MAGNETIC AND FLUORESCENT REVERSE NANOASSEMBLIES

Abstract

Disclosed are magnetic and fluorescent nanoassemblies having reverse architectures. Especially, the nanoassemblies include an organic fluorescent inner core and magnetic nanoparticles contacting the surface of the fluorescent core. The nanoassemblies may further be coated by a polymer adsorbed at its surface, the polymer being optionally functionalized. Also described is a process for manufacturing the nanoassemblies, as well as use of the nanoassemblies, especially for multimodal imaging; in vitro and/or in vivo diagnostics through multimodal imaging; ex vivo sensing and/or extraction; and/or therapy.

Claims

1-15. (canceled)

16. A nanoassembly comprising one organic fluorescent inner core comprising fluorescent organic molecules, and magnetic nanoparticles contacting the surface of said fluorescent inner core; provided that the nanoassembly does not comprise silica, functionalized silica, doped silica, grafted silica or a combination thereof.

17. The nanoassembly according to claim 16, wherein the fluorescent organic molecules are compounds of Formula I, ##STR00013## wherein: L represents a spacer selected from (i), (ii) and (iii) ##STR00014## X represents O, CH.sub.2 or NR.sup.8, wherein R.sup.8 represents H, alkyl or alkene; X represents O or NR.sup.9, wherein R.sup.9 represents H, alkyl or alkene; n represents an integer selected from 1, 2, 3 and 4; R.sup.1 represents CO.sub.2H or P(O) (OH).sub.2; and R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.7 represent each independently an optionally functionalized group selected from alkyl and ester and poly(ethylene glycol).

18. The nanoassembly according to claim 16, wherein the magnetic nanoparticles are superparamagnetic nanoparticles selected from the group consisting of -Fe.sub.2O.sub.3 and Fe.sub.3O.sub.4.

19. The nanoassembly according to claim 16, having a hydrodynamic diameter ranging from 20 to 700 nm.

20. The nanoassembly according to claim 16, wherein the fluorescent inner core comprises a number of organic fluorescent molecule ranging from 110.sup.4 to 110.sup.7.

21. The nanoassembly according to claim 16, comprising a number of magnetic nanoparticles ranging from 110.sup.2 to 110.sup.6.

22. The nanoassembly according to claim 16, comprising at least one polymer adsorbed at the surface of the nanoassembly.

23. The nanoassembly according to claim 22, wherein the polymer is an ionic polymer.

24. The nanoassembly according to claim 22, wherein the polymer is a polyelectrolyte.

25. The nanoassembly according to claim 22, wherein the polymer is of Formula II, ##STR00015## wherein: m represents a positive integer ranging from 20 to 150; x, y and z represent each independently a percentage of m, ranging from 0% to 100% of m, wherein x+y+z is equal to 100% of m; X represents COOH, alkyl, aryl, CO-alkyl, poly(ethylene glycol), or -poly(ethylene glycol)-OMe; Y represents (CO)O-L-R.sup.8, (CO)S-L-R.sup.8, (CO)NH(-L-R.sup.8) or (CS)NH(-L-R.sup.8), wherein: L.sup.1 represents a spacer selected from alkyl, alkene, aryl, arylalkyl, poly(ethylene glycol) and poly(propylene glycol) linking groups having 1 to 150 chain atoms, wherein the linking group can be optionally interrupted or terminated by one or more O, S, NR.sup.9, CO, NHCO, CONH or a combination thereof, wherein R.sup.9 is H or alkyl; and R.sup.8 represents a reactive group selected from N.sub.3, amino, alkylamino, COOH, amide, maleimide, alkene, alkyne, SH, OH, ester, activated ester, activated carboxylic acid, halo, nitro, nitrile, isonitriles, acrylamide, aldehyde, ketone, acetal, ketal, anhydride, glutaric anhydride, succinic anhydride, maleic anhydride, thiocyanate, isothiocyanate, isocyanate, hydrazide, hydrazine, hydrazone, ether, oxide, cyanate, diazo, diazonium, sulfide, disulfide, sulfoxide, sulfone, sulfonic acid, sulfinic acid, sulfate, sulfenic acid, amidine, imide, imidate, nitrone, hydroxylamine, oxime, hydroxamic acid, thiohydroxamic acid, allene, ortho ester, sulfite, enamine, ynamine, urea, pseudourea, semicarbazide, carbodiimide, carbamate, imine, acetylene, olefin, polyene, alkylacrylate, oxetane, ammonium, oxonium, phosphonium, sulfonium, positively charged and neutral metal complex; and Z represents (CO)O-L.sup.2-R.sup.10, (CO)S-L.sup.2-R.sup.10, (CO)NH(-L.sup.2-R.sup.10) or (CS)NH(-L.sup.2-R.sup.10) wherein: L.sup.2 represents a single bond or a spacer selected from alkyl, alkene, aryl, arylalkyl, poly(ethylene glycol) and poly(propylene glycol) linking groups having 1 to 150 chain atoms, wherein the linking group can be optionally interrupted or terminated by one or more O, S, NR.sup.9, CO, NHCO, CONH or a combination thereof, wherein R.sup.9 is H or alkyl; optionally additionally comprising a residue of a reactive group through which L.sup.2 is bonded to R.sup.10; and R.sup.10 represents a bioactive group selected from amino acid, peptide, protein, antibody, affinity protein, enzyme, polysaccharide, dextran, benzylguanine, lipid, lipid assembly, fatty acid, nucleoside, nucleotide, oligonucleotide, hapten, aptamer, ligand, antibiotic, substrate, biotin, avidin, streptavidin, synthetic polymer, poly(ethylene glycol), poly(propylene glycol), polymeric microparticle, nanoparticle, fluorophore, chromophore, radioisotope, macrocyclic complexes of radioisotope, and combinations thereof.

26. The nanoassembly according to claim 22, wherein the polymer is poly(acrylic acid).

27. A process for manufacturing nanoassemblies according claim 16, comprising injecting a solution of fluorescent organic molecules into a dispersion of magnetic nanoparticles under stirring.

28. The process according to claim 27, comprising a subsequent step of addition of at least one polymer.

29. The process according to claim 28, further comprising a subsequent step of functionalization of the polymer by a bioactive group.

30. A pharmaceutical composition comprising nanoassemblies according to claim 16, in combination with at least one pharmaceutically acceptable vehicle.

31. Use of nanoassemblies according to claim 16, to extract and detect molecules and/or biological targets from physiological medium.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0282] FIG. 1 is a schematic representation of the fluo@mag nanoassemblies of the invention and of their process of manufacturing.

[0283] FIG. 2 is a schematic representation of the fluo@mag@polymer nanoassemblies of the invention and of their process of manufacturing.

[0284] FIG. 3 is a TEM image of fluo@mag@PAA nanoassemblies after synthesis. Inlet: zoom-in with a 20 nm scale bar.

[0285] FIG. 4 is the evolution of the transverse relaxivities R2 of fluo@mag@PAA nanoassemblies (D.sub.H=150 nm; black dot labels) and dispersed iron oxide nanoparticles coated with PAA (mag@PAA: D.sub.H=7 nm; white square labels) as a function of frequency. Vertical bars represent measure errors.

[0286] FIG. 5 is the evolution of longitudinal relaxivities R1 of fluo@mag@PAA nanoassemblies (D.sub.H=150 nm; black dot labels) and dispersed -Fe.sub.2O.sub.3: nanoparticles coated with PAA (mag@PAA: D.sub.H=7 nm: white square labels) as a function of frequency. Vertical bars represent measure errors.

[0287] FIG. 6 UV-vis absorption spectra in water of the fluorescent organic nanoparticles (fluo absorption) and -Fe.sub.2O.sub.3 nanoparticles (-Fe.sub.2O.sub.3 absorption) and emission spectra (.sub.exc=450 nm) of the fluorescent organic nanoparticles (fluo emission) of fluo@mag@PAA nanoassemblies (fluo@mag@PAA emission).

[0288] FIG. 7 is fluorescence confocal laser scanning microscopy images of HEK cells incubated with fluo@mag@PAA nanoassemblies at: a) a 3 M concentration of fluorescent molecules with no staining agent (.sub.exc=488 nm, .sub.em>510 nm), b) a 1 M concentration of fluorescent molecules and post-treated with a nucleus staining agent (Hoechst 33342) (.sub.exc=405 nm, .sub.em>510 nm) (objective63. NA 1.4).

[0289] FIG. 8 is TEM images of internalized nanoparticles in HEK cells (70 nm-thin section). a) General view. b) Zoom-in view. c) and d) Endosomes trapping the fluo@mag@PAA nanoassemblies.

[0290] FIG. 9 is T2*-weighted MR images of mouse's liver, a) before the injection, b) 25 min after the injection, c) 230 min. after the injection. The white circle corresponds to a water magnetic resonance phantom.

[0291] FIG. 10 is T2*-weighted MR images of mouse's spleen. a) before the injection, b) 25 min after the injection. The white circle corresponds to a water magnetic resonance phantom.

[0292] FIG. 11 is TEM images of mouse's liver tissue after intravenous injections of the fluo@mag@PAA nanoassemblies of the invention.

[0293] FIG. 12 Comparative emission spectra (4, =350 nm) and fluorescence decays (.sub.em=340 nm, .sub.em=440 nm) in water of 7-amino-4-methylcoumarine (AC). fluo@mag@PAA-AC nanoassemblies issued from the surface functionalization of fluo@mag@PAA with AC. and mag@PAA-AC issued from the surface functionalization of mag@PAA with AC.

EXAMPLES

[0294] The present invention is further illustrated by the following examples.

[0295] Material

[0296] All chemical reagents and solvents were purchased from commercial sources (Aldrich, Acros, SDS) and used as received. Spectroscopic grade solvents purchased from Aldrich were used for spectroscopic measurements.

ABBREVIATIONS

[0297] C. Celsius degree

[0298] cm centimeter

[0299] DAPI 4,6-diamidino-2-phenylindole

[0300] D.sub.H Hydrodynamic diameter

[0301] DLS Dynamic Light Scattering

[0302] DMEM Dulbecco/Vogt modified Eagle's minimal essential medium

[0303] DMSO Dimethylsulfoxide

[0304] emu 1 emu=10.sup.3 A m.sup.2=10.sup.3 J T.sup.1 features the unit of the magnetic moment

[0305] ENH Contrast magnetic resonance enhancement

[0306] G Gauss

[0307] h hour

[0308] HBSS Hank's Balanced Salt Solution

[0309] HEK Human Embryonic Kidney cell

[0310] K Kelvin

[0311] Kg kilogram

[0312] Wavelength

[0313] LSM Laser Scanning Microscope

[0314] M mole per liter

[0315] MDa Mega Dalton

[0316] MDA Cell derived from metastatic site in human mammary gland/breast

[0317] MHz Mega Hertz

[0318] min minute

[0319] mL milliliter

[0320] MRI Magnetic Resonance Imaging

[0321] Ms saturation magnetization

[0322] MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

[0323] ms millisecond

[0324] nm nanometer

[0325] NMR Nuclear Magnetic Resonance

[0326] NPs Nanoparticles

[0327] OCT Embedding matrix for cryotomy

[0328] PAA Poly(acrylic acid)

[0329] PBS phosphate buffered saline

[0330] R1 longitudinal relaxivity

[0331] R2 transverse relaxivity

[0332] RPMI Roswell Park Memorial Institute medium

[0333] s second

[0334] T Tesla

[0335] T1 Longitudinal relaxation time

[0336] T2 Transverse relaxation time

[0337] TE echo time

[0338] TEM Transmission Electron Microscopy

[0339] THF Tetrahydrofuran

[0340] TR repetition time

[0341] M micromole per liter

[0342] UV ultraviolet

[0343] v frequency

[0344] Part AMagnetic-Fluorescent Nanoasemblies

[0345] A.1. Synthetic Procedures

[0346] A.1.1 Fluorescent Organic Molecules

[0347] Fluorescent molecules (la) and (Ib) were obtained as described in Faucon et al., J. Mater. Chem. C., 2013, 1, 3879-3886.

[0348] Stock solutions of the fluorescent molecules (Ia) and (Ib) in spectrophotometric grade THF were prepared at concentrations ranging from 0.05 to 1 wt %.

[0349] A.1.2. Magnetic Nanoparticles

[0350] Nitrate-Stabilized Magnetic Nanoparticles.

[0351] Maghemite -Fe.sub.3O.sub.3 nanoparticles were prepared according to a procedure described by Massart et al (IEEE Trans. Magn., 1981, 17, 1247-1248). Briefly, Fe(II) and Fe(III) salts were mixed in dilute hydrochloric acid. Quick alkalinization of the medium by adding concentrated ammonia enables the coprecipitation of magnetite Fe.sub.3O.sub.4 nanoparticles which were separated. The acidification of -Fe.sub.3O.sub.4 with nitric acid, followed by chemical oxidation with ferric nitrate at 80 C., yielded -Fe.sub.3O.sub.3 nanoparticles. After magnetic decantation, the red precipitate was dispersed in nitric acid (pH=1.2) since nitrate ions act as stabilizing counter-ions of the positively charged surface of the colloidal dispersion (=+25 mV).

[0352] Magnetite Nanoparticles.

[0353] The synthesis was performed according by adapting Santoyo et al. (Chem. Mater. 2011, 23, 1379-1386). Two solutions of iron salts were prepared: FeCl.sub.3, 6H.sub.2O, 1 mol.Math.L.sup.1 in HCl 2 mol.Math.L.sup.1 (10 mL) and FeCl.sub.2, 4H.sub.2O, 2 mol.Math.L.sup.1 in HCl 2 mol.Math.L.sup.1 (2.5 mL). After thorough deoxygenation with nitrogen for 2 h, both solutions were mixed and heated up to 70 C. under argon atmosphere under vigorous stirring. A solution of tetrapropylammonium hydroxide (1 mol.Math.L.sup.1) was then injected at a 0.7 mL.Math.min.sup.1 rate, and vigorous stirring was continued for an extra 20 min. The resulting nanoparticles were washed twice by magnetic decantation and dialyzed against Millipore water until neutral pH was obtained. In order to obtain a stable dispersion of magnetite in acidic media, the nanoparticle were sonicated in 2 M HNO.sub.3 for 15 min and then washed one time by magnetic decantation with 0.1 M of HNO.sub.3. A colloidal dispersion is obtained after stirring the nanoparticles for few days which was eventually filtered through 0.2 m to remove larger nano-objects. After the previous dialysis step, one drop of amine was added to provide a stable dispersion of magnetite nanoparticles Fe.sub.3O.sub.4 in neutral water, which was eventually filtered through 0.2 m to remove larger nano-objects.

[0354] A.1.3. Fluo@mag Nanoassemblies Formation

[0355] A 0.1 wt % stock solution of fluorescent molecules in THF (50 L) was injected into a 0.006 wt % solution of maghemite nanoparticles (2.5 mL) stirred by means of a vortex.

[0356] When a nitric acid solution of magnetic nanoparticles was used, formation of hybrid nanoparticles, dubbed fluo@mag and comprising fluorescent molecules and iron oxide nanoparticles, is instantaneous.

[0357] A.2. Physico-Chemical Characterizations

[0358] A.2.1. Nanoassembly Characterizations

[0359] Methods.

[0360] The hydrodynamic diameter and size distribution of the nanoassemblies were determined by dynamic light scattering (DLS) by means of a nanoparticle size analyzer Zetasizer Nano ZS ZEN 3600 (Malvern Instruments) equipped with a 4 mW HeNe laser, operating at 633 nm, and a photomultiplier detector collecting back-scattered light at an angle of 175. Measurements were carried out at 20 C. on aqueous solutions of nanoassemblies. For each sample, intensity measurements were carried out in a multi-acquisition mode implying automatically adjusted correlograms, and averaged measurements on 3 acquisitions. Nanoparticle mean sizes and distribution widths were obtained by fitting each correlogram with a Cumulants algorithm.

[0361] Measurements of surface potential were carried out by means of a Zetasizer Nano ZS ZEN 3600 (Malvern). The samples were placed in plastic cells. Several measurements were realized for each sample according to a predefined operating procedure.

[0362] The nanoassembly morphology was investigated by transmission electron microscopy (TEM, Hitachi HF2000-FEG). Solutions of nanoassemblies were deposited onto holey carbon coated copper grids (300 mesh) for all compounds except for the fluo@mag nanoparticles prepared from naked -Fe.sub.2O.sub.3 nanoparticles dispersed in dilute nitric acid, which were deposited onto holey carbon-coated gold grids (300 mesh).

[0363] Results.

[0364] DLS and TEM measurements of fluo@mag nanoassemblies invariably yielded a mean diameter of 90-100 nm with a quite narrow size distribution. The colloidal suspension of nanoassemblies was stable in dilute nitric acid over months due to the negatively charged surface (=33 mV) as measured by zetametry. TEM imaging revealed a raspberry-like assembly where -Fe.sub.2O.sub.3 nanoparticles contacted an organic nanosphere resulting from the self-aggregation of fluorescent molecules upon phase separation in aqueous solution.

[0365] 2.2. Photophysical Measurements

[0366] Methods.

[0367] UV-visible absorption spectra were recorded using a Varian Model Cary 5E spectrophotometer, using an integrating sphere DRA 2500. Corrected emission spectra were obtained using Jobin-Yvon. Inc spectrofluorimeter (Fluorolog 2). Fluorescence quantum yields .sub.f in solution were determined from Coumarine 540 A in EtOH (.sub.f=0.38). Fluorescence intensity decays were measured at 580 nm using a monochromator (Hamamatsu MCP R3809U photomultiplier) by the time-correlated single-photon counting method (TCSPC) with a femtosecond laser excitation at 450 nm provided by a Spectra-Physics setup (Titanium-Sapphire Tsunami laser pumped by a doubled YAG laser (Millennia), pumped itself by a two-laser diode array, and doubling LBO crystals). Light pulses at 900 nm were selected by optoacoustic crystals at a repetition rate of 4 MHz, and then doubled at 450 nm.

TABLE-US-00003 TABLE 1 Photophysical characteristics of fluo@mag nanoassemblies dispersed in water. .sub.max(abs) .sub.max(em) .sub.l (f.sub.i).sup.1 <.sub.S>.sup.2 [nm] [nm] [ns] [ns] 443 604 0.77 (34%), 0.24 (48%), 0.39 0.08 (14%), 0.04 (4%) .sup.1After laser excitation at 450 nm and detection at 580 nm. .sup.2The intensity averaged time constant <.sub.S> and normalized time fractional amplitude f.sub.i are calculated from global analysis using a multiexponential fit I(t) = .sub.ia.sub.iexp(t/.sub.i) with a.sub.i pre-exponential factor. The intensity-averaged time constant <.sub.s> is defined as <.sub.s> = .sub.i f.sub.i.sub.i with f.sub.i = a.sub.i.sub.i/.sub.j a.sub.j .sub.j.

[0368] Results.

[0369] The steady-state emission spectrum of the resulting fluo@mag nanoassemblies appeared very similar to that of fluorescent nanospheres in terms of energy (5 nm hypsochromic shift) and intensity (.sub.f=0.01). Such a strong similarity stems from the main contribution of the fluorescent molecules comprised in the core, isolated from the interface. Partial emission quenching of the fluorescent nanospheres occurred via vibrational coupling between water and fluorescent molecules at the interface. For fluo@mag nanoassemblies, such quenching operates from electron transfer from the peripheral fluorescent molecules to the contacting iron oxide nanoparticles, which extends only to a few nanometers, with no effect on the fluorescent molecules inside the core. Shortening of the fluorescence decay was indeed observed by time-resolved fluorescence measurements. The longer lifetime constant, assessed at 1.49 ns for the fluorescent nanospheres, decreased to 0.08 ns for the fluo@mag nanoassemblies. These results emphasize the adopted approach of reverse architectures, insulating fluorescent molecules from iron oxide nanoparticles, known as strong emission quenchers.

[0370] 2.3. Magnetic Measurements

[0371] Methods.

[0372] Temperature dependent magnetization experiments on the colloidal suspension of fluo@mag nanoparticles ([Fe]6.510.sup.5 mol.Math.L.sup.1) were collected with a Quantum Design MPMS-5S SQUID magnetometer working at the temperature of 298 K and in the magnetic field range 0-2 T. The samples were diluted enough to avoid magnetic dipole-dipole interactions. The hysteresis loops were performed at 298 K in ZFC samples.

[0373] Results.

[0374] The magnetization curve as a function of the applied magnetic field at 300 K shows very small coercivity, typical of superparamagnetic nanoparticles. The saturation magnetization values Ms were found to be 22 emu.Math.g.sup.1 for fluo@mag nanoassemblies. The reduced M.sub.S values relative to bulk maghemite (75-80 emu.Math.g.sup.1) could be due to an increase in the surface anisotropy induced by chemisorption of the fluorescent molecules at the surface. Taking into account the iron mass determined from elemental analyses, fit of the magnetization curve provides a size distribution peaking at 7.29 nm (width =0.25) in fair agreement with the mean diameter of the initial nanoparticles (D.sub.H=8.1 nm). Such values tend to indicate that -Fe.sub.2O.sub.3 nanoparticles still keep their superparamagnetic behaviour when adsorbed.

[0375] Part BPolymer Nanoassembly Systems

[0376] Coating the fluo@mag nanoassemblies with an appropriate polyelectrolyte (i.e. polymer) leads to stable colloidal solutions of nanoassemblies undergoing neither aggregation nor dissociation in aqueous media, which is suitable for in vitro and in vivo applications. Their subsequent lyophilization yields fine powders for a long-term storage which can be re-dispersed in media of various chemical natures and ionic strengths with no loss of the structural and functional properties.

[0377] B.1. Synthetic Procedures

[0378] The protocols below describe the stabilization process of the fluorescent and magnetic fluo@mag nanoassemblies by using the PAA polyelectrolyte (M.sub.w=1.8-2.2 kDa) as a polymer. Lyophilization of the stabilized fluo@mag@PAA nanoassemblies for biological uses in various media of interest is described.

[0379] B.1.1. Synthesis of fluo@mag@PAA Nanoassemblies

[0380] With Maghemite Nanoparticles.

[0381] A solution of fluorescent compound (Ib) dissolved in THF (50 L, 0.1 wt. %) was added under vigorous stirring to a solution of maghemite nanoparticles in nitric acid (2.5 mL, 0.006 wt. %, pH=1.2). After a few seconds, the magnetofluorescent nanoassemblies (Ib)-fluo@maghemite were formed. Poly(acrylic acid) (2.1 kDa, 5 mg) was added as powder, ammonium hydroxide (1 mol.Math.L.sup.1) was added dropwise under stirring until pH=9 was reached. The resulting translucent solution was allowed to stir for a further 30 min and dialyzed using a Spectra Por membrane (Standard Grade Regenerated Cellulose; cut-off: 8-10 kDa) against Millipore water (600 mL) over 24 h until the final pH solution of the fluo@mag@PAA nanoassemblies (1) reached a value of 7.

[0382] With magnetite nanoparticles. A solution of compound (Ib) dissolved in THF (50 L, 0.1 wt. %) was added under vigorous stirring to a solution of magnetite nanoparticles in water (2.5 mL, 0.006 wt. %). After a few hours (usually two hours), the magnetofluorescent nanoassemblies (Ib)-fluo@magnetite formed. Polyacrylic acid (2.1 kDa, 5 mg) was added as a powder and then, ammonium hydroxide (1 mol.Math.L.sup.1) was added dropwise under stirring until pH=9 was reached. The resulting translucent solution was allowed to stir for a further 30 min and dialyzed using a SpectraPor membrane (Standard Grade Regenerated Cellulose; cut-off: 8-10 kDa) against Millipore water (600 mL) over 24 h until the final pH solution of the fluo@mag@PAA nanoassemblies (2) reaches a value of 7. The suspension of nanoassemblies was finally stored at 4 C.

[0383] B.1.2. Lyophilization

[0384] In order to store the resulting fluo@mag@PAA nanoassemblies over a long period of time in the solid state, volumes (1 mL to 3 mL) of nanoparticle solutions were placed in glass vials such that the height of the liquid was not higher than 1 cm. The solution was allowed to freeze using liquid nitrogen. Lyophilization was performed over 9 h to 12 h to yield a dark red power that was stored at 18 C. under nitrogen atmosphere.

[0385] B.1.3. Re-Dispersion Procedures

[0386] Nanoassembly redispersion was easily performed by adding the adequate solvent (water, physiological media, alcoholic solvents such as ethanol) to the lyophilized sample. No ultrasound treatment was required since the nanoassemblies undergo no aggregation upon redispersion. Iron concentration in the range of 0.6-310.sup.3 mol.Math.L.sup.1, even in the range of 0.6-710.sup.3 mol.Math.L.sup.1, could be obtained depending of the added amount of re-dispersing solvent.

[0387] B.2. Physico-Chemical Characterization

[0388] The characterizations of fluo@mag@PAA nanoassemblies (1) in terms of size, surface potential, composition, photophysical and relaxivity properties are described. The stability of the colloidal suspensions as a function of time and the nature of solvent was checked by DLS, TEM and UV-vis absorption measurements (UV-visible absorption spectra were recorded using a Agilent Model Cary 5E spectrophotometer, equipped with an integrating sphere DRA 2500). The composition (number of fluorescent molecules in the core and magnetic nanoparticles of the shell) has been determined by both mass spectrometry and magnetic sedimentation.

[0389] DLS and TEM measurements of fluo@mag@PAA nanoassemblies (1) yield a mean diameter of 120-150 nm. DLS measurements, performed in water, generally display slightly larger diameters due to the first water solvation sphere. The nanoassemblies are stable in water over a period of 5 months at room temperature and over almost a year at 4 C. The size distribution varies little after lyophilization and redispersion in various media (water, HBSS, PBS, ethanol) as demonstrated by DLS and UV-vis absorption measurements, in accord with parallel DLS and TEM analyses. The fluo@mag@PAA nanossembly (1) cohesion, ensured by the PAA coating, is also demonstrated by means of ultra-sounds which do not dissociate the nanoassemblies. Mass spectrometry analyses and magnetic sedimentation, enabling the composition determination, show convergent data: the core was made of 10.sup.5 organic molecules (in agreement with previous results involving single nanoparticle photoabsorption measurements), surrounded by a shell of 10.sup.4 maghemite nanoparticles. Relaxivity measurements demonstrate large cooperativity effects between the vicinal iron oxide nanoparticles assembled in the magnetic shell. The threefold increase in the r.sub.2/r.sub.1 ratio observed for the nanoassemblies allows for highly contrasted T2-weighted MRI. Finally, steady-state and time-resolved fluorescence measurements show that the fluorescence signal is not affected by the surrounding media as expected from the protecting role played by the maghemite nanoparticle shell.

[0390] B.2.1. Size Characterizations

[0391] Size characterizations (DLS and TEM measurements) of the fluo@mag@PAA nanoassemblies (1) were carried out after formation, after the lyophilization-redispersion process and after having been subjected to ultra-sounds. After redispersion, no agglomeration is observed by DLS even for solutions with concentrations higher than those of the initial solution before lyophilization. Moreover no nanoassembly dissociation is observed after ultra-sounds.

[0392] The assemblies characterized by TEM, were deposited on holey carbon grids and dried at room temperature. Observations (FIG. 3) were performed with a MO-Jeol MET 1230, working at a 80 kV voltage.

[0393] B.2.2. Colloidal Stability

[0394] The stability of fluo@mag@PAA nanoassemblies (1) was studied as a function of time and in distinct solutions with various pHs and ionic strengths.

[0395] DLS and TEM measurements of nanoassemblies dispersed in Millipore water were conducted right after the synthesis and after a few months. The results feature the high stability of the nanoassembly structure (TEMFIG. 3).

[0396] UV-vis absorption analyses were also performed at 450 nm as a function of time to follow the colloidal stability. In the case of unstable dispersions of nanoparticles, sedimentation is usually observed with a loss of absorbance. Absorbance decreases by less than 2% after 11 days, proving the highly stable colloidal character of the nanoassemblies, be they placed in water or redispersed in HBSS or PBS buffer.

[0397] Measurements of the hydrodynamic diameter distribution of fluo@mag@PAA nanoassemblies were also performed in various media and for various durations of storage at room temperature to assess colloidal stability. The previously lyophilized nanoassemblies were redispersed in pure water or in a physiological medium composed of PBS and 10% of bovin serum, with no significant change in the distribution of hydrodynamic diameter. A slight increase in the mean diameter is observed when the nanoassemblies were dispersed in a medium composed of bovin serum only (100%), due to non-specific adsorption of proteins on the nanoassembly surface. After 24 h. neither decantation nor precipitation was further observed, despite the slight increase in the hydrodynamic diameter. Consequently, the nanoassemblies exhibit unique stability in highly concentrated serum (up to 100%).

[0398] The nanoassemblies are stable over a large pH range (3-12) due to a significant surface potential <31 mV, causing efficient electrostatic repulsions between the nanoassemblies. As a consequence, no aggregation could also be observed in media of varying ionic strength up to 0.3 mol.Math.L.sup.1, which is beneficial for biological applications dealing with media usually with high ionic strength.

[0399] B.2.3. Composition Determination

[0400] Studies were conducted to determine the amount of iron oxide NPs on the surface of the organic core by means of mass spectrometry and magnetic sedimentation respectively.

[0401] Titrations of iron content of the nanoassemblies may also be performed by resorting to ICP-AES measurements (Inductive Coupled Plasma-Atomic Emission Spectrometry).

[0402] Mass spectrometry experiments were realized according to the method described in Doussineau et al., Rapi. Commun, Mass Spectrom, 2011, 25, 617-623. The molar mass distributions for the fluorescent nanospheres and the fluo@mag@PAA nanoassemblies (1) were recorded. The noticeable increase in weight (namely 86 MDa) when going from fluorescent organic nanospheres to fluo@mag@PAA nanoassemblies (1) can be mainly correlated to the amount of iron oxide nanoparticles, assessed around 210.sup.4 nanoparticles per nanoassembly. These measurements also confirm the amount of fluorescent molecules per organic core, valued to be around 10.sup.5 fluorescent molecules.

[0403] Magnetic decantation experiments were carried out to analyze the time-evolution of the normalized absorbance at 552 nm and 446 nm. Magnetic sedimentation was performed with solutions of maghemite nanoparticles dispersed in dilute nitric acid (pH=1.2) and solutions of fluo@mag@PAA nanoassemblies (1) dispersed in water.

[0404] Comparative measurements of the magnetic sedimentation of single maghemite nanoparticle dispersions and fluo@mag@PAA nanoassemblies (1) dispersions show stronger sedimentation for the latter in accord to their higher magnetic content. Modeling the absorbance decay indicated that the magnetic shell of the nanoassembly comprised 10.sup.4 maghemite nanoparticles, in agreement with the data obtained by mass spectrometry.

[0405] B.2.4. Relaxivity Measurements

[0406] Relaxivity measurements (.sup.1H NMR longitudinal T1, and transverse T2 relaxation times) were conducted for the fluo@mag@PAA nanoassemblies (1) in diluted solutions.

[0407] Measurements of .sup.1H longitudinal T1 and transverse T2 relaxation times at different frequencies v at room temperature were performed on diluted solutions of samples (at v=200 and 300 MHz), in the range of 10 kHzv212 MHz for T1 and 15 MHzv60 MHz for T2. Proton T1 and T2 relaxation time measurements were performed on aqueous solutions with different concentrations of nanoassemblies at room temperature (300 K) under an applied magnetic field of 4.7 T using a Tecmag Apollo spectrometer operating at 200 MHz. The rates observed for both longitudinal and transverse relaxations showed a linear dependence according to the nanoparticle concentration. Using the inversion-recovery and spin echo (Carr-Purcell Meiboom Gill) sequences to measure T1 and T2, the longitudinal r.sub.1 and transverse r.sub.2 relaxivities were calculated from the slope of the plot of 1/T1 and 1/T2 curves vs nanoparticle concentration. FIGS. 4 and 5 feature the transverse and longitudinal relaxivities respectively, as a function of frequency, of fluo@mag@PAA nanoassemblies (1) and dispersed iron oxide nanoparticles coated with PAA.

[0408] Higher values of transverse relaxivity r.sub.2 are obtained for fluo@mag@PAA nanoassemblies (1) compared to those of dispersed iron oxide nanoparticles. The longitudinal relaxivity r, varies in the opposite trend. The high values of T2 and the large increase in the r.sub.2/r.sub.1 ratio for the nanoassemblies, related to the T2 weighted MRI, demonstrate the very positive additive effects between the iron oxide nanoparticles at the surface of the nanoassemblies. Such additivity does not exist for isolated iron nanoparticles serving as a reference.

[0409] B.2.5. Photophysical Properties

[0410] Steady-state absorption and emission measurements (FIG. 6), time-resolved fluorescence measurements, and wide-field fluorescence microscopy of fluo@mag@PAA nanoassemblies (1) dispersed in various media were realized to characterize their emission intensity and spectral range.

TABLE-US-00004 TABLE 2 Photophysical characteristics of fluo@mag@P nanoassemblies (1) dispersed in water. .sub.max(abs) .sub.max(em) .sub.l (f.sub.i).sup.1 <.sub.S>.sup.2 [nm] [nm] [ns] [ns] 443 604 0.7 (29%), 0.2 (52%), 0.31 0.04 (19%) .sup.1After laser excitation at 450 nm and detection at 580 nm. .sup.2The intensity averaged time constant <.sub.S> and normalized time fractional amplitude f.sub.i are calculated from global analysis using a multiexponential fit I(t) = .sub.ia.sub.iexp(t/.sub.i) with a.sub.i pre-exponential factor. The intensity-averaged time constant <.sub.s> is defined as <.sub.s> = .sub.i f.sub.i.sub.i with f.sub.i = a.sub.i.sub.i/.sub.j a.sub.j .sub.j.

[0411] No significant effect of the presence of the PAA coating on the emission properties could be observed compared to the non-stabilized fluo@mag nanoparticles. The fluorescence signal is also insensitive to the nature of the surrounding solutions. This permits accurate detection of the nanoparticles in biological media, requesting no change in the fluorescence intensity or color unless specifically expected. Efficient fluorescence signal is detected upon exciting the fluo@mag@PAA in the absorption band of the fluorescent core. The red-shifted emission compared to the maghemite nanoparticle absorption band avoids deleterious emission reabsorption by the maghemite nanoparticles (FIG. 6), although a small decrease in the emission intensity of the core compared to that of neat fluorescent nanospheres could be noticed. This effect is due to electron transfer from the core to iron (III) ions of the maghemite nanoparticles. Such an emission quenching is actually much stronger for the normal or doped architectures usually reported in literature, where each peripheral fluorescent unit is in direct contact with the iron oxide nanoparticles.

[0412] B.3. In Vitro Experiment

[0413] Two different lines of human cells (HEK 293 and MDA-MB-468) are used as well-known model cells for in vitro studies to investigate the uptake of nanoassemblies and their cytotoxicity effects. Fluorescence microscopy and TEM imaging are performed to characterize the fluorescence properties and the arrangement of the nanoassemblies inside the cells.

[0414] Main Results.

[0415] Cell uptake of the nanoassemblies operates within 6-8 h. Cytotoxicity MTT assays reveal that cell viability is not hampered following cell culture with a 15 mol.Math.L.sup.1 Fe concentration, similar to those used for in vivo MRI experiments. Fluorescence microscopy imaging of live cells shows mobile orange bright spots inside the cells, proving no significant aggregation of the internalized nanoparticles. Comparison experiments with fluorescent organic nanospheres prove that the nanoassemblies do not dissociate within the cells since high structural stiffness is brought by the PAA coating. These results are corroborated by 2D TEM imaging of cells showing the intact structure of the nanoassemblies and the circular arrangement of the iron oxide nanoparticles around the organic core.

[0416] B.3.1. Procedures

[0417] Cell Incubation.

[0418] The cells were incubated for at least 24 h on an Ibidi 8-well plate at 37.7 C. in a 5% CO.sub.2 atmosphere. 5000 cells were incubated for experiments running over 72 h. MDA cells were grown in RPMI media containing 10% of fetal bovin serum and 1% of penicillin and streptomycin. HEK cells were grown in DMEM media (high glucose, with glutamine) containing 10% of fetal bovin serum and 1% of penicillin and streptomycin.

[0419] Nanoassembly Internalization.

[0420] Internalization was performed by adding to the cell medium a solution of fluo@mag@PAA nanoparticles (1) dispersed in water (10-20 L) such that the final concentrations of fluorescent molecules and iron were about 110.sup.6 mol.Math.L.sup.1 and 210.sup.5 mol.Math.L.sup.1. The suspension was incubated for 6 h to ensure efficient cell uptake of the nanoassemblies, which could already be observed after 3 h only.

[0421] B.3.2. Cell Viability

[0422] MTT assay: 5000 cells were grown in the appropriate culture medium (200 L) for 24 h. They were then incubated for various periods of time with a solution of fluo@mag@PAA nanoassemblies (1) dispersed in Millipore water. The cell viability was evaluated by using MTT assays. A solution of MTT (20 L; 5 mg.Math.mL.sup.1) was added to the cells which were incubated for a further 2 h-2 h 30. The supernatant solution was removed and DMSO (200 L) was added to dissolve the colored oxidized product. Absorbance read-out at 570 nm provides viability of the incubated cells by comparison with reference cells.

[0423] The MTT assays reveal almost no cell cytotoxicity for nanoassembly solutions with a 15 mol.Math.L.sup.1 iron content (cell viability>95%) which is the common concentration used for all in vitro and in vivo experiments. For very large concentrations of iron (300 mol.Math.L.sup.1), only the HEK cells exhibit significant mortality in agreement with an increase in the oxidative stress caused by the large excess of iron (300-500 g.Math.mL.sup.1).

[0424] B.3.3. Fluorescence Microscopy of Internalized Nanoassemblies in Live Cells

[0425] Fluorescence microscopy was performed in the confocal mode by means of a LSM working in the inverted mode (Nikon AIR Si, oil-immersion objective Plan Apo, 60, 1.4, .sub.exc=488 nm), or in the wide-field mode by means of an inverted microscope (Nikon Eclipse Ti, oil-immersion objective Plan Apo, 60, 1.4, 4, =482 nm).

[0426] Internalized nanoassemblies (1) can clearly be distinguished as spots on FIG. 7a without undergoing significant aggregation inside the cells. No spots can be distinguished inside the nuclei, indicating that the nanoassemblies stay in the cytoplasm and do not penetrate into the nuclei (FIG. 7b).

[0427] TEM Imaging of Internalized Nanoassemblies: [0428] Addition of HEK cells in agar solution at 45-50 C.; [0429] Fixation for 2 h at 4 C. of small pieces of agar gel using a phosphate 0.1 M solution and 3% glutaraldehyde; [0430] Washing with phosphate buffer and Millipore water, [0431] Post-staining using osmium tetraoxide (1%) in Millipore water for 1 h and repeated washings using Millipore water, [0432] Dehydratation using an ethanol bath for 1 h with increasing concentration in ethanol (30%, 50%, 70%, 85%, 95%, 100%); [0433] Dehydratation using a 100% ethanol bath (2 h and overnight at 4 C.); [0434] Exchange of ethanol with propylene oxide; [0435] Inclusion in EPON resine; [0436] Polymerization for 1 day at 55 C. and 60 h at 72 C.; [0437] Staining after slicing for 30 min in uranyl acetate and washing using Millipore water.

[0438] TEM imaging (FIG. 8) reveals the embedment of contrasted iron oxide nanoparticles in cell endosomes after cell uptake. Remarkably, the maghemite nanoparticles are organized following a disk-like arrangement recalling the spherical core-shell structure of the nanoassemblies. Given the strong fluorescence detectable in live cells, these TEM images show that the nanoassemblies keep their integrity after internalization with no dissociation of the magnetic shell from the organic core thanks to the cohesive PAA coating.

[0439] B.4. In Vivo Experiments

[0440] In vivo Magnetic Resonance Imaging was performed on a series of 5 mices. After mice euthanasia, the extracted organs were sliced for fluorescence microscopy imaging using one- and two-photon excitation to minimize tissue auto-fluorescence. In order to gain higher spatial resolution in the imaging of the nanoassemblies, transmission electron microscopy measurements (TEM) were performed on ultra-thin slices of liver (70 nm-thick).

[0441] B.4.1. Magnetic Resonance Imaging on Small Rodents

[0442] Procedure. T2 weighted MRI experiments were performed on 5 female BALB/C mice (5-week old) by means of a MRI spectrometer BioSpect 4.7 T (Bruker). The echo time TE and the repetition time TR of the sequence (T2* mode) were set at 5 ms and 300 ms respectively. A concentration of 13 mol Fe/kg mouse was chosen to perform these analyses.

[0443] The mouse was anesthetized using isofluorane gas at a concentration allowing for 30 heartbeats per minute. Images of the liver were recorded as references before injecting the nanoassemblies. A suspension of fluo@mag@PAA nanoassemblies (1) in HBSS buffer (250 L, 1.3 mol.Math.mL.sup.1 Fe concentration) was injected in the mouse caudal vein. MRI was performed after 20-30 min. An average spleen ENH of 13% and liver ENH of 49% was obtained.

[0444] Results.

[0445] MRI imaging has been performed in order to observe magnetic cooperativity between the iron oxide nanoparticles on the surface of the nanoassembly. As shown on FIGS. 9 and 10, a negative contrast has been obtained by T2* imaging with iron concentrations (13 mol Fe/kg) lower than those commonly used with individual iron oxide nanoparticles (average concentration in the literature: 40-70 mol Fe/kg). No contrast evolution was observed after 3 h 50 after injection

[0446] T2-weighted MRI experiments are focused on the main organs (spleen and liver) responsible for most of the nanoassembly clearance through the recruitment of macrophages for 150 nm-sized nanoparticles. A clear decrease in the T2* contrast in the liver and spleen can be observed 20-25 min. after the injection. These observations feature efficient uptake of the nanoassemblies. They also demonstrate the efficient role of the fluo@mag@PAA nanoassemblies as MRI contrast agents despite the low Fe concentration used. They are to be linked to the high r.sub.2/r.sub.1 ratio of the longitudinal r.sub.1 and transverse r.sub.2 relaxivities (see Point B.2.4.).

[0447] B.4.2. Fluorescence Microscopy of Sliced Tissues

[0448] Tissue sectioning procedure. The mice were sacrificed 24 h after the injection. Five organs (liver, spleen, kidney, lung and heart) were harvested for ex vivo analyses in order to assess the biodistribution of the fluo@mag@PAA nanoassemblies (1). They were successively immersed in a bath of zinc-containing fixatives (Pharmingen, France) at 4 C. for 48 h, and 4 wt. % paraformaldehyde20 wt. % sucrose solution at 4 C. for 24 h. They were eventually frozen in OCT (Tissue-Tek) by using vapors of liquid nitrogen. Zinc-containing fixatives avoid fainting of the fluorescent molecules during the fixing process. The frozen organs were stored at 80 C. for 30 days before use. They were cut in 10 m-thick sections at 20 C. using a cryotome cryostat (Leica Biocut 2030). The sections were immediately deposited on superfrost glass slides, let dry for 2 h before being treated with Prolong (containing or not DAPI as a cell nucleus staining agent) and protected with an upper coverslit to allow for microscopy imaging. The sections were left at 4 C. for at least 72 h before observation.

[0449] Instrumentation.

[0450] Fluorescence microscopy was performed in the confocal mode by means of two kinds of LSM working in the inverted mode: [0451] one-photon fluorescence imaging: Nikon A 1R Si equipped with Ar.sup.+ and HeNe lasers as excitation sources (use of an oil-immersion objective Plan Apo, 60, 1.4, .sub.esc=488 nm)collaboration IRSUN/Nantes; [0452] two-photon fluorescence imaging: Zeiss LSM 780 equipped with a tunable Ti:sapphire femtosecond laser (Mai Tai, 710-920 nm) and a 32-channel QUASAR GaAsp detector allowing for multispectral imaging. All images were acquired upon excitation at 830 nm and using two detection channels (blue centered at 450 (35) nm and red centered at 654 (104) nm)collaboration KU Leuven/Belgique.

[0453] Results.

[0454] Fluorescence microscope imaging clearly distinguished fluo@mag@PAA nanoassemblies as bright emitting spots in the sliced tissues with no penetration in the cell nuclei. The fluo@mag@PAA nanoassemblies efficiently responded to two-photon excitation, allowing for higher localization resolution and NIR investigations in the tissue transparency window. In agreement with the clearance process, the non-functionalized fluo@mag@PAA nanoassemblies mostly accumulated in the liver, and in the spleen to a lesser extent. Very few nanoassemblies were found in the kidneys responsible for the clearance of smaller nanoparticles (a few nm-sized) while none of them was found in the lung and heart.

[0455] Especially, despite the strong autofluorescence displayed by highly pigmented tissues like liver or spleen, emission of the nanoassemblies was clearly distinguished under one- or two-photon excitations. The nanoassemblies appear as localized small and very bright spots with a fluorescence signal centered at 580 nm, neatly red-shifted compared to the 520 nm-centered autofluorescence. The spot dimensions, close to those of a single nanoassembly, taking into account light scattering, let suggest the absence of nanoassembly aggregation or dissociation in vivo. Fluorescence microscope imaging reveals that the nanoassemblies do not enter the hepatocyte cell nuclei. The nanoassemblies exhibit no significant bleaching under normal observation conditions in contrast with the tissue auto-fluorescence, which should enable accurate localization of the nanoassemblies when properly functionalized with targeting agents.

[0456] B.4.3. Transmission Electronic Microscopy of Sliced Tissues

[0457] Procedure and Reagents

[0458] Sodium cacodylate buffer 0.2 mol.Math.L1, pH=7.4 (storage over 1 year500 mL). Trihydrate sodium cacodylate (21.4 g), dihydrate calcium chloride (25 mg), and hexahydrate magnesium chloride (50 mg) were dissolved in distilled water (450 mL). The pH mixture was adjusted upon adding a 0.5 mol.Math.L1 HCl solution under magnetic stirring. Distilled water was eventually added to adjust the total volume to 500 mL.

[0459] Tissue fixation. The tissues were primarily fixed with glutaraldehyde (2.5 wt. %/vol) for 2 h to preserve their structure. The tissues were subsequently washed with cacodylate buffer for 10 min and postfixed with 2% tetraoxide osmium for 1 h. They were washed twice in two baths of cacodylate buffer for 5 min each.

[0460] Dehydration. Tissue dehydration was performed as follows: 50% ethanol for 10 min, 70% ethanol for 10 min. 90% ethanol for 15 min. 100% ethanol2 changes for 15 min each, propylene oxide3 changes for 15 min each.

[0461] Resin embedding. Resin infiltration was performed using a succession of a 1:2 mix of resin:propylene oxide for 30 min, a 1:1 mix of resin:propylene oxide for 30 min, and a 2:1 mix of resine:propylene oxide for 30 min. All samples were placed in fresh resin at ambient temperature overnight or at 4 C. for 1-2 days. Polymerization was performed in an oven at 60 C. for 48 h.

[0462] Sample (block) cutting. All blocks were cut as 70 nm-thin sections using a cryo-ultramicrotome (model Reichert Ultracut S) mounted with a diamond knife (Diatome). The sections were placed on the dark side of a holey carbon-coated copper grid (Delta Microscopies).

[0463] Post-staining. The grids were floated in a saturated solution of uranyle acetate in 50% ethanol for 25 min at room temperature and rinsed briefly twice over distilled water.

[0464] They were left on a drop of lead citrate for 10 min, washed twice with 0.1 mol.Math.L.sup.1 sodium hydroxyde and finally twice with distilled water.

[0465] Results.

[0466] So far, evidence of the fluo@mag@polymer nanoassemblies' uptake by liver macrophages was demonstrated by in vivo MRI and fluorescence histology fluorescence microscopy experiments. In order to gain higher spatial resolution in the imaging of fluo@mag@polymer nanoassemblies, transmission electron microscopy measurements were performed on ultra-thin slices of liver (70 nm-thick). TEM imaging clearly demonstrated the integrity of the administrated nanoassemblies in mice through intraveinous injections since the raspberry-like structure of the nanoassemblies is kept integer (FIG. 11). This important observation proves the high cohesion between the magnetic shell and the fluorescent core after in vivo injection, allowing for accurate multimodal cross diagnostics.

[0467] Part CFunctionalized Nanoassemblies

[0468] C.1. Folic Acid Functionalization

[0469] The fluo@mag nanoassemblies may be coated with biologically-active molecules like folic acid (FA) speeding-up the cell internalization cell internalization of nanoparticles. Folic acid was chosen for its complexing carboxylic unit toward the external layer of iron oxide nanoparticles.

[0470] Complexation of the bimodal fluo@mag nanoassemblies with FA could be realized provided that the resulting nanoassemblies fluo@mag@FA are stored in the dark at 4 C. Steady-state and time-resolved fluorescence measurements performed on the resulting architecture fluo@mag@FA confirmed the tight FA grafting at the surface of the nanoassemblies.

[0471] Synthetic Procedure.

[0472] A solution of fluorescent compound (Ib) dissolved in THF (50 L, 0.1 wt. %) was added under vigorous stirring to a solution of maghemite nanoparticles in nitric acid (2.5 mL, 0.006 wt. %, pH=1.2). After a few seconds, the magnetofluorescent fluo@mag were formed. Folic acid (>5 eq, 1 mg) was added to the mixture, followed by the dropwise addition of ammonium hydroxide (1 mol.Math.L.sup.1) under magnetic stirring until pH=8 was reached. The resulting translucent solution was allowed to stir for a further 30 min. and dialyzed using a Spectra Por membrane (Standard Grade Regenerated Cellulose; cut-off: 8-10 kDa) against Millipore water (600 mL) over 3-4 h to remove the excess of folic acid. Once the fluo@mag nanoparticles started agglomerating, the dialysis was performed in the presence of trisodium citrate salt (50 mmol.Math.L.sup.1) for a few hours. A final dialysis step was performed against Millipore water (600 mL) to remove the excess of citrate ions. The resulting functionalized fluo@mag@FA nanoassemblies were stored at 4 C. to avoid decomplexation of the folic acid units over time.

[0473] Photophysical Characterizations.

[0474] Complexation of FA on fluo@mag nanoparticles could be detected using steady-state and time-resolved fluorescence measurements. Strong emission fluo@mag quenching of the blue fluorescent FA was observed after complexation to the fluo@mag nanoparticles due to fast electron transfer from the FA excited state to the iron oxide nanoparticles. By contrast, no significant blue emission quenching could be detected when folic acid was simply mixed with a suspension of iron oxide -Fe.sub.2O.sub.3 due to partial complexation only. These results thus demonstrated the cooperative complexing behaviors of ensembles of iron oxide nanoparticles with regard to isolated maghemite nanoparticles. Compared to the tight PAA coating on fluo@mag nanoparticles, FA decomplexation however occurred over time. Detection of a strong blue signal thus features uncomplexed FA and has been harnessed to show the release of folic acid in live cells after incubation for 72 h. These results open the possibility to use the fluo@mag and fluo@mag@PAA nanoassemblies as drug cargos when properly functionalized.

[0475] Fluorescence Microscopy Imaging of Live Cells.

[0476] HEK cells were incubated with folic acid-coated fluo@mag nanoassemblies (fluo@mag@FA) and observed by wide-field fluorescence microscopy. The fluo@mag@FA were efficiently internalized in HEK cells. They provided blue emission after 72 h of incubation as a result of the decomplexation of folic acid whereas no such operates after 20 h only. Green emission instead of an orange one was related to the disassembling process of fluo@mag nanoassemblies since there was no PAA coating, known to ensure tight cohesion.

[0477] C.2. Aminocoumarin Functionalization

[0478] The fluo@mag@PAA nanoassemblies (1) could be covalently grafted with biologically-active molecules like aminocoumarine, serving as antiobiotics. The aminocoumarine displays an amino function, amenable to covalently bind the fluo@mag@PAA surface through amide bond formation.

[0479] The surface of nanoassemblies (1) was covalently linked to fluorescent probe 7-amino-4-methylcoumarin (AC) through amide bond formation. Steady-state and time-resolved fluorescence measurements performed on the resulting architecture fluo@mag@PAA-AC allowed to confirm the tight AC grafting at the surface of the nanoassemblies and showed the superiority of fluo@mag@PAA over fluo@mag in terms of surface binding stability of biologically active molecules. Such a functionalization represents the prelude of further grafting of more complex active biomolecules.

[0480] Synthetic Procedure.

[0481] Dilute nitric acid (pH=1.2) was added to a solution of fluo@mag@PAA assemblies, dispersed in Millipore water (0.7 mole Fe, <1 mole PAA, 2.5 mL) until pH=4 was reached. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (10 eq. 1.4 mg, 10 mol) and N-hydroxysuccinimide (10 eq, 1.1 mg, 10 mol) were added to the mixture under vigorous stirring using a vortex. The nanoassemblies were stirred for an extra 2 h before adding ammonium hydroxide (1 mol.Math.L.sup.1) until pH=9 was reached. Then, 7-amino-4-methylcoumarin (40 eq, 7.0 mg, 40 mol) was added to the nanoparticle dispersion and the resulting solution was stirred for a further 16 h. The resulting translucent solution was dialyzed using a Spectra Por membrane (Standard Grade Regenerated Cellulose; cut-off: 8-10 kDa) against Millipore water (600 mL) over 72 h until no more blue fluorescent emission was observed in the dialysis bath. The functionalized fluo@mag@PAA-AC nanoassemblies were stored at 4 C.

[0482] Photophysical Characterizations.

[0483] Steady-state and time-resolved fluorescence experiments were also performed to characterize the surface grafting of 7-amino-4-methylcoumarin, displaying a fluorescence signal at 441 nm (FIG. 12). Strong emission quenching was detected (decrease in the fluorescence signal and fluorescence decay) for the AC immobilized on the surface of fluo@mag@PAA nanoassemblies, compared to water solutions of AC only. This quenching effect was ascribed to efficient energy transfer from the AC excited state to the fluorescent organic core due to spectral overlap between the AC emission spectrum and the fluorescent core's absorption spectrum. The fast component observed in the fluorescence decay for fluo@mag@PAA-AC stems from the close proximity of AC toward the iron oxide nanoparticles and consequently to the covalent binding of AC. (FIG. 12b). No such quenching effect was indeed observed when AC was immobilized on the surface of mag@PAA nanoparticles dispersed in water. This experiment definitely shows that covalent attachment of AC to the fluo@mag@PAA nanoassemblies has been achieved since the distance between an energy donor and an energy acceptor must be shorter than 10 nm for efficient energy transfer. Finally no leakage of the covalently linked AC was detected over a period of two weeks. This experiment represents a first step toward the bioconjugation of biologically-active objects proteins for targeted bioimaging and drug delivery.

[0484] C.3. Maleimide Functionalization

[0485] Bioconjugation of fluo@mag@polymer requires the introduction of reactive groups known to react efficiently and specifically with biological entities. Maleimide derivatives are especially interesting due to their specific reactivity with thiol groups, present in a minor amount, compared to amino groups, in proteins and antibodies. The introduction of maleimide derivatives proceeded through peptide coupling providing covalent amide linkage between the polymer shell and the maleimide units. The grafting of maleimide was evidenced through fluorescence measurements as the resulting emission spectra underwent a slight hypsochromic shift (.sub.max10 nm) of their emission maxima compared to those before coupling.

[0486] Maleimide Derivatives.

[0487] The synthesis of the maleimide derivatives is based on Richter et al (Chem. Eur. J. 2012, 18, 16708-16715). The following maleimide derivatives were coupled to fluo@mag@polymer nanoassemblies (1),

##STR00012## [0488] wherein x ranges from 1 to 10, preferably from 3 to 5.

[0489] Maleimide-Functionalized Ananoassemblies.

[0490] Dilute nitric acid (pH=1.2) was added to a solution of fluo@mag@polymer assemblies, dispersed in Millipore water (4 mole Fe, 10 mL) until pH=4 was reached. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (4.2 mg, 40 mol, 10 eq.) and N-hydroxysuccinimide (3.6 mg, 40 mol, 10 eq.) were added to the mixture under vigorous stirring using a vortex. The nanoassemblies were stirred for an extra 1 h before adding ammonium hydroxide (1 mol.Math.L.sup.1) until pH=9 was reached.

[0491] Then, a trifluoroacetic maleimide salt derivative, as presented herein above, (13.5 mg, 40 mol, 40 eq.), was added to the nanoparticle dispersion, and the resulting solution was stirred for a further 16 h. The resulting translucent solution was dialyzed using a SpectraPor membrane (Standard Grade Regenerated Cellulose; cut-off: 8-10 kDa) against Millipore water (600 mL) over 24 h. The functionalized nanoassemblies were stored at 4 C.

[0492] In order to avoid non-specific interactions, a mixture of maleimide derivatives and commercially available aminoPEG (from 10 to 3000 Da, preferably around 1500 Da) (1/1) could be coupled in the last step of the above reaction.

[0493] C.4 Biotin Functionalization

[0494] The fluo@mag@polymer were functionalized by covalent conjugation with biotin that is a very common ligand used in biology to complex various entities of biological interest coupled to streptavidin. As biotin and streptavidin form an affinity complex with a high stability constant (K.sub.d=10.sup.8), the following results demonstrate that fluo@mag@polymer functionalized with biotin can readily be used to recognize streptavidin-conjugated biological entities.

[0495] Procedure.

[0496] Dilute nitric acid (pH=1.2) was added to a solution of fluo@mag@polymer assemblies (1), dispersed in Millipore water (2 mole Fe, 5 mL) until pH=4 was reached. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (2.8 mg, 20 mol, 10 eq.) and N-hydroxysuccinimide (2.4 mg, 20 mol, 10 eq.) were added to the mixture under vigorous stirring using a vortex. The nanoassemblies were stirred for an extra 1 h before adding ammonium hydroxide (1 mol.Math.L.sup.1) until pH=9 was reached. Then, biotin-X-cadaverin (1 mg, 2 mol, 1 eq.) was added to the nanoparticle dispersion and the resulting solution was stirred for a further 16 h. The resulting translucent solution was dialyzed using a SpectraPor membrane (Standard Grade Regenerated Cellulose; cut-off: 8-10 kDa) against Millipore water (600 mL) over 24 h. The biotin-functionalized nanoassemblies were stored at 4 C.

[0497] In order to avoid non-specific interactions, a mixture of biotin-X-cadaverine and commercially available aminoPEG (from 10 to 3000 Da, preferably around 1500 Da) (1/1) can be coupled in the last step of above reaction.

[0498] Detection, Extraction and Quantification.

[0499] The possibility of bio-functionalized nanoassemblies to serve as sensing agents has been tested by performing the well-known biotin/streptavidin association. The biotin-functionalized nanoassemblies were lyophilized and then redispersed in appropriate buffer and streptavidin conjugated to Alexa Fluor 488 was added. After stirring for 1 h at room temperature, dialysis was performed to remove the excess of fluorescent streptavidin.

[0500] Fluorescence emission measurements of the dialyzed solution displayed a narrow emission signal around 525 nm typical of Alexa Fluor 488 attached to streptavidin, along with the broad red fluorescence characteristic of the core (>600 nm). This signal allowed to unambiguously prove the association of fluorescent streptavidin with the biotin attached to the nanoassembly. Assuming that the emission of fluorophore Alexa Fluor 488 did not change upon biotin-streptavidin association, an amount of about 100 biotins could be inferred from measuring the fluorescence signal intensity coming from Alexa Fluor 488.