Gel formulations for guiding radiotherapy

Abstract

The present invention describes an X-ray contrast composition for local administration, wherein the X-ray contrast composition exhibits contrast properties and wherein at least 60% of an administrated amount of said X-ray contrast composition remains more than 24 hours within 10 cm from an injection point when the X-ray contrast composition is administrated to a human or animal body.

Claims

1. An imaging contrast composition for local administration, wherein the imaging contrast composition exhibits contrast properties and wherein at least 60% of an administrated amount of said imaging contrast composition remains more than 24 hours within 10 cm from an injection point when the imaging contrast composition is administrated to a human or animal body, wherein the imaging contrast composition is a liquid before administration and having the ability to transform into a gel after administration, that increases in viscosity by more than 1,000 centipoise (cP) after administration into a human or animal body, and wherein the imaging contrast composition is an X-ray contrast composition comprising an iodinated derivative of sucrose acetate isobutyrate (SAIB) or an iodinated derivative of sucrose acetate isobutyrate (SAIB) doped into sucrose acetate isobutyrate (SAIB), wherein the structure of the iodinated derivative of sucrose acetate isobutyrate (SAIB) is at least one selected from the following: ##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005## ##STR00006##

2. The imaging contrast composition according to claim 1, wherein the X-ray contrast composition has a viscosity of less than 10,000 centipoise (cP) at 20 C.

3. The imaging contrast composition according to claim 1, wherein the X-ray contrast composition exhibits gel-formation in response to a temperature in the range of 35 to 40 C., in response to hydration, in response to an ion-concentration in the range of 1 M to 500 mM, in response to a pH in the range of 6 to 8 and/or in response to contacting with an initiator.

4. The imaging contrast composition according to claim 1, wherein the X-ray contrast composition also comprises; radioactive compounds, paramagnetic compounds, fluorescent compounds or ferromagnetic compounds, or any mixture thereof, and/or wherein the X-ray contrast composition also comprises at least one pharmaceutical substance.

5. The imaging contrast composition according to claim 1, wherein the iodinated derivate of sucrose acetate isobutyrate (SAIB) is solubilized in a mixture of ethanol and sucrose acetate isobutyrate (SAIB).

6. The imaging contrast composition according to claim 1, wherein the X-ray contrast composition comprises a pharmaceutical substance or particle that contains a pharmaceutical substance.

7. The imaging contrast composition according to claim 1, wherein the iodinated derivate of sucrose acetate isobutyrate (SAIB) is solubilised in a mixture of ethanol and sucrose acetate isobutyrate (SAIB) and contains a pharmaceutical substance or a particle that contains a pharmaceutical substance.

8. The imaging contrast composition according to claim 1, for use in radio therapy, imaging, diagnostics, treatment and/or quality rating of radio therapy, for use as a tissue marker and/or for use as a controlled drug release composition.

9. A kit comprising a syringe, a needle used for injection into a body or surgical related procedures, adapted to an open end of said syringe, and the imaging contrast composition according to claim 1.

10. The kit according to claim 9, wherein one of the surgical related procedures is biopsy.

11. A method of recording an X-ray image of the body of a mammal, comprising the steps of a. providing an X-ray contrast composition comprising an organic X-ray agent in a gel-forming system, wherein the X-ray contrast composition is a liquid before administration and having the ability to transform into a gel after administration, that increases in viscosity by more than 1,000 centipoise (cP) after administration into a human or animal body, and wherein the organic X-ray agent comprises an iodinated derivative of sucrose acetate isobutyrate (SAIB) or an iodinated derivative of sucrose acetate isobutyrate (SAIB) doped into sucrose acetate isobutyrate (SAIB), wherein the structure of the iodinated derivative of sucrose acetate isobutyrate (SAIB) is at least one selected from the following: ##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012## b. administering the X-ray contrast composition to a predetermined location of the mammal, and c. recording X-ray-based images of at least a part of the body of the mammal which comprises the predetermined location.

12. The method according to claim 11, wherein the X-ray contrast composition is parenterally administered to a predetermined location of the body of said mammal, and wherein an X-ray image of at least a part of the body of the mammal including the predetermined location is recorded.

13. A method of joint radiotherapy and X-ray imaging of a target tissue in a mammal, comprising the steps of a. providing an X-ray contrast composition comprising an organic X-ray agent in a gel-forming system, wherein the X-ray contrast composition is a liquid before administration and having the ability to transform into a gel after administration, that increases in viscosity by more than 1,000 centipoise (cP) after administration into a human or animal body, and wherein the organic X-ray agent comprises an iodinated derivative of sucrose acetate isobutyrate (SAIB) or an iodinated derivative of sucrose acetate isobutyrate (SAIB) doped into sucrose acetate isobutyrate (SAIB), wherein the structure of the iodinated derivative of sucrose acetate isobutyrate (SAIB) is at least one selected from the following: ##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018## b. administering the X-ray contrast composition to a predetermined target tissue of the mammal, c. recording X-ray-based images, of at least a part of the body of the mammal which comprises the target tissue, thereby providing a definition of the target tissue, and d. using the definition of the target tissue obtained in c) to direct external beam radiotherapy to the target tissue.

14. A method for directing local administration of a pharmaceutically active agent to a target tissue in a mammal, comprising the steps of a. providing an X-ray contrast composition comprising an organic X-ray agent and an active pharmaceutical agent in a gel-forming system, wherein the X-ray contrast composition is a liquid before administration and having the ability to transform into a gel after administration, that increases in viscosity by more than 1,000 centipoise (cP) after administration into a human or animal body, and wherein the organic X-ray agent comprises an iodinated derivative of sucrose acetate isobutyrate (SAIB) or an iodinated derivative of sucrose acetate isobutyrate (SAIB) doped into sucrose acetate isobutyrate (SAIB), wherein the structure of the iodinated derivative of sucrose acetate isobutyrate (SAIB) is at least one selected from the following: ##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024## b. administering the X-ray contrast composition to a predetermined target tissue of the mammal, c. recording X-ray-based images, of at least a part of the body of the mammal which comprises the target tissue, thereby providing a definition of the target tissue, and d. using the X-ray contrast composition in b) for delivery of the active pharmaceutical agent to a predetermined target tissue of the mammal.

15. The method according to any one of claims 13 and 14, wherein the target tissue comprises tumor cells.

16. The method according to any one of claims 11 to 14, wherein the X-ray contrast composition comprises the feature of exhibiting contrast properties and wherein at least 60% of an administrated amount of the X-ray contrast composition remains more than 24 hours within 10 cm from an injection point when the X-ray contrast composition is administrated to the mammal.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Illustrates various mechanisms of gel-formation including thermo-, ion-, pH-, enzymatically-, initiator- and hydration responsive gel-forming systems.

(2) FIG. 2. Illustrates various thermo responsive gel-forming systems which can exhibit an inverse sol-gel transition.

(3) FIG. 3. Illustrates various ion sensitive gel-forming systems which form gels in high salt concentration.

(4) FIG. 4. Illustrates various pH sensitive gel-forming systems which form hydrogels at specific pH intervals.

(5) FIG. 5. Illustrates various enzymatically sensitive gel-forming systems which form hydrogels in presence of specific enzymes.

(6) FIG. 6. Illustrates the use of sucrose acetate isobutyrate (SAIB) as a hydration sensitive gel-forming system. SAIB dissolved in organic solvent such as ethanol have a low viscosity suitable for injection trough thin needles. Upon hydration the ethanol diffuses out of the matrix resulting in a highly viscous hydrophobic gel suitable for encapsulation of contrast agents.

(7) FIG. 7. Illustrates various iodo-SAIB derivates which may be used for x-ray attenuation.

(8) FIG. 8. Illustrates a synthetic scheme for the synthesis of 2-(2,4,6-triiodophenoxy)acetic acid (3)

(9) FIG. 9. Illustrates a synthetic scheme for the synthesis of 6,6-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)

(10) FIG. 10. Illustrates CT-contrast of iodinated gels with 10-, 25-, or 50 w % (8) ((w % is the weight of the atom/molecule (in this case iodine) divided by the total weight of the material times 100)) and a negative control containing MQ-H.sub.2O were visualized in a clinical CT-scanner at different energies; 80-, 100-, 120- and 140 kV, all 200 mAs, 2 mm (col 400.6 mm).

(11) FIG. 11. Illustrates AuNP synthesis and characterization. A) Synthetic scheme for the synthesis of PNIPAM-coated AuNPs using a seeding approach; B) AuNP characterization by UV-Vis; C) AuNP characterization by DLS; D) AuNP characterization by -potential.

(12) FIG. 12. Illustrates the enhanced stability of PNIPAM coated AuNPs. A) UV-Vis of PNIPAM coated AuNPs before (stock)/after lyophilization and re-suspension in anhydrous EtOH (concentration of AuNP in the range of 1.0-5.0 mg Au/mL); B) DLS of PNIPAM coated AuNPs before (stock)/after lyophilization and re-suspension in anhydrous EtOH (concentration of AuNP in the range of 1.0-5.0 mg Au/mL).

(13) FIG. 13. Illustrates the accumulative release of PNIPAM.sub.3500- and PEG.sub.5000 coated AuNPs from gels composed of SAIB/EtOH/PLA (75:20:5)+3.0 w % PNIPAM.sub.3500 or PEG.sub.5000 coated AuNPs.

(14) FIG. 14. Illustrates a ultrasonography image of Formulation B (SAIB/8/EtOH (55:25:20)) (250 L) in vitro. Gel present at the bottom of a glass beaker under water.

(15) FIG. 15. Illustrates MicroCT images of Formulation B (SAIB/8/EtOH (55:25:20)) (200 L) administered by subcutaneous injection to healthy NMRI mice. A) CT-image recorded 24 h p.i.; B) CT-image recorded 48 p.i.

(16) FIG. 16. A) MicroCT image of SAIB/8/EtOH (65:15:20) injected s.q. in immunocompetent mice; B) MicroCT image of SAIB/8/EtOH (50:30:20) injected s.q. in immunocompetent mice; C) Ex vivo visualization of SAIB/8/EtOH (50:30:20) present in the s.q. compartment 14 w p.i. and D) Gel implants composed of SAIB/8/EtOH (50:30:20) removed after 14 w implantation in immunocompetent mice.

(17) FIG. 17. A) Series of MicroCT images of SAIB/8/EtOH (50:30:20) injected s.q. in mice. MicroCT scans recorded with short time intervals to monitor the gelation kinetics of the iododinated gel; B) Gelation kinetics of SAIB/8/EtOH (50:30:20) (50 L) implanted s.q. in immunocompetent mice and C) 14 w degradation profiles of iododinated gels composed of SAIB/8/EtOH (65:15:20) or SAIB/8/EtOH (50:30:20) after s.q. implantation (50 L).

(18) FIG. 18. Illustrates a CT-image of Formulation B (SAIB/8/EtOH (55:25:20)) administrated intratumoral to a companion dog (American Staffordshire terrier, 9 years, 34 kg) with a mast cell tumor present between the front legs.

EXAMPLES

Example 1

Iodo-SAIB Gel Formation and CT-Contrast In Vitro

(19) Materials

(20) Chemicals were purchased from Sigma-Aldrich Inc. (Brndby, Denmark) unless otherwise stated. 2-(2,4,6-triiodophenoxy)acetic acid (3) and 6,6-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8) was synthesized in two and four steps, respectively, as outlined in FIG. 7 and FIG. 8.

(21) Synthesis

2-(2,4,6-triiodophenoxy)acetic acid (3)

(22) 2,4,6-triiodophenol (1) (10.00 g, 21.2 mmol) was dissolved in dry DMF (75 mL) under N.sub.2-atmosphere. To this solution, tert-butyl bromoacetate (4.20 mL, 28.46 mmol) and K.sub.2CO.sub.3 (8.79 g, 63.6 mmol) were added and the stirred overnight at rt. The solvent was removed in vacou and the remaining yellow oil re-dissolved in EtOAc (150 mL) and washed with MQ-H.sub.2O (3150 mL). The organic phase was dried with MgSO.sub.4, filtrated and concentrated in vacou to give tert-butyl 2-(2,4,6-triiodophenoxy)acetate (2) as a light yellow oil which was used in the next step without further purification. 2 was dissolved in CH.sub.2Cl.sub.2 (60 mL) and trifluoroacetic acid (30 mL) was added. The mixture stirred for 1 h at rt after which the solvent was removed in vacou to give a white solid. The crude product was re-crystallized from EtOH to give 2-(2,4,6-triiodophenoxy)acetic acid (3) as fine white needles (9.58 g, 85% (2 steps)). .sup.1H-NMR (300 MHz, MeOD): 6.58 (s, 2H), 2.95 (s, 2H). MALDI-TOF MS (DHB+Na): Chemical Formula: C.sub.8H.sub.5I.sub.3NaO.sub.3, calculated mass; 552.83. found: 553.08 (M+Na.sup.+).

6,6-TBDPS-Sucrose (5)

(23) Sucrose (4) (3.00 g, 8.76 mmol) was dissolved in dry pyridine (54.0 mL) under N.sub.2-atmosphere. To this solution tert-butyldiphenylchlorosilane (TBDPS-Cl) (2.51 mL, 9.64 mmol) and a catalytic amount of DMAP (107.5 mg, 0.88 mmol) were added and the mixture heated at 70 C. for 3 h. After cooling to rt, TBDPS-Cl (2.51 mL, 9.64 mmol) was added and the mixture stirred overnight at rt. The solvent was removed in vacou and the crude product purified by flash chromatography using a stepwise gradient starting from; i) EtOAc, ii) EtOAc/Acetone/H.sub.2O (100:100:1) and iii) EtOAc/Acetone/H.sub.2O (10:10:1) as eluent to give 6,6-TBDPS-Sucrose (5) as a white solid (4.66 g, 65%). R.sub.f=0.40 (EtOAc/Acetone/H.sub.2O (100:100:1)). MALDI-TOF MS (DHB+Na): Chemical Formula: C.sub.44H.sub.57NaO.sub.11Si.sub.2, calculated mass; 841.08. found: 841.81 (M+Na.sup.+).

6,6-TBDPS-isobutyric-Sucrose (6)

(24) 6,6-TBDPS-Sucrose (5) (3.00 g, 3.66 mmol) was dissolved in dry pyridine (45.0 mL) under N.sub.2-atmosphere. To this solution isobutyric anhydride (15.00 mL, 90.4 mmol) was added and the mixture stirred at rt overnight. Additional isobutyric anhydride (5.0 mL, 15.06 mmol) and a catalytic amount of 4-dimethylaminopyridine (DMAP) (50 mg, 0.41 mmol) were added and the mixture heated to 70 C. for 6 h. The solvent was removed in vacou and the crude product purified by flash chromatography using hexane:EtOAc (5:1) as eluent to give 6,6-TBDPS-isobutyric-Sucrose (6) as clear viscous oil (4.54 g, quantitative). R.sub.f=0.48 (hexane:EtOAc (5:1). MALDI-TOF MS (DHB+Na): Chemical Formula: C.sub.68H.sub.94NaO.sub.17Si.sub.2, calculated mass; 1262.62. found: 1262.22 (M+Na.sup.+).

6,6-OH-isobutyric-Sucrose (7)

(25) 6,6-TBDPS-isobutyric-Sucrose (6) (217.2 g, 0.175 mmol) was dissolved in THF (940 mL) and stirred at RT. Glacial acetic acid (42.1 g, 0.701 mol) was added to the flask followed by addition of tetrabutyl-ammonium fluoride trihydrate (TBAF.3H.sub.2O) (221.1 g, 0.701 mol) in THF (692 mL). The solution was stirred at RT for 15 h after which heptanes (2085 mL) and phosphate buffer (0.5M, 211 mL) (H.sub.2KPO.sub.4 (177.2 g) and HK.sub.2PO.sub.4 (343.3 g) in MQ-H.sub.2O (6544 mL)), pH 7.0) was added. The organic phase was collected and washed with additionally two portions of phosphate buffer (0.5M, 2111 mL). The crude product purified by flash chromatography using a gradient starting from hexanes:EtOAc (7:3) then hexanes:EtOAc (6:4) as eluent to give 6,6-OH-isobutyric-Sucrose (7) as clear viscous oil (106.1 g, 79%). R.sub.f=0.21 (hexane:EtOAc (3:1). .sup.1H-NMR (300 MHz, DMSO-d.sub.6): 5.75 (d, J=6.1 Hz, 1H), 5.50 (d, J=3.6 Hz, 1H), 5.40 (d, J=7.7 Hz, 1H), 5.31 (t, J=7.4 Hz, 1H), 5.18 (t, J=9.8 Hz, 1H), 4.87 (t, J=5.5 Hz, 1H), 4.70 (dd, J=10.4, 3.7 Hz, 1H), 4.29 (d, J=11.9 Hz, 1H), 4.11 (dd, J=12.0, 5.5 Hz, 1H), 3.69-3.44 (m, 4H), 2.64-2.49 (m, 6H), 1.13-0.96 (m, 36H). MALDI-TOF MS (DHB+Na): Chemical Formula: C.sub.36H.sub.58NaO.sub.17, calculated mass; 785.83. found: 785.82 (M+Na.sup.+).

6,6-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8)

(26) 6,6-OH-isobutyric-Sucrose (7) (800 mg, 1.05 mmol) was dissolved in dry DMF (10.0 mL) under N.sub.2-atmosphere. To this solution a pre-mixed mixture of 2-(2,4,6-triiodophenoxy)acetic acid (3) (1.67 g, 3.15 mmol), EDC.HCl (622 mg, 3.15 mmol) and DMAP (769 mg, 6.29 mmol) in dry DMF (10.0 mL) were added and the reaction stirred at rt overnight. The solvent was removed in vacou and the remaining yellow oil re-dissolved in CH.sub.2Cl.sub.2 (40 mL) and washed with MQ-H.sub.2O (340 mL). Organic phase was dried with MgSO.sub.4, filtrated and reduced in vacou to give light yellow oil. Final purification was achieved by flash chromatography using hexane:EtOAc (5:1) as eluent to give 6,6-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8) as white foamy solid (1.56 g, 83%). R.sub.f=0.31 (hexane:EtOAc (5:1). .sup.1H-NMR (300 MHz, MeOD): 8.05 (s, 2H), 8.04 (s, 2H), 5.68 (d, J=3.7 Hz, 1H), 5.56 (d, J=7.3 Hz, 1H), 5.54-5.48 (m, 1H), 5.43 (t, J=7.2 Hz, 1H), 5.37 (t, J=9.8 Hz, 1H), 5.03 (dd, J=10.2, 3.7 Hz, 1H), 4.70-4.06 (m, 12H), 2.73-2.45 (m, 6H), 1.36-1.04 (m, 36H). MALDI-TOF MS (DHB+Na): Chemical Formula: C.sub.52H.sub.64I.sub.6NaO.sub.21, calculated mass; 1809.47. found: 1809.59 (M+Na.sup.+).

(27) Gel Preparation

(28) Three sucrose acetate isobutyrate (SAIB)-based formulations (600 mg each) with increasing amounts of 6,6-(2,4,6-triiodophenoxy)acetoxy-isobutyric-sucrose were prepared as outlined in the table below.

(29) TABLE-US-00001 6,6-(2,4,6-triiodophenoxy)- Formulation SAIB acetoxy-isobutyric-sucrose (8) EtOH A 420 mg 60 mg 120 mg SAIB/8/EtOH (70:10:20) B 330 mg 150 mg 120 mg SAIB/8/EtOH (55:25:20) C 180 mg 300 mg 120 mg SAIB/8/EtOH (30:50:20)

(30) SAIB-solution (90 w/w in EtOH) was weighted off and mixed with 8 and anhydrous EtOH (see table above). The mixtures were homogenized on a ball-mill homogenizer for 60 min (30 s.sup.1) and centrifuged for 20 s at 5000 RPM to remove air bubbles from the formulations. All formulations were homogenous clear solutions with increasing viscosity as a function of the concentration of 8all injectable trough 25 G hypodermic needles.

(31) Iodinated gels (500 L) from formulation A-C were prepared by injection into MQ-H.sub.2O (5.0 mL) containing plastic vials at 37 C. The aqueous solutions were replaced three times and the gels stored at 37 C. for 12 days prior to CT-visualization and HU-contrast measurements in a clinical CT-scanner.

(32) CT-Contrast of Iodinated Gels In Vitro

(33) The three formed iodinated gels with 10-, 25-, or 50 w % 8 and a negative control containing MQ-H.sub.2O were visualized in a clinical CT-scanner at different energies; 80-, 100-, 120- and 140 kV, all 200 mAs, 2 mm (col 400.6 mm). The obtained contrast in Hounsfield unit (HU) plotted as a function of energy is illustrated in FIG. 10 and listed in the table below. Excellent contrast ranging from 1.300-10.500 HU was observed dependent on the w % of 8 and the applied energy.

(34) TABLE-US-00002 w % iodine (before Formulation injection) 80 kV 100 kV 120 kV 140 kV A 4.26 w % 2500 HU 1800 HU 1500 HU 1300 HU B 10.65 w % 5000 HU 4500 HU 3500 HU 3000 HU C 21.30 w % 10500 HU 8800 HU 6200 HU 5900 HU

(35) As may be understood from above, according to one specific embodiment of the present invention, the X-ray contrast composition is a liquid before administration into a human or animal body and having an iodine concentration of more than 1.5 w % before injection, such as 2-30 w %, such as 3-25 w %, such as 4-25 w %.

Example 2

Synthesis and Improved Properties of PNIPAM-Coated AuNP

(36) Materials

(37) Chemicals were purchased from Sigma-Aldrich Inc. (Brndby, Denmark) unless otherwise stated. HAuCl.sub.43H.sub.2O was purchased from Wako Chemicals GmbH (Neuss, Germany) and SH-PNIPAM (MW 3500, PDI=1.24) was purchased from Polymer Source (Dorval, Canada).

(38) AuNP Synthesis, PNIPAM Coating and Particle Characterization

(39) All glassware was cleaned with aqua regia prior to use. Trisodium citrate (10 mL, 38.8 mM) was rapidly injected into a refluxing solution of HAuCl.sub.4*3H.sub.2O (100 mL, 1.0 mM) under vigorous stirring. An immediately color change from light yellow to wine red was observed and the reflux was continued for 15 min after which the solution was cooled to rt. The obtained AuNP-seeds (20 mL) were added to a boiling solution of HAuCl.sub.4*3H.sub.2O (2500 mL, 0.296 mM) under vigorous stirring. Subsequently, trisodium citrate (11.2 mL, 38.8 mM) was added and the mixture refluxed for 30 min resulting in a clear color change from wine red to purple. Additional trisodium citrate (100 mL, 38.8 mM) was added as stabilizer and the mixture heated for additional 1 h. The AuNP solution was cooled to rt and SH-PNIPAM.sub.3500 (40 mg, 11.4 mol) (6 molecules pr/nm.sup.2 AuNP surface area) dissolved in EtOH (5.0 mL) was added. The reaction mixtures stirred overnight at rt (FIG. 11a). The PNIPAM-coated AuNPs was extensively washed with MQ-H.sub.2O and up-concentrated to approx. 2.3 mL (theoretically 65 mg AuNP/mL) by centrifugation (4.500 RPM, 45 min/cycle). The AuNP-seeds, the citrate stabilized AuNPs and the purified up-concentrated PNIPAM-coated AuNP were all characterized by UV-Vis (FIG. 11b), DLS (FIG. 11c) and the -potential was measured (FIG. 11d). The [Au]-concentration of the up-concentration PNIPAM-coated AuNPs were determined by ICP-MS using a Au.sup.3+-standard (1000 mg/mL) in 5% HCl spiked with 0.5 ppt Ir as internal standard. Up-concentrated PNIPAM-coated AuNPs were dissolved in aqua regia and diluted with 5% HCl to theoretically 666 ppt Au.sup.3+. The concentration of the PNIPAM-coated AuNPs was determined to 64 mg Au/mL. The PNIPAM coated AuNPs were stored at 5 C. until further use.

(40) Lyophilization of PNIPAM Coated AuNP and Stability in Organic Solvent

(41) PNIPAM coated AuNPs (see synthesis above) were diluted to 1.0-, 2.5- or 5.0 mg Au/mL (5004 each) with MQ-H.sub.2O and snap-frozen in liquid nitrogen for 2 minutes. The samples were lyophilized overnight (p<6.010.sup.2 mbar) to form dark colored shiny powders. The lyophilized PNIPAM coated AuNPs were re-dissolved in EtOH (0.50 mL) and vortexed for a few seconds. The particles completely re-dispersed within seconds to give dark colored solutions. The particle morphology was evaluated by UV-Vis (FIG. 12a) and DLS (FIG. 12b). No sign of aggregation or instability was observed for the PNIPAM-coated AuNPs neither during lyophilization or EtOH solubilization. The lyophilized powder could easily be stored and weighted off at a later time-point.

Example 3

Controlling Particle Retention in SAIB Gels Based on Particle Hydrophobicity

(42) Chemicals were purchased from Sigma-Aldrich Inc. (Brndby, Denmark) unless otherwise stated. HAuCl.sub.43H.sub.2O was purchased from Wako Chemicals GmbH (Neuss, Germany), SH-PNIPAM (MW 3500, PDI=1.24) was purchased from Polymer Source (Dorval, Canada) and MeO-PEG.sub.5000-SH was purchased from Rapp Polymere GmbH (Tuebingen, Germany).

(43) AuNP Synthesis, PEG.sub.5000 Coating and Particle Characterization

(44) PEGylated AuNPs (PEG.sub.5000) were prepared as outlined for the PNIPAM coated AuNP in Example 2 using SH-PEG.sub.5000 as particle coating polymer. PEGylated particles were characterized by UV-Vis (=539 nm) and DLS (59.70.9 nm) and the concentration determined by ICP-MS (82.6 mg Au/mL).

(45) In Vitro Release of AuNP from SAIB/EtOH/PLA Gels

(46) Formulations (1000 mg each) consisting of SAIB/EtOH/PLA (75:20:5)+3.0 w % PNIPAM.sub.3500 or PEG.sub.5000 coated AuNP was prepared as outlined in the table below.

(47) TABLE-US-00003 PNIPAM .sub.3500.sub. PEG .sub.5000.sub. Formulation SAIB EtOH PLA AuNP AuNP D 750 mg 200 mg 50 mg 30 mg E 750 mg 200 mg 50 mg 30 mg

(48) The gel components were mixed and homogenized by a ball homogenizer (45 min, 30 s.sup.1) to give a clear homogenous solution. AuNPs (PNIPAM.sub.3500 or PEG.sub.5000) were transferred into anhydrous EtOH, mixed with the gel solution and vortexed. In vitro release study was carried out by injection of the formulations (3200 L each) into MQ-H.sub.2O (10.0 mL for PNIPAM-AuNP) or PBS-containing (for PEG-AuNP) glass vial at 37 C. Small aliquots (1.0 mL) were removed as a function of time and replaced with fresh aqueous solutions. The amount of released AuNPs was measured by correlating the UV-Vis absorbance with a standard curve based on the corresponding particles (FIG. 13). A burst release (20%) of the encapsulated hydrophilic PEGylated particles was observed within the first few hours whereas the more hydrophobic PNIPAM coated AuNP remained encapsulated in the SAIB-amorphous glass matrix due to the enhanced hydrophobic interactions with the gel matrix.

Example 4

Iodo-SAIB Gel Formation with PNIPAM-Coated AuNP In Vitro

(49) Materials Chemicals were purchased from Sigma-Aldrich Inc. (Brndby, Denmark) unless otherwise stated. HAuCl.sub.43H.sub.2O was purchased from Wako Chemicals GmbH (Neuss, Germany) and SH-PNIPAM (MW 3500, PDI=1.24) was purchased from Polymer Source (Dorval, Canada). 6,6-(2,4,6-triiodophenoxy)acetoxy-isobutyric-sucrose (8) was synthesized as described in Example 1.
AuNP Synthesis, PNIPAM Coating and Particle Characterization

(50) PNIPAM coated AuNPs were prepared as described in Example 2.

(51) Gel Preparation

(52) A formulation consisting of SAIB/8/EtOH (55:25:20)+3.0 w % PNIPAM-AuNP was prepared as outlined in the table below.

(53) TABLE-US-00004 6,6-(2,4,6- triiodophenoxy)- acetoxy-isobutyric- PNIPAM- Formulation SAIB sucrose (8) EtOH AuNPs F 165 mg 75 mg 60 mg 9 mg SAIB/8/EtOH (55:25:20) + 3.0 w % PNIPAM-AuNP

(54) SAIB-solution (90 w/w % in EtOH) was weighted off and mixed with 8 (see table above). The mixture were homogenized on a ball-mill homogenizer for 60 min (30 s.sup.1) and centrifuged for 20 s at 5000 RPM to remove air bubbles from the formulations. PNIPAM coated AuNPs (141 L, 64 mg AuNP/mL) was diluted with MQ-H.sub.2O (1659 L) and lyophilized to give a shinny powder. The lyophilized PNIPAM-coated AuNPs was re-dispersed anhydrous EtOH (52.8 uL) and mixed with the other gel components.

(55) In Vitro Release of AuNP in MQ-H.sub.2O

(56) An iodinated gel (200 L) with 3.0 w % PNIPAM-coated AuNPs (Formulation F) were prepared by injection into a MQ-H.sub.2O (10.0 mL) containing glass vial at 37 C. Small aliquots (1.0 mL) were removed as a function of time and replaced with fresh MQ-H.sub.2O. The amount of released AuNPs was measured by correlating the UV-Vis absorbance with a standard curve based on the PNIPAM-coated AuNPs. No release of PNIPAM-coated AuNPs was observed throughout the experiment. Formulation F was a homogenous dark colored solution injectable trough 25 G hypodermic needles.

Example 5

Visualization of Iodo-SAIB Gels Using Ultrasonography In Vitro

(57) Materials

(58) Chemicals were purchased from Sigma-Aldrich Inc. (Brndby, Denmark) unless otherwise stated. 6,6-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8) was synthesized as described in Example 1.

(59) Gel Preparation

(60) A formulation consisting of SAIB/8/EtOH (55:25:20) (350 mg) was prepared as described in Example 1 (Formulation B). The iodo-SAIB gel (250 L) was injected into MQ-H.sub.2O (500 mL) in a glass beaker and the gel was allowed to set for 5 days prior to visualization by ultrasonography. Ultrasound imaging of the iodo-SAIB gel was conducted by an Ultrasound Scanner (BK Medical, Herlev, Denmark) with the following settings: Res/Hz 2/21 Hz, B Gain 83%, Dynamic range 80 dB, Noise reject 10, Noise cutoff 32. The iodo-SAIB gel was clearly visible using ultrasonography as illustrated in FIG. 14.

Example 6

Iodo-SAIB Gels as Injectable CT-Contrast Agent In VivoVisibility Study in Immunocompetent Mice

(61) Materials

(62) Chemicals were purchased from Sigma-Aldrich Inc. (Brndby, Denmark) unless otherwise stated. 6,6-(2,4,6-triiodophenoxy)acetoxy-isobutyric-sucrose (8) was synthesized as described in Example 1. Healthy female NMRI (Naval Medical Research Institute) mice were purchased from Taconic (Borup, Denmark).

(63) Gel Preparation

(64) A formulation consisting of SAIB/8/EtOH (55:25:20) (900 mg) was prepared as described in Example 1 (Formulation B).

(65) Animal Setup

(66) Formulation B (SAIB/8/EtOH (55:25:20)) was administrated to healthy female NMRI mice (n=3) by subcutaneous injection (2004 each) under anaesthesia.

(67) MicroCT Imaging of Injectable Iodo-SAIB Gels

(68) The iodinated gels were visualized over time by computed tomography (CT). Images were acquired with a MicroCAT II system (Siemens Medical solutions, Malvern, USA). Excellent CT-contrast was achieved using Formulation B (SAIB/8/EtOH (55:25:20)) as illustrated in FIG. 15A-B (CT-images recorded 24 h p.i and 48 p.i.)

Example 7

Iodo-SAIB Gels as Injectable CT-Contrast Agent In VivoLong Term Stability and Visibility Study in Immunocompetent Mice

(69) Materials

(70) Chemicals were purchased from Sigma-Aldrich Inc. (Brndby, Denmark) unless otherwise stated. 6,6-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8) was synthesized as described in Example 1. Healthy female NMRI (Naval Medical Research Institute) mice were purchased from Taconic (Borup, Denmark).

(71) Gel Preparation

(72) Formulation consisting of a) SAIB/8/EtOH (65:15:20) (750 mg) and b) SAIB/8/EtOH (50:30:20) (750 mg) were prepared as described in Example 1.

(73) Animal Setup

(74) Both formulations; a) SAIB/8/EtOH (65:15:20) and b) SAIB/8/EtOH (50:30:20) were administrated to healthy female NMRI mice (n=28 mice) by subcutaneous injection (50 L each) under anesthesia.

(75) MicroCT Imaging of Injectable Iodo-SAIB Gels and Post-Implantation Visualization

(76) The iodinated gels were visualized over time by computed tomography (CT). Images were acquired with a MicroCAT II system (Siemens Medical solutions, Malvern, USA). Excellent CT-contrast was achieved using both formulations: a) SAIB/8/EtOH (65:15:20) and b) SAIB/8/EtOH (50:30:20) as illustrated in FIG. 16A-B. The obtained CT-contrast was found the scale with the formulated amount of iodo-SAIB (8) in the formulation. After 14 w of implantation the animals were sacrificed and the gels removed from the s.q. compartment (FIG. 16C-D). The iodinated gels were well-defined gels that could easily be removed and transferred without disruption of the gels. They were furthermore soft enough to be deformed using a scalpel.

(77) Gelation Kinetics of Injectable Iodo-SAIB Gels

(78) The gelation kinetics of the iodinated gels composed of SAIB/8/EtOH (50:30:20) was monitored by running multiply micro-CT scans within the first few hours of injection (FIG. 17A). Based on these images the total volume of the iodinated gel as a function of time was calculated as illustrated in FIG. 17B. Gelation of the iodinated gel is caused by efflux of EtOH from the gel matrix which takes place within the first two hours p.i. causing a rapid increase in the viscosity of the iodinated gel and an increase of CT-contrast by approximately 35% due to contraction of the gel.

(79) Degradation Profile of Injectable Iodo-SAIB Gels Over 14 w

(80) The degradation profile of iodinated gels composed a) SAIB/8/EtOH (65:15:20) and b) SAIB/8/EtOH (50:30:20) were monitored by microCT scanning over a period of 14 w. Based on these images the total volume of the iodinated gels as a function of time were calculated as illustrated in FIG. 17C. No difference in degradation profile between the two formulations was observed and a steady-state degradation profile was observed for both formulations. A volume loss, with a 95% confidence interval, of 0.09176 L/day was observed for both formulations after the initial EtOH efflux phase.

Example 8

Iodo-SAIB Gels as Injectable CT-Contrast Agent In VivoVisibility Study in Canine with Spontaneous Tumor

(81) Materials

(82) Chemicals were purchased from Sigma-Aldrich Inc. (Brndby, Denmark) unless otherwise stated. 6,6-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8) was synthesized as described in Example 1.

(83) Gel Preparation

(84) A formulation consisting of SAIB/8/EtOH (55:25:20) (350 mg) was prepared as described in Example 1 (Formulation B).

(85) Animal Setup

(86) Formulation B (SAIB/8/EtOH (55:25:20)) was administrated to a companion dog (American Staffordshire terrier, 9 years, 34 kg) with a mast cell tumor present between the front legs. The iodo-SAIB gel was administrated by intratumoral injection (500 L) using a 25 G needle.

(87) CT Imaging of Injectable Iodo-SAIB Gels in Canine

(88) The iodo-SAIB gel was visualized computed tomography (CT). Images were acquired with a Single slice Siemens CT-scanner (Siemens Medical solutions, Malvern, USA). Excellent CT-contrast was achieved using Formulation B (SAIB/8/EtOH (55:25:20)) as illustrated in FIG. 18 (CT-image recorded 24 h p.i.).