HYPERBRANCHED POLYGLYCEROL SULFATES WITH HYDROPHOBIC CORES

Abstract

The present invention relates to the field of drug carrier systems and polymer therapeutics and diagnostics. It provides derivatized unimolecular polyols with a hydrophobic core moiety and a polyanionic, preferably, sulfated hydrophilic shell, N in particular to unimolecular hyperbranched sulfated polyglycerol micelles having a hydrophilic shell and a hydophrobic core. These may be used for the supramolecular encapsulation and transport of hydrophobic guest molecules into biological systems. The micelles can be applied as a drug carrier system for therapy and for diagnosis. They may also be therapeutic agents on their own. The invention also relates to preparation of said micelles.

Claims

1. A unimolecular sulfated polyglycerol micelle having a hydrophilic shell and a hydrophobic core.

2. The micelle of claim 1 having the formula P(OSO.sub.3.sup.M.sup.+).sub.n, wherein P is polymeric polyglycerol wherein a number n of hydroxyl groups is substituted by sulfate groups OSO.sub.3.sup.M.sup.+, wherein M.sup.+ is a cationic inorganic or organic counter ion to the anionic sulfate group.

3. The micelle of claim 2, wherein P is a polymeric polyglycerol comprising repeated units of glycerol with the formula (ROCH.sub.2).sub.2CHOR on a multifunctional starter molecule which is a polyhydroxy compound having 1 to 1,000 hydroxyl groups, wherein R independently is H or a further glycerol unit.

4. The micelle of claim 1, wherein the degree of sulfation of the hydroxyl groups of the polyglycerol is 30% to 100%.

5. The micelle of claim 1, wherein the hydrophobic core comprises mono- and/or bi-aromatic polyether moieties, wherein the aromatic moiety is selected from the group comprising phenyl, functionalized phenyl, naphthyl, functionalized naphthyl, biphenyl, and functionalized biphenyl derivatives.

6. The micelle of claim 1, wherein the hydrophobic core comprises bi-aromatic moiety.

7. The micelle of claim 1, wherein the hydrophobic core component has a molecular weight of 250 to 100,000 g/mol, and/or wherein the hydrophilic shell component has a molecular weight of 1000 to 1,000,000 g/mol.

8. The micelle of claim 1, wherein the core and/or the shell has an average degree of branching of 0 to 67 %.

9. The micelle of claim 1, supramolecularly encapsulating a guest molecule.

10. The micelle of claim 9, wherein the guest molecule is a therapeutic effector molecule or a diagnostic effector molecule.

11. A pharmaceutical composition comprising the micelle of claim 1, and a biologically acceptable carrier.

12. A method of treating a patient having a condition or disease selected from the group comprising inflammation, arthritis, otitis media, otitis externa, cancer, autoimmune disease, fibrosis, cartilage defect, osteonecrosis, osteochondritis, cardiovascular disease or sepsis, any disease amenable to therapy by inhibition of NG-kappaB and/or AP-1 and/or TGF-beta synthesis, comprising administering the patient the micelle of claim 1.

13. A method of diagnosing a disease or condition in a patient, comprising administering to the patient the micelle of claim 1.

14. A method of delivering a guest molecule to a group of cells selected from the groups of active cells, proliferating cells, osteochondral cells and cartilage cells, comprising administering the pharmaceutical composition of claim 11 to a biological system comprising said cells, wherein the micelle supramolecularly encapsulates a guest molecule.

15. A method for preparing the micelle of claim 1, comprising steps of a) preparing the micelle's core-shell structure, and b) sulfating the micelle; c) optionally, loading a guest molecule into the micelle by solid uptake or by an amorphous solid film

16. The micelle of claim 2, wherein n is 10 or more

17. The micelle of claim 1, wherein the degree of sulfation of the hydroxyl groups of the polyglycerol is 60-99%.

18. The micelle of claim 1, wherein the hydrophobic core comprises biphenyl, naphthyl or derivatives thereof.

19. The micelle of claim 8, wherein the micelle comprises a highly branched sulfated polyglycerol.

20. The micelle of claim 9, wherein the guest molecule comprisers a hydrophobic guest molecule.

Description

LEGENDS

[0051] FIG. 1. Hydrodynamic particle size by intensity of sulfated copolymers with 1 mg mL.sup.1 in 150 mM PBS.

[0052] FIG. 2. Structures of pyrene (4) and ICC dye (5) used for encapsulation studies and cellular transport studies.

[0053] FIGS. 3. TC of 4 and 5 in sulfated block copolymers b2S and b3S determined by UV measurement in MeOH based on =45 300 cm.sup.1 M.sup.1 for pyrene and =132 130 cm.sup.1 M.sup.1 for the ICC dye.

[0054] FIG. 4: Mean fluorescence at 590 nm (Cy3 dye detection) is displayed for A2780 (4 h: light gray, 24 h: dark gray columns) and A549 (4 h: black, 24 h: white columns) cells incubated with copolymers b2S and b3S, free ICC dye, and PBS control.

[0055] FIG. 5: Confocal laser scanning fluorescence microscopy images from QGP-1 cells after incubation with (a) b2S+ICC dye, (b) b3S+ICC dye, (c) free ICC dye, and (d) control PBS for 4 h. The merged pictures (a-d) show the ICC dye in red, stained nuclei in blue (DAPI), and the actin cytoskeleton (Alexa Fluor 488 Phalloidin label) in green; (e-h) present ICC dye channel for (e) b2S+ICC dye, (f) b3S+ICC dye, (g) free ICC dye, and (h) control PBS visualized as intensity grey values.

[0056] FIG. 6: Scheme 1. Synthesis of core-shell type, block copolymers b1 to b3 including steps: (i) KOtBu, NMP, 120 C., 1 h; (ii) comonomer 1, 2,3 in THF/NMP, 120 C., addition over 18 h; (iii) glycidol in THF, 120 C., addition over 18 h.

[0057] FIG. 7: Scheme 2. General mechanism for the synthesis of statistical, unsulfated copolymers as precursor for PGS rl to r3 including steps: (i) KOtBu, NMP, 120 C., 1 h, and (ii) glycidol/comonomer 1, 2, 3 mixture in THF/NMP, 120 C., addition over 20 h. The scheme presents the synthesis of the block copolymer precursor for PGS r.1.

[0058] FIG. 8: Scheme 3. Sulfation of hydroxyl terminated polymer architectures: (i) SO.sub.3-complex, e.g., NH.sub.3SO.sub.3, NMP, 90 C., 3 d; (ii) neutralization with NaOH to pH 10-12.

[0059] FIG. 9: Structure of DOX (6) used for encapsulation/complexation studies.

[0060] FIG. 10: Core-shell structures of the invention. Potential linkers are shown.

EXAMPLES

[0061] Materials

[0062] All chemicals were purchased from Acros Organics and used as received if not otherwise indicated. Glycidol was dried over CaH.sub.2 and distilled under reduced pressure. The purified monomer was stored at 4 C. under inert atmosphere and only used up to two months. All solvents were purchased from Sigma-Aldrich and used without further purification. Dry tetrahydrofuran (THF) was obtained from a solvent purification system. Dry N-methyl-2-pyrrolidone (NMP) and potassium tert-butoxide (KOtBu) were used and stored under AcroSeal conditions and used as received. Monomers 2 and 3 were synthesized according to published procedures. Indocarbocyanine dye (ICC) was obtained from mivenion GmbH.

[0063] Block Copolymerization

[0064] The batch reactor was extensively dried at 140 C. under reduced pressure. After cooling to 65 C., 1,3,5-Trihydroxybenzene (THB), KOtBu, and dry NMP were added under inert conditions. The temperature was increased to 120 C. whereas butanol was distilled and the initiator homogeneously dissolved. The glycidol derivative 1, 2 or 3 (see Table 1) was mixed with THF/NMP, and slowly added to the reactor over a period of 18 h using a precision dosing pump. Subsequently, a solution of glycidol in THF was added to the reactor over 18 h and additionally stirred for another 2 h. After cooling to ambient temperature, the polymer mixture was diluted with methanol and cation exchange resin (Dowex Monosphere 650C UPW, Supelco, Belefonte, USA) was added. After stirring for 3 d, the turbid solution was filtered and remaining solvent was evaporated under reduced pressure. Purification of a partial batch amount was performed by dialysis in saturated aqueous NaC1 solution. .sup.1H NMR spectra are available in FIG. 51.

[0065] PPhGE-block-hPG: M.sub.w=4.3 kDa, PDI=2.78; M.sub.w, Aggregate=126 kDa, PDI =1.91; .sup.1H NMR (700 MHz, DMSO-d.sub.6, ): 7.5-6.5 (m, 5H, Ar H), 4.9-4.2 (m, 1H, OH), 4.0-3.0 (m, 5H, hPG backbone).

[0066] PNpGE-block-hPG:M.sub.w=1.6 kDa, PDI=3.55; M.sub.w,Aggregate=130 kDa, PDI=1.75; .sup.1H NMR (700 MHz, DMSO-d.sub.6, ): 8.5-7.2 (m, 7H, Ar H), 4.9-4.2 (m, 1H, OH), 4.2-3.0 (m, 5H, hPG backbone).

[0067] PbPhGE-block-hPG: M.sub.w=2.4 kDa, PDI=1.23; M.sub.w, Aggregate=105 kDa, PDI=1.81; .sup.1H NMR (700 MHz, DMSO-d.sub.6, ): 7.7-6.8 (m, 9H, Ar H), 5.0-4.2 (m, 1H, OH), 4.2-3.0 (m, 5H, hPG backbone).

[0068] Statistical Copolymerization

[0069] The batch reactor was extensively dried as described. Trimethylolpropane (TMP) was melted at 65 C. under reduced pressure. KOtBu and NMP were added under inert conditions and the temperature was increased to 120 C. A defined monomer mixture of glycidol with 1, 2 or 3 (see Table 1) in THF was prepared and slowly added to the reactor over a period of 20 h whereas the THF was immediately distilled from the reaction mixture. After complete monomer addition and stirring for additional 2 h, the polymer mixture was diluted with methanol and cation exchange resin was added. After stirring for 3 d, the polymer solution was filtered and solvent was evaporated under reduced pressure. Purification of a partial batch amount was performed by dialysis in water for 3 d. .sup.1H-NMR spectra are available in FIG. S1.

[0070] PPhGE-co-hPG: M.sub.w=5.1 kDa, PDI=1.75; .sup.1H NMR (700 MHz, DMSO-d.sub.6, ): 7.4-6.8 (m, 5H, Ar H), 5.1-4.2 (m, 1H, OH), 4.2-3.0 (m, 5H, hPG backbone).

[0071] PNpGE-co-hPG: M.sub.w=5.6 kDa, PDI=1.83; .sup.1H NMR (700 MHz, DMSO-d.sub.6, ): 8.5-6.8 (m, 7H, Ar H), 5.2-4.2 (m, 1H, OH), 4.2-3.0 (m, 5H, hPG backbone).

[0072] PbPhGE-co-hPG: M.sub.w=5.8 kDa, PDI=1.87; .sup.1H NMR (700 MHz, DMSO-d.sub.6, ): 7.7-6.8 (m, 9H, Ar H), 5.0-4.2 (m, 1H, OH), 4.2-3.0 (m, 5H, hPG backbone).

TABLE-US-00001 TABLE 1 Polymerization of hydrophobically derivatized block and statistical composite copolymers including monomer feed ratios and characterization. Como- M.sub.n M.sub.n, Aggr. Initiator nomer Glyicdol I.sub.r, target [kDa] [kDa] I.sub.r, measured DS d [nm] Entry [mmol] [mmol] [mmol] [%] (PDI) (PDI).sup.a [%].sup.b [%].sup.c SD.sup.d Copolymer b1 5.00 66.6 718 9.3 4.3 126 2.7 99 88 3 PPhGE-block- (2.78) (1.91) hPGS (b1S) b2 2.13 21.2 287 7.4 1.6 130 5.3 84 143 8 PNpGE-block- (3.55) (1.75) (21 1) hPGS (b2S) b3 3.93 35.4 574 6.2 2.4 105 6.2 64 136 5 PbPhGE-block- (1.23) (1.81) (18 1) hPGS (b3S) r1 0.97 13.4 144 9.3 5.1 n.d. 6.7 96 222 10 PPhGE-co- (1.75) (8 1) hPGS (r1S) r2 0.95 10.6 143 7.4 5.6 n.d. 6.2 91 222 11 PNpGE-co- (1.83) (10 1) hPGS (r2S) r3 0.94 8.8 143 6.2 5.0 n.d. 5.7 93 237 8 PbPhGE-co- (1.87) (21 2) hPGS (r3S) .sup.aMolecular weight of amphiphilic core structure determined by GPC in organic solvent NMP relative to PEG standard. n.d. indicates not detectable. .sup.bComonomer incorporation was determined from .sup.1H NMR integrals. .sup.cDS was determined by elementary analysis. .sup.dHydrodynamic diameters of the aggregated copolymers measured after intensity; small particles included as secondary species in brackets (occurrence < 2.5%).

[0073] Sulfation of Polymer Architectures

[0074] Sulfation of the polymer architectures was performed as follows: 120 mg of each copolymer was dissolved in NMP and 1.5 to 1.8 equivalents SO.sub.3-complex per hydroxyl group were added. The samples were sonicated for 10 min and subsequently stirred at 90 C. for 3 d. After cooling to room temperature the polymer mixture was precipitated into diethyl ether and stirred for 30 min. The solvent was decanted and purification was performed by dialysis in saturated aqueous NaC1 solution for 3 d with solvent exchange twice a day. .sup.1H NMR spectra are available in Figure S2. The degree of sulfation (DS) was determined by combustion analysis (see Table 1 and Table S1).

[0075] Encapsulation of Guest Molecules

[0076] A stock solution of the guest molecules (4 and 5) with 5 mg mL.sup.-1 in THF or dichloromethane (DCM) was prepared. 200 L, of the stock solution was transferred into a small vial and the solvent was evaporated. In the meantime, a stock solution of each synthesized copolymer with 1 mg mL.sup.1 in in Dulbecco's phosphate buffered saline (DPBS, 1, Sigma-Aldrich, Germany) was prepared. After evaporation of the solvent, 3 mL of each polymer solution and 3 mL of pure PBS was added to the dried dyes. After excessive stirring for 24 h, the samples were filtered through 0.45 m Minisart RC 15 syringe filters (Sartorius Stedim Biotech, Germany).

[0077] Instrumentation

[0078] NMR spectra were recorded on a Joel ECX 400 MHz or a Bruker Avance 700 MHz spectrometer as indicated. Spectra were recorded in ppm and referenced to indicated, deuterated solvents. Molecular weight distributions were determined by means of GPC coupled to a refractive-index detector (RI) obtaining the complete distribution (M., M.sub.p, M.sub.w, dispersity). The unsulfated copolymers were measured in NMP with 0.05M LiBr (1.5 mg mL.sup.1) as mobile phase with a flow rate of 0.8 mL min.sup.1 on a GPC (Thermo Separation Products, Thermo Scientific, Waltham, USA) consisting of a solvent delivery system with pump P-100 and an auto sampler AS-100. Two 30 cm columns (PPS: Polymer Standards Service GmbH, Germany; Gram 100 , 1000 with 7 m particle size) were used to separate the samples. The columns were operated in a column oven at 70 C. The calibration was performed by using linear calibration standard (PSS GmbH, Germany). Aqueous samples (sulfated architectures) were measured under highly diluted conditions (10 mg mL.sup.1) from a GPC consisting of an Agilent 1100 solvent delivery system with pump, manual injector, and an Agilent differential refractometer. Three 30 cm columns (PPS GmbH, Germany; Suprema 100 , 1000 , 3000 with 5 and 10 m particle size) were used to separate aqueous polymer samples using water with 0.05% NaNO.sub.3 as the mobile phase at a flow rate of 1 mLmin-1. The columns were operated at ambient temperature with the RI detector at 50 C. The calibration was performed by using linear pullulan calibration standard (PPS GmbH, Germany). WinGPC Unity from PSS was used for data acquirement and interpretation. Particle size was determined by DLS in UV-transparent low-volume disposable polystyrene cuvettes on a Zetasizer Nano ZS (Malvern Instruments Ltd., UK) equipped with a 4 mW He-Ne laser (=633 nm). The samples were measured at a concentration of 1 mg mL.sup.1 in DPBS at pH 7.4. Prior to the measurement all samples were filtered through 0.45 m Minisart RC 15 syringe filters. Each sample was equilibrated for 1 min at 25 C. and measured with 10 scans for 15 s (173 back-scattering). All stated values are the mean of at least 3 independent measurements and standard deviation is indicated with SD. UV/Vis spectra were recorded on a SCINCO S-3100 UV/Vis spectrometer equipped using UV Disposable Cuvettes 220 nm-900 nm (BrandTech Scientific, Inc., Essex, USA). The transport capacities were determined as follows: 1 mL of aqueous polymer solution in PBS was freeze dried. The dried polymer-dye-complex was suspended in MeOH to extract the dye from the polymer scaffold and extensively stirred for 18 h. Subsequently the suspensions were filtered through 0.45 pm Minisart RC 15 syringe filters (Sartorius Stedim Biotech, Germany).

[0079] Cell Preparation for Fluorescence Microscopy and FACS Analysis

[0080] For confocal laser scanning microscopy, the epithelial human pancreatic cancer cell line QGP-1, the human lung carcinoma epithelial cell line A549 and the ovarian carcinoma cell line A2780 were routinely propagated as follows: DEMEM medium, with 10% fetal calf serum (FCS), 2% glutamine, and penicillin/streptomycin (all from PAN Biotech) added. Cells were seeded into medium at 110.sup.5 cells mL.sup.1, cultured at 37 C. with 5% CO.sub.2, and split 1:5 two times a week. For cytochemistry, cells were seeded at 510.sup.4 cells mL.sup.1 in a 24-well culture plate on glass coverslips (Sigma), and cultured at 37 C. for 24 h. Thereafter, cells were incubated with normal culture medium or medium containing 10.sup.6 M of dye-labeled test substances for QGP-1 cells and 5.010.sup.7 M of dye labeled test substances for A2780 and A549 cells at 37 C. for 4 or 24 h. Afterwards, cells were fixed with cold acetone, rinsed and covered with Alexa Fluor 488 Phalloidin (1:300, Molecular Probes) only for QGP-1 cells. 4,6-diamidino-2-phenylindole (DAPI, Abcam) was used for nuclear counterstain. Con-focal images were acquired at room temperature with a 631.4 NA HC PL APO CS2 oil objective suited in a confocal microscope (TCS-SP8, DMI6000 stand; Leica). Images of different groups were acquired with the same laser and detector settings using the Leica LAS AF software. The fluorescence detection was performed sequentially for each channel set with the acousto optical beam splitter between 500 and 530 nm for the Alexa Fluor 488 and between 590 and 630 nm for the ICC dye. Alexa Fluor 488 was excited using the 488-nm Argon laser line, whereas the ICC dye was excited with a 561-nm diode-pumped solid-state laser. DAPI was excited with an UV laser (405 nm) and fluorescence detection was set with the acousto optical beam splitter between and 420 and 470 nm. For FACS analysis, the three different cell lines were routinely propagated as described. The cells were cultured 410.sup.4 cells mL.sup.1 cells were cultured in 48-well-plates at 37 C. for 24 h. Thereafter, cells were incubated with normal culture medium or medium containing 10.sup.6 M test substance for 4 or 24 h. Thereafter, cells were washed with PBS, detached with 200 uL, per well accutase (PAA), and centrifuged with 350 g, at 4 C. for 5 min. Supernatants were removed and cells were suspended in 50 L PBS with 0.5% bovine serum albumin (Roth). Cells were immediately analyzed in a FACS Calibur analysis instrument (Becton-Dickinson: FL2 BP 585/42 nm).

[0081] Results and Discussion

[0082] Synthesis

[0083] The monomer to initiator ratio for all synthesized copolymers was adjusted to yield a hydrophilic PG matrix of M.sub.n=10,000 g mol.sup.1 with a hydrophobic contribution of M.sub.n=2,000 g mol.sup.1 to yield an overall targeted molecular weight of 12,000 g mol.sup.1. 1,3,5-Trihydroxybenzene (THB) was applied as an initiator for the block copolymerization to obtain a star-like hydrophobic core with an additional central benzene moiety (Scheme 1). Trimethylolpropane (TMP) was used as an initiator for the statistical copolymerization (Scheme 2).

[0084] Synthesis of block copolymers

[0085] The amphiphilic block copolymers were successfully synthesized by a one-pot, two-step procedure (see Scheme 1). After the synthesis of the hydrophobic core structure, a PG shell was added to provide sufficient solubility in aqueous media. With the presented strategy, it was possible to obtain more defined core shell structures as the monomers were added stepwise.

[0086] In comparison to post-synthetic modification protocols, where secondary hydroxyl groups were functionalized all over the scaffold, the hydrophobic moieties were exclusively located in the core in this new approach.

[0087] The amphiphilic structures formed large aggregates in buffered solution. Although the hydrophilic outer shell had about 5-fold the molecular mass of the hydrophobic core, it was not enough to prevent aggregation (see Table 2). Therefore, a unimolecular transport system could not be expected. The main species present in the particle size distribution ranged between 113 and 160 nm for the OH-terminated copolymers 1.1 to 1.3. The aggregation was not only present in DLS but also visible in the GPC measurements (see Table 1). Even the highly polar, organic solvent NMP could not prevent aggregation. Large aggregates with M.sub.n, >100 kDa were detected by the RI-detector referenced to PEG, in all block copolymers.

[0088] The amount of incorporated comonomer into the hydrophobic core determined by NMR was below the targeted content for bl and b2. Although the monomer was added slowly to the initiator, small amounts were consumed by side reactions, e.g., self-initiation, and therefore not incorporated into the core structure. The reduced values, however, can also be explained by a strong shielding of the inner core protons as well as reduced signals from aggregation. Due to heavy aggregation at higher concentrations, the NMR characterization was performed under high dilution. From experiences in NMR characterization of polymers, the concentration was too low for an adequate NMR concentration.

[0089] Synthesis of Statistical Composite Polymers

[0090] The statistical composite polymers were synthesized by simply feeding the batch reactor with a premixed monomer mixture of glycidol with monomer 1, 2, and 3. The same comonomer to glycidol ratio was applied as for the block copolymers bl to b3 (Scheme 2). Trimethylolpropane (TMP) was used as initiator.

[0091] The monomer feed was slowly added to the reaction vessel and copolymers r1 to r3 were obtained as brownish, turbid wax after work up. The molecular weights determined by GPC in NMP were in the same range but below the targeted molecular weights. No aggregate peaks were detected in the GPC measurement for the statistical copolymers, but particle sizes were determined by DLS to be between 235 and 409 nm. These sized aggregates are usually filtered on the pre-column of the GPC equipment.

[0092] The hydrophobic comonomers were incorporated into the PG matrix similarly to the block copolymers but were again below the targeted values, especially for rl. Assuming comparable reactivity to the other monomers and a homogeneous incorporation into the scaffold, the resonance signal was probably reduced by shielding and aggregation phenomena similar to the block copolymers. This effect was more pronounced because of the aggregate size determined by DLS.

[0093] Polyglycerol Sulfate Derivatives with Hydrophobic Cores

[0094] All the investigated copolymer species were sulfated to introduce an electrostatic repulsion to the polymer surface and include a targeting function. Preliminary encapsulation experiments with pyrene otherwise indicated heavy aggregation during loading, which caused the complexes to precipitate even at low concentrations. Therefore, all copolymer species were sulfated using an excess of SO.sub.3 complex, e.g., sulfamic acid (NH.sub.3SO.sub.3), in NMP as seen in Scheme 3.

[0095] Numerous SO.sub.3 complexes are known from literature to be used in sulfation of hydroxyl groups, e.g., SO3 complexes with pyridine (SO.sub.3.Math.Pyr), trimethylamine (SO.sub.3.Math.TMA), triethylamine (SO.sub.3.Math.TEA), N,N-dimethylformamide (SO.sub.3.Math.DMF), or chlorosulfuric acid and sulfamic acid. Here, the sulfation was performed in NMP using the SO.sub.3 complex NH.sub.3SO.sub.3, a modified procedure from literature. The lower reactivity than in other SO.sub.3 complexes was compensated by increasing both reaction temperature and time. The degrees of sulfation (DS) are presented in Table 1. Although the sulfation of all copolymers was conducted according to the same protocol, the DS for polymers b2S and b3S decreased. This reduction may be a result from aggregation during the reaction. Although all samples were sonicated prior to heating and extensively stirred throughout the reaction, aggregation could not be prevented during functionalization. Therefore, a certain number of hydroxyl groups were not sulfated. Its amphiphilic character and the accessibility of the sulfate groups is in line with the discussed assumption.

[0096] The sulfated copolymers were investigated by DLS to determine the particle sizes under buffered conditions prior to dye encapsulation (see FIG. 1). All architectures except b1S showed two distinct particle sizes. However, the occurrence of the peaks indicated that the polymers were mainly aggregated. The PGS copolymers showed similar hydrodynamic sizes depending on the nature of the architecture, which were either block or statistical copolymers. The block copolymers were around 1393 nm and the statistical around 2277 nm in size.

[0097] Copolymer b1S had a significant deviation from the related structures which can be explained by an insufficient amphiphilicity.

[0098] Encapsulation of Guest Molecules

[0099] The encapsulation properties of the sulfated copolymers were investigated using pyrene (4) and indocarbocyanine dye (5, ICC, Cy3) as presented in FIG. 2. Both dyes were encapsulated using the previously described film method. (Kurniasih et al., New J. Chem., 2012, 36, 371-379; Fleige, et al., in Nanocarriers, 2013, vol. 1, pp. 1-9).

[0100] The ICC dye (5) was investigated due to an applicable emission pattern with >600 nm, which has been already exploited in several cellular uptake studies with ICC labeled polyglycerol sulfates due to its tissue permeating properties. In the previously conducted assays, however, the ICC was covalently attached to the PGS scaffold which included numerous functionalization steps. The inventors wanted to investigate the potential use in therapeutic applications with the supramolecular carriers of the invention. Two of the investigated architectures showed transport capacities (TC) in the low M range. FIG. 3 presents the TC for b2S and b3S measured by UV absorbance based on extinction coefficients of =45 300 cm.sup.1 M.sup.1 for 4 and =132 130 cm.sup.1 M .sup.1 for 5.

[0101] Both TCs of b3S with 0.424 and 0.182 mg g.sup.1 copolymer, and of b2S with 0.390 and 0.144 mg g.sup.1(4 and 5), are lower compared to previously investigated architectures, but high enough for the cellular uptake studies. Kurniasih et al. Macromol. Rapid Commun., 2010, 31, 1516-1520; Fleige, et al., in Nanocarriers, 2013, vol. 1, pp. 1-9; Steinhilber et al., MRS Proceedings, 2012, 1403). Regarding the hydrodynamic sizes before and after dye encapsulation, both conjugates showed completely different loading structure relationships (see Table 2). The biphenyl derivatized structure b3S had similar particle sizes to those in the unloaded architecture. The amphiphilicity induced by hydrophobic core seems to successfully stabilize the supramolecular aggregates.

[0102] The naphthyl derivatized architecture showed a similar particle size to the unloaded structure upon loading with pyrene, but the hydrodynamic diameter increased by roughly 25% upon loading with ICC dye. In this case, neither the electrostatic repulsion nor the amphiphilic core shell structure could stabilize the aggregates. In both polymer dye complexes, the surface charge prevented precipitation. Although the degree of sulfation (DS) decreased for b2S and b3S, the lower DS combined with sufficient amphiphilic properties was able to stabilize the aggregates without a guest molecule. The achieved loading is mainly affected by the overall low hydrophobic content within the polymer architecture (1:5). A distinct structure activity relationship was determined, as both block copolymers which contain a bi-aromatic moiety b2S and b3S were able to carry a guest molecule.

[0103] No dye encapsulation was detected for the copolymers r1S to r3S. Although the same comonomer incorporation as for the block copolymers was achieved, the statistical distribution over the whole scaffold obviously reduced the hydrophobicity below the required threshold. From a previous study, the inventors were aware that hydrophobic core functionality is important for encapsulation properties and can influence the maximum loading capacity. Here, a higher core functionalization, similar to the synthesized random composite polymers r1S to r3S, resulted in a decrease of loaded guest molecule, whereas lower functionalization showed higher capacities. In general, the hydrophobic ratio is crucial for the encapsulation of guest molecules as a distinct amphiphilicity has to be introduced to the PG scaffold.

TABLE-US-00002 TABLE 2 Hydrodynamic particle sizes of unloaded and loaded core shell architectures. d Pyrene (4) ICC (5) Carrier SD.sup.a [nm] TC [mg/g] d.sub.loaded [nm] TC [mg/g] d.sub.loaded [nm] b2S 143 8 0.390 133 3 0.144 187 6 (21 1) (22 2) b3S 136 5 0.424 144 3 0.182 131 3 (18 1) (24 2) (23 2) .sup.aHydrodynamic diameters of the aggregated copolymers measured after intensity; small particles included as secondary species in brackets (occurrence < 2.5%). Standard deviations are included with SD.

[0104] Cellular Uptake of Host/Guest Complexes

[0105] The dye loaded block copolymers b2S and b3S were investigated for their cellular uptake properties into the QGP-1 pancreatic carcinoma cell line, A549 human lung carcinoma epithelial cell line and A2780 ovarian carcinoma cell line. Previous studies showed an enhanced cellular uptake of dye labeled dPGS (Licha et al., Bioconjugate Chem., 2011, 22, 2453-2460; Biffi et al., PLoS One, 2013, 8, 1-9). The cellular uptake was compared after 4 h and 24 h by utilizing fluorescence activated cell sorting (FACS) according to a standardized protocol (Reichert et al., Small, 2011, 7, 820-829). The uptake into highly proliferating A2780 and A549 cells increased with incubation time and was similar for the cross-comparison of the normalized values for both nanocarriers b2S and b3S (see FIG. 4). The amount of dye which is endocytosed into the cell is approximately 6-fold for A2780 cells after 4 h and 3-fold after 24 h compared to the free dye. The cellular uptake into A549 is even more pronounced and about 6- to 7-fold after 4 and 24 h, respectively.

[0106] The results obtained by FACS analysis show that the supramolecular PGS-dye complexes b2S and b3S are endocytosed significantly better than the free dye. This is a first indication for the transport potential of the synthesized carriers.

[0107] Further characterization of the cellular uptake and transport properties were performed by confocal laser scanning microscopy (CFM) after incubation times of 4 and 24 h into all three cell lines. The merged pictures from the uptake into QGP-1 cells (FIG. 5) indicated an increased and controlled uptake into the perinuclear region of the living cells after 4 h. In comparison, the free dye did not endocytose into QGP-1 cells. CFM pictures from the uptake into A2780 cells showed a controlled uptake of the polymer-dye complex. Compared to the free ICC dye which is randomly distributed over the whole cell nuclei, the complex is accumulated in the perinuclear region. The CFM pictures of the A549 cells present a similar uptake and transport behaviour. However, there was hardly any uptake of the free dye visible. The present study confirmed the uptake of PGS into the perinuclear region of the cell. In the previous studies, however, the ICC dye was covalently attached to the PG scaffold and therefore could not be liberated.

[0108] Complexation of Doxorubicin

[0109] A complex of the guest doxorubicin (DOX) with a micelle of the invention was prepared to combine both anti-inflammatory properties of the host molecule with the high anti-carcinogenic potential of the complexed drug. Furthermore, the host-guest complex can be applied as a depot for slow release of the drug at the target tissue, which increases bioavailability. In consequence, smaller dosages or extended intervals of drug administration are possible.

[0110] The complexation properties of the sulfated copolymers b2S and b3S which showed encapsulation of dye molecules was investigated using the water-soluble drug doxorubicin (6, DOX) as presented in FIG. 9. The free base DOX was prepared by transforming doxorubicin hydrochlorid (DOXHC1) according to a published method (Fleige, et al., in J. Controlled Release, 2014, 185, 99-108). DOX.Math.HC1 was dissolved in dimethylformamide (DMF) and stirred with 2.5 equ. TEA for 18 h. The encapsulation procedure was performed as described. In comparison, the same amount of DOX was dissolved in PBS without sulfated amphiphilic copolymer. Because of complex formation, the solutions were centrifuged for 30 min at 16 800 g to remove the polymeric drug complexes from the free DOX. The supernatant was investigated by UV/Vis spectroscopy to determine the remaining free drug in solution based on the extinction coefficient of =10 645 cm.sup.1 M .sup.1 6 (Hussain, et al., in Biomacromolecules, 2014, 14, 2510-2520). The amount of complexed DOX was recalculated from the amount dissolved in PBS without sulfated copolymer. UV/Vis measurements were conducted in dilutions of 1:2 and 1:9 because of the large UV absorbance of DOX in PBS. Both copolymers showed average complexation capacities (CC) of 134 and 129 mg g.sup.1 copolymer for b3S and 50.0 and 54.0 mg g copolymer for b2S, with respect to the dilution (see Table 3). The deviation in the capacity depending on the solution was acceptable within the instrumental measurement accuracy.

TABLE-US-00003 TABLE 3 Complexation capacities of polymer-drug complexes for core shell architectures b2S and b3S measured in dilution of 1:2 and 1:9. Absorbance [a.u].sup.a CC of DOX [mg/g] (6) Carrier 1:2 1:9 1:2 1:9 PBS 1.12 0.323 n/a b2S 0.797 0.218 50.0 54.0 b3S 0.249 0.0714 134 129 .sup.aThe absorbance of the different solutions was measured in (high) dilution of 1:2 and 1:9 with PBS. The CC was recalculated from the remaining DOX in the supernatant compared to the solution without sulfated copolymer.

[0111] The CCs showed a similar trend compared to the transport capacities determined for the dyes 4 and 5. The biphenyl derivatized copolymer b3S with a lower DS of 64% showed a 2.5-fold higher complexation compared to the naphthyl derivatized copolymer b2S with a DS of 84%. Large polymer-drug aggregates were formed in the aqueous complexation process. However, the amphiphilic core shell architectures were again able to prevent the large aggregates from precipitation, especially for b3S.