Patent Grant
10465030
The present invention relates to functionalizable ligands, nanoparticles complexed with said ligands, preferably nanocrystals complexed with ligands, and their use for bio-imaging.
Colloidal semiconductor nanoparticles, named “quantum dots” (QDs), are crystalline objects that exhibit specific fluorescence properties. Their absorption cross section is very large, they are bright and their emission spectra have a small full width half maximum, and a peak wavelength that is tunable as a function of their composition, their size and their shape (in the range of a few nanometers to few tens of nanometers). They are also far more resistant to photobleaching than traditional organic dyes. These unique features make them very attractive for diverse applications in the field of medical and biological imaging, such as individual proteins monitoring, multi-color immunostaining, stem cells tracking, fluorescence acquisition cell sorting, or optically assisted surgery.
Functionalization of QDs surface presents many advantages. Especially, a second imaging agent may be coupled at the surface of the QD, to enable bimodal imaging. On another hand, functionalizing QDs' surface by coupling bio-targeting moieties is interesting for bio-imaging. Functionalization by a therapeutically active molecule may also be interesting.
QD syntheses provide colloidal solutions of fluorescent nanocrystals capped with ligands. Functionalization of QD may thus be performed by functionalizing ligands presents at their surface.
Typical QD syntheses provide QDs capped with hydrophobic ligands, while the use of QDs in live-cell imaging requires their complete solubility in water as well as an excellent compatibility with biological media. To make the QDs water-soluble, one method is to perform cap exchange, consisting in the replacement of original ligands by hydrophilic ones, bearing a chemical function able to bind to the nanocrystal surface (Chan et al. Science 1998, 281:2016 and Mattoussi et al. J. Am. Chem. Soc. 2000, 122:12142). Cap exchange results in small and stable QDs. The non-specific interactions of the QD with cell membranes or with biomolecules in general depend mainly on the moieties that are adsorbed on the QD surface. Ligand exchange provides a versatile method to control the size, the nature of the ligand as well as its affinity for the QD surface (ligands that are too strong can indeed dissolve the QD, while ligands that are not strong enough can detach from the QD surface).
Ligand desorption is a strong limitation for the use of QDs in bio-imaging. This desorption, favored in high diluted conditions, causes indeed a loss of colloidal stability and functionality, as well as an increase in aggregation and non-specific adsorption. As a consequence, continued efforts have been made to improve the affinity of passivating ligands for the QD surface. The design of these replacing ligands is also guided by further needs for biological applications of quantum dots, namely: small size; stability over a large pH range, at elevated salt concentrations and in a cellular medium; low non-specific adsorption; and possible functionalization afterwards.
To match the above-mentioned criteria, especially limitation of ligand desorption and provision of small and stable QDs, the Applicant developed a multidentate zwitterionic ligand L2, issued from the copolymerization of a bidentate monomer and a monozwitterionic monomer (Scheme 1) (Giovanelli et al. Langmuir 2012, 28, 15177-15184).
The structure of the lateral chains of L2 was evidenced to enhance affinity of this ligand toward QDs and thus avoid ligand desorption while keeping a small size of particle.
Derivatives of L2, L2-NH2 and L2-PEG-NH2 were also proposed to introduce functionalizable amine later chains (scheme 2):
Especially, QD functionalization was performed for L2-NH2 by conjugation with fluorescein. L2-PEG-NH2-capped QDs were also conjugated via their amine function, with either biotin or avidin by peptidic coupling after capping QD.
Therefore, the Applicant provided a first generation of hydrophilic ligands having a very good affinity for QD and being functionalizable. However, with this first generation of ligands, functionalization requires to use at least three different monomers to form the ligand: an anchoring monomer, an hydrophilic monomer and a functionalizable monomer. Therefore, the resulting ligand is quite complex. Moreover, it was evidenced that functionalization of this type of ligand modifies its anchoring properties and therefore modifies the stability of the QD.
Therefore, there is a need for more simple ligands which remain functionalizable and for which functionalization does not modify anchoring properties.
The Applicant surprisingly showed that anchoring moieties of L2 ligand may be labile. If there is a sufficient number of anchoring functions in the ligand, a part of them is not linked to the surface of the QD and they may be used for functionalizing the ligand, without decreasing the anchoring stability. This presents the advantage of not requiring a modification of the ligand by a third functionalizable monomer. There is only the need to control the length of the ligand to ensure that there is a sufficient number of anchoring monomers to achieved both anchoring and functionalization.
Moreover, the Applicant evidenced that the ligand of the invention, which may be synthesized by radical polymerization in presence of CTA (chain transfer agent), bears one functionalizable end. Indeed, it was surprisingly shown that upon CTA polymerization, ligands bearing at the first end a hydrogen atom and at the other end the CTA were predominantly obtained, whereas numerous possibilities were expected for ends of the polymeric chain.
By varying the CTA to obtain a functionalizable end of the ligand and by selecting the anchoring moiety of the anchoring monomer, orthogonal functionalization of the ligand may be envisaged to introduce 2 types of functional groups on the ligand and thus on the QD. In this case, a first functional group may be introduced at one end of the ligand, while a second functional group may be introduced on some on the anchoring moieties on lateral chains.
Therefore, the Applicant herein provides a new ligand of QDs, which is a copolymer obtained from at least 2 monomers, said monomers being:
The functionalizable ligand of the invention presents the advantage that even coated on QDs, functionalizable end groups of the polymer are accessible to conjugation. Therefore, the ligand may be functionalized after complexation of QD.
This invention thus relates to a nanomaterial comprising:
According to one embodiment, in the nanomaterial of the invention, the nanoparticle is a nanocrystal and the nanocrystal is a 0D, 1D, or 2D nanocrystal, preferably a nanosheet, a nanorod, a nanoplatelet, a nanoplate, a nanoprism, a nanowall, a nanodisk, a nanoparticle, a nanowire, a nanopowder, a nanotube, a nanotetrapod, a nanoribbon, a nanobelt, a nanoneedle, a nanocube, a nanoball, a nanocoil, a nanocone, a nanopiller, a nanoflower, or a quantum dot.
According to one embodiment, in the nanomaterial of the invention, the ligand is of formula (II):
According to one embodiment, in the nanomaterial of the invention, the ligand is of formula (I-e′):
According to one embodiment, in the nanomaterial of the invention, the ligand is of formula (I-f′):
The invention further relates to a ligand which is a copolymer of general formula (I):
H—P[(A)x-co-(B)y]n-L-R
According to one embodiment, the ligand of the invention is of general formula (II):
According to one embodiment, the ligand of the invention is of formula (I-e):
According to one embodiment, the ligand of the invention is of formula (I-f):
According to one embodiment, in the ligand of the invention, L represents an alkylene linking group having 1 to 20 chain atoms and optionally interrupted or terminated by —O— or —S—.
According to one embodiment, in the ligand of the invention, R represents —COOH or —NH2.
The invention also relates to a water-soluble composition comprising nanomaterials according to the invention.
The present invention further relates to the use of a nanomaterial according to the invention or of the water-soluble composition according to the invention for bioimaging, biotargeting, medical imaging or biosensing.
The invention also relates to a method for manufacturing the ligand according the invention comprising polymerizing an anchoring monomer A and a hydrophilic monomer B in presence of an initiator and a chain transfer agent (CTA), wherein anchoring monomer A and a hydrophilic monomer B are as defined above.
The present invention further relates to a method for manufacturing the nanomaterial according to the invention comprising:
In the present invention, the following terms have the following meanings:
The present invention relates to a ligand which is a copolymer obtained from at least 2 monomers, said monomers being:
In one embodiment, the material for which the first moiety MA has affinity, is selected from the group comprising metals, semiconductors, oxides, lanthanides or mixtures thereof.
In one embodiment, the material is preferably selected from gold, iron, silver, copper, aluminum, platinum, lead, palladium, iron oxide, titanium dioxide, cerium oxide, metal chalcogenide, metal pnictide, cadmium, zinc, magnesium, mercury, gallium, indium, thallium, cobalt, tin or mixtures thereof.
In a specific embodiment, the material for which the first moiety MA has affinity, is under the form of particles, preferably nanoparticles. According to one embodiment, nanoparticles may be nanocrystals.
Especially, the present invention relates to a ligand which is a copolymer obtained from at least 2 monomers, said monomers being:
According to one embodiment, the functional group is selected from the group comprising —NH2, —COOH, —OH, —SH, —CHO, ketone, halide; activated ester such as for example N-hydroxysuccinimide ester, N-hydroxyglutarimide ester or maleimide ester; activated carboxylic acid such as for example acid anhydride or acid halide; isothiocyanate; isocyanate; alkyne; azide; glutaric anhydride, succinic anhydride, maleic anhydride; hydrazide; chloroformate, maleimide, alkene, silane, hydrazone, oxime and furan.
According to an embodiment, the bioactive group is selected from the group comprising avidin or streptavidin; antibody such as a monoclonal antibody or a single chain antibody; sugars; a protein or peptide sequence having a specific binding affinity for an affinity target, such as for example an avimer or an affibody (the affinity target may be for example a protein, a nucleic acid, a peptide, a metabolite or a small molecule), antigens, steroids, vitamins, drugs, haptens, metabolites, toxins, environmental pollutants, amino acids, peptides, proteins, aptamers, nucleic acids, nucleotides, peptide nucleic acid (PNA), folates, carbohydrates, lipids, phospholipid, lipoprotein, lipopolysaccharide, liposome hormone, polysaccharide, polymers, polyhistidine tags, fluorophores.
In an embodiment of the invention monomers A and B are methacrylamide monomers.
In one embodiment, the first moiety MA having affinity for the surface of a nanocrystal has preferably affinity for a metal present at the surface of a nanocrystal or for a material E present at the surface of a nanocrystal and selected in the group of O, S, Se, Te, N, P, As, and mixture thereof.
Examples of metal present at the surface of a nanocrystal include, but are not limited to, gold, iron oxide, titanium dioxide, cerium oxide, metal chalcogenide, metal pnictide, cadmium, zinc, magnesium, mercury, aluminium, gallium, indium, thallium, copper, cobalt, platinum, silver, tin, lead and mixtures thereof.
In one embodiment, the ligand of the invention is a copolymer having a plurality of monomers including monomer A and monomer B. In one embodiment, said ligand is a random, alternate or block copolymer. In another embodiment, the ligand is a statistic copolymer. In another embodiment, said ligand is a random or block copolymer consisting essentially of monomer A and monomer B. In one embodiment of the invention, said ligand is a multi-dentate ligand.
According to one embodiment, preferred monomers A are those described in the following table:
According to one embodiment, preferred monomers B are those described in the following table:
According to one embodiment, preferred -L-R are those described in the following table:
Commonly used nomenclature for a copolymer comprising a total of n monomers, x % of said monomers being monomer A and y % of said monomers being monomer B is: P[Ax-co-By]n. When the extremities of the copolymer are specific, they may be indicated on either side of P[Ax-co-By]n, namely under the form: R1—P[Ax-co-By]n—R2. This nomenclature is used hereafter for the copolymer of the invention.
According to one embodiment, the ligand of the present invention is a copolymer of general formula (I):
H—P-[(A)x-co-(B)y]n-L-R
In a preferred embodiment of the invention, R represents —COOH or —NH2.
In a specific embodiment of the invention, L represents an alkylene linking group having 1 to 20 chain atoms, preferably 1 to 12 chain atoms, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 chain atoms, and optionally interrupted or terminated by —O— or —S—.
In one embodiment, L is of formula —S—(CH2)n1—, wherein n1 is a positive integer ranging from 1 to 20, preferably 1 to 5, more preferably 1, 2 or 3.
In a specific embodiment of the invention, L represents an alkylene linking group having 3 to 20 chain atoms, preferably 3 to 12 chain atoms, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 chain atoms, and optionally interrupted or terminated by —O— or —S—.
In one embodiment, L is of formula —S—(CH2)n1—, wherein n1 is a positive integer higher than 2, preferably 3 to 12 chain atoms, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 chain atoms.
In another embodiment, L represents an ethylene glycol derivative, preferably of formula —[CH2—CH2—O]n2—(CH2)n3—S— wherein n2 and n3 are each independently positive integers and n2+n3 is higher than 2; preferably n2+n3 is ranging from 3 to 230. In one embodiment, L represents an ethylene glycol derivative, preferably of formula —[CH2—CH2—O]n2—(CH2)n3—S— wherein n2 and n3 are each independently positive integers and n2+n3 is ranging from 3 to 120; preferably is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 and 120.
In one embodiment, L represents an ethylene glycol derivative, preferably of formula —[CH2—CH2—O]n2—(CH2)n3—S— wherein n2 and n3 are each independently positive integers and n2+n3 is ranging from 3 to 50; preferably is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50.
In another embodiment, L represents an ethylene glycol derivative, preferably of formula —[CH2—CH2—O]n2—(CH2)n3—S— wherein n2 and n3 are each independently positive integers ranging from 1 to 20, preferably 1, 2, 3, 4, 5, 6 7, 8, 9 or 10, more preferably n2 is equal to 8 and n3 is equal to 2.
In another embodiment, L represents a poly(ethylene glycol) derivative having a molecular weight less than 10 000 g/mol; preferably less than 5 000 g/mol; more preferably less than 1 000 g/mol.
In one embodiment, L represents a poly(ethylene glycol) derivative having a molecular weight ranging from 300 g/mol to 1 000 g/mol. In one embodiment, L represents a poly(ethylene glycol) derivative having a molecular weight ranging from 44 g/mol to 300 g/mol.
In one embodiment, L represents a poly(ethylene glycol) derivative having a molecular weight of about 400 g/mol.
In one embodiment, L represents a poly(ethylene glycol) derivative having a molecular weight of about 300 g/mol.
In another embodiment, -L-R represents —S—(CH2)n3—[CH2—CH2—O]n2—COOH wherein n2 and n3 are each independently positive integers and n2+n3 is higher than 2; preferably n2+n3 is ranging from 3 to 230; more preferably ranging from 5 to 115: more preferably n2 is equal to 8 and n3 is equal to 2.
In another embodiment, -L-R does not represent —S—(CH2)n3—COOH wherein n3 is ranging from 1 to 11.
In another embodiment, -L-R does not represent —S—(CH2)n3—NH2 wherein n3 is ranging from 2 to 11.
In one embodiment of the invention, n is ranging from 1 to 1000, from 1 to 499, from 1 to 249, from 1 to 99, from 5 to 75, from 5 to 50, from 10 to 50, from 10 to 30, from 5 to 35, from 5 to 25, from 15 to 25.
In one embodiment, x and y are each independently a percentage of n, preferably a percentage selected in the group of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50% of n.
According to an embodiment, the ligand of the present invention is a copolymer of general formula (II):
In one embodiment of the invention, R2, R3, R5 and R6 are H.
In one embodiment of the invention, R1 and R4 are alkyl groups, preferably methyl.
In one embodiment of the invention, said first moiety MA having affinity for the surface of a nanocrystal and in particular affinity for a metal present at the surface of a nanocrystal includes, but is not limited to, a thiol moiety, a dithiol moiety, an imidazole moiety, a catechol moiety, a pyridine moiety, a pyrrole moiety, a thiophene moiety, a thiazole moiety, a pyrazine moiety, a carboxylic acid or carboxylate moiety, a naphthyridine moiety, a phosphine moiety, a phosphine oxide moiety, a phenol moiety, a primary amine moiety, a secondary amine moiety, a tertiary amine moiety, a quaternary amine moiety, an aromatic amine moiety, or a combination thereof.
In one embodiment of the invention, said first moiety MA having affinity for the surface of a nanocrystal and in particular affinity for a material E selected in the group of O, S, Se, Te, N, P, As, and mixture thereof, includes, but is not limited to, an imidazole moiety, a pyridine moiety, a pyrrole moiety, a thiazole moiety, a pyrazine moiety, a naphthyridine moiety, a phosphine moiety, a phosphine oxide moiety, a primary amine moiety, a secondary amine moiety, a tertiary amine moiety, a quaternary amine moiety, an aromatic amine moiety, or a combination thereof.
In one embodiment of the invention, said first moiety MA having affinity for the surface of a nanocrystal is a dithiol moiety, preferably a propane-1,3-dithiol, more preferably a propan-1-yl-1,3-dithiol moiety.
In one embodiment of the invention, said second moiety MB being hydrophilic includes, but is not limited to, a zwitterionic moiety (i.e. any compound having both a negative charge and a positive charge, preferably a group with both an ammonium group and a sulfonate group or a group with both an ammonium group and a carboxylate group) such as for example an aminocarboxylate, an aminosulfonate, a carboxybetaine moiety wherein the ammonium group may be included in an aliphatic chain, a five-membered cycle, a five-membered heterocycle comprising 1, 2 or 3 further nitrogen atoms, a six-membered cycle, a six-membered heterocycle comprising 1, 2, 3 or 4 further nitrogen atoms, a sulfobetaine moiety wherein the ammonium group may be included in an aliphatic chain, a five-membered cycle, a five-membered heterocycle comprising 1, 2 or 3 further nitrogen atoms, a six-membered cycle, a six-membered heterocycle comprising 1, 2, 3 or 4 further nitrogen atoms, a phosphobetaine wherein the ammonium group may be included in an aliphatic chain, a five-membered cycle, a five-membered heterocycle comprising 1, 2 or 3 further nitrogen atoms, a six-membered cycle, a six-membered heterocycle comprising 1, 2, 3 or 4 further nitrogen atoms, a phosphorylcholine, a phosphocholine moiety, and combinations thereof or a PEG moiety or poly(ether)glycol moiety.
An example of a suitable poly(ether)glycol moiety is —[O—CH2—CHR′]n—R″, wherein R′ can be H or C1-C3 alkyl, R″ can be H, —OH, C1-C6 alkyl, C1-C6 alkoxy, aryl, aryloxy, arylalkyl, or arylalkoxy and n can be an integer in the range of 1 to 120, preferably of 1 to 60, more preferably of 1 to 30.
In another embodiment of the invention, said second moiety MB being hydrophilic is not a PEG moiety.
In one embodiment of the invention, said second moiety MB being hydrophilic is a sulfobetaine group.
According to an embodiment, the ligand of the present invention is a copolymer of general formula (III):
In one embodiment, each of x′ and x″ is independently a percentage of n, preferably a percentage selected in the group of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50% of n, with the condition that at least one of x′ and x″ is not 0%.
In one embodiment, each of y′ and y″ is independently a percentage of n, preferably a percentage selected in the group of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50% of n, with the condition that at least one of y′ and y″ is not 0%.
In one embodiment of the invention, R2′, R3′, R2″, R3″, R5′, R6′, R5″ and R6″ are H.
In one embodiment of the invention, R1′, R1″, R4′ and R4″ are alkyl groups, preferably methyl.
In one embodiment of the invention, said moiety MA comprises said moieties MA′ and MA″. In one embodiment of the invention, said moiety MB comprises said moieties MB′ and MB″.
In one embodiment of the invention, said x is equal to x′+x″. In one embodiment of the invention, said y is equal to y′+y″.
In one embodiment of the invention, said first moieties MA′ and MA″ having affinity for the surface of a nanocrystal and in particular affinity for a metal present at the surface of a nanocrystal include, but is not limited to, a thiol moiety, a dithiol moiety, an imidazole moiety, a catechol moiety, a pyridine moiety, a pyrrole moiety, a thiophene moiety, a thiazole moiety, a pyrazine moiety, a carboxylic acid or carboxylate moiety, a naphthyridine moiety, a phosphine moiety, a phosphine oxide moiety, a phenol moiety, a primary amine moiety, a secondary amine moiety, a tertiary amine moiety, a quaternary amine moiety, an aromatic amine moiety, or a combination thereof.
In one embodiment of the invention, said first moieties MA′ and MA″ having affinity for the surface of a nanocrystal and in particular affinity for a material E selected in the group of O, S, Se, Te, N, P, As, and mixture thereof, include, but is not limited to, an imidazole moiety, a pyridine moiety, a pyrrole moiety, a thiazole moiety, a pyrazine moiety, a naphthyridine moiety, a phosphine moiety, a phosphine oxide moiety, a primary amine moiety, a secondary amine moiety, a tertiary amine moiety, a quaternary amine moiety, an aromatic amine moiety, or a combination thereof.
In one embodiment of the invention, said first moiety MA′ having affinity for the surface of a nanocrystal is a dithiol moiety and said first moiety MA″ having affinity for the surface of a nanocrystal is an imidazole moiety.
In one embodiment of the invention, said second moieties MB′ and MB″ being hydrophilic include, but is not limited to, a zwitterionic moiety (i.e. any compound having both a negative charge and a positive charge, preferably a group with both an ammonium group and a sulfonate group or a group with both an ammonium group and a carboxylate group) such as for example an aminocarboxylate, an aminosulfonate, a carboxybetaine moiety wherein the ammonium group may be included in an aliphatic chain, a five-membered cycle, a five-membered heterocycle comprising 1, 2 or 3 further nitrogen atoms, a six-membered cycle, a six-membered heterocycle comprising 1, 2, 3 or 4 further nitrogen atoms, a sulfobetaine moiety wherein the ammonium group may be included in an aliphatic chain, a five-membered cycle, a five-membered heterocycle comprising 1, 2 or 3 further nitrogen atoms, a six-membered cycle, a six-membered heterocycle comprising 1, 2, 3 or 4 further nitrogen atoms, a phosphobetaine wherein the ammonium group may be included in an aliphatic chain, a five-membered cycle, a five-membered heterocycle comprising 1, 2 or 3 further nitrogen atoms, a six-membered cycle, a six-membered heterocycle comprising 1, 2, 3 or 4 further nitrogen atoms, a phosphorylcholine, a phosphocholine moiety, and combinations thereof or a PEG moiety or a poly(ether)glycol moiety, wherein if MB′ is a PEG moiety, then MB″ is not a PEG moiety and inversely.
In one embodiment of the invention, said second moiety MB′ being hydrophilic is a sulfobetaine group and said second moiety MB″ being hydrophilic is a PEG moiety or a poly(ether)glycol moiety.
In one embodiment, the ligand of the invention is a copolymer synthesized from at least 2 monomers, said monomers being:
In one embodiment of the invention, RA comprising the first moiety MA can have the formula -LA-MA, wherein LA can be a bond or an alkylene, alkenylene, or arylene linking group having 1 to 50 chain atoms and can be optionally interrupted or terminated by —O—, —S—, —NR7—, wherein R7 is H or alkyl, —CO—, —NHCO—, —CONH— or a combination thereof and MA corresponds to the first moiety as described here above.
Preferably, LA is —C(═O)—NH—(CH2)m—NH—C(═O)—(CH2)p—, wherein m is an integer ranging from 1 to 20, preferably from 1 to 10, preferably from 1 to 5, preferably 2, 3, 4 and p is an integer ranging from 1 to 20, preferably from 1 to 10, preferably from 1 to 6, preferably 3, 4, 5.
In a preferred embodiment, -LA-MA is:
wherein m, p and MA are as defined above, preferably m is equal to 3 and p is equal to 4.
In a preferred embodiment, MA is a dithiol moiety and -LA-MA may be represented by:
wherein m, p and MA are as defined above, preferably m is equal to 3 and p is equal to 4.
In a preferred embodiment, -LA-MA is:
wherein m, p and MA are as defined above preferably m is equal to 3 and p is equal to 4.
In a preferred embodiment, -LA-MA is:
In one embodiment of the invention, RB comprising the second moiety MB can have the formula -LB-MB, wherein LB can be a bond or an alkylene, alkenylene, or arylene linking group having 1 to 50 chain atoms and can be optionally interrupted or terminated by —O—, —S—, —NR7—, wherein R7 is H or alkyl, —CO—, —NHCO—, —CONH— or a combination thereof and MB corresponds to the second moiety as described here above.
Preferably, LB is —C(═O)—NH—(CH2)q—, wherein q is an integer ranging from 1 to 20, preferably from 1 to 10, preferably from 1 to 5, preferably 2, 3, 4.
In a preferred embodiment, -LB-MB is:
wherein q and MB are as defined above, preferably q is equal to 3.
In a preferred embodiment, -LB-MB may be represented by:
wherein q is as defined above and MB is a zwitterionic moiety, preferably q is equal to 3.
In one preferred embodiment, -LB-MB may be represented by:
wherein q is as defined above, preferably q is equal to 3
In another preferred embodiment, -LB-MB may be represented by:
wherein q is as defined above, preferably q is equal to 3,
In a preferred embodiment -LB-MB is:
wherein q is as defined above.
In a preferred embodiment -LB-MB is:
In a specific embodiment, the ligand of the invention is of formula (I-a):
wherein n, x, y, L, R, LA, MA, LB and RB are as defined above.
In a specific embodiment, the ligand of the invention is of formula (I-b):
wherein n, x, y, L, R, m, p, MA, LB and MB are as defined above.
In a specific embodiment, the ligand of the invention is of formula (I-c):
wherein n, x, y, L, R, q, MB, LA and MA are as defined above.
In a specific embodiment, the ligand of the invention is of formula (I-d):
wherein n, x, y, L, R, m, p, MA, q and MB are as defined above.
In a specific embodiment, the ligand of the invention is of formula (I-e):
wherein n, x, y, L and R are as defined in formula (I) above; or a reduced form thereof.
In another specific embodiment, the ligand of the invention is of formula (I-f):
wherein n, x, y and L are as defined in formula (I) above; or a reduced form thereof.
In one embodiment of the invention, the ligand has a molecular weight from about 1,000 g/mol to about 200,000 g/mol, preferably from about 1,000 g/mol to about 100,000 g/mol, preferably from about 1,000 g/mol to about 50,000 g/mol, from about 2,000 g/mol to 50,000 g/mol, more preferably from about 2,000 g/mol to about 10,000 g/mol, from about 2,000 g/mol to 10,000 g/mol.
In one embodiment of the invention, the ligand has a polydispersity index inferior to 10, preferably inferior to 9, 8, 7, 6, 5, 4, 3, 2, 1.
In one embodiment of the invention, the ligand has a ratio of monomers A/B in number from about 1/99 to about 99/1. In a particular embodiment, the ligand has a ratio of monomers A/B in number from about 1/99 to about 50/50, preferably from about 3/97 to about 40/60, more preferably from about 10/90 to about 50/50. In another particular embodiment, the ligand has a ratio of monomers A/B in number from about 50/50 to about 99/1, preferably from about 40/60 to about 90/10. In another embodiment, the ligand has a ratio of monomers A/B in number from about 1/99 to about 75/25, preferably from about 5/95 to about 50/50, more preferably from about 5/95 to about 25/75. In a specific embodiment, the ligand has a ratio of monomers A/B in number of about 50/50. In a specific embodiment, the ligand has a ratio of monomers A/B in number of about 20/80.
According to one embodiment, the synthesis of the ligand may be performed in presence of monomer A and monomer B with a ratio of molar amounts of A to B ranging from 1/99 to 99/1, preferably from 5/95 to 50/50, preferably from 10/90 to 50/50; preferably from 10/90 to 30/70, more preferably 20/80.
In another embodiment, of the invention, the ligand is a copolymer synthesized from at least 3 monomers, said monomers being:
According to an embodiment, the ligand of the present invention is a copolymer of general formula (IV):
H—P[(A)x-co-(B)y-co-(C)z]n-L-R
According to an embodiment, the ligand of the present invention is a copolymer of general formula (V):
In one embodiment of the invention, R2, R3, R5, R6, R9, and R10 are H.
In one embodiment, R1, R4, and R8 are alkyl groups, preferably methyl.
According to one embodiment, said third moiety MC being hydrophobic includes, but is not limited to, alkyl, aryl, alkylaryl, arylalkyl, alkenyl groups.
According to one embodiment, preferred monomers C are those described in the following table:
In one embodiment of the invention, RC comprising the third moiety MC can have the formula -LC-MC, wherein LC can be a bond or an alkylene, alkenylene or arylene linking group having 1 to 8 chain atoms.
In one embodiment of the invention, said first moiety MA is not a dihydrolipoic acid (DHLA) moiety.
In another embodiment of the invention, said first moiety MA is not an imidazole moiety.
In one embodiment, when B comprises a monomer comprising a second moiety MB which is a PEG moiety, then B further comprises at least one monomer comprising a second moiety MB which is not a PEG moiety.
In one embodiment of the invention, when the first moiety MA is an imidazole moiety, the second moiety MB is not a PEG moiety.
In one embodiment of the invention, when the first moiety MA is an imidazole moiety, the second moiety MB is not a sulfobetaine moiety. In a specific embodiment of the invention, when the first moiety MA is an imidazole moiety, the second moiety MB is not —N+(Me)2-(CH2)2—SO3− or —N+(Me)2-(CH2)3—SO3−.
In one embodiment of the invention, when the first moiety MA is an imidazole moiety, the second moiety MB is not a carboxybetaine moiety. In a specific embodiment of the invention, when the first moiety MA is an imidazole moiety, the second moiety MB is not —N+(Me)2-(CH2)2—COO−.
In one embodiment of the invention, when the first moiety MA is an imidazole moiety, the second moiety MB is not a phosphobetaine moiety. In a specific embodiment of the invention, when the first moiety MA is an imidazole moiety, the second moiety MB is not —O—P(O2−)—P—(CH2)2—N+(Me)3.
In one embodiment of the invention, when the first moiety MA is a dithiol group, and B comprises a monomer comprising a second moiety MB′ which is a PEG moiety, then B further comprises at least one monomer comprising a second moiety MB″ which is not a PEG moiety.
According to one embodiment, preferred combinations of monomers A and B are described in the following table:
According to one embodiment, preferred ligands comprise combinations of monomers A and B as described in the table above and -L-R is —S—(CH2)2—(O—CH2—CH2)8—COOH, —S—(CH2)12—COOH or any -L-R described hereabove.
The present invention further relates to a method of manufacturing of the ligand of the invention.
In one embodiment, the manufacturing process of the ligand of the invention comprises polymerizing an anchoring monomer A and a hydrophilic monomer B in presence of an initiator and a chain transfer agent (CTA).
According to one embodiment, the synthesis of the ligand may be performed by any radical polymerization known by those skilled in the art. According to one embodiment, the synthesis of the ligand may be performed by controlled radical polymerization such as RAFT polymerization (Reversible Addition-Fragmentation chain Transfer), ATRP (Atom Transfer Radical Polymerization), NMP (Nitroxide Mediated Polymerization) or iodine transfer polymerization. According to another embodiment, the synthesis of the ligand may be performed by non-controlled radical polymerization.
According to one embodiment, the synthesis of the ligand may be performed in presence of a monomer A and monomer B and a solvent such as for example acetic acid, THF, water or a mixture thereof.
In a preferred embodiment, the solvent is acetic acid.
In one embodiment, the polymerization is performed at a temperature ranging from 40° C. to 100° C., preferably from 50° C. to 70° C., more preferably at about 60° C.
In an embodiment, the polymerization is performed for a period of time ranging from 0.5 to 24 hours, preferably from 12 to 18 hours.
In one embodiment, the polymerization of monomers A and B is initiated by an initiator such as for example a diazoinitiator, preferably azobisisobutyronitrile (AIBN), 2,2′-azobis(2-methylpropionamidine); a peroxide, preferably acyl peroxides, acetyl peroxides, benzoyl peroxides, alkyl peroxides, hydroperoxides, acyl alkylsulfonyl peroxides, dialkyl peroxydicarbonates, diperoxyketals, ketone peroxides; a perester; an azo; a disulfide; a tetrazene; a persulfate compounds. Alternatively, the polymerization of monomers A and B is initiated by a redox reaction.
In one embodiment, the polymerization of monomers A and B is performed in presence of a chain transfer agent (CTA) comprising a moiety -L-R, wherein L and R are as defined above. In one embodiment, the chain transfer agent (CTA) does not comprise a moiety -L-R which is —S—(CH2)2—COOH.
In one embodiment, the polymerization of monomers A and B is performed in presence of a chain transfer agent (CTA), wherein the CTA is a substituted alkylthiol or a disulfide. Preferably, the alkylthiol or the disulfide is substituted by a group selected from COOH, NH2, PEG, DNA, RNA, PNA, polysaccharide, peptide or any functional group or any bioactive group as defined herein.
In one embodiment, the chain transfer agent (CTA) is not 3-mercaptopropionic acid (MPA).
In one embodiment, the chain transfer agent (CTA) is not a RAFT agent; preferably is not a chain transfer agent selected from trithiocarbonate, dithiocarbamate or dithiobenzoate.
In one embodiment, the chain transfer agent (CTA) is not dibenzyl carbonotrithioate.
In one embodiment, the molar quantity of the initiator is ranging from 0.1 to 15%, preferably from 0.1% to 10%, in moles relative to the molar amount of monomers.
In one embodiment, the molar quantity of the chain transfer agent is different from 0 and is ranging up to 50%, preferably up to 20%. one embodiment, the molar quantity of the chain transfer agent is ranging from 10−6% to 20%, from 10−5% to 20%, from 10−4% to 20%, from 10−3% to 20%, from 10−2% to 20%, from 0.1 to 15%, from 1% to 15%, from 2% to 15%, from 5% to 15% in moles relative to the molar amount of monomers. In another embodiment, the molar quantity of the chain transfer agent is 10% in moles relative to the molar amount of monomers. In another embodiment, the molar quantity of the chain transfer agent is 5% in moles relative to the molar amount of monomers.
The invention further relates to a functionalizable or functionalized material. According to one embodiment, the invention relates to a mono-functionalizable or mono-functionalized material. According to another embodiment, the invention relates to a bi-functionalizable or bi-functionalized material.
In an embodiment, the functionalizable/functionalized material of the invention comprises a material and at least one ligand according to the invention. In one embodiment, the material is selected from the group comprising metals, semiconductors, oxides, lanthanides or mixtures thereof. In one embodiment, the material is preferably selected from gold, iron, silver, copper, aluminum, platinum, lead, palladium, iron oxide, titanium dioxide, cerium oxide, metal chalcogenide, metal pnictide, cadmium, zinc, magnesium, mercury, gallium, indium, thallium, cobalt, tin or mixtures thereof.
In a specific embodiment, the material is under the form of particles. In one embodiment, the particles of the invention have the shape of a sphere, a cube, a tetrahedron, a rod, a wire, a platelet, a tube, a cube, a ribbon, or mixture thereof. According to an embodiment, particles are microparticles or nanoparticles, preferably nanoparticles. According to one embodiment, microparticles are microspheres. According to one embodiment, nanoparticles are nanocrystals, preferably under the form of quantum dots.
An object of the invention is thus a nanomaterial comprising a nanocrystal complexed with at least one ligand of the invention.
In one embodiment, said nanocrystal is a 0D, 1D, or 2D nanocrystal.
In one embodiment, said nanocrystal may be for instance a nanosheet, a nanorod, a nanoplatelet, a nanoplate, a nanoprism, a nanowall, a nanodisk, a nanoparticle, a nanowire, a nanopowder, a nanotube, a nanotetrapod, a nanoribbon, a nanobelt, a nanoneedle, a nanocube, a nanoball, a nanocoil, a nanocone, a nanopiller, a nanoflower, or a quantum dot.
In one embodiment, said nanocrystal is inorganic. In another embodiment, said nanocrystal is organic.
In one embodiment, said nanocrystal is a semiconductor material, a ceramic material, a magnetic material or a metallic material.
In one embodiment, said nanocrystal is a semi-conductor selected from group IV, group IIIA-VA, group IIA-VIA, group IIIA-VIA, group IA-IIIA-VIA, group IIA-VA, group WA-VIA, group VIB-VIA, group VB-VIA, or group IVB-VIA.
In one embodiment, said nanocrystal is a material MxEy, wherein:
In one embodiment, said material MxEy comprises cationic elements M and anionic elements E in stoichiometric ratio, said stoichiometric ratio being characterized by values of x and y corresponding to absolute values of mean oxidation number of elements E and M respectively.
In one embodiment, said nanocrystal is a material selected from Si, Ge, CdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, HgS, HgSe, HgTe, PbS, PbSe, PbTe, CuInS2, CuInSe2, AgInS2, AgInSe2, CuS, Cu2S, Ag2S, Ag2Se, Ag2Te, InN, InP, InAs, InSb, In2S3, Cd3P2, Zn3P2, Cd3As2, Zn3As2, ZnO, AN, AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, FeS2, TiO2, Bi2S3, Bi2Se3, Bi2Te3, MoS2, WS2, VO2, and alloys and mixtures thereof.
In one embodiment, said nanocrystal is a metallic material such as gold, silver, copper, aluminum, iron, platinum, lead, palladium, iron oxide, titanium dioxide, cerium oxide, metal chalcogenide, metal pnictide, cadmium, zinc, magnesium, mercury, gallium, indium, thallium, cobalt, tin or mixtures thereof.
In one embodiment, said nanocrystal presents a heterostructure, which means that the nanocrystal of the invention is partially coated by at least one layer of inorganic material.
A semiconductor nanocrystal is capable of emitting electromagnetic radiation upon excitation (i.e., the semiconductor nanocrystal is luminescent) and includes a “core” of one or more first semiconductor materials, and may be surrounded by a “shell” of a second semiconductor material. A semiconductor nanocrystal core surrounded by a semiconductor shell is referred to as a “core/shell” semiconductor nanocrystal. The surrounding “shell” material will preferably have a bandgap energy that is larger than the bandgap energy of the core material and may be chosen to have an atomic spacing close to that of the “core” substrate.
In one embodiment, the nanocrystal of the invention presents a core/shell structure, i.e. the nanocrystal comprises a core and a shell of semiconducting material.
In one embodiment, the nanocrystal of the invention has a core/shell structure, i.e. the core is totally coated by at least one layer of inorganic material.
Preferably, said quantum dots are core/shell type I quantum dots. Said type I quantum dot can have a band alignment between the core and the shell such that the exciton created in the shell is transferred in the core where it recombines radiatively.
In another embodiment, the nanocrystal of the invention comprises a core totally coated by a first layer of inorganic material, said first layer being partially or totally surrounded by at least one further layer of inorganic material.
In one embodiment, said core and said at least one layer of inorganic material have the same composition or do not have the same composition.
In one embodiment, said core and said at least one layer of inorganic material may be a semi-conductor from group IV, group IIIA-VA, group IIA-VIA, group IIIA-VIA, group IA-IIIA-VIA, group IIA-VA, group IVA-VIA, group VIB-VIA, group VB-VIA, or group IVB-VIA.
In another embodiment, said core and said at least one layer of inorganic material may comprise a material MxEy, wherein:
In another embodiment, said core and said at least one layer of inorganic material may be composed of a material from Si, Ge, CdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, HgS, HgSe, HgTe, PbS, PbSe, PbTe, CuInS2, CuInSe2, AgInS2, AgInSe2, CuS, Cu2S, Ag2S, Ag2Se, Ag2Te, InN, InP, InAs, InSb, In2S3, Cd3P2, Zn3P2, Cd3As2, Zn3As2, ZnO, AN, AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, FeS2, TiO2, Bi2S3, Bi2Se3, Bi2Te3, MoS2, WS2, VO2, and alloys and mixtures thereof.
In one embodiment, the core and/or the shell can be a semiconductor material including, but not limited to, those of the group II-VI (ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like) and III-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and the like) and IV (Ge, Si, and the like) and IV-VI (PbS, PbSe) and I-III-VI-2 (CuInS2, CuInSe2, AgInS2, AgInSe2, CuGaS2, CuGaSe2, AgGaS2, AgGaSe2,) and L2-VI (Ag2S, Ag2Se) and IV-VI-2 (SnS2, SnSe2) and II-V (Cd3P2) and oxydes (ZnO) materials, and an alloy or a mixture thereof.
In another embodiment, the nanocrystal of the invention presents a heterostructure comprising a metallic material and semiconductor material.
According to one embodiment, the functionalizable/functionalized material of the invention comprises:
According to one embodiment, the material complexed by the ligand of the invention is a nanoparticle, leading to a functionalizable/functionalized nanomaterial.
According to one embodiment, the nanomaterial of the invention comprises:
According to an embodiment, the nanomaterial of the invention comprises:
According to an embodiment, the nanomaterial of the invention comprises:
In a preferred embodiment, the nanomaterial of the invention comprises a ligand of formula (I) wherein R is an activated function such as for example and activated ester or an activated carboxylic acid, preferably an activated ester, more preferably N-hydroxysuccinimide ester.
In one embodiment of the invention, the nanomaterial of the invention comprises a nanocrystal complexed by the ligand of formula (II):
In a specific embodiment, the nanomaterial of the invention comprises a nanocrystal complexed by the ligand of formula (I-e′):
wherein n, x, y, L and R are as defined in formula (I) above.
In a further embodiment, the nanoparticle of the invention comprises a nanocrystal complexed by the ligand of formula (I-f′):
wherein n, x, y and L are as defined in formula (I) above.
In a preferred embodiment, in formula (I-f), L represents an alkylene linking group having 1 to 20 chain atoms and optionally interrupted or terminated by —O— or —S—. More preferably, L is of formula —S—(CH2)n1, wherein n1 is a positive integer ranging from 1 to 20, preferably 1 to 5, more preferably 1, 2 or 3. In another embodiment, L represents an ethylene glycol derivative, preferably of formula —[CH2—CH2—O]n2—(CH2)n3—S— wherein n2 and n3 are each independently positive integers ranging from 1 to 20, preferably 1, 2, 3, 4, 5, 6 7, 8, 9 or 10, more preferably n2 is equal to 8 and n3 is equal to 2.
In another embodiment of the invention, the nanomaterial of the invention comprises a ligand of formula (I) wherein R is a bioactive group, preferably a protein or an antibody.
Preferred embodiments relative to the ligand of formula (I) of the invention described above apply to the ligand of formula (I) comprised in the nanomaterial of the invention.
According to one embodiment, the nanomaterial of the invention comprises a ligand of formula (I) wherein the number of ligand per dot is ranging from less than 100; preferably less than 50 ligands per dots; more preferably less than 30 ligands per dots. In one embodiment, the number of ligand per dot is about 20 ligands.
According to one embodiment, the nanomaterial of the invention comprises a ligand of formula (I) wherein the number of fluorophores per dot is ranging from less than 100; preferably less than 20; preferably is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fluorophores per dot; preferably is ranging from more than 0 to 1. In one embodiment, the number of fluorophores per dot is 0.5.
Another object of the invention is a method for complexing at least one ligand of the invention to at least one material, preferably at least one nanoparticle, more preferably at least one nanocrystal, comprising:
In one embodiment, said weakly binding ligand or molecule may be MPA, or trimethylsilylating agents, or trialkyl oxonium salts (Meerwein's salt), or any monothiol, or amine ligands.
In one embodiment, said first step is performed in basic chloroform and leads to the precipitation of the nanocrystal complexed to the intermediate ligand and to the homogeneous dispersion of the nanocrystal into water at room temperature. In another embodiment, said first step is performed in ethanol and leads to the precipitation of the nanocrystal complexed to the intermediate ligand.
In one embodiment, said second step is performed in an aqueous medium such as 20 mM aqueous NaCl at about 50° C. to 75° C., preferably 60° C., overnight, to remove the weak intermediate ligand and replace it by the ligand of the invention.
In another embodiment, said method may further comprise an ultrafiltration step and then an ultracentrifugation step in an aqueous sucrose gradient.
In the case wherein R represents a functional group in the ligand of formula (I), the process for manufacturing the nanomaterial of the invention may further comprise a subsequent step of bio-conjugation to introduce a bioactive group at the end of the polymeric chain of the ligand.
In a further embodiment, some of the MA moieties of the anchoring monomers A of the ligand of the invention may be functionalized by bioactive groups, once the ligand has been complexed to the nanocrystal.
In an embodiment, the process for manufacturing the nanomaterial of the invention comprises:
By “some of the MA moieties of the anchoring monomers” it is referred to less than 70% of the total number of MA moieties.
The nanomaterial of the invention may be obtained by the complexation of the ligand of formula (I) of the invention with a nanocrystal. Once complexed with the nanocrystal, the ligand of formula (I) may be modified, especially by activation of functional group R present at one end of the ligand. Moreover, the ligand of formula (I) may be modified by conjugation of a bioactive group to the functional group or activated functional group present at one end of the ligand, leading to functionalized ligand of formula (I).
According to one embodiment, the nanomaterial of the invention is activated by modifying the terminal end R of the ligand of formula (I) under the form of a terminal N-hydroxysuccinimide ester. Such activation enables the conjugation of bioactive groups comprising an amino group.
Another object of the invention is a water-soluble composition comprising at least one quantum dot, nanoplatelet or quantum dot having its lateral dimensions larger than its thickness, complexed with at least one ligand of the invention.
Another object of the invention is the use of said quantum dot, nanoplatelet or quantum dot having its lateral dimensions larger than its thickness, complexed with at least one ligand of the invention for bioimaging, biotargeting, medical imaging, biosensing.
Thus, it is readily apparent that said complexes find use in a variety of assays where other, less reliable, labeling methods have typically been used, including, without limitation, fluorescence microscopy, fluorescence histology, fluorescence cytology, fluorescence pathology, cell labeling, flow cytometry, western blotting, Fluorescence Resonance Energy Transfer (FRET), immunocytochemistry, Fluorescence In Situ Hybridization (FISH) and other nucleic acid hybridization assays, signal amplification assays, DNA and protein sequencing, immunoassays such as competitive binding assays and ELISAs, immunohistochemical analysis, protein and nucleic acid separation, homogeneous assays, multiplexing, high throughput screening, chromosome karyotyping, and the like.
The present invention will be better understood with reference to the following examples. These examples are intended to representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.
AIBN: azobisisobutyronitrile;
APMA.HCl: N-(3-aminopropyl)methacrylamide hydrochloride;
CTA: Chain Transfer Agent;
DCC: dicyclohexylcarbodiimide;
EDC: 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride;
LA: 5-(1,2-dithiolan-3-yl)pentanoic acid, also known as lipoic acid;
MES buffer: 2-(N-morpholino)ethanesulfonic acid aqueous buffer;
MFA: N-methylformamide;
MPA: 3-mercaptopropionic acid;
NHS: N-Hydroxysuccinimide;
PEG: poly(ethylene glycol);
QD(s): Quantum Dot(s);
SA: streptavidin;
SPP: 3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt.
Streptavidin was purchased from Biospa; APMA.HCl was purchased from Tebu-bio; SPP and 3-[3-methacrylamidopropyl-(dimethyl)ammonio]propane-1-sulfonate), from Raschig GmbH (RaleMer SPP); all other chemicals used therein were purchased from Sigma-Aldrich. All of these purchased chemicals were used without further purification unless otherwise specified. Chromatography on silica was carried out on Kieselgel 60 (230-240 mesh, Merck) and analytical TLC was performed on Merck precoated silica gel (60 F254) 1H NMR spectrum was recorded on a Bruker Avance DPX 400 spectrometer at 400.13 MHz. Chemical shifts (δ) are expressed in ppm and coupling constant (J) in hertz. Absorption measurements were carried out with a Cary 5E UV-vis-NIR spectrophotometer (Varian).
Synthesis of monomer A (5-(1,2-dithiolan-3-yl)-N-(3-methacrylamidopropyl)-pentanamide)
To a suspension of APMA.HCl (2 g, 11.2 mmol) in dichloromethane (20 mL) was added triethylamine (2.5 mL, 17.9 mmol). Methanol (2 mL) was introduced to obtain complete solubilization. A solution of LA (2.76 g, 13.4 mmol) in dichloromethane (5 mL) was then added, followed by NHS (1.58 g, 13.8 mmol) in one portion. The reaction mixture was cooled down to 0° C. with an ice bath and a solution of DCC (3.00 g, 14.4 mmol) in dichloromethane (10 mL) was injected dropwise. The medium was warmed up to room temperature and further stirred overnight. A pale yellow solution containing a white precipitate was obtained. The solution was washed by a 0.1 M aqueous HCl solution (2×50 mL), deionized water (1×50 mL) and a 0.2 M aqueous NaOH solution (2×50 mL). The organic phase was separated, dried over MgSO4, filtrated and concentrated under reduced pressure. The crude residue was purified by chromatography on silica (eluent: hexane/ethyl acetate 1/4, then hexane/acetone 1/1) to give A (2.88 g, 8.71 mmol, 78%) as a pale yellow solid. R1=0.37 (hexane/acetone 1/1); 1H NMR (CDCl3, 400 MHz): δ 7.03 (sl, 1H); 6.87 (sl, 1H); 5.72 (s, 1H); 5.29 (s, 1H); 3.53-3.39 (m, 1H); 3.29-3.20 (m, 4H); 3.14-3.01 (m, 2H); 2.43-2.35 (m, 1H); 2.18 (t, J=8.0 Hz, 2H); 1.92 (s, 3H); 1.88-1.80 (m, 1H); 1.68-1.55 (m, 6H); 1.48-1.33 (m, 2H).
Synthesis of the Ligand
The polymerization step consisted of the radical copolymerization of two methacrylamides: one containing the precursor of a dithiol anchoring function (monomer A, obtained as described above), the other including a sulfobetaine group (monomer B, SPP commercially available).
Due to mismatching monomers' solubilities, the solvent used for these polymerizations was acetic acid. Various A/B molar ratios have been tested between 10/90 and 50/50.
The amounts of initiating agent AIBN (2,2′-Azobis(2-methylpropionitrile)) and of CTA were chosen in various molar equivalents relative to the total amount of monomers, in order to form various length chains. Various monomer/CTA molar ratios have been tested between 10/1 and 40/1. The monomers conversion rates were determined by 1H NMR in D2O (classically over 90%).
Characterization of the products by NMR confirmed the MA/MB molar ratio by comparison of the acrylamide peaks between the initial state of the polymerization (
A general procedure is given for the synthesis a ligand with x=2 and n=10.
Monomer B (SPP, 1.17 g, 4 mmol, 4 equiv.), monomer A (5-(1,2-dithiolan-3-yl)-N-(3-methacrylamidopropyl)pentanamide, 331 mg, 1 mmol, 1 equiv.) and CTA (0.5 mmol, 0.5 equiv.) were dissolved in acetic acid (20 mL) in a sealed septum flask. The mixture was degassed with argon and then heated at 60° C. A solution of AIBN (82 mg, 0.5 mmol, 0.5 equiv.) in acetic acid (2 mL) under argon atmosphere was further added in one portion. The mixture was stirred overnight at 60° C. Acetic acid was evaporated under reduced pressure. To remove residual acetic acid traces, the residue was dissolved in 20 mL of deionized water and evaporated under reduced pressure (two times). The residue was then dissolved in 20 mL of deionized water and extracted three times with 20 mL of dichloromethane. The aqueous phase was precipitated in 9-fold excess of ethanol. The precipitated polymer was separated by centrifugation (50 mL centrifuge tubes, 2500 rpm, 10 min), washed 2 times with ethanol and then dried overnight under vacuum. The polymer was obtained as a yellow-brown solid (950 mg, 61%).
In the case wherein the ligand is obtained by polymerization with “mercapto-carboxilic acid containing compounds” as CTA, the ligand is referred to as “ligandCOOH”. Especially, in the case wherein the ligand is obtained by polymerization with mercaptoproprionic acid as CTA, the ligand is referred to as “ligand-(CH2)2COOH” and in the case wherein the ligand is obtained by polymerization with a CTA comprising a PEG moiety, the ligand is referred to as “ligand-PEG-COOH”.
In the case wherein the ligand is obtained by polymerization with “mercapto-amino containing compounds” as CTA, the ligand is referred to as “ligandNH2”.
CdSe/CdS/ZnS QDs Synthesis
600-nm-emitting CdSe/CdS/ZnS QDs were synthesized using slight modifications of previously published procedures. CdSe cores were synthesized by reaction of trioctylphosphine selenide and cadmium oleate in octadecene, oleylamine and trioctylphosphine oxide. Three monolayers of CdS shell, followed by two monolayers of ZnS, were grown using cadmium oleate, zinc oleate and sulfur diluted in octadecene following the SILAR (Successive Ionic Layer Adsorption and Reaction) procedure.
Ligand Exchange: Standard Procedure
“Classical” biphasic cap exchange with CdSe/CdS/ZnS core/shell QDs solubilized in chloroform did not succeed. The poor solubility of the ligand of the invention in chloroform and the low partition coefficient between the two solvents could explain the difficult phase transfer of the QDs.
To overcome this problem, a two-step process was chosen. A first exchange was performed using pure mercaptopropionic acid (MPA), on as-synthesized QDs precipitated in ethanol. The QDs were kept overnight at 60° C. in order to have QDs surface was saturated by MPA. The excess of MPA was removed and QDs were dispersed in DMF. The MPA were then deprotonated using a large excess of tert-butoxide. The QDs became then instable in organic solvents and were precipitated and washed with ethanol. QDs were then suspended in a sodium tetraborate (pH 9, 10 mM) water-based buffer. To this homogeneous dispersion, an aqueous solution of previously reduced ligand of the invention (by NaBH4) was added to perform the second ligand exchange. The aqueous medium was kept overnight at 60° C. to move from the weak intermediate QD covered by MPA to QD-ligand. The polymer excess was removed by Vivaspin® filtration. The QD-ligand did not show any change in quantum yield after the ligand exchange.
General Procedure
CdSe/CdS/ZnS core/shell QDs in hexane (0.2-2 nmol respectively for 650-550 nm QDs) were precipitated with ethanol (0.5 mL) and centrifuged (13000 rpm, 5 min). The supernatant was removed. The QDs were dispersed in 3-mercaptopropionic acid (MPA) (0.2 mL). The mixture was sonicated to obtain a homogenous dispersion. The QDs dispersion was stored at 60° C. overnight to perform first cap exchange. The QDs were centrifuged (13000 rpm, 2 min) and the MPA phase was discarded. The QDs were dispersed in DMF (0.2 mL) under sonication. 2 mg of potassium tert-butoxide were added and QDs dispersion was sonicated (1 min). The mixture was centrifuged (13000 rpm, 2 min). The uncolored DMF phase was discarded. The precipitated QDs were washed twice with ethanol (2×0.5 mL EtOH). The QDs were dispersed in sodium tetraborate (pH9 10 mM). Typically, at this step, QDs colloidal dispersion was clear. 200 μL of aqueous solution of the ligand of the invention (10 mg/mL), previously reduced 30 min with NaBH4 (1 mg/mg of polymer), were added to QDs dispersion. The aqueous QDs dispersion was stored at 60° C. overnight to perform the second cap exchange. The excess of free ligand and reagents were removed by three washing by membrane ultrafiltration (Sartorius Vivaspin®500 μL disposable filter—cutoff 100 kDa) at 13000 rpm in 20 mM aqueous NaCl. QDs-ligand were finally taken up in 20 mM aqueous NaCl.
In the case wherein the ligand is a ligandCOOH, as in example 1, resulting coated QDs are referred to as “QDs-ligandCOOH”.
In the case wherein the ligand of the invention is obtained by polymerization with “mercapto-carboxilic acid containing compounds” as CTA (for example: mercaptopropionic acid or O-(2-Carboxyethyl)-O′-(2-mercaptoethyl)heptaethylene glycol), an acidic function is advantageously introduced at one extremity of the ligand (respectively “ligand(CH2)2COOH” and “ligand-PEG-COOH”).
Ligand exchange using these ligandCOOH has been studied in order to provide biocompatible coated QDs presenting carboxylic acidic function, QDs-ligandCOOH. First, reactive N-hydroxysuccinimide (NHS) esters thereof (“QD-ligandNHS”) were prepared and purified. Then, these activated QDs have been used to functionalize QDs with protein (for example: streptavidin or antibodies).
Washing Before Freeze-Drying: Standard Procedure
QDs-ligandCOOH in 20 mM aqueous NaCl were washed three times by membrane ultrafiltration at 13000 rpm using a Sartorius Vivaspin®500 μL disposable filter (cutoff 100 kDa) in pure water. QDs-ligandCOOH were finally taken up in pure water for freeze-drying.
Activation of the Carboxylic Acidic Function of the Polymer on QDs: Standard Procedure
Freeze-dried QD-ligandCOOH (2.5 mg) were dispersed in pure water (50 μL) at room temperature. In parallel, EDC (5 mg, 30 μmol) and NHS (5 mg, 44 μmol) were dissolved in MES buffer 0.2 M pH 5.5 (1 mL). 20 μL of this solution are added to the dispersion of QD and immediately after 180 μL of MFA are added. The reaction was stirred overnight at room temperature before precipitation in acetonitrile (1 mL). The colored precipitate obtained after centrifugation (13000 rpm, 2 min) was washed twice in acetonitrile (1 mL) before drying under vacuum. QDs-ligandNHS were conserved under inert atmosphere at −18° C.
Biomolecules Functionalization of QDs:
Several experiments were carried out starting from either QDs-ligand-COOH or QDs-ligand-NHS.
QDs-ligand-(CH2)2—COOH represents quantum dots coated with a ligand copolymer wherein the end carboxylic function is from 3-mercaptopropionic acid. QDs-ligand-(CH2)2—NHS correspond to the corresponding dots after the reaction with N-hydroxysuccinimide.
QDs-ligand-PEG-COOH represents quantum dots coated with a ligand copolymer wherein the end carboxylic function is from a CTA comprising a PEG moiety; especially from (2-carboxyethyl)-O′-(2-mercaptoethyl)heptaethylene glycol. QDs-ligand-PEG-NHS corresponds to the corresponding dots after the reaction with N-hydroxysuccinimide.
Standard Procedure from QDs-ligandNHS
Dried QDs-ligandNHS (2.5 mg) were dispersed in 120 μL streptavidine or antibody solution (10 mg/mL in aqueous NaHCO3 0.2 M pH 8.4). Protein excess was eliminated by ultracentrifugation on sucrose gradient (40%-10%). QDs-proteins/QDs-antibody were finally taken up in 0.2M aqueous NaHCO3.
Characterizations Biotin Test
First the quantum dots functionalized by streptavidin were tested with biotin-coated agarose beads in order to evaluate the achievement of the functionalization. The results are shown Table 1 and
In this test, the high affinity between streptavidin and biotin leads to fluorescence.
The results of Table 1 show that when quantum dots are coated with a ligand comprising a PEG moiety (QDs-ligand-PEG-COOH), their functionalization with streptavidin leads to fluorescent agarose beads (+).
In the case wherein quantum dots are coated with a ligand comprising a —S—(CH2)2—COOH end obtained from mercaptopropionic acid (QDs-ligand-(CH2)2—COOH), the reaction between the resulting quantum dots and the agarose beads does not lead to fluorescent beads.
These results show that when the ligand is ended by —S—(CH2)2—COOH, the functionalization with streptavidin fails whereas in the case wherein the ligand comprises a PEG moiety, the functionalization of the QDs-PEG-COOH is successfully achieved.
Without willing to be bound by a theory, the Applicant thinks that the size of the spacer affects the functionalization of the coated-quantum dots. The skilled human in the art knows that carboxylic function has good affinity for the surface of dots. Thus, increasing the size of the spacer L would have favored the coiling of the end of the ligand chain on the dots surface, preventing the functionalization by a protein, a fluorophore or an antibody.
Unexpectedly, these experiments show that an enough long spacer is required to implement the functionalization of the QDs-NHS with a protein or an antibody.
Number of Fluorophores by Quantum Dot
Functionalized quantum dots were analyzed by HPLC with an absorbance detector at 254 nm or a fluorescence detector (emission parameters: Lexc.=630 nm and LF=650 nm). The number of fluorophores grafted on a quantum dots is calculated by the ratio between the absorption band of QDs (at 350 nm) and the absorption band of fluorophores (at 650 nm).
The results are shown in the following table:
These results show that when quantum dots are coated with a ligand comprising a -PEG-COOH moiety and then functionalized with a fluorophore (QDs-PEG-F), the functionalization reaction is successfully carried out and the average number of flurophores per dot is 0.5.
In the case wherein quantum dots are coated with a ligand comprising a —(CH2)2-COOH end and then functionalized with a fluorophore (QDs-F), the reaction of functionalization fails.
In conclusion, these results confirm that the design of the ligand end is important. Especially, a too short spacer L does not allow the functionalization of the ligand-coated QDs.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13195737 | Dec 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/076655 | 12/4/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/082656 | 6/11/2015 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20050277739 | Yang et al. | Dec 2005 | A1 |
| Entry |
|---|
| Sun et al. Functionalization of quantum dots with multidentate zwitterionic ligands: impact on cellular interactions and cytotoxicity. J. of Materials Chemistry B, 2013, vol. 1, pp. 6137-6146. (Year: 2013). |
| Yildiz et al. Hydrophilic CdSe—ZnS core-shell quantum dots with reactive functional groups on their surface. Langmuir 2010, vol. 26, No. 13, pp. 11503-11511. (Year: 2010). |
| Chan, W et al., “Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection”, Science, vol. 281, No. 5385, Sep. 1998, pp. 2016-2018. |
| Mattoussi, H et al., “Self-Assembly of CdSe—ZnS Quantum Dot Bioconjugates Using an Engineered Recombinant Protein”, Journal of the American Chemical Society, vol. 122, No. 49, Nov. 2000, pp. 12142-12150. |
| Giovanelli, E et al., “Highly enhanced affinity of multidentate versus bidentate zwitterionic ligands for long-term quantum dot bioimaging”, Langmuir, vol. 28, No. 43, Sep. 2012, pp. 15177-15184. |
| Chen, X et al., “Novel zwiterrionic copolymers with diohydrolipoic acid: synthesis and preparartion of nonfouling nanorods”, Macromoelcules, vol. 46, No. 1, Dec. 2012, pp. 119-127. |
| Yildiz, I et al., “Hydrophilic CdSe—ZnS core shell quantum dots with reactive functional groups on their surface”, Langmuir, vol. 26, No. 13, Jul. 2010, pp. 11503-11511. |
| European Search Report, from corresponding European Appln. No. EP13195737, dated Apr. 24, 2014. |
| International Search Report, from corresponding PCT Appln. No. PCT/EP2014/076655, dated Jan. 8, 2015. |
| Number | Date | Country | |
|---|---|---|---|
| 20160304648 A1 | Oct 2016 | US |
