h at room temperature. The solvent was evaporated, the oily residue was dissolved in 0.5 ml of ultrapure water, and then alkalised using 5M sodium hydroxide solution to pH>11 against a universal indicator paper. Thus prepared aqueous solution of linker-modified GuL (compound 5) was used for the next stage of the synthesis without purification.

[0092] 3.3. Formation of Dextran Nanoparticles with Attached Targeting Agent Glu-CO-Lys.

##STR00014##

[0093] 854 mg of PAD (comprising 410.2 μmol CHO) was dissolved in 8.54 ml of ultrapure water to obtain a 10% (w/v) solution. The aqueous solution of linker-modified GuL (compound 5) was added to that mixture. In thus prepared reaction mixture, 0,5M NaOH solution was used to establish pH of 11.00, and the mixture was stirred at 30° C. for 60 minutes, resulting in modified polyaldehyde dextran (compound 6). After that time, 4.78 ml of 2% (w/v) ultrapure water solution of 1,10-diaminodecane dihydrochloride was added, and thus obtained reaction mixture was stirred at 30° C. for 10 minutes, with pH controlled and adjusted to 10 every 20 minutes. After the end of the reaction, 0.5M HCl solution was used to bring the pH to 7.4. Afterwards, 3.18 ml of 1% (w/v) ethanol solution of sodium borohydride was added. The reduction reaction was carried out at 37° C. for 60 minutes. After the end of the reaction, the pH was brought to 7.4 with 0.5M HCl solution. The final product 8 was purified by dialysis in one hundred-fold volume of ultrapure water for 48 h, with water changed six times. Water was removed from thus purified nanoparticles by lyophilisation.

[0094] 3.4. DOTA Chelator Attachment to Nanoparticles Containing the GuL Targeting Agent

##STR00015##

[0095] 100 mg of nanoparticles lyophilisate (compound 8) was dissolved in 2.0 ml of 0.1M phosphate buffer of 8.0. Afterwards, 0.5 ml of DOTA-NHS suspension in ultrapure water, containing 18.5 mg of the chelator, was added. Thus prepared reaction mixture was stirred at room temperature for 90 minutes. The product was purified through dialysis against one hundred-fold volume of 10 mM acetate buffer with pH of 5.0 for 48 hours, with the buffer solution changed six times. Water was removed from thus purified nanoparticles (compound 9) by lyophilisation.

EXAMPLE 4

[0096] Obtaining Nanoparticles with 2.5% Aldehyde Group Substitution with the GuL Targeting Agent at 97.5% Substitution with the DAD Folding Agent (BCS 319)

[0097] 4.1. Oxidation of Dextran to Polyaldehyde Dextran (PAD)

[0098] Dextran Oxidation Reaction:

[0099] 5.00 g of dextran was dissolved in 100 ml ultrapure water. 0.66 g sodium periodate was added. The oxidation reaction was continued overnight in the dark at room temperature. The product was purified through dialysis for 72 hours in one hundred-fold volume of ultrapure water, with the water changed at least twice. The water was removed by evaporation at 40° C.

[0100] Determination of Aldehyde Groups in PAD:

[0101] 100 μl of 0.8 mM hydroxylamine hydrochloride solution, 300 μl of 0.6 M acetate buffer with pH of 5.8 and 20-100 μl of PAD were added to a 2 ml tube, and then ultrapure water (0-80 μl) was added up to a total volume 500 μl. The assay was conducted for three different PAD volumes (20, 60 and 100 μl). A control sample was prepared: 100 μl of 0.8 mM hydroxylamine hydrochloride solution, 300 μl of 0.6 M acetate buffer with pH of 5.8 and 100 μl of ultrapure water were added to a tube. The samples were mixed, incubated at 95° C. for 15 minutes, and then incubated at room temperature for 5 minutes. 500 μl of 0.05% TNBS solution was added to every sample. The samples were mixed, incubated in the dark at room temperature for 60 minutes. Once the incubation was completed, the sample absorbance was measured at wavelength of 500 nm. 300 μl of 0.6 M of acetate buffer with pH 5.8 mixed with 200 μl of ultrapure water was used as the blank sample. Such assays determined a content of aldehyde groups of 480.3 μmol/1 g PAD.

[0102] 4.2. Reaction of Glu-CO-Lys(OBu.sup.t).sub.3NH.sub.2 with the Linker PEGs.

##STR00016##

[0103] 5.20 mg (0.01025 mmol) of the linker (compound 1) was dissolved in 0.25 ml of anhydrous methylene chloride. Afterwards, 5.00 mg (0.01025 mmol) of α,α-urea of glutamic acid and lysine in the form of tert-butyl triesters (compound 2) and 2 μl of DIPEA were added. The reaction was carried out for 24 h at room temperature. After that time, 75 μl of TFA was added, and the mixing was continued over the next 24 h at room temperature. The solvent was evaporated, the oily residue was dissolved in 0.25 ml of ultrapure water and then alkalised using 5M sodium hydroxide solution to pH>11 against a universal indicator paper. Thus prepared aqueous solution of linker-modified GuL (compound 5) was used for the next stage of the synthesis without purification.

[0104] 4.3. Formation of Dextran Nanoparticles with Attached Targeting Agent Glu-CO-Lys.

##STR00017##

[0105] 854 mg of PAD (containing 410.2 μmol CHO) was dissolved in 8.54 ml of ultrapure water to obtain a 10% (w/v) solution. The aqueous solution of linker-modified GuL (compound 5) was added to that mixture. In such prepared reaction mixture, 0.5M NaOH solution was used to establish pH of 11.00, and the mixture was stirred at 30° C. for 60 minutes, resulting in modified polyaldehyde dextran (compound 6). After that time, 4.90 ml of 2% (w/v) ultrapure water solution of 1,10-diaminodecane dihydrochloride was added, and thus obtained reaction mixture was stirred at 30° C. for 10 minutes, with pH controlled and adjusted to 10 every 20 minutes. After the end of the reaction, a 0.5M HCl solution was used to bring the pH to 7.4. Afterwards, 3.14 ml of 1% (w/v) ethanol solution of sodium borohydride was added. The reduction reaction was carried out at 37° C. for 60 minutes. After the end of the reaction, the pH was brought to 7.4 using 0.5M HCl solution. The final product 8 was purified by dialysis in one hundred-fold volume of the ultrapure water for 48 h, with water changed six times. Water was removed from thus purified nanoparticles by lyophilisation.

[0106] 4.4. DOTA Chelator Attachment to Nanoparticles Containing the GuL Targeting Agent

##STR00018##

[0107] 100 mg of nanoparticles lyophilisate (compound 8) was dissolved in 2.0 ml of 0.1M phosphate buffer of pH 8.0. Afterwards, 0.5 ml of DOTA-NHS suspension in ultrapure water, containing 18.5 mg of the chelator, was added. Thus prepared reaction mixture was stirred at room temperature for 90 minutes. The product was purified through dialysis against one hundred-fold volume of 10 mM acetate buffer with pH of 5.0 for 48 hours, with the buffer solution changed six times. Water was removed from thus purified nanoparticles (compound 9) by lyophilisation.

EXAMPLE 5

[0108] Producing Nanoparticles with 1% Aldehyde Group Substitution with the GuL Targeting Agent at 99% Substitution with the DAD Folding Agent

[0109] 5.1. Oxidation of Dextran to Polyaldehyde Dextran (PAD)

[0110] Dextran Oxidation Reaction:

[0111] 5.00 g of dextran was dissolved in 100 ml ultrapure water. 0.66 g sodium periodate was added. The oxidation reaction was continued overnight in the dark at room temperature. The product was purified through dialysis for 72 hours in one hundred-fold volume of ultrapure water, with the water changed at least twice. The water was removed by evaporation at 40° C.

[0112] Determination of Aldehyde Groups in PAD:

[0113] 100 μl of 0.8 mM hydroxylamine hydrochloride solution, 300 μl of 0.6 M acetate buffer with pH of 5.8 and 20-100 μl of PAD were added to a 2 ml tube, and then ultrapure water (0-80 μl) was added up to total volume 500 μl. The assay was conducted for three different PAD volumes (20, 60 and 100 μl). A control sample was prepared: 100 μl of 0.8 mM hydroxylamine hydrochloride solution, 300 μl of 0.6 M acetate buffer with pH of 5.8 and 100 μl of ultrapure water was added to a tube. The samples were mixed, incubated at 95° C. for 15 minutes, and then incubated at room temperature for 5 minutes. 500 μl of 0.05% TNBS solution was added to every sample. The samples were mixed, incubated in the dark at room temperature for 60 minutes. Once the incubation was completed, the sample absorbance was measured at the wavelength of 500 nm. 300 μl of 0.6 M of acetate buffer with pH 5.8 mixed with 200 μl of ultrapure water was used as a blank sample. Such assays determined a content of aldehyde groups of 480.3 μmol/1 g PAD.

[0114] 5.2. Reaction of Glu-CO-Lys(OBu.sup.t).sub.3NH.sub.2 with the Linker PEGs.

##STR00019##

[0115] 5.20 mg (0.01025 mmol) of the linker (compound 1) was dissolved in 0.25 ml of anhydrous methylene chloride. Afterwards, 5.00 mg (0.01025 mmol) of α,α-urea of glutamic acid and lysine in the form of tert-butyl triesters (compound 2) and 2 μl of DIPEA was added. The reaction was carried out for 24 h at room temperature. After that time, 75 μl of TFA was added, and the mixing was continued over the next 24 h at room temperature. The solvent was evaporated, the oily residue was dissolved in 0.25 ml of ultrapure water, and then alkalised using 5M sodium hydroxide solution to pH>11 against a universal indicator paper. Thus prepared aqueous solution of linker-modified GuL (compound 5) was used for the next stage of the synthesis without purification.

[0116] 5.3. Formation of Dextran Nanoparticles with Attached Targeting Agent Glu-CO-Lys.

##STR00020##

[0117] 2135 mg of PAD (containing 1025.5 μmol CHO) was dissolved in 21.35 ml of ultrapure water to obtain a 10% (w/v) solution. The aqueous solution of linker-modified GuL (compound 5) was added to that mixture. In thus prepared reaction mixture, 0.5M NaOH solution was used to bring the pH to 11.00, and the mixture was stirred at 30° C. for 60 minutes, resulting in modified polyaldehyde dextran (compound 6). After that time, 12.45 ml of 2% (w/v) ultrapure water solution of 1,10-diaminodecane dihydrochloride was added, and thus obtained reaction mixture was stirred at 30° C. for 10 minutes, with pH controlled and adjusted to 10 every 20 minutes. After the end of the reaction, a 0.5M HCl solution was used to bring the pH to 7.4. Afterwards, 8.84 ml of 1% (w/v) ethanol solution of sodium borohydride was added. The reduction reaction was carried out at 37° C. for 60 minutes. After the end of the reaction, the pH was brought to 7.4 using 0.5M HCl solution. The final product 8 was purified by dialysis in one hundred-fold volume of ultrapure water for 48 h, with water changed six times. Water was removed from thus purified nanoparticles by lyophilisation.

[0118] 5.4. DOTA Chelator Attachment to Nanoparticles Containing the GuL Targeting Agent

##STR00021##

[0119] 100 mg of nanoparticles lyophilisate (compound 8) was dissolved in 2.0 ml of 0.1M phosphate buffer of pH 8.0. Afterwards, 0.5 ml of DOTA-NHS suspension in ultrapure water, containing 18.5 mg of the chelator, was added. Thus prepared reaction mixture was stirred at room temperature for 90 minutes. The product was purified through dialysis against one hundred-fold volume of 10 mM acetate buffer with pH of 5.0 for 48 hours, with the buffer solution changed six times. Water was removed from thus purified nanoparticles (compound 9) by lyophilisation.

EXAMPLE 6

[0120] Inhibition of PSMA Receptor by Nanoparticles with Attached GuL Targeting Agent

[0121] A specificity study of nanoparticles with attached GuL targeting agent embedded on the linker towards the PSMA receptor was performed. An enzymatic in vitro assay was conducted to investigate the decrease in the PSMA activity caused by the blocking of the PSMA active site by the GuL. The study was conducted for the following nanoparticles: [0122] BCS 0277-10% substitution of aldehyde groups with the GuL targeting agent [0123] BCS 0290-30% substitution of aldehyde groups with the GuL targeting agent [0124] BCS 0319-2.5% substitution of aldehyde groups with the GuL targeting agent

[0125] for various concentrations of nanoparticles solution used for the analysis, i.e. 16 μg, 4 μg, 1.6 μg, 0.4 μg, 0.16 μg.

[0126] The results are presented in FIG. 1, illustrating the fluorescence drop which reflects the decrease in the enzyme activity. In this way the PSMA inhibition by nanoparticles with an attached GuL targeting agent was established.

[0127] The tests have shown that the greater the binding of nanoparticles (GuL content), the lower the fluorescence representing the PSMA enzyme activity. The tendency confirming an increasing amount of bound GuL targeting agent for 30%, 10% as well as 2.5% substitution of the aldehyde groups with the GuL targeting agent was observed. At the same time, the analysis of the results for various values of nanoparticle solution concentrations shows that the presented method permits a quantitative determination of the GuL agent and definition of the minimal nanoparticle concentration required for the inhibition to occur.

[0128] The tests are conclusive in proving that, once attached to the nanoparticle structure, the GuL targeting agent placed on the linker has a high affinity for the PSMA receptor present on the surface of prostate cancer cells.

EXAMPLE 7

[0129] Affinity of the Nanoparticles with a GuL Targeting Agent for the PSMA Receptor

[0130] The nanoparticles with a GuL targeting agent deposited on the linker were tested for affinity to the PSMA receptor through measurement the degree of its binding on the surface of the LNCaP cells (prostate cancer cell line) exhibiting high overexpression of the PSMA receptor.

[0131] The nanoparticles were labelled with radioactive Lutetium and then incubated at 50 μg/ml concentration with LNCaP on a multiwell plate. The nanoparticle binding capacity and internalisation to cells was determined through the measurement of gamma radiation.

[0132] The method is characterised by high sensitivity of the measurement.

[0133] The results for the following nanoparticles are presented: [0134] BCS 0290-30% substitution of aldehyde groups with the GuL targeting agent [0135] BCS 0318-5% substitution of aldehyde groups with the GuL targeting agent [0136] BCS 0319-2.5% substitution of aldehyde groups with the GuL targeting agent

[0137] The results shown in Table 1 suggest that all the tested nanoparticles exhibit high PSMA receptor overexpression. The tests show that nanoparticles with 2.5% to 5% aldehyde group substitution with the GuL targeting agent have a significantly higher level of affinity for the PSMA receptor.

TABLE-US-00001 TABLE 1 Aldehyde Binding Nano- group on the Internali- Complete particles substitution % surface sation binding 290 30% 25.95% 7.52% 33.47% 318  5% 29.96% 2.78% 32.74% 319 2.5%  46.64% 10.40%  57.04%

EXAMPLE 8

[0138] Testing the Significance of the GuL Targeting Agent Linker for the Specificity of Nanoparticle Binding to the PSMA Receptor

[0139] The GuL targeting agent is attached through a linker—a PEG.sub.5 (BocNH-PEG5-NHS) molecule, which is responsible for increasing the access of the targeting agent to the PSMA receptor. Studies have been carried out to confirm the superiority of the GuL-linker molecule on the surface of the nanoparticle over the GuL molecule attached to the nanoparticle without a linker. The results presented in FIG. 2 illustrate PSMA inhibition by nanoparticles with GuL without the linker (408) and with the linker (277) for various quantities of the targeting agent, i.e. 8000 ng, 800 ng, 80 ng and 8 ng.

[0140] On the basis of the performed tests, it was found that the decrease in fluorescence reflects the degree of the nanoparticle binding with the GuL targeting agent to the PSMA receptor protein. The results obtained confirm the specificity of the binding of nanoparticles by the targeting agent attached to the linker. They also indicate that the targeting agent with the linker increases the efficiency of the attachment process and the potency of the obtained nanoparticles in relation to the receptor when compared to a targeting agent without a linker.

Abbreviations

[0141] DOTA—1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [0142] DTPA—pentetic acid [0143] NOTA—1,4,7-triazacyclononane-1,4,7-triacetic acid [0144] DOTA-NHS—1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and N-hydroxysuccinimide monoester [0145] DOTA-buthvlamine—1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-(4-aminobuthyl)acetamide [0146] DOTA-maleimide—1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide [0147] DOTA-SCN—2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid [0148] PET—Positron Emission Tomography [0149] PET/MRI—Positron Emission Tomography and Magnetic Resonance Imaging [0150] NHS—N-hydroxysuccinimide [0151] SulfoNHS—N-hydroxysulfosuccinimide sodium salt [0152] PFP—pentafluorophenol [0153] TFP—2,3,5,6-tetrafluorophenol [0154] STP—2,3,5,6-tetrafluoro-4-hydroxybenzenesulfonic acid sodium salt [0155] SCN—thiocyanate [0156] PAD—polyaldehyde dextran [0157] DAD—diaminodecane [0158] DIPEA—diisopropylethylamine [0159] TFA—trifluoroacetic acid [0160] GuL or Glu-CO-Lys—α,α-urea of glutamic acid and lysine [0161] Glu-CO-Lys(OBu).sub.3NH.sub.2—α,α-urea of glutamic acid and lysine in the form of tert-butyl triesters