EP3558392A1 - Procédé pour la fonctionnalisation de nanoparticules - Google Patents

Procédé pour la fonctionnalisation de nanoparticules

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Publication number
EP3558392A1
EP3558392A1 EP17816711.0A EP17816711A EP3558392A1 EP 3558392 A1 EP3558392 A1 EP 3558392A1 EP 17816711 A EP17816711 A EP 17816711A EP 3558392 A1 EP3558392 A1 EP 3558392A1
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EP
European Patent Office
Prior art keywords
nanoparticles
solution
advantageously
comu
aunps
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP17816711.0A
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German (de)
English (en)
Inventor
Davide Bonifazi
Valentina CORVAGLIA
Riccardo MAREGA
Stéphane Lucas
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Universite de Namur
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Universite de Namur
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Publication of EP3558392A1 publication Critical patent/EP3558392A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to methods for functionalizing nanoparticles with organic molecules such as antibodies or proteins.
  • the functionalized nanoparticles obtained according to methods of the present invention can be used in a wide variety of medical and biological applications.
  • Conjugates of polyethylene glycol (PEG) and inter alia Cetuximab were prepared by antibody reaction with NHS-PEG-OPSS (with OPPS referring to ortho-pyridyl disulphide or ortho pyridyldithio). More specifically, NHS-PEG-OPSS reacts with the amine groups of antibodies to yield antibody-PEG conjugates through amide bond formation. Purified antibody conjugates were then combined with mPEG-SH and assembled onto the surface of the gold nanoparticles. The ability of the prepared nanoparticles to produce an antibody-dependent cellular cytotoxicity (ADCC) effect in vivo was subsequently investigated.
  • ADCC antibody-dependent cellular cytotoxicity
  • WO 2007/122259 describes the functionalization of nanoparticles with spacers such as polyethylene oxide (PEO) using EDC/NHS chemistry as well, the spacer itself being linked to a protein in a stereo-specific manner, thereby ensuring controlled orientation of the particle-bound protein.
  • spacers such as polyethylene oxide (PEO) using EDC/NHS chemistry
  • An objective of aspects of the present invention is to provide an improved method for functionalizing nanoparticles. It is an object to provide such methods which allow a faster and more efficient functionalization of nanoparticles compared to prior art methods. It is also an object to provide such methods which are easier to implement and which are more cost effective. It is a further object to provide such methods using more versatile chemical compounds. It is hence an object of the present invention to provide methods for functionalizing nanoparticles, which are less laborious, more environmentally friendly, and/or more economical compared to prior art methods.
  • Figure 1 schematically represents a prior art synthetic pathway to functionalize gold nanoparticles with Cetuximab antibodies
  • Figure 2 schematically represents a synthetic pathway according to aspects of the present invention to functionalize gold nanoparticles with Cetuximab antibodies
  • Figure 3 schematically represents the molecular structure of COMU
  • Figure 4 reports the stability assessment of COMU in PBS, evaluated by
  • Figure 5 schematically represents a synthetic pathway towards the formation of a dipeptide Fmoc-NH-Gly-lle-OtBu in the presence of COMU as coupling agent and DIEA (N,N-Diisopropylethylamine) in PBS as solvent;
  • Figure 6 shows optical photographs taken at different time intervals after the introduction of COMU into the reaction mixture described in Figure 5.
  • Figure 7 shows steady-state UV-Visible absorbance profiles (full lines) and differential plots dAbs/dA (dashed lines) of different materials before ( Figures 7a, 7b, 7d, 7f) and after (Figures 7c, 7e, 7g) bioconjugation reaction with COMU for 16h: for Cetuximab ( Figure 7a), AuNPs@PPAA ( Figure 7b), Ctxb-AuNPs@PPAA ( Figure 7c), commercial 5 nm AuNPs ( Figure 7d), commercial 5 nm Ctxb-A
  • FIG. 8 shows thermogravimetric analysis (TGA) characterization of TGA
  • Figure 9 shows fluorescence revelation (Aexcitation 488 nm, Aemission 500 nm) of polyacrylamide gel after electrophoresis of different aliquots pooled at different reaction times from the bioconjugation reaction mixture ( Figure 9a); and fluorescence volume% repartition between the bands located in the deposition area and belonging to the free Cetuximab, as obtained by software editing of the gel revelation (top and bottom of Figure 9b, respectively);
  • Figure 10 shows a comparison of the (bio)chemical properties of Ctxb-
  • AuNPs@PPAA bioconjugates obtained after 16h and 4h reaction time with COMU: TGA of Ctxb-AuNPs@PPAA ( Figure 10a) with temperature-modulated plots (full lines) and differential plot (dashed lines); summary of TGA and atomic absorption spectroscopy (AAS) results ( Figure 10b); and Enzyme-Linked Immunosorbent Assay (ELISA) on EGFR+ cells (A431 ) or EGFR- cells (MDA-MB-453) ( Figure 10c);
  • Figure 11 shows a comparison of chemical composition of commercial available and prepared Nh -bearing AuNPs as assessed by XPS analysis (Figure 11a).
  • Methods for functionalizing nanoparticles according to aspects of the present invention comprise the steps of:
  • first solution comprising an organic molecule (or biological molecule) dissolved (or diluted) in phosphate buffered saline (or PBS), the organic molecule comprising a carboxyl group; providing a second solution, said second solution comprising 1 -Cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate
  • COMU phosphate buffered saline
  • the organic molecule comprises a carboxyl group. More particularly, the organic molecule comprises at least one carboxyl group. Otherwise stated, the organic molecule comprises one or more carboxyl group (i.e. the organic molecule comprises one, two, three, or more carboxyl groups).
  • the organic molecule is an antibody, a protein, carboxylated biotin, or carboxylated DNA, advantageously an antibody or a protein, advantageously an antibody.
  • the antibody is Cetuximab (throughout the description also referred to or denoted as Ctxb or mAb).
  • COMU refers to 1 -Cyano-2-ethoxy-
  • COMU can also be referred to by its alternative names, i.e. as 1 -[1 -
  • COMU (depicted in Figure 3)
  • 3 (interchangeably) be referred to by three names, i.e. 1 -Cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate, 1 -[1 - (Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino]-uronium
  • COMU is commercial available, and can for example be supplied by Iris
  • phosphate buffered saline is denoted or referred to as PBS.
  • the step of stirring the third solution is performed at a temperature between 15°C and 40°C, advantageously between 15°C and 30°C, advantageously between 20°C and 25°C, advantageously at room temperature.
  • the step of stirring the third solution is performed for a time ranging between 10 and 30 minutes, advantageously between 10 and 20 minutes, advantageously for 15 minutes.
  • the step of stirring the third solution is performed at a temperature between 15°C and 40°C, advantageously between 15°C and 30°C, advantageously between 20°C and 25°C, advantageously at room temperature; and for a time ranging between 10 and 30 minutes, advantageously between 10 and 20 minutes, and advantageously for 15 minutes.
  • the step of stirring the fourth solution is performed at a temperature between 15°C and 40°C, advantageously between 15°C and 30°C, advantageously between 20°C and 25°C, advantageously at room temperature.
  • the step of stirring the fourth solution is performed for a time ranging between 2 and 8 hours, advantageously between 2 and 6 hours, advantageously between 3 and 5 hours, advantageously for 4 hours.
  • the step of stirring the fourth solution is performed at a temperature between 15°C and 40°C, advantageously between 15°C and 30°C, advantageously between 20°C and 25°C, advantageously at room temperature; and for a time ranging between 2 and 8 hours, advantageously between 2 and 6 hours, advantageously between 3 and 5 hours, advantageously for 4 hours.
  • COMU is used as coupling agent (or coupling additive, or coupling reagent) in PBS buffered solution for the functionalization of Nhb-bearing nanoparticles with organic molecules (comprising a carboxyl group) via the formation of an amide bond.
  • aspects of the present invention involve the anchoring of organic molecules to amino-bearing nanoparticles via an amide bond by using COMU as coupling agent. Only one type of coupling agent is used.
  • carbodiimides and succinimidyl esters such as EDC (1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide) and/or NHS (/V-hydroxysuccinimide), using organic solvents, such as DMF (dimethylformamide) and MNP (2-methyl-2- nitrosopropane); or ii) EDC/HOBt (hydroxybenzotriazole) in a buffer of MES (2-(/V- morpholino)ethanesulfonic acid).
  • FIG. 1 an example of a prior art synthetic pathway for the preparation of bioconjugates between gold nanoparticles (AuNPs) and Cetuximab antibodies is schematically shown.
  • the AuNPs used are coated with a layer of plasma-polymerized allylamine (PPAA), denoted as AuNPs@PPAA.
  • PPAA plasma-polymerized allylamine
  • the amino groups present in the polymeric shell of AuNPs@PPAA are exploited for amide bond formation with carboxyl groups located on Cetuximab, through carbodiimide chemistry using EDC, NHSS (N- hydroxysulfosuccinimide) as coupling agents and MES buffered solutions as solvent.
  • Bioconjugates of antibodies coupled to the nanoparticles, Ctxb-AuNPs@PPAA are thereby formed.
  • Cetuximab is d by:
  • Figure 1 describes an amide coupling reaction known in the art to produce antibody-functionalized gold nanoparticles.
  • the chemical reaction involves first the activation of the Cetuximab carboxylic moieties by using EDC and NHSS followed by the subsequent amide bond formation in the presence of amine-terminating nanoparticles.
  • the functionalization is performed in MES buffer (pH 7) at room temperature, for 16 hours.
  • FIG 2 is a synthetic pathway for the synthesis of bioconjugates between gold nanoparticles (AuNPs) and Cetuximab antibodies according to an embodiment of aspects of the present invention.
  • the AuNPs used are coated with a layer of plasma- polymerized allylamine (PPAA), denoted as AuNPs@PPAA.
  • PPAA plasma- polymerized allylamine
  • aspects of the present invention use COMU as one and only coupling agent with PBS as reaction media (or solvent). Bioconjugates of antibodies coupled to the nanoparticles, Ctxb- AuNPs@PPAA, are thereby formed.
  • an organic molecule is covalently linked to the coupling agent COMU in PBS solution as reaction media (or solvent). More particularly, COMU activates the carboxylic moieties present on the organic molecule (for example on Cetuximab antibodies as depicted in Figure 2) prior to the amide coupling reaction towards the amine-terminating gold nanoparticles.
  • the bioconjugation in aspects of the invention is already completed after stirring the mixture for 2 to 8 hours, even after stirring for only 4 hours (at a temperature between 15°C and 40°C, advantageously at room temperature).
  • no additional, separate steps of activating the nanoparticles and/or the organic molecule are needed prior to performing the coupling reaction.
  • the time for completing the coupling reaction is significantly reduced, compared to prior art methods.
  • the number and amount of chemical reagents used is significantly reduced as well.
  • methods of aspects of the invention are simplified, requiring less complex reactions (based on relative easy and simple chemical synthesis), and are performed under more simple reaction conditions.
  • the methods of aspects of the invention are also faster and less costly.
  • COMU is known to be highly stable and highly soluble in almost all organic solvents. Indeed, for amide coupling reactions in the art up to now, both in solution and solid phase, coupling agents (as for example COMU), are only being dissolved in organic solvents like dimethylformamide (DMF) and N-Methyl-2-pyrrolidone (NMP).
  • DMF dimethylformamide
  • NMP N-Methyl-2-pyrrolidone
  • COMU also has compatibility for reactions with PBS, i.e. COMU is not deactivated by PBS at all. More particularly, COMU unexpectedly appears to be stable and soluble in water and in (aqueous) PBS solution, contrary to most of the coupling agents used in prior art methods for functionalizing nanoparticles.
  • reaction mixtures formed by coupling reactions using COMU as coupling agent can thus be considered as being less toxic than the reaction mixtures formed by coupling reactions performed in the art using for example EDC and NHS. This can be beneficial for the formed (suspension of) functionalized nanoparticles obtained after purification, for their (its) further use in a variety of biological and medical applications, such as in cancer treatment.
  • the surface of the nanoparticles added to the third solution comprise amine functions (or NH2-functions, or amino functionalities).
  • the nanoparticles added to the third solution are amino-bearing (NH2-bearing) nanoparticles.
  • the nanoparticles added to the third solution comprise a surface or shell or coating exohedrally exposing amine functions.
  • “functionalizing nanoparticles” refers to functionalizing the surface of nanoparticles with organic molecules (resulting in “functionalized nanoparticles”). More particularly, the amino-bearing surface of the nanoparticles is functionalized with organic molecules. In other words, bioconjugates (or conjugates) are formed between the (amine functions exposed on the surface of the) nanoparticles and the organic molecules.
  • the nanoparticles added to the third solution are metal nanoparticles or metal-based nanoparticles, the surface of said nanoparticles comprising amine functions.
  • the nanoparticles added to the third solution are metal nanoparticles, the surface of said nanoparticles comprising amine functions.
  • metal nanoparticles refer to nanoparticles comprising (or consisting of) a metal as such.
  • metal-based nanoparticles refer to nanoparticles comprising (or consisting of) metal alloys, metal oxides, metal nitrides, or metal carbides.
  • the metal of the metal nanoparticles or of the metal based-nanoparticles is selected from the group of transition metals, alkali metals, alkali earth metals, and lanthanides. More advantageously, the metal of the metal nanoparticles or of the metal-based nanoparticles is selected from the group comprising gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iron (Fe), chromium (Cr), manganese (Mn), cobalt (Co), nickel (Ni), copper (Cu), silicon (Si), zirconium (Zr), yttrium (Y), indium (In), iridium (Ir), gallium (Ga), gadolinium (Gd), and titanium (Ti).
  • the metal of the metal nanoparticles is gold
  • the metal nanoparticles are gold nanoparticles.
  • bare (or naked) nanoparticles are coated with a layer of plasma-polymerized allylamine (an amine-functionalized polymer, denoted as PPAA).
  • bare (or naked) metal nanoparticles, or bare (or naked) metal-based nanoparticles are coated with a layer of PPAA.
  • Suitable methods for coating the nanoparticles with PPAA will be apparent for those skilled in the art.
  • polymer-coated AuNPs can be synthesized through step-wise and layer-by-layer plasma vapor deposition of Au (core) and plasma- polymerized allylamine (PPAA), leading to a shell exohedrally exposing amino functionalities, as described by N. Moreau et. Al. in Plasma Process. Polym., 2009, 6, S888.
  • the thickness of the layer of plasma-polymerized allylamine coated onto the bare (or naked) nanoparticles is lower than or equal to 50 nm.
  • nanoparticles coated with a layer of plasma-polymerized allylamine are purified, and then isolated by lyophilisation.
  • the purification is performed by discontinuous diafiltration against H2O in centrifugal concentrators through regenerated cellulose membranes of 10,000 gmol "1 as molecular weight cut-off (MWCO), or by tangential flow filtration with similar membranes in terms of MWCO.
  • compositions of) the organic molecules comprising a carboxyl group are purified, and then isolated by lyophilisation.
  • the purification is performed by discontinuous diafiltration against H2O in centrifugal concentrators through regenerated cellulose membranes of 5,000 or 10,000 gmol " 1 as molecular weight cut-off (MWCO), or by tangential flow filtration with similar membranes in terms of MWCO.
  • the shape of the nanoparticles added to the third solution comprises a sphere, a rod, a prism, a disk, a cube, a cage, a frame, or a mixture thereof. More advantageously, the shape of the nanoparticles added to the third solution is a sphere (or spherical nanoparticles are added to the third solution).
  • the nanoparticles added to the third solution have at least one dimension comprised between 1 nm and 100 nm.
  • the shape of the nanoparticles added to the third solution is a sphere and the diameter of the (bare or naked) nanoparticles is comprised between 1 nm and 100 nm, advantageously between 4 nm and 40 nm, advantageously between 4 nm and 30 nm, advantageously between 4 nm and 20 nm, advantageously between 4 nm and 10 nm.
  • the phosphate buffered saline has a molar concentration comprised between 5 mM and 0.5 M.
  • the phosphate buffered saline has a pH comprised between 6.4 and 7.6, advantageously between 6.6 and 7.5, advantageously between 6.8 and 7.4.
  • the mass concentration of organic molecule in the first solution is comprised between 0.5 mgmL "1 and 2.0 mgmL "1 .
  • the mass concentration of COMU in the second solution is comprised between 0.5 mgmL “1 and 2.5 mgmL "1 .
  • the COMU to organic molecule weight ratio in the third solution is comprised between 0.5:1 to 2:1.
  • the COMU to organic molecule molar ratio in the third solution is comprised between 177:1 to 710:1 .
  • the nanoparticles are added to the third solution in an amount comprised between 0.2 and 2 mg nanoparticles per mg organic molecule.
  • the pH in the fourth solution is comprised between 6.0 and 8.0, advantageously between 6.6 and 7.4, advantageously between 6.8 and 7.2, advantageously the pH in the fourth solution is 7.0.
  • performing the step of stirring the fourth solution yields a colloidal suspension of nanoparticles functionalized with organic molecules, advantageously with antibodies, i.e. bioconjugates are formed between the nanoparticles and the organic molecules, advantageously the antibodies.
  • performing methods for functionalizing nanoparticles according to aspects of the present invention is yielding a colloidal suspension of nanoparticles (surface) functionalized (or bioconjugated) with organic molecules, advantageously with antibodies.
  • the solution is purified so as to obtain purified (surface) functionalized (or bioconjugated) nanoparticles in suspension. More particularly, by performing the step of purification a purified colloidal suspension of functionalized (or bioconjugated) nanoparticles is obtained.
  • purification of the fourth solution is performed by discontinuous diafiltration against H2O in centrifugal concentrators (with MWCO of 300,000 g/mol) or by tangential flow filtration with similar membranes in terms of MWCO.
  • molecular weight cut-off or
  • MWCO refers to the lowest molecular weight solute (in Daltons) in which 90% of the solute is retained by the used membrane.
  • the purified functionalized (or bioconjugated) nanoparticles are isolated from the suspension by lyophilization (or freeze-drying), or the suspension is supplemented with Arabic gum prior to lyophilization.
  • a first solution is provided, said first solution comprising antibody Cetuximab dissolved (or diluted) in PBS; a second solution is provided, said second solution comprising COMU dissolved (or diluted) in PBS; the first solution is added to the second solution (so as) to form a third solution; the third solution is stirred at room temperature for 15 minutes; nanoparticles, advantageously gold nanoparticles, are added to the third solution (so as) to form a fourth solution, the surface of said nanoparticles comprising amine functions; the fourth solution is stirred at room temperature for 4 hours.
  • COMU is used as coupling agent in PBS buffered solution for the functionalization of Nhb-bearing nanoparticles, advantageously gold nanoparticles with Cetuximab antibodies via an amide bond.
  • colloidal suspension comprising functionalized nanoparticles (or colloidal nanoparticle suspension) obtainable by methods according to the invention.
  • the present invention provides functionalized nanoparticles obtainable by methods according to the invention.
  • the present invention provides functionalized nanoparticles, or a colloidal suspension comprising functionalized nanoparticles, obtainable by methods according to the invention for use in therapy (for use in treatment by therapy) or for use in in vivo diagnostics (for use in in vivo diagnostic methods), advantageously for use in a method for treatment of cancer or for use in a method of diagnosis in vivo of cancer
  • nanoparticles or a colloidal suspension comprising functionalized nanoparticles, obtainable by methods according to aspects of the invention can be employed in a wide variety of medical and biological applications.
  • the obtained (suspension of) nanoparticles can be used inter alia as sensor, in imaging, or in drug- delivery.
  • the formed (suspension of) nanoparticles can as well be applied in cancer targeting in vivo or in cancer treatment.
  • nanoparticles functionalized with appropriate biological molecules can for example also be used in order to enhance the efficiency of radio and proton therapy for improving cancer treatment.
  • functionalized nanoparticles can allow for targeting in vivo localization of the nanoparticles, for directing the nanoparticles to specific sites within a body, or for following the movement of specific proteins or RNA molecules in living cells.
  • antibodies targeting for example the epidermal growth factor receptor (EGFR), a membrane protein overexpressed in several kinds of solid tumours, can be conjugated to gold nanoparticles bearing amine-functions and the resulting bioconjugates can be efficiently employed to assess EGFR targeting in vivo, for imaging photothermal treatment, and for drug delivery.
  • EGFR epidermal growth factor receptor
  • antibody-conjugated nanoparticles obtainable by methods according to aspects of the invention can offer increased selectivity and sensitivity in the diagnosis of cancer using imaging techniques such as magnetic resonance imaging (MRI), positron emission tomography (PET), and computed tomography (CT).
  • imaging techniques such as magnetic resonance imaging (MRI), positron emission tomography (PET), and computed tomography (CT).
  • the organic molecules advantageously antibodies, proteins, carboxylated biotin, or carboxylated DNA, advantageously antibodies or proteins, advantageously antibodies, can be radiolabeled with therapeutic markers (or radio-isotopes, radio-nuclides, or radio-emitters).
  • the biomolecules can for example be radioiodinated with 125 l or presenting a chelate of 89 Zr.
  • biomolecules can be tagged with fluorescent dyes (i.e. cyanine-based derivatives).
  • Radioiodination for instance of antibodies
  • Radioiodination can for example be performed using the lodo-Gen kit from Pierce Thermo Scientific (using the Thermo Scientific Pierce ® lodination Reagent, see e.g. R. Marega et. al. in J. Mater. Chem. 2012, 22, 21305).
  • 89 Zr radiolabelling can for example be performed by reacting Ctxb according to the method reported in the literature (see e.g. L. Karmani, Nanomedicine 2014, 9, 1923).
  • wt% refers to the percentage of the component under consideration by total weight of the structure under consideration.
  • nanoparticles As starting material for the nanoparticles, either commercial available (i.e. from AC Diagnostics, Inc.) or prepared Nhb-bearing gold nanoparticles were used.
  • the prepared Nhb-bearing gold nanoparticles were obtained through step-wise and layer-by-layer plasma vapor deposition of Au (core) and plasma-polymerized allylamine, as described by N. Moreau et. Al. in Plasma Process. Polym., 2009, 6, S888, forming gold nanoparticles comprising a coating exohedrally exposing amine functions (denoted as AuNPs@PPAA).
  • the bioconjugations were performed with a reaction time of 16 hours, by employing discontinuous diafiltration against H2O (MWCO 300,000 gmol "1 ) for product purification and subsequent isolation through lyophilisation.
  • This procedure yielded bioconjugated materials (Ctxb-AuNPs@PPAA) at the 5 mg scale, with yields of w/w of around 40 to 70% considering the employed AuNPs mass, starting from the prepared Nhb-bearing gold nanoparticles.
  • the bioconjugation reaction is much less efficient, i.e. 5 nm and 10 nm Ctxb-AuNPs, with yields of w/w around of 10 to 30%. This is probably due to material loss inside the polyetheresulphone (PES) membrane of the diafiltration tubes.
  • PES polyetheresulphone
  • the obtained bioconjugated materials were initially analysed by UV- visible spectroscopy, aiming at identifying the changes in the AuNPs upon the introduction of the peptide moiety of Cetuximab.
  • This qualitative assessment is based on the different UV- visible profile of the starting materials (i.e. plasmonic Au peak at around 520 nm for the AuNPs, and aromatic amino acids absorption at 280 nm for Cetuximab), and on the possibility of removing the physisorbed Cetuximab from the AuNPs upon the diafiltration process. Therefore, the contemporaneous presence of both of the aforementioned absorption features in all sets of bioconjugates represents an initial evidence that the AuNPs bioconjugation occurred.
  • Figure 7 shows the steady-state UV-Visible absorbance profiles (full lines) and differential plots dAbs/dA (dashed lines) of different materials before and after bioconjugation reaction with COMU for 16h.
  • the AuNPs plasmonic peak and the position of the aromatic amino acid absorbance of the protein moiety are indicated by black arrows and dashed arrows, respectively.
  • Ctxb-AuNPs@PPAA was always isolated with higher yields, it was used as the material to perform the subsequent characterization steps of the compositional assessments (by thermogravimetric analysis (TGA) and atomic absorption spectroscopy (AAS)) and size distribution assessments (by CPS Disc Centrifuge from CPS Instruments).
  • Thermogravimetric analysis is a pivotal characterization technique for hybrid organic-inorganic nanomaterials, being a bulk assessment of solids (on the sub-mg scale) that allows for the discrimination of the weight losses due to residual solvent or moisture (in the range of 50°C to 100°C), organic moiety (in the range of 100°C to 600 °C) and for the determination of the residual weight after the thermal scan due to the inorganic part (see for example E. Mansfield et. al. in Anal. Chem. 2014, 86, 1478).
  • TGA represents an ideal characterization technique for the precise assessment of AuNPs bioconjugates composition, which will lead to more accurate product handling for all the subsequent biological investigations where the exact quantification of the biologically active moieties (Ctxb and Au amount) is mandatory for data comparison and rationalization.
  • Figure 8 shows the TGA characterization of Ctxb-AuNPs@PPAA obtained after 16h of reaction with COMU, i.e. TGA of Cetuximab (Figure 8a), AuNPs@PPAA (Figure 8b) and Ctxb-AuNPs@PPAA (Figure 8c) are reported as temperature-modulated plots (full lines) and differential plots dW/dT (dashed lines).
  • TGA of Cetuximab depicted in Figure 8a shows a decomposition occurring in the range between 100°C and 800 °C, with a residual weight at 950 °C close to 0.
  • AuNPs@PPAA materials typically shows decomposition between 100°C and 500°C, as depicted in Figure 8b, due to the polyallylamine moiety (around 30 wt%, thus approximately 5.0 ⁇ allylamine mg "1 of AuNPs@PPAA).
  • Ctxb-AuNPs composition is 40 wt% Au, 17 wt% polyallylamine, and 43 wt% Cetuximab (2.8 nmol Cetuximab/mg of Ctxb- AuNPs@PPAA). This initial results attested the feasibility of substituting the carbodiimide- mediated procedure with the use of COMU and PBS as reagent and medium according to aspects of the present invention.
  • Ctxb-AuNPs@PPAA showed a similar dose-dependent binding (range 0.01 to 0.5 mg/mL of total material, around 0.004 to 0.2 mg of Au), regardless of the overall reaction time, as shown in Figure 10c.
  • DHLA dihydrolipoic acid
  • Short thiols such as mercapto-undecanoic acid
  • thiolated polyethylene glycol derivatives range between 0.2 to 1 nnr 2 , according to the molecular weight (cf. e.g. H. Schuwirth et. al. in ACS Nano 2013, 7, 1 129; X. Xia et. al. in ACS Nano 2012, 6, 512).
  • plasma-polymerized materials can easily chemisorb on Au by exceeding the monolayer structure, and thus exceed the aforementioned densities.
  • one way to express the polymer molecular weight is based on the weight of its monomeric constituent (about 60 gmol "1 for allylamine, which is 10 times less than the DHLA moiety molecular weight);
  • TGA reveals an overall higher amount of organic moiety for the commercial AuNPs (around 45 wt%, cf. graphs in the left part of Figure 11 b and Figure 11 c) compared to the amount detected in the prepared AuNPs@PPAA (25-30 wt%, cf. Figure 8b).
  • the corresponding Ctxb-bioconjugates show different degrees of Ctxb loading, as seen by the weight loss values resulting from TGA of the commercial bioconjugates (around 90 wt% for 5 nm commercial AuNPs, and around 60 wt% for 10 nm commercial AuNPs, right traces in Figure 11 b and Figure 11 c, respectively), and that of the prepared AuNPs@PPAA bioconjugates (Ctxb-AuNPs@PPAA, around 45 wt%, cf. Figure 8c).
  • Table 1 summarizes all the physicochemical properties of representative samples for both commercial available and prepared AuNPs before and after the bioconjugation with Cetuximab, based on the above results obtained from XPS, TGA and fluorescamine assay.
  • Table 1 Summary of physicochemical properties of commercial and prepared AuNPs, and the related Ctxb bioconjugates obtained by using COMU.
  • BE energy
  • Antibody e.g. Cetuximab
  • Cetuximab is obtained from a commercial source (e.g. for Cetuximab, Erbitux ® 2 or 5 mg mL -1 solutions of 20 to 100 mL by Merck & Co) or results from a process of radiolabelling (for example, radioiodination of antibodies can be performed using the lodo-Gen kit from Pierce Thermo Scientific, using the Thermo Scientific Pierce ® lodination Reagent); the antibody is either dissolved or diluted in phosphate buffer saline (PBS, with molarity in the range 5 mM - 0.5 M and pH between 6.6 and 7.4) to final concentrations ranging between 0.5 - 2.0 mgmL 1 (solution 1 );
  • PBS phosphate buffer saline
  • solution 3 is obtained by adding solution 1 to solution 2 with COMU to antibody (e.g. Cetuximab) weight ratios between 0.5:1 to 2:1 (molar ratios ranging from 177:1 to 710:1 , respectively), and is kept under stirring at room temperature for 15 minutes;
  • AuNPs either prepared (as set out in section B above) or from a commercial source (from AC Diagnostics, Inc.) and with Au amounts ranging between 0.2 and 2 mg Au/mg antibody (e.g.
  • solution 4 (pH around 7);
  • solution 4 is stirred at room temperature for a time ranging between 2 and 8 hours, after which it is purified by either discontinuous diafiltration against H2O in centrifugal concentrators or by tangential flow filtration.
  • the purified material can be maintained in suspension, lyophilized or supplemented with Arabic gum prior to lyophilization.
  • the carboxylic moiety of the organic molecule does not require any additional or separate activation prior to the coupling reaction, due to the high coupling efficiency of COMU together with its solubility in PBS buffered solution.
  • the solution is stirred at 25 °C for 4 hours, after which it is purified by discontinuous diafiltration against H2O in centrifugal concentrators with MWCO of 300,000 g mol "1 ; the product is isolated through freeze-drying, obtaining a pinkish red powder.
  • the cost to bioconjugate 5 mg of nanoparticles is 80.5 cents, meaning 16.1 cents per mg/nanoparticle.
  • EDC chemistry HCI (1 g 19.90 €), NHSS (5g 18.00 €) and MES (25g 47.90 €)
  • the cost for 5 mg of nanoparticles is 410 cents, meaning 82 cents per mg/nanoparticle.
  • the cost for performing methods according to aspects of the present invention is thus substantially reduced, compared to the prior art methods for functionalizing nanoparticles.
  • the present invention thus provides an optimization of the bioconjugation reaction between organic molecules and nanoparticles, both in terms of efficiency and chemical reagents involved, compared with known methods in the art. More particularly, methods of aspects of the invention allow a faster and more efficient functionalization of nanoparticles presenting amine functions with organic molecules comprising a carboxyl group (such as antibodies, proteins, carboxylated biotin, or carboxylated DNA) using non-explosive and less toxic chemical compounds. Methods of aspects of the invention provide the functionalization of nanoparticles performed under simpler reaction conditions, reducing the number of reagents and reaction steps. As a consequence, methods of aspects of the invention do not require high costs. Moreover, the methods are based on relative easy and simple chemical synthesis and the followed purification is easy to perform as well, hence allowing the production of the bioconjugates nanoparticles to scale up to industrial level.
  • a carboxyl group such as antibodies, proteins, carboxylated biotin, or carboxylated DNA

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  • Bioinformatics & Cheminformatics (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

La présente invention concerne des procédés pour la fonctionnalisation de nanoparticules avec des molécules organiques comprenant un groupe carboxyle, telles que des anticorps ou des protéines. Les nanoparticules fonctionnalisées obtenues selon les procédés de la présente invention peuvent être utilisées dans une grande variété d'applications médicales et biologiques.
EP17816711.0A 2016-12-21 2017-12-12 Procédé pour la fonctionnalisation de nanoparticules Withdrawn EP3558392A1 (fr)

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