Polymeric Nanoparticles for Enhancing HIFU-Induced Ablation

Abstract

In the field of medical therapy, more in particular in the field of ablation therapy using ultrasound, such as high intensity focused ultrasound (HIFU), devices and methods are disclosed for enhancing the ablation effect of HIFU. More in particular, a polymeric particle is disclosed, including a polymer entrapping a liquid perfluorocarbon for use in high frequency ultrasound (HIFU) ablation therapy in a human or animal body, wherein the HIFU is focused in a focal region, wherein the ablation effect of the HIFU in the focal region is enhanced by administering the particles to the human or animal body, and the liquid perfluorocarbon does not undergo a phase change from liquid to gas during exposure to the HIFU.

Claims

1. A polymeric particle, comprising: a polymer entrapping a liquid perfluorocarbon for use in high intensity focused ultrasound (HIFU) ablation therapy in a human or animal body, wherein the HIFU is focused in a focal region, wherein an ablation effect of the HIFU in the focal region is enhanced by administering the particle to the human or animal body, wherein the liquid perfluorocarbon does not undergo a phase change from liquid to gas during exposure to the HIFU.

2. The particle according to claim 1, wherein the polymer comprises poly(lactic-co-glycolic) acid (PLGA), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), Polydimethylsiloxane (PDMS), or their copolymers.

3. The particle according to claim 1, wherein the particle is a nano-particle.

4. The particle according to claim 1, wherein the intensity of the high frequency ultrasound in the focal region is between 1 and 10,000 Watt.

5. The particle according to claim 1, wherein the liquid perfluorocarbon is a perfluoro crown ether.

6. The particle according to claim 5, wherein the perfluoro crown ether is selected from the group consisting of perfluoro-15-crown-5-ether, perfluoro-12-crown-4-ether and perfluoro-18-crown-6-ether.

7. The particle according to claim 1, additionally comprising a metal chelate.

8. The particle according to claim 7, wherein the metal chelate is a rare earth metal chelate.

9. The particle according to claim 8, wherein the rare earth metal chelate is gadolinium chelate.

10. The particle according to claim 9, wherein the gadolinium chelate is gadoteridol.

11. The particle according to claim 1, wherein the particle comprises a detecting agent.

12. The particle according to claim 1, wherein the particle comprises a therapeutic agent or a targeting agent.

13. The particle according to claim 1, wherein the particle has a diameter between 100 and 300 nanometer.

14. The particle according to claim 1, which is essentially surfactant free or surfactant free.

15. The particle according to claim 11, wherein the detecting agent is a fluorescent or luminescent agent.

16. The particle according to claim 15, wherein the fluorescent or luminescent agent is a dye or a radionuclide.

17. The particle according to claim 12, wherein the therapeutic agent or targeting agent is a drug, a receptor ligand or an antibody.

18. The particle according to claim 13, wherein the particle has a diameter between 100 and 250 nanometer.

19. The particle according to claim 18, wherein the particle has a diameter of 200 nanometer.

20. A polymeric particle, comprising: a polymer entrapping a liquid perfluorocarbon for use in high intensity focused ultrasound (HIFU) ablation therapy in a human or animal body, wherein the HIFU is focused in a focal region, wherein an ablation effect of the HIFU in the focal region is enhanced by administering the particle to the human or animal body, wherein the liquid perfluorocarbon does not undergo a phase change from liquid to gas during exposure to the HIFU, wherein the polymer comprises poly(lactic-co-glycolic) acid (PLGA), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polydimethylsiloxane (PDMS), or their copolymers, wherein the liquid perfluorocarbon is a perfluoro crown ether, wherein the perfluoro crown ether is selected from the group consisting of perfluoro-15-crown-5-ether, perfluoro-12-crown-4-ether and perfluoro-18-crown-6-ether.

21. The particle according to claim 20, additionally comprising a metal chelate.

22. The particle according to claim 21, wherein the metal chelate is a rare earth metal chelate.

23. The particle according to claim 22, wherein the rare earth metal chelate is gadolinium chelate.

24. The particle according to claim 23, wherein the gadolinium chelate is gadoteridol.

25. A polymeric particle, comprising: a polymer entrapping a liquid perfluorocarbon for use in high intensity focused ultrasound (HIFU) ablation therapy in a human or animal body, wherein the HIFU is focused in a focal region, wherein an ablation effect of the HIFU in the focal region is enhanced by administering the particle to the human or animal body, wherein the liquid perfluorocarbon does not undergo a phase change from liquid to gas during exposure to the HIFU, wherein the polymer comprises poly(lactic-co-glycolic) acid (PLGA), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polydimethylsiloxane (PDMS), or their copolymers, wherein the particle additionally comprises a metal chelate.

26. The particle according to claim 25, wherein the liquid perfluorocarbon is a perfluoro crown ether.

27. The particle according to claim 26, wherein the metal chelate is a rare earth metal chelate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1: Temperature mapping data of a tissue phantom without (control) and with particles. The color scale shows the temperature in degrees Celsius.

[0040] FIG. 2: Five mg of PLGA-PFCE-Gd particles were injected in tissue ex vivo, followed by 50 Watt HIFU ablation. Areas with and without nanoparticles are circled. The ablation size is clearly enhanced by the nanoparticles.

[0041] FIG. 3: Gel phantoms containing the particles indicated in Table 4. Particles were dispersed homogeneously in the gel. MR temperature mapping shows that the particles comprising PFCE (with and without Gd) enhanced heating to a value far above empty particles (PLGA only, no perfluorocarbon) or empty gel. The temperature changes are shown in table 5. Empty gel=no particles; PLGA NPs=PLGA nanoparticles with no perfluorocarbon; PLGA-PFCE-Gd NPs=PLGA nanoparticles with perfluoro-crown ether and gadolinium; PLGA-PFCE NPs=PLGA nanoparticles with perfluoro-crown ether.

EXAMPLES

Example 1: HIFU Enhancement in a Tissue Model with Polymeric Particles

[0042] Particles prepared according to example 2 with a high gadolinium content were injected in a sample of chicken breast that served as a tissue phantom (10 mg/ml). HIFU was carried out at 38 W with a 2 second pulse on a Bruker Clinscan system (7 T horizontal bore). The relevant tissue was then sectioned to directly visualize the ablated zone. Temperature changes were also measured in real time using standard MR thermometry sequences. Comparable results were obtained with the same particles comprising medium and low content of gadolinium. Particles without the gadolinium also showed an enhancement of the ablation effect, although this was less than particles with the low gadolinium content.

Example 2: Production of Nanoparticles

[0043] PLGA (0.09 gram) was dissolved in 3 ml dichloromethane in a glass tube. Liquid perfluoro-15-crown-5-ether (890 microliter) was added followed by 50 ml of a solution of Prohance (a 3 mg/ml solution of Gadoteridol) diluted in water. Optionally, additional agents, such as a fluorescent dye, may be added to the fluorocarbon at this stage. If a fluorescent particle was required, 1 mg of IcG or IC-Green (Indocyanine Green, Akorn Pharmaceuticals) was added to the solution.

[0044] We prepared particles with a high, medium and low content of gadolinium. For that purpose, the above mentioned solution of Prohance in water comprised 11.5, 5.75 and 2.85 ml respectively of Prohance added up with water to 50 ml of solution. The entire mixture was then added dropwise into 25 ml of a solution of polyvinyl alcohol in water (20 gram/liter) under constant sonication (Branson Digital Sonifier 250; 3 minute cycle with 60 sec on and 10 sec off and maximum temperature of 20 degrees Celsius and amplitude of 30%; a cuphorn was used) The resulting emulsion was then placed at 4 degrees Celsius and allowed to evaporate with constant stirring for about 12 hours until 24 ml of solution remained. An equal volume of water was then added and the emulsion was centrifuged at 21000 g for 30 minutes at 4 degrees Celsius. The pellet was washed with water twice and the resultant suspension was lyophilized at 60 degrees Celsius, for at least 24 hours, The particles were then placed in sealed tubes and stored at 80 deg Celcius. Unless stated otherwise, the particles used in the experiments described herein are the particles with the highest gadolinium content.

Example 3: Characterisation of Particles

[0045] We found that particles as prepared above were stable for at least a year when kept at 20 degrees Celsius in the dry form. The particles were also stable in solution at working concentrations for at least 3 months at minus 4 degrees Celsius.

[0046] Diameter of particles prepared according to example 2 was determined using dynamic light scattering (DLS) as previously described (Biomaterials. 2010 September; 31(27):7070-7). The particle size ranged from 80 to 500 nm with a sharp peak at 181 nm.

[0047] The particle diameter distribution remained stable for several months. The particles were lyophilised and frozen for storage. However, particles stored as aliquots in water (frozen) were also stable.

[0048] The particles prepared according to example 2 with high and medium gadolinium content, dissolved in water at a concentration of 1 mg/ml appeared to be exceptionally stable under conditions of ultrasound imaging. We measured particle diameter and count rate (indicative of number of particles) before and after exposure to HIFU and low and high intensity ultrasound MI (MI=0.1 and 2.0) for 30 sec. We found that the particles were not destroyed by HIFU or normal ultrasound. We also observed that increasing Gd content improves stability of the particles to ultrasound exposure. We found no change in the diameter, count rate or polydispersity (POI, indicative of the spread of diameter distribution) after exposure to high energy ultrasound for 30 sec.

[0049] It is concluded from the data that the particles as described herein are stable under even the harshest ultrasound conditions and that increasing Gd content improves stability of the particles to ultrasound exposure.

Example 4: Gadolinium Improves Imaging Properties of the Particles

[0050] PIGNPFCE particles were prepared according to example 2 with Gd and tested for ultrasound and MRI (including 1H MRI) visibility. It was found that the addition of gadolinium enhances MRI signal (1H) and can also enhance ultrasound visibility. It is concluded that the addition of gadolinium provides an improvement of the visibility of particles comprising a fluorinated organic compound. Therefore, the particles may be visualized by using normal ultrasound or MRI (both 1H and 19F) after ablation treatment with HIFU, and this visibility may be further enhanced by adding Gd.

Example 5: Alternative Synthesis of Particles

[0051] Particles were made as described previously [Srinivas, M. et al. Customizable, multi-functional fluorocarbon nanoparticles for quantitative in vivo imaging using 19F MRI and optical imaging. Biomaterials 31, 7070-7077 (2010)], with the addition of gadoteridol from ProHance (Bracco Imaging Europe, Amsterdam). Briefly, 1 g polyvinyl alcohol dissolved in 50 ml water only or water and, optionally, ProHance, 1780 l, is added dropwise to 180 mg of PIGA (Resomer RG 502 H, lactide: glycolide molar ratio 48:52 to 52:48; Boehringer Ingelheim, Germany) dissolved in dichloromethane with 8901 PFCE (Exfluor Inc, Texas USA), or alternatively 2321 PFO (Perfluoron, Alcon Inc), on ice, with sonication using a Digital Sonifier 250 (Branson, Danbury, USA) with a cuphorn running at 40% power for 2 minutes in 10 second pulses. Dynamic light scattering was done on a Malvern Zetasizer Nano. Gd content was measured using mass spectrometry.

Example 6: Further Alternative Synthesis of Particles

[0052] PIGA (100 mg, resomer 502H) was dissolved in 3 ml dichloromethane. Perfluoro-15-crown-5 ether (900 l) and Prohance (1.78 ml) were added to the solution of PIGA and a first emulsion was formed by sonication using a microtip having a tip diameter of 3 mm at an amplitude of 40% for 15 seconds (Digital Sonifier s250 from Branson). This first emulsion was rapidly (within 10 seconds) added to a solution of poly(vinyl alcohol) (25 g of water and 100-500 mg of PVA) in a round bottom flask while sonication of PVA-containing flask was started. The entire mixture was sonicated in ice-water bath using a microtip having a tip diameter of 3 mm at an amplitude of 20% or 40% to obtain a second emulsion. The duration of the period from the addition of the first emulsion to the end of the sonication was 3 minutes (Digital Sonifier s250 from Branson).

[0053] After sonication dichloromethane was evaporated at 4 C. or room temperature overnight under stirring to achieve solidification of the beads. The beads were isolated by centrifugation at 27200 g for 35 min in 50 ml centrifugation tubes and resuspended in 25 g of water. The washing step was repeated two more times with resuspention by sonication after second washing (sonication bath, Diagenode Bioruptor). After washing, beads were resuspended in 4 ml of water, frozen with liquid N2 and freeze-dried. The resulting product was a white powder.

[0054] The amounts of the components and the sonication amplitude which were varied are shown in Table 1, together with the properties and the yield of the beads.

TABLE-US-00001 TABLE 1 Radius (DLS; PFCE- Exp. PVA/ Sonication intensity)/ content/ No mg Amplitude nm PDI wt.-% yield/mg 1 100 20% 357 0.49 11 77 2 500 20% 121 0.1 5.3 55 3 100 40% 314 0.39 28 137 4 200 40% 174 0.2 34 189 5 350 40% 146 0.15 39 184 6 500 40% 121 0.123 45 204

[0055] Small beads with narrow particle size distribution were obtained by the process according to the invention (Experiments 5 and 6). It can be observed that a high amplitude (40%) and a large amount of PVA (350 mg or 500 mg) resulted in a desirable combination of a small radius, low PDI, a high PFCE content and a high yield.

Example 7: Preparation of Beads without a Metal Compound

[0056] PLGA (100 mg, resomer 502H) was dissolved in 3 ml dichloromethane (DCM) followed by addition of perfluoro-15-crown-5 ether (900 l). The resulting double phase liquid was rapidly added with a glass pipette to solution of poly(vinyl alcohol) (25 g of water and 100-500 mg of PVA) in a round bottom flask while sonication was started. Care was taken so that the phase of PLGA/DCM and the phase of PFCE were added simultaneously at a constant ratio. The entire mixture was sonicated in ice-water bath using a microtip having a tip diameter of 3 mm at an amplitude of 20% or 40% to obtain an emulsion. The duration of the period from the addition of the double phase liquid to the end of the sonication was 3 minutes (Digital Sonifier s250 from Branson).

[0057] After sonication dichloromethane was evaporated at 4 C. or room temperature overnight under stirring to achieve solidification of the beads. The beads were isolated by centrifugation at 27200 g for 35 min in 50 ml centrifugation tubes and resuspended in 25 g of water. The washing step was repeated two more times with resuspention by sonication after second washing (sonication bath, Diagenode Bioruptor). After washing, beads were resuspended in 4 ml of water, frozen with liquid N2 and freeze-dried. The resulting product was a white powder with a yield of at least 100 mg.

TABLE-US-00002 TABLE 2 Radius (DLS; PFCE- Exp. PVA/ Sonication intensity)/ content/ No mg Amplitude nm PDI wt.-% yield/mg 7 100 20% 354 0.25 15 86 8 100 40% 339 0.24 25 116 9 500 40% 100 0.04 48 154

[0058] Small beads with narrow particle size distribution and a high PFCE content were obtained with a high yield according to the process of the invention (Ex 9).

Experiments 10-12

[0059] Experiment 6 was repeated except that the PLGA was dissolved in a solvent indicated in Table 3.

TABLE-US-00003 TABLE 3 Radius (DLS; PFEC- Exp. PVA/ Sonication intensity)/ content/ yield/ No Solvent mg Amplitude nm PDI wt.-% mg 10 THF 500 40% 171 0.5 15 131 11 Acetone 500 40% 294 0.66 8 90 12 Acetoni- 500 40% 223 0.22 5 95 trile

[0060] Beads obtained are larger and have a broader size distribution than the experiments in which the solvent was dichloromethane.

[0061] Diameter of beads prepared according to examples 1-12 was determined using dynamic light scattering (DLS) as described in Biomaterials. 2010 September; 31 (27):7070-7.

Example 8: Preparation of Beads Using Cup Horn (Experiment 13)

[0062] PLGA (90 mg, resomer 502H) was dissolved in 3 ml dichloromethane.

[0063] Perfluoro-15-crown-5 ether (890 l) was added to the solution of PLGA. 50 ml of an aqueous solution comprising of Prohance with concentration of 3 mg/ml was further added. This mixture was added dropwise to a solution of poly(vinyl alcohol) (20 g/l) in a glass tube while sonication of PVA-containing flask was started. The entire mixture was sonicated in a cup horn at an amplitude of 30% for 3 minutes, with 60 s on and 10 s of cycles (Digital Sonifier s250 from Branson) to obtain a second emulsion. During the sonication the temperature of the cooling water was maintained at 4 C. by a refrigerated circulator.

[0064] After sonication dichloromethane was evaporated at 4 C. overnight under stirring to achieve solidification of the beads. The beads were isolated by centrifugation at 21000 g for 30 min in 2 ml centrifugation tubes and resuspended in 25 g of water. The pellet was washed with water twice and then resuspended in water, frozen at 80 C. and freeze-dried. The resulting product was a white powder with a yield of 50 mg.

[0065] The examples according to experiments 5, 6 and 9 resulted in a much higher yield compared to experiment 13.

Example 9: Nanoparticles Enhance HIFU-Induced Ablation in Tissue

[0066] PLGA-PFCE-Gd nanoparticles as described herein were injected into chicken breast ex vivo. The sample was then subjected to HIFU ablations, using a standard in vivo setting of 50 Watt. Four ablations were carried out for each area i.e. with and without the nanoparticles. The tissue was then sliced (FIG. 2, right panel) to examine the extent of the ablated tissue. It was found that the nanoparticles clearly enhanced the ablated tissue area.

Example 10: Nanoparticles Enhance HIFU-Induced Ablation In Vitro

[0067] Agarose gel phantoms containing homogeneously distributed nanoparticles, or an empty control, were treated at room temperature using MRI-guided HIFU using a standard in vivo setting of 50 Watt. MR thermometry was also carried out (FIG. 3). The aim of this experiment was to measure the effect of the nanoparticles in terms of temperature changes. Table 4 summarizes the properties of the particles used, with diameter and polydispersity (PDI) measured using dynamic light scattering.

TABLE-US-00004 TABLE 4 Particles Diameter (nm) PDI Empty gel (no particles) n/a n/a Empty PLGA 225 8 0.11 PLGA/PFCE/Gd 285 4 0.13 PLGA/PFCE 180 0.14

[0068] The MRI images (FIG. 3) show that the particles clearly enhance the heating effect, well over either empty gel or empty PLGA nanoparticles without any perfluorocarbon.

[0069] Table 5 shows the peak temperatures measured in the gels of FIG. 3.

TABLE-US-00005 TABLE 5 Peak temperatures after 2 or 4 seconds of HIFU. 2 seconds 4 seconds ablation ablation Nanoparticles [degrees C.] [degrees C.] No particles 50 46 Empty PLGA nanoparticles 50 48 PLGA-PFCE nanoparticles 70 70 PLGA-PFCE nanoparticles + Gadolinium 71 75

[0070] It is to be noted that the temperature increase with the nanoparticles containing perfluorocarbon was very high, and this resulted in an underestimation in the peaks of the thermometry scans. It is concluded from the data shown in table 5 that the no particles control as well as the PLGA particles without perfluorocarbon (empty PLGA nanoparticles) show relatively low temperature increases, whereas the perfluorocarbon containing particles caused a much higher increase in temperature.

Example 11: Stability of Particles Under HIFU Conditions

[0071] In this experiment we looked at the effect of high temperature on the nanoparticles, so see if the particles remain intact (i.e. without structural changes) after exposure to high temperature. For this, the particles were incubated at either 50 or 80 degrees C. for 0.5 or 2 hours. This is far in excess of the few seconds of exposure to high temperatures that occurs during HIFU.

[0072] It was found that even in these harsh conditions, no change in particle diameter or polydispersity, as measured by dynamic light scattering, was observed in all conditions (including up to 2 hours at 80 degrees Celsius). This shows that the particles are not damaged or destroyed by HIFU ablation, as the diameter does not change. It was also found that the polydispersity index (POI) never rose above 0.1, indicating a very homogeneous distribution of particle diameters. This, again, shows that the particles do not undergo any structural changes due to high temperature.

TABLE-US-00006 TABLE 6 Stability of PLGA/PFCE/Gd nanoparticles at elevated temperatures. Time Diameter at 50 Diameter at 80 PDI at 50 PDI at 80 [hours] degrees C. [nm] degrees C. [nm] degrees C. degrees C. 0 190 190 0.07 0.07 0.5 200 200 0.07 0.09 2 200 200 0.06 0.07