Compositions and methods for use in medical diagnosis

Abstract

The present invention relates to compositions and methods for use in medical diagnosis and patient monitoring. It more particularly relates to a biocompatible gel comprising nanoparticles and/or nanoparticle aggregates, wherein: i) the nanoparticles and/or nanoparticles of said aggregate comprise an inorganic material comprising at least one metal element having an atomic number Z of at least 25, each of said nanoparticles and nanoparticle aggregates being covered with a biocompatible coating; ii) the nanoparticles' and/or nanoparticle aggregates' concentration is of about or less than 0.5% (w/w); and iii) the apparent viscosity at 2 s.sup.1 of the gel comprising the nanoparticles and/or nanoparticle aggregates is between about 0.1 Pa.Math.s and about 1000 Pa.Math.s when measured between 20 C. and 37 C. The composition of the invention typically allows the delineation and visualization of at least 40% of the target biological tissue when said tissue is observed using an X-ray imaging equipment.

Claims

1. A method for delineating and visualizing at least 50% of a tumor bed in a subject, wherein the tumor bed is the tissue covering the cavity obtained following tumor resection in the subject, and wherein the method comprises a step a) of depositing on the tumor bed in the subject a biocompatible gel comprising nanoparticles and/or nanoparticle aggregates wherein: i) the nanoparticles and/or nanoparticles of the aggregate comprise hafnium (IV) oxide (HfO.sub.2) or comprise gold and hafnium (IV) oxide (HfO.sub.2), each of said nanoparticles and/or said nanoparticle aggregate being covered with a biocompatible coating; ii) the concentration of the nanoparticle and/or nanoparticle aggregates in the biocompatible gel is about or less than 0.5% (w/w); iii) the biocompatible gel comprises cellulose, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, or hyaluronic acid; and iv) the apparent viscosity at 2 s.sup.1 of the biocompatible gel comprising nanoparticles and/or nanoparticle aggregates is between about 1 Pa.Math.s and about 750 Pa.Math.s when measured between 20 C. and 37 C. and the gel is deposited in a layer having a thickness between 100 m and 0.5 cm, wherein the nanoparticles and/or the nanoparticle aggregates of said gel delineate the tumor bed, and a step b) of visualizing at least 50% of the delineated tumor bed in the subject using X-ray imaging equipment.

2. The method according to claim 1, wherein the concentration of the nanoparticle and/or nanoparticle aggregates in the biocompatible gel is between about 0.15% and about 0.5% (w/w).

3. The method according to claim 1, wherein the nanoparticles and/or nanoparticle aggregates comprise hafnium oxide (HfO.sub.2).

4. The method according to claim 1, wherein the nanoparticle or nanoparticle aggregate further comprises at least one targeting agent.

5. The method according to claim 1, wherein the X-ray imaging equipment is a CT scanner.

6. The method according to claim 1, wherein the nanoparticles and/or nanoparticle aggregates comprise hafnium (IV) oxide (HfO.sub.2) and gold.

7. The method according to claim 1, comprising delineating and visualizing at least 70% of the tumor bed in the subject.

8. The method according to claim 1, comprising delineating and visualizing at least 80% of the tumor bed in the subject.

9. The method according to claim 1, wherein the step of delineating and visualizing the tumor bed is performed between 24 hours and two weeks after the step of depositing on the tumor bed in the subject the biocompatible gel comprising nanoparticles and/or nanoparticle aggregates.

10. The method according to claim 1, wherein the biocompatible gel is selected from methylcellulose, carboxymethylcellulose, hydroxyethylcellulose and hydroxypropylcellulose.

11. The method according to claim 1, wherein the nanoparticles comprise gold metal covered with hafnium oxide.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Tumor tissue delineation using clips

(2) From Improving the definition of the tumor bed boost with the use of surgical clips and image registration in breast cancer patients [Int. J. Radiation Oncology Biol. Phys., 78(5): 1352-1355 (2010)]. Tumor bed volume delineation: gross tumor volume (GTV) (red); clinical target volume (CTV) clips=all clips with 0.5-cm margins; planning target volume (PTV) (green)=GTV+CTV clips+0.5-cm lateral and 1-cm superior-inferior margins.

(3) FIG. 2: CT images taken 15 minutes (<30 minutes), 3 days, and 7 days after gel deposition into the cavity left following tumorectomy, showing the tumor bed delineation using a biocompatible hydrogel composed of methylcellulose (4% w/w) comprising nanoparticles and/or nanoparticle aggregates (0.4% w/w) consisting of hafnium oxide. The nanoparticles and/or nanoparticle aggregates have been mixed with the gel prior to the gel deposition into the tumor bed.

(4) FIG. 3: CT images taken 15 minutes (<30 minutes), 3 days and 7 days after gel deposition into the cavity left following tumorectomy, showing the tumor bed delineation using a biocompatible hydrogel composed of methylcellulose (4% w/w) comprising nanoparticles and/or nanoparticle aggregates (0.2% w/w) consisting of hafnium oxide. The nanoparticles and/or nanoparticle aggregates have been mixed with the gel prior to the gel deposition into the tumor bed.

(5) FIG. 4: CT images taken 15 minutes (<30 minutes), 2 days and 7 days after gel deposition into the cavity left following tumorectomy, showing the tumor bed delineation using a biocompatible hydrogel composed of methylcellulose (4% w/w) comprising nanoparticles and/or nanoparticle aggregates (0.4% w/w) consisting of hafnium oxide. The nanoparticles and/or nanoparticle aggregates have been incorporated within the gel at the time of surgery.

EXAMPLES

Example 1: Biocompatible Hafnium Oxide (HfO2) Nanoparticles or Nanoparticle Aggregates, Using Sodium Hexametaphosphate as Coating Agent

(6) A tetramethylammonium hydroxide (TMAOH) solution is added to HfCl.sub.4 solution. Addition of TMAOH solution is performed until the pH of the final suspension reaches a pH comprised between 7 and 13. A white precipitate is obtained.

(7) The precipitate is further transferred to an autoclave and heated at a temperature comprised between 120 C. and 300 C. to perform crystallization. After cooling, the suspension is washed with de-ionized water.

(8) Sodium hexametaphosphate solution is then added to the washed suspension and the pH is adjusted to a pH comprised between 6 and 8.

(9) Sterilization of the nanoparticle or nanoparticle aggregate suspension is performed prior in vitro or in vivo experiments.

Example 2: Synthesis and Physico-Chemical Characterization of Gold Nanoparticles with Different Sizes

(10) Gold nanoparticles are obtained by reduction of gold chloride with sodium citrate in aqueous solution. Protocol was adapted from G. Frens, Nature Physical Science 241 (1973) 21.

(11) In a typical experiment, HAuCl.sub.4 solution is heated to boiling. Subsequently, sodium citrate solution is added. The resulting solution is maintained under boiling for an additional period of 5 minutes.

(12) The nanoparticle's size is adjusted from 15 nm up to 105 nm by carefully modifying the citrate versus gold precursor ratio (see Table 1).

(13) The as-prepared gold nanoparticle suspensions are then concentrated using an ultrafiltration device with a 30 kDa cellulose membrane.

(14) The resulting suspensions are ultimately filtered through a 0.22 m cutoff membrane filter under a laminar hood and stored at 4 C.

(15) Particle size is determined on more than 200 particles, by using Transmission Electronic Microscopy (TEM) and by considering the longest nanoparticle dimension of each particle.

(16) TABLE-US-00001 TABLE 1 Synthesis Samples Particle size (nm) Citrate HAuCl.sub.4 Gold-15 15 2 (1) 20 mL 30 mL 500 mL 0.25 mM Gold-30 32 10 (1) 7.5 mL 40 mM 500 mL 0.25 mM Gold-60 60 10 (1) 2 mL 85 mM 500 mL 0.25 mM Gold-80 80 10 (1) 1.2 mL 43 mM 200 mL 0.30 mM Gold-105 105 25 (1) 1.2 mL 39 mM 200 mL 0.33 mM

Example 3: Biocompatible Hafnium Oxide Nanoparticles' and/or Nanoparticle Aggregates' Incorporation (0.4% w/w) within the Gel Prior to Gel Deposition on the Tumor Bed

(17) A volume of aqueous suspension of biocompatible HfO.sub.2 nanoparticles from example 1 is added to a volume of gel, typically with a polymer (methylcellulose) concentration lying between 3.5% w/w and 4.5 w/w. The volume ratio between the suspension of HfO.sub.2 nanoparticles and the gel being adjusted to reach a final HfO.sub.2 nanoparticle concentration within the gel of 0.4% (w/w). The preparation thus obtained is gently mixed, typically with a magnetic stirrer or a spatula.

Example 4: Biocompatible Hafnium Oxide Nanoparticles' and/or Nanoparticle Aggregates' Incorporation (0.2% w/w) within the Gel Prior to Gel Deposition on the Tumor Bed

(18) A volume of aqueous suspension of biocompatible HfO.sub.2 nanoparticles from example 1 is added to a volume of gel, typically with a polymer (methylcellulose) concentration lying between 3.5% w/w and 4.5 w/w. The volume ratio between the suspension of HfO.sub.2 nanoparticles and the gel being adjusted to reach a final HfO.sub.2 nanoparticle concentration within the gel of 0.2% (w/w). The preparation thus obtained is gently mixed, typically with a magnetic stirrer or a spatula.

Example 5: Assessment by Computed Tomography (CT) of the Quality of the Tumor Bed Delineation Obtained when Using Nanoparticles Embedded in Hydrogel from Example 3

(19) The objective of this experiment was to assess, by CT (Computed Tomography), the quality of tumor bed delineation by nanoparticles (NPs).

(20) The test gel from example 3 was implanted (deposited) into the cavity left by the resection of an EMT-6 ectopic grafted tumor (breast tumor cells) in BALB/cJRj mice.

(21) The CT analysis was performed 15 minutes (<30 minutes), 3 days and 7 days following gel implantation into the cavity left by the resection of the tumor in order to evaluate the volume occupied by the nanoparticles and/or nanoparticle aggregates in the tumor bed over time. For this, a manual segmentation (region of interest (ROI)) was performed around the surgical cavity. Then a thresholding above 120 HU was performed inside the surgical cavity in order to evaluate the presence of nanoparticles or nanoparticle aggregates and to assess both the location and volume occupied by those nanoparticles or nanoparticle aggregates for all mice. FIG. 2 presents the CT images showing more than 50% of cavity delineation as soon as 7 days following surgery and gel implantation.

Example 6: Assessment by Computed Tomography (CT) of the Quality of the Tumor Bed Delineation Obtained when Using Nanoparticles Embedded in Hydrogel from Example 4

(22) The objective of this experiment was to assess, by CT (Computed Tomography), the quality of tumor bed delineation by nanoparticles (NPs).

(23) The test gel from example 4 was implanted (deposited) into the cavity left by the resection of an EMT-6 ectopic grafted tumor (breast tumor cells) in BALB/cJRj mice.

(24) The CT analysis was performed 15 minutes (<30 minutes), 3 days and 7 days following gel implantation into the cavity left by the resection of the tumor in order to evaluate the volume occupied by the nanoparticles or nanoparticle aggregates in the tumor bed over time. For this, a manual segmentation (region of interest (ROI)) was performed around the surgical cavity. Then a thresholding above 120 HU was performed inside the surgical cavity in order to evaluate the presence of nanoparticles and/or nanoparticle aggregates and to assess both the location and volume occupied by those nanoparticles and/or nanoparticle aggregates for all mice. FIG. 3 presents the CT images showing more than 50% of cavity delineation as soon as 7 days following surgery and gel implantation.

Example 7: GEL Preparation

(25) Gel is formed typically with a polymer (methylcellulose) concentration in water lying between 3.5% w/w and 4.5% w/w. The preparation thus obtained is gently mixed, typically with a magnetic stirrer or a spatula.

Example 8: Assessment by Computed Tomography (CT) of the Quality of the Tumor Bed Delineation Obtained when Using Biocompatible Hafnium Oxide Nanoparticles and/or Nanoparticle Aggregates Embedded (0.4% w/w) within the Gel from Example 7, at the Time of Surgery

(26) The objective of this study is to assess, by CT (Computed Tomography), the quality of tumor bed delineation by nanoparticles (NPs).

(27) The test gel from example 7 was implanted (deposited) into the cavity left by the resection of an EMT-6 ectopic grafted tumor (breast tumor cells) in BALB/cJRj mice.

(28) Just before its implantation, a suspension of hafnium oxide nanoparticles from example 1 was added into the gel via a syringe to reach a final concentration of 0.4% w/w.

(29) The CT analysis was performed 15 minutes (<30 minutes), 2 days and 7 days following gel implantation into the cavity left by the resection of the tumor in order to evaluate the volume occupied by the nanoparticles and/or nanoparticle aggregates in the tumor bed over time. For this, a manual segmentation (region of interest (ROI)) was performed around the surgical cavity. Then a thresholding above 120 HU was performed inside the surgical cavity in order to evaluate the presence of nanoparticles and/or nanoparticle aggregates and to assess both the location and volume occupied by those nanoparticles and/or nanoparticle aggregates for all mice. FIG. 4 presents the CT images showing about 80% cavity delineation as soon as 7 days following surgery and gel implantation.

REFERENCES

(30) Customized Computed Tomography-Based Boost Volumes in Breast-Conserving Therapy: Use of Three-Dimensional Histologic Information for Clinical Target Volume Margins. IJROB 75(3):757-763 (2009). Target volume definition for external beam partial breast radiotherapy: clinical, pathological and technical studies informing current approaches. Radiotherapy and Oncology 94:255-263 (2010). Excised and Irradiated Volumes in Relation to the Tumor Size in Breast-Conserving Therapy. Breast Cancer Res Treat 129:857-865 (2011). Guidelines for target volume definition in post-operative radiotherapy for prostate cancer, on behalf of the EORTC Radiation Oncology Group. Radiotherapy & Oncology 84:121-127 (2007). Improving the definition of the tumor bed boost with the use of surgical clips and image registration in breast cancer patients. Int J Radiation Oncology Biol Phys 78(5):1352-1355 (2010). The dynamic tumor bed: volumetric changes in the lumpectomy cavity during breast conserving therapy. Int J Radiation Oncology Biol Phys 74(3):695-701 (2009).