STIMULI - OR BIO- RESPONSIVE COPOLYMERS, THE POLYMERSOMES COMPRISING THE SAME AND THEIR USE IN DRUG DELIVERY

Abstract

The present invention concerns amphiphilic copolymers that may be photo- or redox-cleavable and that may assemble into polymersomes. It also concerns their process of preparation and their use as ding carriers.

Claims

1. A photo- or redox-cleavable amphiphilic copolymer compound of formula (I): ##STR00028## Where: R.sub.1, R.sub.2, R.sub.3 R.sub.4 and R.sub.5 substitution groups on the pyridinium ring are chosen from H, OH, OMethyl, CN, NR.sub.2, NO.sub.2 halogen, or two adjacent R.sub.1, R.sub.2, R.sub.3 R.sub.4 and R.sub.5 are linked together to form one or more aromatic rings fused with the pyridinium ring to which they are attached so as to form an optionally substituted bi-cyclic quinolinium or optionally substituted fused tri-cyclic N-containing heteroaryl (e.g. acridinium), R.sub.6 is H or an alkyl group; R.sub.7 is O, or alkylene; R.sub.8 and R.sub.9 identical or different are independently chosen from H, alkyl, aryl or benzyl, preferably methyl or benzyl; Where the optional substituents are chosen from OH, OMethyl, CN, NO.sub.2, NR.sub.2, halogen; R represents H, phenyl or a methyl group; n.sub.1 and n.sub.3 are identical or different integers comprised between 0 and 5 defining the length of the spacer; n.sub.2 is an integer comprised between 34 and 80 denoting the polymerization degree of ethylene oxide in the hydrophilic block of the amphiphilic copolymer; n.sub.4 is an integer chosen between 18 and 40 denoting the polymerization degree of γ-benzyl-L-glutamate in the hydrophobic block of the amphiphilic copolymer; X.sup.− is a halide or a trifluoromethanesulfonate (OTf.sup.−), phosphate, sulfate, perchlorate or nitrate counterion.

2. The photo- or redox-cleavable amphiphilic copolymer compound of formula (I) according to claim 1 chosen from: ##STR00029## Where X.sup.− is a halide or a trifluoromethanesulfonate (OTf.sup.−), and n.sub.2 and n.sub.4 are defined as in claim 1.

3. An amphiphilic copolymer vesicle, also called polymersome comprising an amphiphilic bilayer, wherein the amphiphilic bilayer of said vesicle comprises a photo-cleavable or redox-cleavable amphiphilic copolymer compound according to claim 1.

4. The polymersome according to claim 3, wherein it further comprises a mineral, i.e. a metal or a semi-metal, or an oxide or other chalcogenide nanoparticle embedded into said polymersome membrane.

5. The polymersome according to claim 4 wherein said mineral, metal or semi-metal, oxide or chalcogenide is chosen with constituent from the periodic table of the elements with an atomic number greater than 21, such as a transition metal (d group), a noble metal, a metal alloy, a semi-metal, an alkali earth or a rare earth such as a lanthanide (f group).

6. The polymersome according to claim 4 wherein said mineral, metal or semi-metal, oxide or chalcogenide nanoparticle is an ultra-small iron oxide nanoparticle (USPIO), a superparamagnetic iron oxide nanoparticle (SPION), a very small iron oxide nanoparticles (VSION), a hafnium-oxide, an iron-bismuth or iron-platinum alloy, a gadolinium oxide, a dysprosium oxide, a bismuth selenide or bismuth telluride, silver, platinum, or a gold nanoparticle or atom cluster.

7. The polymersome according to claim 3 further comprising an active pharmaceutical ingredient (API), said API being encapsulated within either aqueous internal compartment or within hydrophobic membrane of said polymersome.

8. The polymersome according to claim 7, wherein said API is suitable for the treatment of cancers, inflammation, diabetes, bacterial and/or viral infections, orphan diseases.

9. An aqueous suspension comprising polymersomes according to claim 3.

10. A pharmaceutical composition or a medical device comprising an aqueous suspension according to claim 9.

11. A method for the image-guided treatment of a disorder comprising a contrast agent according to claim 16, said method comprising the systemic administration of said compound; the activation of the compound of formula (o) at the targeted site by either an external mean (irradiation, electromagnetic field . . . ) or by an endogenous signal thereby releasing the API at the targeted site; and optionally monitoring the distribution of the mineral, metal or semi-metal, oxide or chalcogenide embedded in polymersomes within the body by a non-invasive bio-imaging technique.

12. The method according to claim 11 wherein the activation is achieved by cleavage of the polymersome membrane induced by UV light, visible photon (photodynamic), ionizing (beta ray, X-ray or gamma) irradiation, ultrasound or endogenous redox activation at the targeted site in the body.

13. The method according to claim 11 wherein: said disorder is a cancer, immune-related diseases, inflammation, diabetes, bacterial and/or viral infections, pediatric or age-related diseases, the polymersome encapsulates an active pharmaceutical ingredient (API), the API is chosen from APIs suitable for the treatment of cancer, immune-related diseases, inflammation, diabetes, bacterial and/or viral infections, pediatric or age-related diseases, such as Sorafenib® Doxorubicin, Monomethyl auristatin E (MMAE), cisplatin, or Combretastatin®, and the activation is carried out by beta rays, X rays or gamma irradiation, or by exploiting exogenous or endogenous redox signals such as redox enzymes, metalloproteases, reactive oxygen species (ROS) like peroxides or reactive nitrogen species (NO⋅).

14. Process of preparation of the copolymer according to claim 1 comprising the step of reacting a compound of formula (II): ##STR00030## with a compound of formula (III)°: ##STR00031## Wherein R.sub.1, R.sub.2, R.sub.3 R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R, X, n.sub.1, n.sub.2 n.sub.3 and n.sub.4 are defined as in anyone of claims 1 to 2.

15. Process of preparation of the suspension of polymersomes according to claim 3 comprising: Optionally mixing a compound of formula (I) with a hydrophobically coated mineral, metal or semi-metal, oxide or chalcogenide nanoparticle suspension and/or an API in an organic solvent miscible with water, Self-assembly by nanoprecipitation of the polymersome vesicles in an aqueous solution (also called “solvent-shift” method), optionally followed by dialysis of the suspension or heating to remove any trace of organic solvent.

16. A contrast agent for a bio-imaging modality, optical endoscopy, ultrasound echography, magnetic resonance imaging (MRI), X-ray scanner also called computed tomography (CT) comprising a photo-cleavable or redox-cleavable amphiphilic copolymer compound according to claim 1 combined with a drug carrier.

17. A contrast agent for a bio-imaging modality, optical endoscopy, ultrasound echography, magnetic resonance imaging (MRI), X-ray scanner also called computed tomography (CT) comprising a polymersome according to claim 3 combined with a drug carrier.

18. A method for the image-guided treatment of a disorder comprising a contrast agent according to claim 17, said method comprising: the systemic administration of said polymersome; the activation of the compound of formula (I) at the targeted site by either an external mean (irradiation, electromagnetic field . . . ) or by an endogenous signal thereby releasing the API at the targeted site; and optionally monitoring the distribution of the mineral, metal or semi-metal, oxide or chalcogenide embedded in polymersomes within the body by a non-invasive bio-imaging technique.

19. The method according to claim 17 wherein the activation is achieved by cleavage of the polymersome membrane induced by UV light, visible photon (photodynamic), ionizing (beta ray, X-ray or gamma) irradiation, ultrasound or endogenous redox activation at the targeted site in the body.

20. The method according to claim 17 wherein: said disorder is a cancer, immune-related diseases, inflammation, diabetes, bacterial and/or viral infections, pediatric or age-related diseases, the polymersome encapsulates an active pharmaceutical ingredient (API), the API is chosen from APIs suitable for the treatment of cancer, immune-related diseases, inflammation, diabetes, bacterial and/or viral infections, pediatric or age-related diseases, such as Sorafenib® Doxorubicin, Monomethyl auristatin E (MMAE), cisplatin, or Combretastatin®, and the activation is carried out by beta rays, X rays or gamma irradiation, or by exploiting exogenous or endogenous redox signals such as redox enzymes, metalloproteases, reactive oxygen species (ROS) like peroxides or reactive nitrogen species (NO⋅).

21. A contrast agent for a bio-imaging modality, optical endoscopy, ultrasound echography, magnetic resonance imaging (MRI), X-ray scanner also called computed tomography (CT) comprising an aqueous suspension according to claim 9 combined with a drug carrier.

22. A method for the image-guided treatment of a disorder comprising a contrast agent according to claim 21, said method comprising: the systemic administration of said suspension of polymersomes; the activation of the compound of formula (I) at the targeted site by either an external mean (irradiation, electromagnetic field . . . ) or by an endogenous signal thereby releasing the API at the targeted site; and optionally monitoring the distribution of the mineral, metal or semi-metal, oxide or chalcogenide embedded in polymersomes within the body by a non-invasive bio-imaging technique.

23. The method according to claim 21 wherein the activation is achieved by cleavage of the polymersome membrane induced by UV light, visible photon (photodynamic), ionizing (beta ray, X-ray or gamma) irradiation, ultrasound or endogenous redox activation at the targeted site in the body.

24. The method according to claim 21 wherein: said disorder is a cancer, immune-related diseases, inflammation, diabetes, bacterial and/or viral infections, pediatric or age-related diseases, the polymersome encapsulates an active pharmaceutical ingredient (API), the API is chosen from APIs suitable for the treatment of cancer, immune-related diseases, inflammation, diabetes, bacterial and/or viral infections, pediatric or age-related diseases, such as Sorafenib® Doxorubicin, Monomethyl auristatin E (MMAE), cisplatin, or Combretastatin®, and the activation is carried out by beta rays, X rays or gamma irradiation, or by exploiting exogenous or endogenous redox signals such as redox enzymes, metalloproteases, reactive oxygen species (ROS) like peroxides or reactive nitrogen species (NO⋅).

25. A contrast agent for a bio-imaging modality, optical endoscopy, ultrasound echography, magnetic resonance imaging (MRI), X-ray scanner also called computed tomography (CT) comprising a medical device according to claim 10 combined with a drug carrier.

26. A method for the image-guided treatment of a disorder comprising a contrast agent according to claim 25, said method comprising: the implantation of said medical device at a disorder site; the activation of the compound of formula (I) at the targeted site by either an external mean (irradiation, electromagnetic field . . . ) or by an endogenous signal thereby releasing the API at the targeted site; and optionally monitoring the distribution of the mineral, metal or semi-metal, oxide or chalcogenide embedded in polymersomes within the body by a non-invasive bio-imaging technique.

27. The method according to claim 25 wherein the activation is achieved by cleavage of the polymersome membrane induced by UV light, visible photon (photodynamic), ionizing (beta ray, X-ray or gamma) irradiation, ultrasound or endogenous redox activation at the targeted site in the body.

28. The method according to claim 25 wherein: said disorder is a cancer, immune-related diseases, inflammation, diabetes, bacterial and/or viral infections, pediatric or age-related diseases, the polymersome encapsulates an active pharmaceutical ingredient (API), the API is chosen from APIs suitable for the treatment of cancer, immune-related diseases, inflammation, diabetes, bacterial and/or viral infections, pediatric or age-related diseases, such as Sorafenib® Doxorubicin, Monomethyl auristatin E (MMAE), cisplatin, or Combretastatin®, and the activation is carried out by beta rays, X rays or gamma irradiation, or by exploiting exogenous or endogenous redox signals such as redox enzymes, metalloproteases, reactive oxygen species (ROS) like peroxides or reactive nitrogen species (NO⋅).

Description

DESCRIPTION OF THE FIGURES

[0097] FIG. 1 illustrates the determination of the half maximal inhibitory concentration (IC50) values on MCF-7 human breast cancer (A, C) and Ovar-3 carcinoma cell lines (B, D) for two anticancer APIs, combretastatin (A, B) and doxorubicin (C, D) either encapsulated in Quino-Ps polymersomes or as free API, showing increase of IC50 by 2 to 3 orders of magnitude.

[0098] FIG. 2 shows the relaxometric measurements, by using 7T MR images of USPIO-embedding polymersomes by increasing Fe concentrations (0 to 1 mM) in saline (0.9% NaCl). (a) T.sub.1 weighted (b) T.sub.2 weighted (c) T.sub.2* weighted MR Images.

[0099] FIG. 3 represents the MRI dynamic susceptibility contrast (DSC) images recorded in living mice at different time points to follow the kinetic uptake and clearance of the magnetic Pico-Ps(Fe) in liver, spleen, kidney, vessels, muscle; (n=3).

[0100] FIG. 4 represents the T.sub.2-weighted MR images of mice i. liver and ii spleen recorded a) before and b-d) after injection of Quino-Ps (Fe) (20% FWR) (a) 0 min (b) 40 min, (c) 48 h, (d) 7 days.

[0101] FIG. 5. Variation of the fluorescence by the treatment of Quino-Ps-DOX with added glutathione (GSH) (10 mM).

[0102] FIG. 6. Calibration curve for the quantification of the doxorubicin release by GSH.

[0103] FIG. 7. TEM analysis of Quino-Ps samples after irradiation by the ID17 biomedical beamline at European synchrotron radiation facility (ESRF, Grenoble, France) (conditions: Cuvette: quartz, d (light path): 1 mm, Beam energy: 120 keV, Bunch length: 48 ps, Bunch repetition rate 5.68 MHz, Dose: 30 Gy).

[0104] FIG. 8. Variation in hydrodynamic diameter (Zavg diameter and PDI) of Quino-Ps (A) and Quino-Ps (Fe) (B) as measured by DLS.

[0105] FIG. 9: In vitro fluorescence signal obtained with varying concentrations of PICO-GNP6Ps.

[0106] FIG. 10: In vivo blood fluorescence signal over time after injection of PICO-GNP6Ps in mice.

[0107] FIG. 11: In vivo fluorescence imaging of PICO-GNP6Ps.

[0108] FIG. 12: The fluorescence spectrum of GNP-modified picolinium NPs (λ.sub.ex=400 nm).

[0109] FIG. 13: The variation of the hydrodynamic size (Z.sub.avg) of GNP-modified picolinium NPs by DLS over 70 days.

Examples of Polymersomes According to the Invention (Therafter Abbreviated Ps)

[0110] 1. Chemical synthesis: preparation of picolinium and quinolinium derived redox probes, their incorporation as linkers between the two blocks of the block-copolymers Representative synthetic pathways are depicted in the schemes 1-4 below for representative compounds of formula (I) of the invention:

##STR00009##

##STR00010##

##STR00011##

(NB: n.sub.1 and n.sub.2 as used in Schemes 1-3 corresponds to n.sub.2 and n.sub.4 respectively in Formula (I)).

##STR00012##

Generals

[0111] Proton nuclear magnetic resonance (.sup.1H NMR) spectra and carbon nuclear magnetic resonance (.sup.13C NMR) spectra were recorded on a Bruker 250 spectrometer (250 MHz and 63 MHz) and on a Bruker AV-500 spectrometer (500 MHz and 125 MHz). Chemical shifts for protons are reported in parts per million (ppm) downfield from tetramethylsilane (TMS) and are referenced to residual proton in the NMR solvent (CDCl.sub.3: δ 7.26). Chemical shifts for carbon are reported in parts per million downfield from tetramethylsilane and are referenced to the carbon resonances of the solvent CDCl.sub.3 (δ 77.16). NMR spectra recorded in other solvent are indicated in the experimental part.

[0112] Data are represented as follows: chemical shift, integration, multiplicity, coupling constants in Hertz (Hz). The following abbreviations are used to set multiplicities: s=singlet, bs=broad singlet, d=doublet, dd=double doublet, ddd=double double doublet, t=triplet, m=multiplet, br m=broad multiplet, bb=broad band.

[0113] All solvents and inorganic reagents were from commercial sources and used without purification unless otherwise noted. Thin layer chromatography (TLC) was performed on aluminium-backed Merck Kieselgel 60 F254 precoated plates. The high-performance liquid chromatography (HPLC) analyses were carried out on Waters device 600 with a normal inverse phase column XTerra®_MS C18 (length: 75 mm, diameter: 4.6 mm, stationary phase: 2.5 μm) using a Waters 2487

[0114] Dual Absorbance Detector (254-365 nm) and an isocratic/linear gradient system of elution (Methanol-ACN—H.sub.2O 7-2-1/H.sub.2O—AcONH.sub.4 10 mM pH 4.6). The volume of injection was 10 μL. The mass analyser was an Agilent from ThermoFisher. The capillary tension was 3.5 kV. The cone tension was 24 V. The temperature of the source was 130° C. and the temperature of desolvatation was 350° C. Data were treated on ThermoQuest.

Synthesis of N-Methyl Picolinium Esters

General Procedure for the Synthesis of Picoline Methyl-Alcohols.

[0115] To a solution of the corresponding pyridine carboxaldehydes (3 mmol) in methanol at 0° C. was added sodium borohydride (1.2 eq.). The mixture was stirred at room temperature (RT) for 1 h before being quenched with HCl (1 M). The solution was then filtered on celite, washed with DCM and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (DCM/MeOH 9/1) to obtain the corresponding alcohol.

Pyridin-2-ylmethanol

[0116] ##STR00013##

[0117] Yellow oil (320 mg, 98%).

[0118] Chemical formula: C.sub.6H.sub.7NO Molecular Weight: 109.12 g.Math.mol.sup.−1

[0119] .sup.1H NMR (CDCl.sub.3, 250 MHz): δ 8.88-8.54 (1H, m), 7.69 (1H, m), 7.29-7.18 (2H, m), 4.75 (2 H, s), 3.16 (1H, bs,).

[0120] .sup.13C NMR (CDCl.sub.3, 250 MHz): δ 159.6, 148.4, 136.9, 122.3, 120.8, 64.3.

[0121] MS (ESI): m/z=110.0 [M+H].sup.+.

Preparation of 5-bromopent-1-yne

[0122] ##STR00014##

[0123] Pent-4-yn-1-yl 4-methylbenzenesulfonate 29: In a solution of pent-4-yn-1-ol (1 eq.) in DCM (10 v), was added triethylamine (2.0 eq) and the solution stirred at 0-5° C. for 10 minute, followed by the addition of tosyl chloride (1.2 eq.) at this temperature. The reaction was left to stir for 10 to 15 h at room temperature. The progress was monitored by TLC in EtOAc/cyclohexane (20:80). The reaction was quenched by the addition of water (20 mL) and the product extracted with DCM 2 to 3 times, dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Solid mass obtained (off white; 95%).

[0124] .sup.1H NMR (CDCl.sub.3, 250 MHz): δ 7.82 (d, 2H), 7.38 (d, 2H), 4.18 (t, 2H), 2.46 (s, 3H), 4.77-4.74 (t, 2H), 2.47 (s, 1H), 2.30-2.24 (dd, 2H), 1.91-1.89 (t, 1H), 1.88-1.82(q, 2H).

5-Bromopent-1-yne

[0125] ##STR00015##

[0126] To a solution of pent-4-yn-1-yl 4-methylbenzenesulfonate (1.0 eq.) in DMSO (8 v) was added LiBr (5.0 eq) (exothermicity observed). The reaction was stirred overnight at room temperature. The reaction was quenched by the addition of water (100 mL), followed by stirring for 10 minutes. The product was extracted using DCM 2 to 3 times, dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Oily mass obtained (colorless; 72% yield).

[0127] .sup.1H NMR (CDCl.sub.3, 250 MHz): 3.59-3.53 (t, 2H), 2.45-2.39 (dd, 2H), 2.13-2.05 (q, 2H), 2.03-2.00 (t, 1H).

Preparation of pent-4-yn-1-yl trifluoromethanesulfonate

[0128] ##STR00016##

[0129] To a solution of pent-4-yn-1-ol (1 eq.) in DCM (10 v), was added pyridine (1.2 eq.) and stirred at 0-5° C. for 5 minutes under inert atm., followed by the addition of triflic anhydride (1.2 eq) at the same temperature. The reaction was stirred for 2 h at room temperature. The reaction was quenched by the addition of water (5 mL) and the product extracted with DCM 2 to 3 times, dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Light brown semi solid 81%).

[0130] .sup.1H NMR (CDCl.sub.3, 250 MHz): 4.62-4.56 (t, 2H), 2.78-2.72 (dd, 2H), 2.30-2.24 (dd, 2H), 2.18-2.12 (m, 3H).

Quaternarization of Various Hydroxymethyl Picolines

2-(Hydroxymethyl)-1-(pent-4-yn-1-yl) pyridin-1-ium

[0131] ##STR00017##

[0132] To a solution of hydroxymethyl picoline (500 mg, 1.00 eq.) in ACN (3 mL), pent-4-yn-1-yl triflate (936 mg, 1.4 equiv.) was added and the solution was stirred at 60-70° C., overnight. Solvent was concentrated in vacuo at 40° C. and the crude product was purified by column chromatography using silica gel (DCM: MeOH). Light brown semi solid, 91%.

[0133] Chemical Formula: C.sub.11H.sub.14NO.sup.+ Molecular Weight: 176.24 g.Math.mol.sup.−1

[0134] .sup.1H NMR (MeOD, 500 MHz): δ 8.90 (d, J=6.3 Hz, 1H), 8.56 (td, J=7.9, 1.5 Hz, 1H), 8.25- (d, J=8.4 Hz, 1H), 7.98 (ddd, J=7.7, 6.2, 1.6 Hz, 1H), 5.03 (s, 2H), 4.68-4.65 (m, 2H), 2.40-2.37 (m, 3H), 2.20-2.14 (m, 2H).

General Method for the Synthesis of Azido Poly(γ-Benzyl-L-Glutamate)

Synthesis of PBLG.SUB.21.-N.SUB.3

[0135] ##STR00018##

[0136] In a glove-box, γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) (2 g, 7.6 mmol) was dissolved in dry DMF (20 mL). The flask was maintained under argon and brought to the fume-hood, where 1-azido-3-aminopropane (36 μL, 0.362 mmol) was added under argon. The reaction mixture was stirred for 18 h at 40° C. The polymer was precipitated by slowly adding the reaction mixture into cold Et.sub.2O (200 mL). The precipitate was filtered, washed with cold Et.sub.2O, and dried in vacuo to provide PBLG.sub.21-N.sub.3 (1.44 g, 86% yield) as a white powder.

[0137] .sup.1H NMR (90/10 CDC.sub.3/TFA, 400 MHz): δ 7.98-7.80 (m, 21H, amide-NH), 7.41-7.22 (m, 105H, aromatic-CH), 5.17-5.05-(m, 42H, Bz-CH.sub.2), 4.65-4.55 (m, 21H, α-CH), 3.40-3.35 (m, 4H, 7-CH.sub.2, 9-CH.sub.2), 2.58-2.39 (m, 42H, γ-CH.sub.2), 2.22-2.04 (m, 21H, β-CHH.sub.2), 2.00-1.86 (m, 21H, β-CHH), 1.82-1.77 (m, 2H, 8-CH.sub.2).

[0138] SEC: Ð=1.13.

Synthesis of PBLG-N.SUB.3 .of Different DPs (PBLG.SUB.20.-N.SUB.3., PBLG.SUB.21.-N.SUB.3., PBLG.SUB.26.-N.SUB.3 .and PBLG.SUB.33.-N.SUB.3.)

[0139] Ring opening polymerization (ROP) of γ-benzyl-L-glutamate N-carboxyanhydride was performed using a 1-azido-3-aminopropane as an initiator to afford α-azido poly(γ-benzyl-L-glutamate) (PBLG) (Scheme 5). Initiator 1-azido-3-aminopropane was synthesized as previously reported (Agut et al, Macromol. Rapid Commun. 2008, 29, 1147-1155). PBLG-N.sub.3 polymers of different degrees of polymerization (DPs) were synthesized by ROP in DMF, the reaction being carried for 18 h out at 40° C. For example, for PBLG.sub.21-N.sub.3, γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) (2 g, 7.6 mmol) was dissolved in dry DMF (20 mL) inside a glove-box to protect the NCA from moisture. The flask was maintained under argon and brought to the fume-hood, where 1-azido-3-aminopropane (36 μL, 0.362 mmol) was added under argon. The reaction mixture was stirred for 18 h at 40° C. The polymer was precipitated by slowly adding the reaction mixture into cold Et.sub.2O (200 mL). The precipitate was filtered, washed with cold Et.sub.2O, and dried in vacuo to provide PBLG.sub.21-N.sub.3 (1.44 g, 86% yield) as a white powder.

[0140] .sup.1H NMR (90/10 CDC.sub.3/TFA, 400 MHz): δ 7.98-7.80 (m, 21H, amide-NH), 7.41-7.22 (m, 105H, aromatic-CH), 5.17-5.05-(m, 42H, Bz-CH.sub.2), 4.65-4.55 (m, 21H, α-CH), 3.40-3.35 (m, 4H, 7-CH.sub.2, 9-CH.sub.2), 2.58-2.39 (m, 42H, γ-CH.sub.2), 2.22-2.04 (m, 21H, β-CHH), 2.00-1.86 (m, 21H, β-CHH), 1.82-1.77 (m, 2H, 8-CH.sub.2). SEC: Ð=1.13. Different DPs were obtained by varying the ratio of monomer to initiator. This provided PBLG.sub.20-N.sub.3, PBLG.sub.21-N.sub.3, PBLG.sub.26-N.sub.3, and PBLG.sub.33-N.sub.3 (Table A). The DPs were determined by .sup.1H NMR spectroscopy in 90:10 CDC.sub.3:trifluoroacetic acid (TFA), based on the integrations of the peaks at ˜3.35 ppm and ˜4.59 ppm, where the peak at ˜3.35 was integrated to 4 as it corresponds to the methylene groups α—to the amide and azide of the initiator, and the peak at ˜4.59 corresponds to the α-carbon of benzyl glutamate. Size exclusion chromatography (SEC) in DMF was used to obtain the molar mass dispersity (Ð) of 1.05-1.13. SEC chromatograms exhibited a bimodal distribution: Such bimodality for polypeptide chains is usually observed and ascribed to coexistence of helical and random coil structures in solution (Lecommandoux et al, Macromol. 2001, 34, 9100-9111; Huesmann et al Macromol. 2014, 47, 928-936), thus PBLG likely undergoes similar conformational equilibrium, explaining bimodality in SEC.

##STR00019##

TABLE-US-00001 TABLE A PBLG-N3 (.sup.a determined by NMR, .sup.b determined by SEC) Entry PBLG.sub.n-N.sub.3 DPa Mn (g/mol)a D.sup.b 1 PBLG.sub.20-N.sub.3 20 4485 1.05 2 PBLG.sub.21-N.sub.3 21 4704 1.13 3 PBLG.sub.26-N.sub.3 26 5800 1.10 4 PBLG.sub.33-N.sub.3 33 7333 1.10

MeO-PEG.SUB.43.-COOH

[0141] ##STR00020##

[0142] Polyethylene glycol monomethyl ether (2 kg.Math.mol.sup.−1) (5 g, 2.5 mmol), TEMPO (2 mg, 0.125 mmol) and KBr (30 mg, 0.25 mmol) were dissolved in 60 mL of DI H.sub.2O. An 8% sodium hypochlorite solution (3 mmol sodium hypochlorite/mmol primary alcohol) was added, the pH of the reaction mixture was adjusted to pH ˜10 and the reaction left to stir for 18 h. TLC showed 50% completion. EtOH (5 mL) was added and the pH reduced to pH ˜3, the mixture of product and starting material was extracted with DCM (×5) dried over MgSO.sub.4, filtered, concentrated in vacuo and precipitated in Et.sub.2O.

[0143] The precipitate was subjected to the above procedure with the same quantities of TEMPO, KBr, NaOCI and H.sub.2O. The reaction was left for 48 h, TLC showed no starting material. EtOH (5 mL) was added and the pH reduced to pH ˜3, extracted with DCM (×5) dried over MgSO.sub.4, filtered, concentrated in vacuo and precipitated in Et.sub.2O to give the product MeO-PEG.sub.43-COOH (4.3 g, 86%).

[0144] .sup.1H-NMR (CDCl.sub.3, 400 MHz): δ 4.15 (s, 2H, CHr-COOH), 3.76-3.73 (m, 2H, PEG-CH.sub.2), 3.69-3.62 (m, 168H, PEG-C.sub.2H.sub.4), 3.55-3.53 (m, 2H, PEG-CH.sub.2), 3.37 (s, 3H, CH.sub.3O).

General Method for the Synthesis of Pico-PEG

[0145] MeO-PEG-COOH (1.15 eq.) and linker (1 eq.) were dissolved dry DCM. EDC-HCl (2 eq) and DMAP (0.2 eq) were dissolved DCM and this was added to the reaction mixture and left to stir for 48 h. The reaction mixture was then precipitated in Et.sub.2O, the precipitate was dried, re-dissolved in DCM and washed with brine. The product was extracted from brine with DCM (×5), dried over MgSO.sub.4, filtered, concentrated in vacuo, dissolved in minimum acetone and precipitated in Et.sub.2O to give the product Pico-PEG (79%-99%).

Pico-PEG

[0146] ##STR00021##

[0147] MeO-PEG.sub.43-COOH (512 mg, 0.2604 mmol) and picolinium linker (58 mg, 0.2264 mmol) were dissolved 8 mL dry DCM. EDC.HCl (87 mg, 0.453 mmol) and DMAP (6 mg, 0.0453 mmol) were dissolved 2 mL dry DCM. (454 mg, 92%).

[0148] .sup.1H-NMR (CDCl.sub.3, 400 MHz): δ 10.25 (dd, J=6.7, 1.1 Hz, 1H, A-aromatic CH), 8.49 (td, J=7.9, 1.1 Hz, 1H, C—CH), 8.13-8.09 (m, 2H, B—CH, D-CH), 5.73 (s, 2H, 5-CH.sub.2), 5.21 (t, J=7.7 Hz, 2H, 4-CH.sub.2), 4.33 (s, 2H, 6-CH.sub.2), 3.76-3.73 (m, 2H, PEG-CH.sub.2), 3.60-3.70 (m, 168H, PEG-C.sub.2H.sub.4), 3.55-3.53 (m, 2H, PEG-CH.sub.2), 3.37 (s, 3H, OCH.sub.3), 2.52 (td, J=6.4, 2.6 Hz, 2H, 2-CH.sub.2), 2.35-2.31 (m, 2H, 3-CH.sub.2), 2.06 (t, J=2.6 Hz, 1H, 1-H).

Block copolymer PEG.SUB.43.-Pico-b-PBLG.SUB.21

[0149] ##STR00022##

[0150] Pent-4-yn-1-yl picolinium triflate (232 mg, 0.1063 mmol) and PBLG-N.sub.3 (200 mg, 42.5 μmol) were dissolved in dry THF (7 mL). Cul (8 mg, 42.5 μmol) and DIPEA (15 μL, 85 μmol) were added sequentially and the reaction mixture was left to stir under argon for 18 h. The reaction mixture was concentrated and purified by silica gel column chromatography (MeOH: DCM, v:v, 1:50.fwdarw.3:20). The product was re-dissolved in minimum DCM and precipitated in Et.sub.2O. The precipitate was dried in vacuo to give the product (210 mg, 71%).

[0151] .sup.1H-NMR (CDCl.sub.3, 400 MHz;): δ 8.35-8.17 (m, 19H, amide-NH). 7.34-7.16 (m, 105H, Bz-aromatic-CH), 5.14-4.95 (m, 46H, Bz-CH.sub.2, 4-CH.sub.2, 5-CH.sub.2), 3.98-3.80 (m, 23H, α-CH, 6-CH.sub.2), 3.79-3.46 (m, 174H, PEG-C.sub.2H.sub.4, 7-CH.sub.2), 3.38 (s, 3H, OCH.sub.3), 3.31-3.35 (m, 2H, 9-CH.sub.2), 2.79-2.01 (broad m, 88H, 2-CH.sub.2, 3-CH.sub.2, R—CH.sub.2, γ-CH.sub.2), 1.86-1.82 (m, 2H, 8-CH.sub.2).

[0152] SEC: Ð=1.09.

Synthesis of N-methyl Quinolinium Esters

General Procedure for the Synthesis of Quinoline Aldehydes

[0153] The appropriate quinoline (1.0 eq.) was introduced to a suspension of selenium dioxide (1.3 eq.) in dioxane, and the mixture was heated at 80° C. for 3 h. After cooling the mixture to rt, the solid was filtered on celite, washed with EtOAc and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (Cyclohexane/EtOAc: 4/1) to obtain the corresponding aldehyde as an orange-red solid.

Quinoline-2-carbaldehyde

[0154] ##STR00023##

[0155] Following general procedure, 2 g (13.96 mmol) of quinaldine was reacted with 2.013 g (18.14 mmol) of selenium dioxide to obtain quinoline-2-carbaldehyde (2.01 g, 92%) Chemical formula: C.sub.10H.sub.7NO Molecular Weight: 157.17 g.Math.mol.sup.−1

[0156] .sup.1H NMR (CDCl.sub.3, 500 MHz): δ 10.24 (s, 1H), 8.32 (d, J=8.5 Hz, 1H), 8.26 (d, J=8.5 Hz, .sup.1H), 8.04 (d, J=8.5 Hz, 1H), 7.91 (d, J=8 Hz, 1H), 7.83 (t, J=10 Hz, 1H), 7.70 (t, J=7 Hz, 1H).

[0157] .sup.13C NMR (CDCl.sub.3, 125 MHz): δ 193.9, 152.8, 148.1, 137.5, 130.63, 130.6, 130.2, 129.4, 128.0, 117.5.

[0158] MS (ESI): m/z=158.1 [M+H].sup.+, 189.9 (hemiacetal).

General Procedure for the Synthesis of Quinoline Methyl-Alcohols

[0159] To a solution of the corresponding aldehydes (1.0 eq.) in methanol at 0° C. was added sodium borohydride (1.2 eq.). The mixture was stirred at rt for 1 h before being quenched with HCl 1M. Water was added and the product was extracted with DCM. The organic layer was washed twice with water and brine, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The product obtained was used without further purification.

Quinolin-2-ylmethanol

[0160] ##STR00024##

[0161] Following general procedure, 0.8 g (5.08 mmol) of quinolone carbaldehyde was reacted with 0.230 g (6.1 mmol) of sodium borohydride to obtain quinolin-2-ylmethanol (0.77 g, 95%).

[0162] Chemical formula: C.sub.10H.sub.9NO Molecular Weight: 159.19 g.Math.mol.sup.−1

[0163] .sup.1H NMR (CDCl.sub.3, 500 MHz): δ 8.14 (d, J=8.5 Hz, 1H), 8.08 (d, J=8.5 Hz, 1H), 7.82 (d, J=8 Hz, 1H), 7.73 (m, 1H), 7.54 (m, 1H), 7.28 (d, J=8.5 Hz, 1H), 4.92 (s, 2H). .sup.13C NMR (CDCl.sub.3, 125M Hz): δ 159.1, 146.9, 137.0, 130.0, 128.8, 127.83, 127.80, 126.5, 118.5, 64.3.

General Procedure for the Synthesis of Brominated Quinolines

[0164] Aqueous HBr (49%) (10 mL) was directly poured into appropriate quinoline alcohols, and the mixture was heated up to reflux for 10-12 h. Solvents were evaporated under reduced pressure and the crude was dried under vacuum in the presence of P.sub.2O.sub.5 to remove trace of water; Brown solid.

2-(Bromomethyl) quinoline hydrogen bromide

[0165] ##STR00025##

[0166] Following general procedure, 10 mL of aqueous hydrogen bromide was added to 1.0 g (6.0 mmol) of alcohol to obtain the desired halogenated compound (1.9 g, quant.).

[0167] Chemical Formula: C.sub.10H.sub.8BrN Molecular Weight: 222.09 g.Math.mol.sup.−1

[0168] .sup.1H NMR ((CD.sub.3).sub.2SO, 500 MHz): δ: 9.18 (d, J=8.27 Hz, 1H), 8.84 (d, J=8.8 Hz, 1H), 8.26 (d, J=10.2 Hz, 1H), 8.04 (d, J=8.7 Hz, 1H), 7.85 (dd, J=8.0, 1.6 Hz, 1H), 7.77 (m, 1H) 5.12 (s, 1H).

[0169] .sup.13C NMR ((CD.sub.3).sub.2SO, 500 MHz): δ 161.1, 159.5, 146.3, 138.8, 134.6, 132.5, 128.1, 127.5, 127.1, 67.1.

General Procedure for Quinolinium PEG Esters

[0170] The brominated quinoline (1.2 eq.) was dissolved into DMF in the presence of K.sub.2CO.sub.3 (2.5 eq.), MeO-PEG.sub.43-COOH (1.0 eq.) was introduced into this solution & mixture was heated at 40° C. for 24-32 h. After cooling the mixture to rt, brine solution was added to the reaction mixture washed with EtOAc (2 to 3 times) and concentrated under reduced pressure. The oily mass was further precipitated by diethyl ether to obtain the corresponding off-white solid.

Quinoline 2-hydroxymethylene PEG Ester

[0171] ##STR00026##

[0172] Following the general procedure, 2.0 g (1 mmol) of MeO-PEG.sub.43-COOH was reacted with the bromo-compound (0.290 g, 1.3 mmol) in the presence of 0.414 g K.sub.2CO.sub.3 (3 mmol) to obtain the desired ester (1.7 g, 85%).

[0173] .sup.1H NMR (CDCl.sub.3, 500 MHz): δ 8.22 (d, J=8.2 Hz 1H), 8.09 (d, J=8.2 Hz 1H), 7.85 (d, J=8.5 Hz 1H), 7.76-7.71 (dd, J=9.4, 6.5 Hz 1H), 7.60-7.54 (t, J=8.24 Hz 1H), 7.5-7.48 (d, J=8.5 Hz 1H), 5.49 (s, 2H), 4.33 (s, 2H), 3.66 (s, 121H, —CH.sub.3 mPEG.sub.2000), 3.39 (s, 3H, —OCH.sub.3 mPEG.sub.2000). .sup.13C NMR (CDCl.sub.3, 500 MHz): δ 174.4, 170.2, 155.6, 147.6, 137, 129.9, 129.2, 127.6, 126.7, 119.5, 71.9, 70.6 (m-PEG-CH.sub.2), 69.7, 67.5, 59.

General Procedure for Preparation of Block Co-Polymers

[0174] Quinoline-PEG esters (1 eq) and pent-4-yn-1-yl trifluoromethanesulfonate (1.2 eq) were dissolved in DCM and stirred at room temperature for 72 h. The mixture was evaporated after that these products used in situ as for preparation of block co polymers. Block co-polymers were synthesized. Alkyne derivatives (1 eq.) and PBLG-N.sub.3 (2.5) were dissolved, respectively, in dry THF (7 mL). Cul (1 eq.) and DIPEA (2 eq.) were added sequentially and the reaction mixture was left to stir under argon for 24-36 h. The reaction mixture was concentrated and redissolved in DCM, this solution was washed by aqueous solution of EDTA (10%) at least two times. The organic solution was dried over Na.sub.2SO.sub.4 and concentrated the mass under vacuum. The desired product was precipitated in diethyl ether. Off-white semi solid mass was obtained (60-75%).

Block Co-Polymer (Quino)

[0175] ##STR00027##

[0176] 100 (130 mg, 55 μmol) and PBLG.sub.20-N.sub.3/PBLG.sub.21-N.sub.3 (100 mg, 22 μmol) were dissolved in dry THF (3.5 mL). Cul (8.3 mg, 4 μmol) and DIPEA (4.2 μL, 5 μmol) were added sequentially and the reaction mixture was left to stir under argon for 24-36 h. Semi-solid off-white color 97.5 mg (75/72%) compounds obtained respectively.

[0177] .sup.1H NMR (CDCl.sub.3 500 MHz): δ

Hydrophobically Coated Very Small Iron Oxide Nanoparticles (VSION)

[0178] Very small iron oxide NPs (VSIONs) of diameters around 4 nm were synthesized by the polyol route with slight modifications to control the nucleation-growth process through the water content of the solvent mixture, as reported in Hemery et al Inorganic Chemistry, 2017, 56(14), 8232-8243. Namely 1.082 g (4 mmol) of FeCl.sub.3.6H.sub.2O and 0.398 g (2 mmol) of FeCl.sub.2-4H.sub.2O were dissolved in 80 g of diethylene glycol (DEG). Separately, 0.64 g (16 mmol) of NaOH pearls was dissolved in 40 g of DEG. Both solutions were stirred overnight under nitrogen flux to prevent oxidation of the Fe(II) species. The solutions were then mixed and stirred for 3 h, before heating the mixture at 210° C. with an oil bath, reflux set-up and mechanical agitation. At first the set-up was open and a flux of nitrogen helped to remove traces of water. When a temperature of 210° C. was reached the set-up was closed, and hot injection of 2.5 mL water (137 mmol) in the vessel through a septum led to a burst of nuclei. The formation of nanoparticles through forced hydrolysis pathway was carried out for 30 min, before letting the system cool down to RT by removing the heating plate. The black sediment was separated magnetically and washed 3× with a mixture of EtOH and EtOAc (1:1 v/v). Possibly present non-magnetic iron oxohydroxides were removed by treatment with 10% nitric acid. 8.6 g of Fe(NO.sub.3).sub.3.9H.sub.2O was then added to the solution as a strong oxidant by heating at 80° C. for 45 min while mechanically stirring. The solution then turned from clear black (magnetite Fe.sub.3O.sub.4) to red (maghemite γ-Fe.sub.2O.sub.3). The IONPs were then washed 2 times with acetone and 2 times with Et.sub.2O before being dispersed in water. The morphology of nanoparticles was investigated by TEM and automated particle sizing led to a diameter range of 4.3±1.1 nm.

[0179] The commercial dispersing agent Beycostat™ NE (NB09, CECA, Puteaux, France) was used to coat the surface of the VSIONs and render them hydrophobic. This surfactant is a mixture of mono- and di-esters of phosphoric acid with poly(ethylene oxide).sub.9 nonylphenyl ether chains. To a mixture of 2.55 mL acidic aqueous VSION dispersion at 18.0 g/L iron oxide (45 mg solid content) and 235 mg of BNE (liquid), 6.5 mL of a 2 M HNO.sub.3 solution was added and the reaction stirred vigorously (400 rpm) with a mechanical stirrer at 60° C. for ˜15 minutes. The VSIONs were settled over a permanent magnet and the supernatant pipetted out. The BNE-coated VSIONs were washed 5× with 10 mL MeOH, before being dispersed in 2.5 mL of THF. The iron oxide content of this dispersion was estimated by dissolving 50 μL of suspension in 5 mL of HCl 5M with help of a sonication bath. The absorbance (optical density) at 350 nm was converted through predetermined calibration, OD.sub.350 nm, 2 mm=0.5043×[Fe].sub.mM+0.0172, into a concentration of 0.91 g/L γ-Fe.sub.2O.sub.3 in THF. DLS measurement with multi-modal (CONTIN) analysis showed peaks at 7.5 and 37.5 nm.

[0180] 2. Self-Assembly Process

Preparation of Self-Assembled Structures for Morphology Studies

Nanoprecipitations of Picolinium Block Co Polymers:

[0181] Block copolymer PEG.sub.44-Pico-b-PBLG.sub.17: 900 μL of water was added fast (in one shot) to a solution (100 μL) of block co-polymer (1 mg) in DMSO. This was dialyzed (3.5 kDa cutoff membrane) against ultrapure water for 18 h. DLS measurement led to Z-Avg=93 nm, PDI=0.17. Micelle morphology was observed by TEM.

[0182] Here Z-Avg represented Z-averaged diameters (2 times R.sub.h) determined by a 2.sup.nd order Cumulant fit of the DLS curves.

[0183] Block copolymer PEG.sub.43-Pico1-b-PBLG.sub.21: 4.5 mL of water was added fast (in one shot) to a solution (500 μL) of block copolymer Pico (5 mg) in THF. It was dialyzed (3.5 kDa cutoff membrane) against ultrapure water for 18 h. DLS measurement led to Z-Avg=143 nm, PDI=0.105. Polymersomes where formed, as evidenced by MALS (ALV ρ-ratio=1.1), TEM and Cryo-TEM images.

[0184] 2.67 mL of water was added quickly to a solution (330 μL) of block copolymer Pico (3.3 mg) in DMSO. The suspension was dialyzed (3.5 kDa) against ultrapure water for 18 h. DLS measurement led to Z-Avg=293 nm, PDI=0.261. Micelles were analyzed by TEM.

[0185] Nanoprecipitation with saline solution 2.7 mL of 0.9% aq. NaCl was added quickly to a solution (300 μL) of block copolymer Pico (3 mg) in THF. The suspension was dialyzed (3.5 kDa cutoff membrane) against 0.9% aq. NaCl for 18 h. Z-Avg=120 nm, PDI=0.118. Polymersomes were analyzed by TEM and MALS (ALV ρ-ratio=1.2).

[0186] Nanoprecipitation with hydrophobic v-Fe.sub.2O.sub.3 VSIONs A suspension (6.6 μL, 5% FWR relatively to polymer) of Beycostat coated 4.3±1.1 nm diameter γ-Fe.sub.2O.sub.3 VSIONs was added to a solution (200 μL) of block copolymer Pico (2 mg) in THF and nanoprecipitation occurred by fast addition of water (1.7 mL). This suspension was kept into a semi-permeable membrane tube (3.5 kDa cutoff) & dialyzed against ultrapure water for 18 h. The nanoparticles were analyzed by DLS: Z-Avg=129 nm, PDI=0.273. Polymersome morphology with iron oxide nanoparticles buried in the hydrophobic membranes were observed by TEM and cryo-TEM.

[0187] Nanoprecipitation with hydrophobic v-Fe.sub.2O.sub.3 NPs in saline solution A suspension (15 μL, 10% FWR relatively to polymer) of Beycostat coated 4.3±1.1 nm diameter γ-Fe.sub.2O.sub.3 VSIONs was added to a solution (220 μL) of block copolymer Pico (2.2 mg) in THF, then 0.9% aq. NaCl (2 mL) solution was added fast into it. This suspension was dialyzed (3.5 kDa cutoff) against 0.9% aq. NaCl for 18 h. DLS analysis led to Z-Avg.=142 nm, PDI=0.201. Polymersome formation was ascertained by MALS (ALV ρ-ratio=1.0) and iron oxide nanoparticles buried in their hydrophobic membrane were observed by TEM.

Nanoprecipitations of Quinolinium Block Co Polymers:

[0188] Block copolymer Quino (1 mg) dissolved in THF (100 μL) and (900 μL) of 0.9% aq. NaCl was added promptly into it for nanoprecipitation. This solution was put in a dialysis bag (15 kDa cutoff) and dialyzed against 0.9% aq. NaCl for 24 h. DLS measurement led to Z-Avg=88 nm, PDI=0.10. TEM pictures suggested the formation of polymersomes. 900 μL Dl water was added promptly into the solution of Block copolymer Qino (1 mg) in THF (100 μL) for nanoprecipitation. This solution was put in dialysis bag (15 kDa cutoff) and dialyzed against Dl water for 18 h. Hydrodynamic size was measured by DLS: Z-Avg=73 nm, PDI=0.231.

[0189] Block copolymer Quino (1 mg/mL) dissolved in THF (100 μL) was added into (900 μL) DI water for nanoprecipitation. This solution was put in dialysis bag (15 kDa cutoff) and dialyzed against ultrapure water for 18 h. DLS measurement led to Z-Avg=59 nm, PDI=0.138. The TEM picture showed small vesicles that might rather be micelles, showing that the addition order is important (with Quino copolymer; vesicles are more likely formed when the aqueous solution is poured into organic polymer solution rather than with the reverse addition order).

[0190] Nanoprecipitation in saline Block copolymer Quino (1.5 mg) was dissolved in THF (100 μL) and (900 μL) of 0.9% aq. NaCl was added quickly into it for nanoprecipitation. This suspension was put in a dialysis bag (15 kDa cutoff) and was dialyzed against 0.9% aq. NaCl for 24 h. DLS measurement yielded a hydrodynamic diameter of Z-Avg=107 nm, PDI=0.130. The morphology was characteristic of polymersomes according to TEM and MALS (ALV ρ-ratio=1.0).

Nanoprecipitation with Hydrophobic v-Fe.sub.2O.sub.3 NPs in Saline

[0191] A suspension (23.4 μL, 5% FWR relatively to polymer) of Beycostat-coated 4.3±1.1 nm diameter γ-Fe.sub.2O.sub.3 VSIONs was added to a solution (100 μL) of block copolymer Quino (1.5 mg) in THF. Then 0.9% aq. NaCl (0.9 mL) was added quickly to the solution. The suspension was dialyzed (3.5 kDa cutoff) against saline for 18 h. Hydrodynamic diameter measured by DLS was Z-Avg=130 nm, PDI=0.120. Morphology according to TEM images consisted of polymersomes loaded with iron oxide NPs in their membrane.

Characterization

GPC

[0192] Gel permeation chromatography (GPC) also called size exclusion chromatography (SEC) was performed on an Ultimate 3000 system from Thermo Fisher scientific equipped with a differential refractive index detector (dRI) detector from Wyatt Technology corporation (Santa Barbara Calif., USA), using DMF with LiBr (1 g/L) as the eluent at a flowrate of 0.8 mL/min. The column set consisted of two KD-803 Shodex™ gel columns (300×8 mm) (exclusion limits 1-50 kDa), maintained at 50° C. Weight-average molar massed (M.sub.W) and mass dispersities (D) were calculated with ASTRA 7.1.0 software from the chromatograms calibrated using a series of polystyrene standards.

DLS

[0193] The hydrodynamic radii and size distribution of the self-assembled copolymers were measured at by dynamic light scattering (DLS) on a Zetasizer™, nano ZS instrument (Malvern company, UK) equipped with a 633 nm helium-neon laser using back-scattering detection at fixed 173° scattering angle on samples equilibrated at 25° C. The measured hydrodynamic sizes of the vesicles as well as the PDI values were obtained by the classical 2.sup.nd order Cumulant fitting procedure of the scattered intensity correlograms (T=25° C., 13 runs of 10-15 s duration times, measurements in triplicates to derive a standard deviation of all values), while the derived countrate in kcps (mean scattered intensity normalized by the attenuator transmission factor) was used as an indication of the concentration of scattering objects (at a constant aggregation number or size of the scattering objects).

SLS

[0194] Multi-angle light scattering measurements (MALS) were performed using an ALV/CG6-8F goniometer, with a 35-mW red helium-neon linearly polarized laser (λ=632.8 nm) and an ALV/LSE-5004 multiple tau digital correlator at 20° C. The data were acquired with the ALV correlator software, the counting time was typically 15 s at each different scattering angles ranging from 30° to 150°, in 5 increments. The radius of gyration (R.sub.g) was determined from a Guinier plot resulting from the measurement of the average scattered intensity at the same angles. In addition, the hydrodynamic diameter (R.sub.h) was obtained by multi-angle measurement of the main decay rate F (by 2.sup.nd order Cumulant fitting of the correlogram) at each angle and plot of F as a function of the square of the scattering vector q (defined by the classical formula as a function of the scattering angle, refraction index of water and wavelength of light), in order to derive the translation diffusion constant of the objects and convert it into a hydrodynamic radius using the Stokes-Einstein formula.

[0195] TEM Transmission electron microscopy (TEM) images were recorded using a FEI Tecnai microscope working at 120 kV equipped with an OSIS Megaview II megapixel camera. TEM samples were prepared by depositing 5 μL of the sample solution (1 mg/mL) onto an Agar Scientific Formvar™/carbon 200 mesh copper grid for 1 min, blotted to remove the excess and followed by staining with a 1.5% uranyl acetate solution for 30 s.

[0196] 3. Drug Encapsulation and Release

Preparation of Drug-Loaded Vesicles

[0197] A solution of doxorubicin hydrochloride (Dox-HCl) in 0.9% of aq. NaCl solution (1 mM) was prepared. It was added rapidly (900 μL) to a copolymer (Quino, 2.1 mg) solution in THF. Solvent and excess of doxorubicin was removed by dialysis against 0.9% of aq. NaCl solution (for 2 mL of solution, 2×3 L brine exchange, using a 15 kDa cutoff dialysis membrane). DLS measurements yielded Z-Avg=93 nm, PDI=0.23.

Nanoprecipitation with Hydrophobic v-Fe.sub.2O.sub.3 NPs

[0198] A suspension (117 μL, 20% feed weight ratio (FWR) iron oxide relatively to polymer, e.g. 0.42 mg) of Beycostat™-coated 4.3±1.1 nm diameter γ-Fe.sub.2O.sub.3 VSIONs was added to a solution (100 μL) of block copolymer Quino (2.1 mg) in THF and a solution of Dox-HCl in 0.9% of aq. NaCl solution (1 mM, 783 μL) was added quickly into it. The suspension was dialyzed (15 kDa cutoff) against 0.9% of aq. NaCl solution for 24 h. DLS measurements led to Z-Avg=141 nm, PDI=0.25. Morphology characteristic of polymersomes was observed by TEM.

Fluorimeter Measurements

[0199] Release of the encapsulated compound: The amount of released dye was followed at RT by fluorescence spectroscopy (Hitachi F7000 spectrometer). Chemically reduced or irradiated samples were placed in low volume Quartz cuvettes (50 μL) and directly analyzed. The λ.sub.ex excitation wavelength was set at 480 nm and the emission λ.sub.em spectra were recorded from 490 to 720 nm. The instrument was used in scan mode, with excitation slit set to 5 nm and emission slit set to 5 nm.

In Vitro Cytotoxicity Studies

[0200] The cytotoxicity of Pico-Ps and Pico-Ps(Fe) as well as of Quino-Ps and Quino-Ps (Fe) were evaluated on cancer and healthy cells by the colorimetric MTT assay using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide as reagent to test the mitochondrial activity of living cells. The cytotoxicity of Pico-Ps and Pico-Ps(Fe) was determined on BWGT3 tumoral hepatocytes and TIB75 healthy hepatocytes chosen as reference cells. Cells were seeded at 2×10.sup.4 cells per well in 100 μL of culture medium and were incubated for 24 h at 37° C. in 5% CO.sub.2-humified atmosphere. Pico-Ps(Fe) and Pico-Ps polymersomes were incubated for 1 h in serum-free RPMI-1640 culture medium at various concentrations (3 wells per condition, repeated twice). Cells were washed with phosphate buffered saline (PBS) and incubated during 1 h in full culture medium. Absorbance at 480 nm was measured in each well. The viability was calculated by reference to the cells without addition of polymersomes. The half maximum inhibitory concentration leading to 50% cell-viability (IC50) was then calculated by nonlinear regression analysis using GraphPad Prism 6 software (La Jolla, Calif., USA).

[0201] Five different concentrations (0, 50, 100, 300, and 500 μg/mL) of Pico-Ps(Fe) and Pico-Ps were prepared by dilution of aliquot samples with cell media, respectively. Pico-Ps(Fe) appeared invariably (but only slightly) more toxic than Pico-Ps after 24 h of incubation with TIB75 cells. In contrast, cell-cultures showed almost negligible cytotoxicity of polymersomes on BWTG3 cells even at higher concentrations (up to 500 μg/mL).

[0202] Also, the cytotoxicity of quinolinium-derived Ps, Quino-Ps and Quino-Ps (Fe) was studied on epithelial cell line BNL 1ME A.7R.1 (American Type Culture Collection TIB-75). Cells were cultured in 25 mL of Dulbecco's Modified Eagle Medium (DMEM), with a high glucose content (4.5 g/L, GlutaMAX), and were supplemented with 10% fetal calf serum (FCS) and 1% penicillin and streptomycin. Cells were placed in 75 cm.sup.2 flasks incubated at 37° C. with 5% CO.sub.2 and sub-cultured by Trypsin-0.05% EDTA (4 mL). Cells were plated at a cell density of 2.0×10.sup.5 cells per well in a 96-well (100 μL/well) plate and incubated for 24 h at 37° C. under 5% CO.sub.2 atmosphere before treatment. After incubation, buffer was replaced by freshly prepared brine (0.9% v/w) and 100 μL of Quino-Ps and Quino-Ps (Fe) were deposited, respectively in three wells, in a triplicated analysis. Cells were incubated further at 37° C. under 5% CO.sub.2 atmosphere. After incubation with the polymersomes, the medium was removed and replaced by 100 μL of the MTT reagent (0.5 mg/mL prepared in the culture medium). After 4 h incubation at 37° C. the reagent was carefully removed and 100 μL of DMSO was added to each well in order to dissolve the colored formazan product. Then the plates were shaken on an orbital shaker for 10-15 minute for dissolving the formazan, and the absorbance was measured by using microplate reader (Infinite®200 PRO series Tecan) at 562 nm (three replicates were read for each samples). The relative cell viability (%, n=3) was calculated (for both Quino-Ps and Quino-Ps (Fe)) as the percentage of living cells of the treated wells compared to the untreated cells. As before, cell-cultures showed almost negligible cytotoxicity at incubated concentration up to 500 μg/mL for the quinolinium probe based polymersomes, with or without VSION loading.

[0203] In another series of experiments, the cytotoxicity of combretastatin and doxorubicin-filled Quino-Ps were compared on MCF-7 human breast cancer and Ovar-3 carcinoma cell lines (FIG. 1). As it can be seen, the encapsulated drugs preserve some of their original biological activity/toxicity, although exhibiting 100-1000 times larger values of IC50 of these two APIs compared to the reference (non-encapsulated) compounds. As no leaching of the encapsulated product was evidenced under similar conditions, the reason of the residual biological activity can be the adsorption of a small fraction of the active compound at the surface of the polymersomes in addition to the main part encapsulated inside their lumen. In both cases, these results demonstrate the efficiency of encapsulation in polymersomes to protect healthy tissues as long as they are not subjected to the external or endogenous signal triggering API release.

In Vivo Biodistribution of the Prepared (Un)Loaded Polymersomes

[0204] Proton relaxivities of polymersome Pico-Ps(Fe) were measured in saline (9% v/w NaCl). Samples were concentrated to reach 1.0 mM.sub.Fe in equivalent iron concentrations (i.e. 0.08 g/L iron oxide) and a series of dilutions were prepared (c.sub.Fe=1.0, 0.5, 0.2, 0.1, and 0.02 mM). The longitudinal relaxation time (T.sub.1) of water protons was measured by saturation-recovery spin-echo sequence (RARE images; echo time TE=13 ms; 10 repetition times TR=15 s, 8 s, 3 s, 1.2 s, 0.800 s, 0.594 s, 0.300 s, 0.144 s, 0.050 s, 0.033 s, repetition time TR=5000 ms, 1500 ms, echo time TE=13 ms) on a 300 MHz MRI scanner (7 T, 30WB, Bruker Biospin) at 25° C., while transversal (T.sub.2) relaxation times were measured by a multi spin-echo train acquisition MSME images (TR/TE=15 s/11 ms, 32 echos; 11 to 32×11 ms). T.sub.2* relaxation times were measured by using a multi echo gradient echo (GRE) sequence (24 echoes TE=4 ms, TR=5 s).

[0205] Fields of view of 3×3 cm.sup.2, a matrix size of 128×64 and a slice with a thickness of 1.5 mm were used for T.sub.1 and T.sub.2 maps. For T.sub.2* mapping, a field of view of 3×3 cm.sup.2, a matrix of 256×192 and a slice with a thickness of 1.5 mm were used. The relaxation rates 1/T.sub.i were plotted against iron concentration, and the corresponding longitudinal and transverse relaxivities r.sub.i were obtained by linear regression 1/T.sub.i=(1/T.sub.i).sub.buffer+r.sub.i C.sub.Fe (FIG. 2). Pico-Ps(Fe) exhibits r.sub.1=0.5 s.sup.−1.Math.mM.sub.Fe.sup.−1 in deionized water and r.sub.1=0.1 s.sup.−1.Math.mM.sub.Fe.sup.−1 in physiological saline, respectively as well as r.sub.2=137 s.sup.−1.Math.mM.sub.Fe.sup.−1 in Dl water and r.sub.2=43 s.sup.−1.Math.mM.sub.Fe.sup.−1 in NaCl, respectively; r.sub.2*=119 s.sup.−1.Math.mM.sub.Fe.sup.−1 in Dl water and r.sub.2*=103 s.sup.−1.Math.mM.sub.Fe.sup.−1 in NaCl were calculated respectively. The low values of r.sub.1 are ascribed to the hydrophobic environment of the VSIONs embedded in the polymersome membranes, impeding any direct “internal sphere” relaxation mechanism of water protons. On the contrary r.sub.2 values are pretty high and close to values of commercial MRI contrast agents such as Resovist®/Cliavist® (discontinued) or Feraheme® (currently on market [Yi-Xiang J Wang, Current status of superparamagnetic iron oxide contrast agents for liver magnetic resonance imaging, World J Gastroenterol. 2015, 21, 13400-13402]) due to the “external sphere” mechanism (i.e. dipolar effect of VSION magnetization working at distance on the water protons) that is enhanced by their clustering within the membranes. Noteworthy, the (r.sub.2*/r.sub.1) values were high enough to use these magnetic polymer vesicles as negative MRI contrast agents resulting into the “darkening” of specific locations of dynamic susceptibility-enhanced (DSC-MRI) (T.sub.2*) weighted-images in tissues where they accumulate (FIG. 2).

[0206] Likewise, the longitudinal and transversal relaxations of Quino-Ps(Fe) were measured by preparing a range of 0.1, 0.2, 0.5 and 1 mM equivalent iron concentrations of suspensions and placing the 5 samples in the MRI spectrometer.

[0207] In order to evaluate the contrast agent capacity/efficacy of Quino-Ps(Fe) polymersomes, a sample was compared to a commercial iron oxide contrast agent (Cliavist) chosen as reliable T.sub.2 contrast reference. The measured relaxivity (r.sub.1, r.sub.2) values of Quino-Ps(Fe) vs. Cliavist® (same as Resovist®) are summarized in Table 1.

TABLE-US-00002 TABLE 1 Comparison between the relaxivity values r.sub.1 and r.sub.2 of iron-based contrast agents, Cliavist ® (Resovist ®) taken as gold standard, and Quino-Ps(Fe) polymersomes. R.sup.2 are the coefficients of determination of the linear fits. Relaxivity Cliavist (mM.sup.−1s.sup.−1) (Resovist) Quino-Ps(Fe) r.sub.1 2.206 0.38 (R.sup.2) (0.99) (0.99) r.sub.2 142.4 108 (R.sup.2) (0.98) (0.90) r.sub.2/r.sub.1 67 284

[0208] Although the longitudinal and transverse relaxivity values r.sub.1 and r.sub.2 of Quino-Ps(Fe) were somewhat weaker than that of the Cliavist®/Resovist® due to the very small core size of VSIONs, the combination of their local hydrophobic environment and clustered state in the membranes lead to a Quino-Ps(Fe) sample that is expected to show superior T.sub.2 MRI contrast agent proprieties in vivo as the r.sub.2/r.sub.1 ratio is as high as 284.

MRI Biodistribution Study on Animal Model

[0209] For recording the dynamic bio-distribution of Pico-Ps(Fe) in mice under in vivo conditions, a specific protocol, developed by the MR imaging facility, was applied (G. Ramniceanu, B-T. Doan et al, Delayed hepatic uptake of multi-phosphonic acid poly(ethylene glycol) coated iron oxide measured by real-time Magnetic Resonance Imaging RSC Advances 2016, 6, 63788-63800). After anesthesia by using 1.5% isoflurane gas in air/O.sub.2 mixture (0.5 L/min and 0.2 L/min, respectively), a 30 G catheter was introduced in the caudal vein, and the mouse was installed in a whole-body 40 mm inner diameter RF birdcage coil (Bruker) with a tube of water as a phantom signal reference. A Dynamic Susceptibility Contrast (DSC) imaging methodology was used for the visualization/capture of Pico-Ps(Fe) magnetic polymersomes by the organs. IntraGateFLASH images were recorded to suppress breath motion artefacts in a kinetic way with 3 min duration and with adapted time resolution according to the uptake and clearance of the Pico-Ps(Fe) up to 11 days: TR/TE=90 ms/3 ms, 52 repetitions. Field of view of 3×3 cm.sup.2, a matrix size of 256×256, for a 117 μm in-plane resolution, and 5 slices distant of 3 mm with a thickness of 1 mm were used. After 3 reference image acquisitions, 100 μL of Pico-Ps(Fe) (c=1.42 mM of Fe in 0.108 M NaCl 0.9% saline) were injected. The biodistribution and clearance were monitored after injection with 3 min time resolution for 1 h, 4 h, 6 h than daily up to 11 days (FIG. 3).

Biodistribution Study of Qui No-Ps(Fe) in Mice by MRI

[0210] The study of the biodistribution of Quino-Ps(Fe) was performed on Balb/c wild type female mice (n=6), aged between 8 to 10 weeks on average. The system was equipped with a temperature-controller set-up, a pneumatic pressure sensor for breathing control, and a face-mask allowing a volatile anesthesia for in vivo imaging of small animals. Mice were anesthetized with 4% isoflurane and kept asleep in the restraining bed during the experiment at a dose of 1.5% and under an oxygen-air mixture (30:70, 0.2 L min.sup.−1). The respiration was monitored throughout the experiments using a plastic sensor positioned at the chest of the mice and a physiological monitor to monitor the respiration. In parallel, a 5 mm saline tube was introduced to each mouse as a reference “phantom” for the quantification of the MRI signal. The mouse was positioned in a radiofrequency coil and inserted into the MRI spectrometer. A bolus of 200 μL of Quino-Ps(Fe) (c=2.5 mM of Fe) suspension corresponding to 16.6 μmol Fe/kg dose) was injected intravenously (caudal vein) via a 0.38 mm internal and 1.09 mm external diameter catheter, adapted to a 30 G needle.

[0211] The kinetic monitoring of the biodistribution in the liver, spleen and kidneys i.e. the capture of the Quino-Ps(Fe) by the reticulo-endothelial system (RES) allowed evaluating their circulation time (FIG. 6). For the MRI analysis, a flash gradient dynamic susceptibility contrast (DSC) echo sequence was used, according to Hoa et al. (Sauramps Medica, 2008, Montpellier, France) with the Intragate (Bruker) method that detects the respiratory and cardiac cycles of the mouse during the sequence. A post-processing operation allowed the elimination of the movements and the reconstruction of images without artefacts. This low 30° pulse angle sequence made possible to use short echo (TE=3 ms) and repetition times (TR=90 ms) while maintaining a weighted T.sub.2* value. The short repetition rate thus allowed high temporal resolution (of 1 min) with good spatial resolution (117×117 μm.sup.2/pixel) in good sensitivity.

[0212] The accumulation of Quino-Ps(Fe) in the organs resulted in decreased MRI signal in T.sub.2* weighted sequence, and thus decreased proton T.sub.2* relaxation time of the surrounding water. The FLASH-Anatomy sequence set (in the Paravision 5.1 software) allowed the selection of five anatomical slices containing the liver, spleen and kidneys. This sequence used a trigger module to synchronize the acquisition with the breathing trays of the animal, and avoid motion artefacts: IntraGateFLASH sequences of the DSC imaging method were applied to these five slices for 3 min. The study was followed for 1 h (1 scan FLASH IG/10 mn), thus points were acquired after 3 h, 6 h and were more spaced for two weeks. In vivo polymersome tracking was performed by 7T MRI bio-imaging. To follow the kinetics of the capture and clearance of the Quino-Ps(Fe) in the target organs, regions of interest were manually designed on the MRI images (FIG. 6). By this way it was possible to quantify the retention, remanence and elimination in various organs, characteristics for the biodistribution of the Quino-Ps(Fe) imaging agent in vivo (G. Ramniceanu, B-T. Doan et al RSC Advances 2016, 6, 63788-63800).

[0213] Before injection of the bolus of Quino-Ps(Fe) (20% FWR iron oxide), the liver appears as an “iso-signal” on the IntraGateFLASH images with respect to the arteries (FIG. 4). During the hepatic capture of the contrast product, one can notice a hyposignal on the liver MRI sections. Looking at the kidneys (i), the MRI contrast seems visually invariable (weak build-up). Considering the spleen (ii), the picture shows significant hyposignal that persists up to 2 weeks after the injection of the nanoparticles.

[0214] As demonstrated by the results reported above, the prepared magnetic polymersomes fulfill the stringent requirements of biocompatibility and stealth properties, parameters that influence diffusion properties and stability under physiological conditions as well as some toxicity issues.

Activation of Polymersomes by Local Electron-Transfer (ET) Using Different Trigger Signals (i.e. Reducing Agents)

[0215] The liberation of doxorubicin was followed by monitoring the fluorescence. Fluorescence spectra were collected by excitation at λ.sub.ex=470 nm wavelength and reading at λ.sub.em=600 nm. In order to quantify the encapsulated drug, a method for the quantitative liberation was needed. Different methods, such as solvolysis, photolysis, addition of redox agents (sodium dithionite, mercaptoethanol, sodium thiosulfate, glutathione) were tested and are discussed as follows.

Glutathione (GSH)-Triggered Drug Release from Quino-Ps-DOX

[0216] In parallel, Quino-Ps-DOX (50 μL) was treated with the natural enzyme glutathione (GSH) in its reduced form, 1 μL at c=10 mM in brine (0.9% (v/w). The concentration of the GSH was set to 10 mM in order to be compatible with the highest intracellular level (FIG. 5).

[0217] In the control (blank) experiment, doxorubicin (Dox) solution (50 μL c=1.0 mmol in brine 0.9% (v/w)) was treated with the GSH solution (reduced form, 1.0 μL c=10 mM in brine 0.9% (v/w)): no change in the fluorescence intensity of doxorubicin was observed, indicating that the observed fluorescence variation is not the consequence of a side reaction of GSH with Dox, and also that Dox appears stable under the experimental conditions.

[0218] The amount of the liberated drug was quantified by fluorescence (λ.sub.ex=470 nm and λ.sub.em=600 nm), by using a calibration curve (FIG. 6) (y=1.79×10.sup.5 (R.sup.2=0.9911)). According to this calibration curve, the cumulative liberation of Dox (after “30 min”) was approximately 58% by considering that initially 1.0 mM concentration (approx. 544 μg) of drug was used for encapsulation.

[0219] Using the calibration curve, the amount of the released drug was calculated as 321 μg. Based on the released amount of drug, a drug loading content (rate of Dox weight over copolymer carrier weight) of 42% and a drug entrapment efficiency (rate of Dox encapsulated over Dox initially added at preparation) of 16% were obtained.

[0220] Samples were activated by X/gamma rays as well. Fast fragmentation was observed by using beam of the ID17 biomedical beamline at ESRF synchrotron (Grenoble, France) (conditions: Cuvette: quartz, d (light path): 1 mm, Beam: 120 keV, Bunch length: 48 ps, Bunch repetition rate 5.68 MHz, Dose: 30 Gy). For gamma rays, a conventional medical irradiator in a hospital was used.

Stability on Storage:

[0221] Samples were stored at 4° C. An aliquot of each sample (1.0 mL) was analyzed by DLS over 3-4 months repeatedly. Both Quino-Ps (FIG. 8A) and Quino-Ps(Fe) (FIG. 8B) samples appeared stable under the storage conditions (at 4° C.) for 4 months, although a slight Zavg increase with small variation in PDI was observed. Same long term shelf-stability was observed for Pico-Ps and Pico-Ps(Fe) as summarized in Table 2.

TABLE-US-00003 TABLE 2 Derived Count Z-Ave PDI Measurement T Rate diameter Width Sample Name Date and Time ° C. (kcps) (nm) (nm) PDI NP5_Pico-Ps 9 Sep. 2016 15:48 25 6064.5 120.1 41.3 0.118 Reproduced NP5_Pico-Ps 9 Sep. 2016 11:55 25 5715.1 118.4 40.4 0.116 Reproduced (Fitrated 0.2 μm) NP5 Pico-Ps 19 Oct. 2017 15:08 25 5346.3 114.4 38.4 0.113 (Sterile, in NaCl 9 g/L) NP5 Pico-Ps 27 Nov. 2017 19:18 25 5323 116.8 41.8 0.128 (NaCl 9 g/L) (Synchrotron irradiation) NP6 Pico-Ps 4 Nov. 2016 11:13 26.2 7985.5 145.3 85.6 0.347 NP6 Pico-Ps 4 Nov. 2016 12:16 25 4333.5 105.1 43.2 0.169 (Filtrated 0.2 μm) INP7 Pico-Ps(Fe) 4 Nov. 2016 11:26 25 18581.3 155 68.7 0.197 INP7 Pico-Ps(Fe) 4 Nov. 2016 12:25 25 15157.9 135.5 48.4 0.128 (Filtrated 0.2 μm) INP7_Pico-Ps(Fe) 28 Nov. 2016 18:51 25 16244.7 146.9 57.4 0.153 (Conventional irradiation) INP7_Pico-Ps(Fe) 8 Dec. 2016 17:19 25 13346.4 139 55.1 0.157 (Synchrotron irradiation) INP7 Pico-Ps(Fe) 19 Oct .2017 14:50 25 13887.5 143 58.7 0.168 1.42 mM Fe NaCl 9 g/L INP7 Pico-Ps(Fe) 27 Nov. 2017 19:50 25 14744 143.8 53.4 0.138 1.42 mM Fe NaCl 9 g/L (2.sup.nd synchrotron irradiation) Long term stability study of Pico-Ps and Pico-Ps(Fe) samples as assessed by DLS up to one year. Z-Ave diameter and PDI correspond to 2.sup.nd order Cumulant analysis of the correlograms, and PDI width is the characteristic broadness of the size distribution calculated by multiplying Z-Ave by the square root of the PDI value. The derived countrate (DCR) is the normalized scattered intensity, which gives indication on the state of aggregation (it increases when colloidal particles are clustered together, or decreases if they are degraded or settle at the bottom of the vial).

Non-Covalent Modification of the Polymersome Bi-Layer by Plasmonic Gold Nanoparticles (GPNP6P) for Fluorescence Tracking, Cancer Diagnosis and Photothermal Therapy.

[0222] Gold nanoparticles (GNPs) were prepared according to the method disclosed by Y. Chen et al Nano Lett. 2017, 17, 6330-6334 (DOI: 10.1021/acs.nanolett.7b03070) and were mixed with the picolinium-derived block co-polymer described above in THF. The nano-precipitation was realized by following the above nanoprecipitation procedure and the samples were analyzed by UV-Vis spectrophotometer. Results are illustrated in FIG. 12) and by DLS (Table 3 below). The hydrolytic stability of the prepared GNP-modified NPs (brine 0.9% w/v), stored at 4° C. was followed by DLS (FIG. 13).

TABLE-US-00004 TABLE 3 The analysis of GNP-modified picolinium NPs by DLS. Cone. of the block Zeta co polymer Hydrodynamic Concentration of potential (mg/mL) size (nm) PDI GNPs (mg/mL) (mV) 1.0 134 0.12 0.125 −3.44

[0223] Further results in respect of GNP6Ps are illustrated in FIGS. 9-11.

[0224] FIG. 9 shows that in the SWIR domain (ex: 808 nm, em: LP 1064 nm), PICO-GNP6Ps fluorescence detection was linear from dilution 1 to 1/256. At the highest dilution prepared (1/256), the detection limit was not reached yet (signal/background ratio=2.65). Further, the solution is well detectable by SWIR fluorescence imaging and will allow efficient detection in vivo in mice. PICO-GNP6Ps were well detectable by SWIR fluorescence imaging with enough sensitivity to consider detection in vivo in mice.

[0225] Pharmacokinetics of PICO-GNP6Ps was carried out in vivo in mice (FIG. 10): It was found that following intravenous injection, PICO-GNP6Ps display a short circulation time in the bloodstream: As soon as 1 h post injection there is almost no more signal circulating in the bloodstream, with a half-life (Diffusion+elimination) of 2.31 min and a pure elimination half-life of 18.48 min.

[0226] The intravenous injection of 200 μL of PICO-GN6Ps in mice led to: [0227] No visible signs of discomfort during injection in healthy mice, [0228] No visible signs of pain or suffering during the awakening phase of the mice, [0229] Normal behavior of the mice up to 48 h post injection.

[0230] As illustrated in FIG. 11, SWIR fluorescence imaging over 48 hours shows a strong liver signal as early as 1 hour. This signal decreases along time but remains significant at 48 h post injection. Some fluorescence signal is also observed in the spleen but no further uptake is identified in vivo by noninvasive fluorescence imaging.