Hafnium(IV) oxide nanoparticles and aqueous compositions thereof

Abstract

The present invention relates to hafnium(IV) oxide (HfO2) nanoparticles nanocrystals comprising or having a diameter of 15 nm or less (≦) stabilized by a plurality of dispersant molecules attached to the surface. The dispersant molecules are composed of catechol or gallol surface adsorbed moieties and oligo(ethylene glycol) moieties. The present invention further relates to compositions of such nanoparticles that are stable colloidal suspensions at physiological pH, and to the use of the compositions in medical treatment and diagnostic applications, particularly in radiotherapy enhancement and as X-ray contrast agents. In yet another aspect, the present invention provides methods of making the compositions and nanoparticles according to the invention.

Description

[Technical field]

[0001] This application claims priority to European Patent Application No. 21210057.2, filed November 23, 2021, and European Patent Application No. 22175125.8, filed May 24, 2022, both of which are incorporated herein by reference.

[0002] Field The present invention relates to hafnium(IV) oxide nanoparticles combined with surface-adsorbed oligo(ethylene glycol) moieties. The present invention further relates to compositions comprising the nanoparticles of the present invention for use as pharmaceuticals or diagnostic agents. [Background technology]

[0003] Colloidal nanocrystals (NCs) have been considered for many biomedical applications, such as bioimaging, drug delivery, and enhanced photothermal and radiotherapy. These NCs are typically hybrids consisting of an inorganic core capped with organic ligands. The ligands determine the interaction between the NC and the solvent and the stability of the nanocolloid. For biomedical applications, control of the NC surface chemistry is important because it plays a role in particle aggregation, cellular uptake, protein repulsion or adsorption, cytotoxicity, circulation time, and targeting approaches. Although surface chemistry is important for all types of NCs (chalcogenides, pnictides, halides, and metal NCs), there is no one-size-fits-all solution. For example, thiolates and thiols have strong binding affinity to Au and CdSe NCs, but interact poorly with metal oxide NCs.

[0004] Metal oxide NCs have been particularly successful in nanomedicine. Three types of inorganic NCs have achieved clinical translation, two of which are oxides, iron oxide and hafnium oxide (Min, Y. Chemical Reviews 2015, 115(19), 11147-11190). These particles are often first synthesized in a non-polar solvent and stabilized by a surfactant (usually with a carboxylate or phosphonate head group and an aliphatic tail). A carboxylic acid (e.g., oleic acid) dissociates on the metal oxide surface, the carboxylate binds to the surface metal site, and the proton binds to the surface oxygen atom. This binding motif is described as NC(XX') because both the proton and the carboxylate are X-type ligands. In a non-polar solvent, the carboxylic acid is quantitatively exchanged with the phosphonic acid in an X-to-X ligand exchange process. In fact, the phosphonic acid is a very strong ligand for the oxidized surface. In contrast, catechol was found to be a rather weak ligand, only able to exchange a small fraction of oleic acid, so there is a clear order of binding strength in non-polar solvents: catechol < carboxylic acids < phosphonic acids.

[0005] In aqueous (or other polar) environments, this order is less clear and is further complicated by various factors, e.g., pH and salt concentration. For example, carboxylic acids are frequently used to stabilize metal oxide NCs in water. These can provide colloidal stability in static systems without competing ligands, but not in phosphate buffered saline (PBS) or cell culture media. Binding affinity increases significantly for multidentate carboxylate ligands, e.g., polymers. In general, reports in the literature are consistent with carboxylic acids being the weakest ligands in water, weaker than phosphonic acid or catechol. The literature is, however, inconclusive as to which phosphonic acid or catechol are the best ligands. Despite the common use of phosphonic acid and catechol in metal oxide NC functionalization, there is no clear consensus on the relative binding affinities. Furthermore, there is usually no direct link between the ligand binding equilibrium and the final colloidal stability of the NCs. Summary of the Invention [Problem to be solved by the invention]

[0006] Based on the above situation, the object of the present invention is to provide means and methods for providing better nanoparticle compositions for pharmaceutical applications. This object is achieved by the subject matter of the independent claims herein and further advantageous embodiments described in the dependent claims herein, the examples, the figures and the general description. [Means for solving the problem]

[0007] One aspect of the present invention relates to nanoparticles comprising nanocrystals comprising or consisting essentially of hafnium (IV) oxide (HfO2) having a diameter of 15 nm or less (diameter < 15 nm), the nanocrystals being stabilized by a plurality of dispersant molecules attached to their surfaces. The dispersant molecule comprises or consists essentially of a surface-adsorbing moiety selected from the group comprising catechol or gallol, and an oligo(ethylene glycol) moiety.

[0008] In another aspect, the invention relates to a composition of the nanoparticles described herein, the composition providing the nanoparticles in a stable colloidal suspension at physiological pH. Another aspect of the invention relates to the use of the nanoparticles and compositions described herein in treatment and diagnostic applications, particularly to enhance radiation therapy and as X-ray contrast agents.

[0009] The present invention also relates to a pharmaceutical composition comprising the nanoparticles or nanoparticle suspension according to the invention and at least one pharma- ceutically acceptable carrier, diluent or excipient. In yet another aspect, the present invention provides methods of making the compositions and nanoparticles according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Terms and Definitions For purposes of interpreting this specification, the following definitions shall apply and whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that the following definitions conflict with any document incorporated herein by reference, the definition set forth herein shall control.

[0011] The terms "comprising," "having," "containing," and "including," as well as other similar forms and grammatical equivalents, as used herein, are intended to be equivalent and open-ended, not to imply that the items following these words are an exhaustive list of such items or are limited to only the recited items. For example, "comprising" components A, B, and C can consist of (i.e., include only) components A, B, and C, or can include not only components A, B, and C, but also one or more other components. Thus, "comprising" and similar forms and grammatical equivalents are intended and understood to include disclosure of embodiments that "consist essentially of" or "consist of."

[0012] When a range of values ​​is listed, it is understood that every value between the upper and lower limits of that range (to one tenth of the unit of the lower limit, unless the context clearly indicates otherwise), and every other listed or subtended value within that listed range, except for any limit expressly excluded within that listed range, is encompassed within the disclosure. When the listed range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0013] Reference herein to a value or parameter with "about" includes (and describes) a variation of that value or parameter itself. For example, "about X" includes the description "X." As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural references unless the context clearly dictates otherwise.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic, and biochemical methods (generally, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th ed., John Wiley & Sons, Inc.) and chemical methods.

[0015] The term "nitrodopamine" refers to a dopamine moiety having a nitro functional group on the dihydroxyphenyl ring of the dopamine moiety. One specific example of nitrodopamine is of the formula: [ka]

[0016] The term "nitroDOPA" refers to a DOPA moiety that has a nitro functional group on the dihydroxyphenyl ring of DOPA (the dihydroxyphenylalanine moiety). One specific example of nitroDOPA is of the formula: [ka]

[0017] The term "mimosine" refers to (2S)-2-amino-3-(3-hydroxy-4-oxopyridin-1-yl)propanoic acid of the following formula: [ka]

[0018] An oligo-ethylene glycol moiety, as used herein, is a group having the formula (CH2-CH2-O)n It refers to a chain described by CH3, where n is an integer from 1 to 15, particularly n is from 2 to 12, and more particularly n is from 2 to 5.

[0019] In the work on which this specification is based, we aimed to clearly establish the order of binding affinities and provide the correct surface chemistry for optimal application in nanomedicine. We chose HfO2NCs as a model system for two reasons: (1) it is a suitable material in nanomedicine and (2) it is compatible with solution nuclear magnetic resonance (NMR) spectroscopy. The latter has proven to be a very powerful tool for the study of nanocrystal surface chemistry. Unfortunately, iron oxide NCs interfere with magnetic fields and cannot be studied with NMR. HfO2NCs are therefore an ideal starting point, and also because their surface chemistry has already been comprehensively studied in non-polar solvents using NMR spectroscopy. First, we 1 H and 31 Using P NMR spectroscopy, we assessed the ligand exchange of natural carboxylic acid ligands against phosphonic acid and catechol ligands. Importantly, we used the same poly(ethylene glycol) ligand chains for all three binding groups, ensuring direct comparison of results. Next, we evaluated the effects of solvent (methanol vs. water) and pH on ligand binding. Furthermore, we used NMR and dynamic light scattering (DLS) to determine the colloidal stability afforded by different ligand types in aqueous and buffered environments, directly correlating this with ligand binding kinetics. Finally, we constructed colloidal stability maps showing which binding groups afford colloidal stability as a function of pH. This practical guide will aid researchers in the design of future surface chemistries.

[0020] Detailed Description of the Invention A first aspect of the present invention relates to a nanoparticle comprising a nanocrystalline core and an organic stabilizer adsorbed to the nanocrystalline core, the organic stabilizer promoting the stability of the particle in aqueous solutions at physiological pH. The nanoparticles are comprised of nanocrystals that include or consist essentially of hafnium (IV) oxide (HfO2) having a diameter of 15 nm or less (diameter < 15 nm) and a plurality of dispersant molecules attached to the surface of the nanocrystals. i. a surface-adsorbing moiety selected from the group including catechol or gallol, particularly a 1,2-hydroxy-4-nitrophenyl moiety; ii. comprising or consisting essentially of an oligo(ethylene glycol) moiety.

[0021] One non-limiting example of such a dispersant molecule is dopamine-MEEAA adduct (MEEAA is 2-[2-(2-methoxyethoxy)ethoxy]acetic acid). [ka]

[0022] In certain embodiments, the dopamine-oligo(ethylene glycol) dispersant molecule may more generally be represented by the general formula: [ka] (wherein n is selected from 1, 2, 3, 4, 5, and 6) It can be written as follows.

[0023] In certain embodiments, the dopamine-oligo(ethylene glycol) dispersant molecule may more generally be represented by the general formula: [ka] (wherein n is selected from 1, 2, 3, 4, 5, and 6) It can be written as follows.

[0024] In certain embodiments, the particles are made of pure hafnium(IV) oxide (HfO2). This is the most chemically readily accessible nanocrystal. However, HfO2 may be considered to be the only part of the particle, e.g., Hf metal may constitute the core and the oxide may constitute the surface of the particle. Higher Hf metal content is expected to result in higher contrast.

[0025] In certain embodiments, the nanocrystals have a diameter of 6 nm or less. In specific embodiments, the diameter is ≦4 nm. In more specific embodiments, the diameter is ≦3.5 nm. The smaller the diameter, the more likely the particle is to diffuse easily in the body and be subject to renal clearance.

[0026] In some embodiments, the nanocrystals have a diameter of 15 nm or less. The inventors believe that particles with a diameter of up to about 15 nm provide the advantages of the present invention for use as CT contrast agents. The stability of the material depends on the length of the dispersant (oligoethylene glycol) chain. Longer chains can stabilize larger particles.

[0027] In certain embodiments, the nanocrystals are characterized by an aspect ratio of 0.5 to 0.9. The particles obtained by the inventors were roughly rice grain shaped. Particles with uniform shape are expected to be more physiologically acceptable.

[0028] In certain embodiments, the dispersant molecules have a molecular mass of 500 g / mol or less. In certain specific embodiments, the dispersant molecules have a molecular mass of 400 g / mol or less.

[0029] In certain particular embodiments, the dispersant molecule is i. a surface-adsorbed moiety selected from nitrodopamine, nitroDOPA, DOPA, dopamine, and mimosine; ii. (CH2-CH2-O) n and in particular consisting of a CH3 moiety, wherein n is an integer selected from 2, 3, 4 and 5.

[0030] In certain particular embodiments, the dispersant molecule has the general formula: [ka] (wherein n is selected from 1, 2, 3, 4, 5, and 6) It is written as:

[0031] In certain particular embodiments, the dispersant molecule has the general formula: [ka] (wherein n is selected from 1, 2, 3, 4, 5, and 6) It is written as:

[0032] In certain embodiments, the nanoparticles comprise additional dispersant molecules, where the additional dispersant molecules are [ka] (In the formula, R dye is a fluorescent dye, optionally covalently linked to a nitrodopamine moiety via a linker having 1 to 25 atoms with an atomic number of 12 or greater. is selected from the group consisting of:

[0033] In certain particular embodiments, the additional dispersant molecule has the general formula: [ka] (wherein n is selected from 1, 2, 3, 4, 5, and 6; R X is selected from the group consisting of dye molecules (particularly fluorescent dye molecules), chemical functional groups that facilitate reaction with dye molecules (particularly chemical functional groups selected from N3, NH2, OH, CCH (ethynyl)) It is written as:

[0034] The term "fluorescent dye" in the present context relates to a small molecule capable of fluorescing in the visible or near infrared spectrum. Examples of fluorescent dye molecules or labels that exhibit visible color include, but are not limited to, fluorescein isothiocyanate (FITC), rhodamine, allophycocyanin (APC), peridinin chlorophyll (PerCP), phycoerythrin (PE), alexa Fluor (Life Technologies, Carlsbad, CA, USA), dylight fluor (Thermo Fisher Scientific, Waltham, MA, USA), ATTO dyes (ATTO-TEC GmbH, Siegen, Germany), BODIPY dyes (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-based dyes), 800CW dyes (indolium-based dyes), etc.

[0035] In certain embodiments, the dispersant is [ka] It is.

[0036] Alternatives to the oligoethylene glycol moiety as used herein include, but are not limited to, oligoglycerol and oligooxazoline chains. In certain embodiments, the density of dispersant molecules on the nanocrystals is between 0.5 and 5 / nm 2 It is. In certain particular embodiments, the nanoparticles contain dye molecules for optical positioning (localization).

[0037] Another aspect of the invention relates to a composition comprising a plurality of nanoparticles according to the aspects and embodiments discussed above. In certain embodiments, the composition is a stable aqueous colloidal suspension. In certain embodiments, the composition has a pH between pH 6 and pH 10. In certain specific embodiments, the composition has a pH between pH 6.5 and pH 8.0. To the inventors' knowledge, this is the first time such ultra-small HfO2 particles have been provided in a stable aqueous solution at physiological pH. In certain embodiments, the compositions are stable at neutral or basic pH values, particularly between pH 6 and pH 8. In certain embodiments, 80% of the nanoparticles have a diameter between 2.0 and 5.0 nm. In certain particular embodiments, 85% of the nanoparticles have a diameter between 2.5 and 4.5 nm.

[0038] A composition can be described in terms of its size heterogeneity by a size distribution or sigma used to fit the distribution to a Gaussian distribution. Exemplary parameters include, but are not limited to, mean diameter=2.6 nm, sigma=0.2 nm, size distribution=7.7%. A composition can be described by its zeta potential. The zeta potential is a function of pH. The inventors have found that for the relevant pH range, the zeta potential is always negative.

[0039] Another aspect of the invention relates to a composition according to the invention of any aspect and embodiment for medical use. Several uses have been described for HfO2 particles, notably as a radiotherapy enhancing agent (radiosensitizer) (Maggiorella et al., Future Oncol. 2012 Sep;8(9):1167-81) and as a computed tomography (CT) contrast agent (McGinnity, Nanoscale, 2016,8, 13627-13637).

[0040] In some embodiments, the concentration (v / v) of the nanoparticles in the composition ranges from 15 μmol / L to 1000 μmol / L. In some embodiments, the concentration (v / v) of the nanoparticles in the composition ranges from 15 μmol / L to 500 μmol / L. In some embodiments, the concentration (v / v) of the nanoparticles in the composition ranges from 15 μmol / L to 250 μmol / L.

[0041] Yet another aspect of the present invention relates to a method for preparing a composition according to the present invention, the method comprising the steps of: a. Providing a suspension of hafnium (IV) oxide (HfO2) nanocrystals stabilized by carboxylate ligands in an aqueous medium; b. adding to the suspension an alkaline solution of dispersant molecules, the dispersant molecules being as described herein: i. a surface-adsorbing moiety having two aromatic OH hydroxide functional groups at neutral pH, the surface-adsorbing moiety being selected from the group including catechol or gallol, particularly a 1,2-hydroxy-4-nitrophenyl moiety; ii. an oligo(ethylene glycol) moiety; The steps consist of Includes. The pH of the alkaline solution of the dispersant molecule is selected such that both aromatic hydroxide functional groups are deprotonated in the alkaline solution. c. The pH of the suspension is then adjusted to a physiological pH; Optionally, separate or isolate the composition. Particularly useful protocols for separation include size exclusion / spin filtration. e. Another optional step includes sonication to resuspend the aggregates.

[0042] One carboxylic acid useful as a stabilizer in the first step of this method is MEEAA, a ligand that binds poorly to the particles but binds well enough to initially disperse the particles in water.

[0043] In one example, the spin filtration step proceeds as follows: NC suspensions containing up to 50 mg of material dissolved in 2 ml of solvent are transferred through a 0.2 μm syringe filter into pre-rinsed Sartorius Vivaspin (30000 MWCO) spin filtration tubes. The suspension is diluted to a volume of 20 ml with Milli-Q water, after which the solution is spun in a centrifuge at 2100 rcf for 30 minutes. A minimum of two spin filtration cycles are performed with Milli-Q water until the filtrate is colorless. The concentrate is collected, evaporated, suspended in H2O, and sonicated for 30 minutes to ensure that all aggregates are resuspended and insoluble matter is minimized.

[0044] Medical treatment Also within the scope of the invention is a method for treating a pathological condition sensitive to radiation therapy, in particular cancer, in a patient in need of such treatment, comprising administering to said patient a composition according to the above.

[0045] Pharmaceutical Compositions, Administration Procedures / Dosage Forms and Salts According to one embodiment of the compounds according to the invention, the nanoparticles or nanoparticle suspensions according to the invention are provided as a pharmaceutical composition, pharmaceutical administration / treatment form or pharmaceutical dosage form.

[0046] In certain embodiments of the invention, the nanoparticles or nanoparticle suspensions of the invention are typically formulated into pharmaceutical dosage forms that provide easily controllable dosing of the drug, providing the patient with a product that is easy and convenient to handle. Similarly, there is provided a dosage form for the prevention or treatment of cancer comprising nanoparticles or a suspension of nanoparticles according to any of the above aspects or embodiments of the invention.

[0047] Additionally, the present invention encompasses pharmaceutical compositions comprising the nanoparticles or nanoparticle suspensions of the present invention and a pharma- ceutically acceptable carrier, hi further embodiments, the composition comprises at least two pharma- ceutically acceptable carriers, such as those described herein.

[0048] Certain embodiments of the invention relate to dosage forms for parenteral administration, such as for subcutaneous, intravenous, intrahepatic or intramuscular injection. Optionally, pharma- ceutically acceptable carriers and / or excipients may be present.

[0049] The dosing regimen for the compounds of the present invention varies depending on known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition and weight of the recipient; the nature and extent of the symptoms; the type of concomitant treatment; the frequency of treatment; the route of administration; the renal and hepatic function of the patient; and the desired effect. In certain embodiments, the compounds of the present invention can be administered in a single daily dose, or the total daily dose can be divided into two, three or four doses administered daily.

[0050] Manufacturing and treatment methods according to the present invention The present invention further encompasses, as a further aspect, the use of the nanoparticles or nanoparticle suspensions specified herein for use in a method for the manufacture of a medicament, as detailed above. In certain embodiments, the medicament is provided for radiotherapy (particularly cancer radiotherapy) or as an imaging agent.

[0051] When alternatives of a single separable feature are referred to in this specification as "embodiments," it is understood that the alternatives can be freely combined to form separate embodiments of the invention disclosed in this specification. The present invention further includes the following items.

[0052] item 1. a. nanocrystals comprising or consisting essentially of hafnium(IV) oxide (HfO2) having a diameter of 15 nm or less; b. a plurality of dispersant molecules attached to a surface of the nanocrystal, each of which comprises: i. a surface-adsorbing moiety selected from the group including catechol or gallol, particularly a 1,2-hydroxy-4-nitrophenyl moiety; ii. an oligo(ethylene glycol) moiety; and a dispersant molecule comprising or consisting essentially of both moieties. Nanoparticles comprising: 2. The nanoparticles according to item 1, wherein the nanocrystals have a diameter of 6 nm or less, in particular a diameter of 4 nm or less, more particularly a diameter of 3.5 nm or less. 3. The nanoparticles according to claim 1 or 2, wherein the nanocrystals are characterized by an aspect ratio of 0.5 to 0.9.

[0053] 4. Nanoparticles according to any one of items 1 to 3, in which the dispersant molecules have a molecular mass of 500 g / mol, in particular a molecular mass of 400 g / mol or less. 5. The dispersant molecule is i. a surface-adsorbed moiety selected from nitrodopamine, nitroDOPA, DOPA, dopamine, and mimosine; ii. (CH2-CH2-O) n a CH3 moiety, wherein n is an integer selected from 2, 3, 4, and 5; and in particular consisting of both parts, In particular, the dispersant molecules are [ka] 5. The nanoparticle according to any one of items 1 to 4,

[0054] 6. The density of dispersant molecules on the nanocrystals is 0.5-5 / nm 2 6. The nanoparticle according to any one of items 1 to 5, 7. Nanoparticles according to any one of items 1 to 6, comprising dye molecules for optical localization. 8. A composition comprising a plurality of nanoparticles according to any one of items 1 to 5. 9. The composition according to item 8, which is a stable aqueous suspension. 10. The composition according to item 9, having a pH between pH 6 and pH 10, in particular a pH between pH 6.5 and pH 8.0. 11. The composition according to item 7 or 8, wherein 80% of the nanoparticles have a diameter between 2.0 nm and 5.0 nm, in particular 85% of the nanoparticles have a diameter between 2.5 nm and 4.5 nm. 12. A composition according to any one of items 8 to 11 for pharmaceutical use. 13. The composition according to any one of items 8 to 11 for use as a radiotherapy potentiator (radiosensitizer).

[0055] 14. The composition according to any one of items 8 to 11 for use as a computed tomography (CT) contrast agent. 15. a. Providing a suspension of hafnium(IV) oxide (HfO2) nanocrystals stabilized by carboxylic acid ligands, in particular MEEAA, in an aqueous medium; b. adding to the suspension an alkaline solution of dispersant molecules, the dispersant molecules being i. a surface-adsorbing moiety comprising two aromatic hydroxide functional groups, said surface-adsorbing moiety being selected from the group comprising catechol or gallol, particularly a 1,2-hydroxy-4-nitrophenyl moiety; ii. an oligo(ethylene glycol) moiety; wherein both aromatic hydroxide functional groups are deprotonated in said alkaline solution; c. adjusting the pH of the suspension to physiological pH; d. Optionally, isolating the composition, particularly by size exclusion / spin filtration. 12. A method for producing a composition according to any one of items 8 to 11, comprising:

[0056] The present invention is further illustrated by the following examples and figures from which further embodiments and advantages can be derived. These examples are meant to illustrate the invention but not to limit its scope. [Brief description of the drawings]

[0057] [Figure 1](A) Solvothermal synthesis of HfO2 nanocrystals starting from 1 equivalent of Hf(O-tBu)4 and 88 equivalents of benzyl alcohol. (B) (Diffusion filtered) 1H NMR spectra of MEEAA-functionalized HfO2 NCs in different solvents. The α and β resonances belong to the residual hydroxy and methyl groups of methanol, respectively. (C) Transmission electron microscopy (TEM) images and data for the synthesized HfO2 NCs. The NC diameter of the roughly spherical NCs was calculated assuming a circular shape after measuring the surface area of ​​at least 150 NCs. Average: 2.64 ± 0.19 nm. Size distribution histograms and a magnified image of a single NC are shown in the bottom left and top right corners, respectively. [Diagram 2] (A) Ligand exchange between MEEAA-functionalized NCs and PA-PEG. (B) 1H NMR reference spectra in MeOD of free ligand as reference and of stepwise titration of MEEAA-functionalized NCs with PA-PEG, equivalents are relative to the total amount of MEEAA present. Resonances * are unidentified impurities. (C) 31P NMR spectrum in MeOD (4096 scans) of stepwise titration of MEEAA-functionalized NCs with PA-PEG. Broadened signals indicate NC attachment. (D) Diffusion-filtered 1H NMR spectrum of MEEAA-functionalized NCs in MeOD after addition of 1.3 equivalents of PA-PEG. Signals arising from bound MEEAA are shown in red and signals arising from PA-PEG are shown as blue stripes. CNC = 1210 μmol.L-1, corresponding to 34 mg of NCs of this size in 0.5 ml MeOD. [Diagram 3] Diffusion-filtered 1H NMR spectrum of a suspension of NCs in MeOD upon addition of 1.6 equivalents of PA-hex-PEG. [Figure 4] (A) and (B) 31P NMR spectra of PA-PEG and PA-hex-PEG functionalized NCs at different volume % DO. (C) Free ligand fraction of PA-PEG and PA-hex-PEG at different volume % DO as determined by peak deconvolution. [Diagram 5](A) Ligand exchange performed between MEEAA-functionalized NCs and nitrodopamine-mPEG. (B) 1H NMR spectra before and after ligand exchange titration performed in DO with nitrodopamine-mPEG. 1.5 equivalents of nitrodopamine-mPEG were added, pH was always kept above 5 during the addition, and the purified nitrodopamine-functionalized NCs spectrum was measured at pH = 7.4. CNC = 128 μmol.L-1, corresponding to 14.4 mg of NCs of this size in 2 ml DO. [Figure 6] Figure 1 shows the effect of pH on ligand binding and aqueous stability for purified NCs functionalized with PA-PEG, PA-hex-PEG, and nitrodopamine-mPEG. (A) Bound and unbound ligand fractions in DO based on NMR peak deconvolution at different pH values. (B) Z-average values ​​of NCs in DLS at different pH values. (C) Zeta potential of NCs at different pH values. All measurements were performed at 25 °C at constant ionic strength (0.01 mol.L-1 NaCl). [Figure 7] Stability of functionalized NCs in different concentrations of phosphate buffered saline (PBS) at pH 7.4 and 25 °C. (A) Colloidal stability of functionalized nanocrystals measured using DLS z-average at different PBS concentrations. (B) Stability of functionalized NCs over time in 2x PBS at pH 7.4 and 25 °C. [Figure 8] Colloidal stability maps for metal oxide nanocrystals functionalized with carboxylic acids, phosphonic acids, and catechol. In this example, we used HfO2 as the NC model system with its respective isoelectric points (IEPs), MEEAA as the carboxylic acid, PA-PEG as the phosphonic acid, and nitrodopamine-mPEG as the catechol. Green on the map correlates with good colloidal stability, areas gradually shifting to red indicate decreased colloidal stability, and red-shaded areas indicate poor colloidal stability. [Figure 9] Figure 2 shows UV-VIS spectra of purified nitrodopamine-mPEG functionalized NCs at different pH values ​​in HO. [Figure 10]The current clinical workflow for sentinel lymph node detection is compared with our proposed workflow, both on the example of a single female breast cancer patient. [Figure 11] (A) Solvothermal synthesis of HfO2 nanocrystals. (B) TEM images of HfO2 NCs. The major and minor diameters of spheroidal NCs were determined by measuring at least 400 particles. The size distribution histogram is shown in one corner of the inset. (C) 1H NMR spectra of nitrodopamine-mPEG functionalized HfO2 NCs in D2O. (D) Volumetric size distribution of HfO2 NCs in PBS determined by dynamic light scattering at 38 °C. (E) Concentration series of HfO2 NCs in PBS measured in CT. Contrast enhancement is expressed in Hounsfield Units (HU). [Figure 12] (A) Functionalization workflow to covalently attach dye to the NC surface. (B) Top: Excitation and emission spectra of IRDye 800CW-DBCO in PBS. Excitation spectrum was recorded following emission at 805 nm. Emission spectrum was recorded with excitation at 730 nm. Bottom: Excitation and emission spectra of NC-dye conjugate in PBS. Excitation spectrum was recorded following emission at 813 nm. Emission spectrum was recorded with excitation at 764 nm. [Figure 13] (A) Coronal and sagittal sections (0.2 mm slab thickness) of CT scans taken at different time points of a mouse subcutaneously injected in the left hind footpad with a NC dose of 0.38 mg NC / g body weight. (B) Volume rendering of the scan 75 min after injection. SLN, skeletal and soft tissues were segmented separately. [Figure 14] Shown is volume rendering of superimposed SPECT and CT scans taken at different time points in a mouse injected subcutaneously in the left hind footpad with a dose of 8.5 MBq of Nanocoll. [Figure 15] 1 shows the full structure of IRDye® 800CW DBCO. EXAMPLES

[0058] Example 1 HfO2-MEEAA model system We synthesized HfO2 nanocrystals (NCs) from hafnium tert-butoxide and benzyl alcohol at 220 °C by a well-established solvothermal process (Lauria et al., ACS Nano 2013, 7(8), 7041-7052) (Figure 1A). The HfO2 surface was functionalized with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (MEEAA, see Figure 1B) to stabilize the nanocrystals in toluene, and all unbound ligands were removed as previously described (De Roo et al., Chem Mater 2018, 30(15), 5485-5492). The nanocrystals have a diameter of 2.64 ± 0.19 nm (μ ± σ) by TEM (Figure 1C) and have a monoclinic crystal structure by X-ray scattering analysis (data not shown). The nanocrystal dispersion in toluene-d8 was 1 The H NMR spectrum showed only broadened resonances, which were assigned to the bound ligand (FIG. 1B). Indeed, the spectral broadening is a typical hallmark of bound ligands due to both homogeneous broadening (T2 relaxation) and inhomogeneous broadening (incomplete solvation of the ligand shell, see Roo, supra).

[0059] The nanocrystals can also be dispersed in ethanol, methanol and water, making them an ideal starting point for our investigations into ligand binding behavior in polar solvents. 1The H NMR spectrum looks significantly different (Figure 1B), with a sharp signal overlapping a broad resonance. We assign the sharp signal to the self-desorbed ligand, which is confirmed by the observation of two sets of resonances in Diffusion Ordered Spectroscopy (DOSY). DOSY separates the (overlapping) NMR resonances according to their diffusion coefficients, allowing us to separate the (fast diffusing) free ligand from the (slow diffusing) NC-bound ligand. We can selectively observe the bound ligand in the diffusion filtered spectrum (Figure 1B), and upon close inspection, we observe that the bound MEEAA resonances in methanol-d4 are slightly sharper than those in toluene-d8, indicating better solvation of the ligand in methanol. Given that all the ligands were bound in toluene, we reason that the solvent clearly plays a role in the adsorption-desorption equilibrium, for example by altering the solubility of the ligand. Indeed, even more of the MEEAA ligand desorbs in DO, but the nanocrystals remain stable at a pH range of 2 to 6. Because NCs rapidly precipitate at pH > 6, MEEAA is not a suitable ligand for many biomedical applications that are typically required to be stable at physiological conditions (pH = 7.4).

[0060] Besides the major MEEAA signal, we 1 Note that we also observed a broad resonance of low intensity in the aromatic region of the H spectrum, which we assigned to the benzoate ligand. Benzoic acid was previously identified as a by-product of nanocrystal synthesis and was found to be adsorbed on the nanocrystal surface. Post-synthesis functionalization of the surface with MEEAA apparently did not remove all of the benzoate from the surface; small amounts remain.

[0061] Example 2 Synthesis and Functionalization of HfO2 Nanocrystals We synthesized HfO2NCs by adapting an established solvothermal process from hafnium(IV) isopropoxide isopropanol adduct and benzyl alcohol at 220 °C (Figure 11A). After synthesis, the NCs were functionalized with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (MEEAA) to stabilize them in toluene, and all unbound ligands were removed as previously described. Importantly, clinically useful HfO2NCs with an average diameter of approximately 8 nm were obtained. 99m To achieve comparable lymphatic drainage to Tc-labeled nanoparticles (i.e., Nanocoll), we choose to synthesize HfO2 NCs of similar size after surface functionalization while remaining below the renal clearance limit. The NCs have long and short diameters of 5.04 ± 3.73 and 2.41 ± 0.85 nm (μ ± 3σ), respectively, by transmission electron microscopy (TEM) (Figure 11B) and are monoclinic (P 21 / c) has a crystal structure.

[0062] Nitrodopamine-mPEG is synthesized and purified as previously reported, and ligand exchange is then performed on the MEEAA-functionalized NCs in water to obtain a purified NC suspension after purification by spin filtration (a form of ultrafiltration). The 1H NMR spectrum of the nitrodopamine-mPEG-functionalized NCs (Figure 11C) shows only broad peaks. The peak broadening is a typical feature of bound ligands and is caused by both inhomogeneous line broadening (corresponding to ligand shell solvation) and homogeneous line broadening (T2 relaxation). The NC solution has a Z-average value of 12 nm in PBS by dynamic light scattering (DLS) (Figure 11D). The Z-average is a single value that represents the average particle size in solution and is highly susceptible to aggregation, and the agreement between the peaks observed in DLS and this low Z-average confirms that the suspension is free of aggregates. Due to the colloidal stability afforded by nitrodopamine-mPEG, highly concentrated NC suspensions could be created in PBS, and Figure 11E shows a concentration series of functionalized NCs in PBS scanned using CT at a tube voltage of 50 kV. A linear increase in X-ray attenuation with NC concentration can be observed, reaching over 6000 HU units, which is far higher than even the densest bones.

[0063] To provide a method for covalently attaching a payload to the NC surface in the dye molecules of the present invention, we synthesized 1-azido-N-(4,5-dihydroxy-2-nitrophenethyl)-3,6,9,12-tetraoxapentadecane-15-amide (nitrodopamine-PEG(4)-N3), see FIG. 10. A one-step nitration reaction starting from dopamine hydrochloride leads to the formation of nitrodopamine hemisulfate. Coupling between nitrodopamine hemisulfate and 15-azido-4,7,10,13-tetraoxapentadecanoic acid succinimidyl ester (NHS-PEG(4)-N3) was performed using N-methylmorpholine (NMM) acting as a non-nucleophilic base. The final nitrodopamine-PEG(4)-N3 ligand was purified using preparative high performance liquid chromatography (HPLC) and fully characterized using electron spray ionization / high resolution mass spectrometry (ESI-HRMS) and NMR spectroscopy. [ka]

[0064] The design of nitrodopamine-PEG(4)-N3 was decided based on the following criteria: the linker should be able to bind strongly to the NC surface, in this case via a nitrocatechol anchor, and should also contain a functional group that can be covalently attached to the payload after functionalization and purification of the NC. We chose to introduce a terminal azide because this group can undergo rapid bioorthogonal copper-free click reaction with any molecule containing a cyclooctyne functionality. The PEG spacer was chosen to be slightly longer than nitrodopamine-mPEG, so as not to significantly increase the solvadodynamic diameter of the NC, while positioning the azide functionality outside the crowded ligand shell to reduce possible steric hindrance during payload coupling.

[0065] We performed the coupling of NCs with dyes using the workflow depicted in FIG. 12A. In a representative functionalization with nitrodopamine-mPEG, we started with MEEAA-stabilized NCs in water and added 1.2 equivalents of deprotonated nitrodopamine-mPEG to perform the exchange, and after purification, the NC-bound organic mass based on TGA and NMR was 18.9 m%, resulting in a ligand density of 1.8 nm-2 and 60 ligands / NC. See SI for calculation methods. For the mixed ligand shell containing both nitrodopamine-mPEG and nitrodopamine-PEG(4)-N3, we aimed to have approximately one azide functional group per NC. With this goal in mind, we created a mixture of 1.18 equivalents of nitrodopamine-mPEG and 0.02 equivalents of nitrodopamine-PEG(4)-N3, which was preactivated and subjected to ligand exchange with MEEAA-functionalized NCs in water. After spin filtration, 1Only a broad conjugation peak could be observed by H NMR, indicating successful purification. Unfortunately, the broadness of the peak and the spectral overlap of nitrodopamine-mPEG and nitrodopamine-PEG(4)-N3 make quantification of azide conjugated to NCs impossible by ERETIC. However, we can perform this quantification in an indirect manner by dye coupling, which is the next step in the workflow. We selected the commercially available IRDye® 800CW DBCO as our dye of choice (see FIG. 15). This dye is frequently used as a near-infrared (NIR) fluorescent tracer and is currently used in multiple clinical trials targeting malignant lesions in breast, pancreas, head and neck, and glioma. This dye absorbs and emits light in the near-infrared spectrum, thus reducing the spectral overlap with tissue autofluorescence. It is also compatible with (pre)clinical grade imaging equipment. One equivalent of IRDye® 800CW DBCO (relative to the amount of azide likely attached to the NCs) was dissolved in endotoxin-free ultrapure water at pH 7 and added to the azide-functionalized NCs, stirred at 30° C. for 2 h, and the suspension was purified again by spin filtration in endotoxin-free water. By analyzing the sample concentration and filtrates using UV-VIS spectroscopy and applying the Beert-Lambert law, we were able to quantify the bound and remaining unbound dye, and thus indirectly measure the dye coupling efficiency. We found that approximately 0.7 dye molecules / NC were covalently bound after coupling. Figure 12B shows the normalized excitation and emission spectra of the freely diffusible dye molecules (top) and the NC-dye conjugate (bottom), both measured in PBS. NIR-dyes typically exhibit strong absorption in the NIR region, but this does not necessarily correlate with strong excitation efficiency in that region. We could observe that, following the 805 nm emission of the free dye, the most efficient region for exciting the molecule is indeed in the visible region. Interestingly, the excitation light spectrum of the NC-dye conjugate appears to be significantly different, following the 813 nm emission, the most efficient excitation region is in the NIR.Although it is beyond the scope of this study to clarify the exact mechanism of this phenomenon, we can hypothesize that the absorbance of nitrodopamine-mPEG in the 300-500 nm region affects the excitation of the conjugated dye. Using DLS, we observed that dye conjugation does not affect NC colloidal stability and does not cause a significant increase in hydrodynamic diameter compared to functionalized mixed shells.

[0066] Example 3 Fluorescence optimization of NC-dye conjugates Next, we determined whether concentration quenching occurs for the fluorescence emission of NC-dye conjugates. To exclude the possibility that nanocrystal concentration affects the fluorescence intensity, we created a concentration series in which the concentration of conjugated fluorescent dye decreases while maintaining equal amounts of NC in each sample. To achieve this, we mixed a suspension of dye-conjugated NCs with a suspension of nitrodopamine-mPEG functionalized NCs and diluted with PBS for each sample. Using an in vivo imaging system, sample fluorescence was visualized with bandpass excitation filters of 710 (± 15) nm and 745 (± 15) nm, respectively, while emission was observed within the ICG window (810-875 nm). A clear trend can be observed from the concentration series, where the fluorescence intensity increases with increasing conjugated dye concentration, reaching an optimum at approximately 28 μmol*L-1. Above this concentration, the detrimental effects of quenching again reduce the fluorescence signal. More specifically, the lowest concentration of conjugated dye, 14.4 μmol*L-1, shows approximately twice the average radiant efficiency (units used to compensate for non-homogeneous excitation light patterns) as the highest concentration, 460 μmol*L-1. This trend and optimal dye concentration was found for both excitation wavelengths, with the only difference being the higher average radiant efficiency of each sample using the 745 (± 15) nm excitation filter (Table 1). Thus, the 745 (± 15) nm filter is the optimal choice for further in vivo experiments. CT scans were performed to show that each fluorescent sample contained equal amounts of NCs.

[0067] [Table 1]

[0068] Example 4 Preliminary in vivo CT optimization For mice, subcutaneous footpad injection is considered the standard injection route to achieve lymphatic drainage with the popliteal lymph node behind the knee as the SLN and the iliac, inguinal, sciatic and renal LNs as the higher echelons. Although the anatomical regions of the LNs are visible in different CT sections, the LNs themselves are difficult or impossible to distinguish from the surrounding tissues. Starting with a NC suspension at a concentration of 291 mg HfO2 / ml in PBS, we slowly injected 50 μL subcutaneously into the left hind leg of the mouse. 50 μL was the maximum tolerated and feasible injection volume in mice at this site, resulting in a dose of 0.38 mg NC / gram body weight for this animal. Remarkably, immediately after injection, strong contrast enhancement in the SLNs (FIG. 13) could be observed, whereas no contrast enhancement was visible in the higher layers. Note also that at this time point, most of the contrast agent is still in the footpad. At 75 minutes after injection, contrast enhancement remains strong in the SLNs, and a slight increase in contrast enhancement can be observed in the iliac LNs, becoming stronger 150 minutes after injection. Mice were scanned longitudinally at several time points over a 24 hour period, during which contrast enhancement appeared to remain stable in the SLNs, while contrast enhancement in the iliac lymph nodes increased and stabilized approximately 4 hours after injection.

[0069] For each time point, we found that the contrast enhancement was mainly in the LN cortex, which is the entry point of lymph into the LN. From this observation, we hypothesized that NCs were not retained long in the medulla of the LN, but quickly exited via the efferent lymphatics to the next LN and eventually entered the bloodstream. Furthermore, we did not observe ipsilateral to contralateral spillover from the NC, indicating that the NCs followed a well-defined ipsilateral drainage route based on their injection location and did not prematurely leak out of the lymphatic system. During our observations over time, the animals did not show signs of pain or discomfort after NC injection and after initial recovery from the first sedation. However, to reduce the drug dose and the total drainage time, we further tested two doses in different mice: 0.19 mg NC / g body weight and 0.28 mg NC / g body weight. From both doses, we observe similar discharge results up to 0.38 mg NC / g body weight. The contrast enhancement for 0.19 mg NC / g body weight is lower than the highest dose, and the contrast enhancement in the SLN is directly visible after injection and can theoretically be used for SLN identification if the scan is performed at the correct time, but a trained eye is required to distinguish it from the background for further scan points. On the other hand, the dose of 0.28 mg NC / g body weight creates strong contrast enhancement in the SLN for all scan points while maintaining the required temporal separation in the appearance of NC in the upper layers, so this dose was selected for the following experiments as a good compromise between increasing contrast enhancement and minimizing NC volume. Furthermore, for further clinical applications, it is also attractive that the contrast enhancement, and therefore the NIR-fluorescence for dual modality probes, remains high enough in the SLN for a longer period of time, since the surgical procedure can take several hours to complete. Also, depending on the SLN location of the malignant lesion to be treated, radiographs may be taken instead of CT to confirm the SLN location. During this initial CT optimization, we were aware of the effects of slight anatomical variations between mice, as would be expected in any type of in vivo experiment using outbred animals.An example of this is the draining behavior of a particular lymph node: for 0.28 mg NC / g body weight, the renal LN became visible at the 240 min scan point, whereas mice receiving a dose of 0.38 mg NC / g body weight show no contrast enhancement in this LN at any time point. It should also be noted that this slight variation in the anatomical location of a particular LN is found between animals, e.g., the ischial LN is generally small in size and located close to the pelvis, and contrast enhancement from the NC in this node can easily be mistaken for signal arising from the pelvic bone instead. In this regard, particular attention should be paid to mice, as this anatomical proximity is more pronounced.

[0070] Example 5 Nanocrystal Formulation Approach From the concentration quenching experiments (Table 1) and preliminary in vivo CT experiments, we learned that there is a mismatch between the ideal dye concentration for CT imaging and the ideal NC concentration. Since soft tissues inherently have values ​​of Hounsfield units in the vicinity of 100-300 in CT, a minimal amount of NCs present in lymph nodes is necessary to visualize them. On the other hand, soft tissues do not show autofluorescence in NIR (with some exceptions), and in combination with highly sensitive fluorescence imaging, very little amount of dye is required. The concentration quenching experiments showed that injection of NC suspensions, where each NC contains one conjugated dye, would be detrimental to the final fluorescence intensity, and furthermore, other in vivo studies using NIR tracers have shown that as little as 1 μg of dye may be sufficient to visualize lymph nodes. 10 To satisfy the needs of both imaging modalities, instead of injecting NC suspensions, where each NC contains a dye molecule, we decided to perform a formulation approach in which the majority of NCs do not have bound dye. Thus, the formulation consists of a mixture of NCs functionalized only with nitrodopamine-mPEG (see FIG. 11A) and NCs conjugated with approximately 1 dye / NC (see FIG. 12), where the injection volume of NCs is still 0.28 mg NC / g body weight, but the concentration of the conjugated dye is only approximately 28 μmol*L-1. To ensure that both dye-free and dye-conjugated NCs show the same lymphatic drainage, a control experiment was performed. In this control experiment, mice were simultaneously injected with dye-free NCs at a dose of 0.28 mg / ml into the right hind footpad and with NCs at a dose of 0.28 mg / ml containing approximately 1 dye / NC into the left hind footpad. We could observe contrast enhancement in both the left and right SLNs simultaneously after injection, which became stronger over time. Accompanying this process, the left SLN showed clear signs of NIR fluorescence detectable through the skin, but the right SLN and limb did not, as expected. From 180 min post-injection onwards, secondary NIR fluorescent signals appeared in the bladder, indicating the onset of renal clearance of NCs.

[0071] Example 6 99m Comparison with Tc-Nanocoll Finally, we compared our NC results with the current clinical standard, where we injected 50 μL of 99m-Tc-Nanocoll subcutaneously into one of the hind footpads in three mice, with each injection having an activity between 6 and 9 MBq. Using a preclinical SPECT scanner, mice were scanned immediately after injection and at 40, 160, 280, and 400 minutes after injection. The time points chosen were less flexible than those for NC with CT, due to the inherently long acquisition time of SPECT. To obtain an acceptable signal-to-noise ratio, we narrowed the field of view to an area covering the popliteal, iliac, ischium, inguinal, and renal LNs, and scanned for 30 minutes. We chose not to scan for longer than 400 minutes, due to the half-life of the injected radioactive tracer being 6 hours. It is important to note that volume renderings for each scan point are shown in FIG. 14 and each SPECT scan is listed along with the CT scan because it is difficult or impossible to obtain anatomical information from a SPECT scan.

[0072] Similar to NC, SLNs become visible immediately after 99mTc-Nanocoll injection. Activity increases after 40 min, but only popliteal LNs are still visible. After 160 min, popliteal and iliac LNs as well as ischial and renal LNs were visible. Furthermore, as can be seen in FIG. 5, ischial LNs are close to the pelvis, which makes it difficult to distinguish LN contrast enhancement from bone in that region. Similar to NC, 99mTc-Nanocoll activity in LNs remains stable over the next 4 h, with activity mostly in the LN cortex. After approximately 24 h, most of the product has decayed.

[0073] Example 7 Clinical Incorporation of Nanocrystals Figure 10 compares the current clinical workflow with how our dual-modality NC can be incorporated with available infrastructure. A single preoperative injection of NC is followed by a preoperative CT scan. The non-radioactive nature of NC and the fast scan time of CT compared to SPECT / CT will allow more flexibility regarding the timing of preoperative injection. Then, once the SLN is identified by CT, intraoperative NIR-fluorescence is used to confirm the location of the SLN and guide its complete resection. Nevertheless, surgery may be performed several hours after the initial injection, which increases the likelihood of visualization up to the upper layers, as we have shown for both dual-modality NC and 99mTc-Nanocoll. This should not be a problem, however, since the preoperative scan is specifically timed to identify which node is the SLN, thus providing the surgeon with the necessary information to remove the correct node. Furthermore, if available in the operating room, a C-arm can be used as a means to intraoperatively confirm the presence of the SLN.

[0074] By removing radioactivity from the equation, we reduce overall patient radiation exposure and eliminate the need for costly SPECT scanners, instead allowing SLNs to be identified using more widely available and rapid techniques such as CT or conventional radiography. A second imaging modality, NIR-fluorescence, is part of the same imaging probe and therefore does not suffer from undesirable tissue extravasation like currently used small molecule dyes.

[0075] Example 8 Competitive Binding of Phosphonic Acids To evaluate the binding strength of phosphonic acids in methanol, we selected (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)phosphonic acid (PA-PEG) as a ligand with a structure comparable to MEEAA (Figure 2A). 1In the H NMR spectrum, the PA-PEG resonance 1 has a chemical shift of 2.05 ppm that is clearly separated from the MEEAA resonance, allowing us to selectively monitor the PA-PEG bond (Figure 2B). Since MEEAA has a methoxy group and PA-PEG does not, resonance f is used to obtain selective information on the MEEAA bond. The other resonances (2-6 and b-e) overlap.

[0076] Starting with MEEAA-stabilized HfO2 nanocrystals in methanol-d4, we sequentially added PA-PEG to 1 The H NMR spectrum is monitored, see FIG. 1B. During the titration, we observe the gradual appearance of a broad resonance around 2.2 ppm, which we assign to resonance 1 of the bound PA-PEG. This is accompanied by a narrower resonance f (FIG. 2B), indicating the removal of MEEAA from the nanocrystal surface. Within the aromatic region, we also observe the desorption of benzoic acid. We conclude that PA-PEG effectively replaces MEEAA and benzoic acid. Given the absence of free phosphonic acid, the exchange is quantitative for most titrations. After the addition of 1.3 equivalents of phosphonic acid, a sharp signal appears in the same region, indicating that free PA-PEG is now present. 31 The same conclusion can be drawn from the P spectrum (Figure 2C), where first the intensity of a broad signal increases and then sharpens after the addition of more than 1 equivalent. 31 P signal appears. Interestingly, the bound PA-PEG resonance still increases slightly in intensity between 1.3 and 1.6 equiv. We reason that the exchange of the final moiety is not quantitative and that the remaining carboxylate ligands are more difficult to remove. This observation is consistent with the binding affinity (ΔG ads ) is not a single fixed value for all MEEAA ligands, but rather a distribution, similar to what has been shown previously for CdSe nanocrystals.

[0077] Unfortunately, the NMR spectrum features many spectral overlaps, with superimposed signals of free and bound ligand. At the end of the titration, the resonances of the free ligand dominate in the spectrum. To gain a deeper insight into the composition of the ligand shell at this point, we turn to the spectrum with the diffusion filter (Figure 2D). Resonances 2-6 overlap with resonances b-e, but the clear presence of resonance f indicates the presence of residual MEEAA on the surface. Based on the diffusion filter profile of bound MEEAA, we assign the red shaded region to the part of the spectrum belonging to MEEAA. The blue patterned region is assigned to PA-PEG. The ratio between the regions gives a rough estimate of the ligand shell composition: 10% MEEAA and 90% PA-PEG, but this estimate ignores the differences in the relaxation behavior of the different resonances. Benzoate is still present on the surface according to the normal 1H spectrum, but the signal-to-noise ratio of these resonances is too low in the spectrum with the diffusion filter due to the fast T2 relaxation (due to their rigidity and closeness to the surface). The above results therefore confirm that the exchange between carboxylate ligands and PA-PEG is not complete in methanol, a conclusion that contrasts with the relative binding affinities of fatty acids and alkylphosphonic acids in non-polar solvents, where the phosphonic acid quantitatively replaces the carboxylic acid with a 1:1 stoichiometry.

[0078] Self-desorption of MEEAA in methanol has already shown that ligand solubility can alter binding affinity. Indeed, ligand binding is an equilibrium process governed by the chemical potentials of the respective species:

number

[0079] Thus, this adsorption-desorption equilibrium depends on the chemical potential (and hence its solubility) of the free ligand. To practically explore this concept, we designed a ligand shell structure that mimics micelles with both hydrophobic and hydrophilic segments. For this, we chose the ligand (6-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-hexyl)phosphonic acid (PA-hex-PEG), see FIG. 3. We hypothesized that this ligand would self-assemble more easily on the nanocrystal surface because the hydrophobic regions would reduce its solubility in polar solvents. With PA-hex-PEG, we performed the same titration experiments as before. The results are largely similar (not shown), showing an exchange of the carboxylate ligand for PA-hex-PEG. However, this time, most of the benzoate ligand is rejected by PA-hex-PEG (although not completely), and the MEEAA resonance is barely detectable in the spectrum with a diffusion filter (FIG. 3). Up to 3% of MEEAA is still present in the ligand shell. We conclude that competitive binding can indeed be manipulated by altering the ligand solubility. Interestingly, benzoic acid appears to have a higher binding affinity than MEEAA, which may be attributed to its higher acidity and lower solubility in methanol.

[0080] Example 9 Purification of Phosphonate-Capped Nanocrystals and Transfer to Water With our ultimate goal of providing a stable surface chemistry for biomedical applications in mind, we attempted to purify our dispersion and disperse it in aqueous media. Precipitation-redispersion cycles are the most common method for purifying nanocrystals. However, the high multifunctionality of the ethylene glycol segments results in colloidal stability in a wide range of solvents, and non-solvents such as hexane do not mix well with methanol. Thus, we choose to purify our dispersion using spin filtration, a form of ultrafiltration. This technique is based on semipermeable membranes that allow small molecules to pass through the pores but not the larger nanocrystals (like dialysis). Separation is performed in a centrifuge to facilitate purification. First, the nanocrystal suspension is placed in a spin filter and further diluted with pure methanol. Dilution does not cause ligand desorption. After filtration, a concentrated dispersion of nanocrystals is collected. The purification is successful, as it was shown that almost all unbound species were removed after three purification cycles. Comparison of the spectra using the diffusion filter before and after spin filtration showed a perfect match, proving that purification did not change the ligand shell composition. An interesting difference was observed between PA-PEG and PA-hex-PEG when the solvent composition was gradually changed from pure methanol-d4 to D2O (Figure 4). PA-PEG is gradually desorbed from the surface when the water content is increased, while PA-hex-PEG remains tightly bound. This further emphasizes our hypothesis that PA-hex-PEG behaves as a micelle mimic, avoiding contact between water and hydrophobic segments. By binding the nanocrystal surface, the PA-hex-PEG ligand creates a hydrophobic inner shell with alkyl-alkyl interactions and a hydrophilic outer shell with ethylene glycol moieties as hydrogen bond acceptors for water molecules. On the other hand, PA-PEG is highly water soluble and therefore its affinity for the hafnium oxide surface is decreased by increasing the water content.

[0081] [ka]

[0082] Example 10 Synthesis and conjugation of catechol We synthesized N-(4,5-dihydroxy-2-nitrophenethyl)-2-(2-(2-methoxyethoxy)ethoxy)acetamide (nitrodopamine-mPEG). See Scheme 2. We chose nitrocatechol instead of unsubstituted catechol because the nitro group reduces the pKa value of catechol and improves the oxidative stability of catechol. Starting from dopamine hydrochloride, a one-step nitration reaction leads to the formation of nitrodopamine hemisulfate. Separately, MEEAA was converted into activated N-hydroxysuccinimide ester (MEEAA-NHS) using N,N'-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS) and 4-dimethylaminopyridine (DMAP). Coupling between nitrodopamine hemisulfate and MEEAA-NHS was performed using N-methylmorpholine (NMM) acting as a non-nucleophilic base. The final nitrodopamine-mPEG ligand was purified using preparative HPLC and fully characterized using ESI-HRMS and NMR spectroscopy.

[0083] When we performed a competitive binding experiment similar to that previously performed (adding nitrodopamine-mPEG to MEEAA-capped HfO2 nanocrystals in methanol-d4), we found that 1 equivalent of nitrodopamine-mPEG was unable to effectively compete for the nanocrystal surface with even 0.4 equivalents of catechol, and a freely diffusing nitrodopamine-mPEG signal could be observed. Such low binding affinity is consistent with many reports of successful surface functionalization with catechol (Okada et al., ChemistrySelect 2018, 3(29), 8458-8461; Dragoman et al., Chemistry of Materials 2017, 29(21), 9416-9428; Amstad et al., The Journal of Physical Chemistry C 2011, 115(3), 683-691; Amstad et al., Nano Letters 2009, 9(12), 4042-4048; Gillich et al., Journal of the American Chemical Society 2011, 133(28), 10940-10950; Xie et al., Adv. Mater. 2007, 19(20), 3163-3166; Bae et al., Bioconjugate Chemistry 2010, 21(3), 505-512), which was completely unexpected. However, in these reports, water or biological buffers were used as the solvent. Therefore, we designed a direct competitive binding experiment in water. Fortunately, MEEAA-stabilized HfO2 nanocrystals are stable in D2O, although a significant portion of MEEAA desorbs. Addition of 1 equivalent of nitrodopamine-mPEG without pH adjustment results in a cloudy suspension at pH=2. Adjusting the pH to pH=5 results in a stable suspension. To allow for systematic stepwise addition of nitrodopamine-mPEG, we prepared a stock solution of nitrodopamine-mPEG containing 2 equivalents of NaOD to doubly deprotonate the catechol. Addition of base changes the color of the solution from pale yellow to a deep burgundy wine color. We added this stock solution in 0.5 equivalent steps to a suspension of MEEAA-stabilized HfO2 and standard1 H NMR and NMR spectra with diffusion filters were recorded. After the addition of 0.5 equivalents, we do not observe a sharp signal belonging to nitrodopamine-mPEG, but we do observe desorbed benzoic acid and desorbed MEEAA. In the spectrum with diffusion filters, we also observe a clear change. In the aromatic region, the broad signal of benzoate is replaced by the broad signal of nitrodopamine-mPEG. The shape of the peak in the region 3-4 ppm also changes. As more nitrodopamine-mPEG is added, the exchange continues, and by the addition of 1.5 equivalents, a sharp (unbound) nitrodopamine-mPEG signal is detected. The suspension remains stable despite a pH of 10.3, and at this point in the titration, further evidence is obtained that the ligand exchange was successful, since MEEAA-stabilized nanocrystals should precipitate at pH>6. We conclude that nitrodopamine-mPEG can quantitatively displace MEEAA from the nanocrystal surface if the pH>5.

[0084] The NCs were purified again using multiple cycles of spin filtration with Milli-Q water as the solvent until the filtrate was nearly colorless. The concentrate was evaporated, redispersed in D2O, and the pH was adjusted to 7.4. Figure 5B shows the normal concentration of the purified suspension. 1 The purity of the sample is remarkable, with only the broadened resonances associated with nitrodopamine-mPEG being observed, including the H NMR spectrum. 1 Based on H NMR and a TGA mass loss of 25.79%, we estimate the nitrodopamine-mPEG density at the nanocrystal surface to be 1.53 nm. -2 We conclude that nitrodopamine-mPEG forms a tightly bound ligand shell on the nanocrystals at physiological pH with no signs of desorption.

[0085] Example 11 pH Dependence of Ligand Binding Since pH appears to play an important role in ligand binding in aqueous environments, we systematically varied the pH from 3 to 10,1 H NMR, 31 P NMR and dynamic light scattering (DLS) were used to assess ligand binding and colloidal stability (Figure 6). From NMR, we were able to determine the bound ligand fraction for the phosphonate ligand. 31 By peak deconvolution of the P resonance, and for nitrodopamine-mPEG, aromatic 1 The H resonance peaks are extracted by deconvolution. From the DLS measurements, we obtained the Z-average value and the zeta potential. The Z-average value is a single value that represents the average particle size and is most affected by aggregation. The zeta potential indicates the degree of electrostatic repulsion between the nanocrystals (zeta potential values ​​above +25 mV or below -25 mV indicate a stable suspension). From FIG. 6, we clearly observe that the bound ligand fraction decreases with increasing pH for both PA-PEG and PA-hex-PEG. With reference to the pKa values ​​of ethylphosphonic acid (pKa1=2.43, pKa2=8.05), it is striking that the bound ligand fraction decreases most rapidly near pKa2. We infer that the second deprotonation of the phosphonic acid causes repulsion between the ligands in the ligand shell, increasing the solubility of the ligand in water. Both of these effects lead to a reduction in the bound ligand fraction. Not surprisingly, the loss of ligand has a detrimental effect on colloidal stability, with the Z-average increasing significantly for pH > pKa2. In summary, PA-hex-PEG behaves slightly better than PA-PEG, but the difference is small. It is plausible that the double anion phosphonate compensates for the short hydrophobic segments.

[0086] A complete loss of colloidal stability would be expected upon ligand desorption. However, no turbidity is visually observed in the samples and the Z-average value remains below 100 nm. Note that the zeta potential drops below -25 mV at pH>8. Steric stabilization is lost as ligand desorption proceeds, but is replaced by electrostatic stabilization, preventing the NCs from becoming completely unstable. The negative charge could come from residual, bound doubly deprotonated phosphonates on the nanocrystal surface or, more likely, from hydroxide adsorption. Indeed, in water, multiple adsorption-desorption equilibria exist simultaneously.

number

[0087] When acid-base equilibrium is also added, the complex pH dependence of the system begins to be realized. According to Figure 6, phosphonic acids keep nanocrystals stable between pH 3 and 8 in static systems. However, in dynamic biological environments (e.g. blood vessels), many competing ligands are present and desorbed ligands are rapidly removed. The equilibrium is adjusted, so that ligands are continuously desorbed from the surface, eventually resulting in loss of colloidal stability. Despite acceptable Z-average values ​​at physiological pH, this dynamic behavior may explain why single phosphonate ligands are not always successful in completely preventing aggregation in physiological media.

[0088] Interestingly, nitrodopamine-mPEG exhibits almost the complete opposite behavior, failing to provide stable colloidal dispersions under acidic conditions, with a very sharp transition around pH=5. This is evidenced by the steep increase in the Z-average value (Figure 6). Between pH 5 and 10, all nitrodopamine-mPEG ligands remain bound, and only at pH=11 does the bound fraction decrease slightly from 100% to 97%. This translates to a superior colloidal stability in the pH range of 5-11. We conclude that for aqueous applications at physiological pH, nitrodopamine-mPEG outperforms PA-PEG and PA-hex-PEG. On the other hand, for aqueous applications at acidic pH, the phosphonate ligands are more suitable. To test whether there is a temperature dependence of the binding dynamics of the functionalized NC system, we performed variable temperature NMR measurements in D2O at pH 7.4 between 25 and 60 °C. 1 H NMR spectra showed no change in the ligand adsorption / desorption equilibrium for both phosphonic acid and nitrodopamine-mPEG, which leads us to tentatively conclude that the results from the pH titrations are also interpreted as physiological temperatures.

[0089] To illustrate the complementary behavior of phosphonate and catechol, we performed a competitive exchange reaction on purified nitrodopamine-mPEG NCs in DO. One equivalent of PA-PEG was added compared to the original amount added to functionalize the NCs with nitrodopamine-mPEG, and NMR measurements were performed at different pH values. The results clearly show that at acidic pH values, partial exchange with PA-PEG occurs, as evidenced by the sharp nitrodopamine-mPEG signal in the aromatic region and the appearance of a methylene triplet around 3 ppm. Nitrodopamine-mPEG NC suspensions, which are normally completely destabilized below pH 5, remained colloidally stable at pH 2.22 due to the mixed catechol-phosphonate ligand shell. The exchange equilibrium mostly shifts back toward nitrodopamine-mPEG as the pH moves toward neutral and basic values.

[0090] Example 12 Stability in phosphate buffered saline (PBS) Finally, we evaluated the stability of NCs functionalized with PA-PEG, PA-hex-PEG, and nitrodopamine-mPEG in phosphate buffered saline (PBS). Stability in PBS is an important prerequisite for biomedical applications, since many in vivo experiments involve the injection of the desired drug or imaging agent in saline or PBS. 1× PBS buffer has a concentration of 137 mmol.L. -1 NaCl, 2.7 mmol.L -1 KCl, 10 mmol.L -1 Na2HPO4 and 1.8 mmol.L -1 KH2PO4, at standard concentrations. When the concentrations are halved or doubled, these buffers are referred to as 0.5x PBS and 2x PBS, respectively. It is clear that PBS contains a relatively high concentration of salt and phosphate ions (which compete for the surface), posing a real challenge to the stability of our functionalized nanocrystals. First, we varied the PBS concentration and immediately measured the Z-average values ​​by DLS (Figure 7A). Up to 1.5x PBS, all ligands keep the nanocrystals colloidally stable. As expected, PA-PEG is the weakest ligand and is unable to prevent the onset of nanocrystal aggregation in 2x PBS solution. Next, the stability of all functionalized NCs was monitored over time in 2x PBS (Figure 7B). Clearly, the PA-PEG-functionalized nanocrystals rapidly aggregate, with precipitation visible after several hours. The colloidal stability of the PA-hex-PEG-functionalized nanocrystals steadily decreases over 24 hours before complete aggregation. Only the nitrodopamine-mPEG functionalized nanocrystals remain completely stable: there is no sign of aggregation over the course of 48 hours, and the suspension remains visually clear for at least one month.

[0091] Example 13 Observations From the above results, it is clear that surface chemistry becomes more complex when going from non-polar to polar (e.g. aqueous) solvents. In non-polar solvents, charged ligands or nanocrystals are thermodynamically unstable, leading to a limited set of binding motifs and clear ligand exchange rules. For example, self-desorption of oleate (deprotonated oleic acid) does not occur in toluene. For the binding motif of PbS(PbX2), removal of the entire Lewis acid PbX2 has been observed in chloroform or coordinating solvents such as THF. Similarly, for HfO2(H,OOCR), desorption of oleic acid is possible by recombination of the carboxylate with the proton. However, these limitations disappear in polar solvents where the charges are stabilized and the proton and carboxylate have independent adsorption / desorption equilibria. See also Eqs. 2-4.

[0092] From the above data, we constructed a colloidal stability map showing which ligands provide colloidal stability in a certain pH range. See FIG. 8. It is also interesting to correlate this stability map with the pKa of the ligands. We take the pKa value of ethylphosphonic acid (pKa1=2.43, pKa2=8.05) as a reference for the PA-PEG and PA-hex-PEG ligands. At pH=3 (where the particles are stable), we calculate that 96% of the phosphonic acid is mono-deprotonated. It can be assumed that the phosphonate binds to the nanocrystal surface in this form. At pH>8 (above pKa2), the bound ligand fraction decreases rapidly and the particles start to aggregate (FIG. 6). The double deprotonation has two consequences: (1) the ligand becomes more soluble in water, and (2) electrostatic interligand repulsions appear instead of stabilizing hydrogen bonds. Both of these effects promote ligand desorption. Furthermore, the isoelectric point of hafnia occurs at pH=8, rendering the surface negatively charged at pH>8, further reducing the binding affinity of negatively charged phosphonate ligands.

[0093] A similar rationale applies to nitro-dopamine-mPEG (pKa1 = 6.6, pKa2 = 11, based on the nitro-dopamine pKa values). At pH = 5 (where the particles are unstable), the inventors calculate that approximately 98% of the ligand is fully protonated and can only interact with the surface via weak hydrogen bonds. Above pH 5, more nitro-dopamine-mPEG begins to lose one proton, can coordinate with the surface metal sites, and provides additional hydrogen bonds to further stabilize the binding state. This is confirmed by comparing the UV-VIS spectrum of nitro-dopamine-mPEG bound to HfO2NC with the reference spectrum of the free ligand. See Figure 9. Between 5 < pH < 11, the inventors find a species that has lost one proton. Above pH > 11, the inventors again observe the appearance of the free ligand in NMR and the doubly deprotonated species in UV-Vis. For the reasons stated above, the doubly deprotonated species has a low binding affinity to the surface. Finally, the carboxylic acid can only lose one proton, and the inventors observe that MEEAA (pKa = 3.4) keeps the NC stable up to pH = 6. The inventors hypothesize that these are less stable than phosphonates and catechols due to the lack of intermolecular hydrogen bonds.

[0094] The stability experiments in buffer show two additional variables, namely competing ligands and salts. In phosphate buffer, a high concentration of phosphate is present, which competes for the surface but does not provide steric stabilization. Phosphonic acids (PA-PEG and PA-hex-PEG) desorb at pH = 7.4 (25% desorption, see Figure 6), so they are in equilibrium and are thus slowly replaced over time. From the inventors' competition experiments, nitro-dopamine-mPEG binds tightly (0% desorption) and cannot be replaced by phosphonic acids. Thus, the high stability of the particles capped with nitro-dopamine-mPEG.

[0095] The above discussion shows that in aqueous media, surface chemistry is a complex interplay between multiple factors. In buffers, there is pH-dependent surface charge, pH-dependent deprotonation of the ligands, and competition by phosphate. Thus, with the pKa of the ligands and the isoelectric point of the nanocrystals available, the colloidal stability map (Figure 8) can be used to begin to predict which ligands will confer colloidal stability at a particular pH.

[0096] Example 14. Synthetic Details (6-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-hexyl)phosphonic acid and (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)phosphonic acid were purchased from SiKEMIA. N-hydroxysuccinimide (≧98%) and dopamine hydrochloride (≧99%) were purchased from Acros Organics. 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (>95.0%) was purchased from TCI Chemicals. Hafnium hafnium(IV) isopropoxide isopropanol adduct (99.99%) was purchased from Fisher Scientific. IRDye 800CW-DBCO was purchased from LiCor. Hafnium(IV) tert-butoxide (99.99%), N,N′-dicyclohexylcarbodiimide (99%), 4-(dimethylamino)pyridine (≧99%), 4-methylmorpholine (99%), sodium nitrite (≧99.0%) and solvents used in the synthesis were purchased from Sigma Aldrich. All purchased reagents were used without further purification. All deuterated solvents were purchased from Sigma Aldrich or Eurisotop.

[0097] Synthesis of MEEAA-NHS. 4 mmol (0.7128 g) of 2-[2-(2-methoxyethoxy)ethoxy]acetic acid and 4.2 mmol (0.4828 g) of N-hydroxysuccinimide were dissolved in 8 ml of dry THF in a pre-dried vial and cooled to 0° C. 4.2 mmol (0.866 g) of N,N′-dicyclohexylcarbodiimide were dissolved in 4 ml of THF in another pre-dried vial and added dropwise to the first mixture by air-free transfer. After stirring the mixture for 15 min at 0° C., 0.2 mmol (0.024 g) of the catalyst 4-dimethylaminopyridine was added. The mixture was stirred overnight at room temperature to obtain a cloudy white mixture. The cloudy solution was transferred to a 50 ml centrifuge tube and centrifuged at 5000 rcf for 5 min, the supernatant was transferred to a flask using a 0.2 μM PTFE syringe filter, and the white solid was washed once with 10 ml of THF to recover the remaining product. After removing the solvent using a rotary evaporator, the viscous liquid was dissolved in 12 ml DCM and the organic phase was extracted four times with MQ water and two more times with brine. The organic phase was dried over MgSO4 and evaporated to dryness using a rotary evaporator. The product was recovered as a colorless viscous liquid in 83% yield.

[0098] 1 H NMR (500MHz, CDCl3): δ 4.5 (s, 2H) δ 3.8-3.76 (m, 2H) δ 3.7-3.66 (m, 2H) δ 3.65-3.6 (m, 2H) δ 3.55-3.51 (m, 2H) δ 3.36 (s, 3H) δ 2.82 (s, 4H). 13 C NMR (500MHz, CDCl3): δ 168.73 (s) δ 166.02 (s) δ 77.24 (s) δ 71.91 (s) δ 71.37 (s) δ 70.607 (s) δ 70.602 (s) δ 66.55 (s) δ 59.06 (s) δ 25.58 (s). Calculated for HRMS 275.26 [M], 292.9 [M+NH4] + Actual value.

[0099] Synthesis of nitrodopamine hemisulfate. 8.753 mmol (1.66 g) of dopamine hydrochloride and 35.219 mmol (2.43 g) of NaNO2 were dissolved in 100 ml MQ water and cooled in an ice bath. 8.33 ml of pre-chilled 20% H2SO4 was added dropwise to the mixture with vigorous stirring, during the addition the mixture turned yellow and cloudy with the formation of brown gas. The mixture was removed from the ice bath and stirred at room temperature for 12 h. After the resulting yellow and cloudy solution was cooled again in an ice bath, the solid was collected by suction filtration using a por4 fritted glass filter. The solid was then washed twice with 50 ml of ice-cold MQ water, once with 50 ml of ice-cold absolute ethanol, and twice with 50 ml of ice-cold diethyl ether. The yellow powder was collected and dried under vacuum overnight, with a final yield of 50%.

[0100] 1 H NMR (500MHz, DMSO-d6): δ 7.46 (s, 1H) δ 6.73 (s, 1H) δ 3.12-2.99 (m, 4H). 13 C NMR (500MHz, DMSO-d6): δ 156.49 (s) δ 145.24 (s) δ 136.31 (s) δ 127.32 (s) δ 111.14 (s) δ 39.15 (s) δ 31.56 (s). HRMS calculated for 296.25 [M], 197.00 [M-H2SO4-H] - Actual value.

[0101] Synthesis of nitrodopamine-mPEG 1.784 mmol (491 mg) MEEAA-NHS and 2.854 mmol (846 mg) nitrodopamine hemisulfate were dissolved in 25 ml dry DMF in a pre-dried flask to give a deep orange solution. The flask was sealed, flushed with argon and cooled in an ice bath. 785 μL N-methylmorpholine was added dropwise to the mixture using air-free method and after stirring for approximately 10 minutes the solution became cloudy. The mixture was stirred at room temperature for 48 hours before being evaporated under vacuum at 40° C. overnight to remove the DMF and give a deep brown liquid. 40 ml 1M HCl was added to the crude product and extracted three times with 40 ml CHCl3, taking care that a deep brown solid formed at the liquid interface during the process and did not enter the organic phase. The organic phase was extracted two more times with 50 ml brine, dried over Na2SO4 and evaporated using a rotary evaporator. The resulting solid was purified by preparative HPLC using a gradient from solvent A (MQ water containing 0.1% TFA) to solvent B (ACN containing 0.1% TFA) and the final product was isolated after lyophilization as a fluffy white to yellow solid in 75% yield.

[0102] 1 H NMR (500MHz, MeOD): δ 7.54 (s, 1H) δ 6.7 (s, 1H) δ 3.93 (s, 2H) δ 3.62(s, 4H) δ 3.61-3.58 (m, 2H) δ 3.57-3.5 (m, 4H) δ 3.35 (s, 3H) δ 3.07 (t, 2H, J = 6.68Hz). 13 C NMR (500MHz, MeOD): δ 173.05 (s) δ 152.4 (s) δ 145.53 (s) δ 141.06 (s) δ 129.39 (s) δ 119.61 (s) δ 113.56 (s) δ 72.97 (s) δ 72.07 (s) δ 71.4 (s) δ 71.39 (s) δ 71.24 (s) δ 59.21 (s) δ 40.47 (s) δ 34.14 (s). Calculated value for HRMS 358.35 [M], 357.35 [MH] - Actual value.

[0103] Synthesis of Nitrodopamine-PEG(4)-N3 0.103 mmol (40 mg) of NHS-PEG(4)-N3 (15-azido-4,7,10,13-tetraoxa-pentadecanoic acid succinimidyl ester) and 0.1648 mmol (48.8 mg) of nitrodopamine hemisulfate were dissolved in 2 mL of dry DMF in a pre-dried flask to give a deep orange solution. The flask was sealed, flushed with argon and cooled in an ice bath. 45.3 μL of N-methylmorpholine was then added dropwise to the mixture using an air-free method. The mixture was stirred at room temperature for 48 h to give a cloudy solution containing a yellow to brown solid. The DMF was evaporated overnight under vacuum at 30 °C. The crude product was then dissolved in 3 ml of Milli-Q water and diluted to 5 mL with 1 mol*L-1 HCl solution with the aim of achieving a pH of approximately 1. The aqueous crude product was extracted three times with 5 ml of CHCl3, taking care that a dark brown solid formed at the liquid interface during the process and did not enter the organic phase. The organic phase was evaporated using rotary evaporation and dried again overnight under vacuum at 30°C to give a brown to yellow sticky solid. After dissolving the resulting solid in 3 mL of a 50 / 50 ACN / Milli-Q water mixture and removing insoluble material with a 0.2 μm syringe filter, the solution was purified by preparative HPLC using a gradient from solvent A (Milli-Q water with 0.1% TFA) to solvent B (ACN with 0.1% TFA) and lyophilization, the final product was isolated as a yellow to brown solid in 70% yield.

[0104] 1H NMR (400MHz, D2O): δ 7.66 (s, 1H), 6.82 (s, 1H), 3.76-3.44 (m, H), 3.07 (t, 2H, J = 6,4Hz), 2,45 (t, 2H, J = 6.11Hz). 13C NMR (100,6MHz, D2O): δ 173.86 (s), 150.5 (s), 142.85 (s), 140.19 (s), 129.4 (s), 118.83 (s), 113.18 (s), 69.58 (s), 69.55 (s), 69.5 (s), 69.48 (s), 69.44 (s), 69.18 (s), 66.71 (s), 50.11 (s), 39.12 (s), 36.04 (s), 32.51 (s). HRMS calculated for 471,2 [M], 470,12 [M - H] - Actual value.

[0105] Synthesis of hafnium oxide nanocrystals NCs were synthesized from hafnium(IV) tert-butoxide (4.8 mmol, 2.26 g, 1.94 mL) and anhydrous benzyl alcohol (40 mL) according to Lauria et al. (ACS Nano 2013, 7(8), 7041-7052). After synthesis, the nanocrystals were collected by adding diethyl ether (17 mL) to the reaction mixture and centrifuging (5000 rcf, 3 min) in a plastic centrifuge tube. The precipitate was washed three times with diethyl ether (17 mL). For functionalization with 2-(2-(2-methoxyethoxy)ethoxy)acetic acid, the precipitate was first dispersed in 17 mL of toluene to obtain a milky cloudy liquid. After addition of 335 μL of 2-(2-(2-methoxyethoxy)ethoxy)acetic acid (0.2885 g, 1.62 mmol) and sonication for 30 min, a clear suspension with some insoluble material was obtained. The insoluble material was removed by centrifugation (5000 rcf, 5 min) and the clear upper layer was transferred to a new plastic centrifuge tube. The NCs were precipitated by adding 1:2 volumes of hexane (isomer mixture) and after centrifugation (5000 rcf, 5 min), the upper organic phase was removed and the NCs were resuspended in toluene. This purification step was repeated three more times before a final resuspension in toluene. The purified NC suspension in toluene remains stable for at least one year. The dispersion in toluene can be dried and dispersed in ethanol, from which the dispersion can be dried again and resuspended in MeOH or water.

[0106] TEM analysis Scanning electron microscopy (TEM) images (of drop-cast suspensions on grids) were obtained on a JEOL JEM-2200FS TEM equipped with a Cs collector.

[0107] Dynamic Light Scattering Analysis Dynamic light scattering (DLS) and zeta potential measurements were performed on a Malvern Zetasizer Ultra dynamic light scattering system in backscattering mode (173°). DLS and zeta potential measurements were performed in glass cuvettes and disposable folded capillary cells, respectively. All measurements were performed in triplicate at 25 °C after equilibrating the system interior for 240 s, and sample concentrations were adjusted to achieve system attenuator values ​​between 9 and 10. DLS data processing was performed with Malvern "ZS Explorer" software using the "generic" analysis model, and zeta potential data processing was performed with the same software using the "monomodal" analysis mode.

[0108] UV-vis analysis UV-VIS spectra were recorded on a PerkinElmer Lambda 365.

[0109] XRD measurement. X-ray diffraction (XRD) was performed on a Bruker D8 Advance equipped with a motorized anti-scatter screen, as well as an Autochanger and Bragg-Brentano θ-θ geometry (goniometer radius 280 mm). The instrument uses Cu Kα radiation (λ = 1.54184 Å) without a Kβ filter. The detector is a 192-channel LynxEye XE-T silicon stripline detector. Samples were prepared by drop-casting NC suspensions onto silicon plates. Measurements were performed in the 2θ range of 15–60° with a step size of 0.02° and a scan speed of 0.5° / min.

[0110] X-ray scattering analysis Pair Distribution Function (PDF) measurements were performed at DESY, Hamburg, Germany, on beamline P21.1 using a Varex 2D detector (2880x2880 pixels and 150x150 μm pixel size) in fast acquisition mode with a sample-to-detector distance of 800 mm. The incident wavelength of X-rays was λ=0.122 Å. Calibration of the experimental setup was performed using nickel standard material.

[0111] Variable temperature NMR measurements Variable Temperature 1 H NMR measurements were recorded on a Bruker Avance III NMR spectrometer operating at 600.13 MHz proton frequency and equipped with an indirect 5-mm BBI probe. The probe is equipped with a self-shielded z-gradient. For experiments performed below 318 K, the temperature was calibrated using a methanol standard accurate to within ±0.2 K. For variable temperature NMR measurements above 318 K, a glycerol standard was used for calibration.

[0112] NMR measurements on PA-PEG functionalized NCs Nuclear magnetic resonance (NMR) measurements on nanocrystal (NC) functionalization with (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)phosphonic acid (PA-PEG) were recorded on a Bruker Avance III NMR spectrometer (titration with PA-PEG and transfer to water) operating at 600.13 MHz proton frequency. The instrument was equipped with a direct observation 5-mm BBFO smart probe ( 31 The instruments were equipped with either a 5-mm 1000-nm NMR (for P NMR) or an indirect 5-mm BBI probe. Both probes were equipped with self-shielded z-gradients. Experiments were performed at 298 K and the temperature was calibrated using a methanol standard accurate to within ±0.2 K.

[0113] All other functionalizations with PA-PEG 1H NMR measurements were recorded on a Bruker Avance III HD NMR spectrometer operating at 600.13 MHz proton frequency at a temperature of 298 K and equipped with a cryogenic QCI-F probe. All other measurements for functionalization with PA-PEG were performed. 31 P NMR measurements were recorded on a Bruker Avance Neo spectrometer operating at 500.13 MHZ proton frequency at a temperature of 298 K and equipped with a BBFO probehead. The probes for both spectrometers are equipped with self-shielded z-gradients. Temperatures were calibrated using a methanol standard accurate to within ±0.2 K.

[0114] NMR measurements on PA-hex-PEG and nitrodopamine-mPEG functionalized NCs Nuclear magnetic resonance (NMR) measurements on nanocrystals (NCs) functionalized with (6-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)hexyl)phosphonic acid (PA-hex-PEG) and N-(4,5-dihydroxy-2-nitrophenethyl)-2-(2-(2-methoxyethoxy)ethoxy)acetamide (nitrodopamine-mPEG) were recorded on a Bruker Avance III HD NMR spectrometer operating at 600.13 MHz proton frequency and equipped with a cryogenic QCI-F probe. 31 P NMR measurements were recorded on a Bruker Avance Neo spectrometer operating at 500.13 MHZ proton frequency at a temperature of 298 K and equipped with a BBFO probehead. The probes for both spectrometers are equipped with self-shielded z-gradients. Temperatures were calibrated using a methanol standard accurate to within ±0.2 K.

[0115] NMR measurements on synthetic ligands Synthetic Ligands 1 H, 13 C{ 1 H}, 31 P{ 1H and 2D NMR measurements were recorded on a Bruker Avance Neo spectrometer operating at 500.13 MHZ proton frequency at a temperature of 298 K and equipped with a BBFO probehead. The probe is equipped with a self-shielded z-gradient. Temperature was calibrated using a methanol standard accurate to within ±0.2 K.

[0116] NMR experimental parameters Quantitative 1D 1 For H measurements, 64k data points were sampled with the spectral width set at 20 ppm and a relaxation delay of 30 s. Concentrations were obtained using the digital ERETIC method. 2 DOSY measurements were performed with double stimulated echo and bipolar gradient pulses (dstebpgp2s). Gradient strength was varied quadratically from 2 to 95% of the probe maximum in 8 steps if intercepts with diffusion filters were required, or in 32 steps if creation of pseudo-2D spectra was required. Gradient pulse duration and diffusion delay were optimized to ensure that the final attenuation of the signal in the final increment was less than 10% relative to the first increment. Diffusion coefficients were obtained by fitting a modified Stejskal-Tanner equation to the signal intensity decay:

number

[0117] where I is the signal intensity, D is the linear diffusion coefficient, γ is the gyromagnetic ratio of the studied nuclei, g is the gradient strength, δ is the pulsed field gradient duration, and Δ is the diffusion delay. A correction factor of 0.6 is applied to δ due to the smoothed rectangular pulse shape used for the gradient pulse. 1D 31 P{ 1 For {H} measurements, 25000 data points were sampled in the zgpg30 pulse sequence with the spectral width set at 270.81 ppm and 4k scans, and the LB was set at 40 Hz during spectral post-processing. 13 C{1 For the {H} measurements, 120480 data points were sampled in the zgpg30 pulse sequence with the spectral width set to 239.49 ppm and a 4k scan.

[0118] Spin filtration purification in MeOH and HO NC suspensions containing up to 50 mg of material dissolved in 2 ml of solvent are transferred to pre-rinsed Sartorius Vivaspin (30000 MWCO) spin filtration tubes through a 0.2 μm syringe filter. The suspensions are diluted to a volume of 20 ml with MeOH or Milli-Q water, and the solutions are then spun in a centrifuge at 2100 rcf for 30 min. For phosphonic acid NC functionalization, three cycles of spin filtration with MeOH and for nitrodopamine-mPEG NC functionalization, a minimum of two cycles of spin filtration with Milli-Q water are performed per sample until the filtrate is colorless. The concentrates are collected, evaporated, suspended in (deuterated) MeOH or (deuterated) H2O, and sonicated for 30 min to ensure that all aggregates are resuspended and insoluble material is minimized.

[0119] Titration with PA-PEG and PA-hex-PEG. A small amount of the purified toluene NC stock suspension was evaporated to give approximately 45 mg of functionalized material. The NC was suspended in 0.5 ml of absolute EtOH and sonicated for 30 min, after which the solvent was evaporated again. The NC was then suspended in 0.5 ml of MeOD and sonicated for 30 min, yielding quantitative 1 H NMR measurements were performed using the digital ERETIC method to determine the MEEAA concentration. The MeOH solvent peak, which partially overlaps with the MEEAA signal, was carefully subtracted from the calculation to ensure accurate concentration determination. Stock solutions containing at least 3 equivalents of PA-PEG or PA-hex-PEG were then created in MeOD. Titrations were performed by adding PA-PEG or PA-hex-PEG in 0.1 equivalent steps, with each addition step flicking the NMR tube and mixing using a vortex for 2 minutes followed by sonication for a few seconds.

[0120] NC functionalization with PA-PEG and PA-hex-PEG. In a typical functionalization, the same method as during titration with PA-PEG or PA-hex-PEG is used, except now 1.5 equivalents of phosphonic acid are added at once to the MEEAA-functionalized NCs, stirred, sonicated for 10 min, and then purified using spin filtration to obtain purified PA-PEG or PA-hex-PEG-functionalized NCs.

[0121] Titration with D2O NCs functionalized with PA-PEG or PA-hex-PEG were purified using spin filtration as described above, and the concentrate was evaporated and resuspended in 500 μL of MeOD. DO was added in a stepwise manner to achieve final DO concentrations of 25, 50, 75, and 100%, respectively. If necessary, the suspension was evaporated between measurements to achieve the desired DO concentration such that the sample volume did not increase by more than 0.8 ml.

[0122] Titration with nitrodopamine-mPEG. A small amount of the purified toluene NC stock suspension was evaporated to give approximately 10 mg of functionalized material. The NC was suspended in 0.5 ml of absolute EtOH and sonicated for 30 min, after which the solvent was evaporated again. The NC was then suspended in 0.5 ml of MeOH and sonicated for 30 min, after which the solvent was evaporated again. The NC was suspended in 0.5 ml of DO and quantitatively purified. 1 H NMR measurements were performed using the digital ERETIC method to determine the MEEAA concentration. 1.5 equivalents of nitrodopamine-mPEG (relative to the amount of MEEAA on the NC) were preactivated in D2O by adding 2 equivalents of NaOD (relative to the amount of nitrodopamine-mPEG required). Preactivated nitrodopamine-mPEG was added in 0.5 equivalent increments, ensuring that the pH remained above about 5 during the addition, and after each addition step, the NMR tube was flicked and then mixed using a vortex for 2 minutes, followed by sonication for a few seconds.

[0123] NC functionalization with nitrodopamine-mPEG. In a typical functionalization, the same method as during the titration with nitrodopamine-mPEG is used, except now 1.5 equivalents of preactivated nitrodopamine-mPEG are added at once to ensure that the pH remains above about 5 throughout the addition. The mixture is stirred and sonicated for 10 min before being purified using spin filtration to obtain purified nitrodopamine-mPEG functionalized NCs. The authors note that this method can be scaled proportionally to larger amounts of NCs, as long as the maximum loading allowed per spin filter is not exceeded.

[0124] Effect of pH on ligand binding For all measurements, 2 ml of solvent was found to be the minimum amount required for the micro pH electrode to be able to measure the pH value. A 5 M NaCl stock solution was used to achieve a sample salt concentration of 0.01 M, and the pH value was adjusted using 0.01 M stock solutions of DCl and NaOD in D2O.

[0125] Phosphonic Acids: Purified PA-PEG and PA-hex-PEG functionalized NCs in MeOH were created using the above method, and the NC suspensions were evaporated and resuspended in DO. 31 P NMR measurements were carried out at several pH values. 31 The amounts of bound and unbound ligand in the P spectra were quantified by multiple peak fitting methods (peak deconvolution).

[0126] Nitrodopamine-mPEG: Purified nitrodopamine-mPEG functionalized NCs in Milli-Q water were created using the above method, and the NC suspension was evaporated and resuspended in D2O. 1 1 H NMR measurements were carried out at several pH values. 1 The amounts of bound and unbound ligand in the H spectra were quantified by multiple peak fitting methods (peak deconvolution).

[0127] Dynamic light scattering stability evaluation For all measurements, purified functionalized NCs in MeOH (for phosphonic acids) or Milli-Q water (for nitrodopamine-mPEG) were created using the methods above, and the NC suspensions were evaporated and resuspended in Milli-Q water. For Z-average and Zeta potential measurements, the NC concentration was adjusted to achieve a system attenuator value between 9 and 10. We found that 2 ml of solvent was the minimum amount required for the micro-pH electrode to be able to measure pH values ​​and perform Z-average and Zeta potential measurements. All measurements were performed in triplicate at 25 °C after equilibrating the inside of the system for 240 s.

[0128] Effect of pH on Z-average value and Zeta potential. A sample salt concentration of 0.01 M was achieved using filtered 5 M NaCl stock solution, and the suspension was sonicated for 15 min and filtered through a 0.2 μM Supor syringe filter to remove dust before starting the titration. The pH value was adjusted using filtered 0.01 M stock solutions of HCl and NaOH in Milli-Q water.

[0129] Stability suspensions in 2x PBS were filtered through a 0.2 μM Supor syringe filter before adjusting the pH to 7.4 using filtered 0.01 M stock solutions of HCl and NaOH in Milli-Q water. The PBS concentration was increased by stepwise addition of filtered 10x PBS stock solution, and the pH was checked after each addition step and readjusted to 7.4 if necessary. Stability over time in 2x PBS measurements were performed in closed quartz cuvettes, which were kept at room temperature and closed conditions throughout the duration of the stability study.

[0130] CT scan General Considerations In vitro and in vivo CT scans were obtained on a Molecubes X-cube tabletop CT scanner using a built-in high-resolution scanning protocol at a tube potential of 50 kV. The resulting scans were reconstructed at voxel sizes of 200, 100 or 50 μm using the scanner's built-in bidirectional reconstruction algorithm, and no further noise reduction steps were applied to the data. Reconstructed data were visualized using the Amide or Horos software packages, and window levels were typically set from -1000 to 1000 HU for each scan unless otherwise stated.

[0131] In vivo CT scanning For in vivo scanning, mice were pre-anesthetized with 5% isoflurane for induction and 2% isoflurane for maintenance in the scanner. After induction, mice were placed in a prone position on a heated scan bed. On average, for whole-body scans, mice received an X-ray dose of approximately 340 mGy per scan. For in vivo experiments, reconstructions were always performed at 200 micron resolution at all scan points.

[0132] CT scan of HfO2NCs concentration series in PBS After scanning and reconstruction, the X-ray attenuation, expressed in Hounsfield unit values, was quantified in each sample tube using a spherical region of interest measurement of 1 × 1 × 1 mm. The median pixel value was taken during quantification to avoid possible influence from outliers. When plotting the X-ray attenuation as a function of NC concentration, the ligand weight (18.9 m%) was pre-subtracted from the NC weight, since the ligand is purely organic and does not contribute significantly to the X-ray attenuation.

[0133] Near-infrared fluorescence imaging NC samples and in vivo lymph node fluorescence were visualized using an IVIS Lumina LT Series III in vivo imaging system. Imaging was performed using 710 (±15 nm) and 745 (±nm) bandpass excitation filters and a bandpass emission filter within the ICG window (810-875) nm. Fluorescent images were typically overlaid with visible light photographs.

[0134] Preparation of catechol-functionalized NCs Nitrodopamine-mPEG functionalized NCs A small amount of the purified MEEAA-functionalized NCs stock suspension in toluene was evaporated to give approximately 20 mg of functionalized NCs. The NCs were resuspended in deuterated benzene and the ligand concentration was quantitatively determined using the digital ERETIC method. 1 The MEEAA concentration was determined by H NMR to give mol MEEAA per mg functionalized NC. A small amount of the purified toluene stock suspension was then evaporated to give approximately 50 mg functionalized NC. The NC was resuspended in 2 ml absolute ethanol and sonicated for 30 min, after which the solvent was evaporated again. The NC was then suspended in 2 ml MeOH and sonicated for 30 min, after which the solvent was evaporated again. Finally, the NC was suspended in 2 ml endodopamine-free ultrapure water and sonicated for 30 min. For the nitrodopamine-mPEG functionalized NC, 1.2 equivalents of nitrodopamine-mPEG based on the MEEAA concentration determined by quantitative NMR were weighed in a separate vial and dissolved in 3 ml endodopamine-free ultrapure water. Two equivalents of NaOH compared to the required amount of nitrodopamine-mPEG were added to the nitrodopamine-mPEG solution to give a deep burgundy wine color. The ligand solution was then quickly added to the NC suspension with vigorous stirring; a brief turbidity during the transition from acidic to basic pH was observed, which is normal; after complete addition of the ligand, a clear red to orange liquid was obtained, which was sonicated for 15 min and vigorously stirred. After sonication and stirring, the pH of the suspension was adjusted to approximately 9 before purification was initiated. The purified nitrodopamine-functionalized NC suspension was obtained after purification by spin filtration.

[0135] Nitrodopamine-mPEG and Nitrodopamine-PEG(4)-N3 functionalized NCs For NCs functionalized with a mixed catechol ligand shell containing roughly 98% nitrodopamine-mPEG and 2% nitrodopamine-PEG(4)-N3 (approximately 1 azide per NC), the same procedure was followed as above, except a mixture of 1.18 equivalents of nitrodopamine-mPEG and 0.02 equivalents of nitrodopamine-PEG(4)-N3 was used.

[0136] Nanocrystal number and ligand density To calculate an approximation of the number of NCs for a given amount of NC weight, we used the material density (9.68 g / cm 3 ) by the molecular weight (210.49 g / mol) to find the molar volume of the material (21.745 cm 3 Start by calculating the .mu.m / mol.

number

[0137] Next, the average NC volume (15.33 nm 3 ) is calculated based on a spheroidal shape, an average major axis radius of 2.52 nm and an average minor axis radius of 1.205 nm.

number

[0138] cm 3 The average NC volume converted to mol HfO2 was then divided by the molar volume and Avogadro's constant to obtain mol HfO2 / NC (7.05*10 -22 Determine the concentration (mol / NC).

number

[0139] Finally, the number of NCs (N NC ) and the NC weight in grams (ligand weight has been subtracted) based on the molecular weight (210.49 g / mol) and HfO2 / NC (7.05*10-22 It is determined by dividing by (mol / NC).

number

[0140] To calculate the ligand density on the NC surface, we calculated the average surface area of ​​the NCs (A NC , 32.42 nm 2 We start by calculating

number

[0141] The amount of ligand in mol (n リガンド , mol) is obtained.

number

[0142] Ligand amount (n リガンド ) by Avogadro's constant to find the number of NCs (N NC ) to obtain Ligand / NC.

number

[0143] Finally, the ligand / NC was applied to the NC surface (A NC ) to get nm -2 The ligand density at

number

[0144] Spin filtration purification A 20 ml Sartorius Vivaspin (30000 MWCO) spin filtration tube was cleaned with 70% ethanol and then pre-rinsed with 20 ml endotoxin-free ultrapure water at neutral pH. The pH 8-9 NC suspension containing a maximum of 50 mg dissolved material was transferred to the spin filtration tube through a 0.2 m syringe filter. The suspension was diluted to a volume of 20 ml with endotoxin-free ultrapure water at neutral pH and the solution was then spun in a centrifuge at 2100 rcf for 20 min. For nitrodopamine-mPEG and mixed catechol ligand shell functionalization, a minimum of 5 cycles of spin filtration were performed with endotoxin-free ultrapure water until the filtrate was colorless. The concentrate was collected from the spin filter and evaporated under vacuum at 35°C. The dried NCs can be stored as a powder at room temperature or resuspended in water or PBS at pH 7.4 at a maximum NC concentration of approximately 300 mg HfO2 / ml. In the latter case, the organic ligand weight (18.9 m%) is subtracted from the functionalized NC weight in the concentration calculation, and the suspension is filtered through a sterile 0.2 m syringe filter and stored in a sterile vial. NC suspensions in water or PBS remain stable for at least 2 months even at high concentrations.

[0145] Dye conjugation to functionalized NCs In functionalizing NCs with mixed catechol ligands, the final product is assumed to have approximately 1 azide per NC. In a typical dye-coupling reaction, 10 mg of mixed catechol-functionalized NCs are first weighed into an HPLC vial and dissolved in 100 μL of endotoxin-free ultrapure water at pH 7. Based on a ligand mass contribution of 18.9 m% (confirmed by TGA mass loss), this corresponds to 8.11 mg of bare nanocrystals, which is then calculated to be 5.4662*10 based on the average major axis of 5.04 nm, the average minor axis of 2.41 nm, and the NC shape of a prolate spheroid. 161.1 equivalents of IRDye-800CW-DBCO (5.4662*1016 molecules, 0.091 μmol, 0.12 mg) were weighed into an HPLC vial on an analytical balance and dissolved in 200 μL of endotoxin-free ultrapure water at pH 7. The dye molecule concentration was calculated using a 1 cm path length and 240000 L*(mol*cm) -1 The dye's extinction coefficient of was determined using UV-VIS spectroscopy applying the Lambert-Beer law with absorbance measured at a wavelength of 774 nm. See Equation 10.

number

[0146] The dye solution was added to the NC and further diluted to a volume of 300 μL to give a clear yellow to green suspension. The reaction mixture was stirred for 2 h at 30° C. protected from light. The NC was then purified by three successive cycles of spin filtration, after which the concentrate was isolated and evaporated under vacuum at 30° C. protected from light. The resulting dark green NC powder, containing approximately 1 dye molecule / NC, was stored in a freezer at −20° C. under argon.

[0147] Formulation preparation The first step is to determine the dye grafting density on the functionalized NCs in order to achieve a formulation with NC and dye concentrations of 292 mg NC / ml and 28 μmol / L, respectively. At this point, 1 mg of dry dye-functionalized NCs is suspended in 4 ml of 1×PBS and measured using UV-VIS with an optical path length of 1 cm and 240000 L*(mol*cm). -1 Using the extinction coefficient of the dye and the Lambert-Beer law for the absorbance measured at the maximum NIR absorption peak (±780 nm), the dye concentration was calculated in mol*L. -1 The dye-functionalized NCs are then mixed with the dye-functionalized NCs powder and sonicated for 15 minutes to achieve a NC concentration of 292 mg NCs / ml and 28 μmol / L conjugated dye. This procedure is taken as a good example: 5.796*10-9 Assume a dye-conjugated NC batch containing mol dye / mg functionalized NCs is available: 0.199 mg of this dye-functionalized powder (18.9 m% organics by weight, 0.161 mg bare NCs) is mixed with 40 μL of nitrodopamine-mPEG NC stock at a concentration of 288 mg NCs / ml to yield the desired NC and dye concentrations.

[0148] subcutaneous footpad injection Prior to subcutaneous injection of PBS solution or NC suspension, mice were anesthetized with 5% isoflurane for induction and reduced to 2% isoflurane for maintenance during the injection procedure. Once anesthetized, mice were placed in a supine position on a heated bed and the hind paw was warmed for approximately 30 seconds using an infrared lamp. The footpad skin was fixed and a 0.5 ml insulin syringe with a 29G needle was inserted subcutaneously in the heel area and advanced approximately 3 mm toward the toe. Throughout the process, the needle was visible through the thin skin layer of the footpad. A maximum volume of 50 μL of PBS solution or NC suspension was then slowly injected while the needle was slowly pulled toward the heel. After completion of the injection, the needle was kept in the footpad for an additional 30 seconds and then removed from the hind paw. Immediately after the needle was removed, a generous layer of 5% xylocaine was applied to the injected hind paw as an analgesic.

Claims

1. a. Hafnium(IV) oxide (HfO) particles with a diameter of 15 nm or less 2 nanocrystals comprising or consisting essentially of said hafnium(IV) oxide; b. a plurality of dispersant molecules attached to the surface of the nanocrystal, each of which comprises: i. a surface-adsorbing moiety selected from the group including catechol or gallol, particularly a 1,2-hydroxy-4-nitrophenyl moiety; ii. an oligo(ethylene glycol) moiety; and a dispersant molecule comprising or consisting of both moieties. Nanoparticles comprising:

2. 2. The nanoparticles according to claim 1, wherein the nanocrystals have a diameter of 6 nm or less, in particular a diameter of 4 nm or less, more particularly a diameter of 3.5 nm or less.

3. The nanoparticles of claim 1 , wherein the nanocrystals are characterized by an aspect ratio of 0.5 to 0.

9.

4. Nanoparticles according to any one of claims 1 to 3, wherein the dispersant molecules have a molecular mass of 500 g / mol, in particular a molecular mass of 400 g / mol or less.

5. The dispersant molecule is i. a surface-adsorbing moiety selected from nitrodopamine, nitroDOPA, DOPA, dopamine, and mimosine; ii. (CH 2 -CH 2 -O) n CH 3 a moiety wherein n is an integer selected from 2, 3, 4, and 5; Nanoparticles according to any one of claims 1 to 3, comprising, in particular consisting of, both moieties.

6. The dispersant molecule is 【Chemical 1】 Nanoparticles according to any one of claims 1 to 3, comprising, in particular consisting of, a compound of formula (I):

7. The nanoparticles comprise further dispersant molecules, the further dispersant molecules comprising: 【Chemistry 2】 (In the formula, R dye is a fluorescent dye) The nanoparticles according to any one of claims 1 to 3, selected from the group consisting of:

8. The density of dispersant molecules on the nanocrystals is 0.5 to 5 / nm 2 The nanoparticles according to any one of claims 1 to 3,

9. The nanoparticles according to any one of claims 1 to 3, which contain dye molecules for optical localization.

10. A composition comprising a plurality of nanoparticles according to any one of claims 1 to 3.

11. 11. The composition of claim 10 which is a stable aqueous suspension.

12. The composition according to claim 11, having a pH of from pH 6 to pH 10, in particular from pH 6.5 to pH 8.

0.

13. 11. The composition according to claim 10, wherein 80% of the nanoparticles have a diameter between 2.0 nm and 5.0 nm, in particular 85% of the nanoparticles have a diameter between 2.5 nm and 4.5 nm.

14. The composition of claim 10 for pharmaceutical use.

15. 11. The composition of claim 10 for use as a radiotherapy potentiator (radiosensitizer).

16. 11. The composition of claim 10 for use as a computed tomography (CT) contrast agent.

17. a. Hafnium(IV) oxide (HfO) stabilized by carboxylic acid ligands, especially MEEAA, in aqueous media 2 ) providing a suspension of nanocrystals; b. adding to said suspension an alkaline solution of dispersant molecules, said dispersant molecules comprising: i. a surface-adsorbing moiety comprising two aromatic hydroxide functional groups, the surface-adsorbing moiety being selected from the group including catechol or gallol, particularly a 1,2-hydroxy-4-nitrophenyl moiety; ii. an oligo(ethylene glycol) moiety; wherein both aromatic hydroxide functional groups are deprotonated in said alkaline solution; c. adjusting the pH of the suspension to a physiological pH; d. Optionally, isolating the composition, particularly by size exclusion / spin filtration; 11. A method for making the composition of claim 10, comprising:

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