Patent Description:
The emergence of single-molecule and super-resolution fluorescence microscopy have been opening a new area of imaging with the promise to visualize biological processes at a molecular level in living cells. For tracking or visualizing single biomolecules in the cytoplasm, the fluorescent emitters need to be very bright. Indeed, the spatio-temporal resolution is directly linked to the number of collected photons. Recently, dye-loaded polymer nanoparticles have achieved very high brightness through the encapsulation of a large amount of fluorescent dye in nanoparticles (<NPL>. Several new strategies, such as fluorescent organic dots with aggregation-induced emission<NUM> or use of salts of fluorophores with hydrophobic bulky counterions<NUM>, had been used to overcome aggregation-caused quenching of the fluorophores encapsulated at very high concentrations.

However, in order to be used in biological environments, single-molecule imaging requires not only high brightness of nanoparticles, but also small probe sizes and an absence of non-specific interactions with biological environments. <NUM> Firstly, in order to increase localization precision, the particle diameter needs to be of the order of the size of the biomolecule (e.g. protein) it is intended to label. Secondly, the size and the surface coating of nanoparticles play a crucial role in defining their intracellular behavior and their influence on the behavior of the labeled biomolecules should be limited. <NUM>,<NUM> Diffusion of nanoparticles in biological environments, such as in cells, can be limited due to structural restrictions and macromolecular crowding effects inside cells. <NUM> Etoc. determined that particles of ≤<NUM> can have Brownian diffusion in the cytosol. <NUM> The inventors also showed in the past that the limit core size, for which the NPs can reach almost all parts of the cytosol, is around <NUM>. <NUM> At the same time, the surface chemistry also has a major effect on the behavior of nanoparticles in biological systems<NUM> and can affect the diffusion and the motion of NPs in the cytosol. <NUM> One reason for this is that the surface chemistry will determine interactions with proteins. In particular, the adsorption of proteins leading to the formation of a protein corona affects all aspects of NP behavior from the cellular uptake to their degradation,<NUM> and as long as there are strong interactions between proteins and the NP surface, nanoparticles are not able to freely move in the cytosol. <NUM> Therefore, the control of the surface properties to reduce (suppress) protein adsorption is essential.

Till now, introducing polyethylene glycol (PEG) is the best known strategy for suppressing protein adsorption on nanoparticles. <NUM> However, PEGylation is not fully adapted to produce nanoparticles having a size similar to that of biomolecules (e.g. protein), since the PEG chains needed for efficiently suppressing interactions with proteins also significantly increase the particle diameter by several nanometers and often by around <NUM>. <NUM>,<NUM>.

For example, <CIT> described a water-dispersible fluorescent particle comprising a mixture of one or more hydrophobic polymers, which are encapsulated in one or more amphiphilic polymers. Said amphiphilic polymers are block copolymers comprising at least one hydrophobic segment and at least one hydrophilic segment, such as PS-b-PEG.

An alternative is the use of zwitterionic (ZI) groups, mimicking the outer surface of the cell membrane. <NUM> The mechanism of suppression of protein adsorption in the case of zwitterions is linked to their very hydrophilic nature and the fact that they contain their own counterion,<NUM>,<NUM> rather than on steric repulsion as in the case of PEG. Therefore, unlike PEG coatings, zwitterionic coatings can be very thin, limiting the increase of particle diameter. Several approaches have been developed to implement zwitterionic, especially carboxybetaine, sulfobetaine and phosphorylcholine, groups on NP surfaces. However, most of zwitterionic nanoparticles developed in the past are inorganic nanoparticles as quantum dots (QDS),<NUM> silica NPs,<NUM> or Au NPs.

<CIT> discloses a composite comprising a core containing one or more magneto-fluorescent nanoparticles and/or one or more quantum dots (semiconductor nanocrystals), and a zwitterionic polymer coating. <CIT> discloses zwitterionic polymer nanoparticles having a diameter of <NUM> to <NUM>. The zwitterionic polymer is a block copolymer bearing a zwitterionic block and a hydrophobic block.

Till now, in the case of polymeric nanoparticles, zwitterionic polymeric particles are mainly obtained from block copolymers bearing a zwitterionic block and a hydrophobic block.

The shortcoming of this approach is that the diameters of these NPs are not satisfactory, which limits their use in in vivo environments. By the way, their synthesis process is often very complex.

Thus, in order to improve in vitro or in vivo detection or tracking of a target biological molecule, it is necessary to provide new families of zwitterionic polymeric nanoparticles having high brightness and controllable smaller size and limited protein surface interactions.

In order to satisfy these requirements, the Inventors of the present invention developed novel luminescent zwitterionic nanoparticles, based on specially designed random hydrophobic polymers, bearing apolar, charged, zwitterionic and reactive groups for bioconjugation.

Contrary to block zwitterionic copolymers known in prior art wherein the zwitterionic block and the hydrophobic block are separated and localized on two ends of the polymer chain, the zwitterionic copolymers forming the nanoparticles of the present invention have both negatively charged groups and zwitterionic groups which are randomly arranged on the polymeric backbone chain among the hydrophobic pendant groups of the copolymer.

Against all odds, it is surprising to observe that this random arrangement of a small amount of zwitterionic groups and negatively charged groups can on the same time further reduce nanoparticle's size and efficiently prevent protein adsorption on nanoparticle's surface while maintaining a high luminescence brightness.

The nanoparticles of the present invention can have a diameter as small as <NUM>-<NUM>, that is particularly suitable for being monitored directly at the single particle level in the cytoplasm of living and fixed cells and tissues.

The first aspect of the present invention is to provide a zwitterionic luminescent polymeric nanoparticle, said nanoparticle comprising at least one luminescent dye and at least one random copolymer, said random copolymer comprising:.

The reactive group is able to chemically react with a corresponding function of a natural or synthetic molecule that recognizes a target biomolecule.

Without being bound by any theory, the nanoparticles of the invention probably have a hydrophobic core made up predominantly of the hydrophobic part of the copolymers and of the dye salt. The excellent resistance to protein adsorption and the high mobility of the particles in the cytosol, on the other hand, suggest a very hydrophilic surface with a high density of zwitterionic groups. Thus, the nanoparticles of the invention are supposed to have a core-shell structure with the hydrophobic parts of the amphiphilic polymers predominantly concentrated in the core, responsible for encapsulation of luminescent dyes, and the charged and zwitterionic parts abundant in the shell, responsible for controlling the interactions.

For a given particle-core size, the nanoparticles of the invention make it possible to reduce by a factor of <NUM> the total size of nanoparticles with respect to that of PEGylated nanoparticles based on the same major monomer. This is due to the smaller size of the particle shell in the case of zwitterionic particles compared to their PEGylated counterparts. The brightness of the particles depends on the number of dyes encapsulated in their core and hence, for a given total size, on the ratio of core to the total particle diameter (core and shell). In consequence, for the same total particle diameter, the brightness of the particles of invention, which have nearly <NUM>-fold larger core diameter because of very thin shell, can be increased by nearly <NUM>-fold. High loading (><NUM> wt%) and quantum yields of the present particles (><NUM>%) have been confirmed. That means the nanoparticles of the invention are ultra-bright compared to non-zwitterionic nanoparticles.

Thus, the nanoparticles of the present invention combined, on the one hand, efficient dye encapsulation and high quantum yields, resulting in high particle brightness, and on the other hand, excellent stability and resistance to protein adsorption.

As used herein, the term "random copolymer" is meant to a copolymer having two or more kinds of monomers which are polymerized into no particular sequence. In a random copolymer, the individual repeating units are randomly distributed along the backbone chain of the copolymer.

The term "repeating unit" is meant to a unit whose repetition would produce the complete polymer chain by linking the repeating units together successively along the chain.

As used herein, the term "pendant group" with respect to a polymer, is meant to a group of molecules attached to the backbone chain of a polymer.

As a consequence, the pendant group of a major monomer is also the major pendant group of the random copolymer polymerized with that major monomer.

The term "backbone" of polymer is referred to a linear chain of a polymer on which all other long chains or short or both, can be considered as pending.

As used herein, the expression "a reactive group which is suitable to be functionalized by a natural or synthetic biologically interesting molecule" is meant to any reactive group for bioconjugation, i.e. to any reactive group which is able to chemically react, for example by mean of a covalent bond, with a corresponding reactive function of a molecule that recognizes a target biomolecule, in particular in cells, tissues or biological fluids, such as for example an antibody, a fragment of antibody, a ligand, an agonist or antagonist of a natural biological molecule, a peptide, an aptamer, an oligonucleotide, a toxin or a chemical drug.

According to the invention, said random copolymer can be a (C<NUM>-C<NUM>)alkyl methacrylate based polymer, an (C<NUM>-C<NUM>)alkyl acrylate based polymer, an acrylamide based polymer, a polyester based polymer, a polyamide (polypetide) based polymer, a styrene based polymer and copolymers thereof.

The term "(C<NUM>-C<NUM>) alkyl methacrylate based polymer" in the context of the present invention is to be understood as a copolymer which is formed by (C<NUM>-C<NUM>) alkyl methacrylate as major monomer and other minor monomers.

In the context of the present invention, the terms "(C<NUM>-C<NUM>)alkyl acrylate based polymer", "acrylamide based polymer", "polyester based polymer", "polyamide based polymer", "styrene based polymer", should be interpreted in the similar way.

Examples of (C<NUM>-C<NUM>) alkyl methacrylate based polymer can be methyl methacrylate based copolymer, ethyl methacrylate based copolymer, propyl methacrylate based copolymer, isopropyl methacrylate based copolymer, or butyl methacrylate based copolymer.

Examples of polyester based polymer can be cited as, but not limited to, polyglycolic acid (PGA) based copolymer, poly(lactide co-glycolide) (PLGA) based copolymer, Polylactic acid (PLA) based copolymer, or polycaprolactone (PCL) based copolymer.

An embodiment of random copolymer comprised in the nanoparticles of the present invention is represented by the formula I below:
<CHM>
Wherein:.

The reactive group R<NUM> is able to chemically react with a corresponding function of a natural or synthetic molecule that recognizes a target biomolecule.

A more particular embodiment of a random copolymer comprised in the nanoparticles of the present invention can be represented by the formula Ia below:
<CHM>
wherein:.

Another embodiment of a random copolymer comprised in the nanoparticles of the present invention is represented by the formula II below:
<CHM>
Wherein:.

According to a particular embodiment, the zwitterionic luminescent polymeric nanoparticle of the invention comprises a random copolymer that is a (C<NUM>-C<NUM>)alkyl methacrylate based polymer, said random copolymer comprising :.

One kind of minor pendant groups comprised in the random polymer of the present invention is negative charged groups chosen from phosphonate, sulfonate and carboxylate.

A random copolymer comprised in the nanoparticles of the present invention has from <NUM> to <NUM>% of repeating units having a negatively charged group as pendant group.

Another kind of minor pendant groups comprised in the random polymer of the present invention is zwitterionic group.

The term of "zwitterionic group" is referred to a chemical group which contains at the same time a part having a positive charge and another nonadjacent part having a negative charge. The examples of zwitterionic groups which can be comprised in above described random copolymer can be chosen from ammoniophosphates, ammoniophosphonates, ammoniophosphinates, ammoniosulfonates, ammoniosulfates, ammoniocarboxylates, ammoniosulfonamides, ammoni-sulfon-imides, guanidiniocarboxylates, pyridiniocarboxylates, pyridiniosulfonates, ammonio(alkoxy)dicyanoethenolates, ammonioboronates, sulfoniocarboxylates, phophoniosulfonates, phosphoniocarboxylates.

A random copolymer comprised in the nanoparticles of the present invention has from <NUM> to <NUM>%, in particular from <NUM> to <NUM>%, more particularly from <NUM> to <NUM>%, of repeating units having a zwitterionic group as pendant group.

The presence of very hydrophilic zwitterionic groups in said random copolymer increases hydrophilicity of copolymer. However, the random copolymer comprised in the nanoparticle of the present invention remains basically hydrophobic due to the major part of hydrophobic pendant groups.

Thanks to the presence of zwitterionic groups and negatively charged groups, the nanoparticle of the invention has a particular reduced size.

The diameter of the nanoparticle of the present invention is varied from <NUM> to <NUM>, particularly from <NUM> to <NUM>, more particularly from <NUM> to <NUM>, still more particularly from <NUM> to <NUM>, still more particularly from <NUM> to <NUM>, still more particularly from <NUM> to <NUM>.

Nanoparticle's diameter and size distribution can be measured according to a conventional method by electron microscopy or dynamic light scattering.

The nanoparticles of the invention comprise also luminescent dyes.

Within the scope of the present invention, said luminescent dyes can be fluorescent dyes or phosphorescent dyes.

Said luminescent dyes can also be luminescent metal complexes.

In a preferred embodiment, said luminescent dye is a fluorescent dye optionally with its counter-ion, said dye and said counter-ion being encapsulated inside the nanoparticle.

Examples of fluorescent dyes can be cited as, but not limited to, rhodamine derivatives, cyanine derivative, fluorescein derivatives, BODIPY derivatives, aza-BODIPY derivatives, coumarines, squaraines, porphyrins or phthalocyanines.

Their suitable counter-ions for ionic dyes can be an inorganic counter-ion or a bulky organic counter-ion.

Examples of inorganic counterion may include, without limitation, chloride, perchlorate, sulfonate, nitrate, tetrafluoroborate, hexafluorophosphate.

The term "bulky organic counterion" as used herein means a large organic anion bearing aromatic and/or aliphatic residues. Examples of bulky organic counterion can be cited, but not limited to tetrakis(pentafluorophenyl)borate, tetrakis(<NUM>-fluorophenyl)borate, tetrakis[<NUM>,<NUM> - bis(trifluoromethyl)phenyl]borate, tetrakis[<NUM>,<NUM> - bis(<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM> - hexafluoro - <NUM> - methoxy - <NUM> - propyl)phenyl]borate, tetrakis[perfluorotert-butoxy]aluminate, or tetraphenylborate.

According to some embodiments, the random copolymer of the present invention can also comprise a third kind of minor pendant group that is a reactive group which is suitable to be functionalized by a natural or synthetic biologically interesting molecule. The reactive group is able to chemically react with a corresponding function of a natural or synthetic molecule that recognizes a target biomolecule.

For example, said reactive group can be a reactive group suitable for "click chemistry", such as azide, tetrazine, alkenyl groups, or other reactive groups which can carry out a cycloaddition, or reactive groups, such as maleimide groups, or active esters, such as pentafluoro phenyl esters or NHS-esters.

This third kind of minor pendant groups makes it possible to combine a natural or synthetic biologically interesting molecule with the luminescent zwitterionic nanoparticles of the invention.

Examples of said natural or synthetic biologically interesting molecule can be, but not limited to, a molecule that recognizes a target biomolecule in cells, tissues or biological fluids, such as an antibody, a fragment of antibody, a ligand, an agonist or antagonist of a natural biological molecule, a peptide, an aptamer, an oligonucleotide, a toxin or a chemical drug.

This kind of luminescent zwitterionic nanoparticles bearing reactive groups is particularly interesting, since they can be easily further functionalized according to destined applications of the nanoparticles.

According to some embodiments, the luminescent zwitterionic polymeric nanoparticle of the present invention can comprise a random copolymer as described before as the only polymeric material.

According to some other embodiments, the luminescent zwitterionic polymeric nanoparticles of the present invention can comprise a random copolymer as described above with another polymer, or comprise at least two kinds of random copolymers as described above.

In a preferred embodiment, the luminescent zwitterionic polymeric nanoparticles of the present invention comprise:.

The mixture of a reactive group-bearing random copolymer with another random copolymer free of reactive group makes it possible to control with more accuracy the quantity of biologically interesting molecules that will be bound to a nanoparticle via these reactive groups, and that will further impact for example the detection sensibility of the nanoparticles, target molecule quantification, or single-molecule tracking.

In a particular embodiment, the zwitterionic polymeric nanoparticle of the invention comprises a first random copolymer and a second random copolymer, the first random copolymer comprising:.

the second random copolymer being free of reactive groups which are suitable to be functionalized by a natural or synthetic biologically interesting molecule and comprising:.

In another particular embodiment, the luminescent zwitterionic polymeric nanoparticles of the present invention comprise:.

This kind of nanoparticles can therefore be functionalized with two kinds of biologically interesting molecules.

The presence of reactive groups on the luminescent zwitterionic nanoparticles allows said nanoparticles to be easily functionalized by a natural or synthetic biologically interesting molecules through a covalent bond to said polymeric chain.

Thus, another aspect of the present invention is to provide a novel functionalized zwitterionic luminescent polymeric nanoparticle.

Said functionalized zwitterionic luminescent polymeric nanoparticle comprises at least one random copolymer comprising:.

In a particular embodiment, said functionalized zwitterionic luminescent polymeric nanoparticles of the present invention can be represented by the formula III below:
<CHM>.

In a particular embodiment, said functionalized zwitterionic luminescent polymeric nanoparticles of the present invention can be represented by the formula IV below:
<CHM>.

Said natural or synthetic biologically interesting molecule is chosen from an antibody, a fragment of antibody, a peptide, an aptamer, an oligonucleotide, a toxin or a chemical drug.

The presence of such natural or synthetic biologically interesting molecule on a nanoparticle confers to said nanoparticle the ability to detect a corresponding biomolecule.

According to the target biomolecule to be detected, a luminescent zwitterionic nanoparticle bearing reactive groups can be functionalized by a corresponding natural or synthetic biologically interesting molecule. For example, in order to detect the expression of a particular protein, the luminescent zwitterionic nanoparticles can be functionalized by an antibody directed to said protein.

These functionalized luminescent zwitterionic nanoparticles of the invention combining small size, ultrahigh brightness, low protein adsorption with target specificity are particularly useful for the application in in vitro or in vivo disease diagnosis, therapeutic treatment or in biological research.

The functionalized nanoparticles of the invention can be used for example as a biosensor for detecting target biomolecules, such as for detecting a particular protein, antibody or peptide.

Thanks to its high brightness, the functionalized luminescent zwitterionic nanoparticles of the invention are more sensitive, easier to use than ELISA test.

Particularly, these functionalized nanoparticles can be used as a contrast agent or medical imaging agent that can be used in in vivo detection, tracking of target molecules or cells for in vivo medical diagnosis, or as a diagnostic agent that can be used in in vitro detection or identification of a biomolecule or a cell expressing said biomolecule.

The present invention concerns also a method for in vitro or in vivo detection or tracking of a target biomolecule by means of a functionalized luminescent zwitterionic polymeric nanoparticle as described above.

Example of said target biological molecule can be, but not limited to, a protein, an antibody, a DNA, a RNA, a siRNA, a microRNA, a toxin.

Thanks to its ultrahigh brightness, the nanoparticle of the present invention can be used in single-molecule tracking.

According to a particular embodiment, the invention concerns a method for in vitro detection or tracking of a target biological molecule, in particular an antigen, in a sample.

Said sample can be a biological sample obtained from biological fluid, from an in vitro cell culture or from a tissue, from plants, from microorganisms, a solution containing biological molecules, an environmental sample, a food sample, a pharmaceutical sample.

By "biological fluid" is meant a liquid contained, excreted or secreted from a living animal or plant, for example: blood, different fraction of blood, lymph, bile, saliva, exudates. In a preferred embodiment of the present invention, the biological fluid is a human or animal origin fluid chosen from serum, inactivated serum, plasma, or blood.

By "tissue" is meant to a human, animal or vegetal tissue. In a particular embodiment of the invention, the sample of a tissue is a sample obtained by biopsy or during surgical operation. In a more particular embodiment, the tissue is a tumoral tissue obtained by biopsy or during surgical operation from a patient suffering from a cancer, or suspected to develop a cancer.

By "environmental sample" is meant to a sample collected from an environment, such as soil, sludge, or waste water.

For the detection, the functionalized luminescent zwitterionic nanoparticles of the invention can be suspended in solution or immobilized on surfaces of microplates.

The present invention concerns also a pharmaceutical composition comprising the functionalized luminescent zwitterionic nanoparticles of the invention for use as a contrast agent, a diagnostic agent or as medical imaging agent.

Said composition can be used as an in vivo diagnostic agent.

The random copolymer contained in luminescent zwitterionic hydrophobic polymeric nanoparticles as described before can be synthesized through free radical polymerization, controlled radical polymerization, condensation, addition ring opening polymerization.

The luminescent zwitterionic hydrophobic polymeric nanoparticles can be obtained by the method of nanoprecipitation.

The present invention provides also a method for preparing a functionalized fluorescent zwitterionic polymeric nanoparticle as described before, said method comprising:.

Another method for preparing a functionalized luminescent zwitterionic polymeric nanoparticle as described before comprises:.

The present invention is exposed more in detail in the following figures and examples.

Methyl methacrylate (<NUM>%, M55909), methacrylic acid (<NUM>%, <NUM>), <NUM>-sulfopropyl methacrylate potassium salt (<NUM>%, <NUM>) and <NUM>-(N-<NUM>-Sulfopropyl-N,N-dimethyl ammonium)ethyl methacrylate (<NUM>%, <NUM>) were purchased from Sigma-Aldrich. Dimethylsulfoxide (DMSO, analytical grade) was obtained from Fisher-Scientific. Milli-Q water (Millipore), acetonitrile (≥ <NUM>%, Sigma-Aldrich) and methanol (≥ <NUM>%, Carlo Erba reagents) were used for preparation of nanoparticles. dichloromethane (≥<NUM>%) from CarloErba and methanol (HPLC grade) from VWR. <NUM>-carboxy- tetramethylrhodamine (TMR from Sigma-Aldrich), phosphate buffered saline (PBS, Fisher Scientific), fetal bovine serum (FBS, Lonza) were used for stability study. Monomers were purified using column chromatography or re-crystallization. Azobis isobutyronitrile (Aldrich, ≥ <NUM>%) was recrystallized twice from ethanol. The other compounds were used as received. R18/F5-TPB was synthesized from rhodamine B octadecyl ester perchlorate (Aldrich, ><NUM>%) and lithium tetrakis(pentafluorophenyl)borate ethyl etherate (AlfaAesar, <NUM>%) through ion exchange followed by purification through column chromatography as described previously.

Synthesis of polymers of invention: The different polymers were synthesized through free radical polymerization. The different monomers were all dissolved in degased DMSO and mixed at the desired ratio. of AIBN were added and the round bottom flask was placed in an oil bath preheated to <NUM>. Once the conversion reached <NUM> %, the reaction was stopped and the polymers reprecipitated twice in methanol and/or water. After drying, the polymers were characterized through NMR and, where possible, size exclusion chromatography. As an example the synthesis of PMMA-SO<NUM>H-ZI is given:.

Stock solutions of copolymers were prepared at a concentration of <NUM>. l-<NUM> in acetonitrile (<NUM> vol. % methanol for ZI polymers). These solutions were diluted at <NUM>. l-<NUM> in the corresponding solvent containing <NUM>, <NUM> or <NUM> wt% of R18/F5-TPB (relative to the polymer). This solution was quickly added to a <NUM>-fold volume excess of water, under shaking (Thermomixer comfort, Eppendorf, <NUM> rpm, at <NUM>) followed by a second dilution in water.

Absorption and emission spectra were recorded on a Cary <NUM> Scan ultraviolet-visible spectrophotometer (Varian) and on a FS5 Spectrofluorometer (Edinburgh Instruments) equipped with a thermostated cell compartment, respectively. The excitation wavelength was set to <NUM> and emission was recorded from <NUM> to <NUM>. QYs were calculated using rhodamine <NUM> in ethanol as reference (QY = <NUM>).

Transmission electron microscopy: 5µl of nanoparticle solution were deposited onto carbon-coated copper-rhodium electron microscopy grids following amylamine glow-discharge. They were then treated for <NUM> with a <NUM>% uranyl acetate solution for staining. The obtained grids were observed using a Philips CM120 transmission electron microscope equipped with a LaB6 filament and operating at 100kV. The acquisition of areas of interest was recorded with a Peltier cooled CCD camera (Model <NUM>, Gatan, Pleasanton, CA). Images were analyzed using Fiji software.

Fluorescence correlation spectroscopy: Measurements were performed on a home-built confocal set-up using excitation at <NUM> using TMR in water as reference. The solution of NPs containing <NUM> wt% dyes were diluted <NUM> times before depositing <NUM>µl on <NUM>-well optical-bottom plates for measurements. NPs stability in presence of salts and proteins were investigated by adding drop-by-drop <NUM> vol. % of <NUM>-fold PBS (10x), FBS or water to the solutions of NPs in low-binding <NUM> Eppendorf tubes. The data were recorded <NUM> after addition and then analyzed using the PyCorrFit software.

HeLa cells were grown in Dulbecco's modified Eagle medium (DMEM, without phenolred, Gibco-Invitrogen), supplemented with <NUM>% fetal bovine serum (FBS, Lonza), L-glutamine, and <NUM>% antibiotic solution (penicillin-streptomycin, Gibco-Invitrogen) at <NUM>° C in humidified atmosphere containing <NUM>% CO<NUM>. Cells were seeded onto a round microscope cover glasses (diameter <NUM>) deposited in <NUM> well plates at a density of <NUM>×<NUM><NUM> cells/well <NUM> before the microinjection.

Microinjection of NPs and cellular imaging: For microinjection experiments, subconfluent HeLa cells plated on glass coverslips were mounted in a Ludin Chamber (Life Imaging Services, Basel, Switzerland). The cells were then placed on a Leica DMIRE <NUM> microscope (<NUM>, <NUM>% CO<NUM>, 100x objective, sCMOS camera, Xenon lamp) and solutions of the different nanoparticles at particle concentrations of <NUM> to <NUM> were microinjected into the perinuclear region of the cells, using a Femtojet/InjectMan NI2 microinjector (Eppendorf). Images sequences were then acquired either on the same setup or on an iMIC microscope (Till Photonics) equipped with a Mutli-LED Spectra X (Lumencor), an Olympus 60x TIRFM (<NUM> NA) objective, and a Flash <NUM> V2+ camera (Hamamatsu) after transfer of the samples.

Live cells were maintained at <NUM> in a <NUM>% CO<NUM> humidified atmosphere using an environmental control system (Life Imaging Services). Time-lapse movies were recorded over <NUM> with a frame rate of <NUM> and binning <NUM>. They were then analyzed using the ImageJ (National Institutes of Health, USA). For single particle tracking, time-lapse movies were recorded over <NUM> with a frame rate of <NUM> for the Imic microscope or <NUM> for the Leica DMIRE <NUM> microscope and a binning <NUM> in order to increase the resolution. With Fiji software, the trajectories were recovered from TrackMate plugin. Then MSD curves were plotted and slopes extracted from the MSD curves with a MATLAB script for particle tracking analysis.

Methacrylate based copolymers bearing sulfonate and zwitterionic (ZI) groups were synthetized according to the method described above in "Materials and Methods". In the following text the polymers are noted PXMA-ZI-x%-SO3H-y%, where XMA stands for the corresponding major monomer (MMA for methyl methacrylate, EMA for ethyl methacrylate, PMA for propyl methacrylate, BMA for butyl methacrylate), x and y correspond to the molar percentage of the zwitterionic and sulfonate groups. By the way, polymers bearing only sulfonate but no zwitterionic groups were also prepared as control.

These polymers were then used to assemble dye-loaded polymer NPs through nanoprecipitation. For this, solutions of the polymers in acetonitrile (with a small amount of methanol) containing different amounts of the salt of a rhodamine B derivative (R18) with a perfluorinated borate (F5-TPB) were added quickly to a large excess of water. The size of the formed NPs was analyzed through transmission electron microscopy (TEM, <FIG>, Table <NUM>).

In the case of polymers bearing only sulfonate but no zwitterionic groups, very small particles were observed: PMMA-SO<NUM>H <NUM>% yielded NPs of about <NUM> and the particle size decreased to about <NUM> for <NUM>% sulfonate. The introduction of <NUM>% ZI on the polymer chains had only a minor influence on the particle size of PMMA-ZI-SO<NUM>H based NPs. They reached <NUM> and <NUM> of diameter, respectively for <NUM>% and <NUM>% sulfonate NPs. The presence of the ZI groups hence did not affect the process of particle formation and the obtained "ZI shell" is thin enough for not influencing the size of NPs. Increasing the hydrophobicity of the alkyl methacrylate monomers on the other hand led to increasing particle size (<FIG>, Table <NUM>) in the order PMMA-ZI <NUM>%-SO<NUM>H <NUM>% < PEMA-ZI <NUM>%-SO<NUM>H <NUM>% < PPMA-ZI <NUM>%-SO<NUM>H <NUM>% < PBMA-ZI <NUM>%-SO<NUM>H <NUM>%, from <NUM> to <NUM>.

The nanoparticles were made fluorescent through the encapsulation of high amounts of dyes (between <NUM> and <NUM> wt% relative to the polymer). Here, the cationic rhodamine R18 was associated to the large and very hydrophobic counterion F5-TPB as this association avoids aggregation-caused quenching and leads to a very hydrophobic dye counterion pair. Dialysis of the dye-loaded NPs over <NUM> showed a release of less than <NUM>% of the used dye, indicating that this approach yielded very efficient encapsulation of the dye salt in the particles, in agreement with previous results showing very efficient encapsulation of dyes with F5-TPB counterion. <NUM> Furthermore, the quantum yield of the particles remained > <NUM>% for a <NUM> wt% loading for all zwitterion bearing polymers, thus ensuring excellent particle brightness.

The stability of the resulting particles in biological media, and in particular the potential of the zwitterionic groups to improve this, was investigated by fluorescence correlation spectroscopy (FCS). The percentage of ZI groups in the polymers was varied from <NUM> to <NUM>% in order to modify the density of ZI on the particle. The particle sizes of NPs obtained from FCS data were in very good agreement with those obtained from TEM (<FIG>, Table <NUM>. The smaller size is associated with the lower amount of dye salt used in FCS experiments. In a first step, the stability of NPs was evaluated through addition of NaCl solutions with increasing concentrations (<FIG>). The stability limit was defined as the last NaCl concentration for which no aggregation of NPs was observed. Particles made from polymers bearing no ZI groups already started to aggregate (and precipitate) as soon as a small amount of salt was added (<FIG>). Addition of ZI groups led to an increase in the particle stability with increasing amount of ZI groups. At <NUM>% of zwitterionic groups the NPs did not show any change in size and no signs of precipitation up to <NUM> NaCl.

The influence of the zwitterionic groups on interactions of the NPs with proteins and other biomolecules was then tested by adding fetal bovine serum (FBS), a complex mixture of salts and biomolecules containing notably numerous proteins (<FIG>). In the case of particles without ZI groups a size increase of about <NUM> was observed, which corresponds to the adsorption of at least a monolayer of proteins and thus the formation of a "hard" protein corona (<FIG>). Starting from <NUM>% of ZI groups the size increase of the NPs upon interaction with proteins was significantly reduced. At <NUM>% of ZI groups no significant increase in particle size was observed, indicating that the surfaces of these particles resisted protein adsorption (<FIG>).

These NPs were then directly microinjected in the perinuclear region of the cytosol of living HeLa cells and their behavior was monitored using epifluorescence microscopy (<FIG>). In all cases, diffuse staining of cytosolic structures was virtually absent. This confirms the efficient and stable encapsulation of the dyes within the NPs and the absence of dye leaching. Maximum projections of the fluorescence intensities over <NUM> gave a general idea of the overall distribution of the particles throughout the cytosol: In the absence of ZI groups, most of the particles remained stuck at the injection point and the few particles in the cytosol had a low mobility, indicating strong interactions of the particles with the cellular constituents. On the other hand, particles bearing <NUM>% ZI groups distributed, in general, well throughout the cytosol. PMMA-based ZI-bearing NPs showed a distribution all over the cytosol and a high mobility, even though there is sometime a "projection" on the nucleus close to the injection point. PEMA-ZI particles showed a homogeneous distribution and high mobility for practically all NPs. This was also observed for PPMA-ZI particles, though here a certain part of the particles remained close to the injection point. In the case of PBMA-based particles, they were more localized at the injection point than particles based on other tested polymers. These results confirmed that <NUM>% ZI groups are sufficient to strongly reduce interactions of the particles with cellular constituents and enable spreading throughout the cytosol. However, these results also confirm that the particle size also has a major influence on intracellular particle diffusion, and only particles below a critical core size of around <NUM> can spread throughout the cytosol due to steric hindrance by cellular structures. <NUM> Here, the PBMA particles had sizes clearly above this threshold, leading to their immobilization. Part of the PPMA particles were also above this threshold, resulting in immobilization of part of the particles.

The high brightness of the particles of the invention enabled monitoring the mobility and diffusion behavior of the NPs at the single particle level and so to better understand the influence of the ZI groups and the type of polymer used on the intracellular behavior of our NPs. An example of a cytoplasmic trajectory of a PEMA-based ZI-bearing NP is represented in <FIG>. Plotting the mean square displacement (MSD) vs lag time for these NPs showed a linear increase up to about <NUM><NUM> with an exponent α of <NUM> (<FIG>). This indicates normal or Brownian diffusion of these particles in the cytosol, which is described, in two dimensions, as MSD = 4DΔtα with D, the diffusion coefficient and α = <NUM>. <NUM> The diffusion coefficients of individual NPs were then extracted from the corresponding MSD curves. <FIG> shows that for PEMA-ZI NPs the distribution of the diffusion coefficients is centered around <NUM><NUM>. s-<NUM>, with a mean D of <NUM><NUM>. s-<NUM>, in good agreement with values obtained for QDs of similar size. <NUM> The distribution shows only a small fraction of NPs with diffusion coefficients below <NUM><NUM>. PMMA-ZI NPs had a similar distribution of diffusion coefficients with a slightly lower mean of <NUM><NUM>. s-<NUM> (<FIG>), in agreement with the slightly larger hydrodynamic size. PPMA-ZI NPs, on the other hand, showed a clearly lower mean diffusion coefficient of <NUM><NUM>. This is probably due to their larger size leading to a restricted cytoplasmic diffusion, as already indicated by their stronger clustering around the injection point. As PMMA-SO<NUM>H <NUM>% NPs without ZI groups remained clustered around the injection point, and hence it was not possible to characterize their diffusion behavior. However, we could show earlier that simple adsorption of Tween <NUM> (T80), a PEGylated surfactant, on such nanoparticles strongly reduces their interaction with proteins and permits their diffusion in the cytosol. <NUM>,<NUM> Interestingly, the mean diffusion coefficient of these NPs was with <NUM><NUM>. s-<NUM> three to four fold lower than those of the PMMA and PEMA ZI NPs, even though their core sizes were very close (<FIG>). One reason for this is certainly the fact that the addition of the PEGylated surfactant increases the NP size by > <NUM>. The large difference in diffusion coefficients further indicates that the ZI groups are more effective in reducing non-specific interactions with intracellular biomolecules and structures than the adsorbed PEG shell.

In the present example fluorescent nanoparticles (NPs) bearing zwitterionic groups have been designed to prevent non-specific interactions and bearing targeting groups to introduce specific interactions with biomolecules. In order to introduce specific interactions, antibodies were used, more specifically cetuximab, which is an antibody against the HER2 receptor. To achieve conjugation of the antibodies to the nanoparticles, it has been relied on copper-free cycloaddition click chemistry between an azide group and a strained alkyne, dibenzylcyclooctyne (DBCO). On the one side, zwitterionic brightly fluorescent nanoparticles with azide groups on their surface have been assembled, through nanoprecipitation of a hydrophobic co-polymer bearing zwitterionic, charged, and azide groups, together with a hydrophobic dye-salt. On the other side, the antibody has been modified in order to introduce reactive DBCO groups. Cellular assays have shown that with the antibody the fluorescent nanoparticles bind specifically to HER2 expressing cells.

This polymer can be represented by the following formula (Ia-<NUM>)
<CHM>.

PEMA-ZI-MAA-Asp(OtBu)-N<NUM> was obtained in <NUM> steps from poly(ethyl methacrylate-co-methacrylic acid-co- <NUM>-(N-<NUM>-sulfopropyl-N,N-dimethyl ammonium)ethyl methacrylate) (PEMA-ZI-MAA). PEMA-ZI-MAA was obtained through free radical polymerization as described above in example <NUM>.

<NUM>H NMR (<NUM>, DMSO-D6) δ, ppm: <NUM> (br, <NUM>), <NUM> (m, <NUM>), <NUM> (br, partial covered with solvent peak), <NUM> (m, <NUM>), <NUM> (br, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>).

In a first step PEMA-ZI-MAA was reacted with Asp(OtBu)-N3 (Tert-butyl <NUM>-amino-<NUM>-((<NUM>-azidopropyl)amino)-<NUM>-oxobutanoate, synthesized according to procedures described by <NPL>. ) in dimethylformamide (DMF, Sigma Aldrich) using benzotriazol-<NUM>-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, TCI) as coupling agent in the presence of N,N-Diisopropylethylamine (DIPEA, Sigma Aldrich) as base. The obtained polymer was purified through precipitation in methanol water mixtures.

In a second step the tert-butyl group was removed through treatment with a <NUM>-to-<NUM> mixture of trifluoroacetic acid (Sigma Aldrich) and dichloromethane (Sigma Aldrich).

After evaporation, and in a third step, the polymer was purified through precipitation and column chromatography.

<NUM>H NMR (<NUM>, DMSO-D6) δ, ppm: <NUM> (br, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (br, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>).

In this polymer, the main monomer is thus ethyl methacrylate, and the molar amounts of COOH groups and zwitterionic groups are respectively <NUM> mol% and <NUM> mol%.

Stock solutions of PEMA-ZI-MAA-Asp(OtBu)-N<NUM> as obtained above in <NUM> were prepared at a concentration of <NUM>/L in acetonitrile with <NUM> vol. % methanol. These solutions were diluted at <NUM>/L in the corresponding solvent containing <NUM>, <NUM>, or <NUM> wt% of R18/F5-TPB which is a fluorescent hydrophobic dye-salt (relative to the polymer). This solution was quickly added to a <NUM>-fold volume excess of phosphate buffer (<NUM>, pH <NUM>), under shaking (Thermomixer comfort, Eppendorf, <NUM> rpm, at <NUM>) followed by a second dilution with the aqueous phase.

Cetuximab (Merck) is a chimeric IgG1 full length antibody directed against the HER2 receptor. The antibody was obtained in its clinical formulation. The buffer exchange of antibody was carried out for borate buffer pH <NUM> via ultrafiltration (MWCO <NUM> kDa, Vivaspin). Concentration of antibody was determined by UV-vis absorbance (ε<NUM>= <NUM>-<NUM> cm-<NUM> for cetuximab mAb), adjusted to <NUM> (<NUM>/mL) and was stored as aliquot at -<NUM>. For experiments, aliquots were thawed and used immediately.

The conjugates were prepared using the modification of a reported protocol. <NUM> Cetuximab (<NUM>, <NUM>µL, <NUM>µmol) was prepared in a borate buffer pH <NUM>. Next, tris(<NUM>-carboxyethyl)phosphine hydrochloride (TCEP, Sigma Aldrich) was added (<NUM>, <NUM>µL, <NUM> eq) and the reaction was incubated at <NUM> for <NUM> under mild agitation (<NUM> RPM). Then, a solution of dibenzocyclooctyne-PEG<NUM>-maleimide (DBCO-PEG<NUM>-maleimide, Sigma Aldrich) in dry DMF (<NUM>) was prepared and added to cetuximab (<NUM>µL, <NUM> eq). Subsequently, the temperature was reduced to <NUM> and the incubation was continued for <NUM>. Afterwards, excess reagents were removed by ultrafiltration (<NUM> kDa MWCO) with PBS buffer (pH <NUM>) to afford the modified antibody-maleimide-PEG<NUM>-DBCO in PBS buffer with yield <NUM>-<NUM>%, as determined by UV-vis.

SKBR-<NUM> cells, a human breast cancer cell line that overexpresses the Her2 (Neu/ErbB-<NUM>) were cultured in Dulbecco's modified Eagle medium (Gibco, DMEM) supplemented with <NUM>% fetal bovine serum (Gibco) and <NUM>% penicillin/streptomycin (<NUM> U/mL, Gibco). The SKBR-<NUM> cell were seeded at a cell density of <NUM> x <NUM><NUM> on <NUM> wells Lab-Tek Chambered Coverglasses (Thermo Scientific) followed by incubation for <NUM> at <NUM> and <NUM>% CO<NUM>. For fluorescence imaging, the medium was discarded and the cells washed with PBS buffer. Then, <NUM>µL of Ab-maleimide-PEG<NUM>-DBCO (<NUM>µg/mL in Optimem, Gibco ) and <NUM>µL of Optimem medium only (control) were added to the cells and incubated for <NUM> at <NUM> and <NUM>% CO<NUM>. Then the cells were washed repeatedly with PBS, and fixed with <NUM> % paraformaldehyde in PBS for <NUM> at <NUM> and <NUM>% CO<NUM>, followed by additions of <NUM>% BSA in PBS was and incubation for another <NUM> minutes at <NUM> and <NUM>% CO<NUM>. Then, fluorescent NPs bearing zwitterionic, charged and azide groups (see <NUM>, <NUM> pM in <NUM>% BSA in PBS solution) were added and the cells were incubated for <NUM> at <NUM> and <NUM>% CO<NUM>. Finally, the cells were washed with <NUM> % BSA in PBS solution and examined through epi-fluorescence microscopy using Nikon Ti-E inverted microscope with a 60x objective (Apo TIRF, oil, NA <NUM>, Nikon). The excitation was provided by light emitting diodes (SpectraX, Lumencor) at <NUM>.

In the present example, fluorescent zwitterionic nanoparticles with reactive groups, allowing for introduction of biologically interesting molecules, were assembled. For this an ethyl methacrylate based polymer, bearing carboxylate and sulfobetaine groups, was synthesized through radical polymerization (<FIG>). In this polymer we then introduced azide groups through reaction with a trifunctional molecule bearing an azide group, an amino group for coupling with COOH groups on the polymer, and a protected carboxylic acid group (Asp(OtBu)-N3, derived from aspartic acid). After deprotection of the carboxylate, a hydrophobic polymer combining ZI groups, COOH groups for the nanoparticle size control during nanoprecipitation, and azide reactive groups (e.g. <NUM> mol% zwitterionic groups, <NUM> mol% COOH, <NUM>-<NUM> mol% N3) was obtained (Formula Ia-<NUM> above).

This polymer was then used to assemble dye-loaded nanoparticles (NPs) through nanoprecipitation: Acetonitrile solutions (containing <NUM>% methanol) of the polymer and <NUM>-<NUM> wt% (relative to the polymer) of the dye salt R18/F5-TPB were quickly added to a <NUM>-fold excess of phosphate buffer at pH <NUM>, followed by further dilution. This resulted in the formation of NPs with sizes between <NUM> and <NUM>, which increased slightly with dye loading as detailed in the following Table <NUM>. These NPs showed a bright fluorescence with fluorescence quantum yields around <NUM>%. Based on the size and the loading of the particles, the number of fluorophores can be estimated to be <NUM>, <NUM> and <NUM> fluorophores per nanoparticles for <NUM>, <NUM>, and 30wt% loading, respectively. The per particle brightness can then be calculated using the formula εxNxQY, where ε is the extinction coefficient of rhodamine (<NUM><NUM>-<NUM>. cm-<NUM>), N the number of dyes per particle, and QY the quantum yield and x the multiplication operator. This results in the case of <NUM> wt% loading in a per particle brightness of <NUM> × <NUM>-<NUM>.

DBCO bearing antibodies, on the other hand, were obtained in a two-step one-pot process (<FIG>). In the first step (tris(<NUM>-carboxyethyl)phosphine) (TCEP) was used to open disulfide links of cetuximab, the antibody. In a second step the antibody was then reacted with a bifunctional reagent bearing maleimide for conjugation to thiol groups on the one side and a DBCO group on the other side, connected by a short oligo(ethylene glycol) linker. Following purification, the presence of reactive DBCO groups through conjugation with fluorophores bearing azide groups has been confirmed by UV-visible absorption measurements.

The antibody/NP system has then been applied to the specific binding of NPs to cells expressing the HER2 receptor. The antibody-NP conjugation was achieved in situ. SKBR-<NUM> cells were cultured in wells on glass coverslips (Labtek) and after <NUM> of culture treated with our DBCO modified antibodies. After fixation of the cells, we then added the azide bearing NPs. As control, the same experiment was performed without addition of the antibodies.

The results of fluorescence microscopy that are reported in <FIG> showed that in the absence of the antibody practically no NPs were detected at the SKBR-<NUM> cells. However, after treatment with the DBCO modified antibodies against the HER2 receptor, addition of the NPs led to a strong fluorescence, especially at the periphery of the cells, probably at the plasma membrane containing the receptor.

Claim 1:
A zwitterionic luminescent polymeric nanoparticle comprising at least one luminescent dye and at least one random copolymer, said random copolymer comprising:
- from <NUM> to <NUM> mol% of repeating units having a pendant group, which is a negatively charged group chosen from phosphonate, sulfonate and carboxylate,
- from <NUM> to <NUM> mol%, in particular from <NUM> to <NUM> mol%, more particularly from <NUM> to <NUM> mol%, of repeating units having a pendant group, which is a zwitterionic group,
- from <NUM> to <NUM> mol% of repeating units having a pendant group, which is a reactive group able to chemically react with a corresponding function of a natural or synthetic molecule that recognizes a target biomolecule,
- from <NUM> to <NUM> mol% of repeating units having a pendant group, which is a hydrophobic group.